Huong Thi Thanh Doan

Development and Evaluation of a Single-Step Duplex PCR for Simultaneous Detection of Fasciola hepatica and Fasciola gigantica (Family Fasciolidae, Class Trematoda, Phylum Platyhelminthes)

ABSTRACT A single-step multiplex PCR (here referred to as a duplex PCR) has been developed for simultaneous detection and diagnosis of Fasciola hepatica and F. gigantica. These species overlap in distribution in many countries of North and East Africa and Central and Southeast Asia and are similar in egg morphology, making identification from fecal samples difficult. Based on a comparative alignment of mitochondrial DNA (mtDNA) spanning the region of cox1-trnT-rrnL, two species-specific forward primers were designed, FHF (for F. hepatica) and FGF (for F. gigantica), and a single reverse primer, FHGR (common for both species). Conventional PCR followed by sequencing was applied using species-specific primer pairs to verify the specificity of primers and the identity of Fasciola DNA templates. Duplex PCR (using three primers) was used for testing with the DNA extracted from adult worms, miracidia, and eggs, producing amplicons of 1,031 bp for F. hepatica and 615 bp for F. gigantica. The duplex PCR failed to amplify from DNA of other common liver and intestinal trematodes, including two opisthorchiids, three heterophyids, an echinostomid, another fasciolid, and a taeniid cestode. The sensitivity assay showed that the duplex PCR limit of detection for each Fasciola species was between 0.012 ng and 0.006 ng DNA. Evaluation using DNA templates from 32 Fasciola samples (28 adults and 4 eggs) and from 25 field-collected stools of ruminants and humans revealed specific bands of the correct size and the presence of Fasciola species. This novel mtDNA duplex PCR is a sensitive and fast tool for accurate identification of Fasciola species in areas of distributional and zonal overlap.

Molecular detection and phylogenetic analysis of Anaplasma bovis from Haemaphysalis longicornis feeding on grazing cattle in Korea

Ticks are vectors of various pathogens that affect humans and animals throughout the world. Anaplasma bovis is one of the most important tick-borne pathogens that cause cattle diseases but there is still very little information available about this agent in Korea. In the present study, 535 Haemaphysalis longicornis tick pools were analyzed from grazing cattle in five Korean provinces. A. bovis was detected in 50 (9.3%) of 535 tick pools using 16S rRNA-based PCR. A. bovis infections were detected for the first time in ticks feeding on cattle in Chungbuk, Geongbuk, and Jeonbuk provinces in Korea. The 50 positive PCR products were sequenced successfully and compared with sequences in GenBank. Phylogenetic analysis of the Korean isolates classified them into four genotypes with nucleotide sequence identities of 99.4-100%. Two of the four genotypes had high similarity (99.8-100%) with known sequences. The other two genotypes have never been identified.

Molecular investigation of tick-borne pathogens in ticks from grazing cattle in Korea.

This study was carried out to identify the tick species that infest grazing cattle and to determine the presence of tick-borne pathogens transmitted by these ticks in Korea. A total of 903 ticks (categorized into 566 tick pools) were collected from five provinces during 2010-2011. The most prevalent tick species was Haemaphysalis longicornis, followed by three Ixodes spp. ticks. The collected ticks were infected with both rickettsial and protozoan pathogens. In all, 469 (82.9%) tick pools tested positive for the Anaplasma/Ehrlichia 16S rRNA gene, whereas 67 (11.8%) were positive for the Babesia/Theileria 18S rRNA gene. Among the rickettsial pathogens, E. canis was detected with the highest rate (22.3%), followed by A. platys (20%), E. chaffeensis (19.4%), E. ewingii (19.3%), Rickettsia sp. (12.4%), A. phagocytophilum (5.5%) and E. muris (0.5%). Among the protozoan pathogens, T. equi was detected with the highest rate (7.2%), followed by T. sergenti/T. buffeli (3.7%) and B. caballi (0.35%). Simultaneous infections with up to seven pathogens were also identified. In particular, ticks infected with rickettsial pathogens were also infected with protozoan pathogens (22 samples). All five provinces investigated infected with tick-borne pathogens.

