In dairy cattle farming, good-quality feed and its appropriate dosage are the basic factor ensuring proper milk yield and cow health in the herd and largely determine profitability. Despite the efforts of farmers and their use of modern methods in the production of feed, the contamination of feed with mycotoxins remains a serious problem. Mycotoxins may already form at the stage of feed plant growth or during the improper transport of feed, but most often contaminate feed during its storage in inappropriate conditions favouring the development of mould fungi, whose metabolites are the main source of fodder contamination. The danger of feed contamination with mycotoxins is that even in low concentrations, they disturb the functioning of the body (
Composition of the total mixed (TMR) and dry mass (DM) daily feed ration for lactating cows (kilograms/cow/day)
Dose component | Amount per cow, daily | |
---|---|---|
kg TMR | kg DM | |
Maize silage | 25.0 | 8.8 |
Haylage | 8.0 | 3.2 |
Ensiled brewer’s spent grain | 8.0 | 2.7 |
Wheat straw | 0.8 | 0.7 |
Ensiled maize grain | 2.5 | 1.7 |
Barley ground grain | 1.5 | 1.3 |
Triticale ground grain | 1.5 | 1.3 |
Ground rapeseeds | 2.7 | 2.2 |
Extracted soyabean meal | 2.0 | 1.7 |
Glycerine | 0.3 | 0.24 |
Vitamin and mineral mixture | 0.2 | 0.18 |
Sodium bicarbonate | 0.2 | 0.2 |
Calcium carbonate | 0.05 | 0.05 |
Total | 52.73 | 24.27 |
Health problems were reported in one herd because many cows had non-specific symptoms of unknown origin: a decreased appetite, diarrhoea, weight loss, lameness, abomasum dislocation, a decreased milk yield, an increased number of somatic cells in milk, and infertility. The toxicological study of this herd’s TMR showed the presence of 32 parts per billion (ppb) ZEN and 769 ppb DON. The mycotoxin content was assessed by the Biomin laboratory (Herzogenburg, Austria) with liquid chromatography combined with tandem mass spectrometry. The average yield for cows in this herd determined in the 305-day lactation period was 6,161–6,725 kg of milk with a fat content of 4.27–4.83% and a protein content of 3.21– 3.28%. Ten cows were selected for which the ZEN content in their blood serum was confirmed at 14.30 ± 3.64 and the DON content at 20.92 ± 5.94 ng/mL. These cows constituted the experimental group (Exp). The plasma concentrations of ZEA and DON were determined by combined separation techniques with the use of immunoaffinity columns and high-performance liquid chromatography (HPLC) with fluorescence detection (27, 44).
The second herd had no health problems. Ten clinically healthy cows were selected as the control group (Con). In addition, standard haematological tests comprising counts of white blood cells, red blood cells, thrombocytes, granulocytes and lymphocytes and determination of haematocrit, haemoglobin, mean corpuscular volume, mean corpuscular haemoglobin and mean corpuscular haemoglobin concentration were performed on selected cows to confirm their general health (45). The average yield in this herd determined in the 305-day lactation was 8,571–8,670 kg of milk with a fat content of 4.51–4.14% and a protein content of 3.14–3.42%.
All selected cows were in the period of 60 +/− 20 days postpartum. The cows were in their second to fourth lactation. The animals’ body condition was rated as good or very good, reflected in body condition scores (BCS) of 3.0–3.25 in Exp cows and 3.5–4.0 in Con cows on a five-point scale (37). Reproductive system monitoring in the herds was carried out regularly at monthly intervals by rectal examination combined with ultrasonography. To cows with no complications during parturition and no signs of inflammation a synchronisation protocol of oestrus and ovulation (presynch-ovsynch protocol) was applied, and artificial insemination (AI) with frozen semen was performed on those animals. Cows with uterine inflammation were properly treated and subsequently subjected to the synchronisation protocol of oestrus and ovulation and AI. Cows with ovarian cycle disturbances were treated individually according to the diagnosed cause. Pregnancy testing was performed routinely approximately 30 to 40 days after insemination by rectal examination combined with ultrasonography. The expected date of parturition was determined by adding 280 days to the day of artificial insemination and was also supported by the pregnancy diagnosis.
The administration of the Mycofix Plus 3E preparation (Biomin, Herzogenburg, Austria) was started in the herd where the presence of mycotoxins was confirmed after the first drawing of blood for testing. The composition of the preparation is not disclosed; the manufacturer only provides general information that the preparation contains specific toxin-eliminating enzymes, adsorbents, carefully selected plants and algae extracts. Mycofix was administered at a dose of 10 g/cow per day for three months as an addition to the feed. A measured dose of the preparation appropriate for the number of cows was added to the fodder cart, which mixed the TMR well with Mycofix, and then the mixture was provided to all cows in the herd.
