ORIGINAL ARTICLES RECENT
DEVELOPMENTS
IN VACCINATION BRUCELLOSIS*
AGAINST
BOVINE
CSlRO Division o f Animal Health, Animal Health Research Laboratory, Private Bag N o . I , P. O., Parkville, Victoria, 3052 SUMMARY: Recent research in vaccination against bovine brucellosis has been directed towards reducing the serological response to vaccination and to developing serological tests better able to distinguish vaccinal titres from those resulting from field infection. In the case of strain 19 vaccination, developments such as reducing the dose of vaccine and improving serological tests have so reduced the serological response as to allow adult vaccination to be carried out in combination with eradication by test and slaughter. B. abortus 45/20 vaccine is being increasingly used as a combined diagnostic aid and protective agent. Progress has been made in identifying antibodies produced in response to this vaccine.
In Australia, 2 types of vaccine are used to protect cattle against infection with Brucella abortus, the living attenuated strain 19 vaccine and the killed 45/20 adjuvant vaccine. Various other vaccines are either in use overseas o r being evaluated. A vaccine that gives good protection without undesirable side effects, such as interference with subsequent diagnostic tests, is not yet available, but efforts towards developing such a vaccine are in progress. Strain 19 The main characteristics of this vaccine are well known. It protects about 70% of vaccinared cattle against a carefully adjusted challenge dose of B. abortus. Only one dose is needed and the resistance to infection lasts several years. Abortion storms occur in strain 19 vaccinated herds. Nevertheless, experience has shown that when most of the female cattle in a heavily infected area are vaccinated, the prevalence of brucellosis is greatly reduced. The vaccine may be used as a n adjunct to eradication by test and slaughter. Stuart et a1 (1959), reporting on the results of I 1 years of vaccination in California, stated that well-vaccinated herds had only one-tenth the number of reactors found in non-vaccinated herds. Vaccination should continue well into the test and slaughter program a n d should o n l y cease when the prevalence has fallen to a low level, say of the order of 0.2%. Vaccination with strain 19 is followed by the production of antibodies. These antibodies are less persistent if the vaccine is given before the animal reaches the age of puberty. In practice vac-
cination is generally restricted to animals aged between 3 and 8 months. Antibodies resulting from vaccination are more reactive in some diagnostic tests than in others. The agglutination test in calves vaccinated at 6 months of age will often remain positive for 18 months, whereas the complement fixation test will have returned to negative in a majority of these animals by 6 months after vaccination although a small proportion, perhaps less than one per cent, of persistant reactors to the C F T d o occur. The socalled supplemental tests soon become negative in S I Y vaccinated cattle a n d are negative in animals (and humans) with non-specific reactions. Examples of these supplemental tests are the mercaptoethanol, the rivanol a n d the heatinactivation tests. Recent progress has resulted from research aimed at modifying vaccination procedures so as to reduce interference with serological tests, o r to develop serological tests that are better at distinguishing reactions due to virulent infection from those resulting from strain 19 vaccination. Pilet and Bonneau (1970) showed that antibody production following strain I 9 vaccination could be suppressed by the simultaneous administration of a n t i - b r u c e l l a s e r u m . D h e n n i n (1973) demonstrated that cattle vaccinated by this method were less resistant to infection than those given strain 19 alone. I t has long been accepted that the younger the calf at vaccination the lower the level of antibodies produced. Redman et a1 (1967) showed that calves vaccinated at 2 o r at 3 months of age developed resistance very similar to those vaccinated between 4 and 8 months of age, and had less antibody production. However, when Deyoe (1974) vaccinated 551
calves at one week or 1 month old he found that they were less resistant to challenge than calves vaccinated at 4 months. Another problem associated with the vaccination of very young calves is the effect of maternal antibody levels. Work at Weybridge, England, (C. Davies, personal communication) demonstrated that calves with passively administered antibodies developed less immunity than those without. Present indications are that 2 months is the lowest practical age limit for vaccination with strain 19. G . Davies (personal communication) reported that a reduction in the dose of strain 19 to 1/20 normal reduced the serological resonse without apparent effect on the resulting immunity. However, a 1000-fold reduction in dose removed the immunising effect. See also review by Manthei (1952). Plommet and Plommet (1975, 1976) and Plommet and Fensterbank (1976) have experimented extensively using strain 19 conjunctivally, firstly in guinea pigs and later in cattle. The effective conjunctival dose for cattle was established at 5 x lo9 to 5 x 1010 viable cells. At this dose level, in calves only insignificant serological reactions developed except in a few animals which developed low Coombs test reactions. In cows, however, doses of 1010bacteria gave transient but significant serum agglutination reactions. The level of immunity induced in cattle by conjunctival vaccination was studied using conjunctival vaccination as a booster 6 months after routine calfhood vaccination and also as 2 conjunctival inoculations 6 months apart. Following challenge in midpregnancy, 1 of 7 non-vaccinated heifers calved normally as compared with 6 of 12, which received calfhood vaccination, 8 of 11 in the group given conjunctival vaccination as a booster following calfhood vaccination and 14 of 19 in the group receiving only 2 conjunctival inoculations. When the groups were compared for evidence of infection, including autopsy about 6 weeks after parturition, the rate of infection in the various groups in the order given above was 717, 11/12, 6/12 and 14/19. The authors suggest alternative applications for conjunctival vaccination: (1) As a booster dose to give greater protection when needed, without serious interference with diagnostic tests or (2) Two conjunctival inoculations 6 months apart should give at least as good protection as the standard method, and could be given at any age without risk of serological response.
