Dissecting the role of transcription factor AP2-M in Babesia asexual replication.

Babesia spp. are obligate intracellular parasites that invade host cells to complete their asexual development and transmission. Here, we identified a transcription factor AP2-M (BXIN_0799) in Babesia sp. Xinjiang (Bxj), a member of the Apicomplexan AP2 family, which regulates gene expression related to red blood cell (RBC) invasion and cell cycle progression. Our genome-wide analysis of (Cut-Tag) data shows that AP2-M specifically recognized DNA motifs in the promoters of target genes. AP2-M target genes included other AP2 gene family members and epigenetic markers, which could modulate gene expression involved in RBC invasion, merozoite morphology, and cell cycle phases, as indicated by RNA sequencing, proteomics, and single-cell RNA sequencing (scRNA-seq) data from an ap2-m gene disrupted strain (AP2-M (-)). We conclude that AP2-M appeared to contribute to the process of red blood cell invasion, maintain merozoite morphology, and cell cycle progression through GS and MS phases.

Establishment of the auxin inducible degron system for Babesia duncani: a conditional knockdown tool to study precise protein regulation in Babesia spp.

BACKGROUND: Babesia duncani is a pathogen within the phylum Apicomplexa that causes human babesiosis. It poses a significant threat to public health, as it can be transmitted not only through tick bites but also via blood transfusion. Consequently, an understanding of the gene functions of this pathogen is necessary for the development of drugs and vaccines. However, the absence of conditional gene knockdown tools has hindered the research on this pathogen. The auxin-inducible degron (AID) system is a rapid, reversible conditional knockdown system widely used in gene function studies. Thus, there is an urgent need to establish the AID system in B. duncani to study essential gene functions. METHODS: The endogenous genes of the Skp1-Cullin-F-box (SCF) complex in B. duncani were identified and confirmed through multiple sequence alignment and conserved domain analysis. The expression of the F-box protein TIR1 from Oryza sativa (OsTIR1) was achieved by constructing a transgenic parasite strain using a homologous recombination strategy. Polymerase chain reaction (PCR), western blot, and indirect immunofluorescence assay (IFA) were used to confirm the correct monoclonal parasite strain. The degradation of enhanced green fluorescent protein (eGFP) tagged with an AID degron was detected through western blot and live-cell fluorescence microscopy after treatment of indole-3-acetic acid (IAA). RESULTS: In this study, Skp1, Cul1, and Rbx1 of the SCF complex in B. duncani were identified through sequence alignment and domain analysis. A pure BdTIR1 strain with expression of the OsTIR1 gene was constructed through homologous recombination and confirmed. This strain showed no significant differences from the wild type (WT) in terms of growth rate and proportions of different parasite forms. The eGFP tagged with an AID degron was successfully induced for degradation using 500 µM IAA. Grayscale analysis of western blot indicated a 61.3% reduction in eGFP expression levels, while fluorescence intensity analysis showed a 77.5% decrease in fluorescence intensity. Increasing the IAA concentration to 2 mM accelerated eGFP degradation and enhanced the extent of degradation. CONCLUSIONS: This study demonstrated the functionality of the AID system in regulating protein levels by inducing rapid degradation of eGFP using IAA, providing an important research tool for studying essential gene functions related to invasion, egress, and virulence of B. duncani. Moreover, it also offers a construction strategy for apicomplexan parasites that have not developed an AID system.

Molecular and microscopic detection of haemoprotozoan diseases in dogs from Haryana, India.

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

Occurrence of Babesia and Anaplasma in ruminants from the Catimbau National Park, Semiarid Region of Northeast Brazil.

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

In vitro evaluation of the automated hematology analyzer XN-31 for rapid diagnosis of equine piroplasmosis.

Equine piroplasmosis (EP) is a protozoal disease affecting equids, caused by Theileria equi and Babesia caballi. EP is conventionally diagnosed using microscopic, molecular, and/or serological methods, which are time-consuming. Consequently, there is a need for faster testing methods. In this study, we evaluated the application of the Sysmex XN-31 automated hematology analyzer, originally a rapid test for detecting malaria in humans, for the diagnosis of EP. The cultured parasites were measured using the XN-31 that had been customized for horse blood samples (XN-31m). The following parameters were evaluated: limit of detection (LoD), limit of quantification (LoQ), linearity, carryover, precision, and correlation with microscopic examination. The XN-31m detected infected red blood cells (RBCs) in approximately 1 minute. The LoD and LoQ for B. caballi were 4.54 infected RBCs/µL and 14.10 infected RBCs/µL, while those for T. equi were 5.80 infected RBCs/µL and 11.44 infected RBCs/µL, respectively. Linearity showed excellent correlation (R2 > 0.99), and carryover never exceeded 0.5%. The coefficient of variation was under 5%. The correlation between the results obtained using XN-31m and microscopic examination was high (R2 > 0.98). In conclusion, the XN-31 analyzer detected B. caballi and T. equi parasites in approximately 1 minute with high sensitivity. The results indicate the potential of the XN-31 analyzer as a fast and user-friendly diagnostic method for EP. IMPORTANCE: In this study, we demonstrated that the automated hematology analyzer, XN-31, can detect red blood cells infected with Babesia caballi and Theileria equi in about 1 minute. We evaluated the diagnostic performance of the XN-31 analyzer for equine piroplasmosis, providing evidence of its potential as a diagnostic tool for this disease.

