Atsuhiko Hasegawa

Positive reactions to common allergens in 42 atopic dogs in Japan.

Clinically important allergens for the diagnosis and treatment of atopic dermatitis vary geographically. In order to identify the most prevalent allergens in atopic dogs in Japan, 42 dogs with a clinical diagnosis of atopy were tested using both in vivo (intradermal skin test (IDST)) and in vitro (antigen-specific IgE assay) allergy tests. Allergens used for IDST included 26 allergen extracts from eight allergen groups: trees, weeds, grasses, house dust mites (HDM), molds, foods, epithelia, and arthropods. Immunodot assay was used to measure antigen-specific IgE against 24 allergens from these eight groups and against fish such as cod and sole. In the 42 dogs, the most common positive allergen reaction was to HDM on both IDST (29/42 dogs or 69%) and in vitro testing (23/42 or 54.8%). The second most frequent positive allergen reaction was to Japanese cedar pollen (21/42 or 50.0% for IDST and 7/42 or 16.7% for in vitro testing). In both tests, less than 20% of dogs had positive reactions to molds or foods. Positive reactions to cat epithelia were frequently found on IDST, but rarely found on in vitro testing. Agreement between the two tests was found in 26 instances: HDM (21 dogs), Japanese cedar pollen (five dogs) and wheat (one dog). In this study, the two most common allergens involved in atopic dermatitis in dogs in Japan were HDM and Japanese cedar pollen.

Prototheca zopfii genotypes isolated from cow barns and bovine mastitis in Japan

This study is the first investigation on Japanese isolates of Prototheca zopfii from bovine mastitis and the cow-barn surroundings by molecular characterization to clarify routes of infection for bovine protothecal mastitis. We performed isolation of Prototheca from cow-barn surroundings (drinking water, sewage and feces) and milk samples from cases of bovine mastitis. Genotypes of the 32 isolates of P. zopfii from cow-barn surroundings and 67 isolates from mastitis were analyzed by genotype-specific PCR assays and restriction fragment length polymorphism (RFLP) assays. All mastitis isolates were identified as P. zopfii genotype 2. Conversely, 29 isolates from cow-barn surroundings were identified as P. zopfii genotypes 1 and 3 isolates as genotype 2, respectively. Given these results, both genotypes of P. zopfii could exist in cow-barn surroundings, but no sites were identified as frequent sources of P. zopfii genotype 2. P. zopfii isolates should thus be further explored with regard to genotype to clarify the reservoir of etiological agents in bovine Prototheca mastitis.

Molecular epidemiology of Arthroderma benhamiae, an emerging pathogen of dermatophytoses in Japan, by polymorphisms of the non-transcribed spacer region of the ribosomal DNA.

In Japan, several isolates of Arthroderma benhamiae, a teleomorphic member of Trichophyton mentagrophytes complex, which were not found by earlier mating studies, have recently been recovered from human and animal dermatophytoses. In the present study, intraspecies polymorphism of A. benhamiae isolated in Japan was investigated using restriction fragment length polymorphisms (RFLP) of the non-transcribed spacer (NTS) region of the ribosomal DNA (rDNA), a method introduced to detect intraspecies polymorphisms of other dermatophyte species, such as T. rubrum. Based on their restriction profiles, there were five DNA types out of eight strains of A. benhamiae isolated in Japan. None of the five DNA types were found among the registered tester strains of A. benhamiae. Therefore, several different strains of A. benhamiae may have been brought into Japan separately.

IgE sensitivity and cross-reactivity to crude and purified mite allergens (Der f 1, Der f 2, Der p 1, Der p 2) in atopic dogs sensitive to Dermatophagoides mite allergens.

The present study investigates IgE-reactivity to crude and purified mite allergens by intradermal skin test (IDST), Immunodot method, and ELISA in atopic dogs sensitive to mite allergens, as well as the allergenic cross-reactivity between Dermatophgoides (D) farinae (DF) and D. pteronyssinus (DP) in dogs by IgE-ELISA inhibition. IDST and Immunodot method for crude mite allergens were performed for atopic dogs and 16 atopic dogs showed sensitivity to mite allergens. Of the 16 dogs, all dogs had anti-DF IgE and 11 had anti-DP IgE. We measured specific IgE to purified major allergens (Der f 1, Der f 2, Der p 1, Der p 2). Of the 16 atopic dogs, six had anti-Der f 1 IgE and seven had anti-Der f 2 IgE. Similarly, of the 16 dogs, six had anti-Der p 1 IgE and seven had anti-Der p 2 IgE. However, eight dogs had no specific IgE to these mite allergens. These dogs may be sensitive to other major mite allergens except Der 1 and Der 2. In the dogs that had both anti-DF and DP IgE, IgE binding to DF was greatly inhibited by DP, and reciprocal inhibition was observed. Based on these data, it appears that there is a strong cross-reactivity between DF and DP in dogs. Similarly, a cross-reactivity between DF and DP in purified allergens was also observed. IDST and Immunodot method are useful methods for the diagnosis of atopic diseases in dogs, and ELISA is a useful method for further investigation of IgE-reactivity for the allergens.

