Survey of tick-borne pathogens in grazing horses in Kyrgyzstan: phylogenetic analysis, genetic diversity, and prevalence of Theileria equi.

Introduction: Tick-borne pathogens (TBP) are an important group of organisms that can affect animals and humans all over the world. Equine piroplasmosis (EP), caused by Theileria equi and Babesia caballi, is considered one of the most important tick-borne diseases and can cause significant clinical symptoms and mortality in horses. Moreover, EP plays a restrictive role in international horse traditions and transportation. Although these species can cause similar symptoms, there are different 18S rRNA genotypes of T. equi (five genotypes) and B. caballi (three genotypes). Besides piroplasma species, Anaplasma and hemotropic mycoplasmas (HM) are known as other important tick-borne pathogens reported in horses. Methods: In this study, we investigated the presence, prevalence, genetic diversity, and phylogenetic analyses of TBPs using PCRs and DNA sequencing in grazing horses in Kyrgyzstan. For these purposes, a total of 311 blood samples were collected from Chuy, Issyk-Kul, Naryn, Osh, Talas, and Jalal-Abad. Results: DNA amplification of TBP revealed that 23 (7.40%) out of 311 samples were found to be positive for T. equi. However, B. caballi, HM, A. phagocytophilum, and A. capra were not detected in this study. The infection rate of T. equi was higher in males (8.11%) than in females (6.35%) (p=0.2880) and in those older than 5 years (9.02%) than in the 1-4 age group (6.35%) (p=0.1950). Phylogenetic analysis of 18S rRNA revealed that A and E genotypes of T. equi have circulated in grazing horses in Kyrgyzstan. Discussion: Information about the genetic diversity of T. equi is important for understanding the population dynamics of the species and developing effective control strategies against this pathogen. This is the first molecular investigation of A. capra in horses in Kyrgyzstan. Although this pathogen has been detected in different hosts in Kyrgyzstan, it was not detected in this study. However, considering the wide host spectrum of A. capra, it is thought that more large-scale studies are needed to understand the effect of horses on the epidemiology of this pathogen.

Uptake of intermittent preventive treatment of malaria in pregnancy and risk factors for maternal anaemia and low birthweight among HIV-negative mothers in Dschang, West region of Cameroon: a cross sectional study.

BACKGROUND: Approximately 32 million pregnant women are at risk of malaria with up to 10,000 maternal deaths and 200,000 neonates at risk annually. Intermittent Preventive Treatment (IPT) with sulfadoxine-pyrimethamine (SP) is recommended by the World Health Organization (WHO) to reduce disease in pregnancy and adverse maternal and newborn outcomes. At least three doses of SP should be taken by pregnant women during antenatal consultation (ANC) beginning from the thirteenth week of pregnancy till parturition. The aim of this study was to assess uptake of IPT during pregnancy and risk factors for maternal anaemia and infant birth weight in Dschang, West region of Cameroon. METHODS: A total of 380 consenting pregnant women at delivery were recruited in a cross- sectional prospective survey between January to December 2021. Data on ANC attendance, total dose of IPT and history of malaria were abstracted from hospital ANC records while socio-demographic characteristics, bed net use and obstetrics history of each participant were also recorded through an interview. Further, blood samples were collected from the intervillous space for assessment of maternal anaemia and microscopic parasitology. Nested PCR based on amplification of the Plasmodium 18S sRNA was carried out to detect submicroscopic infection. IPTp coverage was calculated per WHO recommendation and the prevalence of anaemia and low birth weight were estimated as proportions in the total sample of pregnant women and live births, respectively. Crude and adjusted odds ratios and their 95% confidence intervals were used to estimate associations between pregnancy outcomes considered and risk factors in specific and general models. A p < 0.05 was considered significant. The R software (V4.1.4) was used for all analyses. RESULTS: A majority of pregnant women was aged between 24 and 34 years old (59.2%) and had secondary education (58.8%). Uptake of ≥ 3 IPTp was 64.99% with 77.20% of all who received at least one IPTp doses taking a mix of SP and DP or DP alone in successive ANC contacts. Those with four or more ANC contacts (73.42%) were more likely to have received at least one IPTp. Furthermore, 13.9% of live births had low birthweights (BW < 2500 g) and one in four parturient women with moderate anaemia by WHO criteria. Microscopy (blood smear examination) and PCR-based diagnosis revealed between 0% and 1.57% of parasite-infected placental samples, respectively. Reported malaria in pregnancy predicted maternal anaemia at birth but not birth weight. Only gestational age (< 37 weeks) and bed net use (< 5 months) significantly predicted infant birth weight at delivery. CONCLUSION: The uptake of WHO recommended IPT doses during pregnancy was moderately high. Reported malaria in pregnancy, poor bed net coverage, gestational age less than 37 weeks adversely affect maternal haemoglobin levels at birth and infant birth weight. Asymptomatic and submicroscopic placental parasite infections was found at low prevalence. Together these results highlight the importance of maintaining aggressive measures to prevent malaria in pregnancy and protect the health of mother and baby.

