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Funded Project

Funding Program: IPM Enhancement Grants

Project Title: A Dual Recombinant Vaccine for Brucellosis and Immunocontraception in Feral Swine

Project Director (PD): Nammalwar Sriranganathan [1]

Lead State: VA Lead Organization: Virginia Polytechnic Institute and State University

Undesignated Funding: $29,402

Start Date: May-01-2013 End Date: Apr-30-2014

Pests Involved: Feral swine (Sus scrofa)

Summary: Feral swine (sus scrofa) are a nuisance species across much of the United States costing approximately $1.5 billion annually in crop, property, and environmental damage. This rapidly reproducing species is now considered the second most costly vertebrate pest to control in the US behind only rats and mice (Pimental, 2007). In places where they have become established, especially the Southeastern United States, feral swine cause damage by uprooting crops, destroying fences and farm equipment, predating on young livestock, spreading disease, harming water quality and causing soil erosion. Studies have shown that approximately 70% removal per year of the feral swine in an area is required to maintain same population. Current methods of controlling the population include hunting, poisoning, and trapping, however these methods alone are insufficient (Waithman et al., 1999). In addition to causing massive property and environmental damages, wild pigs also serve as reservoirs for zoonotic and livestock diseases such as brucellosis (Meng et al., 2009). Brucellosis in swine is caused predominantly by the bacterium Brucella suis and there is no approved vaccine against B. suis in the United States. Over $3.5 billion has been spent controlling brucellosis in the United States and the disease is considered eradicated from domestic cattle and swine. However, feral swine are the most abundant remaining reservoir of Brucella with a seropositive rate greater than 50% in some herds (Seleem et al., 2010). Feral swine therefore pose a serious risk to livestock, especially small low-intensity management herds. The aim of this study is to test and refine a novel recombinant live-modified immunocontraceptive vaccine which causes infertility and protects against brucellosis in swine. This to be accomplished by expressing the immunocontraceptive multimeric gonadotropin-releasing hormone (mGnRH) in the attenuated B. suis strain VTRS2 (VTRS2-mGnRH). VTRS2-mGnRH has the potential to confer both disease resistance as well as infertility in feral swine as a novel control method for this invasive nuisance species. This proposal seeks funds to initiate validation and testing of the vaccine strain VTRS2-mGnRH in the mouse model prior to testing in feral pigs. B. suis ”wboA ”leuB, known as VTRS2 is an attenuated rough mutant (”wboA) of the wild-type swine isolate B. suis 1330. The strain also carries a deletion in leuB, a gene required for leucine biosynthesis. This reduces the ability of the strain to grow in a leucine-depleted environment such as the intracellular compartment where the bacteria resides during the course of infection. By complementing leuB on a plasmid DNA vector (pNS4) it is possible to maintain expression of recombinant antigen such as the immunocontraceptive mGnRH without resorting to the use of antibiotic-resistance markers as with conventional bacterial antigen delivery vectors (Rajasekaran et al., 2008). Through these methods we seek to add to the control methods for the feral swine population as well as introduce a candidate vaccine against one of the major zoonotic diseases they carry. Objectives: 1. In vitro characterization of the immunocontraceptive brucellosis vaccine VTRS2-mGnRH To evaluate the immunocontraceptive potential of the strain using an in vitro model, VTRS2-mGnRH will be tested in bacterial culture and macrophage cell culture for mGnRH expression and growth characteristics. Plasmid stability will also be determined by assessing plasmid maintenance in both leucine-deficient and leucine-rich media. The candidate strain VTRS2-mGnRH has already been created and clearance and challenge studies in mice are currently planned for June 2013 to test for protection against brucellosis. a. Confirmation of expression of the GnRH multimer by Western Blot In order to verify that the vaccine strain is expressing the contraceptive immunogen mGnRH, Western blotting will be performed on protein extracts from VTRS2-mGnRH bacterial culture. Once expression is confirmed animal studies will proceed. b. In culture and in vitro growth characteristics of VTRS2-mGnRH To develop a basic knowledge of the novel vaccine strain, culture and in vitro growth curves of strain VTRS2-mGnRH will be determined. Culture growth analysis will allow testing for plasmid stability and will also aid in optimization of extraction protocols by growth to be charted versus time. In vitro growth curves will provide insight as to the attenuation of the strain in mouse and porcine macrophages prior to testing in live animals. This will allow evaluation of the ability of VTRS2-mGnRH to grow in macrophages and more importantly allow determination of mGnRH production. 2.Evaluation of VTRS2-mGnRH as an immunocontraceptive vaccine in the mouse model The ability of VTRS2-mGnRH to compromise fertility in both male and female mice will be tested prior to evaluation in feral swine. Breeding studies will be performed in which either the males or females are vaccinated. Mice will be assessed for breeding success, litter size, reproductive hormone levels, and histopathology of the reproductive tissues. a. Evaluation of the effect of VTRS2-mGnRH in male BALB/c mice To evaluate the effect of the vaccine on the male a breeding study will be performed in which male mice are vaccinated prior to being housed with females for breeding. Males will be tested for the ability to impregnate the female and testosterone levels will be measured. At the end of the study reproductive tissue will be collected. b. Evaluation of the effect of VTRS2-mGnRH in female BALB/c mice To evaluate the effect of the vaccine on the female a breeding study will be performed in which female mice are vaccinated prior to being housed with a male for breeding. Females will be checked for a sperm plug and pregnancy status will be monitored. Breeding efficiency will be calculated and tissues from mothers will be collected. Output: Demonstration of the ability of VTRS2-mGnRH to prevent pregnancy in the mouse model will warrant testing of the vaccine in pigs for use in feral swine. The availability of an orally deliverable immunocontraceptive to wildlife managers will add to the tools at their disposal for reducing the incidence of the disease brucellosis as well as the rapidly growing number of feral swine in the United States.

