Image_1_High-throughput sequencing and characterization of potentially pathogenic fungi from the vaginal mycobiome of giant panda (Ailuropoda melanoleuca) in estrus and non-estrus.PNG

IntroductionThe giant panda (Ailuropoda melanoleuca) reproduction is of worldwide attention, and the vaginal microbiome is one of the most important factors affecting the reproductive rate of giant pandas. The aim of this study is to investigate the diversity of vaginal mycobiota structure, and potential pathogenic fungi in female giant pandas during estrus and non-estrus.MethodsThis study combined with high-throughput sequencing and laboratory testing to compare the diversity of the vaginal mycobiota in giant pandas during estrus and non-estrus, and to investigate the presence of potentially pathogenic fungi. Potentially pathogenic fungi were studied in mice to explore their pathogenicity.Results and discussionThe results revealed that during estrus, the vaginal secretions of giant pandas play a crucial role in fungal colonization. Moreover, the diversity of the vaginal mycobiota is reduced and specificity is enhanced. The abundance of Trichosporon and Cutaneotrichosporon in the vaginal mycobiota of giant pandas during estrus was significantly higher than that during non-estrus periods. Apiotrichum and Cutaneotrichosporon were considered the most important genera, and they primarily originate from the environment owing to marking behavior exhibited during the estrous period of giant pandas. Trichosporon is considered a resident mycobiota of the vagina and is an important pathogen that causes infection when immune system is suppressed. Potentially pathogenic fungi were further isolated and identified from the vaginal secretions of giant pandas during estrus, and seven strains of Apiotrichum (A. brassicae), one strain of Cutaneotrichosporon (C. moniliiforme), and nine strains of Trichosporon (two strains of T. asteroides, one strain of T. inkin, one strain of T. insectorum, and five strains of T. japonicum) were identified. Pathogenicity results showed that T. asteroides was the most pathogenic strain, as it is associated with extensive connective tissue replacement and inflammatory cell infiltration in both liver and kidney tissues. The results of this study improve our understanding of the diversity of the vaginal fungi present in giant pandas and will significantly contribute to improving the reproductive health of giant pandas in the future.</p

Table_1_High-throughput sequencing and characterization of potentially pathogenic fungi from the vaginal mycobiome of giant panda (Ailuropoda melanoleuca) in estrus and non-estrus.DOCX

IntroductionThe giant panda (Ailuropoda melanoleuca) reproduction is of worldwide attention, and the vaginal microbiome is one of the most important factors affecting the reproductive rate of giant pandas. The aim of this study is to investigate the diversity of vaginal mycobiota structure, and potential pathogenic fungi in female giant pandas during estrus and non-estrus.MethodsThis study combined with high-throughput sequencing and laboratory testing to compare the diversity of the vaginal mycobiota in giant pandas during estrus and non-estrus, and to investigate the presence of potentially pathogenic fungi. Potentially pathogenic fungi were studied in mice to explore their pathogenicity.Results and discussionThe results revealed that during estrus, the vaginal secretions of giant pandas play a crucial role in fungal colonization. Moreover, the diversity of the vaginal mycobiota is reduced and specificity is enhanced. The abundance of Trichosporon and Cutaneotrichosporon in the vaginal mycobiota of giant pandas during estrus was significantly higher than that during non-estrus periods. Apiotrichum and Cutaneotrichosporon were considered the most important genera, and they primarily originate from the environment owing to marking behavior exhibited during the estrous period of giant pandas. Trichosporon is considered a resident mycobiota of the vagina and is an important pathogen that causes infection when immune system is suppressed. Potentially pathogenic fungi were further isolated and identified from the vaginal secretions of giant pandas during estrus, and seven strains of Apiotrichum (A. brassicae), one strain of Cutaneotrichosporon (C. moniliiforme), and nine strains of Trichosporon (two strains of T. asteroides, one strain of T. inkin, one strain of T. insectorum, and five strains of T. japonicum) were identified. Pathogenicity results showed that T. asteroides was the most pathogenic strain, as it is associated with extensive connective tissue replacement and inflammatory cell infiltration in both liver and kidney tissues. The results of this study improve our understanding of the diversity of the vaginal fungi present in giant pandas and will significantly contribute to improving the reproductive health of giant pandas in the future.</p

Image_2_High-throughput sequencing and characterization of potentially pathogenic fungi from the vaginal mycobiome of giant panda (Ailuropoda melanoleuca) in estrus and non-estrus.JPEG

