Immune Responses and Efficacy After Administration of a Commercial Brucella abortus Strain RB51 Vaccine to Cattle*

Transcription

1 Immune Responses nd Efficcy After Administrtion of Commercil Brucell bortus Strin RB51 Vccine to Cttle* Steven C. Olsen, DVM, PhD United Sttes Deprtment of Agriculture Bcteril Diseses of Livestock Reserch Unit Agriculturl Reserch Service Ntionl Animl Disese Center 2300 Dyton Avenue Ames, IA ABSTRACT Brucell bortus strin RB51 (SRB51) is newly pproved live vccine to protect cttle ginst brucellosis. The purpose of this study ws to evlute the immunologic responses of cttle to commercilly vilble SRB51 vccine nd to chrcterize the efficcy of the vccine to protect ginst bortion or infection fter midgesttionl chllenge with virulent B. bortus strin 2308 (S2308). All cttle were psture bred, nd pregnncy ws confirmed by rectl plption. Pregnnt cttle were intrconjunctivlly chllenged with colonyforming units (CFUs) of S2308 t 180 dys gesttion. Serologic responses were monitored in ll heifers fter vccintion using the stndrd tube gglutintion test nd dot-blot ssy using killed SRB51 s ntigen. In one study, 3-month-old Hereford heifers were subcutneously inoculted with 10 9 CFU or CFU of commercilly vilble SRB51 vccine or 2 ml of 0.15M sodium chloride *Nmes re necessry to report fctully on vilble dt; however, the USDA neither gurntees nor wrrnts the stndrd of the product, nd the use of the nme by the USDA implies no pprovl of the product to the exclusion of others tht lso my be suitble. (sline). In this study, four of eight nonvccintes, two of four 10 9 CFU SRB51 vccintes, nd two of CFU SRB51 vccintes borted fter midgesttionl chllenge with virulent S2308. The chllenge strin ws recovered t necropsy from mternl or fetl tissue from six of nine nonvccintes, four of four 10 9 CFU SRB51 vccintes, nd seven of 14 heifers vccinted with CFU of SRB51. In seprte study, 6-month-old Hereford heifers were subcutneously inoculted with CFU of SRB51 or sline. Peripherl blood mononucler cells prolifertive responses to γ- irrdited SRB51 were monitored. In this study, three of seven nonvccintes nd one of CFU SRB51 vccintes borted. The S2308 chllenge strin ws recovered from mternl or fetl tissue of five of seven nonvccintes nd seven of 18 SRB51 vccintes. In both studies, cttle vccinted with CFU of SRB51 hd greter (P <.05) ntibody responses to SRB51 t 4 nd 8 weeks fter vccintion thn nonvccintes. SRB51-vccinted cttle hd greter lymphocyte prolifertive responses to killed SRB51 t 10, 12, 14, nd 16 weeks when compred with nonvccintes. The dt presented in this study indicte tht CFU of the commercil SRB51 vc- 183

2 Veterinry Therpeutics Vol. 1, No. 3, Summer 2000 cine ws highly efficcious (P <.02) in preventing Brucell-induced bortion or fetl infection. Considering tht fetl infection nd bortion re the predominnt mechnisms for trnsmission of brucellosis, our dt suggest tht the commercilly vilble SRB51 vccine will be efficcious in preventing bortion nd controlling brucellosis in cttle under field conditions. However, incidence of mternl infection with Brucell t necropsy did not differ (P >.05) mong CFU SRB51 vccintes nd nonvccintes. INTRODUCTION In Februry 1996 new brucellosis vccine, Brucell bortus strin RB51 (SRB51), ws conditionlly licensed by the USDA Animl nd Plnt Helth Inspection Service (APHIS) for use in cttle in the United Sttes. Conditionl pprovl of commercil vccine is grnted under expedited conditions in order to meet n emergency condition, limited mrket, locl sitution, or other specil circumstnces. The conditionl pprovl is only grnted when purity nd sfety of the product hve been demonstrted nd vilble dt re sufficient to give resonble expecttion of efficcy. In this instnce, the decision on efficcy ws bsed on published dt obtined in cttle experiments t the Ntionl Animl Disese Center with the ARS/1 strin of SRB51, the mster seed source for the commercil product. 