The continental distribution of TheileriaandBabesia species in African wild mammals: A systematic review and meta-analysis of molecular prevalence.

Molecular characterization and functional analysis of thioredoxin peroxidase-2 from Babesia duncani.

ABSTRACT Human babesiosis is an emerging infectious disease caused by a bloodborne unicellular parasite called Babesia. One of the major species of Babesia that infects humans is Babesia microti, which is transmitted through tick bites during a blood meal. As a successful pathogen, Babesia has evolved mechanisms to evade and manipulate the human immune system, ensuring effective development and proliferation. One such mechanism is the secretion of extracellular vesicles. While extracellular vesicles have been shown to regulate host–pathogen interactions in related intracellular parasites, their role in modulating neutrophil responses during babesiosis remains unexplored. Neutrophils are a major white blood cell involved in innate immunity and act as first responders, accounting for approximately 70% of all WBCs in circulation. Neutrophils possess mechanisms to protect the host, such as cytokine secretion and the release of neutrophil extracellular traps consisting of the neutrophil’s DNA, which have bound granules, such as myeloperoxidase, in a process known as NETosis. Here, we investigated the effect of Babesia microti – derived EVs using isolated mouse neutrophils and co-culture assays to assess cytokine secretion and NET formation. We found that Babesia microti EVs robustly trigger NETosis and activate the secretion of key inflammatory cytokines. Collectively, these findings reveal a previously uncharacterized role for Babesia microti EVs in modulating host neutrophil activity, providing mechanistic insight into host–parasite interactions during babesiosis, highlighting the importance of EV-mediated signalling in the pathogenesis of B. microti infection.

An epidemiological survey of zoonotic Babesia species in questing ticks in Hokkaido, Japan, using a newly developed PCR-sequencing approach.

Wildlife fleas and ticks in Wisconsin, USA: unrecognized vectors of bacterial pathogens.

Tick-transmitted diseases in dogs are growing more significant in Türkiye and around the world. In areas with tropical and subtropical climates, Hepatozoon, Babesia, Anaplasma, Ehrlichia, and hemotropic Mycoplasma species are among the pathogens that produce both clinical and subclinical illnesses. In this work, the prevalence of Hepatozoon spp., Babesia spp., Mycoplasma haemocanis, Candidatus Mycoplasma haematoparvum, Anaplasma platys, and Ehrlichia canis species in dogs in the southern part of Türkiye’s Central Anatolia Region was examined using molecular techniques. Polymerase chain reaction (PCR) and DNA sequence analysis techniques were used to analyze the isolated DNAs in terms of the 16S rRNA gene of Anaplasma, Ehrlichia, and hemotropic Mycoplasma species, as well as the 18S rRNA gene of Babesia and Hepatozoon species. Of the 140 extracted DNA samples, 32 (22.85%) Hepatozoon spp., 36 (25.71%) M. haemocanis, and 12 (8.57%) contained Ca. M. haematoparvum. Following 16 samples that tested positive for the Hepatozoan genus, it was found that all of them were Hepatozoon canis. Two of the samples were 100% comparable to the H. canis sequences found in GenBank, while the remaining samples were between 98.98% and 99.84% similar. Sequencing of two representative M. haemocanis and one Ca. M. haematoparvum sample revealed that the Ca. M. haematoparvum isolates were 99.19–99.60% similar to the Ca. M. haematoparvum isolates listed in GenBank, but the M. haemocanis samples were 100% identical to other M. haemocanis isolates. Dogs older than one year had considerably higher hemoplasma positivity, and both hemoplasma and Ca. M. haematoparvum positivity rose significantly with mean age (p

