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Aspergillus fumigatus is a common pathogen in poultry and captive wild birds and a emerging opportunistic fungal pathogen in immunocompromised humans. Although invasive aspergillosis is frequently reported in free-ranging wild birds, the incidence and epidemiology of the disease in a natural setting is unknown. We recently reported endemic outbreaks of invasive aspergillosis at white stork nesting sites close to human habitation in Germany with significant subsequent breeding losses. Therefore, we hypothesized that A. fumigatus strains with higher virulence in birds may have evolved in this environment. Sixty clinical and environmental A. fumigatus isolates from six affected nesting sites were genotyped by microsatellite analysis using the STRAf-assay. The isolates showed a remarkable high genomic diversity and, contrary to the initial hypothesis, clinical and environmental isolates did not cluster significantly. Interestingly, storks were infected with two to four different genotypes and in most cases both mating types MAT-1.1 and MAT-1.2 were present within the same specimen. The majority of selected clinical and environmental strains exhibited similar virulence in an in vivo infection model using embryonated hen eggs. Noteworthy, virulence was not associated with one distinct fungal mating type. These results further support the assumption that the majority of A. fumigatus strains have the potential to cause disease in susceptible hosts. In white storks, immaturity of the immune system during the first three weeks of age may enhance susceptibility to invasive aspergillosis.
Keywords: aspergillosis; genotyping; pathogenicity; epidemiology
Aspergillus fumigatus is a ubiquitous, saprophytic mould that can be isolated from a variety of habitats world wide. As a facultative pathogen, it can cause various disease manifestations, including life-threatening invasive aspergillosis (IA) in animals and humans, and has emerged as an increasingly important pathogen for immunocompromised individuals, e.g. transplant recipients and oncological patients (reviewed in Erjavec et al., 2009; Maschmeyer et al., 2007). However, even though the numbers of human cases are increasing, IA is still comparatively rare and affects mainly predisposed individuals. Similarily, aspergillosis is only sporadically observed in other mammalian species (Tell, 2005).
In comparison to mammals, birds are highly susceptible to respiratory infection with A. fumigatus (Tell, 2005). Pulmonary invasive aspergillosis is a common disease in birds causing significant economic losses in poultry industry (Saif et al., 2008). Outbreaks can affect entire flocks with mortality rates of up to 30% (Dyar et al., 1984; Pollock, 2003; Zafra et al., 2008). IA has also been well documented for wild birds kept in captivity (Flach et al., 1990; Saif et al., 2008; Wolff et al., 1992). Recently, we reported for the first time that acute fungal pneumonia can represent a major cause of mortality in free-ranging wild birds before fledging (Olias et al., 2010). Total breeding losses of white storks (Ciconia ciconia) over several successive years have been documented in Germany for several nesting sites since 2005. Of all white stork chicks necropsied in 2007 and 2008 (n=101), 44.6% had infection with filamentous fungi in the lungs with 94.1% prevalence below three weeks of age. Importantly, in 58% of reported fungal infections A. fumigatus has been identified as causative agent. This observation not only emphasized the importance of A. fumigatus as avian pathogen but also suggested an endemic infection. The epidemiology of human IA in hospital settings has been addressed by several groups (Bertout et al., 2001; Chazalet et al., 1998; de Valk et al., 2007a; Rosehart et al., 2002), but only a single study addressed the epidemiology in poultry (Lair-Fulleringer et al., 2003), and recently in captive penguins (Alvarez-Perez et al., 2010). The principal aim of this study was, thus, to investigate whether specific virulent genotypes can be identified in diseased stork chicks at the outbreak sites.
As white storks breed close to human habitation on buildings and man-made structures, and clinical outbreaks of IA in poultry have been attributed to litter contamination (Dyar et al., 1984),we included potential environmental sources of human origin for A. fumigatus in our study. We genotyped 60 clinical and environmental A. fumigatus isolates from six geographically distinct outbreak sites in Germany using a microsatellite assay based on nine short tandem repeat (STR) markers, which has been shown to possess high discriminatory power and reproducibility in epidemiological investigations (Balajee et al., 2008; de Valk et al., 2009; de Valk et al., 2005; de Valk et al., 2007b). Based on the typing results, the virulence of selected strains was analyzed in a new alternative infection model using embryonated chicken eggs (Jacobsen et al., 2010).
