Use of stable isotopes to reveal trophic relationships and transmission of a food-borne pathogen.

Predators in food webs are valuable sentinel species for zoonotic and multi-host pathogens such as Toxoplasma gondii. This protozoan parasite is ubiquitous in warm-blooded vertebrates, and can have serious adverse effects in immunocompromised hosts and foetuses. In northern ecosystems, T. gondii is disproportionately prevalent in Inuit people and wildlife, in part due to multiple routes of transmission. We combined data on T. gondii infection in foxes from Nunavik (northern Québec, Canada) with stable isotope data tracking trophic relationships between foxes and several of their main prey species. Red (Vulpes vulpes) and Arctic fox (Vulpes lagopus) carcasses were collected by local trappers from 2015 to 2019. We used magnetic capture PCR to detect DNA of T. gondii in heart and brain tissues, and enzyme-linked immunosorbent assay to detect antibodies in blood. By linking infection status with diet composition, we showed that infected foxes had a higher probability of consuming aquatic prey and migratory geese, suggesting that these may be important sources of T. gondii transmission in the Arctic. This use of stable isotopes to reveal parasite transmission pathways can be applied more broadly to other foodborne pathogens, and provides evidence to assess and mitigate potential human and animal health risks associated with T. gondii in northern ecosystems.

Epidemiological aspects of the occurrence of anti-Sarcocystis spp. andanti-Toxoplasma gondii antibodies in dogs in the Northern region of Brazil / Aspectos epidemiológicos da ocorrência de anticorpos anti-Sarcocystisspp. e anti-Toxoplasma gondii em cães da região norte do Brasil

We conducted aseroepidemiological study on the occurrence of anti-Sarcocystisspp. and anti-Toxoplasma gondii antibodies in dogs from family farming properties in the municipality of Ji-Paraná, Rondônia.Blood samples were collected from apparently healthy dogs between September 2012 and November 2013. In total, 181 blood serum samples were analyzed using an indirect immunofluorescence assay, among which 57 (31.49%) and 20 (11.04%) were positive for anti-T. gondii and anti-Sarcocystis spp., respectively. Statistical analyses showed that the type of food fed to the dogs was associated with the occurrence of anti-Sarcocystisspp. antibodies. In contrast, age and access to bovine carcasses were the risk factors for anti-T. gondii.The high occurrence of seropositive dogs for Sarcocystis spp. and T. gondii evidences the wide distribution of these agents in the studied area, possibly due to human and animal exposure to these protozoan species. In addition, anti-T. gondii antibodies were directly proportional to dog age. The increase in the number of positive animals with age was statistically significant. Furthermore, high antibody titers (up to 800) against Sarcocystis spp. in dogs suggest the possibility of recent exposure, in addition to environmental contamination by oocysts/sporocysts eliminated by the feces of these animals.

Conduzimos um estudo soroepidemiológico sobre a ocorrência de anticorpos anti- Sarcocystis spp. e anti-Toxoplasma gondiiem cães de propriedades de agricultura familiar no município de Ji-Paraná, Rondônia. Amostras de sangue foram coletadas de cães aparentemente saudáveis, entre setembro de 2012 e novembro de 2013. Ao todo, foram analisados 181 soros sanguíneos por meio do ensaio de imunofluorescência indireta, sendo positivas 57 (31,49%) e 20 (11,04%) amostras para anticorpos anti-T. gondii e anti-Sarcocystis spp., respectivamente. As análises estatísticas demonstraram que o tipo de alimentação fornecida aos cães esteve associado à ocorrência de anticorpos anti-Sarcocystis spp. Em contraste a idade e o acesso à carcaça bovina foram fatores de risco para a presença de anticorpos anti-T. gondii. A alta ocorrência de cães soropositivos para Sarcocystis spp. e T. gondii evidencia a ampla distribuição desses agentes na área estudada, possivelmente devido à exposição humana e animal a essas espécies de protozoários. Além disso, o resultado dos anticorpos anti-T. gondii relacionados a idade do cão mostraram diferença estatística, com aumento significativo no número de animais positivos com a idade. Além disso, altos títulos de anticorpos (até 800) contra Sarcocystis spp. em cães sugerem a possibilidade de exposição recente, além da contaminação ambiental por oocistos/esporocistos eliminados pelas fezes desses animais.