Strain 19 Vaccination and Serological Diagnosis Rose, Lambert and Roepke (1964), Rose and Roepke (1964) and Rose and Amerault (1964) investigated differences in the serological response 552
following strain 19 vaccination and that following natural infection. They found that the predominant antibody produced after vaccination was fast sedimenting in density gradient solution in the ultra-centrifuge, heat liable at 65°C and susceptible to the action of mercaptoethanol, whereas the predominant antibody produced following natural infection was of lower molecular weight and resistant to heat and mercaptoethanol. These studies led to the development of the "supplemental tests", such as the heat inactivation test, the mercaptoethanol test and the rivanol test, which by one means or another remove the effect of the macro-globulin and measure only the content of low molecular weight immunoglobulin. Cattle become negative to these tests following strain 19 vaccination more rapidly than they do to the serum agglutination test (SAT). I t was assumed that the predominant antibody following strain 19 vaccination was macro-globulin (now called IgM) and that following infection the low molecular weight immunoglobulin, IgG. So long as the antibodies present were measured in terms of their reactivity in different serological tests, this explanation sufficed. However, when Beh (1974) measured the actual quantity of antibody present he found that following strain 19 vaccination, levels of IgM fell much more rapidly than did the level of IgC. The behaviour of postvaccinal sera to the various tests becomes more understandable when the relative efficiency of different classes of antibody in serological tests is' taken into consideration. Allan er a1 (1976) demonstrated that in the agglutination tests, IgM is 10 times more effective than lgG, so that the removal or inactivation of small amounts of IgM
...I..
2 ..................................................
.-
/\
. '\
'.-.
"
................................................... --:::2=n"---a 1
2 3 4 5 M o n t h s a f t e r v a c c i na t I on
6
Figure 1. Effect of immunoglobulin levels on serology following strain 19 vaccination. 1 - Positive level for IgM-mediated serology (for example SAT) 2 - Positive level lor IgG-mediated serology (for example supplemental tests) 3 - Duration of positivity in supplemental tests 01 S A T reaction
4 - Duration
Australian VeterinarJ..loiirrial, Vol. 54, December, 197R
will affect the result of the test. This concept is illustrated in Figure 1 . The complement fixation test (CFT) gives results in strain 19 vaccinates that are similar to those given by the supplemental tests. According to Allan et a / (1976) IgM is a more efficient fixer of complement than IgG, (IgG, does not fix complement), but IgM may be at ]east partly inactivated at the temperature used for inactivating serum samples for the CFT. The emergence of the CFT as an effective diagnostic test, with its early return to negative in strain 19 vaccinates, leads us to question the need to restrict S19 vaccination to calves. Worthington et a1 (1973) studied serum antibody titres for a period of 2 years in 99 beef and 22 dairy cows that had been vaccinated as adults with strain 19. SAT titres remained above diagnostic levels throughout, but complement fixaton, mercaptoethanol agglutination and rivanol agglutination titres generally returned to negative levels within 6 months of vaccination. Nicoletti (1976, 1977) used strain 19 vaccination experimentally in cows in large dairy herds in Florida. More than 1,000 cows in infected herds were given the standard dose of strain 19 subcutaneously at approximately 7 to 8 months of pregnancy; subsequently the abortion rate from all causes was less than 1 % . I n more than 700 lactating cows inoculated by various routes, strain 19 could be isolated from the udders of less than 2y0, There was an 80% reduction in the number of reactors removed during the course of the study (6 to 15 months after vaccination). Reducing the dose (up to 1/20 normal) and administering the reduced dose either subcutaneously, intradermally or into the conjunctival sac appeared not to affect immunogenicity, but sharply reduced the level and persistence of antibody production. The author concludes that the problem of titres produced by vaccination can be largely eliminated by using reduced doses of strain 19 and supplemental tests and further, that vaccination of adult cattle is an economical, efficient and practical method of reducing the prevalence of brucellosis in large herds while still allowing the detection of infected animals. Nicoletti and Muraschi (1966), like Jones et (1963), use a complement fixation test based on cold overnight fixation. Cunni ff and Stellar (1968) report that IgG antibody fixes complement . suggests better a t 00 to 4oc and I g M a t 3 7 0 ~This that a test using cold fixation may be preferable for a cattle population where it is important to distinguish between vaccinal reactions and those resulting from natural infection. Plackett et a / (1976) developed the indirect haemolysis test (IHLT) for the diagnosis Of bovine Australian Veterinary Journal, Vol. 54, December, 1978
