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This study presents the first comprehensive analysis of the mitochondrial genome of Babesia ovis, a significant pathogen in ovine babesiosis in Türkiye. The B. ovis mitochondrial genome is a linear monomeric molecule of 6015 bp with an A + T content of 70.5%, featuring terminal inverted repeats (TIRs) at both ends. It encodes three essential proteins (Cox1, Cox3, and Cob) and six fragments of large subunit rRNA genes. Comparative analysis revealed high sequence identity with Babesia sp. Xinjiang (87.5%) and Babesia sp. Dunhuang (87.5%), suggesting a close evolutionary relationship. The study highlights the conservation of mitochondrial gene content across Babesia and Theileria species, emphasizing their utility in phylogenetic studies. The findings also suggest that TIR variability plays a role in mitochondrial genome size differences, influencing species-specific adaptations. This research provides valuable insights into the evolution and functional adaptations of B. ovis and underscores the potential of mitochondrial genomic data in enhancing diagnostic and therapeutic strategies for babesiosis. Further exploration of mitochondrial genomes in Babesia species is essential for understanding their biology and developing effective control measures.

Babesia ovis is a significant tick-borne parasite of sheep, capable of causing both acute disease and long-lasting, low-grade infections. Imidocarb dipropionate (IMDP) is commonly used against babesiosis, yet whether it can completely eliminate B. ovis remains uncertain. In this study, we examined whether the parasite persists after treatment and whether such residual infections can still be transmitted. Three sheep were experimentally infected, treated with IMDP once clinical signs appeared, and then monitored for 180 days by microscopy, nested PCR, and iELISA. Fever and microscopic parasitemia resolved soon after treatment, but nPCR intermittently detected parasite DNA for several weeks. By day 180, all treated sheep were negative by nPCR and microscopy, while two still showed detectable antibodies. Blood collected at this time was transfused into naïve sheep. Two of the three recipients showed nPCR positivity at scattered time points and later seroconverted while showing no clinical signs. In contrast, Rhipicephalus bursa ticks that fed on the treated donors neither acquired the parasite nor transmitted it to recipients, likely because post-treatment parasitemia remained below the acquisition threshold. Overall, these results indicate that IMDP controls clinical disease but may not fully clear B. ovis, allowing silent transmission through blood despite negative routine tests.

Babesia is an intraerythrocytic protozoan parasite which is transmitted by hard ticks of the Ixodidae family. One of the problems associated with protozoan infection is the determination and characterization of the vectors. The aim of the present study was to detect Babesia ovis in the salivary gland of Dermacentor spp. A total of 200 adult Dermacentor ticks (139 D. niveus and 61 D. marginatus) were collected from sheep suspected to be infected with babesiosis in the Ardabil region of Iran from April to September 2015 (active season of ticks); and were identified using standard taxonomic keys. Deoxyribonucleic acid (DNA) was isolated from the salivary glands of ticks and analyzed with the primers derived from the hyper variable V4 region of 18S ribosomal ribonucleic acid (18S rRNA) of the Babesia species using polymerase chain reaction (PCR). Babesia ovis was detected in 5.8% of the D. niveus and 3.3% of the D. marginatus positive samples in the second round of semi-nested PCR. Based on the results obtained from this study, it is concluded that D. niveus and D. marginatus, which are distributed in Ardabil region of Iran, might play a major role in the transmission of infection as a natural vector of B. ovis.

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.