The present study evaluated the diagnostic performance of conventional microscopy and polymerase chain reaction (PCR) based assays for the detection of Babesia infections in dogs, including semi-nested PCR (SN-PCR) targeting the 18S rRNA gene and single-round PCR (SR-PCR) assays targeting the mitochondrial cytochrome b (cytb) and cytochrome c oxidase subunit 1 (cox1) genes for B. gibsoni and B. vogeli, respectively. Mitochondrial sequence variation was further assessed by integrating newly generated sequences from Jabalpur, Madhya Pradesh (central India), with global reference datasets. A total of 100 blood samples from dogs suspected of having haemoprotozoan infections were analysed between June 2022 and May 2023. Microscopic examination of Giemsa-stained smears detected Babesia parasites in 13% of the samples, whereas the 18S rRNA SN-PCR assay identified infections in 29%, comprising B. gibsoni (25%) and B. vogeli (4%). Representative sequences showed 98-99% identity with corresponding GenBank reference sequences. Representative sequences showed 98-99% identity with corresponding GenBank reference sequences. Compared with SN-PCR, microscopy demonstrated moderate sensitivity but perfect specificity, resulting in an overall diagnostic accuracy of 84.0% (p < 0.01). Mitochondrial SR-PCR assays detected B. gibsoni and B. vogeli in 5% and 4% of the samples, respectively. The cytb-based assay showed higher sensitivity and a significant diagnostic association (p < 0.01) than the cox1 assay, whereas the cox1 assay demonstrated lower sensitivity with a non-significant association (p > 0.05). All PCR assays showed 100% specificity and positive predictive value. Bayesian phylogenetic and haplotype analyses indicated that B. gibsoni cytb sequences formed a monophyletic lineage with limited regional structuring, with Indian isolates clustering within a distinct sub-lineage. In contrast, B. vogeli cox1 sequences exhibited low global diversity with a dominant shared haplotype across geographic regions. The 18S rRNA SN-PCR assay showed the highest sensitive method for detecting Babesia infections in dogs. Mitochondrial markers (cytb and cox1) supported species confirmation and comparative phylogenetic assessment, highlighting the complementary value of nuclear and mitochondrial gene targets for molecular surveillance and control of canine babesiosis in India.

The discovery of broad-spectrum antiparasitic agents relies on the ability to evaluate drug efficacy under harmonized in vitro conditions across related species. However, current drug screening pipelines for intraerythrocytic parasites are constrained by the use of species-specific media with distinct nutrient compositions and serum sources, which hinder direct comparison of compound potency. To address this gap, we describe a unified erythrocytic culture system based on DMEM/F12 supplemented with 20% fetal bovine serum (DFS20), which supports robust asexual growth of multiple Plasmodium falciparum strains (3D7, Dd2, HB3, V1/S), Babesia duncani, Babesia divergens (Rouen 87), and Babesia MO1. Parasite proliferation and morphology in DFS20 are comparable to those observed in established species-specific media such as RPMI-1640 for P. falciparum and B. divergens and HL-1/Claycomb/DMEM/F12/SFM for B. duncani, while eliminating reliance on undefined or discontinued proprietary components. Importantly, this standardized medium enables cross-species growth inhibition assays for direct comparison of drug efficacy under identical conditions. Using this platform, we recently screened dihydrotriazines and biguanides targeting the conserved DHFR-TS enzymes and identified potent antifolate candidates with broad-spectrum activity against Babesia and Plasmodium species. For B. duncani, which is uniquely supported by both a continuous in vitro human erythrocyte culture system and a lethal in vivo mouse infection model, integration with the in-culture and in-mouse (ICIM) pipeline enables systematic validation of pharmacodynamics, pharmacokinetics, resistance, and toxicity. This unified DFS20-based system establishes a scalable and reproducible protocol for harmonized drug efficacy evaluation across intraerythrocytic parasites and provides a foundation for the development and prioritization of pan-antiparasitic therapies.Key features• Common culture medium (CCM): DFS20 supports consistent Babesia and Plasmodium growth, enabling standardized, reproducible drug screening across intraerythrocytic protozoan parasites.• Scalable antiparasitic drug screening: SYBR Green I–based in vitro assay adaptable to low-, medium-, or high-throughput compound testing in both drug-sensitive and drug-resistant strains.• Unified red blood cell–based platform that may accelerate efforts to culture challenging or unculturable parasites, thereby expanding opportunities for drug discovery and mechanistic studies.

