The negative impact of certain mycotoxins in dairy cattle

Mycotoxins are generally toxic secondary metabolites produced by toxigenic strains of some genera of molds. In particular, mycotoxins are polyketones compounds resulting from condensation reactions produced under specific physical, chemical and biological conditions that occur when the reduction of the ketone groups in the biosynthesis of the fatty acids, carried out by the molds, is interrupted. These fatty acids are primary metabolites used by molds as an energy source. Mycotoxins are usually formed at the end of the exponential phase or at the beginning of the stationary phase of the mold´s growth. Mycotoxins can cause diseases and disorders in humans and animals, called mycotoxicosis.

Deoxynivalenol (DON) also called vomitoxin, T-2 toxin (T-2), diacetoxyscirpenol (DAS), zearalenone (ZEN) and fumonisins (FB..). Are mycotoxins, among others, produced by toxigenic strains molds of the genus Fusarium. The first three mycotoxins belong to the family of trichothecenes mycotoxins. Fusarium is a genus of mold that is part of the flora of field (fitopathogenic substrates, live plants) and intermediate flora (cereal substrates recently harvested and still wet). This mold grows between 6 and 40 º C with an optimum between 18 and 30 º C. It is aerobic and needs generally, an activity of water (aw) upper to 0.88 to grow and proliferate and upper to 0.91 to produce mycotoxins.

In regard to the temperature, there are cases like Fusarium roseum that requires a minimum of 15 ° C for developing with an optimum between 24 and 27 º C, and instead, one of the mycotoxins that may produces such as zearalenone, will be produced only at temperatures between 10-14 º C. However there are varieties of Fusarium roseum as in the case of Fusarium roseum "gibbosum" and Fusarium roseum "semitectum" that are capable for producing on sorghum substrate at 25 º C, quantities of zearalenone equivalent to those produced at a temperature of 10 º C. Fusarium is one of the groups of molds with more genetic ability to produce mycotoxins when there are the physical, chemical and biological suitable conditions.

The Fusarium contaminates the cereal in the field and later when cereal is subjected to drying process and others, the mold may die but mycotoxin remains in the substrate. Thus, it is not surprising that in the mycological and mycotoxin analysis to be carried out subsequently to the stored grain, the mycotoxin is found but not the Fusarium. On the other hand, also is not surprising to find Fusarium in the stored cereal, or well because the cereal treatment was insufficient to completely kill the mold or as a result of subsequent re-contaminations due for example, vectors transporters such as air and insects.

Aflatoxins (AFB...) and ochratoxin A (OTA) are the principal mycotoxins produced by toxigenic strains of Aspergillus mold. This is a mold that belongs essentially to the storage flora. In general, the minimum temperature required for developing and produce mycotoxins is 10-12 º C. Water activity (aw) needed to initiate his development and to produce mycotoxins is from 0.75 and 0.83, respectively. Aspergillus grows and can produce mycotoxins optimally at 25 ° C with a water activity of 0.95. However, there are strains of Aspergillus flavus that in substrates such as rice, are growing from 6 to 45 º C with an optimum at 37 º C and mycotoxin production is between 11 and 36 º C with a maximum at 30 ° C.

In the present article we will see how some of these mycotoxins can affect dairy cows. Therefore, we will present several cases of contamination in the feed to those animals, that produced adverse effects on them.

We noted that the negative effect of DON in regard to milk production is not sufficiently well studied, because some contradictions exist among authors as we shall see. 

 Although there are over 40 derivatives of trichothecenes, the more significant and important for its toxic effects in animals are, for the moment: vomitoxin or deoxynivalenol (DON), T 2 toxin (T-2) and diacetoxyscirpenol (DAS). The DON belongs to the group B and the two others belong to the group A of trichothecenes mycotoxins.

Trichothecene mycotoxins can be found as natural contaminants in cereals (corn and by-products, barley, sorghum, oats, wheat and by-products, rice, rye and millet), hay and silage.

