Introduction
The protozoan Toxoplasma gondii infects virtually all warm-blooded animals, including birds, humans, livestock and marine mammals (Dubey, Reference Dubey2010). Domestic pigs are considered important in the epidemiology of toxoplasmosis in the USA (Dubey, Reference Dubey2010), but little is known of the role of feral swine.
Feral swine (Sus scrofa) populations in the USA are estimated to exceed five million and their geographic range continues to expand. Feral swine pose a threat to non-biosecure domestic pig facilities by serving as reservoirs for pathogens which may be transmitted to domestic pigs. In a national survey, antibodies to T. gondii were detected in ~20% of feral swine (Hill et al., Reference Hill, Dubey, Baroch, Swafford, Fournet, Hawkins-Cooper, Pyburn, Schmit, Gamble, Pedersen, Ferreira, Verma, Ying, Kwok, Feidas and Theodoropoulos2014). The presence of T. gondii in feral swine is considered a good indicator of contamination in the environment because they are omnivores with a generalist diet, and can become infected by ingesting oocysts while rooting and eating tissues of infected animals. Transmission of T. gondii has been documented in free-ranging domestic pigs through cannibalism (Dubey et al., Reference Dubey, Murrell, Hanbury, Anderson, Doby and Miller1986; Hill et al., Reference Hill, Haley, Wagner, Gamble and Dubey2010). The objective of the present investigation was to isolate and characterize T. gondii from feral swine across the USA.
Materials and methods
Animals and sampled areas
The United States Department of Agriculture's (USDA) Wildlife Services has a task to control feral swine for wildlife damage management purposes and routinely collects sera from a subset for pathogen surveillance. For this study, sera and hearts were collected from 1517 feral swine between September 2012 and October 2017 from 30 states (Table 1). Sex, age (juvenile, sub-adult or adult), date of collection and location information were recorded for each feral swine (Hill et al., Reference Hill, Dubey, Baroch, Swafford, Fournet, Hawkins-Cooper, Pyburn, Schmit, Gamble, Pedersen, Ferreira, Verma, Ying, Kwok, Feidas and Theodoropoulos2014). Samples were submitted for T. gondii testing to the USDA's Animal Parasitic Diseases Laboratory in Beltsville, Maryland as described previously (Hill et al., Reference Hill, Dubey, Baroch, Swafford, Fournet, Hawkins-Cooper, Pyburn, Schmit, Gamble, Pedersen, Ferreira, Verma, Ying, Kwok, Feidas and Theodoropoulos2014).
Serological prevalence of T. gondii in feral swine collected across the USA from 2012–2017
a Age not recorded for two swine. Sex not recorded for three swine.
Serology
Sera were tested for antibodies to T. gondii by the modified agglutination test (MAT) as described by Dubey and Desmonts (Reference Dubey and Desmonts1987). Sera were screened at 1:25, 1:50, 1:100 and 1:200 dilutions or higher.
Isolation by bioassay in mice
A total of 1100 Swiss Webster (SW) mice and 275 INF-γ gene knock-out (KO) mice were used for bioassay and propagation of T. gondii. Myocardium samples (50 g) were homogenized in saline, digested in acidic pepsin, centrifuged and aliquots of homogenates were inoculated subcutaneously into 3–5 outbred albino SW mice, and/or one or two KO mice, which are especially susceptible to toxoplasmosis (Dubey, Reference Dubey2010). Inoculated mice that showed symptoms of toxoplasmosis were terminated and their lungs and brain imprints were examined for T. gondii tachyzoites or tissue cysts, respectively (Dubey, Reference Dubey2010). Survivors were bled 45 days post-inoculation (p.i.) and a 1:25 dilution of serum was tested for T. gondii antibodies by MAT. Mice were euthanized 46 days p.i. and brains of all mice were examined for tissue cysts as described previously (Dubey, Reference Dubey2010). The inoculated mice were considered infected with T. gondii when tachyzoites or tissue cysts were found in their tissues.
