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Ticks and tick-borne diseases have a large impact on animal health and the livelihood of livestock owners, particularly in developing countries. Despite the suitability of Pakistan’s climate for ticks, there is a paucity of systematic work investigating these parasites and the diseases, which they transmit in this country. To better understand the distribution of ticks, the whole country was divided into five agro-ecological zones using Global-Aridity dataset and 108 livestock farms from 9 different districts, covering the semi-arid and the arid agro-ecological zones in Punjab province, were included in the study. Ticks were collected from two randomly selected animals of each ruminant species present at the farm (194 buffaloes, 179 cattle, 80 goats and 18 sheep) and stored in 70% ethanol. Morphological identification of the ticks was subsequently performed using standard taxonomic keys and multikey software, a computer-based polychotomous key. The identification was confirmed by sequencing of a partial fragment from the second internal transcribed spacer (ITS2) and cytochrome oxidase subunit 1 (cox1) genes from randomly selected specimens of each species which proved that the morphological and molecular data are coherent in the identification of the 4 tick species. The prevalence of ticks between the agro-ecological zones was significantly different (P = 0.037). There was no farm found without ticks. In total, 3,807 (12 larvae, 1,231 nymphs, 1,303 females and 1,261 males) ixodid ticks representing four different species were collected: Hyalomma anatolicum (n = 3,021, 79.3%), Rhipicephalus microplus (n = 715, 18.8%), Hyalomma dromedarii (n = 41, 1.0%), and Rhipicephalus turanicus (n = 30, 0.9%). Rh. microplus was the predominant species in the semi-arid zone, whereas Hy. anatolicum was the most abundant tick species in the arid zone. Hy. dromedarii and Rh. turanicus were found only in the arid zone. In all the districts, multiple tick species were found except Multan district, where only Hy. anatolicum was present. The overall proportion of tick-infested ruminants was 78.3% (369/471), and was significantly different (P < 0.001, χ² = 126.9) among animal species. It was highest in cattle (89.9%), followed by buffaloes (81.4%), goats (60%) and sheep (11.1%). The median tick burdens recorded (43 ticks per animal, ranged from 27-67) were significantly different among the animal species (P < 0.001) and were highest in cattle (median = 58), followed by buffaloes (median = 38), goats (median = 19) and sheep (median = 4.5). In large ruminants, older animals carried more ticks than younger animals (buffalo, P = 0.020; cattle, P = 0.002). It was observed that female animals had higher tick burdens than male animals (buffalo, P = 0.002; cattle, P < 0.001; goat, P = 0.014; sheep, P = 0.02). The intensity of infestation was significantly lower in indigenous animals as compared to exotic (P < 0.001) and crossbred cows (P < 0.001), while the difference was not statistically significant between crossbred and exotic cattle (P = 0.11). The majority of the animals were infested with a single tick species (90.5%), while only a few were infested with multiple species (9.5%).
After identification, the ticks were divided into 405 (Hy. anatolicum = 300, Rh. microplus = 89, Hy. dromerdarii = 9, Rh. turanicus = 7) tick pools (semi-arid zone = 113, arid zone = 292) and screened by RLB assay for the presence of DNA of 41 tick-borne pathogens, i.e. Anaplasma, Ehrlichia, Babesia, Theileria and Rickettsia species. Out of total 405 tick pools, DNA from at least one tick-borne pathogen was found in 148 (36.5%) pools. Among the positive pools, 94 (63.5%) had a mixed infection with two or more (ranging from 2 to 5) tick-borne pathogen species with 18 different combinations, whereas 54 (36.5%) pools were infected with single tick-borne pathogen species. The overall prevalence estimates of tick-borne pathogens in Punjab were significantly different (χ² = 90.2, df = 3, P < 0.001), and the prevalence of Ehrlichia spp. (22.2%) was highest, followed by Theileria (9.9%), Anaplasma (7.7%) and Babesia spp. (2.5%). 11 species of Rickettsiales, namely: A. centrale, A. marginale, A. ovis, Anaplasma sp. BL099-6, Ehrlichia sp. (Multan), Ehrlichia sp. ERm58, Ehrlichia sp. Firat, E. mineirensis, Ehrlichia sp. Omatjenne, R. massiliae and R. raoultii, were identified in four tick species (Hy. anatolicum, Hy. dromedarii, Rh. microplus and Rh. turanicus) from two agro-ecological zones in Punjab through genetic analyses of 16S rRNA. Moreover, four Babesia species (B. bigemina, B. bovis, B. caballi, and B. occultans) and three Theileria species (T. annulata, T. ovis, and T. orientalis) were also detected in Hy. anatolicum and Rh. microplus ticks. The most common tick-borne pathogen was a hitherto uncharacterized species, i.e. Ehrlichia sp. (Multan) (18.0%, CI 14.4-22.1), and 16S rRNA gene sequences showed 99% identity to previously described Ehrlichia species. Other common tick-borne pathogens were Ehrlichia sp. ERm58 (16.3%, CI 22.8-20.3), Ehrlichia sp. Firat (16.0%, CI 12.6-20), Theileria annulata (6.7%, CI 4.4-9.6), Anaplasma marginale (5.7%, CI 3.6-8.4), T. orientalis (buffeli) (3.5%, CI 1.9-5.7), E. mineirensis (3.2%, CI 1.7-5.4) and A. centrale (2.7%, CI 1.4-4.8). This study represents the first evidence of the occurrence of Anaplasma sp. BL099-6, Ehrlichia sp. (Multan), Ehrlichia sp. ERm58, Ehrlichia sp. Firat, E. mineirensis, Ehrlichia sp. Omatjenne, R. massiliae, R. raoultii, B. occultans and T. orientalis in Pakistan. Moreover, we report the first detection of A. marginale, E. mineirensis and Ehrlichia sp. Omatjenne in Hy. anatolicum and R. raoultii in Rh. microplus ticks. Our data showed that Hyalomma and Rhipicephalus ticks in Punjab were naturally infected with Rickettsiales, Babesia and Theileria species. Analysis of questionnaire data using univariable and multivariable logistic regression analysis revealed that the absence of rural poultry on farm (P = 0.005, OR = 4.4), not using any acaricides (P < 0.001, OR = 7.5), traditional rural housing system (P = 0.007, OR = 13.1) and grazing (P = 0.003, OR = 12.6) were potential risk factors associated with a higher tick prevalence on livestock farms. Attachment site preferences significantly varied by animal species for Hy. anatolicum (χ² = 140.4, P < 0.001) and Rh. microplus (χ² = 77.6, P < 0.001). It can be concluded that a much broader spectrum of ticks and tick-borne pathogens is present in Pakistan than previously thought, including several potential zoonotic pathogens. In addition, a novel Ehrlichia species with 99% sequence identity to the taxon described previously, was identified. It is expected that the outcomes of this study will be useful in the planning of integrated control strategies for ticks and tick-borne diseases in Pakistan.