From genomics to metabolomics, moving toward an integrated strategy for the discovery of fungal secondary metabolites.

Fungal secondary metabolites are defined by bioactive properties that ensure adaptation of the fungus to its environment. Although some of these natural products are promising sources of new lead compounds especially for the pharmaceutical industry, others pose risks to human and animal health. The identification of secondary metabolites is critical to assessing both the utility and risks of these compounds. Since fungi present biological specificities different from other microorganisms, this review covers the different strategies specifically used in fungal studies to perform this critical identification. Strategies focused on the direct detection of the secondary metabolites are firstly reported. Particularly, advances in high-throughput untargeted metabolomics have led to the generation of large datasets whose exploitation and interpretation generally require bioinformatics tools. Then, the genome-based methods used to study the entire fungal metabolic potential are reported. Transcriptomic and proteomic tools used in the discovery of fungal secondary metabolites are presented as links between genomic methods and metabolomic experiments. Finally, the influence of the culture environment on the synthesis of secondary metabolites by fungi is highlighted as a major factor to consider in research on fungal secondary metabolites. Through this review, we seek to emphasize that the discovery of natural products should integrate all of these valuable tools. Attention is also drawn to emerging technologies that will certainly revolutionize fungal research and to the use of computational tools that are necessary but whose results should be interpreted carefully.

Analysis of the contrast between natural occurrence of toxigenic Aspergilli of the Flavi section and aflatoxin B1 in cassava.

Aflatoxin B1 (AFB1) is a carcinogenic mycotoxin produced by Aspergilli of the section Flavi that may contaminate food, in the field or during storage. Cassava represents an important staple food in sub-Saharan Africa. The analysis of aflatoxigenic fungi in 36 cassava samples obtained from producers in Benin indicated that 40% were contaminated by Aspergilli of the section Flavi. Upon morphological and molecular characterization of the 20 isolates, 16 belonged to Aspergillus flavus, 2 to Aspergillus parvisclerotigenus and 2 to Aspergillus novoparasiticus. This is the first time that this latter species is isolated from food. Although most of these isolates were toxigenic on synthetic media, no AFB1 contamination was observed in these cassava samples. In order to determine the action of cassava on AFB1 synthesis, a highly toxigenic strain of A. flavus, was inoculated onto fresh cassava and despite a rapid development, no AFB1 was produced. The anti-aflatoxin property was observed with cassava from different geographical origins and on other aflatoxigenic strains of the section Flavi, but it was lost after heating, sun drying and freezing. Our data suggest that fresh cassava is safe regarding AFB1 contamination, however, processing may alter its ability to block toxinogenesis leading to secondary contamination.

Roles of cytochromes P450 and ribosome inhibition in the interaction between two preoccupying mycotoxins, aflatoxin B1 and deoxynivalenol.

Mycotoxins are a threat to human and animal health. Climate change increases their occurrence and our dietary exposure. Although humans and animals are concomitantly exposed to several mycotoxins, their combined effects are poorly characterised. This study investigated the interaction between aflatoxin B1 (AFB1), the most potent natural carcinogen, and deoxynivalenol (DON), which is among the most prevalent mycotoxins. AFB1 is associated with hepatocellular carcinoma through its bioactivation by cytochrome P450 (CYP450) enzymes; while DON induces ribotoxic stress leading to an alteration of intestinal, immune and hepatic functions. Analysis of DNA damage biomarkers γ-H2AX and 53BP1 revealed that DON reduces the genotoxicity of AFB1. This effect was mimicked with cycloheximide, another ribosome inhibitor; moreover DOM-1, a DON-derivative lacking ribosome inhibition, did not affect DNA damage. Exposure to DON, alone or in combination with AFB1, decreased the protein levels and/or activities of CYP1A2 and CYP3A4 in a time- and dose-dependent manner. Altogether, these results revealed an original interaction between DON and AFB1, DON inhibiting the genotoxicity of AFB1. The underlying mechanism involves ribosome inhibition by DON and the subsequent impairment of CYP450s, responsible for the bioactivation of AFB1. This work highlights the importance of studying mycotoxins not only individually but also in mixture and of considering food contaminants as part of the exposome.

