Kenji Ohya

Genetic Diversity and Epizootiology of Chlamydophila psittaci Prevalent among the Captive and Feral Avian Species Based on VD2 Region of ompA Gene

To study genetic diversity and occurrence of Chlamydophila psittaci, a total of 1,147 samples from 11 avian orders including 53 genera and 113 species of feral and captive birds were examined using ompA gene based nested PCR. Three types of chlamydiae: C. psittaci (94.12%), C. abortus (4.41%) and unknown Chlamydophila sp. (1.47%) were identified among 68 (5.93%) positive samples (Psittaciformes‐59, Ciconiiformes‐8 and Passeriformes‐1). Based on nucleotide sequence variations in the VD2 region of ompA gene, all 64 detected C. psittaci strains were grouped into 4 genetic clusters. Clusters I, II, III and IV were detected from 57.35%, 19.12%, 10.29% and 7.35% samples respectively. A single strain of unknown Chlamydophila sp. was found phylogenetically intermediate between Chlamydophila species infecting avian and mammalian hosts. Among Psittaciformes, 28 out of 81 tested species including 10 species previously unreported were found to be chlamydiae positive. Chlamydiosis was detected among 8.97% sick and 48.39% dead birds as well 4.43% clinically normal birds. Therefore, it was observed that though various genetically diverse chlamydiae may cause avian chlamydiosis, only a few C. psittaci strains are highly prevalent and frequently associated with clinical/subclinical infections.

The ORF37 (UL24) is a neuropathogenicity determinant of equine herpesvirus 1 (EHV-1) in the mouse encephalitis model

Equine herpesvirus 1 (EHV-1) bacterial artificial chromosome clone (Ab4p BAC) was established based on neuropathogenic strain Ab4p. ORF37 encoding UL24 was replaced with a selection cassette, rpsL-neo gene, to produce an ORF37 deletion mutant, Ab4pORF37. Transfection of RK-13 cells with Ab4pORF37 genome DNA produced infectious virus, indicating that ORF37 is not essential for EHV-1 replication in cell culture. Deletion of ORF37 had no effect on the transcript expression of neighboring genes, ORF36 and ORF38, and the growth activity in MDBK cells. Ab4pDeltaORF37 lost neuropathogenicity in CBA/N1 mice as indicated by the absence of any neurological disorders and death. The growth of Ab4pDeltaORF37 in cultivated neural cells was one order of magnitude lower than that of parental and revertant viruses. These results indicated that the ORF37 is a neuropathogenicity determinant of EHV-1 in the mouse encephalitis model.

Detection of infectious bursal disease virus (IBDV) genome in free-living pigeon and guinea fowl in Africa suggests involvement of wild birds in the epidemiology of IBDV

Infectious bursal disease (IBD) virus (IBDV) serotype 1 is the causative agent of IBD, a highly contagious immunosuppressive disease of young chickens. In this study, we examined IBDV infection in apparently healthy 21 guinea fowls and 20 pigeons obtained in Tanzania and Zambia by virus neutralization test (VNT) and reverse transcription polymerase chain reaction (RT-PCR) for the VP2 hypervariable region (VP2-HVR) of IBDV. Two guinea fowls (9.5%) in Tanzania were RT-PCR and VNT positive for IBDV, and 1 pigeon (5%) in Tanzania was RT-PCR positive and VNT negative. Phylogenetic analysis based on the nucleotide sequences of the PCR products indicated that segment A of IBDV detected from one guinea fowl and a pigeon belonged to the very virulent genotype of European/Asian type, while the other IBDV detected from a guinea fowl belonged to the classical genotype. To our knowledge, this is the first report of detection of the IBDV genome in free-living pigeons and guinea fowls. The detection of IBDV from apparently healthy guinea fowls and pigeons elucidates the role of wild birds in the epidemiology of IBDV.

Development of novel real-time PCR assays for detecting DNA virus infections in psittaciform birds

Viral diseases of psittacine birds are detected presently by PCR. However, conventional PCR methods are not quantitative and the products can sometimes include non-specific products of the same size. To avoid these problems, real-time PCR assays based on the SYBR Green assay system were developed for the detection and quantitation of four virus diseases of psittacine birds: psittacine beak and feather disease, avian polyomavirus infection, psittacid herpesvirus infection, and psittacine adenovirus infection. Up to 1x10(2) copies of virus DNA were detected, indicating that these assays are as sensitive as conventional PCR assays. The assays are specific because they did not amplify any other pathogens including other viruses, bacteria, and fungi in psittacine birds. The assays measured successfully virus loads in clinical samples (blood, feathers, and tissues), showing that these specimens were suitable targets for the detection and quantitation of viral DNA in psittacine birds.

Molecular characterization of infectious bursal disease virus (IBDV) : Diversity of very virulent IBDV in Tanzania

SummaryNucleotide sequences of the VP2 hypervariable region (VP2-HVR) of 14 infectious bursal disease viruses (IBDVs) isolated in Tanzania from 2001 to 2004 were determined. Phylogenetic analysis showed that the isolates diverged into two genotypes and belonged to the very virulent (VV) type. In the phylogenetic tree, strains in one genotype clustered in a distinct group and were closely related to some strains isolated in western Africa, with nucleotide similarities of 96.1–96.8%, while strains in another genotype were clustered within the European/Asian VV type with nucleotide similarities ranging from 97.5 to 99.3%. Both genotypes were widely distributed throughout Tanzania, and had conserved putative virulence marker amino acids (aa) at positions 222(A), 242(I), 256(I), 294(I) and 299(S). Our findings demonstrate for the first time the existence of both African and European/Asian VV-IBDV variants in Tanzania.