Experimental infection of non-immunosuppressed and immunosuppressed goats reveals differential pathogenesis of Babesia aktasi n. sp.

Babesiosis is an acute and persistent tick-borne disease caused by protozoan parasites of the genus Babesia. These hemoparasites affect vertebrates globally, resulting in symptoms such as high fever, anemia, jaundice, and even death. Advancements in molecular parasitology revealed new Babesia species/genotypes affecting sheep and goats, including Babesia aktasi n. sp., which is highly prevalent in goats from Turkiye's Mediterranean region. The objective of this study was to investigate the pathogenesis of B. aktasi infection in immunosuppressed (n=7) and non-immunosuppressed (n=6) goats. These animals were experimentally infected with fresh B. aktasi infected blood, and their clinical signs, hematological and serum biochemical parameters were monitored throughout the infection. The presence of parasites in the blood of immunosuppressed goats was detected by microscopic examination between 4 and 6 days after infection, accompanied by fever and increasing parasitemia. Goats that succumbed acute disease exhibited severe clinical signs, such as anemia, hemoglobinuria, and loss of appetite. However, the goats that survived showed milder clinical signs. In the non-immunosuppressed group, piroplasm forms of B. aktasi were observed in the blood within 2-5 days after inoculation, but with low (0.01-0.2%) parasitemia. Although these goats showed loss of appetite, typical signs of babesiosis were absent except for increased body temperature. Hematological analysis revealed significant decreases in the levels of red blood cells, leukocytes and platelet values post-infection in immunosuppressed goats, while no significant hematological changes were observed in non-immunosuppressed goats. In addition, serum biochemical analysis showed elevated transaminase liver enzymes levels, decreased glucose, and lower total protein values in the immunosuppressed group post-infection. Babesia aktasi, caused mild disease with minor clinical symptoms in non-immunosuppressed goats. However, in immunosuppressed goats, it exhibited remarkable pathogenicity, leading to severe clinical infections and death. In conclusion, this study provides valuable insights into the pathogenicity of the parasite and will serve as a foundation for future research aimed at developing effective prevention and control strategies against babesiosis in small ruminants. Further research is required to investigate the pathogenicity of B. aktasi in various goat breeds, other potential hosts, the vector ticks involved, and its presence in natural reservoirs.

Babesia ovis secreted antigen-1 is a diagnostic marker during the active Babesia ovis infections in sheep.

Ovine babesiosis caused by Babesia ovis is an economically significant disease. Recently, a few B. ovis-specific proteins, including recombinant B. ovis secreted antigen-1 (rBoSA1), have been identified. Immunological analyses revealed that rBoSA1 resides within the cytoplasm of infected erythrocytes and exhibits robust antigenic properties for detecting anti-B. ovis antibodies. This protein is released into the bloodstream during the parasite's development. It would be possible to diagnose active infections by detecting this secretory protein. For this purpose, a rBoSA1-specific polyclonal antibody-based sandwich ELISA was optimized in this study. Blood samples taken from the naturally (n: 100) and experimentally (n: 15) infected sheep were analyzed for the presence of native BoSA1. The results showed that native BoSA1 was detectable in 98% of naturally infected animals. There was a positive correlation between parasitemia level in microscopy and protein density in sandwich ELISA. Experimentally infected animals showed positive reactions from the first or second day of inoculations. However, experimental infections carried out by Rhipicephalus bursa ticks revealed the native BoSA1 was detectable from the 7th day of tick attachment when the parasite began to be seen microscopically. Sandwich ELISA was sensitive enough to detect rBoSA1 protein at a 1.52 ng/ml concentration. Additionally, no serological cross-reactivity was observed between animals infected with various piroplasm species, including Babesia bovis, B. bigemina, B. caballi, B. canis, B. gibsoni, Theileria equi, and T. annulata. Taken collectively, the findings show that the rBoSA1-specific polyclonal antibody-based sandwich ELISA can be successfully used to diagnose clinical B. ovis infections in sheep at the early stage.

