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.

Development and evaluation of specific polymerase chain reaction assays for detecting Theileria equi genotypes.

BACKGROUND: Theileria equi causes equine piroplasmosis, an economically significant disease that affects horses and other equids worldwide. Based on 18S ribosomal RNA (18S rRNA sequences), T. equi can be classified into five genotypes: A, B, C, D, and E. These genotypes have implications for disease management and control. However, no conventional polymerase chain reaction (PCR) assays are available to differentiate the genotypes of T. equi. To overcome this limitation, we developed and evaluated PCR assays specific for the detection of each T. equi genotype. METHODS: A pair of forward and reverse primers, specifically targeting the 18S rRNA sequence of each genotype, was designed. The genotype-specific PCR assays were evaluated for their specificity using plasmids containing inserts of the 18S rRNA sequence of each genotype. Subsequently, the assays were tested on 270 T. equi-positive equine blood DNA samples (92 from donkeys in Sri Lanka and 178 from horses in Paraguay). 18S rRNA sequences derived from the PCR amplicons were analyzed phylogenetically. RESULTS: Each genotype-specific PCR assay accurately targeted the intended genotype, and did not produce any amplicons when 18S rRNA from other T. equi genotypes or genomic DNA of Babesia caballi or uninfected horse blood was used as the template. Previous studies employing PCR sequencing methods identified T. equi genotypes C and D in the Sri Lankan samples, and genotypes A and C in the Paraguayan samples. In contrast, our PCR assay demonstrated exceptional sensitivity by detecting four genotypes (A, C, D, and E) in the Sri Lankan samples and all five genotypes in the Paraguayan samples. All the Sri Lankan samples and 93.3% of the Paraguayan samples tested positive for at least one genotype, further emphasizing the sensitivity of our assays. The PCR assays also had the ability to detect co-infections, where multiple genotypes in various combinations were detected in 90.2% and 22.5% of the Sri Lankan and Paraguayan samples, respectively. Furthermore, the sequences obtained from PCR amplicons clustered in the respective phylogenetic clades for each genotype, validating the specificity of our genotype-specific PCR assays. CONCLUSIONS: The genotype-specific PCR assays developed in the present study are reliable tools for the differential detection of T. equi genotypes.

THE FIRST SURVEY OF BOVINE BABESIA SPECIES INFECTING YAKS (BOS GRUNNIENS) IN MONGOLIA.

Yak (Bos grunniens) farming is an important part of Mongolia's livestock industry. Yaks survive in harsh mountain environments; provide meat, milk, and wool; and serve as a mode of transportation. In Mongolia, yaks are frequently raised alongside other livestock animals such as cattle, Bactrian camels, sheep, goats, and horses. Recently, we demonstrated that Babesia bovis, Babesia bigemina, and Babesia naoakii-parasites with the potential to cause clinical bovine babesiosis-infect not only cattle but also Bactrian camels in Mongolia. However, yaks have never been surveyed for Babesia infections in this country. In the present study, we surveyed yaks in 8 Mongolian provinces: Bayankhongor, Bayan-Ulgii, Khovd, Khovsgol, Omnogovi, Ovorkhangai, Uvs, and Zavkhan. Blood samples were taken and deoxyribonucleic acid (DNA) was extracted from 375 yaks. Furthermore, Giemsa-stained thin smears were prepared from 315 of the 375 blood samples and then examined for the microscopic detection of Babesia parasites. Microscopy revealed that 34 (10.8%) of 315 blood smears were positive for Babesia parasites. All 375 DNA samples were then tested for B. bovis, B. bigemina, and B. naoakii infection using specific polymerase chain reaction assays. We observed that 238 (63.5%) yaks in all surveyed provinces and 8 (2.1%) yaks in 3 provinces (Bayankhongor, Bayan-Ulgii, and Omnogovi) were positive for B. bovis and B. bigemina, respectively. However, all yaks tested were negative for B. naoakii. This epidemiological survey, the first to report Babesia infection in Mongolian yaks, suggests that disease management strategies for yaks in this country should further address bovine babesiosis.

