Validation of a real-time PCR assay for the detection of African swine fever virus in fresh pork meat juice.

African swine fever virus (ASFV) is the etiological agent of African swine fever (ASF), a disease with detrimental effects on the health, welfare, and production of domestic and wild pigs. The ASF laboratory confirmation is based on the analysis of blood, serum and organ samples. However, testing these samples could not be always convenient, economically feasible or possible. This study describes the validation process of a PCR-based assay targeting a portion of p72 gene, used for the molecular detection of ASFV, from meat juice samples obtained from pigs succumbed to ASFV. More specifically, we investigated the capability of a real-time PCR assay to detect ASFV DNA in meat juices obtained from the diaphragmatic muscle along with the correspondent spleens of 55 ASFV-positive pigs and wild boars sampled from confirmed outbreaks in Romania and from 73 ASFV-negative and regularly slaughtered healthy pigs collected in the Abruzzo region (Italy). The test was able to detect viral DNA in both types of samples, with lower Ct values in spleens (mean=21.11, median=20.61) than meat juices (mean=23.08, median=22.40). However, distributions of Ct values were strongly correlated each other (R2= 0.83, P<0.001). Considering the distribution of the observed Ct values in the 55 positive meat juice samples, a 1:10 dilution would be able to detect 90 % of positive samples, whereas a 1:100 dilution would reduce the detectability to 78 % of more contaminated samples. As meat juice could be obtained easily from muscles and considering the potential use of this test on pooled samples, it could represent a tool to aid the investigation of ASFV spread.

Evaluation of cellular and molecular impact of zearalenone and Escherichia coli co-exposure on IPEC-1 cells using microarray technology.

BACKGROUND: The gastrointestinal tract is the primary site of toxin interaction, an interface between the organism and its surroundings. In this study, we assessed the alteration of intestinal mRNA profile in the case of co-occurrence of zearalenone (ZEA), a secondary Fusarium metabolite, and Escherichia coli (E. coli), on the intestinal porcine epithelial cells IPEC-1. We chose this model since the pig is a species which is susceptible to pathogen and mycotoxin co-exposure. RESULTS: After treating the cells with the two contaminants, either separately or in combination, the differential gene expression between groups was assessed, using the microarray technology. Data analysis identified 1691 upregulated and 797 downregulated genes as a response to E. coli exposure, while for ZEA treated cells, 303 genes were upregulated and 49 downregulated. The co-contamination led to 991 upregulated and 800 downregulated genes. The altered gene expression pattern was further classified into 8 functional groups. In the case of co-exposure to ZEA and E.coli, a clear increase of proinflammatory mechanisms. CONCLUSIONS: These results demonstrate the complex effect of single or multiple contaminants exposure at cellular and molecular level, with significant implications that might lead to the activation of pathological mechanisms. A better understanding of the effects of co-contamination is mandatory in developing novel exposure regulations and prevention measures.

Food contaminant zearalenone and its metabolites affect cytokine synthesis and intestinal epithelial integrity of porcine cells.

The intestinal epithelium is the first barrier against food contaminants. Zearalenone (ZEN) is an estrogenic mycotoxin that was identified as a common contaminant of cereal grains and food and feedstuffs. In the present study, we have investigated the in vitro effects of ZEN and some of its metabolites (α-ZOL, ß-ZOL) in concentrations of 10-100 µM on a swine epithelial cell line: Intestinal porcine epithelial cells (IPEC-1). We demonstrated that both ZEN metabolites were more toxic for IPEC cells as resulted from the XTT test, while for doses lower than 10 µM, only ß-ZOL showed a more pronounced cytotoxicity versus epithelial cells as resulted from neutral red assay. ZEN has no effect on TER values, while α-ZOL significantly decreased the TER values, starting with day 4 of treatment. ß-ZOL had a dual effect, firstly it induced a significant increase of TER, and then, starting on day 6, it induced a dramatic decrease of TER values as compared with on day 0. Concerning the cytokine synthesis, our results showed that ZEN has a tendency to increase the synthesis of IL-8 and IL-10. By contrast, α- and ß-ZOL decreased the expression of both IL-8 and IL-10, in a dose dependent manner. In conclusion, our results showed that ZEN and its metabolites differently affected porcine intestinal cell viability, transepithelial resistance and cytokine synthesis with important implication for gut health.

Zearalenone mycotoxin affects immune mediators, MAPK signalling molecules, nuclear receptors and genome-wide gene expression in pig spleen.

