Loading [MathJax]/extensions/MathML/content-mathml.js
info@auctorespublishing.com
+1-(302)-520-2644
+1-(302)-520-2644

Molecular Detection of Aflatoxin Producing Aspergillus Species Isolates in Some Chicken Meat Cuts In Gharbiya Governorate, Egypt

  1. Home
  2. Journals
  3. Nutrition and Food Processing
  4. Archives
Submit Guidelines

Review Article | DOI: https://doi.org/10.31579/2637-8914/076

Molecular Detection of Aflatoxin Producing Aspergillus Species Isolates in Some Chicken Meat Cuts In Gharbiya Governorate, Egypt

  • Shaltout F. A 1
  • Heikal G. I 2
  • Ghanem A. M 3*

1 Food Hygiene and Control Dept. (Meat Hygiene), Faculty of Veterinary Medicine, Benha University, Egypt
2 Food Hygiene Dept., Animal Health Research Institute (Tanta branch), ARC, Egypt
3 Food Security Sector, Egyptian Armed Forces, Egypt.

*Corresponding Author: Ayman Ghanem, Food Security Sector, Egyptian Armed Forces, Egypt

Citation: Shaltout F. A, Heikal G. I and Ghanem A. M. (2022). Molecular detection of aflatoxin producing Aspergillus species isolates in some chicken meat cuts in Gharbiya governorate, Egypt: A Rare Research Report. Nutrition and Food Processing. 5(1); DOI: 10.31579/2637-8914/076

Copyright: © 2022 Ayman Ghanem. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Received: 20 November 2021 | Accepted: 22 December 2021 | Published: 10 January 2022

Keywords: aspergillus species; chicken meat cuts; cpcr; egypt

Abstract

Contamination with fungi and their toxins is considered one of the most dangerous hidden pollutants that threaten the health of the consumer. The presence of mycotoxins in various foods has been recorded, despite their apparent safety for human consumption. Therefore, the current study was conducted to evaluate the prevalence of Aspergillus species by culture method; and aflatoxin producing genes molecularly in total of 75 random samples of chicken cuts represented by wing, breast and thigh (25 of each) that were collected from various groceries and poultries shops located at Gharbiya governorate, Egypt. Results of culture and isolation techniques revealed detection of Aspergillus sp. in 36, 48 and 44% of the examined wing, breast and thigh samples, respectively. Moreover, microbiological identification of the isolated strains showed presence of A. niger, A. flavus, A. fumigatus, A. terreus and A. parasiticus in 16, 13.3, 10.6, 1.3 and 1.3% of the total population of the examined samples. Molecular detection of some aflatoxin production regulating genes (OmtA, Nor1 and Ver1) in ten Aspergillus sp. isolates revealed their detection in 8/10 (80%), 8/10 (80%) and 7/10 (70%) represented by positive bands at molecular weight of 1024, 400 and 537 bp, respectively. Referring to the recorded results, chicken cuts may possess a great silent hazard to the human-being under improper good manufacturing practices and inadequate hygienic conditions during handling and storage.

Introduction

Chicken meat and meat products production in developing countries plays an essential role in supporting food security and poultry meat demands (Wong et al., 2017).

Contamination of meat products with molds can be occur during different preparation stages during slaughtering under bad hygienic conditions using contaminated water or by adding contaminated spices with mold spores or during packing, handling, transportation and storage (Khalalfalla et al., 2017). 

Contamination of meat with Aspergillus species, especially Flavi section, is one of the most hazardous microbial contamination as the majority of Aspergillus species are able to aflatoxins production that can cause diseases associated with aflatoxin poisoning and carcinogenic effects (Leggieriet al., 2021).

Acute aflatoxin poisoning may lead to death as was recorded in Kenya in 2004 (Probst et al., 2007), while chronic poisoning may lead to various recorded mutagens and cancers (Benkerroum, 2020). 

Aspergillus sp. was classified into two groups depending on their toxigenic impacts on food and human health; 1st group includes the aflatoxigenic species such as A. flavus and A. parasiticus, while the 2nd group contains the non-aflatoxin-producing species such as A. tamarii and A. oryzae (Frisvad et al., 2019).

