Molecular Epidemiology And Phylogenetic Analysis Of Hemotropic Mycoplasmas (Mycoplasma Ovis And 'Candidatus Mycoplasma Haemovis') In Sheep And Goats From Parts Of China

Hemotropic mycoplasmas(hemoplasmas,formerly classified as Haemobartonella and Eperythrozoon spp.)are uncultivated,small,pleomorphic,wall-less bacteria that parasitize on the surface of animal erythrocytes,plasma and bone marrow.Hemoplasmas have now been reclassified into the Mycoplasma genus based on 16 S r RNA gene sequence.These pathogens can cause mild to severe hemolytic anemia,icterus,ill-thrift,infertility,and poor weight gain,but death is rare in infected adults.Worldwide,hemoplasmas have been reported to affect livestock,companion animals,wildlife,and humans.With an increasing number of hemoplasmosis clinical cases,many countries have suffered extensive losses of livestock.However,understanding of the molecular epidemiology of hemoplasmas(Mycoplasma ovis and ‘Candidatus Mycoplasma haemovis')is limited in sheep and goats,and the hemoplasma strain/species/variant ‘Candidatus M.haemovis' was poorly studied throughout the world and had never been detected in China until now.In order to investigate the prevalence of hemoplasmas in sheep and goats from part of China,a nested PCR method with high sensitivity and specificity for detection of hemoplasmas in sheep and goats was established,which provides a technical method for rapid detection of hemoplasmas.The nested PCR method was used to investigate the prevalence of hemoplasmas in some areas of China.Positive specimens underwent nucleotide sequencing and phylogenetic analysis.The aim of the present study was to provide a theoretical basis for determining the molecular epidemiology and genetic evolution of hemoplasmas in some regions of China.1.Establishment and application of a nested-PCR assay for detection of hemoplasmas(M.ovis and ‘Candidatus M.haemovis')To establish a nested PCR assay to detect hemoplasmas(M.ovis and ‘Candidatus M.haemovis'),according to the published 16 S r RNA gene sequence of hemoplasmas in Gen Bank(Accession number: AF338268 and AB617737),inside and outside two pairs of primers were designed to establish the nested PCR assay for detecting the disease caused by hemoplasmas.The optimum reaction conditions were screened,and carried out the specificity,sensitivity and reproducibility of test and detection of clinical samples.The results of verification experiments showed that the targeted gene fragment was 506 bp or 489 bp in length and homology of nested PCR product was 97.8%~99.6%,and this detection method had no cross reaction with Eperythrozoon suis,Anaplasma phagocytophilum,Anaplasma ovis,Babesia motasi and Theileria luwenshuni.The sensitivity reached to 0.654 fg·?L-1.This method had good repeatability.The 71.4% positive rate in 77 clinical samples by the nested PCR assay was higher than conventional PCR and the reported nested PCR test results.2.Molecular epidemiology of hemoplasmas(M.ovis and ‘Candidatus M.haemovis')in sheep and goatsIn order to understand the molecular epidemiology of hemoplasmas(M.ovis and ‘Candidatus M.haemovis')in sheep and goats,a total of 1,364 EDTA-anticoagulated blood samples were collected from jugular vein of sheep and goats in Henan,Guizhou,Shanxi,Shaanxi,Yunnan,Qinghai,Heilongjiang provinces and the Inner Mongolia autonomous region.Overall,610 specimens(44.7%,610/1,364)were shown to be hemoplasmas(M.ovis and ‘Candidatus M.haemovis')-positive by nested PCR amplification of the 16 S r RNA gene.The prevalence in goats was 44.1%(379/860)and 45.8%(231/504)in sheep,which was not significantly different.Likewise,there were no significant differences between prevalence in different age groups of goats and sheep.There were some differences in prevalence between grazing and household animals,with a higher prevalence observed in grazing goats and sheep(54.4%,396/728)than household goats and sheep(33.6%,214/636).Male goats and sheep had significantly higher M.ovis infection rates,at 60.0%(138/230),than females(41.6 %,472/1134).Our data showed a high prevalence and widespread distribution of hemoplasmas(M.ovis and ‘Candidatus M.haemovis')in sheep and goats.3.The study of genetic evolution of hemoplasmas(M.ovis and ‘Candidatus M.haemovis')610 positive specimens obtained from nested PCR were tested for hemoplasmas(M.ovis and ‘Candidatus M.haemovis')DNA using a conventional PCR assay that amplified a 1,341 bp fragment of the 16 S r RNA gene.103 positive specimens representative of different hosts,farms/flocks and geographic locations were selected and nucleotide sequences of the nearly complete 16 S r RNA gene together with reference sequences downloaded from Gen Bank were aligned using Clustal X 2.1 with further adjustments made manually as necessary.Then Meg Align and Mega 6.06 software was applied to conduct homology analysis,phylogenetic and molecular evolutionary analysis,respectively.The sequencing results showed that analysis of the 16 S r RNA gene sequences identified M.ovis(n=56)and ‘Candidatus M.haemovis'(n=47)among 103 sequences.After alignment,the obtained 103 16 S r RNA gene sequences formed four genetypes KU983740,KU983746,KU983748 and KU983749.Phylogenetic analysis revealed that M.ovis and ‘Candidatus M.haemovis' genetypes/isolates fell into a group.And the homology of the two 16 S r RNA gene sequences was 97.3%~97.5%.Therefore,we can speculate that M.ovis and ‘Candidatus M.haemovis' may exist as a single species with two copies of 16 S r RNA gene.In addition,the genetypes(KU983740 and KU983746)of M.ovis in sheep and goats in this study fell in a clade with two human genetypes from USA(KF313922 and GU230144)and shared 99.8%-99.9% with them.The results indicate that the M.ovis genetypes obtained in the present study may have potential risk of zoonotic disease and public health significance.