Effect of deoxynivalenol on the levels of toll-like receptors 2 and 9 and their mRNA expression in enterocytes in the porcine large intestine: a preliminary study.

Deoxynivalenol (DON), one of the most prevalent mycotoxins in the world, and is capable of inducing immune disorders in humans and animals. The aim of this study was to determine the effect of feed contaminated with DON on the number of TLR2- and TLR9-positive cells and their mRNA expression in the porcine large intestine. The experiment was conducted on two equal groups of pigs (n=4). The experimental group (E) was administered feed contaminated with DON (1008 µg/kg of feed) for 6 weeks, and the control group (C) was administered non-contaminated feed over the same period of time. A decrease in the expression of TLR2 mRNA was noted in the cecum. The percentage of TLR9-positive enterocytes increased in the ascending colon and decreased in the cecum. The results of this study indicate that DON can modify the local immune response by changing the expression of TLRs.

Immunohistochemical evaluation of apoptosis and proliferation in the mucous membrane of selected uterine regions in pre-pubertal bitches exposed to low doses of zearalenone.

Mycotoxins are toxic secondary metabolites produced by fungi. Those biologically active compounds occur naturally and they include zearalenone (ZEN), a mycotoxin that contaminates plant material, including the ingredients used in the production of commercial dog food. The influence of monotonic, low-dose and long-term exposure to ZEN on pre-pubertal bitches has not been fully explored to date. This paper describes a 42-day experiment performed on clinically healthy female dogs aged approximately 70 days, with estimated body weight of 8 kg. The animals were randomly divided into two experimental groups (EI and EII) and a control group (C) of 10 animals each. Group EI received 50 µg ZEN/kg (of body weight) per os, group EII received 75 µg ZEN/kg BW per os, and the control group was administered placebo. The bitches were ovariohysterectomized at the end of the experiment (at around 112 days of age), and selected sections of the uterine wall were subjected to immunohistochemical analyses (TUNEL and PCNA). A shift towards higher apoptotic (AI) and proliferative index (PI) was observed, in particular in group EI. Higher AI and PI values were noted in the epithelium of all uterine regions analysed and in the uterine glands in the uterine horn proximal to the body of the uterus.

The effect of selected environmental Fusarium mycotoxins on the ovaries in the female wild boar (Sus scrofa).

The contamination of agricultural crops with Fusarium mycotoxins poses one of the greatest problems in food production. Wild boars live in specific habitats and are physiologically sensitive to Fusarium mycotoxins, therefore, they are an interesting model for studies investigating the effects of the discussed toxin, in particular under low-dose exposure. The objective of this study was to determine potential effects of Fusarium mycotoxins ingested with naturally contaminated food on reproductive function based on the proliferation and apoptotic indices of ovarian follicles in female wild boars. The experiment was conducted on 40 wild boars inhabiting north-eastern Poland. The effect of seasonal variations in the quantity and quality of ingested food on the concentrations of Fusarium mycotoxins and their metabolites in the blood of wild boars was analyzed. The observed differences in toxin levels were accompanied by changes in proliferation and apoptotic indices. Proliferation processes were most intense in autumn-winter and were least advanced in winter-spring. The intensity of apoptotic processes was inversely correlated with proliferation.

MicroRNA expression profiles in liver and colon of sexually immature gilts after exposure to Fusarium mycotoxins.

To improve our knowledge of the role of microRNAs (miRs) in responses of the porcine digestive system to two Fusarium mycotoxins, zearalenone (ZEN) and deoxynivalenol (DON), we examined the expression of 7 miRs (miR-9, miR-15a, miR-21, miR-34a, miR-122, miR-125b, and miR-192), previously found to be deregulated in diseased liver and colon cells. In this study, immature gilts were exposed to NOEL doses of ZEN (40 µg/kg/d), DON (12 µg/kg/d), ZEN + DON (40 + 12 µg/kg/d), andplacebo (negative control group) for 7, 14, 21, 28, 35, and 42 days. Before the treatment, expression levels of the selected miRs were measured in the liver, the duodenum, the jejunum, and the ascending and the descending colon of the gilts. Hierarchical clustering of the tissues by their miR expression profiles was consistent with what would be expected based on the anatomical locations and the physiological functions of the organs, suggesting that functions of the miRs are related to the specificities of the tissues in which they are expressed. A subset of 2 pairs of miRs (miR-21+miR-192 and miR-15a+miR-34a), which were assigned to two distinct clusters based on their tissue abundance, was then evaluated in the liver and the ascending and the descending colon during the treatment. The most meaningful results were obtained from the ascending colon, where a significant effect of the treatment was observed, suggesting that during the exposure to mycotoxins, the pathways involved in cell proliferation and survival were disordered. Changes in miR expression in the liver and the descending colon of the treated gilts were smaller, and were associated more with treatment duration than the exposure to ZEN, DON, or ZEN + DON. Further research should focus on identification of genes whose expression is regulated by these aberrantly expressed miRs. This should facili- tate understanding of the miRNA-regulated biological effects of mycotoxins.

