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Background: The 1986 disaster at the Chornobyl Nuclear Power Plant released massive amounts of radioactive material into the local environment. In addition to radiation, remediation efforts and abandonment of military-industrial complexes contributed to contamination with heavy metals, organics, pesticides and other toxic chemicals. Numerous studies have evaluated the effects of this contamination on the local ecology. However, few studies have reported the effect of this contamination on vector-borne pathogens and their hosts. In this manuscript, we characterize tick-borne pathogen presence at two sample locations within the Chornobyl Exclusion Zone, one at the Nuclear Power Plant (NPP) and another 16 km away in Chornobyl City (CC)., Methods: Ticks and whole-blood samples were collected from free-breeding dogs captured at the NPP and CC. Endpoint PCR and quantitative PCR were used to identify tick species and to assess the presence of specific tick-borne pathogens, including Anaplasma phagocytophilum, Borrelia burgdorferi sensu lato, Babesia spp., Bartonella spp., Francisella tularensis and general Anaplasmataceae. A droplet digital PCR assay was developed for Babesia canis and A. phagocytophilum to evaluate their presence in dogs from the two populations. Pathogen prevalences between the two sample populations were compared by calculating Z-scores., Results: Ticks were identified as Ixodes ricinus (n = 102) and Dermacentor reticulatus (n = 4). Overall, 56.9% of I. ricinus ticks were positive for at least one pathogen. A significantly higher prevalence of A. phagocytophilum and B. burgdorferi was found in ticks at the NPP (44.0% and 42.0%, respectively) compared to CC (23.1% and 19.2%, respectively). Babesia spp. (including B. canis and B. caballi) were detected in 8.8% ticks at similar proportions for both populations. Interestingly, we found a significantly lower level of A. phagocytophilum in dogs at the NPP (1.8%) than in dogs at CC (11.7%). In total, 24.3% of dogs were positive for B. canis, evenly distributed across the two populations., Conclusions: The results of this study show contrasting pathogen prevalence in both ticks and dogs at the NPP and CC, which may reflect the differential exposures at the two locations. This work adds an important new component to our understanding of the consequences of prolonged exposure to environmental contamination on the wildlife and ecology within the Chornobyl Exclusion Zone., Competing Interests: Declarations Ethics approval and consent to participate The Chornobyl Nuclear Power Plant authorities under the supervision of licensed veterinarians and veterinary technicians conducted the ethical review and provided permission for the animal study. A letter of support detailing the cooperation between the Clean Futures Fund and the Chornobyl NPP was signed by the NPP acting general director V.A. Seyda and can be provided by TAM upon request. Approval for access to the exclusion zone was handled and granted by the Exclusion Zone Authority. Samples collected for this study were gathered adventitiously while animals were being treated by the medical program and as such are exempt from IACUC approval. Consent for publication Not applicable. Competing interests BAQ receives partial salary support from IDEXX Laboratories, Inc and has been a paid consultant during the development and validation of rapid assays developed by IDEXX Laboratories for veterinary medical applications. The other authors have no competing interests to declare., (© 2024. The Author(s).)

The increasing population of dogs and changes in the climatic conditions have resulted in the emergence and re-emergence of vector-borne diseases in canines. These vectors borne diseases in canines pose a diagnostic challenge to the field veterinarians because of co-infections with several pathogens. Comprehensive data on the prevalence of haemoparasites and haemorickettsiales in pet and community owned dogs from south India are scant. Hence, the present study aims to find and compare the prevalence of these infections in the pet and the community owned dogs of Kerala, a south Indian state. Two hundred and seventy-two pets and 150 community owned dogs were examined by microscopy and polymerase chain reaction (PCR) for infections with different heamoparasites and haemorickettsials from January 2018-November 2020 in the state of Kerala. A high prevalence of Babesia gibsoni infection (42.2-60.0 %) in pet and community owned dogs, followed by Babesia vogeli (5.8-39.3 %), Hepatozoon canis (0.7-28.0 %), Trypanosoma evansi (0.0-27.3 %), Ehrlichia canis (0.3-0.6 %) and Anaplasma platys (0.0-0.6 %) was observed in the present study. Eighty-eight per cent (132/150) of the community owned dogs and 49.2 % (134/272) of the pet dogs were positive for at least one pathogen. Phylogenetic analysis based on the partial nucleotide sequences of 18S rRNA and TRAP gene of B. gibsoni, 18S rRNA genes of B. vogeli and H. canis and RoTat, 1.2 virB9 and 16S rRNA genes of T. evansi, E. canis and A. platys, respectively was carried out. B. vogeli, H. canis, E. canis and A. platys revealed genetic relatedness between the Indian isolates and the isolates from other countries. However, B. gibsoni isolates from the Indian sub-continent were genetically unique compared to other Asian isolates. The clustering of T. evansi isolates from India in two clades viz., livestock origin (cattle, buffalo) and others indicated their genetic variability. The present study summarizes the prevalence of some of the haemoparasites and haemorickettsials in the dog populations of Kerala (south India) and also determined their genetic relationship with the isolates prevalent in dogs in other countries., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2024. Published by Elsevier B.V.)

This study aimed to identify the diversity of Babesia and Theileria species in sheep across various regions of Iran using microscopic and molecular techniques, including species-specific PCR and enzymatic digestion. A total of 373 blood samples were collected from sheep during the tick vector activity period, from 2018 to 2021, in provinces such as Tehran, Alborz, Qazvin, Hamedan, West Azerbaijan, Kerman, and Fars. Results showed that 101 samples (27 %) exhibited piroplasms including 78 samples of Theileria spp. and 23 samples of Babesia spp. A molecular approach using general primers detected piroplasm parasites in 145 samples (38 %). Theileria ovis was notably present in 91 samples (24.39 %), followed by Theileria lestoquardi in 24 samples (6.43 %). Babesia ovis infection was detected in 30 samples (8.4 %). Despite extensive molecular evaluation, no other Babesia species, including Babesia motasi, were identified. Co-infections involving T. ovis and T. lestoquardi (4 samples; 1.07 %) and T. ovis and B. ovis (6 samples; 1.60 %) were observed. No Babesia spp. were detected in Kerman and Fars provinces, although T. ovis and T. lestoquardi were present. Blast analysis of the sequences indicated 100 % intra-species similarity, with inter-species similarities of 83.3 % (B. ovis and T. lestoquardi), 84.4 % (B. ovis and T. ovis), and 96.44 % (T. ovis and T. lestoquardi). In conclusion, B. ovis was the main cause of Babesiosis, while Theileriosis was predominantly attributed to T. ovis and T. lestoquardi. Molecular diagnostics play a key role in accurately distinguishing between these species, particularly in cases of co-infection involving Babesia spp. and Theileria spp., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2024. Published by Elsevier B.V.)

This study aimed to fill a crucial gap in our understanding of Babesia infection in dogs in Mashhad, northeast Iran. We not only investigated the prevalence of Babesia species among dogs but also undertook a comprehensive comparison of clinical, hematological, and clinicopathological findings between infected and non-infected cases, a unique aspect of our research., Materials and Methods: Our research was conducted with meticulous attention to detail. We randomly collected blood specimens from a diverse population of 150 dogs, including owned pets (n = 47), stray dogs (n = 66), and shelter dogs (n = 37), to ensure the reliability and representativeness of our findings. We then used microscopy and PCR to investigate Babesia spp. infection and analyzed various biochemical and hematological variables., Results: The overall prevalence of babesiosis was 15.3 % (23/150) by PCR and 2 % (3/150) by microscopy. Upon microscopic examination, two cases of large Babesia and one case of small-sized Babesia were identified. The sequencing results confirmed that the two dogs testing positive for large-sized Babesia species in this study were both infected with B. vogeli, exhibiting 100 % sequence identity. There was no association between infection and gender, while housing status (k = 37.294, p = 0.000) and age (k = 6.897, p = 0.021) significantly related to infection rate. Among laboratory variables, infection with Babesia spp. showed a remarkable association with Hct (k = 4.749, p = 0.025) and RBC count (k = 14.669, p = 0.000), which were significantly lower in infected dogs compared to non-infected dogs (p < 0.05). Aside from severe non-regenerative anemia observed in all three clinically infected cases, the most clinicopathological changes were observed in one B. vogeli-infected dog, including pancytopenia, azotemia, hyperphosphatemia, hyperkalemia, hypoglycemia, hypocholesterolemia, hyponatremia., Conclusion: This study reveals a higher-than-expected prevalence of canine babesiosis in Northeastern Iran, necessitating further investigation of tick vectors and Babesia spp. distribution. Notably, many infected dogs were asymptomatic, raising concerns about silent spread via carriers. Moreover, the high prevalence of infection in shelters highlights the need for more effective control strategies in these centers., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Background: Ticks represent a significant vector for the transmission of infectious diseases, with the prevalence of tick-borne diseases becoming a prominent global health concern in recent decades. Anaplasma spp., Rickettsia spp., and Piroplasma have been identified as significant pathogens with the potential to impact human and animal health. However, there is a dearth of data concerning the prevalence of these pathogens in the eastern Tibetan Plateau, China., Methods: In this study, a total of 643 Dermacentor silvarum and 314 Haemaphysalis longicornis were identified through the application of morphological and molecular identification techniques on 957 ticks collected from yaks in Zoige County. The assessed of Anaplasma spp., Rickettsia spp., Theileria spp., and Babesia spp. was assessed in 957 ticks and 96 blood samples collected from yaks., Results: Significant discrepancies were observed in the positivity rates for the four pathogens among the tick species and sampling sites. The identification of different species within the four pathogens was based on the analysis of the 16S rRNA of Anaplasma spp., the ompA and ompB genes of Rickettsia spp., and the 18S rRNA of Theileria spp. and Babesia spp. The prevalence ranges of the four pathogens are 9.9-50.2%, 29.5-100%, 16.2-46.4%, and 14.5-58.4%, respectively., Conclusion: In view of the growing zoonotic risks, further investigations into the prevalence of additional pathogens in ticks and animals, including livestock, in the eastern Tibetan Plateau, China, are essential., Competing Interests: The authors declare that the research was conducted without any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Xiang, He, Zhu, Xiao, Pan, Chen, Zheng, Yuan and Hao.)

