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Japanese rock ptarmigans (Lagopus muta japonica) are birds that inhabit only alpine regions of central Honshu Island, Japan, known as the Japanese Alps. The number of these birds has recently declined, and in situ and ex situ national conservation programs for Japanese rock ptarmigans have been initiated. The infections of Eimeria spp. as protozoan parasites of the phylum Apicomplexa, E. uekii and E. raichoi , were frequently reported in the birds. However, the virulence of these Eimeria parasites has not been determined. Here, we analyzed the pathogenicity of these Eimeria parasites using experimental infections of a subspecies model of Japanese rock ptarmigans, Svalbard rock ptarmigans (Lagopus mutus hyperboreus), and evaluated acquired protective immunity against challenge in birds tolerant of low-dose inoculation with Eimeria parasites. Following inoculation with two Eimeria parasites derived from Japanese rock ptarmigans (dose range of 4 × 104 to 4 × 102 for E. uekii and 1.7 × 104 to 4 × 101 for E. raichoi), oocysts were detected at 6–8 days post-inoculation (PI), and the maximum number of oocysts per gram of feces was observed 7–10 days PI and then gradually decreased. The mortality rate and reduction in weight gain of chicks increased following high-dose inoculation of oocysts with abnormal feces (soft and diarrhea). Developmental zoites were detected histopathologically in epithelial tissues and sometimes the lamina propria from the duodenum to the colon. Chicks that survived low-dose inoculation did not show clear clinical symptoms after challenge inoculation. Our results suggest that the pathological characteristics of Eimeria parasites infecting Japanese rock ptarmigans include abnormal feces and reduction in weight gain, resulting in mortality in cases of heavy infection due to high-dose inoculation. These findings provide helpful data for Japanese rock ptarmigan conservation efforts. [Display omitted] • Infection with two Eimeria spp. has been frequently reported in Japanese rock ptarmigans. • Pathological characteristics can be abnormal feces and reduction in weight gain, and mortality in heavy infection. • Immunity acquired by low-dose infection gives tolerance to the challenge inoculation. [ABSTRACT FROM AUTHOR]

Japanese rock ptarmigans, Lagopus muta japonica , are classified as an endangered species in Japan and are found only in the Japanese Alps. The number of birds has decreased in the last half century and cage protection projects have been undertaken as in situ conservation strategies (one of the projects for the recovery plan of Japanese rock ptarmigan) in the mountains. During the period with cage protections, some chicks died and two Eimeria spp., E. uekii and E. raichoi , were identified in the chicks. Here, we examined the soil within the cages and in the surrounding environment to assess potential sources of infection between July to August 2020. We found high numbers of oocysts in the cages, especially at the back sides where the ptarmigan family frequently congregated, but soils in other areas outside the cages were less contaminated or not contaminated at all. The time required for more than 50% of the oocysts to sporulate at 15, 20 and 25 °C for E. uekii was 20, 11, and 5 h, respectively, and 72, 48 and 18 h, respectively, for E. raichoi. Our results cast some doubt that coprophagia by chicks is the source of infection because chicks consumed fresh cecal feces (approximately within 1 h) as far as we know, and instead, the protected chicks might be directly or indirectly infected by oocysts in soils or the environment. [Display omitted] • Cage protection is effective for protecting chicks of Japanese rock ptarmigans. • Soils at the back sides in the cages were highly contaminated with Eimeria spp. • E. uekii can rapidly be sporulated at 15 °C in timber regions. • Protected chicks might be infected by oocysts in soils or the environment. [ABSTRACT FROM AUTHOR]