Pathogenicity of oocysts of T. gondii strains in mice
To determine mouse pathogenicity of the parasite isolates, four T. gondii isolates showing different virulence levels based on initial observation on bioassay in SW mice were selected. For this, four 3–4 months old T. gondii-free cats (Dubey, Reference Dubey1995) were fed tissues of infected mice. Oocysts collected from the faeces of cats (Dubey, Reference Dubey2010) were sporulated in 2% sulphuric acid for a week on a shaker at room temperature, washed, counted and diluted 10-fold from 10−1 to 10−7 to reach an endpoint of ≅1 oocyst. Aliquots from each dilution of oocysts were fed to each of five SW mice and the recipient mice were examined for T. gondii infection. Mice were examined daily for illness for 2 months, and ill mice were euthanized. Survivors were bled and their sera were tested for T. gondii antibodies and their brains were examined for tissue cysts (Dubey, Reference Dubey2010).
Ethical considerations
All experimental procedures were approved by the Beltsville Area Animal Care and Use Committee (Protocol # 15-017, and 15-018), United States Department of Agriculture. Outbred SW and KO mice were purchased from the Jackson Laboratory (Bar Harbor, ME, USA) incompliance with the Institutional Animal Ethics Committee guidelines.
The feral swine were euthanized in the field, often in remote locations, and tissues were transported by the collector to the office, and then shipped by overnight mail. Samples were shipped with ice packs. By the time tissues were received at the USDA laboratory, they often were contaminated with bacteria and not suited for cell culture to isolate T. gondii. A previous study with tissues of naturally infected domestic sows from Iowa indicated that the probability of isolation of T. gondii is very low unless large numbers of mice are used. In this case, of 109 T. gondii isolates obtained from 1000 naturally exposed sows, in most instances only 1 of 10 mice inoculated with sow heart tissue was positive for T. gondii (Dubey et al., Reference Dubey, Thulliez and Powell1995). To increase the probability of isolating parasites and minimizing the number of mice, we decided to use five mice for the bioassay of each feral swine in the current study.
All mice and cats used in the present study were treated humanely and examined twice daily for any signs of illness and were supervised by a veterinarian assigned exclusively to the toxoplasmosis project. Any sick mice were euthanized because our objective was isolation of T. gondii and not testing for mortality. We wanted to collect mouse tissues aseptically for cultivation in cell culture or subpassage to other mice. Cats usually do not become ill within 10 days of ingesting T. gondii infected tissues, even though they can excrete many oocysts (Dubey, Reference Dubey2010). In the present study, cats were euthanized 2–3 days after they started excreting T. gondii oocysts.
In vitro cultivation
Infected mouse tissues were seeded onto CV1 cell culture flasks and tachyzoites were harvested from the medium as previously described (Dubey, Reference Dubey2010).
Genotyping of DNA samples
For successful genotyping of T. gondii strains from asymptomatic naturally infected animals, it is necessary to obtain good quality parasite deoxyribonucleic acid (DNA) with minimal contamination of host tissue. Therefore, parasite isolates from mouse tissues were expanded in cell culture. Genotyping of DNA samples by multilocus polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) markers were carried out following previously reported protocols (Su et al., Reference Su, Shwab, Zhou, Zhu and Dubey2010). Samples with missing data for one to three of the 10 PCR-RFLP markers, but otherwise matching with previously reported genotypes were designated as ‘likely’ of that genotype.
Results
Antibodies to T. gondii were detected in 27.7% (421 of 1517) of feral swine (Table 1). The prevalence of T. gondii antibodies varied only slightly (23.5% to 28.5%) by year from 2012 to 2016. However, seroprevalence was much higher (39.3%) in samples collected in 2017. Among the 734 males and 780 female swine tested, seroprevalence did not differ significantly (26.3% vs 29.2%, χ 2 = 1.62 P > 0.20). Seroprevalence increased significantly with age, with 10.1% of juveniles (n = 129), 16.0% of sub-adults (n = 318) and 38.4% of adults (n = 1068) testing positive (χ 2 = 78.73, P = 0) (Table 1).
