Disease risks associated with free-ranging wild boar in Saskatchewan

Abstract

This study investigated the disease status of Saskatchewan’s feral wild boar population. Whole carcasses, tissue samples, and/or serum from 81 hunter-killed boars from Saskatchewan were submitted to the Canadian Wildlife Health Cooperative (CWHC) between 2009 and 2014. Serological tests were negative for PRRS, H1N1, and H3N2 swine influenza, PCV-2, and TGE/PRCV in 22/22 boars and for Toxoplasma gondii and Mycoplasma hyopneumoniae in 20/20 boars. Of 20 boars whose sera were tested 20 were positive for Actinobacillus pleuropneumoniae, with 7 positive for, among other strains, serotype 14; 16 were positive for Lawsonia intracellularis, 1 was positive and 6 were suspicious for Salmonella spp. Polymerase chain reaction tests were negative for PRRS and PCV2 in 58/58 boars and positive for Torque teno virus in 1/8 boars. Digestion assays were negative for Trichinella spp. in 22/22 boars. The high seroprevalence of A. pleuropneumoniae serotype 14 is noteworthy as this serotype has not been previously reported in North America.

Introduction

Wild boars (Sus scrofa scrofa) were imported into Saskatchewan in the 1990s as part of an agriculture diversification initiative. These animals are difficult to contain; feral populations became established throughout the southern half of the province subsequent to escape from their enclosures as well as possible deliberate releases (1). Feral populations can expand quickly, since wild boars have few natural predators and can have up to 2 litters per year, averaging 4 to 6 piglets per litter (2). Continuing escapes from existing farms may also contribute to population expansion. One of the first populations of free-ranging and reproducing wild boars confirmed in Saskatchewan was in Moose Mountain Provincial Park, which continues to contain one of the largest populations in the province. The current population in the park and adjacent rural municipalities, based on free-ranging boars observed in limited flights over and around the park, is approximately 200 animals (Stolz R, Government of Saskatchewan Ministry of Environment, personal communication, 2014).

Wild boars are potential carriers of viral, bacterial, and parasitic diseases that can affect livestock, wildlife, and humans. These diseases include pseudorabies, porcine circovirus, tuberculosis, brucellosis, and trichinellosis (3). Wild boars are closely related to domestic swine (Sus scrofa domesticus); therefore, transmission of diseases between wild boars and domestic swine is a particular concern. The disease-status of Saskatchewan’s wild boar population is unknown.

The objective of this study was to survey Saskatchewan’s wild boar population for diseases that may pose a risk for humans or domestic livestock, particularly swine.

Materials and methods

Sample collection

Eighty-one hunter-killed wild boars from Saskatchewan were submitted to the Western/Northern node of the Canadian Wildlife Health Cooperative between 2009 and 2013 (CWHC; Saskatoon, Saskatchewan). In 2009, 22 boars were submitted from 3 separate hunts; in 2013, 53 boars were submitted from 7 hunts; and in 2014, 6 boars were submitted from 2 hunts. Depending on feasibility of transport and storage, boars were submitted either as full carcasses or as tissue portions collected by hunters and/or conservation officers. Where possible, blood was collected from the heart or jugular vein as soon as possible after death by hunters and/or conservation officers. Blood was centrifuged and the serum extracted within 24 h of collection at local veterinary clinics and the serum was frozen prior to submission to the CWHC. Carcasses and portions of carcasses were submitted either fresh or frozen depending on weather conditions and time between collection and submission.

Pathology

A complete necropsy was performed by, or under the direct supervision of, a veterinary pathologist on all carcasses submitted whole. All major organs from all 4 pigs submitted from hunt 1 in 2009 and lesions apparent on gross necropsy of all remaining animals were submitted to histopathologic examination.

