Literature review - TiHo Bibliothek elib

Tierärztliche Hochschule Hannover
Comparison between detecting Salmonella spp. by bacteriological method
and Real-Time PCR assay in samples from pig herds
INAUGURAL-DISSERTATION
Zur Erlangung des Grades eines
Doktors der Veterinärmedizin
- Doctor medicinae veterinariae (Dr. med. vet.)
vorgelegt von
Noppadol Somyanontanagul
aus Ratchaburi, Thailand
Hannover 2009
Wissenschaftliche Betreuung: Univ.-Prof. Dr. Thomas Blaha
Außenstelle für Epidemiologie
1. Gutachter: Univ.-Prof. Dr. Thomas Blaha
2. Gutachter: Univ.-Prof. Dr. Karl-Heinz Waldmann
Tag der mündlichen Prüfung: 14.05.2009
To my beloved parents
Publication
NOPPADOL SOMYANONTANAGUL,
THOMAS BLAHA (2008):
HEIKO
NATHUES,
REGINA TEGELER,
Comparison between detecting Salmonella spp. by bacteriological method and Real-Time
PCR assay in samples from pig herds.
In: Oral Proceedings of the 20 th International Pig Veterinary Society Congress, Durban, South
Africa, 2008, 176
Table of contents
Table of contents
1
Introduction
1
2
Literature review
3
2.1
2.1.1
2.1.2
2.1.3
2.1.4
2.1.5
2.1.6
2.1.7
2.1.8
2.1.9
2.1.10
2.1.11
2.1.12
A review of the Salmonella
History
The Genus Salmonella
Taxonomy and nomenclatur
Epidemiology
Porcine Salmonellosis
Treatment
Vaccine
Competitive Exclusion
Salmonella infection in pigs
Dynamics of Salmonella
Salmonella infection in humans
Foodborne outbreaks caused by Salmonella
3
3
3
5
8
9
16
18
21
22
26
28
33
2.2
2.2.1
2.2.2
2.2.3
2.2.4
Detection methods and methods for surveillance of Salmonella
Bacteriological detection methods
Serological detection methods
Molecular detection methods
Methods for surveillance
36
36
44
49
59
2.3
Prevention and Control of Salmonella in pig farm
2.3.1 Prevention and Control of Salmonella
2.3.2 Salmonella control and monitoring programmes in EU
61
61
67
3
Materials and Methods
74
3.1
Investigation on farm
74
3.2
Sample collection and preparation for analysis
3.2.1 Environmental samples
3.2.2 Faecal samples
74
74
76
3.3
81
Bacteriological methods
3.4
Real-Time PCR assay
3.4.1 DNA Extraction
3.4.2 Preparing PCR
84
84
85
3.5
87
Statistical methods
Table of contents
4
Results
89
4.1
4.1.1
4.1.2
4.1.3
4.1.4
4.1.5
Results of Bacteriological method examination
Distribution of Salmonella in faecal samples
Distribution of Salmonella in environmental samples
Proportion of Salmonella isolates from various samples
Results of Salmonella Sero-typing
Results of Salmonella Phage-typing
89
89
89
89
91
95
4.2
4.2.1
4.2.2
4.2.3
4.2.4
4.2.5
4.2.6
4.2.7
4.2.8
4.2.9
Results of real-time PCR assay examination
Result of Ct value (Raw data)
Result of Ct value (Default setting)
Result of Ct value (Modified setting)
Agreement between default and modified setting of real-time PCR assay
The average Ct value of real-time PCR in each type of samples
The standard graph of Ct value from modified setting real-time PCR
Distribution of Salmonella in faecal samples
Distribution of Salmonella in environmental samples
Proportion of Salmonella isolates from various samples
97
97
97
98
99
99
100
102
102
102
4.3
Comparison between bacteriological methods and real-time PCR assay
4.3.1 Comparison between bacteriological methods and real-time PCR assay
4.3.2 Agreement between detecting Salmonella by bacteriological methods
and real-time PCR assay (default setting) in samples from pig herds
4.3.3 Agreement between detecting Salmonella by bacteriological methods
and real-time PCR assay (modified setting) in samples from pig herds
104
104
5
5.1
5.2
5.3
107
108
112
5.4
Discussion
Dynamics of Salmonella
Real-time PCR assay
Agreement between detecting Salmonella by bacteriological methods
and real-time PCR assay in samples from pig herds
Conclusion and recommendation
6
Summary
118
7
Zusammenfassung
120
8
References
122
9
Appendix
172
10
Acknowledgement
185
105
106
114
116
Abbreviations
Abbreviations
$
®
°C
µm
₤
€
AFDA
CDC
CFSPH
CFU
CI
cm2
Ct
DEFRA
DNA
ECDC
EFSA
e.g.
ELISA
et al.
etc.
EU
FHI
FSIS
g
h
i.e.
ISO
l
mg
ml
mm
n
NFSA
No.
PCR
pH
QS
RASFF
RNA
RNase
spp.
U.S .
USDA
= dollar
= registered as trademark
= °celsius
= micrometer
= pound
= euro
= Australian Funeral Directors Association
= Centers for Disease Control and Prevention
= The Center for Food Security and Public Health
= Colony Forming Unit
= Confidence Interval
= square centimeter
= threshold cycle
= Department for Environment, Food and Rural Affairs
= deoxyribonucleic acid
= European Center for Disease Prevention and Control
= European Food Safety Authority
= exampli gratia
= Enzyme-Linked Immunosorbent Assays
= et alii
= et cetera
= European Union
= Norwegian Institute of Public Health
= Food Safety and Inspection Service
= gram
= hour
= id est
= International Standardization for Organization
= litre
= milligram
= milliliter
= millimeter
= number
= Norwegian Food Safety Authority
= number
= Polymerase Chain Reaction
= hydrogenion concentration
= Quality and Safety
= Rapid Alert System for Food and Feed
= ribonucleic acid
= ribonuclease
= species
= United States
= United States Department of Agriculture
Introduction
1 Introduction
Foodborne illness remains a major global health problem. Disease outbreaks resulting from
food borne microbial pathogens are a major public health concern and result in economic loss
for the food industry (ELLINGSON et al. 2004; GOUWS et al. 1998; HEIN et al. 2006).
Salmonellosis in human is one of the leading causes of foodborne bacterial enteritis in many
countries (CÔTÉ et al. 2004; KLERKS et al. 2004; TIRADO and SCHMIDT 2001; WOLFFS
et al. 2006). Salmonellas are widely distributed in nature, gaining entry to almost all aspects
of the human food chain. As with other food borne pathogens, control of human infection
with Salmonella spp. depends primarily on creating and maintaining a high standard of food
hygiene by both the producers and the consumers. Timely intervention of the infection
depends on rapid detection of these pathogens and appropriate preventive measures require a
regular monitoring. Detection of these pathogens has done mainly in frontline diagnostic
laboratories and routine food laboratories. Detailed identification and characterization of this
large group of bacteria is a difficult task requiring a combination of different approaches and
usually left to specialized public health and reference laboratories (NG et al. 1996).
Rapid detection methods are required for the purpose of diagnosis as well as for the
prevention of food contamination and food borne outbreaks (ELLINGSON et al. 2004; HEIN
et al. 2006; NG et al. 1996).
Until now, conventional culture methods have traditionally been considered the “gold
standards” for the isolation and identification of food borne pathogens. However, culture
methods are labor-intensive and time-consuming which are not suitable for routine testing of
large numbers of samples (BOHAYCHUK et al. 2007; FEY et al. 2004; FUKUSHIMA et al.
2006; KLERKS et al. 2004; MYINT et al. 2006; UYTTENDAELE et al. 2003).
Therefore, the rapid, cost-effective and automated diagnosis of foodborne pathogens
throughout the food chain continues to be a major concern for the industry and public health
authorities. Because of these requirements, the Polymerase Chain Reaction (PCR) has become
a powerful tool in microbiological diagnostics during the last decade (GOUWS et al. 1998;
MACIOROWSKI et al. 2000; MALORNY et al. 2004; MYINT et al. 2006; SACHSE 2003).
The second generation of PCR methodologies, the real-time PCR, has the potential to meet all
these criteria by combining amplification and detection in a one-step closed-tube reaction
(MALORNY et al. 2004).
For this reason, the real-time PCR has become a powerful tool for the detection of food borne
pathogens (WOLFFS et al. 2006). Real-time PCR is a rapid, sensitive and specific assay for
the detection of foodborne pathogens such as Salmonella spp. (BOHAYCHUK et al. 2007;
EYIGOR et al. 2002, 2004; KLERKS et al. 2004; KUROWSKI et al. 2002; MALORNEY et
al. 2004; SEO et al. 2004, 2006).
This technique would be a highly valuable tool for the rapid identification of Salmonella
reservoirs in pig herds, especially when questions such as the Salmonella freedom of animal
groups or the efficacy of cleaning and disinfection procedures need to be tested in a very short
1
Introduction
period of time. However, all real-time PCR test for Salmonella spp. have been developed for
food (very little other bacterial contamination) and most of the studies are based on artificial
contaminated samples (ELLINGSON et al. 2004; FEY et al. 2004; HEIN et al. 2006;
KLERKS et al. 2004; MALORNY et al. 2004; SEO et al. 2006; UYTTENDAELE et al. 2003;
WOLFFS et al. 2006). The open question is whether this method is also applicable for
samples from pig herds such as feces, dust, dirt, etc., which are naturally contaminated with
an unknown amount of Salmonella and contain huge amount of other bacterial DNA.
The purpose of this study was to investigate the suitability of the real-time PCR assay for the
detection of Salmonella spp. in samples from pig herds compared to conventional culture
results to determine relative sensitivity and specificity of the real-time PCR. The overall goal
was to study the possibility to use the real-time PCR as a routine laboratory method for
control and monitoring program of Salmonella in pig herds.
2
Literature review
2 Literature review
2.1 A review of the Salmonella
2.1.1 History
The genus Salmonella was named after Daniel Elmer Salmon, an American veterinary
pathologist. Salmon, along with Theobald Smith (1886), discovered the organism that causes
hog cholera, Salmonella enterica var. Choleraesuis (ANONYMOUS 2008b; DURECKO et
al. 2004), when they considered it to be the cause of swine fever (hog cholera). The
importance of the organism as a cause of disease in pigs was neglected when the viral
etiology of swine fever was discovered, and a number of years elapsed before Salmonella
Choleraesuis was recognized as a primary pathogen that was capable of causing several
different disease syndromes (WRAY 2001).
2.1.2 The Genus Salmonella
The genus Salmonella, within the family Enterobacteriaceae, is a morphologically and
biochemically homogenous group of facultatively anaerobic, non-spore forming, oxidasenegative, catalase-positive Gram-negative rod-shaped bacteria; the rods are typically 0.7-1.5 x
2-5µm in size, although long filaments may be formed. Most strains are motile due to peritrichous flagella and ferment glucose with production of both acid and gas. Typically,
Salmonella are non or slow lactose fermenters with some strains fermenting it rapidly
though; adonitol, sucrose, salicin and 2-ketogluconate are not fermented; urea is not
hydrolysed; tryptophan and phenylalanine not deaminated; acetoin not produced; hydrogen
sulphide (H2S) is produced from thiosulphate; lysine and ornithin is decarboxylated. Most
schemes for the detection of the organism are based on these properties (BISPING et al. 1988;
GRIMONT et al. 2000; SCHWARTZ 1999). Some strains produce a biofilm, which is a
matrix of complex carbohydrates, cellulose and proteins. The ability to produce biofilm can
be an indicator of dimorphism, which is the ability of a single genome to produce multiple
phenotypes in response to environmental conditions (ANONYMOUS 2008b).
Figure 2.1: Salmonella (source: http://en.wikipedia.org)
3
Literature review
Many genetic studies have been done on Salmonella species, particularly on serovar
Typhimurium. Its chromosome is very similar to that of Escherichia coli and consists of a
single circular DNA molecule consisting of about 4 x 106 base pairs with a molecular weight
of 4 x 109 and a total length of about 1.4mm. Many of the genes have been mapped
(ANONYMOUS 2008a). According to the biochemical reactions and growth conditions
of Salmonella shown above, different selective plating media have been developed.
Summarize the appearance of Salmonella colonies on a variety of the most frequently used
agar nutrient media (Table 2.1).
Table 2.1: Appearance of Salmonella on most commonly used selective agars
(WALTMAN 2000)
Medium
Bismuth sulphite agar
Brilliant green agar
Brilliant green sulphapyridine
Brilliant green novobiocin
Deoxycholate citrate agar
Gassner agar
Hektoen enteric agar
MacConkey agar
Rambach agar
Salmonella-Shigella agar
Xylose lysine desoxycholate agar
Xylose lysine tergitol 4
Appearance of Salmonella colonies
Black, metallic sheen
Red
Red
Red
Colourless, Black center (H2S production)
Yellow
Blue-green, Black center (H2S production)
Colourless
Crimson with pale borders
Colourless, Black center (H2S production)
Red, Black center (H2S production)
Red, Black center (H2S production)
Typical, Salmonella give colourless colonies with black centers on Salmonella-Shigella (SS)
agar; green, black-center colonies are seen on Hektoen agar; black colonies with a
green background on Xylose lysine tergitol 4 (XLT4) agar; and colonies are red or
fuchsia (crimson with pale borders) on Rambach agar (GRIMONT et al. 2000). Salmonella
grow red with black center on Xylose lysine desoxycholate (XLD) agar; giving yellow
colonies on Gassner; red colonies on Brilliant green agar (BGA) (WALTMAN 2000).
Salmonella can grow within the range 2 to 54°C, although growth below 7°C has
largely been observed only in bacteriological media, not in food, while growth above 48°C is
confined to mutants or tempered strains. The optimum temperature for growth is 37°C. The
natural ecology of most Salmonella strains of concern to public health is the gastrointestinal
tract of warm-blooded animals (BRENNER et al. 2000; COX 1999). The optimum pH for the
growth of Salmonella is within a range of 6.5-7.5, in liquid nutrient media strains can grow at
pH values up to 9.5 and down to 4.05. While growth occurs down to or close to the minimum
pH with non-volatile organic acids such as citric acid or mineral acid such as hydrochloric
acid, growth stops at higher pH values when volatile fatty acids are used. Salmonella grow at
aw (water activity) values between 0.999 and 0.945 in laboratory media, down to 0.93 in
foods, with an optimum of 0.995 (COX 1999).
4
Literature review
2.1.3 Taxonomy and nomenclature
The classification of the Salmonella within this genus has undergone considerable changes in
recent decades (BISPING et al. 1988). Salmonella taxonomy is complicated (TINDALL et al.
2005). Strains of Salmonella are classified into serovars on the basis of extensive diversity of
lipopolysaccharide (LPS) antigens (O) and flagella protein antigens (H) in accordance with
the Kauffmann-White scheme (OIE 2008).
The Kauffman and White classification scheme is a classification system that permits
serological varieties of the genus Salmonella to be differentiated from each other. This
scheme differentiates isolates by determining which surface antigens are produced by the
bacterium. First, the "O" antigen type is determined. "O" antigens are the polysaccharides
associated with the lipopolysaccharide of the bacterial outer membrane. Having found the "O"
antigen group, the "H" antigen is determined. The "H" antigens are proteins associated with
the bacterial flagella (singular; flagellum). Salmonellas exist in two phases; a motile phase
and a non-motile phase. These are also referred to as the specific and non-specific phases.
Different "H" antigens are produced depending on the phase in which the Salmonella is found
(Table 2.2). Non-motile isolates may be "switched" to the motile phase using a Cragie tube bacteria are inoculated down the center of a hollow tube in a semi-solid nutrient agar. Those
bacteria that become motile can then swim out of the bottom of the tube and are recovered
from the agar outside of the tube. Pathogenic strains of Salmonella Typhi carry an additional
antigen, "Vi", so-called because of the enhanced virulence of strains that produce this antigen,
which is associated with a bacterial capsule (ANONYMOUS 2008c; LE MINOR and
POPOFF 1988).
Salmonella nomenclature is not completely standardized. Several synonyms may be used for
the same species or subspecies. Under the classification scheme used by the U.S. Centers for
Disease Control and Prevention (CDC), World Health Organization (WHO) and some
journals, there are now only two species in the genus Salmonella: S. enterica and S. bongori
(CFSPH 2005). There are also more than 2500 serovars within both species, which are found
in a disparate variety of environments and which are associated with many different diseases
(ANONYMOUS 2008b; CFSPH 2005).
Salmonella enterica has 6 subspecies: S. enterica subsp. enterica, S. enterica subsp. salamae,
S. enterica subsp. arizonae, S. enterica subsp. diarizonae, S. enterica subsp. houtenae and S.
enterica subsp. indica. These subspecies are also referred to by a number. S. enteric subsp.
enterica = subspecies I. S. enterica subsp. salamae = subspecies II. S. enterica subsp. arizonae
= subspecies IIIa. S. enterica subsp. diarizonae = subspecies IIIb. S. enteric subsp. houtenae =
subspecies IV. S. enterica subsp. indica = subspecies VI.
However, within this six subspecies are differentiated by genetical, biochemical and, in part,
serological methods (Table 2.3) (ANONYMOUS 2008b; BISPING et al. 1988; CFSPH 2005;
OIE 2008).
5
Literature review
Table 2.2: Antigenic structure of some common Salmonellae (Jay et al. 2005)
O antigens*
Group
H antigens
Serovars
Phase 1
Phase2
1, 2, 12
a
(1, 5)
A
S. Paratyphi A
B
S. Schottmuelleri
1, 4, (5), 12
b
1, 2
S. Typhimurium
1, 4, (5), 12
i
1, 2
S. Hirschfeldii
6, 7 (vi)
c
1, 5
S. Choleraesuis
6, 7
(c)
1, 5
S. Oranienburg
6, 7
m, t
-
S. Montevideo
6, 7
g, m, s,(p)
(1, 2, 7)
C2
S. Newport
6, 8
e, h
1, 2
D
S. Typhi
9, 12, (Vi)
d
-
S. Enteritidis
1, 9, 12
g, m
(1, 7)
S. Gallinarum
1, 9, 12
-
-
3, 10
e, h
1, 6
C1
E1
S. Anatum
* The italicized antigens are associated with phage conversion. ( ) = May be absent.
The nomenclature used throughout this publication follows that devised by Le Minor and
Popoff which divides the bacterial species Salmonella enterica into six subspecies: enterica,
salamae, arizonae, diarizonae, houtenae and indica.
The method of naming serovars of subspecies enterica differs from that used for the other five
subspecies in that the familiar serovar names are assigned to subspecies enterica whilst the
other subspecies are designated by antigenic structure (DEFRA 2006). Serovars in S. enterica
subsp. enterica are referred to by name. The names of these serovars can be shortened from
the full name to the genus and serovar. For example, S. enterica subsp. enterica ser.
Enteritidis can be called Salmonella ser. Enteritidis or Salmonella Enteritidis. Most of the
serovars in the other 5 subspecies of S. enterica, as well as in S. bongori, are referred to by
their antigenic formulas. These formulas include:
6
Literature review
1. The subspecies/species designation (I, II, IIIa, IIIb, IV or VI for S. enterica subtypes; V for
S. bongori)
2. O (somatic) antigens followed by a colon
3. H (flagella) antigens (phase 1) followed by a colon
4. H antigens (phase 2, if present)
Using this convention, a S. enterica subsp. houtenae strain with an O antigen designated 45,
H antigens designated g and z51, and no phase 2 H antigens would be written as Salmonella
serotype IV 45:g,z51: (CFSPH 2005; LE MINOR and POPOFF 1988).
Table 2.3: Terminology and differentiation of the subspecies (subgenera) of the genus
Salmonella (LE MINOR and POPOFF 1988)
Proposed
designation
subsp.
Choleraesuis
short name:
I
subsp.
Salamae
short
name:
II
subsp.
Arizonae
short identity:
IIIa
subsp.
diarizonae short
identity: IIIab
subsp.
Houtenae
short
identity:
IV
subsp.
Bongori
short
identity:
V
subgenus I
subgenus II
subgenus IIIa
monophasic
subgenus IIIb
diphasic
subgenus IV
subgenus V
+
-
-
-
-
-
+
+
+
+
-
- / (+)
+
-
+
Acid from
Lac
Dul
Mucate
Galactouronate
+
+
-
+
+
+
+ / (+)
V
V¹
+
+
+
Utilization of
Mal
d-tartrate
Gel
-
+
- (+)
+
+
- (+)
+
- / (+)
+
-
Previous
designation
Primary
occurrence
warm-blooded
animals
cold-blooded
animals and
environment
ß-galactosidase
(ONPG test)
¹ Monophasic strains: negative, diphasic strains: positive
7
Literature review
2.1.4 Epidemiology
Salmonella has been recovered from the intestines of a wide range of animals
including fish, reptiles, birds, and mammals (COX 1999; WRAY and SOJKA 1977).
Excretion with faeces can result in contamination of water, soil, other animals and
feed. Animals are infected by direct nose-to-nose contact or by contact to the faeces of
infected animals or by feedstuff containing contaminated compounds. The main sources of
salmonellosis in humans are food animals and their products such as raw eggs, poultry meat
and pork (HALD and WEGENER 1999; STEINBACH and HARTUNG 1999; BERENDS et
al. 1998; D‟ AOUST 1997; CLARKE and GYLES 1993).
The reservoir for Salmonella is the intestinal tract of warm- and cold-blooded animals.
Salmonella have mastered virtually all of the attributes necessary to ensure wide distribution,
including abundant reservoir hosts, efficient fecal shedding from carrier animals, persistence
within the environment, and the effective use of transmission vectors (feed, fomites, vehicles,
etc.). Inapparent, long-term carriers that can shed Salmonella in feces continuously or
intermittently, often in high numbers, are common in most host species. Shedding of the
organism can be exacerbated by a long list of stressors, including commingling of pigs,
transportation, concurrent diseases, and food deprivation.
The epidemiology of Salmonella infections in swine is two relatively separate problems:
Salmonella infection of pork carcasses and retail products and infections that cause
salmonellosis in swine. Infection of swine by one or more serotypes is common, but primary
clinical disease caused by serotypes other than S. Choleraesuis or S.Typhimurium is
uncommon. It is important to understand that swine can be infected with a variety of
serotypes that do not cause disease in swine but do represent a source of infection for pork
products.
Extrapolation of epidemiological data from experimental studies where a single serotype with
predetermined dose is administered to naive healthy pigs is not likely to represent field
situations, where there are multiple serotypes, varying doses, intermittent exposures, variable
host resistance, many management variables, and various intercurrent infections and diseases.
Similarly, disease prevalence surveys must be carefully scrutinized to be sure that infection is
not equated with disease, and that a source of infection is not inaccurately implicated
(SCHWARTZ 1999).
In contrast with poultry, where vertical transmission of some serovars may occur from the
layer hen to the egg (BARROW 2000), horizontal transmission is of major importance in
spread of Salmonella in pigs. Pigs get infected with Salmonella through direct contact with
infected pen-mates, via contaminated feed or via the environment. The epidemiology of
Salmonella is complex, because there is a wide range of hosts and vectors, because
Salmonella can survive for a long time in the environment and because of the ability to persist
in the population due to the carrier state.
The number of sources for Salmonella in pig herds is endless (SCHWARTZ 1999).
Salmonella infections within pig herds are often clustered at the pen level (DAVIES et al.
1998) but outbreaks of salmonellosis can typically spread from pen to pen (SCHWARTZ
8
Literature review
1999). Nevertheless, because of the presence of different vectors such as water sources, cats,
insects, rodents, boots, Salmonella may spread to distant pens within the same herd
(BARBER et al. 2002). The role of cats, birds and rodents in the spread of Salmonella
between herds needs to be investigated further.
One of the major triggers for Salmonella excretion in pigs is stress, for instance caused by
transport (ISAACSON et al. 1999; SEIDLER et al. 2001). This might have important
implications concerning food safety, because the number of Salmonella-infected animals can
sometimes increase signficantly during lairage before slaughtering (HURD et al. 2001a;
2001b; 2001c). A high number of Salmonella shedding animals can increase the risk of
carcass contamination. According to Swanenburg et al. (2001a), 20% of the contaminated
carcasses originating from sero-negative herds are caused by Salmonella carrying-pigs,
while 80% is the result of cross-contamination during slaughtering.
Another implication of excreting Salmonella by apparently healthy animals is the
contamination and the survival in the environment including manure to be disposed on the
fields. The study by Guan and Holley (2003) demonstrated that Salmonella spp. could
survive in pig slurry for 14 days at 8°C. Survival is favored in solid manure and at lower
temperatures. Also in water and soil, Salmonella has been shown to survive for 152 and 42-63
days (depending on the temperature), respectively (GUAN and HOLLEY 2003). Boes et al.
(2005) demonstrated that the survival of Salmonella in the soil surface following deposition
of naturally contaminated pig slurry was dependent on the deposition method. Ploughing and
harrowing of soil amended with contaminated pig slurry seemed to be an effective way to
reduce the contamination level of the environment. Letellier et al. (1999a) identified the
water supply as a major source for Salmonella infection in pig herds. In the same study,
it was demonstrated that the environment, including boots, floors, doors but also rodents and
insects contributed to persistent Salmonella infections in pig herds. Also Barber et al.
(2002) suggested that there is a widespread transmission across different ecological
compartments. The latter authors found a correlation between the prevalence of
Salmonella in a herd and Salmonella on pen floors (0.75), in flies (0.89) and on boots (0.79).
2.1.5 Porcine Salmonellosis
The clinical signs of porcine salmonellosis are refer able to septicemia or to enterocolitis, and
this section describes each syndrome separately. Pigs surviving acute septicemia may develop
clinical signs due to bacteremic localization: pneumonia, hepatitis, enterocolitis, and,
occasionally, meningoencephalitis. Pigs initially suffering from enterocolitis may later
develop chronic wasting disease or, occasionally, rectal stricture (SCHWARTZ 1999; WRAY
2001).
The clinical and pathological features of Salmonella infections are extremely variable.
Severity is influenced by serotype, virulence, natural and acquired host resistance, and route
and quantity of the infective dose. Over 200 virulence factors have been associated with
Salmonellae but few have been completely characterized. Generally, those that promote
virulence in pathogenic salmonellae are involved in adhesion, invasion, cytotoxicity, and
resistance to intracellular killing, often working in combination to promote disease. Despite
9
Literature review
distinct differences in clinical signs, many parallels can be drawn between S. Choleraesuis
and S. Typhimurium when discussing pathogenesis (SCHWARTZ 1999; WRAY 2001).
Although large doses (greater than 107) are required to induce disease experimentally,
intraluminal replication may be important with small inoculate from contaminated feed or
water. Disease is facilitated by factors such as peristaltic impairment, interference with
intestinal flora, and elevation of gastric pH (CLARKE and GYLES 1993). Replication to
about 107 organisms/g of intestinal content is required for lesion production in pigs infected
with S. Typhimurium, a finding that probably also applies to other serotypes causing
enterocolitis. Alterations in normal intestinal defenses by antibiotic-induced changes in
normal flora or cold-induced alteration in intestinal motility may reduce the amount of
replication required for disease or increase the ease of Salmonella replication (BOHNHOFF et
al. 1954). Infection with Salmonella Choleraesuis may not require such massive luminal
proliferation as prerequisite for disease, because it is inherently more invasive than other
serotypes, can infect via the pharyngeal tonsil, and regularly causes signs of septicemia 24-72
hours before the onset of diarrhea (SMITH and JONES 1967; CHERUBIN et al. 1974;
WILCOCK 1979; REED et al. 1986).
Most pathogenic S. Typhimurium organisms have type 1 fimbriae and flagella, both of which
appear to be involved in attachment and invasion. Phenotypic shifts may occur in Salmonellae
to produce adhesive pili (ISAACSON 1996). Flagella may also be important in allowing
survival inside macrophages. The ability to invade is a requirement for pathogenesis and is
encoded by a serotype-specific plasmid (HELMUTH and BUNGE 1985). Removal of this
plasmid results in a lack of ability to invade but has no effect on ingestion or killing by
murine macrophages, LPS production, or serum resistance (GULIG and CURTISS 1987).
During the invasion process there is induction of synthesis of new proteins that probably
enhance intracellular survival (FINLAY et al. 1989). Peroxidase-antiperoxidase immune
enzymatic labeling and immunogold labeling techniques have demonstrated that S.
Typhimurium has a low tendency to invade the enteric mucosa and does not have a
predilection for any specific intestinal location, whereas S. Choleraesuis locates preferentially
in the colon on the luminal surface of ileal M cells of Peyer‟s patches (POSPISCHIL et al.
1990). Invasion is by endocytosis by M cells of gut-associated lymphoid tissue as well as
enterocytes. Attachment of the bacteria to epithelial receptors triggers microfilament
controlled uptake, vacuole formation, vacuole transport through the cell cytoplasm, and entry
to the lamina propria via exocytosis through the basement membrane (TAKEUCHI 1967;
TAKEUCHI and SPRINZ 1967). Pass through the epithelium results in mild and transient
enterocyte damage. Salmonellae can synthesize over 30 proteins which are selectively
induced during infection of macrophages, making them facultative intracellular bacteria
(similar to Brucella, Mycobacteria, and Listeria organisms) that can survive within
macrophages and neutrophils in the lamina propria (ROOF et al. 1992a, b). Spread to
mesenteric lymph nodes is rapid, occurring within 2 hours of inoculation of ligated intestinal
loops or 24 hours after oral challenge (REED et al. 1985, 1986). Macrophages are the most
likely disseminators of infection systemically to other sites of infection and pathology.
Concurrent with bacillary spread is the appearance of an acute, predominantly macrophagic,
inflammatory reaction and prominent microvascular damage with thrombosis within the
lamina propria and submucosa. When administered intranasally to esophagatomized pigs, S.
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Literature review
Choleraesuis demonstrated primary colonization of the lung within 4 hours (FEDORKACRAY et al. 1995; GRAY et al. 1995).
Several serovars have been shown to produce enterotoxins, specifically cholera-like toxin
(PRASAD et al. 1990). Very little is known about this toxin as it relates to the pathogenesis of
Salmonella species. A common feature of Salmonella species induced enteritis is severe
damage to intestinal epithelial cells, likely to be the result of a cytotoxin. At least three
cytotoxins have been identified. A wide variety of serovars posses a heat-labile cytotoxin
described by Ashenazi et al., (1988). Another cytotoxin is a low molecular weight membrane
associated toxin, which has not been characterized (REITMEYER et al. 1986). A third toxin,
described by Libby et al. (1990), appears to be present in nearly all Salmonella species,
Shigella and enteroinvasive E.coli. This cloned protein is a 26 kDa cell-associated hemolysin
and its role in virulence is under study.
Roof and Kramer (1989) showed that virulent S. Choleraesuis were able to survive within
porcine neutrophils by inhibiting superoxide anion production and resisting the bactericidal
activity of the cells. Heat shock proteins have been shown to be produced by S.Typhimurium
inside murine macrophages. Mutants that are defective in this ability to produce these proteins
are less virulent in mice and do not survive well in macrophages (FALKOW and
MEKALANOS 1990). Watson et al. (2000) studied the interaction of different Salmonella
serotypes with porcine macrophages in vitro, S. Typhimurium persisted longest and there was
little or no significant difference in induction of pro-inflammatory cytokines by macrophages.
Salmonellas Typhimurium and Dublin damaged macrophages but not S. Choleraesuis and
they concluded that there was no correlation with virulence. Riber and Lind (1999) found a
variation in the bactericidal activity of phagocytes from different pigs against S.
Typhimurium. The phagocytosis of S. Choleraesuis in the lungs of pigs was studied by
Baskerville et al. (1972) during the period 6 h-14 days after intranasal infection. All bacteria
were phagocytosed soon after arrival by polymorphonuclear leucocytes and macrophages;
many were destroyed but some survived and multiplied within the cells. Between days 5-7,
the Salmonella caused necrosis of the phagocytes and were liberated in large numbers and
caused damage of the lung tissues. Later studies showed that the free bacteria did not attach to
or penetrate pulmonary cells and they suggested the damage was caused by toxin
(BASKERVILLE et al. 1973). Matalova et al. (2000) suggested that the presence of living
Salmonella in pig leucocytes delayed the onset of apoptosis. This would lead to the
hypothesis that infection may be prolonged in this situation.
The Lipopolysaccharide (LPS) of Salmonella species is a major determinant of host
specificity and virulence (WRAY 2001). The intact LPS afford resistance to phagocytosis and
killing by macrophages and complement-mediated killing (SAXEN et al. 1987; ROBBINS et
al. 1992). In addition it has been shown that LPS is a major contributor to survival of
Salmonella species in the intestinal tract (NNALUE and LINDBERG 1990). The LPS
component of Salmonella species also contributes to vascular damage and thrombosis.
Endotoxic properties result in fever, disseminated intravascular coagulation, circulatory
collapse and endotoxic shock associated with salmonellosis (TAKEUCHI and SPRINZ 1967).
Motility provided by flagella appears to be important for invasion for some, but not all,
serovars of Salmonella. Regardless of the other contributions the flagella may make, their
presence increases the probability that the organism will come in contact with an epithelial
11
Literature review
cell. It has been shown that strains with polar rather than peritrichous flagella have increased
ability to come in contact with, and potentially invade, epithelial cells (JONES et al. 1992).
A siderophore has been identified in S. Typhimurium called enterobactin (BENJAMIN et al.
1985). This protein does not appear to be necessary for full virulence and the importance of
the protein may be relative to the amount of extracellular growth, which occurs. Interestingly,
pigs infected with S. Choleraesuis have a reduction in serum iron, total-iron binding capacity
and transferrin. The intracellular environment is low in iron and it has been suggesting that S.
Choleraesuis has a nonsiderophore mechanism for scavenging iron (CLARKE and GYLES
1993).
The pathogenesis of the diarrhea typical of enteric salmonellosis and of later stages of
salmonella septicemia has traditionally been attributed to malabsorption and net fluid leakage
by a necrotic, inflamed bowel (SCWARTZ 1999). After ingestion, the pathogens can survive
the acid environment of the stomach by producing acid shock proteins (SMITH 2003).
Consequently, the gastro-intestinal tract can be colonized. Salmonella colonization starts as
high as the stomach where rapid multiplication and even invasion can take place.
Colonization can then spread throughout the small and large intestine, the degree of which
will be determined by the initial Salmonella challenge and the conditions within the gut itself
(BLANCHARD and BURCH 2008). Experimental studies showed that Salmonella could be
isolated daily from faeces during the first 10 days after infection and frequently, but at
irregular intervals, during the next 4 to 5 months (FEDORKA-CRAY et al. 1994).
Salmonella could be isolated in more than 90% of the infected pigs from mesenteric lymph
nodes, tonsils, caecum content or faeces 4 to 7 months after experimental infection
(SCHWARTZ 1999). Under field conditions, where re-infections are likely to occur
frequently (SCHWARTZ 1999), the period of faecal shedding after infection is difficult to
assess. The mechanism by which Salmonella invade the epithelium is partially understood
and involves an initial binding to specific receptors on the epithelial cell surface followed by
invasion. Invasion occurs by the organism inducing the enterocyte membrane to undergo
"ruffling" and thereby to stimulate pinocytosis of the organisms. Invasion is dependent on
rearrangement of the cell cytoskeleton and probably involves increases in cellular inositol
phosphate and calcium. Attachment and invasion are under distinct genetic control and
involve multiple genes in both chromosomes and plasmids (GIANNELLA 2008).
After invading the epithelium, the organisms multiply intracellularly and then spread to
mesenteric lymph nodes and throughout the body via the systemic circulation; they are taken
up by the reticuloendothelial cells. The reticuloendothelial system confines and controls
spread of the organism. However, depending on the serotype and the effectiveness of the host
defenses against that serotype, some organisms may infect the liver, spleen, gallbladder,
bones, meninges, and other organs. Fortunately, most serovars are killed promptly in
extraintestinal sites, and the most common human Salmonella infection, gastroenteritis,
remains confined to the intestine.
After invading the intestine, most Salmonellae induce an acute inflammatory response, which
can cause ulceration. They may elaborate cytotoxins that inhibit protein synthesis. Whether
these cytotoxins contribute to the inflammatory response or to ulceration is not known.
However, invasion of the mucosa causes the epithelial cells to synthesize and release various
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Literature review
proinflammatory cytokines, including: IL-1, IL-6, IL-8, TNF-2, IFN-U, MCP-1, and GMCSF. These evoke an acute inflammatory response and may also be responsible for damage to
the intestine. Because of the intestinal inflammatory reaction, symptoms of inflammation such
as fever, chills, abdominal pain, leukocytosis, and diarrhea are common. The stools may
contain polymorphonuclear leukocytes, blood, and mucus.
Much is now known about the mechanisms of Salmonella gastroenteritis and diarrhea.
Figures 2.2 and 2.3 summarize the pathogenesis of Salmonella enterocolitis and diarrhea.
Only strains that penetrate the intestinal mucosa are associated with the appearance of an
acute inflammatory reaction and diarrhea (Figure 2.4); the diarrhea is due to secretion of fluid
and electrolytes by the small and large intestines. The mechanisms of secretion are unclear,
but the secretion is not merely a manifestation of tissue destruction and ulceration. Salmonella
penetrate the intestinal epithelial cells but, unlike Shigella and invasive E. coli, do not escape
the phagosome. Thus, the extent of intercellular spread and ulceration of the epithelium is
minimal. Salmonella escape from the basal side of epithelial cells into the lamina propria.
Systemic spread of the organisms can occur, giving rise to enteric fever. Invasion of the
intestinal mucosa is followed by activation of mucosal adenylate cyclase; the resultant
increase in cyclic adenosine monophosphate (AMP) induces secretion. The mechanism by
which adenylate cyclase is stimulated is not understood; it may involve local production of
prostaglandins or other components of the inflammatory reaction. In addition, Salmonella
strains elaborate one or more enterotoxin-like substances which may stimulate intestinal
secretion (GIANNELLA 2008).
Mucosal inflammation and necrosis, as well as septicemia, occur in concert with the diarrhea
but perhaps independently of it. Microvascular thrombosis and endothelial necrosis in the
submucosa and lamina propria are consistent early lesions in porcine salmonellosis
(LAWSON and DOW 1966; WILCOCK et al. 1976; JUBB et al. 1993; REED et al. 1986),
probably in response to locally produced endotoxin. Salmonellae are not directly associated
with the damaged vessels but direct the events from the protected intracellular niche of
macrophages in the surrounding submucosa or lamina propria (TAKEUCHI and SPRINZ
1967). Mucosal ischemia as a result of the microvascular thrombosis is probably a major
contributor to the mucosal necrosis so typical of salmonellosis in all species. The second
major contribution to mucosal necrosis is probably from the chemical products of mucosal
inflammation. The systemic signs and lesions of septicemic salmonellosis, in swine almost
exclusively S. Choleraesuis infection, are most commonly attributed to endotoxemia from
bacterial dissemination (SCHWARTZ 1999).
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Literature review
Ingestion of organisms
Colonization of lower intestine
(Ileum and Cecum)
Mucosal invasion
Cytotoxin
Acute inflammation
± Ulceration
Prostaglandin synthesis
Enterotoxin
Cytokines
Activation of adenyl cyclase
↑Cyclic AMP
Fluid production
(Large and small bowel)
Diarrhea
Figure 2.2: Scheme of the Pathogenesis of Salmonella enterocolitis and diarrhea
(GIANNELLA 2008)
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Literature review
Figure 2.3: Invasion of intestinal mucosa by Salmonella spp. (GIANNELLA 2008)
Figure 2.4: Electron photomicrograph demonstrating invasion of guinea pig ileal
epithelial cells by S. Typhimurium. Arrows point to invading Salmonella organisms
(GIANNELLA 2008).
15
Literature review
2.1.6 Treatment
With either septicemic or enteric salmonellosis, the goals of treatment in an outbreak of
salmonellosis are to minimize the severity of clinical disease, prevent spread of infection and
disease, and prevent recurrence of the disease in the herd. With salmonellosis the attainment
of these goals is particularly difficult. Both Salmonella Choleraesuis and S. Typhimurium are
often resistant in vitro to many antibacterial agents used in swine (BARNES and SORENSEN
1975; WILCOCK et al. 1976; BLACKBURN et al. 1984; SCHULTZ 1989; FALES et al.
1990; SCHWARTZ 1997a). During clinical disease, the organism inhabits a protected
intracellular niche inaccessible to many common antibacterials. The use of various antibiotics
to treat enteric salmonellosis is widely advocated (MOREHOUSE 1972; BARNES and
SORENSEN 1975; BLOOD et al. 1979), but much of the information to support this
recommendation has been taken from trials designed to test the prophylactic efficacy of drugs,
not their therapeutic efficacy. Thus, pigs on medicated feed, when inoculated orally with
Salmonellae, have the antibiotic already present in the gastrointestinal tract to interact with
the Salmonellae, resulting in milder disease because of what amounts to a decreased
inoculum. In the few trials designed specifically to test antibacterial drug efficacy against
clinical enteric salmonellosis, such therapy was considered to have little merit (HEARD et al.
1968; GUTZMANN et al. 1976; OLSON et al. 1977; WILCOCK and OLANDER 1978).
In experimental infections, sub-therapeutic levels of tetracyclines have been shown to reduce
Salmonella shedding when the organism was sensitive but had no effect when it was
resistant (CLAUSEN et al. 1998). Apramycin/oxytetracycline was used to treat experimental
Salmonella infections in pigs and shedding of Salmonella was reduced in those pigs on
antibiotics (EBNER and MATHAW 2000). Likewise, Schwartz and Lucas (1994) found that
pigs receiving Aureo sp250 medicated feed and subsequently challenged with Salmonella
Choleraesuis had lower mortalities, less severe clinical illness, better growth rates and feed
conversion than those which received no medication. Wilcock and Schwartz (1992), mention
a number of trials, but the general conclusion was that therapy was equivocal or of little merit
under field conditions. Antimicrobials have also been used to reduce the shedding of
Salmonella by sick or recovered pigs, and once again there is no evidence as to their
efficacy. However, anecdotal information from practitioners suggests that severe Salmonella
infection will respond to appropriate antimicrobial therapy. Ancillary therapy such as the use
of fluids to replace lost electrolytes and to prevent dehydration will assist recovery.
In contrast, vigorous therapy early in the course of septicemia caused by S. Choleraesuis has
been reported to significantly reduce the duration and severity of disease (JACKS et al. 1981).
In that report, therapy was initiated after inoculation but prior to the onset of clinical signs.
Evaluation of efficacy under the field conditions are difficult because of the unpredictability
of the disease and because husbandry changes often accompany the use of antibacterials in an
outbreak. Reports and practitioner communications from the American Midwest, however,
suggest that visibly affected animals respond to aggressive therapy with parenteral
antimicrobials (SCHWARTZ 1991). Septicemic salmonellosis can be treated with a number
of antibiotics including ampicillin, amoxicillin, gentamicin, trimethoprim/ sulfamethoxazole,
third generation cephalosporins, chloramphenicol and fluoroquinolones. Many isolates are
resistant to one or more antibiotics, and the choice of drugs should, If possible, be based o n
susceptibility testing (CFSPH 2005). Mass medication of the population at risk to decrease
16
Literature review
severity of disease and transmission of Salmonellae is also widely practiced. The choice of an
appropriate antimicrobial is aided by antibiograms and previous herd experience. In the
absence of amikacin, gentamicin, neomycin, apramycin, ceftiofur, and trimethoprimsulfonamide are effective in vitro against most isolates (BARNES and SORENSEN 1975;
WILCOCK et al. 1976; MILLS and KELLY 1986; SCHULTZ 1989; EVELSIZER 1990;
FALES et al. 1990, SCHWARTZ 1997b). Anti-inflammatory agents are sometimes
administered to critically ill animals to combat the effects of endotoxin (SCHWARTZ and
DANIELS 1987; SCHULTZ 1989; EVELSIZER 1990; CFSPH 2005). The duration of
therapy for other extra-intestinal infections should be considered based on the site of
infection. In general, 10 to 14 days for bacteremia, 4 to 6 weeks for osteomyelitis, and 4
weeks for meningitis are suggested (CHIU et al. 2004).
The use of antimicrobials for therapy or growth promotion may also disrupt the gut flora,
which often increases the susceptibility of pigs to Salmonella infection. The use of antibiotics
may thus act as a trigger to spread Salmonella infection throughout a herd, which would not
have occurred if the animals remained untreated. This phenomenon has been thoroughly
documented in poultry (SMITH and TUCKER 1978) and is also likely to occur in other
animal species. The EFSA recently gave an opinion on the use of antibiotics to control
Salmonella in poultry (EFSA 2004), which concurred with that of WHO (WHO 1992), i.e.
that Salmonella control should not be based on antibiotics. The emergence of antibiotic
resistance is another serious reason why antibiotics should be used with great care, as
demonstrated by the emergence and fast spread of the multi-resistant S. Typhimurium
definitive phage type (DT) 104 (THRELFALL 2002).
Most Salmonella antimicrobial resistance is plasmidmediated, and removal of selective
pressure of an antimicrobial allows for reversion to susceptibility. Of concern is the recent
emergence of a Salmonella Typhimurium phage type or definitive type 104 (DT 104), isolated
primarily from bovine and human populations, that has chromosomally integrated multiple
antimicrobial resistance (LOW et al. 1997). This isolate has a higher morbidity and mortality
in humans than other S. Typhimurium organisms and is increasing in prevalence in human
and bovine populations. Carrier swine are generally asymptomatic. Although evidence linking
emergence of such isolates to veterinary medical practices is generally lacking, the impact
will affect public health initiatives as well as the availability of therapeutic agents for foodproducing animals. It should be emphasized that Salmonella control programs that rely
strictly on antimicrobials are doomed to failure (SCHWARTZ 1999).
The association between antimicrobial resistance and usage was investigated by Bahnson and
Fedorka-Cray (1999) who found that there was a relationship between tetracycline
resistance and usage but not between any of the other antibiotics. Further studies,
Fedorka-Cray et al. (1999) suggested that Salmonella isolates from lymph nodes were more
likely to be resistant than those from other sources. However, Blaha et al. (1999) could find
no difference in the frequency of resistance between strains from lymph nodes and those
from the environment. They suggested that antimicrobial resistance in Salmonella is much
more complex than just a consequence of antimicrobial usage in animals and further
research is necessary (WRAY 2001).
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Literature review
The use of antibacterial drugs for growth promotion is controversial but Baggesen et al. (1999)
found that tylosin did not promote the shedding of Salmonella in pigs, and Shryock et al.
(1998) found that prolonged feeding of tylosin reduced the duration of Salmonella shedding
in pigs. The use of Flavophospholipol (Bambermycin) has been shown to reduce Salmonella
shedding in pigs and to reduce the level of resistance in enteric micro-organisms (DEALY
and MUELLER 1976; OOSTENBACH 2001). Studies on the productivity and economic
impacts of feed grade antimicrobial use in pork production (ALGOZIN et al. 2001) indicated
that the average daily weight gain and feed conversion ratio improved by 0.9% and 2.3%
respectively and finisher mortality was reduced by 0.29%. Though they considered that pork
producers might be reluctant to produce pigs without the use of antimicrobials they also
suggested that there is a need for carefully controlled field trials to assess their benefit and an
assessment of the risk for human health. In countries where the use of antibiotic growth
promoters has been phased out, disease in pigs increased in prevalence and necessitated
higher usage of antibacterial drugs such as tetracyclines (WRAY 2001).
In developed countries, it is also becoming increasingly accepted that a majority of the
resistant strains of zoonotic Salmonella spp. have acquired that resistance in an animal host
before being transmitted to humans through the food chain (MØLBAK et al. 2002;
THRELFALL 2002). The prevalence of resistant isolates in countries where intensive animal
production is practiced is between 10% and 30%. When herds are held under strong antibiotic
selective pressures, due to the intensive use of antibiotics, the prevalence of resistant strains
rises to between 60% and 90% (HELMUTH 2000). As these bacterial strains are of
considerable potential clinical importance to human health, this is a matter of real concern
(FORSHELL and WIERUP 2006).
In addition to antimicrobial therapy, the successful treatment of salmonellosis relies heavily
on routine husbandry procedures recommended for control of infectious diseases. The
diarrheic pig massively contaminates its environment and is the single most important source
of infection for other pigs. Removal and isolation of sick animals, minimizing exposure to
infective material by scrupulous pen sanitation, frequent cleaning of water bowls, and
restriction of animal or staff movement from potentially contaminated to clean areas are
necessary. Efforts to modify management and environment to decrease stress and increase pig
comfort are essential adjuncts to specific therapy (SCHWARTZ 1999).
2.1.7 Vaccine
Nontyphoid Salmonella serotypes causing gastroenteritis in humans are most often
transmitted through the food chain by contamination of poultry and eggs, pork, beef and dairy
products, and, increasingly in the United States by vegetables and fruits that are irrigated with
Salmonella-contaminated water (MEAD et al. 1999). The question that had been posed by
investigators many years ago is whether vaccination would be a feasible approach when
combined with improved management practices for the control of Salmonella in poultry,
swine, and cattle to lessen the likelihood of Salmonella transmission through the food chain to
humans. In other words, could vaccines to prevent the infection and colonization of animals
with Salmonella contribute to the safety of food? One difficulty in attaining such a goal is the
probably correct assertion that most Salmonella serotypes that colonize animal species and
that are passed through the food chain to humans are essentially members of the normal flora
18
Literature review
of these animals and do not often cause disease. Hence, the design of any efficacious vaccine
to block colonization of or infection by “normal flora” constitutes a difficult task (CHIU et al.
2004).
It is generally accepted that live attenuated, orally administered Salmonella vaccines provide
the best protection against Salmonella infection. The superior protection achieved in
comparison to killed Salmonella bacterins and subunit vaccines is generally attributed to the
ability of live vaccines to stimulate a more effective cell mediated immune response. Oral
administration allows the attenuated mutant to utilize natural routes of infection, which
facilitates the crucial step of antigen presentation to lymphocytes in the gut associate
lymphoid tissue. These events induce the production of secretory IgA on mucosal surfaces
(CLARKE and GYLES 1993).
In many countries, however, inactivated vaccines are the only products available and their
efficacy is equivocal. Linton et al. (1970) considered that immunization with a killed vaccine
conferred only a weak protection against Salmonella infection in general. Davies and Wray
(1997) showed that vaccination of breeding stock on a farm with an inactivated S.
Typhimurium/ S. Dublin vaccine was associated with a reduction of Salmonella from 67%
to12% in weaned pigs and from 52% to 5% in the adult pigs. In Denmark, Dahl et al. (1997a,
b) demonstrated that the use of killed vaccines reduced the clinical impact of S. Typhimurium
infection in pigs but did not reduce subclinical infection.
Recently the development of specific non-reverting mutations to construct both homologous
and heterologous vaccine vehicles, with multiple attenuating mutations, has been achieved
(CHATFIELD et al. 1992). A mutation in the galE region in S. Typhi results in a deficiency
in UDP-glucose-4- epimerase, the enzyme which converts UDP-glucose to UDP-galactose, an
essential component of Salmonella species smooth LPS (LEVINE et al. 1989). In several
large trials on humans this mutant has appeared to be efficacious although a number of doses
have to be administered and some patients develop mild diarrhoea. Because of this success,
this mutation has been employed for many Salmonella serovars including S. Typhimurium
(NNALUE and LINDBERG 1990). However, a galE mutation in S.Choleraesuis does not
reduce virulence in swine and one assumes that this could be the case with other Salmonella
of serogroup C. This is due to the fact that galactose is missing from the oligosaccharide
repeating unit of the O antigen side chain of S. Choleraesuis (NNALUE and STOCKER
1986). The somatic antigens of Salmonella serogroups are the main component of host
specificity and facilitate survival in the gastrointestinal tract and entry onto deeper tissues
(NNALUE and LINDBERG 1990).
Another common attenuation involves the creation of auxotrophic mutants that require
metabolites not available in animal tissues. Aromatic mutants, which have a complete block
in the aromatic biosynthetic path way have a requirement for aromatic metabolites such as
para-aminobenzoate and 2, 3- dihydroxybenzoate. Oral vaccination with aroA, aroD mutants
in mice and calves has been effective in reducing disease and has been shown to be safe
(HOOK 1990; ROBERTSSON et al. 1983; SMITH et al. 1984). Experiments using an aroA
mutant of S. Typhimurium indicated that vaccinated pigs shed Salmonella significantly less
frequently than nonvaccinated pigs (LUMSDEN et al. 1991). Lumsden and Wilkie, (1992)
vaccinated pigs and challenged them, a good antibody response to LPS and killed bacteria
19
Literature review
was obtained, the aroA mutant avirulent in pigs was not shed in the faeces and significantly
reduced the severity of diarrhoea following challenge. Further studies by Lumsden, et al.
(1993) suggested that there may be a genetic basis for the immune response of pigs to
Salmonella because some animal genotypes gave a much better response.
Mutations in global regulatory pathways have also been a popular means of attenuation.
Several studies have utilized strains with deletions in the genes for adenylate cyclase (cya)
and for cAMP-receptor protein (crp). Cyclic AMP and cAMP-receptor protein regulate at
least 200 genes, many of which are required for breakdown of catabolites. Salmonella with
deletion mutations in the cya, crp genes have been shown to be safe and effective in eliciting
protective immunity in mice, chickens and pigs (COE and WOOD 1992; CURTISS and
KELLY 1987; STABEL et al. 1991, 1993). A large study evaluating the safety and efficacy of
a battery of Salmonella Choleraesuis Δcya, Δcrp isogenic mutants in mice indicates that
several of these strains are protective and safe (KELLY et al. 1992). Further studies in pigs of
these mutants showed that they were safe, although in some cases some caused a slight
temperature elevation and increased diarrhoea scores (KENNEDY et al. 1999). When pigs
were immunised orally with a commercial Δcya Δcrp-cdt Salmonella Choleraesuis vaccine
and challenged with S. Typhimurium, there was a reduction in the severity of clinical signs,
the shedding of the challenge strain in the faeces and carriage in the internal organs
(CHARLES et al. 1999). Intra-nasal inoculation with a similar vaccine reduced carriage of S.
Typhimurium in the lymph nodes but did not reduce shedding in the faeces (LETELLIER et
al. 1999b). Similarly Groninga et al. (2000) found that a S. Choleraesuis vaccine provided
some cross protection against experimental S. Derby infection. A recent field study used a live
Salmonella Choleraesuis vaccine to reduce the number of infections with Salmonella (MAES