Materials and methods
Origin of strains
The strains used in this study are a subset of strains obtained from environmental sources and clinical specimens sampled from six nesting sites (ns) in the federal state of Brandenburg, Germany, in 2007 to 2009, denotated as nsA-nsF. The geographical distances between the nesting sites were 3 to 140 miles. An overview of the A. fumigatus isolates obtained and the subset that was subsequently chosen according to their growth pattern (de Hoog et al., 2000) and used in this study for further molecular and virulence analysis is given in Table 1. Environmental strains were isolated from nest material and compost heaps of the nesting sites nsA, nsB, nsC and nsE. The sources were identified by volunteers who had observed parent storks collecting material from those particular compost heaps before entering the nest. For fungal isolation, 20 grams of plant material from each sample were incubated with 20 ml sterile PBS in sterile Whirl-Pak® plastic bags (Nasco, Fort Atkinson, WI) for 1h at 37°C. Subsequently, the fluid was filtered (40 μm cell strainer; BD, Franklin Lakes, NJ) and 2 ml were plated onto solid malt-extract agar (Roth, Karlsruhe, Germany) supplemented with streptomycin sulfate and chloramphenicol (Roth, Karlsruhe, Germany) and further processed as described previously (Olias et al., 2010).
Clinical isolates were isolated post mortem by plating lung material from deceased chicks obtained during aseptic necropsy onto solid malt-extract agar as described previously (Olias et al., 2010). The strains included in this study derived from the lungs of 10 white stork chicks with histologically confirmed pulmonary IA. These birds originate from the above mentioned nesting sites in 2007 and 2008. Additionally, A. fumigatus strains were isolated in 2009 from the lungs of two chicks from nesting site nsA, where outbreaks of IA occurred in four of five successive breeding seasons.
Isolates were initially identified as A. fumigatus by their macroscopic and microscopic morphology (de Hoog et al., 2000) and their ability to grow at 52°C. Additionally, three reference stains (Af 293, ATCC46645; CEA17ΔakuB) were included in this study. An overview of origin and designation of all strains used in this study is given in Table 2.
Genomic fingerprinting by microsatellite assay
Genomic fingerprinting was performed on 60 isolates from the six different nesting sites and three reference stains (Af 293, ATCC46645; CEA17ΔakuB). Fungal DNA was extracted and purified from all isolates as described previously (Olias et al., 2010). For PCR amplification, nine short tandem repeats (STRs) were used comprising the three multiplex panels STRAf2, 3 and 4 (de Valk et al., 2005). Primers were labeled with carboxyfluorescein (FAM), hexachlorocarboxyfluorescein (HEX) or tetrachlorocarboxyfluorescein (TET), respectively, and thermocycling was performed as described (de Valk et al., 2005). The PCR products obtained for the polymorphic loci were mixed with the GeneScan 500 Rox Size Standard (Applied Biosystems, Foster City, CA) and analyzed with the GeneScan program on a ABI 3730XL DNA analyzer (Applied Biosystems), according to the manufacturer's instructions. The exact number of repeats in the obtained PCR products were determined by construction of allelic ladders. Selected PCR products representing suitable positions within the span of alleles of a polymorphic locus were cloned into pDrive (Quiagen, Hilden, Germany). Plasmid DNA was purified and sequenced with universal M13 primers.
The number of repeats of the nine STR loci of all isolates were analyzed by UPGMA clustering using BioNumerics 4.5 software (Applied Maths, Sint-Martens-Latem, Belgium) with the multistate categorical similarity coefficient. Clonal clusters were defined as isolates with the same number of repeat units in all nine loci. Microevolutionary events were defined as changes of less than two repeat units in a single locus of isolates (Balajee et al., 2008).
