Salmonella enterica infections in market swine with and without transport and holding

The objective of this study was to compare, by using identical sample types, the Salmonella enterica prevalences and serovar diversities between pigs necropsied on the farm and those necropsied at the abattoir after transport and holding. We necropsied 567 market weight pigs (>70 kg) from six herds. Pigs were alternately assigned to be necropsied on the farm or at the abattoir. One-half of the group was sent in clean, disinfected trailers to slaughter at a commercial abattoir. After transport (mean distance, 169 km) and 2 to 3 h of holding in antemortem pens, these pigs were necropsied. The 50 pigs remaining on the farm were necropsied the following day. The same sample types and amounts were collected for S. enterica culture at both locations. Results show a sevenfold-higher (P < 0.001) S. enterica isolation rate from pigs necropsied at the abattoir (39.9%; 114 of 286) than from those necropsied on the farm (5.3%; 15 of 281). This difference was also observed for each individual herd. All sample types showed a significantly higher prevalence when comparing abattoir to on-farm collection, respectively: lymph nodes, 9.15 versus 3.6%; cecal contents, 13.6 versus 1.8%; 1 g of fecal matter, 25.2 versus 0.7%. Recovery of additional serovars at the abattoir suggests the pigs are receiving S. enterica from extra-farm sources. This study demonstrates that rapid infection during transport, and particularly during holding, is a major reason for increased S. enterica prevalence in swine. This finding identifies the holding pen as an important S. enterica control point in the pork production chain.     

Preslaughter holding environment in pork plants is highly contaminated with Salmonella enterica

The objective of this study was to determine whether abattoir pens can provide a Salmonella enterica infection source during the 2 to 4 h of preharvest holding. Previous work has suggested that pigs may be getting infected, but little has been reported on the environmental contamination of abattoir holding pens. For 24 groups of pigs studied ( approximately 150 animals/group) at two high-capacity abattoirs, six pooled fecal samples (n, 10 per pool) were collected from each transport trailer immediately after pigs were unloaded. Holding pens were sampled (one drinking water sample and six pooled floor samples consisting of swabs, residual liquid, and feces) prior to entry of study pigs for the routine holding period ( approximately 2.5 h). After slaughter, cecal contents and ileocecal lymph nodes were collected, on the processing line, from 30 pigs in each studied group. All samples were cultured for the isolation and identification of S. enterica by primary enrichment in GN-Hajna and tetrathionate broths, secondary enrichment in Rappaport-Vassiliadis broth, and plating on brilliant green sulfa and xylose-lysine-tergitol-4 agars, followed by biochemical and serological identification. The study pens were highly contaminated with S. enterica; all holding pens sampled had at least one positive sample. Additionally, 33% (8 of 24) of drinking water samples were positive for S. enterica. All 24 groups of pigs had S. enterica-positive cecal contents and ileocecal lymph nodes, including those groups from transport trailers with no positive samples. From pigs, trailers, and pens, 586 isolates representing 36 different Salmonella serovars were isolated. Of the 353 isolates from pigs (109 from ileocecal lymph nodes plus 244 from cecal contents), 19% were identified as belonging to the same serovars as those isolated from the respective pens; 27% were identified as belonging to the same serovars as those isolated from the trailers. Sixteen percent of the unique serovars were isolated from both pigs and pens, suggesting that pens served as the infection source. This study demonstrates highly contaminated abattoir holding pens and watering sources. It also demonstrates that holding pens can serve as an infection source. This study identifies the abattoir holding pens as a significant hazard and a potential control point for Salmonella contamination in the preharvest pork production chain.     

