Detection of Cyclospora cayetanensis in Food and Water Samples: Optimized Protocols for Specific and Sensitive Molecular Methods from a Regulatory Agency Perspective.

Cyclospora cayetanensis is a coccidian parasite of the phylum Apicomplexa that causes cyclosporiasis, a human-specific gastrointestinal disease. Unlike most enteric pathogens, C. cayetanensis does not infect via direct fecal-oral transmission between humans because shed oocysts must be exposed to environmental triggers prior to becoming infectious. The development of specific and sensitive detection methods for C. cayetanensis is crucial to effectively address data gaps and provide regulatory support during outbreak investigations. In this study, new more specific molecular markers for detection of C. cayetanensis were developed based on updated genomic databases of Apicomplexa mitochondrial sequences. Novel alternative reagents and supplies, as well as optimization protocols were tested in spiked produce and agricultural water samples. The selected Mit1C primers and probe combined showed at least 13 mismatches to other related species. The new optimized qualitative real-time PCR assay with modifications to sample processing and replacement of discontinued items produced results comparable to the previously validated methods. In conclusion, the new optimized qualitative Mit1C real-time PCR assay demonstrated an increase in its specificity in comparison to other detection methods previously published, while it showed to be robust and as sensitive as the previous validated method at the FDA. This study has also expanded the array of PCR reagents that can be used to detect C. cayetanensis in produce and agricultural water samples and provided several improvements to the method for detection in agricultural water including replacements for discontinued items and a new dialysis filter for water filtration.

The first Cyclospora cayetanensis lineage A genome from an isolate from Mexico.

BACKGROUND: Cyclospora cayetanensis is a protozoan parasite that causes intestinal illness in humans worldwide. Despite its global distribution, most genomic data for C. cayetanensis has been obtained from isolates collected in the United States, leaving genetic variability among globally distributed isolates underexplored. RESULTS: In the present study, the genome of an isolate of C. cayetanensis obtained from a child with diarrhea living in Mexico was sequenced and assembled. Evaluation of the assembly using a lineage typing system recently developed by the Centers for Disease Control and Prevention revealed that this isolate is lineage A. CONCLUSIONS: Given that the only other whole genome assembly available from Mexico was classified as lineage B, the data presented here represent an important step in expanding our knowledge of the diversity of C. cayetanensis isolates from Mexico at the genomic level.

Editorial for the Special Issue Cyclospora cayetanensis and Cyclosporiasis.

Cyclosporiasis is a foodborne diarrheal illness caused by the parasite Cyclospora cayetanensis [...].

Development and Evaluation/Verification of a Fully Automated Test Platform for the Rapid Detection of Cyclospora cayetanensis in Produce Matrices.

Cyclosporiasis, caused by the coccidian parasite Cyclospora cayetanensis, has emerged as an increasing global public health concern, with the incidence of laboratory-confirmed domestically acquired cases in the US exceeding 10,000 since 2018. A recently published qPCR assay (Mit1C) based on a mitochondrial target gene showed high specificity and good sensitivity for the detection of C. cayetanensis in fresh produce. The present study shows the integration and verification of the same mitochondrial target into a fully automated and streamlined platform that performs DNA isolation, PCR, hybridization, results visualization, and reporting of results to simplify and reduce hands-on time for the detection of this parasite. By using the same primer sets for both the target of interest (i.e., Mit1C) and the internal assay control (IAC), we were able to rapidly migrate the previously developed Mit1C qPCR assay into the more streamlined and automated format Rheonix C. cayetanensisTM Assay. Once the best conditions for detection were optimized and the migration to the fully automated format was completed, we compared the performance of the automated platform against the original "bench top" Mit1C qPCR assay. The automated Rheonix C. cayetanensis Assay achieved equivalent performance characteristics as the original assay, including the same performance for both inclusion and exclusion panels, and it was able to detect as low as 5 C. cayetanensis oocysts in fresh produce while significantly reducing hands-on time. We expect that the streamlined assay can be used as a tool for outbreak and/or surveillance activities to detect the presence of C. cayetanensis in produce samples.

