Daniel Linhares

Evaluation of immune responses to porcine reproductive and respiratory syndrome virus in pigs during early stage of infection under farm conditions

BackgroundPorcine reproductive and respiratory syndrome virus (PRRSV) causes chronic, economically devastating disease in pigs of all ages. Frequent mutations in the viral genome result in viruses with immune escape mutants. Irrespective of regular vaccination, control of PRRSV remains a challenge to swine farmers. In PRRSV-infected pigs, innate cytokine IFN-α is inhibited and the adaptive arm of the immunity is delayed. To elucidate both cellular and innate cytokine responses at very early stages of PRRSV infection, seven weeks old pigs maintained on a commercial pig farm were infected and analyzed.ResultsOne pig in a pen containing 25 pigs was PRRSV infected and responses from this pig and one penmate were assessed two days later. All the infected and a few of the contact neighbor pigs were viremic. At day 2 post-infection, approximately 50% of viremic pigs had greater than 50% reduction in NK cell-mediated cytotoxicity, and nearly a 1-fold increase in IFN-α production was detected in blood of a few pigs. Enhanced secretion of IL-4 (in ~90%), IL-12 (in ~40%), and IL-10 (in ~20%) (but not IFN-γ) in PRRSV infected pigs was observed. In addition, reduced frequency of myeloid cells, CD4-CD8+ T cells, and CD4+CD8+ T cells and upregulated frequency of lymphocytes bearing natural T regulatory cell phenotype were detected in viremic pigs. Interestingly, all viremic contact pigs also had comparable immune cell modulations.ConclusionReplicating PRRSV in both infected and contact pigs was found to be responsible for rapid modulation in NK cell-meditated cytotoxicity and alteration in the production of important immune cytokines. PRRSV-induced immunological changes observed simultaneously at both cellular and cytokine levels early post-infection appear to be responsible for the delay in generation of adaptive immunity. As the study was performed in pigs maintained under commercial environmental conditions, this study has practical implications in design of protective vaccines.

Identification and Complete Genome of Seneca Valley Virus in Vesicular Fluid and Sera of Pigs Affected with Idiopathic Vesicular Disease, Brazil

Numerous, ongoing outbreaks in Brazilian swine herds have been characterized by vesicular lesions in sows and acute losses of neonatal piglets. The complete genome of Seneca Valley virus (SVV) was identified in vesicular fluid and sera of sows, providing evidence of association between SVV and vesicular disease and viraemia in affected animals.

Effect of modified-live porcine reproductive and respiratory syndrome virus (PRRSv) vaccine on the shedding of wild-type virus from an infected population of growing pigs

There are ongoing efforts to eliminate porcine reproductive and respiratory syndrome virus (PRRSv) from regions in the United States swine industry. However, an important challenge for the accomplishment of those efforts is the re-infection of pig units due to the area spread of PRRSv. The objective of this study was to evaluate the effect of PRRS modified-live virus vaccine (MLV) on viral shedding and on dynamics of PRRSv infection in pig populations raised under commercial conditions. The study composed of two rooms of 1000 pigs each. Ten percent of pigs of each room were inoculated with a field isolate of PRRSv. Rooms had separate air spaces and strict scientifically validated biosecurity protocols were adopted to avoid movement of pathogens between rooms. At 8 and 36 dpi (days post inoculation), all pigs of the challenge-vaccine group were inoculated with a MLV vaccine. Pigs of the challenge-control group were placebo-inoculated. Blood and oral fluid samples were collected from each room at 0, 8, 36, 70, 96 and 118 dpi for PRRSv RNA detection using PCR. PRRSv-antibodies were also screened from blood serum samples with a commercially available ELISA test. Additionally, tonsil scraping samples were collected from both groups at 70, 96 and 118 dpi. Moreover, air samples were collected 6 times per week from 0 to 118 dpi and were tested for PRRSv RNA using qPCR assay. There was no difference in the PRRSv infection dynamics measured as duration and magnitude of viremia and seroconversion. Also, there was no difference in the frequency of tonsil scraping samples PRRSv-positive by PCR. However, the challenge-vaccine group had significantly less PRRSv shed compared to the challenge-control group. The challenge-vaccine group had significant less PRRSv-positive oral fluids at 36 dpi. Moreover, the challenge-vaccine group had significant reduction in the cumulative PRRSv shed in the air.

Neonatal Mortality, Vesicular Lesions and Lameness Associated with Senecavirus A in a U.S. Sow Farm.

Summary A 300‐sow farrow‐to‐finish swine operation in the United States experienced a sudden and severe increase in mortality in neonatal piglets with high morbidity followed by vesicular lesions on the snout and feet of adult females and males. Affected live piglets were submitted for diagnostic investigation. Samples tested polymerase chain reaction (PCR) negative for foot‐and‐mouth disease virus, porcine delta coronavirus, porcine epidemic diarrhoea virus, porcine rotavirus types A, B and C, transmissible gastroenteritis virus, and porcine reproductive and respiratory syndrome virus. Senecavirus A (SV‐A) formerly known as Seneca Valley virus was detected by real‐time reverse‐transcription polymerase chain reaction (rRT‐PCR) from serum, skin and faeces of piglets and from serum and faeces of sows. SV‐A was isolated in cell culture from piglet samples. SV‐A VP1 gene region sequencing from piglet tissues was also successful. A biosecurity and disease entry evaluation was conducted and identified potential biosecurity risks factors for the entry of new pathogens into the operation. This is the first case report in the United States associating SV‐A with a clinical course of severe but transient neonatal morbidity and mortality followed by vesicular lesions in breeding stock animals. Veterinarians and animal caretakers must remain vigilant for vesicular foreign animal diseases and report suspicious clinical signs and lesions to state animal health authorities for diagnostic testing and further investigation.