1 6 However, for APHIS to grnt full licensure for the commercil vccine, dditionl scientific dt were required tht demonstrted the efficcy of the commercil vccine. The SRB51 strin is rough mutnt of B. bortus 7 tht does not induce ntibody responses tht rect with conventionl brucellosis surveillnce tests. 5,8 When compred with the B. bortus strin 19 vccine, SRB51 offers similr efficcy in cttle without confounding the bility to detect individuls infected with field strins of B. bortus. 2,3 Becuse the SRB51 vccine is being widely used in the United Sttes nd other countries nd it is recognized s n officil clfhood vccine ginst brucellosis, it ws importnt to evlute the commercil product so tht full licensure of the vccine might be obtined. The purpose of the studies reported here ws to evlute the sfety nd efficcy of commercilly vilble vccine mde from SRB51. MATERIALS AND METHODS Brucell bortus Culture A mster seed stock of SRB51 ws obtined from Dr. Gerhrdt Schurig (Virgini Tech, Blcksburg, VA). After one pssge on tryptose gr the seed stock ws designted ARS/1. For experimentl use in serologic or lymphocyte prolifertion ssys, SRB51 (ARS/1) bcteri were grown on tryptose gr (Difco Lbortories, Detroit, MI) for 48 hours t 37 C. For the dot-blot ssy, SRB51 suspensions ( colony-forming units [CFU]/mL) were inctivted by γ-irrdition ( rds). After irrdition, suspensions were wshed in 0.15M sodium chloride (sline) nd stored in 1 ml liquots t 70 C. For the chllenge portion of the experiment, B. bortus strin 2308 (S2308) (Brucell culture collection, Ntionl Animl Disese Center, Ames, IA) ws grown on tryptose gr for 48 hours t 37 C. The bcteri were hrvested from the gr by spirtion using sline. Suspensions of S2308 were djusted by use of spectrophotometer (Busch nd Lomb, Rochester, NY) nd concentrtions of vible bcteri were determined by plte counts. For vccintion of cttle, commercilly prepred product (Colordo Serum Compny, Denver, CO) derived from ARS/1 ws used ccording to the product outline. The vccine ws diluted in sline to pproximtely 10 9 or CFU bsed on stndrd plte counts on other vils with the sme lot number. After dilution, 184

3 the concentrtion of vible bcteri within the inoculum ws determined by stndrd plte counts. Animls nd Inocultion In two seprte studies, week-old nd 28 5-month-old Hereford heifers, respectively, were purchsed from brucellosis-free herds nd rndomly ssigned to tretments. In the first study, fter cclimtion for 2 weeks, 15 of the 30 heifers were vccinted subcutneously (SQ) with CFU of SRB51, six heifers were SQ inoculted with CFU of SRB51, nd nine heifers were inoculted SQ with 2 ml of sline t 12 weeks of ge. After cclimtion for 4 weeks in the second study, 20 of the 28 heifers were SQ innoculted with CFU of SRB51 nd eight heifers were SQ inoculted with sline t 6 months of ge. All vccintions were dministered in the left cervicl region drined by the superficil cervicl (prescpulr) lymph nodes. Serologic Evlution Blood smples were collected by jugulr venipuncture before vccintion nd t 4, 8, nd 12 weeks fter vccintion. Blood ws llowed to clot for 12 hours t 4 C nd centrifuged. Serum ws divided into 1-mL liquots, frozen, nd stored t 70 C. Serologic titers to Brucell were determined by stndrd tube gglutintion test (STAT). 9 Serologic titers to SRB51 were determined using previously described ntibody dot-blot ssy in which γ-irrdited SRB51 is used s ntigen. 