Cecal enzyme activity in gilts following experimentally induced Fusarium mycotoxicosis.

The objective of the presented study was to examine the influence of Fusarium mycotoxins (zearalenone--ZEN and deoxynivalenol--DON), administered separately and in combination, on the activity of cecal enzymes (ß-glucosidase and ß-glucuronidase) in gilts which were fed fodder con- taminated with these mycotoxins. The activity of ß-glucosidase and ß-glucuronidase varied in the range of 0.170-1.236 µmol · h(-1) · mg(-1) and 8.701-96.704 µmol · h(-1) · mg(-1), respectively. In the first two weeks, the toxins had no significant effect on the activity of ß-glucosidase and ß-glucuronidase in the ascending and descending colon. After week 3 and later on, ZEN and DON administered as a mix- ture led to the highest increase in the activity of both enzymes. Administered separately, DON affected the activity of enzymes more than ZEN. From the third week of the experiment, an increase in the activity of CW ß-glucosidase and ß-glucuronidase was observed.

Evaluation of selected serum biochemical and haematological parameters in gilts exposed per os to 100 ppb of zearalenone.

Zearalenone (ZEN) widely contaminates animal feed of plant origin. The recommended safe concentrations of ZEN in feeds for various animal species are set mainly based on the mycotoxin's hormonal properties (NOEL). Our growing knowledge about biologically active concentrations of ZEN, molecular mechanisms and cells/tissues targeted by ZEN indicates that the harmful effects exerted by this mycotoxin on animals may be far greater than previously believed. This experiment was performed on pre-pubertal gilts divided into a control group (n=9) and an experimental group (ZEN, n=9). The control group received placebo, whereas the experimental group was administered ZEN at a dose of 0.1 mg/kg feed (equivalent to 5 µg/kg BW/day) for 42 days. On days 14, 28 and 42 blood samples were collected from the animals to determine the concentrations of selected zearalenols, serum biochemical and haematological parameters. Conjugated ZEN was found in the blood serum of the experimental gilts. Changes in the analysed biochemical parameters included a transient increase in albumin and cholesterol levels. A statistically significant increase in the concentrations of neutrophilic and acidophilic granulocytes was observed in the white blood cell system. The results indicate that long-term per os exposure of pre-pubertal gilts to low doses of ZEN (below NOEL) has a modulatory effect on liver function and white blood cells.

The effect of T-2 toxin on percentages of CD4+, CD8+, CD4+ CD8+ and CD21+ lymphocytes, and mRNA expression levels of selected cytokines in porcine ileal Peyer's patches.

The immune system is one of the main toxicity targets of the T-2 toxin. In view of scant research data demonstrating the effect of T-2 on cellular and humoral responses in gut-associated lymphoid tissue (GALT), this study set out to investigate the effects of chronic exposure to low doses of the T-2 toxin (200 microg T-2 toxin kg(-1) feed) on percentages of CD4+ and CD8+ T lymphocytes, CD4+/CD8+ double-positive T lymphocytes, CD21+ B cells, and IL-2, IFN-gamma, IL-4 and IL-10 mRNA expression levels in porcine ileal Peyer's patches. The investigated material comprised ileum sections sampled from piglets (aged 8-10 weeks, body weight of 15-18 kg) on days 14, 28 and 42 of the experiment. After 42 days of exposure to T-2, a significant drop in the quantity of the IL-10 product was observed (R = 0.94; S.E. 0.49-0.79; p < 0.001). A gradual decrease in the amount of IL-4 and IFN-gamma cytokine transcripts was found throughout the experiment, but the reported trend was not significant. On experimental days 14 and 42, a significant increase in the percentage of CD8+ T lymphocytes was observed in comparison with the control (p = 0.04 and p = 0.05, respectively), whereas on day 28, a significant decrease in the percentage of the above subpopulation was noted (p = 0.00). The percentage of CD21+ B cells in the experimental group decreased steadily in comparison with the control, and the observed drop was significant on days 28 and 42 (p = 0.06 and p = 0.00, respectively). On days 14 and 28, the percentages of CD4+ and CD8+ T lymphocytes were lower in the experimental animals than in the control group, and the drop reported on day 28 was statistically significant (p = 0.03).

