Nilgai antelope display no signs of infection upon experimental challenge with a virulent Babesia bovis strain.

BACKGROUND: Bovine babesiosis is caused by infection with the protozoal parasite Babesia bovis, which is transmitted by Rhipicephalus (Boophilus) spp. It can cause mortality rates up to 90% in immunologically naive Bos taurus cattle. In south Texas, R. (B.) microplus is known to infest nilgai antelope (Boselaphus tragocamelus); however, their susceptibility to infection with B. bovis and their role in the transmission of the parasite remain unknown. In this study, we challenged nilgai antelope with B. bovis and evaluated their susceptibility to infection. METHODS: Nilgai were needle inoculated with ≈108 B. bovis-parasitized erythrocytes (merozoites) or a homogenate of B. bovis-infected larval ticks (sporozoite) delivered intravenously. Bos taurus beef calves were inoculated in parallel, as this strain of B. bovis is lethal to cattle. Temperature and hematocrit were monitored daily over the course of each study, and whole blood was collected for molecular [polymerase chain reaction (PCR)] and serological [indirect enzyme-linked immunosorbent assay (ELISA)] diagnostic evaluation. Histological sections of nilgai cerebral tissue were examined for evidence of infection. Recipient bovine calves were sub-inoculated with blood from nilgai challenged with either stage of the parasite, and they were monitored for clinical signs of infection and evaluated by a PCR diagnostic assay. Red blood cells (RBCs) from prechallenged nilgai and B. taurus beef cattle were cultured with an in vitro B. bovis merozoite culture to examine colonization of the RBCs by the parasite. RESULTS: Nilgai did not display clinical signs of infection upon inoculation with either the merozoite or sporozoite stage of B. bovis. All nilgai were PCR-negative for the parasite, and they did not develop antibodies to B. bovis. No evidence of infection was detected in histological sections of nilgai tissues, and in vitro culture analysis indicated that the nilgai RBCs were not colonized by B. bovis merozoites. Cattle subinoculated with blood from challenged nilgai did not display clinical signs of infection, and they were PCR-negative up to 45 days after transfer. CONCLUSIONS: Nilgai do not appear to be susceptible to infection with a strain of B. bovis that is lethal to cattle. Tick control on these alternative hosts remains a critical priority, especially given their potential to disseminate ticks over long distances.

A global systematic review and meta-analysis on the babesiosis in dogs with special reference to Babesia canis.

BACKGROUND: Canine babesiosis is a clinically significant tick-transmitted disease caused by several species of the intraerythrocytic protozoan parasite Babesia, which result in a wide range of clinical manifestations, from mild, transient infection to serious disease and even death. OBJECTIVES: The current study aimed to estimate the global prevalence and associated risk factors of Babesia in dogs. METHODS: Multiple databases (PubMed, Scopus, ProQuest, Web of Science and Google Scholar) were searched for relevant literature published from January 2000 up to December 2022. The statistical analyses were performed based on the R software (version 3.6) meta-package. RESULTS: Out of 23,864 publications, 229 studies met the inclusion criteria. The pooled prevalence of canine babesiosis was 0.120 (95% CI; 0.097-0.146). The highest pooled prevalence was found in Europe (0.207, 95% CI; 0.097-0.344). Among several species, Babesia canis was the most prevalent parasite (0.216, 95% CI; 0.056-0.441). The highest pooled prevalence of Babesia in dogs was observed in the summer season (0.097, 95% CI; 0.040-0.174). CONCLUSIONS: Regular screening and appropriate control strategies are recommended for the prevention of transmission of tick-borne disease transmission among dogs.

Dog blood parasite infection in upland and lowland communities of northern Thailand: The role of environment and care of dog owners.

