Secreted subtilisin Sub3 from Microsporum canis is required for adherence to but not for invasion of the epidermis.

BACKGROUND: Microsporum canis is a pathogenic dermatophyte that causes a superficial cutaneous mycosis, mainly in cats and humans. Proteolytic enzymes, including subtilisins, have been postulated to be key factors involved in adherence and invasion of the stratum corneum and keratinized epidermal structures. OBJECTIVES: To evaluate the importance of Sub3 as a M. canis virulence factor using a SUB3 RNA-silenced strain. MATERIALS AND METHODS: The stability of a previously constructed RNA-silenced strain IHEM 22957 was tested in three different ways. The involvement of Sub3 in the adherence process was evaluated using a new ex vivo adherence model of M. canis arthroconidia to feline epidermis. In order to investigate the contribution of Sub3 in epidermal invasion, the pathogenicity of the SUB3 silenced strain was compared with that of the control strain in a guinea pig model of experimental M. canis dermatophytosis. RESULTS: The silenced strain was shown to be stable after four in vitro transfers and after the in vivo experimental infection. This strain has dramatic loss of adherence capacity to feline corneocytes when compared with the parental strain. In contrast, no significant differences were observed at any time during the infection between the control strain and the SUB3 silenced strain, indicating that Sub3 secretion is not required for invasion of epidermal structures. CONCLUSIONS: RNA interference is a useful tool to evaluate pathogenic mechanisms of M. canis. For the first time, a role in pathogenicity could be attributed to a protease of a dermatophyte, namely Sub3 from M. canis, which is required for adherence to but not for invasion of the epidermis.

Effect of sampling method and incubation temperature on fungal culture in canine sinonasal aspergillosis.

OBJECTIVES: To evaluate the most appropriate sampling procedure and the effect of incubation temperature on fungal culture in the diagnosis of canine sinonasal aspergillosis (SNA). METHODS: Sixteen dogs with SNA and 20 dogs with non-fungal nasal disease entered a prospective study. Nasal secretions and mucosal biopsies were collected in all dogs. Fungal plaques were also sampled in dogs with SNA. Each specimen was taken in duplicate from each dog and incubated at room temperature and 37 degrees C. RESULTS: In dogs with SNA, nasal secretions, mucosal biopsies and fungal plaques yielded fungal growth at room temperature in one, one and seven dogs, respectively, whereas fungal growth was obtained at 37 degrees C in three, 12 and 14 dogs, respectively. No specimen collected from any dog with non-fungal nasal disease yielded fungal growth at room temperature or at 37 degrees C. CLINICAL SIGNIFICANCE: The diagnosis of canine SNA is more likely to be confirmed following culture of mucosal biopsies or fungal plaques than nasal secretions sampled blindly with swabs. Incubating cultures at 37 degrees C is more likely to provide a diagnostic outcome than when samples are cultured at room temperature. Fungal culture of nasal specimens has good specificity for the diagnosis of SNA in dogs.

Comparison of the value of measurement of serum galactomannan and Aspergillus-specific antibodies in the diagnosis of canine sino-nasal aspergillosis.

Serology is currently used for the diagnosis of canine sino-nasal aspergillosis (SNA). However, the accuracy of serological testing using commercially available, standardized purified antigen preparations of Aspergillus (CAPurAspAg) has only been poorly documented. The aim of the present study was to assess the diagnostic value of an agar-gel double immunodiffusion (AGDD) test and an anti-Aspergillus IgG ELISA, using CAPurAspAg and the commercially available Platelia test for the detection of serum galactomannan. Sera from 17 dogs with SNA, 18 dogs with a nasal tumour (NT), 11 dogs with lymphoplasmacytic rhinitis (LPR) and 33 control dogs were tested with the 3 methods. AGDD result was positive in 76.5% of dogs with SNA, whereas all sera from dogs with non-fungal nasal disease and control dogs were negative. A positive IgG ELISA result was obtained in 88% of dogs with SNA and in 18% of dogs with LPR. All patients with NT and control dogs had a negative IgG ELISA result. The Platelia test was positive in 24% of dogs with SNA, 11% of dogs with NT, 9% of dogs with LPR and 24% of control dogs. The results of this study suggest that (1) the detection of serum Aspergillus-specific antibodies with AGDD or ELISA, using CAPurAspAg, provides excellent specificity and good sensitivity, (2) the specificity is higher for AGDD (100%) than for ELISA (96.8%) while sensitivity is higher for ELISA (88.2%) than for AGDD (76.5%) and (3) serum galactomannan quantification with the Plateliat test is unreliable for the diagnosis of canine SNA.

Echinococcus multilocularis in Belgium: prevalence in red foxes (Vulpes vulpes) and in different species of potential intermediate hosts.

Echinococcus multilocularis causes a rare but potentially lethal zoonotic infection in humans. This tapeworm is known to be endemic in foxes in several countries of Western and Central Europe. In Western Europe, the common vole (Microtus arvalis) and the water vole (Arvicola terrestris) are considered to be the most important intermediate host species of this cestode whereas the red fox is by far the most important final host. The purpose of this study was to provide data on the prevalences in Wallonia (Southern part of Belgium) both in the red fox and in different potential intermediate hosts. A total of 990 red foxes were examined between January 2003 and December 2004 for the presence of E. multilocularis. The average prevalence was 24.55% (22.38-27.87). Out of 1249 rodents or insectivores belonging to the species Apodemus sylvaticus, Arvicola terrestris, Clethrionomys glareolus, Microtus arvalis, Microtus agrestris and Sorex araneus, only one M. arvalis (out of 914-0.11% (0.003-0.61) and one C. glareolus (out of 23-4.3% (0.1-21.9) were found to be infected. However, the muskrat (Ondatra zibethicus) seems to be a good intermediate host as 11.18% (9.72-12.76) of the animals (n=1718) were found to be infected. A positive correlation was found between the prevalences in foxes and in muskrats in each of the different geological regions. This study indicates that the muskrat is highly sensitive to this zoonotic tapeworm and could perhaps represent a good bioindicator when studying the epidemiology of this parasitic infection in Belgium and in other countries where the muskrat is present.

Ruggedness testing of chromatographic methods: selection of factors and levels.

The first step in a ruggedness test is the selection of factors to be examined and their levels. In this paper, both topics are discussed, thereby completing a strategy described earlier. It is demonstrated, by means of some examples, that depending on the formulation (definition) of a factor, information that is physically more or less meaningful is extracted from the experimental design results. Among others, the inclusion of the compounds of a buffer and of the components of a mixture in a screening design were examined. A general guideline to select the levels of the factors in a ruggedness test was proposed. Some special cases, i.e. asymmetric intervals around the nominal level, were also discussed.