10 Smples were coded such tht serologic evlutions were conducted blindly. Preprtion of Peripherl Blood Mononucler Cells nd Lymph Node Cells for Lymphocyte Prolifertion Assys Five SRB51-vccinted nd five sline-inoculted heifers were rndomly selected t the initition of the 6-months-of-ge vccintion study to evlute prolifertive responses of peripherl blood mononucler cells to SRB51. At 10, 12, 14, nd 16 weeks fter vccintion, blood ws obtined from the jugulr vein of selected heifers nd plced into n cid-citrte dextrose solution. Peripherl blood mononucler cells were enriched by density centrifugtion using Ficoll-sodium ditrizote grdient. Fifty µl of ech cell suspension contining peripherl blood mononucler cells were dded to ech of two seprte flt-bottom wells of 96-well microtiter pltes tht contined 100 µl of RPMI 1640 medium only or 1640 medium contining γ-irrdited SRB51 (10 5 to 10 9 bcteri per well). Cell cultures were incubted for 7 dys t 37 C under 5% CO 2. Microtiter pltes were plced on MicroShker II (Dyntech Lbortories, Inc, Alexndri, VA) every 2 dys during the incubtions nd mixed t n instrument setting of 3.5 for 1 minute. After 7 dys incubtion, cell cultures were pulsed with 1.0 µci of [ 3 H] thymidine per well for 18 hours. Cells were hrvested onto glss filter mts nd counted for rdioctivity in liquid scintilltion counter (1450 Microbet scintilltion counter, Wllc, Inc, Githersburg, MD). Cell prolifertion results were converted to logrithm of the counts per minute nd stimultion indices (counts per minute [cpm] of wells contining ntigen/cpm in the bsence of ntigen) for sttisticl comprisons. Experimentl Brucell Chllenge Animls were rised to dulthood nd psture bred between 14 nd 18 months of ge. Breeding dtes were determined by observtion, nd pregnncy ws confirmed by rectl plption between 60 nd 90 dys gesttion. After trnsfer to biolevel 3 11 continment fcility, pregnnt nimls were intrconjunctivlly chllenged t 180 dys gesttion with 1 185

4 Veterinry Therpeutics Vol. 1, No. 3, Summer CFU S2308 suspended in 100 µl of sline (50 µl/eye). Conjunctivl swbs were obtined from ll cttle t 2 nd 5 dys fter experimentl chllenge exposure to verify persistence of the chllenge strin of B. bortus. Abortion ws defined s premture expulsion from the uterus of the embryo or nonvible fetus. Immeditely fter bortion or 1 week before estimted prturition, cows were euthnized with intrvenous dministrtion of sodium pentobrbitol (Sleepwy, Fort Dodge Lbs, Ft. Dodge, IA). Becuse mny contminting bcteri impir identifiction of Brucell, nonborting cttle were euthnized before prturition to prevent contmintion of fetl or uterine tissue smples. The most common mternl tissues for isoltion of Brucell re reproductive, mmmry, nd lymphoreticulr tissues. In the study reported here, mternl smples obtined t necropsy included bronchil, heptic, internl ilic, mndibulr, protid, prescpulr, retrophryngel, nd suprmmmry lymph nodes; blood; milk from ll four qurters; mmmry glnd tissue from ll four qurters; plcentome or cruncle; spleen; liver; nd vginl swb. Fetl smples obtined included spleen, lung, blood, bronchil lymph node, gstric contents, nd rectl swbs. Swbs nd fluid smples were inoculted directly on tryptose gr pltes contining 5% bovine serum. Tissue smples were triturted in 0.15M NCl using tissue grinder nd plted on tryptose gr contining 5% bovine serum. After incubtion t 37 C nd 5% CO 2, B. bortus bcteri were identified on the bsis of colony morphology nd growth chrcteristics. 