Multi-Mycotoxin Occurrence in Dairy Cattle and Poultry Feeds and …1522071/... · 2021. 1. 21. · metabolites in 16 dairy and 27 poultry feeds, and 24 feed ingredients from Machakos

toxins

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Multi-Mycotoxin Occurrence in Dairy Cattle andPoultry Feeds and Feed Ingredients from MachakosTown Kenya

David Chebutia Kemboi 12dagger Phillis E Ochieng 34dagger Gunther Antonissen 35 Siska Croubels 3 Marie-Louise Scippo 4 Sheila Okoth 6 Erastus K Kangethe 7Johannes Faas 8 Barbara Doupovec 8 Johanna F Lindahl 91011 and James K Gathumbi 1

1 Department of Veterinary Pathology Microbiology and Parasitology Faculty of Veterinary MedicineUniversity of Nairobi PO Box 29053 Nairobi 00100 Kenya kemboidcgmailcom

2 Department of Animal Science Chuka University PO Box 109-00625 Chuka 00625 Kenya3 Department of Pharmacology Toxicology and Biochemistry Faculty of Veterinary Medicine

Ghent University Salisburylaan 133 9820 Merelbeke Belgium GuntherAntonissenUGentbe (GA)phillisemeldaochiengUGentbe (PEO) SiskaCroubelsUGentbe (SC)

4 Department of Food Sciences Faculty of Veterinary Medicine University of Liegravege Avenue de Cureghem 104000 Liegravege Belgium mlscippoulgacbe

5 Department of Pathology Bacteriology and Avian Diseases Faculty of Veterinary MedicineGhent University Salisburylaan 133 9820 Merelbeke Belgium

6 School of Biological Sciences University of Nairobi PO Box 30197 Nairobi 00100 Kenyasheilaokothuonbiacke

7 Independent Researcher PO Box 34405 Nairobi 00100 Kenya mburiajudithgmailcom8 BIOMIN Research Center Technopark 1 3430 Tulln Austria johannesfaasbiominnet (JF)

barbaradoupovecbiominnet (BD)9 International Livestock Research Institute (ILRI) PO Box 30709 Nairobi 00100 Kenya10 Department of Medical Biochemistry and Microbiology Uppsala University SE-751 05 Uppsala Sweden11 Department of Clinical Sciences Swedish University of Agricultural Sciences SE-750 07 Uppsala Sweden Correspondence JLindahlcgiarorg (JFL) jkgathumbiuonbiacke (JKG)dagger Shared first authorship

Received 9 November 2020 Accepted 27 November 2020 Published 3 December 2020

Abstract Mycotoxins are common in grains in sub-Saharan Africa and negatively impact human andanimal health and production This study assessed occurrences of mycotoxins some plant and bacterialmetabolites in 16 dairy and 27 poultry feeds and 24 feed ingredients from Machakos town Kenyain February and August 2019 We analyzed the samples using a validated multi-toxin liquidchromatography-tandem mass spectrometry method A total of 153 mycotoxins plant and bacterialtoxins were detected in the samples All the samples were co-contaminated with 21 to 116 differentmycotoxins andor metabolites The commonly occurring and EU regulated mycotoxins reportedwere aflatoxins (AFs) (70 range 02ndash3185 microgkg) deoxynivalenol (82 range 222ndash1037 microgkg)ergot alkaloids (70 range 04ndash2857microgkg) fumonisins (90 range 324ndash14346microgkg) HT-2 toxin (3range 119ndash138 microgkg) ochratoxin A (24 range 11ndash243 microgkg) T-2 toxin (4 range 27ndash52 microgkg)and zearalenone (94 range 03ndash9104 microgkg) Other unregulated emerging mycotoxins andmetabolites including Alternaria toxins Aspergillus toxins bacterial metabolites cytochalasinsdepsipeptides Fusarium metabolites metabolites from other fungi Penicillium toxins phytoestrogensplant metabolites and unspecific metabolites were also detected at varying levels Except for total AFswhere the average contamination level was above the EU regulatory limit all the other mycotoxinsdetected had average contamination levels below the limits Ninety-six percent of all the samples werecontaminated with more than one of the EU regulated mycotoxins These co-occurrences may causesynergistic and additive health effects thereby hindering the growth of the Kenyan livestock sector

Toxins 2020 12 762 doi103390toxins12120762 wwwmdpicomjournaltoxins

Toxins 2020 12 762 2 of 16

Keywords aflatoxins ergot alkaloids feed safety food safety mycotoxins sub-Saharan Africa

Key Contribution This is the first multi-toxin study done in Kenya and 153 toxins comprising mycotoxinsplant and bacterial toxins were detected in the samples This information provides much-needed inputthat is useful when coming up with mycotoxin mitigation strategies

1 Introduction

Mycotoxins are secondary metabolites produced by fungi and pose a serious problem to human andanimal health when consumed in food and feed These metabolites are produced by molds of differentgenera in particular Aspergillus Fusarium and Penicillium but also Alternaria and Claviceps In thelivestock sector mycotoxins cause reduced feed intake and feed utilization suppression of immunityalter reproduction as well as causing hepatotoxicity nephrotoxicity mortality and subsequently seriouseconomic losses [12] The animal health effects vary from one animal species to the other the type ofmycotoxins duration and levels of exposure [3] In addition some mycotoxins are passed into animalproducts such as milk meat and eggs and thus pose a food safety concern to humans [4ndash9] In Kenyahigh levels of mycotoxins especially aflatoxins (AFs) have been reported in feeds [10ndash14] OverallAFs are the most commonly tested and detected mycotoxins in Africa because of their high toxicityand prevalence in feed and feed ingredients They are also the most regulated in feeds and food inmany countries [3] In Kenya apart from AFs deoxynivalenol (DON) fumonisins (FUM expressed asthe sum of fumonisin B1 (FB1) and fumonisin B2 (FB2)) ochratoxin A (OTA) and zearalenone (ZEN)have also been reported in animal feeds [1113] Regulatory limits have been set for AFs in animalfeed and milk in Kenya but not for other mycotoxins hence there is little monitoring done forthe other mycotoxins in animal feeds This lack of regulation is also present in most sub-Saharancountries with the regulations only addressing AFs except for South Africa where guidance levelsexist for ZEN FUM and DON in animal feeds [15] Worldwide the World Health OrganizationFoodand Agriculture Organization of the United Nations (WHOFAO) through the Codex AlimentariusCommission (CODEX) have set up a regulatory limit for AFB1 in animal feeds which most Africancountries have adopted while the European Union (EU) and the United States of America through theUnited States Food and Drug Agency (USFDA) have also established a regulatory limit for AFs andguidance limits for other mycotoxins [15] And despite their regulation being stricter the EU is a majordestination of trade for most African countries and hence the EU regulatory and guidance values areused for comparison since they may negatively impact trade and in addition they cover a wide varietyof feeds for different species

Little has been done to detect other unregulated fungal metabolites plant toxins and bacterialmetabolites in feeds and feed ingredients in Kenya however they do occur in feeds with eitheradverse beneficial or unknown effects on animal health [16ndash21] Ergot alkaloids are produced byfungi from the genus Claviceps and frequently contaminate cereals Consumption of ergot alkaloidsin feed has a negative impact on the feed intake animal growth and reproduction hence affectinganimal performance [19] Other unregulated metabolites from Aspergillus Fusarium and Penicilliumfungi have also been reported to contaminate feed with studies showing some as emerging mycotoxinshaving a negative impact on animal health and performance [1820] and with some having additiveeffects on other regulated mycotoxins [18] Alternaria mycotoxins are a group of toxins produced byfungi from the genus Alternaria that affect plants such as cereals and oilseeds There are more than70 Alternaria toxins that belong to the chemical groups such as nitrogen-containing compounds steroidsterpenoids pyranones quinines and phenolics with alternariol (AOH) alternariol monomethyl ether(AME) tenuazonic acid (TEA) and tentoxin (TEN) being the major and most studied and havingtoxicological concern [1820] Despite little being known on the toxicological mechanism of most

Toxins 2020 12 762 3 of 16

Alternaria toxins they are hazardous to animal health through cytotoxicity genotoxicity fetotoxicityand teratogenicity [16]

Bacterial metabolites are byproducts from bacteria that contaminate feed and while they maybe considered beneficial since some are antibiotics they may also lead to increased development ofantibiotic-resistant bacteria [20]

Apart from fungal and bacterial metabolites some plant compounds found in the feed may alsohave adverse effects on the animal Phytoestrogens are non-steroidal phenolic plant compounds witha similar structure to estradiol and hence bind with estrogen receptors and may inhibit or promoteestrogenic response Soybean is the major source of these phytoestrogens with dietary phytoestrogenshaving adverse effects on animals [21ndash23]

