Source: CURTISS HEALTHCARE, INC. submitted to
RECOMBINANT ATTENUATED SALMONELLA VACCINES TO REDUCE CAMPYLOBACTER COLONIZATION IN POULTRY
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
SMALL BUSINESS GRANT
Reporting Frequency
Annual
Accession No.
1022543
Grant No.
2020-33610-31987
Cumulative Award Amt.
$100,000.00
Proposal No.
2020-00642
Multistate No.
(N/A)
Project Start Date
Sep 1, 2020
Project End Date
Apr 30, 2023
Grant Year
2020
Program Code
[8.3]- Animal Production & Protection
Recipient Organization
CURTISS HEALTHCARE, INC.
12085 RESEARCH DR
ALACHUA,FL 326156837
Performing Department
(N/A)
Non Technical Summary
Campylobacter is an important foodborne pathogen and the most common cause of acquired foodborne illnesses in humans (some 1.3 million cases per year with over 500 deaths), but also is a leading cause of hospitalization (over 8,000 annually). The majority of human Campylobacter infections are associated with poor handling of raw chicken or consumption of undercooked chicken. Reducing the Campylobacter load at the farm level and reducing contamination at slaughter are therefore important to prevent transmission to humans and is a high priority. One method to reduce Campylobacter load on the farm is to develop vaccines to prevent colonization of chickens. Prevention of infectious diseases by vaccination is more cost-effective than chemotherapy and will reduce the use of antibiotics leading to decreased transmission of drug-resistant pathogens through the food chain to humans. Curtiss Healthcare is developing next generation live synthetic vaccines to address bacterial, viral, fungal and parasitic pathogens. Our technology is centered around the construction of engineered recombinant attenuated Salmonella vaccines (RASVs) strains with high immunogenicity, complete safety and attenuation, and an inability to persist or be shed in the environment after administration. In this application we are proposing to build on our successful preliminary work and construct and characterize RASVs capable of delivering by course spray/oral immunization multiple conserved C. jejuni protective antigens to prevent infection and persistence of Campylobacter in chickens. We will evaluate approximately ten different Campylobacter antigens delivered using RASV technology for their ability to generate an immune response in chickens. The best constructs or combination of constructs will be evaluated in a model of campylobacter colonization in chickens to determine the ability of these vaccines to decrease or eliminate Campylobacter colonization. If successful we would look to develop the most promising constructs for licensure by the USDA.
Animal Health Component
20%
Research Effort Categories
Basic
(N/A)
Applied
20%
Developmental
80%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
3073220109025%
3083220109025%
3113220109025%
7123220110025%
Knowledge Area
311 - Animal Diseases; 712 - Protect Food from Contamination by Pathogenic Microorganisms, Parasites, and Naturally Occurring Toxins; 308 - Improved Animal Products (Before Harvest); 307 - Animal Management Systems;

Subject Of Investigation
3220 - Meat-type chicken, live animal;

Field Of Science
1090 - Immunology; 1100 - Bacteriology;
Goals / Objectives
Campylobacter is an important foodborne pathogen. In Europe, Campylobacteriosis is the foodborne disease most frequently identified and the second in USA, preceded by infections due to Salmonella. The CDC estimates that Campylobacter is not only among the most common causes of acquired foodborne illnesses in humans (some 1.3 million per year with over 500 deaths), but also is a leading cause of hospitalization (over 8,000 annually). Campylobacter is a commensal microorganism of the gastrointestinal tract of many wild animals (birds such as ducks and gulls), farm animals (chickens, cattle and pigs) and companion animals (such as dogs and cats) and it is responsible for zoonoses. The transmission occurs via the fecal-oral route through ingestion of contaminated food and water. The disease varies from a watery diarrhea to a severe inflammatory diarrhea with abdominal pain and fever and can be burdened by complications such as Guillain-Barré Syndrome (GBS), Reactive Arthritis (REA) and irritable bowel syndrome (IBS). Recently, many cases of Campylobacter isolated from human infections, showed resistance to various antibiotics such as tetracyclines and fluoroquinolones. The