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Revisiting Brucellosis in the Greater Yellowstone Area (2017)

Chapter: 9 Remaining Gaps for Understanding and Controlling Brucellosis

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Suggested Citation:"9 Remaining Gaps for Understanding and Controlling Brucellosis." National Academies of Sciences, Engineering, and Medicine. 2017. Revisiting Brucellosis in the Greater Yellowstone Area. Washington, DC: The National Academies Press. doi: 10.17226/24750.
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Suggested Citation:"9 Remaining Gaps for Understanding and Controlling Brucellosis." National Academies of Sciences, Engineering, and Medicine. 2017. Revisiting Brucellosis in the Greater Yellowstone Area. Washington, DC: The National Academies Press. doi: 10.17226/24750.
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Suggested Citation:"9 Remaining Gaps for Understanding and Controlling Brucellosis." National Academies of Sciences, Engineering, and Medicine. 2017. Revisiting Brucellosis in the Greater Yellowstone Area. Washington, DC: The National Academies Press. doi: 10.17226/24750.
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Suggested Citation:"9 Remaining Gaps for Understanding and Controlling Brucellosis." National Academies of Sciences, Engineering, and Medicine. 2017. Revisiting Brucellosis in the Greater Yellowstone Area. Washington, DC: The National Academies Press. doi: 10.17226/24750.
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Suggested Citation:"9 Remaining Gaps for Understanding and Controlling Brucellosis." National Academies of Sciences, Engineering, and Medicine. 2017. Revisiting Brucellosis in the Greater Yellowstone Area. Washington, DC: The National Academies Press. doi: 10.17226/24750.
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Suggested Citation:"9 Remaining Gaps for Understanding and Controlling Brucellosis." National Academies of Sciences, Engineering, and Medicine. 2017. Revisiting Brucellosis in the Greater Yellowstone Area. Washington, DC: The National Academies Press. doi: 10.17226/24750.
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Suggested Citation:"9 Remaining Gaps for Understanding and Controlling Brucellosis." National Academies of Sciences, Engineering, and Medicine. 2017. Revisiting Brucellosis in the Greater Yellowstone Area. Washington, DC: The National Academies Press. doi: 10.17226/24750.
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Suggested Citation:"9 Remaining Gaps for Understanding and Controlling Brucellosis." National Academies of Sciences, Engineering, and Medicine. 2017. Revisiting Brucellosis in the Greater Yellowstone Area. Washington, DC: The National Academies Press. doi: 10.17226/24750.
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Suggested Citation:"9 Remaining Gaps for Understanding and Controlling Brucellosis." National Academies of Sciences, Engineering, and Medicine. 2017. Revisiting Brucellosis in the Greater Yellowstone Area. Washington, DC: The National Academies Press. doi: 10.17226/24750.
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Suggested Citation:"9 Remaining Gaps for Understanding and Controlling Brucellosis." National Academies of Sciences, Engineering, and Medicine. 2017. Revisiting Brucellosis in the Greater Yellowstone Area. Washington, DC: The National Academies Press. doi: 10.17226/24750.
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Suggested Citation:"9 Remaining Gaps for Understanding and Controlling Brucellosis." National Academies of Sciences, Engineering, and Medicine. 2017. Revisiting Brucellosis in the Greater Yellowstone Area. Washington, DC: The National Academies Press. doi: 10.17226/24750.
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Suggested Citation:"9 Remaining Gaps for Understanding and Controlling Brucellosis." National Academies of Sciences, Engineering, and Medicine. 2017. Revisiting Brucellosis in the Greater Yellowstone Area. Washington, DC: The National Academies Press. doi: 10.17226/24750.
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Suggested Citation:"9 Remaining Gaps for Understanding and Controlling Brucellosis." National Academies of Sciences, Engineering, and Medicine. 2017. Revisiting Brucellosis in the Greater Yellowstone Area. Washington, DC: The National Academies Press. doi: 10.17226/24750.
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Suggested Citation:"9 Remaining Gaps for Understanding and Controlling Brucellosis." National Academies of Sciences, Engineering, and Medicine. 2017. Revisiting Brucellosis in the Greater Yellowstone Area. Washington, DC: The National Academies Press. doi: 10.17226/24750.
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Suggested Citation:"9 Remaining Gaps for Understanding and Controlling Brucellosis." National Academies of Sciences, Engineering, and Medicine. 2017. Revisiting Brucellosis in the Greater Yellowstone Area. Washington, DC: The National Academies Press. doi: 10.17226/24750.
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Suggested Citation:"9 Remaining Gaps for Understanding and Controlling Brucellosis." National Academies of Sciences, Engineering, and Medicine. 2017. Revisiting Brucellosis in the Greater Yellowstone Area. Washington, DC: The National Academies Press. doi: 10.17226/24750.