Co-occurrence of different mycotoxins can cause synergistic additive or antagonistic effects withfor example FUM reported to increase the uptake of AFs and subsequently the carry-over to milk [24]Therefore there is a need to regularly monitor the levels of multiple mycotoxins as well as otherbacterial metabolites and plant compounds in animal feeds to have adequate information for effectivemycotoxin management and to safeguard animal and human health

The objective of this study was therefore to assess the natural co-occurrence and levels of fungalmetabolites bacterial metabolites and plant toxins in dairy cattle poultry feeds and feed ingredientsused for animal feed in Kenya

2 Results

A total of 153 toxins comprising mycotoxins plant and bacterial toxins were detected inthe samples All the samples were co-contaminated with between 21 to 116 different mycotoxinsandor fungal metabolites (Figure 1) Further details of the co-occurrence can be found inSupplementary Table S1

Toxins 2020 12 x FOR PEER REVIEW 3 of 17

Bacterial metabolites are byproducts from bacteria that contaminate feed and while they may be considered beneficial since some are antibiotics they may also lead to increased development of antibiotic-resistant bacteria [20]

Apart from fungal and bacterial metabolites some plant compounds found in the feed may also have adverse effects on the animal Phytoestrogens are non-steroidal phenolic plant compounds with a similar structure to estradiol and hence bind with estrogen receptors and may inhibit or promote estrogenic response Soybean is the major source of these phytoestrogens with dietary phytoestrogens having adverse effects on animals [21ndash23]

Co-occurrence of different mycotoxins can cause synergistic additive or antagonistic effects with for example FUM reported to increase the uptake of AFs and subsequently the carry-over to milk [24] Therefore there is a need to regularly monitor the levels of multiple mycotoxins as well as other bacterial metabolites and plant compounds in animal feeds to have adequate information for effective mycotoxin management and to safeguard animal and human health

The objective of this study was therefore to assess the natural co-occurrence and levels of fungal metabolites bacterial metabolites and plant toxins in dairy cattle poultry feeds and feed ingredients used for animal feed in Kenya

2 Results

A total of 153 toxins comprising mycotoxins plant and bacterial toxins were detected in the samples All the samples were co-contaminated with between 21 to 116 different mycotoxins andor fungal metabolites (Figure 1) Further details of the co-occurrence can be found in Supplementary Table S1

The majority of the samples (96) contained more than one of the ten common EU regulated mycotoxins analyzed for with 73 having 5 or more mycotoxins and 13 having 8 out of the 10 common EU regulated mycotoxins Of the samples that were contaminated with AFs 100 were also contaminated with ZEN 98 had FUM 92 had nivalenol (NIV) 89 had DON 87 had DON-3-glucoside (DON-3-gluc) 70 had ergot alkaloids 6 had T-2 toxin (T-2) and 4 had HT-2 toxin (HT-2) Of the feeds contaminated with fumonisin B1 (FB1) 25 also had OTA

Figure 1 Number of samples co-contaminated with a given range of metabolites

Figure 1 Number of samples co-contaminated with a given range of metabolites

The majority of the samples (96) contained more than one of the ten common EU regulatedmycotoxins analyzed for with 73 having 5 or more mycotoxins and 13 having 8 out of the10 common EU regulated mycotoxins Of the samples that were contaminated with AFs 100 werealso contaminated with ZEN 98 had FUM 92 had nivalenol (NIV) 89 had DON 87 had

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DON-3-glucoside (DON-3-gluc) 70 had ergot alkaloids 6 had T-2 toxin (T-2) and 4 had HT-2toxin (HT-2) Of the feeds contaminated with fumonisin B1 (FB1) 25 also had OTA

The most commonly occurring and EU-regulated mycotoxins in the different types of feed andfeed raw ingredients are presented in Table 1 Fusarium mycotoxins ZEN (94 range 03ndash9104 microgkg)FUM (90 range 324ndash116587 microgkg) DON (82 range 222ndash1037 microgkg) and NIV (73range 99ndash144 microgkg) had the highest occurrence with AFs (70 range 02ndash3185 microgkg) and ergotalkaloids (70 range 04ndash2857 microgkg) also having a high occurrence OTA (24 range 11mdash243 microgkg)T-2 (4 range 27ndash52 microgkg) and HT-2 (2 range 119ndash138 microgkg) occurred at a lower incidenceand level

Aflatoxin B1 (AFB1) was the most prevalent amongst the AFs contaminating 69 (range 05ndash134microgkg)of all the samples The other detected AFs were AFG1 (58 range 02ndash123 microgkg) AFB2 (45range 04ndash221 microgkg) AFG2 (31 range 05ndash285 microgkg) and AFM1 (22 range 04ndash11 microgkg)

FB1 (90 range 324ndash83456 microgkg) was the most prevalent FUM Other FUMs were indescending prevalence fumonisin B2 (FB2) (85 range 167ndash33131 microgkg) fumonisin B4 (FB4)(78 range 51ndash12834 microgkg) fumonisin B3 (FB3) (73 range 103ndash9483 microgkg) fumonisin A2 (FA2)(66 range 24ndash1756 microgkg) and fumonisin A1 (FA1) (54 range 16ndash2804 microgkg)

Ergocristinine (42) was the most prevalent ergot alkaloid with other ergot alkaloids beingchanoclavin ergocristine ergocryptine ergometrine ergometrinine ergosin ergosinin ergotamineergotaminine ergocornine and ergocryptinine (Figure 2)Toxins 2020 12 x FOR PEER REVIEW 7 of 17

Figure 2 Occurrence of ergot alkaloids in feed and feed ingredients (n = 67) in Machakos (Kenya) in February and August 2019 period

Occurrence levels of other secondary fungal bacterial plant and unspecified metabolites in the feed and feed ingredients are shown in the Supplementary Figures S1ndashS11

The occurrence of common mycotoxins as per the type of feed is presented in Table 1 Dairy feed was contaminated with multiple mycotoxins in order of predominance FUM (100 range 524ndash21713 μgkg) ZEN (100 range 39ndash1402 μgkg) AFs (94 range 15ndash3185 μgkg) DON (94 range 661ndash567 μgkg) NIV (94 range 159ndash1021 μgkg) DON-3-gluc (88 range 81ndash617 μgkg) ergot alkaloids (63 range 06ndash2857 μgkg) OTA (56 range 2ndash243 μgkg) T-2 (13 range 27ndash44 μgkg) and HT-2 (6 mean 119 μgkg)

AFB1 was the most prevalent of the AFs occurring in 94 of the dairy feed samples (range 13ndash134 μgkg) with 813 being above the East African Community (EAC) and EU Commission limit of 5 μgkg The overall mean of all the samples (135 μgkg) was also above the limit Other AFs were AFG1 (88 range 02ndash123 μgkg) AFB2 (81 range 091ndash221 μgkg) AFG2 (44 range 27ndash285 μgkg) and AFM1 (38 range 16ndash11 μgkg) All other mycotoxins occurred at levels below the EU maximum guidance levels in dairy feeds

A similar occurrence pattern was observed in poultry feed samples ie DON (range 282ndash1037 μgkg) DON-3-gluc (range 38ndash457 μgkg) FUM (range 637ndash26848μgkg) and ZEN (range 52ndash8734 μgkg) occurred in all the poultry feed samples Other frequently detected mycotoxins were NIV (96 range 121ndash1055 μgkg) AFs (93 range 05ndash89 μgkg) ergot alkaloids (81 range 11ndash1132 μgkg Ochratoxin A (OTA) (19 range 25ndash106 μgkg) T-2 (4 range ltLODndash52 μgkg) and HT-2 (4 range ltLODndash138 μgkg) had low occurrence in the poultry feeds

Of all the poultry feed samples 74 had levels above the EAC regulatory limit of 50 μgkg for AFs in adult poultry feed Aflatoxin B1 (93 range 05ndash388 μgkg) was the most prevalent of the AFs and 148 of the samples were contaminated with AFB1 above the EAC regulatory limit of 50 μgkg for adult poultry feed

Of the EU regulated mycotoxins the highest level of FUM (116587μgkg) was reported in maize grains however the mean occurrence level of FUM was lower than for both dairy and poultry feed samples HT-2 and T-2 were not detected in the feed ingredients The levels of the other EU regulated mycotoxins were ZEN (83 range 03ndash9104 μgkg) ergot alkaloids (63 range 04ndash248 μgkg) DON (54 range 222ndash9961 μgkg) AFs (29 range 02ndash994 μgkg) DON-3-gluc (29 range 2ndash634 μgkg) and OTA (8 range 02ndash11 μgkg) Similarly AFB1 and AFG1 were the most prevalent AFs occurring in 25 of all feed ingredients samples in the range of 09 to 498 μgkg and 02 to 349 μgkg respectively