et al. 2001). Twelve groups of c. 380 pigs were randomly allocated to either vaccination at 3
and 16 weeks or no vaccination. In the vaccinate only 0.6% of the 334 lymph nodes were
positive, whereas 7.2% of 321 of those of the controls were positive at slaughter.
A S. Choleraesuis strain which has been cured of the 50 Kb virulence plasmids has been
shown to be safe and efficacious in swine (KRAMER et al. 1992). The non-specific mutation
was obtained by repeated passage through porcine neutrophils. The plasmid free variant lacks
the ability to invade Vero cell monolayers and porcine neutrophils as well as having increased
resistance to killing by H2O2 and phagocytic killing by porcine neutrophils (ROOF et al.
1992c). Kramer and Vote (2000) found that the reduced virulence of granulocytes selected S.
Enteritidis vaccine was limited to the species of animal in which the granulocyte selection
was made.
Proven means of attenuation of serotype Typhimurium for mice did not yield a protective
vaccine when used to attenuate serotype Choleraesuis. This included using aro, galE, and
cyacrp mutations (KELLY et al. 1992; NNAULE et al. 1987). In an attempt to attenuate
serotype Choleraesuis some years ago, Kelly and colleagues discovered the cdt locus adjacent
to crp and found that a serotype Choleraesuis strain with the ∆cya and ∆crp-cdt double
mutations was avirulent and immunogenic in mice (KELLY et al. 1992). In the United States,
there are three licensed serotype Choleraesuis vaccines for swine. The Arco serotype
Choleraesuis vaccine was derived by chemical mutagenesis, but the basis of attenuation is not
very well understood. The Nobl vaccine was passaged through neutrophils and lost its
virulence plasmid, which is the principal attenuating defect (ROOF and DOITCHINOFF
1995). Argus-SC, which is distributed by Bayer but was originally developed by Upjohn, has
20
Literature review
the ∆cya ∆crp-cdt mutations. In studies at Upjohn, young pigs were immunized with the
Bayer Argus-SC vaccine and challenged, nonvaccinated and challenged, or not challenged
(KENNEDY et al. 1999). There was significant morbidity after challenge in animals that were
not vaccinated and a high degree of diarrhea. Since the animals were about 6 weeks of age,
there was no mortality, but the pigs continued to shed the challenge strain a week after
challenge. There was also a significant increase in body temperature compared to the control
nonvaccinated, nonchallenged pigs. Pigs vaccinated with the Nobl vaccine, which lacks the
virulence plasmid, had higher temperatures and diarrheal scores with more Salmonella shed in
feces after challenge than was the case in pigs immunized with the ∆cya ∆crp-cdt vaccine.
Both groups of immunized pigs performed better than the nonimmunized challenged pigs and
showed better weight gains. None of these vaccines for swine have been introduced in Europe
(except Germany) or other parts of the world.
Two stable rough mutants of Salmonella were studied as live oral vaccines by Trebichavsky
et al. (1997). The SF1591 mutant of S.Typhimurium (Ra chemotype) protected 8 germ-free
piglets against subsequent infection with virulent smooth S. Typhimurium LT2, whereas a
deep-rough mutant of Salmonella Minnesota mR595 (Re chemotype) did not protect 7
experimentally infected piglets. Cytokine and leukocyte profiles in the ilea of genotobiotic
piglets colonized for 1 week with either rough mutants alone or with rough mutants followed
by S. Typhimurium LT2 were also investigated. The ileal mucosae of piglets associated with
strain SF1591 alone were not inflamed. Villi contained activated macrophages and
enterocytes expressed transforming growth factor beta (TGF-β). Subsequent infection of
piglets with S. Typhimurium LT2 resulted in immigration of αβ T cells and immunoglobulin
A (IgA) response. In contrast, the ileal mucosae of piglets associated with strain mR595 alone
expressed heat shock proteins and inflammatory cytokines but not TGF-β. Acellular villi
contained numerous γδ T cells but not αβ T cells.
The use of vaccines should be considered as part of an overall strategy to control Salmonella
on the farm and their use should be in conjunction with other measures. Since many
Salmonella infections are sub-clinical, the use of vaccines in carrier animals will be to reduce
Salmonella shedding in finishers and thus reduce carcass contamination. Field trials are
necessary to determine whether this can be achieved economically (WRAY 2001).
2.1.8 Competitive Exclusion
To prevent the Salmonella carrier-state in pigs, new intervention strategies need to be
investigated. Competitive exclusion (CE) is one approach which has been used successfully
with poultry (BAILEY 1987; BAILEY et al. 1992; BLANKENSHIP et al. 1993) and further
information on the subject will be found in Wray and Wray, (2000). A mucosal competitive
exclusion culture from swine (MCES) was developed by Fedorka-Cray et al. (2000a).
Following application in suckling pigs and subsequent challenge with S. Choleraesuis, 28% of
the gut tissues were positive from the MCES treated pigs versus 79% positive tissues from the
control pigs. A 2-5 log10 reduction of Salmonella in the caecal contents or ileocolic junction
was observed in the MCES treated pigs when compared with the controls. These data indicate
that use of MCES may be a useful approach for control of Salmonella in swine. In contrast,
Anderson et al. (1999) found that CE did not reduce carriage of S. Typhimurium in lymph
nodes, although was a reduction of Salmonella shedding in the faeces.
21
Literature review
Letellier et al. (2000) assessed various treatments to reduce S. Typhimurium carriage in
swine. They found that acidification of drinking water, egg yolk immunoglobulins and an
endotoxin vaccine had no effect although a live attenuated S. Choleraesuis vaccine and the use
of bambermycin reduced the number of Salmonella in the mesenteric lymph nodes.
Another form of competitive exclusion has been described by Lovell and Barrow (1999) who
found that infection of gnotobiotic piglets with one strain of Salmonella prevented
colonisation of the intestine 24 hours later by a different Salmonella strain. This phenomenon
was also observed with isogenic mutants but not all mutants were as inhibitory.
Bacteriophage lysate was used by Lee and Harris (2001) to reduce the dissemination of
Salmonella in experimentally infected pigs, and its use reduced the numbers of Salmonella in
the large intestine by two logs as compared with the controls. However, much more extensive
studies are necessary to determine whether its use under field conditions is practical (WRAY
2001).
2.1.9 Salmonella infection in pigs
Pigs are an important reservoir of Salmonella for humans. Infection of man follows either
through direct contact or more frequently conclude from pork and pork products (FEDORKACRAY et al. 2000b). Infected pigs remain healthy carriers in most of the cases and as a
consequence are of great importance to public-health. The detection of Salmonella in pigs is
difficult, because infection does not commonly result in clinical symptoms. However,
subclinical Salmonella infections in pigs are an important food safety problem because of the
transmission route of Salmonella through the food chain to humans (MALORNY and
HOORFAR 2005).
The prevalence of Salmonella in the intestine of individual pigs from different sources is
extremely variable (GRAY et al. 1995, 1996a, 1996b). Individual animals may remain as
carriers for up to 36 weeks (WOOD and ROSE 1992). In Germany it is estimated that 7.3% of
all farmed pigs are carriers of Salmonella (HARTUNG 2001). Salmonella are a major cause
of economic losses in the pig production chain, resulting in millions of dollars in lost income
to the pork industry (SCHWARTZ 1990).
Salmonella has the potential to colonize the gut of the pigs. Most often pigs are healthy
carriers of Salmonella. With the exception of infections with S. Choleraesuis and some types
of S. Typhimurium, Salmonella infections usually produce no severe disease in pigs.
Salmonella Typhimurium is frequently associated with Salmonella infections of pigs
(DAUBE et al. 1998; VAN DER WOLF et al. 1999). The major contamination sources of pig
carcasses are pig (faeces, pharynx and stomach) and environment related (contact surfaces
and handling by workers) (BORCH et al. 1996; BOTTELDOORN et al. 2003).
Salmonella in pigs can be divided in two groups. The first group consists of the host-adapted
serotype Choleraesuis and is usually associated with acute septicemia and enterocolitis. The
second group consists of all other serotypes, which have broader host ranges and are
associated with systemic, enteric, or unapparent symptoms. In 1995, the United States
Department of Agriculture (USDA) National Animal Health Monitoring System conducted a
22
Literature review
national study of U.S. pork producers in 16 states. Salmonella was found in 17.5% of the 988
pens sampled (USDA 1997). Some of the more frequent Salmonella enterica serotypes from
nonclinical finishing swine were serotype Derby, serotype Agona, serotype Typhimurium,
serotype Brandenburg, and serotype Mbandaka. Serotype Choleraesuis was, after serotype
Derby, the second-most-isolated serotype from clinical swine (MALORNY and HOORFAR
2005).
In Europe, the diversity of isolated Salmonella serovars in slaughter pig lymph nodes was big
and in total 87 different serovars were isolated in the European Union. The five most
frequently isolated Salmonella serovars from lymph nodes in the European Union were
respectively in decreasing order S. Typhimurium, S. Derby, S. Rissen, S. 4,[5],12:i:- and S.
Enteritidis (Table 2.4). All these serovars, with the exception of S. Rissen, are frequent causes
of Salmonella infections in humans within the European Union. S. Typhimurium and S. Derby
serovars were highly predominant in lymph nodes; S. Typhimurium being the most common
serovar, detected in 40.0% of the Salmonella positive slaughter pigs and reported by all 24
Salmonella-positive Member States. S. Derby accounted also for an important proportion of
positive lymph nodes (14.6%) and was reported by 20 Salmonella-positive Member States.
Together 30 different serovars were reported from the surface of the slaughter pig carcasses
by the 13 Member States that carried out the test. The five most frequently isolated serovars
from carcasses were respectively in decreasing order S. Typhimurium, S. Derby, S. Infantis, S.
Bredeney and S. Brandenburg (Table 2.4). The former three serovars are frequent causes of
Salmonella infections in humans within the European Union. S. Typhimurium was the most
common serovar isolated on the surface of the slaughter pig carcasses and detected in 49.4%
of the Salmonella positive carcasses. The second most common serovar was S. Derby (24.3%
of the positive carcasses). S. Typhimurium and S. Derby were also the most commonly
reported ones in terms of the number of Member States, in total 10 of the 13 participating
Member States (EFSA 2008).
Table 2.4: Top-ten Salmonella serovars from lymph nodes and carcass swabs in the
slaughter pigs, in the EU and Norway, 2006-2007 (EFSA. 2008)
Serovars
S. Typhimurium
S. Derby
S. Rissen
S. 4,[5],12:i:S. Enteritidis
S. Anatum
S. Bredeney
S. Infantis
S. London
S. Brandenburg
Lymph nodes (%)
40.00
14.62
5.81
4.92
4.85
2.42
1.96
1.88
1.27
1.19
Serovars
S. Typhimurium
S. Derby
S. Infantis
S. Bredeney
S. Brandenburg
S. Reading
S. Enteritidis
S. Kedougou
S. 4,[5],12:i:S. Agona
Carcass swabs (%)
49.35
24.29
3.36
2.07
1.81
1.55
1.29
1.29
1.29
1.03
In Thailand, The National Salmonella and Shigella Center (NSSC) reported Salmonella
serovars isolated from animals (Table 2.5). The five most frequently isolated serovars in 2005
23
Literature review
were respectively in decreasing order S. Rissen, S. Anatum, S. Stanley, S. Amsterdam and S.
Weltevreden and the five most frequently isolated serovars in 2006 were respectively in
decreasing order S. Weltevreden, S. Corvallis, S. Enteritidis, S. Newport and S. Stanley
(NSSC 2005, 2006).
Table 2.5: Top-ten Salmonella serovars from animals in Thailand (NSSC. 2005,2006)
Serovars
2005 (%)
Serovars
2006 (%)
25.34
19.12
S.Rissen
S.Weltevreden
12.38
13.15
S.Anatum
S.Corvallis
11.59
12.35
S.Stanley
S.Enteritidis
9.63
6.37
S.Amsterdam
S.Newport
8.06
5.58
S.Weltevreden
S.Stanley
4.13
4.38
S.Enteritidis
S.Brunei
3.54
3.59
S.I 4,12:i:S.Typhimurium
3.14
3.19
S.Schwarzengrund
S. Virchow
1.77
3.19
S.Altona
S. Javiana
1.57
3.19
S.Kerefeld
S. Amsterdam
Transmission of Salmonella between hosts is thought to occur via the fecal-oral route of
exposure. They are carried asymptomatically in the intestines or gall bladder of many
animals, and are continuously or intermittently shed in the feces. They can also be carried
latently in the mesenteric lymph nodes or tonsils; these bacteria are not shed, but can become
reactivated after stress or immunosuppression (CFSPH 2005). A number of studies have
reproduced experimental infection by the oral route and, during acute disease; pigs will shed
up to 106 S. Choleraesuis g-1 faeces (SMITH and JONES 1967) or 107 S. Typhimurium g-1
faeces (GUTZMANN et al. 1976). Most authors report successful disease reproduction with
dose of 108-1011 Salmonella (GRAY et al. 1995, 1996a, 1996b). Fomites and mechanical
vectors (insects) can spread Salmonella. Further studies in pigs indicated that Salmonella
could be isolated from tonsils, mesenteric lymph nodes and intestine contents already 2 hours
after oral infection (BLAHA 2001). However, aerosol experiments in chickens and mice have
shown that infections with Salmonella can be regularly achieved via this route (CLEMMER
et al. 1960; DARLOW et al. 1961), giving support to the role that aerosol may also play in the
transmission and dissemination of Salmonella in other hosts. Further studies indicated that the
upper respiratory tract may be equally important in transmission and that the tonsils and lungs
may be important sites for the invasion and dissemination of Salmonella in pigs (FEDORKACRAY et al. 1995; GRAY et al. 1996a, b; DICKSON et al. 2002).
Weaned piglets are often infected by Salmonella. In general, S. Typhimurium tends to cause
disease in young pigs from 6 to 12 weeks of age. Disease from this serovar is rare in adult
animals, but infection is frequent. S. Chloeraesuis causes disease among a wider range of
ages. Mortality tends to be higher in younger than in older pigs, while morbidity is often equal
regardless of age. Disease from S. Chloeraesuis in adult pigs is not common. However, if a
susceptible population is exposed, the animals will be significantly affected (WILCOCK and
SCHWARTZ 1992). The occurrence of salmonellosis in suckling pigs is rare, presumably
24
Literature review
because of lactogenic immunity, but infection is not uncommon (FEDORKA-CRAY et al.
2000b; WILCOCK et al. 1976; GOOCH and HADDOCK 1969; SCHWARTZ 1999).
Clinical porcine salmonellosis can be separated into two syndromes: one involves S.
Typhimurium, which is associated with enterocolitis, while the other involves S. Choleraesuis
and is usually associated with septicaemia. Septicaemic pigs are inappetent, lethargic and
febrile, with temperatures of up to 41.7 °C. Respiratory signs may consist of a shallow, moist
cough and diaphragmatic breathing. Clinical signs first appear after 24-36 h of infection
(REED et al. 1986). In most outbreaks, morbidity is high and mortaliity is variable but
generally less than 10% (WILCOCK and SCHWARTZ 1992; REED et al. 1986). Diarrhoea
is normally not a feature of S. Choleraesuis infection until at least the fourth or fifth day of
infection. It may last from 5 to 7 days after onset if chronic reinfection is not occurring. Gross
lesions include colitis, infarction of gastric mucosa, swollen mesenteric lymph nodes,
splenomegaly, hepatomegaly and lung congestion. Random white foci of necrosis are often
observed on the liver (WILCOCK and SCHWARTZ 1992; REED et al. 1986). The
septicaemic form of porcine salmonellosis is usually caused by S. Choleraesuis although
other serovars may occasionally cause acute disease. The clinical signs and post-mortem
findings are well described in the standard text books and will not be considered further
(WRAY 2001).
Enterocolitis in pigs is typically associated with S. Typhimurium infection and occasionally
with S. Choleraesuis infection. In contrast to the septicaemic disease, the initial sign of
infection is often watery, yellow diarrhoea. Infected pigs show inappetence, fever and
lethargy. Mortality is usually low. However, morbidity can be high within a few days of
infection (WILCOCK and SCHWARTZ 1992). Major gross lesions found at necropsy are
focal or diffuse necrotic colitis and typhlitis. Mesenteric lymph nodes are greatly enlarged.
Intestinal lesions develop as red, rough mucosal surfaces, which may also have gray-yellow
debris. Colon and caecal content are bilestained and scant, often with black or sand-like gritty
material on the surface. Intestinal necrosis may be seen as sharply delineated button ulcers,
often associated with resolving lesions (WILCOCK and SCHWARTZ 1992; WOOD and
ROSE 1992; WILCOCK and OLANDER 1978). In case of S. Typhimurium enterocolitis, the
liver and spleen are not enlarged except by terminal congestion (WILCOCK and
SCHWARTZ 1992).
The microscopic lesion that is most often associated with S. Choleraesuis in pigs is the
paratyphoid nodule. This lesion can be viewed in the liver as clusters of histiocytesamid foci
of acute coagulative hepatocellular necrosis and corresponds to the white foci seen grossly
(LAWSON and DOW 1966). Other lesions may include fibrinoid thrombi in venules of
gastric mucosa and cyanotic skin and glomerular capillaries. Swelling of histiocytes and
epithelial cells, typical of gram-negative sepsis, as well as hyperplasia of reticular cells of the
spleen and lymph nodes, is often observed (FEDORKA-CRAY et al. 2000b; WILCOCK et al.
1976, WILCOCK 1978). Histopathological examination reveals necrosis of cryptic and
surface enterocytes, which may be local or diffuse. The lamina propria and submucosa
contain macrophages and lymphocytes, with neutrophils observed only in the very early
stages of disease. It is not uncommon to see lymphoid atrophy or regenerative hyperplasia
associated with this disease (FEDORKA-CRAY et al. 2000b; REED et al. 1986, WILCOCK
et al. 1976).
25
Literature review
2.1.10 Dynamics of Salmonella
The infected swine are an important reservoir and source for introduction and transmission of
Salmonella on-farm. Excretion of Salmonella in feces onto the pen floor is sufficient to serve
as a source for infection of other swine in the same pen or even in the same building (WOOD
et al. 1989; FEDORKA-CRAY et al. 1994, 2005; WEIGEL et al. 1999; HURD et al. 2001a;
2002). According to Friendship (1992), the most important means by which an infectious
agent enters in a herd is by direct pig-to-pig spread, after the introduction of a carrier animal.
However, Berends et al. (1996) suggested that contamination of endemic flora in finishing
sites was the predominant source of infection of finishing pigs. Infection from breeding farms
appears more important when pigs from various sources are mixed on finishing farms.
Baggesen et al. (1997) isolated Salmonella from feces, pens, dust, equipment, and slurry
during their studies in twelve pig farms, with different levels of Salmonella infection. Ghosh
(1972) and Davies et al. (2000a) studied breeding herds where carriers were found frequently.
In both studies the introduction of Salmonella in the studied herds was attributed to breeding
animals. Also, Letellier et al. (1999a) in their study found breeding animals as the source for
the introduction and dissemination of Salmonella into herds.
Although horizontal transmission of Salmonella occurs through fecal-oral or aerogenous
transmission, other vectors must be considered when discussing introduction and
dissemination of the organism in the farms. The number of potential sources of Salmonella
infection is seemingly endless. Observed sources of contamination include rodents, insects,
birds, other animals, humans and contaminated feed and feedstuffs (CLARKE and GYLES
1993; MCCHESNEY et al. 1995). Rodent fecal samples have been shown to contain up to 105
CFU of Salmonella (HENZLER and OPITZ 1992). During an investigation of Salmonella
contamination, which involved 23 pig farms, Davies and Wray (1997) found a wide range of
animals, including rats, mice, cats and birds to be infected. Cats and birds were associated
with contamination of feed and grain stores, and rodents were involved in the perpetuation of
infection in specific buildings on the farm. Flies and dust can also act as mechanical vectors
that spread Salmonella throughout the environment (GREENBERG et al.1970; KHALIL et al.
1994; OLSEN and HAMMACK 2000). Cockroaches are found in almost any place. Much
anecdotal evidence exists of cockroaches being a health risk and a vehicle for the spread of
infectious organisms, including Salmonella (BENNET 1993). Cockroaches are only one part
of the insect flora of domestic and rural environments. Beetles (lesser mealworm) are also
frequently found in farms, and recent studies have shown that they are important reservoirs
for Salmonella (STEELMAN and WALDROUP 2000).
Animal feed is a recognized source of pathogenic microorganisms for farm livestock
(LINTON and JENNET 1970; DAVIES and HINTON 2000). Harris et al. (1997) described a
Salmonella prevalence of 2.9% in feeds and feed ingredients taken from farm environments.
Feed trucks have also been implicated as a source for feed and feedstuffs contamination
(FEDORKA-CRAY et al. 1997a). Feed containing ingredients of animal origin is a potential
source of Salmonella infection to herds, but it should be emphasized that ingredients of
vegetable origin can also be a source of Salmonella-contaminated feed. Australian Funeral
Directors Association (AFDA) survey of animal and plant protein processors demonstrated
that 56.4% of the animal protein and 36% of the vegetable protein products taken from 124
processors were positive for Salmonella (MCCHESNEY et al.1995). However, the most
26
Literature review
frequent Salmonella serotypes isolated from feed are rarely the most prevalent in animals.
Even so, the potential for contamination exists, and the consequences can be serious
(DAVIES and HINTON 2000).
Water is not as likely a source of infection unless surface water is used for consumption or
pigs have access to recycled lagoon water (SCHWARTZ 1999). However, rodents and birds
can contaminate the water, increasing the chance of the spread of infection within the herd.
Salmonella have also been shown to form biofilms on glass and chlorinated polyvinyl
chloride (CPVC) pipes (JONES and BRADSHAW 1996). This could enable the organisms to
effectively colonize water pipe lines in the farms, constituting a potential of maintenance and
dissemination of Salmonella.
Although wild birds are recognized as carriers of Salmonella, evidence suggests that infected
birds are rarely found. Usually, the Salmonella-contaminated environment is regarded as the
main source of infections among wild birds, with the organisms being acquired during food
gathering and drinking (MURRAY 2000).
Farm environments (facilities and equipment) may become persistently contaminated with
Salmonella following the introduction of the bacteria in the herd. Environmental and
management practices may contribute to the dissemination and maintenance of Salmonella
within herds. Davies and Wray (1996) compared two methods of disposal of calf carcasses
artificially contaminated with Salmonella Typhimurium for their capacity to eliminate the
bacteria and spread to the surrounding environment. It was found that the frequency of
Salmonella isolations decreased more rapidly from the burial pit than from the decomposition
pit (4 months compared with 6 months to disappear). During this study, a large population of
blowfly larvae developed within 2 weeks in the decomposition pit and these were Salmonellapositive. Salmonella were also found in wild bird droppings in the vicinity of the pit for a
period of 4 weeks after loading the pit. Nearby drainage ditches were Salmonella-positive,
initially close to the pit but, after 3 weeks, up to 12 meters along the drain.
As mentioned, Salmonella may persist in the environment for long periods, and cleaning and
disinfection routines procedures may not always be efficient in eliminating the contamination.
Bacteria of the genus Salmonella are hardy, surviving freezing and desiccation very well, and
persisting for weeks, months, or even years in a suitable organic substrate (SCHWARTZ
1999). The temperature range for growth of Salmonella is between 5.5 and 45°C (DOYLE
and MAZZOTTA 2000). Modern intensive livestock production has created problems of
excreta disposal, and Salmonella may survive for long periods in infected feces and slurry,
where their survival is dependent on a number of factors, especially the serotype and the
climatic conditions (WRAY 1994). Gray et al. (1995) found that Salmonella Choleraesuis
survives in dry feces for at least thirteen months post-shedding, demonstrating the importance
of cleaning organic matter from the environment. Davies and Wray (1997) found high levels
of Salmonella persisting in pig pens after disinfection. Gebreyes et al. (1999) detected
Salmonella in drag swabs of floors from barns after cleaning and disinfection, and before pig
placement in 82% of the studied cases. In some of the studied cases, finisher pigs shed a
Salmonella serotype that had been found on drag swabs.
27
Literature review
In three persistently infected herds, Dahl et al. (1997a, b) studied the effects of moving pigs to
clean and disinfected facilities, at different ages before Salmonella Typhimurium had been
detected either serologically or bacteriologically. No detectable infection was observed at
slaughter either serologically or bacteriologically in the moved groups of pigs, whereas a
proportion of the pigs raised at the same time in the continuous systems on the farms were
found to be infected. Fedorka-Cray et al. (1997b) were also able to raise piglets free of
infection with Salmonella enterica serovars up to six weeks of age, by removing the piglets
from infected herds to isolation facilities when they were weaned at 10-21 days of age. The
results of these studies demonstrate that the environment plays a critical role in the
Salmonella infection epidemiology in swine herds. As mentioned, an important risk factor for
introducing disease to a swine herd is direct exposure to infected animals. However, humans
may act as mechanical vectors transmiting pathogens among groups of pigs. They are
believed to be another important risk factor for the introduction and dissemination of
pathogens in swine herds (MOORE 1992; FREINDSHIP 1992). Amass et al. (2000)
conducted a study evaluating the efficacy of boot baths in biosecurity protocols in swine
farms. Results of this study demonstrated that most of the on-farm washing and disinfection
of boots are not efficacious for eliminating the contamination with pathogens.
An interesting study was reported by Letellier et al. (1999a), showing the complexity of
Salmonella epidemiology in swine herds. The objective of this study was to identify, in herds
known to be positive for the presence of Salmonella, possible sources of contamination, and
evaluate the prevalence and distribution of Salmonella at the different levels in an integrated
swine production system. In the herds studied, many environmental samples such as water
taken in the pen, boots, floors, doors, rodents or rodent‟s nests were found to be contaminated.
Although it was not concluded that these positive samples were the source of the infection for
swine, there is no doubt that they could be involved in subsequent contamination if
appropriate measures are not taken. Flies were positive for Salmonella on the highly
contaminated farms and as mentioned before, they may be involved in the dissemination of
Salmonella in the environment as carrier of microorganisms (MORSE and DUNCAN 1974;
KHALIL et al. 1994). It is also important to note that in these studied herds, dead animals
were considered as possible sources of contamination, and that boots of animal caretakers
were also found positive for Salmonella indicating that a particular attention should be given
to improve the disinfectant of boots to avoid the dissemination of Salmonella, in agreement
with Amass et al. (2000) and Blaha (2000).
2.1.11 Salmonella infection in humans
Salmonellosis is one of the most common causes of food borne diarrheal disease worldwide.
Most of these infections are zoonotic and are transmitted from healthy carrier animals to
humans through contaminated food. The main reservoir of zoonotic Salmonella is food
animals, and the main sources of infections in industrialized countries are animal-derived
products, notably fresh meat products and eggs. In developing countries, contaminated
vegetables, water, and human-to-human transmission are believed to contribute to a
comparatively larger proportion of the human cases than those in industrialized countries
(WEGENER et al. 2003). However, the incidence of human salmonellosis increased in most
industrialized countries in the 1980s and 1990s. Rapid spread of a limited number of
successful Salmonella clones in different sectors of food animal production (swine, broiler
28
Literature review
chickens, and particularly layer hens) has been suggested as the most important cause of this
increase (THONS 2000).
In Europe, Salmonella trends have been well-documented over the past 20 years
(SCHLUNDT et al. 2004). Salmonellosis remained the second most commonly reported
human zoonoses in spite of a decrease in incidence over the last three years in European
Union (EU). The reported data supported the notion that the major sources of human
Salmonella infections are eggs, and meat from pigs and poultry.
In 2006, a total of 160,649 confirmed cases of human salmonellosis were reported in the EU
(Table 2.6). The EU incidence was 34.6 cases per 100,000 populations, ranging from none to
235.9 cases per 100,000 populations. Germany accounted for 32.7% of all reported cases,
whereas incidence was greatest in the Czech Republic. In 2006, there was a 7.6% decrease in
incidence from 2005, and this was part of a significant, decreasing trend over the past three
years. As in previous years, S. Enteritidis and S. Typhimurium were the most frequently
reported serovars (Table 2.7).
The highest numbers of reported human cases were for age groups 0-4 years and 5-14 years.
A seasonal peak in the number of cases during the late summer and autumn was generally
observed in all Member state (MS), and S. Enteritidis demonstrates a much more prominent
peak than the other serovars (EFSA 2007).
In Thailand, investigation during the last decade revealed that Salmonella spp. is one of the
most common organisms causing infectious diarrheal disease. The National Salmonella and
Shigella Center of the national institute (Department of Medical Sciences, Ministry of Public
Health; NSSC) has been working as the national reference laboratory for detection,
identification and characterization Salmonella and Shigella. Parts of the annual reports for
2005 and 2006 are showed in table 2.8 (NSSC 2005, 2006).
In EU, a wide range of foodstuffs was tested for Salmonella, but the majority of samples were
from various types of meat and products thereof. As in previous years, MS reported
Salmonella findings most frequently from investigations of poultry meat, followed by those of
pig meat.
In 2006, the average proportion of Salmonella positive samples in fresh broiler meat was
5.6% in EU, but also some very high Salmonella frequencies (up to 67.6%) were reported by
MS. Salmonella positive samples were found in moderate proportions in pig meat (on average
1.0%, range 0-11.5%) (EFSA 2007).
Currently, Salmonella more commonly refers to Salmonella contaminated pork as one of the
many sources of food-borne diseases in man. Zoonotic Salmonella infections are
transmissible from pig to man and cause human disease (BLAHA 2000).
29
Literature review
Table 2.6: Reported salmonellosis cases in humans in 2006 (EFSA 2007)
Country
Austria
Belgium
Cyprus
Czech Republic
Denmark
Estonia
Finland
France
Germany
Greece
Hungary
Ireland
Italy
Latvia
Lithuania
Luxembourg
Malta
Netherlands
Poland
Portugal
Slovakia
Slovenia
Spain
Sweden
United Kingdom
EU Total
Cases
4,787
3,630
99
25,102
1,662
453
2,574
6,339
52,575
912
9,752
422
5,164
866
3,557
308
63
1,667
13,362
415
8,784
1,519
5,117
4,056
14,055
167,240
Confirmed cases
4,787
0
99
24,186
1,662
453
2,574
6,339
52,575
825
9,389
420
5,164
781
3,479
308
63
1,667
12,502
387
8,242
1,519
5,117
4,056
14,055
160,649
Cases/100,000
57.9
0
12.9
235.9
30.6
33.7
49.0
10.1
63.8
7.4
93.2
9.9
8.8
34.0
102.2
67.0
15.6
10.2
32.8
3.7
152.9
75.8
11.7
44.8
23.3
34.6
Table 2.7: Top-ten Salmonella serovars from humans source in EU (EFSA 2007)
Serovars
S. Enteritidis
S. Typhimurium
S. Virchow
S. 4,5,12:I:S. Infantis
S. Newport
S. Hadar
UNNAMED
S. Stanley
S. Derby
2006 (%)
59.6
16.7
1.7
1.5
1.2
1.2
0.8
0.8
0.7
0.6
Serovars
S. Enteritidis
S. Typhimurium
S. Hadar
S. Virchow
S. Infantis
S. Agona
S. Newport
S. Stanley
S. Bovismorbificans
S. Derby
30
2005 (%)
69.1
12.8
2.1
1.0
0.8
0.6
0.6
0.5
0.5
0.5
Literature review
Table 2.8: Top-ten Salmonella serovars from humans in Thailand (NSSC 2005, 2006)
Serovars
S. Enteritidis
S. Stanley
S. Cholerasuis
S. Rissen
S. Weltevreden
S. I.4,5,12:i:S. Corvallis
S. Anatum
S. Typhimurium
S. Kedougou
2006 (%)
18.46
10.68
7.95
7.64
6.89
4.30
4.16
3.86
3.28
2.73
Serovars
S. Stanley
S. Enteritidis
S. Weltevreden
S. Rissen
S. Corvallis
S. Cholerasuis
S. Anatum
S. Typhimurium
S. Virchow
S. I.4,5,12:i:-
2005 (%)
12.43
10.98
9.02
8.91
5.72
4.82
4.80
2.64
2.29
2.21
In different epidemiological studies pork has been described as a source for human
salmonellosis (MØLBAK and HALD 1997; DELPECH et al. 1998; PONTELLO et al. 1998;
MURASE et al. 2000). In Denmark, the Netherlands and Germany it was estimated that 15–
20% of all human cases of salmonellosis were associated with the consumption of pork
(BORCH et al. 1996; BERENDS et al. 1998; STEINBACH and KROELL 1999).
The incubation period ranges from five hours to seven days, but clinical signs usually begin
12 hours to 36 hours after ingestion of a contaminated food. Shorter incubation periods are
generally associated with either higher doses of the pathogen or highly susceptible people
(FORSHELL and WIERUP 2006).
In humans, salmonellosis varies from a self-limiting gastroenteritis to septicemia. Whether the
organism remains in the intestine or disseminates depends on host factors as well as the
virulence of the strain. Asymptomatic infections can also be seen. All serovars can produce all
forms of salmonellosis, although a given serotype is often associated with a specific syndrome
(e.g. Salmonella Choleraesuis tends to cause septicemia) (CFSPH 2005).
Infection with Salmonella in humans is usually presented by acute fever, diarrhoea and
abdominal cramps. Although chronical carriage of non-typhoidal Salmonella species is rare,
faecal shedding can last 4 to 7 weeks after infection in adults and in children, respectively
(BUCHWALD and BLASER 1984). In immunocompromised people, such as young
children, elderly, cancer and HIV-patients and pregnant women, bacteraemia and extraintestinal infections may occur including meningitis, septic arthritis, osteomyelitis,
cholangitis, pneumonia and endo-arteritis, occasionally resulting in death (HOHMANN
2001; MC CABE- SELLERS and BEATTIE 2003; CHIU et al. 2004).
In addition to clinical disease, there is the hazard of therapeutic failure due to antimicrobial
resistance of the causative Salmonella strains. Antimicrobial therapy is essential in the
treatment of severely ill people who are at risk for extra-intestinal infection and the
antimicrobial drugs of choice are fluoroquinolones, trimethoprim -sulfamethoxazole,
ampicillin and third-generation cephalosporins (HOHMANN 2001).
31
Literature review
Infections with S. Typhimurium, known for its potential resistance to ampicillin,
chloramphenicol, streptomycin, sulphonamides and tetracycline (ACSSuT), are occurring
with increasing frequency in animals as well as in humans in different European countries
and in the United States (POPPE et al. 1998). Multiresistant strains were first isolated from
cattle in the United Kingdom, but subsequently spread through the rest of Europe and the
United States (THRELFALL et al. 1997). Simultaneously with the use of antimicrobial
agents in human and veterinary medicine, the spectrum of resistance has increased and
resistance to trimethoprim, fluoroquinolones and third-generation cephalosporins are regularly
noticed (WIUFF et al. 2000; RABSCH et al. 2001). This is a major concern in human
medicine, because these antimicrobial agents are essential for clinical use (HOHMANN
2001).
In EU 2006, European Food Safety Authority (EFSA) reported the majority of S. Enteritidis
isolates from humans were fully sensitive to all antimicrobials tested. Compared to 2005,
there was an increase in resistance to nalidixic acid, sulphonamids and ampicillin to 14.8%,
8.0% and 8.1%, respectively. The resistance to ciprofloxacin remained at low level (0.6%) in
EU. From the S. Typhimurium isolates 39.7% were resistant to more than 4 of the
antimicrobials tested, and there was an increased in resistance to sulphonamids and
streptomycin to 59.7% and 51.9%, respectively. The resistance to ciprofloxacin in S.
Typhimurium isolates was 0.7%. Nalidixic acid is an indicator for increasing resistance to
fluoroquinolones (e.g. ciprofloxacin), which are antimicrobials regarded as critically
important for treatment of human cases. In Salmonella isolates from broiler meat, high
proportion resistance (50.8%) was observed to nalidixic acid and the resistance to
ciprofloxacin was 4.6% in EU. In isolates from pig meat these resistance levels were clearly
lower (3.8% and 0.7%, respectively) (EFSA 2007).
Human Salmonella infections also have a significant economical impact. The estimated
costs associated with human salmonellosis are nearly $1 billion (range, $0.6 to $3.5 billion)
annually in the United States (MALORNY and HOORFAR 2005). A study in The
Netherlands showed that the costs associated with human gastroenteritis during 1999 can be
estimated at € 345 million (ranging between € 252 and € 531 million). The costs for
Salmonella infections were estimated at € 4 million or about 1% of the total costs (VAN
DEN BRANDHOF et al. 2004). These figures include direct medical and non-medical costs
and indirect costs such as absence from school or work. In a study in England, it was
concluded that hospital costs were the highest for Salmonella compared to other
infectious intestinal diseases. Total costs for Salmonella infections were estimated to be ₤
46.4 million, the costs for all infectious intestinal diseases were ₤ 742.8 million (ROBERTS
et al. 2003).
32
Literature review
2.1.12 Foodborne outbreaks caused by Salmonella spp.
Foodborne diseases are among the most serious health problems affecting public health and
development worldwide (WHO 1984). Industrialization, mass food production, decreasing
trade barriers, and human migration have disseminated and increased the incidence and
severity of foodborne diseases worldwide (GOMEZ et al. 1997; KAFERSTEIN et al. 1997;
TODD 1997). Salmonellae are among the most common bacterial food borne pathogens
worldwide (TODD 1997). They cause an estimated 1.4 million cases of food borne disease
each year in the United States alone.
In June 2006, the British Broadcasting Corporation (BBC) reported that the Cadbury
chocolate manufacturer withdrew a number of products when products contaminated with
Salmonella caused up to 56 cases of salmonellosis. The problems had been traced to a leaking
pipe at a Cadbury plant in Herefordshire in January 2006, though the announcement was not
made until June (ANONYMOUS 2008d).
The U.S. Government reported that 16.3% of all chickens were contaminated with Salmonella
in 2005, and in the late 1990s as many as 20% were contaminated. In the mid to late twentieth
century, Salmonella enterica serovar Enteritidis was a common contaminant of eggs. This is
much less common now with the advent of hygiene measures in egg production and the
vaccination of laying hens to prevent Salmonella colonization. Many different Salmonella
serovars also cause severe diseases in animals other than human beings (ANONYMOUS
2008d).
In EU 2006, twenty-two Member states (MS) and three non-MS reported a total of 3,131 food
borne outbreaks of human salmonellosis, which constituted 53.9% of the total number of
reported outbreaks in the EU and in the reporting non-MS (Table 2.9).
In 2006, Germany, Austria, Slovakia, Spain and Poland accounted for 78.0% of the
Salmonella outbreaks, reporting 908, 453, 452, 338 and 292 outbreaks respectively. Germany
reported 330 general and 578 household outbreaks, involving 4,851 persons of which seven
died. The majority of Salmonella outbreaks in Austria were small household outbreaks
(83.9%), with 2 – 8 cases. In total, the non-MS, Norway, Romania and Switzerland reported
14 general and 6 household outbreaks caused by Salmonella. S. Enteritidis was the
predominant Salmonella serovar associated with outbreaks (Table 2.9) and accounted for
29.8% of all reported outbreaks, 47.0% of all hospitalizations and 25.5% of all deaths in 2006.
For 37.9% of the outbreaks caused by Salmonella, no serovar was specified. In outbreaks
caused by S. group D, S. Enteritidis or S. Stanley involving more than 25 human cases,
relatively large proportions of cases required hospitalization (30.4%, 19.6% and 24.2%,
respectively). In two S. Enteritidis outbreaks in Hungary and France, three out of four cases
required hospitalization. Slovakia reported 451 S. Enteritidis outbreaks affecting 1,849
persons and Spain reported S. Enteritidis as the cause of 100 general and 63 household
outbreaks, involving 1,724 persons and causing one death (EFSA 2007).
33
Literature review
Table 2.9: Salmonella serovars reported for foodborne outbreaks, 2006 (EFSA 2007)
Outbreaks
Human cases
Serovars
No.
Salmonella spp.
S. Enteritidis
S. Typhimurium
S. group D
S. group B
S. group C
S. Infantis
S. Hadar
S. Kentucky
S. Paratyphi B
S. Virchow
S. Abony
S. Ajiobo
S. Bovismrbificans
S. Give
S. Montevideo
S. Muenchen
S. Newport
S. Paratyphi A
S. Saintpaul
S. Stanley
Total
1,188
1,729
129
26
12
6
5
4
4
4
4
2
2
2
2
2
2
2
2
2
2
3,131
% of total
37.9
55.2
4.1
0.8
0.4
0.2
0.2
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
100.0
No.
6,697
13,853
1,088
207
98
24
48
33
8
25
138
6
161
4
55
52
34
59
8
12
95
22,705
No. admitted
Deaths
192
2,714
149
63
0
0
9
1
2
1
2
2
13
1
0
5
0
7
0
1
23
3,185
6
14
3
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
23
Phage type data were provided for 25.2% of all S. Enteritidis outbreaks. Phagetype
information was only provided for a subset of the outbreaks reported by Austria, Belgium,
Germany, Slovakia and the United Kingdom. The five most commonly reported phage types
were S. Enteritidis PT4, PT8, PT21, PT6 and PT6a; accounting for 120, 110, 92, 32 and 23
outbreaks respectively.
For the 129 outbreaks caused by S. Typhimurium, phagetypes were provided for 20.2%.
Phagetype information was reported for the majority of S. Typhimurium outbreaks in Austria
(25 outbreaks) and Norway (1) and from Slovakia (1), Sweden (1) and the United Kingdom
(2). Nine different phage types were reported, and the most common phagetypes were DT104
(7 outbreaks), DT120 (4) and DT193 (3) (EFSA 2007).
In February 2007, the U.S. Food and Drug Administration (FDA) issued a warning to
consumers not to eat certain jars of Peter Pan peanut butter or Great Value peanut butter due
to risk of contamination with Salmonella Tennessee (ANONYMOUS 2008d).
34
Literature review
In March 2007, around 150 people were diagnosed with salmonella-poisoning after eating
tainted food at a governor's reception in Krasnoyarsk, Russia. Over 1,500 people attended the
ball on March 1 and fell ill as a consequence of ingesting salmonella-tainted sandwiches
(ANONYMOUS 2008d).
In December 2007, about 150 people were sickened by salmonella-tainted chocolate cake
produced by a major bakery chain in Singapore (ANONYMOUS 2008d).
In Sweden, 51 domestic cases with Salmonella Stanley were reported in July and August
2007. Domestic cases of this serotype are unusual in Sweden. The majority of cases were
adults. An outbreak investigation was initiated in July involving the Swedish Institute for
Infectious Disease Control, the Swedish Food Safety Authority and the county medical
officers. An Enter-net alert was issued but did not reveal anything out of the ordinary in other
countries. The case control study performed pointed strongly towards alfalfa sprouts. The
cases had eaten alfalfa sprouts from various food stores or restaurants throughout Sweden.
Most of the product was traced to a large scale sprout producer who had imported alfalfa
seeds through a wholesaler in Denmark from an Italian seed producer. The same seeds had
also been sold to other sprout growers in Sweden. There were no longer any sprouts or seeds
of the implicated batches in the grower's stock but samples (unpasteurized seeds) were taken
from another bag of seeds of the same batch and brand and tested positive for Salmonella but
for another serotype, S. Mbandaka. An alert was issued through the Rapid Alert System for
Food and Feed (RASFF) on 31 August 2007 and the sprouts were withdrawn from the
Swedish market. The grower had heat treated the seeds before sprouting but it did not seem to
have been efficient. Later, four cases with S. Mbandaka from May and June were recognized
to be infected with S. Mbandaka having the same molecular typing patterns as the S.
Mbandaka isolated from the sprouts and two of the cases remembered eating sprouts
(WERNER et al. 2007).
In Norway, an alert was raised when four domestic cases with Salmonella Weltevreden were
reported in October 2007. Domestic cases of this serotype are unusual in Norway. An
outbreak investigation was initiated involving the Norwegian Institute of Public Health (FHI),
the Norwegian Food Safety Authority (NFSA), and the municipal medical officers and an
urgent inquiry was sent to the former Enter-net network through ECDC. In response to the
inquiry, 19, 19 and 8 cases were reported in Norway, Denmark and Finland respectively. The
demographic characteristics were comparable: the cases were adults and predominantly
female.
On 23 October 2007, a Salmonella isolate obtained from a major Danish alfalfa sprout
producer was serotyped as S. Weltevreden. The Danish food authorities issued an alert
through RASFF on the same day. The isolate was later shown to have the same molecular
typing patterns as the isolates from the case-patients from Denmark, Norway and Finland. S.
Weltevreden was also verified in the sprouts sold in Finland and Norway.
The seeds for growing the alfalfa sprouts had been imported to Denmark in July and August
2007. The Danish producer had then exported part of the batch of seeds to a Norwegian
alfalfa sprout producer in September. The batch of seeds used in Denmark and Norway was
35
Literature review
traded via retailers in Germany and the Netherlands to Denmark, and originated from Italy.
The seeds used in Finland came from the same Dutch supplier. The alfalfa sprouts were
recalled and withdrawn in Denmark on 18 October, in Norway on 23 October, and in Finland
on 28 October 2007 (EMBERLAND et al. 2007).
As of July 8, 2008, from April 10, 2008, the rare Saintpaul serotype of Salmonella enterica
caused at least 1017 cases of salmonellosis food poisoning in 41 states throughout the United
States, the District of Columbia, and Canada. As of July 2008, the U.S. Food and Drug
Administration suspects that the contaminated food product is a common ingredient in fresh
salsa, such as raw tomato, fresh jalapeño pepper, fresh serrano pepper, and fresh cilantro. It is
the largest reported salmonellosis outbreak in the United States since 1985. New Mexico and
Texas have been proportionally the hardest hit by far, with 49.7 and 16.1 reported cases per
million, respectively. The greatest number of reported cases have occurred in Texas (384
reported cases), New Mexico (98), Illinois (100), and Arizona (49). There have been at least
203 reported hospitalizations linked to the outbreak, it has caused at least one death, and it
may have been a contributing factor in at least one additional death. The Center for Disease
Control and Prevention (CDC) maintains that "it is likely many more illnesses have occurred
than those reported." If applying a previous CDC estimated ratio of non-reported
salmonellosis cases to reported cases (38.6:1), one would arrive at an estimated 40,273
illnesses from this outbreak (ANONYMOUS 2008d).
2.2 Detection methods and methods for surveillance of Salmonella
2.2.1 Bacteriological detection methods
Microbiological culture techniques have been the predominant diagnostic tool to detect
Salmonella infections for a long time and they are still considered as the “gold standard” for
Salmonella diagnosis (FUNK 2003).
Isolation of Salmonella from the pork production chain is a prerequisite for the estimation of
the prevalence of infection at primary production and the frequency of contamination of pork
products. Isolation can only be performed by the use of bacteriological examination. Isolation
of Salmonella is also necessary for the characterisation of the different serovars involved, for
assessing the extent of antimicrobial resistance, for tracing of infections, e.g. in outbreaks, and
for risk assessment, e.g. of different types of products. The result of the isolation and
subsequent studies and estimations will, however, influenced by the bacteriological method
applied, as no diagnostic method has a sensitivity of 100%.
Salmonella can be readily isolated from samples originating from pigs or pig herds showing
clinical signs of salmonellosis. In such cases, the pigs excrete high numbers of bacteria in
their faeces; these can be detected directly, without any enrichment, by plating on selective
agars. However, clinical infection in pigs is rare compared to the occurrence of subclinical
infection. The latter is characterised by animals being infected without showing any signs of
illness and these animals may be presented for slaughter, becoming a source of contamination
for the slaughter plant and products.
36
Literature review
Subclinically infected animals typically exhibit intermittent excretion of low numbers of
Salmonella in their faeces; this challenges methods of bacteriological isolation. Salmonella
can, however, be isolated from intestinal lymph nodes, reflecting a localised intestinal
infection, a previous exposure to Salmonella or, possibly, spread from internal organs as a
consequence of generalised infection.
The sensitivity of the bacteriological methods depends on both the isolation media used and
the matrix of the samples (type and amount) that are investigated (EFSA 2006).
The major advantage of bacteriological isolation is that the specificity of the culture methods
is considered to be 100% as the final result of bacteriological diagnosis is the identified
isolate (NOLLET 2008).
Standard methods for the isolation of Salmonella, e.g. ISO 6579:2002 (Table 2.10), have been
developed and evaluated in relation to the analysis of food and feed. As the matrix has
considerable influence on the performance of the method due to e.g. levels of competitive
flora, methods developed for analysis of food cannot be assumed to be appropriate for
analysis of materials from primary animal production, e.g. faeces. In recent years, efforts have
been made to develop and evaluate a standard bacteriological method for the isolation of
Salmonella from samples from primary animal production (EFSA 2006).
Table 2.10: Flow chart of the standard ISO 6579 methods recommended for analysis of
samples (EFSA 2006)
PREENRICHMENT
SELECTIVE
ENRICHMENT
ISOLATION
CONFIRMATION
Detection of Salmonella from
Detection of Salmonella in
food and feed
animal faeces
Buffered peptone water (BPW). Incubation 18 h. at 37°C ±1°C
0,1 ml of culture onto MSRV
0,1 ml of culture 1 ml of culture
Incubation for 24 ± 3 h
+ 10 ml RVS
+ 10 ml MKTTn at 41.5°C ±1°C
Incubation for
Incubation for
Additional incubation 24 ± 3h.
24 ± 3 h. at
24 ± 3 h. at
at 41.5°C ±1°C for negative
41.5°C ±1°C
37°C ± 1°C
samples
XLD medium and second agar of choice.
Incubation for 24 ± 3 h. at 37°C ± 1°C
From each plate test a characteristic colony.
If negative, test four other marked colonies
Nutrient agar, incubated for 24 ± 3 h. at 37°C ± 1°C
Biochemical and serological confirmation
37
Literature review
2.2.1.1 Pre-enrichment media
In a first step, samples are diluted in a pre-enrichment medium, followed by homogenization
in a stomacher for 1 minute. The reason for stomaching is that Salmonella spp. are not
homogeneously distributed in faeces, especially when only low numbers of Salmonella spp.
are shed. Funk et al. (2000) however stated that stomaching faecal samples did not increase
the recovery rate of Salmonella. Several pre-enrichment media have been proposed
(D´AOUST 1981). Lactose broth (LB) was perhaps the first to receive widespread use. But
there has been concern that LB, because of the fermentation of lactose and resulting acidity,
would allow the pH to fall to a level that is inhibitory or lethal to Salmonella (HIKER 1975).
Buffered peptone water (BPW) has been used without a fermentable sugar and with greater
buffering capacities. Fricker (1987) and Juven et al. (1984) found that BPW was more
appropriate than LB for isolating Salmonella. The pH of the LB cultures after incubation
ranged from 4.8-5.5, whereas the range for BPW was 5.8-6.4, respectively. BPW has been the
pre-enrichment broth of choice for use in conjunction with Rappaport-Vassiliadis enrichment
media (WALTMAN 2000). Pre-enrichment is usually carried out in buffered peptone water
(BPW). It stimulates the growth of Salmonella, but also of the accompanying flora. In
addition, pre-enrichment includes resuscitation of sub-lethally injured bacteria (HOORFAR
and BAGGESEN 1998). In a study by Jensen et al. (2003), it was demonstrated that the
addition of novobiocin (22μg/ml) in the pre-enrichment step increased the detection of
Salmonella in naturally contaminated pig faecal samples, probably because of a suppression
of the competitive micro-organisms.
In contrast to clinical salmonellosis, where the number of bacteria excreted is high enough
6
7
(10 -10 CFU/g faeces) to be detected by direct plating, subclinical salmonellosis requires
different steps of selective and non-selective enrichment (Figure 2.5). Nietfeld et al. (1998b)
concluded that delayed secondary enrichment (DSE), where samples incubated into a first
enrichment broth were held for 5 days at room temperature before second enrichment,
increased the recovery rate of Salmonella. O‟Carroll et al. (1999) and Davies et al. (2000b)
agreed with the previous study. O‟Carroll et al. (1999) suggested thereby that DSE could be
very useful to compensate for the damaging effects to the bacteria during storage of samples.
38
Literature review
DAY 1: sample 1:9 dilution in buffered peptone water (+ novobiocine)
DAY 2: suspension of 100 μl on MSRV or DIASSALM agar or in 10 ml
RV or TT
DAY 3: plating on XLD, BGA, XLT-4
DAY 4: identification of typical colonies biochemical confirmation in TSI,
indole and lysine
DAY 5: final result
phenotypic characterization: serotyping phage typing antimicrobial
resistance genotyping characterization: PCR, RFLP, PFGE
Figure 2.5: Schematic presentation of the different steps needed for bacteriological
isolation of Salmonella spp. from faeces or organic tissues (NOLLET 2008)
2.2.1.2 Selective-enrichment broth
Pre-enrichment is followed by selective enrichment in one or more steps. Different selective
enrichment media are available (NOLLET 2008). Selective-enrichment broths are formulated
to selectively inhibit other bacteria while allowing Salmonella to multiply to levels that may
be detected after plating (CHAUNCHOM 2003). The most commonly used media for a first
enrichment step are Rappaport-Vassiliadis (RV) broth, tetrathionate broth (TT), modified
semisolid Rappaport-Vassisliadis (MSRV) and Diagnostic Semi-solid Salmonella agar
(DIASSALM). Xylose-lysine-desoxycholate (XLD), xylose-lysine-tergitol 4 (XLT-4) or
brilliant green agar (BGA) are usually used as a selective isolation step (NOLLET 2008).
Selenite enrichment media: Leifson (1936) formulated the first selenite enrichment,
commonly known as selenite F (SF). Selenite is reduced by bacteria, which increases the pH
and reduces the toxicity of selenite. North and Bartram (1953) modified SF by adding cystine
39
Literature review
(selenite-cystine (SC)). Stokes and Osborne (1955) modified SF by changing the carbohydrate
source from lactose to mannitol and adding sodium taurocholate and brilliant green (selenite
brilliant green (SBG)). In a comparative study, Waltman et al. (1995) compared SF, SC, and
SBG with tetrathionate and RV enrichment broths. The tetrathionate and RV enrichment
results were better than the results from the use of selenite enrichment. Other disadvantages
that have resulted in the reduced use of selenite enrichment media because selenium is
considered a hazardous chemical, which has been shown to reduce fertility and produce
congenital defects, is considered to be embryotoxic and teratogenic and has produced an
increase in hepatocellular carcinomas and adenomas in animals (ANDREWS 1996; GOYER
1996).
Tetrathionate enrichment media: In 1923, Muller described a selective enrichment broth that
contained iodine and sodium thiosulphate, which he combined to form tetrathionate
(CHAUNCHOM 2003). Kauffmann (1935) modified the tetrathionate brilliant green (TBG)
enrichment broth of Muller by adding ox bile and brilliant green (MKTT). Different
formulations of tetrathionate enrichment have contributed to the often confusing comparative
studies. Waltman et al. (1995) compared three tetrathionate formulations for the recovery of
Salmonella from environmental samples. They found no significant differences in the three
media. Several studies have shown that tetrathionate enrichment is better than selenite
enrichment (WALTMAN et al. 1995; D‟AOUST et al. 1992).
Rappaport-Vasiliadis enrichment media: Rappaport et al. (1956) described an enrichment
medium based on the ability of Salmonella to:

