INFECTION AND IMMUNITY, Nov. 1978, p. 486-491 0019-9567/78/0022-0486$02.00/0 Copyright i 1978 Americn Society for Microbiology Vol. 22, No. 2 Printed in U.S.A. Brucell Antigen Preprtions for In Vitro Lymphocyte Immunostimultion Assys in Bovine Brucellosis JOHN M. B. KANEENE,'* R. K. ANDERSON,2 D. W. JOHNSON,' AND C. C. MUSCOPLAT' Deprtment of Lrge Animl Clinicl Sciences, College of Veterinry Medicine, University ofminnesot, St. Pul, Minnesot 55108,' nd Division of Epidemiology, School of Public Helth, University ofminnesot, Minnepolis, Minnesot 554552 Received for publiction 16 June 1978 Three Brucell ntigen preprtions, Brucell bortus soluble ntigen, B. bortus strin 45/20 enriched protein ntigen, nd B. melitensis enriched protein ntigen, were compred in terms of their bility to induce specific in vitro lymphocyte immunostimultion responses. Lymphocytes were prepred from peripherl blood of cttle with different exposure experiences to B. bortus orgnisms. Lymphocytes were processed by the Ficoll-ditrizote technique, nd results were ssyed for [3H]thymidine incorportion into DNA by liquid scintilltion spectrometry. The three Brucell ntigen preprtions were compred both t their optiml concentrtions of protein nd on n equl-dry-weight bsis. The results were evluted in terms of specific lymphocyte immunostimultion responses induced by ech preprtion nd the degree of correltion with infection. B. bortus soluble ntigen-induced lymphocyte immunostimultion response correlted best with infection sttus followed by B. bortus 45/20 nd B. melitensis enriched protein ntigens. The implictions of these findings re discussed nd hypothesis is proposed. In n erlier report (6) we described the development of Brucell bortus soluble ntigen (BASA). This ntigen ws used in lter studies (5, 7) to evlute brucell infection sttus in cttle by in vitro lymphocyte stimultion test. The findings in these lter studies (5, 7) confirmed our erlier observtions tht BASA induces significnt lymphocyte stimultion responses (LSR) in lymphocytes from B. bortusinfected cttle. These specific LSR correlted with the infection sttus of the niml, nd when these LSR were compred to those induced by the sme BASA in lymphocytes from B. bortus strin 19-vccinted cttle, they were significntly different. Our findings tht BASA induced significnt lymphocyte stimultion in lymphocytes from infected cttle were in greement with reports on lymphocyte stimultion test tht used lymphocytes from humns infected with brucellosis (1, 10, 11) nd different Brucell ntigens. Becuse of the potentil dignostic impliction of the lymphocyte stimultion test in brucellosis in nimls nd humns nd becuse of the criticl importnce of getting well-stndrdized ntigen preprtion, BASA ws reevluted nd compred with 23 other Brucell preprtions. Severl Brucell ntigen preprtions (totl of 24) were investigted for their bility to induce specific LSR in lymphocytes from cttle with different exposure experiences to B. bortus. Three of the 24 preprtions, BASA, B. bortus 45/20-enriched protein ntigen (B45/20PA), nd B. melitensis enriched protein ntigen (BMPA), induced significnt LSR. These three preprtions were lter criticlly compred in terms of their bility to induce specific LSR in lymphocytes from B. bortusinfected cttle. This