Detection And Identification Of Piroplasm Infection In Ticks By Reverse Line Blot(RLB), Piroplasm Detection By RLB And 16S Microbiome Examination In Ixodes Persulcatus

Piroplasmosis is caused by generally host-specific haemoprotozoan parasites of the genus Babesia and Theileria(phylum Apicomplexa, order Piroplasmida), which are etiological agents of babesiosis and theileriosis, respectively. Ticks are harmful blood-sucking ectoparasites and are main transmitting vectors of piroplasmosis. Ticks can cause direct parasitic damage to hosts due to direct effect of attachment and blood sucking or through the bacterial, protozoan or viral diseases that they transmit. Piroplasmosis leads to clinical disorders in domestic and wild animals in the tropics and subtropics. The economic impacts of this disease include lowered meat and milk production, abortions, lowered fertility, control measure costs, and a general impact on the global animals and animal products trade industry causing economic importance of several billions US dollars. In this study, RLB assay was developed to investigate the presence of piroplasm species in ticks collected from grass from different provinces of China. The PCR amplification was performed using RLB primers that target the hypervariable region 4(V4 region) of 18 S rRNA gene for all members in the genera of Theileria and Babesia. Specific probes for piroplasms species were designed based on V4 region of 18 S rRNA gene. After calculating the copy number, serially diluted plasmids of the different piroplasm species were used to access the sensitivity of the RLB. Sampling was performed from March to June, 2015. The result showed that the RLB assay was highly specific and could distinguish piroplasma species, the sensitivity of developed RLB assay could be up to detecting 102 copies/μl of recombinant plasmid DNA. Both single and mixed infections were recognized in the investigated ticks collected from grasslands, and T. annulata(33/408, 8.1%) and Babesia sp. Xinjiang(30/408, 7.4%) were the most frequently identified pathogens. Ticks collected from animals were used to assess potential threat of transmitting ovine and bovine Babesia and Theileria species to animals by RLB method. The result revealed that T. annulata(118/652, 18.1%) was the dominant piroplasm species and the co-infection of T. luwenshuni + T. sinensis(18/652, 2.8%) was frequently observed. Many tick species were associated with a broader pathogen range than previously reported. Moreover, 25 Ixodes persulcatus ticks from Heilongjiang Province, China, were screened for the presence of piroplasm species by RLB and bacteria by Illumina sequencing based on the variable region 4 of 16 S rRNA gene. The result showed that no Theileria or Babesia species was detected in I. persulcatus ticks through RLB. Two hundred genera of bacteria were detected via high-throughput sequencing of I. persulcatus samples, some of which are of potential pathogenic importance and some are environmental contaminant. Tick-associated bacteria pathogenic to human and animals are of interest in this study, and Rickettsia sp. and Borrelia sp. were the detected bacteria harmful to human and animal. It can be concluded that the piroplasms species-specific RLB assay could be as a potential clinical application in the simultaneously detection and differentiation of Babesia and Theileria species. Meanwhile veterinary practitioners and stakeholders should be made aware of the existence of a high prevalence of T. annulata in China and the vector competence of ticks need to be further clarified by using experimental transmission test. Awareness should be raised concerning I. persulcatus tick bites carrying multiple pathogens in Heilongjiang Province.