9 Cttle were considered to be infected if single colony of B. bortus ws recovered from ny smple obtined t necropsy. ANALYSIS Serologic dt were converted to the logrithm of the titer for nlysis. Serologic responses of clves in both studies were compred over ll times using two-wy nlysis of vrince model. Differences between tretments in prolifertive responses to γ-irrdited bcteri t ech smpling time were compred by generl liner model procedure (SAS Institute, Inc, Cry, NC). Mens for individul tretments were seprted by use of lest significnt difference procedure (P <.05). Fisher s exct test ws used to compre the incidence of bortion or S2308 infection fter S2308 chllenge. RESULTS No clinicl illness or dverse rections were noted in ny heifer t ny time fter vccintion with 10 9 or CFU of SRB51. Serologic Evlution Cttle vccinted with SRB51 in both studies remined negtive on the stndrd tube gglutintion test t ll times fter inocultion. Dotblot titers of SRB51-vccinted nd unvccinted clves did not differ in either study before vccintion. Clves vccinted t 12 weeks of ge with CFU of SRB51 hd greter (P <.05) ntibody titers on the dot-blot test t 4 nd 8 weeks but not 12 weeks fter vccintion when compred to nonvccintes (Figure 1). Dot-blot titers of clves inoculted with 10 9 CFU of SRB51 t 12 weeks of ge did not differ t ny smpling time from titers of nonvccintes. In the second study, heifers vccinted t 6 months of ge with CFU of SRB51 hd greter (P <.05) dot-blot titers t 4, 8, 12, nd 16 weeks fter vccintion when compred with titers of nonvccintes (dt not shown). Lymphocyte Prolifertion Assys Heifers vccinted with CFU of SRB51 t 6 months of ge hd greter (P <.05) prolifertive responses nd stimultion indices to γ-irrdited SRB51 bcteri t 10, 12, 14, nd 186

5 Dot-blot Titers Control CFU RB CFU RB Weeks fter Vccintion 16 weeks fter vccintion s compred to responses of nonvccintes (Figure 2). b Figure 1. Serologic responses of cttle vccinted t 12 weeks of ge with 10 9 or colony-forming units of SRB51 (n = 15), or sline (n = 9), to γ-irrdited SRB51 in dot-blot ssy. Responses re presented s men titer ± SEM. Mens with different superscripts re significntly different ( P <.05). Log CPM 10 1 Controls RB Weeks fter Vccintion Figure 2. Prolifertive responses of peripherl blood mononucler cells from cttle vccinted t 6 months of ge with CFU of SRB51 (n = 5/smpling time) or sline (n = 5/smpling time) when exposed to 10 8 CFU of γ- irrdited SRB51 t 10, 12, 14, or 16 weeks fter innocultion. Cells were incubted t 37 C nd 5% CO 2 for 7 dys nd pulsed for 18 hours with [ 3 H] thymidine. Results re expressed s men log 10 cpm ± SEM. Denotes mens of SRB51-vccintes significntly different ( P <.05) from responses of nonvccintes t tht smpling time. c c c Experimentl Chllenge nd Bcteriologic Evlution SRB51 Vccintion t Twelve Weeks of Age Rectl plption indicted tht 14 of CFU SRB51 vccintes, four of six 10 9 CFU SRB51 vccintes, nd nine of nine nonvccintes were pregnnt. After intrconjunctivl chllenge of pregnnt heifers with S2308, the chllenge strin ws recovered from conjunctivl swbs t both 2 nd 5 dys fter chllenge in ll but four heifers. In two heifers vccinted with CFU of SRB51 nd one nonvccinted heifer, S2308 ws recovered only t 2 dys, wheres in the remining heifers vccinted with CFU of SRB51 swbs were culture positive for S2308 only t 5 dys fter chllenge. At 5 weeks fter chllenge, two SRB51 vccintes were lost becuse of stem line pipe brek t the biocontinment fcility. At 7 weeks fter chllenge, one nonvccinte hd to be euthnized becuse of lmeness. Four of the remining eight nonvccinted cttle borted between 7 nd 9 weeks fter chllenge. Two of the four heifers in the 10 9 CFU SRB51 tretment borted between 8 nd 9 weeks fter chllenge. One heifer in the SRB51 tretment ws found t necropsy to not be pregnnt. Two of the remining 11 heifers vccinted with CFU of SRB51 borted between 8 nd

