[국내논문]Detection and molecular characterization of Hepatozoon canis, Babesia vogeli, Ehrlichia canis, and Anaplasma platys in dogs from Metro Manila, Philippines원문보기
Adao, Davin Edric V.
(Institute of Biology, College of Science, Natural Sciences Research Institute, University of the Philippines Diliman)
,
Herrera, Charles Michael T.
(Institute of Biology, College of Science, Natural Sciences Research Institute, University of the Philippines Diliman)
,
Galarion, Luiza H.
(Institute of Biology, College of Science, Natural Sciences Research Institute, University of the Philippines Diliman)
,
Bolo, Nicole R.
(Institute of Biology, College of Science, Natural Sciences Research Institute, University of the Philippines Diliman)
,
Carlos, Rhodora S.
(Carlos Veterinary Clinic)
,
Carlos, Enrique T.
(Makati Dog and Cat Hospital)
,
Carlos, Sixto S.
(Makati Dog and Cat Hospital)
,
Rivera, Windell L.
(Institute of Biology, College of Science, Natural Sciences Research Institute, University of the Philippines Diliman)
The study of canine vector-borne diseases in the Philippines started in the 1970s but only gained interest in the past decade. Characterization of such diseases in the Philippines remains incomplete, thus, it is necessary to obtain additional information on the prevalence and diversity of canine tic...
The study of canine vector-borne diseases in the Philippines started in the 1970s but only gained interest in the past decade. Characterization of such diseases in the Philippines remains incomplete, thus, it is necessary to obtain additional information on the prevalence and diversity of canine tick-borne diseases in the country. In this study, blood samples were obtained at two veterinary clinics in Metro Manila, Philippines from 114 dogs suspected of having canine tick-borne pathogens. Polymerase chain reaction (PCR) was performed on whole blood DNA extracts followed by sequencing, and the following pathogens were detected: Hepatozoon (H.) canis (5.26%), Babesia (B.) vogeli (5.26%), Ehrlichia (E.) canis (4.39%), and Anaplasma platys (3.51%). Additionally, a set of multiplex PCR primers were developed to detect H. canis, Babesia spp. (B. canis and B. vogeli), and E. canis in canine blood. Multiplex and conventional single-reaction PCR results for the 114 dog blood samples were similar, except for one H. canis sample. Multiplex PCR is, therefore, a useful tool in screening infected dogs in veterinary clinics. This study's results, together with those of previous studies in the country, show that canine vector-borne pathogens are an emerging veterinary concern in the Philippines.
The study of canine vector-borne diseases in the Philippines started in the 1970s but only gained interest in the past decade. Characterization of such diseases in the Philippines remains incomplete, thus, it is necessary to obtain additional information on the prevalence and diversity of canine tick-borne diseases in the country. In this study, blood samples were obtained at two veterinary clinics in Metro Manila, Philippines from 114 dogs suspected of having canine tick-borne pathogens. Polymerase chain reaction (PCR) was performed on whole blood DNA extracts followed by sequencing, and the following pathogens were detected: Hepatozoon (H.) canis (5.26%), Babesia (B.) vogeli (5.26%), Ehrlichia (E.) canis (4.39%), and Anaplasma platys (3.51%). Additionally, a set of multiplex PCR primers were developed to detect H. canis, Babesia spp. (B. canis and B. vogeli), and E. canis in canine blood. Multiplex and conventional single-reaction PCR results for the 114 dog blood samples were similar, except for one H. canis sample. Multiplex PCR is, therefore, a useful tool in screening infected dogs in veterinary clinics. This study's results, together with those of previous studies in the country, show that canine vector-borne pathogens are an emerging veterinary concern in the Philippines.
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제안 방법
Further identification of Anaplasma spp. was conducted by sequencing larger parts of the16S rRNA gene using the primer pairs fD1/EHR16SR and EHR16SD/Rp2 [12]. PCR amplification was performed using previously published protocols for Hep-F/Hep-R [16], PIRO-A1/PIRO-B [5], and EHR16SD/EHR16SR [34].
The primers Hep-F/Hep-R and PIRO-A1/PIRO-B target the18S rRNA genes of their respective CVB pathogens while primers EHR16SD, EHR16SR, fD1, and Rp2 amplify the 16S rRNA gene of Anaplasmataceae. PCR was performed thrice on positive samples and sent to the Philippine Genome Center for sequencing to rule out false positives. Sequences were aligned using the Clustal W feature of BioEdit 7.