Cyclospora cayetanensis: A Perspective (2020-2023) with Emphasis on Epidemiology and Detection Methods.

Cyclospora cayetanensis infections are prevalent worldwide, and the parasite has become a major public health and food safety concern. Although important efforts have been dedicated to advance toward preventing and reducing incidences of cyclosporiasis, there are still several knowledge gaps that hamper the implementation of effective measures to prevent the contamination of produce and water with Cyclospora oocysts. Some of these data gaps can be attributed to the fact that access to oocysts is a limiting factor in C. cayetanensis research. There are no animal models or in vivo or in vitro culture systems to propagate the oocysts needed to facilitate C. cayetanensis research. Thus, researchers must rely upon limited supplies of oocysts obtained from naturally infected human patients considerably restricting what can be learnt about this parasite. Despite the limited supply of C. cayetanensis oocysts, several important advances have happened in the past 3 years. Great progress has been made in the Cyclospora field in the areas of molecular characterization of strains and species, generation of genomes, and development of novel detection methods. This comprehensive perspective summarizes research published from 2020 to 2023 and evaluates what we have learnt and identifies those aspects in which further research is needed.

Development of a targeted amplicon sequencing method for genotyping Cyclospora cayetanensis from fresh produce and clinical samples with enhanced genomic resolution and sensitivity.

Outbreaks of cyclosporiasis, an enteric illness caused by the parasite Cyclospora cayetanensis, have been associated with consumption of various types of fresh produce. Although a method is in use for genotyping C. cayetanensis from clinical specimens, the very low abundance of C. cayetanensis in food and environmental samples presents a greater challenge. To complement epidemiological investigations, a molecular surveillance tool is needed for use in genetic linkage of food vehicles to cyclosporiasis illnesses, estimation of the scope of outbreaks or clusters of illness, and determination of geographical areas involved. We developed a targeted amplicon sequencing (TAS) assay that incorporates a further enrichment step to gain the requisite sensitivity for genotyping C. cayetanensis contaminating fresh produce samples. The TAS assay targets 52 loci, 49 of which are located in the nuclear genome, and encompasses 396 currently known SNP sites. The performance of the TAS assay was evaluated using lettuce, basil, cilantro, salad mix, and blackberries inoculated with C. cayetanensis oocysts. A minimum of 24 markers were haplotyped even at low contamination levels of 10 oocysts in 25 g leafy greens. The artificially contaminated fresh produce samples were included in a genetic distance analysis based on haplotype presence/absence with publicly available C. cayetanensis whole genome sequence assemblies. Oocysts from two different sources were used for inoculation, and samples receiving the same oocyst preparation clustered together, but separately from the other group, demonstrating the utility of the assay for genetically linking samples. Clinical fecal samples with low parasite loads were also successfully genotyped. This work represents a significant advance in the ability to genotype C. cayetanensis contaminating fresh produce along with greatly expanding the genomic diversity included for genetic clustering of clinical specimens.

Molecular Detection of Cyclospora cayetanensis in Two Main Types of Farm Soil Using Real-Time PCR Assays and Method Modification for Commercial Potting Mix.

Cyclospora cayetanensis is a foodborne protozoan parasite that causes outbreaks of diarrheal illness (cyclosporiasis) with clear seasonality worldwide. In the environment, C. cayetanensis oocysts are very robust, and contact with contaminated soil may serve as an important vehicle in the transmission of this organism, and it is considered a risk factor for this infection. The present study evaluated a flotation concentration method, previously shown to provide the best detection results when compared with DNA isolation directly from soil samples, in two main types of farm soil, silt loam soil and sandy clay loam, as well as in commercial potting mix samples inoculated with different numbers of C. cayetanensis oocysts. The flotation method was able to detect as few as 10 oocysts in 10 g of either type of farm soil without modifications, but needed an extra wash and samples of reduced size for the processing of the commercial potting mix to be able to detect 20 oocysts/5 g. A recently modified real-time PCR method for the detection of C. cayetanensis based on a mitochondrial gene target was also evaluated using selected samples of each type of soil. This comparative study confirmed that the concentration of oocysts in soil samples by flotation in high-density sucrose solutions is a sensitive method that can detect low numbers of oocysts in different types of soil.