Endogenous interleukin 12 (IL-12) regulates granuloma formation induced by eggs of Schistosoma mansoni and exogenous IL-12 both inhibits and prophylactically immunizes against egg pathology.

Morbidity in humans infected with Schistosoma mansoni results primarily from the deposition of parasite eggs in portal areas where they induce a granulomatous response. In mice infected with this helminth granuloma formation is a CD4+ T helper (Th) cell-dependent process that is associated with a strong Th2 cytokine response which appears to evolve through a Th0 phase. In this report, we asked whether endogenously synthesized or exogenously induced interferon (IFN)gamma through its suppression of Th2 cell expansion exerts a regulatory role on egg pathology. Depletion of IFN-gamma or natural killer cells resulted in a marked enhancement of granuloma formation around intravenously injected eggs and was associated with increased Th2 and decreased Th1 and interleukin (IL)12 mRNA expression. Similar changes occurred when egg-injected mice were treated with neutralizing monoclonal antibodies specific for IL-12 indicating a role for this cytokine in the regulation of the granulomatous response. In contrast, treatment with exogenous rIL-12 profoundly inhibited primary granuloma formation while increasing IFN-gamma, IL-2, IL-10, and IL-12 pulmonary mRNA levels and suppressing IL-4, IL-5, IL-6, and IL-13 mRNA expression. Cytokine depletion studies indicated that the effects of IL-12 could be attributed primarily to increased IFN-gamma. Importantly, IL-12 also inhibited secondary granuloma formation in mice presensitized with eggs demonstrating a role for the cytokine in reversing established Th2-type responses. Moreover, mice sensitized with eggs in combination with IL-12 to precommit them toward a Th1 response developed only minimal granulomas upon subsequent egg challenge. The latter findings suggest that simultaneous vaccination with antigen plus IL-12 may provide a strategy for the prevention of schistosome egg pathology as well as other diseases stemming from Th2 cytokine production.

Beneficial effects of Saccharomyces cerevisiae RC016 in weaned piglets: in vivo and ex vivo analysis.

Probiotics represents an alternative to replace antibiotics as growth promoters in animal feed and are able to control enteric bacterial diseases and to improve gut immunity. Saccharomyces cerevisiae RC016 showed previously inhibition/coagregation of pathogens) and mycotoxins adsorbent ability (aflatoxin B1, ochratoxin A and zearalenone). The aim of this work was to evaluate beneficial properties of S. cerevisiae RC016 in a non-inflammatory in vivo model in weaned piglets and in an intestinal inflammation ex vivo model induced by the mycotoxin deoxynivalenol (DON). Secretory immunoglobulin A (s-IgA) levels, intestinal cytokines, goblet cells and production parameters were evaluated in a pig model. For the in vivo assays, twelve pigs were weaned at 21 days and assigned to two groups: Control (n=6) and Yeast (n=6). Animals received yeast strain for three weeks. After 22 days the small intestine was recovered for determination of goblet cells and s-IgA. For the ex vivo assay, jejunal explants were obtained from 5 weeks old crossbred piglets and treated as follow: (1) control; (2) treated for 3 h with 10 µM DON used as an inflammatory stressor; (3) incubated with 107 cfu/ml yeast strain; (4) pre-incubated 1 h with 107 cfu/ml yeast strain and then treated for 3 h with 10 µM DON. CCL20, interleukin (IL)-1ß, IL-8 and IL-22 gene expression was determined by qPCR. Oral administration of S. cerevisiae RC016 increased s-IgA, the number of goblet cells in small intestine and all the growth parameters measured. In the ex vivo model, the cytokine profile studied showed a potential anti-inflammatory effect of the administration of the yeast. In conclusion, S. cerevisiae RC016 is a promising candidate for feed additives formulation to improve animal growth and gut immune system. This yeast strain could be able to improve the gut health through counteracting the weaning-associated intestinal inflammation in piglets.

