Single tracheal inoculation of Aspergillus fumigatus conidia induced aspergillosis in juvenile falcons (Falco spp.).

Aspergillosis is a common and life-threatening respiratory disease in raptors with acute and chronic courses. Among raptors, gyrfalcons (Falco rusticolus) and their hybrids are often declared to be highly susceptible with juvenile individuals being the most susceptible. However, species- and age-specific experimental studies are lacking and minimal infective doses (IDs) for Aspergillus spp. conidia are unknown.Therefore, 8-week-old, healthy gyr-hybrid falcons (F. rusticolus X F. cherrug) (N = 18) were experimentally infected with Aspergillus fumigatus using a single intratracheal inoculation with varying dosages of conidia (102 to 107 conidia). Over 28 days, clinical signs were monitored as well as haematological and serological parameters. Following euthanasia, necropsy, histopathology, bacteriology, and mycology were performed. Re-isolated fungi were compared to the inoculum using microsatellite length polymorphisms. During the trial, clinical signs and dyspnoea correlated significantly with the ID. Necropsy revealed fungal lesions in the upper and lower airways of 10/18 inoculated falcons, but not in the control birds. In 9/18 inoculated falcons, fungal granulomas were confirmed in histopathology and A. fumigatus was re-isolated from these granulomas. Except one nasal isolate all re-isolated fungal strains were identical to the inoculum strain. Based on mycology and histopathology a minimal ID of 50% was calculated to be MID50% (±S.E.) = 104.52±0.67 for a single tracheal inoculation of A. fumigatus conidia. This study demonstrates for the first time that a single exposure is able to cause acute aspergillosis in juvenile falcons.

Timing of predisposing factors is important in necrotic enteritis models.

Since the ban of antimicrobial growth promotors, the importance of necrotic enteritis in broilers increases. Reliable and reproducible infection models are required for pathogenesis studies and product screening. Two major predisposing factors in necrotic enteritis models are fishmeal supplementation to feed and Eimeria infection. However, many unsolved issues regarding these predisposing factors still exist. Therefore, the influence of timepoint of fishmeal administration (onset on day 8 or day 18), timing of coccidiosis challenge (day 15 or day 19) and strain of coccidiosis challenge (field strain vs. commercial vaccine) on the induction of necrotic enteritis lesions was investigated. The birds were inoculated with Clostridium perfringens three times per day for four consecutive days (day 17 until day 20) and were scored for the presence of necrotic enteritis on days 22, 23, 24, 25 and 26. Supplementation of the diet with fishmeal from day 8 onwards increased the likelihood of necrotic enteritis compared to supplementation from day 18 onwards. Birds challenged on day 19 with coccidiosis were more likely to have necrotic enteritis on scoring days 23 and 24 compared to birds challenged on day 15. Differences on other scoring days were less pronounced. Finally, the strain of coccidiosis challenge had little influence on the induction of necrotic enteritis. Findings of this study can help researchers to set up successful necrotic enteritis infection models.

T-2 toxin impairs antifungal activities of chicken macrophages against Aspergillus fumigatus conidia but promotes the pro-inflammatory responses.

