Anaplasma Marginale Infection Research Articles

Factors associated with perceived Anaplasma marginale infection and clinical anaplasmosis cases on beef operations in California

Bovine anaplasmosis is a tick-borne disease caused by Anaplasma marginale in the United States. The objective of this study was to use a survey tool to generate information for beef operations in California on anaplasmosis prevention and control management, including to what extent management activities were informed by perceived herd-level exposure to A. marginale infection or occurrence of clinical anaplasmosis cases. We mailed 2,621 questionnaires with questions on Anaplasma status, herd demographics, anaplasmosis control and prevention measures, and environmental factors to beef ranchers in California in October 2020. Survey-weighted chi-square tests were used to compare management differences according to perceived Anaplasma infection status. Generalized estimating equations (GEEs) were used to analyze whether region of California, management practices, or environmental factors were associated with reported clinical cases of anaplasmosis in the previous five years. A total of 466 questionnaires describing 749 herds were obtained and used in this study. Use of management measures, including deliberate exposure of calves to ticks, vaccination for Anaplasma, infection control through antibiotics in feed, maintaining a completely closed herd, blood testing for Anaplasma on all herd additions, and taking no anaplasmosis control and prevention measures, were significantly different between herds with or without perceived A. marginale infection based on producers’ self-declared status. The overall perceived prevalence for Anaplasma infection and reported clinical cases of anaplasmosis at the herd level was 26.0 % (95 % CI: 24.3–27.7 %) and 17.1 % (95 % CI: 15.6–18.6 %) respectively, with the highest perceived infection and case numbers reported in the Central Coast region. In the GEE model, higher odds of reporting clinical cases of anaplasmosis in the previous five years were observed in cattle located in the Central Coast region, cattle within a large herd, cattle that are treated with tick/fly control, cattle in a completely closed herd, and cattle receiving Anaplasma vaccine. Anaplasma infection and bovine anaplasmosis status may be underestimated in beef herds in California based on previous study results. Changing needles between cattle after injections and conducting blood testing for Anaplasma on herd additions are important Anaplasma management measures that are infrequently implemented in beef herds in California. The results show a need for producer education to improve producers’ awareness of bovine anaplasmosis and implement proper measures for disease control and prevention.

RPA-CRISPR/Cas12a assay for the diagnosis of bovine Anaplasma marginale infection

Anaplasma marginale infection is one of the most common tick-borne diseases, causing a substantial loss in the beef and dairy production industries. Once infected, the pathogen remains in the cattle for life, allowing the parasites to spread to healthy animals. Since clinical manifestations of anaplasmosis occur late in the disease, a sensitive, accurate, and affordable pathogen identification is crucial in preventing and controlling the infection. To this end, we developed an RPA-CRISPR/Cas12a assay specific to A. marginale infection in bovines targeting the msp4 gene. Our assay is performed at one moderately high temperature, producing fluorescent signals or positive readout of a lateral flow dipstick, which is as sensitive as conventional PCR-based DNA amplification. This RPA-CRISPR/Cas12a assay can detect as few as 4 copies/μl of Anaplasma using msp4 marker without cross-reactivity to other common bovine pathogens. Lyophilized components of the assay can be stored at room temperature for an extended period, indicating its potential for field diagnosis and low-resource settings of anaplasmosis in bovines.

Exploring the relationship between flumethrin resistance and Anaplasma marginale infection in Rhipicephalus (Boophilus) microplus ticks of cattle.

The study explores the relationship between flumethrin resistance and Anaplasma marginale infection in Rhipicephalus (Boophilus) microplus of cattle in South Gujarat, India. Adult Immersion Test (AIT) was used to assess flumethrin resistance and polymerase chain reaction (PCR) to confirm A. marginale infection. Species-specific PCR resulted in the amplification of 576bp of msp5 gene of A. marginale in 17.69% (49/277) groups of ticks, and subsequent digestion with EcoRI cleaved it into two distinct segments. Navsari district, noted level Ι resistance [resistance factors (RF) = 1.78-3.34], and A. marginale prevalence was 16.67, 15.38, 23.08, 15.38, and 11.76% in Navsari, Jalalpore, Gandevi, Chikhli, and Vansda sub-districts, respectively. Similarly, Vyara and Dolvan sub-districts of Tapi observed level Ι resistance (RF = 1-3.63), with A. marginale positivity of 21.43 and 22.22%, while Valod and Songhad demonstrated susceptibility, with 14.29 and 12.50% of A. marginale, respectively. Moving to Surat, the Mahuva, Bardoli, Mandvi, Palsana, and Kamrej sub-districts observed the level Ι resistance (RF = 1.94-2.89), coupled with 14.29, 17.65, 20, 20, and 21.43% of A. marginale, respectively. Lastly, in Valsad district, Dharampur, Kaparada, Valsad, and Umbergaon noted level Ι resistance (RF = 1.67-1.81), and corresponding A. marginale positivity rates of 18.18, 19.23, 15.00, and 20.00%. The scatter plot unveiled a significant moderate positive correlation between RF and A. marginale positivity% (p = 0.0362), characterized by a Pearson correlation coefficient (r) of 0.4963. The covariance (1.1814) highlighted fluctuations, while the coefficient of determination (r2) (0.2463) clarified that 24.63% of the variability in A. marginale positivity% could be attributed to the RF.

