Molecular Epidemiology and Phylogeny of Theileria ovis and Theileria lestoquardi in Sheep and Goats from Southern Punjab, Pakistan.

Background: Theileria spp. are responsible for ovine and caprine theileriosis, leading to significant morbidity and mortality in small ruminants. The present study aims to investigate Theileria spp. infections in small ruminants from Southern Punjab in Pakistan, and genetic characterize revealed Theileria spp. isolates. Methods: A total of 93 sheep and 107 goats were sampled between May and August 2022. Blood smears were examined microscopically, and PCR amplification targeting the 18S rRNA gene was performed to detect Theileria spp. Additionally, specific PCR assays targeting 18S rRNA and ms1 partial sequences were used to identify Theileria ovis and T. lestoquardi, respectively.  Results: The prevalence of Theileria spp. was significantly higher using PCR (13.5%) compared to microscopic screening (5%). Sheep showed a higher prevalence rate (19.4%) compared to goats (8.4%) (p = 0.024). Young sheep aged ≤ 1 year were more commonly infected with Theileria spp. (41%) compared to older sheep (p = 0.006). The prevalence of Theileria spp. was higher in sheep-only herds (37.3%) compared to goat-only herds (18%) or mixed-species herds (8.1%) (p = 0.015). The prevalence rates of T. ovis and T. lestoquardi were 9% and 2.5%, respectively, with four animals (2 goats and 2 sheep) showing co-infection. Phylogenetic analysis revealed that our T. ovis 18S rRNA sequence clustered with previously reported sequences from sheep in Turkey, China, Spain, and goats in Tanzania. The obtained T. lestoquardi ms1 partial sequence formed a distinct cluster from other T. lestoquardi isolates in Pakistan and neighboring countries.  Conclusion: Theileria spp. co-circulation in Pakistani small ruminants, particularly the presence of T. ovis and T. lestoquardi, highlights the need for attention from animal health decision-makers due to their financial and health impacts.

Molecular evidence of Borrelia theileri and closely related Borrelia spp. in hard ticks infesting domestic animals.

Ticks pose significant threats to hosts by transmitting Borrelia spp., which are grouped into Lyme borreliae, relapsing fever borreliae (RF), and reptiles- and monotremes-associated borreliae. The RF borreliae encompass a group of Borrelia species predominantly transmitted by soft ticks, but some of its members can also be transmitted by hard ticks. Information on the detection and genetic characterization of tick-borne RF borreliae, including Borrelia theileri, is notably rare in Asia, particularly in Pakistan. Herein, we employed molecular techniques to detect borreliae in hard ticks collected from domestic animals in Khyber Pakhtunkhwa, Pakistan. Ticks were subjected to morphological analysis, followed by DNA extraction and PCR amplification of partial fragments of borrelial 16S rRNA and flaB genes. A total of 729 ticks were collected from 264 hosts, with Haemaphysalis cornupunctata (12.9%; 94/729) being the most prevalent, followed by Hyalomma anatolicum (11.7%; 85/729), Rhipicephalus microplus (10.0%; 73/729), Haemaphysalis kashmirensis (9.1%; 66/729), Haemaphysalis bispinosa (8.5%; 62/729), Rhipicephalus sanguineus (8%; 58/729), Haemaphysalis montgomeryi (6.2%; 45/729), Rhipicephalus turanicus (5.5%; 40/729), Hyalomma dromedarii and Ixodes kashmirensis (4.4%; 32/729 each), Rhipicephalus haemaphysaloides (4.1%; 30/729), Haemaphysalis sulcata and Hyalomma scupense (3.8%; 28/729 each), Haemaphysalis danieli (2.9%; 21/729), Hyalomma kumari (2.6%; 19/729), and Hyalomma isaaci (2.2%; 16/729). Based on 16S rRNA detection of Borrelia spp., only R. turanicus yielded positive results, resulting in an overall infection rate of 0.3% (2/160), while using flaB-based detection, four tick species including R. microplus, R. turanicus, Ha. sulcata, and Ha. cornupunctata showed positive results, yielding an overall infection rate of 6.9% (11/160). The amplified DNA fragments of borrelial 16S rRNA and flaB in R. turanicus from goats shared maximum identities of 100 and 99.40% with Borrelia theileri, respectively. Amplified borrelial flaB fragments in R. microplus from cows and sheep displayed 100% identity with B. theileri, while flaB fragments in Ha. cornupunctata and Ha. sulcata from goats revealed identities of 99.32 and 99.75% with undetermined RF Borrelia spp., respectively. Phylogenetic analysis revealed clustering of B. theileri from R. microplus and R. turanicus with the same species, while Borrelia spp. from Ha. cornupunctata and Ha. sulcata with undetermined RF Borrelia spp. Notably, this research marks the first documentation of B. theileri in R. turanicus and the identification of RF Borrelia spp. in Ha. cornupunctata and Ha. sulcata.

