Development of a simple, rapid in vitro cellular assay for bovine tuberculosis based on the production of gamma interferon.

Whereas in vitro assays of antibody responses to infectious agents are commonly used for routine diagnostic purposes, diagnostic tests for cellular responsiveness are confined to in vivo intradermal tests. This paper describes a simple and rapid in vitro cellular assay for bovine tuberculosis. The assay system is based on the detection of gamma interferon (gamma IFN), which is released in response to specific antigen. This assay can be carried out with whole blood samples thus avoiding the time-consuming task of isolating lymphocytes. A simple bioassay has been developed to quantitate the amount of gamma IFN produced, but this will eventually be replaced with an enzyme-linked immunoassay using monoclonal antibodies specific for bovine gamma IFN. The application of this system to bovine tuberculosis and other infectious diseases may provide a convenient in vitro cellular assay for routine diagnostic purposes.

Discrimination between sheep antibodies to Brucella melitensis and to Brucella ovis.

When preparations containing smooth Brucella abortus lipopolysaccharide (LPS) were used as antigen in an ELISA, strong positive reactions were obtained with sera from sheep infected with Brucella melitensis or with Brucella ovis. Oxidation of the LPS with sodium metaperiodate greatly reduced the extent of the cross-reactions with antisera to B. ovis, with little effect on the reactions with antisera to smooth B. melitensis. Periodate oxidation of hot saline extract (HSX) antigen of B. ovis markedly reduced its reactivity in ELISA with anti-B. ovis sera and eliminated cross-reactivity with anti-B. melitensis sera. The reactivity of HSX was maintained after treatment with proteinase K. A simple ELISA system, in which replicate samples from a single serum dilution were tested in parallel against both B. ovis HSX antigen and periodate-oxidised smooth phase B. abortus LPS, was evaluated. It was found to discriminate well between antibodies induced by vaccination or virulent infection with B. melitensis strains and those induced by infection with B. ovis.

Purification of a major Mycobacterium bovis antigen for the diagnosis of bovine tuberculosis.

A Mycobacterium bovis antigen has been purified from culture filtrate by chromatofocusing. This antigen is a major component of culture filtrate and cell extracts and shows a considerable degree of micro-heterogeneity in electric charge and molecular weight. Studies with monoclonal and polyclonal antibodies raised against the purified antigen show that some of its antigenic determinants also occur in higher molecular weight species in culture filtrate and particularly in whole cell preparations. Immunoblotting and ELISA studies, using sera from M.bovis-infected animals, showed that this antigen is one of the most immunoreactive components of M. bovis, recognized by the majority of animals with detectable antibody response to M. bovis. The specificity of the purified antigen is far superior to that of the crude culture filtrate, with very few false positive results. The purified antigen also elicits strong in vivo and in vitro cell-mediated responses. The amino acid compositions of two variants of this antigen have been determined and found to be similar to that of MPB-70.

An ELISA for the detection of anergic tuberculous cattle.

An enzyme-linked immunosorbent assay (ELISA) for bovine antibody to antigens in unheated Mycobacterium bovis culture filtrate was standardised against a reference serum from an experimentally infected cow. Two Northern Territory herds with a total of 561 cattle were tested. All cattle reacting in the caudal fold tuberculin test, those giving strong reactions in the ELISA and those with visible lesions of tuberculosis were subjected to a detailed bacteriological examination. Of the 19 cattle which yielded isolates of M. bovis, only 4 were positive to the tuberculin test. Serum samples from 5 cattle gave ELISA values greater than 7.0 units. None of these 5 reacted in the tuberculin-test and 2 had no visible lesions. Of the 10 remaining cattle from which M. bovis was isolated, 3 had ELISA values between 6.5 and 7.0 units and were also without visible lesions. The ELISA values for the remaining 7 infected cattle ranged down to 4.6 units. Forty cattle yielded no M. bovis on culture of their tissues. They included 7 which were reactors in the tuberculin test and 23 with ELISA values of 7.0 units or more. The evident low specificity and sensitivity of the ELISA make it of little value as an alternative to the tuberculin test, but it can detect some anergic cattle at the cost of increasing the number of false positive reactors. This may be acceptable in some circumstances and would justify the use of the ELISA as a complement to the tuberculin test or to an in vitro assay of T-cell immunity. In the 2 Northern Territory herds described, the removal of 5 of the anergic cattle would have required a cull of 28 animals of 5% of the total. A cut off value of 6.5 units would have eliminated 3 more, but at the cost of culling 80 animals or nearly 15% of the cattle. Even so, 7 cattle from which M. bovis was isolated would have remained undetected by either test.