Dogs play an important role as hosts and reservoirs for many zoonotic diseases. Ehrlichiosis, babesiosis and hepatozoonosis are a group of canine vector-borne diseases that can be transmitted via ectoparasites from dog to dog and also from dog to humans. This study focused on three main blood parasites of dog (i.e., Babesia spp., Ehrlichia spp. and Hepatozoon spp.) among two different landscape types of eight villages of Santhong Sub-district, Nan Province, Thailand. In this study, 149 dogs were surveyed and blood samples were collected. Blood parasite infections in dogs were assessed using molecular detection approach. Babesia canis vogeli, Babesia gibsoni, Ehrlichia canis and Hepatozoon canis were detected with prevalence of infection at 10.7%, 8.1%, 3.4% and 0.7%, respectively. In terms of landscape type, prevalence of overall blood parasites, particularly Babesia spp. infections were higher in dogs living in upland forested areas (28.3%) compared to dogs from lowland agricultural areas (12.3%). Data obtained from the questionnaires on perceptions of dog owners showed that dogs raised all the time outside owner's house, and those dogs whose owners have never bathed and cleaned were more likely to be exposed to blood parasites. As infected dogs could play an important role as reservoirs of the blood parasites, attitude of dog owners may affect public health in terms of zoonotic disease transmission. Effective control measures and surveillance program of arthropod vectors and blood parasite infection in dogs still need to be advocated to minimize zoonotic disease transmission.

Molecular survey of Piroplasmida, Hepatozoon spp. and Anaplasmataceae in anemic and thrombocytopenic dogs from Uruguay.

Canine tick-borne diseases, such as babesiosis, rangeliosis, hepatozoonosis, anaplasmosis and ehrlichiosis, are of veterinarian relevance, causing mild or severe clinical cases that can lead to the death of the dog. The aim of this study was detecting tick-borne protozoan and rickettsial infections in dogs with anemia and/or thrombocytopenia in Uruguay. A total of 803 domestic dogs were evaluated, and 10% were found positive (detected by PCR) at least for one hemoparasite. Sequence analysis confirmed the presence of four hemoprotozoan species: Rangelia vitalii, Babesia vogeli, Hepatozoon canis and Hepatozoon americanum, and the rickettsial Anaplasma platys. The most detected hemoparasite was R. vitalii, followed by H. canis and A. platys. This is the first report of B. vogeli in Uruguay and the second report of H. americanum in dogs from South America. The results highlight the importance for veterinarians to include hemoparasitic diseases in their differential diagnosis of agents causing anemia and thrombocytopenia.

Detection of tick-borne pathogens in Rhipicephalus bursa ticks collected from the autochthonous Garrano breed of horses in Portugal.

The Garrano is a semi-feral horse breed native to several mountains in the northern Iberian Peninsula. Despite being endangered, this unique breed of pony has managed to survive in the wild and continues to be selectively bred, highlighting their remarkable resilience and adaptability to harsh environments. Wildlife plays a critical role in the survival of tick vectors in their natural habitats and the transfer of tick-borne pathogens, as they can serve as reservoir hosts for many agents and amplifiers for these vectors. The semi-feral lifestyle of the Garrano horses makes them particularly vulnerable to exposure to numerous tick species throughout the year. Therefore, the aim of this study was to investigate the occurrence of Anaplasma, Ehrlichia, Babesia, Theileria, and spotted fever rickettsiae in the Garrano horse ticks to obtain a knowledge of circulating agents in this host population. The collected ticks (n = 455) were identified as Rhipicephalus bursa. DNA specimens were organized in pools of 5 ticks, for molecular screening. Pools PCR results confirmed the presence of Candidatus Rickettsia barbariae (n = 12 for the ompB gene, n = 11 for the ompA gene and n = 6 for the gltA gene), Babesia bigemina (n = 1), Babesia caballi (n = 3), Theileria equi (n = 15) and Theileria haneyi (n = 1).These results confirm the circulation of an emerging rickettsial spotted fever group member, Candidatus R. barbariae, in R. bursa ticks. Our findings demonstrated that Candidatus R. barbariae co-circulates with B. bigemina and T. equi, which are vectored by R. bursa. We are reporting for the first time, the detection of T. haneyi among R. bursa ticks feeding in the Garrano horses in Portugal. Surveillance studies for tick-borne infections are essential to provide information that can facilitate the implementation of preventive and control strategies.

A conserved motif in the immune-subdominant RAP-1 related antigen of Babesia bovis contains a B-cell epitope recognized by antibodies from protected cattle.

