Anaplasma marginale (A. marginale), Anaplasma ovis (A. ovis) and Theileria ovis (T. ovis) are among the most commonly reported intracellular tick borne pathogens that infect ruminants across the globe causing huge economic losses. This study aims to report the prevalence and phylogenetic evaluation of these three pathogens infecting sheep and goats (n = 333) that were enrolled from Fort Munro region in Pakistan by using msp1b, msp4 and 18S rRNA genes for A. marginale, A. ovis and T. ovis respectively. Results revealed almost similar infection rates in sheep and goats with an overall prevalence of 11% for A. marginale, 28% for A. ovis and 3% for T. ovis. Concurrent infection was also recorded, however, the number of animals infected with two pathogens (n = 24; 7.2%) was higher than infection with three pathogens (n = 2; 0.6%). Risk factor analysis revealed that sheep reared in small herds had higher A. marginale (P = 0.03) and A. ovis (P = 0.04) infection rates compared to those from large herds. In addition, it was observed that bucks (P ≤ 0.05) and tick-free goats (P ≤ 0.05) exhibited higher A. ovis infection rates than nannies. Phylogenetic analysis of all three pathogens showed that Pakistani isolates were clustered together and were closely related to previously deposited Pakistani isolates as well as with those that were reported from worldwide countries. In conclusion, we are reporting that Pakistani sheep and goats have A. marginale, A. ovis and T. ovis mediated infections and control measures should be taken against them to improve the productivity of the livestock sector.

This study investigates the molecular prevalence and phylogenetic characteristics of two prominent blood-borne pathogens,Toxoplasma gondii(T.gondii) andPlasmodiumspp., in common quails (Coturnix coturnix) sampled from both wild (N = 236) and farmed (N = 197) populations across four districts (Layyah, Dera Ghazi Khan, Lahore, and Multan) in Punjab, Pakistan, during the hunting seasons from 2021 to 2023. Additionally, the impact of these pathogens on the complete blood count (CBC) of the hosts is examined. Out of 433 quails tested, 25 (5.8%) exhibited amplification of the internal transcribed spacer (ITS-1) gene forT.gondii, while 15 (3.5%) showed amplification of theCytochrome bgene forPlasmodiumspp. A risk factor analysis indicated that the prevalence of both pathogens was not confined to specific sampling sites or bird sexes (P > 0.05). District-wise analysis highlighted that hens were more susceptible to bothT.gondiiandPlasmodiumspp. infections than cocks. Wild quails exhibited a higher susceptibility toT.gondiicompared to farmed birds. Significant CBC variations were recorded in infected birds as compared to uninfected ones. BLAST analysis of generated sequences has confirmed the identity of recovered PCR amplicons asT.gondiiandPlasmodium relictum. Phylogenetic analysis revealed that Pakistani isolates clustered with those reported from various countries globally. This study provides the first documentation ofT.gondiiandPlasmodiumsp. infections in Pakistani quails, underscoring the need for detailed investigations across different regions to enhance our understanding of infection rates and the zoonotic potential of these parasites.

Theileria annulata (T. annulata) and Anaplasma marginale (A. marginale) are among the most extensively reported tick borne pathogens and are associated with huge economic losses worldwide. A total of 298 cattle blood samples were screened to report the presence of these two pathogens. The samples were collected from apparently healthy cattle (Achai, n = 155, Jersy, n = 88 and crossbred, n = 55) in Bajaur district of Khyber Pakhtunkhwa (KPK) during June and July of 2022. A total of 31 out of 298 cattle (10.4%) were found infected with T. annulata as PCR amplified a 156 base pair fragment from Tams-1 gene of T. annulata from their blood. While 16/298 animals (5.4%) were found infected with A. marginale as they amplified a 382 base pair fragment specific for msp5 gene of this bacterium. Three animals (1%) were found co infected. Cattle susceptibility to T. annulata infection was significantly higher than A. marginale infection (P < 0.001). Phylogenetic analysis revealed that Pakistani isolates of both detected pathogen clustered together and were closely related isolates from worldwide countries. Prevalence of T. annulata varied significantly among the sampling sites (P = 0.05) while no such association was observed for A. marginale among the tested cattle. Epidemiological data analysis revealed that none of the studied risk factors was found associated either with the prevalence of T. annulata or A. marginale (P > 0.05) among enrolled cattle. In conclusion, our study has revealed a relatively higher prevalence of T. annulata than A. marginale in cattle from the Bajaur district in KPK. This information is important for improving the productivity of the livestock sector, which is one of the main sources of income in the country. It is recommended that this data be taken into account for the development and implementation of effective tick control programs, as well as for the improvement of livestock management practices to prevent and manage TBDs in Pakistan.

Raising small ruminants is the main source of income for farmers in Pakistan especially in rural areas of Dera Ghazi Khan in Punjab. Despite having large sheep population, the prevalence of intra-erythrocytic protozoa, Theileria (T.) lestoquardi, has never been reported from this area. This study was conducted to fill this knowledge gap and 333 blood samples of apparently healthy small ruminants (168 sheep and 165 goats) along with their epidemiological data were collected from Dera Ghazi Khan district during August till November 2022. The polymerase chain reaction (PCR) analysis amplified a 785 base pair amplicon specific for the Merozoite surface antigen (ms 1–2) gene of T. lestoquardi in 2 out of the 168 (3.3%) sheep blood samples, while no goat blood sample out of 165 was found to be infected with T. lestoquardi. DNA sequencing confirmed the presence of Theileria lestoquardi in both samples and phylogenetic analysis revealed that these amplicon resembled the partial ms 1–2 gene sequences detected in small ruminants from Pakistan, India Iran and Egypt. All the studied epidemiological factors (age, sex, breed, size of herd, dogs with herd, composition of herd, size of herd and Tick burden on sheep) were not found associated with the prevalence of T. lestoquardi. In conclusion, this study reports a low prevalence of T. lestoquardi infection in the Dera Ghazi Khan District of Punjab, Pakistan. The data generated from this work will help pave the way for the prophylactic detection and control of ovine and caprine theileriosis in the region.