Study of mRNA Expression by Real Time PCR of Cpkk1, Cpkk2 and Cpkk3, three MEKs of Cryphonectria parasitica,in Virus‐free and Virus‐infected Isogenic Isolates

Cpkk1 and Cpkk2 are two previously characterized Mitogen‐activated protein kinase kinases (MEK) from Cryphonectria parasitica. For the characterization of the third MEK, primers designed to a conserved region of the known fungal MEK sequences were used in a PCR reaction to amplify genomic DNA from C. parasitica. The sequence of the resulting amplicon was compared to known sequences in the database using a Blast search. Results of the sequence comparison indicated that the initial fragment obtained encoded for a new MEK from C. parasitica, that had highest homology to Pbs2 from Saccharomyces cerevisiae. By inverse PCR we obtained a genomic fragment spanning the entire coding sequence of this MEK, which was named Cpkk3. The cDNA of Cpkk3 was obtained by compiling the sequences of RT‐PCR products resulting from the amplification of purified mRNA. TaqMan^® Probes were designed to analyse the expression of Cpkk1, Cpkk2 and Cpkk3 mRNA through RT‐Real Time PCR. This protocol allowed the expression of Cpkk3 to be successfully compared to the expression of Cpkk1 and Cpkk2, two previously cloned C. parasitica MEKs. No variation in expression was associated with the presence of a virus after 2 days of growth in standard conditions whereas an increase in the expression level of all the three MEKs was shown after 4 days of growth.     

Phage Therapy To Reduce Preprocessing Salmonella Infections in Market-Weight Swine

Contamination of meat products with food-borne pathogens usually results from the carcass coming in contact with the feces of an infected animal during processing. In the case of Salmonella, pigs can become colonized with the organism during transport and lairage from contaminated trailers and holding pens, resulting in increased pathogen shedding just prior to processing. Increased shedding, in turn, amplifies the likelihood of carcass contamination by magnifying the amount of bacteria that enters the processing facility. We conducted a series of experiments to test whether phage therapy could limit Salmonella infections at this crucial period. In a preliminary experiment done with small pigs (3 to 4 weeks old; 30 to 40 lb), administration of an anti-Salmonella phage cocktail at the time of inoculation with Salmonella enterica serovar Typhimurium reduced Salmonella colonization by 99.0 to 99.9% (2- to 3-log reduction) in the tonsils, ileum, and cecum. To test the efficacy of phage therapy in a production-like setting, we inoculated four market-weight pigs (in three replicates) with Salmonella enterica serovar Typhimurium and allowed the challenged pigs to contaminate a holding pen for 48 h. Sixteen naïve pigs were randomly split into two groups which received either the anti-Salmonella phage cocktail or a mock treatment. Both groups of pigs were comingled with the challenged pigs in the contaminated pen. Treatment with the anti-Salmonella phage cocktail significantly reduced cecal Salmonella concentrations (95%; P < 0.05) while also reducing (numerically) ileal Salmonella concentrations (90%; P = 0.06). Additional in vitro studies showed that the phage cocktail was also lytic against several non-Typhimurium serovars.The U.S. Centers for Disease Control and Prevention report approximately 40,000 culture-confirmed cases of salmonellosis each year in the United States, which result in approximately 400 deaths (5). Many Salmonella outbreaks are associated with meat and poultry (20), with contamination usually resulting from the carcass coming into contact with the feces of a Salmonella-infected animal during processing (22).There is an association between pork products and Salmonella, as swine are generally considered to be the second largest reservoir of the organism among food animals after poultry. Although infections in adult swine are normally asymptomatic, once colonized, pigs can shed the organism in the feces for weeks and sometimes months (7).While a great deal of research has been done on developing on-farm anti-Salmonella intervention strategies, these methods are confounded by the fact that Salmonella prevalence in pigs often increases once the animals leave the farm as a result of (i) stress-induced reactivation of preexisting infections (14), (ii) new infections from contaminated transport trailers and processing facility holding pens (12, 15, 24, 31), or (iii) both. Consequently, animals with no history of previous Salmonella infection can begin shedding the organism just prior to processing, which is highly problematic in terms of food safety.We hypothesized that phage therapy could be developed as an effective means to counteract transport- and lairage-associated increases in Salmonella colonization in swine. Phage therapy has the advantage of being natural, nontoxic, and relatively inexpensive and could be used just prior to slaughter, unlike many antibiotics (18, 28). Here we describe a series of experiments demonstrating that treating market-weight pigs with an anti-Salmonella phage cocktail prior to their comingling with Salmonella-infected pigs in a highly contaminated environment resulted in reductions in Salmonella colonization. We further show that the phage cocktail could be effectively microencapsulated, making feed or water delivery possible.     