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.

Molecular Prevalence and Genetic Diversity Based on Msp1a Gene of Anaplasma ovis in Goats from Türkiye.

Anaplasma ovis is a tick-borne obligated intraerythrocytic bacterium that infects domestic sheep, goats, and wild ruminants. Recently, several studies have been carried out using 16S rRNA and msp4 genes to identify the genetic diversity of A. ovis. Instead of these genes, which are known to be highly stable among heterologous strains, Msp1a, which is accepted as a stable molecular marker to classify A. marginale strains, was used in A. ovis genetic diversity studies. The genetic diversity of A. ovis strains according to the Msp1a gene has not been extensively reported. Therefore, the purpose of this study was to examine the genetic diversity of A. ovis in goats specifically using analysis of the Msp1a gene. Blood samples were taken from the vena jugularis to the EDTA tubes from 293 randomly selected goats (apparently healthy) in the Antalya and Mersin provinces of Mediterranean region of Türkiye. The Msp1a gene of A. ovis was amplified in all DNA samples through the use of PCR, using a specific set of primers named AoMsp1aF and AoMsp1aR. Among the amplified products, well-defined bands with different band sizes were subjected to sequence analysis. The obtained sequence data were converted into amino acid sequences using an online bioinformatics program and the tandem regions were examined. The Msp1a gene of A. ovis was amplified in 46.1% (135 out of 293) of the goats. Through tandem analysis, five distinct tandems (Ao8, Ao18, Tr15-16-17) were identified, and it was found that three of these tandems (Tr15-16-17) were previously unknown and were therefore defined as new tandems. The study also involved examination of ticks from goats. It was observed that the goats in the area were infested with several tick species, including Rhipicephalus bursa (888/1091, 81.4%), R. turanicus (96/1091, 8.8%), Dermacentor raskemensis (92/1091, 8.4%), Hyalomma marginatum (9/1091, 0.8%), and R. sanguineus s.l. (6/1091, 0.5%). This study provides important data for understanding the genetic diversity and evolution of A. ovis based on tandem repeats in the Msp1a protein.

Small Ruminant Piroplasmosis: High Prevalence of Babesia aktasi n. sp. in Goats in Türkiye.

Small ruminant piroplasmosis is the hemoparasitic infection of sheep and goats caused by Babesia and Theileria species responsible for clinical infections with high mortality outcomes. The disease is transmitted by ixodid ticks and prevalent in the tropical and subtropical regions of the world, including Türkiye. A prevalence survey, using molecular methods, is conducted in this study to determine the frequency of newly defined Babesia aktasi n. sp. and other tick-borne piroplasm species in small ruminants in Turkiye. A total of 640 blood samples from sheep (n = 137) and goats (n = 503) were analyzed by nested PCR-based reverse line blot (RLB) hybridization. The results show that 32.3% (207/640) of apparently healthy, small ruminants are infected with three Theileria and two Babesia species. Babesia aktasi n. sp. was the most prevalent species in goats, with 22.5% of samples being positive, followed by B. ovis (4%), T. ovis (2.8%), T. annulata (2.6%), and Theileria sp. (0.6%). None of the sheep samples were positive for Babesia aktasi n. sp.; however, 51.8% were infected with T. ovis. In conclusion, the findings reveal that B. aktasi n. sp. is highly prevalent in goats, but absent in sheep. In future studies, experimental infections will determine whether B. aktasi n. sp. is infectious to sheep, as well as its pathogenicity in small ruminants.

Discovery of a Novel Species Infecting Goats: Morphological and Molecular Characterization of Babesia aktasi n. sp.

