The endogenous development and pathogenicity of Eimeria anseris (Kotlan, 1932) in domestic goslings.

Twenty-one, 25-day-old, artificially reared, coccidia-free goslings (Anser cygnoides var. domestica) were inoculated orally with 0.5 × 104, 1 × 104, or 100 × 104 sporulated oocysts of Eimeria anseris and sacrificed at intervals from 24 to 216 h post-inoculation (HPI). Nine uninfected goslings served as negative controls. Parts of the visceral organs from goslings, including the intestines, kidneys, and liver, were fixed, sectioned, and observed microscopically. The results revealed that two generations of meronts occurred in the life cycle of E. anseris. The first generation of meronts developed at 24-96 HPI and the second generation at 90-128 HPI. Each meront contained 4-10 merozoites. Development of gamonts began at 128 HPI and mature oocysts appeared at 168 HPI. Developmental stages presented mainly in the epithelial cells of crypts and lamina propria in the posterior parts of the jejunum and ileum. Parasites localized mostly in the cytoplasm and occasionally in the nuclei of host cells. Histological lesions were pronounced in the jejunum and ileum. Desquamation and necrosis of the epithelium of intestine and crypts, infiltration of inflammatory cells, and hemorrhage and mucosal edema were associated with aggregates of endogenous stages. The infected goslings mainly showed severe diarrhea, depression, anorexia, and emaciation, suggesting that E. anseris is highly pathogenic in goslings.

Differential susceptibility to the Withering Syndrome agent and renal coccidia in juvenile Haliotis rufescens, Haliotis discus hannai and the interspecific hybrid.

Withering Syndrome (WS) is a pathogenic chronic disease caused by the intracellular rickettsial-like bacterium "Candidatus Xenohaliotis californiensis" (WS-RLOs), which affects many abalone species. The renal coccidium (Margolisiella haliotis) has often been observed concurrent with WS infection. The red abalone Haliotis rufescens is a very susceptible species to WS and is also infected by the coccidium M. haliotis. In contrast, the Japanese abalone Haliotis discus hannai is not infected by these parasites. Interspecific hybridization is a method for improving important traits in animal husbandry. The objective of this study was to determine susceptibility to WS-RLO and M. haliotis infection in the hybrid generated from a cross between red and Japanese abalones. Juveniles from both species and the interspecific hybrid were challenged by exposure to effluent from red abalone adults infected with both parasites. The animals were analyzed by histology at 130days post-challenge. A 33% prevalence WS-RLOs was observed in the red abalone H. rufescens, whereas a 20% prevalence was observed in the hybrid. Infections were graded on a scale of 0-3. Of these red abalones infected, 53% presented grade 1 infection intensity, 10% had grade 2 infections, and 50% had grade 3 infections. However, the hybrids only presented intensities at the extremes of the scale; of those infected 33% showed grade 1 infections and 66% had grade 3 infections. The coccidium prevalence was 7% in red abalone individuals and 13% in the hybrid abalone. In contrast, the Japanese abalone did not present infections with either parasite. As with the prevalence, the infection intensities for the coccidium were higher in the hybrid abalone; of those infected 25% had grade 2 infections, and 75% had grade 3 infections, but the red abalone presented only grade 2 infection intensities. Therefore, the hybrid did not inherited non-susceptibility or resistance characteristics of the parental H. discus hannai and possessed biological conditions that could foster development of both parasites. Development of a culture based on this hybrid abalone should consider its susceptibility to infection by coccidian, WS-RLOs and the potential for developing the WS disease.

Microenvironment pH modified solid dispersion of Toltrazuril as a new strategy to improve the treatment of experimental Apicomplexan infection.