Toxoplasma gondii infection in wild omnivorous and carnivorous animals in Central Europe - A brief overview.

Toxoplasmosis is an important zoonosis caused by the intracellular protozoan Toxoplasma gondii. This parasite is known to infect almost all warm blooded animals, and meat containing tissue cysts is one of the main sources of infection for omnivorous an carnivorous animals. Over recent years, increasing numbers of omnivorous and carnivorous animals have been drawn to urban or suburban areas by easy access to food or safe shelter, and the presence of wild animals has became more natural to urban residents. However, infected animals can act as intermediate hosts to T. gondii and contribute to the transmission of disease to humans and domestic animals, as well as other wild animal species. This extensive spread of the parasite in the natural environment can be attributed to geographic location, landform or local climate. The present paper summarizes the data available on the prevalence of T. gondii infection among wildlife from Poland, Germany, Slovakia, Czechia, Austria and Hungary. The findings highlight the importance of conducting studies on the presence of the parasite in wildlife, where the data is limited or outdated.

INFECTION PRESSURE IS NECESSARY, BUT NOT SUFFICIENT BY ITSELF, TO EXPLAIN TOXOPLASMA GONDII SEROPREVALENCE IN INTERMEDIATE HOST SPECIES.

Parasite infection pressure is suggested to be a strong driver of transmission within ecosystems. We tested if infection pressure drives seroprevalence in intermediate host species for Toxoplasma gondii. We defined Toxoplasma infection pressure to intermediate host species as the combined influence of cat abundance, environmental conditions, and its prevalence in the cat population. We sampled and tested 2 species of rodent and collated information on Toxoplasma seroprevalence in koalas, wallabies, kangaroos, and sheep. All species were sampled using equivalent methods, within a 2-yr period, and from adjacent regions of low and high Toxoplasma infection pressure. The seroprevalence of Toxoplasma in kangaroos scaled with infection pressure, but we observed no statistical difference in seroprevalence for any other species between these 2 regions. Within the region of low infection pressure, Toxoplasma seroprevalence did not differ between species. However, within the region of high Toxoplasma infection pressure, we observed large variation in seroprevalence between species. Our results demonstrate that infection pressure is not sufficient by itself, but merely necessary, to drive Toxoplasma seroprevalence in intermediate host species. Where Toxoplasma seroprevalence in an intermediate host species is already low, further reducing infection pressure will not necessarily further decrease seroprevalence in those species. This has important ramifications for the mitigation of parasite infections and suggests that reductions in Toxoplasma infection pressure, intended to reduce infections, may be most effective and applicable to species that are known to experience high rates of infection.

Recent aspects on epidemiology, clinical disease, and genetic diversity of Toxoplasma gondii infections in Australasian marsupials.

BACKGROUND: Toxoplasma gondii infections are common in humans and animals worldwide. Among all intermediate hosts of T. gondii, captive marsupials from Australia and New Zealand are highly susceptible to clinical toxoplasmosis. However, most free-range marsupials establish chronic T. gondii infection. Infected marsupial meat may serve as a source of T. gondii infection for humans. Differences in mortality patterns in different species of kangaroos and other marsupials are not fully understood. Lifestyle, habitat, and the genotype of T. gondii are predicted to be risk factors. For example, koalas are rarely exposed to T. gondii because they live on treetops whereas wallabies on land are frequently exposed to infection. METHODS: The present review summarizes worldwide information on the prevalence of clinical and subclinical infections, epidemiology, and genetic diversity of T. gondii infecting Australasian marsupials in their native habitat and among exported animals over the past decade. The role of genetic types of T. gondii and clinical disease is discussed. RESULTS: Fatal toxoplasmosis has been diagnosed in captive Australasian marsupials in Argentina, Chile, China, Germany, Hungary, Japan, Spain, Turkey, and the USA. Most deaths occurred because of disseminated toxoplasmosis. Genetic characterization of T. gondii strains isolated from fatal marsupial infections identified Type III as well as atypical, nonclonal genotypes. Fatal toxoplasmosis was also diagnosed in free-ranging wombats (Vombatus ursinus) in Australia. Genetic characterization of DNA amplified directly from host tissues of subclinical culled kangaroos at slaughter identified many mixed-strain infections with both atypical and recombinant genotypes of T. gondii. CONCLUSIONS: Most Australasian marsupials in their native land, Australia and New Zealand, have high prevalence of T. gondii, and kangaroo meat can be a source of infection for humans if consumed uncooked/undercooked. The genotypes prevalent in kangaroos in Australia and New Zealand were genetically distinct from those isolated or genotyped from most macropods in the USA and other countries. Thus, clinical toxoplasmosis in marsupials imported from Australia is most likely to occur from infections acquired after importation.