Ancillary/Diagnostic testing

Testing protocols varied depending on year of submission and the samples available for testing. In 2009, all submitted boars were tested for Trichinella sp. and subsets from each hunt were tested by polymerase chain reaction (PCR) for porcine reproductive and respiratory syndrome (PRRS) virus, porcine circovirus 2 (PCV-2), and Torque teno virus (TTV). In 2013 and 2014, all boars with tissues available were tested by PCR for PRRS and PCV-2 and all boars with serum available were tested for antibodies to H1N1 and H3N2 swine influenza virus (SIV), PRRS, and transmissible gastroenteritis virus (TGEV)/porcine respiratory corona virus (PRCV). In 2013 boars with serum available were also tested for antibodies to Actinobacillus pleuropneumoniae, Mycoplasma hyopneumoniae, Lawsonia intracellularis, Salmonella spp. and Toxoplasma gondii. In 2014, boars were also tested for influenza A virus by PCR. In all years, feces, when available, were tested via routine fecal flotation for parasites.

Serology

Serological testing for antibodies to PRRS, SIV H1N1, SIV H3N2, and TGEV/PRCV was performed at Prairie Diagnostic Services laboratory (PDS, Saskatoon, Saskatchewan). Samples were tested using commercially available enzyme-linked immunosorbent assay (ELISA) kits: PRRS X3 Ab Test, SIV H1N1 Ab Test, SIV H3N2 Ab Test (Idexx, Westbrook, Maine, USA) and Swinecheck^® TGE/PRCV Recombinant Kit (Biovet, St. Hyacinthe, Quebec), according to manufacturer’s instructions. Serum samples were sent to the Diagnostic Service of the University of Montreal (Montreal, Quebec) where they were tested for antibodies against M. hyopneumoniae and Salmonella spp. using commercially available kits: HerdChek (Idexx) and Diakit Salmonella Swine (Maxivet Laboratories, St. Hyacinthe, Quebec) as well as for L. intracellularis by an indirect fluorescent antibody test (4) and A. pleuropneumoniae serotypes 1 to 15 using a MultiAPP screening ELISA. Each positive sample was then tested with serotype-specific ELISA as described (5).

Serum samples were also sent to the Veterinary Diagnostic Laboratory at Colorado State University, Fort Collins, Colorado, USA, where they were tested for T. gondii antibodies using the modified agglutination test (MAT) for IgG as previously described (6). Sera reacting at greater than 1:40 were considered positive.

Molecular diagnostics

All PCR tests were performed at PDS laboratory on pooled lung, spleen, lymph node, and, when available, tonsil. The PRRS PCR was performed using a commercial kit (Tetracore, Rockville, Maryland, USA) according to manufacturer’s instructions. The PCV-2 PCR and Influenza A virus matrix gene PCR were performed as previously described (7,8). The Torque teno virus PCR was performed using a conventional nested PCR assay based on methods previously described (9), with some modifications. The DNA was extracted from pooled tissue samples using a commercial kit (Qiagen DNeasy Blood & Tissue Kit Cat. #69506; Qiagen, Toronto, Ontario). Samples were eluted to 200 μL with Qiagen elution buffer. The nested PCR protocol used 20 pmol primer pairs and 4 μL of template as described (9), but was modified to a 50 μL reaction mix with the following master mix concentration changes: 25 μM dNTPs, 25 μM MgCl₂, 5 U Taq DNA Polymerase (ThermoFisher Scientific, Waltham, Massachusetts, USA). The amplification was done by using 35 cycles of 94°C for 60 s, 52°C for 30 s, 72°C for 30 s. The master mix and cycling conditions were the same for both the primary and secondary reactions. The nPCR product was analyzed using the Qiagen QIAxcel gel system.

Parasitology

Feces were submitted to PDS for fecal floatation following the routine semi-quantitative method. In 2009 samples of diaphragm and tongue were submitted to the Canadian Food Inspection Agency (CFIA; Winnipeg, Manitoba) for Trichinella testing using a digestion assay (10).

