In July 2009, some farms of breeding Muscovy ducks on the peak of egg laying suffered the decrease of hatching rate and the quality of the eggs showing low mortality and no evident respiratory symptoms. The swelling and congestive ovary was visible after autopsy. This study was brought out for the diagnosis of these cases. The virus was isolated and identified by the methods of virus culture in chicken embryo, physical and chemical properties test, hemagglutinin test, NDV (Newcastle diseases Virus) interference test, electron microscope observation, pathogenicity test and the gene sequence analysis. The results indicated the virus showed the characters of inducing dwarf embryo after inocubation, the sensibility to lipid solvent and the hemagglutination capacity after pancreatic enzyme treatment, the typical morphology of coronavirus, the interference to NDV replication and the homology among 84.7% - 99% of the particial N gene sequences to the reference IBV (Avian infectious bronchitis virus) strains. The strain was identified as IBV isolate and this study confirmed the pathogenicity of IBV to Muscovy ducks.
Amino Acid Sequence ; Animals ; Chick Embryo ; Coronavirus Infections ; veterinary ; virology ; Ducks ; virology ; Female ; Infectious bronchitis virus ; classification ; genetics ; isolation & purification ; Molecular Sequence Data ; Phylogeny ; Poultry Diseases ; virology ; Sequence Alignment

Echinococcus granulosus is the causative agent of cystic echinococcosis with medical and veterinary importance in China. Our main objective was to discuss the genotypes and genetic diversity of E. granulosus present in domestic animals and humans in western China. A total of 45 hydatid cyst samples were collected from sheep, humans, and a yak and subjected to an analysis of the sequences of mitochondrial cytochrome b (cytb) gene. The amplified PCR product for all samples was a 1,068 bp band. The phylogenetic analysis showed that all 45 samples were identified as E. granulosus (genotype G1). Ten haplotypes were detected among the samples, with the main haplotype being H1. The haplotype diversity was 0.626, while the nucleotide diversity was 0.001. These results suggested that genetic diversity was low among our samples collected from the west of China based on cytb gene analysis. These findings may provide more information on molecular characteristics of E. granulosus from this Chinese region.
Animals ; Animals, Domestic/parasitology ; Base Composition ; Base Sequence ; Cattle/*parasitology ; China ; Cytochromes b/*genetics ; DNA, Helminth/genetics ; Echinococcosis ; Echinococcus granulosus/classification/*genetics ; Genetic Variation ; Haplotypes/genetics ; Humans ; Mitochondria/genetics ; Molecular Sequence Data ; Phylogeny ; Polymerase Chain Reaction/*veterinary ; Sequence Alignment ; Sequence Analysis, DNA ; Sheep/*parasitology ; Tibet

Amplification of the 16S rRNA gene from a blood sample obtained from a dog in southeastern Brazil was used to confirm a naturally acquired Ehrlichia (E.) canis infection. Following isolation and culturing of the new bacterial strain called Uberlandia, partial sequences of the dsb and p28 genes were obtained. The dsb partial sequence of the novel strain was 100% similar to dsb gene sequences of E. canis obtained from different geographic areas around the world. Conversely, the p28 partial sequence for the E. canis Uberlandia strain differed at several nucleotides from other sequences available in GenBank. To confirm the antigenic profile of the Uberlandia strain, an indirect immunofluorescence assay against E. canis antigens was performed using dog sera collected from two different areas in Brazil (Uberlandia and Sao Paulo). The results suggest that both antigens were able to identify animals seropositive for E. canis in Brazil since these Brazilian strains appear to be highly conserved.
Animals ; Antigens, Bacterial/blood/*diagnostic use ; Bacterial Outer Membrane Proteins/genetics/metabolism ; Bacterial Proteins/*genetics/metabolism ; Base Sequence ; Brazil ; Dog Diseases/diagnosis/*microbiology ; Dogs ; Ehrlichia canis/*genetics/*immunology/isolation & purification ; Ehrlichiosis/diagnosis/microbiology/*veterinary ; Fluorescent Antibody Technique, Indirect/veterinary ; Male ; Molecular Sequence Data ; Polymerase Chain Reaction/veterinary ; RNA, Ribosomal, 16S/genetics/metabolism ; Sequence Alignment/veterinary

