In two studies conducted from October 1999 to March 2000 and December 2000 to April 2001, adult black bears (Ursus americanus) were orally inoculated with 1.4–3.1X1010 colony-forming units (CFU) of Brucella abortus strain RB51 (SRB51, n=12) or 2 ml of 0.15 M NaCl solution (saline, n=11). We did not detect a difference (P\u3e0.05) in antibody titers to SRB51 in serum obtained before vaccination, at 8 wk after vaccination, or at necropsy at 21 or 23 wk after vaccination between SRB51-vaccinated and nonvaccinated bears. The SRB51 vaccine strain was recovered from tissues obtained at necropsy from one of six SRB51-vaccinated bears in study 1, but none of the six SRB51-vaccinated bears in study 2. Vaccination of black bears with SRB51 did not appear to influence (P\u3e0.05) reproductive performance

Gidlewski, T.

Goff, Jesse

Olsen, Steven C.

Rhyan, Jack

Stoffregen, W. C.

English

DigitalCommons@University of Nebraska

University of Nebraska - Lincoln DigitalCommons@University of Nebraska - Lincoln USDA National Wildlife Research Center - Staff Publications U.S. Department of Agriculture: Animal and Plant Health Inspection Service 2004 Safety Of Brucella Abortus Strain Rb51 In Black Bears Steven C. Olsen US Department of Agriculture, solsen@nadc.ars.usda.gov Jack Rhyan US Department of Agriculture T. Gidlewski US Department of Agriculture Jesse Goff US Department of Agriculture W. C. Stoffregen US Department of Agriculture Follow this and additional works at: https://digitalcommons.unl.edu/icwdm_usdanwrc  Part of the Environmental Sciences Commons Olsen, Steven C.; Rhyan, Jack; Gidlewski, T.; Goff, Jesse; and Stoffregen, W. C., "Safety Of Brucella Abortus Strain Rb51 In Black Bears" (2004). USDA National Wildlife Research Center - Staff Publications. 1056. https://digitalcommons.unl.edu/icwdm_usdanwrc/1056 This Article is brought to you for free and open access by the U.S. Department of Agriculture: Animal and Plant Health Inspection Service at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in USDA National Wildlife Research Center - Staff Publications by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. 429Journal of Wildlife Diseases, 40(3), 2004, pp. 429–433q Wildlife Disease Association 2004SAFETY OF BRUCELLA ABORTUS STRAIN RB51 IN BLACK BEARSSteven C. Olsen,1,5 Jack Rhyan,2 T. Gidlewski,3 Jesse Goff,4 and W. C. Stoffregen11 Bacterial Diseases of Livestock Research Unit, National Animal Disease Center, Agricultural Research Service, USDepartment of Agriculture, Ames, Iowa 50010, USA2 National Wildlife Research Center, Animal and Plant Health Inspection Service, Veterinary Services, US Departmentof Agriculture, Fort Collins, Colorado 80521, USA3 Pathology Laboratory, Animal and Plant Health Inspection Service, Veterinary Services, US Department ofAgriculture, Ames, Iowa 50010, USA4 Metabolic Diseases of Livestock Research Unit, National Animal Disease Center, Agricultural Research Service, USDepartment of Agriculture, Ames, Iowa 50010, USA5 Corresponding author (email: solsen@nadc.ars.usda.gov)ABSTRACT: In two studies conducted from October 1999 to March 2000 and December 2000to April 2001, adult black bears (Ursus americanus) were orally inoculated with 1.4–3.131010colony-forming units (CFU) of Brucella abortus strain RB51 (SRB51, n512) or 2 ml of 0.15 MNaCl solution (saline, n511). We did not detect a difference (P.0.05) in antibody titers to SRB51in serum obtained before vaccination, at 8 wk after vaccination, or at necropsy at 21 or 23 wkafter vaccination between SRB51-vaccinated and nonvaccinated bears. The SRB51 vaccine strainwas recovered from tissues obtained at necropsy from one of six SRB51-vaccinated bears in study1, but none of the six SRB51-vaccinated bears in study 2. Vaccination of black bears with SRB51did not appear to influence (P.0.05) reproductive performance.Key words: Biosafety, Brucella, RB51, serology, Ursus americanus, vaccine.INTRODUCTIONIn 1996, a new official calfhood vaccinefor cattle, Brucella abortus strain RB51(SRB51), was conditionally approved bythe Animal and Plant Health InspectionService branch of the US Department ofAgriculture. The SRB51 strain, a labora-tory-derived, lipopolysaccharide O–sidechain–deficient mutant (Schurig et al.,1991) of B. abortus strain 2308, inducesprotection in cattle and bison (Bison bison)against challenge exposure with virulent B.abortus strains (Cheville et al., 1993; Che-ville et al., 1996; Olsen et al., 2003). Be-cause SRB51 is being considered as a vac-cine for bison in the Greater YellowstoneArea (GYA, USA), studies to evaluate thesafety of this vaccine in representativenontarget