Introduction: Babesia bovis, a tick-borne apicomplexan parasite causing bovine babesiosis, remains a significant threat worldwide, and improved and practical vaccines are needed. Previous studies defined the members of the rhoptry associated protein-1 (RAP-1), and the neutralization-sensitive rhoptry associated protein-1 related antigen (RRA) superfamily in B. bovis, as strong candidates for the development of subunit vaccines. Both RAP-1 and RRA share conservation of a group of 4 cysteines and amino acids motifs at the amino terminal end (NT) of these proteins. Methods and results: Sequence comparisons among the RRA sequences of several B. bovis strains and other Babesia spp parasites indicate a high level of conservation of a 15-amino acid (15-mer) motif located at the NT of the protein. BlastP searches indicate that the 15-mer motif is also present in adenylate cyclase, dynein, and other ATP binding proteins. AlphaFold2 structure predictions suggest partial exposure of the 15-mer on the surface of RRA of three distinct Babesia species. Antibodies in protected cattle recognize a synthetic peptide representing the 15-mer motif sequence in iELISA, and rabbit antibodies against the 15-mer react with the surface of free merozoites in immunofluorescence. Discussion and conclusion: The presence of the 15-mer-like regions in dynein and ATP-binding proteins provides a rationale for investigating possible functional roles for RRA. The demonstrated presence of a surface exposed B-cell epitope in the 15-mer motif of the B. bovis RRA, which is recognized by sera from protected bovines, supports its inclusion in future subunit epitope-based vaccines against B. bovis.

The prevalence of selected vector-borne diseases in dromedary camels (Camelus dromedarius) in the United Arab Emirates.

Vector-borne diseases (VBDs) affecting dromedary camels (Camelus dromedarius) have considerable importance in the United Arab Emirates (UAE) because of the consequences associated with production decline and economic losses. Our study aimed to determine the prevalence of selected VBDs in camels in the UAE and identify risk factors. This research is currently affected by the low number of epidemiological molecular surveys addressing this issue. Blood samples were obtained from 425 dromedary camels from different locations across the UAE. Whole genomic DNA was isolated, and PCR screening was done to detect piroplasmids (Babesia/Theileria spp.), Trypanosoma spp., and Anaplasmataceae spp. (Anaplasma, Ehrlichia, Neorickettsia and Wolbachia spp.). Amplicons were sequenced, and phylogenetic trees were constructed. Trypanosoma sequences were identified as T. brucei evansi, whereas Anaplasmataceae sequences were identified as A. platys-like. All camels were negative for Babesia/Theileria spp. (0%); however, 18 camels were positive for T. b. evansi (4%) and 52 were positive for A. platys-like (12%). Mixed infection with T. b. evansi and A. platys-like was found in one camel. Statistical analyses revealed that camels with a brown coat colour were significantly more prone to acquire the A. platys-like strain compared with those having a clearer coat. A similar finding was observed when comparing urban moving camels with desert indoor and urban indoor camels. Continuous disease surveillance is required to ensure and maintain the good health status of the camels in the UAE. Nonetheless, the risk of disease outbreak remains if the misuse of drugs continues.

Epidemiological assessment of Anaplasma marginale, Babesia bigemina, and Babesia bovis infections in Colombian creole cattle breeds: A molecular survey in northeastern Colombia.

