Occurrence and species distribution of pathogenic Mucorales in unselected soil samples from France.

Mucormycosis is a life-threatening invasive fungal disease that affects a variety of patient groups. Although Mucorales are mostly opportunistic pathogens originating from soil or decaying vegetation, there are currently few data on prevalence of this group of fungi in the environment. The aim of the present study was to assess the prevalence and diversity of species of Mucorales from soil samples collected in France. Two grams of soil were homogenized in sterile saline and plated on Sabouraud dextrose agar and RPMI agar supplemented with itraconazole or voriconazole. Both media contained chloramphenicol and gentamicin. The plates were incubated at 35 ± 2 °C and checked daily for fungal growth for a maximum of 7 d. Mucorales were subcultured for purity. Each isolate was identified phenotypically and molecular identification was performed by ITS sequencing. A total of 170 soil samples were analyzed. Forty-one isolates of Mucorales were retrieved from 38 culture-positive samples. Among the recovered isolates, 27 Rhizopus arrhizus, 11 Mucor circinelloides, one Lichtheimia corymbifera, one Rhizopus microsporus and one Cunninghamella bertholletiae were found. Positive soil samples came from cultivated fields but also from other types of soil such as flower beds. Mucorales were retrieved from samples obtained in different geographical regions of France. Voriconazole-containing medium improved the recovery of Mucorales compared with other media. The present study showed that pathogenic Mucorales are frequently recovered from soil samples in France. Species diversity should be further analyzed on a larger number of soil samples from different geographic areas in France and in other countries.

Molecular monitoring of fungal communities in air samples by denaturing high-performance liquid chromatography (D-HPLC).

AIMS: To describe a new molecular technique for the assessment of fungal diversity in the air. METHODS AND RESULTS: Air samples were collected every week in a henhouse in France during a 15-week period. After air sampling, the collecting membrane was diluted, and the liquid was used for subsequent cultivation and molecular analysis: PCR-temperature temporal gradient electrophoresis (TTGE), which has already been used for the identification of fungal species in air samples and PCR-denaturing high-performance liquid chromatography (D-HPLC), a new technique for the analysis of complex microbial populations. D-HPLC profiles were reproducible from run-to-run, and several fungal organisms could be identified at the species level by sequencing. CONCLUSIONS: PCR-D-HPLC enabled the identification of fungal species (both Ascomycota and Basidiomycota) that may be encountered in air. The new technique allowed the detection of more fungal species than did the PCR-TTGE technique. However, some fungal species were detected only by PCR-TTGE, suggesting that PCR-D-HPLC and PCR-TTGE are complementary. SIGNIFICANCE AND IMPACT OF THE STUDY: PCR-D-HPLC represents a considerable saving in time over currently available procedures for detection and identification of fungal organisms in air. However, the fungal diversity detected by PCR-D-HPLC or by PCR-TTGE was lower than that revealed by culture.

Effects of conidia of various Aspergillus species on apoptosis of human pneumocytes and bronchial epithelial cells.

Aspergillus species can cause mycoses in human and animals. Previously, we demonstrated that A. fumigatus conidia from a human isolate inhibited apoptosis in human pneumocytes and bronchial epithelial cells. In the current study, we studied the effects of A. fumigatus conidia non-human origin and A. flavus, A. nidulans, A. niger and A. oryzae conidia on human cells apoptosis. Human pneumocytes or bronchial epithelial cells were simultaneously exposed to apoptotic inductors and aspergilli conidia. The cell cultures were analyzed by flow cytometry, immunoblotting, and examination of nuclear morphology. Similar to A. fumigatus conidia, A. flavus conidia inhibited cellular apoptosis while A. nidulans, A. niger and A. oryzae conidia did not affect apoptosis. We further studied the species specificity of conidia: there were no differences in the inhibition of apoptosis by A. fumigatus conidia from either human or bird isolates. In order to determine whether the inhibition of apoptosis by conidia is limited to certain strains, the effect on human cell apoptosis of different A. fumigatus human clinical isolates and A. fumigatus of environmental origin was evaluated. All A. fumigatus isolates inhibited apoptosis; an anti-apoptotic factor was released by conidia. For TNF-induced apoptosis, the anti-apoptotic effect of conidia of all isolates was found to be associated with a reduction of caspase-3 in human cells. The results suggest that suppression of apoptosis may play a role in reducing the efficacy of host defense mechanisms during infection with Aspergillus species.