species needed to be conducted.In the studies reported here, antibody re-sponses, tissue localization and clearance,and reproductive performance were as-sessed in black bears (Ursus americanus)after oral exposure to SRB51.MATERIALS AND METHODSTwo studies were conducted from October1999 to March 2000 and December 2000 toApril 2001 at a private facility in South Dakota,USA (428529N, 938639W). Each study used dif-ferent bears with no animals used in both stud-ies. In October (study 1) or December (study2), adult black bears were sedated with 500 to1,000 mg of tiletamine hydrochloride and zo-lazepam hydrochloride (Telazolt; Fort DodgeAnimal Health, Fort Dodge, Iowa, USA) ad-ministered intramuscularly by pneumatic dart(Pneudartt, Williamsburg, Pennsylvania, USA).After attainment of lateral recumbency, ear tagswere placed for identification, and blood wasobtained via jugular venipuncture for serologicanalysis. Bears were then orally inoculated byplacing the inoculum into the pharyngeal re-gion and holding the head vertically until thebear was observed to swallow. In study 1, bearsreceived either 2 ml of 0.15 M NaCl (saline;n56) or 2 ml of saline containing B. abortusstrain RB51 (SRB51; n56) orally. In study 2,only bears in the SRB51 treatment group(n56) were anesthetized and inoculated orally.However, control bears (n55) were identifiedat the time of vaccination for comparison withreproduction in SRB51-inoculated bears. Fol-lowing oral inoculation, bears were grouped bytreatment, with SRB51-vaccinated bearshoused separately from nonvaccinated bears.The SRB51 inoculum in both studies wasprepared by dilution of lyophilized SRB51 vac-cine (Colorado Serum Company, Denver, Col-orado, USA) to approximately 131010 colony-forming units (CFU) on the basis of viabilitycounts of other vials with the same lot number.Briefly, reconstituted vaccine was serially dilut-ed in saline and plated on tryptose agar plates430 JOURNAL OF WILDLIFE DISEASES, VOL. 40, NO. 3, JULY 2004containing 5% bovine serum, and standardplate counts were conducted to determine vi-ability. Standard plate counts of the inoculumdetermined the SRB51 dosage administered.At 8 wk after oral inoculation in study 1,bears in both SRB51 and control treatmentswere anesthetized with Telazol, as previouslydescribed, and blood was obtained via jugularvenipuncture for serologic analysis.NecropsyAt 23 wk after oral inoculation in study 1,SRB51-inoculated bears and their cubs andcontrol bears were anesthetized in their denswith Telazol via pneumatic dart or intramus-cular injection. In a similar manner, SRB51-in-oculated bears and their cubs in study 2 wereanesthetized in their dens at 21 wk after inoc-ulation. After sedation, blood was obtained forserology via jugular venipuncture. All SRB51-inoculated bears and their cubs in both studieswere then euthanized with intravenous injec-tions of sodium pentobarbitol (Sleepaway, Ft.Dodge Labs, Ft. Dodge, Iowa) and necropsied.Control bears were only sedated in study 1, andall were allowed to recover from anesthesia.Maternal tissues collected at necropsy fromSRB51 vaccinates for both bacteriologic andhistologic evaluation included: mammary gland,liver, lung, spleen, kidney, and uterus. Maternalsamples obtained for bacteriologic examinationonly included vaginal, conjunctival, nasal, andrectal swabs; blood; tonsil; and hepatic, parotid,popliteal, prescapular, medial retropharyngeal,mediastinal, internal iliac, mandibular, mesen-teric, and supramammary lymph nodes. Bac-teriologic samples from cubs included blood,spleen, lung, liver, kidney, mesenteric lymphnode, and cerebrospinal fluid. Histologic tech-niques also were used to examine cardiac sam-ples from both sows and cubs and fetal liver,spleen, and kidney samples. All tissues collect-ed for histologic evaluation were fixed in neu-tral buffered 10% formalin embedded in par-affin, sectioned at 5 mm, and stained with he-matoxylin and eosin. Special stains (Congo red,silver, acid-fast, or Prussian blue; Sheehan andHrapchak, 1980) were performed on sectionsin which examination of hematoxylin and eosinsections suggested that additional histologictechniques were warranted.Bacteriologic evaluationTissue samples were triturated in saline andplaced on tryptose agar plates containing 5%bovine serum and a selective medium forSRB51 (RBM) that contains antibiotics, includ-ing rifampicin (Hornsby et al., 2000). The an-tibiotics in RBM media minimized growth ofcontaminants without inhibiting growth ofSRB51, thereby enhancing the ability to detectsmall numbers of SRB51 within samples.Following incubation of plates at 37 C and5% CO2 for 7 days, SRB51 