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Prevalence of zoonotic parasites in feral cats of Central Virginia, USA

Felis catus, the domestic cat, is the definitive host for parasites that may result in adverse health outcomes in humans. Prevalence data of zoonotic parasites in feral cats, which are free‐roaming domestic cats that are born and live in the wild, are limited. The objective of this study was to assess seroprevalence of Toxoplasma gondii antibodies and copro‐prevalence of potentially zoonotic parasites in feral cats and to evaluate risk factors for seropositivity and faecal excretion of parasites. In this cross‐sectional survey, 275 feral cats at Trap‐Neuter‐Release clinics in Central Virginia were tested for parasites via faecal flotation, direct immunofluorescence assay (faeces) and modified agglutination testing (serum). Toxoplasma gondii seroprevalence was 22.35% (95% CI: 17.47–27.86). Faecal prevalence of T. gondii‐like oocysts was 1.04% (95% CI: 0.13–3.71), Toxocara cati 58.85% (95% CI: 51.54–65.89), Ancylostoma spp. 18.75% (95% CI: 13.49–25.00), Giardia duodenalis 5.73% (95% CI: 2.89–10.02) and Cryptosporidium spp. 3.33% (95% CI: 1.37–7.24). Female cats were more likely than males to excrete faecal Ancylostoma spp. eggs (OR 2.88; 95% CI 1.34–6.17). Adults were more likely than immature cats to be seropositive (OR 2.10; 95% CI: 1.11–3.97) and to excrete faecal Ancylostoma spp. eggs (OR 2.57; 95% CI: 1.10–5.99). However, immature cats were more likely than adults to excrete T. cati eggs (OR 6.79; 95% CI: 3.31–13.90) and to excrete one or more potentially zoonotic species (OR 4.67; 95% CI: 2.28–9.55) in faeces. Results of this study have implications for human and animal health and highlight the importance of collaboration between public health, medical and veterinary communities in preventive efforts.

produce the most oocyst numbers of all felids and are believed to contribute most oocysts contaminating the environment (Dubey, 2010). Toxoplasmosis results after ingestion of infectious T. gondii oocysts from an environment contaminated with cat faeces or through ingestion of tissue cysts in raw or undercooked meat of an intermediate host. Toxoplasmosis is characterized by symptoms ranging from mild/flulike to encephalitis and death; infection during pregnancy can result in miscarriage or developmental foetal abnormalities (Jones, Parise, & Fiore, 2014). Toxoplasmosis is reported to be the second deadliest food-borne disease in the USA, costing an estimated 3.3 billion dollars annually (Scallan et al., 2011;USDA, 2014). Foods that have been implicated as sources of T. gondii parasite stages originating from cats include raw produce, undercooked meat, raw seafood and unpasteurized goat's milk (Hussain, Stitt, Szabo, & Nelan, 2017). Wildlife, including game species, may be infected with T. gondii from cats, representing a source of food-borne exposure for people who hunt and consume game animals. For example, studies detected T. gondii seroprevalences of 56.3% in elk in the Central Appalachians, 58.8% of white-tailed deer in Northeast Ohio and 27.7% of feral pigs in North Carolina (Ballash et al., 2015;Cox et al., 2017;Sandfoss, DePerno, Patton, Flowers, & Kennedy-Stoskopf, 2011). Water may be contaminated with T. gondii oocysts shed in the faeces of infected cats. One of the largest documented outbreaks of toxoplasmosis in people was thought to be caused by oocyst run-off from free-roaming domestic and wild cats into a municipal reservoir in British Columbia, Canada (Bowie et al., 1997).
Toxoplasmosis has been targeted for public health action as 1 of 5 neglected parasitic infections in the USA by the Centers for Disease Control and Prevention (CDC) (CDC, 2016).
The ascarid, Toxocara cati, a zoonotic parasite acquired from cats, causes toxocariasis after ingestion of infective T. cati eggs excreted in cat faeces, either on contaminated raw produce, soil or water, or (less frequently) through ingestion of encysted larvae in undercooked meat (Woodhall, Eberhard, & Parise, 2014). After ingestion, parasite larvae migrate through the body of an infected individual, damaging organs, such as lungs, liver, eyes and central nervous system, and may result in permanent debilitating disease including blindness (CAPC, 2016a;Woodhall et al., 2014). Toxocariasis is considered a neglected parasitic infection by the CDC (Woodhall et al., 2014). Although thought to be one of the most common parasitic infections in the USA, the true importance of toxocariasis in humans is not known because of underdiagnosis, in part due to lack of commercially available specific immunological tests but also because symptoms can be vague, resulting in undertesting (Woodhall et al., 2014).
Giardiasis is prevalent in children in developing areas of the world and can result in growth stunting and impaired cognitive development (CDC, 2015b;Feng & Xiao, 2011). Cryptosporidiosis is frequently linked to outbreaks associated with recreational water use in the USA and can cause gastrointestinal disease in people, with potentially severe health outcomes in immunocompromised individuals (CDC, 2015a).
The estimated 60-100 million feral cats which roam outdoors in the USA are a source of environmental contamination with parasites that pose a risk for transmission to humans (Roebling et al., 2014). One cat infected with zoonotic parasites can excrete millions of eggs, cysts or oocysts into the environment, and in many cases, the infective parasite stages are extremely resistant, persisting for months to years in soil and/or water (CAPC, 2016a; Dabritz & Conrad, 2010;Harhay, Horton, & Olliaro, 2010). Cats produce an estimated 1.2 million tons of faeces in the USA each year (Torrey & Yolken, 2013). Feral cats, which are born and live in the wild, do not receive the same level of husbandry and veterinary care as do most owned cats, including routine faecal parasite tests and antiparasitic medications. Furthermore, cats roaming Impacts • We examined the copro-prevalence of potentially zoonotic parasites, seroprevalence of T. gondii antibodies and factors predicting parasite excretion and seropositivity in a feral cat population in Central Virginia.
Immature stages of these parasites are excreted in cat faeces, persist in the environment and can be inadvertently ingested by humans and other animals, resulting in negative health outcomes.
• Several species of parasites were identified, with more than half of the faecal samples testing positive for T. cati eggs and almost one-fourth of the cats testing positive for T. gondii antibodies. Immature cats were significantly more likely than adults to excrete T. cati and one or more potentially zoonotic parasite species in the faeces (p < 0.05).  ;Lilly & Wortham, 2013). Considering the lack of data from feral cats, the objective of this study was to assess the prevalence of potentially zoonotic parasites, specifically the seroprevalence of antibodies against T. gondii and copro-prevalence of T. cati and Ancylostoma spp. eggs, G. duodenalis cysts, and Cryptosporidium spp. and Toxoplasma-like oocysts, in feral cats in Virginia. Sex and age were evaluated as risk factors for seropositivity and excretion of faecal parasites. The hypothesis was that the feral cats would have high parasite copro-prevalence and T. gondii seroprevalence and that age and sex would predict faecal excretion of parasite stages as well as T. gondii seroprevalence.