majority of human Campylobacter infections are associated with poor handling of raw chicken or consumption of undercooked chicken. There is a high prevalence of Campylobacter in both live birds and on carcasses and findings from epidemiological studies, and detection of identical genotypes in both poultry and human infections support the idea that transmission to humans in predominantly the result of the consumption of undercooked contaminated chicken. Reducing the Campylobacter load at the farm level and reducing contamination at slaughter are therefore important to prevent transmission to humans and is a high priority and it has been predicted that decreasingCampylobactercolonization of poultry by 2-log10will reduce human infections by 30-fold. Curtiss Healthcare is developing next generation live synthetic vaccines to address bacterial, viral, fungal and parasitic pathogens. Our technology is centered around the construction of engineered recombinant attenuated Salmonella vaccines (RASVs) strains with high immunogenicity, complete safety and attenuation, and inability to persist or be shed in the environment after administration. These RASVs are engineered to display regulated delayed attenuation in vivo, regulated delayed in vivo synthesis of protective antigens specified by codon-optimized DNA sequences and regulated delayed lysis in vivo. The lysis phenotype permits RASVs to deliver in lymphoid tissues a bolus of protective antigen(s) and confers complete biological containment. These cost-effective vaccines are manufactured under special conditions that enable them to display after oral administration the capabilities of a wild-type strain to survive host defense stresses and efficiently colonize effector lymphoid tissues before manifesting attenuation to preclude disease symptoms and to synthesize protein antigens to induce protective immune responses. In addition, since these vaccines are based on a Salmonella delivery platform they have the added benefit of induction of cross-protective immunity to some Salmonella serotypes which like Campylobacter are transferred via the fecal-oral route and responsible for zoonoses. RASVs stimulate mucosal, systemic and cellular immunities and can be inexpensively manufactured by fermentation with frozen or lyophilized preparation that can be reconstituted at the time and place of use. Our goal is to build on our successful preliminary work and construct new RASVs to deliver by course spray/oral immunization multiple conserved C. jejuni protective antigens to prevent infection and persistence of Campylobacter in chickens. If successful we would look to develop the most promising constructs for licensure by the USDA as part of a Phase 2 SBIR applicationTo achieve our goal of developing a vaccine for use in chickens to prevent/reduce campylobacter colonization we propose the following objectives:Objective 1: Complete cloning of the Cj0427 antigen into our two proprietary plasmid vectors and characterize the stability and inducible protein expression in our proprietary c12341 Salmonella vector (see table 1).Objective 2: Further characterization of the additional nine constructs already cloned into our two proprietary plasmid backbones (see table 1) will be performed to further optimize protein expression and where necessary obtain clones with optimized signal sequences to facilitate secretion of antigen. We expect this will result in approximately 20 plasmid constructs to be evaluated. This includes a prelminary evaluation of the constructs for their ability to be scaled-up in a suitable manufacturing process.Objective 3: We have identified antigens that are conserved among Campylobacter jejuni isolates and can be expressed stably in our RASV system (see table 1). We now aim to evaluate these selected antigens in our c12341 Salmonella vector backbone and assess their ability to generate an immune response in chickens. Following vaccination with the selected vaccine candidates (see table 1) we will evaluate the sera and mucosal tissue from chickens for antibody responses against the vaccinated antigens. Constructs with the best responses will then be evaluated in combination to look for interference.Objective 4: Based on the results from the objectives listed above we will evaluate the two most promising combinations of antigens in a model of Campylobacter colonization in chickens. The results from these studies