Genetic analyses of rabies virus glycoprotein and nucleoprotein gene sequences reveal the emergence of multiple lineages in animals in Arkhangai province, a central region of Mongolia.

Genetic characterizations of rabies viruses circulating in carnivore and non-carnivore animals were investigated for the first time in Arkhangai province, a central region of Mongolia. Also, glycoprotein gene of the rabies virus was sequenced for the first time in Mongolia. The nucleotide sequences of the glycoprotein and nucleoprotein genes were analysed, revealing the presence of multiple lineages in this area. Of particular concern are the lineages identified in carnivores, which might emerge to spread throughout Mongolia, further facilitating transboundary transmission to neighbouring countries, including China and Russia.

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.

Identification and Characterization of Rhipicephalus microplus ATAQ Homolog from Haemaphysalis longicornis Ticks and Its Immunogenic Potential as an Anti-Tick Vaccine Candidate Molecule.

Although vaccines are one of the environmentally friendly means to prevent the spread of ticks, there is currently no commercial vaccine effective against Haemaphysalis longicornis ticks. In this study, we identified, characterized, localized, and evaluated the expression patterns, and tested the immunogenic potential of a homologue of Rhipicephalus microplus ATAQ in H. longicornis (HlATAQ). HlATAQ was identified as a 654 amino acid-long protein present throughout the midgut and in Malpighian tubule cells and containing six full and one partial EGF-like domains. HlATAQ was genetically distant (homology < 50%) from previously reported ATAQ proteins and was expressed throughout tick life stages. Its expression steadily increased (p < 0.001) during feeding, reached a peak, and then decreased slightly with engorgement. Silencing of HlATAQ did not result in a phenotype that was significantly different from the control ticks. However, H. longicornis female ticks fed on a rabbit immunized with recombinant HlATAQ showed significantly longer blood-feeding periods, higher body weight at engorgement, higher egg mass, and longer pre-oviposition and egg hatching periods than control ticks. These findings indicate that the ATAQ protein plays a role in the blood-feeding-related physiological processes in the midgut and Malpighian tubules and antibodies directed against it may affect these tissues and disrupt tick engorgement and oviposition.

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.

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.

Inhibitory effect of naphthoquine phosphate on Babesia gibsoni in vitro and Babesia rodhaini in vivo.

BACKGROUND: Drug resistance and toxic side effects are major challenges in the treatment of babesiosis. As such, new drugs are needed to combat the emergence of drug resistance in Babesia parasites and to develop alternative treatment strategies. A combination of naphthoquine (NQ) and artemisinin is an antimalarial therapy in pharmaceutical markets. The present study repurposed NQ as a drug for the treatment of babesiosis by evaluating the anti-Babesia activity of naphthoquine phosphate (NQP) alone. METHODS: An in vitro growth inhibition assay of NQP was tested on Babesia gibsoni cultures using a SYBR Green I-based fluorescence assay. In addition, the in vivo growth inhibitory effect of NQP was evaluated using BALB/c mice infected with Babesia rodhaini. The parasitemia level and hematocrit values were monitored to determine the therapeutic efficacy of NQP and the clinical improvements in NQP-treated mice. RESULTS: The half maximal inhibitory concentration of NQP against B. gibsoni in vitro was 3.3 ± 0.5 µM. Oral administration of NQP for 5 consecutive days at a dose of 40 mg/kg of body weight resulted in significant inhibition of B. rodhaini growth in mice as compared with that of the control group. All NQP-treated mice survived, whereas the mice in the control group died between days 6 and 9 post-infection. CONCLUSION: This is the first study to evaluate the anti-Babesia activity of NQP in vitro and in vivo. Our findings suggest that NQP is a promising drug for treating Babesia infections, and drug repurposing may provide new treatment strategies for babesiosis.

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.

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.

Host range and geographical distribution of Babesia sp. Mymensingh.

