Fungal Contamination of Poultry Litter: A Public Health Problem

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1 This article was downloaded by: [b-on: Biblioteca do conhecimento online IPL] On: 24 October 2012, At: 07:58 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: Registered office: Mortimer House, Mortimer Street, London W1T 3JH, UK Journal of Toxicology and Environmental Health, Part A: Current Issues Publication details, including instructions for authors and subscription information: Fungal Contamination of Poultry Litter: A Public Health Problem C. Viegas, E. Carolino a, J. Malta-Vacas a, R. Sabino b, S. Viegas a & C. Veríssimo b a Higher School of Health Technologies of Lisbon IPL, Lisbon, Portugal b Nacional Institute of Health Dr. Ricardo Jorge URSZ, Infectious Diseases Department, Lisbon, Portugal Version of record first published: 24 Oct To cite this article: C. Viegas, E. Carolino, J. Malta-Vacas, R. Sabino, S. Viegas & C. Veríssimo (2012): Fungal Contamination of Poultry Litter: A Public Health Problem, Journal of Toxicology and Environmental Health, Part A: Current Issues, 75:22-23, To link to this article: PLEASE SCROLL DOWN FOR ARTICLE Full terms and conditions of use: This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. The publisher does not give any warranty express or implied or make any representation that the contents will be complete or accurate or up to date. The accuracy of any instructions, formulae, and drug doses should be independently verified with primary sources. The publisher shall not be liable for any loss, actions, claims, proceedings, demand, or costs or damages whatsoever or howsoever caused arising directly or indirectly in connection with or arising out of the use of this material.

2 Journal of Toxicology and Environmental Health, Part A, 75: , 2012 Copyright Taylor & Francis Group, LLC ISSN: print / online DOI: / FUNGAL CONTAMINATION OF POULTRY LITTER: A PUBLIC HEALTH PROBLEM C. Viegas 1, E. Carolino 1, J. Malta-Vacas 1, R. Sabino 2,S.Viegas 1, C. Veríssimo 2 1 Higher School of Health Technologies of Lisbon IPL, Lisbon, Portugal 2 Nacional Institute of Health Dr. Ricardo Jorge URSZ, Infectious Diseases Department, Lisbon, Portugal Although numerous studies have been conducted on microbial contaminants associated with various stages related to poultry and meat products processing, only a few reported on fungal contamination of poultry litter. The goals of this study were to (1) characterize litter fungal contamination and (2) report the incidence of keratinophilic and toxigenic fungi presence. Seven fresh and 14 aged litter samples were collected from 7 poultry farms. In addition, 27 air samples of 25 litters were also collected through impaction method, and after laboratory processing and incubation of collected samples, quantitative colony-forming units (CFU/m 3 )and qualitative results were obtained. Twelve different fungal species were detected in fresh litter and Penicillium was the most frequent genus found (59.9%), followed by Alternaria (17.8%), Cladosporium (7.1%), and Aspergillus (5.7%). With respect to aged litter, 19 different fungal species were detected, with Penicillium sp. the most frequently isolated (42.3%), followed by Scopulariopsis sp. (38.3%), Trichosporon sp. (8.8%), and Aspergillus sp. (5.5%). A significant positive correlation was found between litter fungal contamination (CFU/g) and air fungal contamination (CFU/m 3 ). Litter fungal quantification and species identification have important implications in the evaluation of potential adverse health risks to exposed workers and animals. Spreading of poultry litter in agricultural fields is a potential public health concern, since keratinophilic (Scopulariopsis and Fusarium genus) as well as toxigenic fungi (Aspergillus, Fusarium, and Penicillium genus) were isolated. Exposure to certain fungi is known to produce adverse human health effects through three specific mechanisms: (1) generation of a harmful immune response such as allergy or hypersensitivity pneumonitis, (2) direct infection by fungal organism, and (3) toxicirritant effects from mold by-products such as mycotoxins (Norred 1993; Bondy and Pestka 2000; Bush et al. 2006). Soil