Mycological examination of poultry feed with special reference to Aflatoxin and Ochratoxin interaction in poultry farms in El- Behaira Province

   Moulds are capable of reducing the nutritional value of feed stuff as well as elaborating several mycotoxins. Mycotoxin-contaminated feed has adverse effects on animal health and productivity. Also, mycotoxins may be carried over into meat and eggs when poultry are fed with contaminated feed. In a point prevalence study of feed stuff used for poultry nutrition in El Behaira were analyzed for fungal flora, natural incidence of Aflatoxin  and Ochratoxin.

   the mycological examination of 100 poultry feed samples revealed that Penicillium, Fusarium, Alternaria ,Aspergillus Flavus,Aspergillus Ochraceus ,Aspergillus Candidus,Aspergillus Niger, and Mucor were the dominant mould groups with a percentages 40, 23, 36, 30, 33, 8, 40, 30 % respectively.

    Samples were examined for the detection of total aflatoxin (TA) and ochratoxin A (OTA). Overall incidence of TA was recorded as 80% (n=80/100). The OTA was detected in  83% (n=83/1oo). However the co-occurance of TA and OTA was 63% (n=63/100).

 (2/80) samples (2.5%) contain TA  above the permissible limit (20ppb) while OTA was (34/83) samples (41%) at a rate of (6-10) ppb and (3) samples (3.6%) above (10ppb) above the acceptable limit. all samples collected in summer were contained aflatoxin and ochratoxin (100%), and aflatoxin was at low level below the permissible limit  while ochratoxin at high level 30 samples above 5 ppb.

 On  the  other  hand,  in  winter  season  comparatively  lower incidence  and  levels  of  aflatoxins  than that detected  in summer season in which 30/50 samples (60%) were contained aflatoxin at low level while 33/50 samples (66%) contained ochratoxin with 5 samples at high level above the permissible limit.

This study indicates the need for continuous assessment of the mycological and mycotoxins status of poultry feed, in order to feed poultry for optimal performance ensuring food safety and to ensure safe poultry for human consumption.

Keywords  Aflatoxin, Ochratoxin, Poultry feed,Mould

       Mycotoxins are often found as natural contaminants in raw ingredients of poultry feed (Khan et al. 2011). Poultry are highly susceptible to myco-toxicoses caused by aflatoxins AFand ochratoxins (OTA) (Anjum et al. 2011). The AF and OT are the major components of secondary metabolites group produced by several toxigenic fungi        

     Aflatoxicosis results in severe economic loss in the poultry industry that affecting ducklings, broilers, layers, turkeys and quails (CAST 2003). In poultry, AF impairs most of the important production parameters including weight gain, feed intake, feed conversion efficiency, processing yield, egg production, and male and female  reproductive performance (Hussain et al.2010). As a common rule, poultry should not get more than 20 μg/kg TA in the feed.                                                                                        

    Aflatoxin contamination in feed induces immune-supression in poultry, thus the birds become more susceptible to several diseases (Dhanasekaran et al. 2009).

     Toxigenic Aspergillus flavus isolates generally    produces aflatoxins B1 and B2, whereas A. parasiticus produces aflatoxins B1, B2, G1 and G2 (Davis and Diener 1983). The major hosts of A. flavus among food and feed commodities are cereal grains, peanut, cotton seed and protein sources such as rapeseed meal, cotton seed meal, soyabeen meal, sunflower meal, corn gluten meal, copra meal, and palm kernel meal (Anjum et al. 2012).

    Ochratoxins are another group of mycotoxins that are produced by several species of Aspergillus and Penicillium. these include certain members of Aspergillus ochraceus group and Penicillium verrucosum type I and II (Reverberi et al. 2010). The OTA is constituted of three compounds; these are Ochratoxin A, B or C. Among these three, ochratoxin A (OTA) is the most harmful one.

   Ochratoxins are considered as powerful  nephrotoxins, carcinogens, teratogens, and immune-toxins in rats, humans and likely in poultry (Frisvad 1995 Romani et al. 2000 Bozzo et al. 2008).

     Ochratoxin is highly stable during feed storage and feed preparation procedures, thus it may endanger humans as the final consumers of contaminated food of plant or animal origin. Among the feed of animal origin, poultry is frequently consumed (Schiavone et al. 2008). Poultry diets are based on cereals and cereal by-products up to 50-60% on a dry matter basis, and these raw materials are the preferred substrates for Penicillium and Aspergillus growth (Petzinger and Weindenbach 2002). 

   Different feed ingredients that are used in poultry feeds are likely to be contaminated with AF and OT producing fungi due to suitable environments for fungal growth like improper harvesting and storage, unhygienic method of processing and production, poor methodology of consumption and utilization. Therefor regular monitoring of AF and OT in poultry feeds is an important precondition to check toxins build up in poultry feeds.

