Search

Thus far, Entamoeba species have been classified based on morphology such as the number of nuclei in mature cysts and their hosts. Using recently developed molecular tools, ruminant Entamoeba spp. are currently classified into four species/genotypes: E. bovis and Entamoeba ribosomal lineages (RL) 1, 2, and 4. However, the distribution or pathogenicity of ruminant Entamoeba has not been well documented. In the present study, we examined a total of 25 fecal and seven environmental samples collected from six farms in Japan from 2016 to 2017 by the floatation method and PCR and sequencing analyses. Consequently, we detected Entamoeba cysts in 18 of 25 cattle samples and four of the seven environmental samples, including soil and drinking water, by microscopic examinations. In sequential examinations, Entamoeba-positive cattle were found to shed cysts without any clinical symptoms for more than 8 months. By PCR for molecular identification, isolates in ten cattle and one soil sample were successfully sequenced and formed a cluster of E. bovis, which was separated from those of other Entamoeba species/genotypes such as RL1-4 in phylogenetic analysis. To our knowledge, this is the first report about E. bovis in Japan, and our results may implicate that E. bovis is not pathogenic. [ABSTRACT FROM AUTHOR]

Bovine intranuclear coccidiosis is caused by the protozoans Eimeria alabamensis and Cyclospora spp. Here, we characterized the disease and genetically identified the causative species in Japanese black calves with chronic and refractory watery diarrhea. Histologic examinations revealed atrophy of the jejunal villi and numerous parasites in the nucleus of epithelial cells in the jejunum. Based on molecular analyses using 18S ribosomal RNA gene-specific primers that we designed, the parasites were found to be formed in the same cluster as Eimeria subspherica in the phylogenetic tree, which was separated from those of other related Eimeria spp. These results constitute the first report of E. subspherica as a cause of bovine intranuclear coccidiosis. [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]

Cystoisospora suis is a pathogen that causes diarrhea in pigs and can lead to serious disease. Species identification, especially by histopathological examination, is often difficult because of morphologically similar parasites such as Eimeria species. In this study, we used histopathological, bacteriological, virological, and parasitological methods to identify the cause of the disease in two piglets with severe diarrhea. Villous atrophy, diffuse necrosis, and flattening of mucosal epithelial cells were found in the ilea of examined piglets, and coccidian parasites were found in the cytoplasm of the epithelial cells. In some merozoites in the meronts, the presence of two nuclei indicated type 1 merozoites, characteristic of C. suis. According to Cystoisospora-specific PCR targeting the rRNA internal transcribed spacer 1 (ITS1) gene, the sequences of the products were 98.5% similar to those of C. suis. Escherichia coli (O149 serogroup) exhibiting a virulence factor profile (LT, STb, and EAST1 as toxins and F4 as a colonization factor) was detected in one piglet. No other bacteria or significant enteric viruses were found. Co-infection with C. suis and E. coli could imply aggravation of the disease, although further study is needed to assess the pathogenicity of this interaction. This study is the first to clarify by molecular analysis the sequences of C. suis detected in piglets in Japan. [ABSTRACT FROM AUTHOR]

Abstract: Objective: To evaluate the presence of gastrointestinal parasites on cattle in Indonesia because the prevalence of parasites varies between countries depending on the terrain surrounding livestock farms and investigations in Indonesia have never been performed. Methods: Fecal samples from cattle at 35 farms in 7 districts in West Java, Indonesia, has been examined using the floatation or sedimentation methods, and a immunofluorescence assay and experimentally inoculation to mice for Cryptosporidium or Giardia.spp. Results: 153 of 394 examined cattle (38.8%) were infected with gastrointestinal parasites. The prevalence of Eimeria spp., Nematoda spp. (including Oesophagustomum and Bunostomum-like), Fasciola gigantica and Paramphistomum spp. was 22.4%, 11.2%, 12.5% and 3.8%, respectively. Cryptosporidium andersoni (C. andersoni) was also found in two samples. One isolate of this parasite was confirmed to be transmitted to mice, in contrast to the isolates from other countries. Conclusions: although this survey is preliminary, the results shows that the infection of gastrointestinal parasites in Indonesia was not high, but these infected cattle could be as a potential source leading to economic losses in livestock production. [Copyright &y& Elsevier]

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: 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]