0 [11]. Sequences were then uploaded onto the nucleotide BLAST website (National Center for Biotechnology Information, USA) to determine the most similar sequences and confirm their species identity.
All multiplex PCR experiments were performed with KAPA2G Fast multiplex kit according to the manufacturer’s protocol with 0.25 µM of each multiplex primer.
25 µM of each multiplex primer. The optimum annealing temperature was obtained using gradient PCR (58, 59, 60, 61, and 62℃ in each) on DNA extracts of E. canis, B. canis, and H. canis. Single reaction PCR was performed on DNA extracts of E.
The multiplex PCR primers were able to amplify their respective targets at all temperatures used in the gradient PCR experiment with lower limits of detection of 4.5 ng/µL, 19.1 ng/µL, and 2.4 ng/µL for H. canis, B. canis, and E. canis DNA, respectively (data not shown).
대상 데이터
A total of 114 canine blood samples were obtained from the Makati Dog and Cat Hospital in Makati City, Philippines and the Carlos Veterinary Clinic in Parañaque City, Philippines from 2013-2014.
KR261620-KR261622), and uncultured Anaplasma sp. (GenBank accession nos. JN862824 and JX402624) with E. canis (GenBank accession no. U26740) as an outgroup.
Consensus phylogenetic tree based on the maximum likelihood (ML) tree using partial 18S rRNA gene sequences (517 unambiguously aligned nucleotide positions) of 15 Hepatozoon specimens and 1 outgroup species (Sarcocystis arctosi). The bootstrap consensus tree was constructed using the HKY model and inferred from 1,000 replicates. The bootstrap values of the three phylogenetic methods used are shown in the order ML/neighbor joining (NJ) and maximum parsimony (MP).
Consensus phylogenetic tree based on the maximum likelihood (ML) tree using full 16S rRNA gene sequences (819 unambiguously aligned nucleotide positions) of 30 Anaplasma specimens and 1 outgroup species (Ehrlichia canis). The bootstrap consensus tree was constructed using the TrN model and inferred from 1,000 replicates. The bootstrap values of the three phylogenetic methods used are shown in the order ML/neighbor joining (NJ) and maximum parsimony (MP).
Oklahoma dog and Babesia gibsoni). The bootstrap consensus tree was constructed using the GTR+G model and inferred from 1,000 replicates. The bootstrap values of the three phylogenetic methods used are shown in the order ML/neighbor joining (NJ) and maximum parsimony (MP).
platys infections in dogs in Metro Manila, Philippines are present in low prevalence. In this study, H. canis, B. vogeli, E. canis and A. platys were found in 5.26%, 5.26%, 5.26%, and 3.51%, respectively, of 114 dogs admitted at the Makati Dog and Cat Hospital and the Carlos Veterinary Clinic. Phylogenetic analyses and BLAST results confirm the identification of these with high bootstrap support.
성능/효과
The phylogenetic tree of partial H. canis 18S rRNA gene was constructed with sequences from H. canis (GenBank accession nos. EU289222 and KJ634654; National Center for Biotechnology Information), H. americanum (GenBank accession no. AF176836), H. catesbianae (GenBank accession nos. AF176837 and AF130361), H. felis (GenBank accession nos. KM435071, KC138533, and KC138534), and Sarcocystis arctosi (GenBank accession no. EF564590) as outgroup. The phylogenetic tree of Babesia spp.
2. Consensus phylogenetic tree based on the maximum likelihood (ML) tree using partial 18S rRNA gene sequences (343 unambiguously aligned nucleotide positions) of 56 Babesia specimens and 2 outgroup species (Babesia sp. Oklahoma dog and Babesia gibsoni). The bootstrap consensus tree was constructed using the GTR+G model and inferred from 1,000 replicates.
후속연구
The high analytical sensitivity and specificity of the primers are a good compliment for other diagnostic methods such as observation of symptoms, serology, and microscopy since these have known issues in these areas. However, additional tests are necessary to determine the clinical sensitivity and specificity of the primers in larger sample populations due to the low prevalence of CVBDs in the current sample population.
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