experiment ws conducted in two prts. The first prt (prt A) compred the three ntigen preprtions by using optiml concentrtions of ech. The concentrtion tht induced mximum specific LSR for ech preprtion (optiml concentrtion) ws determined first. The second prt (prt B) compred the three preprtions on n equl-dry-weight bsis. The three preprtions were compred in terms of inducing minimum stimultion index (SI) of -3.0 in lymphocytes from cttle infected nd those not infected with B. bortus. MATERIALS AND METHODS Animls for prt A. AniInls for prt A included diry cttle of the following exposure experiences to brucell: 5 nturlly infected with field strins of B. bortus; 7 B. bortus strin 19 shedders (previously dult vccinted); 20 clfhood vccinted (nonshedders); nd 9 nonexposed controls (Tble 1 nd 2). Animls for prt B. Animls for prt B included 18 diry cttle tht were nturlly infected with B. 486
VOL. 22, 1978 COMPARISON OF 3 BRUCELLA ANTIGEN PREPARATIONS 487 TABLE 1. Prt A-comprison of different Brucell preprtions, on optiml concentrtion bsis, for bility to induce LSR in lymphocytes from B. bortus-infected nd nonexposed control cttle Animsd BASA B45/20PA BMPA Group no. lb 1 2_1 Type of Brucell isolted flo. i~~~2 2 1 2 1 21 14.4 45.7 5.5 15.4 3.8 24.7 B. bortus biotype 1 51 26.9 20.0 4.0 8.7 11.3 8.1 B. bortus biotype 1 57 8.6 9.9 3.2 7.5 4.2 5.9 B. bortus biotype 2, nturlly infected 74 13.7 18.9 6.7 9.3 13.4 15.6 B. bortus biotype 1, nturlly infected 611 27.0 11.8 10.1 9.1 12.6 16.0 B. bortus biotype 1, nturlly infected 2 33 8.7 6.9 1.6 4.7 1.6 3.1 38 7.4 3.1 4.6 3.9 5.3 3.2 337 7.4 4.9 3.4 1.4 2.5 1.4 B. bortus strin 19, 438 3.9 5.6 2.1 3.9 3.0 6.9 dult vccintes 561 3.0 3.6 2.9 2.1 2.1 1.5 (shedders) 1429 13.4 8.4 8.4 6.7 10.9 7.8 6004 3.9 4.7 6.1 4.1 3.0 6.8 3 130 1.0 0.9 1.1 1.0 0.6 0.4 131 0.7 0.8 0.7 0.2 0.7 0.7 132 0.9 0.7 0.2 0.1 1.0 0.2 133 0.4 0.3 0.3 0.8 1.1 0.3 142 1.2 0.8 0.4 0.7 0.7 0.5 Nonexposed controls 144 1.3 0.3 0.8 0.4 1.2 1.4 145 0.3 1.0 0.7 1.3 0.4 0.7 146 0.2 1.1 1.0 0.6 1.0 0.2 147 0.4 0.2 0.4 0.2 0.3 1.1 Results re expressed s SIs. b 1, First test; 2, second test within 4 months. bortus biotype 1 field strin nd 5 nonexposed controls (Tble 3). Coliection of blood nd preprtion of lymphocyte suspensions. Peripherl blood ws collected nd processed to obtin lymphocyte suspensions s described in our erlier report (6). Culture medium, mitogen, nd ntigens. Tissue culture medium RPMI 1640 (Biolbs, Chicgo, Ill.) ws used in the sme concentrtion s in our erlier study (6). Concnvlin A (Miles-Yed, Rehovot, Isrel) ws used s mitogen, t concentrtion of 2 rg/culture. Culture conditions, lbeling with [3H]thymidine, hrvesting, nd counting were identicl to those previously described (6). Three Brucell preprtions were used s specific ntigens. (i) BASA ws prepred s previously reported (6). BASA is prepred from smooth strin of B. bortus, nd.the optiml stock hd 1 ng of endotoxin per ml by the limulus lyste ssy. B45/20PA nd BMPA re from rough strins of B. bortus nd B. melitensis, respectively. BMPA ws prepred by cold sline extrction (4) nd contined >90% protein s determined by the Lowry method (8), with no detectble lipopolyscchride; B45/20PA ws prepred by cold sline extrction (D. T. Bermn, personl communiction). The remining 21 preprtions