Tick‐borne pathogens, transmitted by ticks, infect humans and animals worldwide. The brown dog tick, Rhipicephalus sanguineus sensu lato, is a significant vector of a number of pathogens, including Ehrlichia canis, Rickettsia and Anaplasma species. In Ghana, there is limited information on the pathogens carried by Rh. sanguineus s.l. As such, Rh. sanguineus ticks taken from domestic dogs in Kumasi were screened for tick‐borne pathogens, including Coxiella burnetii, Rickettsia, Babesia, Theileria, Anaplasma, Ehrlichia and Hepatozoon species. A total of 204 ticks collected from 56 infested dogs were morphologically identified as Rh. sanguineus s.l. From the 88 pools screened, 36 (40.9%) were positive for pathogen DNA. The pathogens identified were Rickettsia africae (5 pools), Ehrlichia canis (10 pools) and uncultured Anaplasma sp. (21 pools) with maximum likelihood estimates as 2.48% (95% CI: 0.93, 5.38%), 5.22% (95% CI: 2.69, 9.15%) and 11.20% (95% CI: 7.32, 16.29%), respectively. There was no association between the detection of a pathogen and the tick sex or dog breed, age or sex. This study provides important baseline data on the circulation of tick‐borne pathogens in Rh. sanguineus s.l. ticks in Kumasi, with implications for both veterinary and human health. The presence of uncultured Anaplasma sp. suggests a wider diversity of tick‐borne bacteria with unknown pathogenicity. There is a need for integrated tick control, improved diagnosis and additional epidemiological studies to mitigate the impact of tick‐borne diseases in Ghana.

Egress from host cells is fundamental for the spread of infection by apicomplexan parasites, including Babesia species. These tick-borne pathogens represent emerging zoonoses, but treatment options are limited. Here, using microscopy, transcriptomics and chemical genetics, we identified signalling, proteases and gliding motility as key drivers of egress by Babesia divergens. We developed reverse genetic tools in B. divergens to perform a knockdown screen of putative mediators of egress, identifying kinases and proteases involved in distinct steps of egress (aspartyl protease (ASP) 3 and kinases cGMP-dependent protein kinase (PKG) and calcium-dependent protein kinase (CDPK4)) and invasion (ASP2, ASP3 and PKG) of red blood cells. Inhibition of egress stimulates additional rounds of intracellular replication, indicating that exit from the replication cycle is uncoupled from egress. Chemical genetics validated PKG, CDPK4, ASP2 and ASP3 as druggable targets in Babesia spp. and identified promising compounds for babesiosis treatment. Taken together, egress in B. divergens more closely resembles egress in Toxoplasma gondii than in the more evolutionarily related Plasmodium spp.

bjective: to develop a simple and user-friendly technology for the detection of DNA targets for the diagnosis of socially significant diseases based on a colorimetric aggregation assay using gold nanoparticles. Materials and Methods: Gold nanospheres were synthesized by liquid-phase chemistry methods. The physicochemical parameters of the nanoparticles were controlled using absorption spectroscopy, dynamic light scattering, and electron microscopy. Rational design of specific genomic target sequences and oligonucleotides included in the test system was carried out considering thermodynamic constants and using genomic databases. Analytical validation of the test system was performed using a model for the detection of Babesia species DNA — the causative agent of piroplasmosis in humans and animals — by cross-comparison of characteristics with polymerase chain reaction (PCR) with electrophoretic detection in real-time format. Results: A pilot version of the test system for specific detection of up to 10 ng of DNA in blood samples was developed, based on visual discrimination of the color of the reaction mixture with an analysis duration of approximately 30 minutes. The size effect of nanoparticles in the range from 15 to 60 nm on the minimum detection limit of DNA targets was established. Conclusion: This study presents the results of the development and analytical validation of a pilot express test system for DNA diagnostics based on gold nanoparticle labeling and visual color discrimination of the reaction mixture.