represents the main reservoir of fungi (Chmel et al. 1972). Some soil fungi are potential pathogens to both humans and animals (Ajello 1980). Soils rich in keratinous materials are more conducive to growth and occurrence of keratinophilic fungi (Mercantini et al. 1980; 1986), which may be considered a potential pathogen (Rippon 1982). In fact, the richness in keratin residues constitutes a permanent or occasional reservoir for keratinophilic fungi and a source of potential infection for humans and animals (Filipello 1986). Various investigators have examined keratinophilic fungi distribution in soil, and these fungi have attracted the attention of dermatologists and mycologists due to their association with human and animal mycoses (Papini et al. 1998; Rippon and Garber 1969; Mirocha et al. 1972; Mitra et al. 1982). In addition, fungi produce secondary metabolites, like mycotoxins, in response to environmental The authors are grateful to the Portuguese Ministry of Agriculture, Portuguese Ministry of Health, and poultry farmers. This study was funded by the Portuguese Authority for Work Conditions. Address correspondence to Carla Viegas, Higher School of Health Technologies of Lisbon IPL, Portugal. carla.viegas@ estesl.ipl.pt 1341

3 1342 C. VIEGAS ET AL. changes. Mycotoxins may be pro-inflammatory, immunosuppressive, or carcinogenic (Bondy and Pestka 2000; Jarvis 2003). The different chemical groups of mycotoxins include aflatoxins, fumonisins, ochratoxins, rubratoxins, and trichothecene toxins, all with different biologic properties (Norred 1993; Clark and Snedeker 2006: Jarvis 2003; Chung et al. 2003). In Portugal there is an increasing amount of industry of large facilities that produce whole chickens for domestic consumption. Although some studies examined microbial contaminants associated with the various stages related with poultry and meat products processing (Buys et al. 2000; Borch and Arinder 2002), only a few investigations reported on fungal contamination of poultry litter (Anbu et al. 2004, Kotimaa et al. 1991). The material used for poultry litter (bedding materials such as sawdust, wood shavings, straw, and peanut or rice hulls) varies but normally is constituted by pine shavings or sawdust of eucalyptus and other various types of wood. In some regions material used includes rice hulls, peanut hulls, coffee, sugar cane, straw, hay, grass, and processed paper (Fernandes 2004). Litter is a major contributive factor to fungal contamination in poultry farms (HSE 2008; Just et al. 2009; Williams 2009). Further, spreading litter is one of the tasks that normally involve higher exposure of poultry workers to dust (Whyte 2010) and fungi and their metabolites, including volatile organic compounds (VOC) and mycotoxins (Milner 2009; Tsapko et al. 2011). After being used and removed from poultry farms, litter rich in keratinous materials is plowed into agricultural soils. This practice may potentially be dangerous for the soil environment, as well as for both humans and animals (Anbu et al. 2004). Thus, analysis and identification of mycoflora of poultry litter is significant not only to recognize eventual occupational exposure of poultry workers to fungi and their metabolites, but also to evaluate the extent of dissemination of keratinophilic fungi at poultry sites. Bearing this in mind, the goal of this study was to characterize litter fungal contamination and to determine the incidence of keratinophilic and toxigenic fungi. MATERIALS AND METHODS Poultry Farms The selected poultry farms were located in the Lisbon district, Portugal, and the litter samples were collected between January and May Selected farms were dedicated to broiler chicken production, where birds are bred to reach slaughter weight as rapidly as possible. Day-old chicks were transferred from hatcheries to the growing farms, where they are housed in single-story sheds. Farm staff monitor the condition of the birds daily, adjust feed and water equipment as necessary, and administer vaccines. In some cases, the same workers also catch and transport mature chickens to slaughterhouse. After depopulation the manure-removal contractor accumulated manureandloadeditontotrailersfortransport to fields