Hisashi Inokuma

First molecular detection of Ehrlichia canis and Anaplasma platys in ticks from dogs in Cebu, Philippines

Ehrlichia canis infection of dogs in the Philippines has been detected by serological and peripheral blood smear examination methods, but not by molecular means. Anaplasma platys infection in dogs has not yet been officially reported, although it is suspected to occur in the country. Thus, sensitive and specific molecular techniques were used in this study to demonstrate the presence of both E. canis and A. platys in the Philippines. A total of 164 Rhipicephalus sanguineus ticks was collected from 36 dogs. Seven tick samples were found positive with E. canis and one sample with A. platys. To further characterize these pathogens, molecular analyses based on citrate synthase and heat-shock operon genes were also performed. Philippine strains were found to be not divergent from strains from other countries. The present results are the first molecular detection and analyses of E. canis and A. platys in ticks from dogs in the Philippines.

Molecular survey of bovine vector-borne pathogens in Cebu, Philippines

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.

Molecular detection and characterization of Babesia bovis, Babesia bigemina, Theileria species and Anaplasma marginale isolated from cattle in Kenya

BackgroundInfections 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.MethodsNested 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.ResultsB. 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 50xa0% 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.ConclusionsThe 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.

High Genetic Diversity of Anaplasma marginale Detected from Philippine Cattle

ABSTRACT A total of 658 cattle in 6 provinces in the Philippines were screened for Anaplasma marginale infection by using a diagnostic heat-shock operon (groEL) gene-PCR assay. The screening-positive samples were further tested using the major surface antigen protein 1a (Msp1a) gene-PCR assay. Screening PCR results showed 130 cattle (19.8%) were positive for the A. marginale infection. Subsequent amplification using the Msp1a gene only showed 93 samples (14.1%) to be positive. In addition, 37 tandem-repeat structures, including 20 novel structures, and 41 distinct genotypes were identified. Interestingly, multiple infections of 4 different genotypes were also observed in A. marginale-infected cattle. The present study demonstrated the prevalence and characterization of diverse genotypes of A. marginale in the Philippine cattle.

A clinical case of neosporosis in a 4-week-old holstein friesian calf which developed hindlimb paresis postnatally

A 4-week-old female Holstein Friesian calf presented with hindlimb paresis. Neurologic examination of spinal reflexes revealed depressed or absent reflexes of the hindlimbs. Menace responses on both sides disappeared on examination of cranial nerves. The calf was finally diagnosed with Neospora caninum infection by pathological findings including nonsuppurative inflammation associated with cysts in the cerebrum and spinal cord. High levels of antibody against recombinant surface antigen 1 of N. caninum (NcSAG1) were detected by ELISA from both serum and cerebrospinal fluid (CSF) samples. This result suggests that detection of antibodies against N. caninum by NcSAG1-ELISA in serum and CSF could be useful for the clinical diagnosis of neosporosis in calves with acquired neurological signs.

First molecular detection and phylogenetic analysis of Anaplasma phagocytophilum from a clinical case of canine granulocytic anaplasmosis in Japan

Anaplasma phagocytophilum DNA was detected from a dog with canine granulocytic anaplasmosis (CGA) in Japan. Phylogenetic analysis of the DNA using 16S rRNA, gltA, and groEL sequences revealed that the strain was nearly identical to A. phagocytophilum detected from Apodemus agrarius (black-striped field mouse) in China and Korea. To our knowledge, this is the first report of molecular detection and phylogenetic analysis of A. phagocytophilum from a clinical case of CGA in Japan.