Introduction
The protozoan Toxoplasma gondii infects virtually all warm-blooded animals, including birds, humans, livestock and marine mammals (Dubey, Reference Dubey2010). Humans become infected postnatally by ingesting tissue cysts from undercooked meat of infected animals, or by consuming food or drink contaminated with T. gondii oocysts. In the USA, white-tailed deer (Odocoileus virginianus, WTD) are harvested by hunters and infected meat that is consumed by humans or other hosts can result in T. gondii infection. Cases of clinical toxoplasmosis including ocular manifestations have been documented in humans who consumed undercooked venison (Sacks et al., Reference Sacks, Delgado, Lobel and Parker1983; Ross et al., Reference Ross, Stec, Werner, Blumenkranz, Glazer and Williams2001; Schumacher et al., Reference Schumacher, Kazmierczak, Moldenhauer, Hanhly, Montoya, Press, Letzer, Elbadawi, Smiley and Davis2018; Gaulin et al., Reference Gaulin, Ramsay, Thivierge, Tataryn, Courville, Martin, Cunningham, Désilets, Morin and Dion2020).
Deer are popular game animals in several countries (Aubert et al., Reference Aubert, Ajzenberg, Richomme, Gilot-Fromont, Terrier, de Gevigney, Game, Maillard, Gibert, Dardé and Villena2010). In the USA, the WTD population is estimated at approximately 30 million, with nearly 6 million deer harvested annually (Adams and Ross, Reference Adams and Ross2013). Viscera are removed from most harvested deer in the field to preserve the meat, and the viscera is left on site or buried in a shallow grave. Tissues of these deer may be scavenged by mesocarnivores, which can then become infected.
Few states have strict rules with respect to disposal of the carcases of deer hit by vehicles. Bobcats (Lynx rufus) and cougars (Puma concolor) scavenge on deer, and if they eat infected tissues they can excrete T. gondii oocysts in the environment. Since deer–vehicle accidents are common in the USA (Adams and Ross, Reference Adams and Ross2013), there are many opportunities for felids to become infected.
Since deer are herbivores, ingestion of food or water contaminated with oocysts is likely the main mode of post-natal transmission. Thus, WTD are considered an excellent indicator of contamination of the environment by T. gondii oocysts among other hosts, including feral chickens, wild swine and many other species wild ungulates. The objective of the present investigation was to estimate seroprevalence, isolate and characterize T. gondii from WTD across the USA.
Materials and methods
Animals and sampled areas
The United States Department of Agriculture's Wildlife Services (WS) removes deer from select areas where populations are not naturally regulated due to the lack of predators and hunting restrictions. Samples are opportunistically collected from these deer by WS’ National Wildlife Disease Program (NWDP). For this study, sera and hearts were collected from 914 WTD from October 2012 through March 2019 in 19 states (Table 1) following NWDP protocols (Cervid Health Procedures Manual, June 2015). Sex, age group (juvenile or adult), date of killing and location information were recorded for most WTD; not all data were available for all WTD (Table 1). Samples were submitted for T. gondii testing to the USDA's Animal Parasitic Diseases Laboratory (APDL) in Beltsville, Maryland.
Table 1. Prevalence of T. gondii in WTD (O. virginianus) collected across the USA from 2012 to 2019

a No samples received in 2014.
b Age and sex not recorded for four WTD.
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. Deer with positive reaction at 1:25 dilution were considered infected with T. gondii.
Isolation by bioassay in mice
The heart was selected as the organ for isolation of T. gondii because of convenience and because in food animals it is most commonly infected with T. gondii (Dubey, Reference Dubey2010). 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 Swiss Webster (SW) mice, and/or one or two interferon gamma gene knock out (KO) mice, which are especially susceptible to toxoplasmosis (Dubey, Reference Dubey2010). Inoculated mice that became ill were euthanized and tissue imprints of lungs and brains were examined for T. gondii tachyzoites or tissue cysts, respectively (Dubey, Reference Dubey2010). Survivors were bled at the earliest on 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. or later 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 detected in their tissues.
