Source: UNIV OF MINNESOTA submitted to
INFECTION OF PIGS WITH SALMONELLA AND LAWSONIA INCREASES PUBLIC HEALTH RISK
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
EXTENDED
Funding Source
Reporting Frequency
Annual
Accession No.
1005423
Grant No.
2015-67017-23076
Project No.
MINV-63-116
Proposal No.
2014-05705
Multistate No.
(N/A)
Program Code
A1331
Project Start Date
Feb 1, 2015
Project End Date
Jan 31, 2020
Grant Year
2015
Project Director
Isaacson, R.
Recipient Organization
UNIV OF MINNESOTA
(N/A)
ST PAUL,MN 55108
Performing Department
Veterinary and Biomedical Sci
Non Technical Summary
Salmonellosis is the most common bacterial food borne illness and current interventions have had little effect on the prevalence of this pathogen in swine or on the incidence of disease in humans. This probably is the result of the emergence of new strains of S. enterica and the lack of known pathogen control points during animal production. Our preliminary data has shown that pigs co-infected with S. enterica serovar Typhimurium and L. intracellularis shed higher levels of S. enterica and for a longer period of time. Our hypothesis is that an L. intracellularis infection of pigs increases the risk of salmonellosis in humans. The goals of this project are to determine the duration and quantity of S. enterica shed by pigs co-infected with L. intracellularis and to determine if vaccination with an L. intracellularis specific vaccine mitigates S. enterica shedding. A third goal is to quantify microbiome changes in the intestinal tract in response to infections with these two pathogens to identify new targets that could be exploited to reduce shedding of S. enterica. There are 3 specific aims in this project: 1) to determine the duration and quantity of S. enterica shed by pigs infected with S. enterica or S. enterica and L. intracellularis. 2) to determine the effect of vaccination against L. intracellularis on shedding S. enterica, and 3) to determine intestinal microbiome changes in pigs that are singly and dually infected and with or without vaccination.
Animal Health Component
25%
Research Effort Categories
Basic
90%
Applied
10%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
7123510110050%
7123520110050%
Goals / Objectives
Salmonella enterica is a major food borne pathogen and is frequently carried by swine. Epidemiologic evidence and our preliminary data indicates that carriage of S. enterica by pigs is enhanced by infection with the pig pathogen Lawsonia intracellularis. The overall goal of the current study is to develop an novel intervention to control S. enterica in pigs by vaccinating them againstL. intracellularis.There are three specific aims of this study. 1) to determine the duration and quantity of S. enterica shed by pigs infected with S. Typhimurium or S. Typhimurium and L. intracellularis, 2) todetermine the effect of vaccination against L. intracellularis on shedding S. Typhimurium, and 3) todetermine intestinal microbiome changes in pigs that are singly and dually infected and with or without vaccination.
Project Methods
In the first specific aim, we will be confirming thatL. intracellularisenhances shedding ofS. entericahow long during the growth of pigs shedding remains elevated. To perform this work, pigs will be challenged orally withL. intracellularisandS. enterica. At intervals of time fecal samples will be collected and the amount ofS. entericain each sample will be determined usin a modified most probable number analysis tool. The results will be compared to pigs only challenged with S. enterica. In the second specific aim, we will use a vaccine used to protect pigs fromL. intracellularis and determine whether it protects pigs fromS. enterica. To do this work, pigs will be vaccinated and then challenged with one or both pathogens. Fecal samples will be collected as in the first specific aim and the amount ofS. enterica in each sample determined using the most probable number tool. The results will be compared to non-vaccinated but challenged pigs. In the third specific aim, the fecal samples collected in specific aims 1 and 2 will be evaluated to determine the compositions of their microbiomes. Microbiome analysis will used standard techniques that include extraction of total DNA from each fecal sample and using polymerase chain reaction to amplify the 16S rRNA genes in the samples. These PCR products will then be subjected to high yield next generation DNA sequencing. The DNA sequences will be used for taxonomic assignments using the Ribosomal DNA Project data base. Identification of enriched or depleted microbiomes will use MetaStats.

Progress 02/01/16 to 01/31/17

Outputs
Target Audience:Data from this project was presented at the Conference of Research Workers in Animal Diseases which represents a group of scientists that work in the field of animal diseases. Some of the data also was presented at the the Western Canadian Association of Swine Veterinarians (Saskatoon, Sask). This group was a mix of practioneers and researchers. The work also was pesented at the 2016 Leman Conference. This conference is for swine practioneers, regulators, and researchers. Changes/Problems:No major problems were encountered. We have decided to perform some additional in vitro challenge work using IPEC (pig intestinal epithelia cells) to determine if the reason whyLawsoniaaffectsSalmonella? is due to suppression of host inflammatory responses. What opportunities for training and professional development has the project provided?In addition to the graduate student assigned to this project, several other graduate students and a veterinary student were involved in the sample sampling aspects of the project and gave them experience handling pigs. How have the results been disseminated to communities of interest?See the list of abstracts. This work was presented at three scientific meetings (Leman Conference, Conference of Research Workers in Animal Diseases, and the Western Canadian Association of Swine Veterinarians. What do you plan to do during the next reporting period to accomplish the goals?The third specific aim was to measure gut microbiome differences in pigs challenged withSalmonella, SalmonellaandLawsoniawith or without vaccination. Fecal samples have already been collected and are being sequenced to measure if vaccination leads to the prevention of microbiome changes previously observed after challenges withSalmonella, Lawsonia,?or both.