Development and Single Laboratory Evaluation of a Refined and specific Real-time PCR Detection Method, Using Mitochondrial Primers (Mit1C), for the Detection of Cyclospora cayetanensis in Produce.

Regulatory methods for detection of the foodborne protozoan parasite Cyclospora cayetanensis must be specific and sensitive. To that end, we designed and evaluated (in a single laboratory validation) a novel and improved primer/probe combination (Mit1C) for real-time PCR detection of C. cayetanensis in produce. The newly developed primer/probe combination targets a conserved region of the mitochondrial genome of C. cayetanensis that varies in other closely related organisms. The primer/probe combination was evaluated both in silico and using several real-time PCR kits and polymerases against an inclusivity/exclusivity panel comprised of a variety of C. cayetanensis oocysts, as well as DNA from other related Cyclospora spp. and closely related parasites. The new primer/probe combination amplified only C. cayetanensis, thus demonstrating specificity. Sensitivity was evaluated by artificially contaminating cilantro, raspberries, and romaine lettuce with variable numbers (200 and 5) of C. cayetanensis oocysts. As few as 5 oocysts were detected in 75%, 67.7%, and 50% of the spiked produce samples (cilantro, raspberries, and romaine lettuce), respectively, all uninoculated samples and no-template real-time PCR controls were negative. The improved primer/probe combination should prove an effective analytical tool for the specific detection of C. cayetanensis in produce.

Seroepidemiological study of Toxoplasma gondii in equids in different European countries.

Toxoplasmosis, caused by the obligate intracellular protozoan Toxoplasma gondii, is a worldwide parasitic zoonosis. A cross-sectional study was carried out to determine the exposure to T. gondii in equids in Europe. Serum samples from 1399 equids (1085 horses, 238 donkeys, and 76 mules/hinnies) bred in four European countries (Italy, Spain, United Kingdom [UK], and Ireland) were collected during the period of 2013-2021. The overall seroprevalence of T. gondii was 18.9% (95% Confidence Interval [CI]: 16.9-21.0) by using the modified agglutination test (MAT) at a cut-off of 1:25. Seropositivity by country was 27.1% in Italy, 16.6% in Spain, 12.0% in UK and 7.0% in Ireland. Anti-T. gondii antibodies were detected in 12.8% of the horses, 43.7% of the donkeys, and in 28.9% of the mules/hinnies. A Generalized Estimating Equation (GEE) analysis was carried out to study the associations between seropositivity and explanatory variables related to individuals, herds, and management measures on these herds, selected based on the bivariate analysis. The risk for being seropositive for T. gondii was 5.3 and 2.7 times higher in donkeys and mules/hinnies than in horses, respectively. In addition, significantly higher seropositivity was observed in horses from herds that used disinfectants less than once a week (13.9%; p = 0.038, odds ratio [OR] = 1.6; 95% CI: 1.03-2.62) compared with those from herds that performed weekly disinfection of the facilities (9.4%). This is the first large-scale seroepidemiological study on T. gondii comprising horses, donkeys, and mules/hinnies in Europe and the first report of T. gondii exposure in horses from Ireland and UK. We found a widespread distribution of T. gondii among equid populations in different European countries. The seroprevalence found in these species, especially in donkeys and mules/hinnies, highlights the potential risk of human infection through the consumption of their raw/undercooked milk or meat.

Seroprevalence and genetic characterization of Toxoplasma gondii in domestic pigs intended for human consumption in Cuba.

