Population genetics of Toxoplasma gondii: new perspectives from parasite genotypes in wildlife

Toxoplasma gondii, a zoonotic protozoal parasite, is well-known for its global distribution and its ability to infect virtually all warm-blooded vertebrates. Nonetheless, attempts to describe the population structure of T. gondii have been primarily limited to samples isolated from humans and domesticated animals. More recent studies, however, have made efforts to characterize T. gondii isolates from a wider range of host species and geographic locales. These findings have dramatically changed our perception of the extent of genetic diversity in T. gondii and the relative roles of sexual recombination and clonal propagation in the parasite's lifecycle. In particular, identification of novel, disease-causing T. gondii strains in wildlife has raised concerns from both a conservation and public health perspective as to whether distinct domestic and sylvatic parasite gene pools exist. If so, overlap of these cycles may represent regions of high probability of disease emergence. Here, we attempt to answer these key questions by reviewing recent studies of T. gondii infections in wildlife, highlighting those which have advanced our understanding of the genetic diversity and population biology of this important zoonotic pathogen.     

The role of rodents and other wildlife in the epidemiology of swine toxoplasmosis

Two swine production units and their contiguous wildlife populations were used for this study. These herds were located 7 miles apart in the major swine producing area of south central Georgia. Herd A had a Toxoplasma gondii antibody prevalence of 27.6% in all ages of swine sampled, with an increased incidence of 13.6% over a 5-month period. This herd was maintained under a semi-range condition. The swine in Herd B were maintained exclusively in concrete floored, enclosed buildings. This herd had a 0.85% T. gondii prevalence. Rodents and a few other wildlife and domestic species were trapped in or around both herds. These animals were examined for Toxoplasma antibodies using the same test procedure utilized for swine sera, the indirect immunofluorescent (IIF) test. Additionally, rodent tissues were homogenized and suspensions prepared for interperitoneal (I/P) inoculation into CF₁ laboratory mice.Rodents and wildlife species examined were: Mus musculus, Peromyscus leucopus, Rattus norvegicus, Sigmodon hispidus, Procyon lotor, and Didelphis marsupalis. Feral and domestic animals other than swine that also were tested for the presence of T. gondii antibodies included two cats, two horses, and a dog. The overall prevalence of T. gondii antibodies in all non-porcine species examined was 67% for those animals in and around the premises of Herd A, and 63% for those in and around Herd B.Toxoplasma infectivity of rodents whose tissues were processed and inoculated into laboratory mice correlated well with the results of the IIF test on the serum of these wild rodents. No tachyzoites of T. gondii were found in the peritoneal exudate of laboratory mice post I/P inoculation with wild rodent tissue, with one exception.While there was no significant difference in Toxoplasma infectivity in non-porcine species on these two premises, management practices appeared to be the determining factor in swine infection with T. gondii. Excluding wildlife precluded infection.     

Prevalence of serum antibodies to Toxoplasma gondii in free-ranging cats on Tokunoshima Island,Japan

The prevalence of Toxoplasma gondii infection in free-ranging cats on Tokunoshima Island was assessed by testing 125 serum samples using anti-T. gondii IgG indirect enzyme-linked immunosorbent assay. The overall seropositivity rate was 47.2% (59/125). Seropositivity rates in cats with body weight >2.0 kg (57.4%) were significantly higher than in those with body weight ≤2.0 kg (12.5%, P<0.01). Analysis of the number of seropositive cats by settlement revealed the presence of possibly-infected cats in 17 of 23 settlements, indicating the widespread prevalence of T. gondii on the island. This is the first study to show the seroprevalence of T. gondii in free-ranging cats on Tokunoshima Island. The information revealed in this paper will help to prevent the transmission of T. gondii among cats and also in both wild and domestic animals and humans on the island.     

Sporadic endemicity of zoonotic Paragonimus in raccoon dogs and Japanese badgers from Miyazaki Prefecture,Japan

Paragonimiasis is a zoonotic trematode infection caused by Paragonimus spp. To determine the recent status of Paragonimus infections in wild animals, this study investigated Paragonimus spp. in 39 raccoon dogs and 54 Japanese badgers from March 2019 to January 2021 in Miyazaki Prefecture, and examined metacercariae in freshwater crabs. Triploid P. westermani was found in one raccoon dog (2.6%), and metacercariae were recovered from Eriocheir japonica captured near the infected animal collected. One Japanese badger (1.9%) harbored P. skrjabini miyazakii; this prevalence was lower than the approximately 30% that was reported in the 1970s. Results indicated that zoonotic Paragonimus was sporadically prevalent in wild animals. Further investigation in various animals is awaited to elucidate current wildlife reservoirs for those Paragonimus.     

