Inactivation of viruses in liquid manure

Rev. sci. tech. Off. int. Epiz., 1995,14 (2), 435-445
Inactivation of viruses in liquid manure
B. HAAS *, R. AHL *, R. BÖHM ** and D. STRAUCH **
Summary: The stability of some viruses and methods of virus inactivation in
liquid manure are reviewed. The authors discuss experimental data on the
stability of foot and mouth disease virus, classical swine fever virus, Aujeszky's
disease virus, African swine fever virus, swine influenza virus, porcine
paramyxovirus, bovine virus diarrhoea virus and transmissible gastroenteritis
of pigs virus. Recommendations and practical advice are given for the choice
and application of chemical disinfectants for slurry.
KEYWORDS: Disinfectants - Liquid manure - Virus inactivation - Virus
stability.
INTRODUCTION
The causative agents of nearly all bacterial and viral diseases are shed by infected
animals in either faeces or urine or by other means. Most of this infective material
collects on the floor and thus in t h e m a n u r e (27, 28). Consequently, m a n u r e has a
potential for spreading infectious diseases.
' F a r m y a r d m a n u r e ' is a mixture of excreta and substantial quantities of bedding
material, and is sufficiently dense to be handled as a solid. 'Liquid m a n u r e ' or 'slurry',
however, is a mixture of faeces and urine which may also contain cleaning and rain
water, together with small quantities of bedding material and feed, and must therefore
be handled in a somewhat different manner.
In the past, dung pits filled with farmyard m a n u r e g e n e r a t e d t e m p e r a t u r e s
sufficiently high to destroy pathogens. A t present, farmyard m a n u r e often contains
insufficient bedding material, and the faeces of modern high performance animals seem
to differ significantly in composition and structure from those of farm animals reared at
the beginning of the 20th century. Thus, thermophilic microbiological processes can no
longer be g u a r a n t e e d to develop during storage of dung. However, the addition of
granulated quicklime can still ensure reliable disinfection (3,19,23,26).
By contrast, slurry does not normally generate the type of self-heating processes
which could destroy pathogens. Thus, after an outbreak of a notifiable disease, slurry
must be disinfected by other m e a n s (13, 16, 28). Disinfection of liquid m a n u r e is
generally considered to be difficult. In many cases, no a t t e m p t is m a d e to achieve
disinfection, as this is considered almost impossible under practical conditions. However,
most notifiable diseases could possibly be spread by liquid manure, and it is therefore
* Bundesforschungsanstalt für Viruskrankheiten der Tiere, Paul-Ehrlich-Straße 28,72076 Tübingen,
Germany.
** Universität Hohenheim, Institut für Tiermedizin und Tierhygiene, Garbenstraße 30,70599 Stuttgart,
Germany.
436
i m p o r t a n t to treat slurry after such a disease outbreak. Several chemicals can be
considered effective and relatively innocuous to the environment (25). Making decisions
regarding the treatment of contaminated slurry requires knowledge of the stability of
viruses in liquid m a n u r e , as well as of the capabilities and limitations of chemical
disinfectants. Inactivation of viruses in liquid manure by physical or biological means is
also possible, but is often impractical due to the lack of suitable equipment.
STABILITY OF VIRUSES IN LIQUID MANURE:
EXPERIMENTAL DATA AND OPEN QUESTIONS
Very little information is available on the survival times of viruses in farm effluents.
Some viruses may survive in faeces of various kinds, d e p e n d i n g on seasonal
t e m p e r a t u r e . According to Müller (17), Aujeszky's disease virus may survive for
3-15 weeks, Borna disease virus for 22 days, Marek's disease virus for 7 days, Teschen
disease virus for 3-25 days, African swine fever virus for 60-100 days, and foot and
mouth disease virus for 21-103 days. However, under practical conditions, survival time
is strongly dependent on temperature, p H value and the initial burden of pathogens.