Final Report:

Leveraged Funds N/A

Other Products / Outputs Oral presentation: 2013 VMRCVM Graduate Research Symposium; Developing a Bivalent Immunocontraceptive Vaccine Against Brucellosis in Feral Swine; Garrett Smith

Publications N/A, Awaiting final project completion

Impacts Actual impact is currently unknown as breeding experiments are ongoing. However, of the two overarching goals of the VTRS2-mGnRHb vaccine (protection against Brucella challenge and reduction in litter size), protection against challenge has been demonstrated in the mouse model (see baseline data, appendix). Following successful breeding trials in the mouse model evaluation of VTRS2-mGnRHb in feral swine will be performed at NADC. Dr. Steven Olsens brucellosis research group at the USDA National Animal Disease Center (Ames, IA) has agreed to test the constructs in captive-bred feral swine as well as domestic pigs in an approved Animal Biosafety Level-3 facility capable of housing large mammals. Ideally, pig studies will be similar to those performed in mice and breeding success will be evaluated, as well as protection against virulent brucellosis challenge. If the immunocontraceptive vaccine is able to protect against the disease brucellosis and cause infertility in feral pigs, it has the potential to be a major addition to the available tools to control feral pig populations in the United States. Both feral pigs and the diseases they carry pose a serious threat to agriculture, especially in the Southern US. By complementing existing control methods and refining delivery strategies, VTRS2-mGnRHb could prove to be a valuable and low-cost option for wildlife management to utilize in reducing the numbers of feral swine as well as the costly disease brucellosis.

Outcomes Short-term effects: -By adjusting the codon usage it is possible to express the immunocontraceptive peptide mGnRH in an attenuated live Brucella platform VTRS2. This was demonstrated by Western blot of bacterial culture lysate and by detection of mGnRH-specific antibody in inoculated mouse sera by ELISA. -The resulting vaccine VTRS2-mGnRHb is attenuated compared to virulent B. suis and is protective against virulent B. suis challenge in the mouse model. The challenge study is being repeated to reinforce the existing data. Medium-term effects: -Pending the results of ongoing breeding trials, VTRS2-mGnRHb will ready to be tested in feral swine under controlled conditions.

Activities / Events Experimental research activities conducted at the Virginia-Maryland Regional College of Veterinary Medicine (VMRCVM) Center for Molecular Medicine and Infectious Diseases (CMMID), Virginia Tech, Blacksburg, VA. Activities include: redesign of mGnRH construct for use in Brucella (resulting in mGnRHb), cloning of the mGnRHb construct into Brucella suis VTRS2, and in vitro and in vivo assessment of the resulting candidate vaccine VTRS2-mGnRHb.

Target audience This project is intended to serve any and all communities/geographic locations affected by feral swine, especially those with a high incidence of Brucella suis and risk of transmission to both humans and livestock (especially the Southern United States). The most direct users of the project output would be the federal, state, and local agencies tasked with controlling populations of feral swine and/or reducing the incidence of zoonotic diseases like brucellosis in wildlife reservoirs.