Leading report of molecular prevalence of tick borne Anaplasma marginale and Theileria ovis in yaks (Bos grunniens) from Pakistan.

Domestic yak (Bos grunniens) is an economically important feature of the mountainous region of Gilgit-Baltistan in Pakistan where agriculture is restricted and yaks play multiple roles which includes being a source of milk, meat, hides, fuel and power. However little is known about the parasitic infections in Pakistani yaks. Aim of this research was to report the prevalence and genetic diversity of protozoa parasite (Theileria ovis, 18S rDNA gene was targeted) and an obligate bacterium (Anaplasma marginale, msp-1 gene was amplified) in the blood that was sampled from 202 yaks collected from four districts in Gilgit-Baltistan during January 2023 till January 2024. Results revealed that 6/202 (3%) yaks were of Theileria ovis while 8/202 (4%) were Anaplasma marginale infected. Positive PCR products of both parasites were confirmed by DNA sequencing and their similarity with previously available pathogen sequences was determined by BLAST analysis. Phylogenetic tree indicated that isolates of both parasites displayed genetic. Anaplasma marginale infection varied with the sampling districts and Shigar district had the highest rate of bacterial infection. Cows were significantly more prone to Theileria ovis infection than bulls. Calf and hybrid yaks were more prone to Anaplasma marginale infection. In conclusion, this is the first report that yaks residing the Gilgit-Baltistan region in Pakistan are infected with Theileria ovis and Anaplasma marginale. Similar larger scales studies are recommended in various regions of Gilgit-Baltistan to document the infection rates of these parasites to formulate strategies that will lead to the effective control of these pathogens.

First Molecular Evidence of Anaplasma marginale Infection in Naturally Infected Cattle in Myanmar with Severe Hemolytic Anemia

First Molecular Evidence of Anaplasma marginale Infection in Naturally Infected Cattle in Myanmar with Severe Hemolytic Anemia

An epidemiological survey of vector-borne pathogens infecting cattle in Kyrgyzstan.

Cattle production is a major contributor to the national economy of Kyrgyzstan. Most cattle in Kyrgyzstan are managed via extensive systems and graze in communal pastures. As a result, infestations with ectoparasites are widespread, implying that various vector-borne diseases might be common in cattle. However, methods to control such infectious diseases are not available in Kyrgyzstan because the epidemiology of vector-borne pathogens (VBPs) infecting cattle remains unclear. The present study was therefore designed to survey Kyrgyz cattle for VBPs. We prepared blood DNA samples from 319 cattle in Kyrgyzstan and screened them with specific PCR assays for detecting Babesia bovis, Babesia bigemina, Babesia naoakii, Theileria annulata, Theileria orientalis, Trypanosoma evansi, Trypanosoma theileri, and Anaplasma marginale infections. Our findings indicated that the surveyed cattle were infected with six of the eight pathogens targeted, with the exceptions being B. naoakii and Try. evansi. The most common pathogen was T. orientalis (84.3%), followed by B. bigemina (47.6%), T. annulata (16.6%), A. marginale (11.6%), Try. theileri (7.2%), and B. bovis (2.5%). Additional screening of the B. bovis- and B. bigemina-negative samples with a Babesia genus-specific 18S rRNA PCR identified two positive samples, and sequencing analysis confirmed that each of them was infected with either Babesia major or Babesia occultans. To the best of our knowledge, this is the first report of B. bovis, B. bigemina, B. occultans, Try. theileri, and A. marginale infections in cattle in Kyrgyzstan. Our findings suggest that cattle in Kyrgyzstan are at high risk of infectious diseases caused by VBPs.

