Hemolysis Test for Cattle Vaccinated and Infected with

JOURNAL OF CLINICAL MICROBIOLOGY, OCt. 1982, p. 599-3 95-1137/82/1599-5$2./ Copyright C 1982, American Society for Microbiology Vol. 1, No. 4 Comparison of the Complement Fixation Test and the Indirect Hemolysis Test for Cattle Vaccinated and Infected ith Brucella abortus SHELLEY S. SUTHERLAND,* DEBRA V. LE CRAS, AND ROBERT J. EVANS Animal Health Laboratory, Department of Agriculture, South Perth, Western Australia 151 Received 9 November 1981/Accepted 12 June 1982 The complement fixation test (CFT) and indirect hemolysis test (IHLT) ere applied to sera collected from cattle challenged ith Brucella abortus 544. Of the cattle, 48 ere vaccinated ith either B. abortus 19 or B. abortus 45/2 as calves or as adults. The remaining 12 cattle ere not vaccinated. Of the 27 sera from cattle found to be infected, 9 shoed aberrant reactions to the CFT. The advantages of the IHLT for these cattle ere as follos. After challenge, the titers to the IHLT became positive earlier than or at the same time as the titers to the CFT, they persisted longer than the titers to the CFT, and they failed to sho proone reactions, hich are a problem ith the CFT. An additional advantage as that before challenge and after vaccination ith strain 19, the titers to the IHLT rose later and declined earlier than the titers to the CFT. We concluded that the CFT used in conjunction ith the IHLT improves the detection of infected cattle. Bacteriological evaluations have indicated that the complement fixation test (CFT) is the most accurate serological test for the detection of bovine brucellosis and thus is the definitive test in many countries here eradication schemes are in progress (1, 11). Several limitations of the test, hoever, have been encountered. First, the CFT does not differentiate infected animals from animals recently vaccinated ith Brucella abortus strain 19 (S19) or B. abortus strain 45/2 (S45/2) (5). Second, the test often shos anticomplementary reactions and proones, the latter being the result of immunoglobulin G2-type antibodies to B. abortus blocking immunoglobulin Gl and immunoglobulin M antibodies (9). Third, CFT results are subject to great variations depending on minor differences in technique (1, 1). Finally, the CFT fails to identify some animals hich sho aberrant serological reactions, and therefore, in an eradication program this may cause the reappearance of brucellosis in herds certified to be free of brucellosis (4; S. S. Sutherland, D. V. Le Cras, and A. G. Robertson, Aust. Vet. J., in press). The recently developed indirect hemolysis test (IHLT) (12) as designed to overcome the blocking effect of immunoglobulin G2. The test uses erythrocytes hich are sensitied ith a lipopolysaccharide preparation extracted from S19 and hich are lysed by a specific antibody in the presence of excess complement. This paper reports a comparative study of the CFT and IHLT for animals hich ere vaccinated ith S19 or S45/2 and infected after challenge ith B. abortus and hich shoed aberrant reactions in the CFT. MATERIALS AND METHODS Experimental cattle. Sixty 3- to -month-old female calves from a brucellosis-free herd ere divided into three groups. One group (24 cattle) as vaccinated ith the standard dose of S19 (batch no. 32-1, Commonealth Serum Laboratories, Melbourne, Victoria, Australia), the second (24 cattle) as vaccinated ith to doses of S45/2 (batch no. V59, Philips Duphar Pty Ltd., North Sydney, Ne South Wales, Australia), eeks apart, and the third (12 cattle) as not vaccinated. Half of the first and second groups ere vaccinated as calves at 3 to months of age, and half ere vaccinated as young adults at 14 to 1 months of age. The cattle ere mated at 14 to 1 months of age, and approximately 3 months after mating, they ere examined rectally for pregnancy. At this time, the 4 pregnant animals ere challenged via the conjunctival route ith 15 x 1 viable B. abortus strain 544 (S544) organisms (8), and 14 nonpregnant animals received 15 x 17 viable S544 organisms (, 14). Bacteriology. Milk samples ere collected from cos at parturition for bacteriological examination. Most aborted fetuses and eak calves (calves unable to stand unaided) ere subjected to necropsies, and the abomasum contents, livers, kidneys, meconia, lungs, and spleens ere cultured. All cos ere slaughtered approximately 5 eeks after challenge (2 to 3 eeks after parturition). Samples from the Donloaded from http://jcm.asm.org/ on September, 218 by guest 599

SUTHERLAND, LE CRAS, AND EVANS J. CLIN. MICROBIOL. Animal Nymber I Animal Number 2 co IIF A -A IV:' 3;-------- ---T\ I + ulj I i : : : A -E f S19 25 5 75 1 WEEK -NUMBER Animal Number 3 I- 25 5 75 1 S19 Animal Number 4-3 - -------------- Donloaded from http://jcm.asm.org/ - 25 5 75 1 11 45/2 Animal lnumber 5 3.- -----------------~-~----- ---- - ---- r et - 25 5 75 1 45/2 Animal Number 3. ----------- on September, 218 by guest 25 5' 75 1 25 5 75 1 4 S19 S19 FIG. 1. Antibody levels in the sera of nine infected cattle after vaccination and challenge as measured by the CFT (-) and IHLT (----). The horiontal dashed line divides the positive values from the negative values for both tests.