Impacts
What was accomplished under these goals? During the previous year, aims #1 and #2 were completed. Pigs were challenged with Salmonella or Salmonella and Lawsonia and shedding of Salmonella followed over time by using a most probable number culture system from collected feces. The pig prevalence of Salmonella shedding also was monitored. In preliminary experiments where the experimental sample was tissue (jejunum, ileum, cecum, and colon) and where there was as significant increase in shedding ofSalmonellaby pigs infected with bothSalmonellaandLawsonia, in the current trial, that difference was not observed. This probably is the result of sample differences - tissue samples versus feces. In the second aim we vaccinated pigs with aLawsoniavaccine and comparedSalmonellashedding in singly or dually infected pigs to determine if vaccination reduced shedding of Salmonella. Vaccination caused a significant reduction on the shedding ofSalmonellain pigs also challenged withLawsonia.The vaccine had no effect on shedding if the pigs were only challenged withSalmonella. The pig prevalence of shedding ofSalmonellaalso was significantly reduced in vaccinated and dually challenged pigs. Our overall conclusion was that vaccination againstLawsoniamight be an effective means to reduceSalmonellashedding if pigs are likely infected withLawsoniatoo. Since herd prevalence forLawsoniain US herds is near 90%, it is likely thatSalmonellainfected pigs also will be infected withLawsonia.

Publications

  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2016 Citation: 1. Leite, F.L.L, Connie Gebhart, C., Singer, R., Isaacson, R. Vaccination against Lawsonia intracellularis leads to decreased Salmonella enterica serovar Typhimurium shedding in co-infected animals. Leman Conference, St. Paul, MN, 2016. 2. Leite, F.L.L, Connie Gebhart, C., Singer, R., Isaacson, R. Vaccination against Lawsonia intracellularis leads to decreased Salmonella enterica serovar Typhimurium shedding in co-infected animals. Conference of Research Workers in Animal Diseases, Chicago, IL, 2016.


Progress 02/01/15 to 01/31/16

Outputs
Target Audience:Academic research community, government basic and applied researchers, industry related to animal health and food safety. Changes/Problems:Three changes were made to the protocol. The first was that the determination of length ofS. entericashedding was combined with the vaccination study. This was performed since the length of shedding aspect was as part of both aims 1 and 2. This allows us to be more cost effective. The second change is that we had specified groups being composed of 10 pigs per treatment. However, the animal isolation space available to us does not permit that many pigs per room. Therefore, we have increased the number of groups by 3 fold but reduced group size to 3 pigs each. This allows us to perform the same work but also allows us to measure whether there were pen effects unrelated to the treatments. The final change is that peripheral blood also is being collected and the blood will be used to determine if any of the protocols result in increased production of pro-inflammatory cytokines or antiinflammatory cytokines. While not part of the original proposal, these extra measures with allow us to determine if the innate immune response is partly involved in enhanced shedding ofS. entericain the dualling infected pigs. What opportunities for training and professional development has the project provided?One graduate student (Ph.D.) is assigned to this project and will be the basis for his thesis. He is being trained in microbiology, infectious diseases, microbiome analysis, and animal health/food safety. In addition, 2-3 other students (graduate and veterinary) will be involved in sample collection and the work will contribute to their respective training in microbiology, infectious diseases, and animal science. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?As stated in results, pigs have been received to initiate work on all three specific aims. Pigs are being randomized into various groups for different treatments and vaccinations withLawsonia intracellularisvaccine and challenges withLawsonia intracellularis, Salmonella entericaserovar Typhimurium or both will begin January 22, 2016. Besides determining the length of enhanced shedding ofS. entericawhen co-challenged withL.intracellularis, the initial challenges also will be used to determine if vaccination abborgates enhanced shedding ofS. enterica? and fecal samples will be collected to determine the composition of the fecal microbiome. We anticipate that by the end of the current reporting period that we will have confirmed our initial results thatL.intracellularisenhances shedding of S. enterica. We also will have a first trial to determine if anti-L. intracellularisvaccination reduces shedding ofS. enterica.Finally, fecal samples will have been processed and the first analyses of microbiomes in the groups will be completed.

Impacts
What was accomplished under these goals? Pigs have been received to initiate work on all three specific aims. Pigs are being randomized into various groups for different treatments and vaccinations with Lawsonia intracellularis vaccine and challenges with Lawsonia intracellularis, Salmonella enterica serovar Typhimurium or both will begin January 22, 2016. Besides determining the length of enhanced shedding ofS. entericawhen co-challenged withL.intracellularis, the initial challenges also will be used to determine if vaccination abborgates enhanced shedding ofS. enterica? and fecal samples will be collected to determine the composition of the fecal microbiome.

Publications

  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Borewicz, K.A., Kim, H.B., Singer, R.S., Gebhart, C.J., Sreevatsan, S., Johnson, T., and Isaacson, R.E. Changes in the porcine intestinal microbiome in response to infection with Salmonella enterica and Lawsonia intracellularis. PLOS One, 10(10): e0139106.doi:10.1371/journal.pone.0139106.