will be used to select one of the combinations for further development.Table 1. Campylobacter antigens to be used in this proposal.AntigenGeneFunctionSignalPeptide Stable Protein ExpressionConserved*ReferencePYA3764PG8R17Omp18Cj0113peptidoglycan associated lipoproteinYesYesYesYesUS Patent 9328148 B2Peb1Cj0921cbifunctional adhesin/ABC transporter aspartate /glutamate-binding proteinYesYesYesYesUS Patent 9328148 B2DpsCj1534cDNA starvation/stationary phase protection protein, putative bacterioferritinNoYesNoYesUS Patent 9328148 B2Cj0998cCj0998chypothetical proteinYesYesNoYesUS Patent 9328148 B2CjaACj0982csurface antigen/glutamine ABC transporter substrate-binding protein YesYesYesYesUS Patent 9328148 B2Cj0427Cj0427hypothetical proteinYesTo bemadeTo bemadeYesUS Patent 9328148 B2Peb3Cj0289cmajor antigenic peptide PEB3YesYesYesYes(32)TlyACj0588putative hemolysinNoYesNoYesUS Patent 9328148 B2CadFCj1478couter membrane fibronectin-binding proteinYesYesYesYes(33)FlaACj1339cflagellin A (FlaA)YesYesNoYesUS Patent 9328148 B2*Indicates >95% identify with a consensus sequence constructed using amino acid sequences for the indicated protein fromover 10,000 isolates used in our analysis.
Project Methods
Describe the ways in which the project will be conducted, with emphasis on the general scientific methods and any unique aspects or significant departures from usual methods. Include a description of how the results will be analyzed, evaluated, or interpreted. Our research team will clone selected Campylobacter antigens into our proprietary plasmid vectors. Plasmids will be transformed into the c12341 Salmonella strain and transformants assessed for stability of plasmid maintenance, integrity and antigen synthesis ability when strains. Western blots will be performed using an antibody directed against the his-tag on the proteins. We continue to make refinements to our delivery system and other Salmonella vectors may also be used based on our latest findings. We will then evaluate these vaccine constructs for their ability to generate an immune response in chickens. Following vaccination we will evaluate the sera and mucosal tissue from chickens for antibody responses against the vaccinated antigens. Vaccines will be grown on-site in Curtiss Healthcare laboratories under the Standard Operating Procedure (SOP) used for vaccine preparation. Groups of 3-5 SPF chickens housed in isolators will be vaccinated orally with 107 to 108 CFUs of live vaccine at day 1 and again at day 14. On day 21 (six days after the 14 day boost) blood will be collected from the wing veins of immunized birds to assess the IgY antibody responses to the immunized Campylobacter antigens. Birds will then be humanely euthanized and mucosal samples collected for further analysis. To evaluate mucosal immune responses intestinal samples will be collected. To facilitate our evaluation we will synthesize recombinant antigens. Strains carrying plasmids with the Campylobacter antigens for study will be grown and cultures pelleted by centrifugation. The pellet will be resuspended and an aliquot removed to prepare whole-cell-lysates for western blotting using sera collected from immunized birds. The remaining aliquot will be used to purify protein by passage of cell lysates over a metal affinity column for use in ELISAs. The response of immunized birds will be assessed by western blotting and ELISA as described below. For Western blots whole-cell lysates from vaccine constructs producing the selected antigen will be subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting performed as described using sera collected from immunized chickens. ELISA assays will be performed in triplicate as previously described to determine the IgY responses against the various immunized Campylobacter antigens in chicken sera and IgA, IgY and IgM responses in intestinal washes. Briefly, microtiter plates will be coated with purified recombinant Campylobacter proteins as described above and washed/blocked with blocking buffer. An initial 1:10 dilution of sera and a 1:2 dilution of intestinal washes will be made with each sample. Then a 2-fold serial dilutions 7 times from these initial dilutions of sera and intestinal washes performed and loaded in triplicate on a 96 well Nunc MaxiSorb plate. Biotinylated anti-chicken IgA (Alpha Diagnostic Intl. Inc), IgY (Southern Biotechnology) or IgM (Bioss) antibodies diluted 1:10,000 will used