First detection of Sarcoptes scabiei from domesticated pig (Sus

First detection of Sarcoptes scabiei from
domesticated pig (Sus scrofa) and genetic
characterization of S. scabiei from pet, farm
and wild hosts in Israel
Oran Erster, Asael Roth, Paolo S. Pozzi,
Arieli Bouznach & Varda Shkap
Experimental and Applied Acarology
ISSN 0168-8162
Exp Appl Acarol
DOI 10.1007/s10493-015-9926-z
1 23
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Exp Appl Acarol
DOI 10.1007/s10493-015-9926-z
First detection of Sarcoptes scabiei from domesticated pig
(Sus scrofa) and genetic characterization of S. scabiei
from pet, farm and wild hosts in Israel
Oran Erster1 • Asael Roth1 • Paolo S. Pozzi2
Arieli Bouznach3 • Varda Shkap1
•
Received: 22 March 2015 / Accepted: 30 April 2015
Ó Springer International Publishing Switzerland 2015
Abstract In this report we describe for the first time the detection of Sarcoptes scabiei
type suis mites on domestic pigs in Israel and examine its genetic variation compared with
S. sabiei from other hosts. Microscopic examination of skin samples from S. scabieiinfested pigs (Sus scrofa domesticus) revealed all developmental stages of S. scabiei. To
detect genetic differences between S. scabiei from different hosts, samples obtained from
pig, rabbits (Orictolagus cuniculus), fox (Vulpes vulpes), jackal (Canis aureus) and
hedgehog (Erinaceus concolor) were compared with GenBank-annotated sequences of
three genetic markers. Segments from the following genes were examined: cytochrome C
oxidase subunit 1 (COX1), glutathione-S-transferase 1 (GST1), and voltage-sensitive
sodium channel (VSSC). COX1 analysis did not show correlation between host preference
and genetic identity. However, GST1 and VSSC had a higher percentage of identical sites
within S. scabiei type suis sequences, compared with samples from other hosts. Taking into
account the limited numbers of GST1 and VSSC sequences available for comparison, this
high similarity between sequences of geographically-distant, but host-related populations,
may suggest that different host preference is at least partially correlated with genetic
differences. This finding may help in future studies of the factors that drive host preferences in this parasite.
Keywords
COX1
Sarcoptes scabiei Genetic markers Host preference GST1 VSSC & Oran Erster
[email protected]; [email protected]
1
Division of Parasitology, Kimron Veterinary Institute, 50250 Bet Dagan, Israel
2
Israeli Veterinary Services, 50250 Bet Dagan, Israel
3
Division of Pathology, Kimron Veterinary Institute, 50250 Bet Dagan, Israel
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Introduction
The skin mite Sarcoptes scabiei parasitizes several species of mammals, including humans.
S. scabiei burrow into the skin where they feed and lay eggs. In addition to the damage
caused by the burrowing, mites. S. scabiei infection can trigger multiple reactions such as
allergic reaction, inflammation, innate immune reaction and activation of immune components in the skin. Host immune response combined with development of wounds caused
by extensive scratching, can lead to secondary infection and in extreme cases to loss of
weight and even death (Mullen and OConnor 2002). The single species S. scabiei parasitizes a wide variety of mammalian hosts, from marsupials to primates (Klompen 1992).
Within this species there are host-specific types with distinct preference to a specific host
species (human, canine, bovine, etc.). Although transfer between different hosts occurs (for
example, from dogs to humans), it usually results in a single-generation minor infestation.
This is in contrary to transfer of mites within the same species, which occurs not only
horizontally, but also between mother and offspring (Bornstein and Zakrisson 1993) as
reported for piglets while suckling infested sows (Greves and Davies 2012). Furthermore, a
study of sympatric S. scabiei populations residing on humans and dogs showed that these
populations do not mix and maintain distinct genetic characteristics (Walton et al. 1999).
The use of genetic markers has become a useful tool for identification and classification
arthropods, including attempts to distinguish between different S. scabiei types (Amer et al.
