Performing Department
Animal & Avian Sciences
Non Technical Summary
In the US, salmonellosis is the second most common foodborne disease. One of the major routes of human salmonellosis is believed to be consumption and handling of contaminated poultry and poultry products, as Salmonella colonizes the chicken gut as part of the normal flora. Thus, strategies for controlling the colonization of Salmonella in poultry gut, to reduce the incidence of salmonellosis in humans, are essential. We have developed a lysogenic Salmonella vaccine strain (ST+P13/19) by inserting genes 13 (holing) and 19 (lysozyme) of bacteriophage P22 into downstream of sseA, the chaperone of SPI-2 genes of Salmonella enterica (SE) serovar Typhimutium (LT2). Thus the expression of both genes were controlled by the sseA promoter in intracellular conditions. We found that the intracellular viability of ST+P13/19 was reduced >95% compared to LT2 in chicken macrophage cells, and significantly increased pro-inflammatory cytokines and endosomal toll-like receptors. In this project, we aim to evaluate the role of this non-pathogenic and autolytic ST+P13/19 vaccine strain in outcompeting pathogenic SE colonization and preventing cross-contamination of poultry products in a chick model. To evaluate the protection ability against colonization of various pathogenic SE serovars like Typhimurium and/or Enteritidis in vaccinated Broiler chicks will be performed and the host immunity and gut health during the intracellular lysis of the Salmonella vaccine strain in the chicken intestine will be compared. Outcomes of this project will improve the safety of the poultry products and reduce the human gastrointestinal infection with Salmonella and improve human health.
Animal Health Component
0%
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
In this research project, we hypothesize that using a temperate phage conjugated non-pathogenic Salmonella vaccine strain will enhance the chicken immunity against colonization of Salmonella spp. by stimulation of intracellular lysis. The ultimate goal of this project is to produce a bio-preventive agent for controlling Salmonella shedding in poultry, prevent the cross-contamination of poultry products and control the foodborne salmonellosis in human. We aim to evaluate the role of autolytic ST+P13/19 vaccine strain in outcompeting pathogenic SE colonization and preventing cross-contamination of poultry products through immune responses in a chick model. To achieve our goals, we propose these specific aims: 1) Evaluate the protection ability of colonization of various pathogenic SE serovars Typhimurium and Enteritidis in Broiler chicks; and 2) Evaluate the host immunity and gut health during the intracellular lysis of Salmonella vaccine strain in the chicken intestinal epithelial cells.
Project Methods
For specific Aim-1, we will feed the vaccine strain (ST+P13/19 ) to day old chicks and after three days we will challenge the immunized chicks with either SE serovars Typhimurium, Enteritidis or both and evaluate the protection ability of ST+P13/19 against colonization of these serovars in chicken gut, particularly cecum. To achieve our Aim-2, we will evaluate the host immunity including cytokine production and gene expression, and overall gut health/ microbial homeostasis during the intracellular lysis of this vaccine strain in chicken. The details of the approaches are given bellow:Chicken number and group:A total of 180 chicks will be used for 3 trials. Immunogenicity and colonization ability of Salmonella (both vaccine LT2 and pathogenic serovars, Typhimurium and Enteritidis) in chickens highly varies and to get a statistically significant result, the proposed chicken number is required (60 chicks/ trial). To get significant result, we picked minimal chicks per group (15 chicks/group). Similarly, colonization ability of one-day-old chicks with Salmonella is also highly variable from batch to batch, supplier to supplier, and time of hatching. That is why; we need to perform at-least 3 trials to validate our study, even though we assigned 15 chicks per group.Chicks will be reared in "Avian 12-Cage Isolator Unit" placed in ANSC 0336 with BSL-2 facilities. At first, total 60 chicks will be used for the initial trial with Salmonella enterica serovar Typhimurium LT2 vaccine strain. Chicks will be transported from the source to the animal care facility with CARF's Animal Transport Van. The chicks will be randomly assigned to control and test groups (15 chicks per group). Three cages of the unit will be assigned to each group, so a total of 5 chicks per cage. Stocking density has been calculated and confirms the IACUC guideline. A Completely Randomized Design (CRD) will be employed for this purpose.Vaccine strain culture and vaccination:For this study, we have developed the intracellular autolytic Salmonella Typhimurium LT2 vaccine strain (STLT2-INC-ATL) following the protocol approved by Department of Health and Safety, UMD (IBC#11-65) using the S. Typhimurium LT2 ATCC700720. STLT2-INC-ATL vaccine strain will be cultured on LB agar plates for 24 h under aerobic condition and transferred to PBS to a concentration of 1010 cfu/mL. Aliquot 100 µL of STLT2-INC-ATL (Group B & D: 1010 cfu/mL/chick) suspension will be fed to chicken with oral gavage at day 1 and day 8.Bacterial pathogens challenge and chicken euthanization: S. Typhimurium ATCC14028 and S. Enteritidis ATCC13076 will be cultured on LB Agar plates for 24 h under aerobic condition and transferred to PBS to a concentration of 109 cfu/mL. At the 15th day, a totaling 100 µL of S. Typhimurium or S. Enteritidis (109 cfu/mL/chick) suspensions will be fed to all groups with oral gavage (Table 2). The chicks will be reared thereafter for 2 weeks, given regular tap water and fed with chicken starter diet (mash) without growth promoters or synthetic chemicals. At the end of second, third, and fourth week, 3, 6, and 6 chicks from each group will be euthanized respectively.PBS control for vaccination and bacterial pathogen challenge:PBS served as control for both vaccine and bacterial pathogen challenge will be administrated orally to chicken. During the first week, the same volume (100 µL) of PBS containing no vaccine strain will be fed to chicken in group A and C.Chicken oral administration:Chicken in all groups will be fed with either PBS as control or PBS containing STLT2-INC-ATL vaccine strain through oral administration. For this purpose, chicken will be held gently (one at a time). The 1 mL BD CornwallTM Fluid dispensing syringe will be used. Aliquot of 100 µL PBS or bacterial suspension will be taken into the syringe, and this liquid/suspension will be fed to one chick by needle-dispensing system. Chicken will be checked for any injury related to the gavage procedure and transferred back to their respective cages. Chicken will be kept in close inspection for 24 h for any abnormalities in their eating or drinking behaviors. The oral gavage (gavage no. 22: length 2.54 cm; ball diameter: 1.25 mm) procedure will be performed in biosafety cabinet in ANSC animal research facility Building Room #0336.Sample collection and processing: Each week, starting from week 1, 15 fecal samples from each chick group, totaling 60 samples per week, will be collected in sterile Whirl-Pak bags using sterile spoon and will be carried to BSL-2 lab (Dr. Biswas's lab, Room #3150, Animal Sciences Building) at the ANSC for natural micro-flora analysis and bacterial enumeration. Cecum from euthanized chicken will be separated and the number of Salmonella in the cecum will be quantified for all chicken in each group which will provide information on pathogenic bacterial colonization on chicken gut. End of each week, 0.5 ml (day 7), 1 ml (day 14), 1.5 ml (day 21), and 2 ml (day 28) of blood will be collected from both vaccinated and non-vaccinated groups (Table 2) via the brachial vein. All over the experiment period, body weight of the chicken will be measured each week.Gut microbiome Comparison: Comparative quantification of bacterial loads in the gut microflora of each chick group will be analyzed by short-gun sequencing. The same DNA used for qRT-PCR (cecum fluid) will also be used for short gun sequencing. For short gun sequencing, as described by Salaheen et al (2018) will be used to quantify the bacterial loads in the gut of test and control chicks using SYBR green reagents.