Adrian Benedikt Hehl

Toxoplasma gondii and Neospora caninum are closely related apicomplexan parasites of veterinary and public health importance. T. gondii is a zoonotic pathogen for which cats are the definitive host, whereas N. caninum is a major cause of reproductive losses in cattle, with dogs acting as the definitive host. Data on exposure in pet animals in Thailand remain limited. This study investigated seroprevalence and associated risk factors of T. gondii and N. caninum in pet cats and dogs in Pathum Thani Province, an urban area adjacent to Bangkok. Between June 2020 and July 2021, serum samples were collected from 169 owned animals, including 86 cats and 83 dogs, participating in a mobile sterilization program. Antibodies were detected using the indirect fluorescent antibody test (IFAT), and animal characteristics, behaviors, and environmental factors were obtained via owner questionnaires. Serological evidence of exposure to both parasites was detected. Antibodies against T. gondii were detected in 4.73% (8/169) of animals, including 4.65% (4/86) of cats and 4.82% (4/83) of dogs. For N. caninum, the overall seroprevalence was 10.06% (17/169), with a higher prevalence in dogs (15.66%, 13/83) than in cats (4.65%, 4/86). No significant risk factors were identified for T. gondii or N. caninum infection in either cats or dogs (p > 0.05).

Giardia lamblia (syn. intestinalis , duodenalis ) is the causative agent of Giardia sis, a diarrheal disease of global medical importance, especially problematic in young children living in unhygienic, resource-constrained settings. Diagnostics of potential Giardia infections are generally done through classic light-microscopy stool examination. This is often insufficient, and ELISA-based fluorescence detection using costly proprietary reagents is employed. These reagents are often not affordable in contexts where they are needed the most, and this limits their use to resource-rich settings where Giardia sis is rarely problematic. To address these issues in medical equity while designing novel strategies to investigate the Giardia cyst wall, we report on the development and characterisation of alpaca derived single-domain antibodies, known as nanoantibodies, elicited against G. lamblia enriched cyst-wall preparations. We evaluated the effectiveness and binding capacity of twelve unique E. coli -produced recombinant nanoantibody sequences for Giardia cyst wall detection and provide proof of concept for the effectiveness and versatility of these protein domains.

The sporulation of Toxoplasma gondii oocysts is a critical developmental transition transforming non-infective environmental stages into highly resilient a

The objective was to detect T. gondii and various protozoan oocysts in the feces of sheltered cats in Thailand using two molecular approaches, Sanger sequencing and un-targeted Next-generation sequencing (NGS). A total of 166 fecal samples from shelter cats, in 26 samples, Toxoplasma gondii oocyst–like structures were detected. The harvested oocysts were grouped into nine pooled samples. DNA was extracted from all pooled samples and tested using quantitative PCR (qPCR), employing coc1 and coc2 primers, which are commonly used to amplify Apicomplexan DNA. The sequenced of the qPCR products were analyzed by Sanger sequencing. Sequence from all pooled samples had similarity to Cystoisospora spp. To further characterize the oocyst species, NGS was performed. Bioinformatic analysis was conducted using a de novo assembler to generate scaffolds, which were then aligned against a custom database of coccidian whole-genome references. This analysis revealed the presence of T. gondii DNA in three pooled samples. In addition, DNA from other protozoan parasites—including Eimeria spp., Cystoisospora spp., Besnoitia besnoiti, Hammondia hammondi, and Cryptosporidium parvum—was also detected. These findings indicate that T. gondii is circulating among shelter cats, many of which were formerly stray. Moreover, NGS sequencing provided more comprehensive information on the diversity of coccidian species in cat feces compared with Sanger sequencing of PCR-amplified targets.

