[Frontiers in Bioscience E3, 74-80, January 1, 2011]
Detection of airborne trichothecene-producing Fusarium species in chicken houses
Yaling Wang1, 2, 3, Tongjie Chai2, Guozhong Lu3, Lijun Sun1, Yi Ouyang3, Xiaodong Sun3
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TABLE OF CONTENTS
1. ABSTRACT
One hundred and forty three airborne Fusarium isolates in chicken houses belonging to seven Fusarium species were analyzed by PCR with Tri5 gene as a specific marker of mycotoxin product . The result of Tri5 gene sequence analysis indicates that the PCR amplification products were 89%-96% identical to the previously reported Tri5 genes, which were all amplified from four F. poae isolates. T-2 toxin and DON was measured by immunoaffinity column and high performance liquid chromatography in Tri5-positive F. poae isolates after being cultured at constant and alternating temperatures. The production of T-2 toxin under alternating temperatures was 14 and 53 times higher than those at constant temperature of 8�C and 25�C. No DON was detected under either testing temperature condition. It is concluded that T-2 toxin-producing F. poae isolates were present in poultry houses, and the concentration of T-2 toxin produced by Tri5-positive F. poae isolates was increased under alternating temperatures. The application of Tri5-PCR associated with IMC-HPLC is an effective and accurate method for rapid detection of T-2 and DON mycotoxins.
2. INTRODUCTION
The fungal species of the genus Fusarium are common molds in animal farm environments (1). The species of F. avenaceum (Fr.) Sacc., F. graminearum Schwabe and F. poae (Peck) Wollenw. have been considered to be the most important trichothecene-producing molds. Trichothecenes are a family of sesquiterpenoid mycotoxins. They are usually divided into four types (A-D) according to their characteristic functional groups (2). The epidemiological surveys have revealed that A and B of trichothecenes are widely distributed in cereals and feed as natural pollutants (2), however the macrocyclic trichothecenes rarely occur in food and feed (2). Deoxynivalenol (DON) and T-2 toxin are examples of the most common type-B and type-A trichothecenes, respectively. They are harmful to both human and animal health by being absorbed through the gastrointestinal tract or skin, resulting in alimentary toxic aleucia, articular cartilage degeneration, skin allergies, diarrhea, cardiac and nerve system damage (3, 4). Moreover, inhalation, ingestion and skin contact with mycotoxins and mycotoxin-producing fungi may cause infections, allergies and inflammatory reactions for immunocompromised human and animals (5, 6). Inhalation of toxigenic fungi such as aflatoxin-producing Aspergillus Micheli ex Link and especially trichothecene-producing Fusarium mold may result in more serious damage due to continuous mycotoxin production in vivo (7, 8). In a warm and humid environment, the risk of animal or human exposure to mycotoxins is more severe due to favorable environmental conditions (9, 10).
Traditionally, mycotoxin-producing fungi were detected by a complex process including fungal isolation and identification, fungal liquid growth and mycotoxin extraction and analysis. In recent years, more and more studies focused on the analysis of trichothecene genes, and demonstrated that Tri5 genes of trichothecene-producing species of Fusarium are conserved (11). The first step of trichothecene synthesis is catalysis by trichodiene synthase, which catalyzes farnesyl pyrophosphate isomerization and cyclition. This gene is clustered with the other 10 genes involved in mycotoxin synthesis, including Tri5 (trichodiene synthase), Tri4 and Tri11 (oxygenases), Tri3 (acetyltransferase), Tri6 (a zinc finger transcription factor), and Tri12 (trichodiene efflux pump). These genes are clustered at the Tri5 locus. Some of these genes (Tri3, Tri4, Tri5, Tri6) have been used in the detection of mycotoxins (eg.T-2 toxin) produced by F. sporotrichiodes Sherbakoff (3). Several other putative protein-coding genes (Tri7, Tri8, Tri9, Tri10) have also been found at the Tri5 gene locus (12). Within this gene cluster, the functional changes of the genes involved in trichothecene biosynthesis gene cluster may lead to the production of different toxins (13).
