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Revisiting Brucellosis in the Greater Yellowstone Area (2017)

Chapter: 4 Scientific Progress and New Research Tools

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Suggested Citation:"4 Scientific Progress and New Research Tools." 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:"4 Scientific Progress and New Research Tools." 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:"4 Scientific Progress and New Research Tools." 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:"4 Scientific Progress and New Research Tools." 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:"4 Scientific Progress and New Research Tools." 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:"4 Scientific Progress and New Research Tools." 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:"4 Scientific Progress and New Research Tools." 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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Page 71
Suggested Citation:"4 Scientific Progress and New Research Tools." 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:"4 Scientific Progress and New Research Tools." 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:"4 Scientific Progress and New Research Tools." 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:"4 Scientific Progress and New Research Tools." 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:"4 Scientific Progress and New Research Tools." 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:"4 Scientific Progress and New Research Tools." 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:"4 Scientific Progress and New Research Tools." 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:"4 Scientific Progress and New Research Tools." 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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Page 79
Suggested Citation:"4 Scientific Progress and New Research Tools." 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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Page 80
Suggested Citation:"4 Scientific Progress and New Research Tools." 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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Page 81
Suggested Citation:"4 Scientific Progress and New Research Tools." 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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4 Scientific Progress and New Research Tools The scientific knowledge base of Brucella abortus and B. abortus-induced disease pathogenesis has expanded since the previous 1998 National Research Council (NRC) report was issued. This chapter pro- vides a summary of progress since 1998 in understanding B. abortus infection biology; diagnosis of bru- cellosis in cattle, bison, and elk; and Brucella vaccinology. Coupled with systems biology, there is now the ability to more fully understand the infectious process of B. abortus and enable more rapid discovery of brucellosis vaccines and diagnostics for elk and bison. 1. INFECTION BIOLOGY AND PATHOGENESIS OF B. ABORTUS IN CATTLE, BISON, AND ELK 1.1 Background When Brucella abortus come in contact with mucous membranes in the alimentary or respiratory tracts of the host, they invade by attaching to host epithelial cells and quickly transmigrate across the mu- cosa, where they are engulfed by phagocytic cells. The Brucella-containing phagocytic cells disseminate to regional draining lymph nodes and the blood by the lymphatic system, establishing an intermittent bac- teremia. They can then colonize the placenta, fetus, mammary glands, testes, and regional draining lymph nodes of those tissues or organs; colonization of the placenta and fetus frequently results in abortion. Brucellae primarily replicate within macrophages, neutrophils, dendritic cells, and placental tropho- blasts using different survival strategies; however, the pathogen has the ability to replicate in a wide va- riety of additional mammalian cell types including epithelial cells and endothelial cells. The intracellular growth and survival of Brucella in specialized compartments limits exposure to the host immune respons- es, sequesters the organism from the effects of some antibiotics, and is responsible for the unique features of pathology in infected hosts (Anderson and Cheville, 1986; Myeni et al., 2013; Celli and Tsolis, 2015; de Figueiredo et al., 2015). The pathology in pregnant ruminants is typically divided into three phases: the incubation phase which is before clinical signs are evident, the acute phase during which the pathogen is disseminated among host tissues, and the chronic phase during which massive replication B. abortus oc- curs in the placental trophoblasts resulting in severe necrotizing placentitis and fetal death (Anderson and Cheville, 1986). Chronic infection results from the ability of the organism to persist in the cells of the host, which is variable for cattle, elk, and bison (Qureshi et al., 1996; Olsen, 2010). 1.2 Infection Biology and Molecular Pathogenesis While research on the infection biology and molecular pathogenesis of brucellosis has made tre- mendous progress since the 1998 NRC report, several aspects of the host-Brucella relationship remain to be elucidated (Atluri et al., 2011). For example, it is now known how Brucella enter host cells (Rossetti et al., 2012) and exploit this ability to cross mucosal surfaces (Rossetti et al., 2012, 2013). However, the receptors involved in binding host cells are only partially understood (Castaneda-Roldán et al., 2004; Seabury et al., 2005). The intracellular invasion and survival of Brucella depends largely on its Type IV secretion system although exact targets of its effectors are still elusive (O’Callaghan et al., 1999; Comerci Prepublication Copy—Subject to Further Editorial Revision 65