2014). A combination of mitochondrial markers and microsatellite sequences, enabled to
genetically distinguish between S. scabiei type hominis and S. scabiei type canis, showing
clear differences between neighboring populations with no intermix (Walton et al. 1999).
Previous reports on S. scabiei infestations in Israel described infested ibexes (Capra ibex
nubiana) and other ruminants held in captivity (Yeruham et al. 1996, 1999). To our
knowledge, no reports were made on S. scabiei from domesticated animals or from humans
in Israel, and there is no genetic information regarding different Sarcoptes types in Israel.
Domesticated swine population in Israel is confined to only two regions (South-Negev;
North-Galilee) with annual production of around 200,000 slaughtered pigs. Here, we report
for the first time on S. scabiei detected on domestic pigs (Sus scrofa) in Israel and identify
genetic difference between S. scabiei from pig, fox, jackal, hedgehog and rabbit, collected
in Israel.
Materials and methods
Sample collection and preparation for microscopic analysis
Skin scrapings from domesticated pigs (Sus scrofa) grown in a commercial farm in
northern Israel, were collected during routine health inspection using surgical blades from
infected skin crusts. Samples from infested rabbits (Oryctolagus cuniculus) grown in a
commercial pet animal maintenance facility were collected using surgical blade into a
sterile tube. Mites from fox (Vulpes vulpes), jackal (Canis aureus) and hedgehog (Erinaceus concolor) were collected from skin samples of carcasses examined at the Kimron
Institute post mortem facility. Samples for microscopic examination incubated with 1 M
KOH at 65 °C for 45–60 min. Following incubation, 50–100 ll of the solution were plated
on a microscope slide, covered with a cover slip and examined under 1009 magnification
in a light microscope (Olympus BX-50).
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DNA extraction
Samples (*500 lg tissue) were frozen in liquid nitrogen and were grinded with 300 ll cooled
Cell Lysis Buffer (Epicentre) using a disposable sterile 1.5 ml-tube pestle. When the sample
was homogenized, it was incubated with proteinase K (160 ng/ll) at 66 °C for 60 min. Further
steps were performed using the Epicentre Masterpure kit (Madison, WI, USA) according to the
manufacturer’s instructions. DNA was eluted in a final volume of 100 ll.
PCR and sequencing
Alignments of GenBankTM -annotated sequences were done to design primers for the amplification and cloning of the glutathione-S-transferase class 1 (GST1) and voltage sensitive
sodium channel (VSSC) gene segments. Conserved regions were identified and the following
primers were designed: for GST1: forward primer: 50 -AGTCGTAGTGTATATTTGGTGG30 and reverse primer: 50 -GCTTCAGAATTTCGTTGGATTC-30 . For VSSC: forward primer: 50 -AATTGTTGTACTTTCACTACTCG-30 and reverse primer: 50 -CCTGAAACACGCATACAATCCC-30 . The primers used for cytochrome C oxidase subunit 1 (COX1)
PCR were as follows: forward primer: 50 -GACACCCAGAAGTTTACATTC-30 and reverse:
50 -TATATTTTGATAATGAAT-CTC-30 . PCR mix was prepared as follows: 25 ll DreamTaq green mix (Thermo Scientific, Vilnius, Lithuania), primers in a final concentration of
5 lM each, 80–120 ll DNA template, ddH2O to a final volume of 50 ll. PCR of the GST1
and VSSC genes was performed under the following conditions: 2 min at 94 °C, 35 cycles of
the following steps: 30 s at 94 °C, 30 s at 56 °C, 40 s at 72 °C, and a final step of 5 min at
72 °C. PCR of the COX1 gene segment was performed under the following conditions: 2 min
at 94 °C, five cycles of the following steps: 30 s at 94 °C, 30 s at 42 °C, 40 s at 72 °C, 30
cycles of the following steps: 30 s at 94 °C, 30 s at 50 °C, 40 s at 72 °C and a final step of
5 min at 72 °C. PCR products were purified using GeneJet purification kit (Thermo Scientific, Vilnius, Lithuania). PCR products were either sequenced directly or cloned into pGEMT Easy vector (Promega, Madison, WI, USA) according to the manufacturer’s instructions.