Evidence for widespread Leishmania infantum infection among wild carnivores in L. infantum periendemic northern Spain

Leishmania spp. infection was investigated in tissue samples of wild carnivores from the Spanish Basque Country (BC), by PCR and DNA sequencing. The region is at the northern periphery of Leishmania infantum endemic Iberian Peninsula and infection in the dog (reservoir) or other species has not been previously reported. Leishmania kinetoplast DNA was detected by real-time PCR (rtPCR) in 28% (44/156) of animals. Specifically, in 26% of Eurasian badgers (n = 53), 29% of foxes (n = 48), 29% of stone martens (n = 21) and in 25–50% of less numerous species including genets, wild cats, pole cats, European mink and weasels. Infected animals particularly badgers, were most prevalent in the southernmost province of the BC (Araba) in areas dominated by arable land. Subsequent amplification and sequencing of a fragment of the rRNA internal transcribed spacer 2 (ITS2) from a subset of rtPCR positives samples confirmed the species as L. infantum, showing a high sequence homogeneity with ITS2 sequences of L. infantum from dogs and humans from southern Spain. In summary, this study reports for the first time L. infantum infection in wild carnivores from the BC including in stone martens, pole cats and minks in which infection has not been previously described. It supports the need to study infection in dogs and people in this region and is an example of the value of infection surveillance in wildlife to assess potential risks in the domestic environment and their role in spreading infections in non-endemic areas.     

Toxoplasma gondii antibody prevalence and isolation in free-ranging cats in Okinawa,Japan

Cats are an important host of Toxoplasma gondii from an epidemiological perspective because they are the only definitive hosts that excrete oocysts in their feces. In this study, 201 free-ranging cats in Okinawa were examined for T. gondii infection. Using the latex agglutination test, we detected antibodies against T. gondii in 26.9% (54/201) of the cats. Oocysts of T. gondii were not detected upon microscopic examination of the feces of 128 cats. T. gondii was isolated from the tissues of 9 out of 24 seropositive or pseudo-seropositive cats with a bioassay using laboratory mice. Genotyping for the GRA6 gene revealed that five and four of the isolates were type I and II, respectively.     

Alterations in selected serum biochemistry values of free-ranging Bornean leopard cats (Prionailurus bengalensis borneoensis) captured by box traps

Box-traps for capturing wild cats are widely used by researchers since it is one of the most effective methods for trapping these species. Although they are extensively utilised, the effects on the physiology of trapped felids remain unclear. Researchers frequently make judgements regarding the safety of such capture devices by examination of external injuries but often fail to take into consideration other physiological parameters. To assess the effects of capture events on selected serum biochemistry values of free-ranging Bornean leopard cats (Prionailurus bengalensis borneoensis) six free-ranging leopard cats (four males, two females) were trapped by using box-traps in Sabah, Malaysian Borneo. Blood was collected by jugular venipucture after chemical immobilization with a mixture of tiletamine and zolazepam. Blood was analysed for 17 serum biochemistry parameters. The most consistent and significantly higher value found in both sexes was aspartate aminotransferase (AST), followed by high mean value of alanine aminotransferase (ALT). Both mean values exceeded the upper limit of the reference range for captive leopard cats. These results demonstrate that captured leopard cats by box-traps undergo physical exertion and consequently some type of muscle injury/damage. Researchers and wildlife managers should be aware of the physiological response of trapped felids when using box-traps. Devices that facilitate the prompt removal of leopard cats from the traps would be useful for researchers to avoid further damage while live trapping this species.     