Domestic pigs are considered as one of the main intermediate hosts in the zoonotic transmission of Toxoplasma gondii in many countries. Serological and molecular studies are warranted to better understand the epidemiology and transmission patterns of this parasite worldwide. To date, seroepidemiological information on T. gondii in domestic pigs in Cuba is very scarce and there are no reports of T. gondii genotypes circulating in this country. Here, we aimed to estimate the seroprevalence of T. gondii and provide genetic characterization of the strains circulating in slaughtered pigs intended for human consumption in Central Cuba. Seroprevalence was determined in 450 serum samples from slaughtered pigs in Villa Clara province using ELISA. Anti-Toxoplasma gondii IgG antibodies were detected in 100 animals (22.2%, 95% CI: 18.5-26.2). Conventional PCR of the 529-bp marker of T. gondii was performed in hearts and diaphragm tissues of all ELISA-seropositive pigs. Toxoplasma gondii DNA was detected in four animals. Further genetic characterization of the positive DNA samples was performed by multilocus PCR-RFLP and PCR-sequencing typing tools. Molecular analysis revealed four different genetic profiles that were combinations of type I, II, III and u-1 alleles, suggesting the circulation of non-clonal genotypes of T. gondii in domestic pigs in Cuba. Our results indicate that T. gondii is widely distributed in slaughtered pigs in this country, which might have important implications for public health. To the best of our knowledge, this is the first report on genetic characterization of T. gondii in Cuba. Although preliminary, the results suggest a high genetic diversity of T. gondii in the study region. Further studies based on parasite isolation are needed to definitively identify the genotypes circulating and characterize the virulence of strains detected in pigs in Cuba, and to assess the risk of zoonotic transmission from pork products in this country.

Targeted next generation sequencing of Cyclospora cayetanensis mitochondrial genomes from seeded fresh produce and other seeded food samples.

Cyclospora cayetanensis, a coccidian apicomplexan parasite, causes large outbreaks of foodborne diarrheal disease globally. Tracking the source of C. cayetanensis oocyst contamination in food items is essential to reduce, even prevent outbreaks. We previously showed that a genotyping method based on mitochondrial single nucleotide polymorphism (SNP) profiles had discriminatory power in classifying C. cayetanensis clinical isolates. In food specimens, low level contamination by oocysts and difficulties in DNA extraction present significant challenges in genotyping method development. Here, we report the development of a highly sensitive, custom-designed, targeted sequencing method based on the Illumina AmpliSeq platform; our method was capable of consistently generating near-complete mitochondrial genome sequences of C. cayetanensis from foods with low levels of contamination. To simulate environmentally observed contamination levels in foods, we seeded various food matrices, such as fresh produce and prepared dishes, with known quantities of oocysts, and isolated genomic DNA from washed food samples. Using the Ampliseq Targeted Sequencing method, we obtained near-complete mitochondrial genome sequences of C. cayetanensis from food samples seeded with as low as five to ten oocysts and used the data in downstream analysis. The flexibility of the AmpliSeq platform could potentially allow for more genomic targets to be added to achieve higher discriminatory power. This level of sensitivity in capturing high resolution genome data from contaminated food samples is a critical milestone towards the potential development of a comprehensive genotyping method for C. cayetanensis.

Development of a Molecular Marker Based on the Mitochondrial Genome for Detection of Cyclospora cayetanensis in Food and Water Samples.

Cyclospora cayetanensis is a coccidian parasite that causes diarrheal illness outbreaks worldwide. The development of new laboratory methods for detection of C. cayetanensis is of critical importance because of the high potential for environmental samples to be contaminated with a myriad of microorganisms, adversely impacting the specificity when testing samples from various sources using a single molecular assay. In this study, a new sequencing-based method was designed targeting a specific fragment of C. cayetanensis cytochrome oxidase gene and developed as a complementary method to the TaqMan qPCR present in the U.S. FDA BAM Chapter 19b and Chapter 19c. The comparative results between the new PCR protocol and the qPCR for detection of C. cayetanensis in food and water samples provided similar results in both matrices with the same seeding level. The target region and primers in the protocol discussed in this study contain sufficient Cyclospora-specific sequence fidelity as observed by sequence comparison with other Eimeriidae species. The sequence of the PCR product appears to represent a robust target for identifying C. cayetanensis on samples from different sources. Such a sensitive method for detection of C. cayetanensis would add to the target repertoire of qPCR-based screening strategies for food and water samples.

