Development of simple multiplex real-time pcr assays for foodborne pathogens detection and identification on lightcycler

Most acute intestinal diseases are caused by food-borne pathogens. A fast and simple real-time PCR-based procedure for simultaneous detection of food contamination by any of the five food-borne pathogens: Campylobacter jejuni, Mycobacterium bovis, Enterobacter sakazaki, Shigella boydii, Clostridium perfrigens using multiplex EvaGreen real-time PCR for LightCycler was developed and evaluated. Real-time qPCR showed excellent sensitivity. Tm calling and Melting Curve Genotyping (MCG) were used for analysis of PCR product melting curves. The Melting Curve Genotyping option showed good performance for discrimination of positive samples containing DNA of single pathogen or pathogen mixtures from negative samples

A STUDY ON PLASMA FIBRINOGEN AND HAPTOGLOBIN IN LAMBS WITH EXPERIMENTALLY INDUCED HAEMONCHUS CONTORTUS INFECTION

The present study aims to evaluate the changes in concentrations of some acute phase proteins during Haemonchus contortus infection in lambs. This experiment was performed using 12 three-month-old healthy lambs, randomly allocated into 2 equal groups: G1 (uninfected control animals) and G2 (lambs infected with H. contortus). Each lamb of G2 group was orally inoculated with 4000 infective third stage larvae (L3) of H. contortus by placing a probe. Blood samples were collected on post infection days zero, 4, 7, 11, 14, 21, 28 and 32. After sampling, the blood was centrifuged, and the separated plasma was used for the quantitative determination of haptoglobin (Hp) and fibrinogen (Fb). The most pronounced changes occurred in Hp levels, which increased and attained the highest values on post infection days 4, 7, and 11. A peak concentration occurred on post infection day 7 when Hp increased by 45.96% vs the initial level, and by 44.08% vs the control level on the same day. There were no significant changes in Fb concentrations throughout the study

Proteomics and metabolomics characterizing the pathophysiology of adaptive reactions to the metabolic challenges during the transition from late pregnancy to early lactation in dairy cows

The transition from late pregnancy to early lactation is a critical period in a dairy cow's life due to the rapidly increasing drain of nutrients from the maternal organism towards the foetus and into colostrum and milk. In order to cope with the challenges of parturition and lactation, comprehensive adaptive reactions comprising the endocrine and the immune system need to be accomplished. There is high variation in this coping ability and both metabolic and infectious diseases, summarized as \ue2\u80\u9cproduction diseases\ue2\u80\u9c, such as hypocalcaemia (milk fever), fatty liver syndrome, laminitis and ketosis, may occur and impact welfare, productive lifespan and economic outcomes. Proteomics and metabolomics have emerged as valuable techniques to characterize proteins and metabolite assets from tissue and biological fluids, such as milk, blood and urine. In this review we provide an overview on metabolic status and physiological changes during the transition period and the related production diseases in dairy cows, and summarize the state of art on proteomics and metabolomics of biological fluids and tissues involved in metabolic stress during the peripartum period. We also provide a current and prospective view of the application of the recent achievements generated by omics for biomarker discovery and their potential in diagnosis. Biological significance: For high-yielding dairy cows there are several \ue2\u80\u9coccupational diseases\ue2\u80\u9c that occur mainly during the metabolic challenges related to the transition from pregnancy to lactation. Such diseases and their sequelae form a major concern for dairy production, and often lead to early culling of animals. Beside the economical perspective, metabolic stress may severely influence animal welfare. There is a multitude of studies about the metabolic backgrounds of such so called production diseases like ketosis, fatty liver, or hypocalcaemia, although the investigations aiming to assess the complexity of the pathophysiological reactions are largely focused on gene expression, i.e. transcriptomics. For extending the knowledge towards the proteome and the metabolome, the respective technologies are of increasing importance and can provide an overall view of how dairy cows react to metabolic stress, which is needed for an in-depth understanding of the molecular mechanisms of the related diseases. We herein review the current findings from studies applying proteomics and metabolomics to transition-related diseases, including fatty liver, ketosis, endometritis, hypocalcaemia and laminitis. For each disease, a brief overview of the up to date knowledge about its pathogenesis is provided, followed by an insight into the most recent achievements on the proteome and metabolome of tissues and biological fluids, such as blood serum and urine, highlighting potential biomarkers. We believe that this review would help readers to be become more familiar with the recent progresses of molecular background of transition-related diseases thus encouraging research in this field

