Katsuya Hirai

Proposal of Chlamydia pecorum sp. nov. for Chlamydia strains derived from ruminants.

Chlamydia pecorum sp. nov. is proposed as the fourth species of the genus Chlamydia on the basis of the results of a genetic analysis of Chlamydia strains that were isolated from cattle and sheep which had various diseases, including sporadic encephalitis, infectious polyarthritis, pneumonia, and diarrhea. The levels of DNA-DNA homology between C. pecorum and strains of C. psittaci, Chlamydia pneumoniae, and Chlamydia trachomatis were less than 10%. Several DNA probes were used to identify C. pecorum. The C. pecorum strains were distinguished from C. psittaci strains by the results of immunological assays, including an immunofluorescence antibody assay performed with monoclonal antibodies and an immunoblot analysis of the immunological specificity of the major outer membrane protein. Species identification was based on results obtained from DNA analyses and serology. The type strain of C. pecorum is strain ATCC VR628.

Isolation of Coxiella burnetii from Dairy Cattle and Ticks, and Some Characteristics of the Isolates in Japan

Coxiella burnetii was isolated from raw milk (36/214, 16.8%) and uterus swab samples (13/61, 21.3%) originating from dairy cattle with reproductive disorders, aborted bovine fetus samples (2/4, 50%), mammary gland samples (4/50, 8%) originating from healthy dairy cattle, and tick samples (4/15, 26.7%) originating from 2 pastures. Fifty‐nine strains had various degrees of pathogenicity, high (8; 13.6%), moderate (28; 47.5%) and low (23; 39%), for guinea pigs. The results of isolation suggested a high prevalence of Coxiella infection in dairy cattle with reproductive problems in Japan. Twelve strains (7, 2 and 3 strains from cattle, ticks and humans, respectively) and the reference Nine Mile strain of phases I and II were propagated in both yolk sacs of embryonated hen eggs and Buffalo green monkey (BGM) cell cultures. Protein profiles of these strains were similar to those of the reference strain of phase I. Lipopolysaccharide (LPS) profiles of 12 strains were similar to those of the reference strain of phase I and different from those of the reference strain of phase II. The LPS profiles of 12 strains suggested that these strains are associated with an acute form of Q fever.

Sequence and phylogenetic analyses of highly virulent infectious bursal disease virus

SummaryThe nucleotide sequences of the genome segment A and B encoding the precursor polyprotein (NH2-VP2-VP4-VP3-COOH) and VP1 were determined for a highly virulent strain of infectious bursal disease virus (IBDV). The precursor polyprotein and VP1 coding regions of highly virulent OKYM strain consisted of 3 039 nucleotides (1 012 deduced amino acids) and 2 640 nucleotides (879 deduced amino acids), respectively. Comparison of the deduced amino acid sequences of the highly virulent IBDV (HV-IBDV) with other serotype 1 and 2 sequences revealed 17 amino acid residues which were conserved only in the HV-IBDV. Among the 17 unique amino acid differences, 8 were in VP1, 4 were in VP2, 3 were in VP3 and 2 were in VP4. Although it is impossible to predict the effect of the unique amino acid residues without detailed knowledge of the three-dimensional structure and function of the proteins, they could affect the virulence of HV-IBDV. Alignment of the nucleic acid sequences of precursor polyprotein, VP1, VP2, VP3 and VP4 coding regions followed by distance analysis allowed the generation of phylogenetic trees. The same tree topology was obtained for the nucleotide sequence of precursor polyprotein, VP2, VP3 and VP4. On the other hand, the tree topology of VP1 was quite different from that obtained for the nucleotide sequence of precursor polyprotein, VP2, VP3 and VP4. These findings indicate that not a genetic recombination but a genetic reassortment may play an important role in the emergence of HV-IBDV.

Phylogenetic Analysis of the Genus Chlamydia Based on 16S rRNA Gene Sequences

The phylogenetic relationships among Chlamydia spp. were investigated by comparing 16S rRNA gene sequences. In this analysis we used 14 strains of Chlamydia psittaci, including seven feline isolates, two avian isolates, two human isolates, one bovine isolates, one ovine isolate, and one koala isolate; and nine strains of Chlamydia trachomatis, including six human isolates, two swine isolates, and one mouse isolate. A phylogenetic analysis of the 16S rRNA gene sequences of these organisms and seven previously published sequences revealed eight genetic groups which formed two clusters. The first cluster was composed of C. pecorum, Chlamydia pneumoniae, and C. psittaci and included three genetic groups (one group containing avian, human, and ovine strains, one group containing feline strains, and one group containing guinea pig strains). The second cluster was composed of C. trachomatis and also included three genetic groups (one group containing human strains, one group containing swine isolates, and one group containing rodent strains). The strains in each genetic group exhibited similar genetic distances. The results of the phylogenetic analysis agreed with the results of previous genomic DNA, ompA gene allele, and biotyping studies. Therefore, the genetic groups based on genetic distances may be considered a criterion for species identification.

Coxiella burnetii isolates cause genogroup-specific virulence in mouse and guinea pig models of acute Q fever.

