Objective. Equine aspergilloses are rare but potentially life-threatening infections. In horses, Aspergillus fungi usually develop in guttural pouches, which are paired air-filled ventral diverticuli of the Eustachian tubes. An experimental model of guttural pouch aspergillosis was recently described (Greppi et al. 2017) but the pathogenesis of this condition remains largely unknown. The objective of the present study was to estimate the association between the fungal contamination of stables in France and the risk of guttural pouch aspergillosis (GPA) for the horses living in these stables. 

Methods. We examined 4 stables in Normandy and Ile-de-France regions, France (figure 1). Three out of these stables reported at least one case of GPA in the five last years in. In each stable a limited number of representative horse stalls (from 4 to 6) were selected and the following environmental parameters were collected in July 2015 and February 2016: temperature, relative humidity and intensity of air flows. For the evaluation of fungal contamination, surface samples were collected using cotton swabs and indoor air samples (100 L) were collected using an impactor sampler (Air Strategie® Bio-impactor). To select the growth of thermophilic fungi, Petri dishes filled with Sabouraud dextrose agar were incubated at 37°C for 3 days. Fungal colonies were subcultured and identified according to their macroscopic and microscopic appearance. For each positive sample, we estimated the level of fungal contamination by counting the number of colony forming units (cfu). We used the Mann-Whitney test to compare cfu values between the horse stalls. We also used the Wilcoxon signed rank test. A p-value of less than 0.05 was considered statistically significant.

Results. Aspergillus spp. (A. fumigatus, A. flavus, A. niger and A. nidulans) and Mucorales were systematically detected in horse stables. The highest levels of fungal contamination occurred in winter just after the litter has been renewed. Interestingly, fungal contamination differed between the stables where a case of GPA had already been reported and the stable where GPA had never been observed. The mean number of A. fumigatus colonies (from surface and air samples) was significantly higher in stalls just after removing of the litter (p=0.014), and in stalls belonging to a stable where a case of GPA had already been reported (p=0.008 and p=0.08 before and after renewing the litter, respectively). 

Conclusion. The results of the present study should be interpreted with caution because of the lack of power of some statistical tests but they are in accordance with a potential association between the level of fungal contamination in the environment and the risk of aspergillosis in horses.

7 / Fungal contamination in horse stables and risk of equine aspergillosis: results of a pilot study in France