Preparation of conidia suspensions for infection experiments
Malt agar slant plates were inoculated with conidia and incubated at room temperature for seven days. Conidia were suspended in sterile phosphate-buffered saline (PBS) containing 0.1% Tween 20 (AppliChem GmbH, Darmstadt, Germany) and filtered through a 40 µm cell strainer (BD Bioscience, Heidelberg, Germany). After counting the number of conidia, the suspension was diluted with PBS to the desired concentration and used within a few hours. PBS alone served as negative control for infection experiments.
Fertilized chicken eggs of the White Leghorn breed were obtained from a local producer and stored at 8°C for a maximum of seven days prior to incubation. Incubation was performed at 37.6°C and 50-60% relative humidity in a specialized incubator (BSS 300, Grumbach, Germany). From the fourth day of incubation onwards, the eggs were turned four times a day until infection on day 10. Vitality was assessed daily by candling. Infection of embryonated eggs was performed as described previously (Jacobsen et al., 2010). Briefly, after wiping the shell with a disinfectant (Braunol, Braun, Melsungen, Germany) the shell was perforated at the blunt end and one longitudinal side using a dentist drill. An artificial air chamber at the longitudinal side was then formed by applying negative pressure at the hole in the blunt end. After perforation of the shell membrane 0.1 ml inoculum containing 103 or 104 conidia was applied via the artificial air chamber onto the chorioallantoic membrane (CAM) using a 1 ml syringe. The holes were then sealed with paraffin. Survival was monitored for up to eight days by candling. Twenty eggs per group were infected and experiments were repeated twice. Survival data were plotted as Kaplan-Meyer curves and tested for significant differences by the log rank test using Graph Pad Prism Version 5.00 for Windows (GraphPad Software, San Diego, California, USA).
Genomic fingerprinting and mating type
To determine whether: (a) multiple distinct isolates infected individual chicks, (b) clinical isolates were related to each other, and (c) clinical isolates could be traced to an environmental source, we performed microsatellite assays based on nine short tandem repeat (STR) markers. Sixty clinical and environmental isolates from six geographically distinct outbreak sites in Germany were used. Twenty-nine of the 60 isolates originated from post mortem samples of 10 stork chicks, and two to four isolates per chick were analyzed. Based on microsatellite analyses, identical isolates were identified in two cases: (a) Two identical isolates (D08-L-2b and -L-2c) were found in the lung of a chick from nesting site D. The third isolate from this chick (D08-L-2a), however, was genetically distinct. (b) At nesting site A in 2008, chick 1 harboured a strain (A08-L-1b) which was identical to an environmental isolate (A08-C-2b) found in a compost heap near the nesting site in the same year. (Fig. 1).
Two additional strains isolated from this chick (A08-L-1a and A08-L-1c) differed only by one repeat in locus 3A from each other but were not related to the third clinical isolate or identical environmental strain. One clinical isolate of 2009 and one environmental isolate of 2007 from the same nesting site differed only in a single allele change in locus 3C to clinical isolates C08-L-1c and C08-L-1a, respectively, from nesting site C in 24 miles distance. Isolates from nsB, nsE and nsF had unrelated genotypes.
Among environmental isolates, mating types MAT-1.1 and MAT-1.2 were equally distributed with 51.6% of the isolates being MAT-1.1 and 48.4% MAT-1.2 (Tab. 3). MAT-1.1 was found more often than MAT-1.2 in clinical isolates (58.6% and 41.4%, respectively); however, from six of the 10 chicks which harboured more than one genetically distinct isolate, strains of both mating types were isolated (Fig. 1).
