**2. Methodology**

### **2.1 Data collection**

Convenience sampling coupled with snowball sampling methods was used to identify farmers willing to participate in the research. Questionnaires were used to get information from the farmers. The information required from the farmers included the following: plot size in acres, number of cattle owned by the farmer, number of cows that were being milked, age, breed, lactation stage, milking method, volume of milk produced on the farm per day, volume of milk produced by each cow per day, number of milking per day, amount of feed given to each cow per day and also if the farmer had any knowledge on aflatoxins. A total of 14 farmers participated in this study with farm size of 8.5 hectares and above. Most of them were milking cows ranging between 20 and 250, and a few had less than 10 cows. The cows that were being milked were 25 months old and above, and the common breeds were the Jersey, Holstein and crossbreed (Holstein/Jersey) across all milking stages. Majority of the farmers were milking by hand getting a volume of 100 to over 200 litres per farm per day with each cow giving an average of 6–10 litres.

**39**

5 × 10<sup>−</sup><sup>5</sup>

, 5 × 10<sup>−</sup><sup>4</sup>

linearity will have an *r*

**2.4 Statistical analysis**

the standard curve.

Statistics 25.

equipped with a C18 4 × 3 mm2

, 5 × 10<sup>−</sup><sup>3</sup>

and 5 × 10<sup>−</sup><sup>2</sup>

curves was determined using correlation regression (*r*

*2*

*Aflatoxin Occurrence in Dairy Feeds: A Case of Bulawayo, Zimbabwe*

stored in the freezer at −20°C until time for analysis [22].

**2.3 Sample preparation for HPLC analysis**

A total of 96 feed samples which consisted of dairy feed concentrates (CN), mixed ration (MR), brewers spent grain (BSG) and grass (GR) were collected from 13 farms during the dry season (August–October 2016) and the rainy season (January–March 2017). Samples were collected in sterile polythene ziplock bags which were sealed and transported in cooler boxes to the laboratory where they were ground to a fine powder using IKA® M20 universal batch mill (Germany) and

Aflatoxins from feeds were extracted using the immunoaffinity extraction method

,

mg/ml. Aflatoxin detection and quantification

ID security guard cartridge (Phenomenex, Torrance,

*2*

value close to 1. Aflatoxin concentration of the samples

). A curve with good

[23] using Easi-Extract® aflatoxin immunoaffinity columns (R-Biopharm Rhone Limited, Glasgow G20 OXA, Scotland). Extraction was carried out according to the manufacturer's protocol with some modifications as follows: a portion of 50 g of the sample was mixed with 5 g of sodium chloride (NaCl) in a laboratory blender followed by 100 ml of methanol: water (80:20 v/v) and blended for 5 minutes. The mixture was filtered through a fluted filter paper (Whatman No.1) into a clean vessel. A volume of 2 ml of the filtrate was then diluted with 14 ml phosphate buffer saline (PBS) solution and passed through an immunoaffinity column. The column was washed with 20 ml of PBS and the aflatoxins finally eluted with 1 ml methanol (LiChrosolv®, Merck, Germany) into a glass cuvette and diluted with 1 ml of distilled water and then stored at −20°C prior to analysis. Aflatoxin B1, B2, G1 and G2 standards (Trilogy Analytical Laboratory, Washington, USA) were diluted using acetonitrile (LiChrosolv®, Merck, Germany) to give the following concentrations: 5 × 10<sup>−</sup><sup>6</sup>

were done using HPLC (Shimadzu FCV-20H2) with operation conditions as given in the KOBRA® cell instruction manual as follows: derivatisation using KOBRA ® cell at 100 μA setting, with an analytical column Inertsil ODS-3 V 5 μm, 4.6 × 150 mm

CA, USA). Mobile phase was modified from the recommended water: methanol (60:40) to a working condition of 55:45 with 119 mg/litre of potassium bromide (KBr) and 1 ml/litre of 65% nitric acid added at a flow rate of 1.0 ml/minute, and fluorescence detector is set at 362 nm for excitation and emission 425 nm (AFB1 and B2) and 455 nm (AFG1 and G2). Injector was an auto sampler which injected 100 μl of

sample, and elution of the aflatoxins was in the order (AF) G2, G1, B2 and B1. Calibration curves for each aflatoxin, AF (B1), B2, G1 and G2, were constructed using standard solutions which were diluted with acetonitrile to give the following concentrations: 0.005, 0.05, 0.5, 5 and 50 μg/kg. The limit of detection for all the standards was 0.005 μg/kg. The linearity of the standard

was calculated by measuring the area of the peak and then interpolating from

Descriptive statistics was used to show the distribution of aflatoxins in the different feeds and one-way ANOVA used for significance testing using IBM SPSS

*DOI: http://dx.doi.org/10.5772/intechopen.88582*

**2.2 Sample collection**

## **2.2 Sample collection**

*Aflatoxin B1 Occurrence, Detection and Toxicological Effects*

diseases [7–9].

potent of them all [13, 14].

dairy farmers was assessed.