to detect the various antibody isotypes. Plates will be washed, and streptavidin-horseradish peroxidase solution will be added. Color development will be detected using ABTS containing 0.03% H2O2 in citrate buffer, pH 4.35. After color development, the reaction will be stopped with 1% SDS solution and OD405 measured using a microplate reader. Data from Western blotting and ELISA will be collected and the strength of the immune response to various antigens compared. Antigens showing the best immune response including a strong mucosal response will be ranked and the best ones considered for further studies. Based on the results from our initial evaluation of the immune response in chickens we will evaluate the two most promising combinations of antigens in a model of Campylobacter colonization in chickens. The results from these studies will be used to select one of the combinations for further development and be the subject of a phase 2 SBIR application. The Campylobacter jejuni Challenge model will be run at Southern Poultry Research Group (SPRG) in Georgia. The goal of the experiment is to determine if experimental challenge with Campylobacter jejuni will lead to differences in cecal Campylobacter jejuni culture log counts at 35 days of age in broiler chickens that are left unvaccinated versus those vaccinated with one of our constructs. Briefly, groups of 20 to 30 birds will be housed in separate HEPA filtered isolation rooms. Male broilers will be utilized to ensure they have the least disease challenge possible while still using a commercial strain of broilers. On Study Day 0, chicks will be vaccinated with Curtiss Healthcare Campylobacter vaccine via oral gavage or given a saline placebo. On Study Day 11 birds will receive a booster vaccination via oral gavage or saline placebo. On Study Day 14, all trial birds in all isolation rooms will receive a challenge via oral gavage with approximately 105 colony forming units (CFU)/mL Campylobacter jejuni isolate JB. At termination on Study Day 35, all birds will be humanely euthanized, and ceca will be collected from all birds for Campylobacter jejuni culture. Ceca will be placed into sterile plastic sampling bags (Fisher) for Campylobacter jejuni isolation, culturing, and analysis using a Campylobacter jejuni Enumeration Procedure (Serial Dilution Method). Suspected Campylobacter isolates will be confirmed by Gram stain. Data from enumeration studies will recorded and statistical analysis of the results will be performed. All animal experiments conducted at Curtiss Healthcare will be conducted in compliance with and approved by the University of Florida Institutional Animal Care and Use Committee and the Animal Welfare Act under protocol. Animals will be monitored at least twice daily during these experiments for signs of morbidity or motality. All results will be analyzed using the most appropriate statistical test from the SAS program to evaluate the relative significance or lack thereof of results obtained.Describe theEfforts that will be used to cause a change in knowledge, actions, or conditions of a target audience. Include a description of how the output(s) will beEvaluated and/or quantified for its impact on the intended audience(s).If successful we expect our work will result in a lead candidate vaccine suitable for further efficacy and safety evaluations. This candidate vaccine would be expected to be safe in chickens and result in at least a 2-log drop in the colonization of birds in an experimental setting. While this Phase 1 SBIR is not expected to demonstrate formal safety of our vaccines from a regulatory perspective we will be monitoring vaccinated birds for morbidity and mortality and any signs will be noted and factored into our evaluation. However, based on other studies our system has proven to have an excellent safety profile and we do not expect vaccine candidates to exhibit any safety issues. For efficacy we will be challenging vaccinated birds with relevant Campylobacter isolates and evaluating them for a reduction in Campylobacter colonization via standard microbiological plating methods. These studies will be powered to demonstrate statistical differences between groups. Our long-term goal is to develop a vaccine that will achieve licensure by regulatory authorities throughout the world. Such a product would have a defined package label describing the safety and efficacy of the product.

