Feed-Based Multi-Mycotoxin Occurrence in Smallholder Dairy Farming Systems of South Africa: The Case of Limpopo and Free State

Abstract

Mycotoxin contamination of feed does not only cut across food and feed value chains but compromises animal productivity and health, affecting farmers, traders and consumers alike. To aid in the development of a sustainable strategy for mycotoxin control in animal-based food production systems, this study focused on smallholder farming systems where 77 dairy cattle feed samples were collected from 28 smallholder dairy establishments in the Limpopo and Free State provinces of South Africa between 2018 and 2019. Samples were analyzed using a confirmatory UHPLC-MS/MS (Ultra-high performance liquid chromatography-tandem mass spectrometry) method validated for simultaneous detection of 23 mycotoxins in feeds. Overall, mycotoxins assessed were detected across samples with 86% of samples containing at least one mycotoxin above respective decision limits; up to 66% of samples were found to be contaminated with at least three mycotoxins. Findings demonstrated that deoxynivalenol, sterigmatocystin, alternariol and enniatin B were the most common mycotoxins, while low to marginal detection rates were observed for all other mycotoxins with none of the samples containing fusarenon-X, HT-2-toxin and neosolaniol. Isolated cases of deoxynivalenol (maximum: 2385 µg/kg), aflatoxins (AFB₁ (maximum: 30.2 µg/kg)/AFG₁ (maximum: 23.1 µg/kg)), and zearalenone (maximum: 1793 µg/kg) in excess of local and European regulatory limits were found. Kruskal-Wallis testing for pairwise comparisons showed commercial feed had significantly higher contamination for deoxynivalenol and its acylated derivatives, ochratoxin A and fumonisins (FB₁ and FB₂), whereas forages had significantly higher alternariol; in addition to significantly higher fumonisin B₁ contamination for Limpopo coupled with significantly higher enniatin B and sterigmatocystin for Free State. Statistically significant Spearman correlations (p < 0.01) were also apparent for ratios for deoxynivalenol/fumonisin B₁ (r_s = 0.587) and zearalenone/alternariol methylether (r_s = 0.544).

Keywords: UHPLC-MS/MS; dairy cattle; dairy feed; multi-mycotoxins; smallholder farming.

Figure A1

Heat map based on the pairwise Spearman correlation coefficients (rho) between the measured mycotoxin concentrations overall feeds assessed. Darker hues of red indicate a stronger negative correlation while darker hues of blue indicate stronger positive correlations. Unshaded correlations were not significant. ** Correlation is significant at the 0.01 level (2-tailed bivariate). * Correlation is significant at the 0.05 level (2-tailed bivariate).

Figure 1

Graphical representation of multi-mycotoxin incidence rates, inclusive of trace amount detections (

Figure 2

(A), Upper limit toxin concentrations for identified mycotoxins in overall dairy feeds from the two different feed classes: compound feeds vs forages. Bars labelled with toxin name followed by a double Asterix (**) showed significant differences of mean ranks by Kruskal–Wallis test at p ≤ 0.05. (B), Frequencies of identified mycotoxins in same populations by feed class. (C), Upper limit toxin concentrations for identified mycotoxins in overall dairy feeds from the two different sampling seasons: October 2018 and April 2019. Bars labelled with toxin name followed by a double Asterix (**) showed significant differences of mean ranks by Kruskal–Wallis test at p ≤ 0.05. (D), Frequencies of identified mycotoxins in same populations by sampling season. Nivalenol (NIV), deoxynivalenol (DON), 3- acetyl deoxynivalenol (3-ADON), 15- acetyl deoxynivalenol (15-ADONs), zearalenone (ZEN), aflatoxin G₂ (AFG₂), aflatoxin G₁ (AFG₁), aflatoxin B₂ (AFB₂), aflatoxin B₁ (AFB₁), diacetoxyscirpenol (DAS), alternariol (AOH), fumonisin FB₁ (FB₁,) fumonisin FB₂ (FB₂), ochratoxin A (OTA), alternariol monomethyl ether (AME), sterigmatocystin (STERIG), roquefortine C (ROQ-C), enniatin B (ENN B).

Figure 3

(A), Upper limit toxin concentrations for identified mycotoxins in overall dairy feeds from the two different provinces assessed: Limpopo vs Free State. Bars labelled with toxin name followed by a double Asterix (**) showed significant differences of mean ranks by Kruskal–Wallis test at p ≤ 0.05. (B), Frequencies of identified mycotoxins in same populations by province representative of agro-ecological zone. (C), Upper limit toxin concentrations for identified mycotoxins in overall dairy feeds from the five towns assessed: Groblersdal, Jane Furse, Njhakanjhaka, Phutaditjaba and Harrismith. Bars labelled with toxin name followed by a double Asterix (**) showed significant differences of mean ranks by Kruskal–Wallis test at p ≤ 0.05. (D), Frequencies of identified mycotoxins in same populations by town. Nivalenol (NIV), deoxynivalenol (DON), 3-acetyl deoxynivalenol (3-ADON), 15-acetyl deoxynivalenol (15-ADONs), zearalenone (ZEN), aflatoxin G₂ (AFG₂), aflatoxin G₁ (AFG₁), aflatoxin B₂ (AFB₂), aflatoxin B₁ (AFB₁), diacetoxyscirpenol (DAS), alternariol (AOH), fumonisin FB₁ (FB₁,) fumonisin FB₂ (FB₂), ochratoxin A (OTA), alternariol monomethyl ether (AME), sterigmatocystin (STERIG), roquefortine C (ROQ-C), enniatin B (ENN B).

Figure 4

Stacked frequencies (%) of mycotoxins in feeds aggregated by feed class and province. Note: AFs = Sum of AFB₁, AFB₂, AFG₁ and AFG₂/DONs = Sum of DON, 3ADON, 15 ADON/FUMs = Sum of FB₁, FB₂. Zearalenone (ZEN), alternariol (AOH), ochratoxin A (OTA), alternariol monomethyl ether (AME), sterigmatocystin (STERIG), roquefortine C (ROQ-C), enniatin B (ENN B), fumonisins (FUMs), deoxynivalenols (DONs), aflatoxins (AFs).