Phylogenetic analysis
PCR-amplified sequences were compared to annotated sequences using BLAST (http://
blast.ncbi.nlm.nih.gov/Blast.cgi) and were aligned using the Geneious software (Biomatters, Auckland, New Zealand). Alignments were done using the MUSCLE alignment
(Edgar 2004). Phylogenetic trees were constructed using the Geneious software Tree
Builder. The method used was Neighbor-joining, according to the Tamura-Nei model
(1993). Determination of the Percentage of Identical Sites (PIS) was done by calculating
the percentage of columns (positions) in the alignment for which all sequences are identical, a feature included in the Geneious software package.
Results
Sample collection and identification
Skin samples were collected using skin scrapings from routine inspection of domestic pigs
grown in a commercial farrow to weaning farm of about 400 sows in Northern Israel.
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Examined animals, one boar used in natural insemination and a few sows, suffered from
scaled skin and pruritus. Infestation was suspected on the basis of characteristic skin
damage and scratching behavior of the infested animals (Fig. 1a). On the basis of clinical
symptoms, clinically affected animals (gilts, piglets) and all the breeders (sows and boars)
were treated with an acaricide of the Avermectin family according to the manufacturer’s
recommendations immediately after samples collection. Skin samples were prepared as
described in the Materials section, and were examined for the presence of mites. Microscopic examination revealed that all stages of S. scabiei were present, suggesting that this
was an established infestation, rather than a temporal transfer from a different host species
(Mullen and OConnor 2002, Fig. 1b). The S. scabiei samples from the pig skin were
compared to samples obtained from S. scabiei—infested rabbit (Oryctolagus cuniculus),
collected from a commercial pet maintenance facility. Sample from both host species
included all developmental stages, and were morphologically indistinguishable (Fig. 1b,
c). Samples obtained from fox, jackal and hedgehog carcasses examined in the KVI post
mortem facility, were also morphologically indistinguishable.
Fig. 1 Detection of Sarcoptes scabiei type suis on domestic pigs (Sus scrofa) in Israel. a Hair loss (arrows,
top panel) and traces of burrows (arrows, bottom panel) in infected body parts. b Adult (top) and hatching
larva (bottom) from pig. c Adult (top) and all developmental stages (bottom) from rabbit. A Adult, N Nymph
(six legged), L Hatching larva. d Amplification of voltage sensitive sodium channel (VSSC) and glutathioneS-transferase class 1 (GST1) gene segments. Primer specificity was established using the tick Rhipicephalus
bursa DNA as negative control
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Molecular characterization of Sarcoptes scabiei GST1, VSSC and COX1
In order to establish whether S. scabiei isolates from different hosts have genetic variation,
DNA was extracted from skin samples of each host, and was used in a comparative PCR
analysis. Sequences from three genetic markers were used to characterize the samples:
voltage sensitive sodium channel (VSSC), glutathione-S-transferase class 1 (GST1), and
cytochrome C oxidase subunit 1 (COX1). Specificity of the PCR was ascertained by using
tick DNA as a negative control (Rhipicephalus bursa) for the GST1 and VSSC reactions
(Fig. 1d). Comparison of 467 bp VSSC sequences from 11 samples, from different hosts,
resulted in 96.5 % overall percentage of identical sites (PIS), with one clade containing
SsVSSC from pig and hedgehog, and the other with SsVSSC sequences from jackal, fox,
dog, rabbit and human (Fig. 2a). Alignment of S. scabiei type suis only showed 99.6 %
identical sites. The PIS within the non-pig, non-hedgehog clade was 97.7 % (Table 1).