were sonic extrcts (20 of them) nd whole cells suspended in broth medi nd were ll from B. bortus. Optiml concentrtions per culture for ech specific ntigen. A concentrtion of 4.4 jg of protein per culture ws optiml for BASA, 2.0,Lg/culture ws optimnl for BMPA, nd 2.0 jug/culture ws optiml for B45/20PA. Procedures for prt A. Ech niml in prt A (Tbles 1 nd 2) ws bled t lest twice within 4- month period, nd blood ws processed s described bove. After cell dilutions were mde, suspensions of cells from ech nimnl (200 pl) were dded in triplicte to wells of microtitrtion culture pltes, using n utomtic dispenser. Concnvlin A, BASA, B45/20PA, nd BMPA were dded in triplicte to wells of microtitrtion culture pltes, using n utomtic dispenser. Concnvlin A, BASA, B45/20PA, nd BMPA were dded, t the optiml concentrtions stted bove, to the pproprite wells of the pltes. Conditions of culture, concentrtion of [methyl-3h]thymidine (Schwrz/Mnn, Orngeburg, N.Y.), termintion of cultures, hrvesting, nd liquid scintilltion counting were s previously reported (6). Procedures for prt B. Ech niml in prt B (Tble 3) ws bled twice within period of 1 month. The procedures were the sme s in prt A except tht BASA, B45/20PA, nd BMPA were used t the sme concentrtion, 4.4 jg (dry weight) of protein per culture. A concentrtion of 4.4,ug (dry weight) of protein per culture for ech preprtion ws used becuse it ws found to be effective in the three preprtions in our erlier preliminry dose response studies. A con-
488 KANEENE ET AL. TABLE 2. Prt A-comprison of different Brucell preprtions, on optiml concentrtion bsis, for bility to induce LSR in lymphocytes from B. bortus strin 19, cl/hood-vccinted cttle Ani- BASA B45/ BMPA Time post- Group 20PA vccintion Group ml when first no. 1b 2b 1 2 1 2 testedc 4 159 2.5 2.6 1.4 1.1 1.6 1.4 164 1.9 1.2 1.1 0.9 2.2 1.7 170 0.3 2.2 0.4 1.2 2.0 1.7 3 weeks 171 1.3 2.1 1.2 1.4 2.1 1.1 172 0.7 1.6 0.8 0.9 2.4 1.3 5 185 0.2 0.4 1.8 1.2 2.1 2.2 186 2.0 0.7 1.9 0.9 1.2 0.5 1-3 months 187 0.8 3.7 1.9 2.6 2.1 2.8 6 190 1.9 1.5 2.0 1.8 2.2 2.4 191 0.9 1.4 1.3 0.7 0.8 1.5 192 0.4 0.7 1.4 1.6 1.1 1.7 3-6 months 194 2.9 2.2 1.1 1.4 1.3 1.3 7 874 2.5 1.4 1.2 1.2 2.1 0.7 895 2.1 1.7 1.2 1.3 3.3 2.7 907 2.2 0.6 1.0 1.3 2.1 1.4 908 0.7 0.9 0.4 0.8 0.6 0.7 918 2.3 2.4 1.2 0.9 1.1 0.9 612 months 924 1.1 0.9 1.0 0.8 1.4 1.6 929 0.6 0.5 0.7 0.9 2.0 1.8 942 0.8 0.9 0.6 0.9 0.9 0.8 Results re expressed s SIs. 1, First test; 2, second test within 4 months. c No Brucell isolted. TABLE 3. INFECT. IMMUN. centrtion s low s 2 ug of protein per culture ws not very stimultory in the cse of BASA, nd concentrtions bove 4.4,ug of protein per culture were slightly inhibitory in the cses of BMPA nd B45/20PA. RESULTS Expression ofresults. Results for both prts of the study re expressed in two wys: (i) difference in counts per minute = men counts per minute of triplicte cultures without mitogen or ntigen substrcted from men counts per minute of triplicte cultures with mitogen or ntigens; (ii) SI = men counts per minute of triplicte cultures with mitogen or specific ntigen/men counts per minute of triplicte cultures without either mitogen or specific ntigen. Results of prt A. Results of prt A re given in Fig. 1 nd 2 nd Tbles 1 nd 2. The results were evluted by using the