A sero-epidemiological survey of Toxoplasma gondii infection in free-range and caged chickens in northeast China

Acquisition of Toxoplasma gondii infections is mainly through ingestion of parasite-contaminated food. T. gondii oocyst distribution in the living environment of human and livestock is directly linked to the prevalence of the parasite infection in humans and domestic animals. In this study, we investigated the sero-prevalence of T. gondii infection in free-range as well as caged chicken in northeast China. The purpose of this study was to investigate the potential prevalence of T. gondii oocysts in the environments. Sera of 308 free-range chickens and 210 caged chickens collected in three areas in northeast China were tested for anti-T. gondii antibodies with ELISA assays. The infection rates of free-range and caged chickens were 34.7% and 2.8% respectively, indicating that the parasite is widely distributed in the environment and poses threatens to the health of people living in those areas.     

Cats and Toxoplasma: Implications for Public Health

Cats are popular as pets worldwide because they are easy to care for and provide companionship that enriches the lives of human beings. Little attention has been focused on their potential to contaminate the environment with zoonotic pathogens. One such pathogen, the protozoan parasite Toxoplasma gondii, rarely causes clinical manifestations in cats or immunocompetent humans; however, it can have serious adverse effects on human foetuses and immunocompromised patients. Many human infections are believed to be acquired from eating undercooked or raw meat, such as pork and lamb ( Tenter et al. Int. J. Parasitol., 30, 2000, 1217 ; Dubey et al. J. Parasitol. 91, 2005, 1082 ). However, the prevalence of T. gondii infection in human populations that do not consume meat or eat it well‐cooked suggests that the acquisition of infection from the environment, via oocysts in soil, water or on uncooked vegetables, is also important ( Rawal. Trans. Royal Soc. Trop. Med. Hyg., 53, 1959, 61 ; Roghmann et al. Am. J. Trop. Med. Hyg., 60, 1999, 790 ; Chacin‐Bonilla et al. Am. J. Trop. Med. Hyg., 65, 2001, 131 ). In the past 20 years, two changes occurred that significantly increased the size of the cat population in the USA. Pet cat ownership grew from 50 million to 90 million animals, and animal welfare activists created feeding stations for abandoned and free‐roaming cats. As many cat owners allow their cats to deposit faeces outside and cats maintained in colonies always defecate outside, ample opportunity exists for T. gondii oocysts to enter the environment and be transmitted to humans. Prevention efforts should focus on educating cat owners about the importance of collecting cat faeces in litter boxes, spaying owned cats to reduce overpopulation, reducing the numbers of feral cats and promoting rigorous hand hygiene after gardening or soil contact.     

Comparison of nested PCR and real-time PCR for the detection of Toxoplasma gondii in biological samples from naturally infected cats

There are different protocols of molecular diagnosis methods available including DNA extraction methods to diagnose of Toxoplasma gondii, being necessary to perform comparative studies in biological samples. The aim of this study is to compare real-time PCR (rtPCR) and nested PCR (nPCR) to evaluate the detection of T. gondii in naturally infected cats. Biological samples of Toxoplasma-seropositive cats were assayed for detection of T. gondii DNA - extracted by both the lysis buffer and proteinase K (LB proteinase K) method and the acid guanidinium thiocyanate (GuSCN) method - using rtPCR and nPCR. T. gondii DNA was detected by nPCR in 43.6% and 40.8% of the samples from which it was extracted by the LB proteinase K and the GuSCN method, respectively. With rtPCR these figures fell significantly to 33.8% and 14.1%. Despite of nPCR showed higher sensitivity, the agreement observed between two PCRs was good; this agreement, however, was affected by the DNA extraction method used, LB proteinase K method showed better results.     

Evaluation of a Direct Agglutination Test for Detection of Antibodies Against Toxoplasma gondii in Cat,Pig and Sheep Sera

The protozoan parasite Toxoplasma gondii is the causative agent of the zoonosis toxoplasmosis. In sheep and goats, it is one of the most prevalent causes of infectious abortion. Also in pregnant women, a primary infection can result in miscarriage. Humans acquire the infection either by ingestion of oocysts excreted by cats, the definitive host of the parasite, or by eating raw or undercooked meat from latently infected animals (Dubey & Beattie 1988). In Sweden, toxoplasmosis is a notifiable disease, and cases of clinical disease in humans as well as animals must be reported. In both veterinary and human medicine serological assays based on detecting the humoral antibody response of the host against the parasite are used as diagnostic tools. So far, solid phase assays, such as the indirect fluorescent antibody test (IFAT) and the enzyme-linked immunosorbent assay (ELISA), have been widely used to diagnose T. gondii infection in many species including cats, pigs and sheep (Dubey & Beattie 1988). However, both IFAT and ELISA require appropriate anti-species specific immunoglobulins (Ig) that must be carefully evaluated for each species prior to use. This makes these assays complicated and time consuming. Consequently, alternative, simpler methods that do not require specific antisera would be of great value. The direct agglutination test (DA), which is based on the principle that formalin-treated organisms agglutinate in the presence of specific IgG antibodies, is such an assay (Fulton & Turk 1959). The DA-test is widely used in human medicine as a screening test for T gondii infection but it has not yet been thoroughly evaluated for use in veterinary medicine (Uggla & Buxton 1990).     