| Cats
Feral cats (n = 275; 131 females, 144 males) were sampled from July through November 2016. The cats were humanely trapped in standard metal live traps by the general public and brought to monthly feral Trap-Neuter-Release (TNR) clinics sponsored by Operation Catnip, a nonprofit organization based in Richmond, Virginia. Each cat was given an identification number that was placed on its history chart, faecal sample bag and blood tubes. The cats were anaesthetized, physically examined, treated with the insecticide fipronil/(s)methoprene (9.8/11.8%; 0.5 ml topically; FRONTLINE®Plus for cats, Merial Inc., Duluth, GA) and antiparasitic ivermectin (

| Sample collection
Blood and faecal samples were collected from cats during recovery from surgery. Faecal samples were collected from 192 (69.8%) of the cats, either by digital rectal palpation or directly from the trap if available. Faeces were unavailable from 83 (30.2%) of the cats.
Faecal samples were placed into individual plastic re-sealable bags and stored in a cooler on ice for transport and then stored at 4°C. All faecal samples were analysed within 48 hr of collection.
Blood samples were collected from 264 (96.0%) of the cats via venipuncture of a jugular or medial saphenous vein. Blood was not collected from 11 (4.0%) of the cats due to inability to readily collect an adequate volume or because rapid recovery from anaesthesia precluded safe handling of the cat. Blood was collected into a syringe, transferred into serum separator tubes and allowed to clot for 30 min at room temperature before placing into a cooler with ice for transport and then centrifuged at 1,500× g for 10 min at 4°C. Serum was then transferred into microcentrifuge tubes and stored at 4°C until analysis was performed.

| Sample analysis
Individuals conducting serological and faecal examinations knew the identification numbers on the samples and that samples were obtained from cats, but were blinded to cat age and sex. Faecal samples were processed by centrifugal flotation in a 33% zinc sulphate solution as previously described (Zajac & Conboy, 2012). Processed samples were examined microscopically for parasites, first scanning using the 10× objective and then using the 20× or 40× objectives if necessary to confirm identification of smaller parasite cysts and oocysts. Parasite identifications were based on published descriptions, and all parasites identified were recorded (Zajac & Conboy, 2012).
To evaluate for the presence of Cryptosporidium spp. oocysts, fresh faeces remaining after faecal flotation (n = 180) were stored at room temperature in 10% formalin until further analysis using a commercially available direct immunofluorescence assay (DFA) kit (MERIFLUOR® Cryptosporidium/Giardia, Meridian Bioscience Inc., Cincinnati, OH). Testing procedures and interpretation were performed according to the manufacturer's instructions (Anon, Meridian Bioscience, Inc., 2017). Samples were considered positive if 4-6-µm round-to-oval apple green oocysts were observed using a microscope equipped with epifluorescent and differential interference contrast optics.

| Statistical analysis
Target sample size of n = 323 was determined a priori for a 95% CI and alpha of 0.05 based on anticipated seroprevalence (30%-60%) for T. gondii antibodies (Elmore et al., 2010;Nutter, Dubey et al., 2004). Epi Info™ 7 (Centers for Disease Control and Prevention, Atlanta, GA) was used to perform unconditional logistic regression to assess age and gender as risk factors for seroprevalence of T. gondii and copro-prevalence of zoonotic parasites. Applicable test assumptions were met for all statistical procedures. Statistical findings for which p-values were <0.05 were considered significant.