6 Veterinry Therpeutics Vol. 1, No. 3, Summer 2000 TABLE 1. Recovery of Brucell bortus from Tissue Obtined t Necropsy After Midgesttionl Intrconjunctivl Chllenge of Nonvccinted nd B. bortus strin RB51 (SRB51) Vccinted Cttle with Colony-Forming Units (CFU) of B. bortus Strin 2308 Number Recovery of S2308 Tretment N Pregnnt Abortions Mternl Tissue Fetl Tissue Study 1 (3 months of ge) Sline 9 9 4/8 * 6/9 4/8 * 10 9 CFU SRB /4 4/4 2/ CFU SRB /11 7/14 2/11 Study 2 (6 months of ge) Sline 8 7 3/7 5/7 3/ CFU SRB /16 7/17 1/16 Overll Sline /15 10/15 7/ CFU SRB /26 12/26 3/ CFU SRB /4 4/4 2/4 Excludes one heifer euthnized becuse of lmeness t 7 weeks fter chllenge. Excludes two heifers lost to stem line pipe brek t 5 weeks fter chllenge nd one heifer found t necropsy to not hve been pregnnt. Excludes one heifer found t necropsy to not hve been pregnnt. Excludes ll nonpregnnt heifers, euthnized heifers, heifers lost premturely becuse of unrelted cuses, nd one SRB51 vccinte tht ws culture negtive for Brucell on eye swbs fter chllenge nd did not seroconvert on the stndrd tube gglutintion test. The overll incidence of bortion or fetl infection in CFU SRB51 vccintes is reduced (P <.02) when compred to overll dt for nonvccintes. weeks fter chllenge (Tble 1). All nonvccintes nd SRB51 vccintes demonstrted seroconversion on the stndrd tube gglutintion test by 4 weeks fter chllenge. In most nimls tht did not bort, stndrd tube gglutintion titers were negtive t necropsy, wheres nimls tht borted demonstrted high titers (>800) t necropsy. For regultory purposes, cttle with stndrd tube gglutintion titers of 1:100 or greter re clssified s rectors. The S2308 chllenge strin ws recovered t necropsy from mternl nd/or fetl tissue of six of the nine nonvccinted cttle including ll nimls tht borted. In heifers vccinted with 10 9 CFU of SRB51, the S2308 chllenge strin ws recovered from mternl tissue of ll heifers nd from fetl tissue obtined from the two bortions. In the CFU SRB51 group, the S2308 chllenge strin ws recovered from mternl nd fetl tissue from both bortions nd mternl tissue of five other heifers including the nonpregnnt heifer. In ll culturepositive SRB51 vccintes tht did not bort two heifers in the 10 9 CFU nd five heifers in the CFU tretment S2308 ws recovered in low numbers only from mndibulr or protid lymph nodes. SRB51 ws not recovered t necropsy from ny smple. 188

7 SRB51 Vccintion t Six Months of Age After psture breeding, 17 of 20 SRB51 vccintes nd seven of eight nonvccintes were determined to be pregnnt by rectl plption. After intrconjunctivl chllenge with S2308, the chllenge strin could be recovered from conjunctivl swbs t both 2 nd 5 dys in ll but four heifers. In two SRB51 vccinted heifers, S2308 ws not recovered from conjunctivl swbs t either time point. In two other SRB51 vccintes, S2308 ws recovered from conjunctivl swbs only t 5 dys fter chllenge. With the exception of four heifers, ll vccintes nd control heifers seroconverted on the stndrd tube gglutintion test by 4 weeks fter chllenge. Three of the heifers tht did not seroconvert [one nonvccinte nd two SRB51- vccintes] hd S2308-positive eye swbs t 5 dys fter chllenge. The remining heifer tht did not seroconvert, n SRB51 vccinte, hd negtive eye swbs for S2308 t both 2 nd 5 dys fter chllenge. All SRB51 vccintes tht did not bort hd trnsient seroconversion on the stndrd tube gglutintion test nd were seronegtive t necropsy. Three of seven heifers in the control group borted