Seroprevalence and Risk Factors for Toxoplasma gondii Infection in Owned Domestic Cats in Australia

Abstract

Ongoing surveillance of Toxoplasma gondii seroprevalence and exposure risks in owned cats is important to identify effective mechanisms to decrease the prevalence of this global zoonotic parasite. We aimed to determine the seroprevalence of T. gondii and risk factors for seropositivity in owned domestic cats in Australia. Sera, signalment data, postcode, and completed owner-questionnaires surveying diet composition and lifestyle factors were collected for cats presenting to 18 veterinary clinics across Australia. T. gondii-specific IgG was measured by enzyme-linked immunosorbent assay. Data were analyzed using univariable and multivariable logistic regression to evaluate risk factors associated with positive T. gondii IgG serology. Among 417 cats, T. gondii seroprevalence was 39%. More than two-thirds of cats tested (69%) had outdoor access and 59% were fed a diet containing raw meat. Univariable analyses identified, age (>1 year, p < 0.001), a diet containing any raw meat (p = 0.001), raw kangaroo (p = 0.008), raw chicken (p = 0.012), or raw beef (p = 0.017), and hunting (p = 0.049) as risk factors for T. gondii infection. Age (>1 year, odds ratio [OR]: 7.15) and feeding of raw meat (OR: 2.23) remained significant risk factors (p < 0.001) in multivariable analyses. T. gondii seroprevalence did not differ between cats domiciled in urban and semiurban or rural areas. Pet cats in Australia are commonly infected with T. gondii. Feeding raw meat to cats, a common practice in Australia, is associated with T. gondii infection, highlighting the need for education about the health implications for cats from feeding a diet containing raw meat.

Introduction

Toxoplasma gondii is a significant human, mammalian, and avian parasite with worldwide occurrence. Wild and domesticated members of the Felidae family, the only definitive hosts, are the source of infectious sporozoites in oocysts shed in their feces. One cat may shed between three million and 810 million oocysts for a period of ∼8 days during primary infection (Dabritz and Conrad 2010). Ingestion of oocysts in contaminated food or water is the major source of infection for livestock, wildlife, and marine mammals. Carnivorous and omnivorous intermediate hosts, including humans, may also become infected with T. gondii from ingestion of encysted bradyzoites in the meat or organs of other intermediate hosts (Dubey et al. 2009).

T. gondii is recognized as a major foodborne pathogen estimated to infect a third of the global human population (Montoya and Liesenfeld 2004, Andreoletti et al. 2007). During primary infection, sporozoites or bradyzoites differentiate into tachyzoites and replicate within multiple organs. Infection is contained by the host immune response, resulting in the transformation of tachyzoites into bradyzoites within tissue cysts and latent infection, which may persist life-long (Sibley and Ajioka 2008). Although primary infection is usually asymptomatic, infection during pregnancy can have serious consequences, including abortion, and ocular and neurological deficits in the fetus (Montoya and Liesenfeld 2004).

Infection with atypical strains of T. gondii, or reactivation of latent infection, for example, in immunocompromised hosts, can result in severe systemic clinical disease in both definitive and intermediate hosts (Andreoletti et al. 2007, Brennan et al. 2016). In addition, latent infection has been linked to human psychiatric disorders, including schizophrenia, where a causal relationship with T. gondii has been established (Burgdorf et al. 2019). To reduce the risk to human and animal health posed by T. gondii, it is important to understand the region-specific prevalence and population structure of T. gondii in the definitive host, and the factors that increase likelihood of exposure to the parasite. Seropositivity for IgG is widely recognized as a proxy of latent T. gondii infection in healthy definitive and intermediate hosts (Brennan et al. 2016).

Domestic cats are an important reservoir of T. gondii. The prevalence of T. gondii-specific IgG in owned cats varies considerably between regions, being lowest in Japan (6–16%) (Nogami et al. 1998, Salman et al. 2018) and highest in Northern Europe (50–63%) (Jokelainen et al. 2012, Must et al. 2015). Common risk factors for T. gondii infection in owned cats include increasing age, outdoor access, hunting, and a diet containing raw meat (Jokelainen et al. 2012, Must et al. 2015, Salman et al. 2018).