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9 Remaining Gaps for Understanding and Controlling Brucellosis 1. INTRODUCTION Critical knowledge gaps remain that limit understanding, detecting, and preventing the transmission of B. abortus in the Greater Yellowstone Area. Research to fill those knowledge gaps is needed in disease ecology, economics, immunology, vaccines and their delivery mechanisms, animal and pathogen ge- nomics, and diagnostics. Research funding and expertise will need to be expanded to include other disci- plines (such as disease ecology and epidemiology) for addressing gaps in the immediate term while also examining immunology, vaccines, and genomics for applications that would address gaps over the longer term. In this chapter, the committee provides an overview of some of the remaining gaps and research areas to pursue. 2. DISEASE ECOLOGY Due to agency and jurisdictional boundaries and different mandates, there have been no major anal- yses of the epidemic across the tri-state region of Idaho, Montana, and Wyoming. A major analysis is re- quired to better understand the factors that are potentially driving the spatial spread of the disease in some regions but not others. A tri-state approach may aid in understanding the roles of land-use and predation on brucellosis in elk as well as the potential for transmission from elk to cattle and bison (both domestic and wild). Understanding the past and modeling the future spatial dynamics of brucellosis in elk will also require additional assessments of how elk populations are connected to one another. Future land-use and changing human demographics in the Greater Yellowstone Area (GYA) are un- derstudied and are likely to be important components of this system. From 1970-1999, the GYA has ex- perienced a 58% increase in population and a 350% increase in exurban housing (Gude et al., 2006). These developments are disproportionately located on highly productive soils and lands close to water, which are also important wildlife winter ranges throughout the GYA. Many of these newer homeowners are less likely than previous owners to allow public hunting, and this has created areas where elk are dif- ficult for hunters to access and are “out of administrative control” (Haggerty and Travis, 2006). Although elk populations have declined within Yellowstone National Park (YNP), most of the surrounding elk pop- ulations have been stable or increasing such that some are 5-9 times larger than they were in the 1970s and 80s. Hunting license fees and the remittance of half of federal taxes on arms and ammunition (per the Pittman-Robertson Act of 1937) are the primary source of support for most state game and fish agencies, although the number of hunters has decreased in many regions (Winkler and Warnke, 2013; Schorr et al., 2014). These factors have potentially created a dynamic that may be detrimental to wildlife winter ranges across the GYA, and also limited the funds and management tools available to state wildlife agencies. The relative proportion of ranchlands that turn-over from livestock production to subdivisions or amenity owners that restrict hunting access is likely to be critical to the future ecological dynamics of wildlife winter ranges in the GYA and is an important aspect for future research. There are benefits and drawbacks for expanding the existing boundaries of the designated surveil- lance areas (DSAs). Expanding the boundaries would expand the surveillance area and the increased scru- tiny would decrease the likelihood that cattle may be infected outside the DSA and inadvertently moved Prepublication Copy—Subject to Further Editorial Revision 153

Revisiting Brucellosis in the Greater Yellowstone Area to other regions and states, which could result in significant detection delays and expensive clean-up campaigns. Boundary expansion would thus be a potentially beneficial option for nationwide protection. However, DSA expansion would increase testing and management costs for state agencies and stigmatize some ranchers within the DSA which are at a relatively low risk. Current boundaries are primarily based on judgement rather than quantitative optimization and are established independently by each state. To date, there has been no assessment of the risks of cattle being infected outside of DSAs. To determine the best way to draw DSA boundaries, a quantitative risk assessment could be used that combines infor- mation on elk densities, seroprevalence, and cattle locations. It is unknown how unfed elk populations can maintain brucellosis at seroprevalence levels that are similar to or exceed those of elk on the supplemental feedgrounds in Wyoming. Scavengers may play a role by removing infectious fetuses from the landscape, and scavenging rates appear to be faster on sup- plemental feedgrounds than elsewhere. To determine if scavenging is an important factor in reducing cat- tle risk of infection or elk to elk transmission, studies need to be conducted on the potential negative ef- fects of coyote control efforts by the U.S. Department of Agriculture (USDA) Wildlife Services as well as the lack of any hunting regulations on coyotes in any of the three GYA states. Host-parasite systems are almost never isolated, and the amount of cross-species transmission that occurs between elk and bison is unknown. Recent estimates only provide the number of transition events for the currently available isolates rather than estimating actual transmission rates per unit time in differ- ent locations (Kamath et al., 2016). This level of resolution is difficult to achieve and is unavailable for any wildlife system. It is an important area for future research that could be addressed by combining dis- ease dynamic models into the phylogenetic framework (Stadler et al., 2014). In addition, little work has been done on the potential effects of other pathogens on brucellosis dynamics (Hines et al., 2007; Ezenwa and Jolles, 2015). Serological tests are one of the best ways available to monitor population-level trends of brucellosis in wildlife species. Sample collection kits have been given to hunters in Idaho, Montana, and Wyoming. Combined with research-related captures, information from those kits provide more complete data from a broad region. Older wildlife individuals are generally more likely to test positive on serological assays due to having a longer timespan for potential exposures to occur and for detectable antibody levels to de- velop after exposure. Extreme shifts in elk age distributions could be responsible for an approximate dou- bling in the raw seroprevalence (Cross et al., 2007). Serological assays differ in sensitivity and specificity, and therefore seroprevalence data need to be standardized to account for