First Molecular-Based Confirmation of Dermacentor marginatus and Associated Rickettsia raoultii and Anaplasma marginale in the Hindu Kush Mountain Range.

Ticks of the genus Dermacentor Koch, 1844 (Acari: Ixodidae) are poorly known systematically due to their habitation in harsh topographic environments and high mountains. Dermacentor ticks are diversely distributed in the Palearctic, Nearctic, and Oriental regions. There is no available information on the occurrence of Dermacentor marginatus in Pakistan; thus, the current investigation aimed the first morphological and molecular confirmation of this species and associated Anaplasma marginale and Rickettsia raoultii. Ticks were collected from goats (Capra hircus) and morphologically identified. Genomic DNA was extracted from 18/26 (69.23%) tick specimens, including 11 males and 7 females (1 unfed and 6 fed females). Extracted DNA was subjected to PCR for the amplification of genetic markers like 16S rDNA and cox1 for ticks, 16S rDNA for Anaplasma spp., and gltA and ompB for Rickettsia spp. A total of 26 D. marginatus ticks composed of 19 males (73.07%) and 7 females (26.9%) [1 (3.84%) unfed and 6 (23.07%) fed females] were collected from goats. According to amplicons via BLAST analysis, the 16S rDNA sequence showed 97.28-98.85% identity and the cox1 sequence showed 95.82-98.03% identity with D. marginatus. Additionally, the 16S rDNA sequence for Anaplasma sp. was detected in D. marginatus that showed 100% identity with Anaplasma marginale. Rickettsial gltA and ompB sequences for Rickettsia sp. showed 100% identity with Rickettsia raoultii. In phylogenetic analysis, ticks' 16S rDNA and cox1 sequences clustered with the same species. In phylogenetic analysis, A. marginale based on 16 rDNA clustered with A. marginale, while gltA and ompB sequences clustered with R. raoultii. This is the first study on the genetic characterization of D. marginatus and associated A. marginale and R. raoultii in Pakistan. The northern areas of Pakistan, which need to be explored in terms of ticks and associated pathogens due to their zoonotic threats, have been neglected due to the inaccessible climatic conditions.

Molecular Survey of Rickettsia raoultii in Ticks Infesting Livestock from Pakistan with Notes on Pathogen Distribution in Palearctic and Oriental Regions.