VOL. 1, 1982 cc a o F Fcc F co - 3j a COMPARISON OF TESTS FOR BRUCELLOSIS IN CATTLE 1 Animal Number 7 cranial and caudal quarters of each udder, from the kidneys, livers, spleens, uteri, and from the submaxillary, retropharyngeal, mesenteric, internal iliac, and WVUi supramammary lymph nodes ere collected aseptically from each animal for culture. The method of culture as that described by Alton + et al. (1). The clinical results of challenge (see Table 1) *,1 - i J have been described elsehere (15). Serology. Blood samples ere collected from all animals 1 eek before vaccination and eekly thereafter for 8 eeks. The interval beteen sampling as then increased to 1 month, and sampling continued 25 5 75 1 until challenge. Blood samples ere obtained 1 eek 45/2 before challenge and eekly thereafter for 4 eeks 45/2 until slaughter. All sera ere subjected to the IHLT and CFT. The IHLT as performed as described by Plackett et al. (13). Essentially, the method as as OG 2 RECIPROCAL OF TITRE follos. Bovine erythrocytes free of J antigen ere collected in Alsever solution (2) and stored at 4 C for up to eeks. The erythrocytes ere sensitied by being mixed ith an equal volume of alkali-treated lipopolysaccharide. Each batch of complement used as checked against unsensitied cells for guinea pig serum hemolysins. The test as performed in microtiter plates (Flo Laboratories, Inc., McLean, Va.) ith.25 ml of inactivated serum (5 C for 3 min),.25 ml of antigen-sensitied cells, and.25 ml of the complement solution. Plates ere incubated at 37 C for min ith constant mechanical shaking. The test as performed ith serum dilutions of 1:4 to 1:128. + The test as considered positive hen there as 5% - hemolysis at a.3 log2 dilution (1:8). The CFT as done by the Australian standard method (2). This method closely resembles that described previously (1). Complement fixation at a is 51is1 7 dilution of 3 log2 (1:8) as regarded as a positive 4I5 4 4 reaction; this is the level recommended by the Austra- 45/2 lian Bureau of Animal Health (3). Serum samples ere not titrated in the CFT and IHLT to endpoints, but ell beyond the point of significance for each test. Titers to both tests ere OG 2 RECIPROCAL OF TITRE expressed as log2 of the reciprocal of the last dilution at hich a positive reaction occurred ithin the dilutions 1:4 to 1:128. Serological classification of animals. For the purpose of this study, cattle found to be infected at parturition or necropsy ere classified as normally or aberrantly Animal Number 9 reacting animals by their responses to the CFT after challenge. Cattle classified as normal shoed positive titers to the CFT 8 eeks after challenge and mainrn tained these titers until slaughter. Infected animals hose reactions to the CFT differed from this pattern ere classified as aberrant reactors; they did not sho positive titers to the CFT until 8 to 2 eeks after + challenge and did not maintain these titers until slaugh- - ter. The serological responses of cattle in hich no r. infection as found ere not classified as normal or aberrant since culture methods cannot be relied on to. prove. t the absence of infection and so any such conclu- 25 5O W5 * 1 sion ould not be valid. RESULTS Of the cattle in the trial, 27 ere shon after challenge to be infected ith S544. Of these I11 45/2 LOG 2 RECIPROCAL OF TITRE FIG. 1-Continued 27, 18 gave by definition a normal reaction to the CFT, hereas the other 9 cattle shoed aber- Donloaded from http://jcm.asm.org/ on September, 218 by guest