Molecular analyses have been used to confirm aflatoxin productivity of Aspergillus species isolates. omtA, nor1 and ver1 genes are from the commonly used genes encoded aflP, aflD and aflM toxins detection in food items (Sohrabi and Taghizadeh, 2018) yield an accurate, rapid and reliable records of toxigenic aspergillus species especially in food chain (Sadhasivam et al., 2017). 

Therefore, the main target of the current study was to investigate the presence of toxigenic aspergillus species in some chicken meat cuts collected from Gharbiya Governorate markets, Egypt.

Material and Methods

Collection of samples

A total of seventy-five random samples of raw chilled chicken wing, chicken thigh and chicken breast (25 of each) were collected from different local poultries shops and different supermarkets at Gharbia governorate, Egypt. Samples were taken aseptically in polyethylene bags and were transferred to the laboratory in ice box for mycological examination.

Preparation of samples (ISO, 2017)

Twenty-five grams from each sample were carefully and aseptically homogenized in blinder after mixing with 225 ml of sterile peptone water 0.1% to form a dilution of 1:10, from which tenth fold serial dilutions were prepared. 

Determination of Aspergillus species 

culture of the prepared samples was performed according to ISO (2008), where 0.1ml of the previously prepared serial dilutions was spreaded by mean of sterile L-shape glass rod over two Petri-dishes contained solidified Dichloran Rose Bengal agar with chloramphenicol (DRBC) then were incubated at upright position at 25Oc for 5 -7 days.

Identification of isolated strains was performed according to pitt and hocking (2009) macroscopically and microscopically as recorded in Table (1).

Molecular Detection Of Some Aflatoxin Producing Genes Of Some Isolated Aspergillus Strains By Cpcr

Oligonucleotide primers used in cpcr

Three pairs of omtA, nor1 and ver1 primers were prepared and collected from Metabion (Germany). Their special sequence and amplify certain products as were be displayed in Table (2). 

Mycological DNA was extracted following QIAamp DNeasy Plant Mini kit Catalogue no. 69104

Preparation of master mix and thermal profile was adapted according to the manufacturer instructions (Emerald Amp GT PCR mastermix (Takara) Code No. RR310A).

Table 1: Morphological character of Aspergillus species (Pitt and Hocking, 2009)
Table 2: Oligonucleotide primers sequences

Results

As recorded in Table (3); Aspergillus sp. was detected in 32(42.6%) of the total examined samples. In detail, breast samples recorded the highest contamination level (48%); followed by thigh and wing samples, respectively.

Table 3: Prevalence of Aspergillus species in the examined chicken meat cut samples (n= 25 of each).

%* prevalence in relation to the number of each sample (25)

%** prevalence in relation to the total number of samples population (75).

Regarding with the genus identification, A. niger had the highest detection levels (16%) in the examined samples (Table, 4). 

Table 4: Prevalence of identified aspergillus sp. in the examined chicken meat cuts (n= 25 of each)

Referring to the obtained results of molecular detection of some aflatoxin producing genes as recorded in Table (5) and Figs (1 to 3); omtA, nor1 and ver1 genes were detected in 8/10 (80%), 8/10 (80%) and 7/10 (70%) of the examined A. flavus isolates, respectively. presence of these genes indicated the producibility of the examined strain for aflatoxins P, D and M, respectively. 

Table 5: Prevalence of aflatoxin producing genes in A. flavus isolates from the examined samples (n= 10)
Figure 1: Agarose gel electrophoresis of cPCR of omtA (1024 bp) gene of A. flavus.

Lane L: 100 bp ladder as molecular size DNA marker.

Lane P.: Control positive A. flavus for omtA gene.

Lane N.: Control negative.

Lanes 1, 3, 4, 5, 6, 7, 9 and 10: Positive A. flavus for omtA gene.

Lanes 2 and 8: Negative A. flavus for omtA gene.

Figure 2: Agarose gel electrophoresis of cPCR of nor1 (400 bp) gene of A. flavus.

Lane L: 100 bp ladder as molecular size DNA marker.

Lane P.: Control positive A. flavus for nor1 gene.

Lane N.: Control negative.

Lanes 1, 3, 4, 5, 6, 7, 9 and 10: Positive A. flavus for nor1 gene.

Lanes 2 and 8: Negative A. flavus for nor1 gene.

Figure 3: Agarose gel electrophoresis of cPCR of ver1 (400 bp) gene of A. flavus.