IntroductionThe giant panda (Ailuropoda melanoleuca) reproduction is of worldwide attention, and the vaginal microbiome is one of the most important factors affecting the reproductive rate of giant pandas. The aim of this study is to investigate the diversity of vaginal mycobiota structure, and potential pathogenic fungi in female giant pandas during estrus and non-estrus.MethodsThis study combined with high-throughput sequencing and laboratory testing to compare the diversity of the vaginal mycobiota in giant pandas during estrus and non-estrus, and to investigate the presence of potentially pathogenic fungi. Potentially pathogenic fungi were studied in mice to explore their pathogenicity.Results and discussionThe results revealed that during estrus, the vaginal secretions of giant pandas play a crucial role in fungal colonization. Moreover, the diversity of the vaginal mycobiota is reduced and specificity is enhanced. The abundance of Trichosporon and Cutaneotrichosporon in the vaginal mycobiota of giant pandas during estrus was significantly higher than that during non-estrus periods. Apiotrichum and Cutaneotrichosporon were considered the most important genera, and they primarily originate from the environment owing to marking behavior exhibited during the estrous period of giant pandas. Trichosporon is considered a resident mycobiota of the vagina and is an important pathogen that causes infection when immune system is suppressed. Potentially pathogenic fungi were further isolated and identified from the vaginal secretions of giant pandas during estrus, and seven strains of Apiotrichum (A. brassicae), one strain of Cutaneotrichosporon (C. moniliiforme), and nine strains of Trichosporon (two strains of T. asteroides, one strain of T. inkin, one strain of T. insectorum, and five strains of T. japonicum) were identified. Pathogenicity results showed that T. asteroides was the most pathogenic strain, as it is associated with extensive connective tissue replacement and inflammatory cell infiltration in both liver and kidney tissues. The results of this study improve our understanding of the diversity of the vaginal fungi present in giant pandas and will significantly contribute to improving the reproductive health of giant pandas in the future.</p

Molecular characterization and multi-locus genotypes of <i>Enterocytozoon bieneusi</i> from captive red kangaroos (<i>Macropus Rfus</i>) in Jiangsu province, China

<div><p><i>Enterocytozoon bieneusi</i> is the most common pathogen of microsporidian species infecting humans worldwide. Although <i>E</i>. <i>bieneusi</i> has been found in a variety of animal hosts, information on the presence of <i>E</i>. <i>bieneusi</i> in captive kangaroos in China is limited. The present study was aimed at determining the occurrence and genetic diversity of <i>E</i>. <i>bieneusi</i> in captive kangaroos. A total of 61 fecal specimens (38 from red kangaroos and 23 from grey kangaroos) were collected from Nanjing Hongshan Forest Zoo and Hongshan Kangaroo Breeding Research Base, Jiangsu province, China. Using the nested PCR amplification ITS gene of rRNA of <i>E</i>. <i>bieneusi</i>, totally 23.0% (14/61) of tested samples were PCR-positive with three genotypes (i.e. one known genotype, CHK1, and two novel genotypes, CSK1 and CSK2). Multi-locus sequence typing using three microsatellites (MS1, MS3, and MS7) and one minisatellite (MS4) revealed one, five, two, and one types at these four loci, respectively. In phylogenetic analysis, the two genotypes, CHK1 and CSK1, were clustered into a new group of unknown zoonotic potential, and the novel genotype CSK2 was clustered into a separate clade with PtEb and PtEbIX. To date, this is the first report on the presence of <i>E</i>. <i>bieneusi</i> in captive red kangaroos in Jiangsu province, China. Furthermore, a high degree of genetic diversity was observed in the <i>E</i>. <i>bieneusi</i> genotype and seven MLGs (MLG1-7) were found in red kangaroos. Our findings suggest that infected kangaroo may act as potential reservoirs of <i>E</i>. <i>bieneusi</i> and be source to transmit infections to other animal.</p></div

Phylogenetic relationship among the ITS loci of <i>E</i>.<i>bieneusi</i> isolates.

<p>Phylogenetic relationship of the genotypes of <i>E</i>. <i>bieneusi</i> identified in this study and known genotypes previously published in GenBank as inferred by a neighbor-joining analysis of ITS sequences based on genetic distances calculated by the Kimura 2-parameter model. A similar topology tree was also performed by maximum parsimony analysis, with the exception that the CSK2 genotype grouped together with genotypes PtEb and PtEbIX, with 99% bootstrap value. The numbers on the branches are percent bootstrapping values from 1000 replicates, with more than 50% shown in the tree. Each sequence is identified by its accession number, genotype designation, and host origin. Genotypes marked with black circles and open circle are novel and known genotypes identified in this study, respectively.</p

Demonstrate the distribution of ClyA-Apxr in Apxr-OMVs.

<p>Lane M: protein marker. Lane 1: the OMVs-empty. Lane 2: proteinase K plus EDTA treated Apxr-OMVs. Lane 3: untreated Apxr-OMVs. Lane 4: proteinase K treated Apxr-OMVs. Lane 5: EDTA treated Apxr-OMVs.</p

Prevalence and distribution of <i>E</i>. <i>bieneusi</i> genotypes in Nanjing Hongshan Forest Zoo and Hongshan Kangaroo Breeding Research Base.

<p>Prevalence and distribution of <i>E</i>. <i>bieneusi</i> genotypes in Nanjing Hongshan Forest Zoo and Hongshan Kangaroo Breeding Research Base.</p

Lung Histopathology from normal control.

<p>H&E, Magnification 400 X.</p

Western blot of JC8031 recombinant strain reacted with corresponding mouse anti-sera.

<p>Lane M: protein marker. Lane 1: JC8031- pBAD. Lane 2: JC8031- pBAD -Apxr.</p

Transmission electron microscope image of wt-OMVs.

<p>The bar indicates 100 nm.</p