Surveillance for T. gondii exposure among owned cats has been performed infrequently in Australia (Hartley and Munday 1974, Watson et al. 1982), despite an estimated owned cat population of 3.9 million, or 16 cats per 100 people (Animal Medicines Australia 2016). Serological studies of urban stray cats (unowned, free-roaming and dependent on humans for food) and nonurban feral (unowned, unsocialized, and independent of humans) cats (Felis catus) across Australia reveal T. gondii seroprevalences ranging from 20% to 39% in Victoria (Coman et al. 1981, Sumner and Ackland 1999), 30% in Western Australia (Jakob-Hoff and Dunsmore 1983), and 85–96% in Tasmania (Fancourt and Jackson 2014). The aim of this study was to determine the seroprevalence of T. gondii and risk factors for infection among owned cats in Australia.

Materials and Methods

Using a cross-sectional study design, sera were collected prospectively from owned cats presenting to veterinary hospitals over a 4-month period (June 1–September 30, 2015). The study was approved by the Animal Care and Ethics Committee at the University of Sydney (protocol no. 2015/768). Information obtained from medical records included age, sex, breed, desexing, and health status. Owners consenting to inclusion of their cats in the study completed a questionnaire seeking information about their cat's domicile, diet, and lifestyle factors. Information collected included postcode, environment (outdoor access and proportion of time spent outdoors), diet composition (types of commercial food, raw meat, and/or home-cooked diets), and hunting habits. Abode (major city, inner regional, outer regional, remote, or very remote) was determined from postcode using the Accessibility/Remoteness Index of Australia (ARIA https://www.adelaide.edu.au/hugo-centre/services/aria). Cats were included in the study if written consent, a serum sample, and a completed owner survey were obtained.

After centrifugation and separation, samples were stored at −20°C and shipped to the Department of Clinical Sciences in the College of Veterinary Medicine and Biomedical Sciences, Colorado State University, CO. A previously reported T. gondii IgG enzyme-linked immunosorbent assay (ELISA) was performed to detect T. gondii-specific IgG in each serum sample (Vollaire et al. 2005). In short, MicroELISA (Immulon 1b; Thermo Fisher Scientific, Waltham, MA) plates were sensitized with an optimal dilution of a lysate of T. gondii RH strain tachyzoites. All sera, including the positive and negative controls, were assayed at 1:64. Goat anti-cat IgG (peroxidase labeled, heavy chain specific; SeraCare, Milford, MA) was used with a TMB chromogenic substrate (SureBlue™ TMB; SeraCare). When new reagents are used, the assay cutoff level for a positive result is determined using 20 known T. gondii positive feline sera of varying titers and 20 known negative feline. This assay is commercially available in an American Association of Veterinary Laboratory Diagnosticians-certified laboratory (Specialized Infectious Diseases Laboratory, Veterinary Diagnostic Laboratories, Colorado State University, Fort Collins, CO; www.dlab.colostate.edu).

Test samples were assayed in triplicate. Positive-control, negative-control, enzyme-control, and substrate control wells were included on each plate. Data analysis was conducted using binary data. A univariable and multivariable logistic regression analysis was performed to determine the association of seropositivity with the lifestyle factors collected in the survey that included age (greater than or less than 12 months), sex, neuter status, raw meat in the diet (yes or no), types of raw meat in the diet, and whether the cat had been observed to hunt (yes or no). Significance was set at a p-value of <0.25 for univariable analysis and <0.05 for multivariable analysis. Age and sex were considered potential confounders and were included in the model even if nonsignificant.

Results

Sera and survey data from 417 cats from 18 veterinary hospitals across four states and one territory in Australia met the inclusion criteria (Table 1). The median age of all cats was 10 years (range 3 months–21 years). There were 209 males (11 entire and 198 neutered) and 209 females (14 entire and 195 neutered). Data on abode were available for 380 cats, of which 83% were from a major city, 9% inner regional, 7% outer regional, and 2% were in a remote location. A high proportion of cats (n = 283, 69%) had outdoor access, and of these 48% (n = 135) roamed freely. Of cats with outdoor access, 165 (58%) were housed inside at night, whereas the remainder had outdoor access during the day and at night. Litter trays were provided indoors for 87.5% of cats (365 of 417), although 37% of these cats (134 of 365) also had outdoor access.

Table 1.