Bovine babesiosis represents a serious threat to the cattle industry in the tropics and subtropics. Although several Babesia species infect cattle, only B. bovis, B. bigemina and B. divergens are known to cause clinical babesiosis. However, our recent study demonstrated that the newly discovered Babesia sp. Mymensingh might be a virulent species capable of causing clinical babesiosis in cattle. The objective of this study was to determine the host range and geographical distribution of Babesia sp. Mymensingh on a global scale. A total of 2,860 archived DNA samples from 2,263 cattle in Sri Lanka (n = 672), the Philippines (n = 408), Vietnam (n = 460), Uganda (n = 409), Brazil (n = 164) and Argentina (n = 150); 419 buffalo in Sri Lanka (n = 327) and Vietnam (n = 92); and 127 goats and 51 sheep in Vietnam were screened using a Babesia sp. Mymensingh-specific PCR assay. Babesia sp. Mymensingh infection was detected in cattle, buffalo, sheep and goats. Cattle of all countries surveyed in this study except Brazil were found to be infected with Babesia sp. Mymensingh. The highest positive rates were recorded in cattle from the Philippines (11.3%) and Vietnam (9.6%), followed by Argentina (4.7%), Sri Lanka (1.5%) and Uganda (1.0%). Buffalo were found to be infected with this parasite in Sri Lanka (1.2%) and Vietnam (10.9%). Unexpectedly, Babesia sp. Mymensingh was also detected in sheep (2.0%) and goats (1.3%) from Vietnam. These findings were confirmed by PCR amplicon sequencing. In conclusion, our present findings indicate that Babesia sp. Mymensingh, which infects cattle, buffalo, sheep and goats, is endemic in Asia, Africa and South America.

Discovery of a new Theileria sp. closely related to Theileria annulata in cattle from Sri Lanka.

Theileria annulata is a haemoprotozoan parasite that causes a cancer-like illness known as tropical theileriosis in cattle. In the course of analyzing the genetic diversity of T. annulata in Sri Lanka, we observed that merozoite-piroplasm surface antigen (tams1) and surface protein (tasp)-like gene sequences obtained from bovine blood DNA samples, which were PCR-positive for T. annulata, were conserved but shared low identity with T. annulata GenBank sequences. Moreover, the 18S rRNA sequences from the Sri Lankan samples contained ten unique single-nucleotide polymorphisms compared with all known T. annulata sequences. The cytochrome b (cob) gene sequences isolated from the Sri Lankan samples were highly conserved and shared low identity scores with similarly conserved T. annulata sequences from GenBank. Phylogenetic analysis showed that the Sri Lankan tams1-like, tasp-like, 18S rRNA, and cob sequences clustered together and formed sister clades to the common ancestors of all known T. annulata and Theileria lestoquardi sequences. These findings demonstrated that the Sri Lankan cattle were not infected with T. annulata but with a new Theileria sp. (designated as Theileria sp. Yokoyama) closely related to T. annulata.

Screening the Medicines for Malaria Venture Pathogen Box against piroplasm parasites.

Diminazene aceturate (DA) and imidocarb dipropionate are commonly used in livestock as antipiroplasm agents. However, toxic side effects are common in animals treated with these two drugs. Therefore, evaluations of novel therapeutic agents with high efficacy against piroplasm parasites and low toxicity to host animals are of paramount importance. In this study, the 400 compounds in the Pathogen Box provided by the Medicines for Malaria Venture foundation were screened against Babesia bovis, Babesia bigemina, Babesia caballi, and Theileria equi. A fluorescence-based method using SYBR Green 1 stain was used for initial in vitro screening and determination of the half maximal inhibitory concentration (IC50). The initial in vitro screening performed using a 1 µM concentration as baseline revealed nine effective compounds against four tested parasites. Two "hit" compounds, namely MMV021057 and MMV675968, that showed IC50 < 0.3 µM and a selectivity index (SI)> 100 were selected. The IC50s of MMV021057 and MMV675968 against B. bovis, B. bigemina, T. equi and B. caballi were 23, 39, 229, and 146 nM, and 2.9, 3, 25.7, and 2.9 nM, respectively. In addition, a combination of MMV021057 and DA showed additive or synergistic effects against four tested parasites, while combinations of MMV021057 with MMV675968 and of MMV675968 with DA showed antagonistic effects. In mice, treated with 50 mg/kg MMV021057 and 25 mg/kg MMV675968 inhibited the growth of Babesia microti by 54 and 64%, respectively, as compared to the untreated group on day 8. Interestingly, a combination treatment with 6.25 mg/kg DA and 25 mg/kg MMV021057 inhibited B. microti by 91.6%, which was a stronger inhibition than that by single treatments with 50 mg/kg MMV021057 and 25 mg/kg DA, which showed 54 and 83% inhibition, respectively. Our findings indicated that MMV021057, MMV675968, and the combination treatment with MMV021057 and DA are prospects for further development of antipiroplasm drugs.