Phylogenetic analysis of black queen cell virus genotypes in South Korea.

The black queen cell virus (BQCV), a picorna-like honeybee virus, was first isolated from queen larvae and pupae of honeybees found dead in their cells. BQCV is the most common cause of death in queen larvae. Phylogenetic analysis of two Apis cerana and three Apis mellifera BQCV genotypes collected from honeybee colonies in different regions of South Korea, central European BQCV genotypes, and a South African BQCV reference genotype was performed on a partial helicase enzyme coding region (ORF1) and a partial structural polypeptide coding region (ORF2). The phylogeny based on the ORF2 region showed clustering of all the Korean genotypes corresponding to their geographic origin, with the exception of Korean Am str3 which showed more similarity to the central European and the South African reference genotype. However, the ORF1-based tree exhibited a different distribution of the Korean strains, in which A. cerana isolates formed one cluster and all A. mellifera isolates formed a separate cluster. The RT-PCR assay described in this study is a sensitive and reliable method for the detection and classification of BQCV strains from various regions of Korea. BQCV infection is present in both A. cerana and A. mellifera colonies. With this in mind, the present study examined the transmission of honeybee BQCV infections between A. cerana and A. mellifera.

Seroprevalence of Toxoplasma gondii and Trichinella spiralis infections in wild boars (Sus scrofa) in Korea.

Toxoplasma gondii and Trichinella spiralis are important zoonotic pathogens with worldwide distributions. In Korea, several outbreaks of human toxoplasmosis and trichinellosis due to the consumption of infected wild animals have been reported. The purpose of this study was to determine the seroprevalence of T. gondii and T. spiralis infections in wild boars killed in Korea from December 2009 to October 2011. A total of 521 wild boars hunted in eight provinces were examined for antibodies to T. gondii and T. spiralis by using commercial ELISA kits. Overall, 25.1% of serum samples from individual boars were seropositive for T. gondii and 1.7% were seropositive for T. spiralis. Seropositive for T. gondii was found in the boars in all the eight provinces investigated and for T. spiralis in four provinces. This is the first report on the seroprevalence of T. gondii and T. spiralis infections in wild boars in Korea. The consumption of undercooked wild boar meat may expose humans to a high risk of infection.

The ribosomal transcription units of Haplorchis pumilio and H. taichui and the use of 28S rDNA sequences for phylogenetic identification of common heterophyids in Vietnam

BackgroundHeterophyidiasis is now a major public health threat in many tropical countries. Species in the trematode family Heterophyidae infecting humans include Centrocestus formosanus, Haplorchis pumilio, H. taichui, H. yokogawai, Procerovum varium and Stellantchasmus falcatus. For molecular phylogenetic and systematic studies on trematodes, we need more prospective markers for taxonomic identification and classification. This study provides near-complete ribosomal transcription units (rTU) from Haplorchis pumilio and H. taichui and demonstrates the use of 28S rDNA sequences for identification and phylogenetic analysis.ResultsThe near-complete ribosomal transcription units (rTU), consisting of 18S, ITS1, 5.8S, ITS2 and 28S rRNA genes and spacers, from H. pumilio and H. taichui from human hosts in Vietnam, were determined and annotated. Sequence analysis revealed tandem repetitive elements in ITS1 in H. pumilio and in ITS2 in H. taichui. A phylogenetic tree inferred from 28S rDNA sequences of 40 trematode strains/species, including 14 Vietnamese heterophyid individuals, clearly confirmed the status of each of the Vietnamese species: Centrocestus formosanus, Haplorchis pumilio, H. taichui, H. yokogawai, Procerovum varium and Stellantchasmus falcatus. However, the family Heterophyidae was clearly not monophyletic, with some genera apparently allied with other families within the superfamily Opisthorchioidea (i.e. Cryptogonimidae and Opisthorchiidae). These families and their constituent genera require substantial re-evaluation using a combination of morphological and molecular data. Our new molecular data will assist in such studies.ConclusionsThe 28S rDNA sequences are conserved among individuals within a species but varied between genera. Based on analysis of 40 28S rDNA sequences representing 19 species in the superfamily Opisthorchioidea and an outgroup taxon (Alaria alata, family Diplostomidae), six common human pathogenic heterophyids were identified and clearly resolved. The phylogenetic tree inferred from these sequences again confirmed anomalies in molecular placement of some members of the family Heterophyidae and demonstrates the need for reappraisal of the entire superfamily Opisthorchioidea. The new sequences provided here supplement those already available in public databases and add to the array of molecular tools that can be used for the diagnosis of heterophyid species in human and animal infections.