Results

Animals sampled

Carcasses or tissue samples from 81 wild boars were submitted: 22 boars from 3 hunts in 2009, 53 boars from 7 hunts in 2013, and 6 boars from 2 hunts in 2014. Of these, 79 boars were killed in or near Moose Mountain Provincial Park, and 2 were killed near Melfort, Saskatchewan. Two boars were submitted as full carcasses with serum samples, 36 as full carcasses without serum samples, 21 as tissue samples without serum, 13 as tissue and serum samples, and 9 as serum samples alone. Feces were available from 4 boars in 2009, 44 boars in 2013 and 6 boars in 2014. Fourteen of the boars were identified as adults, 31 as immature and 36 were not classified according to age; 27 were male, 48 were female and gender was unspecified for 6 animals. Sixteen of the females were pregnant with an average of 5.8 fetuses per pregnant sow (range: 1 to 10 fetuses). Body weight was recorded for 35 animals; average body weight was 55.5 kg (range: 24 to 120 kg). Body condition score was recorded for 32 animals; 69% (22/32) were in good to excellent body condition, 25% (8/32) were in moderate body condition and 6% (2/32) were in thin body condition.

Pathology

All 38 boars submitted as complete carcasses had a full postmortem examination. One immature female had fibrous pleural adhesions and a 2-cm subcutaneous abscess in the left flank adjacent to a healing skin wound from which 4+ hemolytic Escherichia coli and 4+ non-hemolytic E. coli were cultured on routine bacterial culture. No significant gross pathological lesions were found in the other boars.

Histopathology on tissues from 4 of the boars submitted in 2009 identified 1 boar with 2 small granulomas in the lungs, 1 containing fungal spores identified as Haplosporangium spp. based on histological appearance.

Serology and PCR

The results of serological and PCR testing are shown in Table 1. Except for TTV, L. intracellularis, Salmonella spp. and A. pleuropneumoniae, all tests were negative. One animal was positive for TTV, while all 20 boars that were tested for A. pleuropneumoniae were positive; 35% were seropositive for serotype 14. All wild boars seropositive for A. pleuropneumoniae serotype 10 were either suspect or positive for serotype 13. Boars were frequently positive for > 1 serotype with 70% (14/20) positive for 2 or more serotypes. Serotyping results are shown in Table 2.

Table 1.

Percentage of wild boar from Saskatchewan testing positive (number positive/number tested) for selected disease agents in 2009, 2013, and 2014

	Year
	2009	2013	2014	Overall
PCR on pooled lung, spleen, and lymph node
PRRSV	0% (0/8)	0% (0/44)	0% (0/6)	0% (0/58)
PCV-2	0% (0/8)	0% (0/44)	0% (0/6)	0% (0/58)
Torque teno virus	12.5% (1/8)	ND	ND	12.5% (1/8)
Influenza virus	ND	ND	0% (0/6)	0% (0/6)
Trichinella spp.	0% (0/22)	ND	ND	0% (0/22)
Serology
PRRSV	ND	0% (0/20)	0% (0/2)	0% (0/22)
H1N1 Influenza virus	ND	0% (0/20)	0% (0/2)	0% (0/22)
H3N2 Influenza virus	ND	0% (0/20)	0 (0/2)	0 (0/22)
TGEV/PRCV	ND	0% (0/20)	0 (0/2)	0 (0/22)
Toxoplasma gondii	ND	0% (0/20)	ND	0 (0/20)
Actinobacillus pleuropneumoniae	ND	100% (20/20)	ND	100% (20/20)
Mycoplasma hyopneumoniae	ND	0% (0/20)	ND	0% (20/20)
Lawsonia intracellularis	ND	80% (16/20)	ND	80% (16/20)
Salmonella spp.	ND	5% (1/20) positive; 30% (6/20) suspicious	ND	5% (1/20) positive; 30% (6/20) suspicious

PCV-2 — Porcine circovirus 2; PRRSV — Porcine reproductive and respiratory syndrome virus; TGEV/PRCV — Transmissible gastroenteritis virus and porcine respiratory coronavirus; ND — testing not done.

Table 2.

