Recipient Organization
UNIV OF MINNESOTA
(N/A)
ST PAUL,MN 55108
Performing Department
Veterinary Biomedical Sciences
Non Technical Summary
Bacterial infections have traditionally been treated with various antibiotics in swine farms. L. intracellularis is among the three key bacterial pathogens that can cause severe and deadly enteric diseases in swine that have been associated with high or moderately high antibiotic use by the World Organization of Animal Health (OIE Report, 2015). A potential growing problem is the emergence of resistance to antibiotics authorized for use in pigs. A relatively effective modified live vaccine is available against L. intracellularis. There are, however, considerable limitations that prevent the widespread use of this vaccine. First, production of the vaccine is costly due to its intracellular nature and requirement for a unique atmosphere for growth. And because this is a live attenuated bacterial vaccine, there's a need for an antibiotic free window for vaccination, which poses a challenge since antibiotic usage is necessary to control L. intracellularis and often concurrent, infections with other bacterial pathogens. Therefore, we aim to develop a new generation of a virus-based vaccine that can overcome these limitations and will provide effective protective immunity against L. intracellularis infections of swine.
Animal Health Component
10%
Research Effort Categories
Basic
45%
Applied
10%
Developmental
45%
Goals / Objectives
Lawsonia intracellularis causes proliferative enteropathy, a disease in swine that can significantly affect the health and productivity of this domestic livestock and can result in devastating economic losses to producers. An effective modified live vaccine is available against L. intracellularis. However, there are considerable limitations that prevent the widespread use of this bacterial vaccine. Here, we plan to develop a live virus-based vaccine that is based on our novel Pichinde virus (PICV) trisegmented reverse genetics system, which can overcome some of the limitations of a modified live bacterial vaccine. Toward this end, we have initiated efforts to clone and express different predicted immunogenic proteins of L. intracellularis for which their complete genomes have only recently been sequenced. Based on these exciting recent results, we plan to develop a serires of PICV-based vaccine vectors that encode three of the most immunogenic proteins of L. intracellularis, namely the LI0447, LI0461, and LI1158 protein antigens, as monovalent, bivalent and trivalent vaccines in order to assess their ability to protect swine from L. intracellularis infection. If successful, these live virus-based vaccines will serve as a new generation of vaccines for L. intracellularis that can improve the health and productivity of swine, which is essential to the nation's food supply.
Project Methods
Using the rP18tri reverse genetics system, we plan to clone and generate recombinant PICV viruses that encode a single copy, two copies or three copies of the L. intracellularis genes (LI0447, LI1158, and LI0461) as monovalent, bivalent and trivalent vaccines, respectively. We next plan to assess the infectivity of these recombinant vaccine viruses in several swine cell lines available in our laboratory (e.g., PK15, ST, PPK, and jejunal enterocytes JPEC). Virus growth curves will be conducted in some of these swine cells as outlined (Dhanwani, 2015). The expression levels of the different bacterial antigens from the PICV-based vaccine infected cells will be detected by Western blotting and/or IFA with convalescent (and normal) sera collected from pigs that were known to be infected with these bacteria (from previous controlled challenge trials and available in our laboratory). The vaccines will be purified and concentrated via ultra-centrifugation and sequenced to verify that no mutations have been inadvertently introduced into the bacterial antigens. Serial passaging of the vaccine stocks will also be conducted in cell culture (up to 15 passages) in order to show the stability of the PICV-based vaccines as assessed by sequencing of the bacterial antigens and the titers of viral vaccines over time. Vaccination studies will be carried out as per our previously published paper (Vannucci, 2013b). Briefly, thirty 3-week-old pigs from a high health status farm with no recent enteric history will be purchased. The monovalent, bivalent and trivalent vaccinated pigs will receive a dose of 1.5x106 pfu of the vaccines intramuscularly at 4 weeks-of-age. After vaccination, fecal samples will be collected from vaccinated and unvaccinated pigs daily for 5 days to evaluate potential PICV vectored vaccine shedding. Samples will be tested by RT-PCR and virus isolation will be attempted from positive samples. Pigs will also be monitored daily for any clinical signs, and body weight and temperature recorded weekly. Blood will also be collected at day of vaccination (as pre-immune sera) and 4 weeks post vaccination (at day of challenge) in order to prepare sera necessary for Western blotting analysis against bacterially expressed his-tagged L. intracellularis antigens and to isolate peripheral blood mononuclear cells (PBMC) for swine IFN-g ELISPOT to determine immune responses to the 3 specific bacterial antigens of interest. At 4 weeks post vaccination (28 DPV), all animals will be challenged orally (via oral gavage) with 50 ml each of pathogenic L. intracellularis (about 107 Lawsonia per ml). Pigs will continue to be monitored daily for any clinical signs, and body weight and temperature recorded weekly. Blood will be collected at day of challenge (prior to oral gavage) and 3 weeks later (at day of necropsy) to determine immune responses. Specifically, IPMA will be performed to measure the levels of anti- L. intracellularis specific IgG responses at day 0, 28 (before challenge) and day 56 (at necropsy) as previously described (Guedes, 2002). Positive serum samples (titer ≥ 30) will be tested to endpoint dilution and titers will be reported as the reciprocal of the dilution. Additionally, feces will be collected at the same time and analyzed by rtPCR. Absolute quantification will be calculated using a standard curve for L. intracellularis and expressed as copy numbers of the aspA gene of L. intracellularis per gram of feces. All pigs will be euthanized with a lethal dose of pentobarbital on day 21 post challenge, which is the peak of infection in this model, and evaluated for typical PE lesions. Intestinal samples from terminal jejunum, terminal ileum and proximal cecum will be collected. Two histologic sections will be prepared: one section will be stained by hematoxylin and eosin and the other by immunohistochemistry (IHC) using the streptavidin method with polyclonal antibodies specific for L. intracellularis. Samples taken at necropsy will include serum (for testing humoral immune responses; Guedes, 2002), Peyer's Patches (for IFN-g assay), intestinal washes (for local mucosal IgA assay) and formalin fixed tissues. Blood will also be collected from each pig to isolate PBMCs for swine IFN-g ELISPOT (Guedes and Gebhart, 2010). Briefly, swine IFN-g will be measured after exposure of PBMCs to the 3 respective bacterial antigens by ELISPOT to quantify IFN-gproducing cells.