Progress 09/01/20 to 04/30/23

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported 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? Nothing Reported

Impacts
What was accomplished under these goals? In addition to χ12341Salmonellavector we chose to explore the use of these alternative delivery vehicles, since we have noted some advantages of them in other vaccine programs including our Salmonella food safety program. We have also constructed a new plasmid capable of dual expression of two high-priority antigens. This allowed us to create one vaccine expressing two different antigens as opposed to a bivalent mixture. Furthermore, we constructed various vaccine candidates fused to the ClyA protein that facilitates protein expression and incorporation into Outer Membrane Vesicles (OMVs) to improve immunogenicity. We targeted Peb1, O988c, FlaA, Cja, Dps, TlyA, PorA, and Omp18. To enhance functionality of these antigens, we fused them to the vesicle-associated toxin ClyA of Escherichia coli. The Peb1 and O988c were individually fused ClyA. The FlaA, Cja and Dps was constructed as an expression operon in which FlaA was fused to the ClyA; similarly, TlyA, PorA and Omp18 were constructed as expression operon and TlyA was fused to the ClyA. We confirmed expression of the proteins using antiHis antibodies since each protein was his tagged. The plasmids containing the above constructs were introduced into Salmonella Typhimurium lysis strains χ12341, χ11534 and non-lysis CS17 (?Cya & Crp). Each strain was tested for expression of the antigens and stability during in vitro passages. Based on these results we produced batches for four different vaccine candidates and tested their efficacy in vaccination challenge model. To improve the challenge model, we have created a frozen seed culture. We have also systematically analyzed the in vitro culture conditions for Campylobacter and developed an optimized diluent with reducing agents containing oxygen-scavenging additives to ensure that the first and last chicken in each challenge gets the same quantity of Campylobacter challenge agent. We developed a Real Time quantitative PCR (RT-qPCR) to eliminate the bird-to-bird variability. Efficacy testing of vaccine candidates. We conducted three clinical studies in which we tested the efficacy of the vaccine candidates in broiler chickens. Study 1: Five (5) vaccine candidates (designated as C198, C199, C200, C201 and C202) were tested either singly or in combination. Ninety-six (96) male birds were assigned to four (4) treatment groups with two (2) cages per treatment and twelve (12) birds per cage. The group 1 served as non-vaccinated challenge control; Group 2 (C198), 3(C199 +201) and 4 (200+202) were vaccinated at study day (DOT) 0 by spray and D7 by oral gavage. On DOT14 all chickens were challenged with C. jejuni strain JB (a recent chicken isolate by oral gavage. On DOT 28 and 35, five (5) birds per cage were humanely euthanized. Both ceca and spleen/liver samples from each of the birds from each treatment group were aseptically removed and placed into individual sterile plastic sampling bags for C. jejuni isolation, culturing, and analysis. There were no differences (100% positive) in C. jejuni prevalence between any vaccine and the challenge control at either 28 or 35 days. On day 35, the mean of T4 (C200 and C202) was significantly lower than that of T1 and T2, while T3 was intermediate. The T4 (C200 + C202) had the lowest number at 10 Log 7.71 CFU/g , TG1, 2 and 3 had 10 Log 8.98 CFU/g, 8.82 CFU/g and 8.54 CFU/g, respectively. Prevalence of C. jejuni in liver/spleen was lowest in the untreated control at both day 28 and 35. Study #2: In this study, three treatments evaluating four different combinations of constructs were evaluated for reduction in C. jejuni colonization of ceca and internal organs. The vaccines were coarse sprayed (0.25 ml/chick) at day old and then gavaged on day 7 per experimental design to simulate a drinking water boost. On day 14, all chicks were challenged by oral gavage with the C. jejuni JB Strain at 5.0x 105 CFU/chick. On day 29 and 35, five birds per cage at each time were euthanized and ceca; liver/spleen were removed for C. jejuni enumeration by 10 fold dilution. There