Scot.
moo. J .• 1976 21: 129
LABORATORY DIAGNOSIS OF HUMAN BRUCELLOSIS T. A. McAllister Department of Bacteriology, Royal Hospital for Sick Children, Glasgow
To say that brucellosis is a complex and isolated Brucella suis from a sow's fetus and difficult disease would be a gross under- in 1924 Meyer and Shaw named the genus statement. It is primarily an animal disorder Brucella in honour of David Bruce. In the (zoonosis) and its protean manifestations U.K. the disease is almost entirely caused by concern two great professions and many Brucella abortus from infected cattle, the disciplines therein. This paper deals with a exceptions being a few cases produced by single animal species, a perverse primate with import of soft Italian cheese rich in Br. two distinguishing characteristics-(a) it melitensis (Lancet, 1975). drinks when not thirsty and (b) it reproduces all the year round. In humans the disease Laboratory diagnosis nowadays is largely occupational, affecting The main diagnostic procedures are summarthose in close contact with cows and their ised in Table 1. Strenuous efforts should be products. Clinical diagnosis is fraught with made to obtain a positive culture as this difficulties and laboratory tests play an provides the only irrefutable evidence of important part in diagnostic back-up. They Brucella infection. In the long-established will, of course, only be requested if there is a case the intracellular habitat of the organism strong awareness of the possibility of the and the possible exposure to antibiotics in infection and must always be viewed in the the preceding weeks makes blood culture unrewarding but Br. abortus can be grown light of clinical findings and history. from more than 50 per cent of cases in the acute stage (Payne, 1975). If available, boneHistory Undulant fever (gastric fever) has been recog- marrow and liver biopsies should also be nised along the Mediterranean littoral for cultured. For optimum growth Br. abortus requires centuries. In 1887 Colonel (later Sir) David Bruce, RAMC, isolated an organism which the presence of 5 to 10 per cent CO 2 in the he termed Micrococcus melitensis from a presence of7.5 per cent bovine serum at 37°C. post-mortem spleen and in 1897 Bang isolated Samples should be grown in liver infusion from cows a similar organism which he called broth or on liver agar for 6 weeks with subBacillus abortus. The British forces stationed cultures at weekly intervals. The presence of in Malta (Melita) suffered greatly and Bruce erythritol greatly facilitates growth. Castanwas invited to lead the Mediterranean Fever eda's method, using a combined solid and Commission to investigate the disease. He liquid medium in the presence of 5 to 10 per and his l l-man team, who might be termed cent CO 2 , is recommended as it provides its tntrepidemioiogists, spent a thorough two own in-built system of subculture (Topley & years researching the condition with little Wilson, 1975). Br. abortus is differentiated success. They could find no evidence of from Br. melitensis and Br. suis by differential spread of the disease by insects, air, sewerage, susceptibility tests using the aniline dyes, dust or water. The credit for supplying the thionin, fuchsin, methyl violet and pyronin last piece in the jig-saw goes to Dr Zammit and is itself subdivided into 9 biotypes based of the Malta Health Board, shown with other on cultural, biochemical, metabolic, seromembers of the commission in Figure 1. He logical and bacteriophage results (Topley & chose the island goats as experimental animals Wilson, 1975). The laboratory may provide indirectevidence and discovered high antibody levels in their blood and milk and subsequently isolated of Brucella infection by performing serologiBrucella melitensis from 10 per cent of them. cal tests as listed in Table I. The techniques The source and spread of the organisms were are well described by Kerr et al. (1968) but soon confirmed by others. In 1914 Traum interpretation may be very difficult (Payne,
McAllister
Figure 1. Mediterranean Fever Commission, 1904. . . Standing: Dr T. Zammit, Capt Crawford Kennedy, RAMC, Major J. ~. Weir, RAMC . Seated: Major J. O. McNought, RAMC, Dr J. W. H. Eyre, Col David Bruce, RAMC, Major T. McCulloch, RAMC, Staff Surgeon E. M. A. Clayton, RN
Table I. Laboratory diagnosis of human brucellosis. Positive blood, marrow, biopsy culture Direct Indirect
Serology
Skin test Biopsy
agglutination (IgM and IgO) complement fixation (lgO) Coombs' (AHG) test (monovalent IgO and 19A) brucellin (delayed type hypersensitivity) chronic granuloma
1975). Tests are usually performed for Br. abortus and Br. melitensis in parallel as they