This study aims to construct a eukaryotic expression system for envelope gene of Jaagsiekte sheep retrovirus, observes its localization in 293T cells, and investigates the potential in inducing malignant transformation of NIH3T3 cells. By RT-PCR, the full-length cDNA of envelope gene of Jaagsiekte sheep retrovirus (exJSRV-env) was amplified from the extract of naturally infected sheep lung. The clone of target gene was sub-cloned into eukaryotic expression system pEGFP-C1, and validated by PCR, restriction endonuclease, and sequencing. Bioinformatic analysis concerning biological function and cellular localiza tion of exJSRV-env was also performed. The recombinant clone of exJSRV-env was transfected into 293T cells and NIH3T3 cells by Lipofectamine LTX. The expression and celluar localization in 293T cells were validated by confocal microscopy. Soft agar colony formation assay was employed to test the anchorage-independent growth of NIH3T3. DNA sequencing and restriction enzyme digestion with Kpn I and Hind III indicated the correct construction of the recombinant plasmid, which was named pEGFP-C1/exJSRV-env. Amino acid sequence alignment of exJSRV-env with reference sequences found 85%-100% homogeneity. A YRNM motif was discovered at the cytoplasmic tail of envelope gene, which is exclusively found in exogenous viruses. Phylogenetic tree analysis showed that our clone of exJSRV-env clustered closely with pathogenic exogenous Jaagsiekte sheep retroviruses. Fluorescence microscopy indicated typical membrane localization of exJSRV-env protein. NIH3T3 cells transfected with exJSRV-env lost contact inhibition, and acquired colony forming ability in soft agar. This study indicated that envelope protein of Jaagsiekte sheep retrovirus can induce malignant transformation of mouse fibroblast cell NIH3T3. Discoveries of this study provide a basis for further structural and functional research on Jaagsiekte sheep retrovirus envelope protein.
Amino Acid Sequence ; Animals ; Betaretrovirus ; chemistry ; classification ; genetics ; physiology ; Cell Transformation, Viral ; Green Fluorescent Proteins ; genetics ; metabolism ; Mice ; Molecular Sequence Data ; NIH 3T3 Cells ; Phylogeny ; Retroviridae Infections ; veterinary ; virology ; Sequence Alignment ; Sheep ; Sheep Diseases ; virology ; Transformation, Genetic ; Tumor Virus Infections ; veterinary ; virology ; Viral Envelope Proteins ; chemistry ; genetics ; metabolism

Species identification using DNA sequences is the basis for DNA taxonomy. In this study, we sequenced the ribosomal large-subunit RNA gene sequences (3,037-3,061 bp) in length of 13 Chinese Theileria stocks that were infective to cattle and sheep. The complete 28S rRNA gene is relatively difficult to amplify and its conserved region is not important for phylogenetic study. Therefore, we selected the D2-D3 region from the complete 28S rRNA sequences for phylogenetic analysis. Our analyses of 28S rRNA gene sequences showed that the 28S rRNA was useful as a phylogenetic marker for analyzing the relationships among Theileria spp. in ruminants. In addition, the D2-D3 region was a short segment that could be used instead of the whole 28S rRNA sequence during the phylogenetic analysis of Theileria, and it may be an ideal DNA barcode.
Animals ; Base Sequence ; China ; DNA, Ribosomal/chemistry/genetics ; Molecular Sequence Data ; Phylogeny ; RNA, Ribosomal, 28S/genetics ; Ruminants ; Sequence Alignment ; Sequence Analysis, DNA/veterinary ; Theileria/*classification/genetics/isolation & purification ; Theileriasis/*parasitology

