Adrian P. Ybañez

Molecular analyses of a potentially novel Anaplasma species closely related to Anaplasma phagocytophilum detected in sika deer (Cervus nippon yesoensis) in Japan

An Anaplasma species closely related to Anaplasma phagocytophilum detected in sika deer in Hokkaido, Japan was molecularly analyzed using 16S rRNA, citrate synthase (gltA), and heat-shock operon (groEL) gene sequences. Genome walking was performed to determine its complete gltA and groEL sequences (1233 bp and 1650 bp, respectively). Percent identities to the closest A. phagocytophilum sequences from the US and European strains were 98.6-98.8%, 76.5%, and 80.3-80.8% for 16S rRNA, gltA, and groEL genes, respectively. For deduced amino acid sequences, percent identities to the closest A. phagocytophilum sequences were 66.7% and 97.6% for gltA and groEL genes, respectively. Phylogenetic analyses revealed divergence from any known A. phagocytophilum strain. The lower identities and the divergent phylogenetic position of the Anaplasma sp. detected from sika deer in Japan with established A. phagocytophilum strains provide evidence of its potential novelty.

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.

PCR detection of Babesia ovata from cattle reared in Japan and clinical significance of coinfection with Theileria orientalis.

ABSTRACT 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.

The Phylogenetic Position of Anaplasma bovis and Inferences on the Phylogeny of the Genus Anaplasma

ABSTRACT The present study aimed to determine the complete citrate synthase (gltA) and heat-shock protein (groEL) gene sequences of Anaplasma bovis and to infer phylogenetic relationships within the genus Anaplasma. Multiple alignments from single and concatenated sequences of the 16S rRNA, gltA and groEL genes of the genus Anaplasma were subjected to phylogenetic analyses. Percent identities of A. bovis nucleotide sequences were found highest with A. phagocytophilum in gltA (65.4%) and groEL (79.8%). Single gene phylogenetic tree results assumed similar phylogenetic positions within the genus Anaplasma, except for A. bovis. However, consensus and concatenated sequence phylogenetic trees showed similar results, revealing 2 subgroups within the genus.

Specific Molecular Detection of Anaplasma sp. Closely Related to Anaplasma phagocytophilum in Ixodid Ticks and Cattle in a Pastureland in Hokkaido, Japan

Recent molecular analyses of the Anaplasma sp. closely related to Anaplasma phagocytophilum (previously believed to be A. phagocytophilum) in Japan have clarified its distinct phylogenetic position. PCR methods relying on 16S rRNA- and P44/MSP2-based primers designed to detect this species have low sensitivity and specificity. In this study, a highly sensitive and specific nested PCR method using newly designed primers based on heat-shock operon gene (groEL) was developed to detect this species. The method was later used in an epidemiological study testing DNA samples from 85 Ixodid ticks (collected by flagging) and 50 cattle from the same pastureland in Nakaosobetsu, Hokkaido, Japan. Results revealed prevalence rates of 2.4% (2 of 85) in ticks and 2% (1 of 50) in cattle. The present study also reported the first molecular detection of the Anaplasma sp. closely related to A. phagocytophilum in Japan in H. douglasii, and established a new reliable PCR method that detects this Anaplasma sp. closely related to A. phagocytophilum in Japan.

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.

Diversity of Babesia bovis merozoite surface antigen genes in the Philippines.

Babesia bovis is the causative agent of fatal babesiosis in cattle. In the present study, we investigated the genetic diversity of B. bovis among Philippine cattle, based on the genes that encode merozoite surface antigens (MSAs). Forty-one B. bovis-positive blood DNA samples from cattle were used to amplify the msa-1, msa-2b, and msa-2c genes. In phylogenetic analyses, the msa-1, msa-2b, and msa-2c gene sequences generated from Philippine B. bovis-positive DNA samples were found in six, three, and four different clades, respectively. All of the msa-1 and most of the msa-2b sequences were found in clades that were formed only by Philippine msa sequences in the respective phylograms. While all the msa-1 sequences from the Philippines showed similarity to those formed by Australian msa-1 sequences, the msa-2b sequences showed similarity to either Australian or Mexican msa-2b sequences. In contrast, msa-2c sequences from the Philippines were distributed across all the clades of the phylogram, although one clade was formed exclusively by Philippine msa-2c sequences. Similarities among the deduced amino acid sequences of MSA-1, MSA-2b, and MSA-2c from the Philippines were 62.2-100, 73.1-100, and 67.3-100%, respectively. The present findings demonstrate that B. bovis populations are genetically diverse in the Philippines. This information will provide a good foundation for the future design and implementation of improved immunological preventive methodologies against bovine babesiosis in the Philippines. The study has also generated a set of data that will be useful for futher understanding of the global genetic diversity of this important parasite.