Distribution of cysts and tachyczoites in calves and pregnant cows inoculated with toxoplasma gondii oocysts

Sixteen calves and 6 cows were each inoculated with 100 000 infective oocytes of the GT-1 strain of Toxoplasma gondii. Cattle were necropsied between 3 and 287 days post-inoculation (DPI) and their tissues were inoculated into mice or fed to Toxoplasma-free cats for the detection of Toxoplasma in bovine tissues. Ten to 10 000-fold more T. gondii were recovered from small intestine and mesenteric lymph nodes of calves at 3 and 6 DPI than from lungs and liver, and the number of T. gondii in bovine tissues was reduced 1000-fold between 6 and 8 DPI. By using the pepsin digestion technique, or feeding tissues to Toxoplasma-free cats, it was demonstrated that T. gondii encysted in bovine tissues as early as 11 DPI and persisted as late as 287 DPI. More Toxoplasma gondii cysts occurred in livers than in any other bovine tissue. Of the 6 cows inoculated at 95–155 days after breeding, 5 delivered normal calves and T. gondii was isolated from only one of these calves. One cow was barren. Toxoplasma gondii was not isolated either by mouse inoculation or by feeding cats tissues from 2 cows killed 132 and 190 DPI. Toxoplasma gondii was not isolated in mice inoculated with tissues of cows killed 98 and 109 DPI, but cats fed on bovine tissues shed T. gondii oocysts. The organism, however, was isolated in mice inoculated with the mesenteric lymph nodes of 1 of the 2 cows killed 162 and 168 DPI, and from the small intestine of the other. Cats fed tissues of these cows later shed T. gondii oocysts.     

Exposure of client-owned cats to zoonotic vector-borne pathogens: Clinic-pathological alterations and infection risk analysis

Zoonotic Vector-Borne Diseases (VBDs) represent a relevant health issue for pets and humans. Italy is a major epidemiological hub for feline VBDs, because of suitable conditions for vector biology and disease transmission patterns. The present study investigated the exposure to major zoonotic arthropod-borne pathogens of cats in Italy, along with the evaluation of clinic-pathological features and a risk factor analysis. Out of 167 examined cats, 52 (31.1%) were seropositive for at least one vector-borne pathogen, being positivity for Bartonella henselae the most recorded (18%). Also, various cats seroreacted for Rickettsia felis (10.8%) and Rickettisa typhi (4.2%), Leishmania infantum (3%), Anaplasma phagocytophilum (2.4%) and Ehrlichia canis (2.4%). Forty-six cats were tested also for antibodies against D. immitis and two (4.3%) scored positive. The statistical analysis showed a positive association between flea infestation and seropositivity to B. henselae, other than an association between the administration of monthly ectoparasiticide treatments and seronegativity for Rickettsia spp.; seropositive cats were older than negative animals and the lifestyle (i.e. indoor vs outdoor) was not correlated with exposure to vector-borne pathogens. The majority of seropositive cats appeared clinically healthy or showed aspecific clinical signs. Around 80% of seropositive cats had one or more biochemical and/or complete blood count abnormalities. The present data confirm the endemicity of zoonotic feline VBDs in Italy and indicate that awareness on arthropod infections and transmitted pathogens should be kept high and possible implemented, towards the protection of animal and human health with adequate surveillance plans.     

Giardia infection of cats and dogs in New Zealand

Giardia intestinalis is a pathogenic protozoan which infects humans and a wide range of animal hosts, including cats and dogs⁽¹⁾. However, the status of animals in New Zealand with respect to Giardia infection has not received much attention, so we undertook a preliminary study of cats and dogs in Palmerston North and Hamilton to determine the prevalence of infection, as indicated by the presence of cysts in faeces.     

