Isolation and Genetic Analysis of Bovine Viral Diarrhea Virus from Infected Cattle in Indiana

Species and biotype distribution was determined in 44 bovine viral diarrhea virus- (BVDV-) positive samples submitted to the Animal Disease Diagnostic Laboratory (ADDL) in Indiana during 2006–2008. BVDV RNA was detected in the 5′-untranslated region and Npro region using reverse transcriptase PCR followed by sequencing analysis of the PCR product. Additionally, cases were classified into one of six categories according to history and/or lesions: acute symptomatic, hemorrhagic, respiratory distress, reproductive, persistent infection (PI), and mucosal disease (MD). Of 44 BVDV-positive samples, 33 were noncytopathic (ncp), 10 were cytopathic (cp), and one presented both ncp and cp biotypes. Sequencing analysis demonstrated that all samples belonged to BVDV-1a, BVDV-1b, or BVDV-2. The most common isolate was ncp BVDV-1b, (44%) followed by ncp BVDV-2a (24%). Among the six categories, respiratory clinical signs were the most common (36%) followed by PI (25%) and MD (16%)

Understanding the Reduction of Porcine Epidemic Diarrhea Virus, Porcine Reproductive and Respiratory Syndrome Virus, and Seneca Valley Virus 1 RNA in Inoculated Feed and the Environment Following Thermal Processing

Pelleting of feed has been demonstrated to be an effective mitigation strategy for porcine epidemic diarrhea virus (PEDV) contaminated feed but has not been evaluated for other endemic swine viruses like porcine reproductive and respiratory syndrome virus (PRRSV) or Seneca Valley virus 1 (SVV1). Therefore, the objective of this experiment was to evaluate the efficacy of pelleting to inactivate PEDV, PRRSV, and SVV1 inoculated feed. Ten replicates were conducted in the Cargill Feed Safety Research Center at Kansas State University (K-State) using a pilot scale mixer, bucket elevator, pellet mill (including conditioner and die), and cooler. First, a virus negative batch of gestation feed was run through all equipment to simulate a commercial feed mill, then a positive batch of feed inoculated with all three viruses was run through all feed manufacturing equipment. Feed was conditioned to a minimum of 180°F with a 30 sec retention time; all feed was cooled for 10 min. Feed and environmental samples were taken from each piece of equipment following both the negative and positive batch. Samples were analyzed via PCR at the K-State Veterinary Diagnostic Laboratory. A four-room bioassay was conducted to evaluate the infectivity of the feed samples. Feed from the mixer and bucket elevator had greater quantities of SVV1, PEDV, and PRRSV RNA (P \u3c 0.05) than the other sampling locations. Similarly, environmental samples from the mixer and bucket elevator had greater SVV1 detection (P \u3c 0.05) than those collected from the conditioner, pellet die, and cooler. Pelleting reduced viral RNA (P \u3c 0.05) for all viruses in both feed and environmental samples. Although SVV1 and PEDV RNA were still detectable following pelleting, no pigs inoculated with the pelleted feed showed signs of SVV1 or PEDV clinical infection. Interestingly, PRRSV RNA was not detectable in pelleted feed samples. However, one pig showed signs of replicating PRRSV virus on d 7 of the bioassay which suggests a greater sensitivity when utilizing a bioassay compared to PCR alone. Overall, pelleting reduced the quantity of detectable viral RNA and reduced the risk of infectivity; yet small quantities of viral RNA remaining in the feed and environment following pelleting may increase the risk of re-contamination

Evaluating the Distribution of Porcine Epidemic Diarrhea Virus, Porcine Reproductive and Respiratory Syndrome Virus, and Seneca Valley Virus 1 Inoculated Feed After the Use of Physical or Chemical Mitigants to Flush a Feed Manufacturing Facility

Contaminated feed is a route of virus transmission between feed mills and swine farms. To reduce the risk of transmission, an understanding of the virus distribution and mitigation strategies are needed. The objective of this study was to evaluate the distribution of porcine epidemic diarrhea virus (PEDV), porcine reproductive and respiratory syndrome virus (PRRSV), and Seneca Valley virus 1 (SVV1) inoculated feed in the environment and feed of a feed mill before and after the use of chemical mitigants. A 50-lb batch of feed was run through a mixer and bucket elevator followed by a batch inoculated with PEDV, PRRSV, and SVV1. Following the virus-inoculated batch, a flush treatment of either 1) ground corn (GC); 2) GC + 1.5% liquid formaldehyde (LF; SalCURB LF Liquid, Kemin, Des Moines, IA); 3) GC + 1.5% LF + 25% abrasive material (SalCURB; Shell & Bone Builder, Iowa Limestone Company, Urbandale, IA); 4) double flush – GC + 25% abrasive material followed by GC +1.5% LF (Shell & Bone Builder; SalCURB); or 5) dry formaldehyde (SalCURB F2 Dry, Kemin, Des Moines, IA) was utilized, followed by 3 virus-free batches of complete feed. Feed and environmental samples were collected from each piece of equipment following every batch. Dust samples were collected after manufacturing from the inoculated, flush, and final batches from non-feed contact surfaces. Non-feed contact surfaces were considered those where dust would accumulate during manufacturing but would not be included in the final diet. The surfaces included the grates of the mixer, the top of the discharge bin following the bucket elevator, and the floor surrounding the same discharge bin. Samples were analyzed via a triplex PCR at the Kansas State University Veterinary Diagnostic Laboratory. A treatment × batch × location interaction was not observed (P \u3e 0.05) in feed or the environment for any of the viruses. A flush treatment × batch interaction was observed for SVV1 where greater quantities of viral RNA (P \u3c 0.05) were present in the positive batches and the ground corn flush than in those batches which used chemical mitigants or the post-flush batches. A lower quantity of viral RNA (P\u3c 0.05) in dust was observed in the last batch of feed compared to the inoculated batch for all viruses; however, SVV1 RNA was still detectable in the dust following the last batch in all treatments. A batch effect (P \u3c 0.05) was observed in all sample matrices for PEDV and PRRSV as viral RNA decreased after the implementation of the flush regardless of treatment. The use of chemical mitigants and the implementation of a flush batch reduced the quantity of viral RNA for PEDV, PRRSV, and SVV1. However, viral presence was still observed in feed and the dust on non-feed contact surfaces which could be a source of contamination if re-introduced into finished feed