Lane L: 100 bp ladder as molecular size DNA marker.

Lane P.: Control positive A. flavus for ver1 gene.

Lane N.: Control negative.

Lanes 1, 3, 4, 5, 6, 9 and 10: Positive A. flavus for ver1 gene.

Lanes 2, 7 and 8: Negative A. flavus for ver1 gene.

Discussion

Chicken meat and meat products comply an important source of human protein supplement all over the world because they provide good source of digestible protein, low cholesterol fat, essential amino acids, minerals, and different types of vitamins and minerals.

In Egypt, as well as human population increasing, demand of animal proteins also is increasing represents a serious challenge in which poultry industry plays an essential role in filling nutrition gap as a rapid and more economic source of proteins (Shaltout et al., 2015).

Mold contamination of meat and meat products have been considered a serious source of food spoilage resulting in different organoleptic changes in flavor, color, texture, odor referred mainly to the fungal deterioration especially in poor developing countries due to lack of hygienic measures during processing and handling (Lorenzo et al., 2018). 

Presence of mold in foods may be referred to the rapid, easy disperse and wide spread of the fungal spores which are abundant in the environment introducing food chain through dust, water, workers and equipment. Their presence in food samples is a serious public health concern as these fungi may be associated with the production of mycotoxins (Benedictet al., 2016).

Aspergillus species represents an important mycotic infection in public health concern as a human pathogen and as toxin-producing food contaminant. It releases a lot of spores which found in air, water, soil, plant debris, manure and animal feed. As fungal spore's growing, it secretes digestive enzyme and mycotoxins leading to food spoilage and human mycotoxicosis (Richardson and Rautemaa-Richardson, 2019).

Referring to the recorded results in Table (3), Aspergillus sp. was prominently detected in breast samples other than wings and thighs samples, which came in agree with the previously recorded results of Darwish et al. (2016) and Shaltout et al. (2019) who found that the examined breast samples were more contaminated with fungal infection than wing and thigh samples. While the current prevalence of aspergillus species in the examined samples came lower than those recorded by Hassan (2019) who found Aspergillus sp. in all the examined samples (100%) collected from Gharbiya governorate, Egypt. Moreover, Abuzaid et al. (2020) also detected A. flavus and A. niger in 40 and 80% of the examined sausage samples of chicken origin, respectively. 

Referring to the obtained results of the microbiological identification of Aspergillus sp. isolates as recorded in Table (4), they came in agree with the previously reported results by Darwish et al. (2016) who found that A. niger was the predominant detected strain, followed by A. flavus and A. parasiticus in the examined samples of chicken cuts collected from Zagazig city, Egypt.

Some mold species can cause respiratory infections representing a significant risk for individual with severely weakened immune system (OSHA, 2010). Presence of   mold in high incidence indicate bad hygienic measures adopted during handling, preparation and processing (El-Abbasy, 2007).

Mycotoxins are have been defined as naturally occurring secondary fungal metabolites produced in meat and meat products by direct growth of toxigenic molds such as Aspergillus species which produce Aflatoxins and Ochratoxins which threat public health due to their carcinogenic, hepatotoxic, nephrotoxic, teratogenic and mutagenic effects in human and animals (Agriopoulou et al., 2020).

Aflatoxins are produced by a polyketide pathway that pass through about twenty-seven enzymatic reactions which have been regulated by sets of genes including nor-1, ver-1 and omtA have been shown to be involved in this process. aflD (nor-1) encodes a norsolorinic acid ketoreductase needed for the conversion of the 10-keto group of Norsolorinic Acid (NOR) to the 10-hydroxyl group of Versicolorin A (VERA) (Zhou and Linz, 1999). aflM (ver-1), predicted to encode a ketoreductase, is involved in the conversion of VERA into Sterigmatocystin (ST) (Henry and Townsend, 2005); aflP (omtA) codes for O-methyltransferase, which is one of the main genes responsible for transforming ST into O-methylsterigmatocystin (OMST) that is the precursor for aflatoxin production (Yabe  et al., 1989).

Many other previous studies recorded detection of these genes in their Aspergillus isolates of food origin by various PCR techniques; Manonmani et al. (2005), Rodrigues et al. (2009), and Hassan et al. (2015), who conducted several studies investigating the aflatoxigenicity of Aspergillus sp., could detect different genes in their Aspergillus isolates.

