The occurrence of aflatoxigenic <i>Aspergillus</i> spp. in dairy cattle feed in Southern Brazil

Variane, Augusto Carlos Favaretto; Santos, Fabiane Cristina dos; Castro, Fausto Fernandes de; Barbosa-Tessmann, Ione Parra; Santos, Geraldo Tadeu dos; Pozza, Magali Soares dos Santos

doi:10.1016/j.bjm.2018.05.005

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Brazilian Journal of Microbiology

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Food Microbiology • Braz. J. Microbiol. 49 (4) • Oct-Dec 2018 • https://doi.org/10.1016/j.bjm.2018.05.005 copy

The occurrence of aflatoxigenic Aspergillus spp. in dairy cattle feed in Southern Brazil

Authorship SCIMAGO INSTITUTIONS RANKINGS

ABSTRACT

The presence of mycotoxins or related fungi in animal feed is a major problem for animal and human health. Silage and concentrated feed samples were collected from 21 dairy farms in the Western part of Paraná state in Southern Brazil. Water activity and pH of all samples were measured, and each sample was analyzed to check for the presence of aflatoxigenic Aspergillus. Water activity was observed to be lower in the concentrated feed samples. The pH was lower in the silage samples, indicating fermentation processes. Two silage samples and four concentrated feed samples were contaminated with Aspergillus spp. Seven isolates of Aspergillus spp. were obtained and their potential to produce aflatoxins was evaluated. Four of the isolates, two from the silage samples and two from the concentrated feed samples, produced the aflatoxins B1, B2, G1, and G2 in culture media. These isolates were identified as Aspergillus parasiticus and Aspergillus nomius. The presence of aflatoxigenic isolates of Aspergillus spp. in silage and concentrated feed samples is a matter of concern, because of the risk of aflatoxin production and contamination of the animal feed.

Keywords:
Feed; Dairy; Aspergillus

Farm	Lactating cows feeding^a a Combined = grazing and feeding on the trough during the milking process.	Dry cows feeding^a a Combined = grazing and feeding on the trough during the milking process.	Concentrated feed	Concentrated feed storage	Silage^b b H.G. = humid grain.	Silage silo size H(D) × L × W ^c c H(D) × L × W = Height (Depth) × Length × Width, in meters.	Silage silo type	Silage storage time^e e W, M, and Y = weeks, months, and years.	Silage silo cover	Silage additive
1	Combined	Combined	Formulated	Silo	H.G.	1.5 × 3 × 2	Trench	3 W	Canvas	-
2	Combined	Combined	Formulated	Silo	H.G.	NM^d d NM = not measured.	Surface	6 M	Canvas	-
3	Combined	Combined	Formulated	Bag	H.G.	1 × 3.5 × 2	Trench	1 W	Canvas	-
4	Combined	Combined	Commercial	Bag	H.G.	1.8 × 5 × 2	Trench	1 Y	Canvas	-
5	Combined	Combined	Commercial	Bag	H.G.	1.9 × 4.5 × 2	Trench	1 Y	Canvas	-
6	Combined	Combined	Commercial	Silo	H.G.	2 × 4.5 × 2	Trench	1 M	Canvas	-
7	Combined	Combined	Commercial	Silo	H.G.	1.6 × 5 × 2	Trench	3 M	Canvas	-
8	Combined	Combined	Formulated	Silo	H.G., Tifton	2 × 3.5 × 2	Surface	2 M	Canvas	-
9	Combined	Grazing	Formulated	Silo	H.G., Hay, or Oat	2 × 4 × 2	Trench	1 M	Canvas	-
10	Combined	Combined	Commercial	Bag	H.G., Tifton	1.6 × 4.5 × 2	Trench	2 M	Canvas	Inoculants
11	Combined	Combined	Formulated	Silo	H.G.	1 × 5 × 2	Trench	2 M	Canvas	Inoculants
12	Combined	Combined	Commercial	Bag	H.G.	1.8 × 3 × 2	Surface	2 W	Canvas	-
13	Combined	Combined	Commercial	Bag	H.G.	1.5 × 3.5 × 2	Trench	8 M	Canvas	-
14	Combined	Combined	Commercial	Silo	H.G.	1.6 × 4 × 2	Surface	1 M	Canvas	Inoculants
15	Combined	Combined	Wheat brain	-	H.G.	1.2 × 3 × 2	Trench	2 W	Canvas	-
16	Combined	Combined	Commercial	Silo	H.G.	1.5 × 3 × 2	Trench	2 W	Canvas	-
17	Combined	Combined	Formulated	Silo	H.G.	2 × 4.5 × 2	Trench	6 M	Canvas	Inoculants
18	Combined	Combined	Commercial	Bag	H.G.	1.9 × 4 × 2	Surface	3 W	Canvas	-
19	Combined	Combined	Formulated	Bag	H.G.	1.5 × 4 × 2	Trench	2 M	Canvas	-
20	Combined	Combined	Formulated	Bag	H.G.	1 × 3 × 2	Trench	1 M	Canvas	-
21	Combined	Combined	Commercial	Bag	H.G., Tifton	NM^d d NM = not measured.	Trench	1 W	Canvas	-

