Use of multi-trait and random regression models to identify genetic variation in tolerance to porcine reproductive and respiratory syndrome virus

Background: A host can adopt two response strategies to infection: resistance (reduce pathogen load) and tolerance (minimize impact of infection on performance). Both strategies may be under genetic control and could thus be targeted for genetic improvement. Although there is evidence that supports a genetic basis for resistance to porcine reproductive and respiratory syndrome (PRRS), it is not known whether pigs also differ genetically in tolerance. We determined to what extent pigs that have been shown to vary genetically in resistance to PRRS also exhibit genetic variation in tolerance. Multi-trait linear mixed models and random regression sire models were fitted to PRRS Host Genetics Consortium data from 1320 weaned pigs (offspring of 54 sires) that were experimentally infected with a virulent strain of PRRS virus to obtain genetic parameter estimates for resistance and tolerance. Resistance was defined as the inverse of within-host viral load (VL) from 0 to 21 (VL21) or 0 to 42 (VL42) days post-infection and tolerance as the slope of the reaction-norm of average daily gain (ADG21, ADG42) on VL21 or VL42. Results: Multi-trait analysis of ADG associated with either low or high VL was not indicative of genetic variation in tolerance. Similarly, random regression models for ADG21 and ADG42 with a tolerance slope fitted for each sire did not result in a better fit to the data than a model without genetic variation in tolerance. However, the distribution of data around average VL suggested possible confounding between level and slope estimates of the regression lines. Augmenting the data with simulated growth rates of non-infected half-sibs (ADG0) helped resolve this statistical confounding and indicated that genetic variation in tolerance to PRRS may exist if genetic correlations between ADG0 and ADG21 or ADG42 are low to moderate. Conclusions: Evidence for genetic variation in tolerance of pigs to PRRS was weak when based on data from infected piglets only. However, simulations indicated that genetic variance in tolerance may exist and could be detected if comparable data on uninfected relatives were available. In conclusion, of the two defense strategies, genetics of tolerance is more difficult to elucidate than genetics of resistance.</p

Mild hypothermia delays the development of stone heart from untreated sustained ventricular fibrillation - a cardiovascular magnetic resonance study

<p>Abstract</p> <p>Background</p> <p>'Stone heart' resulting from ischemic contracture of the myocardium, precludes successful resuscitation from ventricular fibrillation (VF). We hypothesized that mild hypothermia might slow the progression to stone heart.</p> <p>Methods</p> <p>Fourteen swine (27 ± 1 kg) were randomized to normothermia (group I; n = 6) or hypothermia groups (group II; n = 8). Mild hypothermia (34 ± 2°C) was induced with ice packs prior to VF induction. The LV and right ventricular (RV) cross-sectional areas were followed by cardiovascular magnetic resonance until the development of stone heart. A commercial 1.5T GE Signa NV-CV/i scanner was used. Complete anatomic coverage of the heart was acquired using a steady-state free precession (SSFP) pulse sequence gated at baseline prior to VF onset. Un-gated SSFP images were obtained serially after VF induction. The ventricular endocardium was manually traced and LV and RV volumes were calculated at each time point.</p> <p>Results</p> <p>In group I, the LV was dilated compared to baseline at 5 minutes after VF and this remained for 20 minutes. Stone heart, arbitrarily defined as LV volume <1/3 of baseline at the onset of VF, occurred at 29 ± 3 minutes. In group II, there was less early dilation of the LV (p < 0.05) and the development of stone heart was delayed to 52 ± 4 minutes after onset of VF (P < 0.001).</p> <p>Conclusions</p> <p>In this closed-chest swine model of prolonged untreated VF, hypothermia reduced the early LV dilatation and importantly, delayed the onset of stone heart thereby extending a known, morphologic limit of resuscitability.</p

The Flagellar Regulator fliT Represses Salmonella Pathogenicity Island 1 through flhDC and fliZ

Salmonella pathogenicity island 1 (SPI1), comprising a type III section system that translocates effector proteins into host cells, is essential for the enteric pathogen Salmonella to penetrate the intestinal epithelium and subsequently to cause disease. Using random transposon mutagenesis, we found that a Tn10 disruption in the flagellar fliDST operon induced SPI1 expression when the strain was grown under conditions designed to repress SPI1, by mimicking the environment of the large intestine through the use of the intestinal fatty acid butyrate. Our genetic studies showed that only fliT within this operon was required for this effect, and that exogenous over-expression of fliT alone significantly reduced the expression of SPI1 genes, including the invasion regulator hilA and the sipBCDA operon, encoding type III section system effector proteins, and Salmonella invasion of cultured epithelial cells. fliT has been known to inhibit the flagellar machinery through repression of the flagellar master regulator flhDC. We found that the repressive effect of fliT on invasion genes was completely abolished in the absence of flhDC or fliZ, the latter previously shown to induce SPI1, indicating that this regulatory pathway is required for invasion control by fliT. Although this flhDC-fliZ pathway was necessary for fliT to negatively control invasion genes, fliZ was not essential for the repressive effect of fliT on motility, placing fliT high in the regulatory cascade for both invasion and motility

Crosstalk between Virulence Loci: Regulation of Salmonella enterica Pathogenicity Island 1 (SPI-1) by Products of the std Fimbrial Operon