Flavonoids isolated from the flowers of Pulsatilla flavescens and their anti-piroplasm activity.

Pulsatilla species are known as "Yargui", and their flowers are traditionally used in Mongolia as a tonic and for the treatment of inflammatory diseases. By chemical investigation of P. flavescens flowers, 21 flavonoids, including a new chalcone C-glucoside, chalconaringenin 2'-O-ß-D-glucopyranosyl-5'-ß-D-glucopyranoside, and two new flavanone C-glucosides, (2R)- and (2S)-naringenin 8-ß-D-glucopyranosyl-4'-O-ß-D-glucopyranoside, were isolated. The absolute configurations of the seven flavanone glucosides were elucidated by ECD spectra. For the isolated compounds, inhibitory activity against Babesia caballi and Theileria equi, which cause fatal diseases in horses, was estimated. Although most of the isolated chalcone and flavanone derivatives did not show any anti-piroplasm activity, all the isolated flavone and flavonol derivatives showed moderate effects against B. caballi and/or T. equi.

Development of unstable resistance to diminazene aceturate in Babesia bovis.

Diminazene aceturate (DA) is commonly used in the treatment of bovine babesiosis caused by Babesia bovis. In this study, we attempted to develop resistance in B. bovis in vitro to DA and clofazimine (CF, a novel antibabesial agent) using short- and long-term drug pressures. In the short term, we found that 6.7 ±â€¯2 (0.54 ±â€¯0.16 µM)-, 12.9 ±â€¯8.6 (1.05 ±â€¯0.7 µM)-, and 14 ±â€¯5.9 (1.14 ±â€¯0.48 µM)-fold increases in the half-maximal inhibitory concentration (IC50) of DA were demonstrated on B. bovis cultivated with 0.04 µM of DA pressure for 4, 8, and 12 days, respectively, as compared to that on parental culture (0.08 ±â€¯0.0065 µM) before drug pressure was initiated. However, in B. bovis cultivated with 0.04 µM of DA pressure after 16 days, the parasites could not tolerate 0.8 µM of DA. In the long term, 7.6 ±â€¯3.5-, 20.5 ±â€¯0.1-, and 26.8 ±â€¯5.5-fold increases in the IC50 of DA were demonstrated on parasites from subcultures at days 8, 3, and 5 post-cultivation, respectively, in a drug-free medium, where these subcultures were obtained from B. bovis cultivated with DA pressure with changing doses for 30, 60, and 90 days, respectively. However, the second and third times, no increase was demonstrated on B. bovis from these subcultures at days 15 and 30 post-cultivation in a drug-free medium. In addition, in B. bovis cultivated with drug pressure after 90 days, the parasites tolerate up to 0.64 µM DA. All findings demonstrated that DA resistance in B. bovis is unstable and lost within 15 days of drug withdrawal. However, treatment with subtherapeutic doses of DA in cattle might result in the development of resistance in B. bovis, which may not even respond to subsequent treatments with high doses of DA. Thus, if the bovine babesiosis caused by B. bovis is unresponsive to DA, treatment with other antibabesial agents might be recommended.

