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Furthermore, mixed infections with multiple microorganisms further complicate and limit the detection potential of traditional diagnostic methods. The present study investigated the tick-borne microorganisms in bovine ticks in Pakistan, employing a high-throughput microfluidic real-time PCR based technique.<\/jats:p>\n              <\/jats:sec><jats:sec>\n                <jats:title>Methods<\/jats:title>\n                <jats:p>Ticks were collected from clinically healthy cattle (<jats:italic>n<\/jats:italic>\u00a0=\u00a0116) and water buffaloes (<jats:italic>n<\/jats:italic>\u00a0=\u00a088) from 30 villages across six districts located in five agro-ecological zones (AEZs) of Pakistan from September to November 2017. The microfluidic real-time PCR was used to test the genomic DNA of individual ticks for the presence of 27 bacterial and eight parasitic microorganisms. Phylogenetic methods were used to assess the genetic relationship of DNA sequences determined herein.<\/jats:p>\n              <\/jats:sec><jats:sec>\n                <jats:title>Results<\/jats:title>\n                <jats:p>PCR detected DNA of at least one microorganism in each of 221 ticks tested (94.4%, 221\/234). DNA-based detection inferred that single pathogens\/endosymbionts were the most common (43.4%, 96\/221) followed by double (38.9%, 86\/221), triple (14.5%, 32\/221), quadruple (2.3%, 5\/221) and quintuple (0.9%, 2\/221) mixed infections. Piroplasms (<jats:italic>Babesia<\/jats:italic>\/<jats:italic>Theileria<\/jats:italic> spp.) were the most prevalent (31.6%, 74\/234), followed by <jats:italic>Ehrlichia<\/jats:italic> spp. (20%, 47\/234) and <jats:italic>Anaplasma marginale<\/jats:italic> (7.7%, 18\/234). <jats:italic>Anaplasma phagocytophilum<\/jats:italic>, <jats:italic>A. ovis<\/jats:italic>, <jats:italic>A. centrale<\/jats:italic>, <jats:italic>Babesia ovis<\/jats:italic>, <jats:italic>Borrelia<\/jats:italic> spp., <jats:italic>Rickettsia<\/jats:italic> spp., <jats:italic>R. massiliae<\/jats:italic>, <jats:italic>Bartonella<\/jats:italic> spp. and <jats:italic>Hepatozoon<\/jats:italic> spp. were also detected. Endosymbionts such as <jats:italic>Francisella<\/jats:italic>-like (91.5%, 214\/234) and <jats:italic>Coxiella<\/jats:italic>-like (1.3%, 3\/234) organisms were also detected in ticks. The highest diversity of microorganisms was detected in <jats:italic>Hyalomma anatolicum<\/jats:italic> ticks (test-positive for 14\/14 microorganisms), followed by <jats:italic>Rhipicephalus microplus<\/jats:italic> (4\/14), <jats:italic>Hy. hussaini<\/jats:italic> (3\/14) and <jats:italic>Rh. annulatus<\/jats:italic> (2\/14). Ticks collected from cattle carried significantly more frequently piroplasms (41.2%, 54\/131; <jats:italic>P<\/jats:italic>\u00a0&lt;\u00a00.05) than those from buffaloes (19.4%, 20\/103). However, the overall prevalence of microorganisms did not vary significantly among ticks from the two host species as well as across different AEZs.<\/jats:p>\n              <\/jats:sec><jats:sec>\n                <jats:title>Conclusions<\/jats:title>\n                <jats:p>To our knowledge, this is the first study to investigate a wide range of tick-borne microorganisms in bovine ticks using a high-throughput diagnostic method from different AEZs in Pakistan. These findings will aid in establishing the distribution patterns and the control of tick-borne pathogens of bovines in Pakistan.<\/jats:p>\n              <\/jats:sec>","DOI":"10.1186\/s13071-019-3862-4","type":"journal-article","created":{"date-parts":[[2020,1,3]],"date-time":"2020-01-03T16:03:11Z","timestamp":1578067391000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":98,"title":["Bovine ticks harbour a diverse array of microorganisms in Pakistan"],"prefix":"10.1186","volume":"13","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-7714-0752","authenticated-orcid":false,"given":"Abdul","family":"Ghafar","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8660-730X","authenticated-orcid":false,"given":"Alejandro","family":"Cabezas-Cruz","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Clemence","family":"Galon","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Dasiel","family":"Obregon","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Robin B.","family":"Gasser","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sara","family":"Moutailler","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8888-0046","authenticated-orcid":false,"given":"Abdul","family":"Jabbar","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2020,1,3]]},"reference":[{"key":"3862_CR1","volume-title":"Tick-borne diseases of humans","author":"J Keirans","year":"2005","unstructured":"Keirans J, Durden L. 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