was no significant difference between treatments or days with respect to Campylobacter prevalence. Study #3: In this study different combinations of live Curtiss Healthcare vaccines were evaluated for efficacy against Campylobacter jejuni challenge strain, J.B. Treatments included a challenge control, Curtiss Vaccines 200/202, Curtiss Vaccines 210/211, and Curtiss Vaccines 200/202/210/211. Each treatment was represented by three replicate floor pens of 25 male Ross broiler chicks. At hatch (DOT 0), vaccines were applied at a dose of 0.25mL per chick and boosted on DOT 7 by gavage (0.1mL/chick). All chicks in all groups were orally gavaged with Campylobacter jejuni JB strain (1.4 x 105 CFU/mL) on DOT 14. On DOT 35, ceca and pooled liver/spleen samples were collected from five birds in each group. On DOT 43, only ceca samples were collected from five birds in each group. Campylobacter prevalence in the ceca on each collection day is found in Table 1. All groups had 100% prevalence in the samples collected on DOT 35. By DOT 42, the combination with Curtiss Vaccines 200/202/210/211 had numerically lower prevalence in the ceca relative other groups (P=0.24), Figure 1. On day 35, enumeration in the ceca was numerically lower in all vaccinated groups relative to the challenged control (Table 2, Figure 2). At this time, the Curtiss Vaccine 200/202/210/211 had the numerically lowest outcome (5.31 log10 CFU/g) relative to the challenge control (5.99 log10 CFU/g) (P=0.23). By day 43, overall Campylobacter colonization of the ceca had increased and all groups were numerically similar. Overall, the Curtiss vaccine 200/202/210/211 had the numerically lowest ceca campylobacter enumeration. Liver/spleen samples were evaluated for Campylobacter jejuni prevalence on DOT 35. The Curtiss vaccine 200/202/210/211 had 7% positives compared to the untreated challenge control at 20% (P=0.27). The Campylobacter jejuni prevalence (100%) on DOT 35 demonstrates a successful uniform challenge for all groups with expected animal-to-animal variations. This demonstrates that our approach to further optimization of the challenge material and culturing methods were successful and reliable. Vaccine candidates c200 and 202 consistently reduced the cecal Campylobacter loads in the ceca of vaccinated chickens when compared to control animals 3 weeks after challenge. Further optimization of vaccine dose, and timing of vaccination may further improve the efficacy of these vaccines. Overall, results are encouraging that one or more of these vaccines or vaccine combinations are effective in lowering C. jejuni colonization in chickens which is likely in reducing foodborne campylobacteria infection in human.

Publications


    Progress 09/01/21 to 08/31/22

    Outputs
    Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported 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?We will further tests our candidates and select the most effective candidate which then will be tested for licesing.

    Impacts
    What was accomplished under these goals? Since our last report we made significant progress in three areas: Challenge development and optimization: In our previous experiments we noticed that there was significant variation (up to 6-7 Log10 CFU/gram of tissue) in the quantity of C. jejuni recovered from the non-vaccinated challenged chickens. This was attributed to this to challenge material and re-isolation procedures since we demonstrated that C. jejuni highly sensitive environmental factors. We addressed these by establishing a frozen challenge material using special medium and also employing special medium during recovery from the tissues. We also developed a quantitative PCR (qPCR) method. We validated both the challenge material and the quantitation methods by using spiked samples which were tested by culture and qPCR methods. We also tested our methodologies in a small challenge experiment. Our results showed that our qPCR method and culture method were correlated well qualitatively. The limit of detection for qPCR was 4log10 CFU/gram and worked well cecal samples and small intestinal samples in which there were large number of C. jejuni but not for liver and spleen samples suggesting C. jejuni load were below