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273 Brucella strains from Africa have been examined for identification and typing in our laboratory, since 1976. Of these, 213 were isolated from Senegal, 30 from Togo, 12 from Morocco, 10 from Rwanda, 7 from Guinea Bissau and 1 from Niger. 272 strains were from cattle. Most of them (260) were from native animals that showed hygromas. 12 strains from Morocco were isolated from aborted calves of the "Pie Noire" dairy breed. One strain was from a human case of Brucellosis in Rwanda. All the strains were classified as members of B. abortus species by the recommended methods of the Subcommittee on Taxonomy of the genus. Of them, 269--including the human strain--were biotype 3/6. The others--2 from Morocco, 1 from Senegal and 1 from Rwanda--were biotype 1. Comment is needless about the 12 strains isolated from imported "Pie Noire" cattle. Their characteristics are identical with those of the B. abortus same biotypes isolated out of Africa. On the other hand, three unusual characteristics distinguish the 260 strains from native cattle and the human one from the main group of B. abortus: their growth characteristics, their mean oxidative profile and--for 219 of them--a negative oxidase test. The unusual behaviour of native B. abortus strains is discussed from an epidemiological and taxonomical point of view.

In a collaborative investigation in which 4 laboratories took part, the Rose Bengal Test (RBT), Serum Agglutination Test (SAT) and 4 different Complement Fixation (CFT) techniques were evaluated in selected cattle for the diagnosis of bovine brucellosis, by comparing the results they gave with the bacteriological examination of a selection of lymph nodes taken from the same animals at slaughter. The RBT correctly classified all but 1 of 79 culture-positive cattle, but was more often positive in culture-negative animals than the other tests. The RBT may be most useful as a screen test. 11% of the culture-positive cattle had SAT titres below 100 iu and almost 4% of them had less than 30 iu. However, the SAT was more effective in cattle vaccinated with the 45/20 vaccine. The CFT, in 1 of 4 techniques used, identified all of the culture-positive cattle at a serum dilution of 1/4 or above and was considered to be far superior to the SAT as a diagnostic test, except perhaps in cattle vaccinated with the 45/20 vaccine.

In the course of eight successive Brucella abortus experiments, 394 cows were followed by clinical, serological and bacteriological examinations. Cultures were made from vaginal swabs and colostrum at delivery and from 10 pools of lymph nodes or organs sampled at slaughter, about 6 weeks after calving. From these cows, 346 had been experimentally challenged with 1.5 X 10(7) B abortus strain 544 via the conjunctiva, 3 congenitally infected and 45 bought from known naturally infected herds. Males were more numerous (64%) in aborted foetuses and less numerous (34%) in premature live calves than females, the sex ratio being normal in calves born at term. Recovery of Brucella infection occurred between calving and slaughter in 13/230 cows (5.7%). It appeared that localization, spread and level of infection in different lymph nodes and organs, duration and outcome of pregnancy, and agglutination titers were strongly correlated. Comparisons between experimentally and naturally infected cows evidenced the fact that both types of infection were very similar. Confidence for field application of experimental results was thus reinforced.

A survey of incidences of brucellosis was carried out in 1378 lactating animals (590 buffaloes; 290 cows; 471 goats; and 108 sheep). Individual reactors to MRT were further identified with blood serum tube and quick agglutination tests.--Recorded incidences of brucellosis in the present investigation were found to be low, 1.4% in cows, 1.3% in goats, 1.2% in buffaloes and 0.9% in sheeps. The present investigation supports the wide use of MRT for screening antibodies for brucellosis in lactating animals which can be followed by SAT.--All those animals which responded to tube agglutination test were also positive for blood serum quick agglutination test.