Production and characterization of monoclonal antibodies specific for Mycobacterium bovis.

A series of monoclonal antibodies (MAbs), specific for Mycobacterium bovis and BCG strains, were tested extensively for cross-reactivity to a wide range of mycobacterial species using ELISA, Western blotting and dot-blot analysis. The MAbs bound specifically to M. bovis and BCG and showed limited cross-reactivity with some strains of M. tuberculosis. All these MAbs recognized a 22 kDa protein previously termed MPB70, and by competitive ELISA analysis appeared to detect at least three M. bovis-specific determinants on the MPB70 molecule.

Cloning of a species-specific antigen of Mycobacterium bovis.

A DNA library from a virulent strain of Mycobacterium bovis was constructed in the expression vector lambda gt11, and the library was probed with antisera to M. bovis. Clones expressing M. bovis antigens were isolated and characterized by using M. bovis-specific monoclonal antibodies that recognize a 22,000-molecular-weight protein (MPB70). MPB70 is a major protein antigen of the vaccine strain of M. bovis BCG and of virulent M. bovis, the causative agent of bovine tuberculosis. Of 32 clones selected by using polyclonal affinity-purified anti-M. bovis sera, 5 were recognized by the anti-MPB-70 monoclonal antibodies, and one monoclonal antibody, SB10, recognized all 5 clones. Characterization of these clones showed that one clone containing a 253-base-pair insert expressed a polypeptide bound by all of the MPB70-specific monoclonal antibodies. Western blots (immunoblots) showed that this cloned protein was recognized by sera from M. bovis-infected cattle, although not all cattle with bovine tuberculosis produced antibodies reactive to this clone. DNA sequencing of the clone showed that it coded for 84 amino acids from positions 17 to 114 of the 161-amino-acid protein, with a 16-peptide deletion between positions 79 and 94. Apart from this deletion, there were seven other variations between the cloned sequence and that deduced from M. bovis BCG MPB70.

Vaccination against bovine brucellosis.

Four experiments are summarized. Initially an attempt was made to modify strain 19 calfhood vaccination so as to eliminate the persistent serological reactions which interfere with eradication programmes. Later the project broadened into a search for an effective method of vaccination that could be applied when required, to all ages of cattle, thus allowing calfhood vaccination to be safely stopped. In the first experiment, reducing the age at vaccination with strain 19 to 1 month practically eliminated the serological response to vaccination, but the resulting immunity was not satisfactory. However, vaccination at 1 month followed by a booster consisting of a reduced dose of strain 19, given conjunctivally 1 year later, stimulated an immunity at least equal to that given by conventional calfhood vaccination. The vaccination of pregnant cows with either of 2 reduced dose levels of strain 19 gave better immunity than calfhood vaccination with the full dose. Uterine strain 19 infections were unacceptably frequent in cows given 6 X 10(9) c.f.u. of strain 19 in early pregnancy, but no such infections were found in 9 cows given 3 X 10(8) c.f.u. Vaccinal antibody titres declined rapidly in the latter group. Vaccination of mature, non-pregnant heifers with 3 X 10(8) c.f.u. of strain 19 produced immunity at least as good as that produced by calfhood vaccination, with a serological response greatly reduced in the majority of cattle. However, a small proportion of vaccination cattle developed high titres persisting for at least 7 months.(ABSTRACT TRUNCATED AT 250 WORDS)

Vaccination of cattle against brucellosis using either a reduced dose of strain 19 or one or two doses of 45/20 vaccine.