Pathogens detected in ticks (Ixodes ricinus) feeding on red squirrels (Sciurus vulgaris) from city parks in Warsaw.

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

Clinical pathology and molecular examination of Babesia spp. infection in dogs; Mashhad, Northeast Iran.

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

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.

First molecular sequencing of Babesia gibsoni in ticks, Iraq.

Background: Tick is one of the most important ectoparasites distributed worldwide and plays an obvious role in the transmission of different infections to humans and animals as dogs. Aim: This study conducted to molecular demonstration of Babesia gibsoni in ticks of stray dogs and phylogenetic analysis of study isolates to detect their identity to global isolates. Prevalence of ticks in dogs, identification of tick species, and their relationship to some risk factors were aimed, also. Methods: A total of 97 stray dogs were inspected grossly to detect and collect ticks that existed in different body parts. After collection, all ticks were examined morphologically to identify their species, and then molecularly by the polymerase chain reaction (PCR) assay to detect B. gibsoni in different species of ticks. Local B. gibsoni isolates were sequenced, documented in the National Center For biotechnology information (NCBI) database, analyzed phylogenetically, and compared with the global GenBank-NCBI isolates. Results: In the current study, ticks were detected in 43.3% of dogs, and were shown to be varied in number and distribution among different body parts of each dog. Concerning its distribution, ticks were observed significantly on the abdomen, ear, and perineal region. In relation to risk factors, ticks were increased significantly in dogs <6 months old in comparison to older dogs, males more than females; and in rural areas more than dogs of sub-urban and urban areas. Based on morphology, different tick species were seen including Hylaomma anatolicum (86.12%), R. sanguineus (11.99%), and Rhipicephalus turanicus (1.89%). Targeting the 18S rRNA gene, PCR assay reported 3.79% positive ticks to B. gibsoni that were seen in R. sanguineus (13.16%) and H. anatolicum (2.56%). Based on phylogenetic analysis data of five local B. gibsoni isolates, this study demonstrated their close relations to the global NCBI-BLAST B. gibsoni Iraqi isolate (ID: MN385424.1). Conclusion: This represents the first Iraqi study that demonstrated molecularly B. gibsoni in different species of ticks that infected stray dogs.

DETECTION OF BABESIA CF. ODOCOILEI, BABESIA CAPREOLI, AND ANAPLASMA PHAGOCYTOPHILUM IN CERVIDS OF THE SCOTTISH HIGHLANDS, UNITED KINGDOM.

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

Prevalence of co-infections with Ehrlichia spp. or Theileria spp. in dogs naturally infected with babesiosis in the Eastern Cape province.

BACKGROUND: Canine babesiosis and ehrlichiosis are tick-borne infections of great significance in South Africa. Theileriosis in dogs in South Africa is still poorly understood. Co-infection with multiple tick-borne diseases has been documented and is perceived as a common occurrence in South Africa. OBJECTIVES: The main objective of this study was to determine the prevalence of co-infections with Ehrlichia canis or Theileria equi in dogs with babesiosis in the Eastern Cape province. There is a lack of data on canine tick-borne disease distribution in this region. Possible associations of population characteristics and haematological and biochemistry measures with a co-infection of E. canis or T. equi in these dogs were also investigated. METHOD: The study population included 150 dogs naturally infected with babesiosis that presented to the Mdantsane State Veterinary Clinic between January 2021 and November 2021. Quantitative polymerase chain reaction was used to confirm the Babesia spp. that the dogs were infected with and to identify co-infections. Association with co-infection for the following parameters were evaluated: sex, breed, age, duration of illness, leukocyte count, band neutrophil count, monocyte count, platelet count, ARC, and serum globulin concentration. Positive and negative predictive values of monocytosis, leukopenia, band neutrophilia, thrombocytopenia, and non-regenerative absolute reticulocyte count for co-infection were also calculated. RESULTS: Babesia rossi was identified in 149/150 samples and B. vogeli in only 1/150 samples. A co-infection prevalence of 2.0% (3/149; 95% CI: 0.4-5.7) with B. rossi and E. canis was found. No other co-infections were reported. No investigated variables showed significant associations with co-infections. Monocytosis, in particular, was not associated with co-infection. CONCLUSION: Co-infection with other tick-borne diseases in dogs with babesiosis is uncommon in the Eastern Cape province. These findings raise the possibility that B. rossi may have a protective effect against other tick-borne diseases.

