Pascal Arné

Aspergillus fumigatus in Poultry.

Aspergillus fumigatus remains a major respiratory pathogen in birds. In poultry, infection by A. fumigatus may induce significant economic losses particularly in turkey production. A. fumigatus develops and sporulates easily in poor quality bedding or contaminated feedstuffs in indoor farm environments. Inadequate ventilation and dusty conditions increase the risk of bird exposure to aerosolized spores. Acute cases are seen in young animals following inhalation of spores, causing high morbidity and mortality. The chronic form affects older birds and looks more sporadic. The respiratory tract is the primary site of A. fumigatus development leading to severe respiratory distress and associated granulomatous airsacculitis and pneumonia. Treatments for infected poultry are nonexistent; therefore, prevention is the only way to protect poultry. Development of avian models of aspergillosis may improve our understanding of its pathogenesis, which remains poorly understood.

Aspergillus and aspergilloses in wild and domestic animals: a global health concern with parallels to human disease

The importance of aspergillosis in humans and various animal species has increased over the last decades. Aspergillus species are found worldwide in humans and in almost all domestic animals and birds as well as in many wild species, causing a wide range of diseases from localized infections to fatal disseminated diseases, as well as allergic responses to inhaled conidia. Some prevalent forms of animal aspergillosis are invasive fatal infections in sea fan corals, stonebrood mummification in honey bees, pulmonary and air sac infection in birds, mycotic abortion and mammary gland infections in cattle, guttural pouch mycoses in horses, sinonasal infections in dogs and cats, and invasive pulmonary and cerebral infections in marine mammals and nonhuman primates. This article represents a comprehensive overview of the most common infections reported by Aspergillus species and the corresponding diseases in various types of animals.

Clinical, mycological and pathological findings in turkeys experimentally infected by Aspergillus fumigatus

Experimental aspergillosis was induced in 1-day-old turkeys by intra-air-sac inoculation of a spore suspension of a 3-day-old Aspergillus fumigatus culture (CBS 144.89) containing 107 spores. Ten additional poults were used as controls. Infected and non-infected animals were closely observed at least twice a day for the appearance of clinical signs and were sequentially sacrificed at days 1, 2, 3, 5 and 7 post-inoculation. In the infected group, most lung tissues and air sac swabs were culture positive from day 1 to day 5. At 1 day post-inoculation, air sac membranes were multifocally and moderately to severely thickened by an oedema and covered by an exudate. A small number of germinating conidia were present in the superficial exudate, already giving rise to small radiating hyphae. Lung lesions were mild, dominated by a diffuse congestion and a mild heterophilic infiltration. From 2 to 3 days post-inoculation, air sac membranes were more severely affected and several granulomas were observed. Both granulomas and exudates were rich in germinated conidia and hyphae. Pulmonary lesions consisted in a diffuse pneumonia. Five days post-inoculation, air sac membrane lesions progressed to a severe, multifocal, heterophilic and granulomatous inflammation. Seven days post-inoculation, a reduction of the severity of the diffuse pneumonia was detected. Concomitantly, the fungal elements were mainly observed as fragmented tubules in the cytoplasm of multinucleate giant cells. The present study demonstrated that healthy turkey poults might be able to withstand exposure to 107 A. fumigatus spores.

Extended-Spectrum β-Lactamase CTX-M-15-Producing Klebsiella pneumoniae of Sequence Type ST274 in Companion Animals

ABSTRACT Screening of extended-spectrum β-lactamase (ESBL)-producing Gram-negative bacteria in companion animals living in the Paris area in France identified a high rate of CTX-M-15-producing Klebsiella pneumoniae. Those isolates were recovered during the 2010-2011 period from both infections and asymptomatic colonizations. Sequence typing revealed that most of these isolates belonged to sequence type ST274. Interestingly, the blaCTX-M-15 gene was located on a specific and novel plasmid scaffold. These findings highlight that companion animals may be reservoirs for CTX-M-15-producing K. pneumoniae evolving separately from the human reservoir of CTX-M-15 producers.

Multiple-locus variable-number tandem repeat analysis for molecular typing of Aspergillus fumigatus

BackgroundMultiple-locus variable-number tandem repeat (VNTR) analysis (MLVA) is a prominent subtyping method to resolve closely related microbial isolates to provide information for establishing genetic patterns among isolates and to investigate disease outbreaks. The usefulness of MLVA was recently demonstrated for the avian major pathogen Chlamydophila psittaci. In the present study, we developed a similar method for another pathogen of birds: the filamentous fungus Aspergillus fumigatus.ResultsWe selected 10 VNTR markers located on 4 different chromosomes (1, 5, 6 and 8) of A. fumigatus. These markers were tested with 57 unrelated isolates from different hosts or their environment (53 isolates from avian species in France, China or Morocco, 3 isolates from humans collected at CHU Henri Mondor hospital in France and the reference strain CBS 144.89). The Simpson index for individual markers ranged from 0.5771 to 0.8530. A combined loci index calculated with all the markers yielded an index of 0.9994. In a second step, the panel of 10 markers was used in different epidemiological situations and tested on 277 isolates, including 62 isolates from birds in Guangxi province in China, 95 isolates collected in two duck farms in France and 120 environmental isolates from a turkey hatchery in France. A database was created with the results of the present study http://minisatellites.u-psud.fr/MLVAnet/. Three major clusters of isolates were defined by using the graphing algorithm termed Minimum Spanning Tree (MST). The first cluster comprised most of the avian isolates collected in the two duck farms in France, the second cluster comprised most of the avian isolates collected in poultry farms in China and the third one comprised most of the isolates collected in the turkey hatchery in France.ConclusionsMLVA displayed excellent discriminatory power. The method showed a good reproducibility. MST analysis revealed an interesting clustering with a clear separation between isolates according to their geographic origin rather than their respective hosts.