following clssifiction: SI - 3.0 ws indictive of infection nd SI < 1.0 ws indictive of negtive response, wheres SI < 1.0-3.0 ws indictive of suspicious response. The dt in prt A demonstrte the following points. (i) All of the three specific brucell preprtions clssified ll smples from group 1 nimls s infected. (ii) BASA clssified ll smples from group 2 s infected; B45/20PA nd BMPA clssified four out of Prt B-comprison of different Brucell preprtions, on equl-dry-weight bsis, for bility to induce LSR in lymphocytes from B. bortus-infected nd nonexposed control cttle' BASA B45/20PA BMPA Group Animl no. Type of Brucell isolted lb 2b 1 2 1 2 8 A 1 7.3 8.6 5.5 3.4 8.1 3.3 Eli 4.2 3.2 13.0 1.0 1.5 9.2 E12 5.0 4.7 1.7 4.7 4.0 3.7 G15 3.1 9.7 5.7 9.0 2.3 3.9 H16 4.9 8.2 3.9 3.2 3.1 4.6 H17 21.9 34.7 14.3 10.1 5.2 6.3 H18 5.3 4.8 2.8 6.6 3.7 7.1 H19 4.1 10.7 21.4 5.5 3.4 2.7 B. bortus biotype 1 H20 12.7 20.4 6.6 7.1 3.1 9.6 field strin (ntu1 H21 16.3 25.9 10.1 15.1 7.2 3.4 rel infecte u- 122 7.2 3.1 4.7 3.5 3.9 3.0 rlly infected) I24 2.8 4.7 3.2 6.8 3.0 3.4 I26 11.7 21.6 4.3 3.4 3.2 4.9 J27 21.7 36.0 12.0 11.6 8.7 9.3 J28 8.8 3.3 7.9 9.7 10.2 12.4 L30 6.7 3.9 6.2 3.1 6.9 7.2 UM25 42.7 45.4 13.2 4.3 20.8 12.3 UM57 30.1 27.2 9.6 5.1 6.7 3.2 9 UM973 0.6 0.9 0.2 1.0 0.8 1.0 UM974 1.0 0.9 0.3 1.0 0.6 0.9 UM975 0.8 0.3 0.5 0.3 0.8 0.6 Nonexposed controls UM977 0.7 0.5 0.9 0.7 0.9 1.0 UM978 0.6 0.4 0.9 1.0 1.0 0.7 Results re expressed s SIs. b 1, First test; 2, second test within 1 month.
VOL. 22, 1978.~ -NBASA [-B 45/20 PA 24-7*. - BMPA - 20 F B bortus Field Strin _l- S Br bortus Strin 19 Shedders 16 C Nn-exposed Controls neo co csem l Z 12 o 8- U) z U 04 C F S C F S C F S C GROUPS FIG. 1. Comprison of in vitro LSR induced by the optiml dose of ech of three Brucell ntigen preprtions in lymphocytes from B. bortus-infected nd nonexposed control cttle. Prt A of the study. SEM, Stndrd error of the men. 24 20 16 x - 12 w U, 8 Z 4 0 (- /-BASA Q-B 45120PA E-BMPA A - 3 Week Post-Vc C * 3-36 D -0-12 I-SEM Months Post-Voc A B C D A B C D A B C D GROUPS FIG. 2. Comprison of in vitro LSR induced by the optiml dose of ech of three Brucell ntigen preprtions in lymphocytes from B. bortus clfhoodvccinted diry cttle. Prt A of the study. SEM, Stndrd error of the men. seven smples on the first testing nd five out of seven on the second testing s infected. (iii) All three preprtions clssified ll smples from group 3 s negtive (Tble 1). (iv) Among the clflfood-vccinted cttle (Tble 2), ll three preprtions correctly clssified the nimls of groups 4, 5, 6, nd 7 s not infected on the first testing, but BASA nd BMPA clssified 1 out of 20 s infected on the second testing. Results of this prt were lso evluted by using difference in counts per minute (Fig. 1 nd 2). All three ntigens induced the highest LSR in smples from B. bortus field strin-infected cttle, nd the next highest LSR ws in smples COMPARISON OF 3 BRUCELLA ANTIGEN PREPARATIONS 489 from strin 19 shedders (Fig. 1). Among the clfhood-vccinted cttle (nonshedders), the difference in LSR mong the three ntigens ws not significnt (Fig. 2). Results ofprt B. Results of prt B re given in Fig. 3 nd Tble 3. BASA clssified ll smples from ll 18 nimls of group 8 s infected. B45/20PA did not detect 2 out of 18 on the first