The main problem is the gastroenteric syndrome. In general, the toxicological properties of these mycotoxins, it depends on the affected animal species, are: 1. Vomiting, diarrhoea, tachycardia. 2. Bleeding, edema, necrosis of skin tissues. 3. Hemorrhages of the epithelial mucosa of the stomach and intestine. 4. Hematopoietic tissue destruction. 5. Decrease in circulating white cells and platelets. 6. Hemorrhagic meninges (brain). 7. Nervous system disorder. 8. Rejection of the feed. 9. Necrotic lesions in different parts of the mouth. 10. Pathological degeneration of cells in the bone marrow, lymph nodes, and intestine.

The systems and organs affected are the digestive system, nervous, circulatory and skin. The trichothecene mycotoxins have potent immunosuppressive activity.

Despite all the undesirable effects mentioned above and as we mentioned, it depends on the animal species involved, in the case of dairy cows some of the problems caused by some of these metabolites, as is the case of DON, are otherwise nature. 

3.1 .- Nonlactating Hol stein dairy cows, were fed diet contaminated with 1500 ppb (micrograms/kg) of vomitoxin (DON) for 3 weeks; after and during 6 weeks were fed diet with 6400 ppb and then all the cows were returned to eat the diet less contaminated (1500 ppb) for 3 weeks.

No toxicities were noted or decrease in weight gain and perhaps the consumption of diet by animals that were feeding the highest DON contamination was slightly lower (Trenholm et al, 1985).

3.2 .- Dairy cows that were fed diet contaminated with 66000 ppb of DON for 5 days, showed no changes in the daily feed intake or milk production, concentrations of calcium, phosphorus, sodium, potassium, magnesium and nitrogen in milk, were not altered, in comparison with a control group.

In the milk was not found DON but if deepoxideoxinivalenol (DOM-1) (metabolite of DON) in concentrations of 26 nanograms/ml. The 20% of ingested DON was found in the urine and feces as DOM-1 (96%) and DON (4%) (Côte et al, 1986). We should note that the time consumption of contaminated food was very short. The metabolite DOM-1 is significantly less toxic than DON, we have no data which is the percentage of low toxicity.

3.3 .- Holstein dairy cows were fed diets contaminated with DON at concentrations of 0 (control), 6000 and 12000 ppb in the concentrate (on dry matter) for 10 weeks. Decreases in milk production were not observed but there was a significant decrease in fat content in the milk. The daily feed intake was not affected and there was no DON or DOM-1 residues in milk, analyzing it with a method based on HPLC (high performance liquid chromatography resolution) - MS (mass spectrometry) with a detection limit of 1 microgram/ml.

The daily intake of DON was 0.59 (the control was slightly contaminated with DON), 42 and 104 mg, approximately and respectively. Diets were formulated with the nutritional requirements needed for milk production of 25 liters/day and a fat content of 3.8%. Each cow fed about 9 Kg. per day of concentrate (on dry matter) (Charmley et al, 1993).

Given these daily intakes (mg) of DON and taking into account the milk production before mentioned, the complete diet intake could be about 16 kg of dry matter, therefore DON contaminations in the complete diet would be 2600 and 6500 ppb on dry matter.

3.4 .- The authors Obremski et al, 2009, indicated in a recent article, that DON in a concentration higher than 1000 ppb in the feed for cows, can decrease appetite, according to the references Swanson et al, 1987 and Weaver et al , 1980.

They also indicate that according to U.S. data collected from more than about 40000 dairy cows, DON decreased milk production and increased somatic cell count on it.

In addition, they report that contaminations with 2600-6500 ppb of DON can cause reduced milk production of around 13% and refer to this Charmley et al, 1993, who are the authors cited in 3.3. - anterior. We believe that there must be a bibliographic reference error, seen that does not match the arguments presented by these authors.

3.5 .- A total mixed ration contaminated with Fusarium mycotoxins, of which the major contaminant was the DON at a concentration of 3500 ppb (on dry matter), was fed for 63 days to 12 mid-lactation Holstein dairy cows with an average milk production of 36 Kg/day. The parameters for the daily feed intake, live weight, milk production and milk composition were not affected, however, metabolic parameters and immune function were severely affected (Korosteleva et al, 2009).