Anaplasmosis and babesiosis are globally distributed arthropod-borne diseases known for causing substantial economic losses due to their high morbidity and mortality rates. This study aims to assess the frequency and epidemiological features associated with the infection of Anaplasma marginale, Babesia bigemina, and Babesia bovis in three Creole cattle breeds (Chino Santandereano (Chino), Casanareño (CAS), and Sanmartinero (SM)) in northeastern Colombia. Between June 2019 and March 2020, a total of 252 Creole cattle were sampled, with Chino, CAS, and SM accounting for 42.8%, 29.5%, and 29.5% of the samples, respectively. Blood samples were subjected to molecular analysis to detect the DNA of A. marginale, B. bigemina, and B. bovis, using species-specific primers. Additionally, Packed Cell Volume (PCV), total serum proteins, and body condition were evaluated. Molecular analyses revealed the presence of B. bigemina, A. marginale, and B. bovis in 83.7% (211/252; 95% CI = 79.1%-88.3%), 59.9% (151/252; 95% CI = 53.8%-66.1%), and 40.9% (103/252; 95% CI = 34.7%-46.9%) of the samples, respectively, with 69% (174/252; 95% CI = 57.8%-80.3%) exhibiting coinfections. Notably, in infected animals, no significant alterations in PCV, total serum proteins, or body condition were observed. Multivariate analyses indicated a statistically significant association between the frequency of A. marginale infection and the breed and season, with a higher frequency in SM during the rainy season (P < 0.05). To our knowledge, this is the first molecular survey that evaluates multiple arthropod-borne pathogens in Colombian Creole breeds. The results revel a high frequency of B. bigemina and A. marginale infections, coupled with a notable frequency of coinfections, all without significant alteration in the PCV, total serum proteins and body conditions. Our findings enhance the understanding of the epidemiological aspects of arthropod-borne pathogens in Colombian Creole breed and contribute to the improvement of sanitary programs for these animals.

Effectiveness of Two New Endochin-like Quinolones, ELQ-596 and ELQ-650, in Experimental Mouse Models of Human Babesiosis.

Endochin-like quinolones (ELQs) define a class of small molecule antimicrobials that target the mitochondrial electron transport chain of various human parasites by inhibiting their cytochrome bc1 complexes. The compounds have shown potent activity against a wide range of protozoan parasites, including the intraerythrocytic parasites Plasmodium and Babesia, the agents of human malaria and babesiosis, respectively. First-generation ELQ compounds were previously found to reduce infection by Babesia microti and Babesia duncani in animal models of human babesiosis but achieved a radical cure only in combination with atovaquone and required further optimization to address pharmacological limitations. Here, we report the identification of two second-generation 3-biaryl ELQ compounds, ELQ-596 and ELQ-650, with potent antibabesial activity in vitro and favorable pharmacological properties. In particular, ELQ-598, a prodrug of ELQ-596, demonstrated high efficacy as an orally administered monotherapy at 10 mg/kg. The compound achieved radical cure in both the chronic model of B. microti-induced babesiosis in immunocompromised mice and the lethal infection model induced by B. duncani in immunocompetent mice. Given its high potency, favorable physicochemical properties, and low toxicity profile, ELQ-596 represents a promising drug for the treatment of human babesiosis.

Characterization and LC-MS/MS based proteomic analysis of extracellular vesicles separated from blood serum of healthy and dogs naturally infected by Babesia canis. A preliminary study.

Canine babesiosis is a rapidly spreading tick-borne disease in Europe, which entails protozoan parasites invading red blood cells. Small extracellular vesicles (EVs) (< 200 nm) were isolated from the serum of 15 healthy and 15 by Babesia canis naturally infected dogs aimed to distinguish EV characteristics and protein profiles. There were no significant differences (P = 0.05) observed in the mean sizes and concentrations of serum EVs between the healthy and canine babesiosis groups. Despite a higher number of Canis lupus proteins detected in EVs from serum of diseased dogs, there were no statistically significant differences (P < 0.05) in the number of protein IDs between the experimental groups. We successfully identified 211 Canis lupus proteins across both experimental groups, of which 147 Canis lupus proteins were validated as being EV-associated. This data set is accessible via the ProteomeXchange PXD047647. EVs isolated from serum of B. canis infected dogs were Cd9+, Cd63+, Cd81+, and Cd82+. Furthermore, 73 Canis lupus proteins were validated as EV-associated and specific for EVs isolated from serum of B. canis-infected dogs. These were predominantly membrane and cytosolic proteins, and innate and adaptive immune system-related proteins, especially those involved in adhesion and proteoglycan mechanisms like integrins. Enrichment was also observed for proteins involved in vascular and cellular responses, including signalling pathways such as VEGF, VEGFR, and the LKB1 network. When only blood-related sites of EV expression were evaluated, the origins of EV proteins were mostly cells of immune system. These were dendritic cells, neutrophils, B cells, monocytes and platelets. In general, proteins were enriched in pathways that collectively regulate various cellular processes, including immune responses, communication, signal transduction, membrane trafficking, and apoptosis. Serum EVs and their protein cargo may have an important role in both the invasion of B. canis and the host's response to the parasitic infection, nevertheless, additional experimental research is warranted. The overall count of identified EV proteins of parasitic origin, meeting cut off criteria of two peptides and 1 % FDR, was relatively low.

