The goal of this study was to evaluate survival of important viral pathogens of livestock in animal feed ingredients imported daily into the United States under simulated transboundary conditions. Eleven viruses were selected based on global significance and impact to the livestock industry, including Foot and Mouth Disease Virus (FMDV), Classical Swine Fever Virus (CSFV), African Swine Fever Virus (ASFV), Influenza A Virus of Swine (IAV-S), Pseudorabies virus (PRV), Nipah Virus (NiV), Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), Swine Vesicular Disease Virus (SVDV), Vesicular Stomatitis Virus (VSV), Porcine Circovirus Type 2 (PCV2) and Vesicular Exanthema of Swine Virus (VESV). Surrogate viruses with similar genetic and physical properties were used for 6 viruses. Surrogates belonged to the same virus families as target pathogens, and included Senecavirus A (SVA) for FMDV, Bovine Viral Diarrhea Virus (BVDV) for CSFV, Bovine Herpesvirus Type 1 (BHV-1) for PRV, Canine Distemper Virus (CDV) for NiV, Porcine Sapelovirus (PSV) for SVDV and Feline Calicivirus (FCV) for VESV. For the remaining target viruses, actual pathogens were used. Virus survival was evaluated using Trans-Pacific or Trans-Atlantic transboundary models involving representative feed ingredients, transport times and environmental conditions, with samples tested by PCR, VI and/or swine bioassay. SVA (representing FMDV), FCV (representing VESV), BHV-1 (representing PRV), PRRSV, PSV (representing SVDV), ASFV and PCV2 maintained infectivity during transport, while BVDV (representing CSFV), VSV, CDV (representing NiV) and IAV-S did not. Notably, more viruses survived in conventional soybean meal, lysine hydrochloride, choline chloride, vitamin D and pork sausage casings. These results support published data on transboundary risk of PEDV in feed, demonstrate survival of certain viruses in specific feed ingredients (“high-risk combinations”) under conditions simulating transport between continents and provide further evidence that contaminated feed ingredients may represent a risk for transport of pathogens at domestic and global levels.

Porcine reproductive and respiratory syndrome virus (PRRSV) contains the major glycoprotein, GP5, as well as three other minor glycoproteins, namely, GP2a, GP3, and GP4, on the virion envelope, all of which are required for generation of infectious virions. To study their interactions with each other and with the cellular receptor for PRRSV, we have cloned each of the viral glycoproteins and CD163 receptor in expression vectors and examined their expression and interaction with each other in transfected cells by coimmunoprecipitation (co-IP) assay using monospecific antibodies. Our results show that a strong interaction exists between the GP4 and GP5 proteins, although weak interactions among the other minor envelope glycoproteins and GP5 have been detected. Both GP2a and GP4 proteins were found to interact with all the other GPs, resulting in the formation of multiprotein complex. Our results further show that the GP2a and GP4 proteins also specifically interact with the CD163 molecule. The carboxy-terminal 223 residues of the CD163 molecule are not required for interactions with either the GP2a or the GP4 protein, although these residues are required for conferring susceptibility to PRRSV infection in BHK-21 cells. Overall, we conclude that the GP4 protein is critical for mediating interglycoprotein interactions and, along with GP2a, serves as the viral attachment protein that is responsible for mediating interactions with CD163 for virus entry into susceptible host cell.

We describe B-cell linear epitopes detected by Pepscan in the Nsp2 and all of the structural proteins of a US PRRSV strain, using sera of 15 experimentally infected pigs. The Nsp2 was found to contain the highest frequency of immunodominant epitopes (n = 18) when compared to structural proteins. Ten of these 18 Nsp2 peptides were reactive with 80 to 100% of the examined sera. In the structural proteins, epitopes consistently recognized by immune sera were located at gp2 (n = 2), gp3 (n = 4), gp5 (n = 3), M (n = 2) and N (n = 2). Overall, the highest degree of immunogenicity and conservation was exhibited by two epitopes identified in the C-terminal end of the M protein (ORF6). The antibodies recognizing the immunodominant epitopes of each protein were detected as early as days 7 to 15 pi and remained detectable until the end of the experiment (day 90 pi). These findings have direct implications for PRRSV differential diagnostics and eventual eradication as the identified epitopes may represent serologic marker candidates for differential (DIVA) PRRSV vaccines, derived from infectious cDNA clones.