Antibodies to Actinobacillus pleuropneumoniae (APP) serotypes present in 20 wild boars from Saskatchewan surveyed in 2013

Serotype	Positive	Suspect
APP 1 (9,11)	0	0
APP 5a,5b	0	0
APP 2	0	0
APP 3 (6,8,15)	5	0
APP 7 (4)	1	0
APP 10	4	0
APP 12	15	1
APP 13	1	3
APP 14	7	1

Parasitology

On routine fecal floatation, no parasites were seen in 93% (50/54) of the boars with fecal samples available. Four percent (2/54) had 1+ Trichuris spp. eggs, 2% (1/54) had 1+ coccidia and 2% (1/54) had eggs consistent with Metastrongylus sp.

Discussion

Low sample sizes for many tests meant we could not detect disease if present at low prevalence, but there are several significant findings in our study. First, there was serological evidence of exposure to A. pleuropneumoniae in all 20 wild boars tested. Actinobacillus pleuropneumoniae is a common cause of severe pneumonia in domestic pigs and has a significant economic impact on the swine industry. Its prevalence in wild boars in North America has not been previously reported. Although A. pleuropneumoniae is common in wild boars in Europe, it is rarely associated with clinical signs (11,12).

Because A. pleuropneumoniae is an obligate pathogen of the porcine respiratory tract and there are no other known natural hosts (12) there are only 2 ways these boars could have become infected: contact with infected domestic swine, which would require close contact between the 2 species, or infection in the original escaped individuals. The high prevalence of serotype 14, which is common in Europe but has not been previously reported in North America (13,14), supports the second theory. Introduction of this serotype to local domestic swine could be problematic as A. pleuropneumoniae vaccines are generally serotype specific (15) and current North American vaccines are not tested for protection against serotype 14. The continued circulation of this bacterium in a small isolated population could facilitate evolutionary drift, potentially enabling the emergence of new strains. This is a concern as A. pleuropneumoniae already displays significant genetic diversity, which has prevented the development of an effective vaccine able to provide cross-protection among strains (15). All boars seropositive for serotype 10 were also positive or suspect for serotype 13. This likely reflects a common pattern of cross-reactivity seen with an atypical strain of serotype 13 that has been previously described in North America (13).

The risk of transmission of serotype 14 or other A. pleuropneumoniae strains to domestic swine is unknown. In domestic herds A. pleuropneumoniae transmission is generally through direct contact and aerosols over distances less than 2.5 m (16). Since most commercial swine production in Saskatchewan occurs in barns, close contact with wild boars is unlikely. There have been suggestions that A. pleuropneumoniae can travel more than 400 to 500 m depending on dominant winds, but transmission at such distances has not been documented (16). If such long distance aerosol transmission is possible, transmission from nearby wild boars to domestic swine could pose a risk to commercial producers. The difference in the predominant serotypes between Canada’s domestic swine population and these wild boars suggests that thus far there has been minimal or no transmission of A. pleuropneumonia between wild boars and domestic swine; however, the presence of antibodies that are likely against the atypical strain of serotype 13, which has only been described in Canada and USA may, at least in part, be due to cross contamination with domestic pigs.

The high seroprevalence of L. intracellularis, a common and economically important cause of severe diarrhea in domestic swine, in these wild boars is a concern as wild boars have been suggested as a potential reservoir of this pathogen in Europe (17). The prevalence of this pathogen in wild boars, to the authors’ knowledge, has not been reported in North America, although wild boars are commonly positive in Europe and Asia on serology or PCR of feces and intestinal mucosa (18). Further studies are needed to determine the risk of transmission to domestic swine as seroprevalence indicates exposure but does not indicate whether there is ongoing infection or shedding of the bacterium. European studies indicate that infected wild boars may intermittently shed the bacterium for upwards of 12 wk after infection (17).

The moderate seroprevalence of Salmonella is a concern as this organism is an important zoonotic pathogen and may pose a threat to humans exposed to boar feces. Again, further testing is necessary as seroprevalence only indicates exposure.

The absence of serological evidence of exposure to PRRS is consistent with low PRRS seroprevalence in wild boar in the USA, where it ranges from 0% to 2%, even in areas with high PRRS prevalence amongst domestic swine (19). Due to low PRRS seroprevalence and apparent resistance to PRRS infection, wild boars are not thought to be significant reservoirs of PRRS (3). Similarly, the absence of serological evidence of exposure to TGE/PRCV is consistent with low seroprevalence to these viruses reported in wild boars in various locations in Europe and the USA (20,11) and indicates that transmission of this disease to domestic pigs from wild boar is unlikely.