Three groups, each of 14 mature Jersey heifers, were vaccinated. They were mated about 2 months later and those that became pregnant were challenged at about 6.5 months of pregnancy by the conjunctival application of virulent Brucella abortus. Group 1 heifers received 2 doses of B. abortus 45/20 vaccine 2 months apart. Only 5 of the 14 heifers became pregnant, and of these 5 only one resisted challenge. Group 2 heifers received only one dose of 45/20 vaccine, 5 of the 10 challenged resisted infection. Group 3 heifers received 3 x 10(8) cfu of strain 19. Six of the 10 heifers challenged resisted infection. All of 5 non-vaccinated control cattle became infected. It appeared advantageous to give only one dose of 45/20 rather than 2 as presently recommended. A single dose of 45/20 vaccine induced resistance to virulent B. abortus approximately equal to that given by the reduced dose of strain 19. One dose of 45/20 vaccine stimulated transient serological positivity in 2 of 28 heifers whereas the reduced dose of strain 19 gave rise to persistent titres in 2 of 14 vaccinated heifers.

The role of the differential complement fixation test, using rough and smooth brucella antigens, in the anamnestic test.

To assess the ability of the differential complement fixation test to distinguish vaccinal reactors from infected cattle, approximately 1,000 heifers were tested by the complement fixation test (CFT) using rough and smooth brucella antigens, before the injection of 45/20 vaccine and at 3 and 6 or 10 weeks after vaccination. Before vaccination 91.5% of heifers were negative to the rough antigen but 0.6% were positive with high titre (greater than or equal to 128). By 10 weeks after injection of 45/20 vaccine 97.6% of heifers were positive to the rough CF antigen, at greater than or equal to 8, a majority reaching greater than or equal to 128. Nineteen pre-vaccinal reactors to the standard CFT were killed and Brucella abortus was isolated from the tissues of 14. Twenty-six post-vaccinal reactors were killed and B. abortus was isolated from the tissues of 8. In the 22 B. abortus infected animals the differential CFT classified 9 correctly as infected, 5 incorrectly as vaccination reactions and 8 as inconclusive. The differential CF was ineffective in distinguishing titres resulting from vaccination with 45/20 vaccine from those due to infection.

Failure of a single dose of Brucella abortus strain 19 vaccine to protect cattle when given early in calfhood.

Groups of heifer calves were vaccinated subcutaneously with the standard dose of strain 19 at the age of 3 to 5 weeks or 5 months. Twelve months after primary vaccination a group of the younger vaccinates were revaccinated with a small dose of strain 19 via the conjunctival sac. The effectiveness of vaccination was assessed by challenge with virulent B. abortus after 5 to 6 months of pregnancy. Animals vaccinated at 3 to 5 weeks, but given no intraconjunctival boost, were not effectively protected, but the group receiving the boost were at least as resistant as those which received the subcutaneous dose at 5 months. The intraconjunctival boost caused only slight and transient serological responses in a few animals.

The indirect haemolysis test (IHLT) for bovine brucellosis - comparisons with the complement fixation test (CFT) in vaccinated and experimentally infected cattle.

Cattle were vaccinated with Brucella abortus strain 19 subcutaneously in doses ranging from the normal (2.0 ml) dose of standard vaccine down to 1/400 of the normal dose, and via the conjunctival sac with 1/2 of 1/20 of the normal dose. Under all vaccination regimes serum antibody titres in the complement fixation test (CFT) rose more rapidly, reached higher levels, declined more slowly and involved a greater proportion of animals, than titres in the indirect haemolysis test (IHLT). The interval between the first positive serological test and parturition was determined for 54 pregnant cows infected with a virulent field strain (VRI 3) of B. abortus. On average the CFT titre rose to 1/4 43 days, and 1/8 33 days, before parturition, while the IHLT reached a 2/8 reaction 31 days before parturition.

Vaccination of pregnant cows with low doses of Brucella abortus strain 19 vaccine.

Two groups, each of 9 Jersey cows, were vaccinated subcutaneously with reduced doses of Brucella abortus strain 19 vaccine (measured by the number of bacteria in the vaccine dose) early in their second pregnancy. Ten weeks later they were challenged, along with a similar group of non-vaccinated cows, by conjunctival instillation of a virulent strain of B. abortus biotype 1. Cows in Group 1 received 1/20th and those in Group 2 received 1/400th the recommended dose of strain 19. Marked reduction in the serological response to vaccination was seen only in Group 2. Four cows in Group 1 excreted strain 19 after parturition, one of them aborted and another calved prematurely with heavy infection of the placenta and foetus with strain 19 in both cases. Resistance to challenge was similar in both vaccinated groups, and higher than previously demonstrated after conventional calfhood vaccination with strain 19. It is concluded that pregnant cows can be effectively vaccinated by the subcutaneous administration of a dose of strain 19 vaccine containing approximately 3 x 10(8) organisms without undue interference with subsequent serological tests or inconvenience resulting from persistence of strain 19 infection.