**2. Methodology**

**2.1 Data collection**

age of 6–10 litres.

the genus *Aspergillus* [6], and their presence in animal feedstuffs has become a potential health hazard to both animals and humans [7]. Toxic effects of aflatoxins in ruminants include liver damage, diminished growth efficiency, diminished milk production and quality and impaired resistance to infectious

In dairy farming, depending on the farming system adopted, the diet consists of the concentrates, alternative protein sources as well as forage; hence the animals are exposed to more than one type of mycotoxins [4]. Although there are more than 20 aflatoxins known, only four of these occur naturally, namely, aflatoxins (AF) B1, B2, G1 and G2, based on their fluorescence under UV light (blue or green) [10–12]. The most abundant aflatoxin in cow feeds and rations is aflatoxin B1 and is also the most

Animals differ in their sensitivity to mycotoxin toxicity [15] with ruminants

Convenience sampling coupled with snowball sampling methods was used to identify farmers willing to participate in the research. Questionnaires were used to get information from the farmers. The information required from the farmers included the following: plot size in acres, number of cattle owned by the farmer, number of cows that were being milked, age, breed, lactation stage, milking method, volume of milk produced on the farm per day, volume of milk produced by each cow per day, number of milking per day, amount of feed given to each cow per day and also if the farmer had any knowledge on aflatoxins. A total of 14 farmers participated in this study with farm size of 8.5 hectares and above. Most of them were milking cows ranging between 20 and 250, and a few had less than 10 cows. The cows that were being milked were 25 months old and above, and the common breeds were the Jersey, Holstein and crossbreed (Holstein/Jersey) across all milking stages. Majority of the farmers were milking by hand getting a volume of 100 to over 200 litres per farm per day with each cow giving an aver-

being more resistant than the monogastrics [16] mainly because they have microorganisms in their rumen which play significant roles in the deactivation and degradation of the aflatoxins as well as alteration of the binding of the aflatoxins to some essential nutrients [17, 18]. However, aflatoxins are poorly degraded by ruminants as most of the rumen microbiota are inhibited by AFB1 concentration of 10 μg/ml [16]. The aflatoxins will get to the bioconversion sites of nutrients and xenobiotics like the intestinal epithelium, liver and kidneys unaltered [16]. In the liver, AFB1 is bio-transformed to AFM1 which enters the circulatory system or is conjugated to glucuronic acid. The conjugated AFM1 is excreted through the biliary system, and the one in circulation may be excreted through urine and milk. It has been shown that AFM1 retains some carcinogenic activity resulting in its reclassification by IARC as a group 1 carcinogen [19–21]. Consumption of AFB1-contaminated feed by lactating cows results in its metabolism into AFM1 subsequently secreted into milk thereby making milk a source of aflatoxin contamination in humans. In this study the extent of aflatoxin contamination of feeds used in different feeding systems adopted by

**38**

A total of 96 feed samples which consisted of dairy feed concentrates (CN), mixed ration (MR), brewers spent grain (BSG) and grass (GR) were collected from 13 farms during the dry season (August–October 2016) and the rainy season (January–March 2017). Samples were collected in sterile polythene ziplock bags which were sealed and transported in cooler boxes to the laboratory where they were ground to a fine powder using IKA® M20 universal batch mill (Germany) and stored in the freezer at −20°C until time for analysis [22].

### **2.3 Sample preparation for HPLC analysis**

Aflatoxins from feeds were extracted using the immunoaffinity extraction method [23] using Easi-Extract® aflatoxin immunoaffinity columns (R-Biopharm Rhone Limited, Glasgow G20 OXA, Scotland). Extraction was carried out according to the manufacturer's protocol with some modifications as follows: a portion of 50 g of the sample was mixed with 5 g of sodium chloride (NaCl) in a laboratory blender followed by 100 ml of methanol: water (80:20 v/v) and blended for 5 minutes. The mixture was filtered through a fluted filter paper (Whatman No.1) into a clean vessel. A volume of 2 ml of the filtrate was then diluted with 14 ml phosphate buffer saline (PBS) solution and passed through an immunoaffinity column. The column was washed with 20 ml of PBS and the aflatoxins finally eluted with 1 ml methanol (LiChrosolv®, Merck, Germany) into a glass cuvette and diluted with 1 ml of distilled water and then stored at −20°C prior to analysis. Aflatoxin B1, B2, G1 and G2 standards (Trilogy Analytical Laboratory, Washington, USA) were diluted using acetonitrile (LiChrosolv®, Merck, Germany) to give the following concentrations: 5 × 10<sup>−</sup><sup>6</sup> , 5 × 10<sup>−</sup><sup>5</sup> , 5 × 10<sup>−</sup><sup>4</sup> , 5 × 10<sup>−</sup><sup>3</sup> and 5 × 10<sup>−</sup><sup>2</sup> mg/ml. Aflatoxin detection and quantification were done using HPLC (Shimadzu FCV-20H2) with operation conditions as given in the KOBRA® cell instruction manual as follows: derivatisation using KOBRA ® cell at 100 μA setting, with an analytical column Inertsil ODS-3 V 5 μm, 4.6 × 150 mm equipped with a C18 4 × 3 mm2 ID security guard cartridge (Phenomenex, Torrance, CA, USA). Mobile phase was modified from the recommended water: methanol (60:40) to a working condition of 55:45 with 119 mg/litre of potassium bromide (KBr) and 1 ml/litre of 65% nitric acid added at a flow rate of 1.0 ml/minute, and fluorescence detector is set at 362 nm for excitation and emission 425 nm (AFB1 and B2) and 455 nm (AFG1 and G2). Injector was an auto sampler which injected 100 μl of sample, and elution of the aflatoxins was in the order (AF) G2, G1, B2 and B1.

Calibration curves for each aflatoxin, AF (B1), B2, G1 and G2, were constructed using standard solutions which were diluted with acetonitrile to give the following concentrations: 0.005, 0.05, 0.5, 5 and 50 μg/kg. The limit of detection for all the standards was 0.005 μg/kg. The linearity of the standard curves was determined using correlation regression (*r 2* ). A curve with good linearity will have an *r 2* value close to 1. Aflatoxin concentration of the samples was calculated by measuring the area of the peak and then interpolating from the standard curve.

#### **2.4 Statistical analysis**

Descriptive statistics was used to show the distribution of aflatoxins in the different feeds and one-way ANOVA used for significance testing using IBM SPSS Statistics 25.
