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United States Department of Agriculture

Agricultural Research Service

Research Project: Strategies to Control and Prevent Bacterial Infections in Swine
2013 Annual Report

1a.Objectives (from AD-416):
Objective 1: Identify the transmission, genetic, and pathogenic mechanisms of the organisms associated with PRDC, concentrating on the bacterial pathogens and their interactions with each other and select swine viruses. Subobjective 1.1: Identify potential virulence factors of H. parasuis through comparative genomics. Subobjective 1.2: Bacterial response to host conditions. Subobjective 1.3: Evaluate ability of PRDC bacterial pathogens to inhibit Influenza A virus vaccine efficacy and/or exacerbate Influenza A virus-associated disease. Objective 2: Identify potential candidates for novel diagnostic assays, vaccines, and biotherapeutics for bacterial pathogens associated with PRDC. Subobjective 2.1. Develop PCR, ELISA, and/or other assays for detection of bacterial pathogens associated with PRDC. Subobjective 2.2. Identify, develop and/or test the efficacy of potential vaccine candidates to control bacterial pathogens associated with PRDC. Subobjective 2.3. Identify potential biotherapeutic candidates to control bacterial pathogens associated with PRDC. Objective 3: Investigate emerging and potential zoonotic bacterial pathogens that could impact the swine industry and design measures to diagnose, prevent, control and eliminate the threat posed to the swine industry. Subobjective 3.1. Evaluate the relationship between highly pathogenic Asian strains of PRRSV and S. suis infection in swine. Subobjective 3.2: Identification of measures that may prevent, control, or eliminate livestock-associated methicillin-resistant Staphylococcus aureus (MRSA) Sequence Type 398 (ST398) in swine.

1b.Approach (from AD-416):
Use comparative genomic methods, microarray analysis, and co-infection studies to explore pathogenic mechanisms of bacteria associated with the porcine respiratory disease complex and their interactions with each other and swine viruses. Assess the usefulness of selected genes or proteins identified in comparative genomic analyses for DNA-based identification and classification, serological detection of infection, and potentially as vaccine candidates. Strategies for improved heterologous protection will be tested using live attenuated vaccines, as will the use of immunomodulators, such as granulocyte colony stimulating factor (G-CSF), for therapeutic, prophylactic, and metaphylactic use to prevent and combat infectious disease and thus reduce antimicrobial usage to treat clinical and subclinical disease. Investigate emerging and potential zoonotic bacteria that could impact the swine industry. Investigations will focus on pathogen strain characteristics and differences, interactions of bacterial and viral pathogens with the swine host and the microbial ecosystems of the pig. Pathology of both zoonotic and endemic bacterial pathogens of swine will be utilized for the purpose of understanding disease pathogenesis and developing effective diagnostic assays and strategies to control these pathogens and diseases in swine and potentially in humans.

3.Progress Report:
We completed virulence testing for the 10 Haemophilus parasuis (HPS) isolates we have sequenced. This will provide meaningful data on which to base genomic comparisons. These efforts support subobjective 1.1. We developed assays to examine the transcriptional response of virulent and avirulent isolates HPS after exposure to serum and complement. These efforts support Subobjective 1.2. We identified a conserved gene in HPS, called wza, that produces a protein that is part of the capsule. A PCR assay has been developed and shown to be specific to HPS, and we have begun evaluating the assay in a blinded fashion and have initiated the cloning of the gene for development of a quantitative assay. We have screened convalescent and naive sera for reactivity to HPS P2 and P5 proteins and concluded that these are not optimal diagnostic targets given the reactivity of naive sera to these proteins and numerous differences between P2 and P5 proteins in different HPS isolates. These efforts support subobjective 2.1. We completed the construction of a novel suicide vector that will allow us to make markerless mutations in H. parasuis, including an AroA mutant to test as a vaccine candidate. The genome sequence data files of an identified virulent (Nagasaki) and avirulent (D74) strain of HPS have been annotated and pseudogene candidates identified in order to introduce a gene encoding fluorescent protein for the purpose of marking a strain. A recombinant Ad5 vector expressing the Bsp22 protein has been generated and pigs were immunized with the vector to test for immunogenicity. These efforts support Subobjective 2.2. We completed in vitro assays evaluating the function of neutrophils isolated from pigs that had been given granulocyte-colony stimulating factor (G-CSF), a compound that enhances immunity against bacterial infections by increasing the number of circulating neutrophils, and found the administration of the compound does not alter cell function. We continued experiments evaluating protection provided by the administration of G-CSF. These efforts support subobjective 2.3. We examined a number of Streptococcus suis strains for virulence and effects of coinfection with other bacteria and viruses. These efforts support subobjective 3.1. To begin experimentally addressing mechanisms contributing to colonization, carriage, and competitive exclusion of livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) strains, we have begun constructing mutants in our MRSA isolates that contain a gene encoding fluorescent protein. We have also performed qPCR to evaluate expression of genes that potentially lead to biofilm formation and tested a collection of strains for production of secreted proteases and nucleases when grown as a biofilm. We found certain strains to be the most sensitive to both inhibition of biofilm formation and dispersal of biofilms by DNaseI, and Proteinase K to both inhibit biofilm formation and disperse mature biofilms in all LA-MRSA strains. These experiments demonstrate the sensitivity is not due to differences in gene expression, secreted proteases, or extracellular nucleases. These efforts support subobjective 3.2.