Evaluation of Flinders Technology Associates cards for collection and transport of samples for detection of Porcine reproductive and respiratory syndrome virus by reverse transcription polymerase chain reaction

Blood, tissue and oral fluid samples collected from experimentally infected animals and field cases were used to evaluate the safety, diagnostic sensitivity and specificity of Flinders Technology Associates (FTA) cards for Porcine reproductive and respiratory syndrome virus (PRRSV) reverse transcription polymerase chain reaction (RT-PCR) diagnostics. The analytical sensitivity of PRRSV RT-PCR from serum and oral fluids in FTA cards was reduced, although the virus could still be detected at concentrations of 101 and 103 TCID/ml, respectively. The sensitivity and specificity of PRRSV RT-PCR detection from serum, blood, and tissue samples in cards collected from experimentally infected animals were 100%. Sensitivity for oral fluids was 45% (95% CI: 19.97–73.01) compared to fresh. For field samples, sensitivity was 89% (95% CI: 77.35–95.63) and 100% (95% CI: 80.00–100) for serum and lung samples, respectively. The sensitivity was the same for samples stored in cards at room temperature or at 4ºC, and tested overnight or after 14 days. Cards inoculated with PRRSV-positive samples did not yield replicating virus after cell culture. In conclusion, FTA cards proved to be a safe, simple, and sensitive alternative method to transport serum, blood, and tissue samples for PRRSV RT-PCR diagnostics; however, a significant decrease in RT-PCR sensitivity should be expected from oral fluid samples.

Serological and Molecular Detection of Senecavirus A Associated with an Outbreak of Swine Idiopathic Vesicular Disease and Neonatal Mortality

ABSTRACT We performed a longitudinal field study in a swine breeding herd that presented with an outbreak of vesicular disease (VD) that was associated with an increase in neonatal mortality. Initially, a USDA Foreign Animal Disease (FAD) investigation confirmed the presence of Senecavirus A (SVA) and ruled out the presence of exotic agents that produce vesicular lesions, e.g., foot-and-mouth disease virus and others. Subsequently, serum samples, tonsil swabs, and feces were collected from sows (n = 22) and their piglets (n = 33) beginning 1 week after the onset of the clinical outbreak and weekly for 6 weeks. The presence of SVA RNA was evaluated in all specimens collected by reverse transcriptase quantitative PCR (RT-qPCR) targeting a conserved region of the 5′ untranslated region (5′-UTR). The serological response (IgG) to SVA was evaluated by the weekly testing of sow and piglet serum samples on a SVA VP1 recombinant protein (rVP1) indirect enzyme-linked immunosorbent assay (ELISA). The rVP1 ELISA detected seroconversion against SVA in clinically affected and non-clinically affected sows at early stages of the outbreak as well as maternal SVA antibodies in offspring. Overall, the absence of vesicles (gross lesions) in SVA-infected animals and the variability of RT-qPCR results among specimen type demonstrated that a diagnostic algorithm based on the combination of clinical observations, RT-qPCR in multiple diagnostic specimens, and serology are essential to ensure an accurate diagnosis of SVA.

Novel Senecavirus A in Swine with Vesicular Disease, United States, July 2015.

To the Editor: Senecavirus A (SVA; formerly known as Seneca Valley virus [SVV] belongs to the genus Senecavirus, family Picornaviridae (1,2). SVA was first isolated in 2001 as a contaminant of the PER.C6 cell line and designated as SVV-001 (1,3). Since its discovery, SVA has been infrequently detected in swine with idiopathic vesicular disease (IVD) (4–6), which clinically resembles foot-and-mouth disease, swine vesicular disease, vesicular exanthema of swine, and vesicular stomatitis. The virus has also been retrospectively detected in previous cases with various clinical conditions in the United States during 1988–2001 (7). However, the clinical significance of SVA in swine could not be determined (7,8).

Full-Length Genome Sequences of Senecavirus A from Recent Idiopathic Vesicular Disease Outbreaks in U.S. Swine

ABSTRACT Since July 2015, vesicular lesions affecting growing pigs and sows accompanied with neonatal mortality have been reported in multiple U.S. states. Senecavirus A has been consistently detected from these cases. The complete genome sequences of 3 recent U.S. Senecavirus A isolates were determined to further characterize this virus.

Economic Analysis of Vaccination Strategies for PRRS Control

Porcine reproductive and respiratory syndrome virus (PRRSv) is a swine-specific pathogen that causes significant increases in production costs. When a breeding herd becomes infected, in an attempt to hasten control and elimination of PRRSv, some veterinarians have adopted a strategy called load-close-expose which consists of interrupting replacement pig introductions into the herd for several weeks (herd closure) and exposing the whole herd to a replicating PRRSv to boost herd immunity. Either modified-live virus (MLV) vaccine or live field-virus inoculation (FVI) is used. This study consisted of partial budget analyses to compare MLV to FVI as the exposure method of load-close-expose program to control and eliminate PRRSv from infected breeding herds, and secondly to estimate benefit / cost of vaccinating sow herds preventatively. Under the assumptions used in this study, MLV held economic advantage over FVI. However, sensitivity analysis revealed that decreasing margin over variable costs below

Systematic Epidemiological Investigations of Cases of Senecavirus A in US Swine Breeding Herds.

47.32, or increasing PRRSv-attributed cost above