detection limit of qPCR. These experiments showed us that C. jejuni colonization is not limited to ceca, it readily colonizes small intestines and internal organs as shown in liver and spleen samples though much lower numbers. We will be further investigating these observations and their relevance to food safety. Construction of new vaccine candidates: For this, we targeted Peb1, O988c, FlaA, Cja, Dps, TlyA, PorA, and Omp18. To enhance functionality of these antigens, we fused them to the vesicle-associated toxin ClyA of Escherichia coli. The Peb1 and O988c were individually fused ClyA. The FlaA, Cja and Dps was constructed as an expression operon in which FlaA was fused to the ClyA; similarly, TlyA, PorA and Omp18 were constructed as expression operon and TlyA was fused to the ClyA. We confirmed expression of the proteins using antiHis antibodies since each protein was his tagged. The plasmids containing the above constructs were introduced into Salmonella Typhimurium lysis strains χ12341, χ11534 and non-lysis CS17 (?Cya & Crp). Each strain was tested for expression of the antigens and stability during in vitro passages. Based on these results we produced batches for four different vaccine candidates and tested their efficacy in vaccination challenge model. Efficacy testing of vaccine candidates: For this we conducted two clinical study in which tested efficacy of the vaccine candidates in broiler chickens. Study 1: Five (5) vaccine candidates (designated as C198, C199, C200, C201 and C202) were tested either singly or in combination. Ninety-six (96) male birds were assigned to four (4) treatment groups with two (2) cages per treatment and twelve (12) birds per cage. The group 1 served as non-vaccinated challenge control; Group 2 (C198), 3(C199 +201) and 4 (200+202) were vaccinated at study day (DOT) 0 by spray and D7 by oral gavage. On DOT14 all chickens were challenged with C. jejuni strain JB (a recent chicken isolate by oral gavage. On DOT 28 and 35, five (5) birds per cage were humanely euthanized. Both ceca and spleen/liver samples from each of the birds from each treatment group were aseptically removed and placed into individual sterile plastic sampling bags for isolation, culturing, and analysis. There were no differences (100% positive) in C. jejuni prevalence between any vaccine and the challenge control at either 28 or 35 days. On day 35, the mean of T4 (C200 and C202) was significantly lower than that of T1 and T2, while T3 was intermediate. The T4 (C200 + C202) had the lowest number at 10 Log 7.71 CFU/g , TG1, 2 and 3 had 10 Log 8.98 CFU/g, 8.82 CFU/g and 8.54 CFU/g, respectively. Prevalence of C. jejuni in liver/spleen was lowest in the untreated control at both day 28 and 35. b) Study #2: In this study, three treatments evaluating four different combinations of constructs were evaluated for reduction in C. jejuni colonization of ceca and internal organs. The vaccines were coarse sprayed (0.25 ml/chick) at day old and then gavaged on day 7 per experimental design to simulate a drinking water boost. On day 14, all chicks were challenged by oral gavage with the C. jejuni JB Strain at 5.0x 105 CFU/chick. On day 29 and 35, five birds per cage at each time were euthanized and ceca; liver/spleen were removed for C. jejuni enumeration by 10 fold dilution. There was no significant difference between treatments or days with respect to Campylobacter prevalence. Below table summarizes the C. jejuni concentrations on cecal samples of control and vaccinated groups. Mean (SE) Campylobacter log10 CFU/g in culture-positive ceca samples by treatment and day. Ceca were collected from 5 birds in each of 2 cages per group on days 29 and 35. Treatment Day 29 Day 35 Total T1. Untreated 5.86 (0.30) 6.13 (0.30) 5.99a (0.21) T2. C200 & C202 5.47 (0.30) 5.52 (0.32) 5.49a (0.22) T3. C210 & C211 5.74 (0.30) 5.23 (0.30) 5.49a (0.21) Total 5.69a (0.30) 5.62a (0.30) 5.65 (0.21) Marginal means with a superscript in common do not differ with a level of significance of 5% over all comparisons. Overall, results are encouraging that one or more of these vaccines or vaccine combinations are effective in lowering C. jejuni colonization in chickens which is likely in reducing foodborne campylobacteria infection in human.