The toxicity of zearalenone (ZEA) was evaluated in swine spleen, a key organ for the innate and adaptative immune response. Weaned pigs were fed for 18 days with a control or a ZEA contaminated diet. The effect of ZEA was assessed on wide genome expression, pro- (TNF-α, IL-8, IL-6, IL-1ß, IFN-γ) and anti-inflammatory (IL-10, IL-4) cytokines, other molecules involved in inflammatory processes (MMPs/TIMPs), as well as signaling molecules, (p38/JNK1/JNK2-MAPKs) and nuclear receptors (PPARγ/NFkB/AP-1/STAT3/c-JUN). Microarray analysis showed that 46% of total number of differentially expressed genes was involved in cellular signaling pathway, 13% in cytokine network and 10% in the inflammatory response. ZEA increased expression and synthesis of pro- inflammatory (TNF-α, IL-8, IL-6, IL-1ß) and had no effect on IFN-γ, IL-4 and IL-10 cytokines in spleen. The inflammatory stimulation might be a consequence of JNK pathway activation rather than of p-38MAPK and NF-kB involvement whose gene and protein expression were suppressed by ZEA action. In summary, our findings indicated the role of ZEA as an immune disruptor at spleen level.

Induction of pro-inflammatory gene expression by Escherichia coli and mycotoxin zearalenone contamination and protection by a Lactobacillus mixture in porcine IPEC-1 cells.

This work investigated the effect of Escherichia coli K88 and zearalenone contamination on pro-inflammatory gene expression (Toll like receptors, cytokines) and signalling molecules and the protective activity of a mixture of Lactobacilli sp. (Lactobacillus plantarum, Lactobacillus acidofilus and Lactobacillus paracasei) in porcine intestinal epithelial cells as part of the local immune system. IPEC-1 cell monolayer was exposed for 1 h to the individual or combined action of E. coli, zearalenone and lactobacilli mixture. Our results showed that TLRs (1-10) and cytokine (IL-1,-6,-8,-10, TNF-α, IFN-γ) genes expressed early (after 1 h of culture) in IPEC-1 cells. E. coli alone increased the TLRs mRNA expression, especially TLR4 and the inflammatory cytokines while ZEA alone showed either no effect or a marginally effect on TLRs, cytokines, and signalling genes when compared to untreated cells. The combined actions of the two contaminants lead to a synergistically up-regulation of key cytokines (IFN-γ, IL-10 and TNF-α) and TLRs (-2,-3,-4,-6, and -10). The live lactobacilli mixture was able to attenuate the pathogen and mycotoxin-induced response by downregulated the majority of inflammatory related genes suggesting that this mixture has an immunomodulatory potential and may be used to lower the inflammatory response.

Exposure to zearalenone mycotoxin alters in vitro porcine intestinal epithelial cells by differential gene expression.

The gut represents the main route of intoxication with mycotoxins. To evaluate the effect and the underlying molecular changes that occurred when the intestine is exposed to zearalenone, a Fusarium sp mycotoxin, porcine epithelial cells (IPEC-1) were treated with 10µM of ZEA for 24h and analysed by microarray using Gene Spring GX v.11.5. Our results showed that 10µM of ZEA did not affect cell viability, but can increase the expression of toll like receptors (TLR1-10) and of certain cytokines involved in inflammation (TNF-α, IL-1ß, IL-6, IL-8, MCP-1, IL-12p40, CCL20) or responsible for the recruitment of immune cells (IL-10, IL-18). Microarray results identified 190 genes significantly and differentially expressed, of which 70% were up-regulated. ZEA determined the over expression of ITGB5 gene, essential against the attachment and adhesion of ETEC to porcine jejunal cells and of TFF2 implicated in mucosal protection. An up-regulation of glutathione peroxidase enzymes (GPx6, GPx2, GPx1) was also observed. Upon ZEA challenge, genes like GTF3C4 responsible for the recruitment of polymerase III and initiation of tRNA transcription in eukaryotes and STAT5B were significantly higher induced. The up-regulation of CD97 gene and the down-regulation of tumour suppressor genes (DKK-1, PCDH11X and TC531386) demonstrates the carcinogenic potential of ZEA.

ω-3 PUFA rich camelina oil by-products improve the systemic metabolism and spleen cell functions in fattening pigs.