Subclinical doses of T-2 toxin impair acquired immune response and liver cytochrome P450 in pigs.

This study was designed to investigate the effect of subclinical doses of T-2 toxin on liver drug-metabolizing enzymes and the immune response. Pigs were offered over a 28-day period either a control diet or diets contaminated with 540, 1324 or 2102microg pure T-2toxin/kg feed. Pigs were immunized with ovalbumin and subsequent humoral and cellular immune responses measured. Monooxygenase and transferase enzyme activities and protein expression were investigated in liver tissue samples. Pigs fed 1324 or 2102microg T-2toxin/kg feed exhibited reduced anti-ovalbumin antibody production without significant alteration to specific lymphocyte proliferation. The livers of pigs exposed to T-2 toxin presented normal cytochrome P450 content, UGT 1A and P450 2B, 2C or 3A protein expression, and glutathione- and UDP glucuronosyl-transferase activities. However, P450 1A related activities (ethoxyresorufin O-deethylation and benzo-(a)-pyrene hydroxylation) were reduced for all pigs given T-2 toxin, with P450 1A protein expression decreased in pigs fed the highest dose. In addition T-2 toxin exposure reduced certain N-demethylase activities. The results of this study confirm the immunotoxic properties of T-2 toxin, in particular toward the humoral immune response. The reduction of monooxygenase activities, even though the liver presented no tissue lesion or lipid peroxidation, suggests possible deleterious interactions of T-2 toxin with these enzymes.

The importance of accounting for sex in the search of proteomic signatures of mycotoxin exposure.

Mycotoxins are natural food and feed contaminants that are toxic to human and animals. Proteomics is an adequate toolbox to investigate the mode of action and the effects of mycotoxins, as these toxicants often alter protein synthesis and degradation, as well as induce changes of important post-translational modifications. For instance, the contaminant deoxynivalenol induces a severe ribosomal stress that affects protein production, whereas the toxin Fumonisin B1 can alter the phosphorylation of a large number of proteins, and patulin is a potent proteotoxic molecule. The response to most mycotoxins is sex-dependent, males being generally more sensitive than females. In addition, for some toxins, the toxic effects observed were different for each sex. Nevertheless, the importance of accounting for a sex-dependent response is often overlooked in toxicology studies involving mycotoxins. Here we review the information that proteomics has provided in pre-clinical studies of mycotoxin exposure as well as the differential response of males and females to these molecules to highlight the need of including male and female individuals when evaluating the impact of mycotoxins in the cell proteome. SIGNIFICANCE: The current trend in mycotoxicology is the combination of several -omics techniques in order to understand the mechanism of action and effects of these toxic natural food contaminants. One of the goals of these experiments is to determine "potential biomarkers" of mycotoxicoses. Nevertheless, the strategy followed in biomarker research must take into account as many possible factors as possible in order to find robust biomarkers for differential diagnosis. Among the factors that can have an influence in the response to mycotoxins, one of the most important is sex. Traditionally, males are preferentially used in research, as they are more sensitive to mycotoxins and their response is not dependent on hormonal levels, thus less variable. However the intrinsic and hormonal differences between sexes makes that results obtained in males are often not directly transferrable to females. In this review, we want to highlight (1) that proteomics has a great potential on mycotoxin research, and (2) the need in taking into account sex differences in proteomic studies, mostly when the discovery of robust biomarkers of mycotoxins response is desired.

Genotoxicity of aflatoxins and their precursors in human cells.

