Comparative chemical genomics in Babesia species identifies the alkaline phosphatase PhoD as a determinant of antiparasitic resistance.

Babesiosis is an emerging zoonosis and widely distributed veterinary infection caused by 100+ species of Babesia parasites. The diversity of Babesia parasites and the lack of specific drugs necessitate the discovery of broadly effective antibabesials. Here, we describe a comparative chemogenomics (CCG) pipeline for the identification of conserved targets. CCG relies on parallel in vitro evolution of resistance in independent populations of Babesia spp. (B. bovis and B. divergens). We identified a potent antibabesial, MMV019266, from the Malaria Box, and selected for resistance in two species of Babesia. After sequencing of multiple independently derived lines in the two species, we identified mutations in a membrane-bound metallodependent phosphatase (phoD). In both species, the mutations were found in the phoD-like phosphatase domain. Using reverse genetics, we validated that mutations in bdphoD confer resistance to MMV019266 in B. divergens. We have also demonstrated that BdPhoD localizes to the endomembrane system and partially with the apicoplast. Finally, conditional knockdown and constitutive overexpression of BdPhoD alter the sensitivity to MMV019266 in the parasite. Overexpression of BdPhoD results in increased sensitivity to the compound, while knockdown increases resistance, suggesting BdPhoD is a pro-susceptibility factor. Together, we have generated a robust pipeline for identification of resistance loci and identified BdPhoD as a resistance mechanism in Babesia species.

A Fluorescence-Based Assay for Measuring Polyamine Biosynthesis Aminopropyl Transferase-Mediated Catalysis.

Polyamines are polycationic molecules that are crucial in a wide array of cellular functions. Their biosynthesis is mediated by aminopropyl transferases (APTs), which are promising targets for antimicrobial, antineoplastic and antineurodegenerative therapies. A major limitation in studying APT enzymes, however, is the lack of high-throughput assays to measure their activity. We have developed the first fluorescence-based assay, DAB-APT, for the measurement of APT activity using 1,2-diacetyl benzene (DAB), which forms fluorescent conjugates with putrescine, spermidine, and spermine, with fluorescence intensity increasing with the carbon chain length. The assay has been validated using APT enzymes from Saccharomyces cerevisiae and Plasmodium falciparum, and the data further validated by mass spectrometry and thin-layer chromatography. Using mass spectrometry analysis, the structures of the fluorescent putrescine, spermidine and spermine 1,2-DAB adducts were determined to be substituted 1,3-dimethyl isoindoles. The DAB-APT assay is optimized for high-throughput screening, facilitating the evaluation of large chemical libraries. Given the critical roles of APTs in infectious diseases, oncology, and neurobiology, the DAB-APT assay offers a powerful tool with broad applicability, poised to drive advancements in research and drug discovery.

Tafenoquine-Atovaquone Combination Achieves Radical Cure and Confers Sterile Immunity in Experimental Models of Human Babesiosis.

Human babesiosis is a potentially fatal tick-borne disease caused by intraerythrocytic Babesia parasites. The emergence of resistance to recommended therapies highlights the need for new and more effective treatments. Here we demonstrate that the 8-aminoquinoline antimalarial drug tafenoquine inhibits the growth of different Babesia species in vitro, is highly effective against Babesia microti and Babesia duncani in mice and protects animals from lethal infection caused by atovaquone-sensitive and -resistant B. duncani strains. We further show that a combination of tafenoquine and atovaquone achieves cure with no recrudescence in both models of human babesiosis. Interestingly, elimination of B. duncani infection in animals following drug treatment also confers immunity to subsequent challenge. Altogether, the data demonstrate superior efficacy of tafenoquine plus atovaquone combination over current therapies for the treatment of human babesiosis and highlight its potential in providing protective immunity against Babesia following parasite clearance.

Babesia duncani, a Model Organism for Investigating Intraerythrocytic Parasitism and Novel Antiparasitic Therapeutic Strategies.

