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Traffic-related analyses are essential to implementing traffic management approaches to create mobility more convenient. In this context, Passenger Car Unit (PCU) values as a uniform measure of vehicles are instrumental in analysis such as traffic capacity estimation, traffic flow model development, level of service determination, etc. These studies always demand a recently calculated set of PCU values since the traffic conditions in arterials often change. PCU values for expressway and three-lane (one-way) road types have not been calculated in the Sri Lankan context, and the available set of values has become somewhat outdated. Hence, this study used Chandra's method to assess the PCU values of ten vehicle categories for four different mid-block road sections in the Colombo district, Sri Lanka. The data relating to the two main variables of this method, categorical speed, and area of vehicles, were collected from field surveys. The results of four-lane arterials were aggregated and compared with the existing PCU values, intending to propose an updated set for general transportation studies in Sri Lanka.

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.

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.

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.

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

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.

application/pdf, Equine piroplasmosis caused by Theileria equi and Babesia caballi is an economically important disease with a worldwide distribution. The objective of the present study was to investigate the seroepidemiology of T. equi and B. caballi in horses reared in various Mongolian provinces. Serum samples prepared from blood collected from horses in 19 Mongolian provinces were screened for antibodies specific to T. equi and B. caballi using enzyme-linked immunosorbent assays based on recombinant forms of T. equi merozoite antigen-2 and the B. caballi 48-kDa merozoite rhoptry protein, respectively. Of 1,282 horses analyzed, 423 (33%) and 182 (14.2%) were sero-positive for T. equi and B. caballi, respectively. Additionally, 518 (40.4%) were positive for at least 1 parasite species, of which 87 (16.8%) were co-infected with both parasites. Both T. equi and B. caballi were detected in all surveyed provinces, and on a per province basis the positive rates ranged from 19.0 to 74.2% and 4.5 to 39.8%, respectively. Theileria equi- and B. caballi-positive rates were comparable between male horses (31.9 and 14.1%, respectively) and female horses (34.5 and 14.3%, respectively). However, the positive rates were higher in the >3-yr-old age group (37.7 and 15.6%, respectively) compared with the 1-3-yr-old age group (19.4 and 10.0%, respectively). These findings confirmed that T. equi and B. caballi infections are widespread among horses all over Mongolia, and that horse age is a risk factor for infection in this country. Our results will be useful for designing appropriate control measures to minimize T. equi and B. caballi infections among Mongolian horses.

application/pdf, Trypanosoma (Megatrypanum) theileri is a cosmopolitan, usually non-pathogenic, trypanosome of cattle transmitted by blood-sucking arthropods, mainly tabanid flies. Several T. theileri strains isolated from domestic and wild ruminants via co-culturing with mammalian feeder cells or blood cells have been characterized morphologically and genetically. Here, we cultured a new trypanosome isolate from a Holstein cow in Hokkaido, Japan, and performed morphological and molecular characterization studies. The new isolate (Obihiro strain) was co-cultivated with Madin–Darby bovine kidney (MDBK) cells in GIT medium supplemented with 10% fetal bovine serum. Trypomastigotes and epimastigotes, but not intracellular parasites, were identified in the culture. Analysis of the V7-V8 region of 18S rRNA sequences showed that the Obihiro strain is positioned within the subgenus Megatrypanum. A dendrogram based on whole internal transcribed spacer rDNA sequence showed that the Obihiro strain clustered in the lineage TthII together with the Japanese isolates of T. theileri, Esashi 9, and Esashi 12, and isolates from Zambia and the USA. T. theileri of the KM strain and a T. theileri-like trypanosome isolated from deer (TSD1 strain) clustered in the lineage TthI, separate from the Obihiro strain. Based on a partial cathepsin L-like protein gene analysis, the Obihiro strain clustered with isolates of the TthIIF genotype, which includes T. theileri from Vietnam, Sri Lanka, and Brazil. Our analyses of the T. theileri Obihiro strain provide relevant insights into its genetic diversity in Japanese cattle and corroborate the host specificity of cattle and deer trypanosomes of the subgenus Megatrypanum. © 2019, Springer-Verlag GmbH Germany, part of Springer Nature.