Zearalenone and deoxynivalenol mycotoxicosis in dairy cattle herds.

Mycotoxin contaminations pose a growing problem in animal production from the economic and toxicological point of view. Clinical symptoms of mycotoxicosis are relatively unspecific, making the disease difficult to diagnose. This study presents a clinical case of dairy cattle infected with natural mycotoxins produced by fungi of the genus Fusarium (zearalenone [ZEA] and deoxynivalenol [DON]) in eastern Poland. In dead and infected cows, the presence of ZEA and DON was determined in the blood serum, significant changes were observed in blood morphological and biochemical profiles, extravasations and bowel inflammations were also observed. The results reported testify to an acute autoimmune process in the intestines as well as immunosuppression.

The effect of experimental, long-term exposure to low-dose zearalenone mycotoxicosis on the histological condition of ovaries in sexually immature gilts.

Farm animals are at risk of exposure to zearalenone (ZEA) in feedstuffs, which may lead to aberrations in their reproductive development, thereby adversely affecting production outcomes. The objective of this study was to determine the effect of long-term (48 days), per os administration of low ZEA doses (50% [20 µg ZEA/kg body weight (bw)] and 100% [40 µg ZEA/kg bw] NOAEL values) on anatomopathological changes in the ovaries of sexually immature gilts. The experiment involved 12 clinically healthy gilts aged 2 months with an initial body weight of about 40 kg and a determined immune status. The animals were randomly divided into two experimental groups (E1, E2) and a control group (C; all n = 4). Group E1 received per os 20 µg ZEA/kg bw for 48 days; group E2 received per os 40 µg ZEA/kg bw for 48 days; and group C received per os placebo for 48 days. Analytical samples of the mycotoxin were administered daily per os in gelatine capsules before morning feeding. Animals were sacrificed at the end of the experiment. The results of anatomopathological examinations of the ovaries in immature gilts subjected to long-term, low-dose ZEA exposure showed that ZEA-induced experimental hyperoestrogenism lowered the proliferative ability of granulosa cells of the ovarian follicle walls and of the connective tissue of the ovarian stroma, in particular at the lower ZEA dose.

The effect of exogenous proteases on selected hematological and serum biochemical parameters in boars.

The increasing interest in enzymatic growth promoters prompted this investigation of the effect of an enzymatic stimulating complex on selected hematological and serum biochemical parameters in boars. The enzymatic complex [five proteases (proteinases-endopeptidases) and two peptidases (exopeptidases) obtained by fermentation from Streptomyces fradiae] was added to diets for three months, at initial doses of 90 (group E1) and 120 (group E2) g/ton feed for the first seven days followed by 40 and 60 g/ton, respectively, in the subsequent weeks. The evaluation was based on selected hematological (RBC, WBC, HCT (Ht), HGB (Hb), MCV, MCH, and MCHC) and biochemical (AST, ALT, AP, Ca, Mg, urea, cholesterol, creatinine, inorganic phosphorus, and oxyglucose) parameters determined before and after the experimental period. The reported values were within physiological norms. Statistical differences were found between experimental groups with respect to WBC, HCT (Ht), MCV, MCH, AP, Ca, Mg, creatinine, and inorganic phosphorus. The study results indicate that the complex of proteolytic enzymes administered to boars caused no negative changes in their metabolic profiles. The statistically different hematological and serum biochemical parameters, while within normal limits, point to an early stage of microcytic anemia and heightened agitation that could be the result of intensified nitrogen metabolism.