EPIDEMIOLOGIC AND PUBLIC HEALTH SIGNIFICANCE OF TOXOPLASMA GONDII INFECTIONS IN BEARS (URSUS SPP.): A 50 YEAR REVIEW INCLUDING RECENT GENETIC EVIDENCE.

Toxoplasma gondii infections are common in humans and animals worldwide. The present review summarizes worldwide information on the prevalence of clinical and subclinical infections, epidemiology, and genetic diversity of T. gondii infections in bears. Seroprevalence estimates of T. gondii in black bears (Ursus americanus) are one of the highest of all animals. In Pennsylvania, seroprevalence is around 80% and has remained stable for the past 4 decades. Approximately 3,500 bears are hunted yearly in Pennsylvania alone. The validity of different serological tests is discussed based on bioassay and serological comparisons. Seroprevalence in grizzly bears (Ursus arctos) is lower than that in black bears. Even polar bears (Ursus maritimus) are infected; infections in these animals are ecologically interesting because of the absence of felids in the Arctic. Clinical toxoplasmosis in bears is rare and not documented in adult animals. The few reports of fatal toxoplasmosis in young bears need confirmation. Viable T. gondii has been isolated from black bears and a grizzly bear. The genetic diversity of isolates based on DNA from viable T. gondii isolates is discussed. Genetic typing of a total of 26 T. gondii samples from bears using 10 PCR-RFLP markers revealed 8 PCR-RFLP ToxoDB genotypes: #1 (clonal type II) in 3 samples, #2 (clonal type III) in 8 samples, #4 (haplogroup 12) in 3 samples, #5 (haplogroup 12) in 3 samples, #74 in 5 samples, #90 in 1 sample, #147 in 1 sample, and #216 in 2 samples. These results suggest relatively high genetic diversity of T. gondii in bears. Overall, T. gondii isolates in bears range from those circulating in a domestic cycle (genotypes #1 and #2) to those mainly associated with wildlife (such as genotypes #4 and #5, together known as haplogroup 12). A patient who acquired clinical Trichinella spiralis infection after eating undercooked bear meat also acquired T. gondii infection. Freezing of infected meat kills T. gondii, including the strains isolated from bears.

Toxoplasma gondii infection in domestic and wild felids as public health concerns: a systematic review and meta-analysis.

Felidae as definitive hosts for Toxoplasma gondii play a major role in transmission to all warm-blooded animals trough oocysts dissemination. Therefore the current comprehensive study was performed to determine the global status of T. gondii infection in domestic and wild felids aiming to provide comprehensive data of interest for further intervention approaching the One Health perspective. Different databases were searched by utilizing particular key words for publications related to T. gondii infecting domestic and wild feline host species, worldwide, from 1970 to 2020. The review of 337 reports showed that the seroprevalence of T. gondii in domestic cats and wild felids was estimated in 37.5% (95% CI 34.7-40.3) (I2 = 98.3%, P < 0.001) and 64% (95% CI 60-67.9) (I2 = 88%, P < 0.0001), respectively. The global pooled prevalence of oocysts in the fecal examined specimens from domestic cats was estimated in 2.6% (95% CI 1.9-3.3) (I2 = 96.1%, P < 0.0001), and that in fecal samples from wild felids was estimated in 2.4% (95% CI 1.1-4.2) (I2 = 86.4%, P < 0.0001). In addition, from 13,252 examined soil samples in 14 reviewed studies, the pooled occurrence of T. gondii oocysts was determined in 16.2% (95% CI 7.66-27.03%). The observed high rates of anti-T. gondii antibodies seroprevalence levels and oocyst excretion frequency in the felids, along with soil (environmental) contamination with oocysts may constitute a potential threat to animal and public health, and data will result of interest in further prophylaxis programs.

