Detection and identification of the Candida species by 25S ribosomal DNA analysis in the urine of candidal cystitis

Candida species in clinical urine samples were identified directly by the newly developed method of PCR analysis on 25S ribosomal DNA (rDNA). Two dogs were referred to the Animal Medical Center, Nihon University School of Veterinary Medicine, Fujisawa, Kanagawa, Japan for the examination of chronic cystitis. Microscopic examination of urine samples from these dogs revealed yeast cells. Urine culture on Sabouraud's dextrose agar at 27 degrees C for 5 days produced white to cream colored colonies. The isolates were identifical to Candida albicans and C. parapsilosis by mycological examination, respectively. The nucleotide sequences of 25S ribosomal DNA from these urine isolates showed 99% similarity to those of a reference strain of Candida albicans or C. parapsilosis. The nucleotide sequences of 25S rDNA obtained directly from urine samples were also identical to C. albicans and C. parapsilosis, respectively. Confirming the results on the isolates cultured from the same urine samples. This PCR analysis method could be available for the direct identification of Candida species in urine samples within 2 days.     

Determination of antigenic proteins of housedust mites in 90 dogs suffering from atopic dermatitis

Housedust mites, Dermatophagoides pteronyssinus (D. pteronyssinus) and Dermatophagoides farinae (D. farinae), are the important causative agents of allergic diseases in human and animals. By using 165 dogs suffering from atopic dermatitis (AD), serum levels of immunogloblin E (IgE) antibody against 25 kinds of allergen including housedust mites were determined. Housedust mites were the most frequent allergen against which 90 of the 165 allergic dogs (54.5%) by IMMUNODOT assay. With the further analysis of immunoblotting assay in the 90 dogs sensitized with housedust mites, antigenic proteins of housedust mites recognized by IgE antibodies were with the apparent molecular masses of 15, 76, 90, 98, and 170-kD. Among them, the 15-kD protein that might be identical to Group 2 antigens (Der f2, Der p2) was prominently observed (52/90). This study indicates that about a half of dogs with AD were sensitized to housedust mites, suggesting that Group 2 antigens of housedust mites may be a major allergen in canine AD.     

Inhibition of appressorial adhesion of Pyricularia oryzae to barley leaves by fungicides

The effects of rice blast fungicides known to inhibit melanisation or penetration on the adhesion of Pyricularia oryzae to the leaf surface of barley were investigated. Adhering appressoria were counted after shaking inoculated leaves vigorously with water. On untreated leaves, appressoria began to adhere at the time when appressorial melanisation was initiated. The number of adhering appressoria then increased gradually, and most were melanised. On chlobenthiazone-treated leaves, appressoria were not melanised and were easily detached from the surface. Similar results were obtained with tricyclazole. However, after treatment with tetrachlorophthalide or pentachlorobenzyl alcohol, which are also known to prevent penetration of P. oryzae, melanised appressoria still remained after shaking in water. It is suggested that appressorial melanisation in P. oryzae is involved in appressorial adhesion. The antipenetrant action of the melanin biosynthesis inhibitors chlobenthiazone and tricyclazole may be ascribed to the lack of adhesive intensity necessary to support the mechanical shearing of the cuticle of rice plants.     

Sensitivity to Protectant Fungicides and Pathogenic Fitness of Clonal Lineages of Phytophthora infestans in the United States

ABSTRACT Since 1991, dramatic changes have occurred in the genetic composition of populations of Phytophthora infestans in the United States. Clonal lineages recently introduced into the United States (US-7 and US-8) are more common now than the previously dominant lineage (US-1). To help determine why these changes occurred, four clonal lineages of P. in-festans common during the early 1990s in the United States and Canada were evaluated for sensitivity to the protectant fungicides mancozeb and chlorothalonil using amended agar assays for isolates collected from 1990 to 1994. No isolate or lineage was resistant to either mancozeb or chlorothalonil. There were significant differences among isolates for degree of sensitivity to one fungicide individually, but there were no significant (P = 0.05) differences among the US-1, US-6, US-7, and US-8 clonal lineages for degree of sensitivity to both fungicides. Therefore, resistance to protectant fungicides cannot explain the rapid increase in frequency of the US-7 and US-8 clonal lineages. Three components of pathogenic fitness (latent period, lesion area, and sporulation after 96 h) were tested for the three clonal lineages that were detected most commonly during 1994 (US-1, US-7, and US-8). All but one of the isolates in this analysis were collected during 1994 and evaluated within 10 months of collection by inoculating detached leaflets of the susceptible potato cultivar Norchip. There were significant differences between the US-1 and US-8 clonal lineages for lesion area and sporulation, and between US-1 and US-7 for latent period. The US-6 clonal lineage was excluded from the pathogenic fitness experiments, because no isolates of this lineage were collected during 1994. Compared with US-7 and US-8, US-1 had the longest latent period and the smallest lesions with the least sporulation. Incorporation of the differences between US-1 and US-8 in computer simulation experiments revealed that significantly more protectant fungicide (e.g., 25%) would be required to suppress epidemics caused by the US-8 clonal lineage compared with US-1. These differences in pathogenic fitness components probably contribute to the general predominance of the "new" clonal lineages (especially US-8) relative to the "old" US-1 lineage.     

Retrospective serological analysis of spontaneous CDV infection in 192 dogs

Spontaneous cases of canine distemper virus (CDV) infection were serologically evaluated. The 192 dogs in which CDV antigen was confirmed from tonsil by immunohistological examination were 2- to 4-months old, of various breeds, and unvaccinated. The prognoses were good in 74 dogs with significantly high levels of anti-CDV passive hemolytic aggregation (PHA) titer. In the other 118 dogs with poor prognoses, anti-CDV PHA titer was not detected. Anti-CDV PHA titer had the most significant association with the prognoses of CDV infection, and could be the most reliable and useful indicator for evaluating such prognoses.     

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.     

Distribution of two groups of melon landraces and inter-group hybridization enhanced genetic diversity in Vietnam

To understand the genetic diversity and differentiation of Vietnamese melon (Cucumis melo L.), we collected 64 landraces from the central and southern parts of the country and assessed molecular polymorphism using simple sequence repeat and random amplified polymorphic DNA markers. The Vietnamese melon was divided into seven cultivar groups, namely “Dua le”, “Dua vang”, “Dua bo”, “Dua gang-andromonoecious”, “Dua gang-monoecious”, “Dua thom”, “Montok”, and the weedy-type melon “Dua dai”. Among these, Dua le, Dua vang, Dua bo, and Dua gang-andromonoecious are cultivated on plains and they formed cluster II along with the reference accessions of Conomon and Makuwa. Based on genetic distance, Dua le and Dua vang were regarded as Makuwa and Dua bo and Dua gang-andromonoecious as Conomon. In contrast, Dua thom and Montok are cultivated in highlands, and they formed cluster III along with landraces from the southern and eastern foot of the Himalayas. Dua gang-monoecious which is commonly cultivated in the southern parts of Vietnam, exhibited the greatest genetic diversity, as explained by its possible origin through the hybridization between Dua gang-andromonoecious and Montok. Genetic differences in melon landraces between plains and highlands and hybridization between these two geographical groups have contributed to the enhancement of genetic diversity in Vietnamese melon.