Viable T. gondii was isolated from 78 feral swine from 21 states (Table 2, Fig. 1). The isolation rate increased with MAT titer; parasites were isolated from 11.7% of 17 feral swine with titers of 1:25, from 25% of 20 with titers of 1:50, from 22.6% of 53 with titers of 1:100 and from 39.8% of 143 with titers of ⩾1:200 (Supplementary Table 1).
Map of USA showing the T. gondii isolates from feral swine.
Toxoplasma gondii isolates from feral swine in the USA from 2012–2017
AL, Alabama; AR, Arkansas; AZ, Arizona; CA, California; FL, Florida; HI, Hawaii; IL, Illinois; IN, Indiana; KS, Kansas; LA, Louisiana; MI, Michigan; MO, Missouri; MS, Mississippi; NC, North Carolina; NY, New York; OH, Ohio; OK, Oklahoma; PA, Pennsylvania; SC, South Carolina; TX, Texas; VA, Virginia; SW, Swiss Webster albino mice; KO, Interferon-γ Knockout mice; ND, Not done.
The SW mice inoculated with tissue digest of hearts from 12 of the 78 infected feral swine showed symptoms of T. gondii infection and a few died or were euthanized between 11 and 27 days p.i. (Table 3).
Isolates of pathogenic T. gondii identified in feral swine collected across the USA from 2012–2017
a Four mice were inoculated with pig hearts.
b One SW mouse from the group fed to cat.
c Three out of four mice died on day 2 – it was no due to toxoplasmosis.
The four cats fed infected mice excreted T. gondii oocysts but remained clinically healthy and were euthanized in good health 7–10 days after feeding infected mouse tissues. Oocysts of two isolates (TgFpLA1 and TgFpLA2, both are genotype #289) were very pathogenic to SW mice; all mice fed their oocysts died/or euthanized of acute toxoplasmosis enteritis or pneumonia and tachyzoites were found in lungs of all infected mice (Table 4). The isolate TgFpHI1 (genotype #24) was mildly pathogenic; mice fed 100 oocysts had signs of acute toxoplasmosis whereas mice fed fewer than 100 oocysts survived and remained asymptomatic. For isolate TgFpMS1 (genotype #5), only a few oocysts were present and low doses (10 and 1 oocysts) were used to challenge mice, all infected mice survived (Table 4).
Pathogenicity of oocysts of four T. gondii isolates derived from feral swine
S, Survived, infected with T. gondii.
Five mice per group. Oocysts were inoculated orally.
a Based-on estimation that the last infective dilution has one infective organism.
b No. of mice dead, and day of death in parenthesis.
Seventy-six of the 78 isolates were genotyped (Table 5); typing results for individual isolates are shown in Supplementary Table 2. The results revealed 15 ToxoDB genotypes, including 43 isolates for genotype #5 (haplogroup 12), 11 isolates for #24, four isolates for genotype #2 (haplogroup 3), two isolates for each of genotypes #3 (haplogroup 2), #4 (haplogroup 12), #216, #221, #289 and #297 and one isolate for each of genotypes #1 (haplogroup 2), #39, #66 (haplogroup 11), #260, #261 and #299. Genotype #5 was the most frequently isolated, accounted for 57.5% (43/76) of the isolates, followed by #24, accounting for 14% (11/76). Genotypes #260, #289, #297 and #299 have not been previously reported. Genotype #289 was mouse-virulent, and originated from each of two feral swine collected concurrently from a location in Louisiana; no other information was available regarding these pigs.
Toxoplasma gondii isolates and genotype by State of feral swine collected from 2012–2017
Two of the 78 isolates were not typed.
