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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.

Recently, we have demonstrated the utility of Eimeria krijgsmanni as a novel mouse eimerian parasite for elucidating the biological diversity. The parasite showed notable infectivity to mice with various levels of immune status and susceptibility to antimicrobial agents including coccidiostat. However, the detailed lifecycle of E. krijgsmanni had not yet been determined and this information was lacking in discussion of previous findings. In the present study, we clarified the morphological characteristics of E. krijgsmanni and its lifecycle in normal mice, and examined the effects in immunodeficient mice and lifecycle stage for challenge infections after the primary inoculation. In immunocompetent mice, the lifecycle consisted of four asexual stages and the sexual sages followed by formation of oocysts during the prepatent periods. Interestingly, the second-generation meronts were detected in all observation periods after the disappearance of the other stages. For the challenge infection of immunodeficient mice, all developmental stages except for the second generation meronts were temporarily vanished. This finding suggests a "rest" or marked delay in development and a "restart" of the promotion toward the next generations. The second generation meronts may play an important role in the lifecycle of E. krijgsmanni.

In the present study, morphological observations on the extracellular structures found on the apical surface of the midgut epithelium, known as the peritrophic membrane (PM) or glycocalyx, are described in Haemaphysalis longicornis females and larvae. These structures have been hypothesized to provide protection to the microvilli of epithelial cells of the digestive tract. Our aim was to determine whether the extracellular structures are important in the digestion of the blood meal and/or as a protection against infection or injury. The PM was detectable in the midgut of engorged larvae by electron microscopy, but not in engorged females. However, a PM-like structure, stainable with toluidine blue, was observed in females by light microscopy. From the results of confocal laser scanning and electron microscopic observations with wheat germ agglutinin (WGA lectin) staining for chitin of the PM, however, the structure was clearly recognized. The structure in the female is likely to be PM because staining with WGA lectin in the presence of GlcNAc indicates the presence of chitin and various morphologies of PM have been reported in insects and ticks. These results show morphologically that different types of PM-like structure are formed in larvae and females of H. longicornis.