potential age and assay effects prior to comparisons across regions and time spans. Epidemiologists have traditionally addressed this is- sue by statistically estimating the effects of age, sex, and other heterogeneities, and then standardized rates to a common population before representing spatial variation (Ahmad et al., 2001; Osnas et al., 2009). Standardization by age, sex, or assay has not yet occurred across the GYA dataset, and there are no strong predictors of why one ranch may be more likely to be infected than another in the same general area. Case-control studies as well as fine scale risk assessments evaluating elk space-use data may help to assess the efficacy of different biosecurity measures. Finally, while one study has examined elk-cattle contact rates (zu Dohna et al., 2014), there have been no studies investigating elk-cattle contact rates in the GYA where such studies are most needed. For example, a study could examine the effects of salt licks on grazing allotments as a factor enhancing elk- cattle contact. The lack of studies may be due to current regulatory rules requiring cattle testing for any known elk-cattle contact. This regulatory stipulation may also reduce the likelihood of producers to come forward and participate in elk-cattle contact studies. 3. ECONOMICS Much information required to calibrate and integrate economic and disease ecology models is una- vailable. More information is needed on the effectiveness and costs of various actions for reducing trans- mission. 154 Prepublication Copy—Subject to Further Editorial Revision

Remaining Gaps for Understanding and Controlling Brucellosis One category of missing information is non-market values associated with GYA wildlife. This in- cludes use values (for example, park visitation, wildlife viewing inside and outside the park, and hunting), and non-use values (for example, conservation of wildlife stocks, and management actions perceived as undesirable, such as mass culls and culls within park boundaries). All of these values need to be specified as functions of bison and elk. One of the most relevant studies in providing such information examined the economic value of various bison and elk management practices for the National Elk Refuge and Grand Teton National Park (Loomis and Caughlan, 2004). However, those values are tied to the particular (ad hoc) management scenarios being analyzed, which impact multiple wildlife populations and make it difficult to identify the various economic values associated with changes in individual wildlife popula- tions. Another relevant study more directly examines hunter demand for elk permits as a function of elk and wolf populations, but only for Northwest Wyoming (Kauffman et al., 2012). Several other types of economic information involving private landowners are also required for a bioeconomic analysis. For instance, surveys are required to acquire information on social and private in- centives for separating cattle from elk, both on private lands (e.g., hazing) as well as public lands (e.g., spatial-temporal grazing decisions). Roberts and colleagues (2012) surveyed ranchers to estimate the costs of implementing various risk-management practices on an average farm in the southern GYA; how- ever, the effectiveness of these practices, along with the associated benefits to ranchers and society, re- mains uncertain. Ruff and colleagues (2016) analyzed the costs and risks at which producers would break even from adopting various practices, but their analysis does not indicate how much effort needs to be applied or the point at which producers would break even from adopting various practices. Moreover, these analyses do not address social incentives for investment in terms of the risk that infected cattle might pose to herds of other producers within and outside the DSA. Economic information is also re- quired about alternative land uses that would pose less or no risk from brucellosis. Economic information is also needed on incentives that might discourage landowners from managing lands as elk habitat/refuge. Lastly, focused research is needed to determine the impact of altering available grazing dates or grazing fees on both cattle grazing and the risk of spreading brucellosis. 4. IMMUNOLOGY Developing new vaccines, especially ones that are effective in elk and that are more effective for cattle and bison than the existing ones, will require an understanding of how host protective immune re- sponses are elicited. Tools to evaluate immune responses will be needed especially for elk and bison as few currently exist. Researchers would find these tools useful, but there is unlikely to be a commercial interest in developing them; thus, funding will need to be sponsored by alternative sources (e.g., govern- ment grants). Other tools, such as methods to redirect immune responses toward protective immunity, are needed to prevent or treat infectious diseases. However, they are less likely to be developed for treating Brucella-infected wildlife or domestic livestock in the near term, but could be considered as part of the long-term research goals. 4.1 Tools to Measure Immune Responses Defining the nature of a protective immune response to virulent field strains of Brucella spp. will be important for designing new vaccines for brucellosis that induce such responses, determining the corre- lates of protection for evaluating potential vaccines, and designing innovative treatments for infected an- imals. The protective immune response for brucellosis involves a type of white blood cell known as a T lymphocyte (or T cell) that is able to produce a soluble product known as cytokine interferon-gamma (IFN-γ). The response may also involve T cells that are able to kill bacteria-infected host cells. These types of T cell responses, known as cellular or cell-mediated responses, are needed because Brucella spp. resides in host cells such as macrophages and trophoblasts (Anderson and Cheville, 1986). IFN-γ acti- vates macrophages to increase their ability to prevent replication of the internalized bacteria (Jiang and Baldwin, 1993). This mechanism of resistance is more effective than antibodies (Pavlov et al., 1982; Prepublication Copy—Subject to Further Editorial Revision 155