The complete coding sequence of Haemonchus (H.) contortus HC29 cDNA was generated by rapid amplification of cDNA ends in combination with PCR using primers targeting the 5'- and 3'-ends of the partial mRNA sequence. The cloned HC29 cDNA was shown to be 1,113 bp in size with an open reading frame of 507 bp, encoding a protein of 168 amino acid with a calculated molecular mass of 18.9 kDa. Amino acid sequence analysis revealed that the cloned HC29 cDNA contained the conserved catalytic triad and dimer interface of selenium-independent glutathione peroxidase (GPX). Alignment of the predicted amino acid sequences demonstrated that the protein shared 44.7~80.4% similarity with GPX homologues in the thioredoxin-like family. Phylogenetic analysis revealed close evolutionary proximity of the GPX sequence to the counterpart sequences. These results suggest that HC29 cDNA is a GPX, a member of the thioredoxin-like family. Alignment of the nucleic acid and amino acid sequences of HC29 with those of the reported selenium-independent GPX of H. contortus showed that HC29 contained different types of spliced leader sequences as well as dimer interface sites, although the active sites of both were identical. Enzymatic analysis of recombinant prokaryotic HC29 protein showed activity for the hydrolysis of H2O2. These findings indicate that HC29 is a selenium-independent GPX of H. contortus.
Amino Acid Sequence ; Animals ; Base Sequence ; Cloning, Molecular ; DNA, Complementary/genetics/isolation & purification ; Glutathione Peroxidase/*genetics/*metabolism ; Goat Diseases/parasitology ; Goats ; Haemonchiasis/parasitology/prevention & control/*veterinary ; Haemonchus/*enzymology/*genetics ; Hydrogen Peroxide/metabolism ; Molecular Sequence Data ; Phylogeny ; RNA, Helminth/chemistry/genetics ; Random Amplified Polymorphic DNA Technique ; Rats ; Rats, Sprague-Dawley ; Sequence Alignment ; Sequence Analysis, DNA

Fecal samples of 2,056 dairy cattle from 14 farms were collected in three geographical regions of China and stained using a modified acid-fast staining technique to identify Cryptosporidium oocysts. A total of 387 (18.82%) positive samples were identified and further analyzed by polymerase chain reaction (PCR) using primers designed to amplify DNA fragments from the small subunit ribosomal RNA. The PCR products were sequenced and the sequences were deposited in the GenBank database under accession numbers EU369377-84 and GU070730-33. Phylogenetic analysis was performed and a distances matrix generated from these sequences confirmed the existence of Cryptosporidium (C.) parvum 'mouse' genotype, C. bovis, C. andersoni, C. hominis, and C. serpentis in cattle. These results represent the first report on the prevalence and genetic identification of Cryptosporidium species, and may contribute to a better understanding of the epidemiology of Cryptosporidium in cattle in China.
Animals ; Base Sequence ; Cattle ; Cattle Diseases/epidemiology/*parasitology ; Chi-Square Distribution ; China/epidemiology ; Cryptosporidiosis/epidemiology/parasitology/*veterinary ; Cryptosporidium/genetics/*isolation & purification ; DNA, Protozoan/chemistry/genetics ; Feces/parasitology ; Female ; Molecular Sequence Data ; Phylogeny ; Polymerase Chain Reaction/veterinary ; Prevalence ; RNA, Ribosomal, 18S/chemistry/genetics ; Sequence Alignment ; Sequence Analysis, DNA