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 ± 2 (0.54 ± 0.16 μM)-, 12.9 ± 8.6 (1.05 ± 0.7 μM)-, and 14 ± 5.9 (1.14 ± 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 ± 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 ± 3.5-, 20.5 ± 0.1-, and 26.8 ± 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., Graphical abstract Image 1, Highlights • Diminazene aceturate (DA) pressure induced resistance development in Babesia bovis. • DA-resistant line tolerated 8 times the IC50 dose determined before drug pressure. • DA resistance was unstable as resistance was lost within 15 days of DA withdrawal. • Treatment with alternative drugs may be essential if B. bovis is unresponsive to DA.

Bovine anaplasmosis caused by Anaplasma marginale represents a serious threat to cattle farming worldwide, especially in the tropics and subtropics. In the present study, archived DNA samples from the blood of cattle (n=437) in the Nuwara Eliya, Galle, Ampara, Polonnaruwa, and Jaffna districts and buffalo (n=327) in the Galle, Polonnaruwa, Mannar, and Mullaitivu districts in Sri Lanka, were screened for A. marginale using a major surface protein 5 (msp5) gene-based PCR assay. The findings showed that 32.7 and 57.5% of cattle and buffalo, respectively, were A. marginale-positive. The rate of positivity differed significantly among geographical regions. In conclusion, the high rates of A. marginale infection in cattle and buffalo highlight the importance of effective control measures 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.

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.

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.

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.

Equine piroplasmosis is an economically significant disease caused by Theileria equi and Babesia caballi, which are tick-borne hemoprotozoan parasites. Infections with these parasite species had never been reported in horses in Indonesia. The aim of the present study was to investigate the prevalence of T. equi and B. caballi in horses reared in parts of Western Java, Indonesia. Blood samples were collected randomly from 235 horses in four different districts (Bandung, Depok, Tangerang, and Bogor) in Western Java, Indonesia. Thin blood smears prepared from the sampled animals were stained by Giemsa and observed under a light microscope. Serum samples prepared from blood were screened by enzyme-linked immunosorbent assays (ELISAs) based on recombinant forms of EMA-2 and BC48 antigens to determine the seroprevalence of T. equi and B. caballi, respectively. DNA samples extracted from the same blood samples were screened by EMA-2 and BC48 gene-based nested polymerase chain reaction (nPCR) assays for T. equi and B. caballi infections, respectively. Of 235 surveyed animals, five (2.1%) and 15 (6.4%) were seropositive for T. equi and B. caballi, respectively, whereas one and four horses were nPCR-positive for T. equi and B. caballi, respectively. All of the surveyed animals were negative for T. equi and B. caballi by microscopy. The T. equi EMA-2 and B. caballi BC48 gene fragments amplified by the nPCR assays were cloned, sequenced, and subjected to bioinformatic and phylogenetic analyses. The T. equi EMA-2 gene sequence from an Indonesian horse was identical to sequences from Florida and Washington strains and clustered together with these sequences in phylogeny. On the other hand, four Indonesian BC48 gene sequences shared 99.8-100% identity scores. This present study is the first to report T. equi and B. caballi in horses in Indonesia. Our findings highlight the need for monitoring horses in Indonesia for clinical piroplasmosis caused by T. equi and B. caballi.