Revisiting Brucellosis in the Greater Yellowstone Area Araya et al., 1989; Araya and Winter, 1990; Oliveira and Splitter, 1995; Goenka et al., 2011). In contrast to T cells, antibodies contribute only moderately to protection in secondary responses (challenge) with B. abortus, and alone are less effective or contribute minimally to protection against a primary infection as suggested from murine studies (Goenka et al., 2011; Vitry et al., 2012). The need for cellular immune responses is also why live vaccines are more effective at protection than killed vaccines (Montaraz and Winter, 1986; Avila-Calderon et al., 2013), since the latter tends to induce immune responses that sup- press the protective IFN-γ response (Zhan et al., 1995). For example, killed whole cell vaccines for Bru- cella have been shown to be ineffective in cattle (Olsen et al., 2005). The commercial vaccine consisting of killed B. abortus rough strain 45/20 is less effective than the living attenuated B. abortus strain 19 (S19) vaccine in conferring protection in cattle (Sutherland et al., 1981; Woodward and Jasman, 1983), which is attributed to the inability of killed vaccines to adequately stimulate cell-mediated immunity needed for protection (Nicoletti and Winter, 1990). Assessing the efficacy of new vaccine candidates in elk and bison will require key reagents such as monoclonal antibodies (mAbs) to measure the types of cells that respond to vaccination and their prod- ucts, such as IFN-γ production by T lymphocytes, which are considered to be a correlate of protective immunity. Because bison and cattle are genetically closely related, many of the bovine-specific mAbs are cross-reactive for bison lymphocytes and has aided in defining bison cell populations (Nelson et al., 2010). However, compared to the mouse model, there are fewer tools to study the immune responses in cattle and even fewer or none for bison and elk. Hence, tools are needed to evaluate all aspects of the in- nate and adaptive immune responses to understand why elk fail to produce robust cellular immune re- sponses compared to the Brucella vaccines and other bacterial vaccines that stimulate appropriate im- mune responses in cattle and bison. This may require the development of species-specific brucellosis vaccines. Developing reagents (e.g., mAbs) is a slow and expensive process, but other techniques (e.g., the use of cytokine mRNA) can provide insights into the host immune response in the absence of mAbs. Also, high-throughput sequencing can evaluate elk or bison mRNA transcripts in response to vaccine candidates or during an infection, which is further discussed later in this chapter. All of these tools require research and development. 4.2 Immunotherapy Post-Exposure The ability to treat cattle post-exposure to brucellosis would be a useful management tool. Use of single antibiotics is not successful, but a combination treatment can eliminate shedding from the milk in up to 71% of animals (Milward et al., 1984, Nicoletti et al., 1985, 1989). However, even when combined with adult vaccination with B. abortus S19, the infection was controlled but not eliminated (Jimenez de Bagues et al., 1991). While combinations of aureomycin plus streptomycin (Kuppuswamy, 1954) or tetra- cyclines plus streptomycin have produced satisfactory results (Giauffret and Sanches, 1974, Marin et al., 1989), it does not result in loss of the lesions characteristic of this disease (Marin et al., 1989). Thus, other approaches will need to be considered and developed for post-infection treatments. Immunotherapy or therapeutic vaccines have been associated with the cancer-causing human papil- loma virus, hepatitis B infections, and the human immunodeficiency virus (HIV) (Jacobson et al., 2016; Lin et al., 2016; Skeate et al., 2016). Therapeutic vaccines may stimulate immune responses to compo- nents of the infectious organism that are not normally recognized by the immune system (known as anti- gens) because their concentration is typically too low or sequestered in particular tissues. Alternatively, they may redirect a protective immune response. This can be done by providing antigens in the context of enhancing agents (known as adjuvants); this may include cytokines such as IL-12 to orchestrate IFN-γ- producing T cell responses (Jacobson et al., 2016, Pennock et al., 2016). Finally, these vaccines could be designed to re-engage immune cells that have undergone immunological exhaustion or been prohibited from expressing their protective potential by regulatory cells and molecules. The latter has been applied in cancer immunotherapy, for example, by blocking the T cell inhibitory molecule CTLA-4 (Callahan et al., 2016). While appealing, it is unlikely that immunotherapeutic vaccines will be a near-term accomplish- ment, but could be considered as part of a research program. 156 Prepublication Copy—Subject to Further Editorial Revision

Remaining Gaps for Understanding and Controlling Brucellosis 5. VACCINES AND DELIVERY MECHANISMS New vaccines and methods of delivery are needed to improve the efficacy of current and future bru- cellosis vaccines. B. abortus vaccines need to be engineered to not cause disease or abortion in wildlife, while still retaining sufficient persistence to stimulate long-term protection. While live Brucella vaccines have been shown to be necessary to induce protective immune responses in cattle, there is evidence that this can be mimicked by taking genes that code for components of Brucella and placing them into another microbe, which acts as a vector for the Brucella genes and mimics a live vaccine, but which may be safer than live-attenuated whole Brucella organisms (Dorneles et al., 2014). Live vaccines can be rendered less virulent by removing genes as well (Chen and Elberg, 1969). Other types of vaccines, that also have a high safety quotient, include DNA and subunit vaccines that deliver a portion of the Brucella genome or physical components of Brucella rather than the whole organism. Delivering vaccines in bait to wildlife is appealing, and conventional live vaccines B. abortus strains RB51 and S19 are effective when given oral- ly and are safe in wildlife. It is possible that alternative vaccines could also be delivered this way. Finally, designing new vaccines with particular components either deleted or new components added would help distinguish vaccinated animals from actual infected animals (known as DIVAs [Distinguish Infected from Vaccinated Animals]). 