I survive relatively high osmotic pressures (using magnesium chloride).

II multiply at relatively low pH (pH 5.2).

III survive in malachite green (106 mg/l).

IV grow with minimal nutritional requirements (5 g peptone /l).
Vassiliadis et al. (1970, 1976) modified the medium by reducing the concentration of
malachite green, which made the medium suitable for incubation at 43°C. Several studies
have shown that RV enrichment was better than either tetrathionate or selenite enrichment
broths (WALTMAN et al. 1995; OBOEGBULEM 1993; SCHLUNDT and MUNCH 1993;
BAGER and PETERSON 1991). RV enrichment medium has been approved for isolating
Salmonella from raw, highly contaminated food and animal feeds, replacing the use of
selenite enrichment media (JUNE et al. 1996). Goossens et al. (1984) developed a semi-solid
medium based on RV enrichment broth (modified semi-solid Rappaport-Vasiliadis or
MSRV). The MSRV plate is incubated at 41.5°C and the motility of Salmonella further
selects and differentiates Salmonella from other microorganisms. Several studies have shown
the advantages of the MSRV culture method (DAVIES and WRAY 1994; READ et al. 1994;
ASPINALL et al. 1992). The type of enrichment media may be of influence not only on the
number of positive samples, but also on the serovars of Salmonella recovered from naturally
contaminated faecal samples from pigs (ROSTAGNO et al. 2005). One should consider using
a combination of methods to increase the reliability of the results.
40
Literature review
Once the colonies of typical colour and morphology are identified, confirmation should be
performed based on biochemical tests using triple sugar iron agar (TSI), indole broth and
lysine broth.
2.2.1.3 Plating media
Plating media should be judged not only on their ability to selectively grow Salmonella, but
also on their ability to differentiate colonies of Salmonella from those of other bacteria. Many
selective plating media are available for the isolation of Salmonella. The plates are incubated
at 35-37°C for 20-24 h and grown colonies suspected to represent Salmonella are taken for
further investigations (WALTMAN 2000).
Brilliant green (BG) agar was developed by Kristensen et al. (1925) and later modified by
Kauffmann (1935). The selectivity of the agar derives from the presence of the brilliant green
dye and the presence of lactose and sucrose (BPLS), which is the basis for the differential
capabilities of the media. Almost all Salmonella fail to ferment either lactose or sucrose and
their colonies appear pink to red, with a reddening of the media. Several investigators have
proposed the use of BPLS (WALTMAN et al. 1995; MALLINSON 1990).
Rambach agar (RAM) was developed based on the finding that Salmonella produce acid from
propylene gycol (RAMBACH 1990). The selective ability results from the presence of bile
salts. The differential characteristics involve the fermentation of propylenegycol, Salmonella
appearing as crimson-red colonies, and a chromogenic detection system for the production of
ß-D galactosidase by other Enterobacteriaceae, which after reaction with the chromogenic
substance, X-Gal (5-bromo-4-chloro-3-indole-ß-D-galactopyranoside), results in the
formation of blue-green colonies. But some serovars of Salmonella do not produce the
characteristic red colonies, e.g. S. Pullorum, S. Gallinarum, S. Abortus, and S. Dublin
(PIGNATO et al. 1995a, b; KUHN et al. 1994).
Other plating media work next to the fermentation of sugars (xylose, saccharose) and phenole
red with desoxycholate and iron ammoniumcitrate (XLD). Apart from the change in pH
Salmonella result in colonies with a black centre (WALTMAN 2000).
The DIN EN ISO 12824:1997 (1998) regulates the use of BPLS agar and another agar chosen
by the laboratory; the new DIN EN ISO 6579:2002 (2003) rules the use of XLD agar.
2.2.1.4 Biochemical identification
Typical Salmonella colonies found on the selective agar should be confirmed by biochemical
tests. The principal biochemical tests by which Salmonella can be identified are given in
Table 2.11 The (+) signs indicate that this factor or reaction must be seen in Salmonella, (-)
indicates the reaction should not appear. Testing all these reactions requires considerable
resources. Therefore it makes sense to use composite media such as triple sugar iron agar
(TSI) which is prepared in tubes for further use. It contains glucose, lactose and sucrose, a
H2S detection system and an indicator (JONES et al. 2000).
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The medium is inoculated by stabbing the loop into the centre of the agar down to the base of
the reaction tube. The loop is pulled back and the rest of the inocular is distributed on the
slope of the agar surface. The tube is inoculated at 37°C for 24 h. When glucose not lactose or
sucrose is fermented by the microorganisms a change of colour from red to yellow can be
observed. The colour fermentation depends on the amount of glucose which is converted.
However, the reaction under the aerobic conditions on the slant reverts and becomes alkaline
(red) because of protein breakdown in the medium. Under anaerobic conditions inside the
agar at the bottom of the tube, the medium remains acid (yellow) and H 2S production is
characterized by blackening of the medium (JONES et al. 2000). Some Proteus spp. and other
species may give a similar reaction, but can be distinguished by their ability to hydrolyse
urea. Urea agar in another reaction tube should always be used in parallel. The typical
reactions of 6 different strains of Enterobacteriaceae in TSI are show in Table 2.12.
The significance of the choice of matrix for bacteriological examination is demonstrated by
comparing the sensitivity of isolation from different matrices. In subclinically infected herds,
slaughter pigs present chronic infections with only intermittent excretion, with a low level of
Salmonellae in faeces (WILCOCK and SCHWARZT 1992). Therefore, the examination of
individual faecal samples from pigs has a poor sensitivity. For faecal samples the sensitivity
has been reported to vary between 9% (swabs) and 78% (25g faeces) (FUNK et al. 2000) and
10-80% (HURD et al. 2001a). However, when used on a herd basis, where all animals are
included in the examination, as done in the Nordic control programmes (excl. Denmark), the
herd sensitivity increases, especially when repeatedly applied. In such cases negative faecal
culture of the whole herd (individual samples on adult pigs and pooled pen samples on young
pigs/finishers), performed twice, with one month interval, will ensure, with a higher
confidence, that all animals are free from Salmonella.
A positive correlation has been reported between the prevalence of Salmonella in faecal
samples collected on farm and in ileocaecal lymph node samples, suggesting that the
prevalence of Salmonella spp. at slaughter can be predicted from preslaughter on-farm
sampling and vice versa (BAHNSON et al. 2005). However, Nollet et al. (2004) considered
that the prevalence of infected lymph nodes reflected the Salmonella status on the farm only if
cross-contamination with animals from other farms had not occurred between transported of
pigs from the farm to slaughterhouse or it was negligible.
The number of caecal/intestinal lymph nodes analysed can be expected to influence the
sensitivity of the analysis. In an investigation by Sorensen et al. (2004), only one caecal
lymph node was analysed, whereas in the surveillance at Swedish slaughterhouses at least five
lymph nodes from the same animal are pooled in one sample. The latter procedure can be
expected to be more sensitive, since more lymph nodes are investigated altogether.
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Table 2.11: Biochemical reactions of typical Salmonella spp. (JONES et al. 2000)
Test
Reaction of
Test
Reaction of
typical strain
Fermentation of :
typical strain
Glucose
Nitrate reduction
+
+
Mannitol
Oxidase
+
Maltose
O/F
F
+
Lactose
Urea hydrolysis
Sucrose
Indole
Salicin
H2S production
+
Adonitol
Citrate utilization
+
Dulcitol
Sodium malonate
+
Lysine decarboxylase
Growth on KCN
+
Arginine dihydrolase
MR
+
+
Ornithine decarboxylase
VP
+
Deamination of phenyalanine
Gelatin liquefaction
KCN, potassium cyanide; MR, methyl red; O/F, oxidation/fermentation; VP, Voges Proskauer
Table 2.12: Biochemical reactions on triple sugar iron agar (JONES et al. 2000).
Organism
Butt
Slope
H2 S
AG
A
Enterobacter
AG
A
Escherichia coli
AG
A
+
Proteus
A or AG
NC or ALK
Morganella
A
NC or ALK
Shigella
AG
NC or ALK
+
Salmonella
AG, acid (yellow) and gas formation; A, acid (yellow); NC, no change; ALK, alkaline (red);
+, hydrogen sulphide (black); –, no hydrogen sulphide (no blackening)
It has been suggested that environmental sampling using e.g. a pair of socks, in pig farms, as
used in broiler flocks (SKOV et al. 1999), could likewise have a higher sensitivity compared
to pooled individual faecal samples (BELOEIL et al. 2004a; KORSAK et al. 2003). The use
of such sampling method could be further evaluated as a simpler and more sensitive
alternative to faecal samples, for example, when used to establish the true Salmonella status
of pens or batches of finishing pigs immediately prior to slaughter (as done with poultry). An
additional option is sampling of manure in the lorry at the time of unloading. Fractioned
sampling during unloading represents a large number of pigs at the same time increasing
sensitivity considerably. In the United Kingdom (UK), it was found that the use of pooled pen
floor faeces gave a useful measure of herd prevalence (ARNOLD et al. 2005). In that study
the highest sensitivity (67%) was achieved when the maximum (20) number of individual
pigs contributed to a pool of 25g, assuming the within-pen prevalence was 25%.
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2.2.1.5 Characterization of Salmonella isolates
After isolating Salmonella, further identification of the isolates is possible and antimicrobial
resistance can be determined. Both are very important in epidemiological studies and in
Salmonella surveillance programmes. Salmonella isolates can be subdivided based on
phenotypic characteristics, using serotyping, phage-typing and patterns of antimicrobial
resistance (LIEBANA, 2002; PEREIRA et al. 2007). Serotyping is usually done following the
Kauffman-White scheme which is based on the agglutination of somatic (O) and flagellar (H)
antigens (POPOFF and LE MINOR 1997). Phage-typing distinguishes strains within serovars
based on their susceptibility to a set of phages (RABSCH et al. 2004). Further differentiation
is based on the characterization of the genotype by DNA-based typing techniques. These
techniques include plasmid analysis; polymerase chain reaction (PCR) based methods and
methods based on the analysis of whole genome such as restriction length polymorphism
(RFLP), restriction endonuclease analysis (REA), pulsed field gel electrophoresis (PFGE) and
hybridization techniques (LIEBANA 2002).
2.2.2 Serological detection methods
The serological tests developed for Salmonella spp are based on the detection of antibodies
against the somatic O-antigens. Most tests are indirect enzyme-linked immunosorbent assays
(ELISA) (NOLLET 2008). All available ELISAs are based on Lipo-Poly-Saccharide (LPS)
antigens. LPSantigens are a part of the cell wall of many bacteria but are very specific for
each kind of bacteria. In the case of Salmonella the LPS is specific for each serogroup. LPS is
very immunogenic and therefore, pigs may react to infection with Salmonella by producing
specific antibodies (EFSA 2006).
Nielsen et al. (1995) first described an ELISA to detect Salmonella antibodies. This test,
developed in Denmark, was an indirect anti-LPS ELISA using the antigens O: 1, 4, 5 and 12
from Salmonella Typhimurium and O: 6 and 7 from Salmonella Choleraesuis. Because of the
different antigens included, the test is called “mix-ELISA”. Based on the Danish test, similar
tests were developed in other laboratories (VAN DER HEIJDEN et al. 1998; PROUX et al.
2000; CZERNY et al. 2001; CHOW et al. 2004). All test kits include the O-antigens 1, 4, 5, 6,
7 and 12 found in Salmonella Typhimurium and Salmonella Choleraesuis, Infantis or
Enteritidis, and should therefore cover the most occurring serovars in Western-Europe and the
United States. However, in an international ring trial comparing 12 different ELISA kits
(VAN DER HEIJDEN 2001), major differences were observed in the sensitivity of the tests
but most tests were sufficient in their specificity.
The interpretation of the results of the indirect mix-ELISA is done based on the measurement
of absorbance values. The outcome is expressed in optical density percentages (OD%) which
are calculated using following formula (NIELSEN et al. 1995):
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Different critical values can be used for determining a sample as positive, depending on the
specific aims and on the test kit used. For the Idexx test kit for example, OD 10% is
considered as the scientific value (Herd Check Swine Salmonella Antibody Test Kit, IDEXX
Laboratories, Inc., Maine, USA). OD 40% is the value used in most Salmonella screening
programmes (MOUSING et al. 1997; OSTERKORN et al. 2001). To be more stringent, the
critical value in the Danish Salmonella Surveillance Programme has been lowered to OD 20%
(ALBAN et al. 2002a).
Although the test protocol is very similar, some indirect mix-ELISA test kits do not express
the results in OD%, but as sample on positive (S/P) ratios (IDEXX Inc., Maine, USA). To
compare the S/P results with the Danish OD%, following formula is to be used:
OD % = (s/p ÷ 2.5) x 100
The critical values for positive samples corresponding with OD 10, 20 and 40% are an S/P
value of 0.25, 0.50 and 1.00, respectively.
Harmonization of methods, by agreement on methodology and calibration, of ELISA tests is a
prerequisite if the results of surveillance are to be comparable between countries (VAN DER
HEIJDEN 2001; VAN DER WOLF et al. 2001a). Studies have shown that results of different
commercial ELISA kits may not be interchangeable (MEIJA et al. 2003). It has also been
suggested that international reference samples should be made available to ensure a minimum
level of sensitivity (VAN DER HEIJDEN 2001) and specificity.
As an alternative for serum, fluid recovered from frozen muscle tissue samples (meat juice)
was used for the detection of Salmonella antibodies in an indirect anti-LPS ELISA
(NIELSEN et al. 1998). As this fluid, being a mixture of serum, lymph and intracellular liquid
is already a physiological dilution of serum, the samples only need to be diluted 1:30
compared to a 1:400 serum dilution. This method is more labour-friendly than taking blood
samples at the herd. An additional advantage is that the collection of samples can be done
after recording the identity of the carcass in the slaughterhouse. A disadvantage is that large
variations in the volume of fluid per gram of tissue can be observed, resulting in a slight
dilution of the specific antibodies in the fluid. This leads to a lower OD% for the meat juice
ELISA than for the serum ELISA. The sensitivity and the specificity of the meat juice ELISA
is, according to Nielsen et al. (1998), 80-89% and 91-100% (depending on the cut-off value
used), respectively, when compared to the serum ELISA.
Apart from Norway and Sweden (WAHLSTRÖM et al. 1997; SANDBERG et al. 2002),
Salmonella screening and surveillance programmes are based on the detection of Salmonella
antibodies in serum or meat-juice samples. As the intention of such programmes is to detect
Salmonella infected herds, it is important to know to what extent serological screening
methods and the bacteriological isolation of Salmonella correlate. Many authors have reported
correlations between both diagnostic techniques and they all agreed that serological testing is
a good method for screening at the herd level, but individual correlation is low. Nielsen et al.
concluded already in 1995 that not all pigs do seroconvert despite inoculation and frequent
excretion of Salmonella Typhimurium in the faeces. There seem to be variations among pigs
regarding the time of seroconversion. In addition, it was possible that pigs were culture
45
Literature review
negative but seropositive, which was probably due to the low sensitivity of bacteriological
isolation. Lo Fo Wong et al. (2003) found a correlation coefficient of 0.62 between the
proportion of culture positive faecal samples and seropositive samples in a herd at a cut-off of
OD 10% and of 0.58 at a cut-off of 40%. Sorensen et al. (2004) demonstrated that the odds
for Salmonella culture positive caecal contents, pharynx and carcass surfaces varied from 1.3
to 1.5 for each increase of 10% in herd serology. Casey et al. (2004) found disagreements
between bacteriological isolation of Salmonella in caecal contents and serological detection of
antibodies in muscle tissue, although no statistical evaluation has been done.
Enzyme-Linked Immunosorbent Assay (ELISA) tests detect antibodies against Salmonella
and are therefore indirect tests that measure previous exposure to Salmonella. Therefore, an
animal that is ELISA positive may no longer be infected, in contrast to a pig that is
bacteriologically positive. Likewise, ELISA negative animals can be recently infected if
testing is performed before detectable levels of antibodies have been produced. These facts
have important consequences for the interpretation of test results which are described:
2.2.2.1 Test characteristics
Test characteristics depend on several factors such as:

Technical design
The ELISA test can be designed with a focus on specific serogroups that occur in a region
(e.g. Salmonella Typhimurium and therefore serogroup B) or which are of interest for other
reasons. The inclusion of LPS from different serogroups may influence the sensitivity of the
test. In principal, antibodies against serogroups (O-antigens) that have not contributed LPS to
the test cannot be demonstrated by the test.
However, some cross-reactivity might occur (e.g. between serogroups B and D1). In
Denmark, the ELISA was estimated to detect antibodies against 90-95% of the serovars found
in the field; in The Netherlands this figure was 89% (BAGGESEN et al. 1996; VAN DER
WOLF et al. 1999). Changes in serovar distribution in the field resulting in a change of
serogroup representation will affect the sensitivity of the ELISA and will require repeated
bacteriological investigations.

Cut-off
In the original publication of the method applied in Denmark by Nielsen et al. (1995) a
scientific cut-off of OD%>10 was established. However, another cut-off was adopted by the
Danish National monitoring scheme (MOUSING et al. 1997). Changing the cut-off will affect
both the sensitivity and specificity. When the cut-off is increased from the scientific cut-off of
OD%>10 to OD %> 20 or even OD%>40, as in the Danish Salmonella Monitoring System
(ALBAN et al. 2002b), the sensitivity drops dramatically. With an increase in the cut-off from
OD%>10 to OD %> 40 the apparent prevalence in finishers decreases to about 45% of that
estimated using the OD %> 10 cut-off (23.7 to 10.4 and 24.5 to 11.1) (VAN DER WOLF et
al. 2001a). In sows, the effect is even more dramatic, when the cut-off is set at OD%>40 the
number of sows found positive is only 16 – 17% of the number found positive at an OD%>10
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Literature review
(60.4 to 9.9)(VAN DER WOLF et al. 2001a). Comparable results were found by Nollet et al.
(2005) in Belgium.

Stage of infection
The interval between the peak of the bacteriological and serological response ranges from one
to a few weeks for experimental infections (LO FO WONG et al. 2004) up to approximately
two months under natural conditions (KRANKER et al. 2003). This means that in the early
stages of an infection, pigs will be seronegative, while positive bacteriological results may be
obtained. In later stages of infection when pigs may have cleared themselves of Salmonella,
antibodies may still be present, classifying the pigs as seropositive. Therefore serological
testing provides a measure of historical exposure that may not correlate closely to
microbiological findings at the time of sampling. However, latent carriers and intermittent
shedders that are difficult to detect bacteriologically can be identified immunologically (LO
FO WONG et al. 2004).

Serovar
The stimulation of the immune system varies for different serovars and results in different
antibody responses. Seropositivity tends to be related to the presence of S. Typhimurium
(STEGE et al. 2000). Stege et al. (2000) concluded that in general S. Typhimurium will give a
clear response, whereas for S. Panama or S. Goldcoast the antibody responses were poor or
not detected. However, exhaustive testing for all serovars found in pigs has not been
conducted.

Passive immunity
Under field conditions, piglets ingest colostrum from their dam who might be seropositive,
thus resulting in passive immunity in these piglets. These maternal antibodies persist for about
8 to 10 weeks, and so, for practical purposes, the ELISA is not used to determine whether
piglets under the age of 10 weeks are or have been infected with Salmonella. However, this
issue requires further investigation (KRANKER et al. 2003; VAN DER HEIJEDEN et al.
1998).

