Hideto Fukushi

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.

Absence of Chlamydia psittaci in ocular adnexal lymphoma from Japanese patients

Low-grade malignant lymphomas arising from mucosa-associated lymphoid tissue (MALT) represent a distinct clinicopathological entity of B-cell non-Hodgkin lymphoma (Isaacson & Wright, 1984). MALT lymphoma tends to remain localised and the extranodal sites of involvement include the gastrointestinal tract, salivary gland, thyroid and ocular region. MALT lymphoma often occurs in relation to autoimmune disorders or chronic antigen stimulation, but the pathogenetic mechanisms of this type of lymphoma have not been well defined. Recently, Ferreri et al (2004) reported that 32 (80%) of 40 Italian patients with ocular adnexal lymphoma harboured Chlamydia psittaci DNA. The findings suggested that C. psittaci infection might contribute to the development of these lymphomas. Soon after this, two other groups independently investigated ocular adnexal lymphoma for the presence of C. psittaci DNA in 57 patients from south Florida (Rosado et al, 2005) and 11 patients from the north-eastern USA (Vargas et al, 2005) and found that none of them carried C. psittaci DNA. One explanation for this discrepancy concerns geographic differences in the incidence of C. psittaci infection. However, all of these reports are based on findings from patients in the USA and Europe, and therefore additional surveys evaluating the association between C. psittaci and ocular adnexal lymphomas including MALT lymphoma in other geographical regions of the world would be warranted. There has been no report from Asia thus far. In this study, we looked for C. psittaci DNA in tumour specimens from Japanese patients with primary ocular adnexal lymphoma. Tissues obtained by an incisional biopsy from 24 Japanese patients (12 males and 12 females, mean age 56 years; range 34–73 years) with localised lymphoproliferative conditions of the ocular adnexa, including 18 MALT lymphomas, three nonMALT lymphomas and three reactive ocular lymphoid hyperplasias, were studied. The localities of the tumours were the orbit in 14 cases, lacrimal glands in four cases, and conjunctiva in six cases. DNA was obtained from frozen or paraffinembedded tissues using the phenol/chroroform extraction technique or DNA extraction system according to the manufacturer’s instructions (Takara, Tokyo, Japan). All samples were successfully amplified by polymerase chain reaction (PCR) for human b-globin sequences, yielding a 123-bp amplicon, which indicated that amplifiable DNAs were present. Three independent PCRs were used to detect C. psittaci DNA in this study. First, we used the same method employed in the previous studies (Ferreri et al, 2004; Rosado et al, 2005; Vargas et al, 2005). The touchdown enzyme time release PCR with CPS100 and CPS101 primers produces a 111-bp amplicon (Madico et al, 2000). The primers amplify a highly conserved region encoding the 16S ribosomal RNA gene. The second method was a nested PCR to detect the same DNA region with the first-step primers (5¢-ACGGAATAATGACTTCGG-3¢ and 5¢-TACCTGGTACGCTCAATT-3¢) and the second-step primers (5¢-ATAATGACTTCGGTTGTTATT-3¢ and 5¢-TGTTTTA GATGCCTAAACAT-3¢), generating a 127-bp amplicon. The PCR primers for the third C. psittaci PCR were derived from another DNA region encoding the ompA gene, and the primer sequences were 5¢-GCCTTAAACATCTGGGATCG-3¢ and 5¢-GCACAACCACATTCCCATAAAG-3¢, giving a 248-bp fragment. All three PCRs failed to detect C. psittaci DNA in any of our samples, while positive control reactions using DNA prepared from C. psittaci strain Cal-10 yielded amplicons of expected size. In addition, our samples were negative for Chlamydia pneumoniae and Chlamydia trachomatis DNA sequences. Our results showed no correlation between C. psittaci infection and ocular adnexal lymphoma in Japan, supporting the observation from the USA (Rosado et al, 2005; Vargas et al, 2005). All these studies were based on testing C. psittaci DNA sequences. Given the serological evidence of an association between chronic chlamydia infections (C. pneumoniae and C. trachomatis) and lymphomas (Anttila et al, 1998), serological surveys for C. psittaci may also help to better understand the relationship between C. psittaci and ocular adnexal lymphoma. Although the ‘hit-and-run’ mechanism cannot be fully excluded, the present findings indicate that C. psittaci is unlikely to have played a pathogenetic role in ocular adnexal lymphoma in the Japanese population.

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.

SCID mouse model for lethal Q fever.

ABSTRACT Q fever, a worldwide zoonosis caused by Coxiella burnetii, has many manifestations in humans. Endocarditis is the most serious complication of Q fever. Animal models are limited to acute pulmonary or hepatic disease and reproductive disorders. An appropriate experimental animal model for Q fever endocarditis does not yet exist. In this study, severe combined immunodeficient (SCID) mice infected with C. burnetii showed persistent clinical symptoms and died, whereas immunocompetent mice similarly infected became asymptomatic and survived. The SCID mice examined in this study had severe chronic lesions in their primary organs: the heart, lung, spleen, liver, and kidney. The heart lesions of the SCID mice were similar to those in humans with chronic Q fever endocarditis: they had focal calcification and expanded macrophages containing C. burnetii. The 50% lethal dose of C. burnetii in SCID mice was at least 108 times less than that in immunocompetent mice. The SCID mouse is highly susceptible to C. burnetii, and the immunodeficiency of the host enhances the severity of Q fever. This animal model could provide a new tool for the study of chronic Q fever and Q fever in immunodeficient hosts.

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.

Differentiation of Coxiella burnetii by sequence analysis of the gene (com1) encoding a 27-kDa outer membrane protein

The gene (com1) encoding a 27‐kDa outer membrane protein in 21 strains of Coxiella burnetii from a variety of clinical and geographical sources was sequenced for strain differentiation. The com1 gene was highly conserved among all the strains tested but there were several differences in nucleotide and deduced amino acid sequences. Based on the com1 gene‐specific nucleotides and deduced amino acids, the 21 strains were divided into four groups. Group 1 contained 14 strains originating from ticks, cattle and human cases of acute Q fever. Groups 2 and 3 included 2 and 3 strains, respectively, originating from human cases of chronic Q fever. Group 4 contained 2 strains originating from a human case of acute Q fever and a goat with abortion. The results indicated that the strains originating from ticks, cattle and human cases of acute Q fever differed at the molecular level from those of human chronic Q fever. This study suggests that a sequence analysis of the com1 gene can be used for strain differentiation of C. burnetii.

A hamster model of equine herpesvirus 9 induced encephalitis

An acute and lethal infection of equine herpesvirus 9 (EHV-9), a new type of equine herpesvirus, was established in Syrian hamsters by intranasal inoculation. Clinical symptoms included the loss of body weight, nasal and ocular discharges and apparent neurological symptoms. Both LD50 and ID50 were equal at 33 plaque forming units. Histological and immunohistochemical examination demonstrated that the virus replicated in the olfactory mucosal cells and in the neurons of the olfactory bulbs, cerebrum and mesencephalon. The induction of encephalitis by intranasal but not by other routes of inoculation (i.v., i.p., i.m.) indicated that EHV-9 entered the brain via the olfactory nerve and then spread trans-synaptically to connecting neurons along the olfactory tract. This animal model should be useful for studying the pathogenesis and neurovirulence of this newly discovered neurotropic virus as well as other neurotropic herpesviruses.