The sporulation of Toxoplasma gondii oocysts is a critical developmental transition transforming non-infective environmental stages into highly resilient and infective forms capable of zoonotic transmission. Despite significant advances, the molecular regulation underlying this complex process remains poorly understood. We generated comprehensive RNA-Seq datasets capturing gene expression dynamics at three timepoints during oocyst sporulation, to illuminate essential regulatory mechanisms and metabolic adaptations. Employing stringent variance filtering, unsupervised clustering, and differential expression analyses, we identified transcriptional patterns associated with key developmental transitions, notably the involvement of T. gondii Apetala2 (TgAP2) transcription factors and other DNA-binding proteins. We highlight significant shifts in metabolic pathways essential for oocyst environmental resilience and infectivity, including lipid metabolism, amino acid biosynthesis, and specialized secondary metabolism. Furthermore, we investigated expression profiles of genes associated with structural development of oocyst and sporocyst walls, environmental persistence, and host transmission. Importantly, combining these datasets with prior life cycle datasets covering intermediate and final host stages, we close an important knowledge gap by providing a genome-wide mRNA expression profile spanning the entire T. gondii life cycle. Integration with previous foundational transcriptomic and proteomic work provides refined resolution and identifies novel candidate genes and pathways. Collectively, these findings substantially advance our understanding of T. gondii sporulation in the wider context of the development in the cat intestine, highlighting critical molecular events underpinning parasite transmission and environmental adaptation.

The sporulation of Toxoplasma gondii oocysts is a critical developmental transition transforming non-infective environmental stages into highly resilient and infective forms capable of zoonotic transmission. Despite significant advances, the molecular regulation underlying this complex process remains poorly understood. We generated comprehensive RNA-Seq datasets capturing gene expression dynamics at three timepoints during oocyst sporulation, to illuminate essential regulatory mechanisms and metabolic adaptations. Employing stringent variance filtering, unsupervised clustering, and differential expression analyses, we identified transcriptional patterns associated with key developmental transitions, notably the involvement of T. gondii Apetala2 (TgAP2) transcription factors and other DNA-binding proteins. We highlight significant shifts in metabolic pathways essential for oocyst environmental resilience and infectivity, including lipid metabolism, amino acid biosynthesis, and specialized secondary metabolism. Furthermore, we investigated expression profiles of genes associated with structural development of oocyst and sporocyst walls, environmental persistence, and host transmission. Importantly, combining these datasets with prior life cycle datasets covering intermediate and final host stages, we close an important knowledge gap by providing a genome-wide mRNA expression profile spanning the entire T. gondii life cycle. Integration with previous foundational transcriptomic and proteomic work provides refined resolution and identifies novel candidate genes and pathways. Collectively, these findings substantially advance our understanding of T. gondii sporulation in the wider context of the development in the cat intestine, highlighting critical molecular events underpinning parasite transmission and environmental adaptation.

Sexual reproduction of Toxoplasma gondii, confined to the felid gut, remains largely uncharted owing to ethical concerns regarding the use of cats as model organisms. Chromatin modifiers dictate the developmental fate of the parasite during its multistage life cycle, but their targeting to stage-specific cistromes is poorly described^1,2. Here we found that the transcription factors AP2XII-1 and AP2XI-2 operate during the tachyzoite stage, a hallmark of acute toxoplasmosis, to silence genes necessary for merozoites, a developmental stage critical for subsequent sexual commitment and transmission to the next host, including humans. Their conditional and simultaneous depletion leads to a marked change in the transcriptional program, promoting a full transition from tachyzoites to merozoites. These in vitro-cultured pre-gametes have unique protein markers and undergo typical asexual endopolygenic division cycles. In tachyzoites, AP2XII-1 and AP2XI-2 bind DNA as heterodimers at merozoite promoters and recruit MORC and HDAC3 (ref. ¹), thereby limiting chromatin accessibility and transcription. Consequently, the commitment to merogony stems from a profound epigenetic rewiring orchestrated by AP2XII-1 and AP2XI-2. Successful production of merozoites in vitro paves the way for future studies on Toxoplasma sexual development without the need for cat infections and holds promise for the development of therapies to prevent parasite transmission.