Conclusion

It could be concluded that, breast samples revealed the highest contamination levels with Aspergillus sp.; in addition, A. niger was the prominently detected strain. PCR technique is a unique diagnostic tool for detection and identification of aflatoxigenic Aspergillus strains especially if the field of food safety. So, application of strict hygienic measures, proper use of water supply and food additives from good sources is recommended.

References

  1. Abuzaid, K.E, Shaltout F.A, Salem R and El-Diasty, E.M. (2020). Microbial aspect of some processed meat products with special reference to aflatoxins. Benha Vet. Med. 39; 24-28.c
    View at Publisher | View at Google Scholar
  2. Agriopoulou S, Stamatelopoulou E and Varzakas T. (2020). Advances in occurrence, importance, and mycotoxin control strategies: prevention and detoxification in Foods. Foods (Basel, Switzerland). 9(2); 137
    View at Publisher | View at Google Scholar
  3. Benedict, K, Chiller T. M and Mody, R. K. (2016). Invasive fungal infections acquired from contaminated food or nutritional supplements: A review of the literature. Foodborne Pathogens and Disease. 13(7); 343–349.
    View at Publisher | View at Google Scholar
  4. Benkerroum, N. (2020). Chronic and acute toxicities of aflatoxins: mechanisms of action. Inter. J. Environ. Res. Pub. Hlth. 17(2); 423.
    View at Publisher | View at Google Scholar
  5. Darwish W.S, El Bayomi R.M, Abd El-Moaty, A.M and Gad T.M. (2016). Mould contamination and aflatoxin residues in frozen chicken meat-cuts and giblets. Jap. J. Vet. Res. 64(2); 167-171.
    View at Publisher | View at Google Scholar
  6. El-Abbasy T.M. (2007). Mycological aspects of quail carcasses with a trial to improve their sanitary status. Thesis, Master of Vet. Med. (Meat Hygiene), Zagazig Univ, Egypt.
    View at Publisher | View at Google Scholar
  7. Frisvad J.C Hubka, V Ezekiel, C.N Hong S. B, Nováková A, Chen A.J, Arzanlou M, Larsen T.O, Sklenář F, Mahakarnchanakul W, Samson R.A and Houbraken, J. (2019). Taxonomy of Aspergillus section Flavi and their production of aflatoxins, ochratoxins and other mycotoxins. Studies in Mycology. 93; 1-63.
    View at Publisher | View at Google Scholar
  8. Hassan M.N, El-Sayed A.A and Nada H.M. (2015). Detection of aflatoxins by HPLC and the expression of biosynthetic nor-1 gene of aflatoxin and ocrA gene of ochratoxin. Benha Vet. Med. J. 29(2); 1-10.
    View at Publisher | View at Google Scholar
  9. Hassan, W. (2019). Some studies on effect of turmeric and lemon extracts on fungal contamination of chicken meat. Thesis, Master of Vet. Med. (Meat Hygiene), Benha Univ, Egypt.
    View at Publisher | View at Google Scholar
  10. Henry K.M. and Townsend C.A. (2005). Ordering the reductive and cytochrome P450 oxidative steps in demethylsterigmatocystin formation yields general insights into the biosynthesis of aflatoxin and related fungal metabolites. J. Am. Chem. Soc. 127; 3724-3733.
    View at Publisher | View at Google Scholar
  11. ISO. (2008). International Organization for Standardization. No.21527-1. Microbiology of food and animal feeding stuffs — Horizontal method for the enumeration of yeasts and moulds — Part 1: Colony count technique in products with water activity greater than 0, 95.
    View at Publisher | View at Google Scholar
  12. ISO. (2017). International Organization for Standardization. No.6887-1. Microbiology of the food chain — Preparation of test samples, initial suspension and decimal dilutions for microbiological examination — Part 1: General rules for the preparation of the initial suspension and decimal dilutions.
    View at Publisher | View at Google Scholar
  13. Khalalfalla F.A, Ali,F.H.M and Saif-Alnasr, M.M. (2017). Microbiological quality of retail meats. J. Vet. Med. Res. 24(2); 311-321.