was identified bycolony morphology, growth characteristics (Al-ton et al., 1988), and resistance to rifampicin(Schurig et al., 1991). Isolates were confirmedas SRB51 in a polymerase chain reaction (PCR)procedure with the use of primers specific forSRB51 (Vemulapalli et al., 1999). Briefly, thereaction mixture consisted of heat-killed cells,0.2 mM nucleotide mix (Boehringer Mann-heim, Mannheim, Germany), 13 PCR BufferII (Perkin Elmer, Branchburg, New Jersey,USA), 2.5 U/ml DNA polymerase (AmplitaqGold, Perkin Elmer), and 1.5 mM MgCl com-bined with 0.2 mM per reaction of specificprimers (upstream primer GCAACGGTGTTC-TTCCACTC and downstream primer GTAT-CAGGCTACGCAGAAGG) for B. abortusomp2A as selected from analysis of its se-quence (Ficht et al., 1989). The samples, in-cluding positive and negative controls, were cy-cled (30 sec at 95 C, 30 sec at 44 C, 1 min at72 C) 30 times in a thermocycler (MJ ResearchInc., Watertown, Massachusetts, USA). Prod-ucts were analyzed by electrophoresis on a1.5% agarose gel after staining with ethidiumbromide.Serologic evaluationBlood samples were allowed to clot, and se-rum was separated by centrifugation. Serumwas divided into 1-ml aliquots, frozen, andstored at 270 C. Serologic titers of animals toSRB51 were determined by dot-blot assay (Ol-sen et al., 1997) in which g-irradiated SRB51is used as antigen, and peroxidase-labeled pro-tein A (1:100 dilution; Kirkegaard and PerryLaboratories Inc., Gaithersburg, Maryland,USA), rabbit anti-canine IgG heavy- and light-chain antibodies (1:5,000; Jackson Immunore-search Laboratories, West Grove, Pennsylva-nia), or rabbit anti-goat IgG heavy- and light-chain antibodies (1:10,000 dilution; JacksonImmunoresearch Laboratories) were used assecondary antibodies.StatisticsBecause of logarithmic conversions, titerswith a value of 0 were analyzed with a value of1. Serologic responses of bears in both studieswere compared over all times by an analysis ofvariance procedure with titers to each second-ary antibody evaluated separately (SAS Insti-tute Inc., Cary, North Carolina, USA). Meansfor individual treatments were separated by aleast significant difference procedure (P,0.05).OLSEN ET AL.—SAFETY OF RB51 IN BLACK BEARS 431FIGURE 1. Serologic responses of nonvaccinated (control) bears or bears orally inoculated with 1.4–3.131010 colony forming units of SRB51 to g-irradiated SRB51 in a dot-blot assay in which peroxidase-conjugated protein A, rabbit anti-canine IgG, or rabbit anti-goat IgG were used as secondary antibodies.Antibody responses were determined before vaccination (time 0; n512 for SRB51 and n56 for control treat-ments), 8 wk after vaccination (n56 for SRB51 and control treatments), and at necropsy 21 or 23 wk aftervaccination (n512 for SRB51 and n56 for control treatments). Responses are presented as mean titer6SEM.A 95% confidence interval was predicted fromthe bacteriologic data by a Clopper-Pearsonprocedure (SAS Institute Inc.).RESULTSThe concentration of viable SRB51 bac-teria within the inocula for studies 1 and2 were determined by standard platecounts to be 1.431010 and 3.131010 CFU,respectively.At necropsy in the first study, twin cubswere found with one SRB51-inoculatedbear and one nonvaccinated bear. No oth-er bears in SRB51 or control treatmentswere pregnant or had cubs present at thetime of evaluation. In study 2, three of thefive paired controls had cubs (two tripletsand one set of twins) at the time of nec-ropsy, whereas three of six SRB51-inocu-lated bears had cubs (three sets of twins).At necropsy in study 1, 23 wk after vac-cination, SRB51 was recovered from theinternal iliac, retropharyngeal, and mes-enteric lymph nodes from one of sixSRB51-inoculated bears. This bear had nocubs and was not pregnant at the time ofevaluation. The SRB51 vaccine strain wasnot recovered from fetal tissues obtainedat necropsy from the two cubs of theSRB51 vaccinate. In study 2, SRB51 wasnot recovered from any tissue obtained atnecropsy from any of the six SRB51-inoc-ulated bears or their cubs.Regardless of secondary antibody, titersof bears orally inoculated with SRB51 didnot differ (P.0.05) from dot-blot titers ofnonvaccinates at any sampling time (Fig.1). In both studies, one of six SRB51-in-oculated bears had titers at necropsy thatwere four dilutions greater than the titerof paired serum obtained before vaccina-tion. In both of these bears, the highesttiters were noted when the anti-canine an-tibody was used as the secondary antibody.When compared with the serum obtainedbefore inoculation, the bear that was cul-ture positive for SRB51 at necropsy in432 JOURNAL OF WILDLIFE DISEASES, VOL. 