Kansas beef bulls with chronic anaplasmosis demonstrate satisfactory breeding soundness outcomes at breeding soundness examination.

The objectives of this study were to evaluate the prevalence of chronic Anaplasma marginale infection in beef bulls from eastern Kansas and compare breeding soundness parameters between A marginale-infected and uninfected bulls. We hypothesized that bulls with chronic anaplasmosis would have inferior breeding soundness exam (BSE) outcomes as a result of persistent A marginale infection or the consequence of initial clinical disease compared to uninfected bulls. 535 client-owned beef bulls from eastern Kansas undergoing routine BSE. Complete BSEs were conducted by participating veterinarians according to the second edition of the Society for Theriogenology Manual for Breeding Soundness Examination of Bulls. Blood samples were collected for PCV determination and analysis of A marginale infection status via quantitative PCR and cELISA. Logistic and linear regression methods were used to evaluate factors associated with A marginale infection status and BSE parameters. Prevalence of chronic A marginale infection was 46% (245/535) among bulls. Unsatisfactory BSE outcome was not statistically associated with chronic anaplasmosis in this study population, although more bulls with chronic anaplasmosis had unsatisfactory BSE outcomes (15.0 ± 2.4% vs 12.0 ± 2.2%). Chronic anaplasmosis is prevalent among eastern Kansas breeding bulls; however, no negative association between chronic anaplasmosis and breeding soundness at time of BSE was observed.

Identification of Anaplasma marginale, Babesia bovis and Babesia bigemina resistance alleles in Crioulo Lageano cattle using PCR-SBT and BoLA-DRB3 gene sequencing.

The BoLA-DRB3 gene in cattle is associated with tolerance to several infectious diseases, such as neosporosis, dermatophilosis, leukosis, and mastitis. This study used PCR-SBT and BoLA-DRB3 gene sequencing to determine the association between the presence or absence of Anaplasma marginale, Babesia bovis, and Babesia bigemina infections in 208 Crioulo Lageano cattle and alleles present in the population. The chi-square test and odds ratio analysis were employed to establish the association. Of the BoLA-DRB3 gene alleles present in the population, two alleles were significantly associated with resistance to A. marginale infections: BoLA-DRB3001:01 (p < 0.001; OR = 0.224), which had a frequency of 7.93%, and BoLA-DRB3024:06 (p = 0.007; OR < 0.00001), which had a frequency of 0.72%. Regarding B. bovis infection, the BoLA-DRB3*011:01 allele (p = 0.002; OR = 0.271) had a frequency of 6% in the population and was associated with resistance to the infection. None of the alleles was associated with resistance to infection by B. bigemina. The Crioulo Lageano breed has alleles that may confer resistance against infection by A. marginale and B. bovis.

Biochemical and hematological aspects of Anaplasma marginale infection in dairy cattle, central Goiás, Brazil

Biochemical and hematological aspects of Anaplasma marginale infection in dairy cattle, central Goiás, Brazil

Temporal Dynamics of Anaplasma marginale Infections and the Composition of Anaplasma spp. in Calves in the Mnisi Communal Area, Mpumalanga, South Africa.

Bovine anaplasmosis, caused by Anaplasma marginale, is one of the most important tick-borne diseases of cattle. Anaplasma marginale is known to be present in the Mnisi community, Mpumalanga Province, with frequent cases of anaplasmosis reported. This study investigated the infection dynamics in calves (n = 10) in two habitats in the study area over 12 months. A duplex real-time PCR assay targeting the msp1β gene of A. marginale and the groEL gene of A. centrale confirmed the presence of A. marginale in five calves in a peri-urban area from the first month, but in only two calves at the wildlife-livestock interface and only after six months. These results were confirmed by 16S rRNA microbiome analysis. Over 50 A. marginale msp1α genotypes were detected in the calves along with five novel Msp1a repeats. Calves in the peri-urban area were more likely to be infected with A. marginale than calves in the wildlife-livestock interface. Cattle management, acaricide treatment, and cattle density could explain differences in infection prevalence in the two areas. Our results revealed that most calves were superinfected by distinct A. marginale strains within the study period, indicating continuous challenge with multiple strains that should lead to robust immunity in the calves and endemic stability in the area.

Seasonal Investigation of Anaplasma marginale Infection in Pakistani Cattle Reveals Hematological and Biochemical Changes, Multiple Associated Risk Factors and msp5 Gene Conservation.