Widespread extrahepatic expression of acute-phase proteins in healthy chicken (Gallus gallus) tissues

Acute phase proteins (APP) are plasma proteins that can modify their expression in response to inflammation caused by tissue injury, infections, immunological disorders or stress. Although APP are produced mainly in liver, extrahepatic production has also been described. As a prerequisite to get insight the expression of APP in chicken during diseases, this study investigated the presence of five APP, including alpha1-acid glycoprotein (AGP), Serum Amyloid A (SAA), PIT54, C-Reactive protein (CRP) and Ovotransferrin (OVT) in twenty tissues collected from healthy chicken (Gallus gallus) by quantitative Real Time PCR and immunohistochemistry. As expected, APP gene abundance was higher in liver compared with other tissues. The mRNA coding for CRP, OVT and SAA was detected in all analyzed tissues with a higher expression in gastrointestinal tract, respiratory and lymphatic samples. SAA expression was particularly high in cecal tonsil, lung, spleen and Meckel's diverticulum, whereas OVT in lung, bursa of Fabricius and pancreas. AGP and PIT54 mRNA expression were detected in all tissues but at negligible levels. Immunohistochemical expression of AGP and OVT was variably detected in different organs, being identified in endothelium of every tissue. Positive cells were present in the epithelium of the mucosal layer of gastrointestinal tract and kidney. Lung and central nervous system stained for both proteins. No positive staining was detected in lymphoid tissues and muscle. These results suggest that most tissues can express different amount of APP even in healthy conditions and are therefore capable to mount a local acute phase reaction

Doxorubicin and congo red effectiveness on prion infectivity in golden Syrian hamster

The effect of doxorubicin and Congo Red on prion protein (PrP) infectivity in experimental scrapie was studied to better understand the effect of these compounds in prion diseases and to establish whether a dose-response correlation exists for Congo Red. This was performed in order to test the effectiveness of compounds that may easily be used in human prion diseases. Brain homogenate containing membrane bound PrPSc monomers was used as inoculum and was previously incubated with doxorubicin 10(-3) M and with increasing concentrations of Congo Red ranging from 10(-7) to 10(-2) M. This study shows for the first time that doxorubicin, and confirms that Congo Red, may interact with pathological PrP monomers modifying their infectious properties. Pre-incubation of infected brain homogenate with Congo Red resulted in prolonged incubation time and survival, independently of Congo Red concentration (p<0.05). Doxorubicin and Congo Red effects do not depend upon interaction with PrP amyloid material

BVDV permissiveness and lack of expression of co-stimulatory molecules on PBMCs from calves pre-infected with BVDV

Bovine viral diarrhea virus (BVDV) has been detected in peripheral blood mononuclear cells (PBMCs) of immunocompetent animals, not being clear whether the development of a specific humoral immune response can prevent BVDV infection. The aim of this study was to evaluate the ability of non-cytopathic BVDV to replicate and produce infectious virus in PBMCs from calves pre-infected with BVDV and to elucidate the immunomodulatory effect of BVDV on these cells in an in vitro model. Quantification of virus was by quantitative PCR, while its replicative capacity and shedding into the extracellular environment was evaluated by viral titration. Apoptosis was assessed by flow cytometry analysis of annexin V and propidium iodide, and by expression of caspase-3/7. Flow cytometry was used to analyze the expression of CD14/CD11b/CD80, CD4/CD8/CD25, MHC-I/MHC-II and B-B2 markers. Our results showed that PBMCs from cattle naturally infected with BVDV were more susceptible to in vitro BVDV infection and showed a more severe apoptosis response than those from na\uefve animals. Non-cytopathic BVDV in vitro infection also resulted in a lack of effect in the expression of antigen presentation surface markers. All these findings could be related to the immunosuppressive capacity of BVDV and the susceptibility of cattle to this infection

Field study on plasma haptoglobin concentrations and total milk somatic cell counts in cows with untreated and treated mastitis