3.6 .-  Rations contaminated with DON at concentrations of 0, 2100, 6300 and 8500 ppb were fed to dairy cows for 3 weeks. There were no effects of DON  on daily feed intake, milk production and milk composition, in comparison with the control group (Chase and Stone, 2003)

3.7 .- Finally, it is very important to refer the studies published by scientists at the University of North Carolina (USA) who indicated that, although many scientific studies has shown no cause and effect relationship between DON levels and reduced milk production, field observations have shown that the presence of DON in concentrations above 300 ppb in the diet, are associated with reduced feed intake, lower milk production, elevated milk somatic cell counts, and reduced reproductive efficiency.

The same filed observations have shown that reductions in milk output of 25 pounds per cow were seen, when the levels of DON contamination, were 500 ppb or more in the ration. The authors also admit that the possible presence of other mycotoxins, or factors more toxic than DON, seems likely  (Jones et al, 1994-2007) 

4.1 .- A final ration contaminated with 1200 ppb T-2 toxin (T-2) caused death in dairy cows that were consuming the contaminated feed for at least five months. The source of contamination was a moldy corn that was contaminated with 2000 ppb of T-2 and was 60% contained in the final formula of the ration. The authors said that contamination could be greater due to the recovery percentage of the analytical method used (Hsu et al, 1972).

4.2..- In dairy cows, the T-2 has been associated with feed refusal, production losses, gastroenteritis, intestinal haemorrhages and death. Practical recommendation from field observations may be avoid T-2 in excess of 100 ppb in the total diet for growing or lactating dairy animals (Jones et al., 1994).

T-2 toxin is associated with a marked reduction of the immune response in calves (Mann et al., 1982, Mann et al., 1984).

4.3 .- Feed contaminated with 640 ppb of T-2 produced in dairy cows, rumen and reticulum  ulceration and  haemorrhagic   intestinal  inflammation (Obremski et al, 2009). 

5.1 .- Although diacetoxyscirpenol (DAS) is a mycotoxin with toxic properties similar to those of the T-2 and even more aggressive, we have no data on studies in dairy cows mycotoxicosis about this mycotoxin. Several times the DAS appears together T-2 as contaminants. 

Zearalenone (ZEN) can be found as natural contaminant in cereals and their by-products, sesame seed, canola, hay and silage.

The main syndrome produced by this mycotoxin is the oestrogenic, obviously affecting the reproductive system. Zearalenone inhibits follicular maturation and ovulation by reducing the concentration of FSH (follicle stimulating hormone), given that this mycotoxin (although structurally different) can adopt a similar configuration to that  17-Beta-estradiol and other natural oestrogens that bind with oestrogen receptors, causing hyper-oestrogenism with swelling and  hypertrophy of the vulva, uterus, mammary glands and ovarian atrophy. Can occur vaginal and rectal prolapse (Gimeno and Martins, 2006). 

6.1.1.1 .- The most significant in regard to problems in dairy cows caused by zearalenone, are the studies published by scientists at the University of North Carolina (USA), such as:

From field observations it seems that in dairy cows, ZEN contaminations in the final ration greater than 250 ppb, can already cause estrogenic problems, abortions, reduced feed intake, decreased milk production, vaginitis, vaginal secretions, poor reproductive performance, and mammary gland enlargement in virgin heifers (Jones et al, 1994-2007).  Problems of rectal prolapse in cows to consume feeds contaminated with ZEN, were observed. 

There are six types of fumonisins: B1, B2, B3, B4, A1 and A2. The most frequent and important for their toxicity are the fumonisin B1 (FB1) and the fumonisin B2 (FB2). The latter ones could be found in natural contaminants in the cereal grains (mainly in corn and corn by-products).

The main syndromes produced (according to the specific animal specie) are: neurotoxic (leukoencephalomalacia), nephrotoxic, pulmonary and cerebral edema, hepatotoxic and cardiac lesions. The affected organs are:  brain, lungs, liver, kidneys and heart. These mycotoxins interfere with the sphingosine and sphinganine metabolism, causing a metabolic disturbance of the sphingolipids which are liver constituents and of lipoproteins.

Usually, the studies on the fumonisins toxicity are referred to the concentration of FB1, however, the presence of the fumonisin B2 (FB2) together with the FB1 is very frequent. The  FB2 concentration represents 15 to 35% of the FB1 concentration (Gimeno, 2009).