Alignment of a 670 bp amplified GST1 segment, from 14 sequences showed one clade
containing GST1 sequences of S. scabiei from human, jackal, fox, dog, hedgehog and
rabbit hosts, and a second clade with sequences from pig only (Fig. 2b). The overall
percentage of identical sites (PIS) was 95.2 %, the PIS within the non-pig host group was
96.4 %, and the PIS within the pig host sequences was 99.5 % (Table 1). The COX1
amplified sequence was 632 bp. In spite of repeated attempts, amplification of COX1
products from two swine hosts, two jackal hosts and one rabbit host was unsuccessful. This
was probably due to technical limitations related to low abundance of S. scabiei DNA in
the skin samples. COX1 analysis of 30 sequences showed overall identical sites percentage
Fig. 2 Phylogenetic analysis of Sarcoptes scabiei marker sequences. a Dendrogram of volume-sensitive
sodium channel (VSSC) gene segment sequences. b Dendrogram of glutathione-S-transferase class 1
(GST1) gene segment sequences. c Dendrogram of cytochrome C oxidase subunit 1 (COX1) gene segment
sequences. Alignment was performed using the MUSCEL algorithm (Edgar 2004) and tree construction was
done using Mega 6 (Tamura et al. 2013)
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Table 1 Percentage of identical sites of Sarcoptes scabiei marker sequences from different hosts
Marker
Host
No. of
sequences
Identical
sites (%)a
GST1
All
14
95.2
GST1
Pig (Sus scrofa)
4
99.0
GST1
Human (Homo sapiens), Rabbit (Orictolagus
cuniculus), Fox (Vulpes vulpes), Jackal (Canis
aureus), Dog (Canis lupus familiaris), Hedgehog
(Erinaceus concolor)
10
96.4
VSSC
All
14
96.5
VSSC
Pig, Hedgehog
VSSC
5
99.6
Other
10
97.7
COX1
All
30
83.6
COX1
Pig
2
98.3
COX1
Human
10
89.0
COX1
Rabbit
5
99.0
COX1
Canine (Fox, Jackal, Dog)
9
95.9
GST1 glutathione-S transferase class 1, VSSC volume-sensitive sodium channel, COX1 cytochrome C
oxidase subunit 1
a
Identical sites percentage (PIS) is calculated as the percentage of columns (positions) in the alignment for
which all sequences are identical
of 83.6 %. In the phylogenetic dendrogram, there was one hominis-type cluster, and all
other sequences did not cluster according to their host preference (Fig. 2c). However, the
PIS value increased when examined within each host species, reaching the following: 89 %
in samples from human host, 95.9 % in canine hosts (dog, jackal, fox), 98.3 % in pig host,
and 99 % in rabbit host (Table 1; Fig. 2c).
The following accession numbers were assigned to the sequences obtained in this work:
Sarcoptes scabiei GST Suis1: KP987779, S. scabiei GST Suis2: KP987780, S. scabiei GST
Suis3: KP987781, S. scabiei GST_Canis1: KP987782, S. scabiei GST Canis2: KP987783,
S. scabiei GST Canis3: KP987784, S. scabiei GST Vulpes: KP987785, S. scabiei GST
Oryctolagus1: KP987786, S. scabiei GST Oryctolagus2: KP987787, S. scabiei GST Erinaceus: KP987788, S. scabiei VSSC Canis1: KP974695, S. scabiei VSSC Canis2:
KP974696, S. scabiei VSSC Canis3: KP974697, S. scabiei VSSC Erinaceus: KP974698, S.
scabiei VSSC_Oryctolagus1: KP974699, S. scabiei VSSC Oryctolagus2: KP974700, S.
scabiei VSSC Oryctolagus3: KP974701, S. scabiei VSSC suis1: KP974702, S. scabiei
VSSC suis2: KP974703, S. scabiei VSSC_suis3: KP974704, S. scabiei VSSC Vulpes:
KP974705, S. scabiei COX1 Suis: KP987789, S. scabiei COX1 Oryctolagus1: KP987790,
S. scabiei COX1 Oryctolagus2: KP987791, S. scabiei COX1 Canis: KP987792, S. scabiei
COX1 Erinaceus: KP987793, S. scabiei COX1 Vulpes: KP987794.