40 40 42

1937 31 24

12

34 30

10 50

102030405060708090

100

occurrence

Type of ergot metabolite

Ergot alkaloids

Figure 2 Occurrence of ergot alkaloids in feed and feed ingredients (n = 67) in Machakos (Kenya) inFebruary and August 2019 period

DON-3-glucoside (DON-3-gluc) a mycotoxin conjugate (72 range 20ndash634 microgkg) had a highoccurrence (98) within the pool of samples that were contaminated with DON

Occurrence levels of other secondary fungal bacterial plant and unspecified metabolites in thefeed and feed ingredients are shown in the Supplementary Figures S1ndashS11

The occurrence of common mycotoxins as per the type of feed is presented in Table 1Dairy feed was contaminated with multiple mycotoxins in order of predominance FUM (100range 524ndash21713 microgkg) ZEN (100 range 39ndash1402 microgkg) AFs (94 range 15ndash3185 microgkg)DON (94 range 661ndash567 microgkg) NIV (94 range 159ndash1021 microgkg) DON-3-gluc (88range 81ndash617 microgkg) ergot alkaloids (63 range 06ndash2857 microgkg) OTA (56 range 2ndash243 microgkg)T-2 (13 range 27ndash44 microgkg) and HT-2 (6 mean 119 microgkg)

Toxins 2020 12 762 5 of 16

Table 1 The occurrence of common EU regulated mycotoxins in feed and feed ingredients in Machakos (Kenya) in February and August 2019 period

All Feed and Feed Ingredients (n = 67) Dairy Feed (n = 16) Poultry Feed (n = 27) Feed Ingredients (Cottonseed Soybean MealMaize) (n = 24)

LOD(microgkg)

POSITIVE

GEOMEAN(microgkg)

RANGE(microgkg)

MEAN plusmn SD(microgkg)

POSITIVE

GEOMEAN(microgkg)

RANGE(microgkg)

MEAN plusmn SD(microgkg)

POSITIVE

GEOMEAN(microgkg)

RANGE(microgkg)

MEAN plusmn SD(microgkg)

POSITIVE

GEOMEAN(microgkg)

RANGE(microgkg)

MEAN plusmn SD(microgkg)

AFB1 02 69 23 05ndash134 183 plusmn 237 94 135 13ndash134 312 plusmn 34 93 47 05ndash388 102 plusmn 10 25 03 09ndash498 197 plusmn 172AFB2 006 45 02 04ndash221 34 plusmn 43 81 14 091ndash221 51 plusmn 58 48 02 04ndash44 17 plusmn 1 17 01 12ndash7 34 plusmn 21AFG1 02 58 11 02ndash123 137 plusmn 209 88 56 02ndash123 217 plusmn 298 70 12 06ndash417 67 plusmn 98 25 03 02ndash349 171 plusmn 118AFG2 05 31 06 05ndash285 51 plusmn 6 44 1 27ndash285 88 plusmn 86 33 05 05ndash64 25 plusmn 2 21 04 16ndash96 46 plusmn 29AFM1 01 22 01 04ndash11 26 plusmn 28 38 02 16ndash11 37 plusmn 33 15 01 04ndash06 05 plusmn 01 21 01 05ndash69 29 plusmn 23

AFs 01 70 23 02ndash3185 345 plusmn 515 94 204 15ndash3185 615 plusmn 766 93 62 05ndash89 172 plusmn 205 29 02 02ndash994 389 plusmn 331DON 04 82 644 222ndash1037 3175 plusmn 2249 94 1954 661ndash567 3594 plusmn 1591 100 2713 282ndash1037 3291 plusmn 2032 54 61 222ndash9961 2449 plusmn 3025DON-3-gluc 1 72 57 20ndash634 185 plusmn 137 88 116 81ndash617 221 plusmn 148 100 139 38ndash457 164 plusmn 97 29 12 2ndash634 194 plusmn 21

NIV 002 70 09 04ndash2857 261 plusmn 292 94 328 159ndash1021 511 plusmn 268 96 319 121ndash1055 432 plusmn 225 33 19 10ndash144 502 plusmn 46FA1 06 54 27 16ndash2804 353 plusmn 512 38 17 138ndash832 39 plusmn 238 52 2 33ndash292 142 plusmn 81 67 53 16ndash2804 522 plusmn 704FA2 06 66 47 24ndash1756 312 plusmn 342 75 77 47ndash872 319 plusmn 249 74 59 24ndash1031 245 plusmn 26 50 26 57ndash1756 416 plusmn 486FB1 2 90 1985 324ndash83456 742 plusmn 12236 100 3112 524ndash1494 4879 plusmn 4071 100 3055 384ndash1926 4314 plusmn 3872 71 905 324ndash83456 14744 plusmn 20347FB2 2 85 728 167ndash33131 3254 plusmn 5544 94 91 266ndash6773 1755 plusmn 1583 96 1022 235ndash7288 1729 plusmn 1567 67 429 167ndash33131 7138 plusmn 9065FB3 6 73 326 103ndash9483 1363 plusmn 1971 63 221 264ndash1243 798 plusmn 306 85 367 204ndash243 708 plusmn 518 67 37 103ndash9483 2658 plusmn 2994FB4 6 78 558 51ndash12834 1272 plusmn 2126 75 388 67ndash1248 542 plusmn 349 89 413 55ndash3878 737 plusmn 951 67 1151 51ndash12834 2623 plusmn 3254

FUM 06 90 2646 324ndash11 6587 10511 plusmn 17224 100 4149 524ndash21713 6524 plusmn 5598 100 420 637ndash26848 5979 plusmn 5415 71 1166 324ndash116587 21462 plusmn 26129OTA 1 3 055 119ndash138 129 plusmn 56 56 12 2ndash243 56 plusmn 68 19 04 25ndash106 48 plusmn 3 8 03 02ndash11 06 plusmn 04Ergot 04 73 119 99ndash144 468 plusmn 499 63 17 06ndash2857 569 plusmn 88 81 31 11ndash1132 26 plusmn 324 63 02 04ndash248 59 plusmn 74HT-2 05 24 05 11ndash243 48 plusmn 1 6 06 119ndash119 119 plusmn 0 4 06 138ndash138 138 plusmn 0 ND ND ND NDT-2 07 4 04 27ndash52 41 plusmn 1 13 05 27ndash44 35 plusmn 08 4 04 52ndash52 52 plusmn 0 ND ND ND ND

ZEN 02 94 181 03ndash9104 813 plusmn 1657 100 199 39ndash1402 352 plusmn 407 100 561 52ndash8734 1034 plusmn 1786 83 48 03ndash9104 713 plusmn 1967

AFsmdashTotal aflatoxins AFB1 ndashAflatoxin B1 AFB2mdashAflatoxin B2 AFG1mdashAflatoxin G1 AFG2mdashAflatoxin G2 AFM1mdashAflatoxin M1 DONmdashDeoxynivalenol DON-3-glucmdashDON-3-glucosideErgotmdashErgot alkaloids FA1mdashFumonisin A1 FA2mdashFumonisin A2 FB1mdashFumonisin B1 FB2mdashFumonisin B2 FB3mdashFumonisin B3 FB4mdashFumonisin B4 FUMmdashFumonisin B1 + Fumonisin B2HT-2mdashHT-2 toxin GeomeanmdashGeometric mean of all the samples nmdashnumber Meanmdashmean of only the positives LODmdashLimit of detection OTAmdashOchratoxin A Rangemdashthe range ofpositives SDmdashstandard deviation T-2ndashT-2 toxin ZENmdashZearalenone

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AFB1 was the most prevalent of the AFs occurring in 94 of the dairy feed samples(range 13ndash134 microgkg) with 813 being above the East African Community (EAC) and EU Commissionlimit of 5 microgkg The overall mean of all the samples (135 microgkg) was also above the limitOther AFs were AFG1 (88 range 02ndash123 microgkg) AFB2 (81 range 091ndash221 microgkg) AFG2 (44range 27ndash285 microgkg) and AFM1 (38 range 16ndash11 microgkg) All other mycotoxins occurred at levelsbelow the EU maximum guidance levels in dairy feeds

A similar occurrence pattern was observed in poultry feed samples ie DON (range 282ndash1037 microgkg)DON-3-gluc (range 38ndash457 microgkg) FUM (range 637ndash26848microgkg) and ZEN (range 52ndash8734 microgkg)occurred in all the poultry feed samples Other frequently detected mycotoxins were NIV (96range 121ndash1055 microgkg) AFs (93 range 05ndash89 microgkg) ergot alkaloids (81 range 11ndash1132 microgkgOchratoxin A (OTA) (19 range 25ndash106 microgkg) T-2 (4 range lt LODndash52 microgkg) and HT-2 (4range lt LODndash138 microgkg) had low occurrence in the poultry feeds