B 0 t n e r (5) investigated the inactivation of viruses in slurry at various t e m p e r a t u r e s
between 5°C and 55°C, and found a strong temperature dependence of survival time for
several viruses (Table I). In a n o t h e r set of experiments (C. E i z e n b e r g e r et al,
unpublished findings), the emphasis was placed on the long-term survival of agents of
notifiable diseases under simulated field conditions. Classical swine fever virus survived
in pig slurry for at least 70 days at 17°C, and for 84 days at 4°C. African swine fever virus
survived for at least 84 days at 17°C, and for 112 days at 4°C. Foot and mouth disease
virus survived in bovine slurry for at least 70 days at 17°C, and for 84 days at 4°C
(Tables II, III and IV).
T h e risk of infection associated with the spreading of slurry on farmland cannot be
clearly estimated, and therefore the safest procedure is to attempt the best possible
d e c o n t a m i n a t i o n of infected slurry before spreading. To achieve a b e t t e r risk
assessment, the decimal reduction time (T90) for given pathogens under specific storage
conditions should b e investigated further and used to calculate the necessary
inactivation time (9).
However, B0tner (6) reported a fast initial drop of Aujeszky's disease virus titres
compared to the rate of inactivation during the rest of the observation period, and thus
concluded that inactivation of Aujeszky's disease virus does not follow first-order
kinetics.
D a t a r e p o r t e d by various a u t h o r s on virus inactivation in slurry are not easily
c o m p a r e d , due to the lack of generally-accepted s t a n d a r d m e t h o d s for this type of
experiment. Different conditions in the slurry, and different methods for virus spiking,
reisolation and detection, may lead to different inactivation rates. To avoid problems
with the adsorption of virus to solid slurry particles and the quantitative elution of these
particles, several a u t h o r s used plastic carriers to which virus was adsorbed
(20; C. Eizenberger et al, unpublished findings). By contrast, B 0 t n e r (6) mixed the
viruses with slurry and reisolated them from the supernatant. Whereas Mack et al. (15)
found it necessary to include an elution with beef extract to reisolate more than 12% of
Aujeszky's disease virus, B0tner (6) reported a reisolation rate for this virus of up to
100% by addition of 10% f e t a l calf serum to the adsorption m e d i u m . D u e to
cytotoxicity of non-centrifuged slurry/virus mixtures, supernatants from centrifuged
437
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Inactivationtimesfor animal viruses in slurry at various temperatures
(5)
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438
TABLE II
Survival of classical swine fever virus in pig slurry at various temperatures
(C. Eizenberger et al, unpublished findings)
Titre at 4°C *
Day
0
14
28
42
56
70
84
98
112
126
Titre at 17°C *
1st test
2nd test
Control **
1st test
2nd test
Control **
7.0
5.5
4.5
4.25
3.75
2.75
<2.5
<2.5
<2.5
<2.5
6.75
5.25
4.75
4.25
3.75
3.0
2.75
<2.5
<2.5
<2.5
6.75
5.25
4.0
3.25
2.5
2.0
<1.5
<1.5
<1.5
<1.5
6.5
5.0
4.25
4.0
3.5
2.75
<2.5
<2.5
<2.5
<2.5
6.5
4.75
4.25
3.75
3.25
<2.5
<2.5
<2.5
<2.5
<2.5
6.75
5.25
3.75
2.25
2.0
<1.5
<1.5
<1.5
<1.5
<1.5
* virus titres in log TCID /carrier
** control carriers were closed so that virus was not exposed to slurry
10
50
samples were used for infectivity titrations. This may lead to misinterpretation of the
results, as virus might be adsorbed to slurry particles and thus removed with the pelleted
material. F u r t h e r m o r e , virus bound to solid might be protected against inactivation.
However, B o t n e r (6) showed - by enzyme-linked i m m u n o s o r b e n t assay - that
Aujeszky's disease virus did not concentrate in the sediment. Furthermore, absence of
infectious virus was confirmed by intranasal inoculation of pigs with a non-centrifuged
TABLE III
Survival of African swine fever virus in pig slurry at various temperatures
(C. Eizenberger et al, unpublished findings)
Day
0
14
28
42
56
70
84
98
112
126
Titre at 4°C *
Titre at 17°C *
1st test
2nd test
Control **
1st test
2nd test
Control **
6.25
5.5
4.5
4.5
4.25
3.5
3.75
3.0
2.75
<2.5
6.5
5.5
4.75
4.25
4.0
3.75
3.25
2.75
<2.5
<2.5
6.5
4.5
4.25
4.0
3.5
2.0
1.75
<1.5
<1.5
<1.5
6.25
4.75
4.25
3.75
3.5
3.0
2.75
<2.5
<2.5
<2.5
6.0
5.5
4.0
3.25
3.5
2.75
<2.5
<2.5
<2.5
<2.5
6.25
4.75
3.5
2.5
1.75
<1.5
<1.5
<1.5
<1.5
<1.5
* virus titres in logjQ TCID /carrier .
