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Despite a goat population of approximately 80 million in Pakistan during 2020−2021, the prevalence of vector-borne pathogens in goats remains largely underexplored. This study aimed to assess the molecular prevalence and phylogenetic characteristics of Anaplasma ovis, Anaplasma marginale and Anaplasma phagocytophilum in goat blood samples (N = 239) collected from three districts (Muzaffargarh, Rajanpur, and Dera Ghazi Khan) in Punjab between September 2023 and October 2024. Blood samples were first screened with generic and then with species specific primers. Molecular analyses revealed a prevalence of 39% for Anaplasma spp. and 14% for A. ovis . A. marginale and A. phagocytophilum were not detected. DNA sequencing, by targeting 16S rRNA and msp4 genes, and BLAST analysis confirmed the presence of Anaplasma spp. and A. ovis, respectively. For both screening, bacterial prevalence rates varied significantly across sampling sites (P = 0.01 for Anaplasma spp. and P = 0.04 for A. ovis ). Additionally, the prevalence of Anaplasma spp. significantly differed among goat breeds (P = 0.004), while no association was found between goat sex and bacterial infections (P > 0.05 for both screening). Notably, Anaplasma spp. infection was associated with a significant decrease in red blood cell count and hemoglobin concentration, while A. ovis infection did not affect the complete blood count profile. Phylogenetic analysis revealed that our Anaplasma spp. isolates clustered with those from Iran, Cyprus and China while our A. ovis isolates clustered with those from Pakistan, China, and Sudan. In conclusion, this study reports the presence of Anaplasma spp. and A. ovis in Pakistani goats and recommends large-scale studies across diverse geo-climatic regions to further investigate the epidemiology, genetic diversity and host-parasite interactions for effective control of these infections in local goat populations.

Background Anaplasma ovis is a major cause of small ruminant anaplasmosis, a tick-borne disease mainly affecting small ruminants in tropical and subtropical regions of the world. Due to health and production problems in dairy goat flocks in Corsica, France, and the demonstration of A. ovis infection in some animals, an extensive survey was conducted in the island in spring 2016. The aim of the survey was to determine the prevalence and geographical distribution of A. ovis infections in goats and ticks as well as possible relationships with anaemia and other health indicators. In addition, the genetic diversity of A. ovis was evaluated. Methods Blood and faecal samples were collected in 55 clinically healthy flocks (10 goats per flock) for A. ovis qPCR, haematocrit determination, paratuberculosis ELISA seropositivity and gastrointestinal nematode egg excretion quantification. Ticks were collected, identified and processed for A. ovis DNA detection. Results A high prevalence of A. ovis DNA detection was found at the individual (52.0%) and flock levels (83.6%) with a within-flock prevalence ranging between 0–100%. Rhipicephalus bursa was the only tick species collected on goats (n = 355) and the detection rate of A. ovis DNA in ticks was 20.3%. Anaplasma ovis DNA prevalence was higher in flocks located at an altitude above 168 m, in goats of Corsican/crossbred breed and in goats > 3 years-old. No relationship was found between A. ovis DNA detection at the individual or flock level and haematocrit, paratuberculosis seropositivity or gastrointestinal parasites. Positive A. ovis goat samples were used for amplification of gltA and msp4 genes for species confirmation and strain identification, respectively. Sequence and phylogenetic analysis of these genes confirmed the detection of A. ovis and allowed identification of six different strains of this pathogen (named Corsica 1-6 (COR1-6). While the msp4 sequence of strain COR1 had 100% identity with strains previously reported, COR2 to 6 were found to be novel strains. The strain COR1 was the most represented, corresponding to 94.6% of the msp4 sequences obtained. Conclusions The results showed a relatively high genetic diversity of A. ovis associated with high bacterial prevalence in goats.

Background Ovine anaplasmosis ( sensu stricto ) is a rickettsial blood disease caused by the tick-borne species Anaplasma ovis . The disease is characterized by mild anemia, fever, and icterus. A more severe clinical presentation is possible in non-endemic areas. There is no existing data on the presence of Anaplasma ovis in Bosnia and Herzegovina. However, given the country’s location within the Mediterranean Basin and the recent molecular detection of Babesia ovis , it is plausible that sheep in the region could naturally be infected with this tick-borne pathogen. Methods and results Blood samples from 81 sheep in the Podrinje and Herzegovina areas were examined by PCR. PCR positivity was found in 38 (46.9%) cases indicating a high number of infected sheep. Mixed infections with Babesia ovis and A.ovis were observed in 63.3% of cases. A higher number of positive sheep was recorded in the area of Herzegovina. Phylogenetic analysis of the gltA , groEL , and msp4 genes of A. ovis revealed numerous genotypes and significant genetic variability. This diversity was not related to geographic origin, tick-borne infection status, or sheep breeding practices in Podrinje and Herzegovina. Conclusions The data obtained in this study suggest that the emergence of new genotypes and the high genetic variability of A. ovis are driven by specific local and micro-environmental factors.