Aspergillosis is the most common fungal disease of the avian respiratory tract and is caused primarily by Aspergillus fumigatus. The respiratory macrophages provide important defence against aspergillosis. T-2 toxin (T-2), a trichothecene mycotoxin produced by Fusarium spp. in improperly stored agricultural products, has immunomodulatory effects. We studied the impact of T-2 on the antifungal response of the chicken macrophage cell line HD-11 against A. fumigatus infection. The macrophages were first exposed to 0.5 to 10 ng/ml T-2 for 24 h, and then their viability, antifungal activity, and cytokine expression in response to A. fumigatus conidial infection were determined. The viability of macrophages decreased when exposed to T-2 at concentrations higher than 1 ng/ml. One hour after conidial infection, phagocytosed conidia were observed in 30% of the non-T-2-exposed macrophages, but in only 5% of the macrophages exposed to 5 ng/ml T-2. Seven hours after infection, 24% of the conidia associated with non-T-2-exposed macrophages germinated, in contrast to 75% of those with macrophages exposed to 5 ng/ml T-2. A. fumigatus infection induced upregulation of interleukin (IL)-1ß, CXCLi1, CXCLi2 and IL-12ß, and downregulation of transforming growth factor-ß4 in macrophages. Exposure of A. fumigatus-infected macrophages to T-2 at 1 to 5 ng/ml further upregulated the expression of IL-1ß, IL-6, CCLi2, CXCLi1, CXCLi2, IL-18 (at 1 and 2 ng/ml) and IL-12ß, and further downregulated that of transforming growth factor-ß4 (at 5 ng/ml). In conclusion, T-2 impaired the antifungal activities of chicken macrophages against A. fumigatus conidia, but might stimulate immune response by upregulating the expression of pro-inflammatory cytokines, chemokines and T-helper 1 cytokines.

Germination of Aspergillus fumigatus inside avian respiratory macrophages is associated with cytotoxicity.

Although aspergillosis is one of the most common diseases in captive birds, the pathogenesis of avian aspergillosis is poorly known. We studied the role of avian respiratory macrophages as a first line of defense against avian aspergillosis. The phagocytic and killing capacities of avian respiratory macrophages were evaluated using pigeon respiratory macrophages that were inoculated with Aspergillus fumigatus conidia. On average, 25% of macrophage-associated conidia were phagocytosed after one hour. Sixteen percents of these cell-associated conidia were killed after 4 h and conidial germination was inhibited in more than 95% of the conidia. A. fumigatus conidia were shown to be cytotoxic to the macrophages. Intracellularly germinating conidia were located free in the cytoplasm of necrotic cells, as shown using transmission electron microscopy. These results suggest that avian respiratory macrophages may prevent early establishment of infection, unless the number of A. fumigatus conidia exceeds the macrophage killing capacity, leading to intracellular germination and colonization of the respiratory tract.

Microsatellite typing of avian clinical and environmental isolates of Aspergillus fumigatus.

Aspergillosis is one of the most common causes of death in captive birds. Aspergillosis in birds is mainly caused by Aspergillus fumigatus, a ubiquitous and opportunistic saprophyte. Currently it is not known whether there is a link between the environmental isolates and/or human isolates of A. fumigatus and those responsible for aspergillosis in birds. Microsatellite typing was used to analyse 65 clinical avian isolates and 23 environmental isolates of A. fumigatus. The 78 genotypes that were obtained were compared with a database containing genotypes of 2514 isolates from human clinical samples and from the environment. There appeared to be no specific association between the observed genotypes and the origin of the isolates (environment, human or bird). Eight genotypes obtained from isolates of diseased birds were also found in human clinical samples. These results indicate that avian isolates of A. fumigatus may cause infection in humans.

Cutaneous hyalohyphomycosis in a girdled lizard (Cordylus giganteus) caused by the Chrysosporium anamorph of Nannizziopsis vriesii and successful treatment with voriconazole.

The Chrysosporium anamorph of Nannizziopsis vriesii was associated with dermatomycosis and high mortality in a group of captive giant girdled lizards (Cordylus giganteus). Treatment of one of the infected girdled lizards with voriconazole, which was selected on the basis of in vitro sensitivity testing of the isolate, resulted in resolution of lesions and negative fungal cultures from the skin. Three hours after oral administration of 10 mg/kg, the plasma level of voriconazole exceeded the 0.25-µg/mL minimal inhibitory concentration tenfold. In conclusion, administration of voriconazole at 10 mg/kg of body weight once daily for 10 weeks resulted in clinical cure and was well tolerated. A longer follow-up time and larger studies will be necessary to determine the long-term efficacy and safety of this treatment in giant girdled lizards.

Development of in vitro models for a better understanding of the early pathogenesis of Batrachochytrium dendrobatidis infections in amphibians.