Bovine anaplasmosis is a tick-borne disease caused by an obligate intercellular Gram-negative bacterium named Anaplasma (A.) marginale. In this study, we report the seasonal prevalence, potentially associated risk factors and phylogeny of A. marginale in cattle of three different breeds from Multan District, Southern Punjab, Pakistan. A total of 1020 blood samples (crossbred, n = 340; Holstein Friesian, n = 340; and Sahiwal breed, n = 340) from apparently healthy cattle were collected on a seasonal basis from March 2020 to April 2021. Based on PCR amplification of the msp5 partial sequence, overall, the A. marginale prevalence rate was estimated at 11.1% (113/1020) of the analyzed cattle samples. According to seasons, the highest prevalence rate was observed in autumn (16.5%), followed by winter (10.6%) and summer (9.8%), and the lowest was recorded in the spring (7.5%). The crossbred and Sahiwal cattle were the most susceptible to A. marginale infection, followed by Holstein Friesian cattle (7.9%). Analysis of epidemiological factors revealed that cattle reared on farms where dairy animals have tick loads, dogs coinhabit with cattle and dogs have tick loads have a higher risk of being infected with A. marginale. In addition, it was observed that white blood cell, lymphocyte (%), monocyte (%), hematocrit, mean corpuscular hemoglobin and mean corpuscular hemoglobin concentrations were significantly disturbed in A. marginale-positive cattle compared with non-infested cattle. Genetic analysis of nucleotide sequences and a phylogenetic study based on msp5 partial sequencing demonstrated that this gene appears to be highly conserved among our isolates and those infecting apparently healthy cattle from geographically diverse worldwide regions. The presented data are crucial for estimating the risk of bovine anaplasmosis in order to develop integrated control policies against bovine anaplasmosis and other tick-borne diseases infecting cattle in the country.

Incidences of Rhipicephalus (Boophilus) microplus (Canestrini, 1888) Transmitted Pathogens in Cattle in West Africa

Context and PurposeIn a context of recent introduction of Rhipicephalus (Boophilus) microplus tick species in West Africa, the purpose of the authors is to estimate incidence density of cattle babesiosis either caused by Babesia bigemina or Babesia bovis, and cattle anaplasmosis. Incidence density represents how quickly a disease or a condition is occurring amongst a group of individuals at risk.MethodsThe longitudinal and prospective study design took place in south, centre, east, west and north of Côte d’Ivoire. Cattle have been followed for 12 months. At the end of each month, each animal has been RT-PCR tested for new infection by Babesia bovis, Babesia bigemina, and PCR–RFLP tested for new infection by Anaplasma marginale.ResultsFindings show for the study area that incidence densities of Babesia bovis, Babesia bigemina and Anaplasma marginale infections in Côte d’Ivoire are, respectively, 15.3 new infections [95% CI 13.1–17.88] per 100 cattle, 32.2 new infections [95% CI 28.5–36.3] per 100 cattle, and 25.9 new infections [95% CI 22.5–29.6] per 100 cattle.ConclusionFinally, there is increasing of infection incidence density following the region distance from the coast or elevation.

Anaplasma marginale Infection of Dermacentor andersoni Primary Midgut Cell Culture Is Dependent on Fucosylated Glycans.

Tick midgut is the primary infection site required by tick-borne pathogens to initiate their development for transmission. Despite the biological significance of this organ, cell cultures derived exclusively from tick midgut tissues are unavailable and protocols for generating primary midgut cell cultures have not been described. To study the mechanism of Anaplasma marginale-tick cell interactions, we successfully developed an in vitro Dermacentor andersoni primary midgut cell culture system. Midgut cells were maintained for up to 120 days. We demonstrated the infection of in vitro midgut cells by using an A. marginale omp10::himar1 mutant with continued replication for up to 10 days post-infection. Anaplasma marginale infection of midgut cells regulated the differential expression of tick α-(1,3)-fucosyltransferases A1 and A2. Silencing of α-(1,3)-fucosyltransferase A2 in uninfected midgut cells reduced the display of fucosylated glycans and significantly lowered the susceptibility of midgut cells to A. marginale infection, suggesting that the pathogen utilized core α-(1,3)-fucose of N-glycans to infect tick midgut cells. This is the first report using in vitro primary D. andersoni midgut cells to study A. marginale-tick cell interactions at the molecular level. The primary midgut cell culture system will further facilitate the investigation of tick-pathogen interactions, leading to the development of novel intervention strategies for tick-borne diseases.