An epidemiological survey of vector-borne pathogens infecting cattle in Kyrgyzstan.

Cattle production is a major contributor to the national economy of Kyrgyzstan. Most cattle in Kyrgyzstan are managed via extensive systems and graze in communal pastures. As a result, infestations with ectoparasites are widespread, implying that various vector-borne diseases might be common in cattle. However, methods to control such infectious diseases are not available in Kyrgyzstan because the epidemiology of vector-borne pathogens (VBPs) infecting cattle remains unclear. The present study was therefore designed to survey Kyrgyz cattle for VBPs. We prepared blood DNA samples from 319 cattle in Kyrgyzstan and screened them with specific PCR assays for detecting Babesia bovis, Babesia bigemina, Babesia naoakii, Theileria annulata, Theileria orientalis, Trypanosoma evansi, Trypanosoma theileri, and Anaplasma marginale infections. Our findings indicated that the surveyed cattle were infected with six of the eight pathogens targeted, with the exceptions being B. naoakii and Try. evansi. The most common pathogen was T. orientalis (84.3%), followed by B. bigemina (47.6%), T. annulata (16.6%), A. marginale (11.6%), Try. theileri (7.2%), and B. bovis (2.5%). Additional screening of the B. bovis- and B. bigemina-negative samples with a Babesia genus-specific 18S rRNA PCR identified two positive samples, and sequencing analysis confirmed that each of them was infected with either Babesia major or Babesia occultans. To the best of our knowledge, this is the first report of B. bovis, B. bigemina, B. occultans, Try. theileri, and A. marginale infections in cattle in Kyrgyzstan. Our findings suggest that cattle in Kyrgyzstan are at high risk of infectious diseases caused by VBPs.

Disruption of a DNA fragment that encodes the microneme adhesive repeat domain-containing region of the BBOV_III011730 does not affect the blood stage growth of Babesia bovis in vitro.

Babesia bovis, an intraerythrocytic hemoprotozoan parasite, causes the most pathogenic form of bovine babesiosis, negatively impacting the cattle industry. Comprehensive knowledge of B. bovis biology is necessary for developing control methods. In cattle, B. bovis invades the red blood cells (RBCs) and reproduces asexually. Micronemal proteins, which bind to sialic acid of host cells via their microneme adhesive repeat (MAR) domains, are believed to play a key role in host cell invasion by apicomplexan parasites. In this study, we successfully deleted the region encoding MAR domain of the BBOV_III011730 by integrating a fusion gene of enhanced green fluorescent protein-blasticidin-S-deaminase into the genome of B. bovis. The transgenic B. bovis, lacking the MAR domain of the BBOV_III011730, invaded bovine RBCs in vitro and grew at rates similar to the parental line. In conclusion, our study revealed that the MAR domain is non-essential for the intraerythrocytic development of B. bovis in vitro.

PCR detection of Theileria equi and Babesia caballi in apparently healthy horses in Paraguay.