An indirect haemolysis test (IHLT) for bovine brucellosis.

A simple indirect haemolysis test (IHLT) was developed to avoid the problem of prozones in the complement fixation test (CFT) for bovine brucellosis. It makes use of a sheep or bovine erythrocytes, treated with a crude lipopolysaccharide fraction of Br. abortus, which are lysed by specific antibody in the presence of excess complement (C'). A number of bovine serum which gave large prozones in the warm CFT, and some in which C'-fixation was completely blocked, were found to react to high titre, without prozones, in the IHLT. Following primary vaccination with Br. abortus strain 19, fewer animals gave positive reactions in the IHLT than in the CFT. Following two doses of 45/20 vaccine, however, positive reactions were more frequent in the IHLT than in the CFT. Preliminary studies of serums from animals known to be infected indicate that the IHLT may be of diagnostic value. The test is easy to carry out, especially when bovine erythrocytes are used, since very few bovine serums require preliminary absorption.

A mechanism for prozone formation in the complement fixation test for bovine brucellosis.

Complement fixation (CF) by bovine IgG1 or IgM antibodies to Brucella abortus was inhibited by specific non-complement-fixing antibodies of the IgG2 subclass. This inhibition may account for the appearance of prozones in CF titrations of some antiserums, and for the occurrence of serums which are positive to the Rose Bengal test, but negative to the CF test.

Identification of the amide-linked fatty acids of Acholeplasma axanthum S743 as D(-)3-hydroxyhexadecanoate and its homologues.

The amide-linked fatty acids of the sphingolipids of Acholeplasma axanthum S743 are predominantly hydroxy acids. These acids were shown by gas-liquid chromatography, mass spectrometry, and polarimetry to be the d(-)3-hydroxy fatty acids. The predominant component of the mixture was 3-hydroxyhexadecanoate (beta-hydroxypalmitate, hydroxy [h] 16:0) followed by h 20Delta (Delta = unsaturated), h14:0, h12:0, and h18Delta in decreasing order of concentration. The fatty acid profile indicates that these beta-hydroxy acids possibly arise from elongation of the fatty acids supplied in the growth medium.

Evidence for a phosphoenolpyruvate-dependent sugar phosphotransferase in Mycoplasma strain Y.

The uptake of (14)C-alpha-methyl-d-glucoside (alphaMG) by washed cells of Mycoplasma strain Y was found to be dependent on the supply of metabolic energy. Glycerol or d-mannose, but not l-lactate, would serve as an energy source. Uptake was inhibited by fluoride, iodoacetate, and arsenate, but not by 2,4-dinitrophenol. d-Glucose was inhibitory, presumably by competing for the transport system. The initial product of accumulation had the properties of a phosphate ester of alphaMG. The proportion of free alphaMG in the cells increased with time, until a steady state was reached in which uptake was balanced by the efflux of free alphaMG from the cells. Broken-cell preparations catalyzed a phosphoenolpyruvate-dependent phosphorylation of alphaMG and of d-glucose.

Lipids of a sterol-nonrequiring Mycoplasma.

The lipids of the sterol nonrequiring Mycoplasma strain S743 were found to include both ester glycerophosphatides (phosphatidylglycerol, acylphosphatidylglycerol, and diphosphatidylglycerol) and ceramide glycerophosphate compounds containing N-hydroxyacyl groups. The major phosphosphingolipid was tentatively identified as a hydroxyceramidephosphorylglycerol containing an O-acyl group. These compounds became labeled during growth in the presence of (32)P-orthophosphate, (14)C-glycerol, or (14)C-palmitate. The lipid fraction also contained free long-chain base. (14)C-palmitate was converted to labeled sphinganine. The long-chain base composition of the lipids was modified by growing the organisms in media containing different fatty acids, which were converted to bases containing two more C atoms per molecule. Ninety per cent of the long-chain base from cells grown in medium supplemented with elaidate consisted of monounsaturated C(20) base.