40, NO. 3, JULY 2004study 1 did not demonstrate a significantincrease in titer to SRB51 at 8 wk afterinoculation or at necropsy with any of thesecondary antibodies used in this study.With the exception of small, subacute,multifocal tonsillar abscesses in oneSRB51-inoculated sow in study 1, no grosslesions were noted at necropsy.Histologic evaluation of lung sectionsfrom eight SRB51-vaccinated adult bears(five in study 1 and three in study 2) re-vealed multifocal, perivascular, and, to alesser extent, peribronchiolar accumula-tions of moderate numbers of histiocytes.The cytoplasm of these histiocytes wereexpanded by an amorphous basophilic ma-terial that also contained numerous bire-fringent crystals. In some adult bears, mul-tifocal accumulations of low numbers oflymphocytes were found in interstitial ar-eas of the kidney or were randomly dis-tributed in hepatic parenchyma.Special stains of histologic sections oflung indicated that lesions were compati-ble with pneumoconiosis. The etiology ofthe particulate material in pulmonary sec-tions was not conclusively determined butmost likely was inhaled environmental de-bris. With these exceptions, no significanthistologic lesions were noted in examina-tion of remaining maternal tissues and allfetal tissues.DISCUSSIONThe results of these studies suggest thatoral exposure to 1010 CFU of SRB51 is notassociated with adverse clinical, histologic,or reproductive effects in bears. Althoughbears in both treatments had poor concep-tion rates in the first study, reproductionperformance of bears in both treatmentsin each study were similar.Our data also suggest that in a smallpercentage of bears (one of 12, 8%; 95%confidence interval, 0.2–38.5%), SRB51can persist for up to 23 wk after oral in-oculation. Failure to recover SRB51 frommucosal swabs, mammary gland, supra-mammary lymph nodes, or uterine and fe-tal tissues in any SRB51-vaccinated bear inour studies suggests that if bears are orallyexposed to SRB51, it is unlikely that theywill transmit the strain laterally or verti-cally. When evaluated with the bacterio-logic data, the poor serologic responsesnoted in our studies might indicate SRB51is not well adapted to colonize or replicatein tissues of black bears and therefore doesnot effectively induce immunologic re-sponses or cause clinical effects. Alterna-tively, SRB51 might not efficiently attachand invade mucosal surfaces of black bearsafter oral exposure.Data from others have suggested thatbears in areas with endemic brucellosismight undergo at least transient infectionwith field strains of B. abortus. Grizzly(Ursus arctos horribilis) and black bearssampled in Montana (USA) near Yellow-stone National Park demonstrated positiveresponses on a panel of brucellosis sero-logic tests, whereas samples from bears innorthern Montana, in an area without bru-cellosis, were negative on the same panelof tests (K. Aune, pers. comm.). Althoughthere are no data at this time to determinewhether exposure to B. abortus has any ef-fects on survival or reproduction of bearsin the GYA, our data and studies in otherspecies (Januszewski et al., 2001; Kreegeret al., 2002) suggest that SRB51 is safe inblack bears and is unlikely to be morepathogenic in bears than B. abortus fieldstrains that are currently present in theGYA environment.ACKNOWLEDGMENTSWe thank A. Duit, D. Pringle, A. Jensen, K.Casey, and D. Buffington for their technical as-sistance.LITERATURE CITEDALTON, G. G., L. M. JONES, R. D. ANGUS, AND J. M.VERGER. 1988. Techniques for the brucellosislaboratory. Institut National de la RechercheAgronomique, Paris, France, pp. 17–136.CHEVILLE, N. F., M. G. STEVENS, A. E. JENSEN, F.M. TATUM, AND S. M. HALLING. 1993. Immuneresponses and protection against infection andabortion in cattle experimentally vaccinated withmutant strains of Brucella abortus. AmericanJournal of Veterinary Research 54: 1591–1597.OLSEN ET AL.—SAFETY OF RB51 IN BLACK BEARS 433, S. C. OLSEN, A. E. JENSEN, M. G. STEVENS,M. V. PALMER, AND A. M. FORANCE. 1996. Ef-fects of age at vaccination of efficacy of Brucellaabortus strain RB51 to protect cattle against bru-cellosis. American Journal of Veterinary Re-search 57: 1153–1156.FICHT, T. A, S. W. BEARDEN, B. A. SOWA, AND L. G.ADAMS. 1989. DNA sequence and expression ofthe 36-kilodalton outer membrane protein geneof Brucella abortus. Infection and Immunity 57:3281–3291.HORNSBY, R. L., A. E. JENSEN, S. C. OLSEN, AND C.O. THOEN. 2000. Selective media for isolationof Brucella abortus strain RB51. Veterinary Mi-crobiology 73: 51–60.JANUSZEWSKI, M. C., S. C. OLSEN, R. G. MCLEAN,L. CLARK, AND J. C. RHYAN. 