A novel Babesia sp. infecting goats was discovered based on the molecular findings obtained in the current study, which was conducted in the Mediterranean region of Türkiye. The goal of this study was to isolate this species of Babesia (Babesia sp.) infecting goats in vivo and to assess the genetic and morphological characterization of the parasite. To identify the animal naturally infected with Babesia sp. and isolate the parasite from this animal, field studies were conducted first, and genomic DNA were extracted from blood samples taken from goats (n = 50). The Theileria, Babesia, and Anaplasma species were identified using a nested PCR-based reverse line blotting (RLB) method. The study included one goat that was determined to be infected with Babesia sp. (single infection) in RLB for in vivo isolation. A blood smear was prepared to examine the parasite's morphology, but it was found to be negative microscopically. Following that, a splenectomy operation (to suppress the immune system) was performed to make the parasites visible microscopically in this animal. Parasitemia began after splenectomy, and the maximum parasitemia was determined to be 1.9%. The goat displayed no significant symptoms other than fever, loss of appetite, and depression. During a period when parasitemia was high, blood from this goat was inoculated into another splenectomized goat (Theileria-Babesia-Anaplasma-Mycoplasma spp. free). On the third day of inoculation, 10% parasitemia with high fever was detected in the goat, and on the fourth day, the goat was humanely euthanized due to severe acute babesiosis symptoms. Except for mild subcutaneous jaundice, no lesions were discovered during the necropsy. According to the microscopic measurement results, ring, double pyriform, spectacle-frame-like, and line forms were observed, and it was observed to be between 1.0-2.5 µm (1.38 ± 0.17 to 0.7 ± 0.21-all forms). A phylogenetic analysis and sequence comparison using the 18S rRNA and cox1 genes revealed that this species is distinct from the small ruminant Babesia species (18S rRNA 92-94%, cox1 79-80%) and has the highest similarity to Babesia sp. deer, which has been reported in deer. Furthermore, it was determined to resemble B. venatorum, B. divergens, Babesia sp. FR1 and Babesia sp. MO1 species, all of which are zoonotic. Additional research is needed to clarify the clinical status of this parasite in goats and other hosts (mountain goat, sheep, calf).

Detection of Theileria orientalis Genotypes from Cattle in Kyrgyzstan.

The ikeda and chitose genotypes of Theileria orientalis, which for many years were thought to be benign, cause a disease that results in significant economic losses in the cattle industry. This study was carried out in order to determine the genotypes of T. orientalis in cattle in Kyrgyzstan, and 149 archived DNA samples known to be T. orientalis were analyzed by the PCR amplification of the major piroplasm surface protein (MPSP) gene region. Single-Strand Conformation Polymorphism (SSCP) analysis was performed to uncover the nucleotide changes in the archived DNA samples, and 15 samples showing different band profiles were subjected to sequence analysis. As a result of the sequence analysis, it was seen that the samples belonged to the buffeli and chitose A genotypes. In order to identify mixed genotypes, PCR was performed using primers specific for these genotypes, and buffeli (type 3), chitose (type 1) and buffeli+chitose were found to be positive in 26.2%, 2% and 71.8% of samples, respectively. As a result of this study, we showed the presence of buffeli (type 3) and chitose (type 1) genotypes of T. orientalis in cattle in Kyrgyzstan. Comprehensive epidemiological studies are needed to understand the clinical infections caused by the pathogenic chitose A and to determine the geographical distribution and different genotypes of T. orientalis.

The detection and phylogenetic analysis of Anaplasma phagocytophilum-like 1, A. ovis and A. capra in sheep: A. capra divides into two genogroups.

In this study, the presence, prevalence, and genotypes of Anaplasma phagocytophilum, A. ovis, and A. capra in sheep were investigated based on 16 S SSU rRNA, groEL, and gtlA gene-specific polymerase chain reaction (PCR), respectively. The sequences of the genes were used for detection of the phylogenetic position of the species. Additionally, a restriction fragment length polymorphism (RFLP) were carried out for discrimination of A. phagocytophilum and related variants (A. phagocytophilum-like 1 and 2). The prevalence of Anaplasma spp. was found as 25.8% (101/391), while it was found that A. ovis, A. phagocytophilum-like 1, and A. capra are circulating in the sheep herds in Kyrgyzstan, according to the PCRs, RFLP and the partial DNA sequencing results. The positivity rates of A. phagocytophilum-like 1, A. ovis, and A. capra genotype-1 were 6.9, 22.5, and 5.3%, respectively. A total of 32 (8.2%) sheep were found to be mix infected. Moreover, phylogenetic analyses and sequence comparison with those available in the GenBank showed that A. capra formed two distinct genetic groups (A. capra genotype-1 and A. capra genotype-2). Considering the zoonotic potential of these species, it may be necessary to make changes in the interpretation of anaplasmosis cases in animals and there is a need for further studies to determine the pathogenicity of the species/genotypes circulating in animals.