** control carriers were closed so that virus was not exposed to slurry
50
439
TABLE I V
Survival of foot and mouth disease virus in cattle slurry at various temperatures
(C. Eizenberger et al, unpublished findings)
Titre at 4°C *
Day
0
14
28
42
56
70
84
98
112
126
Titre at 17°C *
1st test
2nd test
Control **
1st test
2nd test
Control **
6.25
5.25
4.75
5.0
4.25
3.0
2.75
<2.5
<2.5
<2.5
6.5
5.0
4.75
4.25
3.5
2.75
2.75
<2.5
<2.5
<2.5
6.75
5.25
4.0
3.75
2.75
<1.5
<1.5
<1.5
<1.5
<1.5
6.5
5.25
4.25
4.25
3.75
<2.5
<2.5
<2.5
<2.5
<2.5
6.25
5.0
4.0
3.75
3.0
2.75
<2.5
<2.5
<2.5
<2.5
6.5
4.75
4.25
3.25
2.5
<1.5
<1.5
<1.5
<1.5
<1.5
* virus titres in l o g TCID /carrier
** control carriers were closed so that virus was not exposed to slurry
10
50
sample. F u r t h e r studies will be necessary to c o m p a r e different m e t h o d s of virus
isolation from slurry, and thus establish and validate standard protocols for a variety of
viruses. The elucidation of inactivation kinetics also requires further work.
METHODS FOR THE INACTIVATION OF VIRUSES IN
LIQUID MANURE
Long-term storage
The question is frequently raised whether slurry can be inactivated by storage over
several months, especially with respect to community slurry tanks in some regions of
G e r m a n y (4, 10, 20). T h e data p r e s e n t e d above - indicating a titre reduction of
approximately 1-2 log units per month at 4°C for some viruses - show that inactivation
would be possible by long-term storage. However, this approach is problematic, as the
minimum time required to obtain a reasonable safety margin would often be six months.
During this period, no new liquid manure would be allowed to enter the storage tanks;
in most cases, this requirement would be difficult to satisfy. For some notifiable diseases,
this would mean that no susceptible animal could be kept on the farm during this period.
Furthermore, the inactivation of the viruses considered will not necessarily follow firstorder kinetics.
10
Physical methods
Pasteurisation
During pasteurisation, slurry is heated to temperatures between 65°C and 100°C for
at least 30 min. These conditions correspond to the r e c o m m e n d a t i o n s of a G e r m a n
working group on sewage sludge. Some authors recommend heating to 70°C (8).
440
Microwave
treatment
Treatment of slurry with microwaves has been proved to be effective against both
viruses and bacteria in laboratory experiments, but this procedure is at an experimental
stage (11,12,14, 22). The equipment required by this method would be light and easily
t r a n s p o r t a b l e , and no chemicals would be n e e d e d . T h e main disadvantages are the
a m o u n t of energy required for sufficient heating and the relatively high cost of
equipment.
Oligolysis
T h e oligolysis m e t h o d is based on the effect of c o p p e r ions on microorganisms.
Spindler (24) demonstrated that Salmonella senftenberg and Escherichia coli could be
reduced significantly by this method. Further experiments by Müller et al. (18) indicated
t h a t oligolysis is still at an e x p e r i m e n t a l stage, and that results from l a b o r a t o r y
experiments do not always apply to conditions in the field. To date, no experiments have
been performed using viruses.
Biological methods
Aerobic thermophilic
stabilisation
In the course of the aerobic thermophilic stabilisation (ATS) (liquid composting)
process - caused by the activity of aerobic thermophilic bacteria in slurry - exothermal
microbial degradation and metabolic processes result in a t e m p e r a t u r e rise and an
increase in p H to values of 8 or more. Provided that the reaction vessel is well insulated,
the air supply is correctly calculated, and the slurry has a sufficient concentration of
organic dry matter, temperatures can be reached which provide for stabilisation and
sanitation of the slurry (27). ATS has been successfully applied for the sanitation of
sewage sludge and slurry (21).