Influence of low doses of deoxynivalenol on histopathology of selected organs of pigs.

Deoxynivalenol is one of mycotoxins that are most frequently determined in animal feed manufactured in Poland. The examination of histopathological lesions concomitant with deoxynivalenol intoxication is difficult because of the common, often synergistic, reaction of this mycotoxin with other toxins, such as zearalenone or ochratoxin A, which has a strong nephrotoxic activity. The possibility of estimating histopathological lesions in the course of intoxication with pure toxin at various doses is therefore of interest. Dosages used in this experiment relate to clinical cases observed in feeding the animals with whole ration feed obtained by processing feedingstuffs contaminated with Fusarium moulds. However, concerning the fact of one-shot administration of clinically pure toxin, the main question was if it was a sufficient dose to cause changes in the histopathological picture of gastrointestinal tract organs. The experiment was carried out on 12 nursery pigs of mixed breed (Polish White Large x Polish White Ear-pendent) with an average body weigh of 35 kg. The experimental nursery pigs were divided into 3 groups: group I (n=4)--control; group II (n=4)--DON administered at a dose of 0.2 mg/kg b.w.; group III (n=4)--DON administered at a dose of 0.4 mg/kg b.w. After slaughter of the animals, macroscopic examination was performed and segments of duodenum, jejunum, ileum, liver and mesenteric lymph nodes were sampled and assigned for histopathological examination. The results obtained equate to the clinically observed signs in swine production involving some nutrient metabolism disturbances in the gastrointestinal tract in the course of deoxynivalenol mycotoxicosis. Histopathological examination of segments of the duodenum, the jejunum, the ileum, the liver and the lymph nodes indicate that the regressive lesions are more expressed in the experimental group treated with the highest concentration of deoxynivalenol.

The evaluation of the efficacy of sodium carbonate as zearalenone destructor in feeding stuffs.

Contamination of feed with zearalenone (ZEA) is still a serious problem in farm animals feeding, especially in gilts, sensitive to this compound. The relative failure of current methods of decontamination and quality control lead us to look for new techniques. The commonly accepted method for breaking down ZEA was performed in controlled temperature and time conditions. Various sodium carbonate doses (0.5 - 4%) were added to feed naturally contaminated with ZEA (ZEA biosynthesis by F. graminearum isolates). These doses were found to be effective in in vitro studies. The addition of 2% sodium carbonate gave the best results in reducing the phytoestrogen in the feed.

Factors determining sensitivity of prepubertal gilts to hormonal influence of zearalenone.

Among large husbandry animals, swine are the most predisposed to zearalenone (ZEA) intoxication, mainly because cereal is an important component of their diet. Studies performed so far (in vivo, in vitro) suggest that ZEA and its metabolites, which may appear due to ZEA biotransformation (especially alpha-zearalenole; alpha-ZOL), can modify signaling cascades of endogenous sex steroids, through either receptor or non-receptor mechanisms. Of all age groups of swine, immature gilts are particularly predisposed to zearalenone intoxication, as manifested by the occurrence of genital tract tissue dysfunction on exposure to ZEA. The intensity of the adverse effects observed at either systemic or local level in gilts, when compared to sexually mature swine females, suggest that specific age-dependent physiological conditions may exist, which determine the high sensitivity of gilts to exogenous estrogen-like compounds, including ZEA.

Evaluation of the degree of pancreas activity in piglets from sows fed enzymatic stimulating complex.

One of the possibilities for estimating pancreas activity is the estimation of zymogene granule content in pancreatic follicular cells. In the present study, the degree of pancreatic activity was measured in piglets from sows receiving enzymatic stimulating complex throughout pregnancy and during the lactation period. The pancreas was collected for ultrastructural examination from 1-day-old and 21-day-old piglets. The enzyme preparation influenced the ultrastructural structure of the piglet pancreas, but the secretory cells in these animals did not confirm a more intensive course of creation and maturation processes. The accumulation of granules in extra-secretory pancreatic cells was observed, with a large volume of these granules and granular crinophagy observed in older piglets. The findings indicate a slow process of granule release, which may be the result of overproduction, lower requirements for enzymes contained in the granules, or both.