brucellosis. Amongst other advantages the IHLT is less sensitive than the CFT to antibodies resulting from vaccination with strain 19 vaccine. Further evaluation of the 1HL-T currently in Progress may well show i t to be superior to the CFT as the definitive test for bovine brucellosis in areas where strain 19 vaccine is used. Progress in research into Vaccination O f cattle with strain 19 is reviewed in a report to the National Academy o f Sciences of USA (Anon, 1977).
Vaccines Prepared from B . abortus Slrain45/20 The B. abortus strain 45/20, which is a dissociated “rough” strain and therefore nonagglutinogenic, was first used as a live vaccine but its use was discontinued after it had been found to revert to the smooth, virulent type during passage in cattle. McEwen and Samuel (1955) were first to use the 45/20 strain as a killed culture in oily adjuvant. A number of commercial vaccines of this type, which we shall call K45/20A, are now available, the best known being “Duphavac”*. The development and characteristics of this type of vaccine have been described and summarised by Morgan and McDiarmid (1968). In this report, unless otherwise specified, DuPhavac is the Product being r e f m e d to. The more efficient type of K45/20A vaccine has the following main characteristics. The vaccine can be used safely on cattle of any age and regardless of the state of Pregnancy. Two doses of Vaccine are required and to obtain maximum protection, cattle should be at least 6 months old when first vaccinated. Persistent local swellings often develop at the site of inoculation, especially after the second dose of vaccine; they vary greatly in size b u t generally are not unacceptable. The lesion in the CarCaSe is likely to cause complaint if not removed. Intramuscular injection of the vaccine produces a less visible lesion than does subCUtaneOUS injection. In beef cattle subcutaneous injection is preferred because the lesion is easier to detect and remove after slaughter. The serological response to vaccination is limited (Roerink 1966). There is very little response to the first injection. After the second dose of vaccine the production of agglutinating antibody is minimal, a few animals showing suspicious reactions to the serum agglutination test (SAT), but even these have mostly disappeared by 4 months after the second dose. A few positive reactions to the Rose Bengal Test (RBT) will occur during this period. The reaction to the CFT is more serious and a small proportion of vaccinated cattle retain titres for 12 to 15 months. The Milk Ring Test (MRT) remains negative Adjuvant
* A\dlinble
ftonl Pitmati hlourc. 4SirlusKoad. 1 a n e c o v c . New South Wale,
553
when lactating cows are vaccinated. CFT reactions in cattle that had previously been vaccinated with strain 19 are more frequent a n d more prolonged. The anti-bovine globulin test (ABGT) gives a moderate reaction after the first injection and a severe and prolonged reaction after the second. In controlled experiments the degree of immunity produced is similar to that produced by strain 19. The duration of immunity conferred by K45/20A vaccine has not been accurately determined and for this reason a n annual booster is recommended. Morgan and McDiarmid (1968) make the point that small differences in adjuvant used by different producers affect the character of the vaccine. The possibility of using K45/20A vaccine as a diagnostic agent, in what is now known as a n anainnestic test, was first put forward by Tacken (1964). The concept is based on the difference between the primary and the secondary serological response. I f the first dose of K45/20A vaccine produces a secondary rather than a primary response, i t is assumed that the animal has previously experienced a specific brucella stimulus and if i t has not been vaccinated previously, this stimulus is considered to have resulted from natural infection. The cattle are tested serologicall y and, if negative, are given a single dose of 1
DEVELOPMENTS
IN VACCINATION BRUCELLOSIS*
AGAINST
BOVINE
CSlRO Division o f Animal Health, Animal Health Research Laboratory, Private Bag N o . I , P. O., Parkville, Victoria, 3052 SUMMARY: Recent research in vaccination against bovine brucellosis has been directed towards reducing the serological response to vaccination and to developing serological tests better able to distinguish vaccinal titres from those resulting from field infection. In the case of strain 19 vaccination, developments such as reducing the dose of vaccine and improving serological tests have so reduced the serological response as to allow adult vaccination to be carried out in combination with eradication by test and slaughter. B. abortus 45/20 vaccine is being increasingly used as a combined diagnostic aid and protective agent. Progress has been made in identifying antibodies produced in response to this vaccine.