Of all the poultry feed samples 74 had levels above the EAC regulatory limit of 50 microgkg forAFs in adult poultry feed Aflatoxin B1 (93 range 05ndash388 microgkg) was the most prevalent of the AFsand 148 of the samples were contaminated with AFB1 above the EAC regulatory limit of 50 microgkgfor adult poultry feed

Of the EU regulated mycotoxins the highest level of FUM (116587microgkg) was reportedin maize grains however the mean occurrence level of FUM was lower than for both dairyand poultry feed samples HT-2 and T-2 were not detected in the feed ingredients The levelsof the other EU regulated mycotoxins were ZEN (83 range 03ndash9104 microgkg) ergot alkaloids(63 range 04ndash248 microgkg) DON (54 range 222ndash9961 microgkg) AFs (29 range 02ndash994 microgkg)DON-3-gluc (29 range 2ndash634 microgkg) and OTA (8 range 02ndash11 microgkg) Similarly AFB1 and AFG1were the most prevalent AFs occurring in 25 of all feed ingredients samples in the range of 09 to498 microgkg and 02 to 349 microgkg respectively

Table 2 shows the occurrence of the common EU regulated mycotoxins in relation to the twosampling periods February and August 2019 Overall samples collected in August 2019 had a higheroccurrence of mycotoxins as compared to samples collected in February 2019

Table 2 The occurrence of common EU regulated mycotoxins as per sampling period

February 2019 (n = 47) August 2019 (n = 20)

POSITIVE GEOMEAN(microgkg) RANGE (microgkg) POSITIVE GEOMEAN

(microgkg) RANGE (microgkg)

AFB1 60 14 05ndash134 90 75 34ndash388AFB2 38 02 04ndash221 60 04 09ndash53AFG1 47 07 02ndash123 85 32 16ndash417AFG2 34 06 05ndash285 25 05 06ndash125AFM1 32 02 04ndash11 ND ND ND

AFs 62 12 02ndash3185 90 10 34ndash890DON 77 416 222ndash1037 95 1799 282ndash7433

DON-3-gluc 62 37 2ndash634 95 145 38ndash619NIV 66 79 99ndash144 95 302 159ndash1021FA1 72 57 16ndash2804 10 05 217ndash439FA2 57 32 53ndash1756 85 118 24ndash1031FB1 85 1459 324ndash83456 100 409 697ndash1926FB2 81 565 167ndash33131 95 1323 283ndash7288FB3 75 353 103ndash9483 70 27 205ndash1727FB4 81 495 51ndash12834 70 773 76ndash3878

FUM 85 192 324ndash116587 100 5624 98ndash26548OTA 6 03 02ndash243 65 14 19ndash106Ergot 77 11 04ndash1545 55 05 06ndash2857HT-2 ND ND ND 10 07 119ndash138T-2 2 04 27 10 05 44ndash52

ZEN 92 144 03ndash9104 100 308 42ndash131

AFsmdashTotal aflatoxins AFB1mdashAflatoxin B1 AFB2mdashAflatoxin B2 AFG1mdashAflatoxin G1 AFG2mdashAflatoxin G2AFM1mdashAflatoxin M1 DONmdashDeoxynivalenol DON-3-glucmdashDON-3-glucoside ErgotmdashErgot alkaloidsFA1mdashFumonisin A1 FA2mdashFumonisin A2 FB1mdashFumonisin B1 FB2mdashFumonisin B2 FB3mdashFumonisin B3FB4mdashFumonisin B4 FUMmdashFumonisin B1 + Fumonisin B2 HT-2mdashHT-2 toxin GeomeanmdashGeometric meanof all the samples nmdashnumber NDmdashNot detected OTAmdashOchratoxin A Positivemdashabove Limit of detectionRangemdashthe range of positives T-2ndashT-2 toxin ZENmdashZearalenone

Toxins 2020 12 762 7 of 16

Other unregulated mycotoxinsmetabolites were also detected Seven Alternaria toxins altersetinAOH AME infectopyron macrosporin TEN and TEA occurred at an incidence between 33ndash66with TEN being the most prevalent

Of the other Aspergillus toxins aside from AFs 3-nitropropionic acid (81) was the mostprevalent with the other toxins including aspochracin A aspulvinone E averantin averufin kojic acidnorsolorinic acid O-methylsterigmatocystin sterigmatocystin viomellein and versicolorin C occurringat between 9 and 67

Enniatins were the most prevalent depsipeptides with enniatin B being the most prevalent at73 and other Enniatins including enniatin B1 enniatin A1 enniatin A and enniatin B2 occurring atbetween 36 and 70 Beauvericin was the least prevalent at 10

Contamination by Fusarium metabolites was between 16ndash99 with moniliformin being the mostprevalent at 99 Other Fusarium metabolites were 15-hydroxyculmorin acuminatum B apicidinantibiotic Y aurofusarin bikaverin butenolid culmorin deoxyfusapyron equisetin fusaproliferinfusapyron fusaric acid fusarinolic acid monocerin rubrofusarin siccanol W493 5-hydroxyculmorinand epiequisetin

Penicillium toxins had an occurrence of between 6ndash99 with flavoglaucin and quinolactacinbeing the most prevalent at 99 Others included 7-hydroxypestalotin andrastin A citreohybridinolcitrinin cyclopenin cyclopenol cyclopeptine dechlorogriseofulvin dihydrocitrinone griseofulvinmycophenolic acid O-methylviridicatin oxaline pestalotin questiomycin A quinolactacin Brugulovasine A secalonic acid D vermistatin verrucofortine verrucosidin viridicatin aurantiamin Acycloaspeptide A phenopyrrozin and penicolinate

Other fungal metabolites had an occurrence of between 3ndash96 and included apicidin D2 chrysoginacuminatum C ascochlorin barceloneic acid bassianolide chlorocitreorosein citreorosein fungerinilicicolin E LL-Z 1272e mollicellin D neoechinulin A NP139 sclerotinin A xanthotoxin cercosporindiplodiatoxin and paspalin Cytochalasins had a low occurrence with cytochalasin H (34) andcytochalasin J (6) being the only ones present

Apart from fungal toxins bacterial metabolites did occur at between 15ndash94 and included cyclo(L-Pro-L-Val) surfactin A and surfactin B Contamination by phytoestrogens was between 21ndash54with abscisic acid coumestrol daidzein daidzin genistein genistin glycitin and glycitein being thephytoestrogens detected There was low contamination with other plant metabolites with lotaustralinbeing the most prevalent at 24 and linamarin (7) and atropine (4) being the other metabolites

Other unspecific metabolites that contaminated the feeds included asperglaucide asperphenamatebrevianamid F cyclo(L-Pro-L-Tyr) emodin endocrocin fellutanine A iso-rhodoptilometrinN-benzoyl-phenylalanine neoechinulin D rugulusovin skyrin and tryptophol and occurred atbetween 34ndash100

3 Discussion

This is the first study in Kenya to document the occurrence of mycotoxins bacterial metabolitesand plant toxins using a multi-toxin detection method The results document the occurrence of153 different toxins and co-contamination of samples by more than one mycotoxin being commonThe observed high occurrence of multiple mycotoxins in feed and feed ingredients corresponds toprevious reports in Kenya [10ndash14] However most of the previous studies have focused on AFswith little done on other mycotoxins The mixture of different Fusarium metabolites occurred in highfrequency which is in line with findings by Ezekiel et al [20] and Streit et al [18] who reportedthat Fusarium metabolites are the most abundant toxins in animal feeds However in our casePenicillium toxins also did occur at a high frequency

In Kenya regulatory limits for mycotoxins in animal feed only exist for AFs [15] howeverguidance limits have been set for DON ergot alkaloids FUM OTA and ZEN by other bodies such asthe EU [1519] Of the regulated mycotoxins ZEN was the most prevalent mycotoxin occurring in 94of all the feed and feed ingredients (range 03ndash9104 microgkg) This reported incidence and contamination