test but clssified ll 18 s infected on the second test. BMPA did not detect 2 on the first test nd only 1 out of 18 on the second test. All three ntigens did not induce LSR in lymphocytes from nonexposed control nimls (group 9). In both prts, concnvlin A induced significnt LSR (Fig. 4), which suggests tht the lymphocytes were immunocompetent. Using the summry of results for prts A nd B given in Tble 4, sensitivity nd specificity for ech Brucell ntigen preprtion were clculted with the following formuls: sensitivity = [true positives/(true positives + flse negtives)] x 100; specificity = [true negtives/(true negtives + flse positives)] x 100; where true positive = isoltion of B. bortus nd true negtive = no isoltion of B. bortus. The results of the clcultions re presented in Tble 5. The results (Tble 5) show tht BASA hd the highest sensitivity, 100%, B45/20PA ws next highest, nd BMPA hd the lowest sensitivity. B45/20PA nd BMPA sensitivities, however, were not sttisticlly different. B45/20PA hd the highest specificity, wheres BASA nd BMPA hd the sme specificity. 24 L m 20 0 X 16 w D z 12 U) 8 z 8 4 ZBASA [JB 45/20 PA *BMPA F Br bortus Field Strin C Non-exposed Controls I SEM L~~ F C F C GROUPS FIG. 3. Comprison of in vitro LSR induced by three Brucell ntigen preprtions (equl-dryweight concentrtion, 4.4 pg/culture) in lymphocytes from B. bortus-infected nd nonexposed control diry cttle. SEM, Stndrd error of the men. Prt B of the study. F C
490 KANEENE ET AL. INFECT. IMMUN. I- 2! U)I z I phi ocytes from cttle from both prts of the study. SE'M Stndrd error of the men. P Pr gen Br Br BALSA 100 100 100 97.1 soluble frction of Pseudomons multocid in- pre en( the F. Br bortus Field Stmoin A 3 Weeks Post-VOc test system. Compring ntigen preprtions t S.Br obortus Strin 19 B1-3- their optiml concentrtions of protein hs the Shedders C 3-8 Months PostVDc C. dvntge of compring the ntigens t their Non-exposed Controls SEM42 best. Some critics point out tht using n optimum concentrtion of ech preprtion does not t5. llow comprison on n equl-weight bsis of the ctive principl(s) in the preprtion. Thus,!O, ; ; ; the ntigen preprtions were lso compred on - 15.,, i t. i the bsis of equl dry weights of protein. The min disdvntge of the equl-weight bsis is 10 T,! /, e, /, 4.tht the concentrtion selected my fvor one preprtion nd be suppressive for the others. In this study 4.4 Aig of protein per culture ws 5-7 / / /! 7 J / /) used. Preliminry dose response studies indicted tht this concentrtion ws not suppressive for either B45/20PA or BMPA nd ws previously optiml for BASA. This study, there- F S C A B C D F C fore, utilized both methods. PART PARTART A B of Results these ntigens were evluted to induce in terms specific of the LSP bility tht FIG. 4. LSR induced by concnvlin A in lym- would correlte with presence or bsence of in- fection. In reviewmg the findings tht BASA-induced LSR exhibited greter sensitivity in detecting FABLE 4. Summry of results for prts A nd B infected nimls, it my be hypothesized tht Flu Flse positives Flse negtives both B45/20PA nd BMPA re comprtively pure preprtions contining >90% Ht Antigen prepn 1st protein by 2nd 1st 2nd the Lowry test (4; Bermn, personl communitesting testing testing testing ction) nd no detectble lipopolyscchride. 