Background: Extensive attention has been devoted to studies of Trypanosoma lewisi in rodents ever since it became recognised as a zoonotic pathogen known as atypical human trypanosomiasis. Regrettably, although T. lewisi infections of small mammals remain significant public health concerns for humans, there is a lack of comprehensive study in Indonesia., Aim: The aim of the study was to detect T. lewisi from rodents residing in the densely populated residential regions along the coastal areas of Banyuwangi Sub District., Methods: A total of 169 rodents were captured across three villages of Kampung Mandar, Lateng and Kepatihan, using rat single live traps. After being euthanized and identified, the blood samples were collected from each rodent via cardiac puncture. Subsequently, the samples were subjected to native (direct blood microscopic examination), microscopic blood smear examination, and molecular analyses utilizing TRYP1S-TRYP1R (623 bp) and LEW1S-LEW1R (220 bp)., Results: The results demonstrated that two species of rodents were successfully captured: Rattus norvegicus (65.68%) and Rattus tanezumi (34.32%). Based on the native and microscopic blood smear examinations, the prevalence of T. lewisi across three villages was 23.08% and 24.26% for molecular analysis employing both primers, respectively. The highest prevalence was found in Kampung Mandar Village (31.18%), followed by Kepatihan (16.67%) and Lateng Villages (15.71%)., Conclusion: Statistical analysis revealed that T. lewisi was more prevalent in R. tanezumi compared to R. norvegicus . In terms of sex, no statistically significant distinction was observed between female and male infected rodents of either species ( p > 0.05), indicating both species can serve as a source of T. lewisi for humans in the surveyed villages., Competing Interests: The authors declare that there is no conflict of interest.

Background and Aim: Toxoplasmosis is caused by the parasite Toxoplasma gondii . Cats are the only known hosts that excrete resistant oocysts. Wild rats serve as crucial reservoirs and intermediate hosts for T. gondii's survival and dissemination. Consuming soil and water containing oocysts can lead to illness. This study aimed to estimate the prevalence of toxoplasmosis in wild rats through molecular detection as an indicator of environmental contamination in Surabaya., Materials and Methods: One hundred rats were collected from the three areas (housing, dense settlements, and traditional markets) and distributed into the five zones: West, East, Central, North, and South of Surabaya. Brain tissue samples were extracted using a Geneaid™ (New Taipei City, Taiwan) DNA isolation kit and analyzed through the loop-mediated isothermal amplification (LAMP) method., Results: The study analyzed brain tissue from 100 wild rats, consisting of 77 Rattus tanezumi and 33 Rattus norvegicus , displaying 30% LAMP positivity. The study revealed that 30% (30/100) of wild rats tested were infected with T. gondii . The molecular prevalence rate in male rats was 32.35% (22/68), compared to females with 25% (8/32). 41.9% of the housing population, 33.3% of traditional markets, and 22.6% of dense settlements had the highest molecular prevalence. The high positive molecular rate at the trapping site can be attributed to cats and dense populations., Conclusion: Thirty percentage wild rats were tested positive for toxoplasmosis in Surabaya, East Java, Indonesia using LAMP method. Implementing strict control and monitoring is crucial in preventing the transmission of diseases from wild rats to humans. It is necessary to carry out further research related to genetic analysis of T. gondii to determine the type of T. gondii that infects animals and humans in Surabaya through bioassay and molecular test., Competing Interests: The authors declare that they have no competing interests., (Copyright: © Puspitasari, et al.)

The genus Mansonella Faust, 1929 includes 29 species, mainly parasites of platyrrhine monkeys in South America and anthropoid apes in Africa. In Malaysia, Mansonella ( Tupainema ) dunni (Mullin & Orihel, 1972) was described from the common treeshrew Tupaia glis Diard & Duvaucel (Scandentia). In a recent classification of the genus Mansonella , seven subgenera were proposed, with M . ( Tup .) dunni as a monotypic species in the subgenus Tupainema . In this study, we collected new material of M . ( Tup .) dunni from common treeshrews in Peninsular Malaysia and redescribed the morphological features of this species. We found that M . ( Tup .) dunni differs from M . ( Cutifilaria ) perforata Uni et al., 2004 from sika deer Cervus nippon (Cetartiodactyla) in Japan, with regards to morphological features and predilection sites in their respective hosts. Based on multi-locus sequence analyses, we examined the molecular phylogeny of M . ( Tup .) dunni and its Wolbachia genotype. Species of the genus Mansonella grouped monophyletically in clade ONC5 and M . ( Tup .) dunni was placed in the most derived position within this genus. Mansonella ( Tup .) dunni was closely related to M . ( M .) ozzardi (Manson, 1897) from humans in Central and South America, and most distant from M . ( C .) perforata . The calculated p-distances between the cox 1 gene sequences for M . ( Tup .) dunni and its congeners were 13.09% for M . ( M. ) ozzardi and 15.6-16.15% for M . ( C .) perforata . The molecular phylogeny of Mansonella spp. thus corroborates their morphological differences. We determined that M . ( Tup .) dunni harbours Wolbachia endosymbionts of the supergroup F genotype, in keeping with all other Mansonella species screened to date., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 The Authors.)