Toxins 2020 12 762 8 of 16

level were higher than what has previously been reported in Kenya by Rodrigues et al (56 maximum167 microgkg) [13] In our study the maximum level of ZEN reported in the dairy feed (1402 microgkg) wasbelow the EU guidance level of 500 microgkg however the maximum level (91042 microgkg) reported in feedraw ingredients was higher than the guidance limit A similar higher occurrence has been reported inSouth Africa (96 maximum 123 microgkg) 3 with lower incidences reported in Ghana (11 maximum310 microgkg) [13] and Nigeria (51 maximum 80 microgkg) [13] In dairy animals high levels of ZEN havebeen reported to cause reduced feed intake reduced milk yield and reproductive disturbances [25]however short-term exposure to this concentration of ZEN in the dairy feed may indicate ZEN maynot cause acute problems but with 100 of the dairy feeds being contaminated this may cause chronicexposure and hence may affect fertility Poultry are more tolerant of ZEN toxicity and currently there isno guidance limit for ZEN in poultry feed in Kenya This reported level of ZEN in poultry feed(100 range 52ndash8734 microgkg) may not singly have an acute impact on poultry health and productivityhowever recurrent exposure may have an impact on fertility

Widespread FUM contamination of animal feed has been reported in Ghana [13] South Africa [31326]Tanzania [27] Sudan [13] and Kenya [13] In this study 90 of all the samples had FUM with a meanof positives of 1051 microgkg and the maximum contamination level was detected in a maize sample(116587 microgkg) Similarly high levels of FUM were reported by Nyangi et al [27] in maize destined foranimal feed in Tanzania The levels of FUM reported were within the EU guidance levels for FUM in dairy(50 mgkg) and poultry feed (20 mgkg) however due to co-occurrence with other mycotoxins it may stillcause a negative impact in poultry and dairy animal health due to synergistic or additive effects

Type-B trichothecenes comprising of DON the conjugate DON-3-glucoside and NIV showed asignificant incidence of contamination with a prevalence of 82 73 and 72 respectively DON isthe type B trichothecene that has received considerable worldwide interest with the EU setting aguidance limit of 5000 microgkg for complementary and alternative feedstuffs for both poultry and dairyanimals Pigs are the most sensitive species with ruminants being less sensitive with a drop in feedintake and a drop in milk yield being the major reported signs in dairy animals [15] In poultryhigh levels of DON have been reported to affect growth rate feed conversion efficiency and causingincreased sensitivity to infectious diseases such as necrotic enteritis at levels below and approachingEU guidance level and when combined with AFs causes additive toxicity Despite the levels inthis study being within the EU guidance limit studies have shown that levels lower than the EUguidance level may affect metabolic immunological and physiological processes in animals [2829]Similarly Makau et al [11] in a study on contamination of dairy feeds (forages and concentrates) inNakuru Kenya reported 63 of the samples had DON contamination with concentrates having asignificantly higher mean level of contamination (8695 microgkg) but with all samples being below theEU guidance limit The high occurrence of DON-3-gluc together with DON (98 co-occurrence)which is a modified mycotoxin that undergoes cleavage by lactic acid bacteria in the digestive tract ofthe mammals releasing DON is of concern since it increases the exposure to DON in the contaminatedfeed [1830] Similar high co-occurrence of DON and DON-3-gluc has been reported by Streit et al [18]On the other hand type A trichothecenes comprising of T-2 and HT-2 had a low occurrence (4 and 3respectively) In poultry T-2 is more toxic than HT-2 and at levels of 04 mgkg and above causes orallesions and decreases performance [29] while in dairy aside from affecting milk yield and reproductiveperformance it also causes immunosuppression and gastroenteritis [15] However the highest level inthis study was below the EU guidance level of 250 microgkg

The high incidence of total AFs (70 range 02ndash3185 microgkg) is in agreement with previous studiesin Kenya by Okoth and Kola [12] on dairy feed (100 occurrence) and Rodrigues et al [13] on animalfeeds and raw materials (78 occurrence) AFB1 was the most prevalent of the AFs occurring in 69of the feed and raw material samples (range 05ndash134 microgkg) Similar findings have been reported bySenerwa et al [31] in compounded dairy feeds in different regions of Kenya and by Makau et al [11] inconcentrates and forages in Nakuru Kenya Both dairy feed and poultry feed had a high occurrence ofboth total AFs and AFB1 however the occurrence was at a higher level in the dairy feed (geomeans

Toxins 2020 12 762 9 of 16

204 and 135 microgkg respectively) compared to poultry feed (geomeans 62 and 47 microgkg respectively)This may be attributed to the raw materials used for the manufacture of dairy concentrates such ascottonseed cake and sunflower-seed cake that are very susceptible to high contamination by AFs [1232]In dairy animals AFB1 at levels of 75 microgkgndash13 mgkg have been reported to affect productivityreproduction cause hepatotoxicity and nephrotoxicity as well as causing immunosuppression [115]Besides the animal health impact there is a carry-over of AFB1 to milk as AFM1 and this poses a healthhazard to humans since AFM1 is a human carcinogen [9] Several studies in Kenya have reported anoccurrence of between 397 and 100 of AFM1 in milk with the highest level being 463 microgkg andmean occurrence levels of between 0003 and 029 microgkg [571432ndash37] Between 104 and 64 ofthe positive milk samples in these studies exceeded the EU regulatory limit of 005 microgkg for milkThis indicates exposure through contaminated feed The carry-over of AFB1 to milk varies fromless than 1 to 62 [3839] with the level of carry-over usually determined by physiological andnutritional factors such as the animal species individual animal variability feeding regimens and typeof diet presence of other mycotoxins stage of lactation and actual milk production [1524] Thereforewith 813 of the samples exceeding the regulatory limit for both AFB1 (mean 312 microgkg) and AFs(mean 615 microgkg) it indicates a high risk of contamination of milk meant for human consumptionand at levels above the EU (005 microgkg) and East African Community (05 microgkg) regulatory limit forAFM1 in milk posing a health hazard to humans In poultry AFs are reported to cause decreasedweight gain poor feed efficiency reduced egg production hepatotoxicity and immunosuppression [29]Carry-over of AFs in poultry products occurs albeit at a smaller level than in milk [4041] Due to thishigh toxicity to both humans and animals and the carry-over to dairy and poultry products EAC hasset up regulatory limits for AFs and AFB1 in the dairy feed (10 microgkg and 5 microgkg respectively) andadult poultry feed (20 microgkg and 50 microgkg respectively) [42] In poultry 148 and 74 exceeded theEAC regulatory limit for AFB1 and AFs respectively indicating a lower risk to animal and humanhealth however the high incidence coupled with co-occurrence with other mycotoxins may increasethe risk of chronic aflatoxins exposure

Consumption of ergot-contaminated feed can have negative effects on feed intake growthand reproduction Long term exposure of ergot alkaloids even of less than 2000 microgkg depresses animalperformance and causes intoxication [19] In cattle consumption of ergot contaminated feed affectsanimal growth (daily intake of 127 g) with chronic exposure reducing reproductive performanceand causing abortion [19] In comparison poultry has a higher tolerance for ergot toxicity withlevels as high as a 4 gkg diet fed to 28-day old broilers having no effect [19] However long termexposure causes loss of appetite increased thirst diarrhea vomiting and weakness [43] Currentlyno regulatory limit for ergot alkaloids exists in Kenya with the EU setting a limit of 01 mgkg in animalfeed [19] With an occurrence of 70 (range 04ndash2857 microgkg) this shows a substantial amount of thefeed was contaminated with ergot alkaloids A total of 12 ergot alkaloids (chanoclavin ergocristineergocristinine ergocryptine ergometrine ergometrinine ergosin ergosinin ergotamine ergotaminineergocornine and ergocryptinine) were reported which was similar to what Ingenbleek et al [44]reported in food processed from wheat in Benin Cameroon Mali and Nigeria except for chanoclavin

OTA is rarely a problem in cattle due to the rumenrsquos ability to break down OTA into less toxicmetabolites with doses used in the experiment as high as 166 mgkg body weight for 5 days notproducing clinical disease [15] With the highest level reported of 243 microgkg it can therefore beconcluded that OTA is not a major problem in dairy cattle in Kenya as previously concluded in areview by Kemboi et al [15] In poultry high levels of OTA cause nephrotoxicity hepatotoxicityneurotoxicity and immunosuppression with the EU setting a limit of 100 microgkg for complementaryand complete poultry feed [45] With an occurrence of 19 and the highest level of 106 microgkg ittherefore indicates that these levels of OTA only may not be a major problem in poultry Similarlya previous study by Rodrigues et al [13] also reported a low level of OTA (mean 2 microgkg) in animalfeeds and raw material samples from Kenya

Toxins 2020 12 762 10 of 16

Concerning the unregulated metabolites data on occurrence and toxicity is rare in mammalsAME aside from being genotoxic has been shown to affect progesterone synthesis in pigs andpostulated to have an impact on reproductive performance in other mammals [18] TEA fed orallyat 125ndash150 mgkg body weightday for 3 weeks causes a significant impact on the weight gain andcauses lesions on chicken tissues [18] Despite all samples having lower levels of TEA compared tothe dose used in the experiment one sample had levels of 7 mgkg and this may have an impact onanimal health