5.1 Alternative Delivery Methods for Vaccines To date, the methods used to vaccinate wildlife are similar to those adopted for cattle (i.e., subcuta- neous [SC] or intramuscular [IM]). Given that the initial exposure to Brucella almost always occurs mu- cosally because animals sniff or lick Brucella-infected aborted fetuses and/or infected placental tissues, delivery of the vaccine via a mucosal surface could result in an immune response that would induce greater protection (Thorne and Morton, 1978; Samartino and Enright, 1993; Belyakov and Ahlers, 2009; Schumaker, 2013). For example, it has been shown that mucosal vaccination can produce a bias for IFN- γ-producing CD8+ T cells (Clapp et al., 2011, 2016). Oral vaccination studies in cattle and pigs have shown the benefits of such an approach (Nicoletti and Milward, 1983; Nicoletti, 1984; Elzer et al., 1998; Edmonds et al., 2001). Oral S19 vaccination proved to be equally as effective as SC vaccination in pro- tecting pregnant heifers from Brucella-induced abortion (Nicoletti and Milward, 1983; Nicoletti, 1984). Administering RB51 vaccine orally protected against abortion and brucellae colonization infection, and provided equivalent results as SC RB51 vaccination (Elzer et al., 1998). Also, wildlife showed no mor- bidity or mortality as a consequence of oral RB51 vaccination (Januszewski et al., 2001). Oral vaccines also have ease of administration, since they are needle-free and do not require trained personnel. Oral vaccines are subject to enzymatic and proteolytic degradation in the gastrointestinal tract, which can compromise the vaccine’s immunogenicity, but drug formulations are available to counter this effect (Sedgmen et al., 2004). Additional engineering design and testing will be needed to orally vaccinate elk, such as developing baits targeted for elk. A mucosal approach has merit both for live, attenuated B. abor- tus vaccines and for vectored or subunit vaccines. To reduce the need to handle animals more than once, an emerging and effective alternative to prime-boost vaccination is the use of microparticles or nanoparticles for sustained vaccine release (Lin et al., 2015). Biodegradable polymers are designed with specific release rates to mimic booster immuniza- tions and induce anamnestic immune responses. This approach was tested using brucellosis-free red deer (Cervus elaphus elaphus) which are closely related to elk. Seven months after vaccination, animals were challenged with B. abortus S19 by the conjunctival route, and 2 weeks later the red deer that were orally vaccinated with RB51 microencapsulated in alginate plus Fasciola hepatica vitelline protein B (VpB) were the only ones that showed significant reduction in splenic colonization (Arenas-Gamboa et al., 2009a). This also demonstrates that microencapsulated S19 is protective against wild-type virulent B. abortus challenge in female red deer (Arenas-Gamboa et al., 2009b). The development of vectors for safely transmitting vaccines to target wildlife populations, especially through feed, has been shown to be possible for vaccines against rabies and could also be considered for brucellosis (WHO, 2017). Prepublication Copy—Subject to Further Editorial Revision 157

Revisiting Brucellosis in the Greater Yellowstone Area 5.2 Alternative Vaccine Approaches Cross-protective vaccines. Several different approaches could be employed in designing new vac- cines for elk and bison. Live vaccines could include a different Brucella species that is cross-protective to B. abortus. While Brucella species vary in their LPS, they retain many of the immunogenic proteins that could cross-protect against B. abortus infections. Reduced incidence of abortion attributed to B. abortus and B. melitensis was observed in Kuwait following a S19 brucellosis vaccination program of cattle (al- Khalaf et al., 1992). Reduced incidence of B. melitensis infections in humans was also observed following vaccination with B. abortus S19 (Vershilova, 1961). When B. neotomae (the strain originally found in desert wood rats) was irradiated and used to vaccinate mice, it also conferred 100 to 1000-fold level of protection against B. abortus, B. melitensis, and B. suis (Moustafa et al., 2011). This study showed that irradiated organisms (which are living but cannot replicate) may be an alternative to strictly live vaccines. Subunit and vectored vaccines. To generate a novel vaccine for cattle, influenza virus was used as a vector to express two immunodominant Brucella proteins: L7/L12 and omp16 (Tabynov et al., 2014). The immune responses to these proteins have been shown to reduce brucellae colonization in mice. Heifers vaccinated subcutaneously and conjunctivally showed 90% and 80% protection against abortion, respec- tively, compared to only 30% in the control heifers. To understand the duration of protective immunity to Brucella as measured by tissue brucellae colonization infection and abortion, a subsequent study used a reduced dose which showed a similar level of protection against abortion (Tabynov et al., 2016). The re- sults from these studies for vectored vaccines suggest a subunit vaccine approach may be feasible for pro- tecting large animals against Brucella-induced abortions. Identifying suitable vaccine candidates has been extensively pursued and tested in mice (Yang et al., 2013). It remains to be determined whether these vaccine candidates can successfully protect livestock and wildlife. DNA vaccines deliver a portion of the pathogen’s genome to the host. DNA vaccination has been successfully adapted and licensed for horses against West Nile virus (Davis et al., 2001). These vaccines have the advantage of being inexpensive to produce, the capacity to be readily ramped up to generate large quantities of vaccine, and are currently being tested in humans (Jin et al., 2015). Bison have been tested using DNA vaccines for brucellosis (Clapp et al., 2011). The bison white blood cells responded in a time-dependent fashion, showing that bison are responsive to DNA vaccines. Bovine calves were tested with DNA and a Semliki Forest RNA virus-vectored vaccines encoding Brucella superoxide dismutase (SOD) (Sáez et al., 2008). The calves responded to both vaccines as evidenced by increased antibody ti- ters, increased T cell proliferative responses, and increased IFN-γ production. Hence, Brucella SOD de- livered by these mechanisms was immunogenic and serves as a proof-of-concept for DNA vaccines and ruminants. Another approach involves using Brucella’s outer membrane vesicles (OMVs), which tend to be highly immunogenic and often contain components that stimulate a protective immune response. Mice vaccinated with rough B. melitensis mutant had a 1000-fold reduction in bacteria following a virulent B. melitensis challenge (Avila-Calderón et al., 2012). While not tested in a natural host for Brucella, the suc- cess of OMVs used in meningococcal vaccine for humans suggests this approach has potential (Carter, 2013). To determine the efficacy of a subunit vaccine approach, additional studies are needed as many vac- cine delivery systems and formulations are available to enhance the immunogenicity of these or other vaccine candidates. These studies will also need to include evaluation of adjuvants as these may selective- ly enhance immunity in elk and bison. Enhancing immunogenicity. Modification of S19 or RB51 vaccines to improve immunogenicity by adding in genes that code for antigens that stimulate immune responses could be considered even though initial attempts to improve RB51’s immunogenicity were successful in mice, but failed in bison and elk (Olsen et al., 2009; Nol et al., 2016). Future studies may show that more potent protective epitopes need to be expressed to confer protection in wildlife. 158 Prepublication Copy—Subject to Further Editorial Revision

