Maintaining Food Safety Standards Regarding Mycotics in the Poultry Industry

1. Maintaining Food Safety Standards Regarding Mycotics in the Poultry Industry Muhammad Kashif Saleemi DVM PhD Assistant Professor Department of Pathology, University of Agriculture, Faisalabad, Pakistan

2. Fungal contamination results in • Downgrading of commodities (Anderson and Thrane, 2006) • Production of mycotoxins Toxigenic fungi Produce a variety of mycotoxins (Aziz-Baumgartner et al., 2004)

3. Mycotoxins in poultry feeds • 2nd most important issue next to feed cost (Smith, 2009) • Mycotoxin (AFs & OTA) contaminated feeds are big risk to animal health • Contaminated feed may result in residues in foods obtained from the animals like milk, eggs, meat(Bennett & Klich 2003; Hussain et al., 2010)

4. Conditions Favorable for Fungal Growth i. Relative humidity over 70%. ii. Temperatures over 26 °C for a period of a few days to weeks. iii. High moisture content of crop (>16%). Other factors • Stress to the affected plant, such as drought, flood, or insect infestation. (Lasramet al., 2009)

5. Fungal Mycobiota of Poultry Feeds/ingredients Fungi (Filamentous Micromycetes) are Ubiquitous in environment Inevitable contaminants of agricultural products Specific parasites for particular commodities Specific fungi (toxigenic)produce specific toxins

6. Important Fungal Species of Agricultural Products Aspergillus Penicillium Fusarium Alternaria(Pitt and Hocking, 1997)

7. Aspergillus Flavi Section (Chelkowski et al., 1991) A. flavus A. parasiticus A. nomius Toxins: Aflatoxins, CPA Circumdati Section(Samson et al., 2004) A. ochraceus A. sclerotium A. alliaceus Toxins: Ochratoxins, CPA etc.

8. Nigri Section (Black Aspergilli)(Bau et al ., 2005) A. niger A. carbonarious A. tubigensis Toxins: Ochratoxins Penicillium(Magnoli et al ., 2003; Saleemi et al., 2012.)P. verrucosum P. crysogenum P. puberlum Toxins: Ochratoxins, Aflatoxins, CPA etc

9. Fusarium(Park et al., 2005) F. verticillioides F. proliferatum F. culmorum F. graminearum Toxins: Fumonisins, DON, DAS, T-2, ZEN Alternaria A. alternria Toxins: Alterneriol

10. Aspergillosis(Brooder Pneumonia/ Mycotic Pneumonia) Aspergillus fumigatus penetrate egg shells under ideal growth conditions and infect embryos Infected embryos green in color during candling (embryo will die), Infected embryos may each with well developed lesions (early chick mortality) Infected eggs if break in hatcher large number of spores will contaminate whole environment and resulted in outbreak in young chicks In Ovo vaccination is important predisposing factor for Aspergillosis due to puncture of egg shell surface Adult infections usually follow inhalation of large number of spores from heavily contaminated feed, litter or environment.

11. Aspergillosis(Brooder Pneumonia/ Mycotic Pneumonia) Dyspnoea, gasping, cyanosis and accelerated laboured breathing is observed Morbidity is less but mortality is high in clinically infected birds A grey white opacity may develop in one or both eyes when there is eye infection Mycelial growth with sporulation green or black material on air sacs, bronchi, lungs and pericardium Pale yellow or gray nodules in the lungs, air sacs or trachea some times these nodules may contain puss.

12. Aspergillosis: Clinical Signs and Lesions

13. European Union Mycotoxin Legislation • Maximum levels for Aflatoxins in Feeds • Feeds Maximum AFB1 (ng/g) • All feed materials 20 • Complete feeds: • Beef cattle, sheep, goat 20 • Dairy animals 5 • Calves and lambs 10 • Pig and Poultry 20

14. Biomin Recent Survey on Mycotoxin Contamination 2017

16. Mycotoxins: Different Types Mycotoxins: Secondary metabolites of toxigenic fungi • More than 300-400 mycotoxins presently identified, with new isolation techniques are used. • Aflatoxin B1 Hepato-toxic, carcinogenic • Ochratoxin A Nephrotoxic carcinogenic • Fumonisins Teratogenic Carcinogenic • Deoxynivalenol Gastroenteritis, • Zerealenone Hyperestrogenism • T2-Toxin Gastric ulcers/Enteritis

17. MYCOTOXINS: Aflatoxins(AF) AFB1 AFB2 AFG1 and AFG2 AFM1: Metabolite of AFB1 present in Milk Major Aflatoxin Producing Fungi Aspergillus flavus: AFB1 and AFB2 Aspergillus parasiticus: B1, B2, G1 and G2 AFB1/AFM1 possible human carcinogen (IARC,2012)

18. OCHRATOXINS: Human Exposure Ochratoxins has three OTA, OTB and OTC Mainly produced by Aspergillus ochraceous OTA is the most important nephrotoxic mycotoxin In Balkan region of Europe, nephropathy is associated with ingestion of OT contaminated food both in human population and domestic animals (Pfohl-Leszkowicz et al., 2007). From Karachi Pakistan a human physician described higher levels of OTA in the blood of persons suffering from urinary bladder disease (Aslam et al., 2006) .