application/pdf, Oriental theileriosis caused by Theileria orientalis is an economically significant disease in cattle farming. The lack of laboratory animal models and in vitro culture systems is a major obstacle in the drive to better understand the biology of this parasite. Notably, research on the sporozoite stage of T. orientalis has rarely been undertaken, although such investigations are of paramount importance for vaccine development based on blocking sporozoite invasion of its host animals. In the present study, we established a mouse-tick infection model for propagating T. orientalis in mice and for producing the sporozoite stage in tick salivary glands. Splenectomized severe combined immunodeficient mice transfused with bovine erythrocytes were infected with T. orientalis. The larval ticks of Haemaphysalis longicornis were then fed on the T. orientalis-infected mice. The piroplasm and sporozoite stages were microscopically observed in the mouse blood and nymphal salivary glands, respectively. The transcriptomics data generated from the piroplasm and sporozoite stages revealed a stage-specific expression pattern for the parasite genes. The mouse-tick infection model and the transcriptomics data it has provided will contribute to a better understanding of T. orientalis biology and will also provide much needed information for the design of effective control measures targeting oriental theileriosis. © 2018 Australian Society for Parasitology

application/pdf, Mongolia is an agriculturally rich country with large livestock populations that contribute significantly to its national economy. However, the export market for live animals and livestock products is often constrained for various reasons including infectious diseases. Babesia bovis and B. bigemina, which are bovine hemoprotozoan parasites, cause severe forms of clinical babesiosis, in cattle. However, a country-wide survey to determine the exposure rates in various provinces in Mongolia was not conducted to determine the risk for infections with these parasite species. Therefore, we investigated the frequency of antibodies to B. bovis and B. bigemina in cattle reared throughout Mongolia. B. bovis-and B. bigemina-specific enzyme-linked immunosorbent assays (ELISAs) were used to screen the serum samples sourced from 1946 cattle in 19 of 21 provinces and a provincial municipality (Ulaanbaatar) in Mongolia. We found 351 (18.0%) samples positive for B. bovis and 435 (22.4%) samples positive for B. bigemina infections. The B. bovis- and B. bigemina-positive rates ranged from 0.8 to 61.5% and 4.0 to 50.6%, respectively, among the surveyed provinces. The positive rates of B. bovis and B. bigemina infections were relatively higher in the provinces located in northernmost, northern, eastern, southeastern, and southern Mongolia. Additionally, the B. bovis- and B. bigemina-positive rates were not significantly different between females (18.2 and 22.2%, respectively) and males (17.2 and 18.8%, respectively) or between the 1–3-year-old (16.2 and 19.4%, respectively) and >3-year-old (17.1 and 20.9%, respectively) age groups. The differential seropositivity for B. bovis and B. bigemina infections among the provinces may reflect the variations in the risk of cattle being infected with these parasite species. The findings of the present study highlight the need for country-wide control measures, including tick control programs, to minimize the rates of B. bovis and B. bigemina infections in Mongolian cattle. © 2018 Elsevier B.V.

Serum and DNA from blood samples collected from Vietnamese yellow cattle (n=101) and cattle imported from Thailand (n=54) at a Vietnamese slaughter house were screened for Babesia bovis and Babesia bigemina infections by enzyme-linked immunosorbent assay (ELISA) and PCR. The positive rates determined by ELISA (B. bovis and B. bigemina) or PCR (B. bigemina) in the Vietnamese cattle were significantly higher than those found in Thai cattle. Some PCR-positive Vietnamese animals were ELISA-negative, whereas all PCR-positive Thai cattle were ELISA-positive, suggesting that the animals were infected in Thailand. Importing Babesia-infected cattle may lead to the introduction of new parasite strains, possibly compromising the development of anti-Babesia immune control strategies in Vietnam.

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.

