Anaplasmosis and theileriosis are considered the most important tick-borne diseases for livestock production worldwide, causing significant economic losses in tropical and subtropical regions. The present study was aimed to develop a multiplex TaqMan® qPCR assay to simultaneously detect Anaplasma marginale and Theileria annulata and to applied it to investigate naturally infected cattle in Cuba. The assay was highly specific, sensible, and efficient; it was more sensitive than a well-established nested PCR and detected 1 DNA copy of each target. Consistent repeatability and reproducibility within and between multiplex qPCR runs was shown. A total of 223 blood samples collected in western Cuba were analyzed for haemoparasites infection in cattle. The multiplex qPCR assay detected A. marginale in 213 samples (95.5%; CI: 95%; 91.9%–97.5%), but all samples were negative for T. annulata. Additionally, the genetic diversity of A. marginale was assessed using 16S rRNA, MSP1a and MSP4 nucleotide and protein sequences. The MSP1a tandem repeats ranged from three to five, and twelve different MSP1a tandem repeats of A. marginale were found, which presented genotypes C, E, and G in the 5ʹUTR microsatellite region. Phylogenetic analysis using the msp4 gene showed that Cuban strains were closely related to others previously reported in Mexico, Brazil and Asian countries. The multiplex qPCR described here proved to be a rapid, specific and cost-effective mean for the simultaneous detection of A. marginale and T. annulata. Further epidemiological studies using this assay will improve the surveillance of the associated diseases in regions where they are endemic.

无性病和虫害病被认为是全世界牲畜生产中最重要的tick传播疾病，在热带和亚热带地区造成重大的经济损失。本研究的目的是开发一种多重TaqMan®qPCR分析方法，以同时检测边缘无浆质和环纹泰勒菌，并将其用于调查古巴的自然感染牛。该测定具有高度特异性，灵敏性和高效性。它比完善的巢式PCR更为灵敏，可检测到每个靶标的1个DNA拷贝。显示了多重qPCR运行之间以及之间的一致的可重复性和可重复性。对古巴西部收集的总共223份血液样本进行了牛血寄生虫感染分析。检测到多重qPCR分析A.边缘无在213个样本（95.5％; CI：95％; 91.9％-97.5％），但所有样品是阴性T.菇。此外，遗传多样性A.边缘无使用评估16S rRNA基因，MSP1a和MSP4核苷酸和蛋白序列。所述MSP1a串联重复介于三至五，和的十二个不同的MSP1a串联重复A.边缘无发现，其中提出基因型C，E，和G在5'UTR微区。使用MSP进行系统发育分析4个基因显示古巴菌株与先前在墨西哥，巴西和亚洲国家报道的其他菌株密切相关。所述多重qPCR描述这里被证明是一种快速，特异和成本效益的均值的同时检测A.边缘无和T.环纹。使用该测定方法进行的进一步流行病学研究将改善对地方病相关疾病的监测。