Equine piroplasmosis (EP) is a tick-borne disease caused by Theileria equi and Babesia caballi in equids, including horses. EP has a global distribution and often leads to a significant socioeconomic impact on the equine industry. Infected animals remain as carriers and become a source of infection for tick vectors, thereby posing an immense challenge in the disease management. Therefore, detection of these carriers is crucial to assess the risk of transmission and to implement appropriate control measures in endemic countries. Paraguay is a tropical country where various tick-borne diseases are common among livestock; however, the status of EP remains unknown in this country. Because the tick vectors capable of transmitting T. equi and B. caballi are endemic in Paraguay, we hypothesised that Paraguayan horses are infected with these parasite species. To test our hypothesis, we prepared blood DNA samples from a total of 545 apparently healthy horses in 16 of the 17 departments of Paraguay and analysed them with specific PCR assays to detect T. equi and B. caballi. The PCR results showed that 178 (32.7%) and 8 (1.5%) of the horses were infected with T. equi and B. caballi, respectively. Among the infected horses, two (0.4%) were infected with both parasite species. Our analyses further indicated that the positive rates of T. equi infection did not differ between horse breeds, males and females, or age groups. We also found that haematological parameters were the same between the non-infected animals and animals with single infections. By contrast, the two horses co-infected with T. equi and B. caballi had haemoglobin and haematocrit values lower than the normal ranges. In conclusion, the present study demonstrated that Paraguayan horses are infected with T. equi and B. caballi and that the rate of T. equi infection is higher than that of B. caballi. Our findings highlight the need to add EP to the list of differential diagnoses when anaemic horses are presented to equine clinics in Paraguay.

Phylogenetic analyses of the mitochondrial, plastid, and nuclear genes of Babesia sp. Mymensingh and its naming as Babesia naoakii n. sp.

BACKGROUND: The recently discovered Babesia sp. Mymensingh, which causes clinical bovine babesiosis, has a wide geographical distribution. We investigated the phylogenetic position of Babesia sp. Mymensingh using its mitochondrial, plastid, and nuclear genes. Based on morphological and molecular data, Babesia sp. Mymensingh is a unique species and we named it as Babesia naoakii n. sp. METHODS: A blood DNA sample from a Babesia sp. Mymensingh-infected cow was subjected to genome sequencing to obtain the sequences of mitochondrial, plastid, and nuclear genes. Six phylogenetic trees were then constructed with (1) concatenated amino acid sequences of cytochrome oxidase subunit I, cytochrome oxidase subunit III, and cytochrome b genes of the mitochondrial genome; (2) 16S rRNA of the plastid genome; (3) nucleotide sequences of the elongation factor Tu gene of the plastid genome; (4) ITS1-5.8S rRNA-ITS2; (5) concatenated nucleotide sequences of 89 nuclear genes; and (6) concatenated amino acid sequences translated from the 89 nuclear genes. RESULTS: In all six phylogenetic trees, B. naoakii n. sp. formed a sister clade to the common ancestor of Babesia bigemina and B. ovata. The concatenated nuclear genes of B. naoakii n. sp. and their translated amino acid sequences shared lower identity scores with the sequences from B. bigemina (82.7% and 84.7%, respectively) and B. ovata (83.5% and 85.5%, respectively) compared with the identity scores shared between the B. bigemina and B. ovata sequences (86.3% and 87.9%, respectively). CONCLUSIONS: Our study showed that B. naoakii n. sp. occupies a unique phylogenetic position distinct from existing Babesia species. Our findings, together with morphological differences, identify B. naoakii n. sp. as a distinct parasite species.

Molecular survey of Babesia, Theileria, Trypanosoma, and Anaplasma infections in camels (Camelus dromedaries) in Egypt.

The one-humped camel (Camelus dromedarius) or dromedary is an economically important domestic animal. However, infectious diseases, including those caused by vector-borne hemopathogens, frequently compromise the health and production of camels. In this study, we examined infections caused by Babesia, Theileria, Trypanosoma, and Anaplasma species in camels in Egypt. We analyzed blood DNA samples from 148 camels reared in six Egyptian governorates (Giza, Asyut, Sohag, Qena, Luxor, and the Red Sea) using pathogen-specific Polymerase Chain Reaction (PCR) assays. Our results indicated that 29 (19.6%), 22 (14.9%), 1 (0.7%), 2 (1.4%), 1 (0.7%), 2 (1.4%), and 28 (18.9%) of the surveyed animals were infected with Babesia bovis, B. bigemina, Babesia sp. Mymensingh, Theileria sp. Yokoyama, Theileria equi, Trypanosoma evansi, and Anaplasma marginale, respectively. We found that a total of 68 (45.9%) animals were infected with at least one of the detected hemopathogens. Sequencing analyses of PCR amplicons confirmed our diagnostic results. This study is the first to report Theileria sp. Yokoyama and Babesia sp. Mymensingh in Egypt. This is also the first report of infection with these two species in one-humped camel. In conclusion, this study found that camels in Egypt are infected with several vector-borne hemopathogens, including novel parasite species.