In Australia, 2 types of vaccine are used to protect cattle against infection with Brucella abortus, the living attenuated strain 19 vaccine and the killed 45/20 adjuvant vaccine. Various other vaccines are either in use overseas o r being evaluated. A vaccine that gives good protection without undesirable side effects, such as interference with subsequent diagnostic tests, is not yet available, but efforts towards developing such a vaccine are in progress. Strain 19 The main characteristics of this vaccine are well known. It protects about 70% of vaccinared cattle against a carefully adjusted challenge dose of B. abortus. Only one dose is needed and the resistance to infection lasts several years. Abortion storms occur in strain 19 vaccinated herds. Nevertheless, experience has shown that when most of the female cattle in a heavily infected area are vaccinated, the prevalence of brucellosis is greatly reduced. The vaccine may be used as a n adjunct to eradication by test and slaughter. Stuart et a1 (1959), reporting on the results of I 1 years of vaccination in California, stated that well-vaccinated herds had only one-tenth the number of reactors found in non-vaccinated herds. Vaccination should continue well into the test and slaughter program a n d should o n l y cease when the prevalence has fallen to a low level, say of the order of 0.2%. Vaccination with strain 19 is followed by the production of antibodies. These antibodies are less persistent if the vaccine is given before the animal reaches the age of puberty. In practice vac-
cination is generally restricted to animals aged between 3 and 8 months. Antibodies resulting from vaccination are more reactive in some diagnostic tests than in others. The agglutination test in calves vaccinated at 6 months of age will often remain positive for 18 months, whereas the complement fixation test will have returned to negative in a majority of these animals by 6 months after vaccination although a small proportion, perhaps less than one per cent, of persistant reactors to the C F T d o occur. The socalled supplemental tests soon become negative in S I Y vaccinated cattle a n d are negative in animals (and humans) with non-specific reactions. Examples of these supplemental tests are the mercaptoethanol, the rivanol a n d the heatinactivation tests. Recent progress has resulted from research aimed at modifying vaccination procedures so as to reduce interference with serological tests, o r to develop serological tests that are better at distinguishing reactions due to virulent infection from those resulting from strain 19 vaccination. Pilet and Bonneau (1970) showed that antibody production following strain I 9 vaccination could be suppressed by the simultaneous administration of a n t i - b r u c e l l a s e r u m . D h e n n i n (1973) demonstrated that cattle vaccinated by this method were less resistant to infection than those given strain 19 alone. I t has long been accepted that the younger the calf at vaccination the lower the level of antibodies produced. Redman et a1 (1967) showed that calves vaccinated at 2 o r at 3 months of age developed resistance very similar to those vaccinated between 4 and 8 months of age, and had less antibody production. However, when Deyoe (1974) vaccinated 551
calves at one week or 1 month old he found that they were less resistant to challenge than calves vaccinated at 4 months. Another problem associated with the vaccination of very young calves is the effect of maternal antibody levels. Work at Weybridge, England, (C. Davies, personal communication) demonstrated that calves with passively administered antibodies developed less immunity than those without. Present indications are that 2 months is the lowest practical age limit for vaccination with strain 19. G . Davies (personal communication) reported that a reduction in the dose of strain 19 to 1/20 normal reduced the serological resonse without apparent effect on the resulting immunity. However, a 1000-fold reduction in dose removed the immunising effect. See also review by Manthei (1952). Plommet and Plommet (1975, 1976) and Plommet and Fensterbank (1976) have experimented extensively using strain 19 conjunctivally, firstly in guinea pigs and later in cattle. The effective conjunctival dose for cattle was established at 5 x lo9 to 5 x 1010 viable cells. At this dose level, in calves only insignificant serological reactions developed except in a few animals which developed low Coombs test reactions. In cows, however, doses of 1010bacteria gave transient but significant serum agglutination reactions. The level of immunity induced in cattle by conjunctival vaccination was studied using conjunctival vaccination as a booster 6 months after routine calfhood vaccination and also as 2 conjunctival inoculations 6 months apart. Following challenge in midpregnancy, 1 of 7 non-vaccinated heifers calved normally as compared with 6 of 12, which received calfhood vaccination, 8 of 11 in the group given conjunctival vaccination as a booster following calfhood vaccination and 14 of 19 in the group receiving only 2 conjunctival inoculations. When the groups were compared for evidence of infection, including autopsy about 6 weeks after parturition, the rate of infection in the various groups in the order given above was 717, 11/12, 6/12 and 14/19. The authors suggest alternative applications for conjunctival vaccination: (1) As a booster dose to give greater protection when needed, without serious interference with diagnostic tests or (2) Two conjunctival inoculations 6 months apart should give at least as good protection as the standard method, and could be given at any age without risk of serological response.
