Comparative Study of Aflatoxin M1 in Livestock Livers from Minna, Nigeria

Livestock in countries, especially developing countries, is vital to livelihood of people and to the economy. In the developing world, livestock species often represent a sole asset base for small-holder animal husbanders and cattle often provide the majority of draught power for crop production. Nigeria has the largest animal resources in the West African region with live animals counting to about 20 million cattles, 39 million sheeps, 48 million goats, 166 million poultry, 6.6 million pigs and 0.7 million camels and donkeys in the fiscal year 2013 (FAOSTAT, 2015) yet it is unable to meet the recommended protein intake of its populace. According to the same data, 2.6 million tonnes of livestock products were produced for consumption in the year 2013 in Nigeria; the breakdown being eggs- 650 000, milk (whole-fresh cow milk)- 570 000, cow meat (from indigenous cattle)- 338 000, goat- 292 649, pig- 254 250, sheep- 172 507, poultry meat- 170 000 and game- 165 000 in tonnage. While commercial production techniques are used extensively in the poultry and aquaculture sectors, livestock production is largely pastoral and domestic. In Minna, Nigeria, livestock is one of the main protein sources in human diet. These results to high exposure risk and public health impact from animal products that may have been contaminated with aflatoxin.

Studies have shown aflatoxin B1 (AFB1 ) residues in the livers of camels, beef calves, buffalo calves and sheep [1,2]. And liver samples containing AFB1 residues showed remarkable changes including fatty degeneration with areas of petechial hemorrhages, congestion, fibrosis and large whitish focus of necrosis as compared to those with medium and lower AFB1 levels. Aflatoxin M1 (AFM1 ) was the first aflatoxin metabolite identified and it is the principal hydroxylated aflatoxin metabolite present in milk of dairy cows fed with feed contaminated with aflatoxin B1 [3]. The metabolite is also present in the milk of human nursing mothers consuming foodstuffs contaminated with the toxin [4,5]. Occurrence of AFM1 in livestock livers is a public health concern, since AFM1 has been classified by the international agency of research on cancer as, possible human carcinogens [6]. However, in all livestock, aflatoxins can cause reduced milk or egg production, embryonic death, teratogenicity, decreased reproductive performance, tumours and suppressed immune system function, and liver damage, even at low levels [7].

Determination of AFM1 in milk, urine and liver are good biomarker of Aflatoxin B1 (AFB1) exposure, as it is correlated to the amount of AFB1 ingested. The carcinogenic potency of AFM1 is ten times lower than AFB1 but this appears warranted if the biological endpoints involved are carcinogenicity and mutagenicity [8]. Metabolism of AFM1 have been studied in vitro using liver microsomes [9]. Cyclopropenoid fatty acids (CPFA), which promote the carcinogenicity of aflatoxin B1, behave similarly with AFM1 [10]. However, newborn animals that consume aflatoxins can be poisoned by the AFM1 metabolite excreted in milk. Parameters contributing or affecting the levels of AFM1 contamination in livestock livers could be the source, type of or composition of livestock feeds [11], ecologic or economic factors on the farm, pre and post farm managements. The European Union and Codex Alimentarius have regulated maximum level of AFM1 not to exceed 0.05 ppb (50 ng/kg) while US regulations allow levels not above 0.005 ppb (500 ng/kg). Therefore, it is very pertinent that the levels of aflatoxin M1 in livestock liver are monitored, with respect to the case study from Minna metropolis. This will enable reduced risk and rapid incidence of aflatoxicoses, new findings and the countries response concerning aflatoxin. Hence, aiding awareness and reducing the national or global burden attributable to aflatoxin M1 as justified in the study, this is the first report establishing the toxin presence and levels in livestock liver from Nigeria and comparing levels in goat and cow liver, as there is no known record of its kind.

 

After sorting written approval of the Veterinary Council of Nigeria Minna Chapter, fresh liver samples were collected into ice packs from 5 abattoirs within 24 hours of slaughter. Twenty five samples each of goat liver were collected from two abattoirs (n=50) while 24 samples of cow liver were collected each from 3 abattoirs (n=72). Samples were froze dried and the stored at -20°C until used for analysis. Prior to sample analysis, sample temperature was brought up to room temperature.