Remaining Gaps for Understanding and Controlling Brucellosis 5.3 DIVA Vaccines Inclusion of a DIVA component in vaccines allows differentiation between vaccinated and infected animals. If the vaccine retains its LPS, this may produce a false positive serological response; thus, a dif- ferent serological test would need to be developed to rapidly distinguish vaccinated from naturally infect- ed animals (McGiven et al., 2015). One approach to distinguish infected from vaccinated animals is to modify a vaccine for a loss of immunoreactivity, as has been done for RB51 via the absence of O-Ag. Another such vaccine was engineered by preventing expression of the immunogenic Brucella protein bp26 gene, thus generating the M1-luc strain. This strain was used to vaccinate bovine calves that were challenged with virulent B. abortus strain 2308 (Fiorentino et al., 2008). It conferred protection against abortion similar to heifers vaccinated with S19. Subunit vaccines such as those previously described also have the advantage of not converting animals serologically, thus, allowing current serological tests to be used to determine prior Brucella exposures and thus qualify as a DIVA. 5.4 Challenge Studies B. abortus is classified as a biosafety level 3 (BSL-3) agent, and thus research on B. abortus requires the use BSL-3/ABSL-3 or higher enhanced containment conditions (BSL-3-Ag for loose-housed ani- mals). B. abortus is also regulated as a Select Agent under the Bioterrorism Act of 2002, which imposes onerous registration requirements. These requirements limit the ability of researchers to conduct studies, as the Select Agent designation requires researchers to have facilities, protocols, reporting requirements, and security clearances to work with the pathogen that are beyond those required for a BSL-3 pathogen, and are beyond the capacity of many institutions to comply. Because of the restriction, the vaccine S19 has been used as a surrogate for transmission studies in bison to understand transmission by a variety of routes (Uhrig et al., 2013). But immune responses that are important for controlling challenge with vaccine strains may differ somewhat from that needed to control infections by virulent field strains. To efficiently conduct research on immune responses to existing or new vaccines or immunotherapeutics, it will be necessary to remove B. abortus from the Select Agent list. A recent proposal by USDA Animal and Plant Health Inspection Service (APHIS) to remove Brucella from the Select Agent list (USDA- APHIS, 2016) to reduce the restrictions on brucellosis research, although supported by the committee, was unfortunately not approved. Standardized infection and abortion challenge methods need to be followed when conducting the vaccine trials to minimize variation among studies. This would require some initial cooperative studies to determine the minimum infection dose 100% (ID100) for B. abortus for each individual to abort its first calf. A virulent B. abortus strain isolated from the GYA will also need to be characterized and tested in vaccine trials to determine whether B. abortus S2308 is the most representative strain to test. To ensure that virulence is maintained with the challenge strains, all groups will need to follow basic microbiology practices for culture and preservation. Addressing these aspects will greatly facilitate the development of more effective brucellosis vaccines to consistently protect >90% of affected wildlife and livestock against abortion and protect >85% against infection. Raising the standards of protection will effectively increase herd immunity to interrupt transmission of B. abortus amongst elk, bison, and cattle. Finally, in assessing vaccine trial outcomes, it will be necessary to address the issue of vaccines that show protection in con- trolled settings while they do not in field settings, and vice versa (Schurig, 2015). 6. GENOTYPING AND GENETICS Since 1998, the accuracy and speed of DNA and RNA sequencing, “-omics” analyses, and bioin- formatics has improved substantially. Variable Number of Tandem Repeats (VNTR) analysis and so- called next generation sequencing (high throughput sequencing) of whole genomes has allowed for a more detailed analysis of the molecular epidemiology of B. abortus isolates from domestic and wild ru- minants (Higgins et al., 2012; Kamath et al., 2016). Research and epidemiological data have substantially Prepublication Copy—Subject to Further Editorial Revision 159

Revisiting Brucellosis in the Greater Yellowstone Area changed the understanding of Brucella transmission from wild to domestic ruminants in the GYA, which strongly suggests that elk are responsible for current transmission events to domestic cattle and are a sig- nificant source of transmission to other elk and to bison (Rhyan et al., 2013; Kamath et al., 2016). 6.1 Pathogen Genotyping and Gene Expression Profiling The profiling of B. canis and Salmonella enterica within their infected hosts resulted in the discov- ery of rapid genetic mutations and adaptations (Gyuranecz et al., 2013; Mather et al., 2013). A profiling analysis of B. abortus genomes in elk and bison in the GYA could reveal genetic differences in subpopu- lations of B. abortus. Functional adaptations within the different Brucella lineages may explain differ- ences in spatial expansion. Sequencing of whole genomes is already routinely used in diagnostic testing and for molecular characterization of pathogen isolates in some laboratories. Diagnostic laboratories have used genetic differences to characterize viral and bacterial pathogens at the sub-species and isolate- specific levels. This could become a routine procedure for analyzing B. abortus isolates from the GYA. User-friendly and rapid formats for molecular characterization and whole genome sequencing will pro- vide the information necessary to control brucellosis by targeting transmission pathways of specific iso- lates. Thus, research on molecular-based diagnostics for B. abortus will be needed for addressing brucel- losis control in the GYA. The use of microarrays to cover whole genome analysis of Brucella gene expression and the use of next generation sequencing may enhance understanding of both host and pathogen gene expression during the infection process (Rossetti et al., 2010; 2011). Brucella and host gene expression and proteome da- tasets have been generated, progressing toward a comprehensive dual analysis of host and pathogen re- sponses (He et al., 2010; Rossetti et al., 2010; Viadas et al., 2010; Weeks et al., 2010; Kim et al., 2013; Rossetti et al., 2013). The use of powerful bioinformatic algorithms has allowed for the analysis of da- tasets to identify candidate genes and biomarkers of Brucella and hosts, identify and predict Brucella an- tigenic proteins, identify components of subunit vaccines, understand gene regulatory networks, charac- terize the Brucella stress responses, and better understand modulation of host immune responses. The use of systems biology is needed to more effectively exploit elk and bison data for the following: (1) model development, (2) causal discovery, such as understanding the genetic basis for innate susceptibility or resistance to brucellosis, (3) prediction of biological activities, such as immune mechanisms that result in protection from disease, (4) improvement in designing in vitro and in vivo experiments to understand the biology of brucellosis, and (5) identification of biomarkers for protective immunity and diagnosis. 