Akio Kiuchi

Detection of feline herpesvirus 1 DNA by the nested polymerase chain reaction.

The thymidine kinase region of feline herpesvirus 1 (FHV 1) genome in ocular/nasal swabs from cats with clinical manifestations of upper respiratory disease was amplified by nested polymerase chain reaction (nested PCR). Two primer pairs were prepared for nested PCR. FHV 1 DNA in ocular/nasal swabs was extracted using instaGene-DNA purification matrix. Nested PCR for the FHV 1 culture supernatants was ten times as sensitive as single PCR. On comparing viral isolation with single PCR and nested PCR for the detection of FHV 1 in ocular/nasal secretions, of 5 samples that yielded infectious virus in cell culture, 3 (60%) were positive in single PCR and 5 (100%) were positive in nested PCR. When 22 ocular/nasal swabs that did not yield FHV 1 were assayed, 3 were negative in both single PCR and nested PCR, 2 were positive in both single and nested PCR and 17 were positive in only nested PCR. Thus, FHV 1 was detected in 19/22 (86.4%) by the nested PCR and in 2/22 (9%) by single PCR. These results show that nested PCR is 4.8 (24 positive samples/5 positive samples) times as sensitive as single PCR.

Phylogenetic Analysis of Field Isolates of Feline Calcivirus (FCV) in Japan by Sequencing Part of Its Capsid Gene

The molecular epidemiology of the infectious disease caused by feline calcivirus (FCV) in Japan was investigated by analysing the phylogenetic relationship among 21 Japanese field isolates, including the F4 strain, and 30 global isolates. Parts of the capsid gene (B–F) of the isolates were amplified by RT-PCR, and the amino acid sequences were compared with those from the global isolates. Thirty-seven and 14 out of a total of 51 isolates were clustered into two distinct genogroups, I and II respectively, by UPGMA and NJ analysis. Seven of the 21 Japanese isolates (33%) fell into group I together with 30 global isolates, while the other 14 Japanese isolates (67%) belonged to group II. The bootstrap repetition analysis of groups I and II formed by the NJ method gave a value of 99.0%. The 14 latter Japanese isolates were clearly separated from the isolates in group I, and they were different from any previously known FCV, forming a new genogroup, which implies that this lineage has been confined to Japan. Comparing the amino acid sequences shared by groups I and II, the amino acid at position 377 in B region was asparagine (Asn or Asp (NH2)) in group I, while it was lysine (Lys) in all the strains in group II. Similarly, the amino acid at position 539 in the F region was alanine (Ala) or proline (Pro) in group I, while it was valine (Val) in group II; glycine (Gly) at position 557 in group I was serine (Ser) in Group II; and phenylalanine (Phe) or leucine (Leu) at position 566 in genogroup I was tyrosine (Tyr) in group II.

IgE reactivity to a Cry j 3, an allergen of Japanese cedar (Cryptomeria japonica) pollen in dogs with canine atopic dermatitis.

The present study investigated IgE reactivity to a new Cryptomeria japonica pollen allergen (Cry j 3) in dogs with atopic dermatitis by using a fluorometric ELISA. Serum samples from 15 dogs that showed IgE sensitivity to crude C. japonica pollen allergen by ELISA were tested for specific IgE to each allergen, individually. All 15 dogs had anti-Cry j 1 IgE, 6 (40%) had anti-Cry j 2 IgE, and 11 (73%) had anti-Cry j 3 IgE. Further, we found that these anti-Cry j 3 IgE reacted to Cry j 3 with immunoblotting analysis. These findings indicate that Cry j 3 may be a major allergen in dogs.

Large-scale survey of adverse reactions to canine non-rabies combined vaccines in Japan.

Abstract Canine non-rabies combined vaccines are widely used to protect animals from infectious agents, and also play an important role in public health. We performed a large-scale survey to investigate vaccine-associated adverse events (VAAEs), including anaphylaxis, in Japan by distributing questionnaires on VAAEs to veterinary hospitals from April 1, 2006 through May 31, 2007. Valid responses were obtained for 57,300 vaccinated dogs at 573 animal hospitals; we obtained VAAEs information for last 100 vaccinated dogs in each veterinary hospital. We found that of the 57,300, 359 dogs showed VAAEs. Of the 359 dogs, death was observed in 1, anaphylaxis in 41, dermatological signs in 244, gastrointestinal signs in 160, and other signs in 106. Onset of VAAEs was mostly observed within 12h after vaccination (n =299, 83.3%). In this study, anaphylaxis events occurred within 60min after vaccination, and about half of these events occurred within 5min (n =19, 46.3%). Furthermore, where anaphylaxis was reported, additional information to support the diagnosis was obtained by reinvestigation. Our resurvey of dogs with anaphylaxis yielded responses on 31 dogs; 27 of these demonstrated collapse (87.1%), 24 demonstrated cyanosis (77.4%), and both signs occurred in 22 (71.0%). Higher rates of animal VAAEs, anaphylaxis, and death were found in Japan than in other countries. Further investigations, including survey studies, will be necessary to elucidate the interaction between death and vaccination and the risk factors for VAAEs, and thus develop safer vaccines. Moreover, it may also be necessary to continually update the data of VAAEs.