    Publications


      Progress 09/01/20 to 08/31/21

      Outputs
      Target Audience: Nothing Reported Changes/Problems:As a result of the Covid-19 pandemic the company was forced to scale-back our R&D operations in 2020 and the first part of 2021. This presented a great challenge in moving the project forward as the majority of the proposed studies require access to a vivarium. One of the antigens we proposed to clone has proven difficult. This may be because expression is toxic. As an alternative we may trying cloning small fragments or alternatively we may remove it from our list of potential candidates. What opportunities for training and professional development has the project provided? Nothing Reported 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 a result of the Covid-19 pandemic the company was forced to scale-back our R&D operations in 2020 and the first part of 2021. While we have made some progress on the proposed studies with regards to the construction of vaccine candidates, the limitations imposed on our vivarium have not allowed us to start the proposed animal studies. We anticipate that we will have greater access to the vivarium in second half of 2021 and are confident that we will make significant progress on the proposed work and that the project can be completed within the requested extension of time. Earlier this year we requested and were granted a no-cost extension for award number 2020-33610-31987. An interim report was submitted to USDA that describes what was accomplished during the pandemic.

      Impacts
      What was accomplished under these goals? As a result of the Covid-19 pandemic the company was forced to scale-back our R&D operations in 2020 and the first part of 2021. While we have made some progress on the proposed studies with regards to the construction of vaccine candidates, the limitations imposed on our vivarium have not allowed us to start the proposed animal studies. We anticipate that we will have greater access to the vivarium in second half of 2021 and are confident that we will make significant progress on the proposed work and that the project can be completed within the requested extension of time. Earlier this year we requested and were granted a no-cost extension for award number 2020-33610-31987. Below is a review of work done as of May 2021 in support of this SBIR grant to develop recombinant attenuated salmonella vaccines to reduce Campylobacter colonization in poultry. During the Covid imposed shutdown we conducted additional bio-informatic analysis and further prioritized our list of antigens to select for the four we feel have the highest potential including Omp18, Peb1, DPS and Cj0988c. The team also conducted further characterization of the additional nine constructs already cloned into the pYA3764 and pG8R18 backbone and several plasmids containing candidate antigens have been moved into alternative Salmonella delivery vehicles. We have chosen to explore the use of these alternative delivery vehicles since we have noted some advantages of them in other vaccine programs. We have also constructed a new plasmid capable of dual expression of two high priority antigens. In addition, as a reagent we have purified protein for four proteins and for two proteins we have been able to immunized chickens and rabbits to generate a polyclonal antisera for use in western blots and ELISA's. The anti-sera from chickens is of good quality and demonstrated the antigenicity of these proteins in birds. Attempts to clone the Cj0427 antigen into the pYA3764 and pG8R18 vectors and obtain stable protein expression in the c12341 Salmonella vector proved difficult. We therefore propose to drop this candidate from our list. Lastly, we have done additional work to improve the Campylobacter colonization model. Campylobacter is highly sensitive to oxygen and exposure to oxygen during challenge or recovery after challenge can lead to wide variation in the model making it difficult to see positive effects of the vaccines. As discussed in our SBIR proposal the JB Campylobacter jejuni isolate that we propose to use in our studies that is found in the cluster associated with carcass contamination is less well lab adapted and more susceptible to oxygen exposure than the traditionally used NCTC strain. To improve the model, we have created a frozen masterseed culture for use in challenge models to ensure batch to batch consistency in the number of CFU's in each challenge dose. We have also developed an optimized diluent with oxygen scavenging additives that will ensure that the first and last chicken in each challenge gets the same titer regardless of time. These same additives can be used during the recovery phase to minimize the effects of oxygen exposure as a result of time and handling on the CFU counts recovered. We believe these modifications will decrease the variability among treated groups and allow for use of smaller number of animals and better statistical power. The addition of these changes is currently being tested in the model by Southern Poultry Research (SPRG) and results are expected in July of 2021. If successful we would implement these changes in the model for use in evaluating vaccine candidates.

      Publications