Microtubules and specialized microtubule-containing structures are assembled from tubulins, an ancient superfamily of essential eukaryotic proteins. Here, we use bioinformatic approaches to analyze features of tubulins in organisms from the phylum Apicomplexa. Apicomplexans are protozoan parasites that cause a variety of human and animal infectious diseases. Individual species harbor one to four genes each for α- and β-tubulin isotypes. These may specify highly similar proteins, suggesting functional redundancy, or exhibit key differences, consistent with specialized roles. Some, but not all apicomplexans harbor genes for δ- and ε-tubulins, which are found in organisms that construct appendage-containing basal bodies. Critical roles for apicomplexan δ- and ε-tubulin are likely to be limited to microgametes, consistent with a restricted requirement for flagella in a single developmental stage. Sequence divergence or the loss of δ- and ε-tubulin genes in other apicomplexans appears to be associated with diminished requirements for centrioles, basal bodies, and axonemes. Finally, because spindle microtubules and flagellar structures have been proposed as targets for anti-parasitic therapies and transmission-blocking strategies, we discuss these ideas in the context of tubulin-based structures and tubulin superfamily properties.

Toxoplasmosis is one of the most common zoonotic parasitic diseases infecting nearly all warm-blooded animals, including poultry (geese, turkeys, chickens, and ducks). It is caused by Toxoplasma gondii (T. gondii), which is an obligate intracellular protozoan belonging to the Apicomplexa group. In Thailand, duck meat production for domestic consumption and international trade is mainly bred and produced in the central and western parts of the country. Free-grazing ducks in integrated duck–rice production have significant benefits in rice cultivation, accounting for the popularity of this farming system in Thailand. However, ducks are considered particularly susceptible to consuming T. gondii oocysts from water contaminated with cat feces due to the fact of their feeding habits of free-grazing and dabbling. Hence, the prevalence of this zoonotic parasite in a large-scale integrated farming context is particularly challenging with respect to the contamination of the food chain of humans and farm animals. In the present study, we examined the overall prevalence of T. gondii infection in slaughtered free-grazing ducks originating from Central and Western Thailand, setting the stage for an in-depth One Health approach to assess and manage the risks of integrated farming practices. A representative sample size of 161 ducks was calculated using a two-stage sampling method. Specifically, serum samples were collected from 217 slaughtered free-grazing ducks originating in six provinces in Central and Western Thailand. Serum antibodies against T. gondii were detected using an indirect fluorescent antibody test (IFAT). The positive control serum samples were prepared from ducks experimentally immunized with T. gondii. Sixty-eight (31.3%) of the two hundred and seventeen ducks were seropositive with T. gondii. Two groups of fattening ducks and spent layers showed similar seropositivity rates at 29% and 32.3%, with the majority of positive samples being found in the low titer. In addition, a wide distribution of positive serum samples was observed in all six provinces in the present study. To the best of our knowledge, this is the first report on a serological prevalence snapshot in commercially produced duck populations that have high interaction with farmed environments in Thailand, revealing a high infection pressure in areas of integrated duck–rice farming. Importantly, contaminated duck meat for commercial use, as well as offal and carcasses from slaughterhouses, completes the transmission of T. gondii from the environment into the food chain of humans and domestic animals. Hence, from a One Health perspective, it is important to clarify whether this transmission chain extends further to the wild, i.e., predator–prey cycles that are independent of duck farming or are self-contained.

Summary paragraph: Sexual reproduction of Toxoplasma gondii , which is restricted to the small intestine of felids, is sparsely documented, due to ethical concerns surrounding the use of cats as model organisms. Chromatin modifiers dictate the developmental fate of the parasite during its multistage life cycle, but their targeting to stage-specific cistromes is poorly described ¹ . In this study, we found that transcription factors AP2XII-1 and AP2XI-2, expressed in tachyzoite stage that causes acute toxoplasmosis, can silence genes necessary for merozoites, a developmental stage critical for sexual commitment and transmission to the next host, including humans. Their conditional and simultaneous depletion leads to a drastic change in the transcriptional program, promoting a complete transition from tachyzoites to merozoites. Pre-gametes produced in vitro under these conditions are characterized by specific protein markers and undergo typical asexual endopolygenic division cycles. In tachyzoites, AP2XII-1 and AP2XI-2 bind DNA as heterodimers at merozoite promoters and recruit the epigenitors MORC and HDAC3 ¹ , which in turn restrict the accessibility of chromatin to the transcriptional machinery. Thus, the commitment to merogony stems from a profound epigenetic rewiring orchestrated by AP2XII-1 and AP2XI-2. This effective in vitro culture of merozoites paves the way to explore Toxoplasma sexual reproduction without the need to infect kittens and has potential for the development of therapeutics to block parasite transmission.