19. FUMONISINS: Human Exposure Fumonisins: produced mainly by Fusarium Fungi Fusariumverticillioidesand F. proliferatum. Fumonisin B1 (FB1) (generally ~70% of the total fumonisin contamination), Fumonisins (FB2) and Fumonisins (FB3) Major contamination occur on maize, maize based products, sorghum etc. (Bulderet al., 2012). Level ranges from 2-36 µg/kg /daily in African countries &15-23 µg/kg bw/daily in Latin American countries (Kimanyaet al., 2014; Torres et al., 2014)

20. Brief Finding of Previous Research Work on Mycotoxins in Poultry First Established Research Group working on mycotoxins in Poultry and livestock since 2006 in Department of Pathology University of Agriculture Faisalabad Pakistan Seven PhD and Thirty M.Phil students completed research on mycotoxins Two Funded Research projects and third in funding process (2006-2015) Avian Toxicologic Pathology Laboratory

21. Toxigenic Fungi–Mycotoxins-Pathology

22. Teratogenic and Carcinogenic Effects of Mycotoxins in Poultry

23. Histopathology of different tissues of poultry induced by mycotoxins

24. Mycotoxins entering the food chain Infection of plants/crops by moulds Entrance of Fungi and Mycotoxins in Food Chain Contaminated feed Toxin in the blood and organs Contaminated food of animal origin Contaminated food of plant origin Milk Three important segments environment, animals and humans.

25. Prevention/Regulation of mycotoxins in foods/feeds • Good agricultural practices i. Early harvesting ii. Proper drying iii. Physical treatment iv. Sanitation v. Proper storage vi. Insect management • Biological control • Chemical control • Decontamination • Breeding for resistance • Effective Legislation • Surveillance and awareness creation(Zain, 2010)

27. AFLATOXINS: Human Exposure Maximum acceptable levels recommended by European Commission in 2010 are; AFB1 (2 µg/kg) and AF (4 µg/kg) for cereals and nuts for direct human consumption) In Kenya (2004) cereals, AF ranges (1–46400 µg/kg), with 7% of samples above 1000 µg/kg (Lewis et al., 2005) AFM1 detected in milk and urine of exposed animals and humans Human breast milk AFM1 carryover from animal (0.1-0.4%) in developing countries (Shephard, 2004; Magohaet al., 2014) Residues of AFB1in poultry meat exposed to mycotoxins contaminated feed (Hussain et al ., 2007; Khan et al., 2012)

28. Aflatoxins: Role in HCC with Biological Vectors • In Guangxi Zhuang PR China, Yehet al.(1989) reported interaction between HBV infection and dietary aflatoxin exposure had a 10-fold higher incidence of HCC • A population-attributable benefit of 65% for reduced primary liver cancer mortality was estimated from a switch of the dietary staple from maize to rice (Chen et al., 2013) • Human exposure to aflatoxins on a global scale have been reviewed that 4.5 billion persons living in developing countries are chronically exposed to largely uncontrolled amounts of the toxin (Jonathan et al., 2004)

29. Situation in Pakistan A variety of acute and chronic hepatic diseases are prevalent in Pakistani population and it can be assumed that these might be associated with the AFB1, OTA and FB1 contamination of foods In Pakistani population cases of HCC are increasing every year and at present it is being considered that only biological vectors (HCV &HBV) are responsible for this lethal disease Pakistani climate predominantly hot and humid strongly favor the proliferation of toxigenic fungi and production of mycotoxins particularly AFB1, OTA and FB1

30. Situation in Pakistan-------------- Aflatoxin M1 has been reported in dairy milk (Muhammad et al., 2011). Aflatoxins residues in poultry meat has been detected at market age birds (Hussain et al., 2010, Khan et al., 2013). Qazi and Fayyaz (2006) speculated that Aflatoxin contaminated foods might be a significant risk factor for Hepatocellular carcinoma Qureshi et al. (1990) postulated AF as an etiological factor of hepatocellular carcinoma in Karachi region

31. Relationship of Mycotoxin Contaminated Foods with Hepatocellular Carcinoma Hypothesis There is an association between AFB1 levels and the development of hepatocellular carcinoma in patients suffering from viral hepatitis.