Ethical considerations
All experimental procedures were approved by the Institutional Animal Care and Use Committee (Protocol no. 15-017), United States Department of Agriculture, Beltsville, Maryland. Outbred SW and KO mice were purchased from the Jackson Laboratory (Bar Harbor, ME, USA) in compliance with the Institutional Animal Ethics Committee guidelines. The WTD were euthanized in the field, often in remote locations and tissues were shipped overnight by the collector with ice packs. Consequently, 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. The number of T. gondii in tissues of naturally infected large animal tissues is low (estimated one tissue cyst per 50–100 g) and the probability of isolation of T. gondii is very low unless large numbers of mice (10 or more) are used (Dubey et al., Reference Dubey, Thulliez and Powell1995; Dubey, Reference Dubey2010). To balance the possibility of isolating parasites and using the minimum number of mice, we decided to use 3–5 mice for the bioassay of each deer sample in the current study.
All mice used in the current study were treated humanely and examined twice daily for any signs of illness. A veterinarian was 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.
In vitro cultivation
Lungs or brain from infected mice were seeded on to CV1 cell culture flasks and tachyzoites were harvested from the medium as previously described (Dubey, Reference Dubey2010).
Genotyping of DNA samples
In our experience, for successful genotyping of T. gondii strains from asymptomatic naturally infected animals, it is necessary to obtain good quality parasite DNA with minimal contamination of host tissue. Therefore, parasites isolated from mouse tissues were expanded in cell culture. Genotyping of DNA samples by multi-locus polymerase chain reaction (PCR)-restriction fragment polymorphism (RFLP) markers were carried out following the previously reported protocol (Su et al., Reference Su, Shwab, Zhou, Zhu and Dubey2010; Su and Dubey, Reference Su and Dubey2020). Samples with missing data for one to three of the 10 PCR-RFLP markers, which otherwise matched with previously reported genotype were designated as ‘likely’ of that genotype.
Results
Antibodies to T. gondii were detected in 36% (329 of 914) of WTD (Table 1). Among 362 male and 548 female WTD tested (sex was unidentified for four), seroprevalence was higher in females (40.0%) than in males (29.8%). Seroprevalence were higher in adults than juveniles (Table 1).
Of the 329 seropositive deer, hearts from 167 were bioassayed (Table 1; Supplementary Table 1). The selection was based on antibody titres and the number of samples received and the availability of mice. Viable T. gondii was isolated from 36 WTD collected in 11 states (Table 2, Fig. 1) (Supplementary Table 1). The rate of isolating viable parasites was positively associated with MAT titres, with the isolation rates of 10.7, 17.9, 33.3, 59 and 50% for MAT titres of 1:200, 1:400, 1:800, 1:1600 and ⩾1:3200, respectively. Tissue cysts were found in brains of all seropositive mice.

Fig. 1. Map of USA showing the T. gondii isolates from WTD.
Table 2. Collection information for T. gondii isolates from WTD (O. virginianus) collected across the USA from 2012 to 2019

AL, Alabama; GA, Georgia; IN, Indiana; KS, Kansas; KY, Kentucky; LA, Louisiana; MI, Michigan; MN, Minnesota; NJ, New Jersey; NY, New York; OH, Ohio.
SW, Swiss Webster albino mice; KO, interferon-γ knockout mice; ND, not done.
a No isolates from FL, Florida; IL, Illinois; PA, Pennsylvania; ME, Maine; NC, North Carolina, WA, Washington; WI, Wisconsin and WY, Wyoming.
The SW mice that were inoculated with digest of hearts from three of the 36 infected WTD had clinical signs of T. gondii infection, and three mice died or were euthanized at 13, 41 and 46 days p.i. (Table 3). All three isolates were from Louisiana but from different counties and hunted on different dates (Table 3).
Table 3. Isolates of pathogenic T. gondii identified in WTD collected across the USA from 2012 to 2019

Thirty-six isolates were genotyped (Table 4); typing results for individual isolates are shown in Supplementary Table 2. The results revealed seven ToxoDB genotypes, including 24 isolates for genotype #5 (haplogroup 12), four isolates for genotype #2 (type III, haplogroup 3), three isolates for genotype #1 (type II, haplogroup 2), two isolates for genotypes #3 (type II, haplogroup 2), one isolate each for #39, #221 and #224. Genotype #5 was the most frequently isolated, accounting for 66.6% (24 of 36) of the isolates.