Relative efficiencies of two air sampling methods and three culture conditions for the assessment of airborne culturable fungi in a poultry farmhouse in France.

Fungal elements represent a significant part of the biological contaminants that could be detected in the air of animal facilities. The aim of this study was to assess the relative efficiencies of two air sampling methods and three culture conditions for the quantification of airborne culturable fungi in a poultry farmhouse in France. Air samples were collected every week throughout a 15-week period. Two devices were simultaneously used-a rotative cup air sampler (CIP 10-M, Arelco, France) and an air sampler based on filtration (AirPort MD8, Sartorius, Germany). Culture of airborne viable fungi was performed on malt extract agar (ME) and dichloran glycerol-18 (DG18) at 25 or 37°C. CIP 10-M and AirPort MD8 were shown to display comparable performances but significant differences were observed between culture conditions for Aspergillus spp. (p<0.01), Scopulariopsis spp. (p=0.02) and unidentified molds (p<0.01).

Mutations in the Cyp51A gene and susceptibility to itraconazole in Aspergillus fumigatus isolated from avian farms in France and China

Azole resistance in the fungal pathogen Aspergillus fumigatus is an emerging problem and may develop during azole therapy in humans and animals or exposure to azole fungicides in the environment. To assess the potential risk of azole-resistance emergence in avian farms where azole compounds are used for the control of avian mycoses, we conducted a drug susceptibility study including A. fumigatus isolates from birds and avian farms in France and Southern China. A total number of 175 isolates were analyzed: 57 isolates were collected in France in avian farms where chemoprophylaxis with parconazole was performed; 51 isolates were collected in southern China in avian farms where no chemoprophylaxis was performed; and 67 additional isolates came from the collection of a mycology laboratory. No resistant isolate was detected, and the distribution of minimum inhibitory concentrations was similar for isolates collected in farms with or without azole chemoprophylaxis. For 61 randomly selected isolates, the full coding sequence of the Cyp51A gene was determined to detect mutations. Nine amino acid alterations were found in the target enzyme, 3 of which were new.

Simple and highly discriminatory VNTR-based multiplex PCR for tracing sources of Aspergillus flavus isolates.

Aspergillus flavus is second only to A. fumigatus in causing invasive aspergillosis and it is the major agent responsible for fungal sinusitis, keratitis and endophthalmitis in many countries in the Middle East, Africa and Southeast Asia. Despite the growing challenge due to A. flavus, data on the molecular epidemiology of this fungus remain scarce. The objective of the present study was to develop a new typing method based on the detection of VNTR (Variable number tandem repeat) markers. Eight VNTR markers located on 6 different chromosomes (1, 2, 3, 5, 7 and 8) of A. flavus were selected, combined by pairs for multiplex amplifications and tested on 30 unrelated isolates and six reference strains. The Simpson index for individual markers ranged from 0.398 to 0.818. A combined loci index calculated with all the markers yielded an index of 0.998. The MLVA (Multiple Locus VNTR Analysis) technique proved to be specific and reproducible. In a second time, a total of 55 isolates from Chinese avian farms and from a Tunisian hospital have been evaluated. One major cluster of genotypes could be defined by using the graphing algorithm termed Minimum Spanning Tree. This cluster comprised most of the isolates collected in an avian farm in southern China. The MLVA technique should be considered as an excellent and cost-effective typing method that could be used in many laboratories without the need for sophisticated equipment.

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.

Assessment of Aspergillus fumigatus burden in lungs of intratracheally-challenged turkeys (Meleagris gallopavo) by quantitative PCR, galactomannan enzyme immunoassay, and quantitative culture

Aspergillus fumigatus remains a major respiratory pathogen in birds and treatment is still difficult. We challenged different groups of few-day-old turkeys via intratracheal aerosolisation with increasing concentrations (10(5) up to 10(8)) of conidia using a MicroSprayer(®) device. The fungal burden was assessed by real-time PCR, galactomannan dosage, CFU counting and histopathological evaluation in order to provide a comparison of these results within each inoculum groups. Significant mortality, occurring in the first 96h after inoculation, was only observed at the highest inoculum dose. Culture counts, GM index and qPCR results on the one hand and inoculum size on the other hand appeared to be clearly correlated. The mean fungal burden detected by qPCR was 1.3log10 units higher than the mean values obtained by CFU measurement. The new model and the markers will be used to evaluate the efficacy of antifungal treatments that could be used in poultry farms.