Revisiting Brucellosis in the Greater Yellowstone Area et al., 2001; de Jong and Tsolis, 2012; Chandran, 2013; Lacerda et al., 2013; Myeni et al., 2013; Ke et al., 2015). After entering the host, Brucella foils host protective responses by evading the so-called innate im- mune responses (Barquero-Calvo et al., 2007; Carvalho Neta et al., 2008; Gorvel, 2008; de Jong et al., 2010; Gomes et al., 2012; Rossetti et al., 2012; von Bargen et al., 2012). Brucella dampens inflammatory responses, relative to what occurs with other pathogens that infect through the gut mucosa (Oliveira et al., 2008; Rossetti et al., 2013). Brucella also restricts proinflammatory immune responses, including matura- tion of cells known as dendritic cells which are crucial for induction of protective adaptive immune re- sponses (Radhakrishnan et al., 2009; Sengupta et al., 2010; Chaudhary et al., 2012; Kaplan-Turkoz et al., 2013; Smith et al., 2013). The curtailed host immune responses together with Brucella’s ability to live inside host cells and adapt to low oxygen tension make it a successful pathogen (Kohler et al., 2002; Kohler et al., 2003; Billard et al., 2007; Al Dahouk et al., 2008; Lamontagne et al., 2009; Barbier et al., 2011; Hanna et al., 2013). Because Brucella can persist inside host cells indefinitely, this contributes to its spread within the host including to placental trophoblasts, fetal lung, male genitalia, skeletal tissues, reticuloendothelial system, and endothelium (Kim et al., 2013; Roop and Caswell, 2013; Xavier et al., 2013). Because, minimal information is available to describe the interaction of Brucella with target cells and tissue, a holistic systems biology analysis of the pathogenesis of brucellosis at the level of the whole host is needed for bison, elk, and cattle (Carvalho Neta et al., 2008; Delpino et al., 2009; Rossetti et al., 2013; Sankarasubramanian et al., 2016). Identification of the most critical components of pathogenesis will enhance the ability to rationally design vaccines, diagnostics, and therapeutics for elk and bison. For- tunately, many of the currently available molecular approaches and methods can be directly applied to in vitro and in vivo research on both the pathogen and the host for a comparative molecular pathogenesis approach. 1.3 Clinical Disease Under natural conditions, a B. abortus infection is usually acquired by contact with the placenta, fe- tus, fetal fluids, or vaginal discharges from infected cows in all three host species of interest (cattle, bison, elk). Studies both before and since 1998 have shown that following infection, clinical manifestations of B. abortus infection in bison are largely similar to those of cattle (Nicoletti, 1980; Davis et al., 1990, 1991; Rhyan et al., 1994; Roffe, 1999a,b; Olsen and Holland, 2003; Olsen et al., 2009; Rhyan et al., 2009; Xavier et al., 2009; Van Campen and Rhyan, 2010; Xavier et al., 2010). Since 1998, it has been con- firmed that elk are also similar in that systemic clinical signs do not usually occur in the acute stages of infections (Thorne and Morton, 1978; Kreeger et al., 2000; Cook et al., 2002; Kreeger et al., 2002; Van Campen and Rhyan, 2010). In the later stages of infection, the primary clinical disease manifestations are fetal or newborn death, weak calves, metritis with retained placentas—although it is now known that the latter does not occur in elk (Rhyan et al., 2009). Abortions usually occur during the second half of gesta- tion, accompanied by mild mastitis and reduced milk production. After the first abortion, subsequent pregnancies are generally normal, with cows occasionally giving birth to weak calves. But because B. abortus infection may persist, organisms can still shed in milk and uterine discharges (Meador et al., 1989). In chronic stages of brucellosis, infertility may occur in both sexes due to metritis in cows or orchi- tis, epididymitis, seminal vesiculitis, and testicular abscesses in bulls with arthritis and hygromas devel- oping after long-term B. abortus infections. 1.4 Pathology and Pathogenesis The pathogenesis of brucellosis has been most extensively studied by in vitro and in vivo experi- ments in non-target hosts, especially the murine model (Cheers, 1984; Tobias et al., 1993; Grillo et al., 2000; Silva et al., 2011). While studies in models have revealed extensive valuable information on the molecular pathogenesis of brucellosis, these models do not reflect the important differences in the infec- 66 Prepublication Copy—Subject to Further Editorial Revision

Scientific Progress and New Research Tools tion biology of brucellosis in elk, bison, and cattle (Olsen and Palmer, 2014). Studies of the molecular pathogenesis in elk, bison, and cattle have been largely limited because of onerous Select Agent require- ments, lack of large animal biocontainment facilities, and costs for large animal experiments. As a conse- quence, few critical studies have been conducted. The principal lesions of all three species occur in adult female and male reproductive tracts—the placenta and testes, respectively—and the fetal respiratory tract (Rhyan, 2013). The gross pathology and histopathology of infected bison, elk, and cattle have been described in varying levels of detail (Thorne and Morton, 1978; Rhyan et al., 1997; Rhyan et al., 2001; Xavier et al., 2009; Xavier et al., 2010; Olsen and Palmer, 2014), but the pathological lesions are more similar than they are different among the three host species (Payne, 1959; Thorne and Morton, 1978; Davis et al., 1990; Samartino and Enright, 1993; Williams et al., 1993; Rhyan et al., 1994, 1997, 2001, 2009; Palmer et al., 1996; Rhyan et al., 1997; Adams, 2002; Xavier et al., 2009; Carvalho Neta et al., 2010; Xavier et al., 2010; Poester et al., 2013; Rhyan, 2013). Nonetheless, there