Most toxogenic species belong to the genera Aspergillus,Penicillium,Fusarium, and Alternaria (Pitt and Hocking 2009). According to several authors mycotoxins such as aflatoxins, zearalenone, T2-toxin, deoxynivalenol, ochratoxin A, fumonisins, and patulin can be considered the most common mycotoxins found in feed and food ( Hussein and Brasel 2001 Firdous 2003).

Most mycotoxicosis cases in poultry are caused by an intake of low concentration of contaminants over a long time that lead to typical chronic symptoms of poor growth, poor feed efficiency, and suboptimal production. however ingestion of higher concentration leads to acute clinical symptoms associated with specific vital organs, the immune system, and other aspects of avian physiology as well as mortality (Mabbett 2004).

For quality control, the identification of the contaminating fungi is essential because it provides data on the potential production of  its mycotoxins and is a helpful indicator to determine feed hygienic quality ( Khosravi et al. 2008).

The aim of the present study was to determine the AF and OT contents in poultry feed  in El-Behaira Province. Samples were collected from poultry farms and samples were  recived in Animal Health Research Institute Damanhour branch.The study includes isolation and identification of mould genera and species, natural levels of aflatoxin,  ochratoxin  and their interaction.

2. Materials and Methods

2.1. Samples

  A total of 100 representative samples (1-2 kg per sample) of poultry ration (starter and grewer) were collected from poultry farms in El- Behaira, then homogenized and divided to obtain a 1 kg of working sample for analysis. Each sample was ground in a laboratory mill and divided into two parts, one part for mycological examination and the other for mycotoxin determination. . The investigation  period was carried out during June 2020 - May 2021.

  For mycological examination feed samples were immediately analyzed upon arrival or they were stored for 2-3 days in paper bags at room temperature (about 25°C). Feed samples intended for mycotoxin analysis were examined immediately. . Sampling procedure followed the principles of the Romer® Guide (Richard 2000)

  Information including date of sample collection, type of feed or ingredients, place, name of feed mill or farm, date of manufacture or purchase of feed, batch number, brand name, number of feed bags out of which sample collected, sample weight, storage conditions, number of birds at poultry farm, breed, age and mortality of flock were recorded.

                                                      isolation and identification of mould from feed samples.

according to (Cruickshank et al. 1975 and Koburger 1970)          

                                                                                                                                                                                                   Ten grams of each milled feed sample was mixed with 90 mL of 0.1% peptone water and      

shaken on a horizontal shaker for 20 minute each sample was cultured on the surface of Sabourad Dextrose Agar (SDA).The culture grown media were incubated at 25 °C for 3- 5 days. Colonies were sub-cultured on freshly prepared SDA media to obtain pure cultures. Pure cultures were also maintained in SDA slants at 5±1 °C. Mould and yeast were identified by their colonial morphology and microscopic characteristics .The fungal isolates were mounted in lacto phenol cotton blue stain solution on slides with cover slips and microscopically examined for spores and vegetative bodies according to the method described by Barnet and Hunter (1972).                                                                              

  The identification was based on colonial features, pigment production and the micro morphology of the spores produced. Cultures were examined at 4 or 5 days intervals from the onset. Some characteristics were also noted on the texture, colour, shape and the production of pigment on the underside. Fungi were identified at genus level according to Samson et al. (2004)  Pitt and Hocking (2009)  Nelson et al. (1983) and Simmons (2009).

2.3. Detection of mycotoxins residues.

 Total aflatoxin and ochratoxin  were detected in  poultry  feed, using immuno-affinity method ( Scot and Trucksess 1997). Series-4 Fluorometer (VICAM) was used in this procedure .This method depend on Sample extraction by using  methanol (80%) and                                      Column chromatography    by passing the filtered extract through AflaTest-p affinity column. Reading of total aflatoxin was obtained after 60 second as part per billion (ppb).

2.4 Detection of aflatoxin and ochratoxin producing ability of Aspergillus flavus and Aspergillus ochraceus isolates.

Aflatoxin and ochratoxin were produced by growing A.flavus and A.ochraceus on sterile polished maize by the method of Shotwell et al. (1966) in which 5 isolated strains of A.Flavus and A. Ochraceaus were growing on potato dextrose agar for 7-10 days at 28°C ,the cultures had a heavy crop of green conidia  was removed  and a uniform suspention of the spores were inculated in each flask containing 50 gm of ground maize and 25 ml of sterile distilled water) which previously autoclaved and placed on shaker and incubated at 28 c for 7- 10 days, it is important that the maize does not remain as a compact mass . then the the mouldy maize was autoclaved to kill the fungus, dried and ground to fine powder  and mycotoxin was detected by Ser,ies-4 Fluorometer (VICAM).   

