Role of milk immunoglobulins in the Brucella milk ring test

Milk immunoglobulins were extracted from the stained cream layer of positive milk ring tests from experimentally inoculated or naturally infected cows. IgA was always found, associated with IgM in most cases (15/17) and with IgG in a smaller number of cases (11/17). An additional incubation at 20 degrees C for 18 h gave clearer positive and negative results and a lower limit of detection than that of the usual milk ring test.     

Comparison of the dot-immunobinding assay with the serum agglutination test, the rose bengal plate test and the milk ring test for the detection of Brucella antibodies in bovine sera and milk.

In this study, Brucella antibodies in bovine sera and milk were detected using the dot-immunobinding assay (DIA), the serum agglutination test (SAT), the Rose Bengal plate test (RBPT) and the milk ring test (MRT). For this purpose, a total of 116 paired blood and milk samples collected at the same time from 56 aborted and from 60 healthy dairy cows was examined. In DIA, a nitrocellulose membrane (NCM) was used as the solid phase. Antigen adsorbed on the NCM was extracted from Brucella abortus S99 by heat treatment. The results obtained by DIA were compared with those of SAT, RBPT and MRT. Of the 116 paired blood and milk samples, 24 were positive and 72 were negative by all tests used. Serum samples of six aborted cows were positive by DIA, SAT and RBPT but the milk samples were negative by DIA and MRT. Serum and milk samples of four aborted cows gave positive reaction only by DIA tests. The remaining six aborted cows were negative only by MRT and two of them were negative by both RBPT and MRT. Four sera of healthy cows were found to be positive only by SAT.     

Comparison of an indirect ELISA with the Brucella milk ring test for detection of antibodies to Brucella abortus in bulk milk samples.

An indirect enzyme-linked immunosorbent assay (ELISA) for the detection of Brucella abortus antibodies in bovine bulk milk samples was evaluated. About 31 individual milk samples from B. abortus infected cows were diluted into bulk milk from a brucellosis free herd. Individual milk samples obtained from 96 negative or positive herds to ELISA or Brucella ring test (BRT), were tested by ELISA. All positive cows were bled and serum samples were tested by the complement-fixation test (CFT) which was considered the definitive test. A herd was considered infected if at least, one cow was positive in the CFT. Four samples were negative in the BRT at the dilution 1:10 but positive in the ELISA. For samples positive in both tests, BRT titers ranged from 1:10 to 1:480 while ELISA titers ranged from 1:10 to 1:3200.Using bulk milk samples, the sensitivity of the ELISA (98.1%) was higher than the BRT (72.2%) but the specificity of BRT (90.5%) was not statistically different (P=1.0) from the ELISA (88.1%). The implications of the results for brucellosis control are discussed.     

Haemolysis in gel test for detecting bovine antibodies to Brucella abortus lipopolysaccharide

Optimal reagent parameters for a haemolysis in gel test for detection of bovine anti-Brucella abortus antibody were established. Using an alkali-treated crude lipopolysaccharide antigen attached to J-negative bovine erythrocytes, 0.125 mg of antigen, 0.1 ml of erythrocytes and 0.4 ml of guinea pig complement were required to perform 15 tests with appropriate serum controls. The haemolysis in gel test and an IgM radioimmunoassay (RIA) procedure were compared for their ability to detect the onset of increased serum antibody levels and antibody levels in sera diluted to extinction. While the RIA was more sensitive in amount of antibody detected, the haemolysis in gel test was equally able to detect initiation of antibody synthesis.     

Hemolysis in gel test for detection of bovine antibodies to Brucella abortus: comparison of antigens and test modifications

Killed Brucella abortus strain 1119-3 cells were treated with hot phenol/water or dimethyl sulfoxide to extract soluble crude lipopolysaccharide antigen. Antigen preparations were characterized with respect to protein and lipopolysaccharide content and were compared for efficacy in the hemolysis-in-gel test (HIGT) for detecting anti-Brucella antibodies. All antigens were equally effective in sensitizing bovine RBC in the HIGT and in detecting the presence of anti-Brucella antibodies. A slide modification of the HIGT was developed and compared with the plate HIGT. Seemingly, both tests were comparable.     