In this research, the concentrations of the TNF-α cytokine, the IL-6 and IL-10 interleukins, and the SAA and Hp APPs were assessed. These indices were analysed in the blood serum of dairy cows suffering naturally acquired mycotoxicosis caused by the consumption of food contaminated with ZEN and DON. The cows for the experimental group were selected from a herd in which various non-specific disease symptoms were observed, such as a decreased appetite, diarrhoea, progressive weight loss, a decreasing milk yield, an increased number of somatic cells in milk, lameness and decreased fertility. These symptoms appeared and gradually intensified, and treatment of symptoms and attempts to adjust the nutritional dose did not bring the expected improvement in the efficiency or health of the cows in the herd. It was recommended to test for the presence of mycotoxins in feed, and the positive test result was something of a surprise, as no link between these non-specific symptoms and mycotoxicosis had been made beforehand. It is generally known that ruminants are more resistant to mycotoxins than other animals because the microbiota of their rumen decompose and deactivate mycotoxins quite effectively, and thus protect them from mycotoxicosis (26, 47). Using their antibacterial, antiprotozoal and antifungal properties, various mycotoxins are able to quantify and modify the rumen bacterial flora (7, 10, 29). It has been documented that exposing animals to fusarium mycotoxins such as ZEN and DON, even at low doses, adversely affects the stability of the gastrointestinal biocenosis, which is an important indicator of animal health (35). The modified rumen microbiota are not able to ensure proper breakdown and use of the food ration or to properly deactivate the mycotoxins that enter further parts of the gastrointestinal tract and are absorbed into blood as in monogastric animals (7, 28). Therefore, the initially observed disease symptoms differ from typical mycotoxin poisoning and resemble malnutrition with accompanying dysbacteriosis, leading to acidosis, slowing down the digestive processes of feed with the consequences of weight loss and mild diarrhoea with undigested fibre in the stool. Long-term consumption of fodder with mycotoxins leads to a decrease in milk production and an increase in the number of somatic cells in milk. There are also cases of laminitis and infectious diseases (6, 12). The reproductive performance of cows in sick herds also decreases as a result of significant metabolic and hormonal changes and the occurrence of a negative energy balance in animals (46).
Most of the described symptoms were observed in the herd from which the experimental cows were chosen. The three-month observation of the herd showed that the administration of Mycofix was associated with a gradual improvement in the health of the cows in the herd and with the gradual disappearance of the symptoms described. After this time, the number of health disorders in the cows was comparable to the number in the herd from which the control cows were picked. The assessed immunological parameters in the first haematological analysis of cows with mycotoxicosis showed that the concentration of all the evaluated cytokines and Hp in the experimental cows was very high and significantly higher than that of the control cows. Undoubtedly, it was a reaction related to the influence of mycotoxins on the lymphatic tissue of the gastrointestinal tract and possibly also on immunologically competent cells in the entire organism of the cow. The effect of mycotoxins is supported by the parameters of the experimental cows exceeding those of the control cows by as much as fifteen times (see Hp results). This suggests the existence of a very strong body immune response caused by the stimulation of the immune system, with simultaneous pro-inflammatory processes (quantitative increases in TNF-α and IL-6) and anti-inflammatory processes (an increase in IL-10 concentration). Studies by other authors carried out in pigs after administration of even low doses of ZEN also showed a simultaneous significant increase in the concentration of pro-inflammatory (IL-4) and anti-inflammatory (IL-10) interleukins in the lymphoid tissue derived from intestinal Peyer’s patches (30). These antagonistic processes often act simultaneously as pro-inflammatory defence mechanisms that protect the animal against pathogens and anti-inflammatory mechanisms that prevent the body’s own tissues from harming themselves through overactivity of the immune system. The increase in the amount of anti-inflammatory IL-10 may result from the increasing number of TCD4+Foxp3+ and TCD8+CD25+ regulatory lymphocytes, which have suppressor functions that inhibit inflammatory responses (3, 4, 18). By secreting IL-10, these lymphocytes function protectively for body tissues because of the inhibitory effect on immunocompetent effector cells, which weakens autoimmune reactions (32, 39). In the event of triggering regulatory processes, as we observed in our research, the tolerance of the immune system to a strong stimulant – mycotoxins – increases, thanks to which it is possible for the bovine immune system to maintain homeostasis. Otherwise, prolonged exposure to fungal toxins could lead to self-damage in tissues and organs (40). It is not known how long the cows studied were exposed to mycotoxins. However, observing the chronicity of the clinical symptoms described above and the specificity of changes in the assessed immunological parameters, we can assume that the consumption of mycotoxins by cows lasted for quite a long time. The activation of regulatory processes initiated protectively as a result of an acute inflammatory reaction, especially the very high level of Hp and normal SAA in experimental cows, proves an ongoing chronic inflammatory process to be in operation. Acute phase proteins characteristic of cattle such as SAA and Hp are mainly produced by hepatocytes in the liver in response to pro-inflammatory cytokines (