Non-technical summary and Results Feral swine are a major nuisance species in the United States, especially in the South. In addition to costing an estimated 1.5-2 billion dollars annually in property and environmental damage, feral swine carry a number of diseases that can be transmitted to both humans and livestock, especially brucellosis. Brucellosis is a highly infectious disease of livestock that can also be transmitted to humans. Massive concerted efforts have been undertaken eradicate the disease in livestock in the United States however wildlife reservoirs such as bison and feral swine still carry the causative agent Brucella. Of the wildlife reservoirs in the United States, feral swine are the most numerous and widespread and therefore pose a risk to both domestic livestock and humans. Feral swine populations are estimated to have quadrupled in the last decade thus increasing their environmental impact and the risk of disease. Current control efforts, predominantly hunting and trapping, are insufficient to reduce the population on their own and additional cost-effective control methods are needed in the face of limited resources available to government agencies tasked with reducing the number of feral pigs. The overall goal of this project was to refine and test the candidate vaccine VTRS2-mGnRHb in the mouse model. VTRS2-mGnRHb is intended to both protect against brucellosis and confer infertility to feral pigs. The vaccine strain is an attenuated strain of Brucella suis, the predominant Brucella species found in feral swine, and was created by laboratory-mutation of a gene (wboA) necessary for full virulence in the host. An additional mutation was generated in the gene leuB which is required for the metabolism of leucine in the nutrient-depleted environment in which Brucella resides in the host. This mutation allows antibiotic resistance-free expression of the immunogenic peptide mGnRH, a synthetic highly immunogenic form of gonadotropin-releasing hormone (GnRH), a major regulatory hormone of the mammalian reproductive system. By expression of mGnRH in the live bacterial vaccine delivery platform, the hypothesis is that vaccination will cause reproductive deficits in the host by eliciting an immune response against host GnRH thus causing dysregulation of reproductive control. To evaluate VTRS2-mGnRH for potential use in feral pigs, first the vaccine strain was evaluated for expression of the mGnRH multimer, originally provided by Dr. Lowell Miller of the USDA National Wildlife Research Center. The original mGnRH, intended to be produced and purified from Escherichia coli for use in human prostate cancer treatment, is now under evaluation for use in wildlife. It was found that the mGnRH is poorly expressed in Brucella because it was originally designed for use in E. coli. This unanticipated problem was circumvented by creation of a synthetically modified version of the mGnRH gene sequence optimized for use in Brucella. The resulting mGnRHb has the same final peptide product as the original mGnRH but is also able to be expressed in Brucella. In this project, the new construct, VTRS2-mGnRHb has been demonstrated to express the mGnRH peptide and elicit an anti-mGnRH immune response in vaccinated mice. Further evaluation of the candidate vaccine in this project involved measurement of the clearance of the vaccine strain from the host. An ideal live Brucella vaccine clears from the host between 4 and 6 weeks. It was found that after 6 weeks 99.99% of the inoculating dose of VTRS2-mGNRHb was cleared from the spleens of vaccinated mice. Demonstration of clearance of the strain within an acceptable timeframe warranted evaluation of the vaccines ability to protect mice against virulent Brucella suis challenge. To do this, vaccinated and unvaccinated (saline control) mice were experimentally infected with virulent B. suis 1330 8 weeks post-vaccination. Additional mice received a booster dose at 8 weeks post-vaccination and were challenged 2 weeks post-booster. It was found that singly vaccinated mice had approximately 0.5 LOG reduced bacteria in their spleens 2 weeks post-challenge (62% reduction compared with unvaccinated controls). Interestingly, protection was not demonstrated in mice receiving a booster. This is thought to have happened due to the initial challenge dose being higher than predicted or as a result of a long period of time elapsing between original vaccination and challenge (8 weeks was used to allow for sufficient clearance). The clearance and challenge studies were performed using USDA funds as part of a cooperative agreement with the National Wildlife Disease Center, (Fort Collins, CO) however their association with the current project demands that the data be included with this analysis. SRIPM funds are being used to repeat these trials to further strengthen proof of protection against brucellosis in the mouse model while breeding studies are being performed to measure the reproductive effects of the vaccine. Upon demonstration of clearance and protection of VTRS2-mGnRHb in the mouse model, studies were initiated to determine if the vaccine causes reproductive deficits in the mouse model prior to testing in pigs. Briefly, groups of both male and female mice were either vaccinated with VTRS2-mGnRHb or received saline as a negative control. 6 weeks post-vaccination all vaccinated mice will receive a booster (July 2, 2014). 2 weeks post-booster 2 rounds of mating will begin, in the first unvaccinated females with be mated with either vaccinated or unvaccinated males. Each morning females will be assessed for signs of mating behavior and any bred females will be removed from the male and monitored for litter size after gestation. Breeding behavior, litter size, and microscopic changes in reproductive organs will be measured. After the mating of unvaccinated females, the vaccinated females will be placed with either the vaccinated or unvaccinated males, allowed to mate, and similarly measured. Results for vaccinated males bred with both vaccinated and unvaccinated females will be compared and vice versa. This project is ongoing and is expected to end August 2014. The start was delayed by the re-engineering process of mGnRH for use in Brucella. Since protection against brucellosis has been shown, if that data is supported by the ongoing repeated challenge experiment and there is a demonstrable difference in reproductive success between vaccinated and unvaccinated mice this project warrants further exploration. Future experiments involve controlled trials in pigs and then field trials to evaluate the efficacy of the vaccine before implementation is considered. Future potential pitfalls include the challenge of cost-effective delivery of the vaccine to feral pigs, however other studies have tested baited delivery systems with good results. In addition to potentially helping reduce the incidence of brucellosis in the United States, VTRS2-mGnRHb has the potential to add to the tools currently available to wildlife control officials to control nuisance feral swine.

Report Appendices Baseline data reporting [PDF]

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