Ovine babesiosis caused by Babesia ovis is a tick-borne disease that affects sheep in enzootic regions of Southern Europe, Middle East, and Asia, resulting in significant economic loss. Despite its clinical relevance, only a few useful serological markers are available for accurate diagnosis. In the present study, we characterized the B. ovis spherical body protein 4 (SBP4) using in silico, molecular, and immunological approaches, and evaluated its preliminary activity with sera naturally infected sheep, suggesting its potential for future serodiagnostic application. The full-length sbp4 gene was identified via genome analysis, revealing a 984 bp intronless open reading frame encoding a 327-amino acid protein with a predicted signal peptide. Codon-optimized for expression in Escherichia coli , the sbp4 gene was cloned and inserted into the pET-29b(+) vector. Expression in Escherichia coli C43(DE3) produced soluble recombinant rBoSBP4 protein, which was purified via affinity chromatography. The ~ 37 kDa protein was confirmed by SDS‒PAGE and recognized by anti-His antibodies. Western blot and indirect ELISA using sera from naturally B. ovis -infected sheep demonstrated that rBoSBP4 is specifically recognized during infection, whereas negative control sera showed no reactivity. The immunization of rabbits with rBoSBP4 also induced a strong antibody response, as determined by ELISA. These results indicate that rBoSBP4 is both immunogenic and antigenically specific, supporting its potential utility in the development of serological diagnostic assays for ovine babesiosis.

Babesia ovis is a tick-transmitted intraerythrocytic apicomplexan parasite that causes ovine babesiosis, a disease with high economic impact in endemic regions. Although Rhipicephalus bursa is a known biological vector of B. ovis , the dynamics of vertical (transovarial) transmission are poorly understood. In the present study, the transovarial transmission of B. ovis was investigated experimentally in four continuous generations of R. bursa under laboratory controlled conditions. A total of 11 sheep were used during the study. An initial stabilate co-infected with B. ovis and Theileria ovis was inoculated into a splenectomized sheep, followed by an infestation with Babesia- free adult R. bursa . Study of dead tick bodies and larval pools by molecular methodology confirmed the elimination of T. ovis and the vertical transmission B. ovis . Rhipicephalus bursa ticks derived vertically from F 1 to F 4 generations kept high levels of infection (98% in F 3 adults), and consistently led to severe clinical babesiosis in both immunosuppressed and immunocompetent sheep. Although all feeding stages became infected while feeding on their hosts, only adult ticks were able to transmit the parasite to vertebrate hosts. Our findings demonstrate that B. ovis can be transmitted vertically through several tick generations, while preserving its ability to cause severe disease, even without selective pressure. The virulent, mono-infected B. ovis strain developed in this study will provide an infectious challenge model for future vaccine and pathogenesis studies under field-relevant conditions. Babesia ovi s est un parasite apicomplexe intraérythrocytaire transmis par les tiques et responsable de la babésiose ovine, une maladie à fort impact économique dans les régions endémiques. Bien que Rhipicephalus bursa soit un vecteur biologique connu de B. ovis , la dynamique de la transmission verticale (transovarienne) est mal comprise. Dans la présente étude, la transmission transovarienne de B. ovis a été étudiée expérimentalement sur quatre générations continues de R. bursa dans des conditions contrôlées en laboratoire. Au total, 11 moutons ont été utilisés au cours de l’étude. Un stabilat initial co-infecté par B. ovis et Theileria ovis a été inoculé à un mouton splénectomisé, suivi d’une infestation par des adultes de R. bursa indemnes de Babesia . L’étude des tiques mortes et des lots de larves par méthodologie moléculaire a confirmé l’élimination de T. ovis et la transmission verticale de B. ovis . Les tiques R. bursa dérivées verticalement des générations F 1 à F 4 ont conservé des niveaux d’infection élevés (98 % chez les adultes F 3 ) et ont systématiquement entraîné une babésiose clinique sévère chez les moutons immunodéprimés et immunocompétents. Bien que tous les stades d’alimentation aient été infectés en se nourrissant de leurs hôtes, seules les tiques adultes ont pu transmettre le parasite à leurs hôtes vertébrés. Nos résultats démontrent que B. ovis peut être transmis verticalement par plusieurs générations de tiques tout en préservant sa capacité à provoquer une maladie grave, même en l’absence de pression sélective. La souche virulente et mono-infectée de B. ovis développée dans cette étude fournira un modèle infectieux pour de futures études de vaccin et de pathogénèse dans des conditions de terrain.