Locations of Participating Veterinary Clinic, Number of Samples Collected in Each Region, and Regional Seroprevalence

Location	No. of veterinary clinics	No. of samples	No. of seropositive (%)
Townsville and Brisbane, North and South Queensland	5	69	25 (36)
Sydney, New South Wales	10	199	98 (49)
Melbourne, Victoria	2	87	25 (29)
Canberra, Australian Capital Territory	1	7	5 (71)
Perth, Western Australia	2	55	9 (16)

Two hundred and forty-seven cats (59%) had been fed raw meat by their owners. The frequency of raw meat feeding (RMF) was daily (37%, n = 92), weekly (18%, n = 44), monthly (6%, n = 15), or occasionally (39%, n = 96). The majority of cats fed raw meat ate meat from more than one species; beef was fed most often (153/247, 62%), followed by chicken (149/247, 60%), and kangaroo (98/247, 40%).

Overall, 162/417 cats (39%) were seropositive for T. gondii. The overall prevalence in different regions ranged from 16% in Western Australia to 49% in New South Wales (Table 1). In univariable analysis, factors significantly associated with seropositivity were age >1 year (p < 0.001), a diet containing raw meat (p = 0.001), raw kangaroo (p = 0.008), raw beef (p = 0.017), or raw chicken (p = 0.012), successful hunting behavior (p = 0.049), sex (p = 0.024) and urban ARIA code (p = 0.124). (Table 2). Other types of raw meat in the diet that were analyzed, including rabbit, pork, and turkey, were not significantly associated with seropositivity. Cats fed raw lamb were significantly more likely to be seronegative (p = 0.0067).

Table 2.

Results of Descriptive and Univariable Logistic Regression Analyses to Identify Risk Factors for Toxoplasma gondii Seropositivity Determined Using an IgG Enzyme-Linked Immunosorbent Assay

Variable	n	No. of seropositive (%)	OR	95% CI	p
Age^a					<0.001
≤12 months	50	4 (8)	1
>12 months	378	153 (42)	8.18	3.24–27.56
Diet composition^a					0.001
No raw meat	171	48 (28)	1
Raw meat	247	109 (44)	1.38	0.91–2.08
Raw meat
Beef
Yes^a	153	69 (45)	1.64	1.09–2.47	0.017
No	264	88 (33)	1
Chicken
Yes^a	149	68 (46)	1.69	1.13–2.55	0.012
No	268	89 (33)	1
Kangaroo
Yes^a	98	48 (49)	1.87	1.18–2.96	0.008
No	318	108 (34)	1
Lamb^a
Yes	34	8 (24)	0.48	0.2–1.05	0.067
No	383	149 (39)	1
Pork
Yes	15	5 (33)	0.82	0.04–8.66	0.723
No	402	152 (38)	1
Other
Yes	11	6 (55)	0.61	0.13–2.16	0.462
No	406	154 (38)	1
Rabbit
Yes	6	1 (16)	0.33	0.02–2.05	0.257
No	411	156 (38)	1
Turkey
Yes	3	1 (33)	0.82	0.04–8.66	0.873
No	413	156 (38)	1
Hunter^a
Yes (often)	50	26 (52)	0.45	0.24–0.86	0.049
Sometimes	183	70 (38)	0.57	0.3–1.07
Never	184	61 (33)	1
Sex^a					0.024
Female entire	14	1 (7)	1
Female spayed	194	77 (40)	8.48	1.64–155.6
Male entire	11	2 (18)	2.89	0.24–67.86
Male castrated	198	77 (38)	8.27	1.60–151.7
Outdoor access					0.591
Yes	287	110 (38)	1.13	0.73–1.74
No	130	52 (40)	1
ARIA code^a					0.124
Rural	68	20 (29)	1
Urban	349	137 (39)	1.54	0.89–2.76

Significant statistical findings (p < 0.25).

CI, confidence interval; OR, odds ratio.

In the multivariate logistic regression analysis, factors that were significantly associated with being seropositive (Table 3) were age >1 year (p < 0.0001, odds ratio [OR]: 7.15; 95% confidence interval [CI]: 2.61–25.75) and feeding raw meat (p < 0.001, OR: 2.23, 95% CI: 1.44–3.49), whereas cats that had been fed raw lamb were significantly less likely to be seropositive (p = 0.007, OR: 0.32; 95% CI: 0.13–0.72).