The fumonisins toxicity depends of the specific animal specie that is affected as above mentioned, and the dairy cows are very resistant to the toxic action of fumonisins. However it is not the same with lactating calves. 

6.2.1.1 .- Jersey cows in the midlactation period, fed for 14 days a final ration contaminated with 75000 ppb of fumonisins B1, B2 and B3 (FB1 + FB2 + FB3). The cows consumed an average of 3 mg of fumonisins/kg body weight/day.

A transient diarrhoea and an increase of serum cholesterol were observed at the beginning of the contaminant feeding period. However, clinical and hematologic changes were not observed in the animals

Fumonisins residues were not detected in any of the milk samples by two analytical laboratories using methods with a detection limit of 5 ng/ml. (Richard et al., 1996)

The dairy cows are resistant to the toxicity of fumonisins. Not so with calves. 

6.2.1.2 .- Ten milk-fed male Holstein calves aged 7-14 days  with an average weight of 43 kg were given fumonisin B1 (1 mg/kg) intravenously, daily for 7 days. The calves had serious problems of nephrotoxicosis and hepatotoxicosis. The concentrations of sphinganine and sphingosine in liver, kidney, lung, heart and muscle were increased. Sphinganine, but not sphingosine, concentration was increased in brains of treated calves. However, there were no problems of leukoencephalomalacia or pulmonary edema (Mathur et al, 2001).

The transmission of fumonisin B1 to those calves under field conditions, can only be through the mother's milk (which is highly unlikely, almost impossible, as we have seen before) or through the placenta and obviously the problems arise at birth. We don´t have, at the moment, scientific data to sustain that theory. 

There are 18 known types of aflatoxins, of which the most toxic are aflatoxin B1(AFB1) and aflatoxin M1 (AFM1). Aflatoxin M1 is the hydroxilated metabolite of Aflatoxin B1.

Other aflatoxins listed in order of toxicity, from most toxic to least toxic, are: aflatoxin G1 (AFG1), aflatoxin M2 (AFM2), aflatoxin B2 (AFB2), and aflatoxin G2(AFG2). Aflatoxin M2 is a metabolic derivative of aflatoxin B2 produced by animals and found in milk and urine, like AFM1.

Aflatoxins are found as natural contaminants in cereals (specifically in corn, wheat, sorghum and rice) and cereal by-products, oilseed meals (cottonseed meal, peanut meal, rapeseed meal, coconut meal, sunflower seed meal, and others), cassava, and a series of other human food sources, mainly, cereals, dry fruits, sausage products, spices, wines, coffee, legumes, fruits and their juices, milk, and dairy products.

Aflatoxins have high carcinogenic, teratogenic, and mutagenic activity. The major toxic effect produced by aflatoxins is the hepatotoxicosis, but also they can produce kidney problems. The most affected organs are: the liver, the kidneys, and the brain.

Aflatoxins are immunosuppressive, since they inhibit phagocytosis and protein synthesis (antibodies are proteins), interrupting DNA, RNA and ribosome protein synthesis, as well. Amino acid absorption is altered leading to the rise of amino acid hepatic retention.

The AFM1 is approximately ten times less toxic than AFB1.

The AFB1, is not only important because it can cause problems in dairy cows but also must be take into consideration that when a cow is feeding of aflatoxin B1 contaminated ration, a part of aflatoxin B1 is biotransformed in AFM1 through a hydroxylation process. The AFM1 is water soluble and this facilitates their excretion through bodily fluids. Thus, this metabolite may appear as a contaminant in milk and can be a serious risk to human health. It is for this reason that throughout the world and especially the European Union have a very strict Legislation in regard to maximum allowed concentrations of AFB1 contamination in feed for dairy cows, and maximum allowed concentrations of AFM1 contamination in milk and milk products for human consumption, especially for children who are the biggest consumers of these foods (Gimeno 2005; Gimeno and Martins, 2006) 

7.1.1.1.- Holstein dairy cows (mid-lactating period) were given doses corresponding to 13 mg of AFB1/cow/day, during a period of 7 days. This corresponds to a final contaminated ration of 433 ppb of AFB1, considering a consumption of 30 Kg (wet matter) of final ration/cow/day. Some cows received AFB1 in its pure form and others in its impure form from Aspergillus parasiticus cultures that contained other aflatoxins and their metabolites. Feed consumption and milk production decreased significantly in these cows. The somatic cell count was not apparently affected, and the concentrations of aflatoxin M1 in milk were between 1.05 and 10.58 ppb (micrograms/Liter). No aflatoxin M1 was found in the milk after 4 days of ceasing the supply of AFB1.  However, the deleterious effects seemed more serious in the cows that received impure aflatoxin in comparison to those which received pure aflatoxin (Applebaum et al., 1982).