| Approval
This study was approved and conducted in accordance with the Virginia Tech Institutional Animal Care and Use Committee (VT IACUC# 16-106).

| Risk factors
Age and gender were significant predictors of seropositivity to T. gondii and faecal excretion of T. cati and Ancylostoma spp.
(  Additionally, some cats excrete numbers of oocysts which may be below the threshold (1,000 oocysts/g) of microscopic detection (Dubey, 2010). Furthermore, cats usually excrete T. gondii oocysts for about a week and are unlikely to excrete oocysts on subsequent exposures, resulting in only about 1% of cats excreting oocysts at any given time (Dubey, 2010;Elmore et al., 2010).

| D ISCUSS I ON
Serology, a measure of parasite exposure, is superior to faecal flotation as an indicator of T. gondii prevalence in cat populations (Dubey, Lappin, & Thulliez, 1995). Due to the short window of faecal excretion, seropositive cats are not likely to also be actively excreting oocysts. Most T. gondii seropositive cats previously excreted millions of oocysts which are extremely resistant in the environment, persisting for months to years, making serology a good indicator of environmental contamination and zoonotic potential (Dabritz & Conrad, 2010). Seroprevalence of antibodies against T. gondii in this study was 22.35%, which was lower than that expected for a freeroaming, outdoor cat population in a relatively humid region. Reports indicate that feline T. gondii seroprevalence ranges from 16% to 80% in the USA, depending on the cat population, geographical region and immunological test used (Dubey, 2010). Seroprevalence of 16% has been documented in drier US climates but is closer to 60% in humid climates (Elmore et al. 2010 was similar to our methodology, but Sabshin et al. used a less sensitive method of detecting faecal T. cati eggs, which may account for the comparatively low copro-prevalence of that study (Zajac & Conboy, 2012). Furthermore, most cats presented to shelters are treated with antiparasitics, which could result in the underestimation of parasite prevalence if egg-producing parasites are removed prior to collection of faecal samples (Sabshin et al., 2012). Sabshin et al. reported that faecal samples were collected within 24 hr of deworming and that 16% of the cats were owned prior to admission to the shelter. Although many cats presented to shelters are stray, some are owner-surrendered and potentially lived indoors and received routine veterinary care, which could also account for lower T. cati prevalence in shelter cats compared to feral cats. The large percentage of free-roaming cats excreting T. cati eggs in our study represents a significant public health concern. Toxocariasis may be associated with significant morbidity in humans, including blindness and neurological symptoms (Rabinowitz & Conti, 2010;Woodhall et al., 2014). A single infected cat may excrete tens of thousands of environmentally persistent faecal T. cati eggs per day (CAPC, 2016a). Age was a significant risk factor for seropositivity to T. gondii, consistent with findings of other studies (Opsteegh et al., 2012;Saevik et al., 2015). Given the greater length of time exposed to the environment, it follows that aged cats are more likely to have been exposed to T. gondii oocysts and tissue cysts than immature cats.
Interestingly, adults were more likely to excrete faecal Ancylostoma spp. eggs, but immature cats were more likely to excrete T. cati and multiple potentially zoonotic parasite species. This may reflect a generally less robust immune system of kittens compared to adults.
In addition, transmammary transmission of T. cati may occur, giving kittens a unique exposure route for this parasite, whereas cats are mainly infected with Ancylostoma spp. through ingestion of paratenic hosts (Bowman, 1999;CAPC, 2016b). Sex was a significant predictor of Ancylostoma spp. faecal excretion, with females being more likely than males to excrete this parasite. One could hypothesize that female feral cats are immunocompromised compared to male cats, considering that on average, they experience multiple pregnancies per year . Immunosuppression is a feature of late pregnancy, and early lactation may be associated with increased parasite egg excretion (Tizard, 2000).
Feral cats in Central Virginia are a source of parasites that may cause adverse health outcomes in animals and humans. Zoonotic diseases such as toxoplasmosis and toxocariasis in humans can be difficult to diagnose, complicated to treat and may result in permanent organ damage and death. Vaccines to prevent these infections do not exist. Thus, optimal health outcomes hinge on preventing exposure to parasites. Prevention can be achieved through interdisciplinary collaboration of policy makers and public health, veterinary and human health professionals. Prevention measures should emphasize public education regarding the risks of zoonotic parasites associated with free-roaming cats as well as efforts to humanely reduce feral cat populations.

ACK N OWLED G EM ENTS
The authors thank Dr. Meriam Saleh for assistance with performance and interpretation of faecal flotations, Dr. Thomas Taetzsch for assistance in record-keeping and sample processing, and Dr.
Oliver Kwok for performing Toxoplasma serology. Also, thank you to Operation Catnip board members and volunteers who made this study possible. The authors declare that there are no conflict of interests and no external funding was received in connection with this study.