Detection of multimycotoxins in camel feed and milk samples and their comparison with the levels in cow milk

Abstract Camel milk has been considered as an important source of nutrients and is commercialized in many countries of the world including the Middle East. This study aimed to investigate the presence of mycotoxins in camel feed and milk samples in comparison with the cow milk. Fumonisins (FUM), ochratoxin A (OTA), and zearalenone (ZEN) were detected in 14%, 39%, and 39% of the tested camel feed samples, respectively. Among the tested camel feed samples, 8.3% and 5.6% were co‐contaminated with OTA+FUM and FUM+ZEN, respectively. In the case of milk samples, 46.15% of camel and 63.63% of cow were found contaminated with aflatoxin M1 (AFM1). In total, 16.2% and 8.1% of the milk samples were simultaneously contaminated with two and three mycotoxins, respectively. Although the levels of individual mycotoxins in the camel feed and milk samples were within the European Union (EU) permissible limits, their co‐occurrence may pose severe risk to human and animal health due to possible additive and/or synergistic toxicities.

into human food chain poses a risk to human health. The occurrence of multimycotoxins, even if the level of each individual compound is within the permissible limits, is an emerging issue due to recent findings on their synergistic or additive toxicities (Kifer et al., 2020;Sobral et al., 2018). Restricted nutrition intake puts certain groups at risk of exposure to mycotoxins and develops other diseases (Valitutti et al., 2017(Valitutti et al., ,2018. Aflatoxin M1 (AFM1), a hepatic metabolite of aflatoxin B1 (AFB1), is a frequently detected mycotoxin in animal milk and other body secretions (Flores-Flores et al., 2015;Hassan, Al-Thani, Atia, et al., 2018;Min et al., 2021). Like the parent toxin (AFB1), AFM1 is also a known carcinogen (toxicity is 10 times lower than AFB1) and is the only regulated mycotoxin in the milk. Many countries around the world follow the European Union (EU) permissible limit of 50 ng/L in milk, while the Food and Drug Administration has 10 times higher permissible limit of 500 ng/L (European In dairy animals such as cows, there are studies concerning the gut microbe degradation and biotransformation of mycotoxins by the liver microsomal enzymes, and such processes lead to a lower release of mycotoxins (1%-6% of the total dietary exposure) in milk (Fink-Gremmels, 2008;Min et al., 2021). However, little is known about the feed-to-milk carryover of mycotoxins in camel.
Keeping up with the aforementioned knowledge gaps, this study has been designed to investigate the levels and types of mycotoxins in camel feed and in milk collected from the feed market and camel farms in Qatar. In parallel, milk samples from cows were also analyzed for the presence of multimycotoxins for the purpose of comparing their levels in these two important dairy animals.

| Sampling
In this study, a total of 36 feed and 37 milk samples were collected from the feed market and camel farms located in Qatar. All the samples were packed separately in sterile airtight bags and transported to the Department of Biological and Environmental Sciences, Qatar University. Based on their nature, the camel feed samples were divided into cereal-/grain-mixed (n = 23), dry fodder (n = 08), and green fodder (n = 05). Milk samples were separated into camel milk (n = 26) and cow milk (n = 11). Before analysis, feed samples were ground to powder using a blender (for grains and dry fodder) or pestle and mortar in the presence of liquid nitrogen (for green fodder).
All samples were preserved in 50-ml tubes at 4°C in fridge prior to the mycotoxins extraction.

| Extraction and analysis of ochratoxin A (OTA)
All the feed samples were extracted and analyzed for the presence of OTA by following the instructions described in ELISA kit (RIDASCREEN ® Ochratoxin A 30/15; R-Biopharm AG). Of the ground feed samples, 2 g was suspended in 5 ml of 1N HCl and was mixed for 5 min. To each tube, 10 ml of dichloromethane was added, and the samples were left for 15 min in ashaker. After centrifugation, the upper phase was removed and the rest of the tube contents were filtered using the Whatman filter paper. To the filtrate, equal volume of 0.13 M of NaHCO 3 was added. After a thorough mixing for 15 min, tubes were centrifuged again. A total volume of 100 µl from the upper phase was diluted in 400 µl of sodium hydrogen carbonate (0.13 M). To the duplicate ELISA wells, 50 µl of the diluted filtrate was added. Microplate reader (Tecan Sunrise™) was used to measure the absorbances at 450 nm. Data were acquired using Tecan Magellan software, and mycotoxin concentrations in the samples were obtained on the basis of calibration curve using RIDA ® Soft Win-Z9996 (R-Biopharam).

| Extraction and analysis of fumonisins (FUM) and zearalenone (ZEN)
The extraction of FUM and ZEN from camel feed was carried out by following the protocol described in ELISA kits, RIDASCREEN ® Fumonisin (R3401) and RIDASCREEN ® Zearalenone (R1401), respectively. Briefly, 5 g of ground feed samples was mixed with 25 ml of 70:30 methanol:water and was shaken and incubated for 3 min.
The tubes were centrifuged, and the supernatant was diluted in 1.3ml dH 2 O for FUM extraction. For ZEN extraction, the supernatant was diluted (1:7) with the buffer provided with the ELISA kit. In both cases, 50 µl of the diluted samples was applied in the ELISA wells.
Absorbance and mycotoxin concentrations were calculated as described in section 2.2.1 above.

| Mycotoxin analysis in milk
All milk samples were skimmed before analysis for the presence of mycotoxins. For this purpose, 5 ml of milk was centrifuged at 3500 × g for 10 min. The layer of fat was scraped off, and fat-free samples were shifted to new tubes. Levels of aflatoxin M1 (AFM1), OTA, ZEN, and FUM were determined using ELISA kits as described above. In each case, 50 µl of the skimmed milk samples was applied to the duplicate wells of the respective ELISA plates. Absorbance and mycotoxin concentrations were calculated as described above (section 2.2.1) using the microplate ELISA reader and RidaWin ® software, respectively.