Table 3.

Multivariable Logistic Regression Analysis to Identify Risk Factors for Toxoplasma gondii Seropositivity Determined Using an IgG Enzyme-Linked Immunosorbent Assay

Variable	n	No. of seropositive (%)	OR	95% CI	p
Age
≤12 months	50	4 (8)	1
>12 months	367	153 (42)	7.10	2.61–25.75	0.001
Diet composition^a
Raw meat in diet
No	170	48 (28)	2.20	1.42–3.49	0.001
Yes	247	109 (44)
Type of raw meat^a
Lamb
Yes	34	8 (24)	0.32	0.13–0.72	0.007
No	383	149 (39)	1

Significant statistical findings (p < 0.05), results are adjusted for sex due to potential confounding factors.

Discussion

At 39%, the seroprevalence of T. gondii in owned cats in Australia is among the highest reported among owned cats worldwide. A meta-analysis of seroprevalence involving 7285 owned and unowned cats from China between 1995 and 2016, found a median IgG seroprevalence of 20.3% and an estimated pooled IgG seroprevalence of 24.5% (Ding et al. 2017). The largest cohort of owned cats in which surveillance for T. gondii has been performed, comprised 12,628 cats from the United States, of which 23% were seropositive for IgG (Vollaire et al. 2005). In that study T. gondii-specific IgM was also measured, and the combined seroprevalence (IgG or IgM positive) was even higher at 32%. Thus, it is likely that the overall seroprevalence in Australian owned cats is even higher, since active infections may be IgM positive and IgG negative (Lappin et al. 1989).

Increasing urbanization and availability of commercial processed formulations of pet food might be expected to result in a decline in T. gondii seroprevalence among owned cats over time. T. gondii seroprevalence among owned Australian cats was investigated previously in a single study of 80 cats in Sydney, in which 42 (52.5%) were seropositive (Watson et al. 1982). Interestingly, seroprevalence of T. gondii among cats from Sydney in our study (n = 199) was the highest of all regions tested, being 49% and similar to that obtained by Watson et al. nearly 40 years ago.

The association of seropositivity with increasing age or age >12 months, as identified in this study, is the most consistent risk factor identified among owned cats worldwide, consistent with the major source of transmission of infection in cats being horizontal through ingestion of encysted bradyzoites in the tissues of intermediate hosts (Salman et al. 2018, Must et al. 2015, Vollaire et al. 2005, Haddadzadeh et al. 2006).

RMF by pet owners was identified in our study as a risk factor for T. gondii exposure, and we found it is a common practice among Australian cat owners, with 59% of owners having fed their cat raw meat previously, and 39% of these feeding their cats raw meat daily. In a previous survey of feeding practices by cat owners in Sydney, 43% of respondents indicated that meat was included in their cats' current diet (Toribio et al. 2009). The much higher frequency of RMF identified in our survey is likely due to the inclusion of questions about previous feeding history as well as contemporaneous practices.

The common practice of RMF identified in this study may be influenced by cultural and regulatory practices. Its popularity among cat and dog owners has substantially increased in a number of countries in recent years, including the United States, United Kingdom, Sweden, and the Netherlands, in part due to the perception that natural ingredients are the best nutritional choice and have health advantages over commercially processed diets, and because of past contamination scares (Hellgren et al. 2019, Morgan et al. 2017, van Bree et al. 2018, O'Halloran et al. 2019, Bischoff and Rumbeiha 2018). However, such diets are largely offal based, are not heat treated before chilling or freezing, and have been associated with disease outbreaks in pets and humans as well as food recalls, from bacterial contamination with Mycobacterium bovis, Listeria, Salmonella, Camplylobacter, and toxigenic Escherichia coli (O'Halloran et al. 2019).

In the United States, regulatory and professional bodies, including the American Animal Hospital Association, American Veterinary Medical Association (AVMA), and the Food and Drug Administration (FDA), actively discourage the feeding of raw animal products to pets, because of concerns over bacterial and parasitic contamination, as well as nutritional imbalances (Freeman et al. 2013). A similar stance on RMF has not been taken by professional and regulatory bodies in Australia and some veterinarians in Australia strongly advocate RMF over processed diets (Lonsdale 2001, Bilinghurst 2001).