share the common antigens A and M in different proportions (20/1 for Br. abortus and 1/20 for Br. melitensis). The levels obtained vary with the stage of the disease process and also with the degree of constancy of exposure to Brucella antigens, e.g. from occupational contact. In some cases with positive cultures the serological responses may be entirely negative. Serological findings are analogous to footprints in the sandvariable in pattern, evanescent and susceptible to various factors such as waves of antibiotics or antigen release. It is important to examine follow-up specimens in each case. The direct 130
agglutination test detects IgM and IgG and in the absence of occupational exposure (vets, abattoir workers) titres greater than 1/80 are regarded as abnormal (some workers prefer a cut-off point of 1/200). An added refinement is to perform the test in the presence of 2mercaptoethanol which destroys IgM, the residual titre being a measure of IgG. The complement-fixation test detects IgG and a titre of 1/16 (some prefer 1/20) is suggestive of active disease. The Coombs' (anti-human globulin) test measures non-agglutinating IgG and 19A (monovalent forms of immunoglobulin) and titres ~ 1/80 are associated with long-standing brucellosis. The brucellin test measures delayed type hypersensitivity by causing an area of induration and oedema 48 hours after intradermal injection of an extract of Brucella. The test is similar to the tuberculin (Mantoux) test but at present it is regarded more as an epidemiological screening procedure than a reliable diagnostic test. A positive reaction (> to mm. induration) denotes past exposure or infection but gives little indication of the degree of activity of the disease (Topley& Wilson, 1975).
Laboratory Diagnosis of Human Brucellosis
The histological appearance of biopsy material is that of a chronic granuloma indistinguishable from that of tuberculosis or sarcoidosis. It is in no sense pathognomonic.
of IgE in response to antigenic stimulus. It might be possible to draw a more acceptable computer-based series of graphs by pooling collected standard data from various public health laboratories.
Immune response and clinical status Figure 2 is an attempt to present in graphic Conclusion form the immune responses at various stages Protection of the human population from the of a classical untreated case of Br. abortus misery of brucellosis requires a two-pronged infection. The top line represents summation attack, namely eradication of the source of of those beneath and the time scale, like the the organism in cows and prevention of its disease, is appropriately vague. For 2 to 3 spread by pasteurization of all retail milk. weeks after the primary stimulus of infection Meantime detection of sufferers by clinical the blood culture may be positive. This is and laboratory skills is indicated in certain followed by a secondary response comprising areas of the country and it is likely that the IgG and IgM, as shown, giving rise to positive need for these will continue for a considerable direct agglutination and complement-fixation number of years. tests. IgM, which is destroyed by 2-mercaptoethanol, usually falls to undetectable levels REFERENCES in 2 to 3 months. IgG, however, may continue Kerr, W. R., McCaughey, W. J., Coghlan, J. D., Payne, D. J. H., Quaife, R. A., Robertson, L., to rise before also levelling off. In longFarrell, I. D. (1968). Techniques and interpretations standing brucellosis the IgG assumes a nonin the serological diagnosis of brucellosis in man Journal of Medical Microbiology, 1, 181 agglutinating form (monovalent IgG) and some IgA is also present. Both of these can Lancet (1975).1 Leading article. Brucellosis. Lancet, 1,436 be detected by the Coombs' test. In acute exacerbations there is a sudden sharp rise in Payne, D. J. H. (1975). Brucellosis. Medicine, 3, 123 both IgG and IgM, as shown. Occasionally veterinary surgeons exhibit a Topley and Wilson (1975). Principles of Bacteriology, Virology and Immunity. 6th Edition. Edited by widespread skin rash when manually removG. S. Wilson and A. Miles. London: Edward ing placentae. This is caused by the production Arnold
Antibody
level -----Delayed type hypersensitivity (Positive Brucellin test)
•
-----Coombs· test (A.H.G.) positive
I
ComplementU~Gitjon test pcslnve
(it.
• Acute exacerbation
~I
Standard agglutination test positive I>
14oJ
,,/ . . . -i;G:I;"M---- .............. ,
Fades _ ....
IIgM and/or 190)
I
I
I
:lgG+19M
I I
Blood Culture
I I
Positive
I
Rash
,1;E,
I I
I I
IgG it 'gG (unaffected by 2ME)
1
IgG+lgA
I several months
,
I
\ ,
IgM,
I IgG monovalent
I I
I
I
---
/
,
'Response to \
/
"," Stimulus
\
1 year onwa,d_
SUBACUTE
CHRONIC
Figure 2. Immune responses at various stages of human Br. abortus infection.