6.2 Host Genetic Characterization and Gene Expression Profiling Significant advances have been made in characterizing host genetics, including sequencing of the bovine genome and substantial progress on sequencing the deer genome (Red Deer, Cervus elaphus, Rocky Mountain elk, Cervus canadensis) (Elsik et al., 2009; Brauning et al., 2015). Mitochondrial DNA sequencing and microsatellite analysis has enabled the bison populations within the GYA to be character- ized into two distinct subpopulations (Halbert and Derr, 2008; Halbert et al., 2012). Genome analysis in cattle and water buffalo shows distinct genome markers that code for susceptibility to infection, protective immune responses, and other characteristics of genetic resistance, and the results have direct application to bison genetics (Adams and Templeton, 1998; Capparelli et al., 2007; Adams and Schutta, 2010; Mar- tinez et al., 2010; Elsik et al., 2016). Obtaining similar data on elk genetics would allow analysis of how genotype affects vaccine efficacy. Given that infected elk are likely to abort their first calf after infection, this is likely to induce some selective pressure on elk to combat the disease more effectively and provide insight into the genetic basis for the increased resistance to B. abortus. Characterization of the elk genome is a priority as it may be useful as an adaptive management tool for elk in the future. Dual gene expres- sion profiling of host and pathogen is a powerful tool to understand brucellosis infection biology (Perez- Losada et al., 2015). To identify protective brucellosis vaccine candidates, a systems biology analysis of dual Brucella and bovine host gene expression data were combined with reverse vaccinology; similar 160 Prepublication Copy—Subject to Further Editorial Revision

Remaining Gaps for Understanding and Controlling Brucellosis technological approaches could be applied to elk and bison (He and Xiang, 2010; Adams et al., 2011a,b; Chiliveru et al., 2015). Bison culling in the GYA has been designed to meet population targets rather than to selectively cull brucellosis-infected individuals, which has given rise to concerns about bison genetics. With currently available technology to rapidly characterize bison genetic markers and pathogen genotype in a small field-based laboratory setting, it is possible to selectively remove based on disease status and specific bi- son genotypes. This would be a significant aid to making informed and targeted culling decisions in the bison management program and could help address the question of whether culling only infected bison will alter the “natural” population genetic structure of GYA bison. 7. DIAGNOSTICS The success of the brucellosis eradication program in the United States is a testament to the success of the diagnostic approaches used in livestock surveillance over the past 80 years. Brucellosis detection by serology and culture has proven to be useful for both population and individual animal regulatory test- ing, and has not changed appreciably since the 1998 report. These diagnostic methods will continue to be useful into the future for contact tracing if a positive cattle herd is identified in the GYA, DSAs, or imme- diately adjacent federal and private lands. There are logistical difficulties in capturing and retaining wild ruminant species for testing purposes that would also need to be addressed. Nevertheless, there are gaps in diagnostic testing for brucellosis in all three ruminant species that play a central role in the GYA. 7.1 Fitness for Purpose The first step in validating assays is “fitness for purpose.” Factors taken into consideration include timeliness of results and actions arising therefrom, population versus individual animal testing, sensitivity and specificity considerations for presumptive versus confirmatory testing, DIVA, determination of infec- tiousness, species-specific applications, and determination of protective immunity. It may be beneficial to have diagnostic assays rapidly confirm results when testing cattle, given the history of success using the current testing algorithm as described in the UM&R (USDA/APHIS, 2003). However, the ability to rapid- ly and accurately identify infected elk and to differentiate infected from vaccinated elk in the field has priority over further development of diagnostic assays for cattle, as diagnosing elk would have a signifi- cant impact on decision making when individual animals and groups of animals have been captured and/or incapacitated. 7.2 Testing Formats Diagnostic testing technology has changed significantly since the 1998 report. The availability of both laboratory-based and “penside” testing formats has increased considerably. Assay formats in com- mon use since the 1998 report include quantitative real time PCR, DIVA diagnostics, highly specific and sensitive competitive inhibition ELISA’s in kit format, chromatographic visualization such as lateral flow immunochromatography, and simplified DNA amplification techniques such as loop mediated isothermal amplification (LAMP). DNA sequencing has increased in speed and accuracy and has exponentially de- creased in cost since 1998, making it a routine part of pathogen characterization in modern diagnostic la- boratories. 7.3 Priorities for Diagnostic Testing Research Improved timeliness of results and enhanced accuracy, along with better differentiation of vaccinat- ed and infected animals using a single test, would enhance serological testing for all ruminant species that are the focus of brucellosis control in the GYA. As noted earlier, the success of the eradication program using currently available diagnostic assays for cattle suggests that improved diagnostics for cattle may not Prepublication Copy—Subject to Further Editorial Revision 161