Haemoparasitic infections are frequently observed in dogs from tropical regions, including India. The present investigation combined microscopic blood smear examination and PCR assays to assess the occurrence of canine tick-borne diseases (CTBD) from suspected dogs in and around Hisar, Haryana. Using the Giemsa-stained peripheral thin blood smear examination, 15 (12.5%) of the 120 dogs were infected with CTBD, with 5.8%, 3.3%, 2.5%, and 0.8% dogs testing positive for Hepatozoon canis, Ehrlichia canis, Babesia vogeli, and Babesia gibsoni, respectively. Using the PCR assay, CTBD was found to be 64.16% (77/120) in examined dogs. Of the 77 PCR-positive canines, 56 were infected with a single haemoparasite, while 21 were infected with two or more species. H. canis was the most abundant tick-borne pathogen, representing 35%, followed by E. canis 25.8%, B. vogeli 20%, and B. gibsoni 2.5%. The most common co-infection was with H. canis along with E. canis (7.5%). The PCR assay was proven to be more efficient for detecting haemoparasites in dogs compared to blood smear examinations. The study suggests that canine tick-borne diseases are common in Haryana and recommends using PCR-based molecular tests in addition to conventional microscopic examination to diagnose these infections for effective treatment and management of infected canines., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Babesiosis and Anaplasmosis are diseases associated with economic losses; ticks and blood-sucking flies are important zoonotic vectors and reservoirs. This study aimed to investigate the presence of anti-Babesia spp. and anti-Anaplasma marginale antibodies using enzyme-linked immunosorbent assay (ELISA), in ruminants at the Catimbau National Park. Blood samples were collected from 119 sheep, 119 goats, and 47 cattle. Rhipicephalus microplus ticks were collected from cattle. ELISA showed seropositivity of 34% (16/47), 20.3% (24/119), and 16% (19/119) for anti-Babesia bovis; 34% (16/47), 15.2% (18/119), and 9% (7/119) for anti-Babesia bigemina; and 34% (16/47), 35.6% (42/119), and 17% (20/119) for anti-A. marginale antibodies in cattle, goats, and sheep, respectively. The information collected using an epidemiological questionnaire showed that mostly are breed in a semi-intensive system, with access to Caatinga vegetation. The circulation of B. bovis, B. bigemina, and A. marginale was confirmed. Thus, based on the prevalence, this suggests this is an enzootic instability area and is prone to outbreaks.

Equine piroplasmosis (EP) is a protozoal disease affecting equids, caused by Theileria equi and Babesia caballi . EP is conventionally diagnosed using microscopic, molecular, and/or serological methods, which are time-consuming. Consequently, there is a need for faster testing methods. In this study, we evaluated the application of the Sysmex XN-31 automated hematology analyzer, originally a rapid test for detecting malaria in humans, for the diagnosis of EP. The cultured parasites were measured using the XN-31 that had been customized for horse blood samples (XN-31m). The following parameters were evaluated: limit of detection (LoD), limit of quantification (LoQ), linearity, carryover, precision, and correlation with microscopic examination. The XN-31m detected infected red blood cells (RBCs) in approximately 1 minute. The LoD and LoQ for B. caballi were 4.54 infected RBCs/μL and 14.10 infected RBCs/μL, while those for T. equi were 5.80 infected RBCs/μL and 11.44 infected RBCs/μL, respectively. Linearity showed excellent correlation ( R 2 > 0.99), and carryover never exceeded 0.5%. The coefficient of variation was under 5%. The correlation between the results obtained using XN-31m and microscopic examination was high ( R 2 > 0.98). In conclusion, the XN-31 analyzer detected B. caballi and T. equi parasites in approximately 1 minute with high sensitivity. The results indicate the potential of the XN-31 analyzer as a fast and user-friendly diagnostic method for EP., Importance: In this study, we demonstrated that the automated hematology analyzer, XN-31, can detect red blood cells infected with Babesia caballi and Theileria equi in about 1 minute. We evaluated the diagnostic performance of the XN-31 analyzer for equine piroplasmosis, providing evidence of its potential as a diagnostic tool for this disease., Competing Interests: Yuji Toya, Mikako Sengoku, and Seiichiro Tsuchiya are employees of Sysmex corp.

Background: Hemoparasitic infections significantly threaten cattle health globally, leading to economic losses and welfare issues for farmers., Aim: This retrospective study aimed to investigate the prevalence and associated factors of hemoparasitic infections in fighting bulls in southern Thailand over an 8-year period., Methods: Laboratory records of 5,518 bulls from the Livestock Animal Hospital, Faculty of Veterinary Science, Rajamangala University of Technology Srivijaya, were reviewed for the period January 2016 to December 2023. Blood samples were analyzed using thin blood smear methods to identify hemoparasites, and packed cell volume was measured using the microhematocrit method. Detailed profiles and demographic data for each animal were recorded., Results: Among the fighting bulls, 323 (5.85%) tested positive for hemoparasitic infections, with five hemoparasites identified: Trypanosoma spp. (TP), Microfilariae, Anaplasma spp. (AP), Babesia spp. (BB), and Theileria spp. (TR). TP was the most prevalent at 2.90%. Bulls aged 1-5 years showed the highest infection rates at 7.04%, with a decline in infections as age increased. Annual trends peaked in 2018 at 8.80%, with significant yearly fluctuations for most parasites ( p < 0.05) except AP and BB. Monthly analyses revealed the highest infection rates in March, particularly for TP, with significant monthly variations for TR ( p < 0.05). Geographical differences in infection rates between the East and West coasts were minor and not statistically significant. However, seasonal variations were significant for BB and TR on the east coast during summer ( p < 0.05). Bulls with PCV below 30% had a higher prevalence of hemoparasitic infections (8.06%) compared to those with PCV above 30% (5.87%), with significant differences in the prevalence of AP and TR infections ( p < 0.05)., Conclusion: This study adds to the understanding of hemoparasite infections in bulls, informing the development of educational materials for farmers and ultimately empowering them to make better herd health decisions., Competing Interests: It is declared by the authors that no conflicts of interest exist.

We describe a case of autochthonous human Babesia divergens infection in an immunocompetent woman in England. The patient had fever, hemolysis, multiorgan failure, and 18% parasitemia. We confirmed B. divergens by 18S rDNA PCR and sequencing. Clinicians should consider babesiosis as a differential diagnosis in patients with unexplained hemolysis.

The European red squirrel (Sciurus vulgaris) is a common host for Ixodes ricinus ticks in urban and rural habitats, however, studies on ticks and tick-borne pathogens (TBPs) of squirrels have not been conducted in Poland yet. Thus, the aims of the current study were to assess and compare the prevalence and abundance of ticks on red squirrels trapped at two sites in the Warsaw area (in an urban forest reserve and an urban park) and using molecular tools, to assess the genetic diversity of three pathogens (Borrelia burgdorferi sensu lato, Rickettsia and Babesia spp.) in I. ricinus ticks collected from squirrels. For the detection of Rickettsia spp. a 750 bp long fragment of the citrate synthase gltA gene was amplified; for B. burgdorferi s.l. 132f/905r and 220f/824r primers were used to amplify the bacterial flaB gene fragments (774 and 605 bp, respectively) and for Babesia spp., a 550 bpfragment of 18S rRNA gene was amplified. In total, 91 red squirrels were examined for ticks. There were differences in tick prevalence and mean abundance of infestation in squirrels from the urban forest reserve and urban park. Three species of B. burgdorferi s.l., Rickettsia spp., and Babesia microti were detected in ticks removed from the squirrels. Our results broaden knowledge of S. vulgaris as an important host for immature I. ricinus stages and support the hypothesis that red squirrels act as a reservoir of B. burgdorferi. Moreover, we conclude that red squirrels may also play a role in facilitating the circulation of other pathogens causing serious risk of tick-borne diseases in natural and urban areas., (© 2024. The Author(s).)