where it is used as fertilizer. Samples Collection, Preparation and Analyses Seven fresh (new) and 14 aged litter samples were collected from 7 poultry farms in sterilized bags (Table 1). Each litter sample (10 g, not oven-dried prior to processing, retaining the natural water content) was diluted in 100 ml sterilized distilled water, agitated for 30 min at 100 rpm, and 0.2 ml of this suspension was spread onto triplicate Petri dishes containing malt extract agar (2%) with cloramphenicol (0.05 g/l) and incubated for 5 7 d at 27.5 C. Fungal identification was carried out by macroscopic and microscopic observation using lactophenol blue staining and atlas identification (Hoog et al. 2000). Isolated fungi were identified to the species level. Results were reported as the average count of the three TABLE 1. Litter Samples Collected Litter type New Aged Pine shavings 2 4 Straw 2 2 Wood shavings with rice hulls 2 6 Wood shavings 1 2 Total 7 14

4 POULTRY LITTER FUNGAL CONTAMINATION 1343 replicas, in colony-forming units per gram of litter (CFU/g). In addition to litter samples, 27 air samples of 25 litters were also collected through impaction method. Air samples were collected at 1 m height with a flow rate of 140 L/min onto malt extract agar (MEA) supplemented with the antibiotic chloramphenicol (0.05%). After laboratory processing and incubation of the collected samples, quantitative (colony-forming units [CFU]/m 3 ) and qualitative results were obtained with identification of the isolated fungal species. Statistical Analysis Statistical analysis was performed using SPSS Significant differences of the total CFU/g between different litter type were tested by the Kruskal Wallis test and tendencies by median values, 25th and 75th percentiles, and interquartile range. The correlations between total CFU per gram and litter age and between litter fungal contamination and air fungal contamination from the analyzed poultry farms were conducted using the Spearman correlation coefficient. RESULTS Twelve different fungal species were detected in fresh, new litter in a total of 805,500 isolates from all the analyzed poultry farms. Penicillium was the most frequent genus found (59.9%), followed by Alternaria (17.8%), Cladosporium (7.1%), and Aspergillus (5.7%). In addition to these, other genera were also isolated, namely, Scedosporium sp., Paecilomyces sp., Absidia sp., Rhizopus sp., Hormographiella sp., Exophiala sp., Fusarium sp., and Syncephalastrum sp. (Table 2). With respect to aged used litter, 19 different fungal species were detected in a total of 2,276,500 isolates. Penicillium sp. was the most frequently isolated (42.3%), followed by Scopulariopsis sp. (38.3%), Trichosporon sp. (8.8%), and Aspergillus sp. (5.5%). Besides these fungal genera, other fungi were also TABLE 2. Most Frequent Fungi Genus Isolated in New and Aged Poultry Litter Fungi, new litter Frequency (n; %) Penicillium sp. 482,500; 59.9 Alternaria sp. 73,000; 17.8 Cladosporium sp. 57,000; 7.1 Aspergillus sp. 20,000; 5.7 Others 173,000; 9.5 Fungi, used litter Frequency (n; %) Penicillium sp. 962,500; 42.3 Scopulariopsis sp. 871,000; 38.3 Trichosporon sp. 200,000; 8.8 Aspergillus sp. 126,000; 5.5 Others 117,000; 5.1 identified: Acremonium sp., Trichoderma sp., Fusarium sp., Arthriniumsp., Absidia sp., Phoma sp., Paecilomyces sp., and Syncephalastrum sp. (Table 2). Scopulariopsis brevicaulis (81.5%) was the most frequently isolated species among Scopulariopsis genera. In the new litter, Aspergillus fumigatus was the most frequent species identified (32.6%) from Aspergillus genus, and A. flavus was also isolated in 9.9% of the samples. Besides these two species, and among Aspergillus genus, others were also found, namely, A. candidus and A. clavatus-nanicus. In the aged litter, Aspergillus versicolor was the most frequent (73.4%) among Aspergillus genus, but A. fumigatus, A. flavus, and A. niveus were also identified. As depicted in Figure 1, Aspergillus species presented different incidences between new and aged litter. Statistically significant differences were not found when comparing total fungal contamination (CFU/g) between different litter types. FIGURE 1. Aspergillus species incidences in new and used litter.