High rate of feline immunodeficiency virus infection in cats in the Brazilian semiarid region: Occurrence,associated factors and coinfection with Toxoplasma gondii and feline leukemia virus

To evaluate the occurrence of feline immunodeficiency virus (FIV) and factors associated with this and to demonstrate occurrences of coinfection with Toxoplasma gondii and feline leukemia virus (FeLV) in cats, a total of 103 blood samples were collected from owned cats, during home visits. To diagnose FIV and FeLV, immunochromatographic kit was used and serological diagnoses of T. gondii, the indirect immunofluorescence test was performed. The occurrence of FIV-seropositive cats was 23.3% (24/103) and the factor associated with infection was male sex. T. gondii seropositivity of 53.4% (55/103) was observed and 75% of FIV cases (18/24) were positive for T. gondii coinfection. Only 0.9% (1/103) was positive for FeLV. It can be concluded that the seroprevalence of FIV in cats in the Brazilian semiarid region is high and that FIV positive cats were also likely to be T. gondii seropositive, while FeLV had very low occurrence in the study region.     

Seroprevalence of antibodies to Encephalitozoon cuniculi and Encephalitozoon intestinalis in humans and animals

The presence of antibodies against Encephalitozoon cuniculi (E. cuniculi) and Encephalitozoon intestinalis (E. intestinalis) was examined in 215 samples from humans and in 488 samples from five different species of domestic and companion animals in Slovakia. The 215 human samples and samples from 90 swine, 123 non-infected cattle (cattle), 24 cattle infected with bovine leukosis virus (BLV-positive cattle), 140 sheep and 111 dogs were examined by the enzyme-linked immunosorbent assay (ELISA). Samples with serum titres 1:200 or higher were considered as positive. Specific anti-E. cuniculi antibodies were found in humans (0.9%), swine (52%), cattle (2%), sheep (9%) and dogs (15%) except for the BLV-positive cattle at the titre of 1:200. The titre of 1:400 was detected only in humans (0.5%). The presence of specific anti-E. intestinalis antibodies at the titre of 1:200 was confirmed in humans (6%), swine (51%), cattle (11%), BLV-positive cattle (13%) and dogs (6%) but not in sheep. The anti-E. intestinalis antibodies reached the 1:400 in humans (1%), swine (4%) and BLV-positive cattle (17%). The presence of specific anti-E. intestinalis antibodies at the titre of 1:600 was observed only in one swine (1%). Significant differences were observed in animals at titres 1:200 and 1:400 (chi-squared test: p < 0.0001) for both pathogens and in humans only for E. cuniculi at the titre of 1:400 (chi-squared test: p < 0.0075).     

Serological survey for antibodies to Encephalitozoon cuniculi and Toxoplasma gondii in pet rabbits in eastern coastal areas of China

Encephalitozoon cuniculi (E. cuniculi) is a microsporidian parasite commonly detected in rabbits and can infect humans and cause encephalitozoonosis. And Toxoplasma gondii is a prevalent parasite distributed worldwide and can infect almost all warm-blooded animals, including humans. The aim of the current study was to investigate the seroprevalence of E. cuniculi and Toxoplasma gondii, and risk factors of infection in pet rabbits reared in eastern coastal areas of China (Tianjin, Shandong, Jiangsu, Zhejiang, Shanghai and Fujian). Total 222 blood samples of pet rabbits were collected from local veterinary hospitals. The seropositivity rates of E. cuniculi were 16.22% (36/222) according to an Enzyme-linked immunosorbent assay (ELISA). Female pet rabbits was significantly higher than that in males (P=0.002), Zhejiang were markedly higher than those in Jiangsu and Shanghai (P=0.017, P=0.022), and cross-breed rabbits were dramatically higher than those in Chinchilla, New Zealand white, Rex (P=0.02, P=0.006, P=0.008). The seroprevalence of T. gondii was 13.06% (29/222) by the method of ELISA. The seroprevalence in Zhejiang was significantly higher than that in Shanghai (P=0.017). No difference in seroprevalence was detected with respect to the gender, age, species, health status, or season. These findings show that E. cuniculi and T. gondii are present and spread in pet rabbits. Therefore, pet rabbits should be considered as an important reservoir of encephalitozoonosis for humans and maybe important implication for public health in eastern coastal areas of China.     