Failure of seroconversion
Some individuals who are unable to react immunologically to the infection will not
seroconvert even though they are truly infected with a serovar that normally would lead to a
raised antibody level. Part of the explanation for this is genetic resistance to infection in some
pigs (VAN DIEMEN et al. 2002). This phenomenon may occur in 1 or 2 % of pigs
(NIELSEN et al. 1995).
2.2.2.2 Sensitivity
The sensitivity of the ELISA test is its ability to detect antibodies against a defined range of
Salmonella serogroups, indicating prior exposure.
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The sensitivity at individual level has been reported to be 80-90% (NIELSEN et al. 1995;
CHOW et al. 2004), but depends on many factors, as described above. In reality the
sensitivity may, however, be lower. For example, for modelling purposes, the minimum
sensitivity of the Danish mix-ELISA was assumed to be as low as 50% (ALBAN et al.
2002b).
Serology can be used as a screening test to determine herd status at a point in time and for
continuous monitoring by repeated sampling. The interpretation of immunological results is
not always straightforward, and, furthermore, bacteriological and immunological results will
often not correspond at either herd or individual level. However, given that the specificity of
the ELISA is high (with the exception of pigs tested in Sweden where specificity was shown
to be lower, as may also be the case in other low prevalence areas), a positive immunological
result will most likely reflect an infection with Salmonella, past or present, in an individual
pig (LO FO WONG et al. 2004).
The general conclusion of several studies (NIELSEN et al. 1995; STEGE et al. 1997;
CHRISTENSEN et al. 1999; SORENSEN et al. 2000) is that immunological assessment was
reliable mainly at herd level and was especially well suited for identifying high prevalence
herds (ALBAN et al. 2002a; CASEY et al. 2004). In high-prevalence herds there was usually
a long-term problem present and the herd status was anticipated to be relatively stable over
time (CHAUNCHOM, 2003; NIELSEN et al. 1995).
In low-prevalence herds, major infection incidents may occur and consequently changes in
the Salmonella status of such herds can be anticipated (VAN DER WOLF et al. 2001c). Using
immunology in such herds will have the drawback of not identifying such changes rapidly
because of the time lag between infection and seroconversion. Van der Wolf et al. (2003)
pointed out the importance of examining recently collected serological samples to confirm the
continuing low prevalence of infection in herds. Several authors have shown that the
Salmonella status changes frequently both within herds as well as in groups within herds over
time (RAJIC et al. 2005; CARLSON and BLAHA 2001). However, in order to identify
Salmonella-free herds, bacteriological examination is necessary in addition to serological
testing (VAN DER WOLF et al. 2003).
2.2.2.3 Specificity
In the case of the ELISA, the purpose of the test is to detect antibodies that indicate current or
previous infection with the Salmonella serogroups that are incorporated into the test. Thus,
the specificity of the ELISA test is defined as its ability to correctly identify as negative, i.e.
not infected, those pigs that do not have antibodies against the Salmonella serogroups
incorporated in the test.
The specificity of an ELISA test has been evaluated by looking at the results of sera from
Specific Pathogen Free (SPF) herds, longitudinal studies in seronegative herds (VAN DER
WOLF et al. 2001c) and the results of the ring trial for Salmonella ELISAs (VAN DER
HEIJDEN 2001). It can be assumed that the specificity of the Salmonella- ELISAs is high at
the scientific cut-off (VAN DER HEIJDEN 2001).
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An exception was found in Sweden where 4% (122 out of 3050) of finishing pigs tested using
the Danish mix-ELISA were found to be positive (OD% 20-70) (LO FO WONG and HALD
2000). As Sweden has a long standing intensive surveillance and control program for
Salmonella in swine, the animals which tested positive could be considered to be Salmonellafree; however, the origin of these antibodies could not be established (WIUFF et al. 2002).
This result shows that care is required when implementing an existing immunological test in a
new geographical area without prior investigation of possible background problems and of the
general bacteriological profile (i.e. bacteriological survey) of a larger number of animals in
that region (WIUFF et al. 2002; DAVIES et al. 2003; HAMILTON et al. 2003). At present
the ELISA is not sufficiently evaluated for use in low prevalence areas.
2.2.3 Molecular detection methods
Standardized diagnostic procedures to detect the presence of S. enterica in food samples (ISO
6579:2002) are mainly based on microbiological culturing methods, which in general require
up to five days until results are obtained (STEWART et al. 1998). In order to reduce the time
demand, alternative techniques like immunological assays (ACHESON et al. 1994; BOLTON
et al. 2000) and molecular methods (BEJ et al. 1994; DE MEDICI et al. 2003; HELLER et al.
2003) have been applied to detect S. enterica in various samples. Especially real-time PCR
methods such as 5‟ nuclease TaqMan® PCR (HOLLAND et al. 1991) have shown promising
results due to the rapid, sensitive and specific detection of S. enterica (BASSLER et al.1995;
HIGGINS et al. 1998; HOORFAR et al. 2000; KLERKS et al. 2004).
2.2.3.1 Polymerase Chain Reaction (PCR) was first described by Kleepe and colleagues in
1971, but was not demonstrated until 1985 by Saiki and colleagues (EDWARDS et al. 2004).
Many advances have been made since then and PCR assays have become much more
common and easier to use. Due to rapid technological advances, many laboratories now use
PCR methods routinely (EDWARDS et al. 2004).
This simple but elegant technique is based on in vitro amplification of selected nucleic acid
sequences. It is carried out in repetitive cycles, each of which consists of three steps:
denaturation, annealing and extension (Figure 2.6). In the denaturation step, the heating
o
temperature is higher than 90 C which separates double-stranded DNA into single strands.
During annealing, the temperature is lowered, thus enabling two specific primers to hybridize
o
to their complementary sequences in the single strands of target DNA. A temperature of 72 C
is used for the extension step. Then, thermostable DNA polymerase catalyzes continuous
additions of nucleotides to the 3‟ ends of two annealed primers and extends two synthesized
DNA strands. Cycles are repeated with double newly generated DNA as the template. Gel
electrophoresis is usually performed to verify the specifically amplified products based on
their sizes (ENTIS et al. 2001).
The PCR assay has been used to identify Salmonella species in food and clinical samples
(ARAJ and DAS CHUGH 1987; RAHN et al. 1992; COHEN et al. 1993). However, obstacles
in the detection of organisms include the presence of substances inhibitory to PCR (ROSSEN
et al. 1992; WILDE et al. 1990) and the inability to detect <103 cfu g-1 of sample without pre-
49
Literature review
enrichment (EHRLICH and SIRKO 1994). Investigators have improved detection methods in
PCR assays by combining it with immunomagnetic separation (WIDJOJOATMODJO et al.
1991; 1992) or by enrichment culture (STONE et al. 1994). Helmuth et al. (1997) compared a
pre-enrichment PCR with culture for Salmonella detection in 1200 samples and found a
sensitivity of 93% and a specificity of 99%, the method providing a fast and reliable screening
method for Salmonella detection. An invA-based PCR was evaluated for the detection of S.
Typhimurium in the organs of experimentally infected pigs by Scholz et al. (2001). The
results correlated with culture and enabled Salmonella to be detected within 48h. In contrast,
Korsak et al. (2001) compared the use of a PCR method and semi-solid agar, (Diasalm) for
the detection of Salmonella for a variety of pigs samples including pork. Though the
specificity was good the sensitivity of the PCR ranged from 0-66.7% for faecal material and
the technique was only reliable for pork meat and animal feeds. Schmid and Bauer (2001)
using a combination of four hours pre-enrichment, immunomagnetic separation and PCR
were able to detect 1 cfu Salmonella/ g spiked pork sample. However, a number of different
PCR assays have been developed to detect Salmonella in faeces but further work is necessary
to produce a standard PCR assay because results in comparative trials have been poor.
Many studies have shown that PCR techniques are significantly more sensitive than
microbiological culture techniques, especially when the organism is present in low numbers
or is not viable (COHEN et al. 1994, 1995, 1996; AMAVISIT et al. 2001). However, more
recent reports also claim that end-point PCR may not be as sensitive as previously thought,
due in part to false positives, and may not be appropriate for Salmonella identification in
clinical settings (AMAVISIT et al. 2001; EWART et al. 2001; ALINOVI et al. 2003). Studies
have also shown that PCR positive results are obtained much more quickly than culture
positive results (STONE et al. 1994), but according to current literature, there are large
discrepancies between culture and PCR results (EWART et al. 2001; ALINOVI et al. 2003;
HYATT and WEESE 2004).Technological advances in PCR methods now allows real-time
detection of genus specific DNA by fluorescence labeling. The combination of PCR and
subsequent detection of amplification products has been described for a wide variety of
applications (SAIKI et al. 1985, 1986, 1989; LOHEY et al. 1989; KARLSEN et al. 1995;
LOHMANN et al. 1992; ZAMMATTEO et al. 1995; SPECTOR and SPECTOR 1985;
LANDGRAF et al. 1991; KELLER et al. 1990; CHARREL et al. 2004). However, all of these
approaches require time-intensive post-PCR handling steps and bear the risk of reaction
mixtures being contaminated by previously handled amplification products (KWOK and
HIGUCHI 1989; CLEWLEY 1989; LONGO et al. 1990). The development of various
methods for the detection of PCR products during amplification in a closed-tube format
solved these problems and provided a means of gaining insight even into the early exponential
phase of the amplification process (HIGUCHI et al. 1993). Real-time PCR was first
demonstrated by Higuchi and colleagues in 1992, and the first commercial platform was
released by Applied Biosystems in 1996 (EDWARDS et al. 2004).
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Literature review
Figure 2.6: Steps of Polymerase Chain Reaction (Source: http://ku-scmicro36bkk.
tripod.com/PCR.html)
2.2.3.2 Real-time Polymerase Chain Reaction (real-time PCR) is the ability to monitor the
progress of the PCR as it occurs (i.e. in real time). Data is therefore collected throughout the
PCR process, rather than at the end of the PCR. This completely revolutionizes the way one
approaches PCR-based quantitation of DNA and RNA. In real-time PCR, reactions are
characterized by the point in time during cycling when amplification of a target is first
detected rather than the amount of target accumulated after a fixed number of cycles. The
higher the starting copy number of the nucleic acid target, the sooner a significant increase in
fluorescence is observed. In contrast, an endpoint assay (also called a “plate read assay”)
measures the amount of accumulated PCR product at the end of the PCR cycle (AB 2008a).
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Literature review
Several papers have currently appeared where real-time PCR applications for pathogen
detection have been reported (FUNG 2000; RIJPENS and HERMAN 2002; CHEN et al.
2000; HEIN et al. 2001; HOORFAR et al. 2000; KIMURA et al. 1999; NOGVA et al. 2000a,
2000b; OBERST et al.1998; SHARMA et al.1999; VISHNUBHATLA et al.2000). The main
advantages of real-time PCR are high sensitivity, high specificity, excellent efficiency
reduced amplicon size, no post-PCR step that reduce risks of cross-contamination
(RODRIGUEZ-LAZARO et al. 2003).
Other advantages of using Real-Time PCR (AB 2008b):
 Traditional PCR is measured at End-Point (plateau), while Real-Time PCR collects
data in the exponential growth phase,
 An increase in Reporter fluorescent signal is directly proportional to the number of
amplicons generated
 The cleaved probe provides a permanent record amplification of an Amplicon
 Increase dynamic range of detection
 Detection is capable down to a 2-fold change.
One of the disadvantages of real-time PCR is the inability to perform antimicrobial testing or
serotyping, which could affect epidemiological studies and the identification of nosocomial
infections. Also, real-time PCR equipment is quite expensive and requires highly trained
technicians to interpret results (SMITH 2006).
2.2.3.2.1 Basic principles of real-time PCR are based on continuous quantification of
specific fluorescent reporter emission signals during the amplification process. The signal
increases in direct proportion to the amount of PCR product accumulated in the reaction
mixture (HIGUCHI et al. 1993; HEID et al. 1996; MORRISON et al. 1998). By detecting the
amount of fluorescence emission at each amplification cycle, it is possible to monitor the
entire kinetics of an ongoing PCR reaction (figure 2.7).
Figure 2.7: Amplification curves of a 5’ nuclease assay (Source: www.biotech.uiuc.edu.)
52
Literature review
2.2.3.2.2 Chemistries used in real-time PCR, variation of the real-time PCR include DNAbinding dyes, such as SYBR® Green (Figure 2.8), or fluorescent oligonucleotides such as the
Scorpion primers (Figure 2.9) (WHITCOMBE et al. 1999), Molecular Beacons (Figure 2.10)
(TYAGI and KRAMER 1996), Fluorescence Resonance Energy Transfer (FRET) probes
(Figure 2.11) (WITTWER et al. 1997) and TaqMan® probes (HEID et al. 1996). The main
TaqMan® feature is the use of three oligonucleotides in the PCR reaction. Two of the primers
(forward and reverse) allow amplification of the product to which a third dual-labeled
fluorogenic oligonucleotide, the TaqMan® probe, will anneal. Upon polymerase
amplification, the 5‟-3‟ exonuclease activity of the Taq polymerase releases a 5‟ fluorocent
tag from the annealed TaqMan® probe, yielding a real-time measureable fluorescence
emission directly proportional to the concentration of the target sequence (Figure 2.10)
(RODRIGUEZ-LAZARO et al. 2003).
Figure 2.8: SYBR® Green I Dye chemistry (Source: http://www.gibthai.com)
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Literature review
Figure 2.9: Scorpion probe (Source: http://www.gibthai.com)
Figure 2.10: Molecular Beacon probe (Source: http://www.gibthai.com)
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Literature review
Figure 2.11: Fluorescence Resonance Energy Transfer (FRET) probe
(Source: http://www.gibthai.com)
Figure 2.12: TaqMan® probe method (Source: http://www.gibthai.com)
Real-time PCR has been developed for rapid Salmonella detection using different fluorescentbased systems for detection of PCR products. Eyigor et al. (2002) used the double-stranded
DNA binding dye SYBR® Green I for real-time monitoring of Salmonella PCR product and
implemented the real-time PCR assay for detection of Salmonella-positive poultry flocks.
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Literature review
Chen et al. (1997) evaluated the 5‟ nuclease real-time PCR (TaqMan®) for the detection of
Salmonella in foods of animal origin. Knutsson et al. (2002) further investigated the
robustness of the 5‟nuclease real-time PCR for optimization in combination with an
enrichment procedure for Salmonella detection. A highly sensitive and specific molecular
beacon assay was developed by Chen et al. (2000) to detect the presence of Salmonella,
however, no applications of molecular beacon based real-time PCR for detection of
Salmonella in foods were reported.
2.2.3.3 Difference between conventional PCR and real-time PCR
Real-time PCR suggests a “kinetic” rather than an “equilibrium” paradigm for PCR
(WITTWER et al. 1997). Conventional PCR is usually considered as a repetitive process
where three reactions occur at three temperatures three times during each cycle. In contrast,
the kinetic paradigm emphasizes temperature transitions (Figure 2.13). Denaturation and
annealing times are often reduced to “zero” and the temperature may always be changing.
Denaturation, annealing and extension occur at different rates, and, depending on the
temperature, multiple reactions may occur simultaneously. The kinetic paradigm is more
correct, theoretically and practically. Sample temperatures do not change instantaneously but
occur as smooth transitions. Most protocols, however, do not use zero second denaturation
times (WITTWER et al. 1991, 1994). The easiest way to monitor PCR during amplification is
with the use of fluorescence technology. Many applications require only a double strandspecific dye such as SYBR® Green I (MORRISON et al. 1998). Using a generic dye
eliminates the cost and problems associated with probe synthesis. However, certain
applications require greater sequence specificity, and a variety of fluorescently-labeled
oligonucleotide probes can be used to monitor the progress of PCR (PRITHAM and
WITTWER 1998). These include exonuclease (TaqMan®) probes and hybridization probes.
FIGURE 2.13: Comparison of equilibrium and kinetic paradigms of PCR (WANG 2006)
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Literature review
TaqMan® probes are used to detect specific sequences in PCR products by employing the 5'->3' exonuclease activity of Taq DNA polymerase. The TaqMan® probe (20-30 bp) is disabled
from extension at the 3' end, and consists of a site-specific sequence labeled with a fluorescent
reporter dye and a fluorescent quencher dye. During PCR, the TaqMan® probe hybridizes to
its complementary single strand DNA sequence within the PCR target. When amplification
occurs, the TaqMan® probe is degraded due to the 5'-->3' exonuclease activity of Taq DNA
polymerase, thereby separating the quencher from the reporter during extension. Due to the
release of the quenching effect on the reporter, the fluorescence intensity of the reporter dye
increases. During the entire amplification process, this light emission increases exponentially,
with the final level being measured spectrophotometrically after termination of the PCR.
Because increase in the fluorescence intensity of the reporter dye is only achieved when probe
hybridization and amplification of the target sequence have occurred, the TaqMan® assay
offers a sensitive method to determine the presence or absence of specific sequences. Figure
2.12 above shows the process of giving out fluorescence by TaqMan® probes. Quantitation
can be realized by real-time PCR. A PCR reaction profile can be thought of as having three
segments: an early background phase, an exponential growth phase (or log phase) and a
plateau. The background phase lasts until the signal from the PCR product is greater than the
background signal of the system. The exponential growth phase begins when sufficient
product has accumulated to be detected above the background, and ends when the reaction
efficiency falls as the reaction enters the plateau phase. During the “log” phase the
amplification course is described by the equation:
n
T = T (E) (1)
n
o
Where T is the amount of target sequence at cycle n, T is the initial amount of target, and E
n
o
is the efficiency of amplification. The maximum efficiency possible in PCR is 2 because
every PCR product is replicated in every cycle. Typical real-time PCR curves monitored on a
light cycler are shown in Figure 2.14. The concentration is different from left to right,
whereby the left has the highest concentration. The cycle where each reaction first rises above
background is depend on the amount of target present at the beginning of the reaction. The
higher the initial concentration, the sooner of the signal from reporter dye suppresses the
background dye. The Ct value is the point where the fluorescence given out by the reporter
dye is stronger than that of the background dye. The Ct value is decided by setting up a
threshold line on a standard curve made by plotting the Ct value with the initial DNA
concentration. The threshold line is a factor that influences the standard curve and this
relationship is shown on Figure 2.15 (WANG 2006).
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Literature review
FIGURE 2.14: Typical real-time PCR result for different dilutions (WANG 2006)
FIGURE 2.15: Threshold line influences the standard curve (WANG 2006)
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Literature review
2.2.4 Methods for surveillance
The purpose of surveillance is to provide information to be used by decision makers. It is vital
to think through the objectives and also put them into writing before designing a surveillance
program. Periodical evaluations shall be done and provision for such evaluation included in a
surveillance system (TOMA et al. 1999).
Surveys and surveillance can describe and quantify a disease status at a given moment or its
behavior over time and in space in a given population. When designing surveillance it is
important that the sample is representative of the target population. The design and especially
the sampling strategy are defined by the objectives of the survey. A key question is the unit of
concern, e.g. individual carcass, animal, herds/certain types of herds or maybe region (TOMA
et al. 1999). The level of detail of a disease spatial distribution will also affect sampling
design; for instance, is it sufficient with an overall estimate of disease frequency in a country
or herd or should smaller units with different prevalences be identified (TOMA et al. 1999).
For example, it has been shown that separate slaughter of seronegative pig herds can lead to a
decrease in the prevalence of Salmonella-contaminated pork after slaughter
(SWANENBURG et al. 2001b). In such cases, surveillance of the end product (carcasses)
could preferably be done on a slaughterhouse basis or stratified using separate slaughter
batches. When a study includes disease information over time the level of detail required will
affect the frequency of sampling, for example when seasonal, annual or other temporal
changes require to be identified (TOMA et al. 1999). Salmonella prevalence can change
rapidly over a short period, as has been shown in low-seroprevalence countries/herds. In
endemic regions, it can be expected that Salmonella infections will occur regularly as long as
Salmonellae are present in the animal environment, in feed and in animals that are brought
into the herd. If such changes are to be detected rapidly, then such herds have to be sampled
frequently (VAN DER WOLF et al. 2001c).
If certain (usually small) herds are not included in the programme, this has to be considered.
If such pigs are slaughtered at the same slaughterhouses they may still be a risk of
contamination. Husbandry systems where pigs are kept outside total confinement (pasture,
free range etc.) are at an increased risk of getting infected with Salmonella (VAN DER
WOLF et al. 2001b). Any population prevalence estimate should be stratified in order to
monitor these kinds of herds separately from total confined housing. Understanding the
characteristics of the test is essential. When estimating the sensitivity and specificity of a test,
it is essential that the study population is representative of the target population (e.g. those
animals to which the test will be applied in the future). This representativeness refers to
attributes of animal being tested; for example age, breed and also environmental factors that
might affect the sensitivity or specificity of the test (DOHOO et al. 2003).
When establishing the sample size needed, it is important that the sensitivity of the test at
individual and herd level is taken into account. This is especially true for Salmonella where
the sensitivity of tests is not high. Failure to consider an imperfect sensitivity of a test will
lead to an underestimation of prevalence and reduced power to detect Salmonella infected
herds. The expected within herd prevalence should also be taken into account. It can be
expected that, as risk management actions are taken, the within herd prevalence decreases and
consequently a larger sample size is needed to identify an infected herd.
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To assess the infection dynamics in a Salmonella infected herd, repeated sampling in different
cohorts (clusters) of animals is required. Point estimates of pre-harvest prevalence in
subclinically infected herds are not reliable as variations occur in Salmonella prevalence
between cohorts within systems and over time (FUNK et al. 2001; BELOEIL et al. 2003;
KRANKER et al. 2003).
Surveillance can also, apart from quantifying disease, be aimed at detecting disease as soon as
possible, by sampling at critical control points. When disease prevalence becomes very low
such sampling may be more appropriate than to quantify disease occurrence. An example of
such surveillance is the Salmonella control in feed where it is crucial to rapidly identify and
eliminate the risk of any Salmonella contaminated batch (HÄGGBLOM, 1994). However,
results of such surveillance are difficult to compare between countries/regions.
Movement of live animals, carcasses and meat between countries may interfere with
surveillance results and it is preferable that knowledge about such exists. Finally, the cost and
what sampling procedures and analysis are practically possible should be considered when
designing surveillance.
Recent modelling studies have shown that focusing control on high prevalence herds (level 2
and 38) may not be the optimal strategy. The greatest public health benefit was obtained from
modest improvements in all farms rather than large improvements in farms with only a high
prevalence (ALBAN and STARK 2005; COOK et al. 2005). Funk et al. (2005) concluded that
further evaluation of the impact of Salmonella serovar present on farms on seroprevalence and
the relationship of on-farm seroprevalence with food safety risk are needed prior to utilising
serology for pre-harvest Salmonella diagnostics in the US swine herd (FUNK et al. 2005). It
should also be emphasized that immunological surveillance regularly has to be supplemented
by bacteriological culture method in order to detect possibly emerging serovars, which might
not be included in the ELISA and therefore not captured in the surveillance (EFSA 2006).
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2.3 Prevention and Control of Salmonella in pig farm
2.3.1 Prevention and Control of Salmonella
Most periodically have seen dramatic and often a continuously ongoing increase in human
outbreaks of salmonellosis originating from infections in animals. Therefore attention has
been increasingly focused on the prevention and control of Salmonella in animal production,
by bodies such as WHO (WHO 1993), World Organization for Animal Health (Office
International des Epizooties – OIE) (WIERUP 1994) and the EU (Dir 92/117/EEC). The need
for global cooperation in the control of salmonellosis was also emphasized (BÖGEL 1991).
Primarily, attention was concentrated on the poultry production. Today the need to control
Salmonella also in swine production is increasingly focused (EFSA 2006).
In 1980, WHO had already formulated three lines of defence against Salmonella, which still
comprise valid strategic approaches to risk mitigation (WHO 1980):
A) the first approach focuses on controlling Salmonella in the food-producing animal (preharvest control)
B) the second approach involves improving hygiene during the slaughter and further
processing of the meat (harvest control)
C) the third approach targets the final preparation of food by educating the food industry and
consumers about good hygiene practices (post-harvest control).
When it comes to control of Salmonella in pork production systems, there is no silver bullet.
Each farm presents its own unique problems. Instead of one magic tool, farm-specific
biosecurity and sanitation procedures need to be developed and implemented for every farm.
This is an ongoing process. Unlike common hog diseases such as Pseudorabies, Salmonella
control has no end in sight. On-farm Salmonella control is likely to become a major part of
permanent pork quality assurance programs throughout the industry. The ultimate goal of
such programs in the pork chain is not the elimination of Salmonella organisms. The goal is to
prevent them from being introduced into herds supplying the food chain (BLAHA 2000).
Pre-harvest control of Salmonella at the farm level has long been considered an important part
of pathogen reduction schemes (USDA 1993), not least because traditional meat inspection
cannot control Salmonella-contaminated carcasses. Indeed, the latter demonstrate how the
industrialization of animal production “opened the door” of the food chain to pathogens likes
Salmonella (FORSHELL and WIERUP 2006). During the last decade, the structure of the
swine industry has changed markedly, with the introduction of large integrated systems and
breeding pyramids akin to those of the poultry industry. Control measures are of increasing
importance because of consumer concerns about food-borne zoonoses. Epidemiological
studies in recent years suggest that Salmonella infection of sucking piglets is much lower than
that of older animals because of lactogenic immunity and that the application of an Integrated
Quality Control (IQC) Systems agreed upon by all staff, can reduce the prevalence of
Salmonella on pig farms. Application of these systems require some knowledge of the
Salmonella prevalence on an individual farm and this can be monitored as indicated earlier
either by serology or culture (WRAY 2001). The main components of an IQC System are:
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Literature review
Biosecurity
The term “Biosecurity” is widely used, but most dictionaries do not carry a definition for this
word. A good definition may be “measures imposed to protect a biological system from attack
by potentially harmful microorganisms that can reduce the level of health of man and
animals” (HARDY 2002).
As indicated previously there are many routes by which Salmonella can be introduced onto a
farm and the organism is often disseminated widely on farms (WRAY 2001). Biosecurity
plays a significant role in the control process. Starting with purchased stock, the aim should
be to source animals from a supplier of known low or no Salmonella status (BLANCHARD
and BURCH 2008). Control measures (Table 2.13) include changes of clothing and boots for
visitors, bird and rodent control, foot-baths containing active disinfectant outside houses,
limiting access to the site by visitors and lorries, etc. Farm size, stocking densities and pig
density within a region all have a negative effect on the Salmonella status of a farm, perhaps
by predisposing to Salmonella spread within and between farms (WRAY 2001).
It is recommended that all farms conduct a critical review of the measures in place to prevent
disease entering their operation. Application of HACCP (Hazard Analysis of Critical Control
Points) procedures will help to identify areas of greatest risk to the business and allow for
development of preventative strategies (BLAHA 2000; HARDY 2002). Staff training is
essential for them to gain a full understanding of biosecurity and the importance to minimize
risk of infection. Regular audits need to be made with appropriate check lists (similar to those
seen in restrooms) to monitor the practical implementation of the preventative actions
imposed. Biosecurity measures should be used to protect the farming system from both
external entry of pathogens and also internal transfer between different parts of the
farm. Biosecurity enhances animal performance, minimizes disease, reduces medication costs
and overall improves quality assurance of the pork chain (Figure 2.16).
Pig farmers often assume that they have adequate biosecurity procedures because they have a
“shower in/shower out” facility. Some of the most obvious sources of infection receive
inadequate attention on many farms (HARDY 2002). These include the following:
A) Dirty sows are moved from the gestation barn to the clean and disinfected farrowing room.
The skin, hair and feet can carry bacterial infections as can the gastrointestinal tract.
B) No hand protective barrier, either physical or chemical, is used when the staff process
litters of pigs, running the risk of spreading infection from one litter to another.
C) In the farm office it is important to clean the computer keyboard, the telephone, the shower
and toilet and the lunchroom, including all surfaces with an appropriate bactericidal product.
D) There should be a disease prevention disinfectant area at all times at entrances to the farm
for all vehicles and foot dips for staff between all rooms and barns on the farm.
E) In the food industry audit procedures are used to check on the sanitizing procedure. Farms
should consider using a long-lasting bactericidal coating product including a fluorescent dye
that can be detected by “black light.” This is similar to the detection of mold on corn.
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Literature review
Profits
Quality Assurance
Reduced Medication
Reduced Zoonoses
Reduced Mortality
Reduced Disease
Improved
Performance
Biosecurity
Figure 2.16: Biosecurity (HARDY 2002)
All-in All-out systems
Effective cleaning and disinfection are important aspects of disease control. It is generally
accepted that farms should operate an All-in All-out (AIAO) policy, with adequate cleaning
and disinfection after the pen is empty. Linton et al. (1970) found that uninfected animals,
which remained in disinfected pens usually stayed free of Salmonella but as the number of
pigs per pen increased a higher prevalence of infection was found. Tielen et al. (1997) found
that Salmonella negative piglets placed in clean accommodation remained free despite
serological evidence of Salmonella in the sows. In Denmark, removal of 10 week old pigs
from breeding farms infected with S.Typhimurium to clean premises appeared to be effective
in prevention of infection at market age (DAHL et al. 1997a, b). Offsite weaning at 10-16
days prevented Salmonella infection in grow to finish pigs moved to a clean environment
(NIETFELD et al. 1998a). Fedorka-Cray et al. (1997b) weaned pigs at 14-21 days and
removed them to clean accommodation where the piglets remained free of Salmonella.
Improved disinfection of weaner and grower pens on several farms produced significant
reduction in the incidence of positive batches (from 80 to 11% on one farm) (DAVIES and
WRAY 1997).
A word of caution should be introduced, however, as other investigators have shown no
benefit in reducing the prevalence of Salmonella species using AIAO management of
finishing pigs compared with conventional farrow-to-finish systems in North Carolina, USA
and higher prevalence of Salmonella in multiple site AIAO systems. In 1995 AIAO was
practised on 42.4 % of operations with finishing accommodation, comprising 51% of national
hog production (DAVIES et al. 1997). A comparison of continuous flow (CF) systems and
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AIAO systems in the US found little difference between the 2 systems when tissues collected
in the abattoir were examined for Salmonella (PROESCHOLDT et al. 1999).
In Sweden, depopulation is used to control Salmonella infection in pigs, but this is not always
successful, and Wahlström et al. (1997) described a farm on which S. Derby was endemic and
they found that it was not possible to eliminate the infection without permanently decreasing
the pig population by 50%. Depopulation has also been used in Denmark to eradicate S.
Typhimurium DT104 from the national pig herd (MØGELMOSE et al. 1999) but it was not
always successful because contamination persisted on some of the farms and the replacement
pigs became infected. A reappraisal of this strategy has resulted in intervention plans being
produced for S. Typhimurium DT 104 infected herds and herds being declared free on
bacteriological and serological testing: so far this strategy has been successful in15 herds
(MØGELMOSE et al. 2001).
In Denmark, a task-force was established to clarify why chronic infected level three herds
were unable reduce their Salmonella problem. Intervention plans were formulated for 78
herds and 12 months after the implementation of the plan, 61 (78%) of herds were assigned to
level one, 12 (15%) herds to level 2 and 5 herds remained at level 3. Of the 61 herds, 45
maintained their status six months in a row. Bagger and Nielsen, (2001) concluded that it is
possible to reduce the prevalence of Salmonella in chronically infected herds by a
combination of intensive advising, enforced implementation of intervention plans and
economic pressure.
In the United Kingdom, intervention strategies to control Salmonella such as feed
acidification and the use of fermented liquid feed were not successful: the most successful
strategy in batch systems was dry cleaning and disinfection with 2% formaldehyde. However
in many cases the beneficial effects were undermined by the introduction of Salmonella
infected pigs and the failure to disinfect other areas and McLaren et al. (2001) concluded that
the use of single interventions was unlikely to be successful. A chronically infected herd was
depopulated, the pens were cleaned and disinfected and then left empty for six months.
However Salmonella was detected in the soil of the sow paddock and in mice six months after
depopulation and on re-population, Salmonella was detected in replacement breeding stock
and though the level of infection remained low initially, within a year Salmonella was
widespread (DAVIES et al. 2001).
Feeding Systems
Many batches of animal feed are contaminated with Salmonella (WRAY and WRAY 2000).
However, Bisping (1993) concluded that the predominating serovars in recent human
outbreaks, Salmonella Typhimurium and Salmonella Enteritidis, are only rarely present in
feed. Also Fedorka-Cray et al. (1997b) demonstrated only very low levels of Salmonella
contamination of the feed, but nevertheless feed might not be excluded as a source of
Salmonella contamination of pigs. This was confirmed by Harris et al. (1997) who found that
Salmonella could be recovered from feed in 46.7% of the pig herds, indicating that feed might
be an important source for infection.
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Salmonella in feed can be reduced by pelleting the feed but process control (temperature) is
needed and recontamination should be avoided. Pelleted feed is associated with better
production results (EISEMANN and ARGENZIO 1999), but also with an increased risk of
Salmonella-infection compared with non-pelleted wet feed (STEGE et al. 2001; LEONTIDES
et al. 2003, BELOEIL et al. 2004b; LO FO WONG et al. 2004). Mikkelsen et al. (2004)
demonstrated that feeding a coarsely grounded meal feed (with the highest percentage of
particles >1000 μm) changed the physicochemical and microbial properties of the stomach
content, which led to a decrease in the survival of Salmonella during passage through the
stomach. Pigs fed the coarsely grounded meal feed had much higher microbial fermentation in
their stomachs, which led to increased numbers of total anaerobic bacteria, higher
concentrations of various organic acids and a lower pH (3.38) in the stomach. A slower
gastric passage rate has already been reported in pigs fed a coarsely (particle size 903 μm)
versus a finely ground feed (particle size 639 μm) (REGINA et al. 1999). Smith (2003)
assumed that, if the gastric emptying time is short as is the case with finely ground feed,
pathogens have less contact with gastric acid. As a result, the pathogens pass through the
stomach and consequently reach the intestines where they can cause damage.
It has been demonstrated before that acidifying the feed, for instance by supplying liquid
feeding of by-products, is associated with decreased Salmonella shedding (VAN DER WOLF
et al. 1999, 2001a). Also the administration of acidified drinking water (2 ml acid mixture
(Selko®, Tilburg, The Netherlands) / l drinking water), with a pH of 3.5-3.6, showed a
significant reducing effect on the Salmonella seroprevalence (VAN DER WOLF et al.
2001b). Kranker et al. (2001) demonstrated that supplying of ready-mixed pelleted feed is
associated with a higher seroprevalence in finishing pigs and also in sows. Beloeil et al.
(2004b) observed a higher prevalence of Salmonella shedding in herds feeding dry feed
compared to wet feed. Trough feeding, where water and dry pelleted feed may soak for
several hours before feeding, is associated with an increased risk for Salmonella infection
because of multiplying of Salmonella bacteria from faecal contamination (VAN DER WOLF
et al. 1999).
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Table 2.13: A summary for Salmonella control (MAFF 2000)
Control point
Unit
Stock
Keeping Salmonella out
For new units – locate well away from
other farms, in particular pig farms and
landfill sites.
Keep clean and tidy.
Perimeter fence/ information signs.
Parking for vehicles.
Provide washing/ disinfection facilities/
footbaths.
Clean and disinfect regularly.
Introduce a Salmonella monitoring
programme.
Operate All in/All out system.
Purchase stock from reliable source.
Isolate/quarantine purchased stock.
Bedding
Water
Train and inform.
Keep „work clothes‟ on site and clean
and disinfect regularly.
Effective control programme.
Restrict entry.
Visitor book.
Provide clean protective clothing.
Reliable source/ Salmonella tested.
Secure, clean storage away from pigs.
Mixing/milling away from pigs.
Clean source, not contaminated.
Mains or tested source.
Animal waste
Careful disposal away from site.
Equipment
Do not share equipment.
Clean and disinfect regularly.
Staff
Pest control
Visitors
Feed
66
Controlling the spread
Keep clean and tidy.
Provide washing/
disinfection facilities/
footbaths.
Clean and disinfect regularly.
Operate All in/ All out
system.
Keep pigs clean.
Segregate groups.
Isolate sick pigs/ infected
groups.
Keep „work clothes‟ on site
and clean and disinfect
regularly.
Check controls effective.
Provide clean protective
clothing.
Check for signs of
contamination.
Check storage secure.
As for „Feed‟.
Check for signs of
contamination.
Enclosed water system.
Store slurry for at least 4
weeks. Cover and compost
manure. Spread on arable
land but away from nearharvest crops.
Clean between sections of
the farm.
Clean and disinfect regularly.
Literature review
2.3.2 Salmonella control and monitoring programmes in EU
Following the EU legislation (EC 2160/2003), all member states should establish control
programmes to reduce the prevalence of zoonosis and zoonotic agents such as Salmonella.
According to the Zoonosis Act of 2003 all EU members are obliged to establish objectives for
reducing Salmonella in breeder and finisher heards in 2007. EU-approved national plans for
surveillance and control of Salmonella in pigs must be implemented in 2009. Different EU
countries have already implemented a Salmonella surveillance programme. The EFSA journal
(2006) reported, existing national Salmonella monitoring and control programmes:
2.3.2.1 The Control of Salmonella in Sweden
Historical background
A general control of Salmonella started in the early 1950-ies following severe Salmonella
epidemics in particular when Salmonella was spread from a slaughterhouse in 1953-54 and
more than 9,000 people were recorded sick and 90 people died (LUNDBECK et al. 1955).
The current control in swine production
The objective of the control is to ensure that all animal products delivered to human
consumption are free from Salmonella. Any finding of Salmonella, irrespective of serovar, in
animals, humans, feed and food is compulsory notifiable, independent of reason for sampling.
All primary isolates are sero and phage typed and primary isolates from animals are tested for
antibiotic resistance. If a veterinarian suspects that an animal is Salmonella infected he/she is
obliged to perform further investigations to clarify this. Furthermore all sanitary slaughtered
animals are tested for Salmonella.
The strategy is to monitor at critical points of the production chain to ensure that no
Salmonella contamination occurs. When Salmonella is isolated, irrespectively of serovar and
in case of live animals whether clinical signs are present, actions are taken to eliminate the
microbe. Infected farms are e.g. subjected to restrictions which include a ban of movement of
animals except for transport to sanitary slaughter. Environment and animals are sampled and
tested by bacteriological method for Salmonella. Salmonella carriers are eventually
slaughtered or destroyed followed by careful cleaning and disinfection. Restrictions are lifted
following two negative samplings of the whole herd. Up and down streams epidemiological
tracing is undertaken and followed up by similar actions. The basic principle of the control is
the non acceptance of Salmonella contaminated animals, feed and food products.
According to an EU approved scheme additional monitoring for Salmonella is done on a
statistical basis since 1995. Annually, approximately 6,000 pigs at slaughter (five
ileocaecal/intestinal lymph nodes per animal), approximately 6,000 carcasses (1,400cm2 is
swabbed) and 4-5,000 scraping from pork/beef at cutting plants are analysed for the presence
of Salmonella. In addition, 59 faecal samples from all elite breeding and multiplier herds are
tested annually and sow pools twice a year. Furthermore, in all herds affiliated to a voluntary
quality assurance program covering about 60-65% of all slaughtered pigs (approximately
1,300 herds in 2004) 10 faecal samples are collected annually. The testing is paid by the
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industry and in case of restrictions different degrees of compensation, governmental or by
means of insurance are in place depending on compensatory preventive actions in place.
Furthermore all sanitary slaughtered animals as well as any suspect animal at normal
slaughter will be tested. If Salmonella is suspected at autopsy or due to clinical signs, samples
for Salmonella are also collected.
Control of feed
In the Swedish Salmonella control programme for food producing animals the control of
animal feed is an essential element. Monitoring and control of feed has been carried out by
the feed industry since the late 1940´s (THAL et al. 1957). In accordance with the Swedish
animal feed legislation feed must be Salmonella negative.
The need to control Salmonella in feed production became the primary objective for an
industry association founded in 1958, comprising most of the Swedish feed companies.
Several of the early guidelines on how to control Salmonella, were developed as industry
recommendations in collaboration with government experts. Since 1991, a Hazard Analysis of
Critical Control Point (HACCP) approach has been employed in the control of feed mills,
with critical control points being monitored weekly (STERNBERG et al. 2005).
The control of Salmonella in commercial feed must be based on several different strategies.
An important part of the programme is the control and quarantine of contaminated raw
materials before ingredients may be incorporated into compounded feed. After a proper heat
treatment, care must be taken not to re-contaminate the feed during cooling, transport or
storage at the farm level. An important point of the control programme is the HACCP-based
process control in the feed mill where the main hazards are identified. The aim is to make sure
that the processing line for feed is not contaminated with Salmonella. Temperatures above
75°C are used in the feed mills during pelleting for at least 30 seconds. Another important
factor is the hygiene of the premises and the need to develop efficient procedures for cleaning
and disinfection, particularly of the processing line (STERNBERG et al. 2005).
2.3.2.2 The Danish surveillance program of Salmonella in pigs and pork production
In 1995, a serological surveillance programme for detection of Salmonella infection in
slaughter-pig herds was implemented. The programme has been adjusted over the years and
revisions have previously been described in Annual Reports 2000-2002. Originally, the
Danish Veterinary and Food Administration (DVFA) was responsible for the administration
of the programme. However, since May 2002, the Danish Bacon and Meat Council (DBMC)
had carried out the daily administration supervised by the DFVA. All data from the
surveillance of Salmonella in pigs are registered in the central Zoonosis Register database,
which is part of the Central Husbandry Register, administered by the DVFA. Surveillance by
serological testing of meat juice (approx. 600,000 meat-juice samples per year) is carried out
in herds producing more than 200 slaughter pigs per year. These results are used to assign the
herds to one of three levels, based on the proportion of seropositive meat-juice samples
collected over the last three months. The sample results are weighted, such that results from
the most recent month are weighted more heavily than those from previous months.
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Level 1 herds are classified as having none or a small proportion of positive samples, Level 2
has a higher proportion of positives, and Level 3 herds have an unacceptably high proportion
of positive samples. Pigs from Level 3 herds must be slaughtered under special hygienic
precautions. It is mandatory to collect pen-faecal samples from herds placed in level 2 or 3 in
order to clarify the distribution and type of the Salmonella infection. With a few exceptions,
all sow herds supplying piglets to slaughter-pig herds in level 2 or 3 are obligated to collect
pen-faecal samples for determining the distribution of Salmonella within the herd, and to
clarify possible transmission of Salmonella from sow herds to slaughter pig herds. Breeding
and multiplying herds are monitored monthly through serological testing of blood samples. If
the set threshold is exceeded, the herd owner is obliged to collect pen-faecal samples.
Monitoring of Salmonella in pork is based on swab samples taken from three designated areas
of chilled half-carcasses at the slaughterhouse. Samples from 5 carcasses are pooled, except in
slaughterhouses slaughtering 50 pigs or less per month in which case, samples are analysed
individually. When determining the prevalence of pooled samples, the loss of sensitivity and
the probability of more than one sample being positive in each pool are taken into
consideration. A conversion factor has been determined on the basis of comparative studies,
as described in the Annual Report 2001. In 2004, 33,890 samples were pooled and the
prevalence of Salmonella was 1.3%. An additional 148 samples were collected from
slaughterhouses with a low production and were analysed individually. Of these, two samples
were found positive for Salmonella.
2.3.2.3 The British Salmonella monitoring programme, “Zoonoses Action Plan”
The Zoonoses Action Plan Salmonella Programme (ZAP) is an industry-owned initiative that
began in June 2002 for pigs supplied to quality assured abattoirs in Great Britain (GB) and in
January 2003 ZAP was extended to producers in Northern Ireland. To implement ZAP,
muscle samples are collected by Meat and Livestock Commission staff from 3 pigs for every
Pig Movement Order received at the abattoir with the intent that at least 15 samples are
collected every 3 months. Samples are linked to their herd of origin via the registered slap
marks. Samples are frozen in containers that facilitate meat juice collection during thawing
whilst being transported to the laboratory. An indirect lipopolysaccharide (LPS) mixSalmonella meat-juice Enzyme-Linked Immunosorbent Assay (MJE) which detects
antibodies against Group B and C1 Salmonella is conducted by a commercial laboratory.
Testing is accredited to the ISO 17025 standard by the UK Accreditation Service (UKAS).
Results from individual samples and the positive and negative controls are converted to a
Sample to Positive Ratio (S/P Ratio) which is interpreted as negative if it is less than or equal
to 0.25 and positive if it is greater than 0.25. From July 2003 all herds where at least 15
samples had been reported in the preceding 3 months were assigned a ZAP level and expected
to act as follows:

ZAP level 3 – 85% or more MJE results were positive; an action plan must be
developed and implemented to reduce to ZAP level 1 within 11 months.

ZAP level 2 – 65% or more but less than 85% of MJE results were positive; an action
plan must be developed and implemented to reduce to ZAP level 1 within 17 months
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Literature review

ZAP level 1 – less than 65% of MJE results were positive; no action required
ZAP level 1 status can only be regained if the prevalence of MJE positive pigs is below 65%
after testing a minimum of 15 samples in a three month period. Farms that fail to return to
ZAP level 1 will be suspended from Quality Assurance schemes, thus losing access to Quality
Assured abattoirs. ZAP aims to assign ZAP levels to at least 85% of herds delivering >500
pigs a year and to 50% farms delivering between 200-500 pigs per year. In the 3 month period
ending in December 2004, 79.9% of 1,533 holdings were allocated to ZAP levels 1-3 and
92% of these farms were in ZAP level 1. In 2004, 22.9% of 153,321 MJ samples were
positive in the ZAP programme. The overall aim of ZAP is to reduce the prevalence of
Salmonella in assured pigs at slaughter by 25% and the GB Food Standards Agency strategy
is to reduce Salmonella in pigs at slaughter by 50% by 2010.
2.3.2.4 The Irish Pig Salmonella legislation
The purpose of the regulations is to reduce any possible risk of public health problems arising
from the consumption of pork and pig meat products and thereby to maintain consumer
confidence in these products.
The following are the main points of this legislation:
• every pig herd in the country is tested on an ongoing basis for the purpose of establishing its
Salmonella status,
• sampling takes place at slaughter plants,
• samples are tested at the Central Veterinary Research Laboratory,
• the test results are sent directly to a centralised database,
• the database centre calculates the up-to-date Salmonella status of the herd and issues to the
herd owner a certificate of categorisation which is valid for five months,
• this certificate indicates whether the herd is Category 1 (i.e. showing the least evidence of
exposure to Salmonella), Category 2 or Category 3 (the worst status),
• at slaughter, pigs from Category 3 herds are slaughtered separately from other pigs and in a
manner that minimises the risk of cross-contamination,
• the head meat and offals of Category 3 pigs may not be sold in the raw state and must be
either heat-treated in an approved manner before being passed fit for human consumption or
destroyed,
• pigs with no valid category certificate will be treated as Category 3 in slaughter plants.
Pig producers’ responsibilities
Producers must ensure that they are in possession of a valid certificate of categorisation for
their herd and to make it available on request at pig slaughter plants. Each producer arranges
with the plant at which his/her pigs are to be slaughtered to have samples taken and forwarded
for testing to the approved laboratory. A set of samples must be taken three times each year at
intervals of not less than 3 months and not more than 5 months.
A set of samples consists of samples from 24 pigs from the herd submitted together. If the
size of the herd is such that fewer than 24 pigs are presented for slaughter on any individual
day, then samples are to be taken from 24 pigs in every 4 month period. If the number of pigs
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Literature review
presented for slaughter is below 24 in a 4 month period, then samples are to be taken from all
pigs slaughtered in that period.
It is the responsibility of herd owners to ensure that the required level of sampling is
undertaken and that a valid certificate of categorisation exists for his/her herd.
Calculation of Salmonella category
When a set of samples is tested the result of the test is expressed as the percentage of the
samples in the set that tested positive for exposure to Salmonella (e.g. if 6 of the 24 samples
are positive, the result is 25%). The initial herd categorisation is based on a simple average of
the first two test results for the herd (e.g. if the first two test results are 25% and 50%, then the
average of these is reported as 37.5%). Thereafter herd categorization will be established by
calculating a weighted average of the three most recent test results as follows:
Test
Most recent
Second most recent
Third most recent
Weighting
0.5
0.3
0.2
Herds are categorized as:

category 1, if the result of this averaging is 10% or less,

category 2, if the average result is more than 10% and not more than 50%,

category 3, if the average result is more than 50%.
Breeding pigs
It is a requirement of the Salmonella legislation that all breeding pigs being introduced into a
herd come from Category 1 herds and that producers maintain a record of the origin of their
breeding animals.
2.3.2.5 The German “QS Salmonella Monitoring Programme”
In September 2001, the German food industry (encouraged and promoted by the government)
has launched a voluntary quality assurance programme for food producers of all sectors (feed
production, animal production, slaughtering, processing, and grocery retail), the so-called
“Quality and Safety System” (in short: “QS System”) (BLAHA 2004). As for pork
production, so far, about 15,000 pork producers are participating in “QS”. This number is low
compared to the about 70,000 pig producers in Germany, but these 15,000 represent about
70% to 75% of the German pig production. Those pork producers and slaughter plants (100%
of the larger slaughter enterprises) that participate in QS have agreed to participate in the QS
Salmonella Monitoring Programme, which was started in early 2002.
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The programme, like the Danish, the British and the Irish programmes, is targeted at
categorising pig herds according to their assessed risk of carrying Salmonella into the
slaughter plant. Sixty meat juice samples per herd (evenly distributed over one year), taken
from slaughter pigs, are serologically tested for antibodies against Salmonella spp. The
assumption is that herds with many positive pigs pose a higher risk to carry Salmonella into
the slaughter plant than herds with no or only few positive pigs. The current cut-off for the
serological test (based on the Danish mixed-ELISA) is 40% OD.
The current “risk categories” are as follows:

