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Babesia odocoilei-like parasites were first reported in 2003, and their virulence and hosts remain unknown. We report three cases of dogs with canine babesiosis in Iwate Prefecture. Since Iwate Prefecture area is an area of Japan where canine babesiosis is not endemic, we suspected that these cases of canine babesiosis were caused by B. odocoilei-like parasites. In the present study, we tried to identify the Babesia species that caused these cases of canine babesiosis. To classify Babesia parasites, the heat shock protein 70 (HSP70) gene was examined. Accordingly, we cloned and analyzed the HSP70 gene sequences of B. odocoilei-like parasites from three Ixodes ovatus ticks. It was determined that the nucleotide sequence of the HSP70 gene of the B. odocoilei-like parasites was not consistent with that of B. odocoilei, which suggests that these parasites were from a different species than B. odocoilei. Second, we identified the Babesia species that infected the three dogs by using the HSP70 gene and 18S rRNA. A partial HSP70 gene of B. odocoilei-like parasites was detected in the three dogs, but that of B. gibsoni was not detected. Additionally, a partial sequence of 18S rRNA of B. odocoilei-like parasites was detected in two dogs. These results demonstrated that two dogs were certainly infected with B. odocoilei-like parasites and that one dog was probably infected with B. odocoilei-like parasites. Therefore, these dogs were diagnosed with canine babesiosis due to the presence of B. odocoilei-like parasites. As there were only three cases, additional cases are needed to confirm our findings., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Background: Infections with Babesia bovis, Babesia bigemina, Theileria species and Anaplasma marginale are endemic in Kenya yet there is a lack of adequate information on their genotypes. This study established the genetic diversities of the above tick-borne hemoparasites infecting cattle in Kenya., Methods: Nested PCR and sequencing were used to determine the prevalence and genetic diversity of the above parasites in 192 cattle blood samples collected from Ngong and Machakos farms. B. bovis spherical body protein 4, B. bigemina rhoptry-associated protein 1a, A. marginale major surface protein 5, Theileria spp. 18S rRNA, T. parva p104 and T. orientalis major piroplasm surface protein were used as the marker genes., Results: B. bovis, B. bigemina, T. parva, T. velifera, T. taurotragi, T. mutans and A. marginale were prevalent in both farms, whereas T. ovis, Theileria sp. (buffalo) and T. orientalis were found only in Ngong farm. Co-infections were observed in more than 50 % of positive samples in both farms. Babesia parasites and A. marginale sequences were highly conserved while T. parva and T. orientalis were polymorphic. Cattle-derived T. parva was detected in Machakos farm. However, cattle and buffalo-derived Theileria were detected in Ngong farm suggesting interactions between cattle and wild buffaloes. Generally, the pathogens detected in Kenya were genetically related to the other African isolates but different from the isolates in other continents., Conclusions: The current findings reaffirm the endemicity and co-infection of cattle with tick-borne hemoparasites, and the role of wildlife in pathogens transmission and population genetics in Kenya.

A nationwide survey of Babesia gibsoni using Haemaphysalis longicornis collected from dogs and cats in Japan was conducted using molecular methods. A total of 1,341 H. longicornis, including 305 females, 14 males, 332 nymphs and 690 larvae (153 pools) from 44 prefectures, were examined by B. gibsoni-targeted PCR. Partial sequence analysis revealed that 12 of 13 positive samples sequenced, including samples from Tottori, Hiroshima, Yamaguchi, Tokushima, Ehime and Oita prefectures (all in western Japan), were identical to B. gibsoni, and 1 sample from Kyoto Prefecture was most closely related to a Babesia species recently detected from feral raccoons in Hokkaido. H. longicornis is a candidate for transmission vector tick of the new Babesia species.