Seasonal survey, risk factor's analysis and genotyping of Theileria annulata infecting cattle in Punjab province, Pakistan.

Theileriosis is one of the most frequently reported tick borne diseases in tropical and subtropical regions and leads to annual economic losses, such as the reduced dairy products and increased casualties. Tropical theileriosis is caused by Theileria annulata and the present study was designed to improve our knowledge of Theileria annulata infection in Pakistani cattle. In order to assess the prevalence of Theileria annulata on cattle from Multan district in the Punjab province (Pakistan) according to seasons and other risk factors, a total of 1020 blood samples (340 samples each from cross, Holstein Frisian and Sahiwal breed) were collected between 2020 and 2022. Based on Tams-1 partial sequence amplification, the overall T. annulata prevalence was estimated at 11.3% (115/1020). The highest prevalence was observed in autumn season (14.1%), followed by winter (12.9%), summer (11.4%) and spring (6.7%) season. Sahiwal cattle were most susceptible to T. annulata infection (13.2%) followed by Crossbred (11.8%) and Holstein Frisian (8.8%). Epidemiological factor analysis revealed that female cattle, cattle rose with other dairy animals at farm, tick infested cattle, and cattle raised with dogs infested with ticks were associated with the prevalence of T. annulata. White blood cells, lymphocyte (%), Monocyte (%) hemoglobin, mean cell hemoglobin, mean corpuscular hemoglobin concentration, and platelet count were significantly affected blood parameters in T. annulata positive cattle of all three breeds. Representative partial Tams-1 sequences of four Pakistani T. annulata isolates revealed a single genotype genetically close to those infecting cattle from neighboring countries like Iran, Turkey and Egypt. Longitudinal survey and phylogenetic positioning of T. annulata is recommended for epidemiological correlation, diagnosis and treatment of theileriosis in such an agricultural region of Pakistan.

Molecular prevalence, associated risk factors and phylogeny of Anaplasma marginale, Theileria ovis and T. lestoquardi in sheep from Pakistan.

Anaplasma marginale, Theileria ovis and Theileria lestoquardi are intracellular pathogens that infect a wide variety of animals and cause enormous economic losses worldwide. The present investigation aims to report the occurrence and the phylogeny of these pathogens infecting sheep (N = 330) from Rajanpur District in Punjab (Pakistan) by using msp1b gene for A. marginale and 18 S rRNA for T. ovis and T. lestoquardi. Results revealed that prevalence rates of A. marginale, T. ovis and T. lestoquardi were 7.3%, 6.1% and 1.2%, respectively. Four (1.2%) and one (0.3%) sheep were found to be co-infected with two and three pathogens, respectively. Risk factor analysis revealed that rams were more infected with A. marginale compared to ewes (P = 0.015). In addition, it was observed that animals located in herds having dogs and those in small herds were found more infected, respectively, with T. ovis (P = 0.001) and T. lestoquardi (P = 0.009). Phylogenetic analysis revealed that Pakistani isolates of each detected pathogens clustered together and were closely related to other Pakistani isolates and those from worldwide countries such as Iran, Iraq, Turkey, Sudan, Tanzania, South Africa, and USA. This is the first molecular study reporting the natural infection of Pakistani sheep with these three pathogens. These data need to be taken into account in order to improve the productivity of the livestock sector which is one of main sources of income in the country.

Prevalence of Anaplasma marginale in cattle blood samples collected from two important livestock regions in Punjab (Pakistan) with a note on epidemiology and phylogeny of parasite.

