Performing Department
Veterinary Medicine
Non Technical Summary
Salmonella infections remain a significant public health concern, with poultry products being a major source of human illness. Our project aims to develop new vaccines to combat Salmonella in chickens, focusing on types that are becoming increasingly problematic but lack effective prevention methods. We are testing two new vaccine candidates that use weakened forms of Salmonella bacteria. These vaccines are designed to be safer, cheaper, and more effective than current options. They can be given to chickens more easily and are expected to provide longer-lasting protection against multiple Salmonella types. Our research will evaluate how safe these vaccines are for both chickens and humans, and how well they protect chickens from Salmonella infection. We believe these vaccines will effectively reduce Salmonella in poultry, which in turn will lower the risk of human illness from consuming contaminated poultry products. If successful, this project could significantly improve food safety in the poultry industry and contribute to better public health outcomes. It addresses critical needs in food safety and has the potential to make a real difference in reducing foodborne illnesses caused by Salmonella.
Animal Health Component
75%
Research Effort Categories
Basic
(N/A)
Applied
75%
Developmental
25%
Goals / Objectives
Salmonella remains a leading cause of foodborne illness worldwide, with poultry products being a primary source of human infections. While vaccination is a proven intervention for reducing Salmonella prevalence in commercial poultry, there is a critical gap in live vaccines targeting serogroup C, which includes emerging serovars of increasing concern. Our research addresses this urgent need by evaluating two novel live attenuated vaccine candidates: CVD 1979 (S. Newport ΔguaBA ΔhtrA ΔaroA) and CVD 1982 (S. Virchow ΔguaBA ΔhtrA ΔaroA), targeting serogroups C2-C3 and C1, respectively. These vaccines offer potential advantages over killed vaccines, including lower production costs, easier administration, and induction of long-term humoral and cell-mediated immunity. This study will assess the safety and efficacy of each vaccine candidate in chickens, laying the groundwork for future bivalent formulations. We hypothesize that these vaccines will prove safe for both humans and chickens, highly immunogenic, and provide broad protection against multiple Salmonella serovars within serogroup C like serovar Infantis, Newport, Muenchen, Kentucky, and Hadar . Successful outcomes will significantly advance Salmonella control efforts in the poultry industry, ultimately reducing the risk of human salmonellosis. This research aligns with critical food safety priorities and has the potential to make a substantial impact on public health.
Project Methods
1A. To determine the safety (LD50) for the two vaccine candidates when administered individually or combined for one-day-old SPF chicks compared to wild type strains.Experiment: To determine the oral LD50 for attenuated vaccine strains compared to wild-type strains in one-day-old specific pathogen-free (SPF) chicks. Six groups of SPF chicks will receive one of the following treatments: S. Newport CVD 1979, S. Virchow CVD 1982, S. Newport wild-type, S. Virchow wild-type, bivalent SN-CVD-1979 + SV-CVD-1982 vaccine (1:1), and PBS. Each group will be subdivided into 4 subgroups of 5 chicks, with each subgroup receiving one of four ten-fold dilutions (106 to 109 CFU/mL) via oral gavage. Wild-type strains will serve as positive controls, while PBS-inoculated chicks will be negative controls. Bacteria will be grown overnight, pelleted, and resuspended in PBS at appropriate concentrations, with exact inoculation numbers determined by viable counts. Chicks will be monitored twice daily for clinical symptoms and mortality, and weighed at 7, 14, and 21 days post-inoculation. Dead chickens will undergo necropsy for gross lesion scoring, with liver and spleen specimens collected for histopathological evaluation and Salmonella re-isolation.1B. To determine the optimal dosage for the two vaccine candidates when administered individually or combined for one-day-old SPF chicks.Experiment: To determine the optimal dose of the vaccine strains, we will assess the humoral immune response elicited by the vaccines. Blood will be collected from groups 1, 2, 3, and 6 at 7, 14, and 28 days of age and serum antibody levels will be measured using in house ELISA to determine the immune response induced by different vaccine doses. Anti-LPS and anti-FliC serum IgY titers in serum samples will be determined as described previously(Nasrin et al., 2021; Tennant et al., 2011).1C. To determine the shedding and tissue colonization rate for the two vaccine candidates when administered individually or combined for one-day-old SPF chicks.Experiment: To evaluate the shedding rate of the vaccine strains compared to the wild type strains and control, cloacal swabs (with fecal material) will