First detection of Theileria equi in free-roaming donkeys (Equus africanus asinus) in Sri Lanka.

Equine piroplasmosis (EP) is a tick-borne disease caused by Theileria equi and Babesia caballi in equids, including horses, donkeys, zebras, and mules. It is globally endemic with significant economic impact on the equine industry. Infected animals may serve as carriers, and they may be a source of infection for ticks, thereby posing a great challenge for disease management. Sri Lanka is a tropical country, where infections by various tick-borne parasites are common among livestock animals. However, infections by T. equi and B. caballi remain unstudied in Sri Lanka. Therefore, in the present study, we conducted an epidemiological survey to investigate the presence of T. equi and B. caballi in apparently healthy free-roaming donkeys. Blood samples were randomly taken from 111 donkeys in Mannar (n = 100) and Kilinochchi (n = 11) districts in Sri Lanka. Thin blood smears were prepared from the blood samples and subjected to microscopic examination. Additionally, blood DNA samples were prepared and screened for T. equi and B. caballi infections using species-specific PCR assays. Our results showed that 64 (57.7%) and 95 (85.6%) of the donkeys were positive for T. equi by microscopy and PCR, respectively. However, all samples were negative for B. caballi. Phylogenetic analysis of the T. equi 18S rRNA sequences detected two distinct genotypes, namely C and D. To our knowledge, this is the first report of T. equi in Sri Lanka and of genotype C in donkeys. The present study highlights the importance of monitoring the shrinking donkey population in Sri Lanka owing to EP caused by T. equi.

Combination of Clofazimine and Atovaquone as a Potent Therapeutic Regimen for the Radical Cure of Babesia microti Infection in Immunocompromised Hosts.

Human babesiosis caused by Babesia microti can be fatal in immunocompromised patients, and the currently used drugs are often ineffective. A recent study found that clofazimine clears B. microti Munich strain in immunocompromised mice. In the present study, we investigated the efficacies of clofazimine and 2-drug combinations involving clofazimine, atovaquone, and azithromycin against B. microti Peabody mjr strain in immunocompromised mice. Treatment with clofazimine alone, clofazimine plus azithromycin, and atovaquone plus azithromycin was ineffective and failed to eliminate the parasites completely, while a 44-day treatment with clofazimine plus atovaquone was highly effective and resulted in a radical cure.

Molecular survey of bovine Babesia species in Bactrian camels (Camelus bactrianus) in Mongolia.

Bovine babesiosis, which is caused by species of genus Babesia, is a leading cause of considerable economic losses to the cattle industry each year. Bovine Babesia species have frequently been detected in non-cattle hosts, such as water buffalo (Bubalus bubalis), from which the parasites can be transmitted by ticks to cattle. Therefore, Babesia infections should be minimized not only in cattle but also in non-cattle carriers. In the present study, we surveyed the Bactrian camels (Camelus bactrianus) in Mongolia for three clinically significant bovine Babesia species, including Babesia bovis, B. bigemina, and Babesia sp. Mymensingh, which had been detected previously in Mongolian cattle. We screened blood DNA samples from 305 Bactrian camels in six Mongolian provinces for these species, using parasite-specific PCR assays. Our findings showed that the Bactrian camels in Mongolia were infected with all three Babesia species surveyed. The overall positive rates of B. bovis, B. bigemina, and Babesia sp. Mymensingh were 32.1%, 21.6%, and 24.3%, respectively, whereas 52.5% of the surveyed animals were infected with at least one parasite species. We also found that the female Bactrian camels and the Mongolian native camel breed had significantly higher Babesia positive rates than the male Bactrian camels and the Hos Zogdort breed. In Mongolia, cattle and Bactrian camels usually share common pasture lands for grazing; furthermore, tick species infesting cattle also infest Bactrian camels. Our findings, together with these observations, suggest that the tick transmission of bovine Babesia species might be possible between cattle and Bactrian camels. Therefore, strategies for the control of bovine babesiosis in Mongolia should include methods to minimize bovine Babesia species infections in Bactrian camels.

