Ana Paula Frederico Rodrigues Loureiro Bracarense

Chronic ingestion of deoxynivalenol and fumonisin, alone or in interaction, induces morphological and immunological changes in the intestine of piglets

Deoxynivalenol (DON) and fumonisins (FB) are mycotoxins produced by Fusarium species, which naturally co-occur in animal diets. The gastrointestinal tract represents the first barrier met by exogenous food/feed compounds. The purpose of the present study was to investigate the effects of DON and FB, alone and in combination, on some intestinal parameters, including morphology, histology, expression of cytokines and junction proteins. A total of twenty-four 5-week-old piglets were randomly assigned to four different groups, receiving separate diets for 5 weeks: a control diet; a diet contaminated with either DON (3 mg/kg) or FB (6 mg/kg); or both toxins. Chronic ingestion of these contaminated diets induced morphological and histological changes, as shown by the atrophy and fusion of villi, the decreased villi height and cell proliferation in the jejunum, and by the reduced number of goblet cells and lymphocytes. At the end of the experiment, the expression levels of several cytokines were measured by RT-PCR and some of them (TNF-α, IL-1β, IFN-γ, IL-6 and IL-10) were significantly up-regulated in the ileum or the jejunum. In addition, the ingestion of contaminated diets reduced the expression of the adherent junction protein E-cadherin and the tight junction protein occludin in the intestine. When animals were fed with a co-contaminated diet (DON+FB), several types of interactions were observed depending on the parameters and segments assessed: synergistic (immune cells); additive (cytokines and junction protein expression); less than additive (histological lesions and cytokine expression); antagonistic (immune cells and cytokine expression). Taken together, the present data provide strong evidence that chronic ingestion of low doses of mycotoxins alters the intestine, and thus may predispose animals to infections by enteric pathogens.

The low intestinal and hepatic toxicity of hydrolyzed fumonisin B1 correlates with its inability to alter the metabolism of sphingolipids

Fumonisins are mycotoxins frequently found as natural contaminants in maize, where they are produced by the plant pathogen Fusarium verticillioides. They are toxic to animals and exert their effects through mechanisms involving disruption of sphingolipid metabolism. Fumonisin B₁ (FB₁) is the predominant fumonisin in this family. FB₁ is converted to its hydrolyzed analogs HFB₁, by alkaline cooking (nixtamalization) or through enzymatic degradation. The toxicity of HFB₁ is poorly documented especially at the intestinal level. The objectives of this study were to compare the toxicity of HFB₁ and FB₁ and to assess the ability of these toxins to disrupt sphingolipids biosynthesis. HFB₁ was obtained by a deesterification of FB₁ with a carboxylesterase. Piglets, animals highly sensitive to FB₁, were exposed by gavage for 2 weeks to 2.8 μmol FB₁ or HFB₁/kg body weight/day. FB₁ induced hepatotoxicity as indicated by the lesion score, the level of several biochemical analytes and the expression of inflammatory cytokines. Similarly, FB₁ impaired the morphology of the different segments of the small intestine, reduced villi height and modified intestinal cytokine expression. By contrast, HFB₁ did not trigger hepatotoxicity, did not impair intestinal morphology and slightly modified the intestinal immune response. This low toxicity of HFB₁ correlates with a weak alteration of the sphinganine/sphingosine ratio in the liver and in the plasma. Taken together, these data demonstrate that HFB₁ does not cause intestinal or hepatic toxicity in the sensitive pig model and only slightly disrupts sphingolipids metabolism. This finding suggests that conversion to HFB₁ could be a good strategy to reduce FB₁ exposure.

The food contaminant deoxynivalenol activates the mitogen activated protein kinases in the intestine: Interest of ex vivo models as an alternative to in vivo experiments

Trichothecenes induce changes in the intestinal barrier function through decreased expression of cell junction proteins and apoptosis of enterocytes. The mitogen activated protein kinases (MAPK) play an important role in the signaling pathways of cell turnover and differentiation. Using ex vivo and in vivo approaches, the purpose of this study was to investigate the ability of low doses of DON to induce histological changes in the intestine and to activate the MAPK ERK 1/2, p38 and JNK. Twelve weaning piglets received during four weeks a control diet or a DON-contaminated diet (2.3 mg DON/kg feed). Six weaning piglets were used to prepare jejunal explants (ex vivo model). Explants were exposed during 4 h to vehicle, 5 or 10 μM DON. Intestinal changes were graded using a histological score. Pigs fed a DON-diet and explants exposed to DON showed a significant decrease in the jejunal score. In both models, the toxin significantly enhanced phosphorylation of ERK 1/2 and p38, whereas the increased phosphorylation of JNK was non significant. Taken together these results indicate that in vivo or ex vivo exposure of intestinal tissue to DON lead to similar intestinal lesions and activation of MAPK. These effects could impair the homeostasis of intestinal tissue in the aspects of barrier function and immune protection. The similarity of the in vivo and ex vivo results provides also strong evidence that the jejunal explant model is a good alternative for toxicological studies in intestinal tissue.