The phylum Apicomplexa contains some of the most serious human and veterinary parasites, including Eimeria magna, Toxoplasma gondii, and many others. Toltrazuril (TOL) has activity against multiple stages of Apicomplexan parasites, but its clinical use is limited by low bioavailability. In present study, we prepared one new formulation named the microenvironment pH modified solid dispersion (pHM-SD), which was composed of three components including Ca(OH)2, TOL, and PVPk30 with the weight ratio of 1:8:8. In vivo evaluation for bioavailability and efficacy of the pHM-SD was conducted following oral administration and hypodermic injection. The performance of the pHM-SD was also contrast to corresponding results of raw material drug and commercial Baycox® to evaluate the advantages for clinical application. The results showed that the bioavailability of prototype TOL and its active metabolites toltrazuril sulfoxide (TOLSO), toltrazuril sulfone (TOLSO2) in rabbits were improved remarkably after oral administration of the pHM-SD. The safety of the pHM-SD via oral administration was adequately verified via the histopathological examination. We subsequently evaluated effects of the pHM-SD on Eimeria magna oocysts and Toxoplasma gondii tachyzoites. In vivo anti-coccidia efficacy further confirmed that the pHM-SD could be used as a strategy to minimize the oocyst exposure. In vitro cytotoxicity and anti-Toxoplasma tests showed that the pHM-SD had little damage to host cells within the concentration of 100 µg/ mL, and the anti-Toxoplasma efficacy was significantly improved compared with TOL. Combined with the above-mentioned experimental results, we conclude that the pHM-SD maybe a promising candidate for providing better clinical outcomes.

Detection and characterization of Hyaloklossia kasumiensis (Apicomplexa: Sarcocystidae) from Nagoya Daruma pond frog, Pelophylax porosus brevipodus.

The Nagoya Daruma pond frog (Pelophylax porosus brevipodus) is an endangered anuran species endemic to the semi-aquatic environment of Japan. On June 11, 2021, four of the six Nagoya Daruma pond frogs captured from the wild and kept in an aquarium died in rapid succession. Pathological and/or parasitological examinations of two of the four carcasses revealed protozoan infection in their kidneys. The pathogen identified as Hyaloklossia kasumiensis (Apicomplexa: Sarcocystidae: Hyaloklossiinae) based on their morphological and molecular characterization. This is a new host record of H. kasumiensis. Mitochondrial cox1 sequence analysis suggest that the present species detected from Nagoya Daruma pond frog belongs to the same lineage as those found in the Tokyo Daruma pond frog (P. p. porosus) from Ibaraki Prefecture, Japan. This particular lineage may be prevalent among Pelophylax porosus ssp. distributed in Japan.

Structural and Biochemical Features of Eimeria tenella Dihydroorotate Dehydrogenase, a Potential Drug Target.

Dihydroorotate dehydrogenase (DHODH) is a mitochondrial monotopic membrane protein that plays an essential role in the pyrimidine de novo biosynthesis and electron transport chain pathways. In Eimeria tenella, an intracellular apicomplexan parasite that causes the most severe form of chicken coccidiosis, the activity of pyrimidine salvage pathway at the intracellular stage is negligible and it relies on the pyrimidine de novo biosynthesis pathway. Therefore, the enzymes of the de novo pathway are considered potential drug target candidates for the design of compounds with activity against this parasite. Although, DHODHs from E. tenella (EtDHODH), Plasmodium falciparum (PfDHODH), and human (HsDHODH) show distinct sensitivities to classical DHODH inhibitors, in this paper, we identify ferulenol as a potent inhibitor of both EtDHODH and HsDHODH. Additionally, we report the crystal structures of EtDHODH and HsDHODH in the absence and presence of ferulenol. Comparison of these enzymes showed that despite similar overall structures, the EtDHODH has a long insertion in the N-terminal helix region that assumes a disordered configuration. In addition, the crystal structures revealed that the ferulenol binding pocket of EtDHODH is larger than that of HsDHODH. These differences can be explored to accelerate structure-based design of inhibitors specifically targeting EtDHODH.