The population of Japanese rock ptarmigan (Lagopus muta japonica), an endangered species with a habitat above the timberline of the southern Japanese Alps, has declined. As one of the recent conservation strategies for this species, cage protection for broods (hens and chicks) has been introduced in their habitats. Two species of Eimeria have frequently been detected in these birds, but little is known about the parasitic circulation in the region, including among birds and in the environment. Here, we conducted histopathology examinations of dead chicks collected under cage protection in 2018, and examined the feces of the hens and chicks of three broods and environmental soils for parasites in 2019 in order to assess the potential sources of infection and pathogenicity. Developmental zoites were found in the epithelial mucosa and/or the submucosa from the duodenum to the colon of all dead chicks. Fecal examination revealed oocysts of E. uekii and/or E. raichoi in all hens and chicks. Oocysts of Eimeria spp. per gram of feces in chicks increased within 2 weeks after hatching and then gradually deceased. Following infection of the chicks, oocysts could accumulate within the cage areas, and oocyst density exceeded more than 1000 oocysts per gram of cage soils. Based on having sporulated morphologies, oocysts could be infective and therefore, be direct or indirect potential sources of infection. However, based on our findings that not all chicks were clinically affected by the infections, other factors such as microbial flora in the chicks established by coprophagy or from the habitat environment, including climate, might be associated with the pathogenicity of Eimeria spp., although further studies are needed to assess these correlations. Image 1 • Cage protection is effective on conservation of Japanese rock ptarmigans. • Hens caring chicks were highly infected with Eimeria spp. and shedded the oocysts. • Potential sources of the infection could be contaminated soils but not coprophagy behaviors of chicks. • Pathogenicity of Eimeria might be also associated with factors like establishment of gut microbiota or habitat conditions. [ABSTRACT FROM AUTHOR]

Abstract Thus far, two types of Eimeria parasites (E. uekii and type B) have been morphologically identified in wild Japanese rock ptarmigans, Lagopus muta japonica. Although high prevalences were reported for these parasites, genetic analyses have not been conducted. We first clarified the phylogenetic positions of two eimerian isolates using genetic analyses of 18S rRNA and mitochondrial cytochrome c oxidase subunit I gene regions. Consequently, of 61 samples examined, 21 and 11 samples were positive for E. uekii and type B, respectively. Additionally, the infection rate increased in the summer. Molecular analyses revealed both Eimeria isolates formed their own clusters; E. uekii was included in clades of chicken Eimeria and type B was include in clades of turkey Eimeria. Based on our findings in this study and previous data, we herein propose type B as E. raichoi. These genetic data will be helpful to conduct detailed classification and understand the impact of these parasites for conservation of endangered Japanese rock ptarmigans. Graphical abstract Image 1 Highlights • Molecular analyses revealed Eimeria isolates from Japanese rock ptarmigans formed own clades. • The species was phylogenetically closely related to virulent chicken Eimeria spp. • Based on morphological data and sequences, Eimeria type B was proposed as a new species E. raichoi. • These parasites could be the key factor for conservation of the endangered birds. [ABSTRACT FROM AUTHOR]

The Japanese rock ptarmigan, Lagopus muta japonica , inhabits the alpine zone of mountainous areas at 3000 m above sea level. Since L. m. japonica is endangered due to a decline in the overall population, controlling infectious diseases such as those caused by protozoan parasites is a critical factor in the conservation of this species. Although Eimeria spp. are considered to have a negative impact on Japanese rock ptarmigan populations, the ecological interactions between the parasites and their hosts have not yet been fully clarified. We therefore conducted seasonal surveys of the prevalence of Eimeria spp. in Japanese rock ptarmigan populations. In addition, we recorded the ambient temperature in ptarmigan habitat and characterized the ability of eimerian isolates to acquire infectivity. Eimeria spp. were detected in 217 of 520 (41.7%) Japanese rock ptarmigan fecal samples in 2006 and in 177 of 308 (57.5%) fecal samples in 2007. Specifically, we observed two types of oocysts characteristic of E. uekii and type B. In adult birds and chicks, infection rates increased towards August (summer) and then decreased as the temperature decreased toward November (winter). Oocyst counts per gram (OPG) of feces peaked in August in adults and chicks, and OPG values were markedly higher in chicks than in adults. Isolated Eimeria spp. oocysts sporulated at temperatures as low as 8 °C and remained viable after being stored at 4 °C for 6 months. Our findings suggest that Eimeria spp. can complete their annual lifecycle in the cold timberline regions inhabited by the host, the Japanese rock ptarmigan, and that Eimeria spp. infection is widespread in the bird populations examined. [ABSTRACT FROM AUTHOR]