Comparative Evaluation of an Easy Laboratory Method for the Concentration of Oocysts and Commercial DNA Isolation Kits for the Molecular Detection of Cyclospora cayetanensis in Silt Loam Soil Samples.

Cyclospora cayetanensis is a protozoan parasite that causes foodborne outbreaks of diarrheal illness (cyclosporiasis) worldwide. Contact with soil may be an important mode of transmission for C. cayetanensis and could play a role in the contamination of foods. However, there is a scarcity of detection methods and studies for C. cayetanensis in soil. Traditional parasitology concentration methods can be useful for the detection of C. cayetanensis, as found for other protozoa parasites of similar size. The present study evaluated a concentration method using flotation in saturated sucrose solution, subsequent DNA template preparation and qPCR following the Bacteriological Analytical Manual (BAM) Chapter 19b method. The proposed flotation method was compared to three commercial DNA isolation kits (Fast DNATM 50 mL SPIN kit for soil (MP Biomedicals, Irvine, CA, USA), Quick-DNATM Fecal/Soil Microbe Midiprep kit (Zymo Research, Irvine, CA, USA) and DNeasy® PowerMax® Soil Kit (Qiagen, Hilden, Germany)) for the isolation and detection of DNA from experimentally seeded C. cayetanensis soil samples (5−10 g with 100 oocysts). Control unseeded samples were all negative in all methods. Significantly lower cycle threshold values (CT) were observed in the 100 oocyst C. cayetanensis samples processed via the flotation method than those processed with each of the commercial DNA isolation kits evaluated (p < 0.05), indicating higher recovery of the target DNA with flotation. All samples seeded with 100 oocysts (n = 5) were positive to the presence of the parasite by the flotation method, and no inhibition was observed in any of the processed samples. Linearity of detection of the flotation method was observed in samples seeded with different levels of oocysts, and the method was able to detect as few as 10 oocysts in 10 g of soil samples (limit of detection 1 oocyst/g). This comparative study showed that the concentration of oocysts in soil samples by flotation in high-density sucrose solutions is an easy, low-cost, and sensitive method that could be implemented for the detection of C. cayetanensis in environmental soil samples. The flotation method would be useful to identify environmental sources of C. cayetanensis contamination, persistence of the parasite in the soil and the role of soil in the transmission of C. cayetanensis.

Genome Sequence Analysis and Characterization of Shiga Toxin 2 Production by Escherichia coli O157:H7 Strains Associated With a Laboratory Infection.

A laboratory-acquired E. coli O157:H7 infection with associated severe sequelae including hemolytic uremic syndrome occurred in an individual working in the laboratory with a mixture of nalidixic acid-resistant (NalR) O157:H7 mutant strains in a soil-biochar blend. The patient was hospitalized and treated with an intravenous combination of metronidazole and levofloxacin. The present study investigated the source of this severe laboratory acquired infection and further examined the influence of the antibiotics used during treatment on the expression and production of Shiga toxin. Genomes of two Stx2a-and eae-positive O157:H7 strains isolated from the patient's stool were sequenced along with two pairs of the wt strains and their derived NalR mutants used in the laboratory experiments. High-resolution SNP typing determined the strains' individual genetic relatedness and unambiguously identified the two laboratory-derived NalR mutant strains as the source of the researcher's life-threatening disease, rather than a conceivable ingestion of unrelated O157:H7 isolates circulating at the same time. It was further confirmed that in sublethal doses, the antibiotics increased toxin expression and production. Our results support a simultaneous co-infection with clinical strains in the laboratory, which were the causative agents of previous O157:H7 outbreaks, and further that the administration of antibiotics may have impacted the outcome of the infection.

Seroepidemiology of Toxoplasma gondii in wild ruminants in Spain.