Aflatoxins are found as food contaminant and some of them demonstrate a carcinogenic effect. The aflatoxins biosynthetic pathway involves 15 successive steps. The aim of this study was to compare the toxicity of aflatoxins and their precursors in three human cell lines. We tested the four aflatoxins and two of their metabolites; three early metabolic precursors and two late biosynthetic precursors. Cyclopiazonic acid, synthesized in parallel with aflatoxins, was also tested. The cytotoxicity and the genotoxicity was evaluated with the γH2AX assay in three human cell lines with different bioactivation capacities. Our results indicated that the most genotoxic chemicals in the three cell lines were in decreasing order sterigmatocystin (ST), aflatoxin B1 (AFB1), aflatoxicol (AFL), aflatoxin G1 (AFG1) and versicolorin A (VERA). Aflatoxin M1 (AFM1) demonstrated genotoxic property in only one cell line. The other tested compounds did not demonstrate any genotoxic activity. Overall, our results suggested different genotoxic mechanisms of action for the tested compounds, involving specific bioactivation pathways. Moreover, some metabolic precursors of aflatoxins demonstrated genotoxic potential equivalent or greater to AFB1. This should be taking into account for the development of new strategies intended to reduce the aflatoxins exposure and for human risk assessment.

Impact of feed restriction and housing hygiene conditions on specific and inflammatory immune response, the cecal bacterial community and the survival of young rabbits.

Limiting the post-weaning intake of the young rabbit is known to improve its resistance to digestive disorders, whereas a degradation of its housing hygiene is assumed to have a negative impact on its health. This study aims at providing insights into the mechanism of digestive health preservation regarding both host (growth and immune response) and its symbiotic digestive microbiota. A 2×2 factorial design from weaning (day 28) to day 64 was set up: ad libitum intake or restricted intake at 70% of ad libitum, and high v. low hygiene of housing (n=105 per group). At day 36 and day 45, 15 animals/group were subcutaneously immunized with ovalbumin (OVA) to assess their specific immune response. Blood was sampled at 36, 45, 57 and 64 days of age to determine total and anti-OVA immunoglobulin type G (IgG) and haptoglobin levels. The cecal bacterial community was explored (18 per group) by 454 pyrosequencing of genes coding for the 16S ribosomal RNA, whereas cecal pH, NH3 and volatile fatty acid (VFA) concentrations were measured to characterize fermentative activity. A 30% reduction in feed intake reduced the growth by only 17% (P<0.001), and improved the feed conversion ratio by 15% (P<0.001), whereas the degradation of hygiene conditions slightly decreased the feed intake in ad libitum fed rabbits (-3.5%, P<0.02). As poor hygiene conditions did not affect weight gain, feed conversion was improved from day 42 (P<0.05). Restricted feeding led to a lower mortality between day 28 and day 40 (P=0.047), whereas degraded hygiene conditions decreased overall morbidity (7.8% v. 16.6%; P<0.01). Both a reduced intake and low hygiene conditions of housing affected microbiota composition and especially dominant genera belonging to the Ruminococcaceae family (P<0.01). Moreover, low hygiene was associated with a higher Ruminococcaceae/Lachnospiraceae ratio (3.7 v. 2.4; P<0.05). Cecal total VFA and pH were increased (+19%; P<0.001) and decreased (-0.1 pH unit; P<0.05), respectively, in feed-restricted rabbits. Neither specific anti-OVA IgG nor haptoglobin was affected by treatments. Total IgG concentrations were the highest in animals raised in poor hygiene conditions after 8 days of restriction, but decreased after 19 days of restriction in high hygiene conditions (-2.15%; P<0.05). In conclusion, the degradation of hygiene conditions failed to induce a systematic specific and inflammatory response in rabbit, but reduced morbidity instead. Our results suggest that the microbiota composition would be a helpful source of biomarkers of digestive health.

Effect of various doses of deoxynivalenol on liver xenobiotic metabolizing enzymes in mice.