Batrachochytrium dendrobatidis, the causal agent of chytridiomycosis, is implicated in the global decline of amphibians. This chytrid fungus invades keratinised epithelial cells, and infection is mainly associated with epidermal hyperplasia and hyperkeratosis. Since little is known about the pathogenesis of chytridiomycosis, this study was designed to optimise the conditions under which primary keratinocytes and epidermal explants of amphibian skin could be maintained ex vivo for several days. The usefulness of the following set-ups for pathogenesis studies was investigated: a) cultures of primary keratinocytes; b) stripped epidermal (SE) explants; c) full-thickness epidermal (FTE) explants on Matrigel™; d) FTE explants in cell culture inserts; and e) FTE explants in Ussing chambers. SE explants proved most suitable for short-term studies, since adherence of fluorescently-labelled zoospores to the superficial epidermis could be observed within one hour of infection. FTE explants in an Ussing chamber set-up are most suitable for the study of the later developmental stages of B. dendrobatidis in amphibian skin up to five days post-infection. These models provide a good alternative for in vivo experiments, and reduce the number of experimental animals needed.

Interaction of Aspergillus fumigatus conidia with Acanthamoeba castellanii parallels macrophage-fungus interactions.

Aspergillus fumigatus and free-living amoebae are common inhabitants of soil. Mechanisms of A. fumigatus to circumvent the amoeba's digestion may facilitate overcoming the vertebrate macrophage defence mechanisms. We performed co-culture experiments using A. fumigatus conidia and the amoeba Acanthamoeba castellanii. Approximately 25% of the amoebae ingested A. fumigatus conidia after 1 h of contact. During intra-amoebal passage, part of the ingested conidia was able to escape the food vacuole and to germinate inside the cytoplasm of A. castellanii. Fungal release into the extra-protozoan environment by exocytosis of conidia or by germination was observed with light and transmission electron microscopy. These processes resulted in structural changes in A. castellanii, leading to amoebal permeabilization without cell lysis. In conclusion, A. castellanii internalizes A. fumigatus conidia, resulting in fungal intracellular germination and subsequent amoebal death. As such, this interaction highly resembles that of A. fumigatus with mammalian and avian macrophages. This suggests that A. fumigatus virulence mechanisms to evade macrophage killing may be acquired by co-evolutionary interactions among A. fumigatus and environmental amoebae.

Designing a treatment protocol with voriconazole to eliminate Aspergillus fumigatus from experimentally inoculated pigeons.

To investigate the efficacy of voriconazole for the treatment of aspergillosis, three groups of six racing pigeons (Columba livia domestica) were inoculated in the apical part of the right lung with 2x10(7) conidia of an avian derived Aspergillus fumigatus strain. The minimal inhibitory concentration of voriconazole for this strain was 0.25 microg/ml. In two groups, voriconazole treatment was started upon appearance of the first clinical signs and continued for fourteen days. The third group was sham treated. The voriconazole-treated pigeons received voriconazole orally at a dose of 10 mg/kg body weight (BW) q12h (group 1) or 20 mg/kg BW q24h (group 2). Sixteen days post-inoculation all surviving pigeons were euthanized. Weight loss, clinical scores, daily mortality, lesions at necropsy and isolation of A. fumigatus were compared between all groups. In both voriconazole-treated groups, a significant reduction in clinical signs and lesions was observed. Administering voriconazole at 10 mg/kg BW q12h eliminated A. fumigatus and administering voriconazole at 20 mg/kg BW q24h reduced A. fumigatus isolation rates. Mild histological liver abnormalities were found in group 1 (10 mg/kg BW q12h), while mild histological as well as macroscopic liver abnormalities were found in group 2 (20 mg/kg BW q24h). In conclusion, voriconazole at 10 mg/kg BW q12h in pigeons reduces clinical signs and eliminates A. fumigatus in racing pigeons experimentally infected with A. fumigatus.