A small proportion of Zebu genetic background in crossbred calves may not be enough to improve resistance against natural bovine Babesia spp. infections.

The main objective of cattle breeders in tropical and subtropical regions is to acquire animals with taurine-productive traits adapted to the broad weather range of these regions. However, one of the main challenges on using taurine genetics in these areas is the high susceptibility of these animals to tick-borne diseases. Consequently, the present study evaluated from 10 November 2021-19 April 2022, the over 13 assessments, the Babesia bovis and Babesia bigemina DNA loads and the IgG anti-B. bovis and anti-B. bigemina levels in Angus (n = 17, 100% Taurine) and Ultrablack (n = 14, ∼82% taurine and 18% Zebu) calves. Data were analyzed using a multivariate mixed model with repeated measures of the same animal including the fixed effects of evaluation, genetic group, sex, Babesia spp., and their interactions. The repeatability values were estimated from the (co)variances matrix and expressed for each species. The correlations between the DNA loads (CNlog) and IgG titers (S/P) values for the two species were also estimated using the same model. Regarding the specific IgG antibody titers for both Babesia spp., no significant differences were observed between the two genetic groups. However, for B. bovis and B. bigemina DNA loads, Ultrablack calves presented significantly higher values than Angus calves. Under the conditions evaluated in this study, our findings suggest that the low percentage of Zebu genetic in the Ultrablack breed was insufficient to improve resistance against babesiosis. Further studies must demonstrate if the low percentages of Zebu genetics in Taurine breeds can modify the susceptibility to babesiosis infections.

Diagnostic performance of a rapid immunochromatographic test for the simultaneous detection of antibodies to Theileria equi and Babesia caballi in horses and donkeys.

BACKGROUND: Equine piroplasmosis is caused by two tick-borne protozoan parasites, Theileria equi and Babesia caballi,, which are clinically relevant in susceptible horses, donkeys, and mules. Moreover, equine piroplasmosis significantly constrains international trading and equestrian events. Rapidly diagnosing both parasites in carrier animals is essential for implementing effective control measures. Here, a rapid immunochromatographic test for the simultaneous detection of antibodies to T. equi and B. caballi was evaluated using samples from horses and donkeys collected in Greece, Israel, and Italy. The results were compared with an improved competitive enzyme-linked immunosorbent assay (cELISA) for detecting antibodies to both parasites using the same panel of samples. METHODS: Blood samples were collected from 255 horses and donkeys. The panel consisted of 129 horses sampled at four locations in northern Greece, 105 donkeys sampled at four locations in Sicily, and 21 horses sampled at two locations in Israel. The rapid test and the cELISA were performed according to the manufacturer's instructions, and the results were subjected to a statistical analysis to determine the sensitivity and specificity of both tests and their association. RESULTS: The immunochromatographic test provided a result within 15 min and can be performed in the field, detecting both pathogens simultaneously. The overall coincidence rate between the rapid test and the cELISA for detecting antibodies against T. equi was 93% and 92.9% for B. caballi. The rapid test's sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) for T. equi were above 91.5%. Sixteen samples were positive for both parasites in the rapid test and eight in the cELISA. Either test had no significant association between T. equi and B. caballi detection. The detection rates of both parasites were significantly higher in Italy than in Greece or Israel and in donkeys than in horses. The agreement for T. equi between the results of both tests was high in Greece (93.8%) and Italy (95.2%) and moderate in Israel (76.2%). For B. caballi, the specificity and NPV of the rapid test were high (94.2% and 98.3%, respectively), although the sensitivity and PPV were moderate (69.2% and 39.1%, respectively) due to the small sample size. However, for B. caballi, the sensitivity was higher with the rapid test. CONCLUSIONS: The rapid test detected T. equi and B. caballi simultaneously in the field, potentially replacing laborious cELISA testing and is recommended for import/export purposes. The test can also be helpful for the differential diagnosis of clinical cases, since seropositivity may rule out equine piroplasmosis since it does not indicate current or active infection.