Toxoplasmosis is a parasitic zoonosis caused by Toxoplasma gondii which infects warm-blooded species worldwide. Humans can be infected through ingestion of tissue cysts from raw or undercooked meat, including game meat. A nationwide large-scale cross-sectional study was conducted to assess exposure to T. gondii in seven wild ruminant species in Spain. A total of 2,040 serum samples from 77 sampling sites randomly distributed in the five bioregions (BRs) covering mainland Spain were tested for antibodies against T. gondii using the modified agglutination test. The overall seroprevalence was 22.0% (449/2,040). Seroprevalence by species in decreasing order was as follows: 39.6% (141/356) in roe deer (Capreolus capreolus), 37.1% (138/372) in fallow deer (Dama dama), 16.6% (92/553) in red deer (Cervus elaphus), 14.0% (26/186) in Southern chamois (Rupicapra pyrenaica), 11.5% (24/209) in mouflon (Ovis aries musimon), 7.8% (27/346) in Iberian wild goat (Capra pyrenaica) and 5.6% (1/18) in Barbary sheep (Ammotragus lervia). Seropositivity was detected in 74.0% (57/77) of the sampling sites. Results indicate widespread but not homogeneous exposure to T. gondii in wild ruminant populations in Spain during the last two decades and highlight differences related to animal species and spatial distribution of these species in this country; this implies potential consequences of T. gondii for animal health, conservation and public health.

Seroprevalence of Toxoplasma gondii and associated risk factors in domestic pigs raised from Cuba.

A cross-sectional study was carried out to determine the seroprevalence of Toxoplasma gondii and associated risk factors in pigs in the largest pork-producing region in Cuba. Serum samples from 420 pigs, including 210 sows and 210 post-weaning pigs, were tested for antibodies against T. gondii using a commercial indirect enzyme-linked immunosorbent assay. Anti-T. gondii antibodies were detected in 56 animals (13.3%, 95% CI: 10.1-16.6). A generalized estimating equations model revealed that the risk factors associated with higher seropositivity in pigs were altitude (higher in farm's location < 250 m above sea level (masl) versus ≥ 250 masl) and age (higher in sows compared to post-weaning pigs). The results indicated that this protozoan parasite is widely distributed on pig farms in the study area, which is a public health concern since the consumption of raw or undercooked pork meat products containing tissue cysts is considered one of the main routes of T. gondii transmission worldwide. Control measures should be implemented to reduce the risk of exposure to T. gondii in pigs in Cuba.

Detection of Cyclospora cayetanensis on bagged pre-cut salad mixes within their shelf-life and after sell by date by the U.S. food and drug administration validated method.

Recently, outbreaks of Cyclospora cayetanensis in the U.S. were linked to the consumption of a variety of salads containing romaine and/or iceberg lettuce, carrots and/or red cabbage. The Bacteriological Analytical Manual (BAM) Chapter 19b method was validated for the detection of C. cayetanensis in carrots, cabbage and romaine lettuce, but has not been previously evaluated in ready-to-eat (RTE) salad mixes. In addition, the only samples available for traceback investigations are sometimes leftovers in bad conditions. This study evaluated the validated BAM method for detection of C. cayetanensis in two different RTE mixed salads (mix 1: romaine and iceberg lettuces, carrots, and red cabbage and mix 2: romaine and iceberg lettuces, carrots, red cabbage, radish, and pea pods) in good condition and after their sell by date. Individual samples (25 g) were seeded with five and 200 C. cayetanensis oocysts. Unseeded produce was used as negative control. The method included washing of the produce, concentration and extraction of C. cayetanensis DNA and molecular detection of C. cayetanensis 18 S rRNA gene. As few as five oocysts were detected in both fresh and after sell by date mix salads. All unseeded samples were negative, and all samples of both salad types seeded with 200 oocysts were positive. In samples seeded with 200 oocysts, average 18 S rRNA C. cayetanensis CT values were significantly higher in fresh salad mix 1 compared to fresh salad mix 2; CT values were significantly higher in the after sell by date salads compared to their respective fresh mixes (p < 0.05). In conclusion, the BAM method was able to detect as few as five oocysts even in after sell by date RTE mix salads. However, the differences in detection observed, highlight the importance of evaluating the performance of the validated C. cayetanensis detection method in different food matrices and conditions, in advance for future outbreak investigations.