Category I

Category II (= medium risk)
: 20% - 40% positive samples,

Category III (= high risk)
: > 40% positive samples.
(= low risk)
: < 20% positive samples,
Since 2002 the number of participating pig producers has steadily increased. By August 2005
about 6,200 pig producers are already categorised. This number is steadily increasing, since
more and more producers out of the 15,000 QS-participants have sampled more than a year.
All data are collected in a central database “Qualiproof” and can be analysed for further
conclusions for improving the programme.
In 2006, throughout Germany, about 67,000 samples per month are taken and tested for
Salmonella antibodies (per year about 800,000).
The present categorisation result is: out of the 6,200 categorised herds, about 80% are
Category I, about 15% are Category II and about 5% are Category III.
There are two planned for the Salmonella control based on the monitoring results:
- separating Category III pigs from Category I and II pigs at slaughter,
- supporting pig producers in reducing the Salmonella load of their pig herds.
2.3.2.6 The Dutch National Salmonella Control Plan
In The Netherlands, a nation-wide Salmonella Monitoring Programme was started on
February 1, 2005. Although the programme is not a governmental program, it is mandatory
for every finishing pig owner and every slaughterhouse, since it is run by the Product Board
for Live stock and Meat (the “PVV”), which has the authority to oblige all pig owners and
slaughterhouses to participate in the programme. The principle of the programme is the
categorisation of finishing pig herds by means of testing randomly selected serum samples
from every herd. Samples are to be collected from finishers within 3 weeks before marketing
or at slaughter. Thirty six samples per year (12 samples per trimester) are the basis for the
categorisation and the cut-off value (measured in OD%) is 40%. The category thresholds for
assigning the pig herds to three categories (low, medium and high risk of introducing
Salmonella species into the slaughter plant) are as follows:

category 1
: ≤ 20% samples with > OD40,
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
category 2
: >20% and < 40% samples with > OD40,