Malaria and babesiosis are global health threats affecting humans, wildlife, and domestic animals, particularly in Africa, the Americas, and Europe. Malaria can lead to severe outcomes, while babesiosis usually resembles a mild illness but can be severe and fatal in individuals with weakened immune systems. Swift, accurate detection of these parasites is crucial for treatment and control. We evaluated a real-time PCR assay for diagnosing five Plasmodium and three Babesia species from blood samples, assessing its sensitivity, specificity, and analytical performance by analyzing 46 malaria-positive and 32 Babesia spp-positive samples diagnosed through microscopy. The limit of detection for Plasmodium species ranged from 30 to 0.0003 copies/µL. For mixed infections, it was 0.3 copies/µL for P. falciparum/P. vivax and 3 copies/µL for P. malariae/P. knowlesi. Babesia species had a detection limit of 0.2 copies/µL. No cross-reactivity was observed among 64 DNA samples from various microorganisms. The assay showed good sensitivity, detecting Plasmodium and Babesia species with 100 % accuracy overall, except for P. falciparum (97.7 %) and B. microti (12.5 %). The low sensitivity of detecting B. microti was attributed to limitations in microscopy for species identification. This technique heavily relies on the proficiency of the examiner, as species within the genus cannot be distinguished under a microscope. Additionally, Babesia can be confused with the early trophozoite stage (ring forms) of Plasmodium parasites. The findings support multiplex qPCR's diagnostic superiority over the gold standard, despite higher costs. It offers enhanced sensitivity, specificity, and detects mixed infections, crucial for effective monitoring and diagnosis of malaria and babesiosis in endemic regions with significant public health challenges., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Objectives: Human babesiosis is an emerging and potentially fatal tick-borne disease caused by intraerythrocytic parasites of the Babesia genus. Among these, Babesia duncani is particularly notable for causing severe and life-threatening illness in humans. Accurate diagnosis and effective disease management hinge on the detection of active B. duncani infections. While molecular assays are available to detect the parasite in blood, a reliable method for identifying biomarkers of active infection remains elusive., Methods: We developed the first B. duncani antigen capture assays, targeting two immunodominant antigens, BdV234 and BdV38. These assays were validated using established in vitro and in vivo B. duncani infection models, and following drug treatment., Results: The assays demonstrated no cross-reactivity with other species such as B. microti, B. divergens, Babesia MO1, or Plasmodium falciparum, and can detect as few as 115 infected erythrocytes/µl of blood. Screening of 1731 blood samples from various biorepositories, including samples previously identified as Lyme and/or B. microti-positive, as well as new specimens from wild mice, revealed no evidence of B. duncani infection or cross-reactivity., Conclusions: These assays hold significant promise for various applications, including point-of-care testing for the early detection of B. duncani in patients, field tests for screening reservoir hosts, and high-throughput screening of blood samples intended for transfusion., Competing Interests: Declarations of competing interest The authors have no competing interests to declare., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Babesia duncani, responsible for human babesiosis, is one of the most important tick-borne intraerythrocytic pathogens. Traditionally, babesiosis is definitively diagnosed by detecting parasite DNA in blood samples and examining Babesia parasites in Giemsa-stained peripheral blood smears. Although these techniques are valuable for determining Babesia duncani, they are often time-consuming and laborious. Therefore, developing rapid and reliable B. duncani identification assays is essential for subsequent epidemiological investigations and prevention and control. In this study, a cross-priming amplification (CPA) assay was developed, combined with a vertical flow visualization strip, to rapidly and accurately detect B. duncani infection. The detection limit of this method was as low as 0.98 pg/μl of genomic DNA from B. duncani merozoites per reaction at 59 °C for 60 min. There were no cross-reactions between B. duncani and other piroplasms infective to humans and mammals. A total of 592 blood samples from patients bitten by ticks and experimental infected hamsters were accurately assessed using CPA assay. The average cost of the CPA assay is as low as approximately $ 0.2 per person. These findings indicate that the CPA assay may therefore be a rapid screening tool for detection B. duncani infection, based on its accuracy, speed, and cost-effectiveness, particularly in resource-limited regions with a high prevalence of human babesiosis., Competing Interests: Declaration of competing interest The authors declare they have no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Ticks parasitize various hosts, including humans, and are known to transmit pathogens that can be harmful not only to animals but also to humans. To evaluate the possible presence of pathogens in ticks, we aimed to collect and identify tick fauna specimens in Lagoa Comprida Municipal Natural Park, an anthropogenic urban area located in Aquidauana, Mato Grosso do Sul, Brazil. A total of 1216 ticks, of which 51.2% were Amblyomma sculptum, 1.2% were Amblyomma dubitatum, and 41% were Amblyomma spp. were collected. These results show that the prevalence of A. sculptum is significantly higher than that of A. dubitatum across all four seasons. Molecular analyses revealed positive samples for the genus Babesia, including the confirmation of Babesia bigemina in an A. sculptum specimen, marking the first record of this relationship. This unexpected finding demands greater attention and deeper analysis in the context of the epidemiology of tick-borne diseases., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Equine piroplasmosis is caused by Theileria equi and Babesia caballi, which are hemoprotozoan parasites. Understanding the epidemiology and genotypes of T. equi and B. caballi is crucial for developing effective control strategies in endemic countries. However, the endemic status of these two parasite species remains uncertain in Kyrgyzstan due to lack of surveys. Our study, therefore, aimed to detect T. equi and B. caballi infections in Kyrgyzstan and identify their genotypes. Blood samples were collected from 226 horses across all seven provinces of Kyrgyzstan, namely Chuy, Issyk-Kul, Naryn, Talas, Jalal-Abad, Osh, and Batken. These blood samples were subjected to DNA extraction, followed by specific PCR assays targeting T. equi and B. caballi. We found that 56 (24.8%, confidence interval (CI): 19.6-30.8%) and 7 (3.1%, CI: 1.5-6.3%) of the tested horses were positive for T. equi and B. caballi infections, respectively. Theileria equi was detected in all surveyed provinces, whereas B. caballi was found in five provinces, except for Talas and Osh. Subsequent genotype-specific PCR assays showed that T. equi-positive horses harbored all five genotypes: A, B, C (also known as Theileria haneyi), D, and E. On the other hand, phylogenetic analysis of B. caballi rap-1 sequences detected the genotypes A and B1. The prevalence of T. equi and B. caballi suggests a potential risk of clinical equine piroplasmosis among horses in Kyrgyzstan, and the observed genotypic diversity underscores the challenges in managing the disease. Our findings emphasize the need for comprehensive control measures to effectively address equine piroplasmosis in Kyrgyzstan., Competing Interests: Declaration of competing interest All authors declare no conflict of interests associated with this study., (Copyright © 2023. Published by Elsevier B.V.)

Babesia bigemina and Theileria annulata are tick-borne protozoans that cause piroplasmosis in cattle, resulting in huge damages to the livestock industry. The prevalence of these infections depends on various intrinsic and extrinsic risk factors. In Pakistan, there is no information regarding the molecular characterization of Babesia bigemina and the risk factors associated with piroplasmosis. This study aimed to molecularly characterize Babesia spp. and Theileria spp. infecting various cattle breeds in Khyber Pakhtunkhwa, Pakistan, and to shed light on risk factors associated with these infections. Altogether, 219 blood samples were collected from various symptomatic cattle breeds, including Holstein Friesian (65.3%; 143/219), Jersey (21.5%; 47/219) and Sahiwal (13.2%; 29/219). Isolated genomic DNA from these blood samples was used in PCR for the amplification of the 18S rRNA fragment of apicomplexan parasites. Obtained 18S rDNA sequences from cattle hosts showed 99.5% identity with B. bigemina, or 100% with T. annulata. Having an overall infection rate of 61.6% (135/219), the highest infection rate was recorded for T. annulata (43.8%; 95/219), followed by B. bigemina (18.3%; 40/219). Phylogenetic analysis of 18S rDNA sequences revealed that B. bigemina clustered with corresponding species reported from Bolivia, and South Africa, while T. annulata grouped with same species from Italy, India, and Turkey. Among the different risk factors, the breed, season, and tick infestation were found to have a significant (P < 0.05) association with the piroplasmid infections. The information obtained in this study can be employed for effective surveillance and control of babesiosis and theileriosis in Pakistan. In addition to confirming our previous molecular detection of T. annulata in cattle, this study provides the first molecular surveillance and phylogenetic position of B. bigemina and associated risk factors in the study region., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Equine Piroplasmosis (EP) and Equine Granulocytic Anaplasmosis (EGA) are diseases that affect horses, transmitted by ixodid ticks, causing a nonspecific febrile syndrome. Equine Piroplasmosis is endemic in Brazil, and most horses are in enzootic stability. Serological and molecular studies carried out on horses in Brazil have shown the presence of Anaplasma phagocytophilum, however, the clinical relevance of this infection has not yet been established. The present study aims to evaluate the importance of Babesia caballi, Theileria equi, and A. phagocytophilum as etiological agents in horses with clinical manifestations suggestive of these diseases in the metropolitan mesoregion of Rio de Janeiro. A total of 45 animals with clinical signs were submitted to DNA extraction followed by qPCR test. Anaplasma phagocytophilum, Neorickettsia risticii and Theileria haneyi were not found in any of the horses with clinical signs, however 62.2% were infected with at least one agent of EP. Theileria equi was the most frequent etiologic agent (35.5%), followed by coinfection (15.5%) and B. caballi (11.2%). These results suggest that A. phagocytophilum has minor clinical importance in the region, while EP is frequently found in symptomatic horses, representing an important differential diagnosis in suspected cases., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Background: Tick-borne pathogens are understudied among domestic animals in sub-Saharan Africa but represent significant threats to the health of domestic animals and humans. Specifically, additional data are needed on tick-borne pathogens in Chad, Africa. Surveillance was conducted among domestic dogs in Chad for selected tick-borne pathogens to measure (1) the prevalence of antibodies against Anaplasma spp., Borrelia burgdorferi, and Ehrlichia spp.; (2) the prevalence of infections caused by Hepatozoon spp., Ehrlichia canis, Anaplasma platys, and Babesia spp.; and (3) associations of pathogens with demographic, spatial, and temporal factors. Blood samples were collected from domestic dogs at three time points (May 2019, November 2019, June 2020) across 23 villages in southern Chad., Results: Of the 428 dogs tested with the IDEXX SNAP 4Dx test in May 2019, 86% (n = 370, 95% CI = 83-90%) were positive for antibodies to Ehrlichia spp., 21% (n = 88, 95% CI = 17-25%) were positive for antibodies to Anaplasma spp., and 0.7% (n = 3, 95% CI = 0.1-2%) were positive for antibodies to Borrelia burgdorferi. Four different pathogens were detected via PCR. Hepatozoon spp. were most commonly detected (67.2-93.4%, depending on the time point of sampling), followed by E. canis (7.0-27.8%), A. platys (10.1-22.0%), and Babesia vogeli (0.4-1.9%). Dogs were coinfected with up to three pathogens at a single time point, and coinfections were most common in May 2019 compared to November 2019 and May 2020., Conclusions: Overall, this study provides new data about the epidemiology of tick-borne pathogens in domestic dogs in Chad, with potential implications for dog and human health., (© 2024. The Author(s).)