Kojic acid and 3-nitropropionic acid that we reported in the feeds and feed ingredients areAspergillus metabolites that have previously been shown to contaminate animal feeds [1820]Their toxicity to animals has not been demonstrated but the presence of a high level of kojicacid indicates deterioration of the cereal component of the feed by Aspergillus since it is a metabolicbyproduct produced during contamination of cereals [20]

Of the Fusarium metabolites reported moniliformin that occurred in 99 of the samples andaurofusarin that occurred in 91 of the samples are toxic to animals In chicken aurofusarin affects eggquality by decreasing vitamins E A total carotenoid lutein and zeaxanthin concentrations as wellas affect the yolk color by increasing susceptibility to lipid peroxidation and the meat quality bydecreasing protein and fat content [182046] In breeding chickens feeding 264 mgkg aurofusarin infeed compromises the immunity of the progeny18 Studies have shown high levels of moniliformin tobe toxic to chicken [47] turkey [47] pigs [48] and sheep [49] Broiler chickens fed feed contaminatedwith moniliform (50 mgkg) to market age had a lower body weight gain poor feed converting rateand higher mortality [47] Despite the low levels when compared to the toxic doses of moniliforminreported in these studies combined with other toxins may be hazardous as a combination with AFsDON and FB1 have been shown to cause additive effects in poultry and pigs [50ndash52]

The reported depsipeptides enniatins and beauvericin have been previously reported in feeds inSouth Africa [26] Nigeria [20] and in samples collected from Europe and America [18] Beauvericinat levels of 25ndash12 mgkg feed show low or no acute toxicity in broiler chicken and ducklings [53]Little studies have shown the toxic effect of enniatins in livestock A study by Fraeyman et al [54]on chronic dietary intake of enniatin B in broiler chicken showed no major impact on intestinalmorphometry and hepatic histology with a limited transfer to liver tissue However enniatin A hasantibacterial antifungal herbicidal insecticidal and ionophore properties [1820]

Emodin is a metabolite produced by Aspergillus as well as the plant rhubarb root at a frequencyof 93 but at low concentration (range 02ndash117 microgkg) and has been experimentally shown to be toxicto chicken One day old cockerels fed feed with 37 mgkg body weight emodin had a loss of appetiteaccumulation of fecal material with acute epidermal irritation around the cloaca general debilitationand mortality within 5 days of ingestion [1855]

Phytoestrogens are non-steroidal phenolic plant compounds with a similar structure to estradioland hence bind with estrogen receptors and may inhibit or promote estrogenic response Soybean isthe major source of phytoestrogens that can have adverse effects on animals [2123] Phytoestrogensmay also compete with ZEN in binding to the estrogen receptors and thereby may counteract theestrogenic activity of ZEN [22] The occurrence of the phytoestrogen in the study may lead to thisinteraction once consumed by an animal

Cyclo (L-Pro-L-Val) was the most prevalent bacterial metabolite contaminating the feeds at afrequency of 94 with surfactant A and B also detected With little studies done on the effects ofthese metabolites and some considered to be beneficial by being antibiotics they may also lead to thedevelopment of antibiotic-resistant bacteria [20]

The high level of co-contamination of the feed and feed ingredients with the mycotoxins andormetabolites is a concern The majority of the samples (96) were contaminated with more thantwo mycotoxins of animal health public health and international trade significance This is similarto findings by Rodrigues et al [13] on animal feeds and raw materials from Kenya but withoutquantification of the levels of co-occurrence of the mycotoxins Makau et al [11] on a study of dairy

Toxins 2020 12 762 11 of 16

forages and concentrates in Nakuru Kenya also reported DON and AFs co-occurrence Elsewhere inSSA multiple mycotoxin occurrence in feeds has been reported in South Africa [326] and in humanfood in Benin Cameroon Mali and Nigeria [2044] This co-occurrence may cause increased risk toanimal and human health A combination of mycotoxins at low concentrations that individually haveno negative effect may in combination negatively affect the animal [56] In dairy FUMs have beenreported to increase the uptake of AFs and subsequently the carry-over to milk and with 98 of feedswith AFs also having FUM there is an increased risk to animal health and food safety OTA even atlower levels together with other mycotoxins including FB1 is the etiology of mycotoxic nephropathy inpigs and chickens reported in South Africa Northern and Eastern Europe [5758] In poultry DON inaddition to AFs shows additive toxicity [28] Despite the individual effects of the other unregulatedmetabolites in poultry and dairy animals not being reported studies have shown that some such asmoniliformin have additive effects when combined with AFs DON and FB1 This occurrence ofmultiple mycotoxins in feed therefore presents a toxicological hazard to both dairy and poultry aswell as to food safety even when the regulated mycotoxins occur at low levels Apart from theseseveral mycotoxins such as AFs OTA and T-2 also cause immunosuppression in farm animals andthis enhances the risk of the animals getting other diseases [1824]

4 Conclusions

The study was conducted to explore the level of contamination of dairy feed poultry feedand feed raw ingredients in Machakos town Kenya This is the first multi-toxin study done inKenya and 153 toxins comprising mycotoxins plant and bacterial toxins were detected in thesamples This information provides much-needed input that is useful when coming up with mycotoxinmitigation strategies However it should be noted that not all of the 153 toxins represent a hazard toanimal health but toxicological interactions may enhance the toxicity of other regulated mycotoxinsand hence representing increased animal health and public health hazard It should also be notedthat the production of mycotoxins is related to environmental conditions and this may contributeto seasonal as well as year to year variation of mycotoxins contamination [59] Machakos countywhere the samples were collected is located in the lower midland agro-ecological zones and receivesbetween 200 and 1200 mm of rainfall yearly with frequent droughts resulting in crop failures [60]this cause stress to plants and may significantly contribute to a higher contamination level in feedduring such seasons Other factors such as poor storage damage to the grains caused by rodents andpests and abiotic factors such as pH of feed and moisture content can also cause variation betweenbatches of feeds in the same season [59] Within the same batch heterogeneous contamination ofsamples by mycotoxins may also occur causing high variations in observed levels of mycotoxins in thesample Of the regulated mycotoxins AFs represent the major challenge to both poultry and dairy dueto the high numbers above the regulatory limit with the more potent AFB1 being the most prevalentThese possess a food safety challenge due to the carry-over of AFB1 from feed to milk and poultryproducts Other mycotoxins especially Fusarium mycotoxins occurred at high incidences but werewithin the guidance limit There is therefore a need for stronger enforcement of regulations to protectanimal health and productivity and ensure food safety

5 Materials and Methods

51 Study Site

The study was undertaken in Machakos town in Machakos County Kenya Machakos waspurposively selected due to previous studies indicating a high prevalence of AFs in food and feedA total of 67 samples (1 kg each) sampled from the top middle and bottom part of each bag comprisingcompounded dairy and poultry feed and feed ingredients were collected from animal feed retail shops(Agrovets) and market stalls dealing with cereal grains The sampling was done by the individualattendants in each of the shops Forty-seven samples comprising of 7 dairy feed 16 poultry feed

Toxins 2020 12 762 12 of 16

and 24 feed ingredients (maize soybean meal and cottonseed cake) were collected in February 2019while in August 2019 20 samples were collected comprising 9 dairy feed and 11 poultry feed but nofeed ingredients

52 Sample Preparation

The samples were milled to fine uniform particle size using a warring blender (Waring ProductsDIV Torrington CT) and subsequently mixed before a subsample was collected for analysisThe samples were stored at minus20 C until analysis

53 Analytical Method

All samples were analyzed for the presence and levels of mycotoxins and other secondarymetabolites by LC-MSMS as described by Sulyok et al [61] This fully validated method enables theaccurate quantification of more than 500 secondary (toxic) metabolites of plants bacteria and fungiincluding all relevant mycotoxins In cooperation with the University of Natural Resources and LifeSciences Vienna (BOKUIFA-Tulln) this method was introduced into the market as Spectrum 380reg byBIOMIN in the year 2014 Briefly 5 g of finely ground sample was weighed into a 250 mL Erlenmeyerflask and extracted for 90 min using 20 mL of acetonitrilewateracetic acid in the ration of 79201 (vvv)The samples were shaken for 90 min using a GFL 3017 rotary shaker (GFL Burgwedel Germany)and subsequently centrifuged for 2 min at 3000 rpm on a GS-6 centrifuge (Beckman Coulter IncBrea CA USA) The extracts were transferred into glass vials using Pasteur pipettes diluted 11 withacetonitrilewateracetic acid (79201) and subsequently analyzed by injecting 5 microL into the LC-MSMSsystem (Applied Biosystems Foster City CA USA)

