Mycotoxins are a class of low molecular weight compounds that are produced by toxigenic strains of moulds that infect food and feed commodities. They can cause serious animal health related problems as they may be hepatotoxic, nephrotoxic [1,2] and may impair immune responses resulting in increased risk to secondary infection [3]. In addition, these toxins also compromise animal performance arising from reduced feed intake, poor feed utilization, loss in weight gain, decreased barrier function, reduced reproductive capacity and even death [4] leading to serious economic losses [5].

Even though several mycotoxins have been detected in various commodities worldwide, those of greatest significance in Africa and other tropical countries are aflatoxins (AF) and fumonisins (FB) [6]. Literature also seems to suggest that in addition to the AF (AFB₁, B₂, G₁ and G₂) and FB (FB₁, FB₂ and FB₃), ochratoxin A (OTA) and the trichothecenes (TH) are also significant. This is not only because of high toxicities of the compounds, but also that their significance rests mainly on the fact that levels above those currently regulated are often encountered in food and feed commodities. It is in such circumstances that monitoring for these mycotoxins in food and feed commodities before they go into the market is imperative, which does not reflect the case in most African countries.

Contamination of compound feeds by mycotoxins and the potential transfer of these toxins into animal products (egg, organs, meat and milk) for human consumption remain a major concern. The health effects of mycotoxins can be categorized as acute or chronic mycotoxicosis. Most exposures to mycotoxins are detrimental but this may vary from one animal species to the other as well as on the level and duration of exposure. In the Republic of South Africa, outbreaks of mycotoxicoses in animals have been reported due to consumption of contaminated feeds. There have been two episodes of aflatoxicosis reported among dogs, one in 1987 [7] and another in the early 2011 that left at least 220 dogs dead and several others affected in the Gauteng region [8]. Monitoring for mycotoxins in feeds or their raw materials is not routinely performed. Due to the recent event of aflatoxicosis outbreak, it has become necessary for the National Department of Agriculture (NDA), to pass a regulation mandating dairy feed and pet food producers to analyze for mycotoxins [9].

In this study, liquid chromatography tandem mass spectrometry (LC-MS/MS) and LC-fluorescence were used to screen for the 11 well known mycotoxins AFB₁, B₂, G₁ and G₂, FB₁, FB₂, OTA, DON, ZEA, T-2, and HT-2 in animal feeds from S. Africa. The aim of this report was to shed more light on the distribution patterns and co-occurrence of mycotoxins in feeds produced and marketed in the country.

In this study, 92 feed samples for different classes of animals were analyzed for AFB₁, B₂, G₁ and G₂, FB₁, FB₂, OTA, DON, ZEA, T-2, and HT-2. The present study is the first survey on multi-mycotoxin occurrence in feeds from S. Africa using an LC-MS/MS. A summary of the prevalence of mycotoxins and associated levels (unadjusted data) detected is presented in Table 4, and chromatograms illustrating a cattle feed sample positive for FB, OTA, DON, ZEA and AFs is presented in Figure 1. The mycotoxin contamination profile clearly demonstrates that AF, FB, OTA, DON and ZEA are common contaminants of S. African compound feeds. All analyzed feed samples were contaminated with mycotoxins, singly or in combination and in order of predominance, FB (100%), DON and ZEA each recovered from 99% of samples, AF and OTA were found in 30% and 4% samples, respectively, meanwhile HT-2 and T-2 were not detected in any of these samples. Except for ZEA and DON, cattle feeds were generally more heavily contaminated (mean levels) with mycotoxins than feeds destined for the other animal species.

Fumonisins were the most dominant mycotoxins (max: 2999 µg/kg; mean: 916 ± 727 µg/kg) being present in all samples. Since 1988 when fumonisins were discovered in S. Africa [14], they have been recognized as the main mycotoxin contaminant in maize and maize-based products including compound feeds. For compound feeds, 11,000 µg/kg is so far the highest contaminated level reported in S. Africa [15]. Subsequently, surveys of S. African commercial feeds for FB have shown maximum contamination levels below 6000 µg/kg [16,17,18,19].

Concentrations of FB₁ were comparatively higher, ranging from 104 to 2371 µg/kg compared to FB₂ where the range was 103 to 673 µg/kg. Higher contamination levels of FB₁ compared to FB₂ in cereal grains and animal feeds are often reported [19,20,21]. Levels of fumonisins were generally found to be slightly higher in cattle feeds than in chicken, swine or horse feeds. This variation could not be directly linked to the different levels of maize included since cattle feed contains a much smaller amount of maize than chicken or swine feed. In the present study all samples contained FB below regulatory levels for animal feed in S. Africa.

Ochratoxin A was the least predominant mycotoxin being present at detectable levels only in cattle feeds with 4 of the 25 samples containing OTA (mean: 9.9 ± 4.9 µg/kg; max. level: 17.1 µg/kg), whereas no OTA was found in feeds for other animals tested (Table 4). Very limited data on OTA contamination of South African animal feeds and feed components is available. In previous surveys on raw materials or finished feeds, neither OTA was found [16,17] nor was it analyzed at all [19]. In the present study, the incidence and levels of OTA were low, however, contrary to the recent study of Stoev et al. [18], finished feeds from S. African swine farms with mycotoxic nephropathy were contaminated with OTA at a much higher incidence rate and in several cases, levels above acceptable limits were found. It therefore follows that OTA seems to be a mycotoxin that could be detected in S. African cattle feeds and regularly in swine feeds. The same mycotoxin has been shown to be a nephrotoxin playing a role supposedly in association with other mycotoxins in the aetiology of mycotoxic nephropathy encountered in some farm animals in S. Africa [18] and in Northern and Eastern Europe [22,23,24]. It is also carcinogenic and has some immunotoxic properties [25,26] thus, increasing animal’s susceptibility to secondary infection. In the country, OTA is regulated in feeds for swine and poultry only but no specific limits are yet to be set for other animals including cattle.