2001. Biosafety ofBrucella abortus vaccine strain RB51 in non-tar-get species of birds and rodents. Journal of Wild-life Diseases 37: 532–537.KREEGER, T. J., T. J. DELIBERTO, S. C. OLSEN, W.H. EDWARDS, AND W. E. COOK. 2002. Safety ofBrucella abortus strain RB51 in non-target un-gulates and coyotes. Journal of Wildlife Diseases38: 552–557.OLSEN, S. C., M. G. STEVENS, N. F. CHEVILLE, ANDG. SCHURIG. 1997. Experimental use of a dot-blot assay to measure serologic responses of cat-tle vaccinated with Brucella abortus strain RB51.Journal of Veterinary Diagnostic Investigation 9:363–367., A. E. JENSEN, W. C. STOFFREGEN, AND M.V. PALMER. 2003. Efficacy of calfhood vaccina-tion with Brucella abortus strain RB51 in pro-tecting bison against brucellosis. Research in Vet-erinary Science 74: 17–22.SCHURIG, G. G., R. M. ROOP, T. BAGCHI, S. BOYLE,D. BUHRMAN, AND N. SRIRANGANATHAN. 1991.Biological properties of RB51; a stable roughstrain of Brucella abortus. Veterinary Microbiol-ogy 28: 171–188.SHEEHAN, D. C., AND B. B. HRAPCHAK. 1980. Theoryand practice of histotechnology, 2nd Edition. C.V. Mosby Company, St. Louis, Missouri, pp. 55,157, 218, and 241.VEMULAPALLI, R., J. R. MCQUISTON, G. G. SCHURIG,N. SRIRANGANATHAN, S. M. HALLING, AND S. M.BOYLE. 1999. Identification of an IS711 elementinterrupting the wboA gene of Brucella abortusvaccine strain RB51 and a PCR assay to distin-guish strain RB51 from other Brucella speciesand strains. Clinical and Diagnostic LaboratoryImmunology 6: 760–764.Received for publication 3 September 2003.

Safety Of \u3ci\u3eBrucella Abortus\u3c/i\u3e Strain Rb51 In Black Bears

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University of Nebraska - Lincoln 
DigitalCommons@University of Nebraska - Lincoln 
USDA National Wildlife Research Center - Staff 
Publications 
U.S. Department of Agriculture: Animal and 
Plant Health Inspection Service 
2004 
Safety Of Brucella Abortus Strain Rb51 In Black Bears 
Steven C. Olsen 
US Department of Agriculture, solsen@nadc.ars.usda.gov 
Jack Rhyan 
US Department of Agriculture 
T. Gidlewski 
US Department of Agriculture 
Jesse Goff 
US Department of Agriculture 
W. C. Stoffregen 
US Department of Agriculture 
Follow this and additional works at: https://digitalcommons.unl.edu/icwdm_usdanwrc 
 Part of the Environmental Sciences Commons 
Olsen, Steven C.; Rhyan, Jack; Gidlewski, T.; Goff, Jesse; and Stoffregen, W. C., "Safety Of Brucella Abortus 
Strain Rb51 In Black Bears" (2004). USDA National Wildlife Research Center - Staff Publications. 1056. 
https://digitalcommons.unl.edu/icwdm_usdanwrc/1056 
This Article is brought to you for free and open access by the U.S. Department of Agriculture: Animal and Plant Health 
Inspection Service at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in USDA 
National Wildlife Research Center - Staff Publications by an authorized administrator of DigitalCommons@University 
of Nebraska - Lincoln. 
429
Journal of Wildlife Diseases, 40(3), 2004, pp. 429–433
q Wildlife Disease Association 2004
SAFETY OF BRUCELLA ABORTUS STRAIN RB51 IN BLACK BEARS
Steven C. Olsen,1,5 Jack Rhyan,2 T. Gidlewski,3 Jesse Goff,4 and W. C. Stoffregen1
1 Bacterial Diseases of Livestock Research Unit, National Animal Disease Center, Agricultural Research Service, US
Department of Agriculture, Ames, Iowa 50010, USA
2 National Wildlife Research Center, Animal and Plant Health Inspection Service, Veterinary Services, US Department
of Agriculture, Fort Collins, Colorado 80521, USA
3 Pathology Laboratory, Animal and Plant Health Inspection Service, Veterinary Services, US Department of
Agriculture, Ames, Iowa 50010, USA
4 Metabolic Diseases of Livestock Research Unit, National Animal Disease Center, Agricultural Research Service, US
Department of Agriculture, Ames, Iowa 50010, USA
5 Corresponding author (email: solsen@nadc.ars.usda.gov)
ABSTRACT: In two studies conducted from October 1999 to March 2000 and December 2000
to April 2001, adult black bears (Ursus americanus) were orally inoculated with 1.4–3.131010
colony-forming units (CFU) of Brucella abortus strain RB51 (SRB51, n512) or 2 ml of 0.15 M
NaCl solution (saline, n511). We did not detect a difference (P.0.05) in antibody titers to SRB51
in serum obtained before vaccination, at 8 wk after vaccination, or at necropsy at 21 or 23 wk
after vaccination between SRB51-vaccinated and nonvaccinated bears. The SRB51 vaccine strain
was recovered from tissues obtained at necropsy from one of six SRB51-vaccinated bears in study
1, but none of the six SRB51-vaccinated bears in study 2. Vaccination of black bears with SRB51
did not appear to influence (P.0.05) reproductive performance.