Equine Piroplasmosis in Asymptomatic Horses of Western Iran: Comparison of Microscopic Examination and Multiplex PCR.

PURPOSE: Piroplasmosis is responsible for anemia, fever, loss of physical activity and even death in equines. In epidemiological studies, accurate diagnostic tests are essential for detecting asymptomatic carriers. This study aimed to investigate the prevalence of infection in asymptomatic horses from Lorestan province, western Iran by developing a multiplex PCR. METHODS AND RESULTS: Blood samples were examined by microscopy and multiplex PCR targeting the SSU rRNA gene of Theileria equi and Babesia caballi. Out of the total of 165 horses, 19 (11.51%) and 31 (18.78%) cases were positive for piroplasms by microscopy and PCR, respectively. The detection rates of both genera were significantly higher in multiplex PCR compared to microscopy (p < 0.0001). Compared with multiplex PCR, the sensitivities of microscopy for the detection of Babesia were only 28.5%. The prevalence of T. equi infection was significantly higher in summer (p = 0.035). The prevalence of B. caballi was significantly higher in males (p = 0.038). CONCLUSION: Findings indicate that the multiplex PCR described here is a sensitive technique for the detection of piroplasm DNA in carriers. Furthermore, asymptomatic carriers must be considered as an important source of infection for equids living in this region.

Transmission risk evaluation of transfusion blood containing low-density Babesia microti.

Background: Babesia is a unique apicomplexan parasite that specifically invades and proliferates in red blood cells and can be transmitted via blood transfusion, resulting in transfusion-transmitted babesiosis. However, detecting Babesia in blood before transfusion has not received enough attention, and the risk of transfusing blood containing a low density of Babesia microti (B. microti) is unclear, possibly threatening public health and wellness. Purpose: This study aimed to determine the lower detection limit of B. microti in blood and to evaluate the transmission risk of blood transfusion containing low-density B. microti. Methods: Infected BALB/c mouse models were established by transfusing infected whole blood with different infection rates and densities of B. microti. Microscopic examination, nested Polymerase Chain Reaction (nested PCR), and an enzyme-linked immunosorbent assay (ELISA) were used to evaluate the infection status of the mouse models. Meanwhile, the nested PCR detection limit of B. microti was obtained using pure B. microti DNA samples with serial concentrations and whole blood samples with different densities of B. microti-infected red blood cells. Thereafter, whole mouse blood with a B. microti density lower than that of the nested PCR detection limit and human blood samples infected with B. microti were transfused into healthy mice to assess the transmission risk in mouse models. The infection status of these mice was evaluated through microscopic examination, nested PCR tests, and ELISA. Results: The mice inoculated with different densities of B. microti reached the peak infection rate on different days. Overall, the higher the blood B. microti density was, the earlier the peak infection rate was reached. The levels of specific antibodies against B. microti in the blood of the infected mice increased sharply during the first 30 days of infection, reaching a peak level at 60 days post-infection, and maintaining a high level thereafter. The nested PCR detection limits of B. microti DNA and parasite density were 3 fg and 5.48 parasites/µL, respectively. The whole blood containing an extremely low density of B. microti and human blood samples infected with B. microti could infect mice, confirming the transmission risk of transfusing blood with low-density B. microti. Conclusion: Whole blood containing extremely low density of B. microti poses a high transmission risk when transfused between mice and mice or human and mice, suggesting that Babesia detection should be considered by governments, hospitals, and disease prevention and control centers as a mandatory test before blood donation or transfusion.

Epidemiology and genetic diversity of Theileria equi and Babesia caballi in Mongolian horses.