Prevalence of bovine Babesia spp., Anaplasma marginale, and their co-infections in Latin America: Systematic review-meta-analysis

Bovine parasitic sadness, comprised of the diseases babesiosis and anaplasmosis, has a large impact on cattle farming in several countries, as it compromises animal productivity. Using systematic review (SR)-meta-analysis (MA) methodology, our objective was to summarize and to investigate study characteristics associated with prevalence of Babesia spp. and Anaplasma marginale infection in Latin American cattle herds. Five electronic databases were used. The inclusion criteria were studies that assessed the prevalence of Babesia spp. and A. marginale in cattle in Brazil, Argentina, Uruguay, Paraguay, Colombia, and Mexico. The reported prevalences were converted to logistic scale and summarized using random effects MA. The heterogeneity was assessed, separately, for papers from Brazil and from the remaining countries. A total of 28 papers were included in this MA with a high heterogeneity (I2 > 95%). The prevalences of A. marginale, Babesia spp., and their co-infections, in Latin America, were 48.9% (95% CI: 30.3–67.8%), 39.8% (95% CI: 24.6–57.2%), and 26.1% (95% CI: 9.1–55.8%), respectively. The prevalence of A. marginale was higher due to the high prevalence of this agent in Mexico (67.1%). In Brazil, the prevalence was 36.6% for A. marginale, 62.6% for Babesia spp., and 8.2% for their co-infections. The North region of Brazil was reported with the highest prevalence for A. marginale (71.9%), while for Babesia spp., the prevalence was considerably divergent between regions, with the highest values also observed in the North region (97.4%), and the lowest in the South region (9.5%). For studies of Babesia spp. in cattle in Latin America, the heterogeneity was mainly explained by the diagnostic method (98.0%), and country (54.8%). When looking at Brazil alone, a similar pattern was observed, but with lower values (i.e., diagnostic method, 31.5%; region, 25.3%; and climate, 12,4%). The evaluated regions presented different levels of prevalence infections, with most of them being classified as high degree of enzootic instability, which can predispose to the occurrence of outbreaks.

Naturally occurring Anaplasma marginale infection in cattle: Molecular prevalence and associated risk factors, haemato-biochemical alterations, oxidant/antioxidant status and serum trace mineral levels

The present investigation was undertaken to map the distribution of Anapalsma species infection in cattle from the Aizawl region of Mizoram, India, in relation to various risk factors, and to study the haemato-biochemical alterations, oxidant/antioxidant status and serum trace mineral levels in cattle with naturally occurring Anapalsma marginale infection. The study was carried out over 31 months from June 2019 to December 2021. A total of 401 cattle blood samples were collected and screened for the presence of Anaplasma spp. by microscopic examination and polymerase chain reaction (PCR). Non-infected clinically healthy cattle (n = 21) served as control. Blood samples were collected to study the haemogram and serum samples were used for the evaluation of biochemical parameters, oxidative stress indices and trace minerals. During the study period, an overall prevalence of 15.71% was recorded for A. marginale infection in cattle. The prevalence of A. marginale infection was highly associated with age, sex, breed and tick infestation status of animals, floor system and management of farms, and season. The mean values of total erythrocyte count (TEC), haemoglobin (Hb), packed cell volume (PCV), total platelet count, total protein, albumin, superoxide dismutase (SOD), glutathione (GSH), total antioxidant capacity (TAC), copper (Cu) and zinc (Zn) were significantly (P < 0.05) lower, whereas the mean values of mean corpuscular volume (MCV), aspartate aminotransferase (AST), total bilirubin, indirect bilirubin, blood urea nitrogen (BUN), creatinine and lipid hydroperoxide (LPO) were significantly (P < 0.05) higher in cattle infected with A. marginale. A negative correlation of TEC with LPO, and a positive correlation with SOD, GSH, TAC, Cu and Zn suggest a possible link between oxidative stress and the haemolytic crisis noticed in bovine anaplasmosis. Incorporation of antioxidants and organ protective drugs as an adjunct therapy may result in better prognosis.