Background: Ticks carry a variety of microorganisms, some of which are pathogenic to humans. The human risk of tick-borne diseases depends on, among others, the prevalence of pathogens in ticks biting humans. To follow-up on this prevalence over time, a Belgian study from 2017 was repeated in 2021., Methods: During the tick season 2021, citizens were invited to have ticks removed from their skin, send them and fill in a short questionnaire on an existing citizen science platform for the notification of tick bites (TekenNet). Ticks were morphologically identified to species and life stage level and screened using multiplex qPCR targeting, among others, Borrelia burgdorferi (sensu lato), Anaplasma phagocytophilum, Borrelia miyamotoi, Neoehrlichia mikurensis, Babesia spp., Rickettsia helvetica and tick-borne encephalitis virus (TBEV). The same methodology as in 2017 was used., Results: In 2021, the same tick species as in 2017 were identified in similar proportions; of 1094 ticks, 98.7% were Ixodes ricinus, 0.8% Ixodes hexagonus and 0.5% Dermacentor reticulatus. A total of 928 nymphs and adults could be screened for the presence of pathogens. Borrelia burgdorferi (s.l.) was detected in 9.9% (95% CI 8.2-12.0%), which is significantly lower than the prevalence of 13.9% (95% CI 12.2-15.7%) in 2017 (P = 0.004). The prevalences of A. phagocytophilum (4.7%; 95% CI 3.5-6.3%) and R. helvetica (13.3%; 95% CI 11.2-15.6%) in 2021 were significantly higher compared to 2017 (1.8%; 95% CI 1.3-2.7% and 6.8%; 95% CI 5.6-8.2% respectively) (P < 0.001 for both). For the other pathogens tested, no statistical differences compared to 2017 were found, with prevalences ranging between 1.5 and 2.9% in 2021. Rickettsia raoultii was again found in D. reticulatus ticks (n = 3/5 in 2021). Similar to 2017, no TBEV was detected in the ticks. Co-infections were found in 5.1% of ticks. When combining co-infection occurrence in 2017 and 2021, a positive correlation was observed between B. burgdorferi (s.l.) and N. mikurensis and B. burgdorferi (s.l.) and B. miyamotoi (P < 0.001 for both)., Conclusions: Although the 2021 prevalences fell within expectations, differences were found compared to 2017. Further research to understand the explanations behind these differences is needed., (© 2024. The Author(s).)

Despite the high global impacts of canine vector-borne diseases (CVBD) due to their wide distribution and zoonotic potential, the current epidemiological situation of CVBD in many tropical and subtropical regions remains unknown. This study examines the seroprevalence and molecular prevalence of Ehrlichia canis and other pathogens causing CVBDs (Leishmania infantum, Dirofilaria immitis, Babesia spp., Anaplasma spp. and Hepatozoon canis) in dogs living on the island of Boa Vista (Cape Verde Republic). Blood samples and infesting ticks were taken from 150 dogs across the island (stray, shelter, and pet dogs). Serum samples were tested using a rapid immunochromatographic test (Uranotest ® Quattro) that detects antibodies against E. canis, L. infantum, Anaplasma spp. and D. immitis antigen. Levels of serum antibodies against E. canis were measured using the immunofluorescence antibody test (IFAT). In addition, tick-borne pathogens in blood samples (Anaplasma spp., Babesia spp., Hepatozoon spp., and Ehrlichia canis) were detected by microscopy observation and/or PCR plus sequencing. The seroprevalence of E. canis was extremely high at 82% (123/150), as revealed by both immunochromatography and IFAT. Most dogs returning a seropositive test result (82.92%; 102/123) had antibody titres > 1:1280 but showed no clinical signs or notable laboratory abnormalities. Of the 123 animals testing seropositive for E. canis, 67 (54.47%) also presented antibodies against Anaplasma spp., and 13 (10.56%) showed the presence of Hepatozoon spp. gamonts in the blood smear. Ehrlichia canis infection was detected in 17.1% (25/146) of dogs tested by direct sequencing of polymerase chain reaction (PCR) products. Co-infections were detected in seven of these dogs: four dogs tested PCR-positive for both E. canis and A. platys, two dogs tested positive for E. canis and Hepatozoon spp., and one dog tested positive for E. canis, A. platys and Hepatozoon spp. Rhipicephalus sanguineus sensu lato was the only tick species found infesting the canine study population. The high prevalence of tick-borne pathogens detected in dogs from Boa Vista Island highlights a need for improved control measures designed to prevent the transmission of these pathogens., (© 2024. The Author(s).)

Outbreaks of suspected tick-borne disease (redwater fever) have been reported in captive deer of the Scottish Highlands. In this pilot study, polymerase chain reaction and amplicon sequencing were used to detect tick-borne pathogens in opportunistically collected blood and spleen samples from 63 (healthy, n = 44; diseased, n = 19) cervids, and 45 questing and feeding ticks ( Ixodes ricinus ) from the outbreak sites in 2021-2022. Potentially pathogenic Babesia species were detected in deer but not identified in ticks, Anaplasma phagocytophilum was detected in both deer and ticks, and Borrelia afzelii was detected in ticks but not in deer. Sequencing confirmed Babesia capreoli and Babesia cf. odocoilei parasitemia in clinically healthy red deer ( Cervus elaphus ), B. capreoli parasitemia in clinically healthy domestic reindeer ( Rangifer tarandus tarandus ), and two cases of B. cf. odocoilei -associated hemolytic anemia in white-lipped deer ( Cervus albirostris ), of which one was fatal despite imidocarb treatment. White-lipped deer appear to be highly susceptible to babesiosis caused by B. cf. odocoilei . This investigation highlights the importance of disease surveillance, including molecular diagnostics, for the detection of emerging tick-borne pathogens in managed populations of cervids.