Chicken coccidiosis is caused by Eimeria spp., particularly Eimeria tenella , and is characterized by watery or hemorrhagic diarrhea, resulting in death in severe cases. Precociously attenuated live vaccines are widely used to control the disease, and these are produced by serially passaging virulent strains through chickens, and the collection of oocysts from feces at progressively earlier time points during oocyst shedding. Sporozoites of the precocious strain rapidly enter the intestinal mucosa, and their subsequent asexual development reduces their growth. However, there have been few detailed genetic or transcriptional analyses of the strains. Here, we used RNA sequencing to gain novel biological insight into the pathogenicity and precocity of E. tenella . We compared the differential transcription in the sporozoites (the initial stage of endogenous development) of virulent and precocious strains by mapping the sequence reads onto the draft genome of E. tenella . About 90% of the reads from both strains were mapped to the genome, and 16,630 estimated transcript regions were identified. Using Gene Ontology slim and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses and the annotation of the estimated transcripts with Blastx, we found that the expression of some genes involved in carbohydrate metabolism were expressed two-fold more strongly in the virulent strain than in the precocious strain. Characteristically, genes related to proteins secreted from the apical complex, proteases, cell attachment proteins, mitochondrial proteins, and transporters were most strongly upregulated in the virulent strain. Interestingly, the expression of genes associated with cell survival, development, or proliferation was strongly upregulated in the precocious strain. These findings suggest that virulent strains survive long before invasion and invade actively/successfully into host cells, whereas proliferative processes appear to affect precocity. [ABSTRACT FROM AUTHOR]

To date, three Entamoeba spp. ( E . suis , zoonotic E . polecki and E . histolytica ) have been identified in pigs, but their pathogenicity and molecular classification have not been fully determined. Examination and pathological analysis of pigs (n = 3) with diarrhoea was conducted and revealed the presence of Entamoeba organisms. We performed a genetic analysis of the isolate using the small-subunit ribosomal RNA (SSU rRNA) gene region to identify the species. A severe ulcerative colitis was observed histopathologically with inflammatory cells, including macrophages and neutrophils, infiltrating the mucous membranes of the cecum and colon. Many Entamoeba trophozoites were found at the erosion site or at ulcerative lesions. Pathogenic viruses or bacteria were not detected. The SSU rRNA sequence of the Entamoeba isolate was found to be completely homologous to that of E . polecki subtype 3. [ABSTRACT FROM AUTHOR]

Highlights: [•] 25,880 Contigs, generated in Eimeria tenella, had 30% homology with others. [•] The contigs were distributed within three categories including biological processes. [•] The detected genes included enzymes associated with metabolic pathways. [•] Most of the enzymes were up regulated in sporulating stage. [•] New contigs may be E. tenella specific or novel. [ABSTRACT FROM AUTHOR]

Abstract: The genus Cryptosporidium includes many common parasites infecting animals and humans, and is a major cause of diarrheal illness worldwide. The biology of gastric Cryptosporidium spp., including replication in the stomach, has not been well documented. This study evaluated the viability of Cryptosporidium andersoni sporozoites in gastric environments after excystation and examined the endogenous development and histopathological changes in the stomachs of infected mice, using a novel type of C. andersoni. Sporozoites were affected by low pH (61.6% viability after 3h at pH2.0). Electron microscopy revealed developmental parasites on the gastric foveolae but not on the surface of the gastric mucosa. Histopathological examinations at 1, 2, 4 and 12weeks p.i. uncovered three different lesions. The gastric mucosa of foveolae filled with parasites was extended and the amount of neutral mucopolysaccharide at the mucosal surface was decreased with the first type of lesion. The gastric mucosa was atrophied, some gastric glands were disrupted and the amount of acid mucopolysaccharide at the mucosal surface was increased with the second type. Finally, the gastric mucosa was slightly extended and goblet cells were present in the gastric mucosa, indicating intestinal metaplasia, in the third type. No parasites were detected in these areas with increased acidic mucin and indications of metaplasia. The results suggest that C. andersoni parasites could not survive in acidic environments for a long period before invading host cells and preferentially develop in neutral sites of the gastric mucosa, resulting in histopathological changes and chronic shedding of oocysts. [Copyright &y& Elsevier]