Based on the typing results, the virulence potential of selected strains was assessed in a novel alternative infection model using embryonated chicken eggs (Jacobsen et al., 2010). In a first set of experiments we compared the virulence of isolates from chicks with corresponding isolates from nesting material and environmental isolates from compost heaps that had served as a source for nesting material. The laboratory strain CEA17ΔakuB which had previously been shown to be virulent in murine models (da Silva Ferreira et al., 2006) and embryonated eggs (Jacobsen et al., 2010), was used as reference strain. The majority of all strains tested showed indistinguishable virulence potential compared to CEA17ΔakuB (Fig. 2 A-C). One clinical isolate from nsE (E07-L-1a; Fig. 2B) and one isolate from nesting material at site F (F07-N-1b; Fig. 2C) caused significantly higher mortality rates than CEA17ΔakuB. Surprisingly, another clinical isolate, F07-L-1a reproducibly displayed strongly reduced virulence (Fig. 2C and D) but normal growth on malt agar (data not shown). Since the chick from which F07-L-1a was isolated harboured two additional, genetically distinct isolates, we compared the virulence of all three isolates (Fig. 2D). In contrast to F07-L-1a, F07-L-1b and -1c were able to kill infected embryos to a similar extent as the reference strain CEA17ΔakuB, albeit with a one or two day delay, respectively.
Finally, we compared the virulence potential of five clinical isolates from two consecutive years from nsA. All five strains were found to have high virulence with two strains (A09-L-1a and -1b) causing mortality significantly faster than the reference strain (Fig. 3).
Hospital-associated outbreaks of aspergillosis in humans have led to several studies investigating whether patients within the same hospital carry similar strains and whether environmental sources could be identified. In general, these studies revealed a high genetic variability of clinical A. fumigatus isolates (Araujo et al., 2009; de Valk et al., 2005; Debeaupuis et al., 1997; Lair-Fulleringer et al., 2003). However, some studies identified identical isolates in several patients and relevant environmental sources (Balajee et al., 2008; Menotti et al., 2005; Warris et al., 2003). The epidemiology of avian aspergillosis has been only scarcely addressed and focused on poultry (Lair-Fulleringer et al., 2003) and captive penguins (Alvarez-Perez et al., 2010), the latter species being highly susceptible to fungal infections in captivity (Flach et al., 1990). We recently identified aspergillosis as a major cause of mortality in free-ranging white stork chicks, leading to major losses in subsequent years (Olias et al., 2010). White storks construct large nests of sticks lined with twigs, grasses and other soft natural or human-made material and these nests often are reused in subsequent years (Hancock et al., 1992). Importantly, adult storks constantly collect further nesting material during the breeding season that is often taken from nearby compost heaps (von Blotzheim et al., 1971; personal observation). Since A. fumigatus is commonly reported on all sorts of biodegrading plant material (Beffa et al., 1998; Ryckeboer et al., 2003), and has also frequently been isolated in high concentrations from bird nests (Apinis and Pugh, 1967; Hubálek et al., 1973), we hypothesized that nesting material and compost heaps are major sources for infection of chicks and that A. fumigatus strains with higher virulence in birds might have evolved in this environment. To test this hypothesis, we combined microsatellite genotyping with subsequent in vivo virulence analysis of selected clinical and environmental isolates.
Consistent with other studies, our results demonstrate a high genetic diversity of the A. fumigatus population at each outbreak site. As fungal spores are easily distributed, we expected high strain diversity within the environmental isolates. Consequently, the environmental strains typed may represent only a small part of the population potentially inhaled by the affected birds. This notion is supported by a recent epidemiologic study by Chazalet et al. (1998) which extrapolates that a patient with nosocomial IA inhales about 5,000 unique genotypes over a period of three months. Thus, the putatively high number of A. fumigatus strains in the environment in combination with the limited number of samples taken for this study can easily explain why we found only one genotype in both, an infected lung and the environment of the same bird. However, if strains with adaptation to the avian host do exist, we would have expected to find the clinical isolate collection to be enriched for certain genotypes, especially amongst chicks of the same nesting site. In contrast to this hypothesis, we failed to find identical or highly similar genotypes amongst clinical isolates from different birds or distinct clustering of environmental isolates. This suggests that even at nesting sites where outbreaks of IA occurred in up to four successive breeding seasons, the composition of A. fumigatus strains as part of the nest's microflora is constantly changing by introduction of conidia by adult storks via plant material, feed, plumage or by air (Hubalek, 1978). However, the absence of cluster formation is in concordance with previous genetic, biochemical and immunological studies that have reported no association of a particular A. fumigatus genotype with pathogenic potential in humans (Bart-Delabesse et al., 1999; Bart-Delabesse et al., 1998; Debeaupuis et al., 1997).