Epidemiological and Public Health Significance of Toxoplasma gondii Infection in Wild Rabbits and Hares: 2010-2020.

Toxoplasmosis is a zoonosis of global distribution, and Toxoplasma gondii infections are common in humans and animals worldwide. Hares and rabbits are important small game species, and their meat is consumed by humans in many countries. Demand for rabbit meat for human consumption is increasing; therefore, toxoplasmosis in rabbits and hares is of epidemiological significance. Viable T. gondii has been isolated from rabbits. The present review summarizes worldwide information on the seroprevalence, parasitological investigations, clinical cases, isolation, and genetic diversity of T. gondii in wild rabbits, free domestic rabbits, hares, and other rabbits from 2010 to 2020. Differences in prevalence, susceptibility, genetic variants, and clinical implications of T. gondii infection in rabbits and hares are discussed. This review will be of interest to biologists, parasitologists, veterinarians, and public health workers. Additional studies are needed to increase our knowledge of genetic variants and the population structure of T. gondii in rabbits and hares and to understand the differences in susceptibility to T. gondii in hares in different areas.

Seroprevalence of Toxoplasma gondii in outdoor dogs and cats in Bangkok, Thailand.

The aim of this study was to estimate the seroprevalence and risk factors associated with Toxoplasma gondii exposure in dogs and cats from Bangkok, Thailand. Blood samples from 318 dogs and 321 cats were tested for T. gondii antibodies by modified agglutination test (cut-off 1:25). Additionally, 18 dogs and 20 cats were longitudinally sampled for T. gondii antibodies during the same study period, between June and July 2019. The overall seroprevalence in dogs and cats was 7.9% (25/318; 95% CI 4.9­10.8%) and 18.7% (95% CI 14.4­23.0%), respectively. For dogs, risk factors identified were being a mixed-breed animal and living totally outdoors, while increasing age was shown to be a risk factor for cats. Seroconversion was not detected and titres from positive animals remained constant over longitudinal study. The present study indicates that there is a prominent presence of T. gondii in urban and peri-urban areas of Bangkok, suggesting that outdoor dogs and cats should be considered as a possible risk factor for humans.

Seroprevalence and risk factors of Toxoplasma gondii infection in wild ungulates that cohabit in a natural park with human-animal interaction in the Mediterranean ecosystem.

The possibility of Toxoplasma gondii transmitted from game meat to humans is of public health concern. Here we determined seroprevalence and risk factors associated with T. gondii in large game ungulates that cohabit in Sierras de Cazorla, Segura and Las Villas Natural Park (SCSV-NP) (Southern Spain), a natural park with high human-animal interaction. Antibodies against T. gondii in 328 wild ungulates were assayed by the modified agglutination test (MAT ≥ 1:25). Antibodies were found in 39 (11.9%, 95% CI: 8.4-15.4) wild ungulates, with seroprevalence levels of 20.8% in wild boars (Sus scrofa) (5/24), 19.0% in fallow deer (Dama dama) (12/63), 13.9% in Iberian ibex (Capra pyrenaica hispanica) (14/101), 7.9% in red deer (Cervus elaphus) (6/76), and 3.1% in mouflons (Ovis aries musimon) (2/64). Significantly higher seroprevalence was observed in fallow deer and wild boars compared to mouflons. Animals living close to urban areas (<2 km) had 4.6-times higher risk compared to those living at >5 km of urban areas. The results indicate high circulation of T. gondii in wild ungulates in SCSV-NP, which is of animal and public health concern. The increased seroprevalence of T. gondii detected in wildlife ungulates living close to urban areas may increase human infection in those areas if meat from infected animals is consumed raw or undercooked.