DON is one of the major mycotoxic contaminant of cereal grains throughout the world. The purpose of this investigation was to characterize the effects of a range of environmentally relevant doses of DON in mice exposed through a subchronic toxicological assay. Animals received 3 days per week for 4 weeks, 0.014, 0.071, 0.355 or 1.774 mg of toxin/kg b.w. All doses, except 0.014 mg/kg, provoked increases in plasma immunoglobulin A whereas there was no change in plasma biochemical parameters such as alkaline phosphatase, electrolytes or other immunoglobulins. Administration of 0.071 or 0.355 mg/kg doses led to increased liver microsomal pentoxyresorufin depentylase and cytosolic glutathione transferase activities. Examining protein modulation, western blot analyses liver fractions from mice receiving these doses revealed increased levels in both P450 2b, GST alpha and pi isoenzymes without any change in P450 1a expression. A significant competitive inhibition of deoxynivalenol on CDNB conjugation in vitro suggests that the mycotoxin is a putative substrate for glutathione S-transferases. These changes in liver xenobiotic metabolizing enzymes are discussed by considering the structural nature of deoxynivalenol and previous reports on similar effects exerted by other trichothecenes. These results suggest that a subchronic exposure to low doses of deoxynivalenol causes changes in the normal liver metabolism of xenobiotics.

Individual and combined effects of low oral doses of deoxynivalenol and nivalenol in mice.

Deoxynivalenol (DON) and nivalenol (NIV) are toxic Fusarium secondary trichothecene metabolites that often co-occur regularly in cereal grains. These compounds were compared for their toxicity towards C57BL/6 mice on several parameters including alteration in plasma biochemistry, immune system reactivity and hepatic drug metabolism capacity. Mice received individual or combined oral doses of each toxin: 0.071 or 0.355 mg/kg of body weight, administrated three days a week for 4 weeks. Food consumption was altered by the single administration of 0.355 mg/kg of NIV, although no noticeable change of body and organ weights or liver protein contents was detected. NIV administration did cause also significant changes in total CO2 and uric acid concentrations in plasma. Individual toxin exposures led to increases in plasma IgA without no detectable change in the ex vivo production of cytokine by splenocytes. The liver ethoxyresorufin O-deealkylase, pentoxyresorufin O-depenthylase and glutathione S-transferase activities were increased in concert with cytochrome P4501a and P4502b subfamily expression. Administration of combinations of DON and NIV resulted in responses similar to that observed using individual doses of each toxin. However, depending on the ratio of toxin doses and biochemical parameters, some responses could be also additive (plasma IgA and hepatic DCNB conjugation) or synergistic (plasma uric acid).

Low response of BALB/c macrophages to priming and activating signals.

Trehalose dimycolate (TDM), a mycobacterial glycolipid, is a powerful macrophage-priming agent. However, its efficiency seems limited in the case of BALB/c mice. Peritoneal macrophages harvested from TDM-treated BALB/c mice did not control BCG growth in vitro as efficiently as similar macrophages from two other mouse strains, (B6 x D2)F1 and C57BL/6, which are respectively Bcgr and Bcgs. BALB/c macrophages elicited by TDM also exhibited a low capacity to produce hydrogen peroxide and, after activation by lipopolysaccharide (LPS), weak cytostatic activity against P815 mastocytoma cells. Finally, alkaline phosphodiesterase, a marker of resident and inflammatory macrophages, was still expressed at a high level in macrophages of BALB/c mice treated with TDM. Low responsiveness of BALB/c macrophages to stimuli was not observed with TDM only; activation for tumor cytotoxicity of thioglycolate-elicited macrophages from BALB/c mice required also higher doses of interferon-gamma, and LPS. L-Arginine-dependent production of nitric oxide was inducible in macrophages from BALB/c mice, but the conditions required for its induction were more stringent. Thus, the reduced antiproliferative effects of BALB/c macrophages may be due to uncomplete induction of NO synthase after suboptimal stimulation.

Experimental ovine salmonellosis (Salmonella abortusovis): pathogenesis and vaccination.