Chromatographic separation was achieved by binary gradient elution of mobile phase A(methanolwateracetic acid 10891 vvv) and mobile phase B (methanolwateracetic 9721 vvv)with both containing 5 mM ammonium acetate and pumped at a flow rate of 1000 microLmin on a GeminiC18-column 150 times 46 mm id 5 microm particle size equipped with a C18 security guard cartridge4 times 3 mm id (both Phenomenex Torrance CA USA) The elution consisted of an initial 2 min at100 mobile phase A and a linear increase of mobile phase B to 50 within 3 min and further to100 within 9 min followed by a hold-time of 4 min at 100 mobile phase B and a 25 min columnre-equilibration at 100 mobile phase A The injection volume of both the samples and the mycotoxinstandard solutions was 5 microL Identification and quantification of each mycotoxin were performed in theSelected Reaction Monitoring (SRM) mode using a QTrap 5500 LC-MSMS system (Applied BiosystemsFoster City CA USA) External calibration was done using multi-analyte working solutions preparedby mixing different mycotoxins working solutions and mobile phase A

54 Data Management and Analysis

Data was entered in Microsoftreg excel and analysis was done using R version 361 The occurrencethe geometric and arithmetic means and the range of each mycotoxin or metabolite was calculatedThe arithmetic mean was calculated for the positive samples and the geometric mean for all thesamples For calculation of geometric mean half the value of the limit of detection (LOD) for sampleswith levels below the LOD was used Comparison of the mycotoxin or metabolite occurrence andlevel was done between the dairy feed poultry feed and feed ingredients as well as between the twosampling periods

Supplementary Materials The following are available online at httpwwwmdpicom2072-66511212762s1 Figure S1 Alternaria toxins Figure S2 Aspergillus toxins Figure S3 Bacterial metabolites Figure S4Cytochalasins Figure S5 Depsipeptides Figure S6 Fusarium metabolites Figure S7 Metabolites from otherfungi Figure S8 Penicillium toxins Figure S9 Phytoestrogens Figure S10 Plant metabolites Figure S11Unspecific metabolites Table S1 Co-occurrence of some common mycotoxins in dairy feed poultry feed and rawmaterials in Machakos Kenya

Toxins 2020 12 762 13 of 16

Author Contributions Conceptualization GA JFL SC and JKG Investigation DCK PEO and GAMethodology JF and BD writingmdashoriginal draft preparation DCK and PEO writingmdashreview and editingeveryone supervision GA JKG JFL SO SC JF BD M-LS and EKK project coordination GA andSC funding acquisition GA SC and JKG All authors have read and agreed to the published version ofthe manuscript

Funding This research was conducted within the ERA-NET LEAP-Agri MycoSafe-South project funded bythe Belgian Federal Science Policy Office (BELSPO) Belgian National Fund for Scientific Research (NFSR)Research Council of Norway (RCN) Kenyan Ministry of Education Science and Technology (MoEST)South Africarsquos National Research Foundation (NRF) BIOMIN Holding GmbH and Harbro Ltd

Acknowledgments This research was conducted within the ERA-NET LEAP-Agri MycoSafe-South projectThe time of JFL was supported by the CGIAR research program on Agriculture for Nutrition and Health

Conflicts of Interest The authors declare no conflict of interest

References

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Kenya Mycotoxin Res 2018 34 289ndash295 [CrossRef] [PubMed]8 Ribelin WE Fukushima K Still PE The toxicity of ochratoxin to ruminants Can J Comp Med Rev Can

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10 Kangrsquoethe EK Langrsquoa KA Aflatoxin B1 and M1 contamination of animal feeds and milk from urbancenters in Kenya Afr Health Sci 2009 9 218ndash226 [PubMed]

11 Makau CM Matofari JW Muliro PS Bebe BO Aflatoxin B1 and Deoxynivalenol contamination ofdairy feeds and presence of Aflatoxin M1 contamination in milk from smallholder dairy systems in NakuruKenya Int J Food Contam 2016 3 6 [CrossRef]

12 Okoth SA Kola MA Market samples as a source of chronic aflatoxin exposure in Kenya Afr J Health Sci2012 20 56ndash61

13 Rodrigues I Handl J Binder EM Mycotoxin occurrence in commodities feeds and feed ingredientssourced in the Middle East and Africa Food Addit Contam Part B 2011 4 168ndash179 [CrossRef] [PubMed]

14 Sirma A Daniel S Lindahl J Makita K Kangrsquoethe E Grace D Aflatoxin M1 survey in dairy householdsin Kenya In Proceedings of the Food Africa Midterm Seminar Helsinki Finland 16 June 2014

15 Kemboi DC Antonissen G Ochieng PE Croubels S Okoth S Kangethe EK Faas J Lindahl JFGathumbi JK A Review of the Impact of Mycotoxins on Dairy Cattle Health Challenges for Food Safetyand Dairy Production in Sub-Saharan Africa Toxins 2020 12 222 [CrossRef] [PubMed]

16 Escrivaacute L Oueslati S Font G Manyes L Alternaria Mycotoxins in Food and Feed An OverviewJ Food Qual 2017 2017 1569748 [CrossRef]

17 Warth B Parich A Atehnkeng J Bandyopadhyay R Schuhmacher R Sulyok M Krska RQuantitation of Mycotoxins in Food and Feed from Burkina Faso and Mozambique Using a ModernLC-MSMS Multitoxin Method J Agric Food Chem 2012 60 9352ndash9363 [CrossRef] [PubMed]

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18 Streit E Schwab C Sulyok M Naehrer K Krska R Schatzmayr G Multi-Mycotoxin ScreeningReveals the Occurrence of 139 Different Secondary Metabolites in Feed and Feed Ingredients Toxins 20135 504ndash523 [CrossRef]

19 Coufal-Majewski S Stanford K McAllister T Blakley B McKinnon J Chaves AV Wang Y Impacts ofCereal Ergot in Food Animal Production Front Vet Sci 2016 3 15 [CrossRef]

20 Ezekiel CN Bandyopadhyay R Sulyok M Warth B Krska R Fungal and bacterial metabolites incommercial poultry feed from Nigeria Food Addit Contam Part Chem Anal Control Expo Risk Assess 201229 1288ndash1299 [CrossRef]

21 Jefferson WN Patisaul HB Williams CJ Reproductive consequences of developmental phytoestrogenexposure Reprod Camb Engl 2012 143 247ndash260 [CrossRef]

22 Hessenberger S Botzi K Degrassi C Kovalsky P Schwab C Schatzmayr D Schatzmayr GFink-Gremmels J Interactions between plant-derived oestrogenic substances and the mycoestrogenzearalenone in a bioassay with MCF-7 cells Pol J Vet Sci 2017 20 513ndash520 [CrossRef]

23 Kriacutežovaacute L Dadaacutekovaacute K Kašparovskaacute J Kašparovskyacute T Isoflavones Molecules 2019 24 1076 [CrossRef]24 Miazzo R Peralta MF Magnoli C Salvano M Ferrero S Chiacchiera S Carvalho ECQ Rosa CAR

Dalcero AM Efficacy of sodium bentonite as a detoxifier of broiler feed contaminated with aflatoxin andfumonisin Poult Sci 2005 84 1ndash8 [CrossRef]

25 Daumlnicke S Winkler J Invited review Diagnosis of zearalenone (ZEN) exposure of farm animals andtransferof its residues into edible tissues (carry over) Food Chem Toxicol Int J Publ Br Ind Biol Res Assoc 201584 225ndash249 [CrossRef] [PubMed]

26 Changwa R Abia W Msagati T Nyoni H Ndleve K Njobeh P Multi-Mycotoxin Occurrence in DairyCattle Feeds from the Gauteng Province of South Africa A Pilot Study Using UHPLC-QTOF-MSMS Toxins2018 10 294 [CrossRef] [PubMed]

27 Nyangi C Aflatoxins and fumonisin contamination of marketed maize maize bran and maize used asanimal feed in Northern Tanzania Afr J Food Agric Nutr Dev 2016 16 11054ndash11065 [CrossRef]

28 Huff WE Kubena LF Harvey RB Hagler WMJ Swanson SP Phillips TD Creger CR Individualand combined effects of aflatoxin and deoxynivalenol (DON vomitoxin) in broiler chickens Poult Sci 198665 1291ndash1298 [CrossRef]

29 Murugesan GR Ledoux DR Naehrer K Berthiller F Applegate TJ Grenier B Phillips TDSchatzmayr G Prevalence and effects of mycotoxins on poultry health and performance and recentdevelopment in mycotoxin counteracting strategies Poult Sci 2015 94 1298ndash1315 [CrossRef]