Table (2):Incidence of aflatoxin and ochratoxin in examined poultry feed.

Table (3):Level of aflatoxin and ochratoxin in examined poultry feed.

            Table(4):Incidence of samples positive for Aflatoxin and/or Ochratoxin but negative         for Aspergillus spp. Isolation.

  Table(5): Toxigenicity of Aspergillus flavus isolates.                                                                        

                                  Table(6): Toxigenicity of Aspergillus Ochraceus isolates.                      

Table(7):Incidence of aflatoxin and ochratoxin in examined samples in summer and winter season.

   The mycotoxins are natural contaminants of poultry feed ingredients as the  most efficient condition of culture,  harvest, storage  and  handling  are  used.  The  prevalence  of  these

toxins  in  feed  samples  varies  depending  on  geographical location and seasons of the year.

    On the other  hand,  A.flavus  and  A.ochraceus  were found to constitute  a  public  health  hazard  due  to  production  of aflatoxins and ochratoxins that cause some degree  of acute toxicity when consumed  in high  amounts and are potential carcinogen. ( FDA 2000). (Bahtnager and Ehrlich2002).The  significant losses to the poultry industry occur  due to  effects of  ochratoxin  A on  performance  and health.  It  causes  a  reduction  in  growth  rate  and  feed consumption,  poorer  feed  conversion  and increased mortality  ( Saleemi et al. 2010) . 

   Fungal contamination of animal feed, with the consequent mycotoxin production  is one of the major threats to human and animal health (Castillo et al. 2004),  For quality control the identification of the contaminating fungi is essential because it provides data on the potential production of its mycotoxins and is a helpful indicator to determine feed hygienic quality (Khosravi et al. 2008).

   In this work,  most tested samples were contaminated with fungi and mycotoxins. table (1) show theincidence of Aspergillus spp in which A.Flavus ,A. ochraceus,A candidus, and A. niger were isolated and their frequencies of occurrence were 30%,33%,8% and40% respectively.  This was in agreement with (Petzinger and Weindenbach 2002) who  reported that Poultry diets are based on cereals and cereal by-products upto 50-60% on a dry matter basis and these raw materials are the preferred substrates for Penicillium and Aspergillus growth. The toxicogenic fungal contamination of the raw materials occurs during the pre-harvest and/or the post-harvest periods and the finished feeds are exposed during production, processing, transportation, and storage .It has been observed that moulds and mycotoxins presence varies depending on the geographical location and the year. Temperature and humidity play important roles not only in the development of fungi but also in mycotoxin production (Saleemi et al. 2010).

  Other four mould genera were isolated  Penicillium, Fusarium Mucor, and Alternaria and their frequencies of occurrence were 40%, 23%,30% and 36% respectively. (Hassan and Omran 1996)  isolated various molds including  A.flavus  and A. ochraceus  from  yellow  corn  and  mixed  feed  during summer  season,  where  high  moisture  content  and  high temperature existed and they detected   significant  levels of OA in samples. While

( Nooh et al. 2014)  recovered many types of fungi such as Aspergillus and Penicillium from Egyptian maize at field and earlier storage.

   Table (2)show the incidence of total aflatoxin (TA) and ochratoxin (OTA)  in poultry feed in which out of (100) samples were examined (80)samples (80%) were positive for (TA) ,(83)samples (83%) were positive for (OTA), (17) samples (17%) were positive for (TA) only and (20) samples (20%) positive for (OTA) only and (63) samples (63%) were positive for both aflatoxin and ochratoxin . Co-occurrence of mycotoxins was determined in about 63% of tested samplesThis agree with ( Binder  et al. 2007) . who reported that concurrent exposure to multiple mycotoxins is more likely in feed stuff. The combined action of mycotoxins can generate an interactive effect such as additivity, synergism, or antagonism. The synergistic interaction causes the most toxic effects in the case of aflatoxins and OTA. Synergistic effects in poultry were observed in the case of aflatoxins and OTA. This interaction lead to reduction of body weight and increased mortality (Constanzo and Murphy 2012) .

  Table (3) show the incidence of aflatoxin in examined feed in which 76  samples (95%) at rate of (1-10)ppb. 2  samples (2.5%) at a rate of (10-20) ppb. and  2 samples (2.5%) above the permissible limit (20ppb). The high incidence and low values of TA were observed in examined feed samples  . This low incidence of aflatoxin could be due to selection of good quality graded samples (Beg et al. 2006).