Background: Canine babesiosis and ehrlichiosis are tick-borne infections of great significance in South Africa. Theileriosis in dogs in South Africa is still poorly understood. Co-infection with multiple tick-borne diseases has been documented and is perceived as a common occurrence in South Africa., Objectives: The main objective of this study was to determine the prevalence of co-infections with Ehrlichia canis or Theileria equi in dogs with babesiosis in the Eastern Cape province. There is a lack of data on canine tick-borne disease distribution in this region. Possible associations of population characteristics and haematological and biochemistry measures with a co-infection of E. canis or T. equi in these dogs were also investigated., Method: The study population included 150 dogs naturally infected with babesiosis that presented to the Mdantsane State Veterinary Clinic between January 2021 and November 2021. Quantitative polymerase chain reaction was used to confirm the Babesia spp. that the dogs were infected with and to identify co-infections. Association with co-infection for the following parameters were evaluated: sex, breed, age, duration of illness, leukocyte count, band neutrophil count, monocyte count, platelet count, ARC, and serum globulin concentration. Positive and negative predictive values of monocytosis, leukopenia, band neutrophilia, thrombocytopenia, and non-regenerative absolute reticulocyte count for co-infection were also calculated., Results: Babesia rossi was identified in 149/150 samples and B. vogeli in only 1/150 samples. A co-infection prevalence of 2.0% (3/149; 95% CI: 0.4-5.7) with B. rossi and E. canis was found. No other co-infections were reported. No investigated variables showed significant associations with co-infections. Monocytosis, in particular, was not associated with co-infection., Conclusion: Co-infection with other tick-borne diseases in dogs with babesiosis is uncommon in the Eastern Cape province. These findings raise the possibility that B. rossi may have a protective effect against other tick-borne diseases., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper: Yolandi Rautenbach reports financial support was provided by Health and Welfare Sector Education and Training Authority (HWSETA). If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Purpose: Tick-transmitted parasites as Babesia gibsoni, Babesia vogeli, Ehrlichia canis, and Hepatozoon canis are major health concern for dogs. Owing to prevalence and infection severity, there is need of sensitive, specific, and affordable test for their simultaneous detection., Methods: Prevalence of B. gibsoni, B. vogeli, E. canis, and H. canis infections was assessed on 719 blood samples by microscopy and multiplex PCR assay targeting 18S rRNA (B. gibsoni & H. canis), ITS1 & 5.8S rRNA (B. vogeli) and VirB9 gene (E. canis). An internal control (canine-actin) was also included to increase the accuracy of assay and effect of associated risk factors with disease prevalence was also studied., Results: Microscopic prevalence of B. gibsoni, B. vogeli, E. canis and H. canis was 5.0%, 0.1%, 1.4% and 1.0%, respectively, whereas with multiplex PCR assay, the corresponding values were 8.9%, 1.1%, 2.6% and 5.1% besides concurrent infections of B. gibsoni & H. canis (0.4%), B. gibsoni & E. canis (0.4%), E. canis & H. canis (0.3%) and B. gibsoni & B. vogeli (0.1%). Analytical sensitivity of developed assay was 0.1pg (B. gibsoni & H. canis), 0.01pg (B. vogeli), and 1.0pg (E. canis). A ″fair″ (B. vogeli & H. canis) to ″substantial″ (B. gibsoni & E. canis) agreement between two tests was observed with data as statistically significant. Breed, sex and location were significantly associated with B. gibsoni infection., Conclusion: The developed multiplex PCR assay offers a potential solution to detect these pathogens simultaneously, aiding in timely diagnosis and effective disease management in suspected dogs., (© 2024. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Severe babesiosis with 9.8% parasitemia was diagnosed in a patient in the Netherlands who had previously undergone splenectomy. We confirmed Babesia venatorum using PCR and sequencing. B. venatorum was also the most prevalent species in Ixodes ricinus ticks collected around the patient's home. Our findings warrant awareness for severe babesiosis in similar patients.

We report a case of autochthonous human babesiosis in Hungary, confirmed by PCR and partial sequencing of the Babesia spp. 18S rRNA gene. Babesiosis should be considered during the differential diagnosis of febrile illnesses, and peripheral blood smears to detect Babesia spp. should be part of the routine clinical workup.

The transplacental transmission of parasites and hemoparasites is crucial for understanding the epidemiology of diseases. This study aimed to assess the prevalence of hemopathogens in bovine fetuses at various gestational periods. Samples were obtained from a slaughterhouse in the state of Minas Gerais, Brazil, and a total of 236 fetuses were collected. DNA extracted from blood samples (145) and organ samples (a pool of brain and spleen) (236) underwent a nested PCR (nPCR) assay to detect Babesia spp., Theileria spp., Trypanosoma vivax, Anaplasma marginale, Anaplasma bovis, Anaplasma phagocytophilum, Ehrlichia minasensis, and hemotropic Mycoplasma spp. Additionally, serological analysis of 145 plasma samples was conducted using the indirect fluorescent antibody test-IFAT to detect IgG against Babesia bovis, Babesia bigemina, A. marginale, and Trypanosoma vivax. The observed prevalence of transplacental transmission was 19.3 %, 6.2 %, 42.7 % and 2.7 %, for A. marginale, B. bigemina, 'Candidatus M. haemobos', and Mycoplasma wenyonii, respectively. The prevalence of A. marginale by gestational trimester was 16 % (13/81) in the second trimester and 23 % (14/60) in the third trimester, with no positive samples in the first trimester. Regarding the species B. bovis and B. bigemina, all evaluated animals tested negative by nPCR, and no serological evidence for B. bovis was found by the IFAT. Babesia bigemina demonstrated an overall seroprevalence of 6.2 % (9/145), with 4.8 % (7/145) in the last trimester and 1.3 % (2/145) in the second trimester of pregnancy. In total, 42.7 % (62/145) of blood samples were positive for 'Candidatus M. haemobos', with 42 % (34/81) in the middle trimester, and 43 % (26/60) in the final trimester of pregnancy. Mycoplasma wenyonni was detected in 2.7 % (4/145) blood samples, all in coinfection with 'C. M. haemobos'. The prevalence by pregnancy trimester was 25 % (1/4) in the first trimester; 1.2 % (1/81) in the second trimester and 3.3 % (2/60) in the third trimester of pregnancy. Hemopathogen DNA was detected in fetus blood samples but not the brain or spleen samples. All the samples were negative for T. vivax, Theileria spp., Anaplasma spp. and Ehrlichia spp. Overall, in this study, approximately 70 % of fetuses were positive for one or more of the studied parasites. No significant associations were observed between pairs of pathogens, except 'C. M. haemobos' and A. marginale., (Copyright © 2024. Published by Elsevier GmbH.)

Colpodella species are close relatives of Apicomplexan protozoa. Although most species of this genus are free-living organisms that feed on other protists and algae, reports indicate their occurence in ticks and human patients, including an individual with a history of tick bite manifesting neurological symptoms. During an investigation of tick-borne pathogens (TBPs) in blood samples of cattle, goats, and in ticks collected on them, Colpodella sp. DNA was detected in a Rhipicephalus bursa tick collected from cattle, while of Theileria sergenti/buffeli/orientalis, Babesia bigemina, Sarcocystis cruzi, Babesia spp., and Rickettsia spp. were molecularly detected in cattle, goats, and ticks in southern Italy. Data herein reported highlight the unprecedented presence of Colpodella sp. in ticks in Italy, raising concern due to the potential pathogenic role of this less known protozoan. This finding advocates for performing routine epidemiological surveys to monitor potential emerging vector-borne pathogens., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The present investigation aimed to quantitatively assess the level of parasitemia in dogs using qPCR.The dogs selected for this study were infected with the haemoprotozoan parasite Babesia gibsoni. In the study, dogs diagnosed with babesiosis were divided into two groups (n = 12) and subjected to distinct treatment strategies. The first group received clindamycin-metronidazole-doxycycline (CMD) therapy, while the second group was treated with a combination of buparvaquone-azithromycin (BPV-AZM). The level of parasitemia in the infected dogs was determined using an absolute quantification-based qPCR method. This assessment was conducted both prior to initiating the treatment and on the 10th day following the commencement of the treatment protocols. On the tenth day after the initiation of treatment, the CMD group exhibited a lower level of parasitemia in comparison to the BPV-AZM group. In the CMD treated groups, the mean parasitemia decreased from 4.9E + 06 to 3.4E + 06, indicating a reduction in parasitic load. Conversely, in the BPV-AZM treatment groups, the mean parasitemia increased from 1.62E + 06 to 2.87E + 06, suggesting an increase in parasitic load. On the 10th day, the CMD-treated group demonstrated a statistically significant decline in the level of parasitemia, with a P-value of ≤0.001. This indicates a strong and significant reduction in parasitic load following the CMD treatment. Therefore, the absolute quantification-based qPCR method could effectively assess the initial treatment response by measuring the level of parasitemia., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

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., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

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 (CMhm) 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., (© 2024. The Author(s).)

Haemoparasitic diseases constitute a significant constraint to economic livestock farming. Diagnostic techniques that are inexpensive, rapid, reliable, and precise are crucial for the management of diseases. In this context, PCR assays are very valuable yet expensive since the samples must be processed before being included in the PCR reaction. Accordingly, the goal of the current study was to lower the PCR costs without jeopardizing the assay's sensitivity and specificity. For that purpose, the alkaline solution was optimized for low cost and quick DNA extraction (blood lysate), and PCR reagents were modified for optimum reaction. In comparison to purified whole blood genomic DNA, the currently developed and optimized blood lysate method was found to be 95.5% less expensive, as well as being equally sensitive and specific for the molecular detection (PCR) of haemoparasites like Babesia, Theileria , Trypanosoma and rickettsiales in cattle, buffaloes, horses, and dogs. The assay was also demonstrated to be quick, less likely to cross-contaminate, and appropriate for use in laboratories with limited resources. Therefore, the currently developed and optimized blood lysate method could serve as a viable alternative to purified whole blood genomic DNA for molecular detection (PCR) of haemoparasites in animals particularly in resource-limited settings.