Seroprevalence of Toxoplasma gondii and Neospora caninum in sheep intended for human consumption in the Rondônia state,Western Brazilian Amazon

The aim of this study was to carry out a seroepidemiological study of anti-T. gondii and anti-N. caninum antibodies in serum samples from sheep intended for human consumption in the Rondônia state, in the Western Brazilian Amazon, and identify possible risk factors associated with seropositivity. The analysis of the 616 sheep serum samples revealed animal-level seroprevalence of T. gondii in the order of 52.4 % and of N. caninum of 60.6 %. Prevalence of co-infections was 33.4 %. The herd-level seroprevalence was 100 % for both the parasites. Applying of multivariate logistic regression analysis, test of the association between seroprevalence of T. gondii infection and the potential predictors showed that feed, fetal malformation, abortion, neurological problems (lambs), presence of rodents were the significant (P < 0.05) predictors. The variables presence of rodents, cats, wild animals and main activity showed statistical association (P < 0.05) with the seroprevalence of N. caninum. Due to the high prevalence of T. gondii and N. caninum found in this study, the absence of specific slaughterhouses for sheep and raw or undercooked sheep meat for human consumption we concluded that sheep can represent an important source of infection for humans.     

Disease threats to the endangered Iberian lynx (Lynx pardinus)

The Iberian lynx, (Lynx pardinus), is the most endangered felid in the world. To determine whether sympatric carnivores are reservoirs of pathogens posing a disease risk for the lynx, evidence of exposure to 17 viral, bacterial and protozoan agents was investigated in 176 carnivores comprising 26 free-living lynx, 53 domestic cats, 28 dogs, 33 red foxes (Vulpes vulpes), 24 Egyptian mongooses (Herpestes ichneumon), 10 common genets (Genetta genetta) and 2 Eurasian badgers (Meles meles) in the areas inhabited by the last two populations of Iberian lynx, both in Andalusia (South-Western Spain).The results indicated that the lynx had low rates of contact with viral pathogens, with one seropositive finding each for feline leukemia virus, parvovirus and canine adenovirus-1, whereas contact with bacteria and protozoa appeared more frequent. Active infections with parvovirus, Ehrlichia spp., Mycobacterium bovis, Leptospira interrogans and Cytauxzoon spp. were confirmed. In contrast, 53% of the domestic cats were exposed to some infectious agent (prevalence range 4.5–11.4%). Antibodies to canine distemper virus and parvovirus were frequently found in dogs (32% and 42%, respectively) and foxes (30% and 12%). Past or present infections with parvovirus, Ehrlichia spp., Chlamydophila spp., M. bovis, Salmonella enterica, L. interrogans, Toxoplasma gondii, and Neospora caninum were also detected in these and other species surveyed.Questionnaires to owners revealed that 14% of the dogs but none of the cats had been vaccinated, and no cat had been neutered. Based on the apparent absence of acquired immunity of the lynx against infectious agents, the frequent detection of agents among sympatric carnivores, and the reported lack of immunocompetence of the Iberian lynx, a disease outbreak among the local abundant carnivores may pose a serious disease risk for lynx conservation.     

Toxoplasmosis Update and Public Health Implications

Toxoplasma gondii has a coccidian life cycle in the intestine of domestic and wild felids that includes a series of asexual and sexual stages and an oocyst stage that is shed in the feces. Oocysts complete their development outside the body, eventually becoming infective for about 350 species of vertebrates including cats and man. The effects of climate on oocyst survival and the physical and biological means of oocyst dispersal are discussed. Infectivity and pathogenicity for livestock species vary. Acute disease results from rapidly multiplying tachyzoites that may be transmitted by carnivorism, transfusion, vertical transmission and other routes. Patent infections may persist for the life of a host as bradyzoites within tissue cysts. Bradyzoites initiate acute infection in other hosts after carnivorism or organ transplantation or in the same host after immunosuppression. Also discussed are: (a) prevalence of T. gondii in livestock as determined by digestion and serological techniques, (b) identification in humans as accomplished by isolation, serological and skin test techniques and (c) identification in cats as accomplished primarily by fecal examinations for oocysts infective for mice. Source of human infections, major outbreaks, treatment, effects on mental health and methods for preventing toxoplasmosis in man and livestock are listed.