7.1.1.2.- Dairy cows were induced with a mammalian infection using Streptococcus agalactiae, Staphylococcus aureus, and Staphylococcus hyicus, during the lactating period. Subsequently they received and oral dose of AFB₁ corresponding to 0.3 mg/Kg of body weight/day during periods of 12 to 14 days. Considering a 550 Kg body weight cow, with a consumption of 30 Kg of final ration (wet matter)/day, this corresponds to an AFB1 contamination in the final ration of 5500 ppb. Clinical signs of mycotoxicosis and mastitis were studied before, during, and after the mycotoxin administration period. The cows suffered from a lack of appetite, weight loss, decreased milk production and significant enzymatic variation during 1 to 3 weeks after ingesting AFB1. There were no signs of acute mastitis, nonetheless, the bacterial count in the milk increased during the consumption of the mycotoxin. There was an increase in the number of test positive to mastitis during the period following the last administration of the mycotoxin. Aflatoxin M1 was found in the milk within 3 to 6 hours after the consumption of AFB1and remains for a period of 72 hours after giving the last doses of mycotoxin. Aflatoxins B1 and M1 were found in the cow's urine 6 hours after the consumption of AFB1 and remain 72 to 120 hours after giving the last dose of the mycotoxin (Brown et al., 1981).

7.1.1.3.- AFB1 concentrations of 2000 to 2400 ppb in final ration given to 2 year old cows during a period of 7 months, caused serious hepatotoxicosis problems, as well as a significant reduction of milk production (Mirocha et al., 1977). 

Ochratoxin A (OTA) can be found as natural contaminant in cereals (mainly in barley and rice), cereal by-products, flour and peanut meal and in a series of human foods such as: green coffee, legumes, cheeses, smoked meats (ham, bacon, sausages), wines and others.

The main syndrome that produce is nephrotoxic but also can produce a liver disorder which is an accumulation of glycogen in hepatic and muscular tissue. The main organs affected are: the liver and kidney. Ochratoxin A is immunosuppressive. 

There is a lack of information on the toxic effects of OTA in dairy cows. This lack of data is probably due to the different capacities of the rumen protozoa microflora to easily metabolize OTA and hydrolyze it into ochratoxin-alpha, which is not toxic and does not degrade. These capacities vary because this microflora is affected by the kind of feed consumed by the cow.                             

There are studies in sheep demonstrating that the type of diet has a large influence on the metabolizing process of certain mycotoxins. Hence, a diet based on 100% hay takes the ruminal fluid to a pH of 7.1 and mycotoxins, such as ochratoxin A (OTA) are hydrolyzed to ochratoxin-alpha (non toxic) in only 0.6 hours. If the percentage of hay is reduced (70% hay) and the percentage of grain or concentrated feed is increased (30%), the pH of the ruminal fluid changes to 6.5, and the hydrolysis of OTA takes more time (1.3 hours). If the final ration contains 100% grain or is a concentrated feed, the hydrolysis takes up to 3.6 hours at a ruminal fluid pH of 5.7 (Xiao et al., 1991; Hohler et al., 1999). This could be applied to dairy cows, as some authors suggest (Muller et al., 1998).

Calves are deprived of these abilities because of non-functioning rumen, therefore, they are more sensitive to mycotoxins and can be affected by problems of nephrotoxicity caused by OTA.

In the same way can occur in dairy cows receiving a diet that carry to rumen fluid to low pH values, as before we have seen, since with these values may be that not only the transformation of OTA to OTA-alpha will be slow but also not occur.

There have been cases where concentrations of 1000 ppb of OTA in the final ration to dairy cows, have caused problems of feed intake reluctance, diarrhoea and nephrotoxicosis (Obremski et al, 2009). 