Phylogenetic Analysis of the Erysipelothrix rhusiopathiae and Erysipelothrix tonsillarum Based Upon 16s rRNA

The nucleotide sequences of 16s rRNA genes of Erysipelothrix rhusiopnthiae and Erysipelothrix tonsillarum were determined. The sequences are almost similar (99.8%) with only three nucleotides mismatched.

Properties of a calicivirus isolated from cats dying in an agitated state

In June 1993, two of five pet cats kept in Yokohama city in Japan suddenly became agitated and died. Feline calicivirus (FCV) was isolated from them. One strain (FCV-S) was isolated from the spinal cord, lung and tonsil of cat 1, another (FCV-B) from the ileum, medulla oblongata and cervical spinal cord of cat 2, and a third (FCVSAKURA) from the oral cavity of one of the three surviving cats which showed no clinical signs. These three strains were equally resistant to pH 3˙0 and serologically similar to each other, but distinct from strain F9. A genetic analysis, using a 208 base pair fragment from region E of the capsid, showed that FCV-Ari had a 70˙4 per cent nucleotide and 77˙3 per cent amino acid homology and FCV-F9 had a 68˙6 per cent nucleotide and 73˙9 per cent amino acid homology with the three strains, indicating that these two strains were genetically distinct from the three new isolates. Unvaccinated cats and cats which had been vaccinated against FCV-F9 developed watery diarrhoea but did not become agitated after the administration of FCV-S. The FCV-S strain did not induce signs of excitability after it was administered orally to specific pathogen-free cats.

Analysis of Conformational and Sequential IgE Epitopes on the Major Allergen Cry j 2 of Japanese Cedar (Cryptomeria japonica) Pollen in Humans by Using Monoclonal Antibodies for Cry j 2

PurposeJapanese cedar (Cryptomeria japonica; CJ) pollinosis is a type I allergy induced by CJ pollen, and Cry j 2 is one of the major allergens in this pollen. In a previous study, we analyzed IgE epitopes on Cry j 2 in humans by using synthetic peptides. The main purpose of this study was to identify B-cell epitopes on Cry j 2 in patients with CJ pollinosis by using monoclonal antibodies (mAbs) for Cry j 2.MethodsWe used ELISA with mAbs for the epitope analysis. Sera samples were collected from 80 patients with CJ pollinosis, and allergenic epitopes for mAbs and human IgE were identified using ELISA with synthetic peptides. The importance of the epitopes for human IgE was analyzed using an inhibition ELISA.ResultsFour independent epitopes (epitope #1, #2, #3, and #4) were identified on Cry j 2 with the use of mAbs. Epitope #3 and #4, corresponding to peptides No. 25 and No. 33, respectively, were newly determined as epitopes for mAbs and human IgE. Inhibition ELISA showed that not only epitope #2 (sequential) but epitope #1 (conformational) may play an important role in the CJ pollinosis.ConclusionsOur results revealed 4 epitopes, including two new ones, on Cry j 2. We also found that inhibition ELISA with appropriate mAbs could be a viable method of evaluating the importance of the conformational and sequential epitopes for human IgE. These results are beneficial for the development of safer and more efficient therapeutic strategies for treating CJ pollinosis.

Methods for Rapid Cloning and Detection for Sequencing of Cloned Inverse PCR‐Generated DNA Fragments Adjacent to Known Sequences in Bacterial Chromosomes

Since the invention of PCR, many adaptation techniques have been developed for sequencing DNA fragments flanking known sequences. Of them, inverse PCR is a matter of interest because of the simplicity of its principle. However, the protocols for inverse PCR introduced so far consist of some time‐consuming procedures, and with them, we cannot “walk” chromosomes too far since the number of suitable restriction enzymes is limited. Our experiments led to confirming simpler technical approaches applicable to the case of bacterial chromosomes, that is, designing two end‐specific “contextual” sequences with which we can quickly detect the desired clones of targeted DNA fragments by simply analyzing PCR products, employing “the minimum value of the desired fragments” as a “discriminating minimum” value to decrease contaminant DNA fragments, and creating a new tandem of two cleaved end fragments of a known sequence (“reordering”) for PCR amplification in combination with cloning of the inverse PCR‐generated DNA. With the improvements, we could both simplify the procedures and broaden the capacity of the inverse PCR in “walking” chromosomes.