Background: Ticks and tick-borne pathogens (TTBP) are a major constraint to livestock production in Pakistan; despite a high prevalence of TTBPs, knowledge on the capacity of Pakistani ticks to carry pathogens and endosymbionts is limited. Furthermore, mixed infections with multiple microorganisms further complicate and limit the detection potential of traditional diagnostic methods. The present study investigated the tick-borne microorganisms in bovine ticks in Pakistan, employing a high-throughput microfluidic real-time PCR based technique. Methods: Ticks were collected from clinically healthy cattle (n = 116) and water buffaloes (n = 88) from 30 villages across six districts located in five agro-ecological zones (AEZs) of Pakistan from September to November 2017. The microfluidic real-time PCR was used to test the genomic DNA of individual ticks for the presence of 27 bacterial and eight parasitic microorganisms. Phylogenetic methods were used to assess the genetic relationship of DNA sequences determined herein. Results: PCR detected DNA of at least one microorganism in each of 221 ticks tested (94.4%, 221/234). DNA-based detection inferred that single pathogens/endosymbionts were the most common (43.4%, 96/221) followed by double (38.9%, 86/221), triple (14.5%, 32/221), quadruple (2.3%, 5/221) and quintuple (0.9%, 2/221) mixed infections. Piroplasms (Babesia/Theileria spp.) were the most prevalent (31.6%, 74/234), followed by Ehrlichia spp. (20%, 47/234) and Anaplasma marginale (7.7%, 18/234). Anaplasma phagocytophilum, A. ovis, A. centrale, Babesia ovis, Borrelia spp., Rickettsia spp., R. massiliae, Bartonella spp. and Hepatozoon spp. were also detected. Endosymbionts such as Francisella-like (91.5%, 214/234) and Coxiella-like (1.3%, 3/234) organisms were also detected in ticks. The highest diversity of microorganisms was detected in Hyalomma anatolicum ticks (test-positive for 14/14 microorganisms), followed by Rhipicephalus microplus (4/14), Hy. hussaini (3/14) and Rh. annulatus (2/14). Ticks collected from cattle carried significantly more frequently piroplasms (41.2%, 54/131; P < 0.05) than those from buffaloes (19.4%, 20/103). However, the overall prevalence of microorganisms did not vary significantly among ticks from the two host species as well as across different AEZs. Conclusions: To our knowledge, this is the first study to investigate a wide range of tick-borne microorganisms in bovine ticks using a high-throughput diagnostic method from different AEZs in Pakistan. These findings will aid in establishing the distribution patterns and the control of tick-borne pathogens of bovines in Pakistan.

Parasitic diseases caused by ticks constitute a barrier on global animal production, mainly in tropical and subtropical regions. As a country with a temperate and subtropical climate, Turkey has topography, climate, and pasture resources, and these resources are suitable for animal breeding and parasite–host–vector relationships throughout the country. This geography restricts the regulations on animal movements in the southeastern and eastern Anatolia because of the close contact with the neighboring states. The livestock resources in Turkey are regulated by strong foundations. Almost 30% of the agriculture-based gross domestic product is provided by the livestock industry. Parasitic diseases arising from ticks are endemic in Turkey, and they have a significant impact on the economy and animal health, particularly for ruminants. The main and economically-important tick-borne diseases (TBDs) suffered by animals include theileriosis, babesiosis, hepatozoonosis, and cytauxzoonosis caused by protozoa, and anaplasmosis and ehrlichiosis caused by rickettsiae. The most common hemoprotozoan and rickettsial agents are Anaplasma marginale, Anaplasma ovis, Anaplasma phagocytophilum, Anaplasma platys, Babesia bigemina, Babesia caballi, Babesia ovis, Cytauxzoon felis, Ehrlichia canis, Hepatozoon canis, Theileria annulata and Theileria equi. These diseases are basically controlled through treatment and measures for tick control. Vaccination can be performed for only tropical theileriosis caused in Turkey. We reviewed the studies published in domestic and international journals to gather epidemiological data regarding the major TBDs suffered by animals in Turkey.