Analysis of the nonstructural and structural polyprotein regions, and complete genome sequences of Israel acute paralysis viruses identified from honeybees (Apis mellifera) in Korea

Phylogenetic trees were constructed for 24 partial nucleotide sequences of the nonstructural polyprotein (ORF1) and structural polyprotein regions (ORF2) of Korean IAPV genotypes, as well as eight previously reported IAPV sequences from various countries. Most of the Korean genotypes formed a distinct cluster, separate from other country genotypes. To investigate this phenomenon in more detail, three complete IAPV genome sequences were identified from different regions in Korea, i.e., Korea1, Korea2, and Korea3. These sequences were aligned with eight previously reported complete genome sequences and various genome regions were compared. The Korean IAPVs were very similar to those from China and Israel, but highly diverged from USA and Australian genotypes. Interestingly, they showed greater variability than the USA and Australian genotypes in ORF1, but highly similar to the Australian genotype in the ORF2 region. Thus, genetic recombination may account for the spatial distance between the Korean IAPV genotypes and those from other countries.

Molecular genotyping of duck hepatitis A viruses (DHAV) in Vietnam

INTRODUCTION The aim of this study was to identify the genetic characteristics and molecular genotyping of duck hepatitis A virus (DHAV) isolated in Vietnam during 2009-2013. METHODOLOGY Thirty duckling livers from outbreaks between 2009 and 2013 in seven provinces were collected and identified by polymerase chain reaction (PCR). Then, VP1 genes of eleven positive samples and two attenuated vaccine strains were sequenced and analyzed. RESULTS Genotypic and phylogenetic analyses indicated that the 13 Vietnamese isolates were classified into two genotypes, DHAV-1 and DHAV-3. The rate of identity and homology was 91%-100% between the 10 Vietnamese and 26 global strains of DHAV-3, and 92%-100% between 3 Vietnamese and 16 strains of DHAV-1. Between the DHAV-3 and DHAV-1 strains, the divergence reached 30%. At the C-terminal of VP1 for the different strains, a hypervariable region was observed, and notably, six of the Vietnamese DHAV-3 strains in this study showed four consistent differences (at positions T184M, Q200H, K207N, and K214R) within this group that were distinct from all other DHAV-3 strains. CONCLUSIONS This is the first report of molecular characterization of DHAVs in Vietnam. At least two genotypes were identified, DHAV-1 and DHAV-3, with diversified clades within and between genotypes. DHAV-3 seemed to be dominant in Vietnam.

Molecular analysis of Newcastle disease virus isolates reveals a novel XIId subgenotype in Vietnam

Eight Vietnamese Newcastle disease virus field isolates from 2008–2015 and 3 vaccine specimens were genotyped based on their full F gene sequences and compared to 80 reference strains representing all 18 genotypes. Three isolates formed a novel subgenotype XIId, identified for the first time in Vietnam; while the others clustered as follows: four in subgenotypes VIId and VIIh; two in Genotype I; and two in Genotype II. Evolutionary distance calculations confirmed the Vietnamese XIId isolates were distinct from XIIa and XIIb by 0.062–0.070; and from other genotypes by 0.089–0.245. This data demonstrated that a novel XIId subgenotype emerged in Vietnam indicating considerable genetic diversity, thus highlighting the need to implement antigenic matching during vaccination against NDVs.