Genetic Analysis of Babesia Isolates from Cattle with Clinical Babesiosis in Sri Lanka.

Bovine babesiosis is a serious threat to the cattle industry. We prepared blood DNA samples from 13 cattle with clinical babesiosis from the Badulla (n = 8), Jaffna (n = 3), and Kilinochchi (n = 2) districts in Sri Lanka. These DNA samples tested positive in PCR assays specific for Babesiabovis (n = 9), Babesia bigemina (n = 9), and Babesiaovata (n = 1). Twelve cattle were positive for B. bovis and/or B. bigemina One cow was negative for the tested Babesia species but was positive for Babesia on microscopic examination; the phylogenetic positions of 18S rRNA and cytochrome oxidase subunit III gene sequences suggested that the cow was infected with Babesia sp. Mymensingh, which was recently reported from a healthy cow in Bangladesh. We then developed a novel Babesia sp. Mymensingh-specific PCR assay and obtained positive results for one other sample. Analysis of gene sequences from the cow with positive B. ovata-specific PCR results demonstrated that the animal was infected not with B. ovata but with Babesia sp. Hue-1, which was recently reported from asymptomatic cattle in Vietnam. The virulence of Babesia sp. Hue-1 is unclear, as the cow was coinfected with B. bovis and B. bigemina However, Babesia sp. Mymensingh probably causes severe clinical babesiosis, as it was the sole Babesia species detected in a clinical case. The present study revealed the presence of two bovine Babesia species not previously reported in Sri Lanka, plus the first case of severe bovine babesiosis caused by a Babesia species other than B. bovis, B. bigemina, and Babesiadivergens.

Molecular epidemiology of Babesia species, Theileria parva, and Anaplasma marginale infecting cattle and the tick control malpractices in Central and Eastern Uganda.

East Coast fever, babesiosis, and anaplasmosis are the major tick-borne diseases affecting cattle productivity in Uganda. The emergence of acaricide-resistant ticks is suspected to have caused a rise in hemoparasites. This study sought to detect and characterize hemoparasites among farms in acaricide-failure hotspots of central as compared to the acaricide-failure naïve areas in Eastern Uganda. Nested PCR assays were performed to determine the prevalences of Babesia bovis, Babesia bigemina, Theileria parva, and Anaplasma marginale in cattle blood samples sourced from randomly selected farms. Randomly selected isolates were sequenced to determine the genetic diversity of the parasites using the following marker genes: B. bovis spherical body protein 4, B. bigemina rhoptry-associated protein 1a, T. parva 104 kDa microneme-rhoptry antigen, and A. marginale major surface protein 5. Furthermore, partially and fully engorged adult ticks were collected for taxonomy, and tick-control practices were assessed using a semi-structured questionnaire. The prevalences of B. bigemina, T. parva, and A. marginale in cattle were 17.2, 65.1, and 22.0%, and 10.0, 26.5, and 3% in the central and eastern region, respectively. Whilst, B. bovis was not detected in the farms involved. The sequences for B. bigemina, T. parva, and A. marginale from the central region showed 99% identity with those from the eastern region. Of the 548 ticks collected, 319, 147, 76, and 6 were Rhipicephalus (Boophilus) decoloratus, Rhipicephalus appendiculatus, Amblyomma variegatum, and Rhipicephalus evertsi evertsi, respectively. The Rhipicephalus ticks were more abundant in the central region, whereas A. variegatum ticks were more abundant in the eastern region. Tick control malpractices were found in both Central and Eastern Uganda, and 42 of the 56 surveyed farms lacked appropriate restraining facilities and so they utilized either ropes or a 'boma' (enclosure). In summary, B. bigemina, T. parva, A. marginale and their co-infections were more prevalent in the central than eastern region; even though, tick control malpractices were observed in both regions. Therefore, an urgent tick and TBD control strategy is needed.