Key words: Biosafety, Brucella, RB51, serology, Ursus americanus, vaccine.
INTRODUCTION
In 1996, a new official calfhood vaccine
for cattle, Brucella abortus strain RB51
(SRB51), was conditionally approved by
the Animal and Plant Health Inspection
Service branch of the US Department of
Agriculture. The SRB51 strain, a labora-
tory-derived, lipopolysaccharide O–side
chain–deficient mutant (Schurig et al.,
1991) of B. abortus strain 2308, induces
protection in cattle and bison (Bison bison)
against challenge exposure with virulent B.
abortus strains (Cheville et al., 1993; Che-
ville et al., 1996; Olsen et al., 2003). Be-
cause SRB51 is being considered as a vac-
cine for bison in the Greater Yellowstone
Area (GYA, USA), studies to evaluate the
safety of this vaccine in representative
nontarget species needed to be conducted.
In the studies reported here, antibody re-
sponses, tissue localization and clearance,
and reproductive performance were as-
sessed in black bears (Ursus americanus)
after oral exposure to SRB51.
MATERIALS AND METHODS
Two studies were conducted from October
1999 to March 2000 and December 2000 to
April 2001 at a private facility in South Dakota,
USA (428529N, 938639W). Each study used dif-
ferent bears with no animals used in both stud-
ies. In October (study 1) or December (study
2), adult black bears were sedated with 500 to
1,000 mg of tiletamine hydrochloride and zo-
lazepam hydrochloride (Telazolt; Fort Dodge
Animal Health, Fort Dodge, Iowa, USA) ad-
ministered intramuscularly by pneumatic dart
(Pneudartt, Williamsburg, Pennsylvania, USA).
After attainment of lateral recumbency, ear tags
were placed for identification, and blood was
obtained via jugular venipuncture for serologic
analysis. Bears were then orally inoculated by
placing the inoculum into the pharyngeal re-
gion and holding the head vertically until the
bear was observed to swallow. In study 1, bears
received either 2 ml of 0.15 M NaCl (saline;
n56) or 2 ml of saline containing B. abortus
strain RB51 (SRB51; n56) orally. In study 2,
only bears in the SRB51 treatment group
(n56) were anesthetized and inoculated orally.
However, control bears (n55) were identified
at the time of vaccination for comparison with
reproduction in SRB51-inoculated bears. Fol-
lowing oral inoculation, bears were grouped by
treatment, with SRB51-vaccinated bears
housed separately from nonvaccinated bears.
The SRB51 inoculum in both studies was
prepared by dilution of lyophilized SRB51 vac-
cine (Colorado Serum Company, Denver, Col-
orado, USA) to approximately 131010 colony-
forming units (CFU) on the basis of viability
counts of other vials with the same lot number.
Briefly, reconstituted vaccine was serially dilut-
ed in saline and plated on tryptose agar plates
430 JOURNAL OF WILDLIFE DISEASES, VOL. 40, NO. 3, JULY 2004
containing 5% bovine serum, and standard
plate counts were conducted to determine vi-
ability. Standard plate counts of the inoculum
determined the SRB51 dosage administered.
At 8 wk after oral inoculation in study 1,
bears in both SRB51 and control treatments
were anesthetized with Telazol, as previously
described, and blood was obtained via jugular
venipuncture for serologic analysis.
Necropsy
At 23 wk after oral inoculation in study 1,
SRB51-inoculated bears and their cubs and
control bears were anesthetized in their dens
with Telazol via pneumatic dart or intramus-
cular injection. In a similar manner, SRB51-in-
oculated bears and their cubs in study 2 were
anesthetized in their dens at 21 wk after inoc-
ulation. After sedation, blood was obtained for
serology via jugular venipuncture. All SRB51-
inoculated bears and their cubs in both studies
were then euthanized with intravenous injec-
tions of sodium pentobarbitol (Sleepaway, Ft.
Dodge Labs, Ft. Dodge, Iowa) and necropsied.
Control bears were only sedated in study 1, and
all were allowed to recover from anesthesia.