Anaplasmosis, caused by intracellular gram-negative bacteria Anaplasma marginale is one of the most frequently reported tick-borne disease (TBDs) in tropical and sub-tropical countries, including Pakistan. In the present study, a total of 428 cattle blood samples were collected to examine the prevalence and phylogenetic origin of A. marginale in two important livestock regions of Punjab Province in Pakistan, i.e. Lodhran and Dera Ghazi Khan Districts. In addition, association between occurrence of A. marginale in cattle blood and selected epidemiological factors has been also investigated. The presence of A. marginale genetic material was confirmed in 9% of the tested blood samples taken from cattle in Lodhran and in 17% from Dera Ghazi Khan. Prevalence of A. marginale was significantly higher in cattle from Dera Ghazi Khan. All the cattle breeds from both districts were equally susceptible to A. marginale infection. We reported higher prevalence of A. marginale in cattle living indoors or with other dairy animals in Dera Ghazi Khan district. However, no such relationship was observed in the Lodhran district. Sequencing of the msp1b gene shows 96-99% similarity of A. marginale in the study area to those reported from other parts of Pakistan, South Africa, and Israel. We recommend that large scale tick and tick-borne disease control strategies must be implemented in both districts.

Molecular survey of Toxoplasma gondii in cattle and buffaloes and phylogenetic position of Pakistani isolates based on ITS-1 gene.

Toxoplasma gondii (T. gondii) is a facultative heterogeneous parasite that belongs to Apicomplexa and can infect almost all warm-blooded animals, including ruminants, birds and humans. To date, no information is available about the molecular investigation of T. gondii in large ruminants from Pakistan. In the present study, prevalence, risk factors and genetic diversity of this parasite were evaluated by using PCR based on ITS-1 gene followed by sequencing of three selected positive PCR products. A total of 310 blood samples from cattle (N = 190) and buffaloes (N = 120) were collected from randomly selected farms located in Rajanpur district in Punjab (Pakistan). The overall infection rates of T. gondii were 12.2% (23/190) and 0% (0/120), respectively, in cattle and buffaloes. All studied epidemiological factors were not found associated with T. gondii infection in cattle. Sequence analysis of our T. gondii isolates infecting cattle revealed only one sequence considered as the most represented genetic variant (GV1) among T. gondii isolates around the world. Phylogenetic analysis revealed that ITS-1 partial sequences of our isolates clustered with those from T. gondii isolates infecting goats and birds from Pakistan and other isolates found in several animal species from different worldwide countries like China, Thailand, Poland, Canada, USA and Brazil. Our report indicates a natural infection with T. gondii of cattle for the first time in Pakistan by using molecular method. This study is important to the design of control strategy against this parasite in order to improve the output of livestock sector which is the main income source of the population in Pakistan.

Prevalence, epidemiology, seasonality, and phylogeny of Anaplasma marginale in blood samples of goats collected from Punjab, Pakistan.

PURPOSE: In Pakistan, a major constrain to goat farming is the tick and tick-borne diseases that results in financial losses to livestock farmers. This study was conducted to report the molecular prevalence of Anaplasma (A.) marginale in goat blood samples collected during four seasons from Khanewal district in Punjab (Pakistan). METHODS AND RESULTS: The mps1 gene of A. marginale was targeted in 900 blood samples that were collected on seasonal basis (n = 225 per season) and 6.6% (61/900) goats were found positive with A. marginale. Anaplasma marginale positive PCR products were sequenced and submitted to the GenBank. Prevalence of A. marginale varied with sampling season (P = 0.002) and it was highest in the summer (11.5%) followed by the autumn (7.6%), spring (5.3%), and winter seasons (2.7%) respectively. Anaplasma marginale prevalence varied significantly between goat breeds during the autumn (p = 0.01) and summer seasons (p = 0.02). Goats more than 2 years old and livestock farms where only goats were kept and dogs were associated with herds were risk factors for ovine anaplasmosis during different seasons. White and red blood cell counts and parameters associated with their counts were affected in A. marginale infected goats while studied serum parameters remained unaffected. CONCLUSION: PCR is a reliable tool for the detection of A. marginale in goat blood samples. A relatively low prevalence of A. marginale in goats of Khanewal district was observed and the parasite prevalence in goats was higher in the summer (May until September) and autumn (October and November) seasons. Control measures are required to prevent tick-borne diseases in ruminants from Pakistan.