are some significant differences in animal behavior, disease expression, and susceptibility to brucellosis. For example, elk normally calve in solitary confined conditions in con- trast to cattle and bison where parturition is a herd event attracting other members to sniff and lick the calf or aborted fetus (Van Campen and Rhyan, 2010), potentially affecting the exposure dose of B. abortus and thus transmission and frequency of disease. Studies on elk have shown they rarely have mastitis or retained placentas compared to cattle and bison, which means they may be less negatively affected by the disease (Rhyan, 2013). Bison may be considerably more susceptible to brucellosis than cattle, as abortions occurred in <2% of pregnant cattle vaccinated with the B. abortus strain 19 (S19) vaccine compared to 58% of pregnant vaccinated bison (Davis et al., 1991). Higher infection and abortion rates also occurred in experimentally challenged, non-vaccinated bison compared to cattle (Olsen, 2010; Olsen and Johnson, 2011). Additional- ly, clearance time of the B. abortus RB51 vaccine is twice as long in bison as compared to cattle, yet less bacterial colonization of the udder and less mastitis is seen in bison than cattle (Cheville et al., 1992; Roffe et al., 1999a,b; Rhyan et al., 2001). 2. DIAGNOSTICS 2.1 Background Diagnostic assays are vital for the identification of brucellosis in humans and animals for clinical, regulatory, and research purposes (Bricker, 2002a). Bacterial and antibody detection have had key roles in the brucellosis eradication program since its inception in 1934, and there have been numerous advance- ments in the area of diagnostics in the past few decades. Multiple diagnostic approaches have been used in both domestic and wild animals. Livestock sur- veillance initially tested all cattle within a vicinity, but has evolved to focus on animals at surveillance nodes where cattle are accessible, such as slaughter and first point testing (e.g., markets, shows, and sales) while maintaining wide area testing in communities with known infections. Whole herd follow-up testing has also been a mainstay in surveillance, trace-back, and eradication programs. All have used serological assays in a tiered approach with initial high-sensitivity assays followed by confirmatory testing using as- says with greater specificity. Isolation of the bacteria through culture and subsequent identification of B. abortus has been im- portant for confirmation (the so-called “gold standard”) in serologically positive animals. However, the success of culture is dependent on a number of variables. For example, in chronically infected animals, B. abortus may only be present in certain lymph nodes and in fewer numbers than in acutely affected ani- mals. Thus, while a culture positive animal is confirmed as infected with brucellosis, false negative cul- ture results can be obtained if inappropriate tissues are collected or if tissues are not properly collected and handled during collection or laboratory processing. Additionally, in an infected population, sera from animals in early stages of infection or with latent infection may not exhibit positive test reactions (O’Grady et al., 2014). Prepublication Copy—Subject to Further Editorial Revision 67

Revisiting Brucellosis in the Greater Yellowstone Area 2.2 DNA-Based Identification of B. abortus Compared to clinical settings, researchers have been able to use a broader repertoire of diagnostic assays in brucellosis research. In particular, multiple DNA amplification and detection methods provide sensitive and rapid identification and quantification of B. abortus in tissues and fluids originating from live and dead animals. Detection of B. abortus by polymerase chain reaction (PCR) is useful for research purposes, where a positive PCR result after experimental infection is definitive evidence of the presence of B. abortus. The specificity of singleplex and quantitative PCR (qPCR) assays used in research are ex- cellent at the genus level, although much lower at the species and subspecies levels without modification of amplification primers and conditions (Bricker, 2002b; Tiwari et al., 2014). However, neither singleplex nor qPCR are a part of routine diagnostic and regulatory program testing. Other means of specifically identifying various Brucella species, strains, and biovars by DNA-based methods have been developed, including multiplex assays targeting multiple genes and genomic regions, restriction fragment length polymorphisms, and the use of tandem repeat sequences. It can be challenging to determine both analytic and diagnostic sensitivity and specificity for multiplex assays, which impacts the decision-making process for accepting these tests for regulatory purposes. Nevertheless, multiplex PCR assays—such as the “AMOS” test, the “Bruce Ladder,” and more recent modifications of these tests—can differentiate multiple Brucella species and biovars, and can differentiate both S19 and RB51 strains from wild type B. abortus (Bricker et al., 2003; López-Goñi et al., 2008; Kang et al., 2011). 