Microbial biotransformation of DON: molecular basis for reduced toxicity

Bacteria are able to de-epoxidize or epimerize deoxynivalenol (DON), a mycotoxin, to deepoxy-deoxynivalenol (deepoxy-DON or DOM-1) or 3-epi-deoxynivalenol (3-epi-DON), respectively. Using different approaches, the intestinal toxicity of 3 molecules was compared and the molecular basis for the reduced toxicity investigated. In human intestinal epithelial cells, deepoxy-DON and 3-epi-DON were not cytotoxic, did not change the oxygen consumption or impair the barrier function. In intestinal explants, exposure for 4 hours to 10 μM DON induced intestinal lesions not seen in explants treated with deepoxy-DON and 3-epi-DON. A pan-genomic transcriptomic analysis was performed on intestinal explants. 747 probes, representing 323 genes, were differentially expressed, between DON-treated and control explants. By contrast, no differentially expressed genes were observed between control, deepoxy-DON and 3-epi-DON treated explants. Both DON and its biotransformation products were able to fit into the pockets of the A-site of the ribosome peptidyl transferase center. DON forms three hydrogen bonds with the A site and activates MAPKinases (mitogen-activated protein kinases). By contrast deepoxy-DON and 3-epi-DON only form two hydrogen bonds and do not activate MAPKinases. Our data demonstrate that bacterial de-epoxidation or epimerization of DON altered their interaction with the ribosome, leading to an absence of MAPKinase activation and a reduced toxicity.

Molecular Detection of Canine Distemper Virus and the Immunohistochemical Characterization of the Neurologic Lesions in Naturally Occurring Old Dog Encephalitis

The current article describes a spontaneous case of old dog encephalitis (ODE) in a 7–year-old, intact, female Miniature Schnauzer dog from Londrina, Paraná, southern Brazil. Unlike conventional distemper encephalomyelitis, ODE is a poorly understood and extremely rare manifestation of Canine distemper virus (CDV) infection. The dog was presented with progressive clinical manifestations consistent with cerebral dysfunction. Briefly, histopathologic lesions were restricted to the forebrain and included chronic multifocal lymphoplasmacytic encephalitis with extensive perivascular cuffing, astrocytosis, and intranuclear inclusions within astrocytes and giant cells, with both intracytoplasmic and intranuclear inclusions. Immunohistochemistry (IHC) was used to identify the antigens of the nucleoprotein (NP) of CDV and to detect cluster of differentiation (CD)3, CD79a, macrophage (MAC) 387, glial fibrillary acidic protein, and vimentin to characterize the neuroparenchymal lesions. By IHC, CDV NP was demonstrated predominantly within neurons and astrocytes. Cells that formed perivascular cuffs and some astrocyte-like cells reacted intensely to vimentin. Reverse transcription polymerase chain reaction assay from brain sections further confirmed a role for CDV in this disease by the amplification and partial sequence analysis of the NP gene. These findings confirmed simultaneous detection of CDV in ODE by IHC and molecular assays. In addition, results of the current study could contribute to the neuropathologic characterization of this rare manifestation of CDV.

Deoxynivalenol alone or in combination with nivalenol and zearalenone induce systemic histological changes in pigs

Deoxynivalenol (DON), nivalenol (NIV) and zearalenone (ZEA) are mycotoxins commonly produced by Fusarium species. The purpose of the present study was to investigate the effects of DON alone and in combination with NIV and ZEA on several parameters including weight gain and histological aspects of pigs submitted to chronic intoxication. Twenty, 5-week-old piglets received for 28 days one of the following diets: a control diet, a diet mono- contaminated with DON (1.5mg/kg), a diet multi-contaminated with DON (2mg/kg)+NIV (1.3mg/kg)+ZEA (1.5mg/kg) or a diet contaminated with DON (3mg/kg)+NIV (1.3mg/kg)+ZEA (1.5mg/kg). Animals fed the multi-contaminated diets presented a significant decrease in weight gain over the total period. The chronic ingestion of the contaminated diets induced a significant increase on histological changes on the intestine, liver and lymphoid organs. In addition, a significant increase on lymphocyte apoptosis was observed in lymph nodes and spleen in the animals receiving the contaminated diets. These data provide a better understanding of the possible effects of Fusarium toxins, alone or in combinations on the morphology of the intestine and lymphoid organs, which would contribute to the risk assessment of these toxins.

Deoxynivanelol and fumonisin, alone or in combination, induce changes on intestinal junction complexes and in E-cadherin expression.