Invasion of intestinal epithelial cells is a critical step in Salmonella infection and requires the expression of genes located in Salmonella pathogenicity island 1 (SPI-1). A key factor for SPI-1 expression is DNA adenine (Dam) methylation, which activates synthesis of the SPI-1 transcriptional activator HilD. Dam-dependent regulation of hilD is postranscriptional (and therefore indirect), indicating the involvement of unknown cell functions under Dam methylation control. A genetic screen has identified the std fimbrial operon as the missing link between Dam methylation and SPI-1. We show that all genes in the std operon are part of a single transcriptional unit, and describe three previously uncharacterized ORFs (renamed stdD, stdE, and stdF). We present evidence that two such loci (stdE and stdF) are involved in Dam-dependent control of Salmonella SPI-1: in a Dam− background, deletion of stdE or stdF suppresses SPI-1 repression; in a Dam+ background, constitutive expression of StdE and/or StdF represses SPI-1. Repression of SPI-1 by products of std operon explains the invasion defect of Salmonella Dam− mutants, which constitutively express the std operon. Dam-dependent repression of std in the ileum may be required to permit invasion, as indicated by two observations: constitutive expression of StdE and StdF reduces invasion of epithelial cells in vitro (1,000 fold) and attenuates Salmonella virulence in the mouse model (>60 fold). In turn, crosstalk between std and SPI-1 may play a role in intestinal infections by preventing expression of SPI-1 in the caecum, an intestinal compartment in which the std operon is known to be expressed

MrkH, a Novel c-di-GMP-Dependent Transcriptional Activator, Controls Klebsiella pneumoniae Biofilm Formation by Regulating Type 3 Fimbriae Expression

Klebsiella pneumoniae causes significant morbidity and mortality worldwide, particularly amongst hospitalized individuals. The principle mechanism for pathogenesis in hospital environments involves the formation of biofilms, primarily on implanted medical devices. In this study, we constructed a transposon mutant library in a clinical isolate, K. pneumoniae AJ218, to identify the genes and pathways implicated in biofilm formation. Three mutants severely defective in biofilm formation contained insertions within the mrkABCDF genes encoding the main structural subunit and assembly machinery for type 3 fimbriae. Two other mutants carried insertions within the yfiN and mrkJ genes, which encode GGDEF domain- and EAL domain-containing c-di-GMP turnover enzymes, respectively. The remaining two isolates contained insertions that inactivated the mrkH and mrkI genes, which encode for novel proteins with a c-di-GMP-binding PilZ domain and a LuxR-type transcriptional regulator, respectively. Biochemical and functional assays indicated that the effects of these factors on biofilm formation accompany concomitant changes in type 3 fimbriae expression. We mapped the transcriptional start site of mrkA, demonstrated that MrkH directly activates transcription of the mrkA promoter and showed that MrkH binds strongly to the mrkA regulatory region only in the presence of c-di-GMP. Furthermore, a point mutation in the putative c-di-GMP-binding domain of MrkH completely abolished its function as a transcriptional activator. In vivo analysis of the yfiN and mrkJ genes strongly indicated their c-di-GMP-specific function as diguanylate cyclase and phosphodiesterase, respectively. In addition, in vitro assays showed that purified MrkJ protein has strong c-di-GMP phosphodiesterase activity. These results demonstrate for the first time that c-di-GMP can function as an effector to stimulate the activity of a transcriptional activator, and explain how type 3 fimbriae expression is coordinated with other gene expression programs in K. pneumoniae to promote biofilm formation to implanted medical devices

Quantitative Analysis of Porcine Reproductive and Respiratory Syndrome (PRRS) Viremia Profiles from Experimental Infection: A Statistical Modelling Approach

Porcine reproductive and respiratory syndrome (PRRS) is one of the most economically significant viral diseases facing the global swine industry. Viremia profiles of PRRS virus challenged pigs reflect the severity and progression of infection within the host and provide crucial information for subsequent control measures. In this study we analyse the largest longitudinal PRRS viremia dataset from an in-vivo experiment. The primary objective was to provide a suitable mathematical description of all viremia profiles with biologically meaningful parameters for quantitative analysis of profile characteristics. The Wood's function, a gamma-type function, and a biphasic extended Wood's function were fit to the individual profiles using Bayesian inference with a likelihood framework. Using maximum likelihood inference and numerous fit criteria, we established that the broad spectrum of viremia trends could be adequately represented by either uni- or biphasic Wood's functions. Three viremic categories emerged: cleared (uni-modal and below detection within 42 days post infection(dpi)), persistent (transient experimental persistence over 42 dpi) and rebound (biphasic within 42 dpi). The convenient biological interpretation of the model parameters estimates, allowed us not only to quantify inter-host variation, but also to establish common viremia curve characteristics and their predictability. Statistical analysis of the profile characteristics revealed that persistent profiles were distinguishable already within the first 21 dpi, whereas it is not possible to predict the onset of viremia rebound. Analysis of the neutralizing antibody(nAb) data indicated that there was a ubiquitous strong response to the homologous PRRSV challenge, but high variability in the range of cross-protection of the nAbs. Persistent pigs were found to have a significantly higher nAb cross-protectivity than pigs that either cleared viremia or experienced rebound within 42 dpi. Our study provides novel insights into the nature and degree of variation of hosts' responses to infection as well as new informative traits for subsequent genomic and modelling studies