BACKGROUND Ticks and tick-borne pathogens (TTBP) are a major constraint to livestock production in Pakistan; despite a high prevalence of TTBPs, knowledge on the capacity of Pakistani ticks to carry pathogens and endosymbionts is limited. 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. METHODS Ticks were collected from clinically healthy cattle (n = 116) and water buffaloes (n = 88) 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. RESULTS 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 (Babesia/Theileria spp.) were the most prevalent (31.6%, 74/234), followed by Ehrlichia spp. (20%, 47/234) and Anaplasma marginale (7.7%, 18/234). Anaplasma phagocytophilum, A. ovis, A. centrale, Babesia ovis, Borrelia spp., Rickettsia spp., R. massiliae, Bartonella spp. and Hepatozoon spp. were also detected. Endosymbionts such as Francisella-like (91.5%, 214/234) and Coxiella-like (1.3%, 3/234) organisms were also detected in ticks. The highest diversity of microorganisms was detected in Hyalomma anatolicum ticks (test-positive for 14/14 microorganisms), followed by Rhipicephalus microplus (4/14), Hy. hussaini (3/14) and Rh. annulatus (2/14). Ticks collected from cattle carried significantly more frequently piroplasms (41.2%, 54/131; P < 0.05) 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. CONCLUSIONS 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.

中文翻译：

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牛tick在巴基斯坦拥有各种各样的微生物。

背景壁虱和壁虱传播的病原体（TTBP）是巴基斯坦畜牧生产的主要限制因素。尽管TTBP的患病率很高，但是关于巴基斯坦tick传播病原体和共生共生体的能力的知识仍然有限。此外，多种微生物的混合感染进一步使传统诊断方法的检测复杂化并受到限制。本研究利用高通量微流实时PCR技术研究了巴基斯坦Pakistan中tick传播的微生物。方法自2017年9月至11月，从巴基斯坦五个农业生态区（AEZ）六个地区的30个村庄的临床健康牛（n = 116）和水牛（n = 88）收集Ti虫。微流实时PCR被用于测试个体壁虱的基因组DNA是否存在27种细菌和8种寄生微生物。系统发生方法用于评估本文确定的DNA序列的遗传关系。结果PCR检测到的221个s虫中的每个tick虫中至少一种微生物的DNA（94.4％，221/234）。基于DNA的检测推断单个病原体/内共生子是最常见的（43.4％，96/221），其次是双重的（38.9％，86/221），三次的（14.5％，32/221），三次的（2.3％，5） / 221）和五元组（0.9％，2/221）混合感染。浆质体（巴贝虫/泰勒虫属）最普遍（31.6％，74/234），其次是埃里希氏菌。（20％，47/234）和Anaplasma marginale（7.7％，18/234）。吞噬细胞无浆膜，羊油曲霉，中央曲霉，羊栖贝氏杆菌，伯雷利亚菌，立克次体菌，马赛菌，巴尔通体属 和Hepatozoon物种 也被检测到。在壁虱中也检出了内共生菌，如Francisella样（91.5％，214/234）和Coxiella样（1.3％，3/234）。最高的微生物多样性出现在无花果透明壁tick（14/14微生物测试阳性）中，其次是细头裂脑R（4.14）。侯赛尼（3/14）和Rh。环（2/14）。从牛身上采集的cks虫携带质子的频率（41.2％，54/131; P <0.05）比从水牛身上采集的cks虫的频率更高（19.4％，20/103）。但是，在两种寄主物种的tick之间以及不同的AEZ之间，微生物的总体流行率没有显着差异。结论据我们所知，这是第一项使用来自巴基斯坦不同AEZ的高通量诊断方​​法调查牛s中各种tick传播微生物的研究。这些发现将有助于建立巴基斯坦牛的tick传播病原体的分布模式和控制。