An hemolysis-in-gel test (HIGT) for bovine antibody against Brucella abortus was developed and evaluated. Sera to be tested were placed in wells in an agarose gel containing guinea pig complement and J-negative bovine erythrocytes coated with lipopolysaccharide prepared from B. abortus biotype 1. After incubation, zones of hemolysis were produced by positive sera. The activity of some positive sera was heat labile, but was restored to heated sera by addition of a crude preparation of the first component of bovine complement. The HIGT was compared with the complement fixation test (CFT), the buffered antigen plate test (BPAT), the standard tube agglutination test (STAT) and the standard plate agglutination test (SPAT), using sera from 1041 brucellosis-free cattle, from 51 cattle infected with B. abortus biotype 1 or biotype 4, and from six heifers vaccinated with strain 19. Three of 1041 sera (0.29%) from brucellosis-free cattle were HIGT-positive, ten (0.96%) were BPAT-positive, and none were positive in the CFT, STAT, or SPAT. The HIGT was more sensitive, and detected infection earlier than the other tests in the case of B. abortus biotype 1 infection, but was less sensitive for biotype 4 infection. In vaccinated heifers, HIGT reactions appeared later than reactions to the other tests, and persisted as long as 565 days. Further studies are needed to standardize the HIGT test conditions and to improve its sensitivity for B. abortus biotype 4 infections. Attempts to determine the effects of LPS antigen prepared from different biotypes of B. abortus are in progress.

Tissues from 104 cows in herd were examined for brucellae. Brucella abortus, strain 19, was isolated from 22 cows, a field strain of B abortus, biotype 1, was isolated from 9 cows, and both strains were isolated from 2 cows.

Thirty cows naturally infected with Brucella abortus were treated by various routes, using free or liposomal streptomycin or a combination of liposomal streptomycin and a long-acting oxytetracycline preparation. Of 21 cows treated with liposomal streptomycin alone, 3 (14%) were culture negative and 3 had 10 or fewer bacterial colonies isolated from tissues obtained at necropsy. Thirteen (62%) cows continued to shed organisms in udder secretions and were considered treatment failures. Of 9 cows that were given a combination of liposomal streptomycin and long-acting oxytetracycline, 5 (56%) were cured, 3 had 10 or fewer colonies on culture plates of tissue after necropsy and only 1 continued to shed B abortus in udder secretions after treatment. Eleven cows were given streptomycin liposomes by intramammary infusion with or without IM administration of long-acting oxytetracycline. The most effective regimen consisted of 2 intramammary infusions of streptomycin liposomes and 2 doses of oxytetracycline administered IM. Of 5 cows treated thusly, 2 were cured and all others had fewer than 10 B abortus colonies isolated from tissues obtained at necropsy.

The serum agglutinin titres of six heifers experimentally inoculated with live Br. abortus strain 45/20 were similar to those produced in six bullocks inoculated with virulent Br. abortus strain 544. However, the agglutinin response appeared slowly and disappeared earlier in the heifers than in the bullocks. It is concluded that Br. abortus strain 45/20 is an unstable strain, could revert to the smooth form and become highly agglutinogenic in non-pregnant heifers. The significance of using an unstable rough strain of Br. abortus vaccine in the diagnosis of bovine brucellosis is briefly discussed.

Each Brucella species is known to have a definite host preference. In this respect, brucellosis in cattle is primarily due to B. abortus. As part of an investigation of the species and biovars responsible for bovine brucellosis in France, 312 strains have been examined since 1978 by the methods recommended by the Subcommittee on the Taxonomy of the genus Brucella. Of these, 264 (85%) were indeed classified as B. abortus members. However the 48 others (15%) had all the characteristics that define the species B. melitensis which usually affects small ruminants. Of these 48 strains, 43 (90%) came from the southern part of France in which B. melitensis infection in sheep and goats is enzootic and where the dissemination of this species by sheep flocks moving to mountain pastures most often accounted for cattle contamination. Evidence that B. melitensis infection in cattle traced to infected small ruminants was also strongly supported by biotyping. Of the 48 strains, 45 (94%) indeed were B. melitensis biovar 3 which is the most common in infected sheep and goats in France. B. melitensis infected cattle, which also constitutes a serious risk for public health, must therefore be considered by veterinary authorities because of epizootiologic implications in eradication and control programmes of sheep and goat brucellosis.

Studies using the in vitro lymphocyte stimulation test (LST) were conducted with cattle in a dairy herd with a high percentage of reactors to several serological tests for brucellosis. Lymphocytes were prepared from peripheral bovine blood by the Ficoll-diatrizoate technique. Lymphocytes were cultured using microtitration culture plates. Brucella abortus soluble antigen, at a concentration of 4.4 microgram/culture, was added to the appropriate wells of microtitration culture plates and incubated for 6 days. The lymphocyte stimulation responses were measured by assaying for [3H]thymidine incorporation into DNA. Seroagglutination tests were conducted simultaneously with the LST, and tissues were collected after slaughter of the cattle for bacteriological culture to isolate B. abortus. All 21 animals studied were serologically negative for anti-brucella antibodies. Two of the 21 animals were classified as infected with Brucella by the LST, and B. abortus biotype 1 was isolated from tissues of these same two animals. The LST exhibited significant sensitivity and specificity in this study, and more observations of this nature might strengthen the application of this assay as an aid in the diagnosis of brucellosis.

