First Detection of Theileria sinensis-like and Anaplasma capra in Ixodes kashmiricus: With Notes on cox1-Based Phylogenetic Position and New Locality Records

SIMPLE SUMMARY: Ixodes species are the main vectors of bacteria and piroplasm for different vertebrate hosts. Research on these unexplored concerns has been neglected in different regions including Pakistan. Recently, we molecularly characterized Ixodes kashmiricus ticks and associated Rickettsia spp.; however, the cox1 sequence and associated Theileria spp. and Anaplasma spp. for this tick are unknown. This study aimed to genetically identify I. kashmiricus based on the cox1 sequence and associated Theileria spp. and Anaplasma spp. A total of 352 ticks including adult females, nymphs and males were collected from small ruminants. The BLAST results and phylogenetic analysis of the cox1 sequence revealed a close resemblance with the Ixodes ricinus complex sequences. The 18S rDNA and 16S rDNA sequences showed maximum identity with Theileria cf. sinensis or Theileria sinensis and Anaplasma capra, respectively, and they phylogenetically clustered with the same species. This is the first report on the cox1 sequence of the I. kashmiricus tick, new locality records, and associated T. sinensis-like and A. capra. In order to determine the epidemiology of Ixodes ticks and their related pathogens, a widespread tick investigation is required. ABSTRACT: Ixodes ticks transmit Theileria and Anaplasma species to a wide range of animals. The spreading of ticks and tick-borne pathogens has been attributed to transhumant herds, and research on these uninvestigated issues has been neglected in many countries, including Pakistan. Recently, we used internal transcribed spacer (ITS) and 16S ribosomal DNA partial sequences to genetically characterize Ixodes kashmiricus ticks and their associated Rickettsia spp. However, the data on its cox1 sequence and associated Theileria spp. and Anaplasma spp. are missing. This study aimed to genetically characterize I. kashmiricus based on the cox1 sequence and their associated Theileria spp. and Anaplasma spp. The I. kashmiricus ticks were collected from small ruminants: sheep (Ovis aries) and goats (Capra hircus) of transhumant herds in district Shangla, Dir Upper and Chitral, Khyber Pakhtunkhwa (KP), Pakistan. Out of 129 examined hosts, 94 (72.87%) (56 sheep and 38 goats) were infested by 352 ticks, including adult females (175; 49.7%) followed by nymphs (115; 32.7%) and males (62; 17.6%). For molecular analyses, 121 ticks were subjected to DNA isolation and PCR for the amplification of the cox1 sequence for I. kashmiricus, 18S rDNA for Theileria spp. and 16S rDNA sequences for Anaplasma spp. The obtained cox1 sequence showed 89.29%, 88.78%, and 88.71% identity with Ixodes scapularis, Ixodes gibbosus, and Ixodes apronophorus, respectively. Phylogenetically, the present cox1 sequence clustered with the Ixodes ricinus complex. Additionally, the 18S rDNA sequence showed 98.11% maximum identity with Theileria cf. sinensis and 97.99% identity with Theileria sinensis. Phylogenetically, Theileria spp. clustered with the T. cf. sinensis and T. sinensis. In the case of Anaplasma spp., the 16S rDNA sequence showed 100% identity with Anaplasma capra and phylogenetically clustered with the A. capra. PCR-based DNA detection targeting the amplification of groEL and flaB sequences of Coxiella spp. and Borrelia spp., respectively, was unsuccessful. This is the first phylogenetic report based on cox1 and new locality records of I. kashmiricus, and the associated T. sinensis-like and A. capra. Significant tick surveillance studies are needed in order to determine the epidemiology of Ixodes ticks and their associated pathogens.