Babesia ovis is a tick‐borne protozoan parasite that poses a significant threat to sheep production, particularly in endemic regions such as Türkiye. It is known to be transmitted by ixodid ticks through both transstadial and transovarial routes. This study was aimed at investigating the natural transovarial transmission potential of B. ovis in Rhipicephalus bursa and Rhipicephalus turanicus ticks under field conditions. A total of 751 adult ticks were collected from 84 small ruminants (41 sheep and 43 goats) in Elazığ Province, Türkiye. Following species identification, 85 fully engorged female R. bursa ( n = 45) and R. turanicus ( n = 40) were incubated under controlled conditions to allow oviposition. DNA was extracted from the female carcasses and their corresponding larval pools and analyzed using nested PCR targeting the 18S rRNA gene of B. ovis . The results revealed that B. ovis DNA was detected in 19.04% (8/42) of R. bursa females and in 7.89% (3/38) of R. turanicus females. Importantly, all larval pools derived from B. ovis ‐positive R. bursa females also tested positive, indicating natural transovarial transmission. In contrast, none of the larval pools from R. turanicus tested positive, despite the presence of B. ovis in the female carcasses. These findings suggest that R. bursa may serve as a competent natural vector and reservoir for B. ovis , whereas R. turanicus seems to possess less ability for vertical transmission of the parasite.

Background Cats are hosts and reservoirs for many haemopathogens such as piroplasms, Rickettsia , hemotropic Mycoplasma , Bartonella , Ehrlichia , and Anaplasma , which are transmitted by various vector arthropods and some of which have a zoonotic concern. Although it is noteworthy that the rate of ownership of companion animals has increased in Türkiye in recent years and that cats account for a large proportion of these animals, there is limited research on the vector-borne infectious agents carried by them. The present study aimed to provide a comprehensive molecular epidemiological data and molecular characterization of feline vector-borne haemopathogens (FVBHs), including piroplasms, anaplasmataceae, rickettsias, haemoplasmas, and Bartonella species in Türkiye. In total, 250 feline blood samples were collected from client-owned cats ( n  = 203) and shelter cats ( n  = 47) brought to the Small Animal Hospital of Selcuk University, Veterinary Faculty. Results Overall, 40 (16%) cats were found to be infected with at least one of the investigated haemopathogens and piroplasm, Mycoplasma spp. and Bartonella spp. prevalence was 1.6%, 11.2%, and 4.8%, respectively. No Anaplasma / Ehrlichia spp. and Rickettsia spp. DNA was detected in the investigated feline samples. Sequence analysis revealed that all four piroplasms belonged to Babesia ovis with a 97.93–99.82% nucleotide sequence identity to 18S rRNA gene sequences from Spain and Türkiye, while some sequenced hemoplasmas were Mycoplasma haemofelis ( Mhf ), Candidatus Mycoplasma haemominutum ( C Mhm) and Mycoplasma wenyonii , and Bartonella spp. were Bartonella henselae and Bartonella koehlerae species. Co-infections with Mycoplasma spp. and Bartonella spp. were also detected in 4 cats (1.6%) in this study, where single infections were predominant. Conclusion This study provides valuable information on zoonotically important feline vector-borne hemopathogens in Türkiye, some of which have received attention under the One Health perspective, and is the first molecular epidemiological study to demonstrate the presence of Babesia ovis , the causative agent of ovine babesiosis, and Mycoplasma wenyonii DNA, the causative agent of bovine haemotropic mycoplasmosis, in cats. Further studies on the roles of such pathogens detected in unspecific hosts and the host specificity of the vectors that transmit them will contribute to the elucidation of this situation.