category 3
: ≥ 40% samples with > OD40.
Slaughterhouses producing more than 10,000 pigs per year are monitored using swab samples
as described by USDA/FSIS and in EU regulation 2001/471/EC, swabbing 300 cm2 or a
destructive method. Slaughterhouses slaughtering less than 150,000 pigs / year sample ten
carcasses every 14 days and all samples are investigated separately. Slaughterhouses
slaughtering more than 150,000 pigs / year sample five carcasses every day which will be
investigated as one pooled sample. Both schemes result in 10 Salmonella cultures every two
weeks for every slaughterhouse.
2.3.2.7 Salmonella control in pigs in the other EU Member States
In Austria, regional programmes (e.g. in Styria) have been implemented, which are a good
basis for the planned national programme (EFSA 2006). In the Austrian Province of Styria the
implementation of Directive 92/117/EEC, which requires measures to be taken for the control
of "food-borne diseases", resulted in the setting-up of a Salmonella surveillance programme
for pork production (WAGNER and KÖFER 2004).
Köfer et al. (2006) reported, the Styrian Salmonella Monitoring Programme for pork
production is based on a representative analysis of the current status, serological meat juice
monitoring and bacteriological tests of carcass halves and parts and has been in operation
since 1999. A total of 34,170 meat juice samples from 3,417finisher herds were tested using
the meat juice SALMOTYPE®-ELISA (Labor Diagnostik, Leipzig, Germany) in the period
from 1999 to 2003. More than 95% of the samples investigated were below the negative cutoff of <20% based on the 5-year average. The mean extinction values for meat juice samples
showed regional differences, which were visualized for epidemiological purposes using the
VETGIS(R) geographical information system (Department of Veterinary Administration,
Graz, Austria). Salmonellae spp. were detected in only 15 cases (0.13%) of a total of 11,330
bacteriologically tested wipe samples from meat-processing plants. The Salmonella isolates
detected included four S. Typhimurium, two S. Enteritidis PT 4, five S. Infantis, one S.
Bredeny, one S. Saintpaul, one S. Brenderup and one S. Livingstone isolates. The proportion
of Salmonella-contaminated pork in the total population estimated from the annual sample
showed a falling tendency. It decreased from 0.48% (CI: 0.23 </= P </= 0.85) in 1999 to
0.14% (CI: 0.07 </= P </= 0.24) in 2003. The contamination of Styrian pork with Salmonella
is extremely low and thus poses a negligible risk of infection to consumers.
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Materials and methods
3. Materials and Methods
3.1 Investigation on farm
Based on the serological results of the Salmonella antibody–ELISA test, 48 finishing pig
farms in the North West of Germany in the period of time from August 2007 – April 2008
were selected. All farms had a high serological prevalence of Salmonella positive pigs (QS
system; category III, > 40% of sero-positive pigs).
3.2 Sample collection and preparation for analysis
According to their serological meat juice antibody status, 48 seropositive finishing pig herds
were selected. Samples were collected approximately 20 samples per herd. All samples were
transported to the Field Station for Epidemiology of University of Veterinary Medicine
Hannover, Foundation in Bakum on the same day. The investigation included real-time PCR
and bacteriological examinations of faecal and environmental samples. The samples were
taken on each sampling day. The preparation and examination in the laboratory were also
considered (Figure 3.1). All Salmonella isolations were sub-typed by phage-typing in
cooperation with the Robert-Koch Institute in Wernigerode.
3.2.1 Environmental samples
Environmental samples such as rodent or cat faeces and swabs from pen wall, floor, chain,
ventilator, feeder, nipple, boot and other equipments were collected by sterile gauze swab
(Figure 3.2-3.6) (Tubular Bandage tg®, Lohmann & Rauscher International GmbH,
Germany), moistened with Buffer peptone water (BPW, Oxoid Ltd., UK.).
After sampling, the swab of each sample was placed into a labeled sterile plastic bag (VWR®
International GmbH, Germany), which was closed and immediately put into sterile box.
At laboratory, 10 grams of each environmental sample was weighed into sterile plastic bag
(Stomacher®400, Seward Ltd., UK). Then, each sample was mixed with 90 ml. of buffer
peptone water (BPW, Oxoid Ltd., UK.) and homogenized by homogenizer (EasyMix®, AES,
France) for 2 minutes. Next the samples were incubated at 37°C in an incubator for 16 hours
(Real-Time PCR assay) or 24 hours (Conventional bacteriological method) (Figure 3.9).
After incubation, 1.5 ml. of the pre-enrichment culture (BPW) was transferred into 2 ml.
reaction tube (Eppendorf®, Germany). Buffer peptone water was used to directly extract
Salmonella DNA for real-time PCR assay. Moreover, the remaining buffer peptone water was
used for the conventional bacteriological method of Salmonella as described below.
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Materials and methods
Sample collection
Faecal and environmental sample
Pre-enrichment step
Pre-enrichment step
BPW, 37°C, 16 hours
BPW, 37°C, 24 hours
DNA-Extraction step
Enrichment step
RV, 42°C, 24 and 48 hours
Real-Time PCR
Rambach and XLD agar
(ABI prism 7500)
37°C, 48 hours
Blood and Gassner agar
37°C, 48 hours
Sero-typing
(Kauffmann-White Scheme)
Phage-typing
(Robert-Koch Institute)
Figure 3.1: Scheme of sample collection on farm and preparation of samples in the
Laboratory for the analysis of Salmonella.
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Materials and methods
3.2.2 Faecal samples
Faeces were collected from rectum (Rectal swab, Cotton-tipped applicators in sterile
container, Heinz-Herenz, Germany) of different growing pigs in each group (Figure 3.7) or
faeces of finishing pigs from the pen floor (taken by means of gauze sock (Tubular Bandage
tg®, Lohmann & Rauscher International GmbH, Germany), which was over disposable
plastic boot) (Figure 3.8).
After sampling, swab was brought into a labeled sterile plastic bag (VWR® International
GmbH, Germany), which was closed and immediately put into sterile box.
At laboratory, the 5 rectal swabs were pooled and gauze sock were weighed into a sterile
plastic bag (Stomacher®400, Seward Ltd., UK.) and each sample was mixed with 90 ml. or
225 ml. of buffer peptone water (BPW, Oxoid Ltd., UK.) respectively and homogenized by
homogenizer (EasyMix®, AES, France) for 2 minutes, and incubated at 37°C in an incubator
for 16 hours (Real-Time PCR assay) or 24 hours (Conventional bacteriological method).
After incubation, 1.5 ml. of the pre-enrichment culture (BPW) was transferred into 2 ml.
reaction tube (Eppendorf®, Germany). Buffer peptone water was used to directly extract
Salmonella DNA for real-time PCR assay (Figure 3.10). Moreover, the remaining buffer
peptone water was used for the bacteriological method of Salmonella as described below.
Figure 3.2: Chain swab
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Materials and methods
Figure 3.3: Floor swab
Figure 3.4: Drive board swab
77
Materials and methods
Figure 3.5: Rodent feaces
Figure 3.6: Drinker Nipple swab
78
Materials and methods
Figure 3.7: Rectal swab
Figure 3.8: Overshoe or gauze sock method
79
Materials and methods
Figure 3.9: Pre-enrichment step, sample was enriched by BPW at 37°C for 24 hours.
Figure 3.10: 1.5 ml. of the pre-enrichment culture (BPW) was transferred into 2 ml.
reaction tube (Eppendorf®, Germany)
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Materials and methods
3.3 Bacteriological methods
All samples were cultured according to ISO 6579, while sero-typing were according to
Kauffmann-White Scheme that was established at the Field Station for Epidemiology in
Bakum. Moreover, Salmonellae were sub-typed by phage-typing in cooperation with the
Robert-Koch Institute in Wernigerode.
For the culture, after pre-enrichment step, samples from the pre-enrichment broths (BPW)
were inoculated into selective broths (Rappaport-Vassiliadis, RV, Oxoid Ltd., UK.) and
incubated at 42°C for 24 hours and 48 hours (Figure 3.11).
After incubation, Rappaport-Vassiliadis broth was taken 1µl. loop and streaked on Rambach
agar plate (Rambach® Agar, Merck KGaA, Germany) and Xylose lysine deoxycholate agar
plate (XLD., Heipha Dr. Müller GmbH, Germany). Plates were incubated at 37°C for 48 h.
After incubation, agars were checked up to suspect colony, crimson with pale border or pink
on Rambach agar, red with black centre on XLD agar (Figure 3.12). Suspect colony having
typical appearance of Salmonella was transferred and streaked over one plate of Blood agar
(Heipha Dr. Müller GmbH, Germany) and Gassner agar (Heipha Dr. Müller GmbH,
Germany), Plates were incubated at 37°C for 24 hours.
After incubation, agars were checked up to suspect colony, milky on Blood agar and yellow
on Gassner agar (Figure 3.13). Suspect colonies were confirmed and sero-typed by using the
slide agglutination Salmonella antiserum test (SIFIN®, Germany) in accordance with the
Kauffmann-White Scheme.
Sero-typing by the slide agglutination method (Figure 3.14), suspected colonies were
transferred onto a slide. After that the slide were drop by test reagent (25µl.), and mixed well,
therefore it become homogenous, slightly milky suspension results (The result could be read
easily by placing slide on a dark surface).
The reaction was read with the naked eye by holding the slide in front of a light source against
a black background and rocking it (tilting it back and forth).
Sodium Chloride (NaCl) solution was used for the negative control. For the evaluation, the
test could only be evaluated if the negative control remained milky-opaque.
Positive: visible agglutination after the sample had been tilted back and forth less than 20
times. In a strongly positive reaction, agglutination (coarsely or finely flocculent) appeared as
soon as the bacterial mass was mixed in. In the weakly positive result, agglutination only
appeared after the slide had been tilted back and forth 10-20 times.
Negative: if the suspension remained milky-opaque, or the reaction began to occur only after
the slide had been tilted back and forth more than 20 times, the result was negative.
After sero-typing, Salmonella colony was transferred into transportation media and sent for
phage-typing to the Robert-Koch Institute (Figure 3.15).
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Materials and methods
Figure 3.11: Enrichment step, samples were enriched by Rappaport-Vassiliadis at 42°C
for 24 hours and 48 hours
Figure 3.12: Typical Salmonella colonies, crimson with pale border or pink on Rambach
agar, red with black centre on XLD agar.
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Materials and methods
Figure 3.13: Typical Salmonella colonies, milky on Blood agar and yellow on Gassner
agar.
Figure 3.14: Sero-typing by the slide agglutination method
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Materials and methods
Figure 3.15: Salmonella colony was transferred into transportation media and sent for
phage-typing to the Robert-Koch Institute
3.4 Real-Time PCR assay
To analyze the pre-enriched faeces and environmental swab samples, the real-time PCR assay
was used after extraction the DNA.
3.4.1 DNA Extraction
DNA extraction was achieved using a commercially available Kit for isolation of genomic
DNA. The PrepMan®Ultra Sample Preparation Reagent (Applied Biosystems, USA.) was
used. The protocol for DNA isolation from sample was followed:
Pre-enriched sample (BPW) was transferred to a sterile microcentrifuge tube (Eppendorf®,
Germany) (Figure 3.10), the tube was centrifuged at maximum speed in a microcentrifuge for
3 minutes. Supernatant was then discarded, and the cell pellet was resuspended in 100 µl. of
PrepMan® Ultra Sample Preparation Reagent. The suspension was mixed and heating by
thermomixer (Eppendorf, Germany) at 95-100°C for 10 minutes, then the tube was briefly
vortexed again. After vortexed, the tube was centrifuged with maximum speed in a
microcentrifuge for 3 minutes.
The 50 µl. supernatant was transferred into a new microcentrifuge tube and divided 10 µl. of
the supernatant (sample DNA) were transferred to a new tube containing 90 µl. of RNase-free
water, then briefly vortexed the tube to mix the contents. The sample DNA was ready for realtime PCR.
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Materials and methods
3.4.2 Preparing PCR
After DNA extraction step, preparing PCR was second step by using the TaqMan®
Salmonella enterica Detection Kit (Applied Biosystems, USA.). Preparing PCR consisted of
creating the plate document and the reaction mix (Figure 3.16).
The procedure for preparing PCR was followed:
Created and set up the plate document with thermal-cycling conditions specified in the
following table:
AmpliTaq Gold® Enzyme
Activation
STEP
Time
Temp.
PCR
Cycle (45 cycles)
Denature
Anneal/Extend
15 seconds
1 minute
95°C
60°C
HOLD
10 minutes
95°C
All reagents were thawed, and the tube was labeled with the target name. Creating the Premix
solution accord to the following table:
Component
2X Environmental Master Mix (EMM)
10X Target Assay Mix (TAM)
Total volume
Volume (µl) for One 30-µl reactions
15.0
3.0
18.0
The solution was mixed by gently pipeted up and down. The 18 µl. of Premix Solution was
transferred into each well to the used, gently pipeted at the bottom of the well.
The 12 µl of unknown sample, negative control and positive control were transferred into
each sample well, gently pipeted up and down to mix the solution. Closed an optical cover to
the plate and made sure reagents were in the bottom of the wells, used a centrifuge with a
plate adapter to briefly centrifuged the plate. After preparing PCR step, the reaction plate was
loaded into the ABI Prism 7500 real-time PCR system (Figure 3.17). Started the run and
waited out for the results.
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Materials and methods
Figure 3.16: Preparing PCR step
Figure 3.17: ABI Prism 7500 real-time PCR system
86
Materials and methods
3.5 Statistical methods
The comparison between 2 type methods of bacteriological results and real-time PCR results
were tested by SAS 9.1 (Statistical Analysis System, Cary, NC, USA.).
Statistic analysis was used Kappa index to estimate the result of real-time PCR assay
compared with conventional culture result.
Contingency table for the evaluation of Cohen's kappa index:
Result of the technique 1
Type
Result of the
technique 2
Total
Positive
Negative
Total
Positive
Negative
a
c
b
d
a+b
c+d
a+c
b+d
N
Kappa index was calculated dividing the subtraction of observed coincidence - expected
coincidence by the substraction of 1 - expected coincidence, being the observed coincidence
(a+d)/N and the expected coincidence [(a+b)x(a+c) + (c+d)x(b+d) ]/NxN.
In general, the following criteria based on the interpretation table:
Kappa index
< 0.00
0.00 – 0.20
0.21 – 0.40
0.41 – 0.60
0.61 – 0.80
0.81 – 1.00
Agreement
Less than chance
Slight
Fair
Moderate
Substantial
Almost perfect
In the contingency table the value of Sensitivity and Specificity was calculated as
 Sensitivity
= a / (a+c)
 Specificity
= d / (b+d)
The correlation coefficients
In probability theory and statistics, correlation, (often measured as a correlation
coefficient), indicates the strength and direction of a linear relationship between two random
variables. In general statistical usage, correlation or co-relation refers to the departure of two
variables from independence. In this broad sense there are several coefficients, measuring the
degree of correlation, adapted to the nature of data.
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Materials and methods
A number of different coefficients are used for different situations. The best known is the
Pearson product-moment correlation coefficient, which is obtained by dividing the covariance
of the two variables by the product of their standard deviations.
Interpretation of the size of a correlation
Several authors have offered guidelines for the interpretation of a correlation coefficient. As
Cohen (1988) himself has observed, however, all such criteria are in some ways arbitrary and
should not be observed too strictly. This is because the interpretation of a correlation
coefficient depends on the context and purposes. A correlation of 0.9 may be very low if one
is verifying a physical law using high-quality instruments, but may be regarded as very high
in the social sciences where there may be a greater contribution from complicating factors.
Along this vein, it is important to remember that "large" and "small" should not be taken as
synonyms for "good" and "bad" in terms of determining that a correlation is of a certain size.
For example, a correlation of 1.0 or −1.0 indicates that the two variables analyzed are
equivalent modulo scaling. Scientifically, this more frequently indicates a trivial result than a
profound one. For example, consider discovering a correlation of 1.0 between how many feet
tall a group of people are and the number of inches from the bottom of their feet to the top of
their heads.
Correlation
Small
Medium
Large
Negative
-0.3 to -0.1
-0.5 to -0.3
-1.0 to -0.5
Positive
0.3 to 0.1
0.5 to 0.3
1.0 to 0.5
In statistical hypothesis testing, the p-value is the probability of obtaining a result at least as
extreme as the one that was actually observed, given that the null hypothesis is true. The fact
that p-values are based on this assumption is crucial to their correct interpretation. More
technically, a p-value of an experiment is a random variable defined over the sample space of
the experiment such that its distribution under the null hypothesis is uniform on the interval
(0, 1). Many p-values can be defined for the same experiment.
p-value
>0.05
0.01 – 0.05
0.001 – 0.01
<0.001
Significance levels
Not significant
Significant
Strong significant
High significant
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Results
4 Results
4.1 Results of Bacteriological method examination
4.1.1 Distribution of Salmonella in faecal samples
Table 4.1.1 shows the distribution of Salmonella in faecal samples, which the percentage of
the bacteriological methods was positive average at 15. 27 % .
Table 4.1.1: Distribution of Salmonella in faecal samples
Sample
Faeces (Gauze sock)
Faeces (Rectal swab)
Total
No. Sample
Culture
No. Positive
28
5
33
175
41
216
% Positive
16.00
12.19
15.27
4.1.2 Distribution of Salmonella in environmental samples
Table 4.1.2 shows the distribution of Salmonella in environmental samples, which the
percentage of the bacteriological methods was positive average at 22. 31 % .
Table 4.1.2: Distribution of Salmonella in environmental samples
Sample
Environmental
No. Sample
Culture
No. Positive
154
690
% Positive
22.31
4.1.3 Proportion of Salmonella isolates from various samples
Table 4.1.3 demonstrates a total of 906 samples of Salmonella test. Overall Salmonella was
isolated in 20.64% (187/906). From faecal samples, number of Salmonella isolations were
16.27% (28/175) and 12.19% (5/41) from pooled faecal samples (gauze sock method) and
rectal swab samples respectively.
Environmental samples reveal that the percentage numbers of Salmonella isolations were
22.31% (154/690). Samples which swab from scale had the highest proportion of the isolates.
The numbers of isolates were not found Salmonella in cat faeces and heater swab samples.
89
Results
Table 4.1.3: Proportion of Salmonella isolates from various samples
Sample
No. Sample
Faeces (gauze sock)
Rectal swab
Pipe
Feeder
175
41
30
72
Culture
No. Positive
28
5
9
16
Separate wall
Ventilator
Floor , walkway
Wall
Drive board
40
29
216
28
72
11
4
54
6
13
27.50
13.79
25.00
21.42
18.05
Load ramp
Carriage
Nipple drinker
Scale
Cat faeces
22
15
24
22
3
2
3
8
11
0
9.09
20.00
33.33
50.00
0
Ceiling
Lamp
Heater
Toy (Ball, Chain)
Boot
24
19
17
23
23
1
6
0
3
2
4.16
31.57
0
13.04
8.69
Rodent faeces
Total
11
906
5
187
45.45
20.64
90
% Positive
16.00
12.19
30.00
22.22
Results
4.1.4 Results of Salmonella Serotyping
4.1.4.1 Distribution of Salmonella serogroups in the farms
Table and Figure 4.1.4.1 shows serogroups of Salmonella which were found the most
frequently. A total of 186 isolates were tested. The serogroup which had the highest
proportion was Salmonella group B (97.85%), group G (1.61%) and group C (0.54%).
Table 4.1.4.1: Distribution of Salmonella serogroups in the farms
Serogroup
B
G
C
No. Sample
182
3
1
% Sample
97.85
1.61
0.54
Distribution of Salmonella serogroup
100
90
80
70
60
% Positive 50
40
30
20
10
0
97.85
1.61
B
G
0.54
C
Serogroup
Salmonella gr.B
Salmonella gr.G
Salmonella gr.C
Figure 4.1.4.1: Distribution of Salmonella serogroups in the farms
91
Results
4.1.4.2 Distribution of Salmonella serogroups in each type of samples
Faecal samples were found contaminated with Salmonella group B in the highest frequency
(17.20%), followed by group G (0.54%). Samples by gauze sock method and rectal swab were
found contaminated with Salmonella group B in the highest frequency 14.52% and 2.69%
respectively. Environmental swab samples such as swab from wall, floor, boot, etc. were
found contaminated with Salmonella group B (16.77%) in the highest frequency, following
by group G (1.08%) and group C (0.54%), while Salmonella group G was found
contaminated in faecal sample, floor swab and drive board swab sample, Salmonella group C
was found contaminated only in floor swab sample (Table 4.1.4.2).
Table 4.1.4.2: Distribution of Salmonella serogroups in each type of samples
Serogroup
Sample
B
n
C
Faeces (gauze sock)
Rectal swab
Pipe
Feeder
27
5
8
16
%
14.52
2.69
4.30
8.60
Separate wall
Ventilator
Floor , walkway
Wall
Drive board
11
5
52
6
12
5.91
2.69
27.96
3.23
6.45
Load ramp
Carriage
Nipple drinker
Scale
Cat faeces
2
3
8
11
1.08
1.61
4.30
5.91
Ceiling
Lamp
Heater
Toy (Ball, Chain)
Boot
1
6
0.54
3.23
3
2
1.61
1.08
Rodent faeces
5
2.69
92
G
n
%
n
1
%
0.54
1
0.54
1
0.54
1
0.54
Results
4.1.4.3 General distribution of Salmonella serogroup
Table and Figure 4.1.4.3 shows general distribution of Salmonella serogroup. There were
97.85%, 1.61% and 0.54% of samples contaminated with Salmonella serogroup B, G and C
respectively.
Table 4.1.4.3: General distribution of Salmonella serogroup
Serogroup
B
C
G
Serotype
S. Typhimurium
S. Derby
S. Goldcoast
S. Kedougou
No. Positive
148
34
1
3
% Positive
79.57
18.28
0.54
1.61
General distribution of Salmonella serogroup
80
79.57
70
60
50
% Positive
40
30
18.28
20
10
0.54
1.61
0
Salmonella spp.
S. Typhimurium
S. Derby
S. Goldcoast
Figure 4.1.4.3: General distribution of Salmonella serogroup
93
S. Kedougou
Results
4.1.4.4 Distribution of Salmonella serotypes were separated by group of samples
Samples from faecal and environment were found contaminated with S. Typhimurium in the
highest frequency. Salmonella Typhimurium was found 15.05% in faecal samples and
64.52% in environmental samples (Table and Figure 4.1.4.4).
Table 4.1.4.4: Distribution of Salmonella serotypes w e re separated by group of
samples
Faeces
Serogroup
Environment
Serotype
S. Typhimurium
S. Derby
S. Goldcoast
S. Kedougou
B
C
G
n
%
n
%
28
4
1
15.05
2.15
0.54
120
30
1
2
64.52
16.12
0.54
1.08
Distribution of Salmonella serotypes
were separated by group of sample
70
64.52
60
50
40
% Positive
30
20
10
16.12
15.05
2.15
0.54 1.08
0.54
0
Faeces
S. Typhimurium
Environment
S. Derby
S. Goldcoast
S. Kedougou
Figure 4.1.4.4: Distribution of Salmonella serotypes were separated by group of
samples
94
Results
4.1.5 Results of Salmonella Phage-typing
Table and Figure 4.1.5 shows phage-types of Salmonella which were found the most
frequently. A total of 174 isolates were tested. The highest proportion of the phage-types
was Salmonella Typhimurium DT193 (66.67%), following by S. Derby DY PT55 (5.75%), S.
Derby DY PT11 (5.17%), S. Derby DY PT51 (4.60%), S. Derby DY PT04 (4.02%), S.
Typhimurium ut (4.02%), S. Typhimurium DT 120 (3.44%), S. Typhimurium DT 097
(1.72%), S. Kedougou (1.72%), S. Typhimurium DT 104 (1.15%), S. Typhimurium DT 208
(0.57%), S. Goldcoast (0.57%), and Salmonella subspecies I (0.57%).
Table 4.1.5: General distribution of Salmonella phage-types
Phage-type
Salmonella Typhimurium DT193
No. Sample
116
% Sample
66.67
Salmonella Derby DY PT55
Salmonella Derby DY PT11
Salmonella Derby DY PT51
Salmonella Derby DY PT04
Salmonella Typhimurium ut
10
9
8
7
7
5.75
5.17
4.60
4.02
4.02
Salmonella Typhimurium DT120
Salmonella Typhimurium DT097
Salmonella Kedougou
Salmonella Typhimurium DT104
Salmonella Typhimurium DT208
6
3
3
2
1
3.45
1.72
1.72
1.15
0.57
1
1
174
0.57
0.57
100.00
Salmonella Goldcoast
Salmonella subspecies I
Total
95
Results
General distribution of Salmonella phage-type
4.6%
4.02%
5.17%
5.75%
66.66%
S. Tm DT193
S. Tm DT104
S. Derby PT11
S. Tm ut
S. Tm DT208
S. Derby PT51
S. Tm DT120
S. Goldcoast
S. Derby PT04
S. Tm DT097
S. ssp. I
Figure 4.1.5: General distribution of Salmonella phage types
Salmonella Typhimurium phage types
1.48%
4.44% 2.22%
5.2%
0.74%
85.92%
S. Tm DT193
S. Tm UT
S. Tm DT120
S. Tm DT097
S. Tm DT104
S. Tm DT208
Figure 4.1.6: Phage types of Salmonella Typhimurium
96
S. Kedougou
S. Derby PT55
Results
4.2 Results of real-time PCR assay examination
4.2.1 Result of Ct value (Raw data)
The average Ct (Threshold cycle) value (Table 4.2.1) for all samples which were positive by
real-time PCR (n=387) was 33.21. Salmonella was detected by cultural examination after 24
hours (n=136), the average Ct value was 28.76. Samples, which were negative after 24 hours
of culturing but positive after 48 hours and real-time PCR positive (n=51), showed an
increased Ct value of 33.31. Those sample, were also negative by cultural examination after
48 hours but positive by real-time PCR (n=200), demonstrated the highest average Ct value of
36.22.
Table 4.2.1: The average Ct value (Raw data)
Culture result
Positive after 24 hours
Positive after 48 hours
Negative after 48 hours
Overall
N
136
51
200
387
Average Ct value
28.76
33.31
36.22
33.21
Minimum
17.60
23.33
0.00
0.00
Maximum
41.00
38.50
44.99
44.99
4.2.2 Result of Ct value (Default setting; cut-off value = 41)
Raw data analysis according to Standard guide protocol of Applied Biosystems, threshold
value setting is at 0.2 (default setting) for Salmonella samples and also for Internal Positive
Control (IPC). The cut-off of Ct value was found at 41.00 (the average Ct value was negative
after 48 hours+ S.D.).
The average Ct (Threshold cycle) value (Table 4.2.2) for all samples positive by real-time
PCR (n=358) was 32.52, which the cut-off was at 41.00. Salmonella was detected by cultural
examination after 24 hours (n=136), the average Ct value was 28.75. Samples, which were
negative after 24 hours of culturing but positive after 48 hours and real-time PCR positive
(n=51), showed an increased Ct value of 33.31. Those sample which were also negative by
cultural examination after 48 hours but positive by real-time PCR (n=171) demonstrated the
highest average Ct value of 35.28.
Table 4.2.2: The average Ct value (Default setting; cut-off value = 41.00)
Culture result
Positive after 24 hours
Positive after 48 hours
Negative after 48 hours
Overall
N
136
51
171
358
Average Ct value
(cut-off = 41.00)
28.75
33.31
35.28
32.52
97
Minimum
17.60
23.33
26.60
17.60
Maximum
41.00
38.50
41.00
41.00
Results
4.2.3 Result of Ct value (Modified setting; Threshold = 1.4)
Modified analysis according to RapidFinder® protocol of Applied Biosystems, threshold
value setting is at 1.4 for Salmonella samples and 1.0 for Internal Positive Control (IPC).
The average Ct (Threshold cycle) value (Table 4.2.3) for all samples positive by real-time
PCR (n=337) was 36.33. Salmonella was detected by cultural examination after 24 hours
(n=134), the average Ct value was 31.77. Samples, which were negative after 24 hours of
culturing but positive after 48 hours and real-time PCR positive (n=51), showed an increased
Ct value of 37.46. Those sample which also were negative by cultural examination after 48
hours but positive by real-time PCR (n=152) demonstrated the highest average Ct value of
39.56.
Table 4.2.3: The average Ct value (Modified setting; Threshold = 1.4)
Culture result
Positive after 24 hours
Positive after 48 hours
Negative after 48 hours
Overall
N
134
51
152
337
Average Ct value
(cut-off = 41.00)
31.77
37.46
39.56
36.33
Minimum
20.92
26.76
29.98
20.92
Maximum
44.98
43.98
44.74
44.98
The average Ct value
Ct value of real-time PCR
45
40
41
38.5
35
30
33.31
28.75
15
35.28
26.6
25
20
41
23.33
17.6
10
5
0
Positive after 24h.
Positive after 48h.
Result of cultural examination
Figure 4.2.2: The average Ct value (Default setting)
98
Negative after 48h.
Results
Ct value of real-time PCR
The average Ct value
50
45
40
35
30
25
20
15
10
5
0
44.98
44.74
43.98
39.56
37.46
31.77
29.98
26.76
20.92
Positive after 24h.
Positive after 48h.
Negative after 48h.
Result of cultural examination
Figure 4.2.3: The average Ct value (Modified setting)
4.2.4 Agreement between default and modified threshold setting of real-time PCR assay
Table 4.2.4 shows the agreement between default and modified threshold setting of real-time
PCR assay. As a result, the agreement between the two types of methods were found to be
almost Perfect (kappa index = 0.8853).
Default setting
Type
Modified
setting
Total
Positive
Negative
Total
Positive
Negative
337
50
387
0
519
519
337
559
906
4.2.5 The average Ct value of real-time PCR (modified setting) in each type of samples
The average Ct value of real-time PCR (modified setting) in each type of samples (Table
4.2.5), the lowest of average Ct value were found in rodent faecal samples (28.45), followed
by scale swab sample (30.35). I n p ig f a e c e s w e r e fo u nd a ve r a g e C t va l u e
3 7 . 2 6 . Heater swab samples were found average Ct value in the highest (40.44).
99
Results
Table 4.2.5: The average Ct value of real-time PCR in each type of samples
Origin
1.Faeces
2.Rectal swab
3.Pipe
4.Feeder
N
56
4
12
24
Average
37.26
A
34.08
A
35.50
A
35.95
A
S.D.
5.11
2.42
4.73
4.35
Minimum
26.22
31.99
27.37
26.88
Maximum
44.67
37.35
43.98
42.61
5.Separate wall
6.Ventilator
7.Floor
8.Wall
9.Drive board
17
11
89
10
22
35.68
37.86
36.34
37.34
37.18
A
A
A
A
A
4.57
4.77
5.29
4.11
5.69
24.91
28.48
25.31
29.03
25.36
44.04
44.06
44.98
42.05
44.45
10.Load ramp
8
38.97
A
2.81
35.59
44.63
11.Carriage
12.Nipple
13.Scale
14.Cat faeces
5
13
11
-
37.94
34.71
30.35
-
A
B
B
-
5.23
6.18
7.05
-
30.49
25.30
22.06
-
41.84
44.74
43.29
-
15.Ceiling
16.Lamp
17.Heater
18.Toy
19.Boot
11
10
5
12
11
39.69
34.94
40.44
36.32
36.47
A
A
A
A
A
2.50
4.16
3.11
4.97
5.38
36.11
30.88
37.21
24.87
28.96
42.52
40.74
44.18
42.03
44.09
20.Rodent faeces
6
28.45
B
9.54
20.92
41.54
4.2.6 The standard graph of Ct value from modified setting real-time PCR
The standard graph of Ct value (Figure 4.2.6), rodent faecal samples were found
contaminated with Salmonella in the highest relative quantity value (≈ 106), followed by
scale swab samples (≈ 105) a nd n i p p l e d r i nk e r s w a b s a m p l e s ( ≈ 104) . Rodent
faeces, scale swab and nipple drinker swab samples were compared with pig faeces (≈ 10 3) by
means of the T-test, rodent faeces, scale swab and nipple drinker swab samples were found
different significance.
100
Results
1000000
Ralative Quantity
100000
10000
Rodent
faeces
Scale
Nipple
Faeces
1000
100
Standards
10
1
Ct value of real-time PCR
Figure 4.2.6: The standard graph of Ct value
Rodent faeces
Rectal swab
Floor
Sep.Wall
Scale
Separate wall
Faeces
Load ramp
Nipple
Toy
Carriage
Heater
Lamp
Feeder
Ventilator
Figure 4.2.7: The standard graph of Ct value in each type of samples
101
Pipe
Boot
Wall
Results
4.2.7 Distribution of Salmonella in faecal samples
Table 4.2.7 shows the distribution of Salmonella in faecal samples which the percentage of
the real-time PCR (modified setting) was positive average 2 7. 77 % .
Table 4.2.7: Distribution of Salmonella in faecal samples
Sample
Faeces (Gauze sock)
Faeces (Rectal swab)
Total
No. Sample
Real-time PCR
No. Positive
56
4
60
175
41
216
% Positive
32.00
9.75
27.77
4.2.8 Distribution of Salmonella in environmental samples
Table 4.2.8 shows the distribution of Salmonella in environmental samples which the
percentage of the real-time PCR (modified setting) was positive average 40. 14 % .
Table 4.2.8: Distribution of Salmonella in environmental samples
Sample
Environmental
No. Sample
Real-time PCR
No. Positive
277
690
% Positive
40.14
4.2.9 Proportion of Salmonella isolates from various samples
Table 4.2.9 demonstrates a total of 906 samples of Salmonella test. Overall Salmonella was
isolated in 37.19% (337/906). From faecal samples, the numbers of Salmonella isolates were
32.00% (56/175) and 9.75% (4/41) from faecal samples (gauze sock method) and rectal swab
samples respectively.
Environmental samples reveal that the percentage numbers of Salmonella isolates were
40.14% (277/690). Samples from rodent faeces showed the highest proportion of Salmonella.
The numbers of isolates were not found Salmonella in cat faeces samples.
102
Results
Table 4.2.9: Proportion of Salmonella isolates from various samples
Sample
No. Sample
Faeces (gauze sock)
Rectal swab
Pipe
Feeder
175
41
30
72
Real-time PCR
No. Positive
56
4
12
24
Separate wall
Ventilator
Floor , walkway
Wall
Drive board
40
29
216
28
72
17
11
89
10
22
42.5
37.93
41.20
35.71
30.55
Load ramp
Carriage
Nipple
Scale
Cat faeces
22
15
24
22
3
8
5
13
11
0
36.36
33.33
54.16
50.00
0
Ceiling
Lamp
Heater
Toy (Ball, Chain)
Boot
24
19
17
23
23
11
10
5
12
11
45.83
52.63
29.41
52.17
47.82
Rodent faeces
Total
11
906
6
337
54.54
37.19
103
% Positive
32.00
9.75
40.00
33.33
Results
4.3 Comparison between bacteriological methods and real-time PCR assay
4.3.1 Comparison between bacteriological methods and real-time PCR assay
Table and Figure 4.3.1 were compared between detecting Salmonella spp. by bacteriological
methods and real-time PCR (modified setting) in 906 samples from 48 finishing pig herds.
Table 4.3.1: Comparison between bacteriological methods and real-time PCR assay
Sample
No.
real-time PCR
Bacteriological method
Faeces (gauze sock)
175
n (+)
56
%
32.00
n (+)
28
%
16.27
Rectal swab
41
4
9.75
5
12.19
Environmental swab
690
277
40.14
154
22.31
Total
906
337
37.19
187
20.64
Comparison between Bacteriological methods
and real-time PCR assay
45
40.14
40
35
30
% Positive
27.77
22.31
25
20
15.28
15
10
5
0
Faeces
Environment
real-time PCR
Bacteriological
Figure 4.3.1: Comparison between bacteriological methods and real-time PCR assay
104
Results
4.3.2 Agreement between detecting Salmonella by bacteriological methods and real-time
PCR assay (default setting) in samples from pig herds
Table 4.3.2 shows the agreement between detecting Salmonella by bacteriological methods
and real-time PCR assay in faecal and environmental samples. Overall, 906 samples were
taken from 48 finishing pig farms. 20.64% (187/906) of samples were found Salmonella
positive by bacteriological methods and 39.51% (358/906) of samples were found
Salmonella positive by real-time PCR assay (default setting).
As a result, the agreement between the two types of methods were found to be Moderate
(kappa index = 0.5695). The real-time PCR resulted in a relative sensitivity of 100% and
specificity of 76.22% (95%C.I.=0.731-0.7933) compared with bacteriological methods, which
the correlation (Pearson´s) was found high significance (r=0.6280; p<0.0001).
Table 4.3.2: Agreement between detecting Salmonella by bacteriological methods and
real-time PCR assay (default setting) in samples from pig herds
Bacteriological method
Type
Real-time PCR
Positive
(default setting)
Negative
Total
Total
Positive
Negative
187
0
187
171
548
719
358
548
906
Figure 4.3.2: Correlation between bacteriological method and real-time PCR assay
105
Results
4.3.3 Agreement between detecting Salmonella by bacteriological methods and real-time
PCR assay (modified setting) in samples from pig herds
Table 4.3.3 shows the agreement between detecting Salmonella by bacteriological methods
and real-time PCR assay in faecal and environmental samples. Overall, 906 samples were
taken from 48 finishing pig farms. 20.64% (187/906) of samples were found Salmonella
positive by bacteriological methods and 37.19% (337/906) of samples were found
Salmonella positive by real-time PCR assay (modified setting).
As a result, the agreement between the two types of methods were found to be Moderate
(kappa index = 0.5999). The real-time PCR resulted in a relative sensitivity of 98.93%
(95%C.I.=0.9746-1.004) and specificity of 78.86% (95%C.I.=0.7587-0.8184) compared with
bacteriological methods, which the correlation (Pearson´s) was found high significance
(r=0.6643; p<0.0001). There were 2 samples from 187 bacteriological positive samples were
found negative by real-time PCR (modified setting). However, those 2 samples tested to be
positive by the real-time PCR (default setting).
Table 4.3.3: Agreement between detecting Salmonella by Bacteriological methods and
real-time PCR assay (modified setting) in samples from pig herds
Bacteriological method
Type
Real-time PCR
Positive
(modified)
Negative
Total
Total
Positive
Negative
185
2
187
152
567
719
337
569
906
Figure 4.3.3: Correlation between bacteriological method and real-time PCR assay
106
Discussion
5 Discussion
Pork and pork products are one of the main for foodborne enteric illness in humans caused by
Salmonella (CHAUNCHOM 2003). The source of infection was traced back to a farm.
Contamination of pork products is related to asymptomatic intestinal carriage of Salmonella
by living pigs arriving at the slaughterhouse (BORCH et al. 1996). While pig farms can not
guarantee that their pigs will always be free of Salmonella. The goal of such programs in the
pork chain is not the elimination of Salmonella. The goal is to prevent them from being
introduced into herds supplying the food chain (BLAHA 2000).
To prevent human disease due to the consumption of Salmonella contaminated pork, many
studies have focused on the epidemiology and the control of Salmonella in finishing pigs
(VAN DER WOLF et al. 1999; LEONTIDES et al. 2003; BELOEIL et al. 2004b; NOLLET et
al. 2004). In order to reduce the occurrence of Salmonella in pork, a decrease of Salmonella
carriage at the farm level is needed. On the other hand, the spread of Salmonella contaminated
slurry on fields and crops may constitute a threat for environmental preservation. Therefore,
efforts undertaken at the farm level to reduce Salmonella shedding contribute to increase both
human food safety and environmental safety.
Prevention of Salmonella must start at the herd level by analyzing the specific ports of entry
(contaminated feed, birds and rodents, which are carriers of a wide variety of Salmonella) by
which Salmonella could enter their herds and the factors that maintain Salmonella on the farm
(contaminated boots, tools, etc.) (BLAHA 2000; SWANENBURG et al. 2001a,b).
Salmonella control in term of Pre-harvest focused on issues such as defining prevalence level,
identifying risk factors and assessing interventions. In order to know how the Salmonella
prevalence in pig herds can be reduced, it is essential to know what the risk factors are for a
high Salmonella prevalence (NOLLET 2008). Timely intervention of the infection depends on
rapid detection of Salmonella (NG et al. 1996).
Until now, conventional culture methods have traditionally been considered the “gold
standards” for the isolation and identification of foodborne pathogens such as Salmonella.
However, culture methods are labour-intensive and time-consuming which are not suitable for
routine testing of large numbers of samples (BOHAYCHUK et al. 2007; FEY et al. 2004;
FUKUSHIMA et al. 2006; KLERKS et al. 2004; MYINT et al. 2006; UYTTENDAELE et al.
2003). For this reason, the real-time PCR has become a powerful tool for the detection of food
borne pathogens (WOLFFS et al. 2006). However, all real-time PCR test for Salmonella spp.
have been developed for food (very little other bacterial contamination) and most of the
studies are based on artificial contaminated samples (ELLINGSON et al. 2004; FEY et al.
2004; HEIN et al. 2006; KLERKS et al. 2004; MALORNY et al. 2004; SEO et al. 2006;
UYTTENDAELE et al. 2003).
The open question is whether this method is also applicable for samples from pig herds such
as faeces, dust, dirt, etc., which are naturally contaminated with an unknown amount of
Salmonella spp. and contain huge amount of other bacterial DNA? In order to answer this
question, the real-time PCR assay for the detection of Salmonella in 906 samples from 48
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Discussion
seropositive finishing pig herds (QS system; Category III) was compared to bacteriological
culture method to determine the relative sensitivity and specificity of the real-time PCR.
5.1 Dynamics of Salmonella
The European Union, national authorities and the pig industry are increasingly interested in
the Salmonella status of pig populations. Subclinical infections in pigs constitute a risk to
human health because at slaughter faecal material from the animal may contaminate its
carcass. Many more pigs are infected subclinically than have clinical salmonellosis, and they
constitute an important source of carcass contamination (ROWE et al. 2003).
In many countries efforts are being made to reduce the prevalence of Salmonella on pig farms
(DAHL et al. 1997a). The development of efficient strategies to control Salmonella species
requires the knowledge of basic epidemiological data, such as the sources, prevalence and
distribution of pathogens in animals and production units (LETELLIER et al. 1999a).
As generally accepted, pigs get orally infected with Salmonella (FEDROCKA-KRAY et al.
1995; SCHWARTZ 1999) and the major source is contaminated environment and direct
contact with Salmonella shedding pen-mates (SCHWARTZ 1999).
Many risk factors for a high Salmonella prevalence in pigs suggest different routes of
infection of pigs. Salmonella enterica is known to survive well in the environment
(SANDVANG et al. 2000), and the direct and/or indirect transmission of Salmonella from the
environment to the pigs is believed to play an important role in the infections of pigs.
This study analyzed data on the proportions of Salmonella detected from faecal and
environmental samples. Overall the proportion of positive Salmonella isolation by
bacteriological method from samples has an upward trend of 15.27% (faecal samples) to
22.31% (environmental samples) and the proportion of positive Salmonella isolation by realtime PCR assay (modified setting) from samples had same an upward trend of 27.77% (faecal
samples) to 40.14% (environmental samples). The result shows that Salmonella is more
upward by the detection of real-time PCR assay than the bacteriological method. The result
might be the cause of following the presence of stressed but active Salmonella cells, which
failed to grow in the culture media. (LENSE et al. 2000).
Published risk factor studies for Salmonella mostly used faecal or serum samples collected at
the herd. Isolation of Salmonella from faeces by bacteriological method is hindered due to the
relative low number of Salmonella organisms in the faeces (DAVIES et al. 2000b).
Salmonella shedding decreased to undetectable levels at the end of finishing period, which
has also reported in other studies (FUNK et al. 2001; KRANKER et al. 2003; BELOEIL et al.
2004b).
As faecal samples have a low sensitivity for detecting infected pigs (HURD et al. 2002), it is
possible that some of the pigs were infected with Salmonella but not shedding at the time of
sampling (NOLLET 2008).
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Discussion
This study found distribution of Salmonella by bacteriological method in faecal samples
which rectal swabs (12.19%) have a lower percentage of the Salmonella positive compared to
pooled faecal samples (gauze sock method) (16.00%). In agreement with real-time PCR
(modified setting) results, rectal swabs (9.75%) have a lower percentage of the Salmonella
positive compared to pooled faecal samples (gauze sock method) (32.00%). However, the
modified setting results were found similarly to default setting results of real-time PCR.
Farzan et al. (2008) also found 11% of Salmonella in rectal swabs and 18% of Salmonella in
pooled faecal samples and similar finding reported in Funk et al. (2000) and Korsak et al.
(2003). In 2001, Hurd et al. reported that the sensitivity of bacteriological culture might vary
between 10% and 80% depending on the sampling and testing protocol used. Different faecal
sample size was compared in another study, and an increased sensitivity was associated with
increased faecal sample size (FUNK et al. 2000).
Also, the variation in Salmonella status between rectal swabs and pooled faecal samples
deserves consideration in a sampling strategy.
As a mentioned, Salmonella may persist in the environment for long periods, and cleaning
and disinfection routines procedures may not always be efficient in eliminating the
contamination. Bacteria of the genus Salmonella are hardy, surviving freezing and desiccation
very well and persisting for weeks, months, or even years in a suitable organic substrate
(SCHWARTZ 1999). The temperature range for growth of Salmonella is between 5.5-45°C
(DOYLE and MAZZOTTA 2000). Davies and Wray (1997) found high levels of Salmonella
persisting in pig pens after disinfection. Gebreyes et al. (1999) detected Salmonella in drag
swabs of floors from barns after cleaning and disinfection, and before pig placement in 82%
of the studies case. In some of studied cases, finisher pigs shed a Salmonella serotype that had
been found on drag swabs.
Proportion of Salmonella, which was isolated by bacteriological method from environment
samples in this study, reveals that the percentage of Salmonella isolates was 22.31%. Swab
samples from scale (50%) had the highest proportion of the isolates. Rodent faeces (45.45%)
had the second highest proportion of the isolates. The numbers of isolates were not found