Babesia ovata is a tick-transmitted hemoprotozoan parasite of cattle. In the present study, we analyzed tick DNA samples (n=1459) prepared from questing ticks collected from various cattle pastures in Hokkaido (Shibecha, Taiki, Otofuke, Memuro, and Shin-Hidaka districts) and Okinawa (Yonaguni Island) prefectures of Japan for B. ovata. When all the tick DNA samples were screened by a previously described B. ovata-specific apical membrane antigen-1 (AMA-1) gene-based polymerase chain reaction (PCR) assay, none of the DNA samples was positive. Therefore, we developed a PCR assay based on the protozoan beta-tubulin (β-tubulin) gene to detect B. ovata from ticks in Japan. In the specificity test, the PCR assay amplified the expected 444-bp target gene fragment from B. ovata DNA. No PCR products were amplified from DNA samples from other blood pathogens, bovine blood, or ticks. In addition, the PCR assay detected 100 fg of B. ovata-genomic DNA extracted from an in vitro culture of the parasites. Subsequently, when all the tick DNA samples were screened by this new PCR assay, 18 were positive for B. ovata. Positive samples were found only in the Yonaguni and Memuro areas. In Okinawa, where all the ticks were identified as Haemaphysalis longicornis, 9.7% of the samples were PCR-positive, while a single tick (Ixodes ovatus) from Memuro was infected with B. ovata. When the nucleotide sequences of the PCR amplicons were phylogenetically analyzed, they formed a separate clade containing a previously described β-tubulin gene sequence from B. ovata (Miyake strain), confirming that the PCR assay had detected only B. ovata from the tick DNA samples. This is the first report that describes the PCR detection of B. ovata in ticks. The findings warrant transmission experiments to evaluate I. ovatus as a potential vector of B. ovata., (Copyright © 2014 Elsevier GmbH. All rights reserved.)

Vector-borne diseases (VBDs) continue to threaten the worldwide livestock industry, but comprehensive epidemiological surveys on such diseases have not been conducted in the Philippines. In the present study, we screened 408 bovine blood samples from 9 areas in Cebu, Philippines, for various VBD pathogens using specific PCR assays. The results revealed prevalences of 54.7, 15.4, 10.0, and 12.0% for Anaplasma spp., Babesia bigemina, Babesia bovis, and Trypanosoma (Tr.) theileri, respectively. In contrast, none of the samples were positive for Trypanosoma (Tr.) evansi, Theileria (Th.) orientalis, and Theileria (Th.) annulata. Mixed infections were observed in 24.2% of the samples tested. Phylogenetic analysis based on the 16S rRNA gene revealed that the Anaplasma spp. sequences from the present study were genetically close either to Anaplasma marginale or Anaplasma phagocytophilum. In addition, B. bovis RAP-1 and Babesia bigemina AMA-1 gene sequences were identical and monophyletic to other known B. bovis and B. bigemina sequences. On the other hand, Tr. theileri cathepsin-L like protein gene sequences shared 97.1-100% identities with those from the USA and Brazil and clustered within a single genotype in the phylogenetic tree. The molecular identification of several VBD pathogens in Cebu cattle calls for the implementation of control measures to prevent the spread of these pathogens to nearby localities or islands, and ultimately, economic losses to the Philippine economy., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Babesia ovata is a tick-transmitted hemoprotozoan parasite that infects cattle. In our study, bovine blood samples (n=2,034) were collected from 10 different countries (Brazil, China, Ghana, Japan, Mongolia, the Philippines, South Africa, Sri Lanka, Thailand and Vietnam) and DNA extracted. The DNA samples were screened using an established and specific polymerase chain reaction (PCR) assay targeting the Apical membrane antigen 1 (AMA-1) gene. Parasite DNA was detected among samples collected from Japan, Mongolia and Thailand. Sequence analyses confirmed that the PCR assay detected only B. ovata AMA-1, and that amplicons from different geographical locations were conserved. Our findings highlight the importance of designing adequate strategies to control B. ovata infection in Japan, Mongolia, and Thailand.

We describe here the clinical significance of coinfection with Theileria orientalis and Babesia ovata in cattle. Anemia status in a herd of dairy cattle in Japan was investigated in relation to infection with these parasites. Our findings indicate that while B. ovata infection might not be the primary cause of anemia in the cattle, it may contribute to the clinical development of anemia in animals coinfected with both B. ovata and T. orientalis.

A nationwide epidemiological survey of Babesia gibsoni infection in non-fighting dogs was conducted using an improved ELISA with recombinant B. gibsoni thrombospondin-related adhesive protein (BgTRAP). A total of 1206 dogs from 27 prefectures were examined and 128 (10.6%) tested positive. In the eastern part of Japan, 39 dogs out of the 559 (7.0%) examined were positive, while 89 dogs out of 647 (13.8%) tested positive in the western part of Japan. Although the percentage of dogs that tested positive was significantly (p=0.0001) lower in the eastern part compared to the western part of Japan, overall these results indicate that B. gibsoni infection of dogs has a widespread geographic distribution throughout the country. A history of tick infestation was identified as a significant risk factor for B. gibsoni infection (p=0.0091), while sex (p=0.9411), age (p=0.0920) and breed (p=0.0549) of dogs were not statistically significant risk factors. These results indicate that tick infestation is the most dominant risk factor for B. gibsoni infection of non-fighting dogs in Japan and suggest that other B. gibsoni transmission routes, such as fighting and transplacental transmission, may be less important.