application/pdf, Babesia bovis BOV57, which is a homolog of the Theileria parva vaccine candidate antigen P67, is expressed in both the tick and blood stages of the life cycle of this parasite. However, the vaccine potential of BOV57 remained to be investigated. In the present study, we generated recombinant BOV57 (rBOV57) and prepared polyclonal antibodies against rBOV57 in mice and rabbits. Indirect immunofluorescence assays conducted with the mouse anti-rBOV57 antibody demonstrated that BOV57 localized at the apical end of paired merozoites in infected bovine red blood cells, whereas the antigen was found in the parasite membrane around the apical end of intraerythrocytic single and extracellular merozoites. In an invasion-inhibition assay, the rabbit anti-rBOV57 antibody potentially inhibited RBC invasion of B. bovis merozoites in vitro. In addition, the invasion inhibition mediated by rabbit anti-rBOV57 antibody resulted in a reduced growth rate of B. bovis in the in vitro culture. These findings indicated that B. bovis BOV57 plays a critical role in the invasion of merozoites into red blood cells, suggesting its potential as a subunit vaccine candidate against B. bovis infection in cattle. Furthermore, we analyzed the genetic diversity of bov57 gene sequences isolated from Sri Lanka, Mongolia, the Philippines, and Vietnam. The bov57 gene sequences derived from Mongolia, the Philippines, and Vietnam were conserved, whereas insertion and/or deletion mutations resulted in sequence diversity among the Sri Lankan samples. In summary, BOV57 is an invasion-related, neutralization-sensitive antigen encoded by the bov57 gene, which displays higher sequence diversity than previously reported. c 2016 Elsevier B.V.

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.

application/pdf, 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.

Equine piroplasmosis caused by

application/pdf, The diseases caused by hemoprotozoan parasites in cattle often result in economic losses. In Sri Lanka, previous studies found that the up-country wet zone, which is located in central Sri Lanka, was characterized by a high rate of Theileria orientalis and a low rate of Theileria annulata compared with the dry zone. In this study, DNA samples were prepared from the blood of 121 cattle in Galle, a coastal district located in low-country wet zone in Sri Lanka, and were PCR-screened for Babesia bovis, Babesia bigemina, T. annulata, T. orientalis, and Trypanosoma theileri. All the parasite species, except B. bovis, were detected among the surveyed cattle. The animals had a high rate of T. orientalis (100%) and a low rate of T. annulata (1.6%), as in the up-country wet zone. Babesia bigemina and Tr. theileri were detected in 19.0% and 20.6% of the animals, respectively, and their infection rates were higher in the animals reared in extensive management systems (32.8% and 27.9%, respectively) than in those managed in intensive/semi-intensive systems (5.0% and 13.3%, respectively). Genotypic analyses found that the T. orientalis mpsp type 5 was predominant similar to up-country wet zone, and that Tr. theileri consisted of seven catl genotypes, including two new genotypes (IL and IM) and four previously detected genotypes (IA, IB, II, and IK). These findings suggest that the hemoprotozoan infection profiles are largely conserved within the wet zone, despite differences in the geography, cattle breeds, and management practices between the up-country and low-country wet zones. © 2019 Elsevier B.V.

Trypanosoma brucei (Tb) is a pathogenic protozoan causing sleeping sickness in humans. Despite little knowledge of how the produced reactive oxygen species (ROS) kills this protozoan, the research on the killing mechanism using chemical compounds and the phagosome in the macrophages has suggested that the protozoan is highly susceptible to the increased oxidative stress. Because the prescribed drug can react with various kinds of molecules and the second produced intermediate compounds, in this study, we clarified the immediate killing effect on Tb in the condition of increased oxidative stress using a low-temperature plasma at atmospheric pressure (LTP) equipment. Results Show the significant growth inhibition of Tb in the LTP-treated medium, the loss of morphological homeostasis with twisted to puffed appearance, and demonstrated the swelled changes on mitochondria and endoplasmic reticulum. In conclusion, this study revealed how the increased oxidative stress kills Tb using LTP technology.

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.

application/pdf, Babesia ovata, which is transmitted by Haemaphysalis longicornis, is an intraerythrocytic protozoan parasite of cattle. Based on its morphology, B. ovata is classified as a large-type Babesia. The developmental stages of B. ovata have been described both in cattle and the tick vector. In infected adult female ticks, the parasite is transovarially transmitted to the tick eggs. The sexual reproduction of B. ovata has been demonstrated in the tick midgut. The diagnostic tools that are currently available for the specific detection of B. ovata in cattle include microscopy and polymerase chain reaction assays. The development of improved molecular and serological diagnostic tools has been constrained by the limited availability of genetic data. B. ovata has been reported in cattle populations in Japan, Korea, China, Mongolia and Thailand. B. ovata was thought to be a benign parasite; however, infections in immuno compromised or Theileria orientalis-infected animals are clinically significant. Thus, control strategies aimed at minimizing the prevalence of B. ovata are vital. The taxonomy of B. ovata is unclear, and the phylogenetic position has not been well defined. Consequently, non?B. ovata species have sometimes been classified as B. ovata. In this review, we provide an outline of the lifecycle, geographical distribution, and control of B. ovata, and critically discuss the taxonomy and phylogeny of this bovine Babesia. c 2016 Elsevier B.V.