Structural and immunological characterization of an epitope within the PAN motif of ectodomain I in Babesia bovis apical membrane antigen 1 for vaccine development.

BACKGROUND: Bovine babesiosis caused by Babesia bovis (B. bovis) has had a significant effect on the mobility and mortality rates of the cattle industry worldwide. Live-attenuated vaccines are currently being used in many endemic countries, but their wide use has been limited for a number of reasons. Although recombinant vaccines have been proposed as an alternative to live vaccines, such vaccines are not commercially available to date. Apical membrane antigen-1 (AMA-1) is one of the leading candidates in the development of a vaccine against diseases caused by apicomplexan parasite species. In Plasmodium falciparum (P. falciparum) AMA-1 (PfAMA-1), several antibodies against epitopes in the plasminogen, apple, and nematode (PAN) motif of PfAMA-1 domain I significantly inhibited parasite growth. Therefore, the purpose of this study was to predict an epitope from the PAN motif of domain I in the B. bovis AMA-1 (BbAMA-1) using a combination of linear and conformational B-cell epitope prediction software. The selected epitope was then bioinformatically analyzed, synthesized as a peptide (sBbAMA-1), and then used to immunize a rabbit. Subsequently, in vitro growth- and the invasion-inhibitory effects of the rabbit antiserum were immunologically characterized. RESULTS: Our results demonstrated that the predicted BbAMA-1 epitope was located on the surface-exposed α-helix of the PAN motif in domain I at the apex area between residues 181 and 230 with six polymorphic sites. Subsequently, sBbAMA-1 elicited antibodies capable of recognizing the native BbAMA-1 in immunoassays. Furthermore, anti-serum against sBbAMA-1 was immunologically evaluated for its growth- and invasion-inhibitory effects on B. bovis merozoites in vitro. Our results demonstrated that the rabbit anti-sBbAMA-1 serum at a dilution of 1:5 significantly inhibited (p < 0.05) the growth of B. bovis merozoites by approximately 50-70% on days 3 and 4 of cultivation, along with the invasion of merozoites by approximately 60% within 4 h of incubation when compared to the control groups. CONCLUSION: Our results indicate that the epitope predicted from the PAN motif of BbAMA-1 domain I is neutralization-sensitive and may serve as a target antigen for vaccine development against bovine babesiosis caused by B. bovis.

Serosurvey for equine piroplasms in horses and donkeys from North-Western Nigeria using IFAT and ELISA.

Equine piroplasmosis is caused by apicomplexan parasites, namely, Babesia caballi and Theileria equi, which are transmitted to equids principally through ticks. To ascertain the exposure of equines to agents of equine piroplasms, we tested serum samples collected from horses (n = 272) and donkeys (n = 170) in North-Western Nigeria for the presence of antibodies against B. caballi and T. equi using IFAT and ELISA. The seroprevalence of T. equi in the horses determined using IFAT and ELISA was 48.89% and 45.96%, respectively, while for B. caballi, it was 6.3% and 0.4%, respectively. For T. equi, the seroprevalence based on IFAT and ELISA results in donkeys was 14.1% and 2.9%, respectively, while for B. caballi, the seroprevalence was 2.4% and 0.6%, respectively, for ELISA and IFAT. Mixed infection detected in the horses using IFAT and ELISA was 5.5% and 0.4%, respectively, while no mixed infection was observed in the donkeys. The seroprevalence of T. equi was significantly (P < .0001) higher than that of B. caballi in both horses and donkeys. Comparatively, the IFAT detected a greater number of piroplasm seropositive animals than ELISA, indicating a difference in their diagnostic accuracy. Findings from this study confirm the existence of equine piroplasms in both horses and donkeys in North-Western Nigeria and highlights the need for robust and effective control measures against the disease.