Table 4. T. gondii isolates from WTD (O. virginianus) bioassayed samples and PCR-RFLP genotype per state collected from 2012 to 2019

a No samples were bioassayed from ME, WI and WY.
Discussion
Nearly one-third of humans have been exposed to T. gondii, but not all develop clinical symptoms of disease. It is unknown how the severity of toxoplasmosis in immunocompetent hosts is linked to the parasite strain, host variability or to other factors. Attention has been focused on the genetic variability among T. gondii isolates from apparently healthy and sick hosts (Grigg and Sundar, Reference Grigg and Sundar2009). Historically, T. gondii was considered clonal with low genetic diversity and grouped into three types, namely I, II and III (Howe and Sibley, Reference Howe and Sibley1995; Sibley and Ajioka, Reference Sibley and Ajioka2008). However, recent studies have revealed a greater genetic diversity of T. gondii, particularly isolates from Brazil (Shwab et al., Reference Shwab, Saraf, Zhu, Zhou, McFerrin, Ajzenberg, Schares, Hammond-Aryee, van Helden, Higgins, Gerhold, Rosenthal, Zhao, Dubey and Su2018). A recent update indicated 279 genetic variants (Su and Dubey, Reference Su and Dubey2020). Severe cases of toxoplasmosis have been reported in immunocompetent patients in association with atypical T. gondii genotypes (Ajzenberg et al., Reference Ajzenberg, Bañuls, Su, Dumètre, Demar, Carme and Dardé2004; Demar et al., Reference Demar, Ajzenberg, Maubon, Djossou, Panchoe, Punwasi, Valery, Peneau, Daigre, Aznar, Cottrelle, Terzan, Dardé and Carme2007; Elbez-Rubinstein et al., Reference Elbez-Rubinstein, Ajzenberg, Dardé, Cohen, Dumètre, Yera, Gondon, Janaud and Thulliez2009; Lorenzi et al., Reference Lorenzi, Khan, Behnke, Namasivayam, Swapna, Hadjithomas, Karamycheva, Pinney, Brunk, Ajioka, Ajzenberg, Boothroyd, Boyle, Dardé, Diaz-Miranda, Dubey, Fritz, Gennari, Gregory, Kim, Saeij, Su, White, Zhu, Howe, Rosenthal, Grigg, Parkinson, Liu, Kissinger, Roos and Sibley2016). However, little is known about the association of genotype and clinical disease in animals and humans in the USA (Dubey, Reference Dubey2010).
In the current study, 36% of WTD were seropositive, and these data are in accord with several regional surveys with seroprevalences of 28.7–74.4% (Table 5). These results are comparable because in all surveys listed in Table 5, the same MAT was used to detect T. gondii antibodies. WTD are the most common wild cervids in the USA and they are spreading from rural to urban areas. WTD have been imported to Europe (Jokelainen et al., Reference Jokelainen, Näreaho, Knaapi, Oksanen, Rikula and Sukura2010). Nothing has been published concerning the genotypes of T. gondii in WTD in Europe, but it would be most interesting to learn whether they may have played any role in introducing or propagating parasite strains otherwise considered endemic to North America.
Table 5. Summary of prevalence of T. gondii in WTD from previous reports in the USAa

a In addition to these reports, Yu et al. (Reference Yu, Shen, Su and Sundermann2013) reported ToxoDB PCR-RFLP genotype #5 for the isolate TgWtdAL from the heart and tongue of a WTD from Alabama.
In the current study, there was relatively a low to moderate diversity of the isolates identified. Genotypes #1, #2, #3, #4, #39 and #221 have previously been identified in animals in the USA, with the first three being most common in animals on farms (Jiang et al., Reference Jiang, Shwab, Martin, Gerhold, Rosenthal, Dubey and Su2018). Genotype #224 was previously reported in dog from Grenada (Dubey et al., Reference Dubey, Tiwari, Chikweto, DeAllie, Sharma, Thomas, Choudhary, Ferreira, Oliveira, Verma, Kwok and Su2013b).