Intranuclear coccidiosis in tortoises - discovery of its causative agent and transmission.

Intranuclear coccidiosis of testudines (known as TINC) is an emerging disease in chelonians. Although endogenous stages were repeatedly detected in various tissues, attempts to find the oocysts in faeces failed, leaving the question of the transmission and classification of the causative agent of TINC unresolved. We recorded small spherical oocysts (∼6-7 µm in diameter) of an eimeriid coccidium in faeces of a leopard tortoise (Stigmochelys pardalis). Sporulated oocysts were used for the experimental oral inoculation of juvenile coccidia-free tortoises representing 5 species (S. pardalis, Testudo graeca, T. hermanni, T. horsfieldii, and Geochelone sulcata). The oocysts' association with TINC was confirmed based on clinical signs, histopathological findings of intranuclear endogenous stages of the coccidium in many organs (including intestine), and by the partial 18S rDNA sequence analysis of the DNA isolated from organs of the experimentally infected animals and from a single naturally infected as well as from all experimentally infected tortoises. Breeding colonies of chelonians should be screened for this pathogen in order to prevent its further spread and unwanted introduction into endangered free-ranging chelonian populations.

Susceptibility to Various Coccidiostats in the Murine Coccidian Parasite Eimeria krijgsmanni.

INTRODUCTION: Murine Eimeria spp. have been used as effective experimental models of disease instead of large mammalian hosts such as cattle. We here examine drug susceptibility of the uncharacterized murine intestinal protozoan parasite, Eimeria krijgsmanni. MATERIALS AND METHODS: The effectiveness of different treatments against infection of E. krijgsmanni was examined for suppression of oocyst shedding: ST mixture ST mixture, pyrimethamine, Ektecin and toltrazuril. RESULTS: ST mixture and pyrimethamine did not suppress oocyst shedding effectively. Although therapeutic efficacy of Ektecin was demonstrated, the dose required was larger than that for cattle and chickens. Oocyst shedding was only completely suppressed completely by continuous administration of toltrazuril. Furthermore, it was confirmed through morphological examination that early developmental stage zoites appeared in host epithelial cells during and following treatment by toltrazuril, and toltrazuril could not eliminate residual zoites in epithelial cells. CONCLUSION: E. krijgsmanni may be relatively resistant to these anti-coccidian agents and might therefore have different characteristics that differ from other coccidia with regard to drug susceptibility.

Novel Characteristics of Mitochondrial Electron Transport Chain from Eimeria tenella.

Eimeria tenella is an intracellular apicomplexan parasite, which infects cecal epithelial cells from chickens and causes hemorrhagic diarrhea and eventual death. We have previously reported the comparative RNA sequence analysis of the E. tenella sporozoite stage between virulent and precocious strains and showed that the expression of several genes involved in mitochondrial electron transport chain (ETC), such as type II NADH dehydrogenase (NDH-2), complex II (succinate:quinone oxidoreductase), malate:quinone oxidoreductase (MQO), and glycerol-3-phosphate dehydrogenase (G3PDH), were upregulated in virulent strain. To study E. tenella mitochondrial ETC in detail, we developed a reproducible method for preparation of mitochondria-rich fraction from sporozoites, which maintained high specific activities of dehydrogenases, such as NDH-2 followed by G3PDH, MQO, complex II, and dihydroorotate dehydrogenase (DHODH). Of particular importance, we showed that E. tenella sporozoite mitochondria possess an intrinsic ability to perform fumarate respiration (via complex II) in addition to the classical oxygen respiration (via complexes III and IV). Further analysis by high-resolution clear native electrophoresis, activity staining, and nano-liquid chromatography tandem-mass spectrometry (nano-LC-MS/MS) provided evidence of a mitochondrial complex II-III-IV supercomplex. Our analysis suggests that complex II from E. tenella has biochemical features distinct to known orthologues and is a potential target for the development of new anticoccidian drugs.