Anaplasma are obligate intracellular Gram-negative rickettsial bacteria of medical and veterinary interest in both tropical and subtropical regions [1]. The disease caused by Anaplasma spp. has been recognized over a century, and is still an important issue worldwide [2, 3]. Since disclosure of zoonotic potential of A. phagocytophilum in 1994, there has been great interest in these bacteria [1, 4]. Until recently, six species have been recognized in the genus Anaplasma: Anaplasma marginale, Anaplasma bovis, Anaplasma phagocytophilum, Anaplasma centrale (A. marginale centrale), Anaplasma platys and Anaplasma ovis [5]. Anaplasma carpa has recently been described and considered as an emerging zoonotic pathogen in China [6]. The members in the genus Anaplasma differ in their cellular tropism, vectors, host range and pathogenicity [5]. Ovine anaplasmosis is caused by A. ovis, which is an obligate intra-erythrocytic pathogen of small ruminants [5, 7]. The causative agent was first described in sheep in 1912, and is widely distributed in Asia, Africa, Europe and the USA [7, 8]. This organism infects sheep, goats and some wild ruminants [9, 10]. Recently, an A. ovis variant was detected in a patient in Cyprus, indicated the zoonotic potential of this agent [11]. The life-cycle of A. ovis involves vertebrates and ticks, and animals can develop persistent infections and serve as reservoir hosts [12]. Currently, the identification and characterization of A. ovis mainly relies on the analysis of 16S rRNA and msp4 genes; however, these genes are highly conserved among heterologous strains [3, 13]. In previous reports, the major surface protein 1a (Msp1a), encoded by the msp1a gene, has been recognized as a stable molecular marker for classifying strains of A. marginale [14]. It has been revealed that A. marginale Msp1a could have evolved on the strength of immune selection pressure and differs among strains due to variable sequences and numbers of tandem amino acid repeats located in the N-terminal region of the protein [15]. The repeated region of A. marginale Msp1a contains the adhesion domain for tick cells and erythrocytes, which is essential for the invasion and transmission of the organism [15]. Previous reports have reported that immunization of cattle with Msp1a induces partial protection when challenged with A. marginale [15, 16]. Recently, Msp1a has also been identified in A. centrale, although attempts on other Anaplasma species have been performed [17]. In this study, we investigated the occurrence of A. ovis in small domestic animals in China, and identified the msp1a gene from A. ovis-positive samples. The A. ovis isolates identified herein were subsequently characterized based on the Msp1a amino acid repeats.

Background Tick-borne diseases cause economically significant losses to animal production globally, and anaplasmosis and theileriosis are associated with the greatest losses. However, the spread of the relevant pathogens in flocks of domesticated animals in southern Egypt is little understood. Accordingly, in this study, we aimed to determine the prevalences of Anaplasma ovis , Theileria ovis , and Theileria lestoquardi in southern Egyptian sheep and goats through blood tests, and to make a molecular characterization of the A. ovis detected in sheep targeting a specific gene. Results We collected blood samples collected from 300 sheep and goats ( n =150 /species) in Luxor Province in southern Egypt, and analyzed them for the presence of A. ovis, T. ovis and T. lestoquardi with screening by conventional and nested PCR targeting the msp4 and msp5, 18S rRNA, and merozoite surface protein genes. For A. ovis 140/300 samples (46.66%) were positive overall, with 90/150 (60%) and 50/150 (33.33%) positive samples in sheep and goats, respectively. Two major surface protein genes of A. ovis, msp4 and msp5, were sequenced using DNA extracted from sheep and goat blood samples, for phylogenetic analysis and genotyping. The msp4 gene sequence revealed no significant genetic diversity, to contrast to data on A. ovis strains from other countries. For T. lestoquardi , 8/150 (5.33%) samples were positive in sheep, but no samples were positive in goats (0%). For T. ovis, 32/150 (21.33%) samples were positive in sheep, but no samples were positive in goats (0%). Sequencing targeting the merozoite surface protein gene for T. lestoquardi and the small subunit ribosomal RNA gene for T. ovis revealed no significant genetic diversity in the study, another contrast to data on A. ovis strains from other countries. Conclusion This study provides valuable data on phylogenetic and molecular classifications of A. ovis, T. ovis and T. lestoquardi found in southern Egyptian sheep and goats. It also represents the first report on detection and molecular characterization of T. lestoquardi in southern Egyptian sheep based on the specific merozoite surface protein gene, thus providing valuable data for molecular characterization of this pathogen in southern Egypt.

The Xinjiang Uygur Autonomous Region (Xinjiang) borders eight countries and has a complex geographic environment. There are almost 45.696 million herded sheep in Xinjiang, which occupies 13.80% of China’s sheep farming industry. However, there is a scarcity of reports investigating the role of sheep or ticks in Xinjiang in transmitting tick-borne diseases (TBDs). A total of 894 ticks (298 tick pools) were collected from sheep in southern Xinjiang. Out of the 298 tick pools investigated in this study, Rhipicephalus turanicus (Rh. turanicus) and Hyalomma anatolicum (H. anatolicum) were identified through morphological and molecular sequencing. In the southern part of Xinjiang, 142 (47.65%), 86 (28.86%), and 60 (20.13%) tick pools were positive for Rickettsia spp., Theileria spp., and Anaplasma spp., respectively. Interestingly, the infection rate of Rickettsia spp. (73%, 35.10%, and 28.56–41.64%) was higher in Rh. turanicus pools than in H. anatolicum pools (4%, 4.44%, and 0.10–8.79%) in this study. Fifty-one tick pools were found to harbor two pathogens, while nineteen tick pools were detected to have the three pathogens. Our findings indicate the presence of Rickettsia spp., Theileria spp., and Anaplasma spp. potentially transmitted by H. anatolicum and Rh. turanicus in sheep in southern Xinjiang, China.