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.

The prevalence of pathogens in ticks collected from humans in Belgium, 2021, versus 2017.

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

High serological and molecular prevalence of Ehrlichia canis and other vector-borne pathogens in dogs from Boa Vista Island, Cape Verde.

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

Significance of Anaplasma phagocytophilum, Babesia caballi, and Theileria equi as etiologic agents in horses with clinical manifestations from the metropolitan area of Rio De Janeiro, Brazil.

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

Population genetics of Babesia vogeli based on the mitochondrial cytochrome b gene.

The current study aimed at population genetic characterization of B. vogeli based on the cytochrome b (cyt b) gene sequences (≥ 685 bp) available in the GenBank. Phylogenetic trees placed all the sequences of B. vogeli in a single large monophyletic clade; however, it was further divided into two subclades (Bv1 and Bv2). Out of seven nucleotide variations observed between Bv1 and Bv2 subclades, four were synonymous (G92A, C170T, T488C and A659G), and three were non-synonymous (G324A, C438A and G465A) resulting in amino acid substitutions at three places (V108I, L146I and V155I). Within different B. vogeli populations, the nucleotide and haplotype diversities were low. The median-joining haplotype network revealed only two haplotypes (Hap_1 and Hap_2). A geographical sub-structuring was noticed in the B. vogeli populations, with moderate genetic differentiation (FST = 0.05000; P < 0.05) and a very high gene flow (Nm = 4.75) between Indian and Chinese populations. Neutrality tests and mismatch distributions for the Indian population and the overall dataset of B. vogeli indicated a constant population size. This study provides the first insight into the genetic characterization, population genetics and haplotype network of B. vogeli based on the cyt b gene.

Babesia bigemina and Theileria annulata infections in cattle: molecular detection, phylogenetic analysis, and assessment of risk factors.

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

Surveillance of tick-borne pathogens in domestic dogs from Chad, Africa.

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

Standardization of quantitative PCR (qPCR) method to detect the level of parasitaemia in Babesia gibsoni infected dogs.

The present investigation aimed to quantitatively assess the level of parasitemia in dogs using qPCR.The dogs selected for this study were infected with the haemoprotozoan parasite Babesia gibsoni. In the study, dogs diagnosed with babesiosis were divided into two groups (n = 12) and subjected to distinct treatment strategies. The first group received clindamycin-metronidazole-doxycycline (CMD) therapy, while the second group was treated with a combination of buparvaquone-azithromycin (BPV-AZM). The level of parasitemia in the infected dogs was determined using an absolute quantification-based qPCR method. This assessment was conducted both prior to initiating the treatment and on the 10th day following the commencement of the treatment protocols. On the tenth day after the initiation of treatment, the CMD group exhibited a lower level of parasitemia in comparison to the BPV-AZM group. In the CMD treated groups, the mean parasitemia decreased from 4.9E + 06 to 3.4E + 06, indicating a reduction in parasitic load. Conversely, in the BPV-AZM treatment groups, the mean parasitemia increased from 1.62E + 06 to 2.87E + 06, suggesting an increase in parasitic load. On the 10th day, the CMD-treated group demonstrated a statistically significant decline in the level of parasitemia, with a P-value of ≤0.001. This indicates a strong and significant reduction in parasitic load following the CMD treatment. Therefore, the absolute quantification-based qPCR method could effectively assess the initial treatment response by measuring the level of parasitemia.

Evaluation of the inhibitory effects of sitamaquine on Babesia infections.

The treatment strategies for either human or animal babesiosis have been established and used for many years. With the rising indications of drug resistance and adverse side effects, finding effective and alternative therapies is urgently needed. Sitamaquine (SQ) is an 8-aminoquinoline that was first synthesized as a part of the collaborative anti-malarial program that led to primaquine. In this study, we evaluated the inhibitory effects of SQ on Babesia spp. in vitro and in vivo. The half-maximal inhibitory concentration (IC50) on in vitro cultured Babesia gibsoni was 8.04 ± 1.34 µM. Babesia gibsoni parasites showed degenerative morphological changes following SQ treatment. The in vivo growth inhibitory effects of SQ were evaluated in BALB/c mice infected with B. microti and atovaquone (ATV)-resistant B. microti strain. Oral administration of SQ at a dose of 20 mg/kg significantly inhibited the growth of B. microti and ATV-resistant B. microti. Meanwhile, SQ also showed inhibitory effects on the growth of B. rodhaini, a lethal rodent Babesia species. All mice infected with B. rodhaini treated with SQ survived, whereas the mice in the control group succumbed to the disease. The results obtained in this study indicate that SQ has potent inhibition effects against Babesia spp., which support SQ as a prospective alternative candidate for babesiosis treatment.