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.

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.

Inhibition of malaria and babesiosis parasites by putative red blood cell targeting small molecules.

Background: Chemotherapies for malaria and babesiosis frequently succumb to the emergence of pathogen-related drug-resistance. Host-targeted therapies are thought to be less susceptible to resistance but are seldom considered for treatment of these diseases. Methods: Our overall objective was to systematically assess small molecules for host cell-targeting activity to restrict proliferation of intracellular parasites. We carried out a literature survey to identify small molecules annotated for host factors implicated in Plasmodium falciparum infection. Alongside P. falciparum, we implemented in vitro parasite susceptibility assays also in the zoonotic parasite Plasmodium knowlesi and the veterinary parasite Babesia divergens. We additionally carried out assays to test directly for action on RBCs apart from the parasites. To distinguish specific host-targeting antiparasitic activity from erythrotoxicity, we measured phosphatidylserine exposure and hemolysis stimulated by small molecules in uninfected RBCs. Results: We identified diverse RBC target-annotated inhibitors with Plasmodium-specific, Babesia-specific, and broad-spectrum antiparasitic activity. The anticancer MEK-targeting drug trametinib is shown here to act with submicromolar activity to block proliferation of Plasmodium spp. in RBCs. Some inhibitors exhibit antimalarial activity with transient exposure to RBCs prior to infection with parasites, providing evidence for host-targeting activity distinct from direct inhibition of the parasite. Conclusions: We report here characterization of small molecules for antiproliferative and host cell-targeting activity for malaria and babesiosis parasites. This resource is relevant for assessment of physiological RBC-parasite interactions and may inform drug development and repurposing efforts.

Absence of Anti-Babesia microti antibody in commercial intravenous immunoglobulin (IVIG).

BACKGROUND: Babesiosis is a worldwide emerging protozoan infection that is associated with a spectrum of disease severity from asymptomatic infection to severe organ damage and death. While effective treatment strategies are available, some immunocompromised patients experience severe acute and prolonged/relapsing illness due in part to an impaired host antibody response. Intravenous immunoglobulin (IVIG) has been used as an adjunctive therapy in some immunocompromised babesiosis patients, but its therapeutic effect is uncertain. We evaluated the presence of Babesia microti antibodies in commercial samples of IVIG. METHODS/PRINCIPLE FINDINGS: The presence of B. microti antibodies in commercial samples of IVIG were tested using an immunofluorescence assay. A subset of samples was then tested for B. microti antibodies using an enzyme linked immunosorbent assay. Out of 57 commercial IVIG samples tested using IFA, and 52 samples tested using ELISA, none were positive for B. microti antibodies. CONCLUSIONS: Commercially available IVIG may not be of therapeutic benefit for babesiosis patients. Additional sampling of IVIG for B. microti antibody and a clinical trial of babesiosis patients given IVIG compared with controls would provide further insight into the use of IVIG for the treatment of babesiosis.

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.

Molecular identification, risk factor assessment, and phylogenetic analysis of tick-borne pathogens in symptomatic and asymptomatic cattle from South-Eastern Iran.

Tick-borne pathogens (TBPs) represent a substantial threat to cattle globally, exerting adverse impacts on production, health, and economic viability. This study delves into the prevalence and implications of TTBPs in cattle sourced from resource-limited smallholder livestock farms situated in southeastern Iran, proximate to Afghanistan and Pakistan. Blood and tick specimens were systematically collected from a cohort of 230 cattle, comprising 150 asymptomatic and 80 symptomatic individuals. Genomic DNA isolated from blood samples underwent rigorous examination for the presence of key TBPs, including Anaplasma marginale, A. phagocytophilum, A. bovis, A. centrale, Babesia bigemina, and Theileria annulata, utilizing multiple genetic markers. Nucleotide sequence analysis facilitated the reconstruction of phylogenetic relationships. The study also evaluated various potential risk factors, such as clinical status, gender, age, breed, tick infestation, and management practices, to elucidate their associations with TTBPs. Among the cattle cohort, a staggering 87.8% (202/230) tested positive for at least one pathogen. Prevalence statistics encompassed A. marginale (72.2%), T. annulata (68.3%), A. phagocytophilum/A. platys-like complex (66.1%), A. centrale (16.7%), B. bigemina (10.0%), and A. bovis (6.1%). Remarkably, mixed infections involving two, three, and four pathogens were detected in 23%, 52.1%, and 2.2% of animals, respectively. Notably, all asymptomatic cattle were positive for at least one TBP. Tick infestation was observed in 62.2% (143/230) of cattle, predominantly caused by Hyalomma anatolicum (82.5%), Rhipicephalus (Boophilus) annulatus (13.1%), and R. sanguineus sensu lato (4.4%). Risk factors linked to TBPs encompassed tick infestation, older age, and crossbred animals. Clinical presentations among symptomatic cattle encompassed fever, anemia, weight loss, anorexia, jaundice, and enlarged superficial lymph nodes. This study underscores the pivotal role of asymptomatic carriers in the propagation of TTBPs within endemic regions. Furthermore, it emphasizes the potential for the implementation of molecular diagnostics to unmask subclinical infections, thereby affording the opportunity for targeted interventions aimed at ameliorating the burden of TTBPs in resource-constrained smallholder dairy farms.

