Burden of Aatoxin in Poultry Feeds in Selected Chicken Rearing Villages of Bishoftu-ethiopia

Background: Aatoxins are major contaminants of feed used in poultry industry that negatively affect animal and human health. In Ethiopia, previous studies on aatoxins mainly considered cattle feed and milk, but scarce information exists for poultry feeds. Method: The aim of this study was to determine the burden of aatoxin in poultry feed in bishoftu.Cross sectional study was conducted from December, 2018 to May, 2019and 33 compound poultry feed samples were randomly collected from chicken rearing villages of Bishoftuand analyzed for G2, G1, B2 , B1 and total aatoxins using HPLC. Results: The result indicated thatfrom a total of 33 samples 31(94%) samples were contaminated with aatoxin. The mean level of aatoxin G2, G1, B2, B1 and total aatoxinswere 18.00 µg/g, 88.5499 µg/g, 13.50µg/g, 70.11µg/g and 190.18µg/g respectively. This study curtained the level of aatoxinin 25 (72.75%) samples for AFT and 22 (66.67%) samples for AFB1 were above the limit of FDA regulatory levels of 20µg/g for poultry feed. Conclusion: The study showed the high contamination of aatoxins in poultry feed. The study warrants the need for preventive strategies of aatoxin contamination including implementation of regulatory legislation in poultry feeds in Bishoftu.


Introduction
A atoxins, a class of mycotoxins produced mainly by A.parasiticus and A. avus, are major contaminants of common feed ingredients used in poultry rations (Smith et al., 1995). There are four major naturally produced a atoxins known as B1, B2, G1, and G2. A atoxin B1 is the most potent toxin of the group and cause liver cancer (Bennett, 2003). Contamination by A atoxin can take place at any point along the food or feed chain under a wide range of climatic conditions (Giray et al., 2007).
In developing countries including Ethiopia, poultry industry offers an opportunity to feed the rising human population and to provide income for farmers (Tadelle et al., 2007). However the emergence of a atoxin poses negative impact in poultry industry (Bakırdere et al., 2012). When a atoxin contaminated feed is consumed by chickens, physiological changes such as weight gain, feed intake, feed conversion e ciency, and reproductive performance are compromised (Denli et al., 2009). A atoxin in poultry also causes changes in biochemical and hematological parameters which enhance susceptibility to diseases (Pravin et al. 2017).
A atoxins are not only dangerous for health, but a atoxins also deteriorate the marketing quality of contaminated products; thus, involving strong economic losses (Patchimaporn et al., 2017;Shashidhara et al. 2003). This includes reduced egg quality, egg production, mortality of chickens, increased veterinary treatment costs and challenges in disposal of contaminated feeds and feed ingredients ( Ethiopia is favorable for the growth of a atoxicogenic fungi and hence a atoxin contamination of grains as supported by different studies (Ayalew A. 2010;Alemayehu et al., 2014). Studies also showed that liver diseases are increasing in Ethiopia and cause the death of many individuals. Most probably a atoxin contamination is the common agent for this disease among others. A atoxin contamination of animal feeds, livestock and animal products are also increasing in the country (Amare et al., 2006).Globally, a atoxin in poultry fed have been reported by different scholars (Ezekiel et al., 2010;Streit et al., 2013, Njobeh et al., 2012,but data on a atoxin levels in poultry feeds remains scarce in Ethiopia; however contaminated food of animal origin are a major source of exposure to the human (Rádulyet al. 2020).Regular monitoring is crucial for ensuring the safety of poultry feeds. In addition awareness of farmers about the risk factors that aggravate contamination of animal feed by a atoxin in Ethiopia is very poor (Mulugeta, 2017, Nakavuma et al., 2020.Therefore the current study investigated the burden of a atoxin levels on poultry feed samples from households of chicken rearing villages of Bishoftu, practicing small-scale private commercial chicken production.

Materials And Methods
Description of the study area The study was conducted in Bishoftu, Oromia Regional State which is found about 47 km south east of Addis Ababa, the capital city of Ethiopia. Bishoftu is located at 8º 43'-8º 45 Based on the secondary data obtained from Bishoftu municipal o ce there were a total of 36 Small to medium scale poultry farm micro-enterprises actively functioning in 2019. Therefore, this number is so relatively small and the sampling fraction exceeds 10%, the nite population correction factor (FPC) was used to calculate the required sample size (n') as follow: (Daniel, 1999) n' = 1/(1/n+1/N) Where, n= the original estimate of the required sample size in an in nite population and it is calculated as follow: Where, Z α =1.96,p = expected contamination (p=50%=0.5), q=1-p=0.5, L 2 =5% = 0.05, 1.96 (CI = 95%) hence, n=384 and N=36= number of poultry farm micro-enterprises actively functioning in 2019 in Bishoftu town. Thus, the desired sample size was n'=33 Sample collection and sampling Poultry feed samples were collected randomly from ve vilages based on the accessibility of small scale poultry farms and sampling. They include Air force (n=3), Kajima (n=7), Hora (n=8), Cheleleka (n=8) and Babogaya (n=8). Since a atoxins occur in heterogeneous fashion in feed, strati ed random sampling was used to make a composite sample consisting of subsamples from every part of a store, sack, or unit of feeds. During sampling the feeds were grouped into 4 parts depending on the layers and height of the bags. Then choose a simple random sample from each group and 100g of sample was weighed and zipped with plastic bag.

