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Abstract Ticks borne diseases (TBDs) are an important disease of animals including ruminants, equine, and dromedaries, they are responsible for significant economic losses to the livestock industry in countries where they occur. Piroplasmosis is one of the TBDs which caused by Theileria (T) spp. or Babesia (B) spp. or both which transmitted by ticks of genera Dermacentor, Hyalomma, and Rhipicephalus. The objective of this study is to use sensitive, fast and specific molecular tools, such as multiplex (m) Polymerase Chain Reaction (PCR) for simultaneous detection of T. equi and B. caballi in equine and examined the presence of T. haneyi infections in Egypt using uiplex (u) PCR. In addition to molecular detection and genetic characterization of piroplasma spp. infecting in camel (Camelus dromedarius) using conventional (c) PCR, mPCR, uPCR and nested (n) PCR followed by sequencing. A total of 155 equine blood samples (79 horse and 76 donkey) and 531 camel blood samples have been collected from different geographical governorates in Egypt. In equine, the microscopical examination result revealed that Theileria spp. were detected in 5 (6.3%) horses and 7 (9.2%) donkeys. The mPCR method revealed the presence of T. equi in 16 (20.3%) horses and 10 (13.1%) donkeys and the presence of B. caballi in one (1.2%) horse. B. caballi was not detected in donkeys. Additionally, T. haneyi was detected in Egypt for the first time in 42 (53.1%) of the horses and 29 (38.1%) of the donkeys tested samples. In camel, piroplasma spp. were detected in 58 (11%) of samples microscopically. While the cPCR detected Babesia/Theileria spp. in 203 (38%) of samples. Multiplex PCR was applied on all piroplasma spp. positive samples by cPCR (203) for simultaneous detection of T. equi, B. caballi, B. bigemina and B. bovis. The multiplex PCR result revealed the presence of T. equi, and B. bovis infection in 84 (41%) and 9 (4%) of samples, respectively. Moreover B. caballi was detected in 11 (5.4%) of samples in mixed infection with T. equi. In addition to B. bigemina mixed infection was detected 2 (1%) of the samples. Furthermore, T. haneyi was detected in 2 (0.9%) of samples using uPCR. Sequencing and phylogenetic analyses of the identified spp. revealed that T. equi, B. caballi, B. bovis, B. bigemina camel Egyptian isolates were closely related to different isolate from equine and cattle from different countries registered in GeneBank. The blast analysis of nPCR sequences revealed that there are three new piroplasma spp. infecting camel as B. vulpes 7 (22%), Babesia sp. 4 (9%) and Theileria sp. 1 (3%), for the first time in camel. Moreover, T. haneyi was detected for the first time in camel. |