First report on molecular epidemiology, seasonality and phylogeny of Toxoplasma gondii infecting goats from Khanewal district in Punjab, Pakistan.

Raising small ruminants is the main source of income for farmers in Pakistan. Economic losses caused by Toxoplasma gondii to small ruminants have been reported worldwide, however reports on molecular detection of T. gondii are lacking in Pakistan despite a large goat population. The current study was carried out from March 2019 till February 2020 to report the seasonal and molecular prevalence of T. gondii in different breeds of goats located in Khanewal district of Punjab, Pakistan. A total of 898 blood goat samples were collected during the four seasons and screened for T. gondii DNA by using PCR based on the amplification of ITS-1 partial sequence. Out of 898 goats, 48 (5.3%) were found positive to T. gondii. The prevalence of T. gondii varied according to season (Chi square test,P = 0.016) and the highest prevalence was observed in goats tested during the summer (8.8%) followed by the spring (5.7%), the winter (4.4%) and the autumn season (2.2%). PCR products positive to T. gondii were confirmed by DNA sequencing and BLAST analysis. Phylogenetic study based on ITS 1 gene provided evidence that the amplified isolates of T. gondii were highly conserved in Pakistani goats. Buck (Fischer exact test, P = 0.002) and farms containing other dairy animals next to goats (Fischer exact test, P = 0.001) and farms with a water supply from pools (Fischer exact test, P = 0.001) were more infected with T. gondii. Infected goats had a reduction on red blood cell count (Two-sample t test, P = 0.01) and hemoglobin concentration (Two-sample t test, P = 0.03) and an increase in the number of monocytes (%) (Two-sample t test, P = 0.05), mean cell hemoglobin (Two-sample t test, P = 0.01) and serum creatinine (Two-sample t test, P = 0.01) as compared to T. gondii uninfected goats. In conclusion, we report a relatively low PCR based prevalence of T. gondii in goats from Khanewal district as previously the serum ELISA test based prevalence of T. gondii in Pakistani goats varied between 19-52%. Control measures should be taken to eradicate T. gondii infection in goats of the study area.

Molecular Epidemiology of Theileria annulata in Cattle from Two Districts in Punjab (Pakistan).

The present study was designed to report the molecular prevalence of T. annulata in cattle blood samples collected from Punjab in Pakistan. A total of 428 cattle blood samples were collected from Districts Lodhran (n = 218) and Dera Ghazi Khan (n = 210). The prevalence of T. annulata was determined by the amplification of a fragment from its cytochrome b gene and parasite prevalence was significantly higher (p = 0.03) in the blood samples of cattle collected from Dera Ghazi Khan (70/210; 33%) as compared to Lodhran (52/218; 24%). Presence of T. annulata was also confirmed by the amplification of a fragment from their 30 kDa gene. The amplified PCR products of both genes were confirmed by DNA sequencing and these partial DNA sequences were submitted to GenBank. Phylogenetic analysis revealed that amplified partial gene sequences resembled previously reported T. annulata sequences in cattle from India, China, Iran, Tunisia, Turkey and Egypt. The incidence of T. annulata infection was higher in Sahiwal cattle (p = 0.04) than the other enrolled cattle breed from Dera Ghazi Khan. Female cattle from Lodhran (p = 0.02), while males (p = 0.02), animals housed in close compounds (p = 0.04), animals with a tick burden (p = 0.005) and farms with only cattle (p = 0.01) in Dear Ghazi Khan were found to be more susceptible to T. annulata infection. We recommend that large-scale tick and tick-borne disease control strategies be implemented in both districts under investigation, especially in Dera Ghazi Khan.

First report regarding molecular epidemiology and novel variant identification of Anaplasma centrale in cattle from Pakistan.