Familial Danish dementia: co-existence of Danish and Alzheimer amyloid subunits (ADan AND A{beta}) in the absence of compact plaques

Familial Danish dementia is an early onset autosomal dominant neurodegenerative disorder linked to a genetic defect in the BRI2 gene and clinically characterized by dementia and ataxia. Cerebral amyloid and preamyloid deposits of two unrelated molecules (Danish amyloid (ADan) and beta-amyloid (Abeta)), the absence of compact plaques, and neurofibrillary degeneration indistinguishable from that observed in Alzheimer disease (AD) are the main neuropathological features of the disease. Biochemical analysis of extracted amyloid and preamyloid species indicates that as the solubility of the deposits decreases, the heterogeneity and complexity of the extracted peptides exponentially increase. Nonfibrillar deposits were mainly composed of intact ADan-(1-34) and its N-terminally modified (pyroglutamate) counterpart together with Abeta-(1-42) and Abeta-(4-42) in approximately 1:1 mixture. The post-translational modification, glutamate to pyroglutamate, was not present in soluble circulating ADan. In the amyloid fractions, ADan was heavily oligomerized and highly heterogeneous at the N and C terminus, and, when intact, its N terminus was post-translationally modified (pyroglutamate), whereas Abeta was mainly Abeta-(4-42). In all cases, the presence of Abeta-(X-40) was negligible, a surprising finding in view of the prevalence of Abeta40 in vascular deposits observed in sporadic and familial AD, Down syndrome, and normal aging. Whether the presence of the two amyloid subunits is imperative for the disease phenotype or just reflects a conformational mimicry remains to be elucidated; nonetheless, a specific interaction between ADan oligomers and Abeta molecules was demonstrated in vitro by ligand blot analysis using synthetic peptides. The absence of compact plaques in the presence of extensive neuro fibrillar degeneration strongly suggests that compact plaques, fundamental lesions for the diagnosis of AD, are not essential for the mechanism of dementia.     

BRI2 interacts with amyloid precursor protein (APP) and regulates amyloid beta (Abeta) production

Transmembrane proteins BRI2 and amyloid precursor protein (APP) co-localize with amyloid beta (Abeta) lesions in sporadic Alzheimer disease and mutations in both precursor proteins are linked to early-onset familial cases of cerebral amyloidosis associated with dementia and/or cerebral hemorrhage. A specific interaction between BRI2 and APP was unveiled by immunoprecipitation experiments using transfected and non-transfected cells. The use of deletion mutants further revealed that stretches 648-719 of APP751 and 46-106 of BRI2, both inclusive of the full transmembrane domains, are sufficient for the interaction. Removal of most of the APP and BRI2 extracellular domains without affecting the interaction implies that both proteins interact when are expressed on the same cell membrane (cis) rather than on adjacent cells (trans). The presence of BRI2 had a modulatory effect on APP processing, specifically increasing the levels of cellular APP as well as beta-secretase-generated COOH-terminal fragments while decreasing the levels of alpha-secretase-generated COOH-terminal fragments as well as the secretion of total APP and Abeta peptides. Determining the precise molecular pathways affected by the specific binding between APP and BRI2 could result in the identification of common therapeutic targets for these sporadic and familial neurodegenerative disorders.     