Epidemiologic and Public Health Significance of Toxoplasma gondii Infections in Venison: 2009-2020.

Toxoplasma gondii infections are common in humans and animals worldwide. The ingestion of food or water contaminated with oocysts excreted by infected cats or ingestion of uncooked or undercooked meat containing tissue cysts of T. gondii are the 2 major modes of transmission of T. gondii. Deer are a popular game. Recently, outbreaks of clinical toxoplasmosis were reported in humans in North America linked to ingestion of undercooked venison. Here, we review prevalence, persistence of infection, clinical disease, epidemiology, and public health risks of T. gondii infections in deer and other cervids for the past decade. Estimates of worldwide serological prevalence are summarized individually for each species of deer, elk, moose, and caribou. Genetic diversity of 112 viable isolates of T. gondii from cervids is discussed, including its public health significance. Prevalence of T. gondii in deer is very high. Any part of a deer, including liver, spleen, and muscles, should be cooked thoroughly before human consumption.

A hotspot of Toxoplasma gondii Africa 1 lineage in Benin: How new genotypes from West Africa contribute to understand the parasite genetic diversity worldwide.

Through international trades, Europe, Africa and South America share a long history of exchanges, potentially of pathogens. We used the worldwide parasite Toxoplasma gondii to test the hypothesis of a historical influence on pathogen genetic diversity in Benin, a West African country with a longstanding sea trade history. In Africa, T. gondii spatial structure is still non-uniformly studied and very few articles have reported strain genetic diversity in fauna and clinical forms of human toxoplasmosis so far, even in African diaspora. Sera from 758 domestic animals (mainly poultry) in two coastal areas (Cotonou and Ouidah) and two inland areas (Parakou and Natitingou) were tested for T. gondii antibodies using a Modified Agglutination Test (MAT). The hearts and brains of 69 seropositive animals were collected for parasite isolation in a mouse bioassay. Forty-five strains were obtained and 39 genotypes could be described via 15-microsatellite genotyping, with a predominance of the autochthonous African lineage Africa 1 (36/39). The remaining genotypes were Africa 4 variant TUB2 (1/39) and two identical isolates (clone) of Type III (2/39). No difference in terms of genotype distribution between inland and coastal sampling sites was found. In particular, contrarily to what has been described in Senegal, no type II (mostly present in Europe) was isolated in poultry from coastal cities. This result seems to refute a possible role of European maritime trade in Benin despite it was one of the most important hubs during the slave trade period. However, the presence of the Africa 1 genotype in Brazil, predominant in Benin, and genetic analyses suggest that the triangular trade was a route for the intercontinental dissemination of genetic strains from Africa to South America. This supports the possibility of contamination in humans and animals with potentially imported virulent strains.

Investigation of the role of stray cats for transmission of toxoplasmosis to humans and animals living in Izmir, Turkey.