2 SUTHERLAND, LE CRAS, AND EVANS TABLE 1. Clinical, bacteriological, and serological results of challenge of cattle ith virulent S544 after vaccination as calves or adults ith S19 or S45/2 No. of cattle No. of abortions No. of infected cattle" Group or neonatal With normal With aberrant Total Pregnant deaths due to Total reactions to reactions to challenge CFT CFT 1 (S19 vaccinated) Calf 12 7 1 4 1 3 Adult 12 11 1 1 2 (S45/2 vaccinated) Calf 12 1j 3 3 (2)' 3 2 Adult 12 job 3 (2) 5 3 3 (control) 12 8 (3) 9 a Found to be infected by bacteriological examination at slaughter. b One died. ' Numbers in parentheses sho numbers of cattle not pregnant during the trial. rant reactions to the CFT. All of the 9 cattle shoing aberrant reactions and 9 of 18 shoing normal responses had previously been vaccinated ith S19 or S45/2 (Table 1). Of the 33 cattle in hich no infection as found, 2 ere pregnant. Of the 2, 2 died early in the trial, 22 had healthy calves (beteen 18 and 25 eeks after challenge), and the remaining 2 had stillborn calves (2 eeks after challenge). No infection as found in the tissues of these to stillborn calves or in the tissues or milk of the dams. Of the nine infected animals shoing aberrant serological reactions, eight ere pregnant, and six had healthy calves (beteen 2 and 22 eeks after challenge) (Table 2). Of the remaining 18 infected cattle shoing normal serological responses, 13 ere pregnant. Of these, eight aborted (beteen 7 and 15 eeks after challenge), three had eakling calves (beteen 12 and 19 eeks after challenge), and one had a healthy calf (2 eeks after challenge). The progressive titers to the CFT and IHLT for the nine cattle ith aberrant reactions are shon in Fig. 1. TABLE 2. In the four cattle vaccinated ith S19 (animals 1, 2, 5, and ), positive titers to the CFT ere present 1 eek after vaccination and ere maintained at that level in one of the animals (animal 2) for 4 eeks. In to of these four cattle, positive titers to the IHLT ere not seen after vaccination, and in the other to cattle, they ere present for at most 2 eeks (Fig. 1). After challenge, the titers to the IHLT in seven of the cattle (animals 1, 3, and 5-9) became positive 1 to eeks earlier than the titers to the CFT (Table 2). In the remaining to cattle, the titers to the to tests become positive simultaneously. Three of the nine cattle had positive titers to the CFT for only 1 to 3 eeks of the 4-eek postchallenge period, and five of the nine animals had negative titers to the CFT at slaughter (Table 2). It as 1 eeks after challenge of animal before a positive titer to the IHLT first occurred, but it as a further eeks (5 eeks before parturition) before a positive titer persisted. The titer to the IHLT then remained positive until slaughter 2 eeks later. Positive titers to the CFT ere seen in this Time before CFT and IHLT became positive and serology at slaughter of nine infected cattle hich shoed aberrant serological reactions to the CFT Animal Vaccination history Calf Incubation period" Titer at slaughter no. Age Strain produced CFT IHLT CFT IHLT 1 Calf S19 Healthy 8 5 - + 2 Calf S19 Healthy 24 24 - - 3 Calf S45/2 Weakling 2 23 - + 4 Adult S45/2 Healthy 12 12-5 Calf S19 Healthy 2 1 + + Adult S19 Healthy 1 1 - + 7 Adult S45/2 Weakling 1 11 + + 8 Adult S45/2 Healthy 1 12 + + 9 Calf S45/2 None 21 2 + + a Weeks after challenge before positive titer. J. CLIN. MICROBIOL. Donloaded from http://jcm.asm.org/ on September, 218 by guest

VOL. 1, 1982 COMPARISON OF TESTS FOR BRUCELLOSIS IN CATTLE 3 animal for only 12 eeks of the 4-eek postchallenge period (Fig. 1). In animals numbered 1 and -9, proones hich complicated the interpretation of the results of the CFT ere present for several eeks, particularly near the time of parturition. No proones ere observed in the IHLT for any of the nine animals. DISCUSSION Vaccination of sexually mature animals ith S19 has been reported to interfere ith serological tests and therefore has not been recommended in recent years (7, 12). In this study, consistent ith other