Anaplasma centrale (A. centrale) is an obligate red blood cell residing tick transmitted rickettsiae that has not been studied extensively for its prevalence in cattle along with its epidemiology. Aim of this investigation was to report the seasonal prevalence, phylogeny and epidemiological parameters associated with the prevalence of A. centrale in cattle breeds enrolled from District Layyah in Punjab, Pakistan. A total of 844 blood samples [Cross breed = 300, Holstein Friesian = 244, Sahiwal breed = 300)] were collected from apparently healthy cattle along with epidemiological data during 2017-18. PCR amplified 426 base pair fragment from 16S rRNA gene of A. centrale in 14.4% (122/844) of cattle. Amplified 16S rRNA partial gene sequence of A. centrale were confirmed by DNA sequencing and deposited to GenBank. Highest A. centrale prevalence was observed in spring (24%) followed by autumn (12.4%) summer (10%) and winter (7.1%) seasons. Sahiwal breed (18.3%) was most susceptible to A. centrale infection followed by cross (12.3%) and Holstein Friesian breed (12.3%). 69/844 (8.2%) of Giemsa stained cattle blood smears were also found positive for Anaplasma spp. Farms where animal use to drink pool water and farms where dogs and other dairy animals were living with cattle had higher A. centrale prevalence. Female cattle and dogs having tick burden were found associated with A. centrale infection. Hematological profile was severely disturbed in A. centrale positive cattle. It is recommended that A. centrale should be screened in cattle, in addition to A. marginale, for the effective control of tick born diseases in Pakistan.

Molecular detection and prevalence of Theileria ovis and Anaplasma marginale in sheep blood samples collected from Layyah district in Punjab, Pakistan.

Theileria ovis and Anaplasma marginale are intracellular pathogens affecting a wide range of animals, causing huge economic losses worldwide. The present study reports the molecular evidence of Theileria ovis and Anaplasma marginale in sheep blood samples (N = 218) collected from Layyah district in Punjab (Pakistan), where economy heavily relies on livestock. A 520 base pair fragment specific for 18S ribosomal RNA gene of Theileria ovis was PCR amplified in 23/218 (10.6%) sheep blood samples, while for Anaplasma marginale, a 265 base pair fragment specific for msp1b gene was generated in 15/218 (6.9%) sheep blood samples. Two blood samples were found co-infected (0.9%) with both parasites. Amplified PCR products of both parasites were confirmed by DNA sequencing and submitted to GenBank. Prevalence of both Theileria ovis (p = 0.3) and Anaplasma marginale (p = 0.4) varied non-significantly among the investigated sheep breeds. Tick burden on dogs present with sheep herds was found associated with Theileria ovis infection in sheep (p = 0.05). It was observed that lambs (p = 0.009), sheep in small herds (p = 0.04), and tick burden on dogs present with sheep herds (p = 0.01) were associated with Anaplasma marginale infection in sheep during the present study. In conclusion, we are reporting a higher prevalence of Theileria ovis than Anaplasma marginale in blood samples of sheep collected from Layyah district. Tick-infested dogs were found to be risk factors for the transmission of both pathogens in sheep, and tick control strategies should be extended to dogs associated with sheep herds in this area.

Complete mitochondrial genome characterization and phylogenetic analyses of the main vector of Crimean-Congo haemorrhagic fever virus: Hyalomma marginatum Koch, 1844.

The Mediterranean tick, Hyalomma marginatum, is the most important vector of Crimean-Congo haemorrhagic fever virus and several pathogens that cause animal and human diseases and economic losses to livestock production. Given the medical and veterinary importance of this tick species, we sequenced and characterized its mitochondrial genome (mitogenome) for the first time. We designed two new primer sets and combined long-range PCR with next generation sequencing to generate complete mitogenomes with deep coverage from 10 H. marginatum adults. The mitogenomes contained 13 protein-coding genes (PCGs), 22 transfer RNAs (tRNAs), two ribosomal subunits, two control regions, and three tick-box motifs. The nucleotide composition of the H. marginatum mitogenomes were A+T biased (79.76%) and exhibited negative AT- and GC- skews across most PCGs. All PCGs were initiated by ATK codons and two truncated termination codons were seen in the COX2 and COX3 genes. All tRNAs exhibited typical cloverleaf structures, except for tRNACys and tRNASer1. A total of 62 polymorphic sites defined ten unique haplotypes. Phylogenetic analyses based on the 13 PCGs of 56 tick species revealed that four Hyalomma species (H. marginatum, H. asiaticum, H. rufipes, and H. truncatum) formed a monophyletic clade with strong support. The results of this study provide a comprehensive resource for further studies on the systematics, population genetics, molecular epidemiology, and evolution of ticks.