131
moo. J .• 1976 21: 129
LABORATORY DIAGNOSIS OF HUMAN BRUCELLOSIS T. A. McAllister Department of Bacteriology, Royal Hospital for Sick Children, Glasgow
To say that brucellosis is a complex and isolated Brucella suis from a sow's fetus and difficult disease would be a gross under- in 1924 Meyer and Shaw named the genus statement. It is primarily an animal disorder Brucella in honour of David Bruce. In the (zoonosis) and its protean manifestations U.K. the disease is almost entirely caused by concern two great professions and many Brucella abortus from infected cattle, the disciplines therein. This paper deals with a exceptions being a few cases produced by single animal species, a perverse primate with import of soft Italian cheese rich in Br. two distinguishing characteristics-(a) it melitensis (Lancet, 1975). drinks when not thirsty and (b) it reproduces all the year round. In humans the disease Laboratory diagnosis nowadays is largely occupational, affecting The main diagnostic procedures are summarthose in close contact with cows and their ised in Table 1. Strenuous efforts should be products. Clinical diagnosis is fraught with made to obtain a positive culture as this difficulties and laboratory tests play an provides the only irrefutable evidence of important part in diagnostic back-up. They Brucella infection. In the long-established will, of course, only be requested if there is a case the intracellular habitat of the organism strong awareness of the possibility of the and the possible exposure to antibiotics in infection and must always be viewed in the the preceding weeks makes blood culture unrewarding but Br. abortus can be grown light of clinical findings and history. from more than 50 per cent of cases in the acute stage (Payne, 1975). If available, boneHistory Undulant fever (gastric fever) has been recog- marrow and liver biopsies should also be nised along the Mediterranean littoral for cultured. For optimum growth Br. abortus requires centuries. In 1887 Colonel (later Sir) David Bruce, RAMC, isolated an organism which the presence of 5 to 10 per cent CO 2 in the he termed Micrococcus melitensis from a presence of7.5 per cent bovine serum at 37°C. post-mortem spleen and in 1897 Bang isolated Samples should be grown in liver infusion from cows a similar organism which he called broth or on liver agar for 6 weeks with subBacillus abortus. The British forces stationed cultures at weekly intervals. The presence of in Malta (Melita) suffered greatly and Bruce erythritol greatly facilitates growth. Castanwas invited to lead the Mediterranean Fever eda's method, using a combined solid and Commission to investigate the disease. He liquid medium in the presence of 5 to 10 per and his l l-man team, who might be termed cent CO 2 , is recommended as it provides its tntrepidemioiogists, spent a thorough two own in-built system of subculture (Topley & years researching the condition with little Wilson, 1975). Br. abortus is differentiated success. They could find no evidence of from Br. melitensis and Br. suis by differential spread of the disease by insects, air, sewerage, susceptibility tests using the aniline dyes, dust or water. The credit for supplying the thionin, fuchsin, methyl violet and pyronin last piece in the jig-saw goes to Dr Zammit and is itself subdivided into 9 biotypes based of the Malta Health Board, shown with other on cultural, biochemical, metabolic, seromembers of the commission in Figure 1. He logical and bacteriophage results (Topley & chose the island goats as experimental animals Wilson, 1975). The laboratory may provide indirectevidence and discovered high antibody levels in their blood and milk and subsequently isolated of Brucella infection by performing serologiBrucella melitensis from 10 per cent of them. cal tests as listed in Table I. The techniques The source and spread of the organisms were are well described by Kerr et al. (1968) but soon confirmed by others. In 1914 Traum interpretation may be very difficult (Payne,
McAllister
Figure 1. Mediterranean Fever Commission, 1904. . . Standing: Dr T. Zammit, Capt Crawford Kennedy, RAMC, Major J. ~. Weir, RAMC . Seated: Major J. O. McNought, RAMC, Dr J. W. H. Eyre, Col David Bruce, RAMC, Major T. McCulloch, RAMC, Staff Surgeon E. M. A. Clayton, RN
Table I. Laboratory diagnosis of human brucellosis. Positive blood, marrow, biopsy culture Direct Indirect
Serology
Skin test Biopsy
agglutination (IgM and IgO) complement fixation (lgO) Coombs' (AHG) test (monovalent IgO and 19A) brucellin (delayed type hypersensitivity) chronic granuloma
1975). Tests are usually performed for Br. abortus and Br. melitensis in parallel as they