Pathogens such as Plasmodium, Babesia, and Theileria invade and multiply within host red blood cells, leading to the pathological consequences of malaria, babesiosis, and theileriosis. Establishing continuous in vitro culture systems and suitable animal models is crucial for studying these pathogens. This review spotlights the Babesia duncani in culture-in mouse (ICIM) model as a promising resource for advancing research on the biology, pathogenicity, and virulence of intraerythrocytic parasites. The model offers practical benefits, encompassing well-defined culture conditions, ease of manipulation, and a well-annotated genome. Moreover, B. duncani serves as a surrogate system for drug discovery, facilitating the evaluation of new antiparasitic drugs in vitro and in animals, elucidating their modes of action, and uncovering potential resistance mechanisms. The B. duncani ICIM model thus emerges as a multifaceted tool with profound implications, promising advancements in our understanding of parasitic biology and shaping the development of future therapies.

Babesia BdFE1 esterase is required for the anti-parasitic activity of the ACE inhibitor fosinopril.

Effective and safe therapies for the treatment of diseases caused by intraerythrocytic parasites are impeded by the rapid emergence of drug resistance and the lack of novel drug targets. One such disease is human babesiosis, which is a rapidly emerging tick-borne illness caused by Babesia parasites. In this study, we identified fosinopril, a phosphonate-containing, FDA-approved angiotensin converting enzyme (ACE) inhibitor commonly used as a prodrug for hypertension and heart failure, as a potent inhibitor of Babesia duncani parasite development within human erythrocytes. Cell biological and mass spectrometry analyses revealed that the conversion of fosinopril to its active diacid molecule, fosinoprilat, is essential for its antiparasitic activity. We show that this conversion is mediated by a parasite-encoded esterase, BdFE1, which is highly conserved among apicomplexan parasites. Parasites carrying the L238H mutation in the active site of BdFE1 failed to convert the prodrug to its active moiety and became resistant to the drug. Our data set the stage for the development of this class of drugs for the therapy of vector-borne parasitic diseases.

Identification of Babesia microti immunoreactive antigens by phage display cDNA screen.

Human babesiosis is a malaria-like illness caused by protozoan parasites of the genus Babesia. Babesia microti is responsible for most cases of human babesiosis in the United States, particularly in the Northeast and the Upper Midwest. Babesia microti is primarily transmitted to humans through the bite of infected deer ticks but also through the transfusion of blood components, particularly red blood cells. There is a high risk of severe and even fatal disease in immunocompromised patients. To date, serology testing relies on an indirect immunofluorescence assay that uses the whole Babesia microti antigen. Here, we report the construction of phage display cDNA libraries from Babesia microti-infected erythrocytes as well as human reticulocytes obtained from donors with hereditary hemochromatosis. Plasma samples were obtained from patients who were or had been infected with Babesia microti. The non-specific antibody reactivity of these plasma samples was minimized by pre-exposure to the human reticulocyte library. Using this novel experimental strategy, immunoreactive segments were identified in three Babesia microti antigens termed BmSA1 (also called BMN1-9; BmGPI12), BMN1-20 (BMN1-17; Bm32), and BM4.12 (N1-15). Moreover, our findings indicate that the major immunoreactive segment of BmSA1 does not overlap with the segment that mediates BmSA1 binding to mature erythrocytes. When used in combination, the three immunoreactive segments form the basis of a sensitive and comprehensive diagnostic immunoassay for human babesiosis, with implications for vaccine development.

Continuous in vitro propagation of Babesia microti.

The currently accepted initiation of Babesia infection describes a sporozoite stage infused into the host, along with other saliva components, by the tick vector. This sporozoite can enter and initiate erythrocyte infection directly. In the particular case of Babesia microti, however, that sporozoite loses the ability to further propagate in vitro once deprived of its natural host. True B. sensu stricto do not require the host collaboration described in this study. Hence it has become a current topic of research involving B. microti (B. sensu lato), a rather unique species that requires host collaboration to maintain an erythrocyte propagation cycle. The main attachment protein is synthesized by this parasite in excess and exported to the host from the erythrocyte infrastructure to immunize the host at all stages of infection. The synthesis of host immune IgM antibody is necessary for the propagation of B. microti, being central to entry into uninfected host erythrocytes. Sequential use of the host immune system then involves complement factor C3b to complete the three-part assembly necessary to initiate the rhoptry sequence for invasion of uninfected erythrocytes and further propagation. These several components must be furnished within the in vitro culture medium and the sequence of these reactions is discussed. The corollary view of the parasite survival versus the host immune defenses is also discussed as it involves the same host factors promoting continuing parasite growth. This is the first description of continuous in vitro propagation of B. microti.