INTRODUCTION: Toxoplasma gondii is a protozoan parasite that has a widespread distribution among mammalians and birds. One of the reasons for the high prevalence may be due to ingesting oocyst disseminated by stray cats' feces. In Turkey, most of the citizens are closely associated with stray cats and they love to pet and feed them on the streets. In this study, we aimed to determine the prevalence of T. gondii DNA in feces of stray cats living in Izmir, Turkey in order to identify the transmission potential to humans and other animals. METHODOLOGY: Feces and blood samples of 465 stray cats were investigated for the presence of T. gondii oocysts by microscopy and for the presence of T. gondii DNA by two real time PCR methods. Furthermore, serum samples were analyzed for anti-T. gondii IgG antibodies using an ELISA. RESULTS: Oocysts were detected in 0.43% of the stray cats by microscopy. T. gondii DNA was detected in 14.37% of the stray cats' feces samples. The seroprevalence rate was 37.84%. In the feces and/or blood PCR positive group, 35.89% of them were seropositive. Among the 176 seropositive cats, T. gondii DNA was detected in feces of 27 cats (15.34%). CONCLUSIONS: This study first time showed the inter relation of T. gondii DNA in feces and blood samples and seropositivity. In sum, over 14% of the stray cats living outdoor may have an important role in transmission of toxoplasmosis to humans in Izmir as well as to other animals.

Vertical transmission of Toxoplasma gondii in naturally infected ewes in the semiarid region of Brazil.

To evaluate transplacental transmission of Toxoplasma gondii in naturally infected ewes, blood samples were collected from 55 pregnant ewes and their offspring, before ingestion of colostrum. From 16 offspring of positive ewes and nine offspring from negative ewes, blood samples were obtained after 48 h and 7, 14, 21, 28, 35, 42, 49 and 56 days after birth. T. gondii antibodies were detected in serum samples using the indirect fluorescence antibody test (IFAT ≥ 64). Four of the 30 positive ewes (13.3 %) had offspring positive for T. gondii before ingesting colostrum (vertical transmission). The colostrum antibody titers decreased every week, and only 20 % (2/10) of the lambs in continued to present detectable antibody titers until day 56 after birth. Therefore, vertical transmission of T. gondii in lambs was indication of occur and is an important route for transferring and maintaining the agent in sheep herds in the Brazilian semiarid region.

Public Health Significance of Toxoplasma gondii Infections in Cattle: 2009-2020.

Toxoplasma gondii infections are common in humans and animals worldwide. The ingestion of food or water contaminated with oocysts excreted by infected cats or ingesting uncooked or undercooked meat containing tissue cysts of T. gondii are the 2 major modes of transmission of T. gondii. Viable T. gondii is more prevalent in pork and lamb than in beef. In the past decade, there have been many articles on the high seroprevalence in cattle, particularly from China. There is a report of an outbreak of acute toxoplasmosis in humans suspected to be linked to the ingestion of Artisan fresh cheese from cow's milk. There are conflicting reports concerning the rate of congenital transmission of T. gondii in cattle, especially from Brazil. In a report from Brazil, viable T. gondii was isolated from the blood of 1 of 60 pregnant cows slaughtered at an abattoir and from 1 fetus. The role of beef in the epidemiology of T. gondii infections is still not clear. Here, we review prevalence, persistence of infection, clinical disease, epidemiology, and public health risks of T. gondii infections in cattle from beef and cow's milk worldwide for the past decade.

Fractional seroprevalence rates in common prey species can cause more than half of feral cats to be exposed to Toxoplasma gondii annually.

Rodents comprise a major component of cat (Felis catus) diets in many ecosystems, and life cycle diagrams of Toxoplasma gondii typically depict small rodents as quintessential intermediate hosts. Counter-intuitively, small rodents often experience a lower T. gondii seroprevalence than do larger sympatric mammals. This observation has repeatedly caused confusion about the relative importance of small rodents to the ecology of T. gondii. To address this confusion, we modified the Reed-Frost epidemic model to develop a simple binomial equation to model T. gondii transmission from prey to feline predators. This equation takes into account variations in prey seroprevalence and the frequency with which they are consumed by felids. Even when T. gondii seroprevalence in prey is < 1%, computation reveals that the risk of feline exposure to T. gondii can easily exceed 50 % annually. For example, if cats eat an average of 1 mouse per day, a seroprevalence of 0.2 % (1/500) in mice will cause 51.9 % of cats to be exposed to T. gondii annually. Our simple equation demonstrates that both prey seroprevalence and the rate at which prey are consumed are of approximately equal importance to the ecology of T. gondii. When inferring the importance of various prey species to the ecology of T. gondii, researchers must consider the predation and dietary habits of felids from within their study system. Our simple binomial equation could also be used to predict T. gondii exposure rates of humans or other carnivorous animals from various dietary sources or be applied to other predator-prey parasite life cycles.