Salmonella in cat feaces and heater swab samples. Real-time PCR (modified setting) found
the percentage of Salmonella isolates at 40.14% in environmental samples. Rodent faeces
(54.54%) had the most proportion of the isolates. Drinker nipple swabs (54.16%) had the
most second proportion of the isolates. The numbers of isolates were not found Salmonella in
cat feaces. However, real-time PCR detected 29.41% of Salmonella in heater swab samples.
Davies and Wray (1997) found a wide range of animals, including rat, mice, cats and birds to
be infected. Cats and birds were associated with contamination of feed and grain stores, and
rodents were involved in the perpetuation of infection in specific buildings on the farm. Flies
and dust can also act as mechanical vectors that spread Salmonella throughout the
environment (GREENBERG et al. 1970; KHALIL et al. 1994; OLSEN and HAMMACK
2000).
Veling et al. (2002) ascribed the lower prevalence to the role of cats as predator of rodents
and birds carrying or excreting Salmonella similarly to this study, which cat faeces were not
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Discussion
found Salmonella. However, Evans and Davies (1996) identified cats as a risk factor because
cats are potential carriers.
Letellier et al. (1999a) reported the complexity of Salmonella epidemiology in swine herds.
The objective of study was to identify, in herds known to be positive for the presence of
Salmonella, possible sources of contamination, and evaluate the prevalence and distribution
of Salmonella at the different levels in an integrated swine production system. In the herds
studied, many environmental samples such as water taken in the pen, boots, floors, doors,
rodents or rodent‟s nests were found to be contaminated. Although it was not concluded that
these positive samples were the source of the infection for swine, there is no doubt that they
could be involved in subsequent contamination if appropriate measures are not taken. Flies
were positive for Salmonella on the highly contaminated farms and as mentioned before, they
may be involved in the dissemination of Salmonella in the environment as carrier of
microorganisms (MORSE and DUNCAN 1974; KHALIL et al. 1994). It is also important to
note that in these studied herds, dead animals were considered as possible sources of
contamination, and that boots of animal caretakers were also found positive for Salmonella
indicating that a particular attention should be given to improve the disinfectant of boots to
avoid the dissemination of Salmonella, in agreement with Amass et al. (2000) and Blaha
(2000).
If the pig farmers uses the same materials (shovels, brooms, buckets, etc.) and wears the same
boots and clothes in the pig units, indirect transmission of Salmonella from pen to pen is
possible (DAVIES et al. 1998). Concordantly to this study, boot, drive board, carriage, scale
swabs were found Salmonella by both detection methods.
On 9 June 2008 the European Food Safety Authority (EFSA) published a survey on
Salmonella levels detected in slaughtered pigs across the European Union in 2006-2007.
Salmonella was estimated on average to be present in one in ten pigs slaughtered for human
consumption (10.3%), according to an EU-wide report from an EFSA Task Force. Levels for
Salmonella detected in pigs varied from 0% to 29% between Member States. Among all
Salmonella detected, Salmonella Typhimurium and Salmonella Derby (which are two
common Salmonella types found in infection cases in humans) were detected in 4.7% and
2.1% of pigs slaughtered for human consumption, respectively. Salmonella is the second most
reported cause of food-borne diseases in humans in Europe with 160,649 people suffering
from Salmonella infections in 2006 (approximately 35 people in every 100,000).
This study found the general distribution of Salmonella serotype in the Northwest of
Germany. There were 79.57%, 18.28%, 1.61% and 0.54% of samples contaminated by S.
Typhimurium, S. Derby, S. Kedogou and S. Goldcoast respectively. Although some of the
serotypes detected, like S. Kedougou and S. Goldcoast, are quite rare, they still hold a food
safety aspect. The S. Typhimurium and S. Derby were the most-frequent serovars was
concordant with earlier studies in other European countries (VAN DER WOLF et al. 1999;
STEGE et al. 2000; OSTERKORN et al. 2001) and the USA (DAVIES et al. 1997; FUNK et
al. 2001).
In 6 pig herds, 2 serovars could be identified within one herd in this study. Similarly to
previous studies by Rowe et al. (2003) and Carlson and Blaha (2001) also found multiple
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Discussion
serovars occurring within one herd. The other study, Farzan et al. (2008) reported only 1
colony per sample was serotyped, so that the difference between pig and pen samples could
be biased. In previous studies, multiple serovars have been recovered from a sample when
more than 1 colony per sample was serotyped (RAJIC et al. 2005). Since the pen samples
were collected from 5 spots on the floor, multiple serovars could have been identified if more
than 1 colony per pooled sample had been serotyped. In addition, since fecal samples were
collected from only 2 pigs per pen, other pigs in the pen might shed other serovars. Therefore,
neither a sample of 2 pigs in each pen nor serotyping of 1 colony per pooled sample truly
represents the Salmonella serovars and phage types that exist on swine farms.
Rowe et al. (2003) reported the finding that more than one serotype was isolated at a given
stage of production from only three of the 30 infected farms may indicate that a horizontal
spread of infection was more common than transmission between stages of production. In
addition, when the weaners on a particular farm were positive it was more likely that the
fatteners and dry sows were also positive, which could be interpreted as a further indication of
horizontal spread. Dahl et al. (1997a) found that horizontal spread was a major source of
Salmonella infection for pigs; they raised finishers free from S. Typhimurium infection by
removing them to clean disinfected facilities, despite the fact that the pigs had been born in
herds with a high level of Salmonella infection in finishing pigs. The importance of horizontal
transmission suggests that control measures such as all-in all-out management systems and
thorough cleaning and disinfection, when carefully implemented, should be effective at
reducing infection levels.
Surveillance of Salmonella serovars and phage types from human and animal sources is
relevant for detecting outbreaks, for identifying sources of infection and for implementing
prevention and control measures. Two major changes in the sero- and phage type distribution
of nontyphoid salmonellosis have occurred during the last decades in many European
countries and the United States of America (RABSCH et al. 2001).
Many isolates of multiple-drug-resistant Salmonella enteric serovar Typhimurium phage type
DT 104 exhibit resistance to a core group of antimicrobials, including ampicillin,
chloramphinicol, streptomycin, sulfonamides and tetracycline (commonly abbreviated as
ACSSuT). Resistance to tetracyclines, streptomycine, and sulfonamides were most common
among S. Derby isolates from pigs (VALDEZATE et al. 2005). It affects humans and pig
industry in every country.
In this study, a total of 174 Salmonella serotypes were phage-typed and found different 13
phage types. S. Typhimurium DT 193 was the most frequently identified phage type from
samples and followed by S. Derby DY PT 55. Similar findings were reported in other
countries. The S. Typhimurium DT 193 was the most prevalent phage type isolated in
England (BURCH 2008), Spain (GARCIA-FELIZ et al. 2007) and India (MURUGKAR et al.
2005).
Moreover, the previous studies in Northwest of Germany, Bode (2007) detected Salmonella in
samples from pig herds. S. Typhimurium DT 208 was the most phage type and S. Derby PT
04 ranked the second. However, Austing (2005) found that 92.1% of isolated Salmonella
strains were identified as S. Typhimurium. Lysotypes S. Typhimurium DT 104 dominated and
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Discussion
followed by S. Typhimurium DT 193. Only a few S. Derby and S. subspecies I were isolated.
The S. Typhimurium DT 193 isolates showed resistance to 11 antimicrobials.
EFSA (2006) reported Salmonella phage types in Germany. The most 2 common phage types
were DT 104 and DT 193. Antimicrobial resistance in Salmonella, the predominating
resistance serovar S. Typhimurium (representing over 70% of all isolates from pigs) was
responsible for the resistance level observed. Multiresistance could frequently be detected
especially in phage types DT 104 as well as DT 193, U 302 and RDNC. However, this study
was not objective to antimicrobial resistance test in Salmonella.
5.2 Real-time PCR assay
Environmental substrates like faeces and dirt have become a major concern with respect to
food safety, since these substrates are suspected to a major role in the introduction of
pathogens in the food chain. For example, Salmonella are frequently found in association with
faeces and environment. Therefore, the detection and quantification of pathogens like
Salmonella that are present in environmental substrates like faeces and dirt, is of high
importance. It will enable risk assessment and pathogen monitoring at different stages in the
pig production chain and ensure food health and safety in the food industry.
Standardized diagnostic procedures to detect the presence of S. enterica in food samples (ISO
6579:2002) are mainly based on microbiological culturing methods, which in general require
up to five days until results are obtained (STEWART et al. 1998). In order to reduce the time
demand, alternative techniques like immunological assays (ACHESON et al. 1994; BOLTON
et al. 2000) and molecular methods (BEJ et al. 1994; DE MEDICI et al. 2003; HELLER et al.
2003) have been applied to detect S. enterica in various samples. Especially real-time PCR
methods such as 5‟ nuclease TaqMan® PCR (HOLLAND et al. 1991) have shown promising
results due to the rapid, sensitive and specific detection of S. enterica (BASSLER et al.1995;
HIGGINS et al. 1998; HOORFAR et al. 2000; KLERKS et al. 2004).
Several papers have currently appeared where real-time PCR applications for pathogen
detection have been reported (FUNG 2000; RIJPENS and HERMAN 2002; CHEN et al.
2000; HEIN et al. 2001; HOORFAR et al. 2000; KIMURA et al. 1999; NOGVA et al. 2000a,
2000b; OBERST et al.1998; SHARMA et al.1999; VISHNUBHATLA et al.2000).
Real-time PCR has been developed for rapid Salmonella detection using different fluorescentbased systems for detection of PCR products. Eyigor et al. (2002) used the double-stranded
DNA binding dye SYBR® Green I for real-time monitoring of Salmonella PCR product and
implemented the real-time PCR assay for detection of Salmonella-positive poultry flocks.
Chen et al. (1997) evaluated the 5‟ nuclease real-time PCR (TaqMan®) for the detection of
Salmonella in foods of animal origin. Knutsson et al. (2002) further investigated the
robustness of the 5‟nuclease real-time PCR for optimization in combination with an
enrichment procedure for Salmonella detection. A highly sensitive and specific molecular
beacon assay was developed by Chen et al. (2000) to detect the presence of Salmonella.
However, no applications of molecular beacon based real-time PCR for detection of
Salmonella in foods was reported.
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Discussion
In this study, the real-time PCR was developed for the detection of Salmonella in samples
from pig herds. The method consist of a pre-enrichment step of the sample in BPW done
overnight followed by an extraction-purification step for the bacterial DNA.
The method using BPW can be easily applied as a routine diagnostic procedure because this
medium is used for pre-enrichment in the horizontal ISO method for the detection of
Salmonella spp. The overnight pre-enrichment period may not be sufficient to guarantee
sufficient outgrowth for PCR samples especially for stressed Salmonella cells as shown in the
artificially contaminated samples subjected to freeze-stress. Stressed cell have an increased
lag time and especially if low numbers of cells are present the lag-time may vary
considerably. When cells are exposed to stress, a restricted enrichment time may decrease the
overall sensitivity of the combined enrichment-PCR assay (UYTTENDAELE et al. 2003).
However, Calvo et al. (2007) reported in real-time PCR analyses, a BPW overnight
incubation of 10 CFU inoculums would produce a positive real-time PCR result at Ct values
between 25 and 28.
However, molecular methods like TaqMan® PCR are limited by the fact that these are
dependent on the suitability of the extracted DNA for PCR. Especially DNA extracted from
faeces and dirt can have co-extracted contaminates like humic and fulvic acids known to
cause problems during PCR amplification. Other component different from humic and fulvic
acids but commonly present in dirt, have also been related to PCR inhibition (KLERKS
2007). To control the effects of inhibiting agents on PCR amplification efficiency, TaqMan®
PCR was improved recently by introduction of a general internal amplification control to
prevent the occurrence of false negative results (HOORFAR et al. 2000; KLERKS et al.
2004). The use of an internal amplification control (IAC) in the reactions helps to control
false negatives resulting from PCR inhibition or reaction malfunction because of either
spoiled reactives or a damaged thermocycler (Calvo et al. 2007). To avoid PCR inhibition in
this study, 1:10 and 1:100 dilutions were used. Similarly to previous studies, Uyttendaele et
al. (2003), Hein et al. (2006) and Calvo et al. (2007) required 1:10 dilution of the samples
because of inhibition.
In this study, the average Ct value for all samples positive by real-time PCR was 36.33
(modified setting) and 32.52 (default setting). Salmonella was detected by cultural
examination after 24 hours, the average Ct value was 31.77 (modified setting) and 28.75
(default setting). Samples were negative after 24 hours but positive after 48 hours of cultural
examination and real-time PCR positive, showed an increased Ct value of 37.46 (modified
setting) and 33.31 (default setting), those samples negative by cultural examination after 48
hours but positive by real-time PCR demonstrated the highest average Ct value of 39.56
(modified setting) and 35.28 (default setting). That is correspondent to a very low bacterial
load in enrichment broth. The Threshold value of modified setting was 1.4 according to
RapidFinder® guide of Applied Biosystems and the cut-off value from default threshold
setting of real-time PCR was found at 41.00.
However, in previous studies were also reported about Ct value. In the report of Knutsson et
al. (2002), expression of results by use of Ct values in the range of 35 to 40 are not as accurate
and precise as expression of results by use of Ct values in the range of 20 to 30, making it
necessary to reevaluate positive samples with Ct values in range of 35 to 40. Malorny et al.
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Discussion
(2004) reported the average Ct value of Salmonella detection in artificial contaminated
samples. The average Ct values in range of 19.76 to 44.42, the relative quantity of Salmonella
genome copies per PCR reaction were found in the range of 10 6 to 1 respectively. However,
Ct value of 45 was not found Salmonella genome by real-time PCR. Similarly to Calvo et al.
(2007); 18.35 to 35.63 of Ct values, Salmonella genome concentration were found in the
range of 106 to 10 respectively.
5.3 Agreement between detecting Salmonella by bacteriological methods and real-time
PCR assay in samples from pig herds
This study found the agreement between detecting Salmonella by bacteriological methods and
real-time PCR assay in faecal and environmental samples. Overall, 906 samples were taken
from 48 finishing pig farms. 20.64% of samples were found Salmonella positive by
bacteriological methods and 39.51% and 37.19% of samples were found Salmonella
positive by real-time PCR (default setting) and real-time PCR (modified setting).
Real-time PCR detected more positive results than bacteriological culture method, as
expected from previous studies (COHEN et al. 1994, 1995, 1996; KLERKS 2007). It has been
shown that bacterial culture of faecal and environmental samples for Salmonella because of
dilution, damage cell, lost of viability, intermittent shedding and carrier stage animals.
The results in this study, it appears to be sensitive to a wide range of serovars. The agreement
between the two types of method was found to be Moderate. The real-time PCR (default
setting) resulted in a relative sensitivity of 100% and specificity of 76.22% comparing with
bacteriological methods, which the correlation (Pearson´s) was found high significant (r =
0.6280; p<0.0001). The real-time PCR (modified setting) resulted in a relative sensitivity of
98.93% and specificity of 78.86% comparing with bacteriological methods, which the
correlation (Pearson´s) was found high significant (r = 0.6643; p<0.0001). However, the
agreement between default setting real-time PCR and modified setting real-time PCR was
found to be almost Perfect.
These results were similar to other published real-time PCR techniques, such as Smith (2006)
reported, horses in the Large Animal Hospital that exhibit diarrheal symptoms were culture
and real-time PCR tested for Salmonella in faecal samples. The results were compared and
found 91.7% of sensitivity and 95.2% of specificity. Bohaychuk et al. (2007) reported a realtime PCR assay for the detection of Salmonella in a wide variety of food and field samples.
When compared with the culture method, the diagnostic sensitive of the PCR assay for the
various samples ranged from 97.1 to 100%, and the diagnostic specificity ranged from 91.3 to
100%. Kappa values ranged from 0.87 to 1.00, indicating excellent agreement of the real-time
PCR assay to the culture method. Kurowski et al. (2002) reported the detection of Salmonella
spp. in faecal specimens by use of real-time PCR assay. Results of real-time PCR assay were
compared with bacterial culture results to determine relative sensitivity and specificity. The
results found in a relative sensitivity of 100% and a specificity of 98.2%. However,
Rodriguez-Lazaro et al. (2003) reported the validation of rapid, real-time PCR assay based on
TaqMan® technology for the unequivocal identification of Salmonella to be used directly on
an agar-grown colony. The results found the Ct value of each PCR reaction with a threshold
fixed at 0.2 Salmonella samples showed average of 15.30 (S.D. of 0.9). The accuracy of this
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Discussion
assay in terms of specificity was 100%. According to the TaqMan® Salmonella enteric
Detection Kit (Applied Biosystems) protocol was reported the sensitivity of the PCR is 1-10
copies of the target DNA per reaction. TaqMan® pathogen Detection Kits are designed to
work on enriched samples and detect down to 1 CFU in 25 grams of food. The specificity of
the TaqMan® Salmonella enteric Detection Kit has demonstrated 100% inclusivity for more
than 50 strains of Salmonella. It has also demonstrated 100% exclusivity for 24 other nonSalmonella strains.
The main advantages of real-time PCR are high sensitivity, high specificity, excellent
efficiency reduced amplicon size, no post-PCR step that reduce risks of cross-contamination
(RODRIGUEZ-LAZARO et al. 2003). One of the disadvantages of real-time PCR is the
inability to perform antimicrobial testing or serotyping, which could affect epidemiological
studies and the identification of nosocomial infections. Also, real-time PCR equipment is
quite expensive and requires highly trained technicians to interpret results (SMITH 2006).
In the pig industry, early detection of Salmonella is required from screening incoming feed,
monitoring equipment and environment, Salmonella-monitoring program, to ensuring the
safety and quality of pigs and pork. Results from real-time PCR with pre-enrichment can be
obtained as soon as the day after submission. Culture results can take 5 to 7 days to be final.
However, it would be very difficult to determine the source of an outbreak without
antimicrobial patterns and serovar information. Real-time PCR assay is rapidly growing and
becoming more popular in routine laboratory settings and can be easily implemented in
accredited laboratory with limited experience in molecular biology. The simple, rapid and
efficient technique, the real-time PCR assay in this study could potentially facilitate
presumptive and monitoring Salmonella in pig herds. While the initial cost of real-time PCR
equipment is high, an outbreak of Salmonella in the pig industry would likely also be high.
The results of this study demonstrated that real-time PCR can be a potential powerful tool to
detection and quantitation of Salmonella in samples from pig herds. Even though the
developed method in this study has not been tested with other parts of pig production chain,
we believe that, with proper optimization, it will be applicable to other parts of pig production
chain as well.
The advance knowledge about how to extract the necessary Salmonella DNA amount for the
detection of Salmonella spp. by means of the real-time PCR method and the development of a
system for detecting Salmonella spp. within pig production chains was demonstrated. The
reduction in time and labour make this highly sensitive and specific real-time PCR assay an
excellent alternative to conventional culture methods. The real-time PCR would be highly
valuable for Salmonella monitoring and control programmes also in pig herds. It offers
decisions in due time such as after cleaning and disinfection. Moreover, it can be used for
decisions for the pig movement between Salmonella controlled herds.
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Discussion
5.4 Conclusion and recommendation
1. Salmonella spp. is the leading cause of worldwide foodborne diseases and the resulting in
economic loss for the food industries. Pork and pork products are one of the main for
foodborne enteric illness in humans caused by Salmonella.
2. To prevent human disease due to the consumption of Salmonella contaminated pork, many
studies have focused on the epidemiology and the control of Salmonella in finishing pigs
3. In the pig industry, early detection of Salmonella is required from screening incoming feed
or weaning pigs, monitoring equipment and environment, Salmonella-monitoring program, to
ensuring the safety and quality of pigs or pork.
4. Until now, conventional culture methods have traditionally been considered the “gold
standards” for the isolation and identification of food borne pathogens. However,
conventional culture techniques for the detection of Salmonella spp. need labour intensive and
time consuming. Therefore, they are not suitable for routine testing of a large number of
samples.
5. The real-time PCR has become a powerful tool for the food borne pathogens detection. The
real-time PCR is a rapid, sensitive and specific method for detecting specific DNA, and it has
become routine method for identifying several bacterial and viral agents for many purposes.
6. The purpose of this study is to investigate the suitability of the real-time PCR assay for the
detection of Salmonella spp. in samples from pig herds compared to conventional culture
results to determine relative sensitivity and specificity of the real-time PCR.
7. In total, 906 samples such as faeces, swabs from pen walls, floors, feeders, boots etc. from
48 Salmonella seropositive finishing pig herds (QS system; Category III) in the Northwest of
Germany were tested.
8. This study found distribution of Salmonella by bacteriological method and real-time PCR
assay in faecal samples which rectal swabs have a lower percentage of the Salmonella
positive compared to pooled faecal samples (gauze sock method).
9. If the pig farmers uses the same materials (carriage, drive board, scale, etc.) and wears the
same boots and clothes in the pig units, indirect transmission of Salmonella from pen to pen is
possible. Concordantly to this study, boot, drive board, carriage, scale swabs were found
Salmonella by both detection methods.
10. The S. Typhimurium and S. Derby were the most-frequent serovars in this study. A total
of 174 Salmonella serotypes were phage-typed and found different 13 phage types. S.
Typhimurium DT 193 was the most frequently identified phage type from samples and
followed by S. Derby DY PT 55.
11. Real-time PCR (default setting) appears to be sensitive to a wide range of serovars,
sensitivity and specificity were calculated as 100% and 76.22% respectively using culture as
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Discussion
the “gold standard”. The results of this study show this molecular sequence correlates
moderate (Kappa-index=0.56). The correlation (Pearson‟s) was found high significant
(p<0.0001).
12. Real-time PCR (modified setting) appears to be sensitive to a wide range of serovars,
sensitivity and specificity were calculated as 98.93% and 78.86% respectively using culture as
the “gold standard”. The correlation (Pearson‟s) was found high significant (p<0.0001). The
results show this molecular sequence correlates moderate (Kappa-index=0.59) with culture
technique and would be a useful addition to routine lab procedures. However, agreement
between two types of default setting and modified setting method of real-time PCR was found
to be almost perfect.
13. The main advantages of real-time PCR are the high sensitivity, high specificity, excellent
efficiency reduced amplicon size, no post-PCR step that reduce risks of cross-contamination.
One of the disadvantages of real-time PCR is the inability to perform antimicrobial testing or
serotyping, which could affect epidemiological studies and the identification of nosocomial
infections. Also, real-time PCR equipment is quite expensive and requires highly trained
technicians to interpret results.
14. The real-time PCR would be highly valuable for Salmonella monitoring as well as control
programmes in pig herds. It offers decisions in due time such as after cleaning and
disinfection. Moreover, it can be used for decisions for the pig movement between Salmonella
controlled herds.
15. Even though the method developed in this study has not been tested with other part of pig
production chain, further studies will be applicable to other part of pig production chain as
wel.
117
Summary
6 Summary
Noppadol Somyanontanagul
Comparison between detecting Salmonella spp. by Bacteriological method and RealTime PCR assay in samples from pig herds
Salmonella spp. is the leading cause of worldwide food borne diseases and the resulting in
economic loss for the food industries. Until now, conventional culture methods have
traditionally been considered the “gold standards” for the isolation and identification of food
borne pathogens. However, conventional culture techniques for the detection of Salmonella
spp. need labour intensive and time consuming. Therefore, they are not suitable for routine
testing of a large number of samples (MYINT et al. 2006). For this reason, the real-time PCR
has become a powerful tool for the food borne pathogens detection. The real-time PCR is a
rapid, sensitive and specific method for detecting specific DNA, and it has become routine
method for identifying several bacterial and viral agents for many purposes (WOLFFS et al.
2006). The purpose of this study was to investigate the suitability of the real-time PCR assay
for the detection of Salmonella spp. in samples from pig herds compared to conventional
culture results to determine relative sensitivity and specificity of the real-time PCR. The
overall goal was to study the possibility to use the real-time PCR as a routine laboratory
method for control and monitoring program of Salmonella in pig herds.
In total, 906 samples such as faeces, swabs from pen walls, floors, feeders, boots etc. from 48
Salmonella seropositive finishing pig herds (QS system; Category III) in the Northwest of
Germany were simultaneously tested by real-time PCR assay and conventional bacteriological
culture method that were established at the Filed station for epidemiology in Bakum. All
samples were enriched in buffer peptone water at 37°C for 18±2 hours. Subsequently, each
enrichment broth was tested simultaneously by the standardized real-time PCR protocol and
by the conventional bacteriological culture method. The real-time PCR was carried out with
the TaqMan® Salmonella enterica Detection Kit according to the manufacture instructions.
Reactions were performed on the ABI Prism 7500 real-time PCR System; the results were
analyzed with SDS version 1.4 (all Applied Biosystems; USA). The bacteriological detection
of Salmonella from the enrichment broth was performed according to DIN-ISO 6579. All
Salmonella isolates were sub-typed by phage-typing in cooperation with the Robert-Koch
Institute in Wernigerode. In the end, the results were analyzed by SAS 9.1 (Statistical
Analysis System, USA.) to determine the Correlation coefficient, Cohen‟s Kappa-index,
relative sensitivity and specificity of the real-time PCR applied to samples from pig herds.
Overall, 337 of 906 (37.19%) samples were positive by real-time PCR and 187 of 906
(20.64%) were positive by conventional culture. The 185 samples positive by conventional
culture were all positive by real-time PCR (modified setting). Therefore, Kappa-index was
calculated as being 0.59, which means that correlation between the two methods is moderate.
The average Ct (Threshold cycle) value for all samples positive by real-time PCR was 36.33.
If Salmonella was detected by cultural examination after 24 hours (n=134), the average Ct
value was 31.77. Samples, which were negative after 24 hours of culturing but positive after
48 hours and PCR positive (n=51), showed an increased Ct-value of 37.46. Those samples
118
Summary
were also negative by cultural examination after 48 hours but positive by real-time PCR
(n=152), demonstrated the highest average Ct-value of 39.56 which is correspondent to a very
low bacterial load in the enrichment broth. The real-time PCR resulted in a relative sensitivity
of 98.93% and specificity of 78.86%, compared with conventional culture, which the
correlation coefficients (Pearson´s) between 2 type of methods was found high significant
(r=0.6643, p<0.0001).
The advance knowledge about how to extract the necessary Salmonella DNA amount for the
detection of Salmonella spp. by means of the real-time PCR method and the development of a
system for detecting Salmonella spp. within pig production chains was demonstrated. The
reduction in time and labour make this highly sensitive and specific real-time PCR assay an
excellent alternative to conventional culture methods. The real-time PCR would be highly
valuable for Salmonella monitoring and control programmes also in pig herds. It offers
decisions in due time such as after cleaning and disinfection. Moreover, it uses for decisions
for pig movement between Salmonella controlled herds.
119
Zusammenfassung
7 Zusammenfassung
Noppadol Somyanontanagul
Methodenvergleich zwischen kultureller Anzucht und real-time PCR zum Nachweis von
Salmonella spp. in Schweinebeständen
Salmonellen stellen weltweit eine der Hauptursachen lebensmittelbedingter Erkrankungen dar
und verursachen gleichzeitig wirtschaftliche Verluste in der Nahrungsmittelindustrie. Die
Isolierung und Identifikation von Krankheitserregern in Lebensmitteln durch die traditionelle
kulturelle Untersuchungsmethode wird als „Gold Standard“ bezeichnet und bis heute
durchgeführt. Der kulturelle Nachweis von Salmonellen ist sehr zeit- und materialaufwendig
und somit für Routineuntersuchungen bei hohem Probenaufkommen nicht immer optimal
(MYINT et al. 2006). Die real-time PCR ist eine schnelle, sensitive und spezifische Methode
zum Auffinden spezifischer DNA und eignet sich daher auch besonders zum Nachweis
lebensmittelbedingter Krankheitserreger als Routinemethode (WOLFFS et al. 2006).
Das Ziel dieser Studie ist die Untersuchung, ob die real-time PCR eine geeignete Methode
zum Nachweis von Salmonellen in Schweinebeständen ist. Im Vergleich mit der kulturellen
Methode soll die Sensitivität und Spezifität der real-time PCR überprüft werden. Das Ziel der
Studie ist die Überprüfung der Verwendung der real-time PCR als Untersuchungsmethode
zur Diagnostik von Salmonellen bzw. für den Einsatz beim Salmonellenmonitoring in
Schweinebeständen zum Auffinden der Eintragsquellen.
Die Untersuchung wurde in 48 Schweinebetrieben im Nordwesten Deutschlands
durchgeführt, die aufgrund des positiven Salmonellenantikörpernachweises nach dem QSSystem in Kategorie III ( hohe Salmonellenantikörperbelastung) eingestuft wurden. Insgesamt
wurden 906 Proben unterschiedlicher Herkunft (Sammelkotproben, Abstriche von
Stallwänden, vom Stallgangboden, den Futterautomaten, Stiefeln usw.) gleichzeitig mittels
kultureller Methode und real-time PCR in der Außenstelle für Epidemiologie in Bakum
untersucht.
Alle Proben wurden in gepuffertem Peptonwasser (Voranreicherung) bei 37°C für 18±2
Stunden angereichert. Anschließend wurden die Proben gleichzeitig nach dem
standardisierten real-time PCR Anweisungsprotokoll sowie mit der konventionellen
bakteriologischen Methode getestet. Die real-time PCR wurde anhand der Herstellerangaben
des TaqMan® Salmonella enterica Detection Kit durchgeführt. Der Untersuchungsprozess
wurde mit dem ABI Prism 7500 real-time PCR System vollzogen. Die Ergebnisse wurden
anhand des SDS version 1,4 (Applied Biosystems; USA) analysiert. Die bakteriologische
Überprüfung auf Salmonellen aus der Voranreicherung wurde gemäß der DIN-ISO 6579
durchgeführt. Die Typisierungen der isolierten Salmonellenstämme erfolgten in
Zusammenarbeit mit dem Robert-Koch-Institut in Wernigerode. Abschließend wurden die
Ergebnisse durch das SAS 9,1 (Statistical Analysis System; USA.) analysiert, um den
Konkordanz-Index Kappa, die relative Sensitivität und Spezifität der real-time PCR und seine
Gültigkeit für die Untersuchung von Schweinebeständen zu ermitteln.
120
Zusammenfassung
Mit der real-time PCR wurden 337 von 906 (37,19%) und mit dem konventionellen Verfahren
187 von 906 (20,64%) als positive Proben ermittelt. Alle 185 positiven Proben aus dem
konventionellen Verfahren wurden auch durch die real-time PCR nachgewiesen. Nach dem
Konkordanz-Index Kappa ergibt sich eine deutliche Korrelation von 0,59 zwischen den
beiden Methoden. Der durchschnittliche Ct-Wert (Threshold Cycle) für alle positiv getesteten
Proben durch die real-time PCR lag bei 36,33. Wurden Salmonellen in den kulturellen
Untersuchungen der Proben nach 24 Stunden (n=134) gefunden, betrug der durchschnittliche
Ct-Wert 31,77. Für Proben, die nach 24 Stunden negativ, aber nach 48 Stunden positiv, sowie
bei der real-time PCR positiv ausfielen (n=51), ergibt sich ein höherer Ct-Wert von 37,46.
Proben, die in der kulturellen Untersuchungen auch nach 48 Stunden ein negatives Ergebnis
bezüglich der Salmonellenkulturen zeigten, aber durch die real-time PCR (n=152) als positiv
erkannt wurden, wiesen den durchschnittlich höchsten Ct-Wert von 39,56 auf, was eine
niedrige bakterielle Konzentration in den flüssigen Medien aufzeigt. Verglichen mit den
traditionellen kulturellen Untersuchungsmethoden führten die real-time PCR-Ergebnisse zu
einer relativen Sensitivität von 98,93% und einer Spezifität von 78,86%, was eine hoch
signifikante (r=0,6643; p<0,0001) Korrelation zwischen den beiden Untersuchungsverfahren
ergibt.
Die neuen Erkenntnisse zum Extrahieren der erforderlichen Salmonellen-DNA für die
Erforschung von Salmonellen in Schweineproduktionsketten durch die real-time PCR wurden
demonstriert. Die Verringerung des Zeit- und Arbeitsaufwands macht die sensitive und
spezifische real-time PCR zu einer hervorragenden Alternative zu dem traditionellen
kulturellen Verfahren. Die real-time PCR ist eine sehr effiziente Methode für die
Durchführung von Salmonellenmonitoring und Salmonellenkontrolle in Schweinebeständen.
Ferner ermöglicht es eine schnellere Entscheidung bezüglich der Reinigung und Desinfektion
der Umgebung und Materialien, eine Trennung von mit salmonelleninfizierten Tieren
innerhalb eines Bestandes und während des Tiertransportes sowie die gezielte Lenkung der
Tierbewegungen zwischen salmonellenkontrollierten Schweinebeständen.
121
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171
Appendix
9 Appendix
Table 9.1: The positive results of real-time PCR and bacteriological method
Farm
1
2
2
3
3
3
3
3
3
3
4
4
4
4
4
4
4
5
5
5
5
5
6
6
6
6
6
6
6
6
7
7
7
7
7
7
7
7
7
7
7
7
7
7
Sample
20
1
7
1
8
1
7
1
3
7
7
8
7
9
7
4
11
7
4
1
7
4
1
7
7
7
8
7
1
7
1
5
7
16
4
6
9
9
1
7
4
6
5
7
Culture 24h
Culture 48h
0
0
0
0
1
0
0
0
1
1
0
0
0
1
0
0
1
0
0
0
1
1
0
0
0
0
0
0
1
1
0
1
0
0
0
1
0
1
1
1
1
1
0
0
Ct (default)
0
0
1
0
1
0
0
0
1
1
0
0
0
1
1
0
1
0
0
0
1
1
1
0
0
0
0
1
1
1
1
1
0
0
1
1
0
1
1
1
1
1
0
0
172
41.02
39.40
33.48
35.01
31.41
41.00
29.41
36.61
29.25
25.83
38.05
39.42
36.71
29.87
36.44
36.51
36.79
39.32
32.93
37.70
26.38
30.27
35.64
31.97
40.17
34.67
36.30
32.12
33.73
31.03
32.32
31.90
29.15
37.38
33.01
26.53
40.67
41.00
29.68
29.93
29.54
33.13
35.19
42.64
Ct (modified)
0.00
0.00
37.92
39.28
35.38
0.00
33.09
42.29
32.64
29.13
43.23
0.00
42.44
33.25
41.93
40.93
41.37
0.00
36.78
43.04
29.66
33.88
39.71
35.65
0.00
39.20
41.49
36.08
38.14
35.03
34.49
33.95
31.15
40.05
35.26
28.48
43.76
43.97
31.74
32.07
31.69
35.34
37.72
0.00
Appendix
Table 9.1: The positive results of real-time PCR and bacteriological method (continued)
Farm
8
8
8
8
8
8
8
9
9
9
9
9
10
11
11
11
12
12
13
13
13
14
14
14
14
14
14
14
15
16
16
16
17
18
18
18
18
18
18
19
19
19
19
20
20
20
20
20
Sample
1
3
1
7
1
7
1
7
7
9
13
4
1
1
7
9
2
7
6
8
7
1
7
4
5
1
1
10
9
3
7
7
7
7
12
4
7
10
4
7
2
4
9
7
9
1
7
3
Culture 24h
Culture 48h
0
0
0
0
0
0
1
1
1
1
0
1
1
1
1
1
1
1
0
0
0
0
1
0
1
0
0
1
0
1
1
0
1
0
1
1
1
0
0
1
1
1
1
1
1
1
1
1
Ct (default)
0
1
0
1
0
0
1
1
1
1
1
1
1
1
1
1
1
1
0
0
0
1
1
0
1
0
1
1
0
1
1
1
1
0
1
1
1
0
0
1
1
1
1
1
1
1
1
1
173
38.46
30.24
39.16
29.58
36.93
37.72
28.69
23.53
36.23
38.28
37.62
40.65
33.41
36.72
36.95
35.26
38.99
38.34
38.56
37.24
38.01
33.50
26.13
36.10
25.80
36.00
29.38
37.90
44.23
24.18
31.53
38.50
24.61
39.87
33.79
23.55
26.34
41.37
37.31
27.92
33.18
30.67
33.72
27.77
22.09
27.52
22.18
29.07
Ct (modified)
43.80
33.73
44.61
33.02
42.05
43.87
31.99
27.03
41.76
44.27
43.29
0.00
37.72
41.57
42.07
39.93
0.00
44.94
44.06
42.05
43.18
37.57
29.32
41.62
29.07
40.86
32.89
44.63
0.00
27.37
35.30
43.40
28.00
0.00
38.36
26.88
32.32
0.00
42.61
31.22
37.35
34.17
38.78
31.40
25.36
31.04
25.47
32.29
Appendix
Table 9.1: The positive results of real-time PCR and bacteriological method (continued)
Farm
20
20
20
20
20
21
21
21
22
22
22
22
22
22
23
23
23
23
23
23
23
23
23
23
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
25
25
25
26
27
27
27
27
Sample
11
13
9
7
7
1
7
7
3
4
7
12
7
11
2
7
9
4
10
7
20
1
7
1
1
7
3
5
1
3
5
7
9
1
7
7
15
7
7
1
7
9
7
3
8
1
7
4
Culture 24h
Culture 48h
1
1
1
1
1
1
0
0
1
1
1
0
0
1
1
1
0
0
1
0
0
1
1
0
0
1
0
0
0
1
0
1
0
0
0
0
0
1
0
0
0
0
0
0
1
1
1
0
Ct (default)
1
1
1
1
1
1
0
0
1
1
1
1
0
1
1
1
0
0
1
0
1
1
1
0
0
1
0
0
0
1
1
1
0
0
0
0
0
1
0
0
0
0
1
1
1
1
1
1
174
27.30
27.85
30.21
22.09
22.45
23.08
32.57
33.02
26.59
28.04
34.00
33.06
30.34
30.90
30.94
27.39
34.08
35.66
35.13
35.13
36.07
32.93
28.47
35.02
37.20
30.18
34.71
35.86
35.15
30.31
31.18
30.11
34.30
36.92
34.43
35.99
35.45
32.62
34.37
33.13
36.85
38.76
36.69
37.60
29.91
30.44
29.16
33.45
Ct (modified)
30.49
31.16
34.04
25.31
25.69
26.22
36.55
36.96
29.89
31.33
38.37
36.93
33.81
34.32
34.48
30.65
38.63
40.51
39.70
39.98
41.54
37.09
32.24
39.89
42.04
33.71
38.90
40.32
39.46
33.78
34.89
33.64
38.38
41.86
38.57
40.79
39.95
36.63
38.97
37.31
0.00
44.45
41.43
43.98
33.32
34.09
32.61
37.40
Appendix
Table 9.1: The positive results of real-time PCR and bacteriological method (continued)
Farm
27
27
27
27
27
27
27
27
28
29
29
29
29
29
29
29
29
29
29
30
30
30
30
30
30
30
30
31
31
31
31
33
33
33
33
33
34
34
34
34
34
34
34
34
34
34
34
34
Sample
5
6
7
7
7
7
5
3
9
8
7
7
9
1
7
7
13
6
5
7
5
5
7
7
7
9
15
7
5
7
7
5
7
4
8
5
1
7
7
9
4
1
4
8
7
1
1
5
Culture 24h
Culture 48h
1
1
0
1
0
1
0
0
0
0
0
0
1
0
0
1
1
1
0
0
1
0
0
0
0
0
0
1
1
1
0
1
1
0
0
0
0
1
0
0
0
1
1
1
0
0
0
0
Ct (default)
1
1
1
1
0
1
0
0
0
1
1
0
1
0
0
1
1
1
0
0
1
1
1
0
0
0
0
1
1
1
1
1
1
1
1
0
1
1
1
1
1
1
1
1
1
0
1
1
175
28.39
29.34
32.69
30.69
39.28
30.31
38.34
34.01
40.81
33.74
33.70
37.37
28.25
35.23
37.86
32.62
29.08
31.98
34.49
36.76
29.71
33.30
35.89
41.00
35.06
44.37
44.34
38.50
32.38
34.35
33.43
30.06
27.01
35.98
34.85
39.74
34.24
30.49
32.35
32.26
35.32
30.27
30.86
31.21
33.78
38.82
35.56
35.57
Ct (modified)
31.67
32.86
36.60
34.63
0.00
34.22
44.04
39.29
0.00
38.40
38.13
43.47
31.60
39.90
44.02
36.98
32.39
36.44
39.39
42.85
33.07
37.55
41.67
0.00
39.99
0.00
0.00
44.98
36.48
38.93
37.28
32.74
29.46
39.97
39.51
0.00
38.22
34.15
36.11
36.21
40.03
33.91
34.45
34.86
38.17
0.00
40.77
40.42
Appendix
Table 9.1: The positive results of real-time PCR and bacteriological method (continued)
Farm
34
34
35
35
35
36
36
36
36
36
36
36
41
41
41
41
42
42
42
42
42
42
42
45
45
45
45
45
45
45
45
45
45
45
45
45
45
45
45
45
45
45
45
45
45
45
45
45
Sample
3
4
1
7
7
12
7
9
1
7
4
1
7
7
7
13
1
5
11
9
18
5
1
1
12
3
17
18
15
16
1
6
1
1
5
4
1
9
1
15
1
3
12
6
19
1
1
8
Culture 24h
Culture 48h
0
0
0
0
1
0
1
0
0
1
1
0
0
0
1
1
0
0
0
0
0
0
0
1
1
0
0
0
0
0
1
0
1
1
0
0
1
0
0
0
0
0
0
0
0
0
0
0
Ct (default)
1
1
0
0
1
0
1
0
0
1
1
0
0
0
1
1
0
0
0
0
0
0
0
1
1
1
0
0
1
1
1
0
1
1
1
0
1
1
1
0
1
0
0
0
0
0
0
0
176
32.68
35.45
36.21
38.89
26.07
44.25
32.48
43.78
37.96
35.47
33.28
35.21
35.57
33.17
27.48
30.87
41.52
41.36
41.19
42.57
34.59
37.19
36.73
24.05
21.94
33.02
33.21
36.11
33.44
33.08
23.97
34.67
24.06
29.39
32.66
35.47
26.20
33.78
33.97
34.64
34.32
35.32
37.72
36.54
36.67
36.28
42.08
35.53
Ct (modified)
36.75
40.45
0.00
43.60
30.13
0.00
36.39
0.00
44.67
40.77
37.36
40.18
40.82
37.62
30.81
34.60
41.52
0.00
0.00
0.00
39.19
42.69
42.50
27.58
25.30
37.19
37.21
41.04
37.54
37.19
27.25
39.31
27.39
32.85
36.53
40.30
29.54
38.08
38.28
36.62
38.85
40.22
44.74
42.18
42.64
41.40
0.00
40.37
Appendix
Table 9.1: The positive results of real-time PCR and bacteriological method (continued)
Farm
45
46
46
46
46
46
46
46
46
46
46
46
46
46
46
46
46
46
46
46
46
47
47
47
47
47
47
47
47
47
47
47
47
48
48
48
48
48
48
48
48
48
48
48
48
48
48
48
Sample
1
8
2
2
2
6
4
7
2
2
2
12
7
1
8
1
12
7
13
8
19
1
5
9
7
1
12
18
15
6
7
7
10
18
15
12
16
17
4
7
9
6
8
5
7
19
1
12
Culture 24h
Culture 48h
0
0
0
1
0
0
0
0
0
0
1
0
1
1
1
1
0
1
1
0
1
0
0
1
0
0
1
1
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Ct (default)
1
0
0
1
0
0
1
1
0
0
1
1
1
1
1
1
1
1
1
0
1
0
0
1
0
0
1
1
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
177
32.84
42.35
42.95
29.11
43.39
43.80
27.88
25.33
43.51
42.73
28.49
33.91
27.36
25.82
25.60
29.65
23.33
30.34
27.37
42.42
25.48
41.35
33.26
29.08
38.80
30.63
24.88
21.43
44.99
30.59
27.16
25.08
41.73
32.34
32.20
36.74
32.87
37.51
32.65
35.81
34.17
36.14
34.36
32.64
32.52
34.81
36.33
34.54
Ct (modified)
36.89
0.00
0.00
32.51
0.00
0.00
31.24
28.77
0.00
0.00
31.99
38.47
30.81
29.19
29.03
33.15
26.76
34.12
30.87
0.00
28.91
0.00
37.25
32.52
44.40
34.22
28.24
24.87
0.00
34.19
30.54
28.42
0.00
36.24
36.11
42.36
37.07
44.18
36.92
40.70
38.81
41.50
39.01
36.58
36.55
39.43
41.95
39.71
Appendix
Table 9.1: The positive results of real-time PCR and bacteriological method (continued)
Farm
48
48
48
48
48
48
48
48
48
48
48
48
48
48
49
49
49
49
49
49
49
49
49
49
49
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
51
51
51
Sample
17
1
16
15
20
7
7
17
11
19
1
18
15
10
1
12
18
5
4
7
20
19
9
18
7
15
7
19
9
17
11
1
18
12
15
18
16
10
17
18
15
18
11
17
10
9
1
1
Culture 24h
Culture 48h
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
1
1
1
1
1
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
1
Ct (default)
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
1
1
1
1
1
1
0
0
0
0
0
0
0
1
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
1
0
1
178
33.25
40.46
35.95
34.90
34.57
36.81
38.57
34.85
35.68
37.58
37.44
39.78
36.39
43.33
30.31
25.28
28.87
21.48
24.90
27.71
23.19
38.13
37.70
34.50
35.26
36.57
37.65
36.80
26.69
43.68
44.14
33.94
33.80
37.78
33.13
32.66
34.06
35.15
37.22
34.85
36.86
36.64
36.28
44.10
44.00
23.26
33.62
24.03
Ct (modified)
37.93
0.00
40.74
39.81
39.28
42.23
0.00
39.70
41.84
0.00
44.34
0.00
42.28
0.00
33.83
28.62
32.27
24.91
28.23
31.12
26.64
44.09
43.32
39.12
40.22
42.52
43.88
42.45
30.05
0.00
0.00
38.40
38.49
0.00
37.22
36.70
38.67
40.15
43.20
40.76
42.45
42.03
41.68
0.00
0.00
26.72
38.02
27.43
Appendix
Table 9.1: The positive results of real-time PCR and bacteriological method (continued)
Farm
51
51
51
51
51
51
51
51
51
51
51
51
51
51
51
51
51
51
51
51
52
52
52
52
52
52
52
52
52
52
52
52
52
52
52
52
52
52
52
52
52
52
52
52
52
53
53
53
Sample
12
16
7
19
20
13
1
10
13
9
7
20
20
16
19
13
10
13
16
10
18
12
7
10
19
12
1
4
1
18
15
7
7
7
9
19
4
4
10
19
19
10
16
16
13
1
4
6
Culture 24h
Culture 48h
1
1
1
1
1
1
0
0
1
0
0
1
1
1
0
1
0
1
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
1
1
1
0
0
0
Ct (default)
1
1
1
1
1
1
0
0
1
0
0
1
1
1
0
1
0
1
1
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
1
1
1
0
0
0
179
32.33
28.27
23.37
26.57
17.80
24.29
36.63
34.47
19.06
37.21
34.27
17.60
17.98
27.54
26.60
23.59
33.13
19.08
27.99
34.02
28.41
28.49
31.93
42.18
30.44
30.19
29.19
28.13
31.24
29.76
34.77
34.51
33.99
29.41
29.52
33.15
30.96
30.85
30.08
31.55
32.32
31.74
27.55
27.46
18.72
35.02
40.07
36.21
Ct (modified)
36.15
31.65
26.82
30.01
21.07
27.73
41.85
39.17
22.38
42.74
38.84
20.92
21.22
30.97
29.98
26.98
37.62
22.39
31.25
38.83
31.83
31.64
35.90
0.00
33.84
33.88
32.74
31.54
35.23
33.27
39.67
40.55
37.18
32.94
33.12
37.35
34.50
34.65
36.08
35.35
37.02
35.59
30.96
30.88
22.06
39.95
0.00
41.58
Appendix
Table 9.1: The positive results of real-time PCR and bacteriological method (continued)
Farm
Sample