application/pdf, Babesia bovis, an intraerythrocytic protozoan parasite, causes severe clinical disease in cattle worldwide. The genetic diversity of parasite antigens often results in different immune profiles in infected animals, hindering efforts to develop immune control methodologies against the B. bovis infection. In this study, we analyzed the genetic diversity of the merozoite surface antigen-1 (msa-1) gene using 162 B. bovis-positive blood DNA samples sourced from cattle populations reared in different geographical regions of Thailand. The identity scores shared among 93 msa-1 gene sequences isolated by PCR amplification were 43.5-100%, and the similarity values among the translated amino acid sequences were 42.8-100%. Of 23 total clades detected in our phylogenetic analysis, Thai msa-1 gene sequences occurred in 18 clades; seven among them were composed of sequences exclusively from Thailand. To investigate differential antigenicity of isolated MSA-1 proteins, we expressed and purified eight recombinant MSA-1 (rMSA-1) proteins, including an rMSA-1 from B. bovis Texas (T2Bo) strain and seven rMSA-1 proteins based on the Thai msa-1 sequences. When these antigens were analyzed in a western blot assay, anti-T2Bo cattle serum strongly reacted with the rMSA-1 from T2Bo, as well as with three other rMSA-1 proteins that shared 54.9-68.4% sequence similarity with T2Bo MSA-1. In contrast, no or weak reactivity was observed for the remaining rMSA-1 proteins, which shared low sequence similarity (35.0-39.7%) with T2Bo MSA-1. While demonstrating the high genetic diversity of the B. bovis msa-1 gene in Thailand, the present findings suggest that the genetic diversity results in antigenicity variations among the MSA-1 antigens of B. bovis in Thailand.

application/pdf, Babesia bovis is an apicomplexan hemoprotozoan that can invade bovine red blood cells (RBCs), where it multiplies asexually. RBC invasion assays using free viable merozoites are now routinely used to understand the invasion mechanism of B. bovis, and to evaluate the efficacy of chemicals and antibodies that potentially inhibit RBC invasion by the parasite. The application of high-voltage pulses (high-voltage electroporation), a commonly used method to isolate free merozoites from infected RBCs, reduces the viability of the merozoites. Recently, a cold treatment of B. bovis in vitro culture was found to induce an effective release of merozoites from the infected RBCs. In the present study, we incubated in vitro cultures of B. bovis in an ice bath to liberate merozoites from infected RBCs and then evaluated the isolated merozoites in RBC invasion and invasion-inhibitions assays. The viability of the purified merozoites (72.4%) was significantly higher than that of merozoites isolated with high-voltage electroporation (48.5%). The viable merozoites prepared with the cold treatment also invaded uninfected bovine RBCs at a higher rate (0.572%) than did merozoites prepared with high-voltage electroporation (0.251%). The invasion-blocking capacities of heparin, a polyclonal rabbit antibody directed against recombinant B. bovis rhoptry associated protein 1, and B. bovis-infected bovine serum were successfully demonstrated in an RBC invasion assay with the live merozoites prepared with the cold treatment, suggesting that the targets of these inhibitors were intact in the merozoites. These findings indicate that the cold treatment technique is a useful tool for the isolation of free, viable, invasion-competent B. bovis merozoites, which can be effectively used for RBC invasion and invasion-inhibition assays in Babesia research. (C) 2016 Elsevier Inc. All rights reserved.