Emerging tick-borne illnesses, such as anaplasmosis, babesiosis, or ehrlichiosis, are caused by obligate intracellular pathogens that have clinically comparable presentations. Diagnostics used in laboratories today are serologic assays and blood smear analyses, which have known diagnostic limits. This study evaluated the performance of a sample-to-answer direct real-time PCR laboratory-developed test for the multiplex qualitative detection of Anaplasma , Babesia , and Ehrlichia DNA in whole-blood specimens. Compared to two standard-of-care (SOC) methods, the DiaSorin tick-borne laboratory-developed test for Anaplasma detection demonstrated a positive percent agreement (PPA) and negative percent agreement (NPA) of 100% (95% CI, 0.80 to 1.0) and 89% (95% CI, 0.74 to 0.97), respectively with a discordant rate of 9.3% against microscopy. After discordant resolution, the NPA increased to 100%. For Babesia , the test demonstrated a PPA of 100% (95% CI, 0.90 to 1.0) and NPA of 100% (95% CI, 0.90 to 1.0). Compared to a SOC PCR method Anaplasma samples showed a PPA of 100% (95% CI, 0.66 to 1.0) and NPA of 100% (95% CI, 0.90 to 1.0). Ehrlichia results showed a PPA of 100% (95% CI, 0.69 to 1.0) and NPA of 100% (95% CI, 0.90 to 1.0). The total percent agreement was 98% (95% CI, 0.95 to 0.99) with a κ statistic of 0.95 (95% CI, 0.90 to 0.99) or almost perfect agreement compared to SOC methods. This laboratory-developed test for detecting Anaplasma , Babesia , and Ehrlichia DNA provides rapid and reliable detection of tick-borne infections without nucleic acid extraction., Importance: This work demonstrates that detection of tick-borne illnesses, such as anaplasmosis, babesiosis, or ehrlichiosis, can be performed directly from whole blood with no extraction. The assay described here has a high positive and negative percent agreement with existing methods and is used as the standard of care. An increasing incidence of tick-borne illness combined with shortage of well-trained technologists to perform traditional manual testing, testing options that can be adapted to various lab settings, are of the utmost importance., Competing Interests: Materials for this study were supplied by DiaSorin Molecular.

Background: Tick is one of the most important ectoparasites distributed worldwide and plays an obvious role in the transmission of different infections to humans and animals as dogs., Aim: This study conducted to molecular demonstration of Babesia gibsoni in ticks of stray dogs and phylogenetic analysis of study isolates to detect their identity to global isolates. Prevalence of ticks in dogs, identification of tick species, and their relationship to some risk factors were aimed, also., Methods: A total of 97 stray dogs were inspected grossly to detect and collect ticks that existed in different body parts. After collection, all ticks were examined morphologically to identify their species, and then molecularly by the polymerase chain reaction (PCR) assay to detect B. gibsoni in different species of ticks. Local B. gibsoni isolates were sequenced, documented in the National Center For biotechnology information (NCBI) database, analyzed phylogenetically, and compared with the global GenBank-NCBI isolates., Results: In the current study, ticks were detected in 43.3% of dogs, and were shown to be varied in number and distribution among different body parts of each dog. Concerning its distribution, ticks were observed significantly on the abdomen, ear, and perineal region. In relation to risk factors, ticks were increased significantly in dogs <6 months old in comparison to older dogs, males more than females; and in rural areas more than dogs of sub-urban and urban areas. Based on morphology, different tick species were seen including Hylaomma anatolicum (86.12%), R. sanguineus (11.99%), and Rhipicephalus turanicus (1.89%). Targeting the 18S rRNA gene, PCR assay reported 3.79% positive ticks to B. gibsoni that were seen in R. sanguineus (13.16%) and H. anatolicum (2.56%). Based on phylogenetic analysis data of five local B. gibsoni isolates, this study demonstrated their close relations to the global NCBI-BLAST B. gibsoni Iraqi isolate (ID: MN385424.1)., Conclusion: This represents the first Iraqi study that demonstrated molecularly B. gibsoni in different species of ticks that infected stray dogs., Competing Interests: The authors have no conflict of interest to disclose.

Blood samples from fifteen captive Indian wolves (Canis lupus pallipes) maintained at Arignar Anna Zoological Park, Vandalur, Chennai were screened for the presence of Babesia spp., Ehrlichia canis and Trypnosoma evansi DNA by PCR. Out of 15 wolf samples, 3 samples were found positive for Babesia spp. The amplified 18S rRNA gene fragments from 3 wolves were sequenced and confirmed as Babesia gibsoni. A maximum likelihood tree was constructed using the three sequences along with other Babesia spp. sequences derived from GenBank adopting HKY nucleotide substitution model based on the Bayesian Information Criterion. The phylogenetic analysis confirmed that the three sequences were of Babesia gibsoni and highly divergent from Babesia canis, B. vogeli and B. vulpes. This might be a possible spill over event of B. gibsoni from community dogs through blood feeding dog ticks. This is the first report and molecular confirmation of B. gibsoni infection in captive Indian wolves., Competing Interests: Declaration of competing interest The authors declare that there are no conflicts of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Piroplasmosis, a tick-borne disease affecting livestock, including camels, is caused by intracellular apicomplexan parasites belonging to the order Piroplasmida. Despite its importance, there's limited research on piroplasmosis among Egyptian camels. This study aimed to fill this gap by investigating tick-borne piroplasmids in camels from Cairo and Giza Governorates. Out of 181 blood samples collected between October 2021 and March 2022 from apparently healthy one-humped camels (Camelus dromedarius), PCR assays revealed a 41.4 % infection rate with various piroplasmids. Detected species included B. bovis (17.7 %), B. bigemina (12.2 %), B. caballi (8.3 %), B. naoakii (11.6 %), B. microti (1.7 %), T. equi (4.4 %), and Theileria spp. (28.7 %). Phylogenetic analysis revealed the first detection of T. equi genotype E in Egypt and identified a novel B. caballi genotype. Additionally, B. microti isolates were identified as the US-type. These findings shed lights on piroplasmosis among Egyptian camels, and provide valuable information for devising effective control strategies, especially B. microti, a pathogen with potential human health risks., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The hard tick clade (Ixodidae) currently comprises 762 species worldwide (266 Prostriata and 496 Metastriata). A quarter of hard ticks are found in the Neotropical region, and 42 species have been documented in Colombia. Ixodidae species are important vectors of pathogens such as bacteria, helminths, protozoa, and viruses. In tick-borne diseases, vertebrate hosts perform an important role in the transmission, maintenance, and spread of pathogens. Colombia ranks sixth among countries with the highest mammal biodiversity, with a total of 548 species, where some of these species may be involved in pathogen transmission cycles with ticks as vectors. This research evaluated the presence of two genera of bacteria (Borrelia and Rickettsia) and the protozoan (Babesia) in ticks and mammals in the Orinoquia region of Colombia, establishing interaction networks. The information comes from 734 mammals (655 wild and 79 domestic), belonging to 59 species. Tick infestation (n = 1,805) was found with 14.85 % (n = 109) of the examined mammals and corresponds to nine tick species confirmed morphologically and molecularly. To detect pathogens 272 ticks were collected while feeding on 96 mammals; samples from 93 mammals were analyzed. The presence of borreliae from the relapsing fever group (RFG) and the Lyme disease group (LDG) were detected. Rickettsia spp. was detected in ticks and mammals, while Babesia bigemina was only detected in ticks. This research is the first to address the prevalence of zoonotic pathogens in domestic and wild mammals infested with hard ticks in the Department of Arauca, Colombia. Considering that reporting cases of infections with Babesia, Borrelia, and Rickettsia in Colombia is not mandatory, their impact on public health cannot be estimated. This highlights the importance of continuously detecting, confirming, and identifying these and other important pathogens within the "One Health" framework, as they have a significant economic and medical-veterinary impact globally., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Questing ticks carry various tick-borne pathogens (TBPs) that are responsible for causing tick-borne diseases (TBDs) in humans and animals around the globe, especially in the tropics and sub-tropics. Information on the distribution of ticks and TBPs in a specific geography is crucial for the formulation of mitigation measures against TBDs. Therefore, this study aimed to survey the TBPs in the questing tick population in Bangladesh. A total of 2748 questing hard ticks were collected from the pastures in Sylhet, Bandarban, Sirajganj, Dhaka, and Mymensingh districts through the flagging method. After morphological identification, the ticks were grouped into 142 pools based on their species, sexes, life stages, and collection sites. The genomic DNA extracted from tick specimens was screened for 14 pathogens, namely Babesia bigemina (AMA-1), Babesia bovis (RAP-1), Babesia naoakii (AMA-1), Babesia ovis (18S rRNA), Theileria luwenshuni (18S rRNA), Theileria annulata (Tams-1), Theileria orientalis (MPSP), Anaplasma marginale (groEL), Anaplasma phagocytophilum (16S rRNA), Anaplasma bovis (16S rRNA), Anaplasma platys (16S rRNA), Ehrlichia spp. (16S rRNA), Rickettsia spp. (gltA), and Borrelia (Bo.) spp. (flagellin B) using genus and species-specific polymerase chain reaction (PCR) assays. The prevalence of the detected pathogens was calculated using the maximum likelihood method (MLE) with 95 % confidence interval (CI). Among 2748 ixodid ticks, 2332 (84.86 %) and 416 (15.14 %) were identified as Haemaphysalis bispinosa and Rhipicephalus microplus, respectively. Haemaphysalis bispinosa was found to carry all the seven detected pathogens, while larvae of R. microplus were found to carry only Bo. theileri. Among the TBPs, the highest detection rate was observed in A. bovis (20/142 pools, 0.81 %, CI: 0.51-1.20), followed by T. orientalis (19/142 pools, 0.72 %, CI: 0.44-1.09), T. luwenshuni (9/142 pools, 0.34 %, CI: 0.16-0.62), B. ovis (4/142 pools, 0.15 %, CI: 0.05 - 0.34) and Bo. theileri (4/142 pools, 0.15 %, CI: 0.05-0.34), Ehrlichia ewingii (3/142 pools, 0.11 %, CI: 0.03-0.29), and Babesia bigemina (1/142, 0.04 %, CI: 0.00 - 0.16). This study reports the existence of T. luwenshuni, E. ewingii, and Bo. theileri in Bangladesh for the first time. The novel findings of this study are the foremost documentation of transovarian transmission of B. bigemina and E. ewingii in H. bispinosa and also provide primary molecular evidence on the presence of E. ewingii and Bo. theileri in H. bispinosa. Therefore, this study may shed light on the circulating TBPs in ticks in the natural environment and thereby advocate awareness among physicians and veterinarians to control and prevent TBDs in Bangladesh., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper, (Copyright © 2024 Elsevier B.V. All rights reserved.)