2.3 Serology Obtaining tissues for culture is often infeasible especially in wildlife populations, and culturing B. abortus is not always successful from infected animals. As a result, antibody detection is used as a proxy for infection (Gilbert et al., 2013). Serological tests reveal past exposure but not necessarily whether an individual is actively infectious, and the interpretation of seropositivity relative to the likelihood of an animal being infected needs to be evaluated relative to the knowledge of the population being tested (Nielsen and Duncan, 1990). In populations where prevalence is high, results are less likely to be false positives and more likely to be accurate indicators of disease (Gilbert et al., 2013). Although some ani- mals may become transiently seropositive yet not infected after exposure, those animals usually do not retain a positive titer for the long term. A positive serological result is an accurate indicator of infection in bison (Clarke et al., 2014). Bru- cella abortus biovar 1 was cultured from all but 3 of 36 seropositive bison (91%), and of the 88 seronega- tive bison, none had positive results of culture from any tissues (Clarke et al., 2014). Furthermore, infect- ed seropositive bison cows likely remain seropositive and infected for a prolonged time, with positive antibody titers to B. abortus remaining remarkably stable over time (Rhyan et al., 2009). The ability of an infected animal to transmit brucellosis varies, depending largely on the reproduc- tive status of that animal. Therefore, predicting “infectiousness” of a particular animal in a known infect- ed population can be difficult. For disease management purposes, all seropositive animals in a known in- fected population would be considered likely to be infected with the potential to be infectious to other animals at various times. The types of serological tests and algorithms for identifying B. abortus infected cattle and bison for regulatory purposes is outlined in the USDA-APHIS Uniform Methods and Rules (UM&R) for brucello- sis eradication, and the Standard Operating Procedures for Submission and Testing of Brucellosis Sero- logical Specimens (USDA-APHIS, 2003, 2014). The currently accepted testing procedures for serology that are most commonly used in approved brucellosis testing laboratories include the Buffered Acidified Plate Antigen (BAPA), Rapid Automated Presumptive (RAP) Examination, Fluorescence Polarization Assay (FPA), and Complement Fixation (CF) test. Under certain conditions, the UM&R also allows for the use of other tests, such as Card Agglutination, Particle Concentration Fluorescence Immunoassay (PCFIA), and IDEXX HerdCheck Milk Antibody ELISA, as well as the various brucellosis milk surveil- lance tests for herd testing. 68 Prepublication Copy—Subject to Further Editorial Revision

Scientific Progress and New Research Tools While the FPA has been much more broadly adopted over the past decade in laboratories approved for brucellosis testing, there have been no new developments for routinely used regulatory tests since the 1998 report. This largely reflects the confidence in the level of validation resulting from decades of use that is the basis for regulatory decision making. As specified in the UM&R, these assays are approved for use in both cattle and bison testing. Cross-reactions are possible in serological assays due to antibodies directed against other bacteria (e.g., E. coli, Salmonella, Francisella and Yersinia spp.). These cross-reactions appear as false-positives and affect the specificity of the diagnostic test. In general, serological tests in cattle have high specificities (>96%), suggesting that false positives are relatively rare in cattle (Nielsen, 2002). Similarly, cross- reactivity does not appear to be a problem in bison (See et al., 2012), and the BAPA, Card, SPT, RIV, and CF tests are of high specificity in elk (Clarke et al., 2015). When the seroprevalence began to increase in some elk herds in Montana, it was suspected that the cause was cross-reactions with Y. enterocolitica O-antigen side chain epitopes (Shumaker et al., 2010). As a result, there was an increased use of the west- ern blot test to rule out potential cross-reactions (Gevock, 2006; Anderson et al., 2009). However, Montana Department of Fish, Wildlife, and Parks noted that three of seven culture positive elk samples for which blood samples are also available were incorrectly identified as Yersinia cross-reactions by the western blot test (Anderson et al., 2009). In recent unpublished work provided to the committee, researchers with the WGFD have demonstrated under experimental conditions that routine tests used for B. abortus diagnosis in cattle and bison (such as RAP, FPA, and others) show cross-reactions using serum from Brucella-negative, Yersinia enterocolitica infected elk. However, elk titers against Y. enterocolitica do not persist beyond an average of 4 months after infection (personal communication, W.H. Edwards, 2015). The lack of persistent titers, together with the relatively few false-positives observed in areas with- out brucellosis, suggest that cross-reactivity observed with Yersinia infected elk may be minimal (Clarke et al., 2015). Lastly, immunoblot testing is in general more difficult to perform and interpret consistently in the diagnostic laboratory, which makes quality management a challenge. For these reasons, the western blot is not the best routine assay for detecting Brucella infected elk. 2.4 Elk Testing and Interpretation The BAPA test is the best alternative among existing and commonly used Brucella diagnostic as- says to screen elk serum for Brucella antibodies, as indicated by data on sensitivity and specificity of var- ious routinely used Brucella antibody detection assays (CT, rivanol, standard plate, CF, and BAPA) (Clarke et al., 2015). Consistent with these data, the United States Animal Health Association passed a resolution in 2011 recommending the use of the BAPA for presumptive testing of elk. A competitive ELISA assay has also been validated for use in elk (Van Houten et al., 2003). The cELISA can differenti- ate S19 vaccinated from unvaccinated but infected elk, and has a reasonable degree of overall accuracy if the purpose of testing is determining seroprevalence in a vaccinated elk herd. However, the cELISA failed to identify approximately 10% of elk from which it was possible to culture B. abortus. All culture positive elk were also positive on conventional Brucella serology assays. Thus, the specificity obtained