From previously published reports, 69 isolates from WTD in the USA have been genotyped (Table 5). Among these isolates, 13 ToxoDB PCR-RFLP genotypes were identified, including #1, #2, #3, #4, #5, #54, #74, #146, #154, #167, #216, #220 and #221. From these genotypes, #5 (haplotype 12) accounted for 42% (29 of 69), was the most frequently isolated. Genotype #4 (also known as haplotype 12) accounted for 16% (11 of 69). Genotypes #1 and #3 together known as type II and haplogroup 2, accounted for 15% (10 of 69). Combining the 36 isolates from our current study with previously reported 69 isolates from WTD, 15 genotypes are identified, including #1, #2, #3, #4, #5, #39, #54, #74, #146, #154, #167, #216, #220, #221 and #224. Among these, 50.4% (53/105) isolates were characterized as genotype #5. Genotypes #1 and #3 (together as type II, haplogroup 2) accounted for 14.3% (15/105) of the isolates. Genotype #4 (haplotype 12) accounted for 10.5% (11/105) and genotype #2 (type II, haplogroup 3) accounted for 9.5% (10/105). The other 10 genotypes were less frequently identified. A recent national survey of feral swine in the USA indicated a similar pattern, in which genotype #5 was dominant and accounted for 57% of 76 isolates genotyped (Dubey et al., Reference Dubey, Cerqueira-Cézar, Murata, Verma, Kwok, Pedersen, Rosenthal and Su2020). Based on these two studies, dominance of genotype #5 suggests sylvatic transmission of T. gondii in wildlife in the USA.
Although bobcats are the most likely hosts involved in the sylvatic cycle of T. gondii in the USA (Dubey, Reference Dubey2010; VanWormer et al., Reference VanWormer, Miller, Conrad, Grigg, Rejmanek, Carpenter and Mazet2014; Verma et al., Reference Verma, Sweeny, Lovallo, Calero-Bernal, Kwok, Jiang, Su, Grigg and Dubey2017), other wild cats may participate in both feral and domestic cycles introducing atypical genotypes to domestic cats, thereby facilitating transmission of potentially more pathogenic genotypes to humans, domestic animals and wildlife (VanWormer et al., Reference VanWormer, Miller, Conrad, Grigg, Rejmanek, Carpenter and Mazet2014). The partition of T. gondii genotypes such as #1, #2 and #3 among domestic animals and #5 in wildlife is mainly due to distinct sylvatic and domestic transmission cycles, though both cycles overlap to a certain degree (Jiang et al., Reference Jiang, Shwab, Martin, Gerhold, Rosenthal, Dubey and Su2018; Shwab et al., Reference Shwab, Saraf, Zhu, Zhou, McFerrin, Ajzenberg, Schares, Hammond-Aryee, van Helden, Higgins, Gerhold, Rosenthal, Zhao, Dubey and Su2018). Recent evidence indicates that the strains of T. gondii prevalent in wildlife 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 domesticated animals (Dubey and Prowell, Reference Dubey and Prowell2013; Crouch et al., Reference Crouch, Mittel, Southard, Cerqueira-Cézar, Murata, Kwok, Su and Dubey2019), indicating a plausible route for introduction of virulent strains of T. gondii from the sylvatic cycle.
Our results revealed low to moderate genetic diversity of T. gondii in WTD in the USA, with genotype #5 (haplogroup 12) identified as the most dominant type in the USA. The contribution of WTD to the epidemiology of T. gondii deserves additional scrutiny. Considering the high rate of exposure of WTD to T. gondii in the USA, results affirm that the environment is highly contaminated with oocysts.
Supplementary material
The supplementary material for this article can be found at https://doi.org/10.1017/S0031182020000451.
Acknowledgements
We would like to thank many WS employees who collected and submitted samples for this project.
Financial support
This research was supported in part by an appointment to the Agricultural Research Service (ARS) Research Participation Program administered by the Oak Ridge Institute for Science and Education (ORISE) through an interagency agreement between the U.S. Department of Energy (DOE) and the U.S. Department of Agriculture (USDA). ORISE is managed by ORAU under DOE contract number DE-SC 0014664. All opinions expressed in this paper are the authors' and do not necessarily reflect the policies and views of USDA, ARS, DOE or ORAU/ORISE.
Conflict of interest
None.
Ethical standards
All experimental procedures were approved by the Institutional Animal Care and Use Committee (Protocol no. 15-017), United States Department of Agriculture, Beltsville, Maryland.