4 BASA 0/29 1/29 0/12 0/12 BASA, on the other hnd, is not purified B45/20PA 0/29 0/29 3/12 2/12 preprtion. The BASA would be expected to BMPA 0/29 1/29 3/12 2/12 hve wider diversity of ntigens thn the other two preprtions. It could be hypothesized tht BASA 0/5 0/5 2/18 0/18 sensitized lymphocytes interct with specific n- BMPA 0/5 0/5 2/18 1/18 tigens in given preprtion. The preprtion with greter ntigenic diversity could interct Prt A, Optiml concentrtion of ech ntigen. with more lymphocytes, nd, hence, the sumrt B, Equl-dry-weight concentrtion of ech nti- mtion of these responses could result in higher SI (SI_ 3.0 = infection). The comprtively purified preprtions my hve limited..ablė Sensitivity specify odiversity of ntigens nd only rable 5. Sensitivity nd specificity of different myitrc ihte,rsligi few lymphocytes Ivle Brucell ntigen preprtions in both prt A nd my interct with them, resultng i SI vlues prt B <3 (= no infection). prsitivityb Investigtors of other infectious diseses reucell nti- Senitity (%) Specificity (%) ported less lymphocyte stimultory effect by gen prepn 1st testi 2nd test- 1st 2nd test- comprtively purified preprtions. Mheslt tetmg ing testing ing wrn et l. (9) reported tht soniclly treted B4' 5/20PA 85.7 93.8 100 100 duced higher lymphocyte trnsformtion thn IPA 85.7 90.9 100 97.1 preprtion pprently free of endotoxin (rel- BM tively purified). Dniel nd Hinz (3) reported tht purified protein derivtive of Mycobcte- DISCUSSION rium tuberculosis elicited greter in vitro lymphocyte trnsformtion thn single protein nn quntittively evluting ctivity ofntigen tigens isolted from the sme purified protein I prtions contining different mounts of derivtive. Cox et l. (2) used three preprtions dotoxin nd protein, it ws recognized tht from coccidioidin for in vitro lymphocyte tests se differences could ffect the kinetics of the nd skin tests. The preprtion tht ws less
VOL. 22, 1978 purified nd hd less protein content, on dryweight bsis, elicited higher LSR thn the other preprtions. The findings in the present study seem to be in greement with the studies cited bove. It is now generlly believed tht the protein component of microbes is primrily responsible for eliciting cell-medited immune responses (3). It is not cler, however, s to how pure nd how diverse the ntigen should be to chieve mximum sensitivity nd specificity in this system. The dt generted in this study nd those reported elsewhere (2, 3, 9) suggest tht proteins my hve to be diversified before significnt LSR cn be elicited consistently. It my lso be hypothesized tht proteins need to be ttched to other components such s lipid (to form lipoproteins) for better orienttion nd presenttion to the lymphocytes, vi the mcrophges. It should lso be mentioned here tht BASA contins endotoxins, s we reported erlier (6). It is possible tht some, though it is impossible to sy how much, of the stimultory ctivity is due to the endotoxin. The findings in these studies led us to suggest the following lines of further reserch. It might be worthwhile to develop n ntigen tht hs greter sensitivity thn B45/20PA nd BMPA nd which my hve greter specificity thn BASA nd BMPA. The clssifiction level for infection (SI - 3.0) might be lowered to 2 or 2.5 for more highly purified