1. Developed an assay to detect newly emerging strains of porcine reproductive and respiratory syndrome virus (PRRSV). PRRSV is a virus that infects pigs and causes reproductive losses and respiratory disease. It is the top disease problem for pig producers in the United States and many other parts of the world. This virus mutates very rapidly and new strains of PRRSV are constantly emerging. Current differentiation of PRRSV strains involves a few selected U.S. veterinary diagnostic laboratories performing techniques that typically take two weeks or more for results. ARS scientists at the National Animal Disease Center in Ames, Iowa, have developed a new assay called a microarray specific for PRRSV that can function as a diagnostic tool that will rapidly identify and differentiate viral strains. Our novel PRRSV microarray recognizes multiple regions throughout the PRRSV genome and as a result, our PRRSV microarray has a greater sensitivity than currently used methods while simultaneously detecting genetic diversity. This test is a faster and more reliable test that can detect newly emerging domestic and foreign strains of PRRSV that threaten the national swine herd.

2. Determination of influenza vaccine efficacy for limiting transmission between pigs and people. Influenza virus spillover from pigs to people is concern for both public health officials as well as swine producers. In 2012 there were more than 300 cases reported in which a particular strain of swine influenza virus was isolated from people, the majority occurring at agricultural fairs in the summer. One method to limit transmission of the virus from pigs to people is to control the virus in pigs through vaccination. ARS scientists at the National Animal Disease Center in Ames, Iowa, evaluated the ability of currently available commercial swine influenza vaccines and experimental vaccines to limit replication and spread of a strain of swine influenza associated with spillover at agricultural fairs in 2012. One commercial vaccine provided significant protection, but it did not prevent transmission of the virus to non-vaccinated pigs in a neighboring pen. One experimental vaccine that is a live-attenuated virus given to pigs in the nose was able to prevent infection. The results from this work will provide pig owners and agricultural personnel experimental data for determining the value of commercial vaccines at limiting infection with this particular strain of swine influenza virus, which should contribute to decreasing the frequency of spillover from pigs to people. It also highlights the need for future work to develop assays that are predictive of a vaccine's effectiveness.

3. Determined that enhanced immune response may limit the need for antibiotic use. Overuse of antibiotics has been blamed for the growing resistance of bacteria to their effects. Neutrophils are white blood cells that circulate in the blood and play an important role in combating infections. Granulocyte-Colony Stimulating Factor (G-CSF) is a substance produced by the body in response to infection that increases the number of circulating neutrophils. The use of immunomodulators like G-CSF is a promising area that enhances the body's own ability to prevent and combat infectious disease while eliminating or reducing the use of antibiotics. ARS scientists at the National Animal Disease Center in Ames, Iowa, completed a series of studies in which pigs were given a G-CSF in a vector which allows it to have longer lasting effects. The vector worked better than predicted resulting in enhanced neutrophil levels in the blood of pigs for up to 3 weeks after a single injection. This novel delivery of G-CSF precludes the need for manufacturing G-CSF and elicits a longer duration of effect in the body than current modalities provide today. These findings will benefit veterinarians and pharmaceutical companies working towards novel methods to reduce antibiotic usage in pigs that develop bacterial infections.