Maternal tissues collected at necropsy from
SRB51 vaccinates for both bacteriologic and
histologic evaluation included: mammary gland,
liver, lung, spleen, kidney, and uterus. Maternal
samples obtained for bacteriologic examination
only included vaginal, conjunctival, nasal, and
rectal swabs; blood; tonsil; and hepatic, parotid,
popliteal, prescapular, medial retropharyngeal,
mediastinal, internal iliac, mandibular, mesen-
teric, and supramammary lymph nodes. Bac-
teriologic samples from cubs included blood,
spleen, lung, liver, kidney, mesenteric lymph
node, and cerebrospinal fluid. Histologic tech-
niques also were used to examine cardiac sam-
ples from both sows and cubs and fetal liver,
spleen, and kidney samples. All tissues collect-
ed for histologic evaluation were fixed in neu-
tral buffered 10% formalin embedded in par-
affin, sectioned at 5 mm, and stained with he-
matoxylin and eosin. Special stains (Congo red,
silver, acid-fast, or Prussian blue; Sheehan and
Hrapchak, 1980) were performed on sections
in which examination of hematoxylin and eosin
sections suggested that additional histologic
techniques were warranted.
Bacteriologic evaluation
Tissue samples were triturated in saline and
placed on tryptose agar plates containing 5%
bovine serum and a selective medium for
SRB51 (RBM) that contains antibiotics, includ-
ing rifampicin (Hornsby et al., 2000). The an-
tibiotics in RBM media minimized growth of
contaminants without inhibiting growth of
SRB51, thereby enhancing the ability to detect
small numbers of SRB51 within samples.
Following incubation of plates at 37 C and
5% CO2 for 7 days, SRB51 was identified by
colony morphology, growth characteristics (Al-
ton et al., 1988), and resistance to rifampicin
(Schurig et al., 1991). Isolates were confirmed
as SRB51 in a polymerase chain reaction (PCR)
procedure with the use of primers specific for
SRB51 (Vemulapalli et al., 1999). Briefly, the
reaction mixture consisted of heat-killed cells,
0.2 mM nucleotide mix (Boehringer Mann-
heim, Mannheim, Germany), 13 PCR Buffer
II (Perkin Elmer, Branchburg, New Jersey,
USA), 2.5 U/ml DNA polymerase (Amplitaq
Gold, Perkin Elmer), and 1.5 mM MgCl com-
bined with 0.2 mM per reaction of specific
primers (upstream primer GCAACGGTGTTC-
TTCCACTC and downstream primer GTAT-
CAGGCTACGCAGAAGG) for B. abortus
omp2A as selected from analysis of its se-
quence (Ficht et al., 1989). The samples, in-
cluding positive and negative controls, were cy-
cled (30 sec at 95 C, 30 sec at 44 C, 1 min at
72 C) 30 times in a thermocycler (MJ Research
Inc., Watertown, Massachusetts, USA). Prod-
ucts were analyzed by electrophoresis on a
1.5% agarose gel after staining with ethidium
bromide.
Serologic evaluation
Blood samples were allowed to clot, and se-
rum was separated by centrifugation. Serum
was divided into 1-ml aliquots, frozen, and
stored at 270 C. Serologic titers of animals to
SRB51 were determined by dot-blot assay (Ol-
sen et al., 1997) in which g-irradiated SRB51
is used as antigen, and peroxidase-labeled pro-
tein A (1:100 dilution; Kirkegaard and Perry
Laboratories Inc., Gaithersburg, Maryland,
USA), rabbit anti-canine IgG heavy- and light-
chain antibodies (1:5,000; Jackson Immunore-
search Laboratories, West Grove, Pennsylva-
nia), or rabbit anti-goat IgG heavy- and light-
chain antibodies (1:10,000 dilution; Jackson
Immunoresearch Laboratories) were used as
secondary antibodies.
Statistics
Because of logarithmic conversions, titers
with a value of 0 were analyzed with a value of
1. Serologic responses of bears in both studies
were compared over all times by an analysis of
variance procedure with titers to each second-
ary antibody evaluated separately (SAS Insti-
tute Inc., Cary, North Carolina, USA). Means
for individual treatments were separated by a
least significant difference procedure (P,0.05).
OLSEN ET AL.—SAFETY OF RB51 IN BLACK BEARS 431
FIGURE 1. Serologic responses of nonvaccinated (control) bears or bears orally inoculated with 1.4–
3.131010 colony forming units of SRB51 to g-irradiated SRB51 in a dot-blot assay in which peroxidase-
conjugated protein A, rabbit anti-canine IgG, or rabbit anti-goat IgG were used as secondary antibodies.
Antibody responses were determined before vaccination (time 0; n512 for SRB51 and n56 for control treat-
ments), 8 wk after vaccination (n56 for SRB51 and control treatments), and at necropsy 21 or 23 wk after
vaccination (n512 for SRB51 and n56 for control treatments). Responses are presented as mean titer6SEM.
A 95% confidence interval was predicted from
the bacteriologic data by a Clopper-Pearson
procedure (SAS Institute Inc.).
RESULTS
The concentration of viable SRB51 bac-
teria within the inocula for studies 1 and
2 were determined by standard plate
counts to be 1.431010 and 3.131010 CFU,
respectively.