Culture 24h
9
13
6
7
7
1
15
53
53
53
53
53
53
53
Culture 48h
0
0
0
0
0
0
0
Ct (default)
0
1
0
0
0
0
0
34.95
34.71
35.43
37.30
36.10
37.01
35.30
Ct (modified)
39.90
40.02
40.47
42.87
40.90
43.99
42.38
Sample: 1 Faeces; 2 Rectal swab; 3 Pipe; 4 Feeder; 5 Separate wall; 6 Ventilator; 7 Floor ,
walkway; 8 Wall; 9 Drive board; 10 Loadramp; 11 Carraige; 12 Drinker Nipple; 13 Scale;
14 Cat faeces; 15 Ceiling; 16 Lamp; 17 Heater; 18 Toy (Ball,Chain); 19 Boot; 20 Rodent
faeces


Culture: 1 = Positive; 0 = Negative
Ct (default) = Threshold at 0.2; Ct (modified) = Threshold at 1.4
Table 9.2: Salmonella phage type in each type of samples
No.
7/1
7/2
7/5
7/6
7/11
7/13
7/14
7/15
7/16
8/3
8/6
8/15
9/2
9/5
9/6
9/7
9/9
10/9
11/2
11/3
11/8
12/1
12/13
Sample
faeces
separate wall
feeder
ventilator
drive board
feaces
floor
feeder
ventilator
pipe
floor
feaces
floor
floor
drive board
scale
feeder
feaces
feaces
floor
drive board
rectal swab
floor
Phagetype
S.Tm,DT193
S.Tm,DT193
S.Tm,DT193
S.Tm,DT193
S.Tm,DT193
S.Derby PT04
S.Derby PT04
S.Derby PT04
S.Derby PT04
S.Tm,DT193
S.Tm,DT120
S.Tm,DT193
S.Tm,DT120
S.Tm,DT120
S.Tm,DT120
S.Tm,DT120
S.Tm,DT120
S.Tm,DT193
S.Kedougou
S.Kedougou
S.Kedougou
S.Tm,DT193
S.Tm,DT193
No.
34/1
34/2
34/4
34/6
34/7
34/8
34/9
34/10
34/13
34/15
34/16
34/17
34/18
35/17
36/3
36/10
36/11
41/9
41/11
45/1
45/2
45/3
45/6
180
Sample
feaces
floor
floor
drive board
feeder
feaces
feeder
wall
floor
feaces
separate wall
pipe
feeder
floor
floor
floor
feeder
floor
scale
feaces
nipple
pipe
ceiling
Phagetype
S.Tm,DT193
S.Tm,DT193
S.Tm,DT193
S.Tm,DT193
S.Tm,DT193
S.Tm,DT193
S.Tm,DT193
S.Tm,DT193
S.Tm,DT193
S.Tm,DT193
S.Tm,DT193
S.Tm,DT193
S.Tm,DT193
S. ssp.I
S.Derby PT11
S.Derby PT11
S.Derby PT11
S.Goldcoast
S.Tm,DT208
S.Tm,DT193
S.Tm,DT193
S.Tm,DT193
S.Tm,DT193
Appendix
Table 9.2: Salmonella phage type in each type of samples (continued)
No.
14/1
14/2
14/5
14/12
14/15
16/3
16/4
16/12
17/9
18/4
18/5
18/8
19/1
19/2
19/7
19/16
20/1
20/2
20/3
20/4
20/5
20/8
20/9
20/10
20/12
20/13
21/9
22/5
22/6
22/8
22/10
22/18
23/1
23/2
23/7
23/9
23/10
23/11
24/2
24/6
Sample
feaces
floor
separate wall
feaces
load ramp
pipe
floor
floor
floor
nipple
feeder
floor
floor
rectal swab
feeder
drive board
floor
drive board
feaces
floor
pipe
carraige
scale
drive board
floor
floor
feaces
pipe
feeder
floor
nipple
carraige
rectal swab
floor
load ramp
mouse
feaces
floor
floor
ventilator
Phagetype
S.Tm,DT193
S.Tm,DT193
S.Tm,DT193
S.Tm,DT193
S.Tm,DT193
S.Tm,DT104
S.Tm,DT193
S.Tm,DT193
S.Tm,DT193
S.Tm,DT193
S.Tm,DT193
S.Tm,DT193
S.Tm,DT193
S.Tm,DT193
S.Tm,DT193
S.Tm,DT193
S.Derby PT55
S.Derby PT55
S.Derby PT55
S.Derby PT55
S.Derby PT55
S.Derby PT55
S.Derby PT55
S.Derby PT55
S.Derby PT55
S.Derby PT55
S.Tm,DT193
S.Tm,DT193
S.Tm,DT193
S.Derby PT51
S.Derby PT51
S.Tm,DT193
S.Tm,DT193
S.Tm,DT193
S.Tm,DT193
S.Tm,DT193
S.Tm,DT193
S.Tm,DT193
S.Tm,DT193
S.Tm,DT193
No.
45/7
45/8
45/10
45/11
45/12
45/15
45/16
45/18
45/20
45/29
46/4.2
46/9
46/10
46/11.3
46/15
46/17
46/18
46/19
46/20
46/21
46/22
46/23
46/26
47/11
47/15
47/16
47/25
49/1
49/2
49/3
49/6
49/7
49/8
49/9
50/9
50/20
51/1
51/3
51/4
51/5
181
Sample
lamp
feaces
feaces
feaces
separate wall
feaces
drive board
feaces
feaces
feaces
rectal swab
feeder
floor
rectal swab
nipple
floor
feaces
wall
feaces
nipple
floor
scale
boot
drive board
nipple
chain
floor
feaces
nipple
chain
separate wall
feeder
floor
mouse faeces
drive board
chain
drive board
feaces
nipple
lamp
Phagetype
S.Tm,DT193
S.Tm,DT193
S.Tm,DT193
S.Tm,DT193
S.Tm,DT193
S.Tm,DT193
S.Tm,DT193
S.Tm,DT193
S.Tm,DT193
S.Tm,DT193
S.Tm,DT193
S.Tm,DT193
S.Tm,DT193
S.Tm,DT193
S.Derby PT51
S.Derby PT51
S.Derby PT51
S.Derby PT51
S.Derby PT51
S.Tm,DT193
S.Derby PT51
S.Tm,DT193
S.Tm,DT193
S.Tm,DT193
S.Derby PT11
S.Derby PT11
S.Derby PT11
S.Tm,ut
S.Tm,ut
S.Tm,ut
S.Tm,ut
S.Tm,ut
S.Tm,ut
S.Tm,ut
S.Derby PT11
S.Derby PT11
S.Tm,DT193
S.Tm,DT193
S.Tm,DT193
S.Tm,DT193
Appendix
Table 9.2: Salmonella phage type in each type of samples (continued)
No.
24/7
24/8
24/14
25/11
26/3
27/4
27/6
27/10
27/12
27/13
27/14
27/15
27/18
27/29
29/7
29/9
29/11
29/15
29/17
29/21
30/6
30/7
30/10
31/9
31/11
31/16
31/22
33/3
33/4
33/14
33/17
Sample
drive board
floor
floor
floor
ventilator
wall
feaces
floor
feeder
separate wall
ventilator
floor
floor
floor
wall
floor
drive board
floor
scale
ventilator
separate wall
separate wall
floor
floor
separate wall
floor
floor
separate wall
floor
feeder
wall
Phagetype
S.Tm,DT193
S.Tm,DT193
S.Tm,DT193
S.Tm,DT193
S.Tm,DT193
S.Tm,DT193
S.Tm,DT193
S.Tm,DT193
S.Tm,DT193
S.Tm,DT193
S.Tm,DT193
S.Tm,DT193
S.Tm,DT193
S.Tm,DT193
S.Tm,DT193
S.Tm,DT193
S.Tm,DT193
S.Tm,DT193
S.Tm,DT193
S.Tm,DT193
S.Tm,DT097
S.Tm,DT097
S.Tm,DT097
S.Tm,DT193
S.Tm,DT193
S.Tm,DT193
S.Tm,DT193
S.Tm,DT193
S.Derby PT04
S.Derby PT04
S.Derby PT04
No.
51/6
51/10
51/11
51/12
51/15
51/18
51/19
51/20
51/22
51/24
51/25
52/4
52/18
52/24
52/25
52/26
53/7
S. Tm = Salmonella Typhimurium
182
Sample
floor
boot
mouse faeces
scale
scale
mouse faeces
mouse faeces
lamp
scale
scale
lamp
floor
feeder
lamp
lamp
scale
scale
Phagetype
S.Tm,DT193
S.Tm,DT193
S.Tm,DT193
S.Tm,DT193
S.Tm,DT193
S.Tm,DT193
S.Tm,DT193
S.Tm,DT193
S.Tm,DT193
S.Tm,DT193
S.Tm,DT193
S.Tm,DT104
S.Tm,DT193
S.Tm,DT193
S.Tm,DT193
S.Tm,DT193
S.Derby PT11
Appendix
Table 9.3: Number of positive samples in each farm
Farm
No. Sample
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
33
34
35
36
41
42
43
44
17
22
15
16
14
15
18
15
12
11
14
13
15
16
14
15
15
16
19
14
14
19
14
18
18
7
32
17
22
26
22
19
18
17
14
15
22
11
10
No. PCR +
(default)
0
2
7
7
5
8
13
7
5
1
3
2
3
7
0
3
1
5
4
10
3
6
10
16
3
1
12
1
10
6
4
5
14
3
5
4
3
0
0
183
No. Culture +
0
1
3
3
2
4
9
3
5
1
3
2
0
5
0
3
1
3
4
10
1
5
6
5
1
1
9
0
6
3
4
4
13
1
3
2
0
0
0
No. PCR +
(modified)
0
1
6
6
4
7
13
7
4
1
3
1
3
7
0
3
1
4
4
10
3
6
10
16
2
1
11
0
10
5
4
4
13
2
5
4
3
0
0
Appendix
Table 9.3: Number of positive samples in each farm (continued)
Farm
No. Sample
45
46
47
48
49
50
51
52
53
Total
48 Farms
31
38
30
36
20
36
26
26
22
906
( 100.00% )
No. PCR +
(default)
25
13
9
28
11
16
23
24
10
358
( 39.51% )
No. Culture +
14
13
4
0
7
2
15
5
1
187
( 20.64% )
PCR (default) = Threshold at 0.2; PCR (modified) = Threshold at 1.4
184
No. PCR +
(modified)
25
13
9
24
11
15
23
24
9
337
(37.19%)
Acknowledgement
10
Acknowledgement
I would like to express my gratitude to my supervisor Prof. Dr. Thomas Blaha, for giving me
the opportunity to study about the interesting Salmonella and Pre-harvest food safety, for his
extensive advice and guidance.
The financial support from Charoen Pokphand Foods Public Company Limited (CPF),
Thailand is gratefully acknowledged. I am bound to President and Chief of Executive Officer,
Mr. Adirek Sripratak; to Executive Vice President, Mr. Somkuan Choowatanapakorn and to
Senior Vice President, Mr. Damnoen Chaturavittawong for their financial support of my study
in Germany.
Thanks to the Field Station for Epidemiology in Bakum, University of Veterinary Medicine
Hannover, Foundation in Germany for my research support.
My gratefulness is extended to Dr.med.vet. H. Nathues, M. Busemann, M. Sieve, R. Tegeler,
S. Schwermann-Jäger, C. Fischer, H. Roter who gave me a lot of help and guidance both in
the laboratory and during the time of writing as well as German language and German
culture during the time we worked together and thank you for their friendship and friendly
atmosphere in the laboratory.
I wish to thank all of Dr.med.vet students and members of the Field Station for
Epidemiology, especially Mrs. Verena Gotter for their helpful and friendly support.
I would like to give special thanks to Dr. Wolfgang Rabsch and all members of the Robert
Koch Institute for Salmonella phage-typing.
I am deeply grateful to Prof. Dr. Dr. h.c. W. Winkenwerder and his family for their advice,
helpful and friendly support. I have furthermore to thank Prof. Dr. Yongyuth Sujjawanich
Chairman of Council, Mahanakorn University of Technology, especially Dr. Bandhit
Thanintharatarn who inspired and gave a great advice in this study.
My special thank is extended to Mrs. Maritta Ledwoch for her attention to us and to pig
farmers for letting me to collect samples in pig herds. I also want to thank Mr. Karl-Josef
Mählmayer for his residence as my home during my research. My counsin, Teeraphat
Thedcharoen looked closely at the dissertation for English style and grammar, correcting both
and offering suggestions for improvement.
I would like to give special thanks to my beloved parents whose encouragement enabled me
to complete this work.
185