Genogrouping of Vaccine Breakdown Strains (VBS) of Feline Calicivirus in Japan

Although prevention of feline calcivirus (FCV) infection by vaccination has been attempted, and isolation of FCV, development of the disease, and a few fatal cases in vaccinated cats have been reported. Fifteen FCV strains isolated from cats that had been vaccinated with commercially available FCV vaccines (F9, FCV-255, and FC-7) were genogrouped. Molecular analysis of viral genomes involved the construction of a phylogenetic tree of capsid genes using the NJ method. Cat anti-F9 serum and rabbit anti-FCV-255 serum were used for virus neutralization tests. Molecular phylogenetic analysis of the amino acid sequences of 15 virus isolates and those of the previously published and GenBank-deposited 9 global and 14 Japanese strains showed that 8 (53%) of the 15 virus isolates as well as the vaccine strains F9 and FCV-255 belonged to genogroup I (GAI), and 7 (47%) belonged to genogroup II (GAII). Of the 8 GAI strains, 2 were isolated from cats that had been vaccinated with an F9 strain live vaccine, 5 from cats vaccinated with an FCV-255-derived vaccine, and 1 from a cat vaccinated with an FC-7-derived vaccine. Of the 7 GAII strains, 5 were isolated from cats that had been vaccinated with the F9 strain live vaccine, 1 from a cat vaccinated with the FCV-255-derived vaccine, and 1 from a cat vaccinated with the FC-7-derived vaccine. These results indicate that more vaccine breakdown strains isolated from the cats vaccinated with the F9 strain-derived vaccine belong to GAII than to GAI, whereas more vaccine breakdown strains isolated from the cats vaccinated with the FCV-255 strain-derived vaccine belong to GAI than to GAII, and that when the FC-7 strain-derived vaccine is used, the vaccine breakdown strains belong almost equally to GAI and GAII. Thus, the genogroups of virus isolates varied with the vaccine strain used (p < 0.05). On the other hand, the neutralizing titres of feline anti-F9 serum and rabbit anti-FCV-255 serum against the 15 isolates were very low, showing no relationships between neutralizing antibody titres and genogroups. The DNA sequence identities between the virus isolates and the vaccine strains were low, at 70.6–82.9%, and no strains were found to have sequences derived from the vaccine strains. Alignment of amino acid sequences showed that the GAI or GAII virus isolates from the F9-vaccinated cats differed at position 428 of the 5’ hypervariable region (HVR) of capsid region of the F9 strain, whereas those from the FCV-255-vaccinated cats differed at positions 438, 453, and 460 of the 5’HVR of capsid region E of the F9 strain. We speculate that these differences influence genogrouping. The amino acid changes within the F9 linear epitopes common to GAI and GAII were noted at positions 450, 451, 457 of 5’HVR of the capsid region E in the isolates from F9-derived vaccine-treated cats, and 449, 450, and 451 of 5’HVR of capsid region E in the isolates from FCV-255-derived vaccine-treated cats, suggesting that these amino acid changes are involved in escapes. These results suggest that alternate vaccination with the F9 and FCV-255 strains or the use of a polyvalent vaccine containing GAII strains serves to inhibit development.

A Case of Cutaneous Sterile Pyogranuloma/Granuloma Syndrome in a Maltese

Cutaneous sterile pyogranuloma/granuloma syndrome (SPGS) is a locally restricted multinodular dermatitis. Affected dogs are typically healthy, but a few show systemic signs. Herein, a case of a dog presenting with generalized ulcerative dermatitis with systemic signs of mild anemia and an increased C-reactive protein level is described. Cutaneous SPGS was diagnosed by histopathology, negative staining causative organisms, and polymerase chain reaction for Mycobacterium spp. Successful treatment was achieved by immunosuppressive drugs, including prednisolone and azathioprine, administered for at least 20 mo. Recurrences of skin lesions were observed when prednisolone and/or azathioprine were discontinued. Long-term management with immunosuppressive agents may be required if the affected dog exhibits severe symptoms of cutaneous SPGS.