Masanobu Onozaki

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.

Vital growth factors of Malassezia species on modified CHROMagar Candida

A comparison of several media, i.e., potato dextrose agar with olive oil (Oil-PDA), modified Dixon agar (mDIX) and variations of Leeming and Notman agar (LNA) for the isolation and growth of Malassezia and Candida species was examined. Since LNA supported the highest growth of Malassezia species its key components, i.e., ox bile, glycerol monostearate, glycerol and Tween 60, were added to CHROMagar Candida. All 7 species of Malassezia grew well on this modified medium (LN-CHROM) after incubation for 4 days at 30 degrees C and development was equal to that observed on LNA. Colonies on LN-CHROM were smooth and from pink to dark purple in color. Furthermore, the use of LN-CHROM did not alter the colony characteristics of Candida species as compared to that found on CHROMagar Candida. The results of the present investigation indicate that the use of LN-CHROM would make possible the simultaneous isolation and identification of Malassezia and Candida species.

Short communication: Molecular typing of Prototheca zopfii from bovine mastitis in Japan

Prototheca zopfii causes bovine mastitis, resulting in reduced milk production and the secretion of thin watery milk with white flakes. Prototheca zopfii has been biochemically and serologically divided into at least 2 genotypes, P. zopfii genotype 1 and P. zopfii genotype 2. The latter is known to be the main causative agent of bovine protothecal mastitis. Prototheca zopfii was later reclassified into 5 varieties: var. zopfii (genotypes 1 and 2), var. 1 (formerly Prototheca blaschkeae), var. 3 (formerly P. moriformis), and var. portoricensis. In this study, the 18S ribosomal DNA sequences of diverse clinical specimens from different areas in Japan were studied to clarify the pathogenicity of P. zopfii var. zopfii. The phylogenetic tree revealed that all genotype 2 isolates were grouped in a cluster of P. zopfii var. zopfii SAG 2021(T) (type strain genotype 2), and were independent from the cluster of the genotype 1 isolates. Thus, all isolates from bovine mastitis in Japan were identified as P. zopfii genotype 2. Therefore, P. zopfii var. zopfii genotype 2 is associated with bovine mastitis.

In vitro susceptibility of Prototheca zopfii genotypes 1 and 2

Prototheca zopfii causes bovine mastitis that leads to reduced milk production. Since P. zopfii isolates from mastitis have been assigned P. zopfii genotype 2, it suggests that this genotype is the etiologic agent of the infection. However, isolates of P. zopfii have not been investigated with regard to their in vitro drug susceptibility. In this study, we examine the susceptibility of genotype 2 strains from bovine mastitis and genotype 1 isolates recovered from cow-barn surroundings. The in vitro susceptibility of ten isolates and the type strain (SAG2063(T)) of P. zopfii genotype 1, and equal number of genotype 2 isolates and the type strain (SAG2021(T)) were assessed by E-test against amphotericin B (AMB), gentamicin (GM), kanamycin (KM) and itraconazole (ITZ). Results showed that P. zopfii genotype 1 isolates are more susceptible in vitro to AMB, GM and KM than those of genotype 2. Moreover, genotype 2 isolates and seven isolates of genotype 1, including the type strain, are not susceptible to ITZ (>10 μg/ml). Thus, drug susceptibility of P. zopfii differs between these two genotypes.

Rapid identification of Prototheca zopfii by nested polymerase chain reaction based on the nuclear small subunit ribosomal DNA

that the Tunisian families are likely linked to the previously identified critical 15q22–15q24 interval. For families PPK3 and PPK4, no other cases could be investigated. Nevertheless, it is noteworthy that the two patients belonging to these families shared a common haplotype between markers D15S534 and D15S650. Parametric multipoint linkage analysis gave the maximum cumulative lod score value of 5.33 for marker D15S650. These results support genetic linkage of the PPK-families to the tested critical interval on 15q22–15q24. It is noteworthy that in family PPK2, individuals IV-1 and IV-2 born to a consanguineous marriage were homozygous for the interval between microsatellite markers D15S213 and D15S988; this segment is likely inherited from a common ancestor. As their mother III-2 was healthy, this suggests that the disease locus should be distal to marker D15S988. In the present study, we provide confirmatory evidence for the location of the punctate PPK in 15q22–15q24 although the precise gene interval should be reconsidered when compared to the study of Gao et al. that suggested that the disease is proximal to D15S988, based on recombinant healthy individuals [5]. To our knowledge, this is the first report of Buschke–Fischer– Brauer’s disease in North African families.