Tafenoquine for Relapsing Babesiosis: A Case Series.

BACKGROUND: Relapsing babesiosis often occurs in highly immunocompromised patients and has been attributed to the acquisition of resistance against drugs commonly used for treatment such as atovaquone, azithromycin, and clindamycin. Tafenoquine, which is approved for malaria prophylaxis and presumptive antirelapse treatment of Plasmodium vivax malaria, has shown activity against Babesia microti in several animal models of acute infection and in a single human case of relapsing babesiosis. Here, we report 5 cases of relapsing babesiosis treated with tafenoquine, including the previous case, and begin to define the conditions for optimal use of tafenoquine in relapsing babesiosis. METHODS: A definitive diagnosis of babesiosis was made by microscopic examination of Giemsa-stained thin blood smears or a real-time polymerase chain reaction (PCR) that targets the parasite 18S rRNA gene. Clearance of B. microti infection was ascertained by use of blood smear and real-time PCR. RESULTS: Tafenoquine was initiated with a loading dose of 600 mg. A weekly maintenance dose consisted of 200 mg or 300 mg; the lower dose was associated with a delayed clearance of B. microti. In 2 cases, all antimicrobial agents but tafenoquine were discontinued prior to clearance of infection. In 2 other cases, clearance was achieved while tafenoquine was administered along with other antimicrobial agents. In 3 of these 4 cases, tafenoquine was used in combination with atovaquone-proguanil. Other agents included atovaquone, azithromycin, and/or clindamycin. In 1 case, tafenoquine was administered alone and failed to prevent relapse. CONCLUSIONS: Tafenoquine can be a useful adjunct for the treatment of highly immunocompromised patients experiencing relapsing babesiosis caused by B. microti.

Confirmed Case of Autochthonous Human Babesiosis, Hungary.

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.

Autochthonous Human Babesiosis Caused by Babesia venatorum, the Netherlands.

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.

Autochthonous Human Babesia divergens Infection, England.

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.

Host-pathogen associations inferred from bloodmeal analyses of Ixodes scapularis ticks in a low biodiversity setting.

Tick-borne pathogen emergence is dependent on the abundance and distribution of competent hosts in the environment. Ixodes scapularis ticks are generalist feeders, and their pathogen infection prevalence depends on their relative feeding on local competent and non-competent hosts. The ability to determine what host a larval life stage tick fed on can help predict infection prevalence, emergence, and spread of certain tick-borne pathogens and the risks posed to public health. Here, we use a newly developed genomic target-based technique to detect the source of larval bloodmeals by sampling questing nymphs from Block Island, RI, a small island with a depauperate mammalian community. We used previously designed specific assays to target all known hosts on this island and analyzed ticks for four human pathogenic tick-borne pathogens. We determined the highest proportion of larvae fed on avian species (42.34%), white-footed mice (36.94%), and white-tailed deer (20.72%) and occasionally fed on feral cats, rats, and voles, which are in low abundance on Block Island. Additionally, larvae that had fed on white-footed mice were significantly more likely to be infected with Borrelia burgdorferi and Babesia microti, while larvae that had fed on white-footed mice or white-tailed deer were significantly more likely to be infected with, respectively, mouse- and deer-associated genotypes of Anaplasma phagocytophilum. The ability to detect a nymph's larval host allows for a better understanding of tick feeding behavior, host distribution, pathogen prevalence, and zoonotic risks to humans, which can contribute to better tick management strategies. IMPORTANCE: Tick-borne diseases, such as Lyme disease, babesiosis, and anaplasmosis, pose significant public health burdens. Tick bloodmeal analysis provides a noninvasive sampling method to evaluate tick-host associations and combined with a zoonotic pathogen assay, can generate crucial insights into the epidemiology and transmission of tick-borne diseases by identifying potential key maintenance hosts. We investigated the bloodmeals of questing Ixodes scapularis nymphs. We found that avian hosts, white-footed mice, and white-tailed deer fed the majority of larval ticks and differentially contributed to the prevalence of multiple tick-borne pathogens and pathogen genotypes in a low biodiversity island setting. Unraveling the intricate network of host-vector-pathogen interactions will contribute to improving wildlife management and conservation efforts, to developing targeted surveillance, and vector and host control efforts, ultimately reducing the incidence of tick-borne diseases and improving public health.