Ticks are hematophagous ectoparasites that transmit different pathogens such as Rickettsia spp. to domestic and wild animals as well as humans. Genetic characterizations of Rickettsia spp. from different regions of Pakistan are mostly based on one or two genetic markers and are confined to small sampling areas and limited host ranges. Therefore, this study aimed to molecularly screen and genetically characterize Rickettsia spp. in various tick species infesting camels, sheep, and goats. All the collected tick specimens were morphologically identified, and randomly selected tick species (148) were screened molecularly for the detection of Rickettsia spp. by amplifying three rickettsial DNA fragments, namely, the citrate-synthase gene (gltA), outer-membrane protein A (ompA), and outer-membrane protein B (ompB). After examining 261 hosts, 161 (61.7%) hosts were found infested by 564 ticks, including 287 (50.9%) nymphs, 171 (30.3%) females, and 106 (18.8%) males in five districts (Kohat, Dera Ismail Khan, Lower Dir, Bajaur, and Mansehra). The highest occurrence was noted for Hyalomma dromedarii (number = 72, 12.8%), followed by Haemaphysalis sulcata (n = 70, 12.4%), Rhipicephalus turanicus (n = 64, 11.3%), Rhipicephalus microplus (n = 55, 9.7%), Haemaphysalis cornupunctata (n = 49, 8.7%), Hyalomma turanicum (n = 48, 8.5%), Hyalomma isaaci (n = 45, 8.0%), Haemaphysalis montgomeryi (n = 44, 7.8%), Hyalomma anatolicum (n = 42, 7.5%), Haemaphysalis bispinosa (n = 38, 6.7%), and Rhipicephalus haemaphysaloides (n = 37, 6.6%). A subset of 148 ticks were tested, in which eight (5.4%) ticks, including four Hy. turanicum, two Ha. cornupunctata, one Ha. montgomeryi, and one Ha. bispinosa, were found positive for Rickettsia sp. The gltA, ompA, and ompB sequences revealed 100% identity and were phylogenetically clustered with Rickettsia raoultii reported in China, Russia, USA, Turkey, Denmark, Austria, Italy, and France. Additionally, various reports on R. raoultii from Palearctic and Oriental regions were summarized in this study. To the best of our knowledge, this is the first report regarding genetic characterization and phylogenetic analysis of R. raoultii from Pakistan. Further studies to investigate the association between Rickettsia spp. and ticks should be encouraged to apprise effective management of zoonotic consequences.

Molecular Detection of Rickettsia hoogstraalii in Hyalomma anatolicum and Haemaphysalis sulcata: Updated Knowledge on the Epidemiology of Tick-Borne Rickettsia hoogstraalii.

Ticks are hematophagous ectoparasites that transmit pathogens to animals and humans. Updated knowledge regarding the global epidemiology of tick-borne Rickettsia hoogstraalii is dispersed, and its molecular detection and genetic characterization are missing in Pakistan. The current study objectives were to molecularly detect and genetically characterize Rickettsia species, especially R. hoogstraalii, in hard ticks infesting livestock in Pakistan, and to provide updated knowledge regarding their global epidemiology. Ticks were collected from livestock, including goats, sheep, and cattle, in six districts of Khyber Pakhtunkhwa (KP) Pakistan. Overall, 183 hosts were examined, of which 134 (73.2%), including goats (number = 39/54, 72.2%), sheep (23/40, 57.5%), and cattle (71/89, 80%) were infested by 823 ticks. The most prevalent tick species was Rhipicephalus microplus (number = 283, 34.3%), followed by Hyalomma anatolicum (223, 27.0%), Rhipicephalus turanicus (122, 14.8%), Haemaphysalis sulcata (104, 12.6%), Haemaphysalis montgomeryi (66, 8.0%), and Haemaphysalis bispinosa (25, 3.03%). A subset of 210 ticks was selected and screened for Rickettsia spp. using PCR-based amplification and subsequent sequencing of rickettsial gltA and ompB fragments. The overall occurrence rate of R. hoogstraalii was 4.3% (number = 9/210). The DNA of Rickettsia was detected in Hy. anatolicum (3/35, 8.5%) and Ha. sulcata (6/49, 12.2%). However, no rickettsial DNA was detected in Rh. microplus (35), Rh. turanicus (35), Ha. montgomeryi (42), and Ha. bispinosa (14). The gltA and ompB fragments showed 99-100% identity with R. hoogstraalii and clustered phylogenetically with the corresponding species from Pakistan, Italy, Georgia, and China. R. hoogstraalii was genetically characterized for the first time in Pakistan and Hy. anatolicum globally. Further studies should be encouraged to determine the role of ticks in the maintenance and transmission of R. hoogstraalii in different hosts.

Argas persicus and Carios vespertilionis Ticks Infesting Ducks, Domestic Fowls and Bats in Pakistan: First Report on Molecular Survey and Phylogenetic Position of Borrelia anserina.