Development of a stable transgenic Theileria equi parasite expressing an enhanced green fluorescent protein/blasticidin S deaminase.

Theileria equi, an intraerythrocytic protozoan parasite, causes equine piroplasmosis, a disease which negatively impacts the global horse industry. Genetic manipulation is one of the research tools under development as a control method for protozoan parasites, but this technique needs to be established for T. equi. Herein, we report on the first development of a stable transgenic T. equi line expressing enhanced green fluorescent protein/blasticidin S deaminase (eGFP/BSD). To express the exogenous fusion gene in T. equi, regulatory regions of the elongation factor-1 alpha (ef-1α) gene were identified in T. equi. An eGFP/BSD-expression cassette containing the ef-1α gene promoter and terminator regions was constructed and integrated into the T. equi genome. On day 9 post-transfection, blasticidin-resistant T. equi emerged. In the clonal line of T. equi obtained by limiting dilution, integration of the eGFP/BSD-expression cassette was confirmed in the designated B-locus of the ef-1α gene via PCR and Southern blot analyses. Parasitaemia dynamics between the transgenic and parental T. equi lines were comparable in vitro. The eGFP/BSD-expressing transgenic T. equi and the methodology used to generate it offer new opportunities for better understanding of T. equi biology, with the add-on possibility of discovering effective control methods against equine piroplasmosis.

Geographic origin of Explanatum explanatum (Creplin, 1847) Fukui, 1929 detected from domestic water buffaloes in Sri Lanka.

The adult stage of Explanatum explanatum has economic importance in the production of ruminants, especially water buffaloes. This species has been widely reported in the Indian sub-continent. Recently, molecular analyses to reveal the dispersal route of this species were performed in Bangladesh, Nepal, and India. In the present study, we focused on E. explanatum distributed in Sri Lanka. A total of 52 flukes were collected from water buffaloes in Sri Lanka and identified as E. explanatum based on the internal transcribed spacer 2 (ITS2) region of nuclear ribosomal DNA. Analysis of the mitochondrial NADH dehydrogenase subunit 1 (nad1) gene from DNA samples detected 18 haplotypes, and five of them were identical to those from the Indian E. explanatum. The pairwise fixation index value indicated that the Sri Lankan population had a comparatively closer relationship with the Indian population than with the Bangladeshi or Nepalese populations. The Sri Lankan population showed significantly lower genetic variability than the Indian population, suggesting that the Indian population was the ancestor of the Sri Lankan population. The movement of host ruminants, including water buffaloes, was probably involved in the introduction of the fluke into Sri Lanka. The results of our study provide useful information for elucidating the geographic origin of E. explanatum distributed in the Indian subcontinent.

Molecular detection of Borrelia burgdorferi (sensu lato) and Rickettsia spp. in hard ticks distributed in Tokachi District, eastern Hokkaido, Japan.