Disruption of bacterial interactions and community assembly in Babesia-infected Haemaphysalis longicornis following antibiotic treatment.

BACKGROUND: A previous study highlighted the role of antibiotic-induced dysbiosis in the tick microbiota, facilitating the transstadial transmission of Babesia microti from nymph to adult in Haemaphysalis longicornis. This study builds on previous findings by analyzing sequence data from an earlier study to investigate bacterial interactions that could be linked to enhanced transstadial transmission of Babesia in ticks. The study employed antibiotic-treated (AT) and control-treated (CT) Haemaphysalis longicornis ticks to investigate shifts in microbial community assembly. Network analysis techniques were utilized to assess bacterial interactions, comparing network centrality measures between AT and CT groups, alongside studying network robustness and connectivity loss. Additionally, functional profiling was conducted to evaluate metabolic diversity in response to antibiotic treatment. RESULTS: The analysis revealed notable changes in microbial community assembly in response to antibiotic treatment. Antibiotic-treated (AT) ticks displayed a greater number of connected nodes but fewer correlations compared to control-treated (CT) ticks, indicating a less interactive yet more connected microbial community. Network centrality measures such as degree, betweenness, closeness, and eigenvector centrality, differed significantly between AT and CT groups, suggesting alterations in local network dynamics due to antibiotic intervention. Coxiella and Acinetobacter exhibited disrupted connectivity and roles, with the former showing reduced interactions in AT group and the latter displaying a loss of connected nodes, emphasizing their crucial roles in microbial network stability. Robustness tests against node removal showed decreased stability in AT networks, particularly under directed attacks, confirming a susceptibility of the microbial community to disturbances. Functional profile analysis further indicated a higher diversity and richness in metabolic capabilities in the AT group, reflecting potential shifts in microbial metabolism as a consequence of antimicrobial treatment. CONCLUSIONS: Our findings support that bacterial interaction traits boosting the transstadial transmission of Babesia could be associated with reduced colonization resistance. The disrupted microbial interactions and decreased network robustness in AT ticks suggest critical vulnerabilities that could be targeted for managing tick-borne diseases.

Feline vector-borne haemopathogens in Türkiye: the first molecular detection of Mycoplasma wenyonii and ongoing Babesia ovis DNA presence in unspecific hosts.

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.

Investigation of Babesia spp. and Theileria spp. in ticks from Western China and identification of a novel genotype of Babesia caballi.

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.

Phylogenetic inferences based on distinct molecular markers reveals a novel Babesia (Babesia pantanalensis nov. sp.) and a Hepatozoon americanum-related genotype in crab-eating foxes (Cerdocyon thous).

Piroplasmids and Hepatozoon spp. Are apicomplexan protozoa that may cause disease in several canid species. The present study aimed to expand the knowledge on the diversity of piroplasmids and Hepatozoon in crab-eating foxes (Cerdocyon thous; n = 12) sampled in the Pantanal of Mato Grosso do Sul State, central-western Brazil. PCR assays based on the 18S rRNA were used as screening. Three (25%) and 11 (91.7%) were positive for piroplasmids and Hepatozoon spp., respectively. Co-infection was found in three C. thous. Phylogenetic analyses based on the near-complete 18S rRNA, cox-1 and hsp70 genes evidenced the occurrence of a novel of Babesia spp. (namely Babesia pantanalensis nov. sp.) closely related to Rangelia vitalii and Babesia sp. 'Coco'. This finding was supported by the genetic divergence analysis which showed (i) high divergence, ranging from 4.17 to 5.62% for 18 S rRNA, 6.16% for hps70 and 4.91-9.25% for cox-1 and (ii) the genotype network (which displayed sequences separated from the previously described Piroplasmida species by median vectors and several mutational events). Also, phylogenetic analysis based on the 18S rRNA gene of Hepatozoon spp. positioned the sequences obtained herein in a clade phylogenetically related to Hepatozoon sp. 'Curupira 2', Hepatozoon sp. detected in domestic and wild canids from Uruguay and Hepatozoon americanum. The present study described Babesia pantanalensis nov sp. and Hepatozoon closely related to H. americanum in crab-eating foxes from Brazil. Moreover, the coinfection by piroplasmids and Hepatozoon sp. for the first time in crab-eating foxes strongly suggesting that this wild canid species potentially acts as a bio-accumulate of hemoprotozoan in wild environment.