Table (3) show the incidence of ochratoxin in examined feed in which 46 samples (55.4%) at rate of (1-5)ppb, 34 samples (41%) at a rate of (6-10) ppb and 3 samples (3.6%) above (10ppb),in this study  the examined samples have high incidence and levels of ochratoxin (OTA) that reach up to the permissible limit. (Akinmusire et al. 2019).

    In Table (4) 50 samples (62.5%) out of 80 samples produced TA at a rate of (1-10)ppb but we could not isolate A. flavus from the samples also 50 samples(62.2%)out of 83 samples contained OTA but A. ochraceus could not be isolated from examined samples ,40 samples (80%) produced ochratoxin at a rate of (1-5)ppb and 10 samples (20%) produced ochratoxin at a rate of (6-10)ppb. these results may be due to that mycotoxins were synthesized by organisms  when metabolically active in moist stored feed in sealed and unsealed silos and the species which synthesized these toxins may have been killed by  heat treatment during the preparation of the feed or may be feed ingredients treated with antifungal additives.

    In table (5) the results of screening of isolated strains of A. flavus for aflatoxins production revealed that 3 isolates out of 5 tested isolates were found to be aflatoxin producers at a rate of above the permissible limit and 2 isolates produced aflatoxin at a rate below the permissible limit but all are toxins producers. The permissible limits are ranged between 15 ppb. (WHO 1975)and 20 ppb ( FAO 1995 and FDA 2000).

 Also the results of screening of isolated strains of A. ochrceaus for ochratoxin production revealed that 4 isolates out of 5 tested isolates were found to be ochratoxin producers at a rate of above the permissible limit and 1 isolates produced ochratoxin at a rate below the permissible limit but all are toxins producers In table (6)    The same  findings  were detected by (  Hassan et al. 2012  and El-Hamaky   et al. 2016).

     In this study, about 50% of the samples were collected in winter season and the other 50 samples were collected in summer. the results revealed that all samples collected insummer were contained aflatoxin and ochratoxin (100%), and aflatoxin was at low level below the permissible limit  while ochratoxin at high level 30 samples above 5 ppb.  On  the  other  hand,  in  winter  season  comparatively  lower incidence  and  levels  of  aflatoxins  than that detected  in summer season in which 30 samples (60%) were contained aflatoxin at low level while 33 samples (66%) contained ochratoxin with 5  samples at high level above the permissible limit( table 7). ( Abidin et al. 2013   Saleemullah et al. 2006 and Anjum et al. 2012) studied the effect of storage conditions on TA production and found a positive relationship between TA level and moisture content. Interactions between available water and temperature are the most important two factors in which fungi be able to germinate, grow and produced  toxins these results also agree with  Marín et al. ( 2012)  and  Nooh et al. (2014). detected incidence rates of OTA in feeds at winter season were lower than that detected in summer season, Where, the OTA residues were detected in all examined samples of  white corn, concentrated feeds and silage  (100%), during summer season. Whereas, 83.3%, 50%, 33.3% and 72% of examined yellow corn, wheat bran, barseem hay and poultry feed samples were contaminated with OTA, respectively.

 In this study, high humid environment and elevated temperature during June to August were conducive for the growth of fungi producing mycotoxins (Sabri et al. 1989). The high incidence of significant  levels of aflatoxins and ochratoxin A  in feed at  summer season rather than winter season warrants  that  there is urgent  need  to  under take  mycotoxins  awareness  creation programs among  different  foods and feed stuffs  in Egypt to secure  the  bad  environmental  condition  that  enhanced mycotoxins  production  in  feed  and  food. 

  In this work, although the levels of the Aflatoxin detected on poultry feed was lower than the regulation limits established, while the ochratoxin detected with high incidence and level and the co-occurrence of them was demonstrated. The scientific literature offers a broad spectrum of information on the effects of individual mycotoxins in various animal species but concurrent exposure to multiple mycotoxins is more likely in feed stuff. while mycotoxins have attracted worldwide attention due to their direct impact on human health due to food contamination . it is also relevant to consider to what extent mycotoxins can be carried over into edible tissues like meat and eggs when poultry are fed with contaminated feed.

Conclusion

   Higher incidence and low  level of TA are detected in poultry feeds On the other hand, OTA contamination is present at high incidence and high levels in poultry feed. However, this situation demands for immediate necessary control measures. Adequate post-harvest drying should be done, and proper storage condition should be maintained. Finally, strict regulations and surveillance programs for testing food and feed for aflatoxin and ochratoxin contamination are highly recommended to improve the health status of the consumers

We suggest that periodic monitoring and the application of the Hazard Analysis and Critical Control Points (HACCP) on the prevention and control of mycotoxins in the animal feed industry are needed in order to guarantee the hygienic quality of feed to ensure health and productivity of poultry as well as prevent human food borne diseases.