Ticks transmit various pathogens, including parasites, bacteria and viruses to humans and animals. To investigate the ticks and the potentially zoonotic pathogens that they may carry, questing ticks were collected in 2017 from 7 sites in Tokachi District, eastern Hokkaido, Japan. A total of 1563 ticks including adults (male and female), nymphs and larvae were collected. Four species of ticks were identified: Ixodes ovatus, Ixodes persulcatus, Haemaphysalis japonica and Haemaphysalis megaspinosa. Of the 1563 ticks, 1155 were used for DNA extraction. In total, 527 individual tick DNA samples prepared from adults (n = 484), nymphs (n = 41) and larvae (n = 2); and 67 pooled tick DNA samples prepared from larval stages (n = 628) were examined using PCR methods and sequencing to detect Borrelia burgdorferi (sensu lato) and Rickettsia spp. The phylogenetic analysis of Borrelia spp. flaB gene sequences showed the presence of the human pathogenic B. burgdorferi (s.l.) species (Borrelia garinii, Borrelia bavariensis and Borrelia afzelii) in I. persulcatus, whereas the non-pathogenic species Borrelia japonica was found only in I. ovatus. In I. persulcatus, B. garinii and/or its closely related species B. bavariensis was detected in both adults and nymphs at a prevalence of 21.9% whereas B. afzelii was found only in adults (1.8%). The prevalence of B. japonica in adult I. ovatus was 21.8%. Rickettsia species were identified through phylogenetic analysis based on gltA, 16S rRNA, ompB and sca4 genes. Four genotypes were detected in the samples which were classified into three species. The prevalence of human pathogenic Rickettsia helvetica was 26.0% in I. persulcatus adults and nymphs, 55.6% in I. persulcatus larval pools, and 1.7% in H. megaspinosa larval pools. The prevalence of "Candidatus R. tarasevichiae" was 15.4% in I. persulcatus adults and nymphs and 33.3% in I. persulcatus larval pools. The prevalence of "Candidatus R. principis" in H. megaspinosa adults and nymphs was 11.1% whereas it was detected in 3.4% of the H. megaspinosa larval pools. These results indicate that most of the risks of Lyme borreliosis and spotted fever group rickettsiosis infection in eastern Hokkaido, Japan, are restricted to I. persulcatus.

Molecular evidence of Babesia caballi and Theileria equi in equines and ticks in Nigeria: prevalence and risk factors analysis.

Babesia caballi and Theileria equi are biological agents responsible for equine piroplasmosis (EP). We conducted a robust and extensive epidemiological study in Nigeria on the prevalence and risk factors of EP. Blood (468, both horses and donkeys) and ticks (201 pools) were screened using polymerase chain reaction (PCR). DNA of equine piroplasms was observed in tick pools with B. caballi amplified in Rhipicephalus evertsi evertsi only [minimum infection rate (MIR) of 7.6%] while T. equi was observed in R. e. evertsi (MIR, 61.6%), Hyalomma dromedarii (MIR, 23.7%) and H. truncatum (MIR, 50.0%). Overall results showed that 196/468 (41.9%) animals were positive for equine piroplasms (both B. caballi and T. equi). The prevalence for T. equi was 189/468 (40.4%) compared to 7/468 (1.5%) for B. caballi. In the horses and donkeys, respectively, the prevalence for T. equi was (39.9%; 112/281) and (41.2%; 77/187) compared with (1.4%; 4/281) and (1.6%; 3/187) due to B. caballi. Our analysis showed that location (Jigawa state), Talon breed, horses used for work and reproduction, unsatisfactory husbandry practices, contact with other mammals are risk factors that associated positivity to T. equi infection in horses, whilst horses kept on intensive management appeared to be less prone to infection. On the other hand, Jangora breed of donkeys and location (Jigawa state) are risk factors to infection with T. equi in donkeys. Findings suggest the persistence of EP in equids and ticks in Nigeria.

Activities of artesunate-based combinations and tafenoquine against Babesia bovis in vitro and Babesia microti in vivo.