Aflatoxin extraction and column chromatography were achieved using AOAC official method 982.24 (2010) with modifications, for quantification by HPLC method [12]. Dried liver tissue were blended until homogenous. About 100 g portion was weighed into 500 ml wide-mouth, glass-stoppered erlenmeyer flask. Citric acid (10 ml) solution was added and mix thoroughly with 30 cm×1 cm glass stirring rod. After 5 min it was stirred again and mixed with 2 g of silica gel, (diatomaceous earth) 200 ml methylene chloride was added and stirred to remove excess solid from rod and the flask was stirred vigorously on wrist-action for 30 min. The mixture was filtered through flow paper into 300 ml erlenmeyer containing 10 g Na2SO4, the filter top was closed and compressed against funnel to obtain its maximum filtrate volume. Flask was gently swirled intermittently for 2 min and content was refiltered through medium flow paper into 250 ml graduate, volume was recorded. Filtrate was evaporated in 500 ml round-bottom flask on water bath to near dryness and saved for column chromatography.

A separating column was set with glass wool inserted, 50 ml methylene chloride was poured in and 10 g of silica gel was added in addition to 3-4 ml methylene chloride and mixed with stainless steel rod, methylene chloride was drained halfway to settle silica, and silica was rinsed off column sides with methylene chloride, one scoop of anhydrous sodium sulphate was added to supernate solvent above silica gel to cap column. Excess methylene chloride was drained to 1 cm above column packing. The concentrated filtrate was then redissolved in 25 ml methylene chloride and added to column, the round-bottom flask was rinsed, methylene chloride was added to the column and the entire solution was drained through column by gravity. When flow rate slowed, anhydrous sodium sulphate was stirred gently, when filtrate reached anhydrous sodium sulphate, column sides were rinsed with methyl chloride and drained similarly. Column was washed with 25 ml toluene- acetic acid (9:1), 25 ml hexane in each and 25 ml hexane-ether-acetonitrile (6:3:1) and washes were discarded. Aflatoxin was eluted with 40 ml methyl chloride-acetone (4:1) and evaporated to near dryness in vacuum. This was however evaporated to as low as 2 ml and was put in amber bottle and stored in the refrigerator for the next stage which is the HPLC analysis.

Aflatoxin extract was analyzed and quantified using High Performance Liquid Chromatography (Agilent technologies 1200). Peak areas of the samples were used to calculate the amount of respective aflatoxin M1 in the samples using the following HPLC parameters: Fluorescent detector (FLD DEABO01608), Wavelength (365 nm and 435 nm for excitation and emission respectively), Retention time (1.5 min), Flow rate (0.2 ml/min), Injection volume (20 μL), Column (C18), Mobile phase of Acetonitrile and Water (65:35) and AFM1 standards (Trilogy analytical laboratory, Washington MO 63090, United States). The limit of detection for AFM1 was 0.001 µgL-1. After the HPLC analysis of spiked and unspiked samples, the percentage recovery was calculated was 92.36 %.

Obtained peak areas from the resulting HPLC chromatograms of the aflatoxin M1 standard stock solution were used to prepare a calibration curve (Appendix 1-3).

Mean values after mathematical analysis, were consequential through one-way analysis of variance (ANOVA) (Appendix 4). Least significant difference between livestocks (Cow and Goat) were compared by a Welch two sample t-test and pairwise multiple comparism of the different locations of sample collection was determined using SPSS Version 17. With a p-value of <0.05 level, mean values amongst treatment groups were considered to be distinctive.

 

High Performance Liquid Chromatographic evidence showed the presence of aflatoxin M1 with different concentrations in 58% goat liver and 83.3% of cow liver samples (Table 1). Mean concentration of AFM1 was higher in in cow liver sample than in goat liver sample (Figure 1) (Appendix 1).

After analyzing AFM1 contamination in liver samples from the five abattoirs, a bi-variant scatter plot (Figure 2) show evidence that cow liver samples had the highest concentration of sample contamination, their maximum levels were significantly higher than that of goat liver samples. Figure 3 then shows that even the mean aflatoxin M1 levels in the cow liver samples from the three abattoirs were higher than those found in goat liver samples from two abattoirs, a broad-spectrum comparative analysis assisted this comparison.

 

Conversion of aflatoxin B1 to aflatoxin M1 takes place in the liver, leading to the elevated levels of AFM1 in the organ [13]. The entrance of aflatoxins in livestock organs or tissues is mainly through their feeds, which must have been contaminated with aflatoxigenic fungi such as Aspergillus flavus or Aspergillus parasiticus either at preproduction or postproduction stages [14]. Aflatoxin M1 occurrence in livestock liver can thereby be managed by controlling level of aflatoxin B1 in feed. Analyses in this study showed unique comparative levels of AFM1 occurrence in goat (58%) and cow (83.33%) livers under study and within the abattoirs where liver sample were collected.