Salmonella enterica subsp. enterica ser. Abortusovis, a sheep-adapted serotype, causes a contagious disease. Abortion is the major symptom and the main source of contamination. Research on this ovine disease may aid farmers, but may also contribute to comparative biological knowledge. Innate resistance partly controlled by the Ity locus, increased resistance to reinfection and humoral and T-cell-mediated immunity were observations gained with a murine model. In ewes, abortion regularly occurs following subcutaneous challenge carried out from the third month of gestation onwards. This ovine model was used to evaluate prevention methods for Salmonella Abortusovis infection. One subcutaneous injection of a live attenuated lyophilized vaccine containing a selected streptomycin-independent reverse mutant was shown to protect ewes against abortion and excretion of Salmonella Abortusovis. This vaccine could be administered simultaneously with other commercial live vaccines such as Brucella melitensis Rev. 1 vaccine. In sheep, application of the vaccine to the conjunctiva (an easy, individual and hygienic route of mucosal vaccination) was followed by lymph node bacterial colonization and a serological response without local or general clinical reactions. The early events of natural infection remain to be explored, as do the mechanisms underlying the host specificity of Salmonella Abortusovis.

Tumor necrosis factor is required for the priming of peritoneal macrophages by trehalose dimycolate.

Trehalose dimycolate (TDM), a glycolipid present in the cell wall of Mycobacterium spp., is a powerful immunostimulant. We have developed an original model of macrophage activation where TDM is injected in vivo to prime peritoneal macrophages. These primed macrophages do not express inducible NO synthase (NOS II), however, they can be fully activated, i.e. induced to express NOS II and to develop a NOS II-dependent antiproliferative activity, following in vitro exposure to low concentrations of LPS. In a previous paper, we have shown that TDM-priming of mouse peritoneal macrophages is mediated by the sequential production of IL-12 and IFN-gamma. In the present paper, we investigated the role of TNF in the priming of macrophages by TDM. By semi-quantitative RT-PCR, we have shown that TDM injection induced transcription of TNF-alpha in peritoneal cells. TNF-mRNA levels peaked 5 hours after TDM injection and remained elevated for at least 32 hours. TNF expression was absolutely necessary for macrophage priming, as injection of an anti-TNF monoclonal antibody, 4 h before and 20 hours after TDM injection, prevented LPS-dependent activation of macrophages in vitro. This result was confirmed by the inability of TDM to prime macrophages from LT-alpha/TNF-alpha knockout (LT/TNFKO) mice. In addition, analysis of LT/TNFKO mice treated with TDM revealed that induction of the IL-12 transcript in their peritoneal cells and expression of a functional NADPH oxidase in macrophages are TNF-independent events.

Pathologic study of an experimental canine arthritis induced with Complete Freund's Adjuvant.

OBJECTIVE: To describe the physiopathological features of an experimental monoarthritis in a canine model. METHODS: Pathological changes of the femorotibial joints, periarticular tissues and lymphoid organs were investigated by histology, immunohistochemistry, flow cytometry and cytokine bioassays after intra-articular administration of Complete Freund's Adjuvant in dogs. RESULTS: An edematous acute synovitis with considerable diapedesis of the neutrophilic granulocytes was observed during a primary response. This was followed by a clinical remission, although a multifocal subacute synovitis was noted with inflammatory infiltrates of mononuclear cells (macrophages > CD8 > CD4). Blood CD14 expression was upregulated and the percentage of CD8+ cells was significantly decreased. Two to four weeks post-induction, a decrease in functional impotency (secondary response) and a pyogranulomatous periarthritis with intramuscular extensions were noted. Focal cartilage erosion was due to adherent granulation tissue (CD4 > CD8 > macrophages). CONCLUSION: In this canine model, experimentally induced arthritis was mainly the result of a cell-mediated immune reaction towards a non-identified antigen.

Classical and alternative pathway haemolytic activities of ovine complement: variations with age and sex.