Farm samples	Water activity^a a Data represent the average of duplicate.			pH^a a Data represent the average of duplicate.			Orange reverse colonies in AFPA medium
	Surface	Depth	Spot	Surface	Depth	Spot
1	0.98	0.97	0.98	7.48	4.87	5.00	-
2	0.98	0.99	0.97	5.23	4.37	5.40	-
3	0.98	0.98	0.98	6.69	5.36	6.92	-
4	0.99	0.98	0.97	5.32	5.28	5.33	-
5	0.98	0.98	0.98	4.84	5.04	5.50	-
6	0.97	0.97	0.97	4.49	5.20	4.46	-
7	0.99	0.98	0.98	6.15	6.08	5.46	-
8	0.97	0.97	0.97	4.70	4.40	5.09	X (Spot)
9	0.97	0.97	0.98	5.10	5.44	6.16	-
10	0.97	0.97	0.98	4.67	4.51	4.98	-
11	0.98	0.98	0.98	5.16	4.81	6.02	-
12	0.98	0.98	0.97	5.43	6.23	5.85	-
13	0.98	0.98	0.97	5.64	6.21	5.41	-
14	0.98	0.98	0.98	5.35	6.98	7.42	-
15	0.98	0.97	0.98	5.51	6.74	6.11	-
16	0.97	0.97	0.97	4.94	6.96	7.26	X (Spot)
17	0.98	0.98	0.98	5.31	4.95	7.34	-
18	0.97	0.98	0.98	6.27	5.25	5.42	-
19	0.98	0.97	0.98	4.55	4.68	6.02	-
20	0.97	0.97	0.98	6.67	4.45	6.80	-
21	0.97	0.95	0.98	5.20	5.20	6.70	-
Average^b b Averages, inside an analysis, followed by different letters are statistically different according to t-test and Duncan's test (α = 0.05). The coefficient of variation for water activity was 0.43% and for pH was 13.18%.	0.98 ± 0.001^a	0.98 ± 0.001^a	0.98 ± 0.001^a	5.46 ± 0.12^ab	5.38 ± 0.01^b	5.90 ± 0.02^a

Farm	Concentrated feed type	Water activity^a a Data represent the average of duplicate.	pH^a a Data represent the average of duplicate.	Orange reverse colonies in AFPA medium
2	Formulated	0.83	5.24	-
3	Formulated	0.69	6.32	X
4	Commercial	0.67	6.84	-
5	Commercial	0.64	6.33	-
6	Commercial	0.69	6.27	-
7	Commercial	0.68	6.83	-
8	Formulated	0.70	6.88	X
10	Commercial	0.69	7.00	-
11	Formulated	0.69	6.36	X
12	Commercial	0.65	7.39	-
13	Commercial	0.67	6.66	-
14	Commercial	0.71	6.27	-
15	Wheat bran	0.72	6.42	X
16	Commercial	0.68	6.70	-
17	Formulated	0.71	6.82	-
18	Commercial	0.67	6.33	-
19	Formulated	0.68	5.86	-
20	Formulated	0.86	6.02	-
21	Commercial	0.67	6.47	X
Average		0.72 ± 0.06	6.27 ± 0.51

Isolates^b b The samples number indicates their origin regarding the number of silage or concentrated samples. The letter after the number indicates their origin regarding Concentrated feed (C) or Silage (S) samples.	Species	GenBank acession number (AN)	GenBank AN of similar sequences (% of identity)	PCR				Fluorescence in CMA^a a The analysis was done in triplicate.	Ammonium hydroxide vapor^a a The analysis was done in triplicate.	TLC
				apa-2	omt-A	ver-1	nor-1			MGA	CMA	YES
UEM 443^c c A. parasiticus UEM 443, positive control. NT = non-tested.	A. parasiticus	MG964332	JQ316518.1 (100%) HQ340110.1 (100%)	+	+	+	+	+	+	B1, B2, G1, G2	B1, B2, G1, G2	B1, B2, G1, G2
UEM 3C	A. nomius	MG964333	MG857640.1 (100%) MG857637.1 (100%)	+	+	-	+	-	-	-	-	B1, B2, G1, G2
UEM 8C1	A. flavus	MG964334	MG734749.1 (100%) KY319338.1 (100%)	+	+	+	+	-	-	-	-	-
UEM 8S	A. parasiticus	MG964335	KY937934.1 (100%) HQ340110.1 (100%)	-	-	-	+	-	-	-	-	B1, B2, G1, G2
UEM 8C2	A. nomius	MG964336	MG857640.1 (100%) MG857637.1 (100%)	+	+	+	+	-	-	-	-	B1, B2, G1, G2
UEM 11C	A. flavus	MG964337	MG734749.1 (100%) KY319338.1 (100%)	+	+	+	+	-	-	-	-	-
UEM 15C	A. tamarii	MG964338	MG857652.1 (100%) LC127424.1 (100%)	-	-	-	-	-	-	-	-	-
UEM 16S	A. parasiticus	MG964339	KY937934.1 (100%) HQ340110.1 (100%)	-	+	+	+	+	+	B1, B2, G1, G2	B1, B2, G1, G2	B1, B2, G1, G2
UEM 21C	Trichoderma longibrachiatum	MG964340	MF144551.1 (100%) KY225659.1 (100%)	NT	NT	NT	NT	NT	NT	NT	NT	NT

Sociedade Brasileira de Microbiologia USP - ICB III - Dep. de Microbiologia, Sociedade Brasileira de Microbiologia, Av. Prof. Lineu Prestes, 2415, Cidade Universitária, 05508-900 São Paulo, SP - Brasil, Ramal USP 7979, Tel. / Fax: (55 11) 3813-9647 ou 3037-7095 - São Paulo - SP - Brazil
E-mail: bjm@sbmicrobiologia.org.br

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[1] Conflicts of interest

The authors declare no conflicts of interest.