Avian metapneumovirus (aMPV) causes upper respiratory tract infections in chickens and turkeys. Although the swollen head syndrome (SHS) associated with aMPV in chickens has been reported in Korea since 1992, this is the study isolating aMPV from chickens in this country. We examined 780 oropharyngeal swab or nasal turbinate samples collected from 130 chicken flocks to investigate the prevalence of aMPV and to isolate aMPV from chickens from 2004-2008. Twelve aMPV subtype A and 13 subtype B strains were detected from clinical samples by the aMPV subtype A and B multiplex real-time reverse transcription polymerase chain reaction (RRT-PCR). Partial sequence analysis of the G glycoprotein gene confirmed that the detected aMPVs belonged to subtypes A and B. Two aMPVs subtype A out of the 25 detected aMPVs were isolated by Vero cell passage. In animal experiments with an aMPV isolate, viral RNA was detected in nasal discharge, although no clinical signs of SHS were observed in chickens. In contrast to chickens, turkeys showed severe nasal discharge and a relatively higher titer of viral excretion than chickens. Here, we reveal the co-circulation of aMPV subtypes A and B, and isolate aMPVs from chicken flocks in Korea.
Animals ; Antibodies, Viral/blood ; Base Sequence ; *Chickens ; Glycoproteins/chemistry/genetics ; Metapneumovirus/immunology/*isolation & purification ; Molecular Sequence Data ; Paramyxoviridae Infections/immunology/*veterinary/virology ; *Phylogeny ; Poultry Diseases/immunology/*virology ; RNA, Viral/chemistry/genetics ; Respiratory Tract Infections/immunology/*veterinary/virology ; Reverse Transcriptase Polymerase Chain Reaction/veterinary ; Sequence Alignment ; Sequence Analysis, DNA ; Serotyping ; Specific Pathogen-Free Organisms ; Turkeys

In order to control the H9N2 subtype low pathogenic avian influenza (LPAI), an inactivated vaccine has been used in Korea since 2007. The Korean veterinary authority permitted the use of a single H9N2 LPAI vaccine strain to simplify the evolution of the circulating virus due to the immune pressure caused by the vaccine use. It is therefore important to determine the suitability of the vaccine strain in the final inactivated oil emulsion LPAI vaccine. In this study, we applied molecular rather than biological methods to verify the suitability of the vaccine strain used in commercial vaccines and successfully identified the strain by comparing the nucleotide sequences of the hemagglutinin and neuraminidase genes with that of the permitted Korean LPAI vaccine strain. It is thought that the method used in this study might be successfully applied to other viral genes of the LPAI vaccine strain and perhaps to other veterinary oil emulsion vaccines.
Animals ; Base Sequence ; Birds ; DNA, Viral/chemistry/genetics ; Hemagglutinin Glycoproteins, Influenza Virus/chemistry/genetics ; Influenza A Virus, H9N2 Subtype/genetics/*immunology ; Influenza Vaccines/genetics/*immunology ; Influenza in Birds/*immunology/prevention & control/virology ; Molecular Sequence Data ; Neuraminidase/chemistry/genetics ; Polymerase Chain Reaction/veterinary ; Republic of Korea ; Sequence Alignment ; Vaccines, Inactivated/genetics/immunology

Peste des petits ruminants virus is a member of Morbillivirus Paramyxoviridae. The complete genome of a Peste des petits ruminants virus (PPRV) isolate, China/Tib/07 was sequenced and molecular characteristics was analyzed. The internal sequences of the virus genome were amplified by RT-PCR with primers designed according to the published data in GenBank, while the sequences of the 3' and 5' ends of the genome were determined by RACE. Amplification products were directly sequenced,assembled and analyzed with DNAStar4.0. Results showed that China/Tib/07 genome consisted of 15 948 nucleotides in length, encoding six structural proteins and two non-structural proteins just like other known PPRV genomes. Phylogenetically, the virus genome shared homology of 91.6%-98.1% with Southwest Asian isolates among PPRV strains and the highest homology of 64.3% with rinderpest virus among morbillivirus members.
Animals ; Base Sequence ; Cercopithecus aethiops ; China ; Genome, Viral ; Molecular Sequence Data ; Peste-des-Petits-Ruminants ; veterinary ; virology ; Peste-des-petits-ruminants virus ; classification ; genetics ; isolation & purification ; Phylogeny ; Sequence Alignment ; Sequence Analysis, DNA ; Sheep ; Sheep Diseases ; virology ; Vero Cells ; Viral Proteins ; genetics