In the multivariate analyses, we did not find an association between consumption of specific types of raw meat and T. gondii infection, perhaps because most owners who fed their cats raw meat fed different types of meat. The three most common meats fed in this study were beef, chicken, and kangaroo, all of which can serve as intermediate hosts for T. gondii. Given that the infectious dose of T. gondii is extremely low, consumption of even a small amount of raw animal products can result in infection and oocyst shedding (Powell and Lappin 2001). Education of pet owners to freeze meat for several days before feeding it would mitigate this risk, since freezing meat for a minimum of 3 days at −10°C or below reliably inactivates T. gondii bradyzoite tissue cysts (El-Nawawi et al. 2008). Our finding that cats that had been fed raw lamb were significantly more likely to be seronegative for T. gondii than those not fed raw lamb was unexpected, since sheep are known intermediate hosts of the parasite. The result could reflect a number of confounding factors such as demographics of households that feed lamb, or the association may be spurious since only a small number of cats were identified as being fed this type of meat.

Data on T. gondii exposure in animal species used for meat production in Australia are scarce. However, seroprevalence in lambs (16%) and sheep (32%) is relatively high (Kiermeier et al. 2008), as it is for kangaroos and other macropods (8–20%) (Johnson et al. 1989, Parameswaran et al. 2009, Mayberry et al. 2014). The 90% seroprevalence detected among chickens from one free-range farm in Australia highlights the increased zoonotic risk of T. gondii infection posed by free-range chicken compared with indoor-raised poultry (Dubey 2010).

There are no recent data on T. gondii in Australian cattle, although surveillance in the 1970s and 1980s showed a low seroprevalence of 0–2% (Munday 1975, Norton et al. 1989). Beef has been described as a “low-risk” meat with regard to T. gondii transmission to humans in the United States, based on failure to detect T. gondii oocysts in feces after naive cats were fed raw beef (Dubey et al. 2005). However, conflicting data from the Netherlands showed that beef was the most important meat-borne source of T. gondii infection (Opsteegh et al. 2011). Also, epidemiological studies in humans, including in the United States, consumption of raw or undercooked beef has been recognized as a significant risk factor for T. gondii exposure (Jones et al. 2009, Cook et al. 2000).

Most cats in this study had outdoor access (69%), but in contrast to previous studies, outdoor access did not confer an increased risk factor of T. gondii infection.

Conclusion

The seroprevalence of T. gondii was high in owned cats in Australia, and was associated with RMF, which was a common practice by their owners. This finding highlights the need for education of pet owners about meat safety to reduce the risk of transmission of this zoonotic parasite.

Footnotes

Acknowledgments

The authors thank the veterinarians, veterinary clinics, and cat owners around Australia who willingly participated in this study and generously gave of their time to recruit and collect samples and fill in questionnaires. They are particularly grateful to the coordinating veterinarians at each practice: Dr. Anne Fawcett (Sydney Animal Hospital Inner West), Dr. Annabelle Fulmer (Maroubra Veterinary Hospital), Dr. Russell Drake (French's Forest Veterinary Hospital), Dr. Clare Bumak (Fig Tree Vet Hospital), Dr. Andy Woodward (Mentone Veterinary Hospital), Dr. Richard Gowan (Melbourne Cat Clinic), Dr. Leah Puk (Paddington Cat Hospital), Dr. Martine van Boeijen (Perth Cat Hospital), Dr. Louise Beveridge (Bedford Veterinary Hospital), Dr. Carl Adagra (Tropical Queensland Cat Clinic), Dr. Barbara Jameson (Vets @ Acacia Gardens), Dr. Jessica Talbot (Cremorne Veterinary Hospital), Dr. Katherine Briscoe (Animal Referral Hospital Homebush), Dr. Catherine Brett (Enfield Veterinary Hospital), Dr. Joanna White (Sydney Animal Specialist Hospital), Dr. Susan Bennett (The Animal Hospital at Murdoch University), and Dr. Susan Jih (The Cat Clinic Brisbane).

Author Disclosure Statement

No conflicting financial interests exist.

Funding Information

This study was funded in part by the Cat Protection Society of NSW, CPS2017_2019.

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