8 .- BIOTRANSFORMATION OF SOME MYCOTOXINS BY RUMEN FLUID ACTION (RUMEN PROTOZOA MICROFLORA  AND RUMEN BACTERIA)

8.1 .- It is considered the rumen fluid (rumen protozoa microflora and rumen bacteria), the first defense system against certain mycotoxins, it has an action on ZEN, OTA, T-2 toxin and DAS, however, this fluid has no action on AFB1, fumonisins and DON (Kiessling et al., 1984; Obremski et al, 2009). However, according to other authors, a part of the AFB1 being consumed through contaminated feed, is transformed in the rumen by the ruminal fluid into aflatoxicol, which is 18 times less toxic than AFB1. This is a reversible process, aflatoxicol can be reverted into AFB1, and it is the reservoir for AFB1 production, thus it is considered dangerous (Santi Devi Upadhaya et al., 2010).

We must emphasize and highlight that the rumen fluid biotransform ZEN in alpha and beta-zearalenol, of which alpha-zearalenol is 3-4 times more estrogenic than ZEN, and therefore can not be considered as a true detoxification reaction.

With respect to DON, some authors indicate that DON anaerobic incubation with rumen fluid of cow, produces the metabolite de-epoxy-deoxynivalenol (DOM-1), which is not toxic (Hedman and Pettersson, 1997) .

For T-2 toxin, DAS and other trichothecene mycotoxins, these biotransformation processes should be irreversible and should arrive in final chemical form DE-EPOXI, that is non-toxic form. If some of the intermediate compounds that are formed in these biotransformations remain as residues, can be as much or more toxic that the original mycotoxin. Thus in the case of the DAS and T-2, they are deacetylated and converted into monoacetoxyscirpenol and HT-2 toxin, respectively, so neither can be considered as a true detoxification reaction since these resulting compounds are toxic ( Kiessling et al., 1984). 

9 .- MAXIMUM TOLERABLE CONCENTRATIONS OF SOME MYCOTOXINS IN THE TOTAL MIXED RATION FOR DAIRY COWS

AFB1: 5-25 ppb (micrograms/kg); ZEN: 250 ppb; DON 250 ppb; T-2: 100 ppb; FB1: 35000 ppb (Gimeno, 2009).

In general, these values differ from those established by the European Union, which are as follows (complete diet containing 12% moisture basis) (Official Journal of the European Union, 2003, Official Journal of the European Union, 2006 ):

AFB1: 5 ppb; ZEN: 500 ppb; DON: 5000 ppb; FB1 + FB2: 50000 ppb.

The value of 5 ppb for aflatoxin B1, is Legislation. The other values are recommendations that have not yet become as Legislation.

As a result of contamination of milk with AFM1, the maximum value of AFB1 contamination should not exceed 5 ppb.

The tolerable maximum value of 25 ppb of AFB1, it is only oriented as a maximum value of security problems in their own cows mycotoxicosis, however we must return to the lowest value if there is milk production. 

10.1 .- As has been noted, there is great disparity between DON concentrations that caused mycotoxicosis in experimental tests and those found in a large number of field observations and also caused mycotoxicosis, the latter being substantially lower concentrations. One of the explanations could be given is that, when the studies are referred to the field observations, the DON mycotoxin can be accompanied by other mycotoxins that are not analyzed and also are contamining the ration. In this case, synergies between mycotoxins and/or cumulative effects can occur, while the problem is attributed exclusively to the  DON mycotoxin found in the analysis. However, this is only a hypothesis. Further studies must be necessaries in order to clarify this situation,

11.1.- There are Anti-Mycotoxins Additives (AMA) that act by enzymatic and/or bacterial processes inside the animal and bio-transform the mycotoxins in derivative compounds which can be in general and not always less toxic or not toxic. This action is similar to the biotransformation by the rumen fluid.

It is also very likely that some of these enzymes and/or bacteria do not biotransformed the AFB1, DON and fumonisins, as happens with rumen fluid.

11.2 .- Finally, it is very important take care and vigilance against these mycotoxins as such as was seen, they can cause serious problems that result in important and significant economic losses. 

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