Equine piroplasmosis is a tick-borne disease caused by Theileria equi and Babesia caballi in horses. Because of its impact on horse industry, control of this disease is crucial for endemic countries. The control of equine piroplasmosis may be influenced by the genotypic diversity of T. equi and B. caballi. Mongolia, a country with a thriving livestock industry, is endemic for T. equi and B. caballi. However, nationwide epidemiological surveys have not been conducted to determine the current status of infections and genetic diversity of these two parasite species. Therefore, the objective of this research was to investigate the infection rates and genotypes of T. equi and B. caballi in horses across Mongolia. Blood samples were collected from 1353 horses in 15 of Mongolia's 21 provinces, and their DNAs were analyzed with T. equi- and B. caballi-specific PCR assays. Additionally, blood smears were prepared from 251 horses, stained with Giemsa, and examined under a light microscope to identify T. equi and B. caballi. The microscopy revealed that 30 (11.9%) and 4 (1.6%) of the 251 horses were positive for T. equi and B. caballi, respectively. By contrast, PCR assays detected the T. equi and B. caballi in 1058 (78.2%) and 62 (4.6%) horses, respectively. Phylogenetic analysis of 18S rRNA sequences from 42 randomly selected T. equi-positive DNA samples detected the genotypes A and E. On the other hand, the rap-1 sequences from 19 randomly selected B. caballi-positive DNA samples occurred in clades representing the genotypes A and B1, as well as in a distinct clade closely related to the genotype A. Our findings confirm the widespread occurrence of T. equi and B. caballi infections in Mongolian horses, highlighting the need for a comprehensive control approach.

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.

Assessment of Babesia spp. prevalence in various domestic animals across Southern Punjab, Pakistan.

Parasitic diseases, notably babesiosis, exert a substantial impact on the global cattle industry, posing challenges to commerce, economies, and human health. This study, conducted in Southern Punjab, Pakistan, aimed to assess the prevalence of Babesia spp. across various livestock species using microscopic and PCR methods. A total of 180 blood samples (60 from each district) were systematically collected from apparently healthy animals, with 36 samples obtained from each domestic animal species, including camel, cattle, buffalo, goat, and sheep, noting that 12 samples were collected from each district for each animal species. Overall prevalence was determined to be 32.8% (59/180), with varying rates among species: 25.0% in cattle, 41.66% in buffalo, 30.55% in goats, 33.3% in sheep, and 33.3% in camels. Microscopic examination revealed slightly varied infection rates among large and small domestic animals (22.2%), while PCR results indicated a 32.8% overall infection rate in both large and small domestic animals, with no statistical significance. District-wise analysis showed regional variations, with Muzaffargarh recording a prevalence rate of 23.33% through microscopic examination, while Lodhran and Bahawalpur recorded 21.67%. PCR results revealed higher rates (38.33%, 26.67%, and 33.33%, respectively), underlining the importance of employing PCR for accurate detection. Examining ruminant types, large ruminants exhibited a 32.4% infection rate, while small domestic animals showed 33.3%, with no significant difference (p=0.897). District-wise prevalence showcased significant variation, with Muzaffargarh demonstrating a 25% prevalence, Lodhran 22%, and Bahawalpur 22%, through microscopic examination. PCR results displayed 38.33%, 27%, and 33.3%, respectively, with no statistical significance. Detailed analysis of individual districts highlighted variations in infection rates among camels, cattle, buffalo, goats, and sheep. The binomial test indicated significant differences through microscopic analysis (P=0.011) but non-significant variations through PCR (P=0.065), emphasizing the precision of PCR. Regional variations in prevalence, notably with Punjab exhibiting the highest frequency (33.87%) and KPK the lowest (13.24%), suggest potential influences from varying veterinary practices and environmental factors. This study underscores the pivotal role of PCR alongside microscopy for accurate babesiosis diagnosis. These findings contribute to the broader understanding of babesiosis prevalence, emphasizing the necessity of advanced molecular techniques for informed control measures.

Expression of sex-specific molecular markers by Babesia bovis gametes.

