Risk factors associated with tick infestations on equids in Khyber Pakhtunkhwa, Pakistan, with notes on Rickettsia massiliae detection

BACKGROUND: Studies on ticks infesting equids are lacking in various parts of the world, including Khyber Pakhtunkhwa (KP), Pakistan. The aim of this study was to investigate the diversity of ticks infesting equids, associated risk factors and rickettsial detection in ticks from equids in KP. METHODS: Inspection of 404 equid hosts from November 2018 to October 2019 resulted in the collection of 550 ticks. Data on tick-associated risk factors were collected from equid owners by means of a questionnaire. After morphological identification, partial DNA sequences of the tick mitochondrial 16S rRNA gene were used for taxonomic confirmation of species. Partial sequences of the gltA and ompA genes were used for Rickettsia detection in ticks. RESULTS: A total of 550 tick specimens were collected on 324 (80.2%) of the equids inspected, of which 161 were horses (50%), 145 (45%) were donkeys and 18 were mules (5%). The ticks were identified as belonging to the following five species: Rhipicephalus microplus (341 specimens, 62% of the total ticks), Rh. haemaphysaloides (126, 23%), Rh. turanicus (39, 7%), Rh. sanguineus (s.l.) (33, 6%) and Hyalomma anatolicum (11, 2%). The most prevalent tick life stage was adult females (279, 51%) followed by adult males (186, 34%) and nymphs (85, 15%). Higher tick infestations were observed on male equids (relative risk [RR] 0.7432, P < 0.0005) and adult equids (RR 1.268, P < 0.0020). Ticks were frequently attached to the axial region of horses (55, 21%), sternum of donkeys (44, 21%) and belly of mules (19, 23%) (P < 0.04). Temporal patterns of tick infestation in association with temperature and humidity were highly significant (P < 0.05). Risk factors, such as animal housing (P < 0.0003), living management (P < 0.006), grazing type (P < 0.01) and location in hilly areas (P < 0.02), significantly enhanced the chances for tick infestation. Tick species analyzed in this study were phylogenetically related to species from Afghanistan, China, South Africa and Taiwan. Partial sequences of the gltA and ompA genes obtained from Rh. microplus and Rh. haemaphysaloides were 100% identical to the spotted fever group pathogen Rickettsia massiliae. CONCLUSIONS: Equids exposed to significant risk factors were infected by one or more of at least five tick species in KP, Pakistan, and some of the ticks harbored the human pathogen R. massiliae. GRAPHICAL ABSTRACT: [Image: see text]