Evolution of the environmental contamination by thermophilic fungi in a turkey confinement house in France.

Fungal species constitute a major part of environmental contaminants in facilities where animals are housed. The present investigation was aimed at describing the relative abundances of fungal species and their concentrations in a turkey confinement house in France. Fungal cultures from poultry feed, litter, and air were undertaken every week throughout the 16-wk period of breeding. The incubation temperature of 40 degrees C was selected to isolate thermophilic fungal species (especially Aspergillus spp. and Candida albicans) that are potentially pathogenic for birds. The 2 species Aspergillus fumigatus and Aspergillus flavus were recovered at a mean of 10.5 and 37.0 cfu/m(3) of air sampled, respectively. Individual samplings yielded concentrations of up to 150.0 cfu/m(3) for A. flavus in the first weeks of the investigation. Other fungal species were recovered at a mean of 18.9 cfu/m(3) (maximum 36.3 cfu/m(3)) in the air. The yeast C. albicans was first detected at wk 4 from litter samples and at wk 7 from poultry feed. Densities of C. albicans remained very high in litter samples (63.2 cfu/g) even after new litter was added at wk 10. To analyze the genetic polymorphism of A. fumigatus, the most pathogenic mold in birds, a total number of 198 isolates (134 from air, 34 from litter, and 30 from feed samples) were genotyped using 2 polymorphic microsatellite markers. More than half (42 out of 73, 57.5%) of the genotypes were detected only once. This finding suggests that the contamination of the breeding environment is not due to a single source and confirms the very high genetic diversity of environmental A. fumigatus isolates. As during the study period, no outbreak of fungal infections occurred; the levels of fungal contaminations reported here do not seem sufficient, at least alone, to trigger fungal infections.

Eumycetoma caused by Cladophialophora bantiana in a dog.

We report a case of eumycetoma due to Cladophialophora bantiana in a 3-year-old male Siberian Husky living in France. The dog presented a tumefaction on the thorax and deformity of the second and third subjacent ribs, which were surgically removed. Macroscopic black granules were visible on the ribs, and direct microscopic examination revealed their fungal origin. Cultures yielded pure colonies of C. bantiana. The identification of the causative agent was confirmed after amplification and sequence analysis of fungal internal transcribed spacers 1 and 2 and 5.8S ribosomal DNA regions. Surgery and antifungal treatment with oral itraconazole associated with flucytosine allowed apparent cure after a 10-month follow-up. Envenomation with pine processionary caterpillars (Thaumetopoea pityocampa) and subsequently intensive corticotherapy were considered as possible predisposing factors. This is, to the best of our knowledge, the first case in which C. bantiana is identified as the causative agent of eumycetoma.

Evaluation of the efficacy of oral lufenuron combined with topical enilconazole for the management of dermatophytosis in catteries.

The efficacy of oral lufenuron, a chitin synthetase inhibitor, combined with topical enilconazole, was evaluated for the management of Microsporum canis infection in 100 cats housed in two catteries in France. The cats were treated with weekly rinses with enilconazole (0.2 per cent) for four weeks and, in each cattery, one group (A) was also treated with micronised griseofulvin (25 mg/kg administered orally twice a day for five weeks) and a second group (B) was treated with 60 mg/kg lufenuron administered orally once on day 0 and again after 30 days. All the cats were examined individually for cutaneous lesions and mycological cultures were made when the treatment began and after 15, 30, 60 and 90 days. In the first cattery, the cats' clinical scores after 30 and 60 days were significantly lower in group B than in group A. In both catteries and both treatment groups, the mean number of fungal colonies decreased rapidly during the first 15 days of treatment, remained stable for the following 45 days but increased from day 60 to the end of the experiment on day 90.