Full-length cDNA cloning of Toll-like receptor 4 in dogs and cats

In the present study, full length of canine and feline Toll-like receptor 4 (TLR4) cDNAs were sequenced, and the expression of canine and feline TLR4 mRNAs in dog and cat tissues were investigated. The full-length cDNA of TLR4 of dog and cat was 2709 bp encoding 637 amino acids and 3113 bp encoding 833 amino acids, respectively. The similarity of canine and feline TLR4 were 83.6% at the nucleotide sequence level and 77.6% at the amino acid sequence level. At the amino acid sequence level, canine and feline TLR4 showed sequence similarities of approximately 62-78% with those of Homo sapiens, Mus musculus, Bos taurus and Equus caballus, respectively. Southern hybridization analyses with TLR4 cDNA probes gave one distinct band in BamHI, EcoRI and HindIII digests of genomic DNA from dogs and cats, respectively, indicating the likely presence of a single TLR4 gene in each species. By RT-PCR analysis, mRNA of canine TLR4 was expressed highly in peripheral blood leukocytes (PBL), moderately in spleen, stomach and small intestine, at low levels in liver, with no expression in kidney, large intestine and skin. On the other hand, mRNA of feline TLR4 was expressed highly in lung, bladder and PBL, moderately in kidney, liver, spleen and large intestine and at low levels in pancreas and small intestine.

Chitin Synthase 2 Gene Sequence of Malassezia Species

Nucleotide sequences of the chitin synthase 2 (CHS2) gene of seven species, Malassezia furfur, M. globosa, M. obtusa, M. pachydermatis, M. restricta, M. slooffiae and M. sympodialis, were analyzed for their phylogenetic relationship. About 620‐bp genomic DNA fragments of the CHS2 gene were amplified from these Malassezia species by polymerase chain reaction (PCR) and sequenced. The CHS2 nucleotide sequences of these Malassezia species showed more than 95% similarity between the species. A phylogenetic analysis of the nucleotide sequences of CHS2 gene fragments of seven Malassezia species revealed that the species were genetically distinct from each other.

Isolation of Aspergillus udagawae from a fatal case of feline orbital aspergillosis

Aspergillus fumigatus is the predominant etiological agent of sino‐orbital aspergillosis in humans and animals. Here we report for the first time A. udagawae, a previously recognised but rare opportunistic pathogen causing fatal orbital aspergillosis in a cat. Identification of this isolate was secured by comparative sequence based analyses of the ITS and the β tubulin region. Antifungal susceptibility testing results revealed that this isolate had high in vitro MIC to amphotericin B (AMB) that correlated with in vivo failure of therapy with AMB.

Molecular heterogeneity in clinical isolates of Malassezia pachydermatis from dogs

Molecular investigation of 16 strains, conventionally identified to be Malassezia pachydermatis, isolated from dogs in Japan was carried out by random amplification of polymorphic DNA (RAPD) and chitin synthase 2 (CHS2) gene sequence analyses. The RAPD band patterns of 13 clinical isolates were identical to that of standard strain of M. pachydermatis (CBS-1879). The other three clinical isolates were different from the standard strain of M. pachydermatis in RAPD patterns, and two of the three isolates were identical. About 620 bp genomic DNA fragments of the CHS2 gene were amplified from the same 16 clinical isolates of M. pachydermatis by polymerase chain reaction (PCR) and sequenced. The phylogenetic analysis of the nucleotide sequences of CHS2 gene fragments of the 16 clinical isolates revealed that the 13 strains were genetically very close to the standard strain of M. pachydermatis and the other two isolates were genetically close to the standard strain of M. furfur rather than M. pachydermatis. The remaining one isolate was phylogenetically distinct from all the seven Malassezia species reported so far.

Cluster Analysis of Human and Animal Pathogenic Microsporum Species and Their Teleomorphic States, Arthroderma Species, Based on the DNA Sequences of Nuclear Ribosomal Internal Transcribed Spacer 1

We performed a cluster analysis of human and animal pathogenic Microsporum species and their teleomorphic states, Arthroderma species, including A. otoe‐related species (M. canis, M. audouinii, M. distortum, M. equinum, M. langeronii, and M. ferrugineum) and M. gypseum complex (A. fulvum, A. gypseum, and A. incurvatum) using DNA sequences of nuclear ribosomal internal transcribed spacer 1 (ITS1). The dendrogram showed the members of A. otae‐related species to be monophyletic and to construct an extremely closely related cluster with a long horizontal branch. This ITS1‐homologous group of A. otae was organized in 6 unique genotypes, while sequences of the members of the ITS1‐homologous group of M. gypseum complex are more diverse. This ITS1‐based database of Microsporum species and their teleomorphic states will provide a useful and reliable species identification system: it is time‐saving (takes two to three days), accurate and applicable even to strains with atypical morphological features or in a non‐culturable state.

First case of feline systemic Cryptococcus albidus infection

This paper, as best as the authors can determine, is the first to describe a documented case of systemic infection caused by Cryptococcus albidus in a cat. The patient had a history of paralysis of the hind legs and had been treated with prednisone for 1 month. Microscopic examination of a fine needle biopsy specimen from a right popliteal lymph node showed granulomatous inflammation with many encapsulated yeast cells. Moreover, microscopic examination of Indian ink preparations of the cerebrospinal fluid revealed encapsulated ovoid yeast cells. Thus this case was diagnosed to be cryptococcosis. However, the cat died after treatment for three days with voriconazole. Isolates recovered from samples of the cerebrospinal fluid, liver and spleen were identified as C. albidus by molecular analysis, as well as through morphologic and biochemical studies. Therefore, this case indicates that C. albidus should be considered as a potential feline pathogen.