32. Specific project aims To determine mycotoxins levels in the blood/serum of hepatocellular carcinoma patient To determine mutation on tumor suppressor p53 gene from blood/serum of HCC patients as well as hepatitis B and C status. To find out the role of aflatoxins in the development of HCC.

33. Host Institute (Part of Fellowship) College of Public Health Sciences (CPHS)Chulalongkorn University Bangkok Thailand (October 2016-January 2017) Laboratory Training and Samples Analysis in Host Institute (Biomolecular Laboratory)

34. RESULTS

35. Mutations in p53 gene after restriction analysis M 2 3 4 5 254bp 250bp 92bp No mutation Wild type 100bp 66bp No mutation Wild type 50bp Lane 1: Marker 50bp Lane 2-4: samples Lane 5: Negative control (Un-cut)

36. Results: Liver Function Tests (Mean ± SD) Values in each column followed different small letters are statistically different P ≤ 0.05.

37. Results and Discussion: Mutation in p53 gene No AFB1 specific mutation at codon 249-50 of exon 7 was detected in these samples collected from Pakistan In current study only few specific primers were used and only one mutation was addressed Samples collected during this study were only 150 sand from specific area However it was observed that in all those samples having AFB1 present ALT, AST and GGT levels were high The results of current study may be considered as preliminary data and it may not be applied community wide.

38. Results: AFB1 Levels by HPLC-FD The aflatoxins B1 was detected in 51(34 %) serum samples out of 150 samples The levels of AFB1 were varied from 0.006 to 1.053 µg/L. The AFB1 was also detected from 5 samples of healthy individuals and its level was 0.002 to 0.034 µg/L. According to FAO AFB1 levels should be less than 0.05 µg/L this maximum acceptable level

39. During Visit to CPHS-CU Bangkok

40. Thanks to Organizers of 1st International Collaborative Mycology Conference

43. Standardization of Laboratory Techniques for Amplification of p53 tumor suppressor gene Standardization of protocols in the Biomolecular Laboratory CPHS Chulalongkorn University DNA Extraction from Human WBCs from already present samples in the laboratory PCR analysis of DNA samples to amplify the required gene tp53 Successfully amplified the p53 gene from these samples Also learnt DNA extraction from filter paper dried blood spots, it was new and interesting for me.

44. Amplification of p53 tumor suppressor gene from different samples of human WBCs and Project Samples DNA extraction and amplification of p53 gene A total of 200 µl of blood was be used for genomic DNA extraction using QIAamp DNA Mini Kit (Cat # 51104) DNA samples were quantified through Nano drop in Pakistan and reconfirmed in CPHS lab DNA amplification of p53 gene by PCR analysis using following primers Primer 1 (up) (5’-CTT GCC ACA GGT CTC CCC AA-3’), Primer 2 (down) (5’-AGG GGT CAG CGG CAA GCA GA-3’). PCR products of 254-bp amplified and obtained on 4% agarose

45. Restriction analysis of p53 gene for mutations As mentioned in my project hypothesis AFB1 induced mutations in p53 tumor suppressor gene at codon 249-250 exon 7 For this mutation detection restriction analysis was performed with amplified p53 gene. Restriction cut through restriction enzyme and these samples were put for 16 hours at 37oC in water bath In case of mutations there must be band size of 153bp, if no mutations wild type samples product size must be small fragments of 66 and 96bp

46. Restriction site Hae III (BsuRI) Restriction Cut Reaction PCR product: 5 µl Buffer: 2 µl Restriction enzyme: 0.2 µl ddH2O: 12.8 µl Total volume: 20.0 µl

47. Aflatoxin B1 detection from serum samples: HPLC The second part of my project was analysis of human serum samples for possible Aflatoxin B1 levels. Whole analysis was performed on High Pressure Liquid Chromatography with florescent detection (HPLC-FD) available in Department of Pathology Laboratory. The samples were already collected and stored in the refrigerator at -20 oC, were extracted by immunoaffinity columns available for AFB1 extraction from serum samples. Conditions: For the analysis of AFB1, each sample was derivatized prior to injection (Anonymous, 2000). A mixture of acetonitrile: methanol: water (22.5:22.5:55) was used as mobile phase with the flow rate of 1.0 mL/min, temperature of column oven was 30 oC. The emission and excitation wavelength were 360 and 440 nm.