At necropsy in the first study, twin cubs
were found with one SRB51-inoculated
bear and one nonvaccinated bear. No oth-
er bears in SRB51 or control treatments
were pregnant or had cubs present at the
time of evaluation. In study 2, three of the
five paired controls had cubs (two triplets
and one set of twins) at the time of nec-
ropsy, whereas three of six SRB51-inocu-
lated bears had cubs (three sets of twins).
At necropsy in study 1, 23 wk after vac-
cination, SRB51 was recovered from the
internal iliac, retropharyngeal, and mes-
enteric lymph nodes from one of six
SRB51-inoculated bears. This bear had no
cubs and was not pregnant at the time of
evaluation. The SRB51 vaccine strain was
not recovered from fetal tissues obtained
at necropsy from the two cubs of the
SRB51 vaccinate. In study 2, SRB51 was
not recovered from any tissue obtained at
necropsy from any of the six SRB51-inoc-
ulated bears or their cubs.
Regardless of secondary antibody, titers
of bears orally inoculated with SRB51 did
not differ (P.0.05) from dot-blot titers of
nonvaccinates at any sampling time (Fig.
1). In both studies, one of six SRB51-in-
oculated bears had titers at necropsy that
were four dilutions greater than the titer
of paired serum obtained before vaccina-
tion. In both of these bears, the highest
titers were noted when the anti-canine an-
tibody was used as the secondary antibody.
When compared with the serum obtained
before inoculation, the bear that was cul-
ture positive for SRB51 at necropsy in
432 JOURNAL OF WILDLIFE DISEASES, VOL. 40, NO. 3, JULY 2004
study 1 did not demonstrate a significant
increase in titer to SRB51 at 8 wk after
inoculation or at necropsy with any of the
secondary antibodies used in this study.
With the exception of small, subacute,
multifocal tonsillar abscesses in one
SRB51-inoculated sow in study 1, no gross
lesions were noted at necropsy.
Histologic evaluation of lung sections
from eight SRB51-vaccinated adult bears
(five in study 1 and three in study 2) re-
vealed multifocal, perivascular, and, to a
lesser extent, peribronchiolar accumula-
tions of moderate numbers of histiocytes.
The cytoplasm of these histiocytes were
expanded by an amorphous basophilic ma-
terial that also contained numerous bire-
fringent crystals. In some adult bears, mul-
tifocal accumulations of low numbers of
lymphocytes were found in interstitial ar-
eas of the kidney or were randomly dis-
tributed in hepatic parenchyma.
Special stains of histologic sections of
lung indicated that lesions were compati-
ble with pneumoconiosis. The etiology of
the particulate material in pulmonary sec-
tions was not conclusively determined but
most likely was inhaled environmental de-
bris. With these exceptions, no significant
histologic lesions were noted in examina-
tion of remaining maternal tissues and all
fetal tissues.
DISCUSSION
The results of these studies suggest that
oral exposure to 1010 CFU of SRB51 is not
associated with adverse clinical, histologic,
or reproductive effects in bears. Although
bears in both treatments had poor concep-
tion rates in the first study, reproduction
performance of bears in both treatments
in each study were similar.
Our data also suggest that in a small
percentage of bears (one of 12, 8%; 95%
confidence interval, 0.2–38.5%), SRB51
can persist for up to 23 wk after oral in-
oculation. Failure to recover SRB51 from
mucosal swabs, mammary gland, supra-
mammary lymph nodes, or uterine and fe-
tal tissues in any SRB51-vaccinated bear in
our studies suggests that if bears are orally
exposed to SRB51, it is unlikely that they
will transmit the strain laterally or verti-
cally. When evaluated with the bacterio-
logic data, the poor serologic responses
noted in our studies might indicate SRB51
is not well adapted to colonize or replicate
in tissues of black bears and therefore does
not effectively induce immunologic re-
sponses or cause clinical effects. Alterna-
tively, SRB51 might not efficiently attach
and invade mucosal surfaces of black bears
after oral exposure.
Data from others have suggested that
bears in areas with endemic brucellosis
might undergo at least transient infection
with field strains of B. abortus. Grizzly
(Ursus arctos horribilis) and black bears
sampled in Montana (USA) near Yellow-
stone National Park demonstrated positive
responses on a panel of brucellosis sero-
logic tests, whereas samples from bears in
northern Montana, in an area without bru-
cellosis, were negative on the same panel
of tests (K. Aune, pers. comm.). Although
there are no data at this time to determine
whether exposure to B. abortus has any ef-
fects on survival or reproduction of bears
in the GYA, our data and studies in other
species (Januszewski et al., 2001; Kreeger
et al., 2002) suggest that SRB51 is safe in
black bears and is unlikely to be more
pathogenic in bears than B. abortus field
strains that are currently present in the
GYA environment.
ACKNOWLEDGMENTS
We thank A. Duit, D. Pringle, A. Jensen, K.
Casey, and D. Buffington for their technical as-
sistance.
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Received for publication 3 September 2003.


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Safety Of \u3ci\u3eBrucella Abortus\u3c/i\u3e Strain Rb51 In Black Bears

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