by using a cELISA, while helpful in differentiating vaccinated from infected animals, was offset by somewhat reduced sensitivity. This is a disadvantage for presumptive testing of individual animals when a high degree of sensitivity is essential. There is no perfect serological test for brucellosis and no single test alone is reliable, thus the use of multiple tests increases the confidence in diagnosis (Nielsen and Duncan, 1990). Brucella abortus S19 vaccine has been known to cause positive test results in many animals, espe- cially those recently vaccinated. The Wyoming Game and Fish Department began vaccinating elk with S19 in 1985 on the Grey’s River supplemental feedground and gradually expanded the program across all of the other feedgrounds except one (Dell Creek). In the supplemental feedgrounds, however, very few elk are identified as vaccine strain positive at 1.5 years or older despite the vaccination of more than 90% of juveniles. In addition, if S19 was creating false positives, one would expect a large fraction of 1.5-year old individuals to be seropositive on conventional serological assays. Instead it appears as though S19 Prepublication Copy—Subject to Further Editorial Revision 69

Revisiting Brucellosis in the Greater Yellowstone Area g i r e induced seroprevalenc gradually increases with the age of vaccination as would be expected for field s ce i h r exposures Therefore, S19 does not appear to ind s. S duce long last ting serologic titers on th elk feedgro cal he ounds (Maichak et al., 2017). A recent public r cation descriibes the use of synthe e etic oligosacc charides rep presenting th O- he polysacchharide side ch hain of Bruce and relat species in an indirect ELISA assay (McGiven et al., ella ted n y 2015). Ini itial validatio data provid proof of principle that synthetic oli on de p t igosaccharide representin the es ng capping M-epitope of the side chain can provide excellent sp ecificity in di M t n e iscriminating antibodies ag g gainst various Br rucella specie as well as Y. enterocolit O:9. The use of synthe oligos als provides a ready es Y tica etic so source of antigen with hout the need for culture of B. abortus. While additi ional validatio data are n on needed to examin analytical sensitivity, di ne s iagnostic sens sitivity, and d diagnostic spe ecificity, the data suggest that a better sero ological assay for multiple species may be available in the near fu y e uture. In 20014, Idaho, Montana, and Wyoming ag M greed to a uniiform testing and interpreta ation algorith for hm serologica testing of elk. Both Rap Automated Presumptiv (RAP) Exa al e pid d ve amination and Fluorescenc Po- d ce larization Assay (FPA plate tests are run in pa A) arallel on eac sample. T interpreta ch The ation algorith (as hm shown in Figure 4-1) uses a tiered approach sim u a milar to testing of cattle for regulatory p g r purposes. How wever, the curren elk testing and interpret nt tation schema is rather complex and highlights th challenges with atic d he s serologica testing of elk for Brucell infection. al la FIGURE 4-1 Greater Yellowstone Are tri-state sch ea hematic for serrological testin of elk. Test abbreviations are as ng follows: RAP – Rapid Automated Pres R A sumptive; FPA – Fluorescenc Polarization Assay; CFT – Complement Fixa- A ce n t tion Test. NOTE: If the initial testi results are not interpretab (i.e., a “no test”), the ma ing ble anual card aggllutination test is run. *If initial RAP and FPA (plate) testin is positive and the FPA tu test is neg A ng a tube gative, submis ssion to the Na ational Veterinary Services Labo y oratory (NVSL for CFT is required if the animal is out L) e tside a known brucellosis en ndemic area. 70 Pre epublication Copy—Subje to Further Editorial Rev ect r vision

Scientific Progress and New Research Tools 3. COMMERCIAL VACCINES IN WILDLIFE B. abortus strain 19 (S19) and B. abortus strain RB51 (RB51) are commercially available live vac- cines against B. abortus that are licensed for use in cattle. A number of studies to evaluate their ability to prevent infection and abortion in elk and bison are reviewed here. 3.1 Vaccination of Elk The S19 vaccine was previously reported to be about 60% effective in preventing abortion in elk when the animals were vaccinated as calves (Thorne et al., 1981; Herriges et al., 1989). This study in- volved the vaccination of >40,000 elk on feedgrounds by the WGFD. However, since 1998, a study found the effective rate to be much lower when a limited number of elk were evaluated in a controlled setting (Roffe et al., 2004). While there were fewer abortions in the vaccinated group relative to the unvaccinated group, the protection rate was considered too low to be efficacious, especially since Brucella was isolated at equal rates from the calves and fetuses in the two groups. There have been questions on whether the number of Brucella organisms used to infect the elk in some studies represents a dose similar to that expe- rienced by elk at feedgrounds that come into contact with aborted fetuses (Roffe et al., 2004). In the field, the estimated exposure would be 4.1  106 live organisms for 10-cm diameter of skin contact (Cook, 1999). The challenge dose used in the above study was only about twice as large, making it a realistic dose although slightly more stringent than a natural infection in the field. In another study, vaccination of feedground elk with S19 delivered via ballistics did not decrease the rate of abortion or still births that occurred following infection. However, if 100% of juveniles were vaccinated, there were fewer abortion events relative to the rate that occurred when none were vaccinated (Maichak et al., 2017). Overall, S19 vaccination is considered to be inadequate for generating protective immunity in elk in the GYA. The RB51 vaccine