An immunochromatographic test (ICT) using recombinant BgSA1 (rBgSA1) for the detection of antibodies against Babesia gibsoni was developed and evaluated. Only the serum samples collected from dogs infected with B. gibsoni were positive in the ICT, but the serum samples from dogs infected with closely related parasites and from healthy dogs were negative. The specific antibodies could be detected in a dog experimentally infected with B. gibsoni at both the acute and chronic infection stages by the ICT. To evaluate the clinical application of the ICT, a total of 94 serum samples collected from domestic dogs in Japan were tested with the ICT and the previously established enzyme-linked immunosorbent assay (ELISA) with rBgSA1. Twenty-one of the tested samples (22.3%) were positive in both the ICT and the ELISA. The concordance between the ELISA and the ICT was found to be 95.8%. These results suggested that the ICT using rBgSA1 is rapid, simple, accurate, and suitable for the diagnosis of B. gibsoni infection of dogs in the field.

The heat shock protein 70 (hsp70) genes of Babesia gibsoni, B. canis canis, B. canis vogeli, and B. canis rossi isolated from infected dogs were cloned by polymerase chain reaction (PCR) and sequenced. In the nucleotide sequence and the predicted amino acid sequence of the gene, the parasites were very similar to each other. The nucleotide sequences of the hsp70 gene had more variety than those of 18S nuclear subunit ribosomal DNA (18S rDNA). A phylogenetic analysis of these sequences and comparisons with sequences from other Babesia and Theileria species revealed that all canine babesial isolates analyzed in the present study were closely related to each other and formed one cluster. Additionally, a phylogenetic analysis of Babesia and Theileria species showed that these parasites could be divided into three groups: group A including canine babesial isolates, B. divergens, B. odocoilei, B. bovis, B. caballi, and B. ovis; group B including Theileria annulata, T. orientalis, and T. cervi; and group C including B. microti and B. rodhaini. These results suggested that a phylogenetic analysis of the hsp70 gene sequence might be helpful in classifying Babesia and Theileria species, and that canine babesial isolates might be closely related to each other, indicating their evolution from the same ancestry.

The clinical usefulness of antibodies against Babesia gibsoni detected by ELISA with recombinant P50 was examined in dogs in an area where B. gibsoni infection was endemic. Only 8 among 14 dogs with acute type B. gibsoni infection without a previous history of infection were positive. This high percentage of false-negative results is thought to be a weak point of ELISA as a diagnostic tool. However, 14 other anemic dogs with a confirmed history of B. gibsoni infection were all positive, thus confirming the higher sensitivity of ELISA in detecting a history of infection.

The prevalence of Babesia (Theileria) equi and B. caballi infections in donkeys in western Xinjiang China was investigated. In total, 93 serum samples were randomly taken from donkeys in the Kashi and Ili areas, and examined for B. equi and B. caballi infections by enzyme-linked immunosorbent assays using recombinant antigens. Of the 93 samples, 9 (9.6%) and 36 (38.7%) samples were positive for B. equi infection and B. caballi infection, respectively. In addition, 2 (2.2%) samples were positive for both B. equi and B. caballi infections. These results indicate that equine babesiosis might be extensively prevalent in donkeys in western Xinjiang.

To determine the distribution of Babesia gibsoni infection in dogs in the eastern part of Japan, an epidemiological survey of dogs suspected of having B. gibsoni infection was attempted using the polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA). Thirty-five of 115 such dogs (30.4%) were positive by PCR and/or ELISA. The 35 positive dogs consisted of 28 Tosa dogs, 4 American Pit Bull Terriers, and 3 mongrel dogs in Aomori, Fukushima, Ibaraki, Gunma, Chiba, Tokyo, Kanagawa, and Nagano Prefectures. The positive dogs had a significantly lower rate of tick exposure and a higher rate of bites by other dogs. Twenty-two of 35 B. gibsoni-positive dogs were infected with hemoplasma, and the rate of infection was significantly higher than that of B. gibsoni-negative dogs.