Detection of Ehrlichia, Anaplasma, and Babesia spp. in dogs of Cebu, Philippines

Background: Ehrlichia, Anaplasma, and Babesia spp. are canine pathogens transmitted by the Rhipicephalus sanguineus tick which can cause varied clinical signs. These pathogens have been investigated in the Philippines, but coinfection has not been reported yet. Aim: The aim of this study was to evaluate the presence of Ehrlichia/Anaplasma and Babesia spp. in Philippine dogs. Materials and Methods: A total of 100 dogs from seven different veterinary establishments in Cebu, Philippines, were examined for Ehrlichia/Anaplasma and Babesia spp. infection using peripheral blood smear examination and polymerase chain reaction (PCR). Inclusion criteria included a history or presence of tick infestation, anemia, and/or thrombocytopenia. Clinical signs were recorded. Statistical analyses were performed between PCR positivity and clinical signs and hematological results. Results: A total of 10 and 18 dogs were found to be positive for Ehrlichia/Anaplasma and Babesia spp., respectively. One animal was PCR positive for both pathogens, which is the first report of coinfection in the country. The most common clinical signs observed include inappetence (89%), lethargy (80%), thrombocytopenia (85%), and anemia (74%). Analyses revealed that inappetence (p=0.044) and weight loss (p=0.028) were found statistically significant with Ehrlichia/Anaplasma infection. Basophil (p=0.001) and eosinophil counts (p=0.000) were also found significantly different between Ehrlichia/Anaplasma spp.-positive and -negative dogs. On the other hand, differential monocyte count (p=0.009) was found significantly different between Babesia spp.-positive and -negative dogs. Conclusion: The present study showed low infection rates of canine ehrlichiosis/anaplasmosis and babesiosis and provided additional evidence for the presence of the pathogens in the area.

Profile and artificial insemination practices of technicians and the artificial insemination success rates in Leyte, Samar, and Biliran, Philippines (2011-2015).

Background: Artificial insemination (AI) is a reproductive biotechnology that may be influenced by several factors, including the profile of the technicians and the practices used. Assessing technician’s profile and their AI practices can be significant in improving AI success rate. Aim: This study aimed to know the profile and current practices used by AI technicians (AITs), to determine the success rates of AI in water buffaloes in Leyte, Samar, and Biliran from 2011 to 2015, and to evaluate the possible association between the parameters investigated. Materials and Methods: A total of 50 AITs from Leyte, Samar and Biliran, Philippines, were interviewed using a fixed questionnaire about their profile and employed AI practices, and 20,455 AI-related records of the Philippine Carabao Center (PCC) at Visayas State University (VSU), Baybay City, Leyte, were screened and analyzed. AI success rates were determined by retrospective analysis of the gathered data. Statistical analysis was performed between the technician profile and practices and the AI success rates. Results: Results revealed that most of the technicians were male, around 31-40 years old, married, college graduates, working under local government units, had other sources of income, and with 1-5 years of continuous AI practice averaging 51-100 inseminations per year. Most of them attended only one basic training seminar, which was conducted more than 3 years ago in PCC in VSU. AI success rates were recorded highest in 2011 and lowest in 2015. Statistical analyses showed that some technician profile parameters (civil status, average AI per year, and the training center) and several practices (checking of soft cervix, rectal palpation, thawing temperature method, straw cutting method, and semen deposition) might have an influence on the success of AI. Conclusion: This study documents the first report on AIT’s profile and their employed AI practices and the AI success rates in Leyte, Samar, and Biliran, Philippines. Selected profile parameters and AI practices may influence AI success rates. AITs should perform more AI services and revisit the employed practices.