30 Broekaert N Devreese M van Bergen T Schauvliege S De Boevre M De Saeger S Vanhaecke LBerthiller F Michlmayr H Malachovaacute A et al In vivo contribution of deoxynivalenol-3-β-d-glucoside todeoxynivalenol exposure in broiler chickens and pigs Oral bioavailability hydrolysis and toxicokineticsArch Toxicol 2017 91 699ndash712 [CrossRef]

31 Senerwa DM Mtimet N Sirma AJ Nzuma J Kangrsquoethe EK Lindahl JF Grace D Direct marketcosts of aflatoxins in Kenyan dairy value chain In Proceedings of the ANH Academy Week Addis AbabaEthiopia 20ndash24 June 2016

32 Mohammed S Munissi JJE Nyandoro SS Aflatoxin M1 in raw milk and aflatoxin B1 in feed fromhousehold cows in Singida Tanzania Food Addit Contam Part B Surveill 2016 9 85ndash90 [CrossRef]

33 Kirino Y Makita K Grace D Lindahl J Survey of informal milk retailers in Nairobi Kenya and prevalenceof aflatoxin M1 in marketed milk Afr J Food Agric Nutr Dev 2016 16 11022ndash11038 [CrossRef]

34 Mulunda F Determination and Quantification of Aflatoxin M1 in Fresh Milk Samples Obtained inGoats and Cattle in Selected Rural Areas of the Limpopo Province South Africa J Hum Ecol 201656 183ndash187 [CrossRef]

35 Kagera I Kahenya P Mutua F Anyango G Kyallo F Grace D Lindahl J Status of aflatoxincontamination in cow milk produced in smallholder dairy farms in urban and peri-urban areas ofNairobi County A case study of Kasarani sub county Kenya Infect Ecol Epidemiol 2018 9 1547095[CrossRef] [PubMed]

36 Oluwafemi F Badmos A Kareem S Ademuyiwa O Kolapo A Survey of aflatoxin M1 in cowsrsquo milkfrom free-grazing cows in Abeokuta Nigeria Mycotoxin Res 2014 30 [CrossRef] [PubMed]

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37 Kuboka MM Imungi JK Njue L Mutua F Grace D Lindahl JF Occurrence of aflatoxin M1 in rawmilk traded in peri-urban Nairobi and the effect of boiling and fermentation Infect Ecol Epidemiol 20199 1625703 [CrossRef] [PubMed]

38 Sumantri I Murti TW van der Poel AFB Boehm J Agus A Carry-over of aflatoxin B1-feed intoaflatoxin M1-milk in dairy cows treated with natural sources of aflatoxin and bentonite J Indones TropAnim Agric 2012 37 271ndash277 [CrossRef]

39 Coppock RW Christian RRG Jacobsen BJ Aflatoxins In Veterinary Toxicology Basic and ClinicalPrinciples Academic Press Cambridge MA USA 2012

40 Herzallah S Aflatoxin B1 residues in eggs and flesh of laying hens fed aflatoxin B1 contaminated diet Am JAgric Biol Sci 2013 8 156ndash161 [CrossRef]

41 Iqbal SZ Nisar S Asi MR Jinap S Natural incidence of aflatoxins ochratoxin A and zearalenone inchicken meat and eggs Food Control 2014 43 98ndash103 [CrossRef]

42 Sirma AJ Lindahl JF Makita K Senerwa D Mtimet N Kangrsquoethe EK Grace D The impacts ofaflatoxin standards on health and nutrition in sub-Saharan Africa The case of Kenya Glob Food Secur 201818 57ndash61 [CrossRef]

43 Bailey CA Fazzino JJJ Ziehr MS Sattar M Haq AU Odvody G Evaluation of sorghum ergot toxicityin broilers Poult Sci 1999 78 1391ndash1397 [CrossRef]

44 Ingenbleek L Sulyok M Adegboye A Hossou SE Koneacute AZ Oyedele AD Kisito CSKJKoreissi Dembeacuteleacute Y Eyangoh S Verger P et al Regional Sub-Saharan Africa Total Diet Study in BeninCameroon Mali and Nigeria Reveals the Presence of 164 Mycotoxins and Other Secondary Metabolites inFoods Toxins 2019 11 54 [CrossRef]

45 European Commission (EC) Setting Maximum Levels for Certain Contaminants in Foodstuffs EC BrusselsBelgium 2006

46 Dvorska JE Surai PF Speake BK Sparks NHC Effect of the mycotoxin aurofusarin on the antioxidantcomposition and fatty acid profile of quail eggs Br Poult Sci 2001 42 643ndash649 [CrossRef]

47 Broomhead JN Ledoux DR Bermudez AJ Rottinghaus GE Chronic Effects of Moniliformin in Broilersand Turkeys Fed Dietary Treatments to Market Age Avian Dis 2002 46 901ndash908 [CrossRef]

48 Harvey R Edrington T Kubena L Rottinghaus G Turk J Genovese K Nisbet D Toxicity ofmoniliformin from Fusarium fujikuroi culture material to growing barrows J Food Prot 2001 64 1780ndash1784[CrossRef] [PubMed]

49 Lamprecht SC Marasas WFO Thiel PG Schneider DJ Knox-Davies PS Incidence and toxigenicityof seedborne Fusarium species from annual Medicago species in South Africa Phytopathology 198676 1040ndash1042 [CrossRef]

50 Kubena L Harvey R Buckley S Edrington T Rottinghaus G Individual and combined effects ofmoniliformin present in Fusarium fujikuroi culture material and aflatoxin in broiler chicks Poult Sci 199776 265ndash270 [CrossRef] [PubMed]

51 Harvey RB Kubena LF Rottinghaus GE Turk JR Casper HH Buckley SA Moniliformin fromFusarium fujikuroi culture material and deoxynivalenol from naturally contaminated wheat incorporatedinto diets of broiler chicks Avian Dis 1997 41 957ndash963 [CrossRef]

52 Harvey B Edrington TS Kubena LF Rottinghaus GE Turk JR Genovese KJ Ziprin RL Nisbet DJToxicity of fumonisin from Fusarium verticillioides culture material and moniliformin from Fusariumfujikuroi culture material when fed singly and in combination to growing barrows J Food Prot 200265 373ndash377 [CrossRef]

53 Jestoi M Emerging fusarium-mycotoxins fusaproliferin beauvericin enniatins and moniliformin A reviewCrit Rev Food Sci Nutr 2008 48 21ndash49 [CrossRef]

54 Fraeyman S Croubels S Devreese M Ducatelle R Rychlik M Antonissen G Chronic Dietary Intakeof Enniatin B in Broiler Chickens Has Low Impact on Intestinal Morphometry and Hepatic Histologyand Shows Limited Transfer to Liver Tissue Toxins 2018 10 45 [CrossRef]

55 Wells JM Cole RJ Kirksey JW Emodin a toxic metabolite of Aspergillus wentii isolated fromweevil-damaged chestnuts Appl Microbiol 1975 30 26ndash28 [CrossRef]

56 Grenier B Oswald I Mycotoxin co-contamination of food and feed Meta-Analysis of publicationsdescribing toxicological interactions World Mycotoxin J 2011 4 285ndash313 [CrossRef]

Toxins 2020 12 762 16 of 16

57 Klaric MS Rasic D Peraica M Deleterious effects of mycotoxin combinations involving ochratoxin AToxins 2013 5 1965ndash1987 [CrossRef] [PubMed]

58 Stoev SD Paskalev M MacDonald S Mantle PG Experimental one year ochratoxin a toxicosis in pigsExp Toxicol Pathol Off J Ges Toxikol Pathol 2002 53 481ndash487 [CrossRef] [PubMed]

59 Gruber-Dorninger C Jenkins T Schatzmayr G Global Mycotoxin Occurrence in Feed A Ten-Year SurveyToxins 2019 11 375 [CrossRef]

60 Kangrsquoethe EK Korhonen H Marimba KA Nduhiu G Mungatu JK Okoth SA Joutsjoki VWamae LW Shalo P Management and mitigation of health risks associated with the occurrence ofmycotoxins along the maize value chain in two counties in Kenya Food Qual Saf 2017 1 268ndash274 [CrossRef]

61 Sulyok M Stadler D Steiner D Krska R Validation of an LC-MSMS-Based Dilute-and-Shoot Approachfor the Quantification of gt 500 Mycotoxins and Other Secondary Metabolites in Food Crops Challenges andSolutions Anal and Bioanal Chem 2020 412 2607ndash2620 [CrossRef]

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