Abstract: Previously, we reported that an isolate of novel type of Cryptosporidium andersoni detected in cattle in Japan contained Type A (identical to C. andersoni reported previously) and Type B (having a thymine nucleotide insertion unlike the Type A) genotypes in the 18S rRNA gene. Here, we conducted an extensive investigation of Cryptosporidium infections in adult cattle in Japan from 2004 to 2007. Consequently, Cryptosporidium sp. were detected in 12 of the 205 cattle examined (5.9%), and partial sequences of the Cryptosporidium oocyst wall protein (COWP) gene in all isolates were identical to those of the previously reported data for C. andersoni whereas two signals were observed in the sequence of the partial 18S rRNA gene in all the isolates. In transmission studies using five of the isolates, they all infected SCID mice. Modified multiplex PCR using DNA of a single oocyst isolated from the infected SCID mice revealed that the partial sequences in the 18S rRNA gene of 40–80% of 10 isolates were identical to the Type A genotype of C. andersoni and those of other samples were identical to the Type B genotype. These results suggested that the C. andersoni novel type is widespread in cattle throughout Japan, and have multiple copies (Types A and B) in the 18S rRNA gene. [Copyright &y& Elsevier]

Abstract: Cryptosporidium oocysts were detected in the feces of cattle in Saga, Japan. Isolates were morphologically large. We attempted to identify the species or genotypes of the isolates by analyzing the partial sequences of the 18S rRNA and Cryptosporidium oocyst wall protein (COWP) genes, and measuring the infectivity in mice. The isolates showed 100% homology with Cryptosporidium andersoni in the COWP gene sequence and it could be transmitted to mice, but in the 18S rRNA gene, there was an additional signal in the ABI sequence chromatogram. To examine the additional signal, we analyzed both the 18S rRNA and the COWP gene sequences of a single oocyst passaged from mice using a modified multiplex PCR that was able to amplify both genes. As a result, it was revealed that two distinct genotypes (Types A and B) of a novel C. andersoni type existed in the 18S rRNA gene, whereas the COWP gene sequences of both oocysts were identical to C. andersoni. Although the sequence of the 18S rRNA gene of Type A was identical to that of C. andersoni, that of Type B had a thymine insertion and was not identical to any sequence registered with GenBank. Here we report that this is a new type of C. andersoni. [Copyright &y& Elsevier]

Abstract: Previously, we reported ‘a novel type’ of Cryptosporidium andersoni detected from cattle in Japan, and showed that the isolate was infective to mice. In the present study, we examined the patterns of oocyst shedding in both immunocompromised and immunocompetent mice, as well as pathological lesions in the infected mice. After oral inoculation with 1×106 oocysts, all five severe combined immunodeficiency (SCID) mice began to shed endogenously produced oocysts on day 6 post-inoculation (p.i.). The number of oocysts per day (OPD) reached 1×106 on day 17 p.i., and an OPD level of 1×106 to 107 was maintained until 91 days p.i. when the mice were sacrificed. In the five immunocompetent mice inoculated with 1×106 oocysts, the pre-patent and patent periods were 6 and 19 days, respectively, and the maximal OPD level was 1.5×105 on average. On histological examinations of infected SCID mice, a large number of parasites were present on the surface of the gastric glands of the stomach, but not in other organs examined. In conclusion, the novel type of C. andersoni, which genetically coincides with C. andersoni reported in other countries, is infective to mice, but susceptibility was lower than that of Cryptosporidium muris infecting rodents from the perspective of infectivity to immunocompetent mice. [Copyright &y& Elsevier]

Abstract: In a previous study, we have developed several chicken monoclonal antibodies (mAbs) against Eimeria acervulina (EA) in order to identify potential ligand molecules of Eimeria. One of these mAbs, 6D-12-G10, was found to recognize a conoid antigen of EA sporozoites and significantly inhibited the sporozoite invasions of host T lymphocytes in vitro. Furthermore, some of these chicken mAbs showed cross-reactivities with several different avian Eimeria spp. and the mAb 6D-12-G10 also demonstrated cross-reactivities with the tachyzoites of Neospora caninum and Toxoplasma gondii. Cryptosporidium spp. are coccidian parasites closely related to Eimeria spp., and especially C. parvum is an important cause of diarrhea in human and mammals. In the present study, to assess that the epitopes recognized by these chicken mAbs could exist on Cryptosporidium parasites, we examined the cross-reactivity of these mAbs with Cryptosporidium spp. using an indirect immunofluorescent assay (IFA) and Western blotting analyses. In IFA by chicken mAbs, the mAb 6D-12-G10 only showed a immunofluorescence staining at the apical end of sporozoites of C. parvum and C. muris, and merozoites of C. parvum. Western blotting analyses revealed that the mAb 6D-12-G10 reacted with the 48-kDa molecular weight band of C. parvum and C. muris oocyst antigens, 5D-11 reacted the 155kDa of C. muris. Furthermore, these epitopes appeared to be periodate insensitive. These results indicate that the target antigen recognized by these chicken mAbs might have a shared epitope, which is present on the apical complex of apicomplexan parasites. [Copyright &y& Elsevier]

