Epidemiological Investigation Of Haemoplasmosis In Pigs&Dogs And Their Co-relation With Vectors In Hainan Island,China

Background:The haemotropic Mycoplasmas such as,Mycoplasma suis,M.parvum,M.haemocanis and Candidatus M.haematoparvum are epierythrocytic prokaryotes and causative agents of porcine eperythrozoonosis and canine haemoplasmosis.Many studies previously reported that haemoplasmosis can feasibly be a vector borne disease.In recent years,infections caused by hemotrophic mycoplasmas in animals and humans are rising within China.The potential causing zoonotic infection by this bacterium is more prevalent in farmers and veterinary doctors,who have easily close contact with farm animals,than in other public.Domestic animals such as pigs,dogs,cats,cattle and birds are economically important animals and disease outbreaks related to them affect huge economical losses.Haemotropic infections in domestic animals from Hainan Island,China have not been previously reported.Objective:The objective of this study was to investigate the prevalence of Mycoplasma spp.in domestic animals in Hainan Island,China,and to identify any correlation with other mycoplasmas/????? associated with domestic animals.We also studied different vectors such as,mosquitoes,flies,ticks to find any correlation with transmission of ????? from animal to other animals in the field.In this study we also focused on molecular detection of bacterial endosymbionts-Coxiella like bacteria by using a PCR technique to identify Coxiella 16S rRNA sequences in R.sanguineus tick samples(65 Adults ticks)collected at Hainan,China.Methods:The study includes PCR amplification and sequencing using universal primers for M.suis,M.parvum,M.haemocanis and Candidatus M.haematoparvum 16S rRNA,23 S rRNA genes as well as species specific Coxiella-like bacteria Cox 16S rRNA,Cox Cap gene and Cox pyr gene.Further phylogenetic analysis was carried out to find any correlation betweenother haemoplasmas from different animal spp.A total 593 pig blood samples were collected and tested for M.suis and M.parvum by end point PCR,real-time PCR and a kit based immuno-chromatographic assay.To identify possible vectors mosquitoes and flies were collected from the local farms positive sites for mycoplasma infection found in pigs at Hainan and DNA samples isolated and tested for the presence of any haemoplasmas by end point PCR using primers specific for M.suis and M.parvum.Also we reared mosquitoes in laboratory and tried adults mosquitoes feed on blood samples with mycoplasma bacteria.On the other hand,end point PCR specifically designed to identify M.haemocanis and Candidatus M.haematoparvum from total of 226 dog blood samples collected from Hainan province were used.The universal primer HBT(16S rRNrA gene)used to amplify 595 bp product.Further based on reference sequence data available at NCBI for other haemoplasma species and species identified in this study used to compare by phylogenetically.To identify possible vectors we collected hard ticks from the same dog farm.Total 65 ticks were collected and whole DNA isolated,further tested for the presence of M.haemocanis and Candidatus M.haematoparvum by using specific and universal primers for 16S rRNA gene by end point PCR.Also ticks and dog blood samples were tested for Coxiella-like endosymbionts by using Cox 16S rRNA,Cox Cap gene and Cox pyr gene,to amplify 532 bp,601 bp and 504 bp PCR product respectively and tried to find correlation by phylogenetic analysis from 16S rDNA sequences.Results:The results showed that 6.4%(15/233)of the pigs from Hainan Island,China were found positive with M.parvum.This study increases an interesting question regarding the probability of different endemic Mycoplasma species present in the Island when compared to the Mainland China(Beijing,Guizhou and Fujian prov:inces with 2.1%(2/95),0.4%(1/218)and 4.2%(2/47),respectively),where only M.suis has been found in this study.Out of three diagnostic tests we used here,we found that real-time PCR and immunochromatography are more sensitive than the end point PCR for detection of Mycoplasma spp.from pig blood samples.However,total of 226 dog blood samples were randomly collected from three selected sites in Hainan,only 2(0.8%)dog blood samples were found to be infected(PCR Positive)with M.haemocanis,40(17.6%)dog blood samples infected with Coxiella endosymbiont,which are endosymbionts of R sanguineus dog hard ticks.Total 65 tested ticks from the same farm also found infected with Coxiella-endosymbionts(PCR positive)and showed interesting results about that the female ticks were found to be more infected with Coxiella compare to male R.sanguineus ticks(male ticks 8/30=26.6%and female ticks 22/35=62.8%).Conclusion:Our present study describes that M.parvum is present in pigs from Hainan.This raises a need for further studies to understand the economic importance,evolution and transmission of the species.Due to less sample size of vectors collected and tested in this study,in future it challenges to identify and control vectors borne haemoplasmosis.This is the first report of M.haemocanis infection in dogs in Hainan,which demands further investigations regarding determine the role of vectors in disease transmission,disease severity,and haemoplasma infections pathogenicity.As in this study we tested different vectors such as mosquitoes,ticks and flies but we were unable to find any probable vectors for haemoplasma transmission,which also implies that there are chances that some other vectors could be the vector to the disease.The ticks and dog blood samples tested here showed the presence of Coxiella endosymbionts,which could transmit the disease from ticks to dogs.Although the study demands further investigations to use species specific primers for identification of Coxiella like bacteria.