The Tri5-PCR method has been applied for the detection and mycotoxin analysis of Fusarium species in grains (14). It has also been used to distinguish between toxin and non-toxin producing Fusarium species (3). T-2 toxin produced by Fusarium species could cause chicken's performance reduction, immune function decline, and blood biochemical changes (15). The task of quick identification of T-2 toxin-producing Fusarium species was always considered to be a key to the control of the harmful effects of molds on chickens and workers. The current study was aimed at applying Tri5-PCR technique to partially amplify the trichodiene gene from the genomic DNA of airborne Fusarium species, to screen for mycotoxin-producing Fusarium isolates and to develop a reliable method for the accurate detection of mycotoxin-producing Fusarium isolates. The toxin products of Tri5-PCR positive isolates and the toxin-producing conditions were further investigated. This study was also performed to determine airborne mycotoxin-producing Fusarium levels in order to control the potential exposure risks to mycotoxins in chicken houses.
3. MATERIALS AND METHODS
3.1. Sample Collection
Samples of airborne fungi were collected in December 2006 from the chicken houses of a poultry farm in Dalian, Liaoning province, China. The air samples were collected by using an Andersen-6 six stage air sampler at an air flow rate of 28.3 L/min, 50 cm above ground, and 2 - 4 min per sample.
3.2. Fungal culture and identification
Fungi were cultured on Rose Bengal Chloromycetin (RBC) medium in an illumination incubator for 3-7 days at 25�C. Fungal colonies were counted according to their morphological characteristics under an Olympus stereo microscope. Pure cultures were isolated and confirmed by transferring conidia to standard Petri dishes containing Potato Dextrose Agar (PDA) and cultured for 3-7 d at 25�C. Pure cultures were preserved by vacuum freezing and drying method. Fusarium strains were cultured on PDA, Saccharose Nirenberg Agar (SNA), and Potato Sucrose Agar (PSA) for accurate identification. The Fusarium species were morphologically identified based on their colonies and reproductive structures according to Leslie and Summerell (16), Gerlach and Nirenberg (17), Nelson (18), Joffe (19) and Booth (20).
3.3. DNA extraction
The Fusarium isolates were grown at 25° C for 6-7 days in three 1.5 mL Eppendorf tubes, each containing 0.5 ml liquid glucose yeast medium (GYM, which consisted of: NH4H2PO4, 1.0g; KCl, 0.2g; MgSO4�7H2O, 0.2 g; glucose, 10.0 g; yeast extract, 5.0 g; CuSO4 solution which consisted of 0.005 g CuSO4�5H2O per litre, 1ml; ZnSO4 solution which consisted of 0.01 g ZnSO4�7H2O per liter, 1 ml; Distilled water, 1000 ml). The genomic DNA was extracted as described by Cenis (21) and the concentration was determined by electrophoresis in 1% agarose gel, staining with ethidium bromide and imaging with a GDS 8000 (UVP, US) transilluminator.
3.4. Tri5 gene amplification
Genomic DNA was quantified by 2% agarose gel electrophoresis. The total reaction volume of the PCR reaction was 25 �L, containing 1� PCR buffer (1.5 mM MgCl2), 0.4 �M each primer HATri/F (5'-CAGATGGAGAACTGGATGGT) and HATri/R (5'-GCACAAGTGCCACGTGAC), 1 U Taq DNA polymerase (TAKARA Biotechnology Dalian Co. Ltd.), 50 �M of each dNTP, and 50 ng template DNA. The PCR was conducted with an initial denaturation step of 94�C for 75 sec, followed by 35 cycles of 94�C for 15 sec, 62�C for 15 sec, and 72�C for 45 sec, with a final extension step of 72�C for 4 min 15 sec.
3.5. Nucleotide sequencing
DNA sequencing was performed with the primers HATri/F and HATri/R in separate reactions using the Applied Biosystems Prism BigDye terminator v3.1 kit. Sequencing of the Tri5 gene fragment was conducted by an Applied ABI PRISM 3730 DNA Sequencer (Shanghai Sangon Biotech Inc). Forward and reverse sequences were aligned and the sequences of Tri5 gene fragment were determined, and then analyzed by BLAST in NCBI (22).
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3.6. Mycotoxin-producing culture of Tri5 positive isolates
The Tri5 positive isolates of F. poae (DF0416, DF0215, DF1040, DF0591) (Table 2) were inoculated in GYM (21) and respectively incubated at constant temperatures (8�C or 25�C) for 15 days, and at alternating temperatures of 8�C and 25�C at 12 h intervals. The Tri5 negative airborne strains were simultaneously inoculated as control.