ZEA was present in 99% of the samples and quantification levels were reached in 47 samples (51%) ranging between 30 and 610 µg/kg. Swine feeds had the highest levels of ZEA (range: 127–170 µg/kg) followed by chicken feeds where the levels ranged from 30 to 610 µg/kg. In horse feeds, the range was 39–46 µg/kg. Generally, we found that the distribution pattern of ZEA was similar and highly correlated with that of DON except that DON levels were generally higher in the respective samples. Such a similarity can best be explained based on the fact that these toxins are produced by the same fungi principally Fusarium graminearum and Fusarium culmorum and that the requirements for production [27,28] are similar.

Deoxynivalenol is one of the mycotoxins that have received considerable interest in S. Africa [29] as incidences of high DON contamination levels have been reported in agricultural commodities [18,20,30,31]. According to the Southern African Grain Laboratory [30], there are contamination levels in the range of 200–13,000 µg/kg found in maize. Griessler [17] found that 90% of raw materials and compound feed samples contained very high concentrations (mean: 1015 µg/kg; max. of 11,022 µg/kg) of the toxin. Our data revealed that the two swine feeds analyzed had the highest DON contamination levels (1343 and 2356 µg/kg) that were above regulatory limit of 1000 µg/kg for S. Africa. The lowest contamination level of 620 ± 386 µg/kg for DON was recovered from feeds for chicken, while slightly higher mean levels of 742 ± 338 and 891 ± 512 µg/kg were obtained from horse and cattle feeds, respectively.

Aflatoxin was present in 30% of the samples. As seen in Table 4, cattle feeds (incidence rate of 52%) were the most contaminated with AF (mean: 14.7±22.8 µg/kg; max: 71.8 µg/kg) and 4 of the 25 samples analyzed had levels that exceeded regulatory limits set for South African cattle feeds. Two of these samples were dairy feeds that had levels that were at least 7 times above the limit of 5 µg/kg regulated. The presence of AF particularly AFB₁ in dairy feed is particularly worrying as levels of 10 µg/kg or even less may result in the appearance of aflatoxin M₁ (AFM₁) in the milk at levels above those now permissible (0.05 µg/kg) in Europe or South Africa [32]. Poultry and horse feeds also contained AF, but contamination levels were comparatively low (max level: 1.8 µg/kg or below) with none detected in swine feeds. AFB₁ and AFG₁ occurred more frequently and at higher levels than AFB₂ and AFG₂. However, in 3 samples, AFG₂ was the only AF detected either singly or together with AFG₁.

In our study, a quantitative difference in mycotoxin contamination between the different feed types was evident and as previously stated, cattle feeds were the most contaminated. Aflatoxin levels were higher in cattle feeds, i.e., having mean levels >20 times higher than the levels recovered from other types of feed and in particular, OTA was only detected in feeds for cattle (Table 4). The most likely reason for this variation may be the use of different feed raw materials for different types of animals and also the level of inclusion (Table 1). Breytenbach, L. in an email [33] stated that in S. Africa, cotton seed oilcake or full fat cotton seed is directed solely for ruminant feed production and the same feed ingredients were consistently found by Reiter et al. [10] and Mngadi et al. [16] to contain high levels of AF. Those raw materials may also contain OTA probably why high levels of these toxins were observed in some cattle feeds.

Mngadi et al. [16] and Stoev et al. [18] provided some data in which several mycotoxins co-occur in feeds within the country. In our study, the co-existence of mycotoxins within the same sample was very common and data revealed that 91 samples contained multiple mycotoxins, with 67% that with three mycotoxins (FB + DON + ZEA), 26% with 4 mycotoxins (FB + DON + ZEA + AF), while 5 samples had 5 mycotoxins (FB + DON + ZEA + AF + OTA). Based on the S. African feed regulations, 13% of samples, mostly for cattle, contained mycotoxin levels regarded as unfit for livestock feeding. Accordingly, 7 feed samples showed unacceptable levels of ZEA and AF and in 4 of these (calf and dairy feeds), co-contamination of ZEA and AF occurred at unacceptable levels. In addition some cattle feed samples also contained extremely high AF levels and the same samples also contained high amounts of OTA.

Such multiple mycotoxin contaminations in feeds is crucial as mycotoxins may toxicologically interact with each other eliciting marked synergistic and additive actions [32] especially between mycotoxins found at high concentrations. These actions provoked by OTA at low levels combined with FB₁, have been the prime suspect for increased incidence of mycotoxic nephropathy in S. African swine [18]. This theory was experimentally tested [34] when swine were fed diets containing OTA and/or FB₁ and the same mycotoxic nephropathy observed under field conditions was enhanced in animals exposed to these mycotoxins in combination.

From the results of this screening of AFB₁, B₂, G₁ and G₂, FB₁, FB₂, OTA, DON, ZEA, T-2, and HT-2 in various kinds of feed, it can be concluded that in South Africa, cattle feeds are probably more contaminated with mycotoxins than feeds for other animal species as they were found to contain the highest mean levels of AF and FB. In addition, OTA contamination only occurred in cattle feeds and in most of the positive samples, this toxin co-existed with other mycotoxins at high contamination levels that could induce some synergistic or additive effects in animals. Because such mycotoxins as AF and OTA can be carried over to animal-derived food products, human exposure to such mycotoxins may be a problem. Feed raw materials and finished feeds should for that reason be routinely monitored in order to identify potential sources of mycotoxins.