Senecavirus A (SVA), an emerging picornavirus of swine, causes vesicular disease (VD) that is clinically indistinguishable from foot-and-mouth disease (FMD) in pigs. Many aspects of SVA interactions with the host and the host immune responses to infection, however, remain unknown. In the present study, humoral and cellular immune responses to SVA were evaluated following infection in pigs. We show that SVA infection elicited an early and robust virus-neutralizing (VN) antibody response, which coincided and was strongly correlated with VP2- and VP3-specific IgM responses. Notably, the neutralizing antibody (NA) responses paralleled the reduction of viremia and resolution of the disease. Analysis of the major porcine T-cell subsets revealed that during the acute/clinical phase of SVA infection (14 days postinfection [p.i.]), T-cell responses were characterized by an increased frequency of αβ T cells, especially CD4 T cells, which were first detected by day 7 p.i. and increased in frequency until day 14 p.i. Additionally, the frequency of CD8 and double-positive CD4 CD8 T cells (effector/memory T cells) expressing interferon gamma (IFN-γ) or proliferating in response to SVA antigen stimulation increased after day 10 p.i. Results presented here show that SVA elicits B- and T-cell activation early upon infection, with IgM antibody levels being correlated with early neutralizing activity against the virus and peak B- and T-cell responses paralleling clinical resolution of the disease. The work provides important insights into the immunological events that follow SVA infection in the natural host. Senecavirus A (SVA) has recently emerged in swine, causing outbreaks of vesicular disease (VD) in major swine-producing countries around the world, including the United States, Brazil, China, Thailand, and Colombia. Notably, SVA-induced disease is clinically indistinguishable from other high-consequence VDs of swine, such as FMD, swine vesicular disease, vesicular stomatitis, and vesicular exanthema of swine. Despite the clinical relevance of SVA-induced VD, many aspects of the virus infection biology remain unknown. Here, we assessed host immune responses to SVA infection. The results show that SVA infection elicits early B- and T-cell responses, with the levels of VN antibody and CD4 T-cell responses paralleling the reduction of viremia and resolution of the disease. SVA-specific CD8 T cells are detected later during infection. A better understanding of SVA interactions with the host immune system may allow the design and implementation of improved control strategies for this important pathogen of swine.

Immunoglobulin Y (IgY

No abstract

Glycoprotein 3 (GP3) is a highly glycosylated PRRSV envelope protein which has been reported as being present in the virions of PRRSV type I, while missing in the type II PRRSV (US) virions. We herein present evidence that GP3 is indeed incorporated in the virus particles of a North American strain of PRRSV (FL12), at a density that is consistent with the minor structural role assigned to GP3 in members of the Arterivirus genus. Two 15aa peptides corresponding to two different immunodominant linear epitopes of GP3 derived from the North American strain of PRRSV (FL12) were used as antigen to generate a rabbit monospecific antiserum to this protein. The specificity of this anti-GP3 antiserum was confirmed by radioimmunoprecipitation (RIP) assay using BHK-21 cells transfected with GP3 expressing plasmid, MARC-145 cells infected with FL12 PRRSV, as well as by confocal microscopy on PRRSV-infected MARC-145 cells. To test if GP3 is a structural component of the virion, (35)S-labelled PRRSV virions were pelleted through a 30% sucrose cushion, followed by a second round of purification on a sucrose gradient (20-60%). Virions were detected in specific gradient fractions by radioactive counts and further confirmed by viral infectivity assay in MARC 145 cells. The GP3 was detected in gradient fractions containing purified virions by RIP using anti-GP3 antiserum. Predictably, the GP3 was less abundant in purified virions than other major structural envelope proteins such as GP5 and M. Further evidence of the presence of GP3 at the level of PRRSV FL12 envelope was obtained by immunogold staining of purified virions from the supernatant of infected cells with anti-GP3 antiserum. Taken together, these results indicate that GP3 is a minor structural component of the PRRSV type II (FL12 strain) virion, as had been previously described for PRRSV type I.

ResumoO leite é um alimento natural e rico em nutrientes, porém a forma como muitas vezes é obtido interfere na sua qualidade microbiológica e nutricional. E a qualidade dos produtos é uma exigência cada vez maior do mercado consumidor. Assim, objetivou-se identificar micro-organismos presentes no leite de tanques resfriadores, na região sul do Rio Grande do Sul, e estabelecer uma correlação entre o manejo dos animais e sua presença no tanque. Foram coletadas amostras de leite em propriedades de seis municípios e realizadas análises microbiológicas para identificação microbiana. As médias das contagens foram: Staphylococcus sp. 5,32x10 6 UFC/mL, S. aureus 1,33x10 5 UFC/mL, enterobactérias 1,82x10 7 UFC/ mL. Houve presença de Escherichia coli (27,8% das propriedades), Streptococcus agalactiae (6,2%), S. dysgalactiae (37,2%), S. uberis (16,8%), Candida sp. (15,7%), Aspergillus sp. (5,8%), Trichosporum sp. (3,6%) e Cryptococcus sp. (1,5%). Identificou-se uma Odds Ratio de 3,2 para S. agalactiae com relação à ordenha manual e de 3,1 quando um único pano era usado para secagem dos tetos. Para S. dysgalactiae houve 55,8% de probabilidade da ocorrência em caso de não secagem dos tetos. Para S. bovis, uma Odds Ratio de 2,8 em propriedades que ordenham seus animais em estábulos de madeira. Houve aumento significativo (p=0,003) na contagem de células somáticas (CCS) de propriedades que realizam ordenha manual em relação àquelas mecanizadas. A ordenha manual aumentou as contagens de S. aureus (p=0,04). A realização de pré-dipping contribuiu para a redução da contagem de Staphylococcus sp. e houve diferença significativa em relação ao grupo que não realizava esta prática (p=0,04). Conclui-se que as técnicas de manejo mais precárias influenciam negativamente na qualidade microbiológica do leite, aumentando os riscos de ocorrência de agentes infecciosos e elevando as contagens de micro-organismos. Palavras-chave: Qualidade microbiológica, leite, Staphylococcus, Streptococcus AbstractMilk is naturally a good provider of a whole range of nutrients, however an inadequate milking may significantly interfere on its nutritional and microbiological quality. The main purpose of this study was