The classical (CH50) and alternative (ACH50) pathway haemolytic activities of sheep complement were measured with microtechniques. Storage of blood at room temperature (instead of 4 degrees C) before centrifugation and usage of sera stored at -70 degrees C were compatible with complement titration. The effects of age and sex were tested in 303 sera obtained from animals aged between 2 weeks and 3.5 years old. ACH50 titres were low during the first 1.5 months of life then increased to reach the level found in adults at the age of 3 months. Conversely, CH50 titres were very high in suckling lambs, decreased up until the age of 3 months and then increased to reach the adult level at 1 year old. In lambs, the haemolytic complement activity was significantly higher in females than in males.

A reverse transcription-polymerase chain reaction method to analyze porcine cytokine gene expression.

A reverse transcription-polymerase chain reaction (RT-PCR) method was developed in order to provide a highly sensitive, rapid, and simple means of simultaneously measuring the expression of porcine cytokines in immune cell populations. Oligonucleotide primers were designed to amplify porcine cytokine cDNA from genes encoding IL-1 alpha, IL-1 beta, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IFN-gamma, TNF-alpha, TNF-beta and the housekeeping genes beta-actin and cyclophilin by PCR. Primers were chosen from different exons to detect for possible genomic DNA contamination of samples. To validate RT-PCR, unstimulated and concanavalin A (ConA) stimulated porcine peripheral blood mononuclear cells (PBMCs) were cultured from 2 h to 72 h, RNA was extracted and reverse transcribed, and cDNA was amplified using the different primer sets. Band intensities of PCR products were quantified by densitometric scanning and values were normalized against cyclophilin. For each of the cytokines, the kinetics of gene expression were similar among PBMCs isolated from different animals and could be grouped into two main patterns. Lymphocyte derived cytokines (IL-2, IL-4, IFN-gamma, and TNF-beta) exhibited low level expression in unstimulated cells and increased expression in ConA-stimulated PBMCs. IFN-gamma and IL-2 mRNA levels peaked at 24 h and returned to baseline by 72 h, whereas IL-4 and TNF-beta mRNA levels did not return to baseline by 72 h. In contrast, substantial mRNA levels for inflammatory cytokines (IL-1 alpha, IL-1 beta, IL-6, IL-8, IL-12, and TNF-alpha) and IL-10 were detected from both unstimulated and ConA-stimulated PBMCs. Results indicate that RT-PCR is a sensitive and convenient method to monitor cytokine mRNA expression in porcine samples.

Changes in performance, blood parameters, humoral and cellular immune responses in weanling piglets exposed to low doses of aflatoxin.

A feeding trial was conducted to evaluate the effect of aflatoxin (AF)-contaminated diets on growth and hematological and immunological parameters. Low doses of aflatoxins (140 and 280 ppb) were included in a corn-soybean diet provided for ad libitum consumption to 36 weanling piglets for a period of 4 wk. A "dose-related" decrease in weight gain was observed in treated animals. This effect was significant (P < 0.05) in the 280 ppb-treated group compared to the control group. Ingestion of AF-contaminated feed at either level had no effect on total red blood cell numbers or on their relative number of lymphocytes, monocytes, neutrophils, basophils, and eosinophils in blood. Likewise, AF did not alter globulin, albumins, or total protein concentrations in serum, nor did AF alter the expression of regulatory cytokines produced by either Th1 (IL-2) or Th2 (IL-4) lymphocyte subsets in phytohemagglutinin-stimulated blood samples. By contrast, AF had a biphasic effect on total white blood cell number; the low dose of AF (140 ppb) decreased the total number of white blood cells, whereas the high dose (280 ppb) had the opposite effect. Consumption of AF also increased the concentration of gamma-globulin in the serum. A reduced immune response induced by Mycoplasma agalactiae in the 280-ppb-treated group was also observed. Cytokine mRNA expression in phytohemagglutinin-stimulated blood cells indicated that AF decreased proinflammatory (IL-1beta, TNF-alpha) and increased anti-inflammatory (IL-10) cytokine mRNA expression. These results demonstrate that low doses of AF depress growth and alter many aspects of humoral and cellular immunity in pigs.