In Serbia, Babesia canis is the predominant cause of canine babesiosis, while B. gibsoni and B. vulpes have been reported only sporadically. Limited awareness of these small Babesia species often leads to misdiagnosis and inappropriate treatment. International reports indicate that American Staffordshire Terriers (ASTs) and related breeds are particularly affected. This study examined 101 ASTs over a two-year period to determine the occurrence of small Babesia species and identify dog- and owner-related risk factors. Collected data included age, sex, body condition score (BCS), clinical signs, presence of scars, history of vector-borne diseases (VBD), living conditions, antiparasitic prophylaxis, and tick exposure. Molecular identification using PCR and Sanger sequencing revealed that 37 dogs were positive for Babesia spp.; among them, 36 were infected with B. gibsoni and one with B. vulpes. Significant risk factors associated with infection included the presence of scars, low BCS, history of VBDs, tick exposure, and living in rural areas. These findings demonstrate a notably high occurrence of B. gibsoni and the presence of B. vulpes in ASTs in Serbia. The results highlight the need for improved awareness, accurate molecular diagnostics, and targeted preventive and therapeutic measures. Additionally, observed risk factors suggest chronic infection and support transmission pathways linked to dog bites and tick exposure.

For the first time, unknown protists were found in the midgut of larvae Hirsutiella zachvatkini (Schluger, 1948) (Trombiculidae), feeding on a bank vole Clethrionomys glareolus in the Pskovskaya Oblast (Russia). The same protozoans were also identified in the tissues of the vole. Generally, these cells were spindle-shaped, around 4 µm in length, exhibiting a violet cytoplasm and a blue centrally located elongated nucleus, as highlighted by staining. Most probably, these cells represent one of the Babesia (Piroplasmida) species in a single-cell stage (e. g., sporozoites), which got into the vole accidentally as a result of a possible tick bite. Alternatively, these cells represent one of the Toxoplasma (Eimeriida) species in the individual cell form (e. g., tachyzoites or sporozoites) that may be encountered in other animals. However, a single instance of the presence of protozoans in trombiculid larvae may suggest that (i) the circulation of protozoans among the population of voles or trombiculids in northwestern Russia is highly restricted and is not of an epidemiological importance and (ii) this path of infection is blind and may cause the subsequent death of both the larva and the protists.

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a medical condition characterized by extreme fatigue lasting at least 6 months. Based upon case reports, patients infected with Babesia or Bartonella spp. have reported a history of chronic fatigue and concurrent neurological symptoms. In this study, 50 study participants reporting fatigue lasting from six months to 19 years and one or more neurological symptoms were selected. PCR assays were used to amplify Babesia and Bartonella spp. DNA from blood and enrichment blood cultures. Using targeted qPCR amplification and DNA sequencing, infection with Babesia spp., Bartonella spp. or both genera was confirmed in 10, 11, and 2 individuals, respectively. Of 50 participants, 12 (24%, 95% CI: 12–36%) were infected with a Babesia species, while Bartonella species infection was documented in 13/50 individuals (26%, 95% CI: 13.8–38.2%). This study provides documentation supporting a potential role for Babesia and Bartonella infection in patients with presentations consistent with ME/CFS. Prospective case–control studies, using highly sensitive direct pathogen detection techniques, are needed to determine whether or the extent to which infection with members of these two genera contributes to or causes ME/CFS.