After three months of administering Mycofix, pro-inflammatory processes were significantly suppressed, which was documented by a decrease in the TNF-α and IL-6 values. In contrast, the level of IL-10, despite its decrease, was still much higher than the level in the control group. This may indicate ongoing anti-inflammatory processes that protect against autoimmune reactions. It is difficult to explain what the cause of the maintenance of anti-inflammatory processes was; it is possible that despite mycotoxins’ limited absorption, they were still affecting the bovine organism. It is also possible that the effect of Mycofix was insufficient or that the dose of this preparation used in our research (10g/cow/day) turned out to be too low, not fully protecting against the penetration of mycotoxins from the gastrointestinal tract into the blood. The limited inactivation of mycotoxins in the rumen and the passage of their small amounts to the distal parts of the gastrointestinal tract may no longer cause any symptoms, but may allow them to affect the gastrointestinal immune system and gut-associated lymphoid tissue. Such an explanation is very probable, because after three months of administration of Mycofix, an elevated value of IL-6, and especially a high concentration of Hp, was maintained, which indicated a chronic, mildly intensified inflammatory process. It is also possible that longer time or a prolonged period of use of the mycotoxin deactivator is required for IL-10 and Hp levels to decrease. It also cannot be ruled out that the increase in the level of cytokines and APP in cows in our studies was a consequence of other comorbidities, because, according to the literature data, these biomarkers are non-specific and may also increase in many other disease entities’ courses (3, 4, 42). In the studies cited, the increase in the level of cytokines only coincided with some disease entities, and only for some may they be markers. Our research confirmed that the increase in the level of cytokines and Hp coexisted with mycotoxicosis in cows. However, the authors want to draw attention to the emergence of various non-specific symptoms in the cows with mycotoxicosis, which are unlikely to be caused by mycotoxins. We can assume that too strongly activated nonspecific immune mechanisms are responsible for at least some of these symptoms. There are studies that have unequivocally shown that cytokines actively participate in and can even cause many diseases, both metabolic and non-metabolic (25, 36). It has been shown that there are close correlations between the increase in IL-6 concentration and disturbance of lipid and protein metabolism, abnormal fatty acid metabolism and even the occurrence of oxidative stress. Dysfunctions in one or all of these processes can lead to metabolic diseases, liver damage and liver failure (25). Similarly, as a pro-inflammatory cytokine, TNF-α in humans is involved in the pathogenesis of many metabolic diseases, mainly chronic ones such as diabetes, contributing to insulin resistance and disorders of lipid metabolism (36). In cows with fatty liver syndrome, a correlation was also described between the increase in TNF-α and insulin resistance as well as disturbance of lipid metabolism (1, 31). In addition, IL-6 and TNF-α can stimulate the breakdown of adipose tissue in the body by lowering feed consumption, inducing insulin resistance and directly initiating lipolysis (23).
Taking into account the above information, it should be emphasised that the level of cytokines and APP in cows during mycotoxicosis is an important descriptor of the state and reactivity of the immune system, and even the intensity of the ongoing inflammatory reaction. However, the value of the studied indicators as markers of mycotoxicosis in cows is limited, mainly because their levels also increase in many other disease entities. In the presented study, there are also too few data to clearly indicate that the increase in the concentration of cytokines and APP may be the cause of medical conditions in cows with mycotoxicosis. Only the close relationship between mycotoxicosis, an increase in cytokines and Hp and the emergence of non-specific clinical symptoms can be observed with certainty. On the other hand, the presented research clearly revealed that despite the administration of the mycotoxin absorbent in the amount recommended by the producer and the recovery of the cows to full health, the levels of IL-10, Hp and IL-6 were still significantly higher than those in the control group. Therefore, the determination of the level of cytokines and APP seems to be a very useful and precise tool for the assessment and application of an appropriate dose of the absorbent or the evaluation of its effectiveness.
Our research shows that the assessment of the level of cytokines and APP in cows with mycotoxicosis ably elucidates the state and reactivity of the immune system and the intensity of any ongoing inflammatory reaction. Simultaneous stimulation of antagonistic pro-inflammatory and anti-inflammatory processes has been shown, and the processes serve to protect the animal against pathogens and prevent autoimmune reactions. A close relationship has also been demonstrated between mycotoxicosis, an increase in the level of cytokines and Hp and the emerging non-specific clinical symptoms. However, the data are inadequate to indicate that upregulation of cytokines and APP provoke diseases in cows with mycotoxicosis. Cytokine and APP level assays may be treated as precise techniques for the assignment of the appropriate dose of mycotoxin absorbent or the investigation of how well it alleviates mycotoxicosis, because despite its use and the disappearance of clinical symptoms in the tested cows, high levels of IL-10, Hp and IL-6 were maintained.