Human zoonotic onchocercosis is caused by Onchocerca dewittei japonica, parasitic in wild boars (Sus scrofa leucomystax) in Japan. Previously, microfilariae longer than those of Onchocerca dewittei japonica were observed in skin snips from wild boars during the study of O. dewittei japonica. Moreover, the third-stage larvae (L3) of these longer microfilariae were obtained from the blackfly Simulium bidentatum after experimental injections. Based on morphometric and molecular studies, similar L3 were found in blackflies during fieldwork in Oita, Japan. However, except for O. dewittei japonica, adult worms of Onchocerca have not been found in wild boars. In this study, we discovered adult females of a novel Onchocerca species in the skin of a wild boar in Oita, and named it Onchocerca takaokai n. sp. Females of this new species had longer microfilariae and differed from O. dewittei japonica in terms of their morphological characteristics and parasitic location. The molecular characteristics of the cytochrome c oxidase subunit 1 and 12S rRNA genes of the new species were identical to those of the longer microfilariae and L3 previously detected, but they differed from those of O. dewittei japonica at the species level. However, both species indicated a close affinity among their congeners and Onchocerca ramachandrini, parasitic in the warthog in Africa, was basal in the Suidae cluster of the 12S rRNA tree., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Hemorrhagic diarrhea in poultry is caused by Eimeria tenella, the most pathogenic avian coccidian parasite, and new approaches to treat the disease are continually being sought. Although eimeripain, a cathepsin B-like cysteine protease from E. tenella, has recently been identified as a novel anticoccidial drug target, its localization during the intracellular development of parasites remains unclear. Here, we demonstrate the expression of eimeripain during asexual and sexual development of E. tenella in vivo. Promature eimeripain was detected only in the early immature second generation of schizonts. In contrast, the mature eimeripain was most strongly detected in the middle-sized immature second generation of schizonts. Both promature and mature eimeripain disappeared depending on the maturation level of second generation of schizonts, but were strongly expressed again in the third generation of schizonts. In the sexual stage, both promature and mature eimeripain were detected in the cytoplasm of micro- and macro-gametocytes and zygotes, but expression became weak in zoites forming oocysts. Collectively, our findings suggest that eimeripain might play a key role in the differentiation of intracellular zoites in the ceca and could be an interesting candidate to develop a novel, effective anti-coccidian drug.

Most causative agents of babesiosis, Babesia parasites, are transmitted transovarially in ixodid ticks. In this study, B. gibsoni, the causative agent of canine babesiosis which has transovarial transmission, was detected in tissues of the vector tick, Haemaphysalis longicornis using a modified quantitative PCR assay. Conventional PCR results showed that the newly designed primer set, which amplifies a 143-bp fragment of rhoptry-associated protein-1 (BgRAP-1) gene in B. gibsoni, was 100 times more sensitive than primers targeting P18 gene encoding 18 kDa protein of B. gibsoni, which was recently renamed as thrombospondin related adhesive protein (BgTRAP) gene, in an artificially generated sample solution containing metagenomic DNA (B. gibsoni DNA extracted from infected dog blood mixed with tick DNA). The TaqMan probe-based quantitative PCR (qPCR) for BgRAP-1 could also detect infected RBCs (iRBCs) at levels of 3.5 × 10(5) to 3.5 × 10(1)/μl, a range that is broader than that of a past SYBR Green-based qPCR method for P18/BgTRAP, which had a detection limit of 3.5 × 10(3) iRBCs/μl. Using this qPCR assay, we attempted to quantify the B. gibsoni burden in tick ovaries and embryonated eggs. Levels of infection were normalized to the copy number of tick's genomic DNA fragment of ribosomal DNA internal transcribed spacer region 2 (ITS2) for the standardization. According to this, low levels of parasite burden were quantified in ovaries and eggs. This detection system is sensitive and is recommended as a tool for elucidating the biological interactions between the vector tick H. longicornis and the parasite, B. gibsoni.

In a survey of pathologic agents in the wild animals of central Japan, we found a Hepatozoon sp. in the lungs of Japanese black bears in Fukui, Shiga, and Gifu Prefectures, Japan. Histopathologic examination of organs and tissues from the 18 bears inspected showed hepatozoonosis in all. Immature and mature meronts were found in all lobules and between alveoli, with a few found between pleura and in connective tissue. In the lungs, inflammatory cells were not found around meronts, merozoites, or tubercles made of macro-phages including zoites, but inflammatory cells were found around degenerating cells, zoites, and tubercles. A Hepatozoon sp. has not been reported as being detected in bears of any species before.