The protective effects of BMSA1 and BMSA5-1-1 proteins against Babesia microti infection.

The intracellular parasite Babesia microti is among the most significant species causing human babesiosis and is an emerging threat to human health worldwide. Unravelling the pathogenic molecular mechanisms of babesiosis is crucial in developing new diagnostic and preventive methods. This study assessed how priming with B. microti surface antigen 1 (BHSA 1) and seroreactive antigen 5-1-1 (BHSA 5-1-1) mediate protection against B. microti infection. The results showed that 500 µg/ml rBMSA1 and rBMSA5-1-1 partially inhibited the invasion of B. microti in vitro by 42.0 ± 3.0%, and 48.0 ± 2.1%, respectively. Blood smears revealed that peak infection at 7 days post-infection (dpi) was 19.6%, 24.7%, and 46.7% in the rBMSA1, rBmSA5-1-1, compared to the control groups (healthy mice infected with B. microti only), respectively. Routine blood tests showed higher white blood cell, red blood cell counts, and haemoglobin levels in the 2 groups (BMSA1 and BMSA5 5-1-1) than in the infection control group at 0-28 dpi. Moreover, the 2 groups had higher serum interferon-γ, tumor necrosis factor-α and Interleukin-17A levels, and lower IL-10 levels than the infection control group throughout the study. These 2 potential vaccine candidate proteins partially inhibit in vitro and in vivo B. microti infection and enhance host immunological response against B. microti infection.

Can herd seroprevalence be used as an indicator of enzootic stability for bovine anaplasmosis? Insights from a case-control field study in Brazil.

Bovine anaplasmosis presents a significant challenge to livestock production in tropical, subtropical, and temperate regions. For many years, the concept of enzootic stability/instability (initially established for babesiosis) and herd seroprevalence as an indicator of outbreak risks have been applied to anaplasmosis. However, this model has never been definitively validated for Anaplasma marginale. The objective of this study was to examine the relationship between herd immunity (seroprevalence) and the occurrence of anaplasmosis outbreaks in Southern Brazil. A case-control study was conducted, categorizing farms into two groups: cases (farms with a history of clinical anaplasmosis) and controls (those without anaplasmosis). Thirteen farms were identified as "cases", while 23 were identified as "controls". A substantial difference in seroprevalence distribution between the two groups was observed. The majority of "control" farms exhibited over 75% of animals with antibodies to A. marginale in both calves and heifers, whereas the majority of "case" farms had a seropositive cattle percentage below 75%. Additionally, twelve months after cattle serology tests, we conducted a prospective follow-up survey to identify any clinical cases of anaplasmosis. Statistical associations (P < 0.05) were found between both retrospective and prospective anaplasmosis outbreaks and the hypothetical threshold of herd seroprevalence (75%). We hypothesize that herd seroprevalence may be an indicator of the risk of occurrence of clinical anaplasmosis. It appears that the epidemiology of cattle anaplasmosis, at least in our conditions, aligns with the well-known model of enzootic stability/instability originally applied to bovine babesiosis.

Enzootic stability of tick fever in Holstein calves grazing in a tropical region, subjected to strategic cattle tick control with fluralaner.