is composed of a mutant strain of B. abortus that lacks the O-polysaccharide side chain. As a result, animals vaccinated with RB51 do not make antibodies to the O-polysaccharide; the presence of such antibodies is used as an indicator of infection in conventional brucellosis diagnostic tests. Given at one of two dosages (109 live organisms for adults and 1010 for calves), RB51 is considered to be as efficacious in preventing infection and abortions in cattle as the S19 vaccine (Cheville et al., 1996; Olsen, 2000; Olsen et al., 2009). In contrast, experimental trials indicate that the RB51 vaccine is ineffective at protecting elk from brucellosis (Cook et al., 2000) in that the RB51 vaccine resulted in only low levels of protection when administered intramuscularly or by biobullets (Cook et al., 2002; Kreeger et al., 2000). Animals given a booster dose of RB51 1 year after the initial RB51 vaccination aborted at a rate equal to or higher than that of unvaccinated animals, demonstrating that RB51 is not efficacious for elk (Kreeger et al., 2002). 3.2 Immune Responses by Elk to Vaccination Since neither S19 nor RB51 vaccines protect elk from infection or abortion, it is likely that the im- mune responses in elk differ from those of cattle. Immune responses are manifested as antibodies and in- terferon (IFN)-γ, a product of the immune system’s T lymphocytes, known as a cytokine, important for controlling brucellosis. IFN-γ production can be measured using a commercially available kit made for measuring IFN-γ of red deer (Olsen et al., 2006). The expressed IFN-γ gene sequence for red deer is iden- tical to elk except for the last amino acid; therefore, the same kit can be used to detect elk IFN-γ (Sweeney et al., 2001). Antibody responses to the vaccine strains of B. abortus were detected in vaccinat- ed elk and an expansion of CD4 T lymphocytes were seen after vaccination, but in vitro tests determined that lymphocyte multiplication in response to bacteria was not greater for vaccinated than unvaccinated elk. In comparison, lymphocyte replication in response to bacteria was detected for cattle and bison cells following vaccination with RB51 (Stevens et al., 1995; Olsen et al., 2002). A similar discrepancy oc- curred when elk were vaccinated with Mycobacterium bovis (BCG), a vaccine that typically induces a Prepublication Copy—Subject to Further Editorial Revision 71

Revisiting Brucellosis in the Greater Yellowstone Area strong IFN-γ response in cattle but not in elk, indicating that this is not peculiar to Brucella alone. To quantitatively and qualitatively evaluate the differences in immune responses between cattle and elk, it is necessary to first understand the elk immune system. To do this, tools are needed to identify and measure the cells and molecules involved in elk immune responses; however, those tools currently do not exist. 3.3 Vaccination of Bison Both B. abortus S19 and RB51 vaccines have variable efficacy in bison (see Table 4-1). When S19 was given to adult pregnant bison, either by needle or ballistically using hollow pellets containing freeze- dried S19 organisms, 50% aborted (Davis et al., 1991). This demonstrated that pregnant bison are more sensitive to abortion with S19 than pregnant cattle. Nevertheless, when bison were challenged with B. abortus strain 2308 (a fully virulent field isolate) in their second trimester of pregnancy having been pre- viously vaccinated with S19, 67% of bison were protected from abortion and 39% were protected from infection while only 4% of nonvaccinated bison failed to abort (Davis et al., 1991). These levels of pro- tection in bison following S19 vaccination are only slightly lower than the range found for cattle. Howev- er, other studies showed that S19 vaccination of bison calves is inadequate (Davis, 1993; Davis and Elzer, 2002). In contrast to S19, RB51 vaccination is safe in male bison, pregnant female bison, and in bison calves (Elzer et al., 1998). However, a number of studies show that RB51 efficacy varies in bison adults and calves. Protection from abortion using RB51 ranged from 0% to 100%, while protection against fetal infection ranged from 0% to 81% as summarized in Table 4-1. Calfhood vaccination of bison with RB51 was shown to provide protection from abortion when bison were challenged with virulent B. abortus S2308 mid-gestation in one study (Olsen et al., 2003). RB51 vaccination also reduced the recovery of S2308 from calf tissues, but not maternal tissues. In contrast, another group did not obtain significant ef- ficacy in RB51-booster vaccinated bison (Davis and Elzer, 1999). Studies have also shown mixed results on the efficacy of RB51 booster doses in pregnant bison. Adult pregnant bison given two doses of RB51 did not abort even though RB51 was present in fetal tissues (Olsen and Holland, 2003). Another study demonstrated that the booster dose resulted in higher IFN-γ (the immune system product associated with protective immunity to Brucella) responses, and none had infected fetuses (Olsen and Johnson, 2012a,b). Adult female YNP bison that had been previously vaccinated with 107 or 109 live RB51 organisms were revaccinated during the first trimester and boosted during the second trimester. An additional group of Kansas bison that had been previously vaccinated with 109 live RB51 organisms was also boosted during pregnancy. While abortion rates were slightly lower than for unvaccinated animals, the investigators concluded that RB51 was not significantly protec- tive and questioned whether vaccination with the standard dose would be more effective (Davis and Elzer, 1999; Elzer et al., 2002). No significant differences in abortion or calf infection rates were seen among animals vaccinated once, left unvaccinated, or vaccinated twice. Thus, RB51 vaccination did not protect against