Anaerobic
digestion
Anaerobic digestion of slurry requires special equipment (digestors). While reliable
inactivation of pathogenic microorganisms by a mesophilic process is not possible under
n o r m a l operating conditions, the t h e r m o p h i l i c process may be suitable for the
sanitation of slurry (4) under certain conditions.
Composting of solid material
If equipment is available for separation of the solid fraction of slurry from the liquid
fraction, the solid fraction can be composted, while the liquid fraction must be treated
by other means (e.g. addition of chemicals) (4).
Chemical methods
Of practical importance for the chemical inactivation of slurry are aldehydes, acids,
oxidising agents and alkalis.
Aldehydes
Aldehydes are economical, biodegradable and have almost no corrosive effect on
equipment and buildings.
T h e main disadvantages of aldehydes are the strong t e m p e r a t u r e d e p e n d e n c e of
inactivation rates and an irritating smell. For t r e a t m e n t of slurry, formaldehyde has
been shown to be very effective.
441
Acids
Acids can either inactivate viruses by p H value alone (using inorganic acids), or also
by the interaction of lipophilic structures with membranes of enveloped viruses (using
organic acids). Inorganic acids have a strong corrosive effect and are therefore rarely
used for the treatment of slurry. The protein content of slurry considerably reduces the
inactivating effect of organic acids.
Oxidising
agents
Oxidising agents can inactivate viruses and other microorganisms. Mixing oxidising
agents with slurry leads to strong foaming, which limits the practical use of these
substances. In special cases w h e r e this can be tolerated (e.g. disinfection of small
volumes), the use of peracetic acid can be recommended (7).
Alkalis
Alkalis are economical and effective, even in solutions with high protein content.
T h e main disadvantage of alkalis is their corrosive effect and, in case of lime, their
volume. Slaked lime and sodium hydroxide are the most i m p o r t a n t alkalis used for
treating slurry.
Environmental considerations play an important role in the choice of disinfectants
for slurry. Some a u t h o r s have examined t h e effect of lime, sodium hydroxide,
formaldehyde and peracetic acid on arable land ( 7 , 1 2 , 1 4 , 1 6 ) . In summary, these
reports showed that despite transient damage to plants, harvests from fields fertilised
with freshly disinfected slurry were generally higher than those from non-fertilised
control fields. T h u s , an incubation time of four days (as r e q u e s t e d by t h e official
recommendations in Germany) should prevent long-term damage to plants (4).
PRACTICAL RECOMMENDATIONS FOR THE TREATMENT OF
LIQUID MANURE
There is a slight possibility that slurry from farms with clinically-healthy animals may
contain certain p a t h o g e n s . To minimise this low risk, an expert group of the
Commission of the E u r o p e a n Communities elaborated interim guidelines in 1978 for
the utilisation of ' n o r m a l ' slurries; t h e s e guidelines were later a m e n d e d (1). T h e
recommendations may be summarised as follows:
- wherever possible, slurry should be utilised on tillage crops (excluding those for
fresh consumption)
- slurry should be stored for at least 60 days in summer or 90 days in winter, before
being spread on pasture
- after application of slurry, a period of 30 days should be allowed before grazing,
preferably with adult or non-susceptible animals.
Further details are given by Strauch (27).
After an o u t b r e a k of a notifiable disease, chemical t r e a t m e n t of slurry may b e
necessary. Several conditions must be met to ensure p r o p e r disinfection by chemical
treatment. Böhm et al. (4) present advice on technical problems which must be solved to
achieve sufficient and reliable inactivation of viruses by chemical methods. If possible,
storage tanks should not b e filled to the limit, to enable the addition of a sufficient
442
amount of chemicals. Chemical disinfectants must be thoroughly dissolved and evenly
distributed in the slurry. Vigorous stirring is necessary before, during and after the
addition of chemicals. Chemicals should be added to the storage tank at several points
simultaneously. As powdery or granular substances are difficult to dissolve in liquid
manure, the application of aqueous suspensions is strongly recommended, unless highperformance (e.g. 100 hp per 500m ) stirring equipment is available. In addition, the
efficacy of t r e a t m e n t with a q u e o u s solutions d e p e n d s on the intensity of stirring.