Discussion
This report is the first to characterize S. scabiei on domestic pigs in Israel, morphologically
and molecularly. Identification of all life stages on the examined animals suggests that the
mites found on the pigs were an established S. scabiei type suis population, and not a
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temporary transfer (Mullen and OConnor 2002). Arlian et al. (1984) demonstrated a
successful transfer of S. scabiei from dogs to rabbits, but a similar transfer from dogs to
rats, guinea pigs, pigs and mice (normal-haired, hairless and nude) was unsuccessful.
Transfer of S. scabiei from pigs to dogs was unsuccessful as well. Even when a successful
transfer occurred, some of the animals did not develop permanent infestation, not even
after a second exposure (Arlian et al. 1984). The authors concluded that although the S.
scabiei types are not strictly host-specific, physiological preferences must play a role in
host selection. Attempts to morphologically distinguish between different S. scabiei
variants relied on different patterns of ventro-lateral scales density (Fain 1978). This
approach requires a large number of individual samples suitable for either high resolution
phase contrast microscopy or electron microscopy (Fain 1978; Arlian et al. 1984). Later
attempts to distinguish between different S. scabiei populations relied on the use of mitochondrial genes and hypervariable microsatellite sequences (Skerratt et al. 2002; Walton
et al. 1997, 2004). In our study, the condition and the number of the individual samples
from which DNA was extracted did not allow high resolution morphological examination.
Recent studies on S. scabiei genetics used several markers, both nuclear and mitochondrial
(Alasaad et al. 2009; Amer et al. 2014; Makouloutou et al. 2015; Zhao et al. 2015). It was
concluded that while ITS-2 sequence variations are not linked to host or geographic
differences, variations in mitochondrial markers may better reflect population differences
stemming from host preference or geographic location. In order to establish whether S.
scabiei variants could be distinguished using a molecular approach, we compared the
sequences of three markers obtained from analyzed samples to annotated sequences from
other hosts. The COX1 analysis did not show a significant linkage between host preference
and genetic variation (Fig. 2c). This is consistent with the findings of Walton et al. (2004),
where COX1 analysis showed genetic linkage to geographic location, but not to the host.
However, differences within samples from the same host did show increased percentage of
identity, compared with the overall PIS (Table 1). Furthermore, the analysis of two other
gene segments, GST1 and VSSC, did suggest a host-related sequence variation (Fig. 2a, b).
The increase of identical sites percentage (PIS) in the GST1 marker from 95.2 % in the
entire set of samples, to 99.5 % in the porcine samples from different origins, may suggest
that genetic difference in this gene reflects discrete populations, as was demonstrated using
S. scabiei microsatellites (Walton et al. 1999, 2004). The small number of available
samples and the fact that they originated from adjacent geographical regions, call for a
cautious interpretation of this analysis. Yet, the results of this report support the aforementioned studies, further demonstrating that S. scabiei from different hosts are genetically
different.
The protein encoded by the GST1 gene was studied for its activity and immunogenicity,
but was not used as a marker for genetic relationship (Dougall et al. 2005; Pettersson et al.
2005). Similarly, the gene encoding VSSC was studied for mutations associated with
acaricidal resistance (Pasay et al. 2006, 2008). The genetic difference described in this
report may be related to the function of these two proteins in acaricidal response and host
immune response. However, further work is required to establish possible connection
between the function of VSSC and GST1, and host preference. This report may offer an
additional use for these two genes as markers for a genetic study of S. scabiei. The limited
number of currently available sequences makes it difficult to generate a comprehensive and
accurate genetic analysis of S. scabiei variants. It is therefore anticipated that as the
number of sequences increases, distinction between different variants will become easier
and more reliable. This may help future studies related to the effects of host preference and
geography on S. scabiei populations.
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