Chlamydophila felis CF0218 Is a Novel TMH Family Protein with Potential as a Diagnostic Antigen for Diagnosis of C. felis Infection

ABSTRACT Chlamydophila felis is a causative agent of acute and chronic conjunctivitis and pneumonia in cats (feline chlamydiosis). Also, C. felis is a suspected zoonotic agent of such diseases as non-Chlamydia trachomatis conjunctivitis in humans, although this is controversial. At present, there is no serodiagnostic system that specifically detects C. felis infection conveniently. Current systems use antigens such as lipopolysaccharide that cross-react with all chlamydia species. In addition, it is difficult to distinguish between cats that are vaccinated with the commercial vaccine against C. felis and cats that are infected with C. felis. Here, we describe a new candidate diagnostic antigen for diagnosis of C. felis infection, CF0218, that was obtained by screening a genomic expression library of C. felis Fe/C-56 with C. felis-immunized serum. CF0218 was a putative transmembrane head (TMH) family protein with bilobed hydrophobic motifs at its N terminus, and orthologues of CF0218 were not found in the Chlamydophila pneumoniae or Chlamydia trachomatis genomes. The recombinant CF0218 was not recognized by antiserum against C. trachomatis, suggesting that CF0218 is C. felis specific. CF0218 transcription during the course of C. felis infection was confirmed by reverse transcription-PCR. By indirect immunofluorescence analysis, CF0218 was colocalized with the C. felis-formed inclusion bodies in the infected cells. The antibody response against CF0218 was elevated following C. felis infection but not by vaccination in experimentally vaccinated and infected cats. These results suggest that CF0218, a novel TMH family protein of C. felis, possesses potential as a C. felis infection-specific diagnostic antigen.

Molecular characterization of avian polyomavirus isolated from psittacine birds based on the whole genome sequence analysis.

Seven avian polyomaviruses (APVs) were isolated from seven psittacine birds of four species. Their whole genome sequences were genetically analyzed. Comparing with the sequence of BFDV1 strain, nucleotide substitutions in the sequences of seven APV isolates were found at 63 loci and a high level of conservation of amino acid sequence in each viral protein (VP1, VP2, VP3, VP4, and t/T antigen) was predicted. An A-to-T nucleotide substitution was observed in non-control region of all seven APV sequences in comparison with BFDV1 strain. Two C-to-T nucleotide substitutions were also detected in non-coding regions of one isolate. A phylogenetic analysis of the whole genome sequences indicated that the sequences from the same species of bird were closely related. APV has been reported to have distinct tropism for cell cultures of various avian species. The present study indicated that a single amino acid substitution at position 221 in VP2 was essential for propagating in chicken embryonic fibroblast culture and this substitution was promoted by propagation on budgerigar embryonic fibroblast culture. For two isolates, three serial amino acids appeared to be deleted in VP4. However, this deletion had little effect on virus propagation.

Molecular phylogeny of equine herpesvirus 1 isolates from onager, zebra and Thomson’s gazelle

Viruses related to equine herpesvirus type 1 (EHV-1) were isolated from an aborted fetus of an onager (Equus hemionus) in 1984, an aborted fetus of Grevy’s zebra (Equus grevyi) in 1984 and a Thomson’s gazelle (Gazella thomsoni) with nonsuppurative encephalitis in 1996, all in the USA. The mother of the onager fetus and the gazelle were kept near plains zebras (Equus burchelli). In phylogenetic trees based on the nucleotide sequences of the genes for glycoproteins B (gB), I (gI), and E (gE), and teguments including ORF8 (UL51), ORF15 (UL45), and ORF68 (US2), the onager, Grevy’s zebra and gazelle isolates formed a genetic group that was different from several horse EHV-1 isolates. Within this group, the onager and gazelle isolates were closely related, while the Grevy’s zebra isolate was distantly related to these two isolates. The epizootiological origin of the viruses is discussed.

A novel budgerigar-adenovirus belonging to group II avian adenovirus of Siadenovirus

Five budgerigars in the same breeding facility died or showed ruffled feathers. To determine the cause, five dead or euthanized budgerigars were examined. Splenomegaly was observed at necropsy in all birds examined. Histopathology of the spleen revealed a slight-to-moderate deletion of lymphocytes and increase of macrophages. Concurrent congestions in several tissues such as liver, lung, kidney, and/or brain and basophilic intranuclear inclusion bodies in the epithelial cells of renal tubules were found in all the birds examined. Psittacine adenoviral DNA was detected in the kidney of one of the five budgerigars by PCR. Sequencing and phylogenetic analysis of the hexon gene revealed that the adenovirus gene detected in the budgerigar was derived from an unknown adenovirus belonging to the genus Siadenovirus. Using a new pair of primers based on the obtained sequence, we confirmed the presence of the newly found adenovirus in all five birds. The newly found unknown adenovirus is designated as Budgerigar Adenovirus 1.

Molecular epidemiology of infectious bursal disease virus in Zambia.

Nucleotide sequences of the VP2 hypervariable region (VP2-HVR) of 10 infectious bursal disease viruses detected in indigenous and exotic chickens in Zambia from 2004 to 2005 were determined. Phylogenetic analysis showed that the viruses diverged into two genotypes and belonged to the African very virulent types (VV1 and VV2). In the phylogenetic tree, strains in one genotype clustered in a distinct group and were closely related to some strains isolated in western Africa (VV1), with nucleotide similarities of 95.7%- 96.5%. Strains in the other genotype were clustered within the eastern African VV type (VV2), with nucleotide similarities of 97.3%- 98.5%. Both genotypes were distributed in the southern parts of Zambia and had a unique conserved amino acid substitution at 300 (E→A) in addition to the putative virulence marker at positions 222(A), 242(I), 256(I), 294(I) and 299(S). These findings represent the first documentation of the existence of the African VV-IBDV variants in both indigenous and exotic chickens in Zambia.