Across the world, ticks act as vectors of human and animal pathogens. Ticks rely on bacterial endosymbionts, which often share close and complex evolutionary links with tick-borne pathogens. As the prevalence, diversity and virulence potential of tick-borne agents remain poorly understood, there is a pressing need for microbial surveillance of ticks as potential disease vectors. We developed a two-stage protocol that includes 16S-amplicon screening of pooled samples of hard ticks collected from dogs, sheep and camels in Palestine, followed by shotgun metagenomics on individual ticks to detect and characterise tick-borne pathogens and endosymbionts. Two ticks isolated from sheep yielded an abundance of reads from the genus Rickettsia, which were assembled into draft genomes. One of the resulting genomes was highly similar to Rickettsia massiliae strain MTU5. Analysis of signature genes showed that the other represents the first genome sequence of the potential pathogen Candidatus Rickettsia barbariae. Ticks from a dog and a sheep yielded draft genome sequences of Coxiella strains. A sheep tick yielded sequences from the sheep pathogen Anaplasma ovis, while Hyalomma ticks from camels yielded sequences belonging to Francisella-like endosymbionts. From the metagenome of a dog tick from Jericho, we generated a genome sequence of a canine parvovirus. Here, we have shown how a cost-effective two-stage protocol can be used to detect and characterise tick-borne pathogens and endosymbionts. In recovering genome sequences from an unexpected pathogen (canine parvovirus) and a previously unsequenced pathogen (Candidatus Rickettsia barbariae), we demonstrate the open-ended nature of metagenomics. We also provide evidence that ticks can carry canine parvovirus, raising the possibility that ticks might contribute to the spread of this troublesome virus.

Anaplasmosis and ehrlichiosis are important tick-borne rickettsial diseases of medical and veterinary importance that cause economic losses in livestock. In this study, the prevalence of Anaplasma ovis, Ehrlichia canis and Ehrlichia chaffeensis was investigated in ticks collected from sheep in various farms in Van province, which is located in the Eastern Anatolian Region of Turkey. The ticks used in this study were collected by random sampling in 26 family farm business in 13 districts of Van province. A total of 688 ticks were collected from 88 sheep and 88 tick pools were created. All ticks identified morphologically as Rhipicephalus bursa. Phylogenetic analysis of Chaperonin and 16S rRNA gene sequences confirmed A. ovis, E. canis and E. chaffeensis in this study. Of the 88 tick pools tested, 28.41% (25/88) were positive for at least one pathogen. Anaplasma DNA was detected in five of the 88 pools (5.68%), E. canis DNA was detected in 19 of the 88 pools (21.59%), and E. chaffeensis DNA was detected in one of the 88 pools (1.14%) of R. bursa ticks. To our knowledge, this is the first report describing the presence of A. ovis, E. canis, and E. chaffeensis in R. bursa ticks collected from sheep in Turkey. Further studies are needed to investigate other co-infections in sheep in Turkey.

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.

Background Ovine anaplasmosis is a tick-borne disease that is caused by Anaplasma ovis in sheep and goats. The pathogen is widely distributed in tropical and subtropical regions of the world. At present, diagnosis of the disease mainly depends on microscopy or nucleic acid based molecular tests, although a few serological tests have been applied for the detection of A. ovis infection. Results Here we describe the identification of an A. ovis protein that is homologous to the A. marginale appendage-associated protein (AAAP). We expressed a recombinant fragment of this protein for the development of an indirect enzyme-linked immunosorbent assay (ELISA) for the detection of A. ovis . Anaplasma ovis -positive serum showed specific reactivity to recombinantly expressed AAAP (rAAAP), which was further confirmed by the rAAAP ELISA, which also demonstrated no cross-reactivity with sera from animals infected with A. bovis or other related pathogens in sheep and goats. Testing antibody kinetics of five experimentally infected sheep for 1 year demonstrated that the rAAAP ELISA is suitable for the detection of early and persistent infection of A. ovis infections. Investigation of 3138 field-collected serum samples from 54 regions in 23 provinces in China demonstrated that the seroprevalence varied from 9.4% to 65.3%, which is in agreement with previous reports of A. ovis infection. Conclusions An A. ovis derived antigenic protein, AAAP, was identified and the antigenicity of the recombinant AAAP was confirmed. Using rAAAP an indirect ELISA assay was established, and the assay has been proven to be an alternative serological diagnostic tool for investigating the prevalence of ovine anaplasmosis of sheep and goats.