Resistance of preimplantation bovine embryos to infection with Brucella abortus

Preimplantation bovine embryos were exposed in vitro to Brucella abortus to determine if the bacteria would adhere to zona pellucida (ZP)-intact embryos or adhere to or infect ZP-free embryos. Brucella abortus was not isolated from ZP-intact or ZP-free groups of embryos after 10 sequential antibiotic-free washings. Brucella abortus was isolated from all groups containing ZP-defective embryos after the exposure period and washing. Detrimental effects on healthy in vitro development of embryos were not observed.     

A modified milk ring test for the recognition of brucellosis in individual lactating cows and heifers

Extract

During the early 1950s, whilst the writer was in practice in Kenya, the need for some simple, practical method of picking out cows infected with brucellosis was recognized. Whatever test was used had to be applicable to as large a proportion of the female herd as possible, had to be simple to perform, requiring little laboratory technique or equipment, and had to be at least as reliable as the whey-plate, or blood-serum tests. The necessity for such a test was brought about by the fact that the only diagnostic laboratory in the country was at Kabete, some 250 miles away. Any material sent there was liable to deteriorate en route and the average delay of ten days before a report could be expected made such a field test desirable. This condition also applies here in Northland at the present time.     

Cellular and humoral responses of Brucella abortus-infected bovine fetuses

Fifty-nine bovine fetuses naturally and experimentally infected with Brucella abortus were studied. Lymphoid hyperplasia in multiple lymph nodes, lymphoid depletion in the thymic cortex, adrenal cortical hyperplasia, and disseminated inflammatory foci composed mainly of large mononuclear leukocytes were present in infected fetuses. Histopathologic changes in naturally infected fetuses were indistinguishable from those infected fetuses inoculated in utero. Fetuses inoculated with 1.0 X 10(3) to 1.0 X 10(5) colony-forming units of strain 2308 B abortus were aborted on postinoculation day (PID) 7 to 19. Fetuses obtained by PID 9 and 10 had increased immunoglobulin concentrations and antibody. Increased cortisol values were present in fetuses obtained as early as PID 6. The initial fetal inflammatory response was composed of large mononuclear leukocytes. In fetuses obtained by PID 9 to 10, moderate numbers of neutrophils mixed with mononuclear leukocytes were present in the inflammatory foci. This shift in the initial inflammatory reaction coincided with the appearance of agglutinating antibody.     

The bovine immune response to Brucella abortus IV. Studies with a double immunodiffusion test for antibody against A2.

A double immunodiffusion test for precipitins against Brucella antigen A2 was developed and applied to a variety of samples. The A2 precipitins were produced by a heifer infected with B. abortus strain 2308, cattle vaccinated with killed B. melitensis strain H38 or live B. abortus strain 19 and by a dog infected with B. canis. Precipitins were also detected in the second International Standard for anti-Brucella abortus serum, in several anti-B. canis sera and at low levels in one anti-B. ovis serum tested. Antisera produced in calves against Yersinia enterocolitica serotype 0:9 had no anti-A2 activity despite titers greater than or equal to 1/1024 and greater than or equal to 1/80 in standard Brucella agglutination and CF tests, respectively. The test for A2 precipitins lacked specificity as weak reactions were obtained with five of 295 sera from brucellosis-free herds. This test was relatively insensitive, detecting precipitins in only 16 of 24 sera from infected cattle and 27 of 54 sera positive by complement fixation and enzyme labelled antiglobulin tests performed with whole cell and smooth lipopolysaccharide antigens, respectively. The A2 precipitins were detected in nine sera from five cattle, in two infected herds, which were negative by agglutination and complement fixation tests.     

Differential reactivity of bovine lymphocytes to species of Brucella

The reactivity of bovine lymphocytes to 4 species of Brucella was tested in thymidine-uptake assays, using long-term cultured lymphocytes and freshly obtained blood mononuclear cells. Lymphocytes were taken from cows that had been challenge exposed with a virulent strain of B abortus at midgestation. The cows were classified retrospectively as being naturally resistant or susceptible to brucellosis. Lymphocytes taken from these cows had 3 patterns of reactivity with species of Brucella: pattern 1 was defined by reactivity with 4 species (B abortus, B canis, B suis, and B melitensis); pattern 2 was defined by reactivity with all these species, except B melitensis; pattern 3 was defined by reactivity with B abortus and B canis, but not with B suis or B melitensis. There was a statistically significant correlation between susceptibility to brucellosis and expression of lymphocyte cross-reactivity with B suis (P less than 0.01) and with B melitensis (P less than 0.001).     