Discussion
The primary objective of the present study was to isolate and genetically characterize T. gondii occurring in feral swine in the USA. We have previously reported seroprevalence of T. gondii in feral swine samples collected between 2006–2013, which was 17.7% by ELISA and 28.4% by MAT (Hill et al., Reference Hill, Dubey, Baroch, Swafford, Fournet, Hawkins-Cooper, Pyburn, Schmit, Gamble, Pedersen, Ferreira, Verma, Ying, Kwok, Feidas and Theodoropoulos2014). The results of this study (27.7% seroprevalence) supplement previously reported data and indicate that T. gondii infection remains high in feral swine in the USA. This prevalence of T. gondii antibodies was like the 23% (4759 of 16 788) seroprevalence detected in wild pigs worldwide (Rostami et al., Reference Rostami, Riahi, Fakhri, Saber, Hanifehpour, Valizadeh, Gholizadeh, Pouya and Gamble2017).
Most isolates from feral swine in the present study were identified as ToxoDB genotype #5, which is the dominant type in North American wildlife (Dubey et al., Reference Dubey, Velmurugan, Rajendran, Yabsley, Thomas, Beckmen, Sinnett, Ruid, Hart, Fair, McFee, Shearn-Bochsler, Kwok, Ferreira, Choudhary, Faria, Zhou, Felix and Su2011; Khan et al., Reference Khan, Dubey, Su, Ajioka, Rosenthal and Sibley2011; Jiang et al., Reference Jiang, Shwab, Martin, Gerhold, Rosenthal, Dubey and Su2018). This contrasts with what has been most frequently derived from domestic pigs in USA, in which the dominant T. gondii genotypes are #1 and #3 (collectively known as type II, haplogroup 2) and #3 (type III, haplogroup 3) (Velmurugan et al., Reference Velmurugan, Su and Dubey2009; Jiang et al., Reference Jiang, Shwab, Martin, Gerhold, Rosenthal, Dubey and Su2018). Importantly, these data substantiate a distinction between transmission among feral and farmed pigs in North America (Jiang et al., Reference Jiang, Shwab, Martin, Gerhold, Rosenthal, Dubey and Su2018). In Europe, the type II T. gondii lineage is dominant in human population, and genotyping data showed that it is also true in wildlife including wild hogs (Richomme et al., Reference Richomme, Aubert, Gilot-Fromont, Ajzenberg, Mercier, Ducrot, Ferté, Delorme and Villena2009; Aubert et al., Reference Aubert, Ajzenberg, Richomme, Gilot-Fromont, Terrier, de Gevigney, Game, Maillard, Gibert, Dardé and Villena2010), suggesting T. gondii population is largely homogeneous and no partition of parasite genotypes in the region. Several studies in China indicated dominance of ToxoDB genotype #9 in domestic pigs (Zhou et al., Reference Zhou, Nie, Zhang, Wang, Yin, Su, Zhu and Zhao2010; Jiang et al., Reference Jiang, Huang, Zhou, Zhang, Su, Deng and Zhu2013; Wang et al., Reference Wang, Liu, Jiang, Zhang, Yuan, He, Su and Yang2016), however, there is limited information regarding genotypes in wildlife for a comparison. Recent data from Brazil indicated high genetic diversity of T. gondii in domestic pigs (Feitosa et al., Reference Feitosa, Vilela, de Almeida-Neto, dos Santos, de Morais, Alves, Nakashima, Gennari, Athayde and Pena2017). But information is still limited to compare the parasites from domestic animal vs wildlife. To better understand the partition of transmission, studies of T. gondii genotypes in domestic animals and wildlife in other regions such as Asia, Africa, Australia and South America are needed.
Pathogenicity of oocysts of four T. gondii strains in mice suggested that the newly identified genotype #289 (isolates TgFpLA1 and TgFpLA2) is highly virulent. However, genotype #24 (isolate TgFpHI1), common in Hawaii, is mildly pathogenic. The genotype #5 (isolate TgFpMS1), prevalent in wildlife in North America, is also mildly pathogenic to mice. This result indicates that, even though most T. gondii strains in the U.S. are not highly virulent, there is a low frequency of highly virulent parasites circulating in wildlife.