Fusariotoxins such as fumonisin B1 (FB1) and deoxynivalenol (DON) cause deleterious effects on the intestine of pigs. The aim of this study was to evaluate the effect of these mycotoxins, alone and in combination, on jejunal explants from piglets, using histological, immunohistochemical and ultrastructural assays. Five 24-day old pigs were used for sampling the explants. Forty-eight explants were sampled from each animal. Explants were incubated for 4 hours in culture medium and medium containing FB1 (100 µM), DON (10 µM) and both mycotoxins (100 µM FB1 plus 10 µM DON). Exposure to all treatments induced a significant decrease in the normal intestinal morphology and in the number of goblet cells, which were more severe in explants exposed to DON and both mycotoxins. A significant reduction in villus height occurred in groups treated with DON and with co-contamination. Expression of E-cadherin was significantly reduced in explants exposed to FB1 (40%), DON (93%) and FB1 plus DON (100%). The ultrastructural assay showed increased intercellular spaces and no junction complexes on enterocytes exposed to mycotoxins. The present data indicate that FB1 and DON induce changes in cell junction complexes that could contribute to increase paracellular permeability. The ex vivo model was adequate for assessing intestinal toxicity induced by exposure of isolated or associated concentrations of 100 µM of FB1 and 10 µM of DON.

Biotransformation approaches to alleviate the effects induced by fusarium mycotoxins in swine.

Mycotoxin mitigation is of major interest as ingestion of mycotoxins results in poor animal health, decreased productivity, as well as substantial economic losses. A feed additive (FA) consisting of a combination of bacteria (Eubacterium BBSH797) and enzyme (fumonisin esterase FumD) was tested in pigs for its ability to neutralize the effects of mono- and co-contaminated diets with deoxynivalenol (DON) and fumonisins (FB) on hematology, biochemistry, tissue morphology, and immune response. Forty-eight animals, allocated into eight groups, received one of eight diets for 35 days: a control diet, a diet contaminated with either DON (3 mg/kg) or FB (6 mg/kg), or both toxins, and the same four diets with FA. Inclusion of FA restored the circulating number of neutrophils of piglets fed the FB and DON + FB diets. Similarly, FA counteracted the minor changes observed on plasma concentrations of albumin and creatinine. In lung, the lesions induced by the ingestion of FB in mono- and co-contaminated diets were no longer observed after addition of FA in these diets. Lesions recorded in the liver of pigs fed either of the contaminated diets with FA were partly reduced, and the increased hepatocyte proliferation was totally neutralized when FA was present in the co-contaminated diet. After 35 days of exposure, the development of the vaccinal response was significantly improved in animals fed diets supplemented with FA, as shown by results of lymphocyte proliferation, cytokine expression in spleen, and the production of specific Ig. Similarly, in jejunum of animals fed diets with FA, occurrence of lesions and upregulation of pro-inflammatory cytokines were much less obvious. The ameliorative effects provided by FA suggest that this approach would be suitable in the control of DON and FB that commonly co-occur in feed.

Leptospirosis in slaughtered sows: serological and histopathological investigation

Swine leptospirosis is recognized world wide as an important cause of reproductive failure. Serum and histo-pathological examinations have been constantly used to diagnose this disease. This study was carried out on slaughtered sows from Northern Parana State to compare serum findings with kidney, liver, ovary and uterus histo-pathological examinations. The microscopic serum-agglutination carried out on 36 animals detected 24 with titles > 100 and 12 negative ( 100 serological titles and five from the negative group. The results showed that all the 16 animals had indicative lesions in the liver or in one of the kidneys. There were no lesions in the ovaries or uterus. Microscopic serum-agglutination title readings between 20 and 80 were detected in animals of the group considered negative with title < 100.

Nivalenol has a greater impact than deoxynivalenol on pig jejunum mucosa in vitro on explants and in vivo on intestinal loops

The mycotoxins deoxynivalenol (DON) and nivalenol (NIV), worldwide cereal contaminants, raise concerns for animal and human gut health, following contaminated food or feed ingestion. The impact of DON and NIV on intestinal mucosa was investigated after acute exposure, in vitro and in vivo. The histological changes induced by DON and NIV were analyzed after four-hour exposure on pig jejunum explants and loops, two alternative models. On explants, dose-dependent increases in the histological changes were induced by DON and NIV, with a two-fold increase in lesion severity at 10 µM NIV. On loops, NIV had a greater impact on the mucosa than DON. The overall proliferative cells showed 30% and 13% decrease after NIV and DON exposure, respectively, and NIV increased the proliferative index of crypt enterocytes. NIV also increased apoptosis at the top of villi and reduced by almost half the proliferative/apoptotic cell ratio. Lamina propria cells (mainly immune cells) were more sensitive than enterocytes (epithelial cells) to apoptosis induced by NIV. Our results demonstrate a greater impact of NIV than DON on the intestinal mucosa, both in vitro and in vivo, and highlight the need of a specific hazard characterization for NIV risk assessment.