Introduction: Tick-borne diseases (TBDs) pose a major hindrance to livestock production in countries with limited resources. Effective prevention and management of TBDs require a thorough understanding of disease vectors and pathogens. However, there is limited information on studies of bovine tick-borne pathogens (TBPs) using molecular methods in Malawi. This study aimed to detect TBPs of cattle populations in southern Malawi, which has the largest cattle population in the country., Methodology: A total of 220 blood samples from apparently healthy cattle were collected in six districts, and were screened for selected TBPs using polymerase chain reaction (PCR)., Results: The overall detection rate of TBPs was 72.3%. Among the detected pathogens, Babesia bigemina had the highest detection rate (34.5%), followed by Anaplasma marginale (23.2%), Anaplasma phagocytophilum (22.3%), Theileria taurotragi (22.3%), Theileria parva (15.5%), Anaplasma bovis (9.6%), Babesia bovis (7.3%), Theileria mutans (4.1%), and Babesia naoakii (2.7%). Among the positive samples, 64.2% were found to be co-infected with two or more TBPs, with the highest number of seven pathogens detected in a single sample. The study documents the existence of A. phagocytophilum, B. bovis, and B. naoakii in Malawian cattle for the first time., Conclusion: The findings herein demonstrate a significant burden of TBPs on cattle in Malawi, which gives a challenge in combating TBDs. The high TBP burden, along with the high co-infection frequencies in Malawian cattle necessitates the urgency to implement effective control strategies to enhance cattle production in the country., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Piroplasm including Babesia spp. and Theileria spp. in cattle can cause illness that affects livestock productivity, resulting in significant production losses, especially in tropical and subtropical regions such as Thailand. This study aimed to investigate the prevalence of bovine piroplasms and to identify these blood parasites based on the 18S ribosomal RNA gene in cattle in the northeastern part of Thailand. Piroplasmid infections among beef and dairy cattle were examined using nested PCR. Furthermore, amplicon DNA was sequenced and analyzed, and a phylogenetic tree was constructed to determine the genetic diversity and relationships of the parasite in each area. A total of 141 out of 215 (65.6%) cattle were positive for infection with Babesia or Theileria. DNA analysis revealed that infection by Babesia bigemina, Babesia bovis, Theileria orientalis, Theileria sinensis, and Theileria sp. were common piroplasms in cattle in this region, with a high sequence shared identity and similarity with each other and clustered with isolates from other countries. This study provides information on the molecular epidemiology and genetic identification of Babesia spp. and Theileria spp. in beef and dairy cattle to provide a better understanding of piroplasm infection in cattle in this region, which will help control these blood parasites. Moreover, this is the first report identifying T. sinensis circulating among Thai cattle., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Babesia ovis , transmitted by Rhipicephalus bursa ticks, is the causative agent of ovine babesiosis, a disease characterized by fever, anemia, hemoglobinuria, and high mortality in sheep. This study investigates whether sheep that survived babesiosis without treatment can serve as a source of infection for B. ovis -free host-seeking R. bursa larvae in a later season. Three donor sheep were experimentally infected with B. ovis , and after six months, persistence of B. ovis was assessed through blood and tick transmission experiments. Blood from donor sheep was intravenously injected into three recipient sheep, while donor sheep were also infested with B. ovis -free R. bursa larvae. Engorged nymphs molted to adults, and new recipient sheep were infested with these ticks. All recipient sheep were monitored for B. ovis for 100 days using microscopic, serological, and molecular approaches. The presence of B. ovis was confirmed in the recipient sheep that received blood, leading to clinical infection in two. However, no B. ovis was detected in recipient sheep infested with ticks. These results suggest that sheep recovering from B. ovis infection do not serve as a source of infection for R. bursa larvae in subsequent seasons., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Firat, Ulucesme, Aktas, Ceylan, Sevinc, Bastos, Suarez and Ozubek.)

Tick-borne diseases in animals are increasing rapidly worldwide, but there is insufficient information about tick-borne diseases infecting dogs in southern Egypt. Thus, in the current study, we detected the presence of Anaplasma marginale (A. marginale) and Babesia canis vogeli (B. canis vogeli) in the blood of dogs. The results revealed that 4/100 (4%) were positive, and a higher infection rate was found in males (75%), than females (25%). The phylogenetic analysis for the major surface protein 4 (msp4) gene in this study was compared with amplicons separate from other reported isolates with alignment by identity 100% with cattle and camels from Egypt, and the phylogenetic analysis for the B. canis vogeli small subunit ribosomal RNA (SSU rRNA) gene in this study identified identity by 99.89% with dogs from Egypt. This report is considered the first report in southern Egypt about A. marginale in dogs based on the sequence analysis of the msp4 gene, providing new data for the classification and identification of A. marginale in dogs compared to A. marginale isolated from other animals in southern Egypt., (© 2024. The Author(s).)

Equine piroplasmosis (EP) is a global worldwide infection, which can lead to the death of animals. Despite the causative agents of EP being well studied, there are no data on the distribution and genetic characteristics of EP agents in any region of Russia. In this study, blood samples from 750 horses from Novosibirsk province, Irkutsk province, and Altai region of Russian Siberia were examined for the presence of EP agents. Theileria equi and Babesia caballi were detected in all examined regions, with mean prevalence rates of 60.4% and 7.2%, respectively. The identified pathogens were genetically characterized by the 18S rRNA gene. The determined T. equi sequences were highly conserved and belonged to genotypes A and E, with genotype E being found in 88.6% of genotyped samples. In contrast to T. equi, B. caballi sequences were genetically diverse. Seven sequence variants of B. caballi were identified, and only two of them matched known sequences from the GenBank database. The determined B. caballi sequences belonged to four distinct branches within genotype A. Mixed infections with several variants of B. caballi or with T. equi and B. caballi were common. The conducted phylogenetic analysis based on all available B. caballi sequences of the 18S rRNA gene (> 900 bp) from GenBank and from this study first demonstrated the presence of five monophyletic clusters within genotype A and three clusters within genotype B. Thus, the genetic study of B. caballi from Siberia has significantly expanded the data on the genetic diversity of this pathogen., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Background: Babesia spp. are protozoan parasites that infect the red blood cells of domesticated animals, wildlife and humans. A few cases of giant pandas (a flagship species in terms of wildlife conservation) infected with a putative novel Babesia sp. have been reported. However, comprehensive research on the morphological and molecular taxonomic classification of this novel Babesia sp. is still lacking. This study was designed to close this gap and formally describe this new Babesia sp. infecting giant pandas., Methods: Detailed morphological, molecular and phylogenetic analyses were conducted to characterise this Babesia sp. and to assess its systematic relationships with other Babesia spp. Blood samples from giant pandas infected with Babesia were subjected to microscopic examination. The 18S ribosomal RNA (18S rRNA), cytochrome b (cytb) and mitochondrial genome (mitogenome) of the new Babesia sp. were amplified, sequenced and assembled using DNA purified from blood samples taken from infected giant pandas. Based on the newly generated 18S rRNA, cytb and mitogenome sequences, phylogenetic trees were constructed., Results: Morphologically, the Babesia sp. from giant pandas exhibited various forms, including round to oval ring-shaped morphologies, resembling those found in other small canine Babesia spp. and displaying typical tetrads. Phylogenetic analyses with the 18S rRNA, cytb and mitogenome sequences revealed that the new Babesia sp. forms a monophyletic group, with a close phylogenetic relationship with the Babesia spp. that infect bears (Ursidae), raccoons (Procyonidae) and canids (Canidae). Notably, the mitogenome structure consisted of six ribosomal large subunit-coding genes (LSU1-6) and three protein-coding genes (cytb, cox3 and cox1) arranged linearly., Conclusions: Based on coupled morphological and genetic analyses, we describe a novel species of the genus Babesia, namely, Babesia ailuropodae n. sp., which infects giant pandas., (© 2024. The Author(s).)