Evaluation of the dermatophyte test medium RapidVet-D.

The performance of the dermatophyte test medium (DTM) RapidVet-D was assessed using hair samples collected from experimentally infected guinea pigs. Three dermatophyte species were included in the study: Microsporum canis, Trichophyton mentagrophytes and Trichophyton equinum. DTM substrates were inoculated with infected hairs and scales, incubated at 18, 21, 24, 27 or 37 degrees C and examined daily for 15 days. The rapidity of colour change was clearly related to the incubation temperature and to the number of infected hairs deposited on the reactive substrates. With the optimum incubation temperature 27 degrees C, a systematic colour change could be observed only a few days post-inoculation: 3 days with M. canis infected hairs, 4 days with T. equinum and 5 days with T. mentagrophytes.

Efficacy of halofuginone lactate in the prevention of cryptosporidiosis in suckling calves.

The efficacy of halofuginone lactate in the prevention of cryptosporidiosis in suckling calves was evaluated in a multicentre, control versus placebo, randomised, double-blind clinical trial. Seventy-eight six- to 48-hour-old calves were treated daily with 120 microg/kg bodyweight of halofuginone lactate administered orally for seven consecutive days, while 80 calves received a placebo. Faecal samples were collected on the first day of dosing and four, seven, 14 and 21 days later, and Cryptosporidium parvum oocysts were counted and faecal indices for diarrhoea were determined after a clinical examination. An analysis of variance for repeated measurements showed a highly significant difference in favour of halofuginone lactate for both the oocyst counts (P=0.0002) and the faecal diarrhoea indices (P=0.0001) throughout the trial. The difference was greatest after seven days, when the mean oocyst count of the placebo group was 2.5 times and its mean faecal index was twice the mean of the halofuginone lactate group. One day after the end of the treatment the calves which received halofuginone lactate excreted 44 per cent fewer C parvum oocysts and 44 per cent fewer of them had diarrhoea. The reduction was even greater (65 per cent) when liquid diarrhoea was assessed, with 32.5 per cent of the calves in the placebo group having liquid diarrhoea compared with 11.5 per cent in the halofuginone lactate group. The treatment was well tolerated and easily administered.

Parallel phylogenies of Pneumocystis species and their mammalian hosts.

The single name Pneumocystis carinii consists of an heterogeneous group of specific fungal organisms that colonize a very wide range of mammalian hosts. In the present study, mitochondrial large subunit (mtLSU) and small subunit (mtSSU) rRNA sequences of P. carinii organisms from 24 different mammalian species were compared. The mammals were included in six major groups: Primates (12 species), Rodents (5 species), Carnivores (3 species), Bats (1 species), Lagomorphs (1 species), Marsupials (1 species) and Ungulates (1 species). Direct sequencing of PCR products demonstrated that specific mtSSU and mtLSU rRNA Pneumocystis sequence could be attributed to each mammalian species. No animal harbored P. carinii f. sp. hominis. Comparison of combined mtLSU and mtSSU aligned sequences confirmed cospeciation of P. carinii and corresponding mammalian hosts. P. carinii organisms isolated from mammals of the same zoological group systematically clustered together. Within each cluster, the genetic divergence between P. carinii organisms varied in terms of the phylogenetic divergence existing among the corresponding host species. However, the relative position of P. carinii groups (rodent, camivore or primate-derived P. carinii) could not be clearly determined. Further resolution will require the integration of additional sequence data.

Prevalence of Cryptosporidium infection in calves in France.

