Background: Masked palm civets are known to play an important role in the transmission of some zoonotic pathogens. However, the distribution and zoonotic potential of Enterocytozoon bieneusi, Giardia duodenalis and Cryptosporidium spp. in these animals remain unclear. Methods: A total of 889 fecal specimens were collected in this study from farmed masked palm civets in Hainan, Guangdong, Jiangxi and Chongqing, southern China, and analyzed for these pathogens by nested PCR and DNA sequencing. Results: Altogether, 474 (53.3%), 34 (3.8%) and 1 (0.1%) specimens were positive for E. bieneusi, G. duodenalis and Cryptosporidium sp., respectively. Sequence analysis revealed the presence of 11 novel E. bieneusi genotypes named as PL1-PL11 and two known genotypes Peru8 and J, with PL1 and PL2 accounting for 90% of E. bieneusi infections. Phylogenetically, PL4, PL5, PL9, PL10 and PL11 were clustered into Group 1, while PL1, PL2, PL3, PL6, PL7 and PL8 were clustered into Group 2. Assemblage B (n = 33) and concurrence of B and D (n = 1) were identified among G. duodenalis-positive animals. Further multilocus genotyping of assemblage B has revealed that all 13 multilocus genotypes in civets formed a cluster related to those from humans. The Cryptosporidium isolate from one civet was identified to be genetically related to the Cryptosporidium bamboo rat genotype II. Conclusions: To the best of our knowledge, this first report of enteric protists in farmed masked palm civets suggests that these animals might be potential reservoirs of zoonotic E. bieneusi and G. duodenalis genotypes.
Cryptosporidiosis is a significant cause of diarrhea in sheep and goats. Among the over 40 established species of Cryptosporidium, Cryptosporidium xiaoi is one of the dominant species infecting ovine and caprine animals. The lack of subtyping tools makes it impossible to examine the transmission of this pathogen. In the present study, we identified and characterized the 60-kDa glycoprotein (gp60) gene by sequencing the genome of C. xiaoi. The GP60 protein of C. xiaoi had a signal peptide, a furin cleavage site of RSRR, a glycosylphosphatidylinositol anchor, and over 100 O-glycosylation sites. Based on the gp60 sequence, a subtyping tool was developed and used in characterizing C. xiaoi in 355 positive samples from sheep and goats in China. A high sequence heterogeneity was observed in the gp60 gene, with 94 sequence types in 12 subtype families, namely XXIIIa to XXIIIl. Co-infections with multiple subtypes were common in these animals, suggesting that genetic recombination might be responsible for the high diversity within C. xiaoi. This was supported by the mosaic sequence patterns among the subtype families. In addition, a potential host adaptation was identified within this species, reflected by the exclusive occurrence of XXIIIa, XXIIIc, XXIIIg, and XXIIIj in goats. This subtyping tool should be useful in studies of the genetic diversity and transmission dynamics of C. xiaoi.
Enterocytozoon bieneusi is a zoonotic pathogen with worldwide distribution. Among the 11 established groups of E. bieneusi genotypes based on phylogenetic analysis of the ribosomal internal transcribed spacer ( ITS ), the human-infective potential and population genetics of the Group 1 genotypes from diverse hosts are well characterized. In contrast, Group 2 genotypes from ruminants have unclear population genetics, leading to poor understanding of their host range and zoonotic potential. In this study, we sequence-characterized 121 Group 2 isolates from dairy cattle, beef cattle, yaks, Tibetan sheep, golden takins, and deer from China at five genetic loci ( ITS , MS1, MS3, MS4 and MS7), comparing with data from 113 Group 1 isolates from nonhuman primates. Except for MS7, most of the genetic loci produced efficient PCR amplification and high nucleotide identity between Groups 1 and 2 of E. bieneusi genotypes. In population genetic analyses of the sequence data, a strong linkage disequilibrium was observed among these genetic loci in the overall Group 2 population. The individual ITS genotypes (I, J and BEB4) within Group 2, however, had reduced linkage disequilibrium and increased genetic exchanges among isolates. There was only partial genetic differentiation between Group 1 and Group 2 genotypes, with some occurrence of genetic recombination between them. Genetic recombination was especially common between genotypes I and J within Group 2. The data presented indicate a high genetic identity between Group 1 and Group 2 genotypes of E. bieneusi , which could be responsible for the broad host range and high zoonotic potential of Group 2 genotypes in China. As there is no effective treatment against E. bieneusi , the One Health approach should be used in the control and prevention of zoonotic transmission of the pathogen.
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