BackgroundBabesia spp. are widespread tick-borne intraerythrocytic protozoa, infecting a broad range of vertebrate hosts. Red foxes are reservoirs of Babesia vulpes, belonging to the Babesia microti-like group (clade I), and play an important role in the epidemiology of canine and wildlife babesiosis. Besides B. vulpes, another species of this genus was molecularly reported in red foxes from Israel and Iraq and provisionally named “Babesia sp. MML-2014”; however, no morphological description of this small Babesia species was provided, preventing a proper species naming.MethodsInfection with piroplasmid species was detected and described by microscopy of stained blood smears in one red fox from Southern Italy. Molecular characterization of the Babesia sp. and differentiation from B. vulpes was performed through PCR amplification of nuclear (18S rRNA, ITS1-5.8S-ITS2) and mitochondrial (cytochrome c oxidase subunit 1, cox1) gene markers, followed by DNA sequencing and phylogenetic analysis. In addition, Ixodes kaiseri ticks collected from the infected fox were screened for piroplasmids by PCR.ResultsSequence comparison of piroplasmids showed 98–99% identity with the undescribed Babesia sp. MML-2014 and phylogenetic analyses confirmed that this taxon belongs to the Western group (clade III) and is differentiated by B. vulpes. Morphological and morphometric analyses further demonstrated that Babesia sp. nov. is a distinct small piroplasm and is characterized by unique Maltese cross forms. Based on the above, we named Babesia banethi sp. nov. as a new taxon. In addition, Babesia sp. nov. DNA was detected in the intestine of one engorged I. kaiseri specimen.ConclusionsThis study provides genetic and morphological findings of B. banethi sp. nov. A morphological description with measurements of the parasite forms in red fox erythrocytes, differential diagnosis supplemented by genetic characterization, and the deposition of the holotype in suitable collections have been made in compliance with the ICZN guidelines.Graphical Supplementary InformationThe online version contains supplementary material available at 10.1186/s13071-025-07179-y.

Ticks are important vectors that transmit viral, bacterial, and protozoan pathogens and cause diseases in vertebrates. The epidemiology of tick-borne pathogens in cats has been less well investigated than in dogs, and the role of cats in this epidemiology is only partially understood. The main tick‑borne protozoa reported in cats include species of Babesia, Hepatozoon, and Cytauxzoon. The aim of this study was to investigate tick-borne blood protozoa in 200 domestic cats in Ankara province of Turkey. Blood and tick samples were collected from each animal. Of 200 cats examined, 6 (3%) were found positive for Hepatozoon spp., and two different haplotypes were identified. Hepatozoon gamonts were observed in 1% of blood smears. In conclusion, the species and haplotypes present in the region were identified and compared with those found worldwide. These data provided molecular characterization of the relevant pathogens and revealed potential risks to domestic cats.

Haemoprotozoan infections, caused by protozoan parasites such as Trypanosoma, Theileria, Babesia, and Hepatozoon species, are of significant veterinary importance due to their economic impact on livestock and companion animals. Accurate and rapid diagnosis is critical for effective disease management and control. Traditional diagnostic methods, including microscopy and polymerase chain reaction (PCR), have limitations such as low sensitivity, time consumption, requirement for skilled personnel, and the need for sophisticated laboratory infrastructure. Loop-Mediated Isothermal Amplification (LAMP) has emerged as a highly sensitive, specific, rapid, and cost-effective molecular diagnostic tool. LAMP amplifies target DNA under isothermal conditions using Bst DNA polymerase and a set of specifically designed primers, enabling visual detection through colorimetric dyes, turbidity, or fluorescence without the need for expensive equipment. This technique has been successfully applied for the detection of Trypanosoma evansi, Theileria annulata, Babesia spp., Babesia gibsoni, and Hepatozoon canis in field samples, demonstrating higher sensitivity and specificity compared to conventional methods. LAMP offers a simple, field-deployable, and reliable diagnostic approach suitable for early detection, epidemiological studies, and disease control programs, particularly in resource-limited settings. Its rapidity, ease of use, and adaptability position LAMP as a promising tool for improving animal health and productivity.