Seroprevalence of Toxoplasma gondii Infection and Associated Factors in Sheep from Jalisco, Mexico.

The objective of the study was to identify the seroprevalence of anti-Toxoplasma gondii antibodies in sheep herds from 3 municipalities from Jalisco, Mexico, as well as estimate the association between seroprevalence and certain factors presents in the farms. In total, 12 sheep farms that maintain only hair breeds were included in the work. From these farms, 336 blood samples were collected, corresponding 324 to ewes and 12 rams. Serum samples were subjected to ELISA test, and the association between the frequency of antibodies and some potential risk factors was estimated. The overall seroprevalence to anti-T. gondii antibodies in the population studied was 17.8% (60/336; 95% confidence interval [C.I.] 14-22), all farms had positive animals, and the seroprevalence of antibodies ranged between 7 to 32%. Seroprevalence in specific municipalities was 18.7% in Lagos de Moreno, 17.8% in Encarnación de Díaz, and 16.9% in San Juan de los Lagos. Seroprevalence in ewes was 17.5% (57/324; 95% C.I. 13-22), and seroprevalence in rams was 25% (3/12; 95% C.I. 6-57), while among breeds it was 17.8% in Pelibuey (20/112; 95% C.I. 11-26), 16.6% in Kathadin (14/84; 95% C.I. 9-26), 15.4% in Blackbelly (13/84; 95% C.I. 8-25), and 23.2% in Dorper (13/56; 95% C.I. 13-36); no differences were observed among breeds (p < 0.05). The presence of cats on the farms was associated with seroprevalence (odds ratio [OR] 2.8; 95% C.I. 1.8-7.3, p < 0.001), as was the absence of a rodent-control program (OR 1.5; 95% C.I. 0.8-3.2, p < 0.05). No other factors were identified as associated with seroprevalence.

CD209 C-Type Lectins Promote Host Invasion, Dissemination, and Infection of Toxoplasma gondii.

Toxoplasma gondii, the causative agent of toxoplasmosis and a major opportunistic parasite associated with AIDS, is able to invade host cells of animals and humans. Studies suggested that the ability of host invasion by the tachyzoite, the infectious form of T. gondii, is essential for the pathogenicity to promote its dissemination to other parts of animal hosts. However, the detailed molecular mechanisms for host invasion and dissemination of the parasites are not clear. On the other hand, viruses and bacteria are able to interact with and hijack DC-SIGN (CD209) C-type lectin on antigen presenting cells (APCs), such as dendritic cells and macrophages as the Trojan horses to promote host dissemination. In this study, we showed that invasion of T. gondii into host cells was enhanced by this parasite-CD209 interaction that were inhibited by ligand mimicking-oligosaccharides and the anti-CD209 antibody. Furthermore, covering the exposures of DC-SIGN by these oligosaccharides reduced parasite burden, host spreading and mortality associated with T. gondii infection. These results suggested that interaction of T. gondii to APCs expressing DC-SIGN might promote host dissemination and infection. Can the blockage of this interaction with Mannan and/or anti-CD209 antibody be developed as a prevention or treatment method for T. gondii infection?

GENOTYPE IDENTIFICATION OF TOXOPLASMA GONDII IN MACROPODS FROM A ZOOLOGICAL PARK IN FLORIDA, USA.