Cryptosporidium species have been found in more than 150 species of mammals, but there has been no report in raccoon dogs. Here we found the Cryptosporidium organism in a raccoon dog, Nyctereutes procyonoides viverrinus, and identified this isolate using PCR-based diagnostic methods. Cryptosporidium diagnostic fragments of the 18S ribosomal RNA, Cryptosporidium oocyst wall protein and 70-kDa heat shock protein genes were amplified from the isolate and sequenced to reveal the phylogenetic relationships between it and other Cryptosporidium species or genotypes reported previously. The results showed that the raccoon dog isolate represented the C. parvum cattle genotype which could be a causative agent in human cryptosporidiosis. [Copyright &y& Elsevier]

Abstract: Cryptosporidium andersoni is a protozoan parasite found in many countries that invades the stomachs of primarily adult cattle. Unlike the isolates of C. andersoni in cattle from other countries, C. andersoni isolates from Japanese cattle can infect mice and were identified as a novel type and later defined as C. andersoni Kawatabi type. The biological characteristics of C. andersoni Kawatabi type have not yet been well documented. In the present study, we assess the infectivity of this type isolate in mice with different immune competence status and age. We found that inoculation of more than 1×104 oocysts is needed to establish infection in mature mice irrespective of immune status. All of the infected immunocompetent mice recovered after a patent period of approximately 20days. In immunodeficient mice, the pre-patent period was prolonged compared with that of 1×106 oocysts, but the pattern and the maximum shedding measured by the number of oocysts per day were almost identical. In neonatal immunocompetent and immunodeficient mice, inoculation with 1×104 to 105 oocysts was also needed to establish infection. Our results indicate that there is a threshold of oocysts needed to establish patent infection in the acidic conditions of the stomach. [Copyright &y& Elsevier]

Attenuated strains of avian Eimeria parasites, generated by the selection of precocious lines through serial passaging in chicks, have been used widely as live vaccines. Detailed morphological transitions including their life cycle depending on the passages remain poorly understood. Here, we showed early development and acceleration of transitions in morphological forms of the asexual schizonts of E. tenella that had been attenuated for virulence by serial passaging. Our results may be helpful in understanding parasitism, facilitating further molecular analyses such as comparative genomic or transcriptomic tests. Unlabelled Image • E. tenella was attenuated by selection of precocious lines through passaging in chicks. • Precocity in 10-times-passaged strains may reflect accelerated development of the asexual schizonts. • Size reduction of the 2nd schizonts (the most pathogenic stage) after an additional 10 rounds of passaging may reflect attenuation. • Our results may be helpful for understanding parasite biology in the context of further molecular studies. [ABSTRACT FROM AUTHOR]

Abstract: Previously, we characterized an undocumented strain of Eimeria krijgsmanni by morphological and biological features. Here, we present a detailed molecular phylogenetic analysis of this organism. Namely, 18S ribosomal RNA gene (rDNA) sequences of E. krijgsmanni were analyzed to incorporate this species into a comprehensive Eimeria phylogeny. As a result, partial 18S rDNA sequence from E. krijgsmanni was successfully determined, and two different types, Type A and Type B, that differed by 1 base pair were identified. E. krijgsmanni was originally isolated from a single oocyst, and thus the result show that the two types might have allelic sequence heterogeneity in the 18S rDNA. Based on phylogenetic analyses, the two types of E. krijgsmanni 18S rDNA formed one of two clades among murine Eimeria spp.; these Eimeria clades reflected morphological similarity among the Eimeria spp. This is the third molecular phylogenetic characterization of a murine Eimeria spp. in addition to E. falciformis and E. papillata. [Copyright &y& Elsevier]