    View at Publisher | View at Google Scholar
  14. Leggieri M.C, Toscano P and Battilani P. (2021). Predicted aflatoxin B1 increase in Europe due to climate change: actions and reactions at global level. Toxins. 13(4); 292.
    View at Publisher | View at Google Scholar
  15. Lorenzo J.M, Munekata P. E, Dominguez R, Pateiro M, Saraiva J.A and Franco, D. (2018). Main groups of microorganisms of relevance for food safety and stability: general aspects and overall description. Innov. Technol. Food Preserv. 53-107.
    View at Publisher | View at Google Scholar
  16. Manonmani H.K, Anand S, Chandrashekar A and Rati, E.R. (2005). Detection of aflatoxigenic fungi in selected food commodities by PCR. Process Biochemistry 40. 2859-2864.
    View at Publisher | View at Google Scholar
  17. Norlia M, Jinap S, Nor-Khaizura M.A.R, Radu S, Samsudin N.I.P. and Azri, F.A. (2019). Aspergillus section Flavi and aflatoxins: occurrence, detection, and identification in raw peanuts and peanut-based products along the supply chain. Front. Microbiol.10; 2602.
    View at Publisher | View at Google Scholar
  18. OSHA
    View at Publisher | View at Google Scholar
  19. Pitt, J.I. and Hocking, A.D. (2009). Fungi and Food Spoilage. 2nd Ed., Springer, Boston, MA. Ch. 4; 19.
    View at Publisher | View at Google Scholar
  20. Probst C, Njapau H and Cotty, P.J. (2007). Outbreak of an acute aflatoxicosis in Kenya in 2004: identification of the causal agent. Appl. Environ. Microbiol. 73(8); 2762-2764.
    View at Publisher | View at Google Scholar
  21. Richardson M and Rautemaa-Richardson, R. (2019). Exposure to Aspergillus in home and healthcare facilities' water environments: focus on biofilms. Microorganisms. 7(1);7-12.
    View at Publisher | View at Google Scholar
  22. Rodrigues P, Venencio A, Kozakiewicz Z and Lima N. (2009). A polyphasic approach to the identification of aflatoxigenic and non-aflatoxigenic strains of Aspergillus section Flavi isolated from Portuguese almonds. Inter. J. Food Microbiol. 129; 187-193.
    View at Publisher | View at Google Scholar
  23. Sadhasivam S, Britzi M, Zakin V, Kostyukovsky M, Trostanetsky A, Quinn E and Sionov E. (2017). Rapid detection and identification of mycotoxigenic fungi and mycotoxins in stored wheat grain. Toxins. 9(10); 302-310.
    View at Publisher | View at Google Scholar
  24. Shaltout F.A, Nasief M.Z, Lotfy L.M and Gamil, B.T. (2019). Microbiological status of chicken cuts and its products. Benha Vet. Med. J., 37: 57-63.
    View at Publisher | View at Google Scholar
  25. Shaltout F.A, Zakaria I.M, Eltanani J. and Elmelegy, A.S. (2015). Microbiological status of meat and chicken received to University student hostel. Benha Vet. Med. J. 29(2): 187-192.
    View at Publisher | View at Google Scholar
  26. Sohrabi N and Taghizadeh M. (2018). Molecular identification of aflatoxigenic Aspergillus species in feedstuff samples. Curr. Med. Mycol. 4(2); 1-6. doi: 10.18502/cmm.4.2.66. PMID: 30324150; PMCID: PMC6181065.
    View at Publisher | View at Google Scholar
  27. Wong J.T, Bruyn J.D, Bagnol B, Grieve H, Li M, Pym R and Alders, R.G. (2017). Small-scale poultry and food security in resource-poor settings: A review. Glob. Food Secur. 15; 43-52.
    View at Publisher | View at Google Scholar
  28. Yabe K, Ando Y, Hashimoto J, Hamasaki T. (1989). Two distinct O-methyltransferases in Aflatoxin biosynthesis. Appl. Environ. Microbiol. 55; 2172-2177.
    View at Publisher | View at Google Scholar
  29. Zhou, R. and Linz, J.E. (1999). Enzymatic function of the Nor-1 protein in aflatoxin biosynthesis in Aspergillus parasiticus. Appl. Environ. Microbiol. 65; 5639-5641
    View at Publisher | View at Google Scholar
a