Prevalence and genotypes of Toxoplasma gondii in feline faeces (oocysts) and meat from sheep, cattle and pigs in Switzerland

Abstract

The protozoan parasite Toxoplasma gondii infects almost all warm blooded animal species including humans, and is one of the most prevalent zoonotic parasites worldwide. Post-natal infection in humans is acquired through oral uptake of sporulated T. gondii oocysts or by ingestion of parasite tissue cysts upon consumption of raw or undercooked meat. This study was undertaken to determine the prevalence of oocyst-shedding by cats and to assess the level of infection with T. gondii in meat-producing animals in Switzerland via detection of genomic DNA (gDNA) in muscle samples. In total, 252 cats (44 stray cats, 171 pet cats, 37 cats with gastrointestinal disorders) were analysed coproscopically, and subsequently species-specific identification of T. gondii oocysts was achieved by Polymerase Chain Reaction (PCR). Furthermore, diaphragm samples of 270 domestic pigs (120 adults, 50 finishing, and 100 free-range animals), 150 wild boar, 250 sheep (150 adults and 100 lambs) and 406 cattle (47 calves, 129 heifers, 100 bulls, and 130 adult cows) were investigated by T. gondii-specific real-time PCR. For the first time in Switzerland, PCR-positive samples were subsequently genotyped using nine PCR-restriction fragment length polymorphism (PCR-RFLP) loci (SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1 and Apico) for analysis. Only one of the cats shed T. gondii oocysts, corresponding to a T. gondii prevalence of 0.4% (95% CI: 0.0–2.2%). In meat-producing animals, gDNA prevalence was lowest in wild boar (0.7%; 95% CI: 0.0–3.7%), followed by sheep (2.0%; 95% CI: 0.1–4.6%) and pigs (2.2%; 95% CI: 0.8–4.8%). The highest prevalence was found in cattle (4.7%; 95% CI: 2.8–7.2%), mainly due to the high prevalence of 29.8% in young calves. With regard to housing conditions, conventional fattening pigs and free-range pigs surprisingly exhibited the same prevalence (2.0%; 95% CI: 0.2–7.0%). Genotyping of oocysts shed by the cat showed T. gondii with clonal Type II alleles and the Apico I allele. T. gondii with clonal Type II alleles were also predominantly observed in sheep, while T. gondii with mixed or atypical allele combinations were very rare in sheep. In pigs and cattle however, genotyping of T. gondii was often incomplete. These findings suggested that cattle in Switzerland might be infected with Toxoplasma of the clonal Types I or III, atypical T. gondii or more than one clonal Type.

Introduction

Toxoplasma gondii is one of the most prevalent zoonotic parasites worldwide. While only felidae can act as definitive hosts and thus shed oocysts in their faeces, almost all warm-blooded animals can serve as intermediate hosts. These, upon primary infection, will first undergo a tachyzoite stage infection by the parasite, followed by the formation of bradyzoite-containing tissue cysts, primarily in brain or muscles (Dubey and Beattie, 1988, Dubey, 1996; reviewed by Tenter et al., 2000). Humans may acquire a T. gondii infection via (1) oral uptake of sporulated oocysts from the environment, (2) consumption of raw or undercooked meat containing tissue cysts or (3) via transplacental transmission of the parasite from the non-immune mother to the foetus. Studies in Europe have shown that 35–58% of women at child-bearing age were seropositive for T. gondii (reviewed by Tenter et al., 2000). In Switzerland, 46% of women at child-bearing age presented a seropositive immune status (Jacquier et al., 1995), but seroprevalences are likely to have decreased in the last decade, following the general trend in Europe (reviewed in Pappas et al., 2009). In fact, two locally restricted studies carried out with women in child-bearing age in Switzerland detected seroprevalences of 35% and 8.2%, respectively (Signorell et al., 2006, Zufferey et al., 2007). In Europe, congenital toxoplasmosis affected approximately 1–10 out of 10,000 newborns, of whom 1–2% suffered from general health problems or even died and 4–27% developed ocular disease (Cook et al., 2000). The European Food Safety Authority (EFSA) has recognized toxoplasmosis as the parasitic zoonosis with the highest human incidence and has recently published a scientific opinion that clearly states the need of representative data on the occurrence of toxoplasmosis and the distribution of the parasite in Europe (EFSA, 2007). Recently, different options of obtaining T. gondii-free meat have been discussed (Kijlstra and Jongert, 2008). It is thus of great importance to collect reliable data on the actual prevalence of T. gondii in meat-producing animals, but also on the prevalence of oocyst-shedding by cats to assess the infection-risk for the human population.

In North America and Europe, mainly three T. gondii clonal lineages dominate, designated as clonal Types I, II and III based on PCR-restriction fragment length polymorphism (PCR-RFLP) and microsatellite typing (Howe and Sibley, 1995, Ajzenberg et al., 2002). These clonal types exhibit different levels of virulence in outbred mice. While clonal Type I always causes a lethal infection, the other two clonal types are far less virulent (Sibley and Boothroyd, 1992). In South America and Asia, however, T. gondii genotypes are predominantly atypical and of mixed clonal Types (Lehmann et al., 2006, Dubey et al., 2007). Such genotypes could arise when a feline host ingests prey infected with T. gondii of more than one of the three clonal Types I, II, or III. In these cases, the clonal types may undergo sexual recombination in the feline gut and the resulting progeny would thus represent a mixture of the two parental genotypes (Su et al., 2002, Saeij et al., 2006). More recently, T. gondii with atypical allele combinations have also been observed in Germany (Herrmann et al., 2010). As yet, nothing is known about the T. gondii genotypes present in the animal populations in Switzerland.

The aim of this study was to assess the prevalence of T. gondii oocyst-shedding in cats, using faecal samples obtained from animal shelters, catteries and routine diagnostic samples at the Institute of Parasitology in Bern, as well as the prevalence of T. gondii gDNA in diaphragm samples of cattle, sheep and pigs. In addition, and for the first time in Switzerland, genotyping of oocysts shed by cats and parasite gDNA detected in muscles of meat-producing animals was carried out. Furthermore, parasite gDNA detected in muscle of meat producing animals of a previous study (Wyss et al., 2000) was subjected to genotyping because at the time of that former study, genotyping was not yet done.