Usually, the equipment available on farms will not achieve sufficiently vigorous stirring
of sediments. Therefore, mobile high-performance stirring equipment should be made
available by veterinary authorities, farmers' associations or other institutions.
3
During exposure of slurry to disinfectants, no fresh slurry must enter the tank. The
disinfection of liquid m a n u r e stored u n d e r occupied stables should generally be
avoided, as this violates basic rules of hygiene. If disinfection of liquid m a n u r e in an
occupied stable is absolutely inevitable, formalin may be used instead of alkaline
chemicals. However, the concentration of formalin gas in the stable may then cause
problems, which must be overcome by ensuring sufficient ventilation.
Regulations for disinfection of liquid manure after outbreaks of notifiable diseases in
G e r m a n y are formulated in a directive of the F e d e r a l Ministry of A g r i c u l t u r e (2).
Valuable advice is given on practical questions, as follows:
- Liquid manure must be disinfected by chemical means if no heat disinfection is
applied.
- Before and during the addition of disinfectants, and for six hours afterwards, the
liquid manure must be thoroughly stirred.
- Stirring, for at least two h o u r s , must be r e p e a t e d daily until the m a n u r e is
considered safe.
- After treatment, the manure should be ploughed into arable land.
- The following chemicals are recommended for disinfection of liquid manure:
3
- C a ( O H ) (slaked lime, lime h y d r a t e ) : 4 0 % solution at a rate of 40-60 1/m ;
exposure time > 4 days; also suitable for use at temperatures between 0 and -10°C.
2
3
- N a O H (sodium hydroxide): 5 0 % , 16-30 1/m , exposure time > 4 days; p H > 12;
also suitable for use at temperatures between 0 and +10°C.
3
- Formalin: 35-37% solution of formaldehyde in water, 25-401/m , exposure time
> 4 days; not suitable at temperatures below +10°C; efficacy reduced below +20°C.
3
- Peracetic acid: 25-401/m , exposure time > 1 h; only suitable in special situations,
due to strong formation of foam; also suitable for use between 0 and +10°C.
The incubation time of 4 days requested in the regulations should be considered the
absolute minimum, and exposure for 7 days would be more advisable.
443
INACTIVATION DES VIRUS DANS LE PURIN. - B. Haas, R. Ahl, R. Böhm et
D. Strauch.
Résumé : Les auteurs traitent de la stabilité de certains virus et des méthodes
d'inactivation de ces virus dans le purin. Ils examinent les données expérimentales
concernant la stabilité des virus de la fièvre aphteuse, de la peste porcine classique,
de la maladie d'Aujeszky, de la peste porcine africaine, de la grippe porcine, de la
paramyxovirose porcine, de la diarrhée virale bovine et de la gastro-entérite
transmissible du porc. Les auteurs font des recommandations et donnent
quelques conseils pratiques concernant le choix et l'application des désinfectants
chimiques des lisiers.
MOTS-CLÉS : Désinfectants - Inactivation des virus - Purin - Stabilité des
virus.
*
* *
INACTIVACIÓN DE LOS VIRUS EN EL ESTIÉRCOL LICUADO. - B. Haas, R. Ahl,
R. Böhm y D. Strauch.
Resumen: Los autores se refieren a la estabilidad de ciertos virus y a los métodos
de inactivación de estos virus en el estiércol licuado. Examinan datos
experimentales acerca de la estabilidad de los virus de la fiebre aftosa, de la peste
porcina clásica, de la enfermedad de Aujeszky, de la peste porcina africana, de la
gripe porcina, de la paramixovirosis porcina, de la diarrea viral bovina y de la
gastroenteritis transmisible del cerdo. Por último, proponen recomendaciones y
criterios prácticos para elegir los desinfectantes químicos y aplicarlos al estiércol
licuado.
PALABRAS CLAVE: Desinfectantes - Estabilidad de los virus - Estiércol
licuado - Inactivación de los virus.
*
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