Retention of ingested porcine reproductive and respiratory syndrome virus in houseflies.

OBJECTIVE: To evaluate retention of porcine reproductive and respiratory syndrome virus (PRRSV) in houseflies for various time frames and temperatures. SAMPLE POPULATION: Fifteen 2-week-old pigs, two 10-week-old pigs, and laboratory-cultivated houseflies. PROCEDURE: In an initial experiment, houseflies were exposed to PRRSV; housed at 15 degrees, 20 degrees, 25 degrees, and 30 degrees C; and tested at various time points. In a second experiment to determine dynamics of virus retention, houseflies were exposed to PRRSV and housed under controlled field conditions for 48 hours. Changes in the percentage of PRRSV-positive flies and virus load per fly were assessed over time, and detection of infective virus at 48 hours after exposure was measured. Finally, in a third experiment, virus loads were measured in houseflies allowed to feed on blood, oropharyngeal washings, and nasal washings obtained from experimentally infected pigs. RESULTS: In experiment 1, PRRSV retention in houseflies was proportional to temperature. In the second experiment, the percentage of PRRSV-positive houseflies and virus load per fly decreased over time; however, infective PRRSV was found in houseflies 48 hours after exposure. In experiment 3, PRRSV was detected in houseflies allowed to feed on all 3 porcine body fluids. CONCLUSIONS AND CLINICAL RELEVANCE: For the conditions of this study, houseflies did not support PRRSV replication. Therefore, retention of PRRSV in houseflies appears to be a function of initial virus load after ingestion and environmental temperature. These factors may impact the risk of insect-borne spread of PRRSV among farms.

Post-transplant upregulation of chemokine messenger RNA in non-human primate recipients of intraportal pig islet xenografts.

BACKGROUND: We have previously shown that pig-to-primate intraportal islet xenografts reverse diabetes, escape hyperacute rejection, and undergo acute cellular rejection in non-immunosuppressed recipients. To gain a better understanding of mechanisms contributing to xenoislet rejection in non-human primates we examined gene expression in livers bearing islet xenografts in the first 72 h after transplantation. METHODS: Liver specimens were collected at sacrifice from seven non-immunosuppressed rhesus macaques at 12, 24, 48 and 72 h after intraportal porcine islet transplantation. Following total RNA extraction, mRNA was quantified using SYBR green real-time reverse transcription polymerase chain reaction (RT-PCR) for species-specific immune response genes. Data were analyzed using comparative cycle threshold (Ct) analysis, adjusted for specific primer-efficiencies and normalized to cyclophilin expression. RESULTS: Porcine insulin mRNA was detected in all liver samples. Cluster analysis revealed differential gene expression patterns at 12 and 24 h (early) compared with at 48 and 72 h (late) post-transplant. Gene expression patterns were associated with histological findings of predominantly neutrophils and only a few lymphocytes at 12 and 24 h and an increasing number of lymphocytes and macrophages at 48 and 72 h. Transcript levels of CXCR3 and its ligands, interferon-inducible protein 10 (IP-10) and monokine induced by IFN-gamma (Mig), significantly increased between early and late time points together with expression of MIP-1alpha, regulated on activation normal T expressed and secreted protein (RANTES) and MCP-1. CCR5 showed only a marginal, non-significant increase. Fas ligand, perforin and granzyme B transcripts were all elevated at 48 and 72 h post-transplant. CONCLUSIONS: Our data suggest that CXCR3, with ligands IP-10 and Mig, is involved in T cell recruitment in acute islet xenograft rejection in non-human primates. Upregulation of RANTES and MIP-1alpha transcripts in the absence of a significant CCR5 increase suggests a possible involvement of other chemokine receptors. MCP-1 expression is associated with T cell and macrophage infiltration. Elevated cytotoxic effector molecule expression (Fas ligand, perforin, granzyme B) indicates T-cell mediated graft destruction by cytotoxic and cytolytic mechanisms within 48 to 72 h after transplantation. These results identify the CXCR3-mediated chemoattractant pathway as an immunosuppressive target in pig-to-primate islet xenotransplantation.

Nitric oxide synthase stimulates prostaglandin synthesis and barrier function in C. parvum-infected porcine ileum.

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.