Analytical Procedure
The a atoxin content in the feeds and feed ingredients was determined at the veterinary drug and animal feed administration and control authority-Ethiopia, following the manufacturer's instructions. All chemicals and reagents were of analytical grade and HPLC standard. (Sigma-aldrich, St. Louis, MO, USA). The working solutions of a atoxins B1, B2, G1 and G2 standards (50 µg/g of each a atoxin in capped amber bottles) were prepared according to the AOAC procedure (AOAC, 2000) Instrumentation: Quantitative analyses of the a atoxins were carried out using a HPLC unit that consisted of a pump and quaternary gradient system. Fluorescence detector was used for the quantitation under the following conditions: 360 nm excitation and 440 nm emission. The analytical column was a ZORBAX SB-C18, 150 x 4.6, 3.5μm particle size. All HPLC analysis were carried out under isocratic conditions using a mobile phase of water: acetonitrile: methanol (60:25:15) and the ow rate was xed at 1.0 ml/ min. The injection volume was 20 μl and it was standard injection with needle wash. Stop time for quaternary pump was 10 min and the temperature of the column was 35°C on both sides (Shimadzu, USA) Sample Preparation: Sample preparation was conducted using the method of AOAC O cial method 950.02 for animal feed. To achieve the maximum particle size reduction and thoroughness of the mix the entire lots of samples were ground through hammer mill and passed through number 14 sieve split sample sequentially in sample splitter. The coarse portions were regrind and Weigh 50gm from each sample for a atoxin estimation.
Extraction: About 20 g of grounded sample was added into a beaker with 2.0 g of NaCl. Then extraction was done with 100 ml methanol/water (4/1, v/v) and 50 mL of n-hexane in a blender jar at high speed for ve minutes. The extract was then passed through a plaited lter. Fourteen milliliter of the puri ed extract was added to 86 mL PBS buffer (pH 7.2). Finally the evenly mixed sample was ltered using a Buchner funnel and a lter paper.
Clean-up: The A aCLEAN TM immunoa nity column was used for cleanup purposes. After opening the column, the storage buffer was drained until the level reaches the upper frit. The sample was passed through a 0.2 µm syringe lter to remove residual turbidity. Then 25 mL of the diluted extract was taken and passed through the A aCLEAN TM column with a ow rate of 2 mL/min (1 drop/sec). Then the sample was allowed to drain through the column until there is no more sample in the column. The column was then washed with 10 mL of distilled water and the residual water removed from the column by applying a gentle pressure of vacuum pump. Finally the elution was done using 1ml of methanol at least 2 times. The rst addition of methanol was let for 5 minutes in order the methanol act on the column to break the a atoxin-antibody bond.

Statistical analysis
For data analysis, Microsoft Excel 2013 and IBM SPSS Statistics version 20 software were used. One-way analysis of variance (ANOVA) was performed to evaluate the levels of total a atoxin mean comparison between the study villages. A P-value of less than 0.05 (P < 0.05) was considered as statistical signi cance.

Results
A total of 33 compound poultry feed samples were analyzed to quantify A atoxin using HPLC. Accordingly the result revealed that, 31(94%) samples were contaminated with a atoxin G2 with the range 0 to 221.43 µg/g, G1: 0 to 921.43µg/g, B2: 0 to142.98 µg/g, B1: 0.667 to 633.94 µg/g and AFT 0.893 to 1919.79µg/g. Moisture content ranges from 7.33 to 11.17 with mean of 9.19 (Table-1). The mean of a atoxin G2, G1, B2, B1 and AFT in the samples from all villages were 17.93µg/g, 91.37µg/g, 13.79µg/g, 72.77µg/g and 195.88µg/g respectively. As summarized in table 2 individual a atoxins as well as total a atoxin concentrations in samples from Air Force and Hora sites are higher than the rest study villages. The ANOVA results revealed that, there was no signi cant mean differences in a atoxin contamination of compound poultry feed among the ve study sites (Air force, Cheleleka, Kajima, Babogaya and Hora) at level of signi cance 0.05.There was no signi cant mean difference in total a atoxin as well as individual a atoxin among the feed samples in the study sites (Table 3.) Overall, the high levels of a atoxin contamination in feed should be of concern for the poultry sector, as a atoxin can have seriously impact on poultry industry (Ortatatli and Oguz, 2001). In addition meat and eggs consumers have suffered from complications arising from the use of infected poultry products. Finally, society as a nal customer must pay exorbitant costs of increased regulation and research, low exports and high imports and treatments (Umaya, 2011).

Conclusion
This study highlights the occurrence of aftoxins in poultry samples from Bishoftu-Ethiopia, where many small to medium -scale private commercial chicken farms exist due to presence of large-scale intensive private commercial farms and agro industries that work on manufacturing of feeds and vend day-old chicks. The survey indicates that 25 (72.75%) samples for AFT and 22 (66.67%) samples for AFB1 were above the limit of FDA regulatory levels of 20µg/g for poultry feed. In these regard feed manufacturers should ensure the ingredients and nished feed are to the expected quality more over farmers in home should consider proper storage of feeds. Therefore the study warrants the need for preventive strategies of a atoxin contamination in poultry feed and recommended involvement of regulatory bodies in the poultry industry in town and increase knowledge of poultry farmers on proper practices of feed handling.