Argasid ticks have the vectorial potential for transmitting disease-causing pathogens to avian hosts, resulting in economic losses that may not be fully estimated. Borrelia species are the responsible agents of borreliosis in poultry, animals and humans. Our previous studies have reported a high prevalence of Argas persicus infesting domestic fowls in Khyber Pakhtunkhwa (KP), Pakistan. However, molecular screening and genetic characterization of Borrelia spp. in A. persicus have been neglected in Pakistan. In this study, we focused on the molecular epidemiology and genetic characterization of Borrelia spp. associated with A. persicus ticks infesting domestic fowls and ducks, and Carios vespertilionis infesting bats in selected districts of KP. Overall, 1818 ticks, including females (415; 23%), males (345; 19%), nymphs (475; 26%) and larvae (583; 32%), were collected from 27 locations in nine districts (Peshawar, Mardan, Swabi, Charsadda, Chitral, Lakki Marwat, Bannu, Bajaur and Hangu) from domestic fowls, ducks and their shelters, and bats. A subset of 197 ticks was selected for DNA extraction and PCR to amplify fragments of the cytochrome c oxidase (cox) gene for ticks and flagellin B (flaB) for the detection and genetic characterization of associated Borrelia spp. Among these, only Borrelia anserina DNA was detected in 40 ticks (27.2%) of different life stages, where highest prevalence was found in female ticks (18; 45%), followed by nymphs (12; 30%), larvae (7; 17.5%) and males (3; 7.5%). Tick infestation in shelters (1081; 77%) was higher than on hosts (323; 23%). The resultant cox amplicons of A. persicus showed 100% identity with the same species reported from Pakistan, China, Iran, Kenya, Kazakhstan, Algeria and Egypt and C. vespertilionis show 100% identity with the species reported from Pakistan, China, Japan, Kenya, Vietnam, Spain, Netherlands, the United Kingdom and Hungry, and clustered with the aforementioned species in the phylogenetic tree. The obtained Borrelia sequences showed 100% identity with B. anserina and revealed a close resemblance to the relapsing fever group and clustered in a monophyletic clade with B. anserina from India, Iran and Brazil in a phylogenetic tree. These results establish the first molecular characterization of B. anserina in A. persicus infesting domestic fowls and ducks in the region, as well as their shelters. To effectively control zoonotic consequences, country-wide surveillance research should be encouraged to screen soft ticks infesting various birds for associated pathogens.

Seasonality, epidemiology and phylogeny of Theileria ovis with a note on hematological and biochemical changes in asymptomatic infected goats from Pakistan.

Caprine theileriosis, caused by Theileria ovis is a serious production issue, especially in the areas that depend on goats and sheep for milk, meat, and other economic benefits. Pakistan has a large goat population, but few reports have been documented from this country regarding PCR-based detection of T. ovis. The molecular prevalence of T. ovis, on a seasonal basis, in various goat breeds enrolled from Muzaffar Garh district of Punjab in Pakistan was determined from October 2018 to September 2019. In this study, 1084 goat blood samples were screened for the detection of T. ovis DNA through PCR-based amplification of 18S rRNA gene. Out of 1084 goats, 12 (1.11%) were infected with T. ovis. The parasite prevalence varied with the sampling seasons (Chi square test, P = 0.008), and the parasite prevalence was highest in goat blood samples collected in summer (2.39%) followed by winter (1.88%). DNA sequencing and BLAST analysis confirmed the presence of T. ovis, and the amplified isolates from the 18S rRNA gene of T. ovis were found to be highly conserved during phylogenetic analysis. Young goats (Fischer exact test, P = 0.022) were found more infected with T. ovis during the winter season. Infected goats had elevated white blood cell counts (Two-sample t-test, P = 0.04), blood urea nitrogen to Creatinine ratio (Two-sample t-test, P = 0.02) and decreased serum Creatinine (Two-sample t-test, P = 0.001) as compared to T. ovis negative goats. We report a relatively low molecular prevalence of T. ovis in goats from the Muzaffar Garh district. However, it is recommended that control measures to eradicate T. ovis infection in goats in this area should be taken.