The absence of detection of PCV-2 contrasts with studies in the USA where PCV-2 seroprevalence in wild boar populations ranged from 42% to 71.7% (21,22). As in the USA, PCV-2 infection is common in domestic swine in Canada with a seroprevalence of 82.4% (318/386) (23). Porcine circovirus 2 transmission primarily occurs via direct contact (24) so the lower prevalence in Saskatchewan’s wild boar compared to that in the USA may reflect lower rates of direct contact with domestic swine as almost all of Saskatchewan’s domestic pigs are housed in indoor confinement facilities, whereas pigs in the southern United States may be housed outdoors or in open barns. The current relatively low wild boar densities in Saskatchewan may also be contributing to the low PCV-2 prevalence as PCV-2 seroprevalence in wild boar tends to be lower in areas with lower population densities (25).

Torque teno virus DNA was found in 1 of 8 boars tested. The TTV detected was of the G2 genotype, which is now classified as Torque teno sus virus 2 (TTSuV2). Torque teno viruses are a recently described group of circular single-stranded DNA viruses that are widespread in domestic pig populations throughout the world including Canada, with detectable TTV DNA in 100% of serum samples of domestic pigs from Saskatchewan (26). To the authors’ knowledge, no previous studies have looked for TTV in wild boar in North America, although wild boar in Europe are commonly infected (27). In Europe wild boar specific TTSuV strains, with higher than expected genotypic diversity, are capable of undergoing recombination events with TTSuV strains in domestic swine allowing the rapid emergence of novel strains (27). The significance of these viruses on the health of domestic pigs is still an active area of research. They are considered non-pathogenic by themselves, but there is increasing evidence that co-infection with other viruses, such as PCV-2, influences the development and outcome of some diseases such as post-weaning multisystemic wasting syndrome (PMWS) (28).

None of the boar tested showed serological evidence of exposure to H1N1 or H2N3 influenza or had detectable Influenza A RNA on PCR. Influenza A viruses in swine are a major concern because swine can be infected with both human and avian influenza A strains and therefore act as a mixing vessels where recombination can occur enabling the emergence of novel strains. Rarely these novel strains may have different host specificities and virulence and may potentially pose a human pandemic threat. Interactions between wild boar and migrating waterfowl may increase the chance of infection with avian influenza strains. Little is known about the circulation of influenza viruses in feral swine and wild boar. Globally there are very few reported cases of detectable influenza virus in wild boar, and seroprevalence varies with the region tested and the year, ranging from 0% to 26% in various studies across Europe and from 0% to 91% in various regions of the USA (29). Since other studies have found significant variation in seroprevalence depending on the time of year tested, continued monitoring of Saskatchewan’s boar population, particularly during waterfowl migrations, is needed.

None of the 20 boars tested had serological evidence of exposure to T. gondii, a zoonotic protozoan parasite. In Europe and the USA wild boar generally have a high T. gondii seroprevalence in comparison to other species (3) and consumption of wild boar has caused outbreaks of toxoplasmosis in humans (3). In the USA reports of toxoplasma seroprevalence in wild boar range from 13% in California to 49% in South Carolina (3). In Saskatchewan, a 6.8% (11/161) seroprevelence of T. gondii in domestic swine has been previously reported (30). It is also present in wildlife; for example, Hwang et al (31) reported a 15.6% (10/64) seroprevalence in skunks in Saskatchewan. In our study, based on an estimated population size of 200 animals and a sensitivity of 82.9% for the T. gondii MAT used (6) the sample size of 20 pigs was adequate, with 95% confidence, to detect disease present within the population at a prevalence of 18% or greater. Since the expected prevalence in these wild boars is likely less than 18%, based on prevalence in domestic swine and other wildlife species, further testing is required.