share the common antigens A and M in different proportions (20/1 for Br. abortus and 1/20 for Br. melitensis). The levels obtained vary with the stage of the disease process and also with the degree of constancy of exposure to Brucella antigens, e.g. from occupational contact. In some cases with positive cultures the serological responses may be entirely negative. Serological findings are analogous to footprints in the sandvariable in pattern, evanescent and susceptible to various factors such as waves of antibiotics or antigen release. It is important to examine follow-up specimens in each case. The direct 130
agglutination test detects IgM and IgG and in the absence of occupational exposure (vets, abattoir workers) titres greater than 1/80 are regarded as abnormal (some workers prefer a cut-off point of 1/200). An added refinement is to perform the test in the presence of 2mercaptoethanol which destroys IgM, the residual titre being a measure of IgG. The complement-fixation test detects IgG and a titre of 1/16 (some prefer 1/20) is suggestive of active disease. The Coombs' (anti-human globulin) test measures non-agglutinating IgG and 19A (monovalent forms of immunoglobulin) and titres ~ 1/80 are associated with long-standing brucellosis. The brucellin test measures delayed type hypersensitivity by causing an area of induration and oedema 48 hours after intradermal injection of an extract of Brucella. The test is similar to the tuberculin (Mantoux) test but at present it is regarded more as an epidemiological screening procedure than a reliable diagnostic test. A positive reaction (> to mm. induration) denotes past exposure or infection but gives little indication of the degree of activity of the disease (Topley& Wilson, 1975).
Laboratory Diagnosis of Human Brucellosis
The histological appearance of biopsy material is that of a chronic granuloma indistinguishable from that of tuberculosis or sarcoidosis. It is in no sense pathognomonic.
of IgE in response to antigenic stimulus. It might be possible to draw a more acceptable computer-based series of graphs by pooling collected standard data from various public health laboratories.
Immune response and clinical status Figure 2 is an attempt to present in graphic Conclusion form the immune responses at various stages Protection of the human population from the of a classical untreated case of Br. abortus misery of brucellosis requires a two-pronged infection. The top line represents summation attack, namely eradication of the source of of those beneath and the time scale, like the the organism in cows and prevention of its disease, is appropriately vague. For 2 to 3 spread by pasteurization of all retail milk. weeks after the primary stimulus of infection Meantime detection of sufferers by clinical the blood culture may be positive. This is and laboratory skills is indicated in certain followed by a secondary response comprising areas of the country and it is likely that the IgG and IgM, as shown, giving rise to positive need for these will continue for a considerable direct agglutination and complement-fixation number of years. tests. IgM, which is destroyed by 2-mercaptoethanol, usually falls to undetectable levels REFERENCES in 2 to 3 months. IgG, however, may continue Kerr, W. R., McCaughey, W. J., Coghlan, J. D., Payne, D. J. H., Quaife, R. A., Robertson, L., to rise before also levelling off. In longFarrell, I. D. (1968). Techniques and interpretations standing brucellosis the IgG assumes a nonin the serological diagnosis of brucellosis in man Journal of Medical Microbiology, 1, 181 agglutinating form (monovalent IgG) and some IgA is also present. Both of these can Lancet (1975).1 Leading article. Brucellosis. Lancet, 1,436 be detected by the Coombs' test. In acute exacerbations there is a sudden sharp rise in Payne, D. J. H. (1975). Brucellosis. Medicine, 3, 123 both IgG and IgM, as shown. Occasionally veterinary surgeons exhibit a Topley and Wilson (1975). Principles of Bacteriology, Virology and Immunity. 6th Edition. Edited by widespread skin rash when manually removG. S. Wilson and A. Miles. London: Edward ing placentae. This is caused by the production Arnold
Antibody
level -----Delayed type hypersensitivity (Positive Brucellin test)
•
-----Coombs· test (A.H.G.) positive
I
ComplementU~Gitjon test pcslnve
(it.
• Acute exacerbation
~I
Standard agglutination test positive I>
14oJ
,,/ . . . -i;G:I;"M---- .............. ,
Fades _ ....
IIgM and/or 190)
I
I
I
:lgG+19M
I I
Blood Culture
I I
Positive
I
Rash
,1;E,
I I
I I
IgG it 'gG (unaffected by 2ME)
1
IgG+lgA
I several months
,
I
\ ,
IgM,
I IgG monovalent
I I
I
I
---
/
,
'Response to \
/
"," Stimulus
\
1 year onwa,d_
SUBACUTE
CHRONIC
Figure 2. Immune responses at various stages of human Br. abortus infection.
131