The initial development of Babesia ovata in the midgut of the vector tick Haemaphysalis longicornis has been demonstrated through in vitro and in vivo studies. Although the research on the partial developmental cycles of B. ovata in the tick midgut was performed in our previous study by using ticks fed on experimentally B. ovata –infected cattle, detailed information on the developmental stages of B. ovata in H. longicornis was limited. This report describes the sequential development of stages of B. ovata in an in vitro study using B. ovata– infected erythrocytes and tick midgut contents. The in vivo study also confirmed the developmental stages in the midgut contents of artificially B. ovata– infected ticks. In this observation, we have recognized the distinct forms of B. ovata developmental stages in the tick midgut; the aggregation forms and ray bodies with shorter spikes and light-stained cytoplasm were shown by Giemsa staining. The similarities and differences of the stages as compared to previous reports have been discussed. [ABSTRACT FROM AUTHOR]

Abstract: Ticks employ a battery of proteases to digest the contents of host blood meals. Host hemoglobin degradation is facilitated by proteolytic networks in the midgut, the first major region of the body where ingested blood comes into contact with the tick’s internal tissues. Our previous studies indicated that HlCPL-A, a cathepsin L-like cysteine protease isolated from the midgut of the ixodid tick Haemaphysalis longicornis, is a potent hemoglobinase, and plays important roles in the digestion of blood acquired from a host. In this paper, we report the effects of silencing of the HlCPL-A gene in H. longicornis using RNA interference (RNAi). We observed that the survival of HlCPL-A-silenced ticks was reduced compared with that of controls during blood digestion, most likely due to the compromised ability of ticks to digest blood. The morphological analysis results of midgut lumen were different between HlCPL-A-silenced ticks and controls, indicating that HlCPL-A plays a crucial role in hemolysis in the midgut of ticks. The expression level was analyzed using quantitative RT–PCR-based endogenous expression approach. Compared to that in malE double stranded RNA (dsRNA)-treated ticks, in the midgut of HlCPL-A dsRNA-treated ticks, some proteases and inhibitors related to the hemoglobin digestive cascade were up-regulated while the others were down-regulated. These results suggest that HlCPL-A is related to the multi-enzyme cascade and protease network for hemoglobin digestion. These findings suggest that the hemoglobin digestive cascade may assemble in the midgut of ticks. [Copyright &y& Elsevier]

Abstract: Inhibitors of proteases play key roles in the biological processes of vertebrate and invertebrate animals, including arthropod parasites. Here, we describe a cDNA that encodes a functionally active chymotrypsin inhibitor of the BPTI/Kunitz family of serine protease inhibitors from the hemocytes of the ixodid tick, Haemaphysalis longicornis, herein called HlChI. HlChI sequence is evolutionarily conserved and contains six cysteine residues and three disulfide bonds with a calculated molecular weight of 9.1 kDa. HlChI-specific mRNA was expressed in all developmental stages of ticks and the expression was up-regulated by host''s blood-feeding processes. Endogenous HlChI was localized mainly in the hemocytes. HlChI potently inhibited bovine pancreatic α-chymotrypsin for hydrolyzing the fluorogenic substrate (IC50 8.32 nM, K d 5.35 ± 1.01 nM) and bovine casein digestion. However, HlChI weakly inhibited bovine pancreatic trypsin and could not affect the porcine elastase activity, suggesting its narrow specificity to chymotrypsin. HlChI was stable over the pH range 2–11 and heating up to 70 °C at pH 8. HlChI was highly stable to 8 M urea and 2% SDS at pH 8.0, when treated for 24 h at 37 °C. However, 0.2 M 2-mercaptoethanol caused complete but reversible inactivation of HlChI. Knockdown of HlChI gene by RNA interference (RNAi) caused death of the feeding ticks, failure of ticks to engorge and significantly reduced body weight gain. RNAi also resulted in significantly decreased egg conversion ratio and fecundity. These results suggest that HlChI is a chymotrypsin-specific inhibitor with high stability and may play regulatory functions in host''s blood-feeding processes and tick reproduction. [Copyright &y& Elsevier]