There are limited reports of the genetic characterization of Toxoplasma gondii infecting captive macropods in North America. A novel genotype, ToxoDB PCR-RFLP genotype 263, was reported from six wallabies at a zoological facility in Virginia, USA, prompting an investigation into the genotypes from T. gondii strains infecting macropods at a zoological park in Florida, USA. Cardiac muscle and/or lung samples from an agile wallaby (Macropus agilis, n = 1), red kangaroos (Macropus rufus, n = 8), red-necked wallaby (Macropus rufogriseus, n = 1), and a tammar wallaby (Macropus eugenii, n = 1) that died between 2014 and 2018 were collected. All 11 cases were confirmed to have died from systemic toxoplasmosis by histopathology and immunohistochemical staining. Multilocus PCR-RFLP genotyping of T. gondii was performed directly on tissue samples or on parasites isolated from myocardium by mouse bioassay. Two cases of toxoplasmosis were identified as the reported novel genotype, ToxoDB PCR-RFLP genotype 263, but no common source of exposure could be identified. Five cases were identified as genotype 2 (type III strain, haplogroup 3), and four cases were identified as genotype 216, which has been previously reported in North American wildlife. There were no overt differences in lesion severity or distribution related to genotype. These results suggest that the premise was contaminated with at least three genotypes of T. gondii causing systemic toxoplasmosis in macropods. The largest cluster of fatal toxoplasmosis in macropods in the study period occurred following severe rainfall flooding of the exhibit, suggesting the transmission of T. gondii by water and pointing out the importance of this transmission mechanism. In summary, our study revealed three T. gondii outbreaks that caused significant loss of macropods within 5 yr in a zoological facility in Florida. More studies are needed to understand transmission and prevention of toxoplasmosis in sensitive zoo animals.

Toxoplasma gondii tkl1 Deletion Mutant Is a Promising Vaccine against Acute, Chronic, and Congenital Toxoplasmosis in Mice.

In this study, we generated a tkl1 deletion mutant in the Toxoplasma gondii type 1 RH (RHΔtkl1) strain and tested the protective efficacies of vaccination using RHΔtkl1 tachyzoites against acute, chronic, and congenital T. gondii infections in Kunming mice. Mice vaccinated with RHΔtkl1 mounted a strong humoral and cellular response as shown by elevated levels of anti-T. gondii-specific IgG, IL-2, IL-12, IFN-γ, and IL-10. All RHΔtkl1-vaccinated mice survived a lethal challenge with 1 × 103 tachyzoites of type 1 RH or ToxoDB#9 (PYS or TgC7) strain as well as 100 cysts or oocysts of Prugniuad strain. All mock-vaccinated plus infected mice have died. Vaccination also protected against cyst- or oocyst-caused chronic infection, reduced vertical transmission caused by oocysts, increased litter size, and maintained body weight of pups born to dams challenged with 10 oocysts on day 5 of gestation. In contrast, all mock-vaccinated plus oocysts-infected dams had aborted, and no fetus has survived. Vaccinated dams remained healthy postinfection, and their brain cyst burden was significantly reduced compared with mock-vaccinated dams infected with oocysts. In vivo depletion of CD4+ T cells, CD8+ T cells, and B cells revealed that CD8+ T cells are involved in the protection of mice against T. gondii infection. Additionally, adoptive transfer of CD8+ T cells from RHΔtkl1-vaccinated mice significantly enhanced the survival of naive mice infected with the pathogenic strain. Together, these data reaffirm the importance of CD8+ T cell responses in future vaccine design for toxoplasmosis and present T. gondii tkl1 gene as a promising vaccine candidate.

Toxoplasma gondii infection in meat-producing small ruminants: Meat juice serology and genotyping.

The consumption of ovine and caprine meat is considered one of the major transmission routes for Toxoplasma gondii infection in humans. The present study aimed at obtaining epidemiological and molecular data on T. gondii infection in small ruminants slaughtered or commercialized in Italy. Meat juices from 227 sheep and 51 goats were analyzed with a commercial ELISA and antibodies were detected in 28.6% sheep and 27.5% goats. A significant difference was highlighted between adult sheep and the other considered categories (young sheep, young and adult goats) concerning the detection of antibodies (94.1%; p-value = .008). Muscles of positives samples were submitted to molecular analysis, and T. gondii DNA was detected in 15 sheep and three goats; sequencing of B1 gene showed that all belonged to Type II. The present study confirmed small ruminants' meat as a possible source of T. gondii infection for consumers eating raw or undercooked meat, particularly in those countries where the consumption of sheep and goats' meat products is a traditional gastronomic habit.

Combining spatial analysis and host population genetics to gain insights into the mode of transmission of a pathogen: The example of Toxoplasma gondii in mice.