5 1344 C. VIEGAS ET AL. TABLE 3. Median Values, 25th and 75th Percentiles, and Interquartilile Range of Total Fungal Contamination, by Litter Type Litter type Median (total CFU/g) 25th Percentile (total CFU/g) 75th Percentile (total CFU/g) Interquartile range (total CFU/g) Pine shavings 200,500 90, , ,000 Straw 285, ,000 1,785,000 1,672,000 Wood shavings 156, , ,250 with rice hulls Wood shavings 830, ,500 3,200,000 2,587,500 FIGURE 2. Scatterplot for total fungal contamination CFU/ gand litter age. However, a tendency was observed for wood shavings litter to present higher CFU values and for wood shavings with rice hulls litter to present lower values (Table 3). Correlation of total fungal load (CFU/g) and litter age showed a weak correlation nonsignificant (Figure 2). Correlation of litter fungal contamination (CFU/g) and air fungal contamination (CFU/m 3 ) shows a significant positive correlation. The results indicated that litter fungal contamination contributes 87.3% to the presence of air fungal contamination (Figure 3). DISCUSSION Microbial pathogens are a significant economic concern to the broiler industry. The main source of poultry pathogens is the mixture of bedding material, chicken excrement, and FIGURE 3. Scatterplot for air fungal contamination (CFU/m 3 ) and litter fungal contamination (CFU/g). feathers that comprises the litter of a poultry house (Dumas et al. 2011). Microorganisms and their metabolites, as components of organic dust, may also exert adverse effects on the respiratory health of workers in poultry buildings. According to the FAO (Williams 2009), one of the potential pollutants and issues related to poultry production is litter (bedding materials such as sawdust, wood, shavings, straw, and peanut or rice hulls) (cited by Williams 2009). Although many studies evaluated the bacterial community composition of poultry litter, few reports were found concerning mycological content. Thus, surfaces sampling, in addition to air sampling, is essential to achieve fungal contamination characterization and evaluation, and may be used to identify contamination sources (Stetzenbach et al. 2004; Klánová and Hollerová 2003). Regarding the studied

6 POULTRY LITTER FUNGAL CONTAMINATION 1345 settings, litter is one of the most contributive factors to fungal contamination in poultry farms (HSE 2008; Just et al. 2009; Williams 2009), and its analysis is crucial to evaluate occupational and public health risks. This may be attributable to exposure to inhalable particles capable of inducing irritation, or allergic or toxic respiratory diseases. Further, adding fresh straw litter during the growth of birds may result in heavy exposure both to inhalable dust and to fungal spores, partly as a consequence of microbial degradation of the previous straw (HSE 2008). Some of the fungal isolates more frequently identified in this study belong to Penicillium and Aspergillus species in fresh, new, and aged litter. Cladosporium sp. was also frequently isolated in the new litter and Scopulariopsis sp. was found in high counts in aged litter. Kotimaa and colleagues (1991) noted Aspergillus, Penicillium, and Cladosporium were the most prevalent genera found in feeding and bedding material. Anbu et al. (2004) reported the most prevalent species isolated in litter plowed into fields were Fusarium solani, Aspergillus flavus, A. nidulans, A. niger, Penicillium citrinum, P. frequentans, Scopulariopsis brevicaulis, A. versicolor, Cladosporium oxysporum, Penicillium purpurogenum, andtrichoderma viridae. Some of the fungal species detected in the present study are considered potential agents of infection in humans and animals (Ponikau et al. 1999; Tosti et al. 1996; Ghannoum et al. 2000; Araújo et al. 2003). Previously, Viegas et al. (2012b) obtained data regarding the same poultry environments through air sampling. Twenty-eight species/genera of fungi were identified with Scopulariopsis brevicaulis (39%), the most commonly isolated species, and Rhizopus sp. (30%), Penicillium sp. (10%), and Aspergillus sp. (9.7%), the most frequently isolated genera. Penicillium sp., Aspergillus sp., and Scopulariopsis sp. genera were also prevalent fungi in the analyzed litter. This situation may be be due to litter spreading normally involving high dust aerosolization (Whyte 2010), and consequently, large spreading of fungi and their metabolites such as VOC and mycotoxins (Milner 2009; Tsapko et al. 2011). Further, particle size is a key factor in poultry dust production since rate of aerosolization, settling velocity, and resuspension rate of airborne particles differ depending on particle size (Pedersen et al. 2000). Kemp et al. (2002) indicated that concentration and type of spore are vital when identifying a species since these factors play an important role in air and surface dispersion. Other studies performed in poultry farms across Europe reported similar