A novel and low-cost cross-priming amplification assay for rapid detection of Babesia duncani infection.

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.

Identification and molecular characterization of a novel Babesia orientalis rhoptry neck protein 4 (BoRON4).

Babesia orientalis, a protozoan parasite transmitted by the tick Rhipicephalus haemaphysaloides, holds significant economic importance along the Yangtze River. Key factors in the host invasion process include rhoptry neck proteins (RON2, RON4, and RON5) and apical membrane antigen 1 (AMA1). However, the intricacies of the interaction between AMA1 and RONs remain incompletely elucidated in B. orientalis. To better understand these crucial invasion components, the RON4 gene of B. orientalis (BoRON4) was cloned and sequenced. RON4 is 3468 base pairs long, encodes 1155 amino acids, and has a predicted molecular weight of 130 kDa. Bioinformatics analysis revealed a unique region (amino acid residues 109-452) in BoRON4, which demonstrates higher sensitivity to epitope activity. The BoRON4 gene was strategically truncated, amplified, and cloned into the pGEX-6p-1 vector for fusion expression. We successfully used the mouse polyclonal antibody to identify native BoRON4 in B. orientalis lysates. Furthermore, the corresponding BoRON4 protein band was detected in the water buffalo serum infected with B. orientalis, while no such band was observed in the control. Additionally, I-TASSER and Discovery Studio software were used to predict the tertiary structures of BoRON4 and its ligands, CH-PKA and CH-complex. These ligands can serve as lead compounds for the development of anti-babesiosis drugs. In conclusion, BoRON4 emerges as a promising candidate antigen for distinguishing water buffalo infected with B. orientalis from their normal counterparts. This study positions BoRON4 as a potential diagnostic antigen for babesiosis in water buffalo, contributing valuable insights to the field of parasitology.

The first study of the prevalence and genetic diversity of Theileria equi and Babesia caballi in horses in Russia.

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.

Acute splenic pathology on CT in patients with babesiosis.

PURPOSE: To better understand the occurrence of splenic disease as a potential manifestation of babesiosis by retrospectively estimating the frequency of acute splenic injury on abdominal and pelvic CT in a cohort of patients with active babesia infection. MATERIALS AND METHODS: In a search of our single institution, suburban teaching community hospital database, 57 patients were found to have positive babesia infection between the years 2021-2023. 29 of these patients underwent abdominal and pelvic CT (22 with and 7 without intravenous contrast), and 3 underwent abdominal ultrasound without any CT. The imaging was reviewed for the presence or absence of splenic abnormalities, and for follow-up imaging. Parasitemia levels at the time of imaging were also reviewed; parasitemia levels < 4% are associated with mild to moderate disease, whereas parasitemia levels > 4% are associated with severe disease. RESULTS: 21/32 (66%) patients who underwent any type of abdominal imaging (ultrasound, MRI, and CT) had splenomegaly. Of the 22 patients who had IV contrast-enhanced CT scans, 6 were found to have splenic infarction (27%). One of these 22 patients had multiple rounded non-peripheral hypoenhancing foci on both CT and MRI which did not meet criteria for infarction, in association with splenomegaly, and which resolved after treatment. 0/6 patients in the splenic infarction group had parasitemia levels greater than 4%, while 4 of the 16 patients (4/16) without infarction had parasitemia levels of greater than 4%. CONCLUSION: Our study showed that splenic disease in patients with babesiosis mostly took the form of splenomegaly, and in a substantial minority of patients as splenic infarction. There were no cases of splenic rupture and perisplenic hematoma in our case series, likely reflecting a limitation of the relatively small study size. Concordant with prior studies, we found no identifiable association between parasitemia levels and the presence of splenic infarction.