abortion and only one-third of all the calves were protected against infection in that study (Elzer et al., 2002). To address whether booster vaccination with RB51 can enhance protection in yearlings, one group of bison heifer calves was vaccinated subcutaneously while a second group was darted (Olsen and Johnson, 2012b). All animals were naturally bred, some of the subcutaneously RB51 vaccinated animals were boosted with RB51, and pregnant bison were challenged with virulent B. abortus S2308. Unvac- cinated controls had an 83% abortion rate compared to 33% for the animals that received a single-dose of RB51, 57% for the darted animals, and none of the twice-vaccinated bison aborted. The results again in- dicate that multiple doses of RB51 has efficacy in bison. However, regardless of vaccination status, 100% of the fetuses/calves had viable wild-type B. abortus in their tissues (Olsen and Johnson, 2012b). 72 Prepublication Copy—Subject to Further Editorial Revision

TABLE 4-1 Summary of RB51’s Efficacy in Bison % Bison # of Age of Primary Vaccine Dose Strain 2308 Time of Protected % Bison Protected Bison/ Vaccine & Booster at 1° & Booster Challenge Dose Challenge (Days Against Against Fetal Study Bison Source Vaccine Group Group Doses Vaccinations Vaccinations (CFUs) (CFUs)* of Gestation) Abortion Infection Olsen et al., Iowa saline 13 - 3-8 months old 0 107 150-180 38% 38% 2003 Iowa RB51 37 1 3-8 months old 1.2-6.1x1010 107 150-180 85% 81% 7 Davis and Colorado saline 19 - adult 0 10 150-180 21% Not determined Elzer, 1999 9 9 7 Kansas RB51 8 2 adult 10 /10 10 150-180 50% Not determined Yellowstone RB51 20 3 adult/1st trimes./ 107 or 109/109/109 107 150-180 50% Not determined National Park 2nd trimes. Elzer et South Dakota - 27 - 6 months old 0 107 mid-gestation 67% 0% al., 2002 10 7 South Dakota RB51 28 1 6 months old 10 10 mid-gestation 75% 0% South Dakota RB51 28 3 6 /12 /18 months 1010/1010/1010 107 mid-gestation 71.4% 32% old Olsen and Brucellosis-free saline 6 - 8-10 months old 0 107 170-180 17% 0% Johnson, 2012b herd Brucellosis-free RB51 7 1 8-10 months old darted with 1.8x1010 107 170-180 43% 0% herd Brucellosis-free RB51 6 1 8-10 months old 2.2x1010 107 170-180 67% 0% Prepublication Copy—Subject to Further Editorial Revision herd Brucellosis-free RB51 5 2 8-10 /23-25 1.1x1010/2.2x1010 107 170-180 100% 0% herd months old Olsen et al., Brucellosis-free saline 6 - 8-11 months old 0 107 170-180 0% 17% 2015 herd Brucellosis-free RB51 5 1 8-11 months old 1.6x1010 107 170-180 80% 40% herd Brucellosis-free RB51 14 2 8-11 /19-22 1.6x1010/2.8x1010 107 170-180 93% 57% herd Olsen et al., Brucellosis-free saline 8 - 10 months old 0 107 170-180 0 0% 2009 herd Brucellosis-free RB51 + sodC, 6 1 10 months old 7.4x1010 107 170-180 33% 0% herd wboA Brucellosis-free RB51 6 1 10 months old 4.26x1010 107 170-180 66% 0% herd NOTE: *Bison were challenged via the conjunctival route with virulent wild type B. abortus strain 2308. 73

Revisiting Brucellosis in the Greater Yellowstone Area Alternative methods for delivering RB51 have been evaluated. Vaccinating bison by darts induced immune responses similar to those achieved by hand vaccination, but neither the dart or hand vaccination method protected the bison from abortion when challenged with B. abortus S2308 (Olsen and Johnson, 2012b). However, bison that were given a booster dose showed protection from abortion. A follow-up study demonstrated that giving a booster dose of RB51 results in a greater IFN-γ response as measured by mRNA transcripts, reduced percentage of abortions, and less bacterial colonization of tissues (Olsen et al., 2015). 4. NEW SCIENTIFIC TOOLS INFORMING BRUCELLOSIS INFECTION BIOLOGY, PATHOGENESIS, AND VACCINOLOGY New molecular tools have recently been developed that link cell biology with genetics and ge- nomics. Bioinformatics has also emerged as an important tool to manage and analyze massive datasets of biological information. The expansion of the “-omics” (fields of study related to the genome, transcrip- tome, proteome, metabolome), genomics tools, and next-generation sequencing technologies now enable in-depth analyses needed to understand cellular function and behavior of B. abortus and its hosts (includ- ing elk, bison, and cattle). 4.1 Brucella Genome More than 30 complete Brucella genomes have been sequenced since 1998, providing a database for comparative analysis of gene structure and homologies, gene expression, regulatory networks, protein synthesis, and metabolic pathways. Gene variations among strains have been identified by comparative genomics and through speciation. The identified variations only partially explain the differences in viru- lence among Brucella species and their specificity for certain host species (He, 2012). Genes of a pathogen can be interrogated by a process known as reverse vaccinology to identify their potential to induce immune responses in their host and this has been applied to the Brucella genomes (He and Xiang, 2010; He, 2012; Gomez et al., 2013a,b; Vishnu et al., 2015). Candidate gene products have been tested for in vivo efficacy, an approach that could be used to tailor brucellosis vaccines for elk, bi- son, and cattle (Ko and Splitter, 2003; Wang et al., 2012; Gomez et al., 2013a; Gomez et al., 2013b). For example, Vaxign (a Web-based vaccinology tool) identified 14 outer membrane proteins that are con- served in six virulent strains of B. abortus, B. melitensis, and B. suis (He and Xiang, 2010). Some of these proteins were shown to induce antibody and T cell responses in immunized mice (Gomez et al., 2013a). This type of information may also be useful for developing new diagnostic tests. Whole genome sequencing and other sequence-based technologies can show evolutionary relation- ships of Brucella relative to geography and host origin. This is a particularly relevant tool for understand- ing the epidemiology of Brucella infections among cattle, elk, and bison in Yellowstone National Park (Beja-Pereira et al., 2009; Higgins et al., 2012; Rhyan et al., 2013; Kamath et al., 2016). Gene expression analysis of Brucella during host adaptation has identified critical factors for viru- lence and long-term survival of Brucella (Kim et al., 2013, 2014). Inactivation or knockout of Brucella genes allows gene function to be identified in pathogenesis and virulence. This could also facilitate en- hanced vaccine development by producing new attenuated strains of the bacteria (O’Callaghan et al., 1999; Rosinha et al., 2002; Ficht, 2003; Arenas-Gamboa et al., 2008, 2009; Kim et al., 2014). 