3.7 DON and T-2 detection
The culture solutions of Tri5 positive and negative isolates in GYM were filtered through a 0.44 �m nitrocellulose filter. DON or T-2 toxin was isolated and purified by using a DON or a T-2 immunoaffinity column (VICAM Co. Ltd, USA), followed by quantification using an HPLC system (HP1100, Agilent Technology Inc) as described by Sui (23) and Li (24 ). The T-2 standard was purchased from Sigma (purity more than 99%).
4. RESULTS
4.1. Airborne Fusarium species
One hundred and forty three airborne Fusarium isolates were aquired from 108 airborne samples collected from poultry houses of a chicken farm in Dalian, China in 2006 (Table 1). Seven Fusarium species were morphologically identified, namely F. poae (Peck) Wollenweber (n = 4), F. graminearum Schwabe (n = 44), F. oxysporum Schlecht. ex Snyder et Hansen (n = 5), F. verticillioides (Sacc.) Nirenberg (n = 29), F. equiseti (Corda) Sacc. (n = 8), F. solani (Martius) Appel et Wollenw. ex Snyder et Hansen (n = 50) and F. nivale (Fr.) Ces. (n = 3). The highest occurrence rate was 34.97% for F. solani, and the lowest was 2.10% for F. nivale (Table 1). The genomic DNAs of 143 Fusarium isolates were successfully extracted.
4.2. Tri5 - PCR assay
The results of Tri5-PCR reaction for the 143 Fusarium isolates show that specific fragments of 260 bp (EF547372) were obtained from all four isolates of F. poae but not from the other Fusarium isolates (Figure 1), suggesting that all F. poae isolates were capable of producing trichothecene. Analysis of 50 BLAST hits of the partially amplified Tri5 gene from F. poae using the nucleotide-nucleotide searching option in GenBank indicates that the sequences of the amplified products had 89% to 96% similarity to the other Fusarium Tri5 genes (Table 2).
4.3. Detection of mycotoxin related to Tri5 gene
All isolates of F. poae were cultured to assay for the production of T-2 toxin and DON by the methods as described in the experimental procedure. The concentrations of T-2 toxin (1.751+/-0.003 μg/ml) produced by the isolates cultured at alternant temperature 8�C and 25�C were 14 and 53 times higher than those treated at constant temperature 8�C (0.121 +/- 0.017 μg/ml) and 25�C (0.033 +/- 0.002 μg/ml) (Table 3, Figure 2, Figure 3, Figure 4, Figure 5). None of the tested Tri5 negative cultures was found to produce T-2 toxin, and DON was not detected in any of the tested fungal cultures. These results suggest that the airborne F. poae isolates are substantial producers of T-2 toxin (1.751 +/- 0.003 �g/ml), and their T-2 toxin producing ability was significantly increased when exposed repeatedly to alternating high and low temperatures.
5. DISCUSSION
The toxicity of trichothecene susquiterpenoids is being increasingly recognized as important in agriculture and the health of immunocompromised human subjects. The traditional method of identifying and detecting environmental toxins produced by Fusarium spp. requires laborious procedures including laboratory culture, morphological examination, and fungal liquid culturing to assay for toxin production. These methods are time-consuming and often inaccurate. The screening of fungal isoaltes with trichodiene synthase encoding sequence by means of hybridization to Tri5 gene cloned from F. poae only reveals its genetic capability for trichothecene synthesization and gives no proof for the actual toxin production (11). Therefore, the technique of Tri5-PCR associated with immunoaffinity -HPLC analysis was applied in this study to assay for rapid determination of airborne trichothecene-producing Fusarium isolates for the detection of toxic metabolites (T-2 toxin and DON). The mass screening of fungal isolates for their genetic capability of trichothecene production could be conducted by the Tri5-PCR method.