Systemic catabolism of Alzheimer's Abeta40 and Abeta42

To better understand the physiologic excretion and/or catabolism of circulating peripheral amyloid beta (Abeta), we labeled human Abeta40 (monomeric, with predominant unordered structure) and Abeta42 (mixture of monomers and oligomers in approximately 50:50 ratio, rich in beta-sheet conformation) with either Na(125)I or (125)I-tyramine cellobiose, also known as the cell-trapping ligand procedure, testing their blood clearance and organ uptake in B6SJLF1/J mice. Irrespective of the labeling protocol, the peptide conformation, and the degree of oligomerization, both Abeta40 and Abeta42 showed a short half-life of 2.5-3.0 min. The liver was the major organ responsible for plasma clearance, accounting for >60% of the peptide uptake, followed by the kidney. In vivo, hepatocytes captured >90% of the radiolabeled peptides which, after endocytosis, were preferentially catabolized and excreted into the bile. Biliary excretion of intact as well as partially degraded Abeta species became obviously relevant at doses above 10 microg. The use of biotin-labeled Abeta allowed the visualization of the interaction with HepG2 cells in culture, whereas competitive inhibition experiments with unlabeled Abeta demonstrated the specificity of the binding. The capability of the liver to uptake, catabolize, and excrete large doses of Abeta, several orders of magnitude above its physiologic concentration, may explain not only the femtomolar plasma levels of Abeta but the little fluctuation observed with age and disease stages.     

Matrix Metalloproteinase 2 (MMP-2) Degrades Soluble Vasculotropic Amyloid-β E22Q and L34V Mutants,Delaying Their Toxicity for Human Brain Microvascular Endothelial Cells

Patients carrying mutations within the amyloid-β (Aβ) sequence develop severe early-onset cerebral amyloid angiopathy with some of the related variants manifesting primarily with hemorrhagic phenotypes. Matrix metalloproteases (MMPs) are typically associated with blood brain barrier disruption and hemorrhagic transformations after ischemic stroke. However, their contribution to cerebral amyloid angiopathy-related hemorrhage remains unclear. Human brain endothelial cells challenged with Aβ synthetic homologues containing mutations known to be associated in vivo with hemorrhagic manifestations (AβE22Q and AβL34V) showed enhanced production and activation of MMP-2, evaluated via Multiplex MMP antibody arrays, gel zymography, and Western blot, which in turn proteolytically cleaved in situ the Aβ peptides. Immunoprecipitation followed by mass spectrometry analysis highlighted the generation of specific C-terminal proteolytic fragments, in particular the accumulation of Aβ-(1–16), a result validated in vitro with recombinant MMP-2 and quantitatively evaluated using deuterium-labeled internal standards. Silencing MMP-2 gene expression resulted in reduced Aβ degradation and enhanced apoptosis. Secretion and activation of MMP-2 as well as susceptibility of the Aβ peptides to MMP-2 degradation were dependent on the peptide conformation, with fibrillar elements of AβE22Q exhibiting negligible effects. Our results indicate that MMP-2 release and activation differentially degrades Aβ species, delaying their toxicity for endothelial cells. However, taking into consideration MMP ability to degrade basement membrane components, these protective effects might also undesirably compromise blood brain barrier integrity and precipitate a hemorrhagic phenotype.     

CD300 heterocomplexes, a new and family-restricted mechanism for myeloid cell signaling regulation

The CD300 family of myeloid immunoglobulin receptors includes activating (CD300b, CD300e) and inhibitory members (CD300a, CD300f), as well as molecules of uncertain function presenting a negative charge within their transmembrane domain (CD300c, CD300d). In this paper, we establish that CD300c is a functional immune receptor able to deliver activating signals upon ligation in RBL-2H3 mast cells. CD300c signaling is partially mediated by a direct association with the immune receptor tyrosine-based activation motif-bearing adaptor FcεRγ. The existence of complementary transmembrane-charged residues in certain CD300 receptors suggested the formation of heterodimers within this family. Indeed, we proved the interaction between CD300b and CD300c in transfected COS-7 cells and demonstrated that it has important functional consequences. Unexpectedly, dimmer formation was dependent on the immunoglobulin domains rather than the charged transmembrane residues. Concordantly, all CD300 members were found to interact with each other, even with themselves, forming both homo- and heterodimers. We found that the combination of CD300 receptors in a complex differentially modulates the signaling outcome, strongly suggesting a new mechanism by which CD300 complexes could regulate the activation of myeloid cells upon interaction with their natural ligands.