BACKGROUND: In 2022, fluralaner was launched on the market for use in the control of the cattle tick Rhipicephalus microplus after showing 100% efficacy in registration trials against the causative agents of cattle tick fever (TFAs). The aim of the present study was to determine whether a strategic control regimen against R. microplus using fluralaner (FLU) in Holstein calves grazing in a tropical region would alter the enzootic stability status of cattle tick fever, triggering outbreaks in these animals up to 22 months age. METHODS: In this study, a group of calves treated with FLU was compared with a control group treated with the regimen currently being used on the farm, which consisted of the fipronil + fluazuron formulation (FIFLUA). In the first experiment, the efficacy of the FIFLUA pour-on formulation was evaluated in a field study. In the second experiment, which lasted 550 days, two experimental groups (n = 30/group) of Holstein calves naturally infested with R. microplus were analyzed. Calves aged 4 to 10 months received either a specific treatment regimen with FLU (experimental group) or FIFLUA (control group). During this period, tick counts, animal weight measurement, feces collection (to determine eggs and oocysts per gram of feces), tick fever monitoring, blood smears (to ascertain enzootic stability of the herd), PCR testing for TFAs and serology (indirect enzyme-linked immunosorbent assay [iELISA]) were performed. All calves were evaluated for signs of tick fever between ages 11 and 22 months. RESULTS: FIFLUA showed an acaricidal efficacy of > 90% from post-treatment days 14 to 35. Regarding treatments against the TFAs, the average number of treatments was similar between groups, but animals treated with FLU had a smaller reduction in packed cell volume on some of the evaluation dates of the second and third treatment against TFAs. In calves aged 10 months in the FLU group, B. bovis was not detected by PCR (0/15 samples), 40% of the samples had antibody titers and 33% (10/30) of the samples had positive blood smears. Regarding B. bigemina, > 86% of the samples in both groups tested positive for B. bigemina DNA and antibodies; there was no difference in the antibody titers between the groups. There were no clinical cases of cattle tick fever in calves aged 11 to 22 months. CONCLUSIONS: In comparison with the control treatment, the strategic control regimen against R. microplus with FLU that was implemented in the present study did not negatively affect the enzootic stability status of A. marginale and B. bigemina in the herd up to 22 months of age. The enzootic stability status of B. bovis was not reached by either group. These results likely represent a characteristic of the local tick population, so further studies should be performed.

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.

THEILERIA EQUI INFECTION IN WORKING HORSES OF PAKISTAN: EPIDEMIOLOGY, MOLECULAR CHARACTERIZATION, AND HEMATOBIOCHEMICAL ANALYSIS.

Theileria equi is 1 of the emerging and prevailing tick-borne hemoprotozoans adversely affecting the equids worldwide, including Pakistan. The current study aimed to investigate the prevalence and molecular characterization of T. equi in working horses (n = 194), the comparative efficacy of different diagnostic tests, associated risk factors, and hematobiochemical analysis. The blood samples of horses were subjected to microscopic examination, cELISA, and polymerase chain reaction (PCR) and the results revealed a prevalence of 9.79, 21.13, and 13.40%, respectively, for T. equi in working horses. The comparison of microscopy and cELISA results with PCR showed that cELISA had higher sensitivity (84.62%), but lower specificity (88.69%) and accuracy (88.14%) in comparison to microscopy (57.69, 97.62, and 92.27%). Molecular characterization of T. equi by phylogenetic analysis revealed a 61% resemblance of study isolates with each other OL662926, OL662925, and 82% similarity with isolate OL662924 while also showing homology with T. equi isolates of South Africa, South Korea, India, Pakistan, and Brazil. The risk factor analysis revealed a significant association (P < 0.05) of tick control status, previous tick history, tick infestation, house hygiene, deworming/vaccination, and the presence of other livestock species with T. equi infection in horses. The hematobiochemical profile revealed a significant (P < 0.05) decrease in red blood cells (RBCs), hemoglobin (Hb), packed cell volume (PCV), white blood cells (WBCs), platelet (PLT), phosphorus, and an increase in lymphocytes, granulocytes, aspartate aminotransferase (AST), glucose, bilirubin, blood urea nitrogen (BUN), and creatinine in T. equi-infected horses. The current study is the first comprehensive report for comparative evaluation of microscopy, cELISA, and PCR, assessment of epidemiological risk factors as well as hematobiochemical variations due to T. equi infection in Pakistan.

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.

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.

Repeated imidocarb treatment failure suggesting emerging resistance of Babesia canis in a new endemic area in north-eastern Germany.

Canine babesiosis has been increasingly diagnosed in various regions of Germany such as north-eastern Germany in recent years. A dog with several relapses of Babesia canis infection after treatment with imidocarb is described. A 9-year-old male Magyar Viszla with B. canis infection was referred after two treatments with imidocarb (dosage 2.1 mg/kg SC) because of lethargy, fever and pancytopenia (additional treatments with prednisolone and doxycycline). Merozoites were detected in the blood smear and imidocarb treatment was repeated. Clinical signs, pancytopenia and a positive B. canis PCR occurred after the 3rd (6 mg/kg SC), 4th (7.7 mg/kg SC) and 5th (7.5 mg/kg SC and doxycycline for 4 weeks in addition) imidocarb injection and thorough tick prevention with isoxazoline and permethrin products. 12 days after the 5th injection, the PCR was negative for the first time. The dog was again presented with fever 35 days after the 5th injection. The B. canis PCR was positive and laboratory examination revealed pancytopenia. Treatment with atovaquone/azithromycin for 18 days was performed and no further relapse occurred for 32 weeks. In the case of suspected imidocarb resistance in B. canis infection, treatment with atovaquone/azithromycin can be an alternative.

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.