results. Rimac et al. (2010) in Zagreb noted the most prevalent fungi were species from the genera Penicillium sp., Fusarium sp., Aspergillus sp., Mucor sp., Rhyzopus sp., Scopulariopsis sp., Mucor sp., Aspergillus sp., Penicillium sp., and Fusarium sp. These fungi were also the predominant genera in other study in agricultural facilities in Ukraine and Poland (Tsapko et al. 2011). Others investigators also concluded that airborne fungi present in poultry facilities include Cladosporium sp., Aspergillus sp., Penicillium sp., and, less commonly, Alternaria sp., Fusarium sp., Geotrichum sp., and Streptomyces sp. (Sauter et al. 1981; Lee et al. 2006). It is noteworthy that only one sample from each litter was taken, and variations in fungal contamination are expected. Nevertheless, the prevalent genera found are common in several studies not only in poultry farms (Kotimaa et al. 1991; Anbu et al. 2004; Rimac et al. 2010; Sauter et al. 1981; Lee et al. 2006), but also in other agricultural settings (Tsapko et al. 2011). Some specific species found in poultry litter and also in environmental samples from the same poultry farms (Viegas et al. 2012b), such as Scopulariopsis brevicaulis, need to be considered with caution, since exposure to this species is associated with cases of occupational allergy (Ponikau et al. 1999) and also is known to produce onychomycosis (Tosti et al. 1996). Contribution from this species to the fungal load in litter and air is high, and data obtained show an enhanced risk to poultry workers and a public health concern. Fusarium sp. was found in new, fresh, and aged litter. An increased diagnosis of onychomycosis in occupational workers

7 1346 C. VIEGAS ET AL. produced by this genus justifies the inclusion of these fungi as possible etiologic agents of Tinea pedis and onychomycosis (Ghannoum et al. 2000; Araújo et al. 2003). With respect to the Aspergillus genus, different species were isolated in both new and aged litter. As in the case of other species, it is conceivable that possible spreading occurs in the poultry environment, possibly by aerosolization of fungal species present onto litter, which may become airborne depending on the occupants and the activities carried out by them, and also depending on number of the chickens and other features related to animals present (Milner 2009; Tsapko et al. 2011; Buttner and Stetzenbach 1993; Scheff et al. 2000). Aspergillus species are strongly linked with exacerbations of asthma and other respiratory allergic diseases (Chaudhary and Marr 2011). More than 80% of Aspergillusrelated conditions such as extrinsic allergic alveolitis, asthma, allergic sinusitis, chronic eosinophilic pneumonia, hypersensitivity pneumonitis, severe asthma with fungal sensitization (SAFS), and allergic bronchopulmonary aspergillosis (ABPA) are most frequently produced by A. fumigatus, the species most frequently found in new litter. In addition, according to the American Industrial Hygiene Association (AIHA 1996) in the Field Guide for the Determination of Biological Contaminants in Environmental Samples, the confirmed presence of the species Aspergillus flavus and Aspergillus fumigatus, requires implementation of corrective measures. Both species were identified in poultry litter, which shows the relevance of this study. It is also noteworthy that Aspergillus fumigatus isolated in litter and air of poultry farms (Viegas et al. 2012b) is one of the saprophytic fungi most widespread in air and capable of inducing severe or sometimes fatal aspergillosis (Yao and Mainelis 2007). In addition, Aspergillus flavus, also isolated in the poultry-farm air, is a well-known producer of potent mycotoxins (aflatoxins). In addition to this species, other isolated genera, like Fusarium and Penicillium, are also mycotoxin producers (Araújo et al. 2003). There is evidence that mycotoxins produce human diseases from inhalation and from ingestion of fungus-contaminated food (Shlosberg et al. 1991; Bondy and Pestka 2000; Norred 1993). Bosch and Munoz 1988) provided data illustrating that increasing human hepatocellular carcinoma rates corresponded to increasing levels of dietary aflatoxins exposure. In air samples collected from the same poultry farms, using molecular biology, toxigenic strains (aflatoxinproducing) belonging to the Aspergillus flavus complex were detected (Viegas et al. 2012a). It is known that use of conventional methods for fungal quantification (fungal culture) may underestimate the results obtained. The incubation temperature chosen might not be the most suitable for every fungal species, resulting in the inhibition of some species and being beneficial for others (Zorman and Jerseck 2008). Regarding Aspergillus genus, the focus of interest is normally the viable spores produced because these possess the ability to infect humans and animals (Samson et al. 2000), and this is the basis for application of