The surface antigen P50 of Babesia gibsoni is an important candidate for the development of a diagnostic reagent for canine piroplasmosis. In order to establish an effective diagnostic method for practical use, the gene encoding truncated P50 (P50t) lacking a signal peptide and C-terminal hydrophobic regions were cloned and expressed in Escherichia coli as a fusion protein with glutathione S-transferase (GST). More than 90% portion of the GST-P50t was expressed as a soluble form, in contrast with GST-P50f (full-length), which was completely expressed as an insoluble form. This result indicates that removal of the hydrophobic signal peptide and C-terminus had dramatically improved its hydrophilicity. The purified GST-P50t was tested in an enzyme-linked immunosorbent assay (ELISA) for detection of antibodies to B. gibsoni in dogs. The ELISA with GST-P50t clearly differentiated between B. gibsoni-infected dog sera and uninfected dog sera. In addition, the ELISA detected no cross-reactivity with sera from dogs experimentally infected with the closely related parasites, B. canis canis, B. canis vogeli, and B. canis rossi. Field serum samples collected from dogs in Japan and China were examined for the diagnosis of B. gibsoni infection by using the ELISA. 14.5% (9/62), 5.8% (7/120), and 5.4% (2/37) of tested samples were positive for dogs from Okinawa, Yamaguchi, and Osaka prefectures, Japan, respectively. On the other hand, 4.8% (2/41) of tested samples were positive for dogs from Nanjing, China. These results suggest that the GST-P50t could be a reliable reagent for practical use in ELISA for the serodiagnosis of canine piroplasmosis caused by B. gibsoni.

A total of 80 free-roaming dogs on Okinawa Island, Japan, were examined for Babesia infection using the polymerase chain reaction (PCR) and sequence analysis. Of 80 samples, 12 were positive in a Babesia genus-specific PCR. Consequent species-specific PCR for B. canis and B. gibsoni revealed that 5 (6.3%) and 7 (8.8%) dogs were infected with B. canis and B. gibsoni, respectively. Sequence analysis of the PCR products revealed that the 18S rRNA gene sequence of B. canis detected from dogs in Okinawa was very close to B. canis vogeli with sequence similarity of 99.94%.

Detection and analysis of Babesia species from ticks recovered from dogs in Japan were attempted by PCR and nucleotide sequence analysis based on the 18S rRNA gene, respectively. A total of 1136 ticks were examined for Babesia DNA by 18S rRNA-based PCR and nucleotide sequencing. Partial sequences of Babesia canis vogeli DNA were detected from six ticks in Aomori, Nara, Hiroshima, Oita, and Okinawa Prefectures; and Babesia gibsoni Asia-1 DNA was also detected in four ticks in Osaka, Hiroshima, Miyazaki, and Okinawa Prefectures. Unique sequences of 1678 bp were also obtained from Ixodes ovatus ticks in Akita and Fukui Prefectures. The sequences were similar to those of Babesia odocoilei (97.7%) and Babesia divergens (97.6%). This is the first report of the detection of DNA belonging to this group in Japan.

In order to identify the alternative effective chemotherapeutic agents for murine babesiosis, some selected drugs were examined for their efficacy against protozoan infection in the mouse-Babesia rodhaini (B. rodhaini) model. Clindamycin was not completely effective for elimination of parasites in a dose of 50 mg or 100 mg/kg BW/day b.i.d. but effective to prolong the life span of hosts, while it completely cured B. rodhaini infections in a dose of 200 mg. On the other hand, a double therapy consisting of 2 treatments with 100 mg clindamycin and 100 mg clindamycin and with 100 mg clindamycin and 100 mg tetracycline; respectively, and a single therapy with 100 mg tetracycline or 200 mg clindamycin, had a possibility to clear away B. rodhaini organisms from hosts. However, almost all the treatment groups, had a relapse of the infection within 10 days post treatment or re-treatment. Cured mice by treatment with clindamycin and clindamycin, or clindamycin and tetracycline showed complete resistance against challenge with B. rodhaini, while mice cured by administration of clindamycin at 200 mg or tetracycline at 100 mg showed incomplete resistance to challenge infection. The present data suggest that the two former chemotherapies can induce effective protective immunity (premunization), but the latter two chemotherapies induce incomplete premunization.

Serological tests were performed to investigate extent of tick-borne diseases in dogs infested with Rhipicephalus sanguineus at a kennel in Okayama Prefecture. Three of 22 dogs (13.6%) were positive for Ehrlichia canis. Two of 19 dogs (10.5%) were positive for Rickettsia japonica. Three of 22 dogs (13.6%) were positive for Babesia gibsoni. None of these animals were positive for Coxiella burnetii or Hepatozoon canis. A microfilaria was detected in a drop smear of hemolymph from an engorged female tick, however, species was not determined. It is possible that these ticks can transmit pathogens to domestic dogs which are rare in Japan.