Although some studies suggest a lower virulence potential of environmental strains when compared to clinical strains (Aufauvre-Brown et al., 1998; Mondon et al., 1996; Peden and Rhoades, 1992), there is growing evidence that the pathogenicity of A. fumigatus is multifactorial, involving networks of genes that have probably evolved as a result of microbial interactions in its primary ecological niche and that most environmental strains have the potential to cause disease in susceptible hosts (Brock, 2009; Askew, 2008; Tekaia and Latgé, 2005).To determine the virulence potential of the clinical and environmental strains isolated in this study, we tested selected strains in an infection model which we have recently shown to be suitable to detect virulence differences between genetically defined strains (Jacobsen et al., 2010). The majority of strains, independent of their origin, demonstrated a virulence potential similar to our chosen reference strain, CEA17ΔakuB. CEA17ΔakuB is a derivative of a clinical strain from an IA patient (d'Enfert, 1996) and has been shown to be virulent in murine models (da Silva Ferreira et al., 2006). Although three clinical stork isolates tested showed increased virulence, the virulence of the other six clinical isolates tested was indistinguishable from that of the environmental strains. Moreover, we found one clinical isolate to be significantly reduced in virulence. This highlights the general problem that culture results from IA specimens may be influenced by colonization, inhaled spores and contaminations during the process of sampling (Rickerts et al., 2007; Spreadbury et al., 1993). Thus, the isolation of a strain from a clinical or post mortem specimen does not necessarily indicate high virulence and the use of complex infection models is necessary to assess the virulence potential of A. fumigatus isolates.
In humans and recently in captive penguins polyclonal A. fumigatus infections have been identified in cases of respiratory IA (Alvarez-Perez et al., 2010; Bertout et al., 2001; Symoens et al., 2001). However, other investigations suggest that only one or two genotypes are usually involved in human pulmonary IA (Bart-Delabesse et al., 1999; Bart-Delabesse et al., 1998; Girardin et al., 1994). In stork chicks, we found at least two and up to four distinct A. fumigatus genotypes in each clinical specimen, consistent with the high genetic diversity of the environmental isolates and the hypothesis that the majority of environmental strains have the potential to cause IA in susceptible hosts. Interestingly, although mating type MAT-1.1 was slightly overrepresented amongst clinical isolates, we commonly found coinfections with strains of both mating types. Furthermore, the virulence of MAT-1.1 and MAT-1.2 strains in embryonated eggs was indistinguishable, suggesting that the virulence potential is not linked to a specific mating type. This contradicts a study which suggested that MAT-1.1 is associated with higher virulence (Alvarez-Perez et al., 2009). The coexistence of both mating types in the same lung is of additional interest in birds, as it has been well documented that A. fumigatus regularily sporulates in the lung and air sac system of chronically and often subclinically infected birds (Ainsworth and Rewell, 1949; Di Somma et al., 2007; Richard et al., 1984). With the recent discovery of a sexual cycle in A. fumigatus (O'Gorman et al., 2009) there is a theoretical possibility that the chronically infected avian respiratory tract might provide an environment for mating.
In summary, our results demonstrate a high genetic diversity of A. fumigatus strains involved in natural outbreaks of pulmonary IA in free-ranging white stork chicks. Although we were able to identify clinical isolates with a higher virulence in an embryonated egg model, the overall data did not support the hypothesis that distinct A. fumigatus strains with a higher virulence are responsible for the outbreaks. Moreover, except for one strain, the genotyped clinical isolates could not be traced to an environmental infection source. Instead, the results of this study strongly support our previous proposal that immune immaturity in the first three weeks of age is a major predisposing factor for IA in white storks (Jovani and Tella, 2004; Olias et al., 2010). Furthermore, stork chicks were commonly found to be coinfected with two or more A. fumigatus strains that had concurrent mating types.