4.2 Host Genomes Substantial progress has been made on assembling the Bison bison bison reference genome (NIH, 2016). A bison reference genome provides fundamental information and can eventually help identify any genetic basis for increased susceptibility of bison to B. abortus. A deer reference genome (red deer, Ca- nadian elk) is also being completed and validated (Brauning et al., 2015). Functional genomics can detect host genes that are either expressed or repressed, and could further reveal the mechanism by which B. 74 Prepublication Copy—Subject to Further Editorial Revision

Scientific Progress and New Research Tools abortus survives. For example, gene silencing (using RNA interference) was used to knockdown specific host genes during Brucella infection in model systems, which allowed scientists to identify the genes con- trolling major infection pathways (Qin et al., 2008; Rossetti et al., 2012). While genetic resistance against brucellosis is a complex polygenic trait in cattle and bison, newer genetic tools can provide the means to better understand the genetic basis for susceptibility to B. abortus in elk and bison and to clone livestock or wildlife for enhanced genetic resistance to B. abortus (Adams and Templeton, 1998; Westhusin et al., 2007; Adams and Schutta, 2010). Brucella and host gene expression and proteome datasets have been generated in the past decade, which will provide future opportunities for a comprehensive analysis of both host and pathogen responses during infection (Rajashekara et al., 2006; Carvalho Neta et al., 2008; Lamontagne et al., 2009; He et al., 2010; Rossetti et al., 2010; Viadas et al., 2010; Weeks et al., 2010; Lin et al., 2011; Wang et al., 2011; Liu et al., 2012; Rossetti et al., 2012, 2013; Karadeniz et al., 2015). To date, datasets have been analyzed to understand gene regulatory networks, characterize Brucella stress responses, and understand modulation of host responses (He et al., 2010; He, 2012; Hanna et al., 2013; Kim et al., 2013, 2014; Karadeniz et al., 2015). 5. CONCLUSION Even though there is now a greater scientific understanding of B. abortus than in 1998, there contin- ue to be major gaps in understanding infection biology and molecular pathogenesis of brucellosis in each host. New tools and reagents are needed to gain a basic understanding of the uniqueness of the elk im- mune system response to Brucella to develop elk specific vaccines. There has been limited progress in understanding Brucella host preference and genetic resistance to brucellosis to manage transmission be- tween domestic animals and wildlife species (Godfroid et al., 2011, 2014), but new molecular and bioin- formatics tools offer greater hope to understand these phenomena (see Chapter 9 on Remaining Gaps for Understanding and Controlling Brucellosis). REFERENCES Adams, L.G. 2002. The pathology of brucellosis reflects the outcome of the battle between the host genome and the Brucella genome. Veterinary Microbiology 90(1-4):553-561. Adams, L.G., and C.J. Schutta. 2010. Natural disease resistance to brucellosis: A review. The Open Journal of Veterinary Science 4:61-71. Adams, L.G., and J.W. Templeton. 1998. Genetic resistance to bacterial diseases of animals. Revue Scientifique et Technique Office International des Epizooties 17(1):200-219. Al Dahouk, S., V. Jubier-Maurin, H.C. Scholz, H. Tomaso, W. Karges, H. Neubauer, and S. Kohler. 2008. Quantita- tive analysis of the intramacrophagic Brucella suis proteome reveals metabolic adaptation to late stage of cel- lular infection. Proteomics 8(18):3862-3870. Anderson, N.J., J.M. Ramsey, and K.D. Hughes. 2009. 2008 Brucellosis Survey Final Report. Montana Fish, Wildlife & Parks, Wildlife Laboratory, Bozeman, MT. Anderson, T.D., and N.F. Cheville. 1986. Ultrastructural Morphometric Analysis of Brucella abortus-Infected Trophoblasts in Experimental Placentitis. American Journal of Pathology 124(2):226-237. Arenas-Gamboa, A.M., T.A. Ficht, M.M. Kahl-McDonagh, and A.C. Rice-Ficht. 2008. Immunization with a single dose of a microencapsulated Brucella melitensis mutant enhances protection against wild-type challenge. Infection and Immunology 76(6):2448-2455. Arenas-Gamboa, A.M., T.A. Ficht, M.M. Kahl-McDonagh, G. Gomez, and A.C. Rice-Ficht. 2009. The Brucella abortus S19 DeltavjbR live vaccine candidate is safer than S19 and confers protection against wild-type challenge in BALB/c mice when delivered in a sustained-release vehicle. Infection and Immunology 77(2):877-884. Atluri, V.L., M.N. Xavier, M.F. de Jong, A.B. den Hartigh, and R.M. Tsolis. 2011. Interactions of the human patho- genic Brucella species with their hosts. Annual Reviews in Microbiology 65:523-541. Barbier, T., C. Nicolas, and J.J. Letesson. 2011. Brucella adaptation and survival at the crossroad of metabolism and virulence. FEBS Letters 585(19):2929-2934. Prepublication Copy—Subject to Further Editorial Revision 75

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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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