Molecular Epidemiology and Risk Factors of Theileria annulata Infection and Rhipicephalus and Hyalomma Tick Infestation in Three Districts of Northern Pakistan.

BACKGROUND: Ticks and tick-borne diseases pose a major threat to livestock productivity in tropical and subtropical regions, by transmitting pathogens of veterinary and public health concern, including Theileria species. Despite the threats posed to the large ruminant industry, integrated studies on the causative agent of tropical theileriosis and its tick vectors from the same infected hosts, along with their associated risk factors, remain limited in northern Pakistan. This study investigated Theileria infection, its associated tick species, and host- and environmental-related risk factors in large ruminants from the region using an integrated approach.

METHODS: A total of 3151 ticks (female: 1630, 51.7%; male: 725, 23%; nymph: 458, 14.5%; larvae: 338, 10.7%) were collected from 900 animals (805 cattle, 89.45%; 95 buffaloes, 10.55%), along with 221 blood samples from symptomatic, tick infested animals (202 cattle, 91.4%; 19 buffaloes, 8.6%) between May 2024 and April 2025. Ticks were identified morphologically, while Theileria infection was assessed by microscopy and PCR targeting the 18S rRNA gene. Phylogenetic analysis was performed in MEGA X using the maximum-likelihood method. Host- and environmental-related risk factors were assessed for their association with tick infestation and Theileria infection.

RESULTS: Two tick genera were identified morphologically: Rhipicephalus (83.59%) and Hyalomma (16.41%). Within Rhipicephalus, Rhipicephalus microplus (70.29%) was the dominant species, followed by Rhipicephalus haemaphysaloides (7.61%) and Rhipicephalus sp. (5.68%). The genus Hyalomma comprised Hyalomma anatolicum (8.82%) and Hyalomma turanicum (7.58%). Tick infestation was significantly higher in cattle (46.33%) than in buffaloes (22.11%). In cattle, infestation rates were 46.49% in Upper Dir, 46.62% in Bajaur and 45.90% in Lower Dir, whereas in buffaloes, the corresponding rates were 24.14%, 23.53% and 18.75%, respectively. Microscopic examination detected Theileria infection in 18.09% (40/221) of blood samples from clinically affected, tick-infested animals (37/202 cattle, 18.31%; 3/19 buffaloes, 15.78%), whereas the detection rate by PCR was 45.24% (100/221) (92/202 cattle, 45.54%; 8/19 buffaloes, 42.11%). By BLAST analysis, the obtained sequence of 18S rRNA showed a maximum identity of 99.62% with a Theileria annulata sequence from Turkey, China and Pakistan, and phylogenetic tree grouped it with corresponding species from Asia and Europe. Risk factors analysis showed that host species was significantly associated with tick infestation, while age, season, and acaricide treatment were significantly associated with infestation in both cattle and buffaloes (p < 0.05). Age, season and acaricide use were significantly associated with Theileria infection in cattle, whereas in buffaloes, only acaricide use was significant. Gender, presence of rural poultry, grazing system and district showed no significant association (p > 0.05) with tick infestation or Theileria infection in either host.

CONCLUSIONS: This study provides an integrated assessment of the causative agent of tropical theileriosis and associated tick species from the same large ruminants, along with their associated risk factors, in the mountainous terrain of northern Pakistan. Given that tick vectors and tropical theileriosis can significantly affect livestock-dependent economies, this study may help guide better preventive measures in the region.