between 6 nd 10 weeks fter S2308 chllenge. One SRB51-vccinted heifer borted 11 weeks fter chllenge. The S2308 chllenge strin ws recovered from mternl or fetl tissue obtined t necropsy from five of seven control heifers. In comprison, S2308 ws recovered t necropsy from mternl tissue of seven of 18 SRB51 vccintes, including one heifer tht ws not pregnnt t necropsy. In four of the SRB51 vccintes tht did not bort, S2308 ws recovered in low numbers only from mndibulr or protid lymph nodes. In two SRB51 vccintes, tissue positive for S2308 t necropsy included the mmmry glnd, suprmmmry lymph node, nd retrophryngel lymph node, in ddition to mndibulr nd/or protid lymph nodes. Only fetl tissue from the SRB51 vccinte tht borted ws culture positive for the S2308 chllenge strin. SRB51 ws not recovered from ny smple obtined t necropsy. Efficcy of SRB51 For nlysis of efficcy of SRB51, nonpregnnt nimls, noninfected nimls, nd nimls lost premturely to other cuses in either study were excluded. When dt from both studies were combined with these exclusions, sttisticl nlysis indicted tht clfhood vccintion with CFU of SRB51 significntly reduces (P <.02) the incidence of bortion or fetl infection with S2308 when compred with controls. However, incidence of recovery of S2308 from mternl tissue t necropsy did not differ (P >.05) mong nonvccintes nd heifers tht were vccinted t 12 weeks or 6 months of ge with CFU of SRB51. DISCUSSION Results of this study suggest tht the commercil SRB51 vccine is cliniclly sfe nd induces immune responses tht protect ginst fetl infections or bortions cused by B. bortus. The limited dt obtined in the studies reported here suggest tht clfhood vccintion with 10 9 CFU of SRB51 does not induce protection ginst brucellosis. Becuse clfhood vccintion with CFU of SRB51 did protect ginst experimentl chllenge with virulent B. bortus strin, our dt suggest tht the current recommended clfhood dosge of SRB51 ( ) is pproprite. Becuse shedding of bcteri t the time of bortion or birth of Brucell-infected fetus is the predominnt method for trnsmission of brucellosis, our dt suggest tht the commercil SRB51 vccine will be highly efficcious under field conditions. 189

8 Veterinry Therpeutics Vol. 1, No. 3, Summer 2000 Efficcy of brucellosis vccines under field conditions hs been found to be greter thn efficcy noted under experimentl conditions 12 nd most likely reflects differences under field conditions in exposure to brucellosis. Therefore, it is nticipted tht protection induced by the commercil SRB51 vccine under field conditions will be greter thn protection from bortion nd infection noted in the experimentl studies reported here. Dt suggest tht long-term protection of cttle ginst Brucell infections is medited through cell-medited rther thn humorl immune responses. 13 Although blstogenic responses do provide evidence of stimultion of cell-medited immunity, others hve found no correltion between the mgnitude of lymphocyte prolifertive responses nd protection ginst Brucell infection. 14,15 Although dt in the studies reported here did not demonstrte significnt reductions in mternl infections, previous studies in our lbortory hve demonstrted tht clfhood vccintion with SRB51 significntly (P <.05) reduces recovery of S2308 t necropsy, 12 weeks fter midgesttionl chllenge, when compred with nonvccintes (i.e., 20% nd 60% S2308 recovery, respectively). 