Vector-borne diseases affecting dogs have worldwide distribution and continue to expand at an alarming rate. Several canine vector-transmitted illnesses including filariasis, babesiosis, ehrlichiosis, and hepatozoonosis are commonly found throughout India. This research titled "Comparative evaluation of blood smear examination and polymerase chain reaction for the diagnosis of canine babesiosis" examined 4304 dogs presented to the Veterinary Clinical Complex at the College of Veterinary Science and Animal Husbandry in Mhow (M.P.) along with private veterinary facilities throughout Indore. The research spanned twelve months from March through February. Blood specimen collection focused on dogs displaying clinical signs including elevated body temperature, deteriorated overall health, lethargy, pallid mucous membranes, and loss of appetite for initial assessment. The gathered blood specimens underwent screening to detect babesiosis through thin blood film analysis following Giemsa staining. Among fifty specimens tested using PCR, 7(14%) produced a characteristic 380 bp amplified product confirming Babesia species infection, while only 2(4%) showed positive results for intra-erythrocytic piroplasms during microscopic evaluation. Every specimen that tested positive through microscopic analysis also demonstrated positive results via PCR testing.

Canine Babesiosis causes a fatal disease marked by haemolytic anaemia. Of the identified dog blood groups, some have been found to affect the host's susceptibility/resistance to certain infections. However, limited information exists on the influence of canine blood types on tolerance of dogs to babesiosis. This study assessed the prevalence of different Dog Erythrocyte Antigen (DEA) 1 blood group in Abeokuta, Nigeria, and evaluated potential relationship between DEA 1 blood types and susceptibility to babesiosis. 200 blood samples (1 mL each) were collected from client-owned dogs. Alvedia® test kit was used to identify DEA 1.1, DEA 1.2 and DEA 1.0 genotypes. Deoxyribose nucleic acid (DNA) was extracted from each sample, and Babesia species detected using Polymerase Chain Reaction (PCR). Data were analyzed for relationship between blood types and Babesia infection using Chi-square. DEA 1 positive dogs accounted for 63.5%, a significantly higher proportion than DEA 1 negative dogs (36.5%) in the study population. Boerboel, Rottweiler, Caucasian, and local breeds showed significantly higher frequency (73.3% to 86.4%) of DEA 1 positive blood types compared to DEA 1 negative types (13.6% to 26.7%). In contrast, German shepherd breed showed no significant difference in frequency of DEA positive (51.2%) and DEA negative (48.8%) dogs. Additionally, no significant sex-based differences were observed in DEA 1 blood type distribution. The prevalence of Babesia species in DEA 1 positive dogs (63.0%) was not significantly different (p > 0.05) from that in DEA 1 negative dogs (60.3%). These findings suggested that the DEA 1 blood group may not influence the tolerance or susceptibility of dogs to Babesia infection.

Avian louse flies (Hippoboscidae: Ornithomyinae) are blood-feeding parasites with largely unexplored vector potential, although they have been implicated in the transmission of pathogens of medical and veterinary relevance, such as West-Nile virus, Babesia and Trypanosoma species. We screened 253 specimens of nine Ornithomyinae species for selected bacteria (Anaplasma, Rickettsia, Bartonella, Borrelia, and Ehrlichia spp.) as well as for trypanosomes and piroplasms. Anaplasma phagocytophilum was detected in a single Ornithomya avicularia specimen, and Haematospirillum jordaniae was identified in three Ornithomya fringillina individuals from the same location, representing the first report of these bacteria in avian louse flies. In addition, eight different Trypanosoma sequences were obtained, belonging to the T. corvi/culicavium and T. bennetti groups. Unexpectedly, a sequence resembling T. theileri was recovered from an Ornithoica turdi specimen. Our findings suggest that the role of louse flies in transmitting the investigated bacterial pathogens is likely minimal, but their involvement in the ecology of Trypanosoma species warrants further study. Consistent with previous work, we propose that louse flies could serve as valuable sentinels for monitoring pathogens in wild bird populations.