ntigens. Improvements in specificity nd sensitivity, long with chnges in interprettion, might provide even greter vlidity nd relibility of LSR results induced by such ntigens. The determintion of clssifiction level for interprettion should be crefully evluted, using only cttle from which brucelle hve been isolted. The specific line of reserch should be to chrcterize BASA further where lipopolyscchride, protein, crbohydrtes, etc., will be seprted nd purified. Lipopolyscchrides nd proteins would then be compred in terms of their stimultory bilities. The protein frction should lso be chrcterized further, nd different proteins should be compred in terms of their stimultory ctivities. COMPARISON OF 3 BRUCELLA ANTIGEN PREPARATIONS 491 ACKNOWLEDGMENTS This reserch ws supported in prt by grnts from the University of Minnesot Experimentl Sttion nd Veterinry Services, APHIS, U.S. Deprtment of Agriculture. We thnk L. M. Jones nd D. T. Bermn, University of Wisconsin, Mdison, for providing the protein ntigens. We lso thnk E. E. Slone nd D. J. Klusner for technicl help. LITERATURE CMD 1. Bscoul, S., M. Perldi, A. L Mrine, nd C. Lcve. 1976. Stimulting ctivity of brucell frctions in humn lymphocyte trnsformtion test. Immunology 31:717-722. 2. Cox, R. A., E. Brummer, nd G. Lecr. 1977. In vitro lymphocyte responses of coccidioidin skin test-positive nd -negtive persons to coccidioidin, spherulin, nd Coccidioides cell wll ntigen. Infect. Immun. 16:751-755. 3. Dniel, T. M., nd C. F. Hinz. 1974. Rectivity of purified proteins nd polyscchrides from Mycobcterium tuberculosis in delyed skin test nd cultured lymphocyte mitogenesis ssys. Infect. Immun. 9:44-47. 4. Jones, L M., R. Diz, nd A. G. Tylor. 1973. Chrcteriztion of llergens prepred from smooth nd rough strins of Brucell melitensis. Br. J. Exp. Pthol. 54:492-507. 5. Kneene, J. M., R. K. Anderson, D. W. Johnson, C. C. Muscoplt, P. Nicoletti, R. D. Angus, D. E. Pietz, nd D. J. Klusner. 1978. Whole-blood lymphocyte stimultion ssy for mesurement of cell-medited immune responses in bovine brucellosis. J. Clin. Microbiol. 7:550-557. 6. Kneene, J. M., D. W. Johnson, R. K. Anderson, R. D. Angus, D. E. Pietz, nd C. C. Muscoplt. 1978. Kinetics of in vitro bovine lymphocyte immunostimultion with Brucell bortus ntigen. Am. J. Vet. Res. 39:235-239. 7. Kneene, J. M., D. W. Johnson, R. K. Anderson, R. D. Angus, D. E. Pietz, nd C. C. Muscoplt. 1978. Specific lymphocyte stimultion in cttle nturlly infected with strins of Brucell bortus nd cttle vccinted with BrucelU bortus strin 19. Am. J. Vet. Res. 39:585-589. 8. Lowry, 0. H., N. J. Rosebrough, A. L. Frr, nd R. J. Rndll. 1951. Protein mesurement with the Folin phenol regent. J. Biol. Chem. 193:265-275. 9. Mheswrn, S. K., E. S. Theis, nd S. K. Du. 1976. Studies on Psteurell multocid. III. In vitro ssy for cell-medited immunity. Avin Dis. 20:332-341. 10. Renoux, G., A. Plt, J. M. Guillumin, nd G. Renoux. 1975. Hemgglutintion pssive, trnsformtion lymphoblstique et migrtion des leucocytes ppliquees nd dignostic des brucellosis. Interntionl Symposium on Brucellosis (II). Dev. Biol. 31:145-156. 11. Swidersk, H., T. Osuch, nd W. J. Brzosk. 1971. Peripherl blood lymphocyte blst trnsformtion test s pplied for the dignosis of brucellosis. Exp. Med. Microbiol. 23:133-138.