The aim of the present study was to determine plasma haptoglobin concentrations in cows in field conditions. Experiments were carried out a total of 106 cows, divided in 5 groups. The 1stgroup comprised 25 cows with low milk somatic cell counts (SCC), without microbiological findings used as controls; the IIndgroup -23 cows with untreated subclinical mastitis with high milk SCC and without microbiological findings; the IIIrdgroup: 13 cows with untreated subclinical mastitis with high milk SCC and with microbiological findings. The IVthgroup consisted of 17 cows with subclinical mastitis, high milk SCC, microbiological findings, treated with tetracycline HCL 200 mg, neomycin 250 mg, bacitracin 2000 IU, prednisolone 10 mg (Mastijet fort\uae, MSD Animal Health), and the Vthgroup - 28 cows with clinical mastitis treated with Mastijet fort\uae, MSD Animal Health. Our results showed that the mean plasma haptoglobin concentrations in the I (control) group was 0.054\ub10.003 g/L. In the IIndgroup (0.288\ub10.04 g/L) Hp was 5.2 times higher that the mean values of controls (P<0.001). Mean Hp concentration of group III was also statistically significantly different (P<0.001) than that of the control group. The value of Hp in group IV (0.215\ub10.04 g/L) was significantly lower (P<0.05) that mean values of IIndgroup but substantially higher (P<0.001) vs controls. The concentration of haptoglobin in cows of the Vthgroup was statistically significantly lower (P<0.001) compared both with control and IIndgroups. In this study, total somatic cell count showed statistically significant differences (P<0.001) between the control and experimental groups

Effects of nucleotides administration on growth performance and immune response of post-weaning piglets

The aim of this study was to assess the effect of nucleotides administration on growth perform- ance and immune response in post-weaning piglets. Twenty-eight male weaned piglets, homo- geneous for age and weight were randomly allocated to two experimental treatments. Treated group (T) was daily orally administered 0.8g/head of a mixture of nucleotides suspended in 2.1 mL water solution; while control group (C) received 2.1 mL saline solution. Body weight (BW) and average daily gain (ADG) were individually recorded weekly, while feed intake (FI), and gain:feed (G:F) were recorded and calculated on pen basis. Faecal score was evaluated every seven days. On day 0, 9, 18 and 27 blood samples were collected to determine IgA, IgG and haptoglobin concentration. At day 28 all piglets were sacrificed, and tissue samples of ileal Peyer\u2019s patches were collected for the evaluation of IL1a, IL1b, IL6, IL10, TNFa, TLR2, TLR4 and PPARc gene expression. Nucleotides supplementation significantly increased BW (17.37 vs. 19.00kg/pig; p 1\u20444 &lt;.01), ADG (.351 vs. .400kg/d; p &lt; .01), and FI (3.96 vs. 4.39kg/d; p &lt; .01), but not G:F (.61 vs. .64; p 1\u20444 .29). Faecal consistency was not different between the experimental groups and no occurrence of diarrhoea was reported. IgA and IgG content in blood was not influenced by the treatment, as well as gene expression of inflammatory cytokines in Peyer\u2019s patches. The present trial shows that nucleotide administration is able to improve growth per- formance of post-weaning piglets, with no effects on inflammatory response and the expression of immune-related genes

Purification, inhibitory properties, amino-acid-sequence and identification of the reactive-site of a new serine proteinase- inhibitor from oil-rape (Brassica napus) seed

A new serine proteinase inhibitor, rapeseed trypsin inhibitor (RTI), has been isolated from rapeseed (Brassica napus var. oleifera) seed. The protein inhibits the catalytic activity of bovine beta-trypsin and bovine alpha-chymotrypsin with apparent dissociation constants of 3.0 x 10(-10) M and 4.1 x 10(-7) M, at pH 8.0 and 21 degrees C, respectively. The stoichiometry of both proteinase-inhibitor complexes is 1:1. The amino acid sequence of RTI consists of 60 amino acid residues, corresponding to an M(r) of about 6.7 kDa. The P1-P1' reactive site bond has been tentatively identified at position Arg20-Ile21. RTI shows no similarity to other serine proteinase inhibitors except the low molecular weight mustard trypsin inhibitor (MTI-2). RTI and MTI-2 could be members of a new class of plant serine proteinase inhibitors