Revisiting Brucellosis in the Greater Yellowstone Area be the highest priority for development. However, if vaccine development for cattle is a research priority, it would be beneficial to couple any new vaccine with a DIVA diagnostic. The same priority would need to be given to coupled vaccine and DIVA diagnostic development for bison and elk. A diagnostic test for wild ruminant diagnostics would need to be sensitive enough to avoid missing true positives, but the specificity would need to be high enough that culling of true negatives would be minimized. The required specificity for bison will likely be greater than for wild elk since genetic consid- erations are not as important for elk. For both of these species, it would be ideal to have field assays capa- ble of achieving the desired level of sensitivity and specificity. Identifying bacterial DNA as a way to identify a B. abortus infection is challenging, particularly in individual animals. While the numbers of bacteria are extremely high in diseased tissues (e.g., aborted fetuses) and maternal birth tissues (e.g., pla- centa), cyclic temporal variation in the level of bacteremia is common. There will be times when bacteria are either not in the bloodstream or are at a level below the sensitivity of current PCR techniques, particu- larly in older individuals. Thus, false negatives are highly likely (Tiwari et al., 2014). Nevertheless, new testing formats for PCR (such as the use of immunomagnetic beads) to concentrate bacteria prior to DNA isolation and PCR amplification can have enhanced analytical sensitivity. This technique has been suc- cessfully adapted to identify Mycobacterium paratuberculosis, a bacterium that can be challenging to identify by PCR from milk and feces (Khare et al., 2004). Similarly, attempting to develop a DNA-based test to determine the infectiousness of an individual will be challenging due to the cyclic nature of the disease. A negative antigen or nucleic acid detection test result at one point in time cannot be interpreted as evidence that an individual will remain noninfec- tious, at least in cattle and likely in bison. Less is known about infection in elk and further studies are needed to examine the possibility that elk could have a stable carrier state in which the bacteria are at a quantity and/or located in tissue that affects transmission. Nevertheless, this is an example where caution is warranted in ensuring that the purpose of testing (for example, determination of infectiousness) is achievable given the profile of the disease pathogenesis and the capabilities of the assay being proposed for use. Elk diagnostic testing will become increasingly important in the future if B. abortus infected elk continue to spread beyond current DSAs and prevalence in elk continues to increase. While assays for testing cattle for Brucella infection have a long history of success in effectively identifying positive cases, none of the current diagnostic assays have optimal characteristics for rapid, sensitive, and specific deter- mination of disease status in elk. This is due to the challenges in handling elk, obtaining specimens, and holding animals until testing is completed. Prioritization will need to be given in developing a suitable assay for serological or antigen/DNA targeted identification of infected elk, optimally in a format capable of being performed “penside” to provide reliable results in the field. 7.4 Challenges in Validating Assays for Wildlife Obtaining positive and negative samples to validate new diagnostic tests for wildlife is difficult. De- termining diagnostic sensitivity and specificity requires a large number of samples from animals with known disease status, preferably along with metadata about age, demography, stage of disease, time of year, and other similar characteristics that can impact test results. Bayesian approaches for assay valida- tion have been used in the past by taking advantage of prior probabilities based on previous test valida- tions, population characteristics, and other variables (Branscum et al., 2005). This approach has value when samples with a known disease status are limited. However, it would be ideal to have a biorepository of samples with associated metadata that could be accessed when a new diagnostic assay for elk or bison shows promise. The preservation of samples (e.g., serum, tissues, B. abortus isolates, DNA, RNA) for future host and pathogen genetic characterization in a biorepository with relevant metadata would be of significant value for research. If such a biorepository were created, it would be important to form a multi- user oversight group to manage acquisition, cataloging, and use of these valuable samples for research and diagnostic test development. 162 Prepublication Copy—Subject to Further Editorial Revision

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Revisiting Brucellosis in the Greater Yellowstone Area Get This Book
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Brucellosis is a nationally and internationally regulated disease of livestock with significant consequences for animal health, public health, and international trade. In cattle, the primary cause of brucellosis is Brucella abortus, a zoonotic bacterial pathogen that also affects wildlife, including bison and elk. As a result of the Brucellosis Eradication Program that began in 1934, most of the country is now free of bovine brucellosis. The Greater Yellowstone Area (GYA), where brucellosis is endemic in bison and elk, is the last known B. abortus reservoir in the United States. The GYA is home to more than 5,500 bison that are the genetic descendants of the original free-ranging bison herds that survived in the early 1900s, and home to more than 125,000 elk whose habitats are managed through interagency efforts, including the National Elk Refuge and 22 supplemental winter feedgrounds maintained in Wyoming.

In 1998 the National Research Council (NRC) issued a report, Brucellosis in the Greater Yellowstone Area, that reviewed the scientific knowledge regarding B. abortus transmission among wildlife—particularly bison and elk—and cattle in the GYA. Since the release of the 1998 report, brucellosis has re-emerged in domestic cattle and bison herds in that area. Given the scientific and technological advances in two decades since that first report, Revisiting Brucellosis in the Greater Yellowstone Area explores the factors associated with the increased transmission of brucellosis from wildlife to livestock, the recent apparent expansion of brucellosis in non-feedground elk, and the desire to have science inform the course of any future actions in addressing brucellosis in the GYA.

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