Toxoplasma gondii is a ubiquitous highly prevalent zoonotic protozoan. Cats are the definitive hosts, while all other warm-blooded animals are intermediate hosts for this parasite. Commensal rodents, being the main prey of cats, are probably the major reservoir for T. gondii in the domestic environment. Rodents can acquire infection after ingestion of oocysts that have sporulated in the environment. However, experimental evidence shows that vertical transmission can be sufficient for the perpetuation of transmission between generations of mice. In natural settings, the relative epidemiological importance of vertical transmission over oral transmission is a matter of debate and raises the question of the possibility of a T. gondii cycle in the absence of cats. In the present study, we took advantage of an extensive survey of commensal rodents in Dakar, Senegal, where the house mouse is the predominant putative reservoir of T. gondii. Mice genotypes and spatial location through GPS referencing of all trapping localizations were investigated in relation to T. gondii infection in eight sites of the city of Dakar and on Goree Island. In each sampling site, the occurrence of over-prevalence zones of T. gondii infection was investigated through Kulldorf's statistic using SaTScan software. Genetic structure and relatedness between mice were investigated within each over-prevalence zone, in order to find clues of transmission between related mice. Within each of the four over-prevalence zones identified across nine sites, infected mice belonged to more than one genetic group. No association between the degree of relatedness and the occurrence of T. gondii infection could be detected. These findings suggest an environmental source of infection for mice associated with localized putative foci of environmental contamination and support an oral route of infection for mice from Dakar rather than a cycle based on vertical transmission. However, further investigations based on a denser sampling in different epidemiological contexts are recommended.

Genotyping of viable Toxoplasma gondii from the first national survey of feral swine revealed evidence for sylvatic transmission cycle, and presence of highly virulent parasite genotypes.

Feral swine are known reservoirs of various pathogens, including Toxoplasma gondii. Here, we report the first national survey of viable T. gondii in feral swine in the USA. We paired serological surveys with parasite isolation and bioassay to evaluate the prevalence and genetic diversity of these parasites. From 2012-2017, sera and tissues from 1517 feral swine across the USA were collected for the isolation of viable T. gondii. Serum samples were initially screened for antibodies to T. gondii, and then the tissues of seropositive feral swine were bioassayed in mice. Antibodies were detected in 27.7% of feral swine tested by the modified agglutination test (1:25 or higher). Antibody positive rates increased significantly with age, with 10.1% of juveniles, 16.0% of sub-adults and 38.4% of adults testing seropositive. Myocardium (50 g) from 232 seropositive feral swine was digested in pepsin and bioassayed in mice. Viable T. gondii was isolated from 78 feral swine from 21 states. Twelve of the 78 isolates were pathogenic to outbred Swiss Webster mice and 76 of the 78 isolates could be propagated further in cell culture and were genotyped. For genotyping, deoxyribonucleic acid extracted from cell culture-derived tachyzoites was characterized by polymerase chain reaction restriction fragment length polymorphism using the genetic markers SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1 and Apico. Genotyping revealed 15 ToxoDB genotypes, including 43 isolates for genotype #5 (haplogroup 12), 11 isolates for #24, four isolates for #2 (haplogroup 3), two isolates for each of genotypes #3 (haplogroup 2), #4 (haplogroup 12), #216, #221, #289 and #297 and one isolate for each of genotypes #1 (haplogroup 2), #39, #66, #260, #261 and #299. Genotype #5 was the most frequently isolated, accounted for 57% (43/76) of the isolates, followed by #24, accounted for 14% (11/76). Genotypes #260, #289, #297 and #299 are new types. Genotype #289 was highly virulent to mice and originated from feral swine collected in Louisiana on the same day at the same location. Genotype #216 was previously demonstrated to be highly virulent to mice. Our results indicate moderate genetic diversity of T. gondii in feral swine in the USA, with the genotype #5 (haplogroup 12) dominant in the continental USA, whereas genotype #24 (10/14) was dominant in Hawaii, suggesting different population structures of the parasites among the two distinct geographical locations.