Two multicentre surveys were conducted in France to estimate the prevalence of Cryptosporidium infection in calves using qualitative ELISA for detection of Cryptosporidium coproantigens and oocysts. The first survey involved 4-12-day-old calves in six dairy-calf distribution centres, collecting calves from seven Administrative Regions (Aquitaine, Bretagne, Franche-Comté, Lorraine, Normandie, Nord, Pays de Loire). For each region, 20 calves were selected every month for 12 consecutive months (October 1995-September 1996). Prevalence of Cryptosporidium infection was 17.9% (Confidence Intervals (C.I.) 95%=[16.1%; 19.8%]) among the 1628 selected calves, of which only 5.3% had diarrhoea. The second survey conducted between November 1995 and May 1996 involved 4-21-day-old calves examined by veterinary practitioners who selected 189 livestock farms of dairy- or suckler-type in ten Administrative Departments (Allier, Cantal, Creuse, Doubs, Ille-et-Vilaine, Maine-et-Loire, Manche, Pas-de-Calais, Saône-et-Loire, Vendée). Cryptosporidia were detected in 105 (55.6%) of the farms. Among the 440 calves examined, of which 398 (90.5%) presented diarrhoea, cryptosporidia were found in 191 animals, i.e. a prevalence of 43.4% (C.I. 95%=[38. 8%; 48.0%]). Breed of calves and type of housing had very little impact on prevalence in this survey. Some regional variations could be noticed, even if cryptosporidia infection is widespread. Monthly variations could be related to seasonal peaks in calving with a lower infection rate during summer.

Fungal flora on cutaneous and mucosal surfaces of cats infected with feline immunodeficiency virus or feline leukemia virus.

OBJECTIVE: To compare cutaneous and mucosal mycoflora in cats infected with FIV or FeLV with that in noninfected cats. ANIMALS: 85 client-owned cats; 24 seropositive for FIV, 10 seropositive for FeLV, 1 seropositive for both viruses, and 50 seronegative for both viruses. PROCEDURE: Cutaneous specimens were obtained from the coat and external acoustic meatus (ear canal) and mucosal specimens from the oropharynx and rectum. Fungi were isolated from specimens, using Sabouraud dextrose agar incubated at 27 or 37 C for cutaneous and mucosal specimens, respectively. RESULTS: Fungal colonies were cultured from at least 1 specimen from 83 of 85 (97.6%) cats. The most common fungal isolates were Aspergillus spp (cultured from 59.3% of all specimens), Penicillium spp (50.0%), Cladosporium spp (44.2%), Scopulariopsis spp (41.8%), and lipophilic yeasts of the genus Malassezia (31.4%). A greater diversity of fungal genera was isolated from retrovirus-infected cats, and Malassezia spp were more commonly recovered from these cats, compared with noninfected cats. Candida albicans, Cryptococcus neoformans, and dermatophytes (eg, Microsporum canis) were rarely isolated from any cat. Significant differences in frequency of isolation of C. neoformans and dermatophytes were not found between infected and noninfected cats. CONCLUSIONS AND CLINICAL RELEVANCE: Cats infected with FIV or FeLV may have a greater diversity of cutaneous and mucosal mycoflora than noninfected cats. However, infected cats may be no more likely than noninfected cats to expose humans to zoonotic fungi such as C. albicans, C. neoformans, and M. canis.

Prominent animal mycoses from various regions of the world.

The mycoses selected for presentation in this section are relatively common diseases of companion animals or livestock in certain areas of the world. Malasseziosis is arguably the most frequent mycosis of dogs (as otitis externa and dermatitis) throughout the world, although its diagnosis is often overlooked. Protothecosis is also geographically widespread, particularly in cattle where severe mastitis is a result of adventitious infection from the environment. In contrast, coccidioidomycosis and pythiosis are geographically limited in their occurrence (coccidioidomycosis by geographic region and pythiosis by climate), but within regions where they do occur, their presence in animals is not unusual. It was our intention to review recent developments in each of these diseases.