Camelina oil-cakes results after the extraction of oil from Camelina sativa plant. In this study, camelina oil-cakes were fed to fattening pigs for 33 days and its effect on performance, plasma biochemical analytes, pro-/anti-inflammatory mediators and antioxidant detoxifying defence in spleen was investigated in comparison with sunflower meal. 24 crossbred TOPIG pigs were randomly assigned to one of two experimental dietary treatments containing either 12% sunflower meal (treatment 1-T1), or 12.0% camelina oil-cakes, rich in polyunsaturated fatty acids ω-3 (ω-3 PUFA) (treatment 2-T2). The results showed no effect of T2 diet (camelina cakes) on feed intake, average weight gain or feed efficiency. Consumption of camelina diet resulted in a significant decrease in plasma glucose concentration (18.47%) with a trend towards also a decrease of plasma cholesterol. In spleen, T2 diet modulated cellular immune response by decreasing the protein and gene expression of pro-inflammatory markers, interleukin 1-beta (IL-1ß), tumor necrosis factor alpha (TNF-α), interleukin 6 (IL-6) and interleukin (IL-8) and cyclooxigenase 2 (COX-2) in comparison with T1 diet. By contrast, T2 diet increased (P<0.05) in spleen the mRNA expression of antioxidant enzymes, catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase 1 (GPx1) by 3.43, 2.47 and 1.83 fold change respectively, inducible nitric oxide synthase (iNOS) (4.60 fold), endothelial nitric oxide synthase (eNOS) (3.23 fold) and the total antioxidant level (9.02%) in plasma. Camelina diet increased also peroxisome-proliferator activated receptor gamma (PPAR-γ) mRNA and decreased that of mitogen-activated protein kinase 14 (p38α MAPK) and nuclear factor of kappa light polypeptide gene enhancer in B-cells (NF-κB). At this level of inclusion (12%) camelina oil-cakes appears to be a potentially alternative feed source for pig which preserves a high content of ω-3 PUFA indicating antioxidant properties by the stimulation of detoxifying enzymes expression and the suppression of spleen pro-inflammatory markers.

Effects of zearalenone and its derivatives on porcine immune response.

Zearalenone (ZEN), a mycotoxin produced by several Fusarium spp., is most commonly found as a contaminant in stored grain and has chronic estrogenic effects on mammals. In this in vitro study, we compared the effects of zearalenone (ZEN) and some of its derivatives: α-zearalenol (α-ZOL), ß-zearalenol (ß-ZOL), and zearalanone (ZAN) on several peripheral blood mononuclear cell (PBMC) parameters: cytotoxicity, proliferation, as well as antibody and cytokine synthesis. The amounts of toxins necessary to inhibit viability, in a dehydrogenase enzyme activity assay (MTT test), by 50% were: 22.7 µM for ZEN, 29.1 µM for α-ZOL, 17.3 µM for ß-ZOL and 26.3 µM for ZAN. The administration of 10 µM toxin induced a decrease in the ConA stimulated proliferation of PBMC by 19.6% for ZAN, 45.4% for ZEN, 43.6% for α-ZOL and 85.2% for ß-ZOL, when compared to the control stimulated cells. Also, ZEN and its metabolites at concentrations higher than 5 µM induced a significant decrease of the IgG, IgA or IgM levels. Concentrations of 5 and 10 µM of ZEN and ZAN significantly decreased the TNF-α synthesis in the supernatant of the stimulated cells; 10 µM of ZAN also decreased IL-8 synthesis. In conclusion, our results show that ZEN and ZEN derivatives altered several parameters of the humoral and cellular immune response. Therefore, our results are clinically relevant as ZEN and its metabolites are frequent contaminants of animal feed and we have shown that intoxicated animals are incapable of inducing an adequate immune response.

Assessment of the potential of a boron-fructose additive in counteracting the toxic effect of Fusarium mycotoxins.

Trichotecenes are mycotoxins produced by Fusarium sp., which may contaminate animal feeds and human food. A feeding trial was conducted to evaluate the effect of a fusarotoxin-contaminated diet, and to explore the counteracting potential of a calcium fructoborate (CFrB) additive on performance, typical health biochemistry parameters and immune response in weaned pigs. A naturally contaminated maize, containing low doses of deoxynivalenol, zearalenone, fumonisins and T-2/HT-2 toxins (1790, 20, 0·6 and 90 parts per billion), was included in a maize-soyabean meal diet, and given ad libitum to eight weaned piglets (two groups: four pigs/group) for a period of 24 d. CFrB was administered to one of the contaminated groups and to another four piglets as a daily supplement, following the manufacturer's recommendation. A decrease in performance was observed in contaminated animals at this concentration of feed toxins, which was ameliorated by the dietary CFrB supplementation. Fusarium toxins also altered the pig immune response by increasing (P < 0·05) the ex vivo peripheral blood mononuclear cell proliferation (111·7 % in comparison with control), the respiratory burst of porcine granulocytes (15·4 % for responsive cells v. 5·1 % for unstimulated cells and 70·95 v. 22·65 % for stimulated cells, respectively), the percentage of peripheral T, CD3(+), CD3(+)CD4(+) and CD3(+)CD8(+) subsets and the synthesis of IL-1ß, TNF-α and IL-8 (123·8, 217·1 and 255·1 %, respectively). The diet containing the CFrB additive reduced these exacerbated cellular immune responses induced by Fusarium toxins. However, consumption of CFrB did not counteract the effect of mycotoxins on biochemistry parameters, and increased plasma IgM and IgG of contaminated pigs.