conventional methodologies in this study. Some of the tasks conducted in the poultry industry, such as catching birds for transport to slaughter, also present an increased occupational risk. The catching of birds is by hand, and birds naturally spread their wings to resist the movement, which is an effective way of generating litter dust and consequently increases exposure to fungi and mycotoxins (HSE 2008; Oppliger et al. 2008). This occurs because although mycotoxins are not volatile, if found in the respirable air, mycotoxins are associated with the presence of mold spores or particles (Bush et al. 2006). The others tasks associated with increased occupational risk are spreading new litter and turnover of litter, and dust aerosolization and consequently aerosolization of fungi and mycotoxins (Whyte 2010). A correlation between litter fungal contamination (CFU/g) and air fungal contamination (CFU/m 3 ) was noted, corroborating the presence of indoor aerosolization of fungi isolated in litter and indicating that litter from analyzed poultry farms is a source of indoor fungal contamination. This finding needs be considered as a public health concern as well as

8 POULTRY LITTER FUNGAL CONTAMINATION 1347 an occupational threat, since for relatively dry materials, like poultry litter, where screens, separators, and box spreaders are used, bioaerosol generation occurs and may result in longer distance transport due to lower particle density and ability of smaller particles to travel (Milner 2009). From the obtained results it was observed that there is a tendency for wood shavings litter to present higher fungi loads and wood shavings with rice hulls litter to have lower loads. Kotimaa and colleagues (1991) found straw and wood shavings presented the worst sources of mold dust. These varying results from new litter fungal load contamination may be attributed to two principal factors: storage conditions (Kotimaa et al. 1991), and fungal contamination of the original environment such as sawmills (Crook and Olenchock 1995; Demers et al. 1997; Dennekamp et al. 1999; Tatken 1987). With respect to aged litter, many factors influence fungal contamination, including animal activity, air temperature, relative humidity, ventilation rate, animal stocking density, animal mass, type of bird, bird age, type of feed, feeding method, time of day, air distribution, relative locations of dust sources, and presence or absence of air cleaning technologies (Whyte 2010; Ellen et al. 2000). These factors may provide a basis as to why correlation between total CFU/g and litter age was not found, but rather a weak negative correlation was noted. It seems that other variables exert on litter fungal load a higher influence than age. CONCLUSIONS Data in this study highlight the need for monitoring fungal contamination in new and aged litter. Litter fungal quantification and species identification play important roles in the evaluation of potential health risks to exposed workers and animals. Spreading of poultry litter in agricultural fields is a potential public health concern, since keratinophilic (Scopulariopsis and Fusarium genera) as well as toxigenic fungi (Aspergillus, Fusarium and Penicillium genera) were detected. Litter is dry and prone to generate particles spreading all the potential pathogenic fungi to nearby rural agglomerates, consequently producing adverse human health effects. REFERENCES Ajello, L Natural habitats of the fungi that cause pulmonary mycoses..medical Mycology, Zbl. Bkt Suppl. 8, edited by J. H. Preusser, New York, NY: Gustav Fisher Verlag. American Industrial Hygiene Association Field guide for the determination of biological contaminants in environmental samples. Fairfax, VA: AIHA. Anbu, P., Hilda, A., and Gopinath, S Keratinophilic fungi of poultry farm and feather dumping soil in Tamil Nadu, India. Mycopathologia 158: Araújo, A., Sousa, M., Bastos, O., and Oliveira, J Onicomicoses por fungos emergentes: Análise clínica, diagnóstico laboratorial e revisão. Ann. Bras. Dermatol. 78: Bondy, G. S., and Pestka, J Immunomodulation by fungal toxins. J. Toxicol. Environ. Health B 3: Borch, E., and Arinder, P Bacteriological safety issues in red meat and ready meat products, as well as control measures. Meat Sci. 62: Bosch, F., and Munoz, N Prospects for epidemiological studies on hepatocellular cancer as a model for assessing viral and chemical interactions. IARC Sci. Publications 89: Bush, R., Portnoy, J., Saxon, A., Terr, A., and Wood, R The medical effects of mold exposure. J. Allergy Clin. Immunol. 17: Buttner, M., and Stetzenbach, L Monitoring airborne fungal spores in an experimental indoor environment to evaluate sampling methods and the effects of human activity on air sampling. Appl. Environ. Microbiol. 59: Buys, E., Nortjé, G., Jooste, P., and Von, A Bacterial population associated

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