Genotype #24 was the second-most frequently isolated type in this study. Ten of the 14 isolates identified in Hawaii belong to #24, which accounted for 71% (10/14) of those isolates (Table 2). Genotype #24 has previously been identified in chickens from Costa Rica and Brazil (Dubey et al., Reference Dubey, Su, Oliveira, Morales, Bolaños, Sundar, Kwok and Shen2006; Ferreira et al., Reference Ferreira, Buery, Moreira, Santos, Costa, Pinto, Baraviera, Vitor and Fux2018), and in bobcats from Mississippi, USA (Verma et al., Reference Verma, Sweeny, Lovallo, Calero-Bernal, Kwok, Jiang, Su, Grigg and Dubey2017), suggesting it is widely distributed in the USA. Bioassays in mice indicate #24 strains are not highly virulent to mice (Tables 3 and 4). Among the four new genotypes identified in this study, two (#260 and #297) were from Hawaii. In addition, genotype #261 was also first identified in Hawaiian geese (Work et al., Reference Work, Verma, Su, Medeiros, Kaiakapu, Kwok and Dubey2016). These results indicate that the T. gondii population in Hawaii differs from those in the continental USA.
Other genotypes, including #1, #2, #3, #4, #39 #66, #216 and #221, have previously been identified in animals in the USA, with the first four being common (Jiang et al., Reference Jiang, Shwab, Martin, Gerhold, Rosenthal, Dubey and Su2018). Among these genotypes, #216 is highly virulent to mice (Dubey et al., Reference Dubey, Choudhary, Kwok, Ferreira, Oliveira, Verma, Marks, Pedersen, Mickley, Randall, Arsnoe and Su2013a, Reference Dubey, Hill, Zarlenga, Choudhary, Ferreira, Oliveira, Verma, Kwok, Driscoll, Spiker and Su2013b, Reference Dubey, Verma, Calero-Bernal, Cassinelli, Kwok, van Why, Su and Humphreys2015).
Recent evidence indicates that wildlife T. gondii strains can also cause clinical disease in humans (Jokelainen et al., Reference Jokelainen, Murat and Nielsen2018; Pomares et al., Reference Pomares, Devillard, Holmes, Olariu, Press, Ramirez, Talucod, Estran, Su, Dubey, Ajzenberg and Montoya2018) and domestic cats (Dubey and Prowell, Reference Dubey and Prowell2013; Crouch et al., Reference Crouch, Mittel, Southard, Cerqueira-Cézar, Murata, Kwok, Su and Dubey2019). It is suggested that partition of T. gondii genotypes among domestic animals and wildlife is mainly due to distinct sylvatic and domestic transmission cycles, though both cycles overlap to a certain degree (Shwab et al., Reference Shwab, Saraf, Zhu, Zhou, McFerrin, Ajzenberg, Schares, Hammond-Aryee, van Helden, Higgins, Gerhold, Rosenthal, Zhao, Dubey and Su2018).
Our results revealed moderate genetic diversity of T. gondii in feral swine in the USA, with genotype #5 (haplogroup 12) dominant in continental USA, whereas genotype #24 (10/14) was dominant in Hawaii, suggesting different population structures of the parasites among the two distinct genographical locations. The T. gondii isolates detected in feral swine generally resembled those found in other wildlife species and were distinct from those that are typically identified in domestic pigs, and include novel genotypes including ones that are highly virulent to mice. The contribution of feral swine as a reservoir of infection deserves additional scrutiny, as well as their potential in disseminating parasites to humans.
Supplementary material
The supplementary material for this article can be found at https://doi.org/10.1017/S0031182019001586.
Financial support
This research received no specific grant from any funding agency, commercial or not-for-profit sectors.
Conflict of interest
None.
Ethical standards
Feral swine were sampled following protocols developed by WS' National Wildlife Disease Program (NWDP) (USDA-APHIS-WS, NWDP, Comprehensive Feral Swine Disease Surveillance Procedure Manual, 2012).