2,3,4 Becuse previous studies in our lbortory hve used SRB51 in log phse growth, one explntion for the difference my be tht the commercil vccine used in the current study ws lyophilized product. It should lso be noted tht the dosge used for vccintion in the current study ws t the minimum recommended clfhood dosge, nd the possibility cnnot be excluded tht higher dosges of the commercil product might enhnce protective responses, lthough t this time there is no study plnned to ddress this. In previous study in our lbortory, cttle vccinted t 7 months of ge with or CFU of SRB51 in log phse did not differ in their protection ginst infection following experimentl chllenge. 4 Dt from previous studies in our lbortory lso hve suggested tht efficcy of SRB51 is greter when clves re vccinted between 6 nd 10 months of ge. This trend is supported by dt presented in this mnuscript tht suggest slightly higher incidence of bortion nd mternl infection in clves vccinted t 12 weeks of ge compred with clves vccinted t 6 months of ge. CONCLUSION The commercilly vilble SRB51 vccine is efficcious in preventing bortions or fetl infections cused by virulent strins of Brucell bortus. ACKNOWLEDGMENTS The uthor thnks Aileen Duit, Allen Jensen, Mrc Knipper, Terry Krusmn, John Lies, Ktie Lies, Drl Pringle, Dennis Weuve, nd Lrry Wright for their technicl ssistnce. This work ws done s prt of officil duties by government employees of the United Sttes Deprtment of Agriculture, Agriculturl Reserch Service, nd therefore copyright resides with the USDA. REFERENCES 1. Cheville NF, Jensen AE, Hlling SM, et l: Bcteril survivl, lymph node chnges, nd immunologic responses of cttle vccinted with stndrd nd mutnt strins of Brucell bortus. Am J Vet Res 53: , Cheville NF, Stevens MG, Jensen AE, et l: Immune responses nd protection ginst infection nd bortion in cttle experimentlly vccinted with mutnt strins of Brucell bortus. Am J Vet Res 54: , Cheville NF, Olsen SC, Jensen AE, et l: Effects of ge t vccintion on efficcy of Brucell bortus strin RB51 to protect cttle ginst brucellosis. Am J Vet Res 57: , Olsen SC, Bricker B, Plmer MV, Jensen AE: Responses of cttle to two dosges of Brucell bortus 190

9 strin RB51: Serology, clernce nd efficcy. Res Vet Sci 66: , Stevens MG, Hennger SG, Olsen SC, Cheville NF: Serologic responses in dignostic tests for brucellosis in cttle vccinted with Brucell bortus 19 or RB51. J Clin Microbiol 32: , Stevens MG, Olsen SC, Cheville NF: Comprtive nlysis of immune responses in cttle vccinted with Brucell bortus strin 19 or strin RB51. Vet Immunol Immunopthol 44: , Schurig GG, Roop RM, Bgchi T, et l: Biologicl properties of RB51: A stble rough strin of Brucell bortus. Vet Microbiol 128: , Olsen SC, Evns D, Hennger SG, et l: Serologic responses of clfhood-vccinted cttle to Brucell bortus strin RB51. J Vet Dign Invest 8: , Alton GG, Jones LM, Angus RD, Verger JM: Techniques for the brucellosis lbortory. Pris, Frnce, Institut Ntionl de l Recherche Agronomique, 1988, pp Olsen SC, Stevens MG, Cheville NF, Schurig G: Experimentl use of dot-blot ssy to mesure serologic responses of cttle vccinted with Brucell bortus strin RB51. J Vet Dign Invest 9: , Biosfety in Microbiologicl nd Biomedicl Lbortories, ed 4. US Deprtment of Helth nd Humn Services, HHS Publiction No , 1999, pp Crwford AB: Experiences in the control of contgious bortion of cttle in the United Sttes. Vet J 100: , Nicoletti P, Winter AJ: The immune response to B. bortus: The cell-medited response to infections in Nielsen K, Duncn JR (eds): Animl Brucellosis, Boc Rton, CRC Press, 1990, pp Chukwu CC: Comprison of the brucellin skin test with the lymphocyte trnsformtion test in bovine brucellosis. J Hyg Lond 96: , Confer AW, Hll SM, Fulkner CB, et l: Effects of chllenge dose on the clinicl nd immune responses of cttle vccinted with reduced doses of Brucell bortus strin 19. Vet Microbiol 10: ,