Search

Cell culture models implicate increased nitric oxide (NO) synthesis as a cause of mucosal hyperpermeability in intestinal epithelial infection. NO may also mediate a multitude of subepithelial events, including activation of cyclooxygenases. We examined whether NO promotes barrier function via prostaglandin synthesis using Cryptosporidium parvum-infected ileal epithelium in residence with an intact submucosa. Expression of NO synthase (NOS) isoforms was examined by real-time RT-PCR of ileal mucosa from control and C. parvum-infected piglets. The isoforms mediating and mechanism of NO action on barrier function were assessed by measuring transepithelial resistance (TER) and eicosanoid synthesis by ileal mucosa mounted in Ussing chambers in the presence of selective and nonselective NOS inhibitors and after rescue with exogenous prostaglandins. C. parvum infection results in induction of mucosal inducible NOS (iNOS), increased synthesis of NO and PGE2, and increased mucosal permeability. Nonselective inhibition of NOS (NG-nitro-L-arginine methyl ester) inhibited prostaglandin synthesis, resulting in further increases in paracellular permeability. Baseline permeability was restored in the absence of NO by exogenous PGE2. Selective inhibition of iNOS [L-N6-(1-iminoethyl)-L-lysine] accounted for approximately 50% of NOS-dependent PGE2 synthesis and TER. Using an entire intestinal mucosa, we have demonstrated for the first time that NO serves as a proximal mediator of PGE2 synthesis and barrier function in C. parvum infection. Expression of iNOS by infected mucosa was without detriment to overall barrier function and may serve to promote clearance of infected enterocytes.

Porcine reproductive and respiratory syndrome (PRRS) continues to be one of the most significant diseases of swine. IDEXX HerdChek PRRS, a commercially available enzyme-linked immunosorbent assay (ELISA), has become the industry standard for the detection of antibodies against PRRS virus (PRRSV). The need to accurately determine the PRRSV serostatus of herds and individual animals has prompted the development of several follow-up assay methods. A highly specific and repeatable blocking ELISA (bELISA) was developed on the basis of the use of an expressed PRRSV nucleocapsid (N) protein as the antigen and a biotinylated monoclonal antibody. Validation of the bELISA used sera from 316 animals experimentally and naturally infected with North American PRRSV and sera from 370 uninfected animals. Receiver operating characteristic analysis of the data calculated a diagnostic sensitivity of 97.8% and a diagnostic specificity of 100%. The between-run coefficient of variation of an internal quality control serum was 4.24%. The bELISA was able to detect seroconversion as well as the IDEXX ELISA and the indirect fluorescent antibody (IFA) assay; kappa values were 0.94 and 0.96, respectively. A collection of 133 serum samples with unexpected positive IDEXX ELISA results was obtained from 4,038 diagnostic samples submitted from farms from which PRRS-negative results were expected. The bELISA identified 97% of the samples as PRRS seronegative, while the IFA identified 100% as seronegative. The anticipated use of the bELISA is as a follow-up test to the IDEXX ELISA for determining the PRRSV serostatus of individual animals with unexpected positive test results from swine herds from which negative results are expected.

We recently demonstrated that pigs infected with porcine encephalomyocarditis virus (EMCV) develop a persistent infection (up to 90 days post-infection (PI)) in the heart and brain that is accompanied by virus-induced pathologic changes, and that EMCV productively infects human cardiomyocytes in vitro, suggesting that EMCV may pose a risk to humans following transplantation of pig tissues to humans (Brewer et al. J Virol 2001; 75: 11621-11629). In this report, we demonstrate that intra-abdominal of myocardial or pancreatic sections from acutely-EMCV infected pigs (2 days PI) in either non-mutant C57BL/6 or C57BL/6-RAG-1-/- mice that lack B or T lymphocytes, resulted in transmission of the virus and acute fatal disease in all mice. In recipient RAG-1-/- mice, fatal EMCV disease occurred within 2 days post-transplantation, and it was accompanied by high virus titers in brain, heart, liver, spleen, kidneys and skeletal muscle, whereas in non-mutant C57BL/6 mice, disease occurred 5 to 6 days post-transplantation and was accompanied by lower virus titers. Transplantation of myocardial or pancreatic tissues from chronically EMCV-infected pigs (21 and 50 days PI) did not induce clinical disease, but resulted in detection of EMCV RNA in the brain of recipient RAG-1-/- mice, no viral RNA was detected in non-mutant C57BL/6 mice. Intra-abdominal transplantation of uninfected porcine myocardial tissues into RAG-1-/- mice followed by intramuscular inoculation with EMCV induced acute clinical disease but did not result in transmission of virus to the xenograft. These results show that EMCV can be efficiently transmitted from pig myocardial and pancreatic tissues to mice, providing a model of pig-to-human viral xenozoonosis that can be used to develop and test prophylactic and therapeutic measures against such infection.

Pentoxifylline, a methylxanthine derivative and nonspecific type 4 phosphodiesterase inhibitor, has been used to improve survival of animals with sepsis and to attenuate lung injury in acute lung inflammation. The purpose of this study was to examine whether pentoxifylline would inhibit the expression of inflammatory cytokines, particularly tumor necrosis factor alpha (TNF), and thereby decrease the pathophysiology of acute porcine pleuropneumonia. E. coli lipopolysaccharide (LPS) and bacterial extracts of A. pleuropneumoniae--induced elevations in TNF mRNA which were fully abrogated by addition of pentoxifylline in both alveolar macrophage and neutrophil cultures. A 30% reduction in the level of LPS-induced interleukin (IL)-1beta mRNA levels also was achieved in macrophages. Pentoxifylline did not affect either IL-1alpha or IL-8 expression in vitro. Pentoxifylline therapy in vivo significantly reduced the number of band neutrophils in swine but did not reduce the pathology associated with pleuropneumonia, including changes in serum zinc, iron, or haptoglobin. Neither did it alter TNF, IL-1, IL-6, or IL-8 expression. Measurement of pentoxifylline and its metabolites in pig sera suggested that efficacious doses of pentoxifylline were probably not achieved in vivo. However, subcutaneous doses of pentoxifylline higher than 25 mg/kg produced transient diarrhea, vomiting, and tremors. These results suggest that pentoxifylline is an effective pharmacological tool for the dissection of cytokine regulation in vitro, but inhibitory concentrations may not be achievable for in vivo pharmacological use in swine.