Histological estimation of the small intestine wall after administration of feed containing deoxynivalenol, T-2 toxin and zearalenone in the pig.

Fusarium spp. moulds are common in moderate climate regions of North America, Asia and Europe. They produce hepatotoxic and nephrotoxic mycotoxins, acting like estrogens, impairing hemopoesis and immunosuppressing. Actively dividing skin cells, lymphatic tissue, haemopoetic tissue and gastrointestinal tissue are the most sensitive for these trichothecenes action. The mucosal membrane of the gastrointestinal tract is the first barrier of the organism contacting with foreign antigens like feed proteins, natural toxins, saprophytic and pathogenic microflora and mycotoxins. The aim of this study was to perform histological estimation of the porcine small intestine after short term intoxication with low doses of deoxynivalenol (DON), T-2 toxin (T-2) and zearalenone (ZEA) obtained from wheat naturally contaminated with Fusarium moulds. Experimental pigs (n=5) were fed for 14 days feed containing DON, T-2 and ZEA (28.9, 11.5 and 33.2 microg kg(-1) of feed). On the last day of the experiment, the animals were euthanised and samples of the jejunum were collected for histological examination. In the experimental pigs, normally developed intestinal villi and crypts were found. However, number of acidophilic granulocytes in the mucous membrane and decreased numbers of goblet cells, increased numbers of endothelial lymphocytes and numerous plasma cells in intestinal epithelium was observed. On the surface of the intestinal epithelium the glycocalyx was poorly developed. The results obtained suggest that short term intoxication with low doses of DON, T-2 and ZEA does not cause significant changes in the histological structure of the small intestine in the pig. However, low concentrations of DON, T-2 and ZEA probably influence enterocytes metabolism and evoke inflammation of the mucous membrane of the small intestine.

Ultrastructural changes of ovarian follicle and corpus luteum after experimental zearalenone mycotoxicosis in bitch.

The aim of the study was to determine the influence of experimental zearalenone (ZEA) mycotoxicosis on the ultrastructure of ovaries in bitches receving zearalenone for 100 days per os in the anestrus phase. Experiment has been conducted on 9 sexually mature clinically healthy bitches. The sexually mature animals aged 1-3 years were in anestrus. The dogs have been divided into three groups: EI and EII, receiving ZEAper os in two dosages (25 microg/kg and 50 microg/kg b.w.) and control animals which received placebo per os. On the last day of experiment, ovariohysterectomy was performed in all the bitches and the uterine samples were submitted to ultrastructural analyses. The study has revealed that long lasting administration of higher dose of zearalenone causes ultrastructural changes in the granular layer of ovarian follicles, which showed due to their biological activity decrease. The changes in the granular layer, atrophy of intercellular connections and the appearance of intercellular spaces can stand for reduced activity of this layer. Changes in morphology of mitochondria, rough endoplasmic reticulum and Golgi apparatus sugest ZEA influence on cell metabolism and secretory processes. It seems therefore that in bitches and other monogastric animals, intoxications induced by this mycotoxin causes ultrastructural changes in granular cells of ovarian follicles.

Histopathological examination of ovaries in bitches after experimental zearalenone mycotoxicosis.

The present study deals with the influence of experimental ZEA mycotoxicosis on histopathological lesions in ovaries of bitches, which were administered zearalenone per os during anestrus phase for one hundred days. The experiment was performed on 9 sexually mature, clinically healthy bitches. The animals assigned into two experimental groups received zearalenone per os at two doses, 25 microg/kg b.w. and 50 microg/kg b.w., respectively: the bitches from control group received placebo per os. On the last day of zearalenone intoxication, the bitches were ovariohystorectomized. Histopathology and immunohistochemistry were performed. The study revealed that zearalenone and its metabolites caused profound regressive lesions: granular cells degeneration and atrophy. Numerous edemas and blood extravasations were also found. The intensity of these changes was significantly dose dependent. Furthermore, in ovarian cells and tissues of both experimental groups, no reaction for PCNA antigen was observed. In conclusion, zearalenone and its metabolites exerts unfavorable effects on the morphology of ovaries in bitches.

Histopathological and immunohistochemical examinations, and changes in proliferation activity of the uterus in bitches following zearalenon micotoxicosis.