Phylogenetic exploration and haemato-biochemical significance of Anaplasma marginale infection in water buffalo (Bubalus bubalis)

During 2017-2020, a total of 8352 buffalo blood samples were screened microscopically for Anaplsma species and 104 positive samples were found. Later on same samples were confirmed positive using genomic DNA PCR technique. For efficiency of PCR technique, a total of 335 blood samples were randomly chosen. Microscopy revealed Anaplasma species in erythrocytes of 1.19% (4 samples) of the total blood samples (n=335). Initially PCR assay was standardized targeting 16S rRNA for detection of genus Anaplasma with expected amplicon size 781 bp. Thereafter, msp1 β gene was targeted for species level identification of Anaplasma (Anaplasma marginale) with expected amplicon size 407 bp. A total of fourteen samples out of 335 samples (4.17%) found positive by PCR. Randomly, two confirmed positive samples were sent for bidirectional sequencing. The sequencing results, submitted in GenBank(MW269530, MW269531), further confirm the Anaplasma marginale infection in buffalo. The study show higher specificity and sensitivity of PCR test above blood smear examination. Erythron of infected animals showed significantly low level of mean Hb, PCV, RBC count, MCH, MCHC and high level of mean MCV, indicating regenerative hypochromic macrocytic anaemia. Infected animals had a significantly low mean level of total protein, albumin and globulin and significantly high level of phosphorus, total bilirubin, direct and indirect bilirubin. The current study provides indication of the association between A. marginale infection and probable liver damage or hepatic fatty degeneration. Nevertheless advanced studies are warranted to explain the precise pathophysiological mechanism of this association.

Evaluation of a recombinant MSP5 based dot-elisa for serodiagnosis of Anaplasma marginale infection in bovines

Evaluation of a recombinant MSP5 based dot-elisa for serodiagnosis of Anaplasma marginale infection in bovines

The study of prevalence and factors affecting Anaplasma maginale infection in domestic goats in Chonburi province, Thailand

The objective of this study was to determine the prevalence and factors affecting Anaplasma marginale infection of goat farming in Chonburi province, Thailand. A total of 182 samples were collected from clinical services records from the Veterinary Teaching Hospital, Rajamangala University of Technology Tawan-ok. Moreover, the potential risk factors of A. marginale infection, including vectors infestation, barn-types, preventive medicine protocols using vector-control agents and/or deworming programs, and the farming areas were statistically analyzed. The results revealed that 24.7% of goats were positive for A. marginale infection. The Nhong Yai district had the highest infection rate at 34.1% (15/44). The Mueng, Chonburi and Sri Racha district goats were infected at the rate of 27.5% (11/40) and 24.3% (9/37), respectively. The potential significant factors affecting A. marginale infection were vectors infestation (P &lt; 0.01) and preventive medicine protocols using vector-control agents and/or deworming programs (P &lt; 0.05). In conclusion, the vectors infestation increases the chance of A. marginale infection. Conversely, appropriate consistent preventive medical health management by application of vector-control agents and/or deworming programs can reduce the likelihood of infection. Our study could serve as a guideline for prevention planning and for further study of livestock rearing behavior concerning infection of blood parasites in nearby areas

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.

English

Anaplasmosis is a hemo-rickettsial disease of cattle and is most prevalent in tropical and subtropical regions of the world including Pakistan. This disease has been placed as one of the most economically important haemoparasitic diseases. The aim of the current study was to determine the molecular characterization and to assess the prevalence of Anaplasma marginale (A. marginale) infection in cattle and associated risk factors in three districts of Khyber Pakhtunkhwa (KP) province of Pakistan viz., Mardan, Kohat and Swat. The blood samples were collected conveniently from 434 tick-infested animals keeping the aseptic measures. A. marginale was identified from blood samples by microscopy and PCR. Sequencing and phylogenetic analysis of the sequenced isolates of this study showed close sequence similarity with the reported strains of USA, Thailand, Uganda, Uruguay, Zimbabwe, Philippines and China. Moreover, multiple sequence alignment of the 16S ribosomal RNA gene sequences of 5 different clones of the A. marginale depicts substantial variation in the genotypes of A. marginale found in different locations of KP. The prevalence of A. marginale infection was non-significantly associated (P &gt; 0.05) with districts, season, breed, age and sex of cattle. The highest prevalence of A. marginale infection was recorded in district Swat (20.30%) followed by Kohat (16.81%) and Mardan (15.00%) districts of KP. The prevalence of infection was highest in exotic breeds and their crosses, adults and female cattle. 10.70, 16.11, 46.70 and 26.70% were the prevalence of infection recorded for winter, spring, summer and autumn season, respectively. This study concludes that A. marginale infection is dominant in district Swat followed by Kohat and Mardan districts of KP province of Pakistan, respectively. © 2021 Friends Science Publishers