BACKGROUND: Bovine babesiosis caused by Babesia bovis is one of the most important tick-borne diseases of cattle in tropical and subtropical regions. Babesia bovis parasites have a complex lifecycle, including development within the mammalian host and tick vector. In the tick midgut, extracellular Babesia parasites transform into gametes that fuse to form zygotes. To date, little is known about genes and proteins expressed by male gametes. METHODS AND RESULTS: We developed a method to separate male gametes from in vitro induced B. bovis culture. Separation enabled the validation of sex-specific markers. Collected male gametocytes were observed by Giemsa-stained smear and live-cell fluorescence microscopy. Babesia male gametes were used to confirm sex-specific markers by quantitative real-time PCR. Some genes were found to be male gamete specific genes including pka, hap2, α-tubulin II and znfp2. However, α-tubulin I and ABC transporter, trap2-4 and ccp1-3 genes were found to be upregulated in culture depleted of male gametes (female-enriched). Live immunofluorescence analysis using polyclonal antibodies confirmed surface expression of HAP2 by male and TRAP2-4 by female gametes. These results revealed strong markers to distinguish between B. bovis male and female gametes. CONCLUSIONS: Herein, we describe the identification of sex-specific molecular markers essential for B. bovis sexual reproduction. These tools will enhance our understanding of the biology of sexual stages and, consequently, the development of additional strategies to control bovine babesiosis.

Babesia negevi infection in dogs and response to treatment.

Canine babesiosis is an important protozoan tick-borne disease associated with anemia and thrombocytopenia and caused by several different Babesia spp. Babesia negevi was first reported to infect dogs in the Middle East in 2020. This study describes the presentation, clinical signs, parasitemia levels quantified by molecular techniques, laboratory findings and treatment of dogs infected with B. negevi following the first description of this species. Clinical findings in the infected dogs, a 3-year old female and two 8-week old male and female pups, included extreme lethargy and pale mucous membranes, anemia and thrombocytopenia found in all three animals. Fever was present in the older female and icterus in the female pup. Babesia parasites resembling B. negevi were detected by microscopy of blood smears from the dogs. PCR of blood targeting the 18S rRNA and cox1 genes confirmed that babesiosis was caused by B. negevi and PCR targeting the Borrelia flagellin gene indicated co-infection with Borrelia persica in two dogs. Treatment of the dogs with imidocarb dipropionate resulted in clinical improvement and initial decrease in the B. negevi parasite load as detected by quantitative PCR in two dogs, however the female pup continued to deteriorate and died. The parasite load in the 3-year old female decreased from 43,451 parasites/µl blood pre-imidocarb dipropionate treatment to 803 parasites/µl within two weeks. In the surviving pup, it decreased from 3,293,538 parasites/µl pre-treatment to 20,092 parasites/µl after two weeks. Babesia negevi DNA was still recovered from blood samples by PCR despite repeated treatment with imidocarb dipropionate one-month post-treatment in the surviving pup and up to seven months post-treatment in the 3-year old female. Only treatment with atovaquone and azithromycin for ten days eliminated B. negevi in both dogs as confirmed by negative PCR two weeks later. In conclusion, treatment with imidocarb dipropionate was helpful for recovery from clinical disease but did not facilitate parasite elimination, and it is therefore recommended to treat canine B. negevi infection with the combination of atovaquone and azithromycin.

Molecular detection of Babesia vesperuginis in bats from Lithuania.

Babesia vesperuginis is an intraerythrocytic protozoan parasite that circulates among bats and ticks in many countries worldwide. However, the distribution of B. vesperuginis in the Baltic region has not been studied. A total of 86 dead bats from eight different species were collected and screened for Babesia spp. using real-time PCR. Overall, 52.3% (45/86) of the bats were found positive for Babesia spp. The prevalence of Babesia spp. in different organs varied, with the highest prevalence observed in heart tissues (37.0%) and the lowest in liver tissues (22.2%). However, the observed differences in prevalence among organs were not statistically significant. Blood samples from 125 bats of nine different species were also analyzed for Babesia spp. prevalence using real-time PCR and nested PCR. The results showed a prevalence of 35.2% and 22.4%, respectively. Moreover, 28.3% (17/60) of the examined blood samples were confirmed positive for Babesia spp. through blood smear analysis. The total of 32 partial sequences of the 18S rRNA gene derived in this study were 100% identical to B. vesperuginis sequences from GenBank. In eight species of bats, Pipistrellus nathusii, Pipistrellus pipistrellus, Pipistrellus pygmaeus, Vespertilio murinus, Eptesicus nilssonii, Eptesicus serotinus, Myotis daubentonii and Nyctalus noctula, Babesia parasites were identified. In E. nilssonii, Babesia spp. was identified for the first time.