BACKGROUND: Babesiosis represents a veterinary and medical threat, with a need for novel drugs. Artemisinin-based combination therapies (ACT) have been successfully implemented for malaria, a human disease caused by related parasites, Plasmodium spp. The aim of this study was to investigate whether ACT is active against Babesia in vitro and in vivo. METHODS: Mefloquine, tafenoquine, primaquine, methylene blue and lumefantrine, alone or in combination with artesunate, were tested in vitro against Babesia bovis. Parasite growth was verified using a SYBR green I-based fluorescence assay. Mice infected with Babesia microti were treated with mefloquine or tafenoquine, alone or in combination with artesunate, and parasitemia was verified by microscopy and PCR. RESULTS: All drugs, except lumefantrine, showed in vitro activity against B. bovis, with methylene blue showing the most potent activity (concentration 0.2 µM). Combination with artesunate led to improved activity, with mefloquine showing a striking 20-fold increase in activity. Tafenoquine (10 mg/kg, base), combined or not with artesunate, but not mefloquine, induced rapid clearance of B. microti in vivo by microscopy, but mice remained PCR-positive. Blood from mice treated with tafenoquine alone, but not with tafenoquine-artesunate, was infective for naive mice upon sub-inoculation. CONCLUSIONS: Tafenoquine, and most likely other 8-aminoquinoline compounds, are promising compounds for the development of ACT for babesiosis.

Clofazimine, a Promising Drug for the Treatment of Babesia microti Infection in Severely Immunocompromised Hosts.

BACKGROUND: Persistent and relapsing babesiosis caused by Babesia microti often occurs in immunocompromised patients, and has been associated with resistance to antimicrobial agents such as atovaquone. Given the rising incidence of babesiosis in the United States, novel drugs are urgently needed. In the current study, we tested whether clofazimine (CFZ), an antibiotic used to treat leprosy and drug-resistant tuberculosis, is effective against B. microti. METHODS: Mice with severe combined immunodeficiency were infected with 107B. microti-infected erythrocytes. Parasites were detected by means of microscopic examination of Giemsa-stained blood smears or nested polymerase chain reaction. CFZ was administered orally. RESULTS: Uninterrupted monotherapy with CFZ curtailed the rise of parasitemia and achieved radical cure. B. microti parasites and B. microti DNA were cleared by days 10 and 50 of therapy, respectively. A 7-day administration of CFZ delayed the rise of parasitemia by 22 days. This rise was caused by B. microti isolates that did not carry mutations in the cytochrome b gene. Accordingly, a 14-day administration of CFZ was sufficient to resolve high-grade parasitemia caused by atovaquone-resistant B. microti parasites. CONCLUSIONS: Clofazimine is effective against B. microti infection in the immunocompromised host. Additional preclinical studies are required to identify the minimal dose and dosage of CFZ for babesiosis.

Molecular epidemiological survey of Babesia bovis, Babesia bigemina, and Babesia sp. Mymensingh infections in Mongolian cattle.

Bovine babesiosis caused by Babesia species is an economically significant disease of cattle. Severe clinical babesiosis in cattle is caused by Babesia bovis, B. bigemina, and the recently discovered Babesia sp. Mymensingh. Mongolia is an agricultural country with a large cattle inventory. Although previous studies have detected active infections of B. bovis and B. bigemina in Mongolian cattle, only a few provinces were surveyed. Additionally, the endemicity of Babesia sp. Mymensingh in Mongolia remains unknown. We screened blood DNA samples from 725 cattle reared in 16 of the 21 Mongolian provinces using B. bovis-, B. bigemina-, and Babesia. sp. Mymensingh-specific PCR assays. The overall positive rates of B. bovis, B. bigemina, and Babesia sp. Mymensingh were 27.9% (n = 202), 23.6% (n = 171), and 5.4% (n = 39), respectively. B. bovis and B. bigemina were detected in cattle in all surveyed provinces; whereas Babesia sp. Mymensingh was detected in 11 of the 16 surveyed provinces. On a per province basis, the B. bovis- B. bigemina-, and Babesia sp. Mymensingh-positive rates were 5.9-52.0%, 9.1-76.3%, and 0-35.7%, respectively. In conclusion, this is the first report of Babesia sp. Mymensingh in Mongolia. In addition, we found that species of Babesia that are capable of causing bovine clinical babesiosis, including B. bovis, B. bigemina, and Babesia sp. Mymensingh, are widespread throughout the country.