| Statistical analysis
The data on the prevalence of mycotoxins in camel feed and milk samples were presented in percentage (%) of the positive samples detected. Further comparisons were made on the basis of the feed nature and sample source. Levels of different mycotoxins in camel feed and milk samples were presented in ranges (minimum-maximum) and their mean values. Analysis of variance (ANOVA) was performed for the comparison between mycotoxins levels in the camel and cow milk samples. SPSS software was used to analyze the data.

| Prevalence of mycotoxins in different types of camel feed
Mycotoxin-associated pathologic outcomes and altered performance in camels are poorly studied; however, like other animals, camel is also susceptible to mycotoxicosis as reported in the natural and experimental exposure in UAE (Osman et al., 2004) and Saudi Arabia (Al-Hizab et al., 2015). In the present study, a total of 36 camel feed samples were collected from the camel feed market and camel farms located in Qatar and analyzed for the occurrence of ochratoxin A (OTA), fumonisins (FUM), and zearalenone (ZEN). Among the cereal-/grain-mixed feed, FUM and OTA were detected in 60.86% and 21.73% of the samples, respectively ( Figure 1). In Saudi Arabia, for testing camel feed, Bokhari (2010) found that 85% of total 40 samples were positive for the OTA contamination. Comparatively, lower OTA contamination in this study might be associated with the nature of samples. In the present investigation, OTA was not detected in green and dry fodder samples. This is presumably due to lesser susceptibility of fodder to ochratoxigenic Aspergillus and Penicillium infection compared with the cereals and grains. ZEN, an estrogenic metabolite of Fusarium spp., was detected in 100%, 80%, and 8.6% of the dry fodder, green grasses, and cereal-/grain-mixed feed samples, respectively. The presence of ZEN in animal feed is frequently reported at different significant levels between grains and forges.
In one of our previous studies, in the marketed feed grains in Qatar,  Likewise, in our findings, ZEN also was not found in the growing forage samples.

| Level of mycotoxins in camel feed in relation to EU permissible limits
There are no specific regulatory limits for mycotoxins in camel feed.
Considering the EU limits in feedstuff for other dairy animals, in this study, the levels of all detected mycotoxins were within the maximum permissible limits. OTA was detected only in the cereal-or grainmixed samples with levels ranging from 0.23 to 9.44 ng/g (Table 1).
These levels are far below 250 ng/g, which is the EU permissible limit

| Mycotoxins in the camel and cow milk samples
Milk is rarely tested for the contamination with mycotoxins other than AFM1. In this work, a significant number of camel (46.15%) and cow milk samples (63.63%) were tested positive for AFM1 contamination ( Figure 3). Yousof and Zubeir (2020) in Sudan reported even lower AFM1 contamination in camel milk (15.6%) compared with a higher (82.2%) occurrence in the cow milk. The lower incidence of AFM1 in camel milk compared with the cow may be associated with (a) lower dietary intake of parent AFB1, as camels are offered less feed concentrate compared with bovine feeding regimes; (b) activity of camel's ruminal microflora that leads to more degradation of AFB1; (c) intestinal morphological differences impeding the absorption of AFB1 in camel; or (d) activity of hepatic microsomal enzymes leading to the biodegradation of AFB1 to other biotransformed metabolites different than AFM1 in camel. In the detailed study, all these hypotheses can be studied one by one to arrive at precise conclusion.
Among the cow milk samples, 81.8% were found contaminated with OTA, which was not detected in any of the camel milk samples.
Relatively less percentage of the camel milk samples were contaminated with FUM and ZEN at 11.53% and 3.84%, respectively. On the contrary, FUM was not detected in the cow milk samples, while ZEN was found comparatively higher (36%) in the samples. The presence of FUM in milk has been reported earlier by Maragos and Richard (1994) in 0.6% of the tested samples and by Gazzotti et al. (2009) in 80% of the samples. Likewise, the occurrence of ZEN in raw cow milk has been reported by El-Hoshy (1999) in 20% of the tested samples.
Overall, a higher percentage of the cow milk samples were contaminated with mycotoxins compared with the camel milk.
AFM1 was detected neither in the camel milk nor in the cow milk samples from farm B (Figure 4) OTA was not detected in any of the camel milk samples, while FUM, ZEN, and AFM1 were found in the ranges of 28-38 ng/L, 50.31 µg/L, and 5.32-12.73 ng/L, respectively ( Figure 5). In accordance with the study outcomes, the occurrence of OTA in the cow milk has been previously reported in France (Boudra et al., 2007), Italy (Pattono et al., 2011), and China (Huang et al., 2014). The levels reported in Italy (70-110 ng/L) were higher, while those in France

| Occurrence of multimycotoxins in feed and milk samples
The  their synergistic or additive toxic impacts create health risk for the exposed subjects (Queiroz et al., 2012;Smith et al., 2016). There are studies where the co-exposure to more than one mycotoxin was proven to result in significant toxicological outcomes (Sobral et al., 2018;Sun et al., 2014).

ACK N OWLED G EM ENTS
This project was made possible by NPRP grant #8-392-4-003 from the Qatar National Research Fund (a member of Qatar Foundation).
The findings achieved herein are solely the responsibility of the authors. The publication of this article was supported by the Qatar National Library member of the Qatar Foundation.

CO N FLI C T O F I NTE R E S T
The authors declare that there is no conflict of interest in this research.

E TH I C A L A PPROVA L
Ethics approval was not required for this research.

DATA AVA I L A B I L I T Y S TAT E M E N T
Data available on request due to privacy/ethical restrictions.