Strain 19 Vaccination and Serological Diagnosis Rose, Lambert and Roepke (1964), Rose and Roepke (1964) and Rose and Amerault (1964) investigated differences in the serological response 552
following strain 19 vaccination and that following natural infection. They found that the predominant antibody produced after vaccination was fast sedimenting in density gradient solution in the ultra-centrifuge, heat liable at 65°C and susceptible to the action of mercaptoethanol, whereas the predominant antibody produced following natural infection was of lower molecular weight and resistant to heat and mercaptoethanol. These studies led to the development of the "supplemental tests", such as the heat inactivation test, the mercaptoethanol test and the rivanol test, which by one means or another remove the effect of the macro-globulin and measure only the content of low molecular weight immunoglobulin. Cattle become negative to these tests following strain 19 vaccination more rapidly than they do to the serum agglutination test (SAT). I t was assumed that the predominant antibody following strain 19 vaccination was macro-globulin (now called IgM) and that following infection the low molecular weight immunoglobulin, IgG. So long as the antibodies present were measured in terms of their reactivity in different serological tests, this explanation sufficed. However, when Beh (1974) measured the actual quantity of antibody present he found that following strain 19 vaccination, levels of IgM fell much more rapidly than did the level of IgC. The behaviour of postvaccinal sera to the various tests becomes more understandable when the relative efficiency of different classes of antibody in serological tests is' taken into consideration. Allan er a1 (1976) demonstrated that in the agglutination tests, IgM is 10 times more effective than lgG, so that the removal or inactivation of small amounts of IgM
...I..
2 ..................................................
.-
/\
. '\
'.-.
"
................................................... --:::2=n"---a 1
2 3 4 5 M o n t h s a f t e r v a c c i na t I on
6
Figure 1. Effect of immunoglobulin levels on serology following strain 19 vaccination. 1 - Positive level for IgM-mediated serology (for example SAT) 2 - Positive level lor IgG-mediated serology (for example supplemental tests) 3 - Duration of positivity in supplemental tests 01 S A T reaction
4 - Duration
Australian VeterinarJ..loiirrial, Vol. 54, December, 197R
will affect the result of the test. This concept is illustrated in Figure 1 . The complement fixation test (CFT) gives results in strain 19 vaccinates that are similar to those given by the supplemental tests. According to Allan et a / (1976) IgM is a more efficient fixer of complement than IgG, (IgG, does not fix complement), but IgM may be at ]east partly inactivated at the temperature used for inactivating serum samples for the CFT. The emergence of the CFT as an effective diagnostic test, with its early return to negative in strain 19 vaccinates, leads us to question the need to restrict S19 vaccination to calves. Worthington et a1 (1973) studied serum antibody titres for a period of 2 years in 99 beef and 22 dairy cows that had been vaccinated as adults with strain 19. SAT titres remained above diagnostic levels throughout, but complement fixaton, mercaptoethanol agglutination and rivanol agglutination titres generally returned to negative levels within 6 months of vaccination. Nicoletti (1976, 1977) used strain 19 vaccination experimentally in cows in large dairy herds in Florida. More than 1,000 cows in infected herds were given the standard dose of strain 19 subcutaneously at approximately 7 to 8 months of pregnancy; subsequently the abortion rate from all causes was less than 1 % . I n more than 700 lactating cows inoculated by various routes, strain 19 could be isolated from the udders of less than 2y0, There was an 80% reduction in the number of reactors removed during the course of the study (6 to 15 months after vaccination). Reducing the dose (up to 1/20 normal) and administering the reduced dose either subcutaneously, intradermally or into the conjunctival sac appeared not to affect immunogenicity, but sharply reduced the level and persistence of antibody production. The author concludes that the problem of titres produced by vaccination can be largely eliminated by using reduced doses of strain 19 and supplemental tests and further, that vaccination of adult cattle is an economical, efficient and practical method of reducing the prevalence of brucellosis in large herds while still allowing the detection of infected animals. Nicoletti and Muraschi (1966), like Jones et (1963), use a complement fixation test based on cold overnight fixation. Cunni ff and Stellar (1968) report that IgG antibody fixes complement . suggests better a t 00 to 4oc and I g M a t 3 7 0 ~This that a test using cold fixation may be preferable for a cattle population where it is important to distinguish between vaccinal reactions and those resulting from natural infection. Plackett et a / (1976) developed the indirect haemolysis test (IHLT) for the diagnosis Of bovine Australian Veterinary Journal, Vol. 54, December, 1978