be collected at 7, 14 and 21 days of age. At 28 days of age, chicks will be euthanized and liver, spleen and ceca will be collected to evaluate tissue colonization. Salmonella prevalence will be estimated by following the National Poultry Improvement Plan (NPIP) standard isolation protocol for Salmonella from cloacal swabs and organs. Salmonella load will be enumerated using the modified 3 tube most probable number (MPN) method with microplate dilutions (Berghaus et al., 2013).Experimental design for Objective 2: To evaluate the efficacy of the monovalent live-attenuated S. Newport CVD 1979 and S.Virchow CVD 1982 vaccine candidates individually in reducing tissue colonization, shedding, and inducing immunity against challenge with homologous field strains of S. Newport and S.Virchow respectively.Experiment: To evaluate efficacy of each live-attenuated vaccine candidate individually, 40 one-day-old SPF chicks will be randomly divided into 4 groups (10 chicks per group), designated as Vac-SN, Novac-SN, Vac-SV, and Novac-SV as summarized in Table 2. Vaccinated groups (Vac-SN and Vac-SV) will be inoculated with vaccine SN-CVD-1979 and SV-CVD-1982, respectively, through oral gavage at 1 day of age and will be boosted at 14 days of age. At the same times, non-vaccinated groups (Novac-SN and Novac-SV) will be given PBS as placebo. On day 28, the animals vaccinated with SN-CVD-1979 and an unvaccinated group (NOVAC-SN) will be challenged with a nalidixic acid resistant strain of Salmonella Newport (SN-Nalres). The other two groups (Vac-SV, and Novac-SV) will be challenged with a nalidixic acid resistant strain of Salmonella Virchow (SV-Nalres). Blood will be collected from vaccinated groups at 1, 7, 14, 21 and 28 days of age and anti-LPS serum antibody levels will be measured using ELISA to evaluate the immune response induced by single and two vaccine doses. Cloacal swabs will be collected on day 7 (35 DOA) after the challenge to detect and quantify bacterial shedding in droppings. Chickens will be euthanized by CO2 on day 7 after the Salmonella challenge. Salmonella prevalence will be estimated by following the NPIP standard isolation protocol for Salmonella from tissues. Salmonella load will be enumerated using the modified 3 tube MPN method with microplate dilutions. Nalidixic acid will be added to XLT4 culture plates to select for the challenge strains.Animal Source and housingSPF chicks will be used for this project to ensure they are free from Salmonella and any maternal antibodies that may interfere with assessment of the vaccine immunogenicity and efficacy. For all animal procedures, we have protocols approved by the Institutional Animal Care and Use Committee of the University of Maryland. Specific pathogen free (SPF) white leghorn chicks will be acquired from Charles River Laboratories, Wilmington, MA and specifically from non-vaccinated breeders for Salmonella. The chicks will be housed in negative pressure isolators (HM1500) with ad libitum access to feed and water at the laboratory animal corridor at Department of Veterinary Medicine, University of Maryland.Bacteriological analysisTo estimate fecal shedding and tissue colonization of challenge strains (nalidixic acid resistant Salmonella), the number of Salmonella colonies per gram of tissues or droppings will be quantified as described previously (1). Briefly, cecal tonsils, liver, and spleen will be collected, weighed, homogenized and diluted with tetrathionate broth in a 1:9 weight/volume ratio. Then ten-fold serial dilutions will be created within a 96-well plate and each dilution will be inoculated onto selective media (xylose lysine tergitol-4) agar plates containing 25 μg/mL of nalidixic acid and incubated at 37°C for 24 hours. Isolates typical for NTS (black colonies) will be counted at 48 hours, and the colony forming units CFU/gram will be calculated and compared between groups. Additionally, for improved sensitivity for detection of Salmonella at low concentrations, tissues will undergo a standard isolation protocol for Salmonella. Briefly, samples will be diluted with tetrathionate in a 1:9 weight/volume and incubated at 37°C for 24 hours, followed by inoculation into Rappaport-Vassalidis broth and incubation at 42°C for 24 hours. Then, 0.1 ml will be plated onto XLT-4 agar containing 25 µg/ml nalidixic acid. Colonies phenotypically consistent with Salmonella will be re-plated onto tryptic soy agar, and confirmed as Salmonella using polyvalent O antiserum and/or using biochemical tests or by PCR.Statistical AnalysisThe goal of the statistical analysis will be to test the hypothesis that the vaccinated groups have reduced log concentration of Salmonella in pooled tissues and fecal shedding. The mean log concentration will be used as the dependent variable. Differences between groups will be tested by including treatment as a fixed effect. Data will be analyzed using a generalized linear model (PROC GLIMMIX, SAS v. 9.4). A P value of ≤0.05 (two tail) will be considered significant.