Porcine circovirus type 2 (PCV2)-infection and re-inoculation with homologous or heterologous strains: virological, serological, pathological and clinical effects in growing pigs.

Long-term PCV2 infection and/or concurrent infection with genotypes PCV2a and PCV2b may play a role in the development of clinical porcine circovirus-associated disease (PCVAD). To evaluate this premise, 24 11-week-old specific pathogen-free (SPF) pigs were randomly assigned to 1 of 4 treatments: negative controls, a single inoculation with PCV2a, single inoculation followed by re-inoculation with a homologous PCV2a strain, or repeated inoculations with heterologous strains (PCV2a, PCV2b). Pigs were evaluated for clinical signs daily through 140 days post inoculation (dpi). Serum samples were collected every other day from dpi 0 through 14 and weekly thereafter. PCV2-inoculated pigs were viremic by dpi 2 and 13 of 18 pigs remained viremic at 140 dpi. No statistical differences in the onset, level, or duration of PCV2 viremia were detected among treatment groups. Anti-PCV2 antibodies were detected between 14 and 28 dpi and were present through 140 dpi without statistical differences in antibody response among treatment groups. In the current study, pigs had extended viremia combined with detectable tissue PCV2 antigen levels despite the presence of high levels of anti-PCV2 antibody; however, no clinical disease was observed

Infection of Cesarean-Derived Colostrum-Deprived Pigs with Porcine Circovirus Type 2 and Swine Influenza Virus

Porcine circovirus type 2 (PCV2) and swine influenza virus (SIV) are important pathogens for porcine respiratory disease complex, which is economically significant worldwide. The pathogenesis of PCV2–SIV coinfection is unknown. In this study, we focused on establishing a challenge model for PCV2 to determine whether SIV influences PCV2 replication and increases the severity of PCV2-associated disease. Cesarean-derived colostrum-deprived pigs were inoculated intratracheally with cell culture medium only (negative control group), PCV2 only, or PCV2 followed 1 wk later with SIV H1N1. Two pigs from each group were necropsied at 12, 21, 28, and 35 d after inoculation. Coinfection with SIV did not increase the number of PCV2 genomic copies in serum or target tissues or the severity of microscopic lesions associated with PCV2 in lung or lymph node. The antibody titer to PCV2 did not differ significantly between PCV2–SIV- and PCV2-infected groups. In conclusion, SIV H1N1 did not influence PCV2 replication in dually infected pigs in this study

Effective Targeted Gene Delivery to Dendritic Cells via Synergetic Interaction of Mannosylated Lipid with DOPE and BCAT

The efficient delivery of plasmids encoding antigenic determinants into dendritic cells (DCs) that control immune response is a promising strategy for rapid development of new vaccines. In this study, we prepared a series of targeted cationic lipoplex based on two synthetic lipid components, mannose-poly­(ethylene glycol, MW3000)-1,2-distearoyl-<i>sn</i>-glycero-3-phosphoethanolamine (Mannose-PEG3000-DSPE) and <i>O</i>-(2<i>R</i>-1,2-di-<i>O</i>-(1′<i>Z</i>-octadecenyl)-glycerol)-3-<i>N</i>-(bis-2-aminoethyl)-carbamate (BCAT), that were formulated with 1,2-dioleoyl-<i>sn</i>-glycero-3-phosphoethanolamine (DOPE) for evaluation as nonviral vectors for transgene expression in DCs. First, we optimized the N/P ratio for maximum transfection and then screened the effects of mannose targeting for further enhancement of transfection levels. Our results indicate that efficient delivery of gWIZ GFP plasmid into DCs was observed for mannose compositions of ∼10%, whereas low transfection efficiencies were observed with nontargeted formulations. Mannose-targeted lipofectamine complexes also showed high GFP expression levels in DCs relative to nontargeted lipofectamine controls. The best transfection performance was observed using 10 mol % Mannose-PEG3000-DSPE, 60 mol % BCAT, and 30 mol % DOPE, indicating that the most efficient delivery into DCs occurs via synergistic interaction between mannose targeting and acid-labile, fusogenic BCAT/DOPE formulations. Our data suggest that mannose-PEG3000-DSPE/BCAT/DOPE formulations may be effective gene delivery vehicles for the development of DC-based vaccines