Herd and individual animal risks associated with bovine tuberculosis skin test positivity in cattle in herds in south west England

The aim of this study was to investigate the cattle—exposure factors associated with the risk of a bovine animal reacting to a bovine tuberculosis (bTB) skin test at a whole herd test. There were 148 study farms enrolled. These were located in six counties of the south west of England in an area considered endemic for bovine tuberculosis (bTB): 24% were restocked after foot and mouth disease (FMD) in 2001; all farms were located within the Randomised Badger Culling Trial (RBCT) area. Data on cattle on these farms were sourced from the bTB Vetnet database from 1996 to 2004 and from the British Cattle Movement Scheme database. Individual animal records were created that included data on whether or not an animal became a reactor at a full-herd bTB test between 1 June 2001 and 19 August 2004, their prior exposure to cattle with bTB (defined by presence at a bTB test where at least one reactor was detected), whether the animal was homebred, the farm history of bTB and the farm restocking status. Data from 144 farms were used, 4 farms had no data.Cattle were more likely to react to the bTB skin test when they had been present at a previous bTB herd test (or tests) where other cattle had reacted to the skin test. This positively correlated with age and the number of bTB tests an animal had had. Cattle on restocked farms were less likely to react to the skin test compared with cattle on continuously stocked farms. These results highlight the likely importance of exposure to infected cattle at a previous test as a source of infection to cattle that subsequently became reactors and suggest that there was a lower risk of exposure to bTB to cattle in newly formed herds.     

Interaction of bovine chorioallantoic membrane explants with three strains of Brucella abortus.

Chorioallantoic membrane (CAM) explants were used to determine the in vitro growth and cytotoxic potential of 3 strains of Brucella abortus. Bovine CAM explants were inoculated with 2 x 10(7) colony-forming units of the pathogenic strain 2308, attenuated strain 19, or the rough strain RB51 of B abortus. After inoculation, the explants were harvested and examined at 2 or 4 hours, 12 or 14 hours, and 24 or 26 hours of incubation. Bacterial growth associated with each explant was determined by counting colony-forming units. The degree of cellular damage in each explant associated either with bacterial growth or bacterial toxins was evaluated by morphometric analysis after trypan blue staining. Significant differences were not detected in the numbers of bacteria of any strain of B abortus in the CAM explants at comparable time intervals. The rate of growth of the bacteria in CAM explants was higher between 2 and 12 hours after inoculation than between 12 and 24 hours after inoculation. Cytotoxic effects associated with strain 2308 were significantly (P less than 0.05) greater than that caused by other strains. Cytotoxic effects associated with strain 19 and rough strain RB51 were similar, and both were significantly (P less than 0.05) greater than the phosphate buffer solution control. Chorioallantoic membrane explants inoculated with a filtrate of heat-killed strain 2308 induced minimal cellular damage, compared with that caused by the viable bacteria. These results indicated that the number of B abortus in trophoblasts was independent of the degree of cellular damage.     

Kinetics of in vitro bovine lymphocyte immunostimulation with a Brucella abortus antigen.

A Brucella abortus-soluble antigen was investigated, using in vitro assay of lymphocyte immunostimulation, to determine which concentration of this antigen and which period of incubation of the lymphocyte cultures would induce maximum specific lymphocyte immunostimulation as an additional method for further study of B abortus infection in cattle. Soluble antigen was prepared from autoclaved cells of B abortus strain 1119-3. Peripheral blood lymphocytes were obtained from cattle infected with B abortus and from healthy control cattle not infected with B abortus. The lymphocytes were prepared by the Ficoll-Hypaque density gradient technique, suspended in RPMI 1640 medium (1.5 X 10(6)/ml), cultured with several dilutions of soluble antigen, and incubated. Prior to termination of incubation, cultures were labeled with 1 muCi of [3H]thymidine and, after harvesting, assayed for [3H]thymidine incorporation in DNA by a liquid scintillation spectrometer. Maximum specific immunostimulation of lymphocytes from B abortus-infected cattle was induced in this assay system with 6 days' incubation and 22 microgram of protein/ml/1.5 X 10(6) lymphocytes, using protein content to express concentration of soluble antigen in this system.