brucellosis. Amongst other advantages the IHLT is less sensitive than the CFT to antibodies resulting from vaccination with strain 19 vaccine. Further evaluation of the 1HL-T currently in Progress may well show i t to be superior to the CFT as the definitive test for bovine brucellosis in areas where strain 19 vaccine is used. Progress in research into Vaccination O f cattle with strain 19 is reviewed in a report to the National Academy o f Sciences of USA (Anon, 1977).
Vaccines Prepared from B . abortus Slrain45/20 The B. abortus strain 45/20, which is a dissociated “rough” strain and therefore nonagglutinogenic, was first used as a live vaccine but its use was discontinued after it had been found to revert to the smooth, virulent type during passage in cattle. McEwen and Samuel (1955) were first to use the 45/20 strain as a killed culture in oily adjuvant. A number of commercial vaccines of this type, which we shall call K45/20A, are now available, the best known being “Duphavac”*. The development and characteristics of this type of vaccine have been described and summarised by Morgan and McDiarmid (1968). In this report, unless otherwise specified, DuPhavac is the Product being r e f m e d to. The more efficient type of K45/20A vaccine has the following main characteristics. The vaccine can be used safely on cattle of any age and regardless of the state of Pregnancy. Two doses of Vaccine are required and to obtain maximum protection, cattle should be at least 6 months old when first vaccinated. Persistent local swellings often develop at the site of inoculation, especially after the second dose of vaccine; they vary greatly in size b u t generally are not unacceptable. The lesion in the CarCaSe is likely to cause complaint if not removed. Intramuscular injection of the vaccine produces a less visible lesion than does subCUtaneOUS injection. In beef cattle subcutaneous injection is preferred because the lesion is easier to detect and remove after slaughter. The serological response to vaccination is limited (Roerink 1966). There is very little response to the first injection. After the second dose of vaccine the production of agglutinating antibody is minimal, a few animals showing suspicious reactions to the serum agglutination test (SAT), but even these have mostly disappeared by 4 months after the second dose. A few positive reactions to the Rose Bengal Test (RBT) will occur during this period. The reaction to the CFT is more serious and a small proportion of vaccinated cattle retain titres for 12 to 15 months. The Milk Ring Test (MRT) remains negative Adjuvant
* A\dlinble
ftonl Pitmati hlourc. 4SirlusKoad. 1 a n e c o v c . New South Wale,
553
when lactating cows are vaccinated. CFT reactions in cattle that had previously been vaccinated with strain 19 are more frequent a n d more prolonged. The anti-bovine globulin test (ABGT) gives a moderate reaction after the first injection and a severe and prolonged reaction after the second. In controlled experiments the degree of immunity produced is similar to that produced by strain 19. The duration of immunity conferred by K45/20A vaccine has not been accurately determined and for this reason a n annual booster is recommended. Morgan and McDiarmid (1968) make the point that small differences in adjuvant used by different producers affect the character of the vaccine. The possibility of using K45/20A vaccine as a diagnostic agent, in what is now known as a n anainnestic test, was first put forward by Tacken (1964). The concept is based on the difference between the primary and the secondary serological response. I f the first dose of K45/20A vaccine produces a secondary rather than a primary response, i t is assumed that the animal has previously experienced a specific brucella stimulus and if i t has not been vaccinated previously, this stimulus is considered to have resulted from natural infection. The cattle are tested serologicall y and, if negative, are given a single dose of 1