ABSTRACT Q fever is a zoonotic disease of worldwide significance caused by the obligate intracellular bacterium Coxiella burnetii. Humans with Q fever may experience an acute flu-like illness and pneumonia and/or chronic hepatitis or endocarditis. Various markers demonstrate significant phylogenetic separation between and clustering among isolates from acute and chronic human disease. The clinical and pathological responses to infection with phase I C. burnetii isolates from the following four genomic groups were evaluated in immunocompetent and immunocompromised mice and in guinea pig infection models: group I (Nine Mile, African, and Ohio), group IV (Priscilla and P), group V (G and S), and group VI (Dugway). Isolates from all of the groups produced disease in the SCID mouse model, and genogroup-consistent trends were noted in cytokine production in response to infection in the immunocompetent-mouse model. Guinea pigs developed severe acute disease when aerosol challenged with group I isolates, mild to moderate acute disease in response to group V isolates, and no acute disease when infected with group IV and VI isolates. C. burnetii isolates have a range of disease potentials; isolates within the same genomic group cause similar pathological responses, and there is a clear distinction in strain virulence between these genomic groups.

Characterization of Immunosuppression in chickens by infectious bursal disease virus.

Chickens were infected with infectious bursal disease (IBD) virus in ovo or at different times posthatching to 6 weeks of age. The B- and T-cell responses in the lymphoid tissues and blood were examined sequentially to 8 weeks of age by using indirect immunofluorescence. The proportion of B-cells was consistently lower in infected birds than in controls, especially in chicks infected as embryos or at 1 day old. The proportion of T-cells increased following these early infections but was slightly lower in spleen and blood of birds infected at 1, 4, and-6 weeks of age. Serum IgM levels dropped significantly after infection, regardless of the time of infection. IgG levels decreased following early infection but increased after infection at 1 week old or more. The results strongly suggest that B-cells are the target for IBD virus infection.

Geographic population structure and sequence divergence in the mitochondrial DNA control region of the Japanese wild boar (Sus scrofa leucomystax), with reference to those of domestic pigs

Mitochondrial DNA (mtDNA) control regions from 40 Japanese wild boars were examined by direct sequencing after amplification by PCR. From the DNA sequences obtained, we found eight haplotypes, whose differences arose via transitions. The geographical distribution of these different haplotypes indicated that wild boar populations inhabited limited areas and that there was some restricted gene flow between local populations. Eight mtDNA haplotypes from Eastern and Western domestic pigs and the Ryukyu wild boar were also analyzed as references to those from Japanese wild boars. The cluster analyses of the control-region sequences showed that those from Japanese wild boards belong to the Asian type as do those from Eastern domestic pigs and the Ryukyu wild boar, which differed from the European type (Western domestic pigs).

In Vitro Attenuation of Highly Virulent Infectious Bursal Disease Virus: Some Characteristics of Attenuated Strains

Some strains of highly virulent infectious bursal disease virus (HV-IBDV) were adapted through serial passage in embryonated eggs. The embryonated egg-adapted HV-IBDV was successfully adapted to grow in chicken embryo fibroblast (CEF) cell cultures showing a cytopathic effect by preparing the CEF cells from the virus-infected embryos. The embryonated egg- and cell culture-adapted strains significantly reduced their pathogenicity to, and did not kill any, young chickens in experimental infection. The bursal lesions of the adapted strain-infected chickens were similar to those observed in classical strain-infected chickens. Cross-virus neutralization analysis showed antigenic diversity between the cell culture-adapted HV-IBDV strains and classical strains. In immunization tests, the adapted strain-immunized chickens showed good protection against the fatal infection of HV-IBDV. Especially, in case of challenge at 3 days postimmunization, the adapted strains showed effective immunogenicity. The adapted strains appear to provide a new and effective live vaccine against HV-IBDV infection.

A seroepidemiological study of the risks of Q fever infection in Japanese veterinarians

The causative agent of Q fever, a widespread zoonotic disease, is the bacteria Coxiella burnetii. Although cases of Q fever have been documented in countries throughout the world, the prevalence of the disease in Japan is not yet known. Q fever is a demonstrated occupational hazard to those employed in zoological professions, but the risk to Japanese veterinarians has not yet been quantified. In order to evaluate the risk to Japanese veterinarians, we performed a serological survey using serum samples from 267 veterinarians. Two control groups consisting of 352 medical workers and 2003 healthy blood donors were also evaluated. The antibody titers of the serum samples were measured by indirect immunofluorescence assay (IFA) using phase II C. burnetii Nine Mile strain as the antigen. The positive rate of IgG antibody was 13.5% in the veterinarians, which was higher than in the blood donors (3.6%, p < 0.001) and medical workers (5.1%, p < 0.001). These findings suggest that Japanese veterinarians have a higher risk of infection by C. burnetii than other members of the Japanese population. An interesting finding of this study was that positive rates of IgG and IgM antibodies in the blood donor group were higher in younger individuals. The IgM antibody positive rate was the highest in females under 30 years old.

Acute cerebellitis caused by Coxiella burnetii

We report a childhood case of severe acute cerebellitis caused by Coxiella burnetii. After 10 days of fever and headache, the patient fell into a drowsy state. Examination of the cerebrospinal fluid (CSF) revealed pleocytosis, an increased level of protein, and negative results in bacterial and viral studies. Magnetic resonance imaging demonstrated a herniated tonsil compressed by the swollen vermis. Administration of minocycline relieved the patients clinical symptoms. C. burnetii was isolated from the CSF obtained during convalescence. Ann Neurol 1999;45:124–127