application/pdf, We screened cattle and goats from the districts of Chama, Monze and Mumbwa in Zambia for animal African trypanosomes, Babesia bigemina and Theileria parva using PCRs; 38.1% of the samples tested positive for at least one of the parasite species. The most common parasite was Trypanosoma vivax (19.8%). Its incidence was significantly higher in goats than in cattle, (P

application/pdf, Babesia bovis is the most virulent Babesia organism, resulting in a high mortality rate in cattle. The genetic diversity of B. bovis merozoite surface antigens (MSAs), such as MSA-1, MSA-2b, and MSA-2c, might be linked to altered immune profiles in the host animals. The present study aimed to develop type-specific PCR assays for Asian msa-1 genotypes, thereby re-analyzing the genetic diversity of msa-1 in Sri Lanka, Mongolia, and Vietnam. Specific primers were designed for nine Asian msa-1 genotypes, which had been detected based on the phylogeny constructed using msa-1 gene sequences retrieved from the GenBank database. Specificity of the type-specific PCR assays was confirmed using plasmids containing the inserts of msa-1 gene fragments that represent Asian genotypes. Furthermore, no amplicons were observed by these PCR assays when DNA samples of Babesia bigemina, Babesia ovata, Theileria annulata, Theileria orientalis, Trypanosoma evansi, Trypanosoma theileri, Anaplasma marginale, and Anaplasma bovis, and non-infected bovine blood were analyzed. In total, 109 B. bovis-positive blood DNA samples sourced from Sri Lanka (44 cattle), Mongolia (26 cattle), and Vietnam (23 cattle and 16 water buffaloes) were then screened by the type-specific PCR assays. The sequences derived from all of the PCR amplicons were phylogenetically analyzed. Out of 109 DNA samples, 23 (20 from cattle and 3 from water buffaloes) were positive for at least one genotype. In agreement with previous studies, five and four different genotypes were detected among the DNA samples from Sri Lanka and Vietnam, respectively. In contrast, four genotypes, including three novel genotypes, were detected from Mongolia. Five DNA samples were found to be co-infected with multiple genotypes. The sequences of the PCR amplicons clustered phylogenetically within the corresponding clades. These findings indicated that the type-specific PCR assays described herein are useful for the determination of genotypic diversity of the B. bovis msa-1 gene in Asia.

application/pdf, A PCR-based survey of hemoprotozoa parasites detected Babesia bigemina, Theileria orientalis and Trypanosoma theileri among cattle and water buffalo in Vietnam, and a new Babesia sp. closely related to Babesia ovata was detected in cattle only. In addition, Theileria annulata and Trypanosoma evansi were not detected in both cattle and water buffalo. Phylogenetic analysis detected T. orientalis MPSP genotypes 3, 5, 7 and N3 in cattle and 5, 7, N1 and N2 in water buffalo. Additionally, water buffalo-derived T. theileri CATL sequences clustered together with a previously reported cattle-derived sequence from Vietnam. This is the first report of a new Babesia sp. in cattle, and T. orientalis MPSP genotype 7 and T. theileri in water buffalo in Vietnam.

application/pdf, Bovine babesiosis is a serious threat to the cattle industry. We preparedblood 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 samplestested positive in PCR assays specific for Babesia bovis (n 9), Babesia bigemina(n 9), and Babesia ovata (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 forBabesia on microscopic examination; the phylogenetic positions of 18S rRNA and cytochrome oxidase subunit III gene sequences suggested that the cow was infectedwith Babesia sp. Mymensingh, which was recently reported from a healthy cow inBangladesh. We then developed a novel Babesia sp. Mymensingh-specific PCR assayand obtained positive results for one other sample. Analysis of gene sequences fromthe cow with positive B. ovata-specific PCR results demonstrated that the animalwas 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 isunclear, as the cow was coinfected with B. bovis and B. bigemina. However, Babesiasp. Mymensingh probably causes severe clinical babesiosis, as it was the sole Babesiaspecies detected in a clinical case. The present study revealed the presence of twobovine 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 Babesia divergens. © 2018 American Society for Microbiology. All Rights Reserved.