Background: In recent years, Babesia and Bartonella species co-infections in patients with chronic, nonspecific illnesses have continued to challenge and change the collective medical understanding of "individual pathogen" vector-borne infectious disease dynamics, pathogenesis and epidemiology. The objective of this case series is to provide additional molecular documentation of Babesia odocoilei infection in humans in the Americas and to emphasize the potential for co-infection with a Bartonella species., Methods: The development of improved and more sensitive molecular diagnostic techniques, as confirmatory methods to assess active infection, has provided increasing clarity to the healthcare community., Results: Using a combination of different molecular diagnostic approaches, infection with Babesia odocoilei was confirmed in seven people suffering chronic non-specific symptoms, of whom six were co-infected with one or more Bartonella species., Conclusions: We conclude that infection with Babesia odocoilei is more frequent than previously documented and can occur in association with co-infection with Bartonella spp., (© 2024. The Author(s).)

Background: Babesiosis is a tick-borne infection caused by piroplasmid protozoa and associated with anemia and severe disease in humans, domestic animals and wildlife. Domestic cats are infected by at least six Babesia spp. that cause clinical disease., Methods: Infection with a piroplasmid species was detected by microscopy of stained blood smears in three sick cats from Israel. Genetic characterization of the piroplasmid was performed by PCR amplification of the 18S rRNA, cytochorme B (CytB) and heat shock protein 70 (HSP70) genes and the internal transcribed spacer (ITS) locus, DNA sequencing and phylogenetic analysis. In addition, Haemaphysalis adleri ticks collected from two cats were analyzed by PCR for piroplasmids., Results: The infected cats presented with anemia and thrombocytopenia (3/3), fever (2/3) and icterus (1/3). Comparison of gene and loci sequences found 99-100% identity between sequences amplified from different cats and ticks. Constructed phylogenetic trees and DNA sequence comparisons demonstrated a previously undescribed Babesia sp. belonging to the Babesia sensu stricto (clade X). The piroplasm forms detected included pear-shaped merozoite and round-to-oval trophozoite stages with average sizes larger than those of Babesia felis, B. leo and B. lengau and smaller than canine Babesia s.s. spp. Four of 11 H. adleri adult ticks analyzed from cat # 3 were PCR positive for Babesia sp. with a DNA sequence identical to that found in the cats. Of these, two ticks were PCR positive in their salivary glands, suggesting that the parasite reached these glands and could possibly be transmitted by H. adleri., Conclusions: This study describes genetic and morphological findings of a new Babesia sp. which we propose to name Babesia galileei sp. nov. after the Galilee region in northern Israel where two of the infected cats originated from. The salivary gland PCR suggests that this Babesia sp. may be transmitted by H. adleri. However, incriminating this tick sp. as the vector of B. galilee sp. nov. would require further studies., (© 2024. The Author(s).)

Babesia spp. and Theileria spp. are tick-borne protozoan parasites with veterinary importance. In China, epidemiological and genetic investigations on many Babesia and Theileria species were still absent in many areas and many tick species. From Aug 2021 to May 2023, 645 ticks were collected from the body surface of domestic animals (camels, goats, sheep, and cattle) using tweezers in seven counties in three provinces including Xinjiang (Qitai, Mulei, Hutubi, and Shihezi counties), Chongqing (Youyang and Yunyang counties), and Qinghai (Huangzhong county). Three tick species were morphologically and molecularly identified (334 Hyalomma asiaticum from Xinjiang, 245 Rhipicephalus microplus from Chongqing, and 66 Haemaphysalis qinghaiensis from Qinghai). A total of three Babesia species and two Theileria species were detected targeting the 18S gene. The COI and cytb sequences were also recovered from Babesia strains for further identification. In R. microplus from Chongqing, Babesia bigemina, the agent of bovine babesiosis, was detected. Notably, in H. asiaticum ticks from Xinjiang, a putative novel genotype of Babesia caballi was identified (0.90%, 3/334), whose COI and cytb genes have as low as 85.82% and 90.64-90.91% nucleotide identities to currently available sequences. It is noteworthy whether the sequence differences of its cytb contribute to the drug resistance of this variant due to the involvement of cytb in the drug resistance of Babesia. In addition, Theileria orientalis and Theileria annulata were detected in R. microplus from Chongqing (12.20%, 31/245) and H. asiaticum from Xinjiang (1.50%, 5/334), respectively. These results suggest that these protozoan parasites may be circulating in domestic animals in these areas. The pathogenicity of the novel genotype of B. caballi also warrants further investigation., (© 2024. The Author(s).)

Ticks are blood-sucking arthropods that can transmit pathogens to their host. As insular ecosystems can enhance tick-host interactions, this study aimed to understand tick diversity, pathogen presence, and their respective associations in the Azores and Madeira archipelagos. Unfed or partially engorged ticks (n = 120) were collected from 58 cats and dogs in the Azores (n = 41 specimens) and Madeira (n = 79 specimens) from November 2018 to March 2019. Vector identification was based on morphology and molecular criteria. For pathogen sequencing, 18S gene fragment for Babesia/Hepatozoon and gltA for Rickettsia were performed. Sequence data was explored using BLAST and BLAST and phylogenetic inference tools. In the Azores, Ixodes hexagonus, I. ventalloi, and Rhipicephalus sanguineus (n = 6; 14.6%, n = 6; 14.6%, and n = 29; 70.7% respectively) were found and in Madeira I. ricinus and R. sanguineus (n = 78, 98.7%; and n = 1, 1.3%; respectively) were identified. Tick COI markers confirmed species highlighting confirmation of R. sanguineus s.s. and genotype A of I. ventalloi. In the Azores Islands, the detected Rickettsia massiliae was linked to R. sanguineus (dogs and cats) and I. hexagonus (dogs), and in Madeira Island, R. monacensis (dogs) and Hepatozoon silvestris (cats) were found associated with I. ricinus. Further, I. ventalloi presence in the Azores expands west its known range, and Hepatozoon silvestris in Madeira may suggest that I. ricinus could have a role as a potential vector. Finally, as R. massiliae and R. monacensis presence underlines public health risks, surveillance by health authorities is crucial as pathogen-tick interactions may drive disease spread, therefore monitoring remains pivotal for disease prevention., (© 2024. The Author(s).)

Ticks play an important role in the transmission of parasitic diseases, especially pathogenic protozoa in canine hosts, and it is very important to determine the role and extent of their infection with these pathogens in order to determine important control strategies. This study assessed the molecular prevalence of three protozoan pathogens including Hepatozoon canis, Leishmania spp. and Babesia spp., in ticks using PCR. A total 300 stray dogs were investigated and 691 ticks (171 male, 377 female and 143 nymph) were detected directly from 45 infested dogs. Species, stage of growth, and gender were determined for each tick. DNA extracted from 224 ticks (26 male, 165 female and 33 nymph). The molecular presence of three protozoan pathogens including Hepatozoon spp. (18S rRNA gene), Leishmania infantum (kinetoplastid minicircle DNA) and Babesia spp. (ssrRNA gene) were investigated using PCR method. One species of ticks, Rhipicephalus sanguineus was identified. Two of the target pathogens, Hepatozoon spp. (7/83; 8.43 %) and Babesia spp. (1/83; 1.2 %), were detected by PCR method. Sequence analysis of the ssrRNA gene of detected Babesia spp. showed a close relationship to the deposited strains of Babesia vulpis in the gene bank. To the best of our knowledge, this is the first study to undertake a phylogenetic analysis of H. canis and Babesia spp. in stray dogs in Alborz province, Iran and the first report about molecular detection of Babesia vulpis from tick infesting dogs in Iran. According to the above results, it seems necessary to implement tick control programs in dogs., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Soft ticks pose significant health risks as vectors of various pathogens. This study explored the spatio-temporal distribution and genetic relationships of the soft tick species Argas persicus infesting domestic hens (Gallus gallus domesticus) across different districts in Pakistan. An examination of 778 hens revealed a notable tick infestation prevalence of 70.82%, with a total of 1299 ticks collected from 551 hens. The overall mean intensity was 2.19 soft ticks per infested chicken, and the overall mean abundance was 1.61 soft ticks per examined hen. Morphological identification confirmed all collected ticks (n = 1210) as A. persicus, comprising 719 males, 333 females, 121 nymphs, and 38 larvae. The Haveli, Muzaffarabad, and Kotli districts had the highest infestation rates, while Bagh had the lowest. Molecular analyses of tick DNA, focusing on 16S rDNA and 12S rDNA sequences, revealed genetic similarities among A. persicus soft ticks from Pakistan and other regions, providing insights into their evolutionary history. Importantly, no Babesia, Rickettsia, or Anaplasma infections were detected in the examined samples. These findings enhance the understanding of soft tick infestation patterns and the genetic diversity of A. persicus in the studied region., Competing Interests: Declaration of competing interest None., (Copyright © 2024 Elsevier B.V. All rights reserved.)