4. Determined a mechanism by which porcine reproductive and respiratory syndrome virus (PRRSV) causes immunosuppression. PRRSV is a virus that infects pigs and causes reproductive losses and respiratory disease. It is the top disease problem for pig producers in the United States and many other parts of the world. The immune response to PRRSV following infection has been characterized as weak and delayed; thus, the infection is not controlled and becomes chronic and vaccines tend to be ineffective. ARS scientists at the National Animal Disease Center in Ames, Iowa completed a study in which pigs were given one of four different PRRSV isolates, each with varying ability to cause disease. Within 2 days of infection all of the pigs had a significant decrease in the number of circulating lymphocytes, a type of white blood cell involved in immunity. While the number of cells in circulation steadily increased over time, by day 10 following infection, the number of circulating lymphocytes was still below normal. In addition, there were significant decreases in the cellularity of the thymus, an organ required for the development of a population of circulating lymphocytes. The decrease in thymus size correlated with the ability of the virus to cause disease and predispose to secondary infections. These results indicate that PRRSV causes significant changes in the population of lymphocytes which may explain the inadequate adaptive immune response elicited following infection. If we can find ways to counteract these effects we can improve the protective effects of PRRSV vaccines.

Review Publications
Brockmeier, S.L., Loving, C.L., Vorwald, A.C., Kehrli, Jr., M.E., Baker, R.B., Nicholson, T.L., Lager, K.M., Miller, L.C., Faaberg, K.S. 2012. Genomic sequence and virulence comparison of four Type 2 porcine reproductive and respiratory syndrome virus strains. Virus Research. 169(1):212-221.

Braucher, D.R., Henningson, J.N., Loving, C.L., Vincent, A.L., Kim, E., Steitz, J., Gambotto, A.A., Kehrli, Jr., M.E. 2012. Intranasal vaccination with replication-defective adenovirus type 5 encoding influenza virus hemagglutinin elicits protective immunity to homologous challenge and partial protection to heterologous challenge in pigs. Clinical and Vaccine Immunology. 19(11):1722-1729.

Nicholson, T.L., Conover, M.S., Deora, R. 2012. Transcriptome profiling reveals stage-specific production and requirement of flagella during biofilm development in Bordetella bronchiseptica. PLoS ONE. 7(11):e49166.

Hester, S.E., Lui, M., Nicholson, T., Nowacki, D., Harvil, E.T. 2012. Identification of a CO2 responsive regulon in Bordetella. PLoS One. 7(10):e47635.

Loving, C.L., Kehrli, M.E., Brockmeier, S.L., Bayles, D.O., Michael, D.D., Schlink, S.N., Lager, K.M. 2013. Porcine granulocyte-colony stimulating factor (G-CSF) delivered via replication-defective adenovirus induces a sustained increase in circulating peripheral blood neutrophils. Biologicals. Available:

Brockmeier, S.L., Register, K.B., Nicholson, T.L., Loving, C.L. 2012. Bordetellosis. In: Zimmerman, J., Karriker, L., Ramirez, A., Schwartz, K., Stevenson, G., editors.Diseases of Swine. 10th edition. Ames, IA: Iowa State University Press. pp. 670-679.

Brockmeier, S.L., Loving, C.L., Mullins, M., Register, K.B., Nicholson, T.L., Wiseman, B.S., Baker, R.B., Kehrli, Jr., M.E. 2013. Virulence, transmission, and heterologous protection of four isolates of Haemophilus parasuis. Clinical and Vaccine Immunology. 20(9):1466-1472.

Loving, C.L., Lager, K.M., Vincent, A.L., Brockmeier, S.L., Gauger, P.C., Anderson, T.K., Kitikoon, P., Perez, D.R., Kehrli, Jr., M.E. 2013. Efficacy in pigs of inactivated and live attenuated influenza virus vaccines against infection and transmission of an emerging H3N2 similar to the 2011-2012 H3N2v. Journal of Virology. 87(17):9895-903.

Last Modified: 4/18/2014
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