None of the 22 boars tested for Trichinella spp. had detectable larvae in their tongues or diaphragms. Trichinella spp. are zoonotic nematodes that encyst in muscle and are spread by the ingestion of uncooked, infected meat. In 1993 there was a Trichinella outbreak affecting 24 people in Ontario following the consumption of infected farmed wild boar from 2 farms near Dufferin, Ontario (32). Globally, of the 8 currently recognized Trichinella spp., T. spiralis is the most common cause of trichinellosis in humans. Pigs are the most common sources of T. spiralis infection as they are easily infected and may have extremely large numbers of larvae in their meat. In Canada, domestic swine are Trichinella-free and Canadian wildlife are not thought to carry T. spiralis (33). Because of this, in Canada T. nativa is the most common cause of human trichinellosis, usually resulting from consumption of under-cooked wild game (33). Fortunately, wild boars are less readily infected with other Trichinella spp. than with T. spiralis and in experimental infections of wild boar there is establishment of only moderate numbers of T. pseudospiralis larvae and low numbers of T. nativa, T. meurelli and T6 larvae (34). Thus, Trichinella infection is of less concern in Canada’s wild boar population than in countries where T. spiralis is present. Based on a 100% sensitivity for digestion assay to detect clinically meaningful larval numbers (10) and an estimated population of 200 animals the 20 animal sample size is adequate, with 95% confidence, to detect Trichinella spp. at a prevalence 15% or greater.

Low numbers of Trichuris sp. eggs were detected in the feces of 4% (2/48) of the boar. T. suis is a common nematode in swine, although its prevalence is decreasing as more swine are kept in indoor confinement facilities. This parasite has a direct lifecycle and the eggs are very hardy so transmission of this parasite from wild boar to domestic swine is possible if there is fecal contamination of feed; however, as this parasite is already widespread in domestic swine consequences of transmission would be minimal.

Low numbers of Metastrongylus spp. eggs were found in the feces of 1 of the boar. Metastrongylus spp. are nematode worms, which parasitize the bronchi and bronchioles. They require an earthworm intermediate host to complete the lifecycle so they are quite rare in domestic swine raised indoors on concrete floors, which is typically the case in Canada. Although prevalence was very low in the current study, Metastrongylus spp. are highly prevalent in wild boar in Europe and the southern USA (35). Transmission of Metastrongylus sp. to domestic swine is very unlikely, given the low prevalence in wild boar and indoor housing of commercial animals.

Tests for other important pathogens of wild boar, such as Brucella suis, pseudorabies hepatitis E virus, Mycobacterium spp. and others were not undertaken because of perceived low risk, budgetary limitations and/or regulatory constraints.

The body condition of most of the boar was good to excellent, and 15 of the females were pregnant with an average of 5.8 fetuses, indicating that this species is well adapted to Saskatchewan and that, unless control measures are instituted, the population will likely continue to expand. That only 15/44 females were pregnant is likely because most of the hunts took place in January, before the normal breeding season, and most of the animals collected that were classified according to age (69%; 31/45) were immature. The presence of a large proportion of immature animals and pregnant animals with normal numbers of fetuses indicates that the harsh Canadian climate is not an obstacle to population growth of these animals.

The main limitation of this study was the small number of boars tested and that other than the 2 boars shot near Melfort, all the boars came from a localized geographical area near Moose Mountain Provincial Park, so extrapolation of disease prevalence in this population may not be appropriate for Saskatchewan as a whole.

The biggest risk identified in this study was serological evidence of A. pleuropneumoniae serotype 14, which has not been previously reported in North America and could pose a threat to domestic swine. Serological evidence of exposure to L. intracellularis and Salmonella sp. is also noteworthy. Based on this assessment, Saskatchewan’s wild boar population has a low prevalence of diseases of concern to humans and domestic animals in comparison to wild boar populations in Europe and the USA. However, increased sample sizes and sampling of wild boar from other areas of the province are needed in order to demonstrate the absence of disease. As well, ongoing surveillance to detect changes in disease prevalence and to detect the introduction of new pathogens is needed, particularly if wild boar populations in the USA continue their current northern expansion and start to overlap with those in Canada.