Early modulation of the cecal microbial activity in the young rabbit with rapidly fermentable fiber: impact on health and growth.

This study aimed at comparing various diets predicted to induce different stimulations of the cecal microbial activity of the young rabbit fed ad libitum from 16 to 70 d of age: i) a diet enriched with rapidly fermentable fiber expected to stimulate the cecal microbial activity (RFF group); ii) a control diet with a standard composition (C group); iii) and the same control diet with tiamulin and apramycin antibiotics, expected to inhibit the microbial activity (C+AB group). A total of 398 rabbits were used from 42 litters and weaned at 28 d of age. An in vivo digestibility trial was performed on 36 rabbits of 42 to 46 d of age housed in individual metabolic cages. The feed intake and growth rates were lower in the RFF group compared with the C+AB group (-15% in ADFI and -11% in ADG, P<0.001), with a lower weight of -183 g at 70 d (P<0.001). No significant difference was found on ADG and final BW between the RFF and the C groups, but the RFF diet allowed a better G:F ratio at postweaning (P<0.01). The digestion of soluble fiber (total dietary fiber minus NDF) was greater for the RFF group. The C+AB diet had a positive effect on the postweaning morbidity rate (P<0.05) but did not affect the mortality rate and the health risk index (morbidity and mortality). Conversely, the RFF diet appeared to reduce the mortality rate compared with the C+AB diet, especially before 41 d of age. Concerning the cecal microbial activity, a supply of RFF in the diet increased the cecal VFA concentrations (+28% vs. C+AB and +22% vs. C, P<0.001) and lowered the pH. The VFA pattern was affected at 45 and 60 d, with a dominance of acetate in the RFF group (+4% vs. C+AB and C groups, P<0.001) instead of butyrate in the C+AB and C groups (-3.6% and -5% vs. C+AB and C, respectively, P<0.001). Antibiotics addition (C+AB group) reduced the VFA concentration, but only after weaning (-25% at 45 d of age) without changing the fermentation pattern. In conclusion, early intake of RFF in young rabbits stimulated the cecal microbial activity, and reduced the voluntary feed intake, leading to a reduced G:F ratio.

Structural and functional development of small intestine in intrauterine growth retarded porcine offspring born to gilts fed diets with differing protein ratios throughout pregnancy.

Protein level in the maternal diet plays a crucial role in fetal programming during pregnancy. Low or high protein level increases the risk of intrauterine growth retardation (IUGR). The aim of this study was to investigate the structural and functional development of the small intestine in piglets from sows fed a control (C, 12.1% protein), a high protein (HP, 30% protein), or a low protein (LP, 6.5% protein) diet during pregnancy. Newborns were classified as IUGR (birth weight ≤1.18 kg) and non-IUGR (birth weight >1.18 kg). The piglets were euthanized on postnatal day (PD)1, PD28 and PD188. The LP diet in non-IUGR neonates resulted in decreased body weight on PD1. The LP and HP diets resulted in both decreased body weight and delayed catch-up growth in the IUGR piglets. The HP and LP-diets increased the length of villi on PD1 in non-IUGRs but not in IUGRs. At birth, the expressions of Ki67 and active caspase 3 in mid-jejunum epithelium of HP and LP non-IUGR neonates were significantly lower as compared to C non-IUGRs whilst in IUGRs the respective expressions were as high as in C non-IUGRs. The postnatal dynamics of brush border enzyme activities and vacuolated enterocytes disappearance showed significant drop in enterocyte maturation in IUGR as compared to non-IUGR neonates. In conclusion, both HP and LP diets led to retarded development of non-IUGR piglets. In IUGR piglets both HP and LP diets resulted in delayed catch-up growth, without adaptive changes in brush border digestive enzymes.