The research was aimed at determining the impact of the experimental oral administration of zearalenon on the uterus in bitches. The study was carried out on 9 sexually mature 1 to 3 year-old bitches that were divided into three groups: two experimental ones and a control group. The experimental animals were orally administered zearalenon at 25 microg/kg BW (body weight) (group DI; n=3) and 50 microg/kg BW (group DII; n=3) throughout 100 days while the control bitches (group K; n=3) were administered placebo. After the period of 100 days, ovariohysterectomy was performed, and the uterine samples were submitted to histopathological and immunohistochemical examinations in order to determine the structure of the wall and the presence of PCNA antigen. In the experimental animals, the histopathological examinations revealed regressive lesions, such as degeneration and atrophia within the endometrium and myometrium, and local circulatory disorders, i.e. edema and extravasation. These lesions were not reported in the controls. In the bitches administered zearalenon, the tissues did not express any proliferative activity as determined by the presence of PCNA. It is assumed that, following the administration of zearalenon for 100 days orally to bitches, disruption of the structures within the wall of the uterus (i.e. degeneration, atrophy, edema, and extravasation within the endometrium and myometrium) develops. This research proves that zearalenon ingested by bitches may lead to dysfunction of the uterus and cause disruption of its structures.

The impact of zearalenone on the level of the selected estrogens in blood serum of sexually immature gilts.

The aim of the study was to evaluate the influence of low dose (LOEL - lowest observed effect level) of zearalenone (200 microg/kg b.w.), applied per os for 7 days (short-term intoxication), on sexual behavior, concentration of the examined xenobiotic and its metabolite and selected estrogens in sexually immature gilts: ovariohysterectomised (group D1) and intact (group D2) animals. Clinical signs of oestrus (reddening, oedema and hyperaemia of the vulva and serorhoea from the reproductive tract--lack of standing reflex) were obserwed in group D1 on day 6 and in group D2 on day 4 of the experiment. Laboratory analyses of blood plasma were carried out determine the presence of zearalenone and alpha-zearalenole. They revealed an increase in the level of alpha-zearalenol before the oestrus, decrease in total amount of both examined substances on day when the oestrus appeared and increase in the level of both examined xenobiotics in the post oestrus period together with the higher share of zearalenone. Medium concentrations of estrone and estradiol within the borders of method determination in the majority of periods examined. Higher levels of estrone (32.0 pg/ml) were found on day 4, in the group D2 and estradiol (6.5 pg/ml) on day 6 in the D2 group. The presents study revealed that zearalenone applied per os at LOEL dose causes the incidence of apparent sexual readiness (without standing reflex) in sexually immature gilts with the somatically immature reproductive system.

Level of zearalenone in blood serum and lesions in ovarian follicles of sexually immature gilts in the course of zearalenone micotoxicosis.

The aim of the study was to determine how a low dose of zearalenone applied orally for eight days influences the level of zearalenone (ZEN) and alpha-zearalenole in blood plasma and causes the occurrence of histopathological changes in the cells of the ovarian follicles in sexually immature gilts. The animals were divided into 2 groups (control, C; n = 4 and experimental, E; n = 4). The gilts from group E were treated daily with zearalenone at a dose of 200 microg/kg b.w. The level of zearalenone and alpha-zearalenole (ZON as the sum of the levels of both zearalenone and alpha-zearalenole) was measured daily. On day eight of the experiment the animals were sacrificed and their ovaries were taken for histopathological examination. The tissue sections obtained were HE- and PAS-stained according to McManus. The presence of PCNA antigen was also estimated. The highest concentration of ZON was noted on day 5 in group E (8.16 +/- 2.49 ng/ml). External estrus symptoms without standing reflex were observed in group E on day 4. In group C there were no pathological changes in the ovaries. In group E, a few ovarian follicles were found, but they were located in the cortical layer. They were filled with a liquid substance rich in protein and without the granulosa layer. There was disintegration with apoptotic-like changes of the PCNA-negative cells in the granulosa layer of single mature follicles. On day 4 the dose of zearalenone caused disturbances in the process of development and maturation of some of the best developed ovarian follicles. This probably occurred through the activation of on apoptosis-like process of the granulosa cells with simultaneous manifestation of estrus without standing reflex.