The test results show that all the Tri5 positive F. poae isolates produced T-2 toxin but not DON. In contrast, the previous studies reported that the F. poae isolates from Norway and Poland were not T-2 toxin producers (25). The presence of toxigenic fungi depends on both extrinsic (environmental and geographic conditions) and intrinsic factors (constituents of matrix) (26). For instance, most Fusarium isolates from high latitude area (Northern US, Northern Canada, Norway, Russia, and Northern China) produced T-2 and HT-2 toxin, while the Fusarium isolates from lower latitude areas, e.g. Egypt and India hardly produced mycotoxin (27). Moreover, Fusarium isolates originating from different matrix, such as soil, feed/food and air, might not be equally toxigenic (28, 29, 30). The current investigation reveals the fact that the F. poae isolates in the air of poultry houses in Dalian, China (Northeastern China) were T-2 toxin-producers.
Our previous work showed that the levels of airborne mycotoxins were closedly related to the presence of toxigenic fungi (1). However, the relationship between airborne T-2 toxin or DON and the concentration of airborne Fusarium species was not investigated in the current study. The molecular mechanisms that regulate the production of T-2 toxin or DON are still not clear, but it is confirmed that the Tri5 gene-positive Fusarium isolates were potentially producers of tricothecene, especially T-2 toxin. It does not suggest that the presence of the T-2 toxin-producing Fusarium species in the air of chicken houses mean the presence of airborne T-2 toxin. On one hand, the occurrence of T-2 toxin-producing Fusarium isolates (4/143) in the air of chicken house was much lower than in the maize fields (13/40) (27). On the other hand, the level of T-2 toxin produced by Tri5 gene-positive Fusarium isolates was significantly influenced by environmental conditions, especially temperature. When Tri5 gene-positive F. poae isolates were cultured under an alternating temperature of 8�C and 25�C every 12 h, the T-2 toxin production level was much higher than at constant temperature (8�C or 25�C). All F. poae isolates cultured at 8�C or 25�C produced little toxin. The concentration of T-2 toxin produced by Tri5-positive F. poae could be increased by repeated high and low temperature alternation. These results indicate that the level of T-2 toxin in the chicken house may be very low because the chicken houses were usually kept at a relatively constant temperature. However, mycotoxins are normally considered to be primarily in the mycelia and spores of the toxigenic fungal strains (31, 32, 33, 34, 35). If airborne fungal spores are inhaled into the bronchi and alveoli, they will be lysed and the human body is thereby exposed to their primary and secondary metabolites (5). Inhalation of toxigenic fungi may result in more serious damage due to continuous mycotoxin production in vivo (36). "Non-specific" granuloma of the paranasal sinuses and orbit in Northern Sudanese was shown to be the result of infection by toxigenic Aspergillus flavus (7). Excretion of aflatoxin by frogs after implantation with toxigenic Aspergillus flavus suggests that A. flavus retains it aflatoxin-producing ability during simulated aspergillosis in frogs (8). Therefore, more attention should be provided to the T-2 toxin-producing Fusarium species found in the chicken house environment. The PCR technique could be used for fast identification of mycotoxin-producing Fusarium species and it would be helpful for developing strategies to avoid or reducing mycotoxin contamination of the chicken raising environment.
6. CONCLUSION
The current study is the first report of screening for toxigenic Fusarium isolates in the air of chicken houses by using Tri5-PCR coupled with IMC-HPLC. Furthermore, the explored method has never been used for detection of T-2 toxin-producing Fusarium isolates in chicken houses. This study takes advantage of PCR technique for the characterization of the toxigenic Fusarium species in the animal raising environment. The Tri5-PCR technique is faster and more accurate than traditional methods such as laborious subculture and microscopic observation of the airborne spores. The T-2 toxin-producing Fusarium isolates can be rapidly detected by Tri5-PCR combined with HPLC and T-2 toxin immunoaffinity column. As an effective and fast detection method, it could be widely applied in future investigations of airborne toxins and toxigenic fungi.
7. ACKNOWLEDGEMENTS
This study was financially supported by the Chinese International Cooperation Program (2009DFA32890) and the National Natural Science Foundation of China as projects No. 30571381 and No. 30771584.
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Key Words: Fusarium; trichothecene; T-2 toxin; Tri5; PCR
Send correspondence to: Tongjie Chai, College of Animal Science and Veterinary Medicine, Shandong Agricultural University, Daizong Str. No. 61, Taian 271018, Shandong Province, PR. China,Tel: 0086-538-8241503, Fax: 0086-538-8241503, E-mail:chaitj117@163.com