studies, the titers to the IHLT in animals vaccinated ith S19 rose later after vaccination and declined earlier than the titers to the CFT (12, 14a). The cattle ith normal reactions to the CFT shoed positive responses to this test ithin a fe eeks of challenge and maintained them until slaughter. In contrast, for the nine cattle ith aberrant reactions to the CFT, several eeks or months elapsed after challenge before significant titers to this test ere present. This meant that for most of the period after challenge, many of the cattle ith aberrant serological reactions to the CFT ould be regarded as uninfected in an eradication program based on the CFT alone. In most of these cattle, the titers to the IHLT rose earlier and lasted longer than the titers to the CFT. This suggests that the IHLT used in conjunction ith the CFT could improve the detection of infected cattle. Using the IHLT as a supplementary test has the advantage of being readily incorporated into a laboratory system in hich the CFT is used as a screen test, and as observed in this study and reported by others (13), it gives positive reactions hen sera in the CFT exhibit the proone phenomenon. ACKNOWLEDGMENTS We are grateful to the Department of Agriculture for the use of their facilities and to the staff of Wongan Hills Research Station for the management of cattle and collection of specimens. We are also grateful for the assistance given to us by the staff of the Brucella laboratory of the Department of Agriculture. LITERATURE CITED 1. Alton, G. G., L. M. Jones, and D. E. Piet. 1975. Bacteriological methods. W.HO. Monogr. Ser. 55:11-3. 2. Anonymous. 1977. Standardised complement fixation test for bovine brucellosis. Aust. Vet. J. 53:394-4. 3. Australian Bureau of Animal Health. 1979. Standard definition and rules. p. 1-9. In Bovine brucellosis and tuberculosis national eradication campaign, vol. 1. Australian Bureau of Animal Health, Canberra, Australia. 4. Christie, T. E., W. R. Kerr, and W. J. McCaughey. 198. Brucellosis eradication in Northern Ireland. Vet. Rec. 82:17-183. 5. Cunningham, B 198. The control and eradication of brucellosis. 1. Serological response in cattle folloing vaccination ith S19 and killed Brucella 45/2 adjuvant vaccine. Vet. Rec. 82:7-11.. Cunningham, B. 1977. A difficult disease called brucellosis, p. 11-2. In R. P. Craford and R. J. Hidalgo (ed.), Bovine brucellosis: an international symposium. Texas A & M University Press, College Station, Tex. 7. Gordon, W. S. 1953. The assessment of brucellosis immunity in cattle, p. 177-181. Proceedings of 15th International Veterinary Congress, Stockholm 1953, vol. 2, Gernandts Boktryekeri, Stockholm. 8. McEen, A. D., F. W. Priestly, and J. D. Patterson. 1939. An estimate of a suitable infective dose of B. abortus for immunisation test of cattle. J. Comp. Pathol. Ther. 52:11-128. 9. McNaught, D. J., R. J. Chappel, G. S. Allan, J. A. Bourke, and B. A. Rogerson. 1977. The effects of IgG, and of antigen concentration on prooning in the complement fixation test for bovine brucellosis. Res. Vet. Sci. 22:194-197. 1. Morgan, W. J. B., I. Davidson, and C. N. 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Vet. Microbiol. 7:15-175. 15. Sutherland, S. S., A. G. Robertson, D. V. Le Cras, G. M. Robertson, J. M. Johnston, and R. J. Evans. 1981. The effect of challenge ith virulent Brucella abortus on beef cattle vaccinated as calves or adults ith either Brucella abortus strain 19 or 45/2. Aust. Vet. J. 57:47-473. 1. U.S. Department of Health, Education, and Welfare. 195. Standardied diagnostic complement fixation method and adaptation to microtest. Public health monograph no. 74. U.S. Department of Health, Education, and Welfare, Washington, D.C. Donloaded from http://jcm.asm.org/ on September, 218 by guest