Seventy two non-vaccinated cattle with various complement fixation (CF), rose bengal (RB) and enzyme-linked immunosorbent assay (ELISA) results at slaughter were examined bacteriologically and serologically. Brucella abortus was recovered from 49 (68.1%) of the cattle and the use of a biphasic culture medium was entirely responsible for the detection of 6 (12.2%) of the culture positive cattle. The supramammary and retropharyngeal lymph nodes were the most rewarding tissues to culture. A comparison of culture results and serological status demonstrated that B. abortus could be isolated from cattle with negative RB and CF tests and that the ELISA was useful in detecting these cattle and infected cattle with low CT titres. The RB test was also useful as it detected all but 4 of the cattle found to be infected.

Forty-five brucella infected cows were studied 15 to 30 days following abortion. Eighteen cows were slaughtered and autopsied for examination of Brucella in the organs and lymph nodes. The remaining 27 cows were treated during two and one-half months and then slaughtered after undergoing a treatment with 0, 1, 2 or 3 intraperitoneal injections of oxytetracycline dissolved in 100 ml of physiological saline. The treatment modified only slightly the natural evolution of antibody titers. The levels of infection were similar for all cows which received no treatment. Cows treated with oxytetracycline had less severe infection than the non treated animals and four were infection-free at slaughter. The level of infection of treated cows was independant of the treatment regime. The advantages of treating non pregnant cows to reduce the level of infection and risk of abortion were discussed.

Thirty Togolese strains of Brucella from cattle were classified as members of B. abortus biotype 3 by the recommended methods of the Subcommittee on Taxonomy of this genus. However two unusual characteristics distinguish the Togolese strains from the main group of B. abortus: their mean oxidative profile altered on four of the conventional substrates (L-asparagine, L-arabinose, D-galactose and D-xylose) and their very slow growth on usual media. These two original characteristics are discussed from an epidemiological and taxonomical point of view.

Cell-mediated immune responses in cattle adult-vaccinated with Brucella abortus strain 19, cattle infected with B abortus field strain, and nonexposed cattle were studied by an in vitro lumphocyte-stimulation test (LST). Lymphocytes were prepared from peripheral bovine blood by the Ficoll-diatrizoate technique, and results were assayed for [3H]thymidine incorporation into DNA by liquid scintillation spectrometry. Serotests and bacteriologic isolation attempts were conducted simultaneously with LST. Lymphocytes from cattle infected with field strains had significantly (P = 0.01) higher specific lymphocyte-stimulation inexposed controls. The LST, the serum standard-tube agglutination test (STT), the Rivanol (RIV) test, and the complement-fixation (CF) test correctly classified cattle from which field strains and strain 19 of B abortus were isolated. The LST was negative in cattle vaccinated with B abortus strain 19 (nonshedding), but the three serotests had many false-positive reactions. The CF test had the least false-positive reaction, followed by the RIV test, and the STT was the least specific. Well before the three serotests became positive, the LST was positive in samples from some cattle during the incubation period of the infection. There was little or no correlation between cell-mediated immune responses (as measured by LST) and serum antibody responses (as measured by STT, RIV test, and CF test) in vaccinated but culture-negative cattle and in some nonvaccinated cattle during the incubation period.

Antibodies specific for Brucella abortus were purified from the serum of hyperimmunized sheep using immunochemical procedures. They were conjugated to fluorescein isothiocyanate and used in a fluorescent antibody (FA) test for B. abortus. The conjugate did not stain any heterologous bacterial or fungal species tested and background fluorescence associated with its use on smears and sections of abortion materials was particularly low. Of 239 cases of abortion examined fluorescent microscopy demonstrated B. abortus in all 12 cases in which the organism was isolated. A few areas of fluorescence typical of B. abortus were also seen in 3 cases from which the organism was not cultured. B. abortus was demonstrated in lymph nodes from 6 of 36 Brucella reactor cows by culture and 7 by the FA test. However, only very low numbers of B. abortus were isolated or seen and sampling errors would have been significant. Use of the FA test allows diagnosis of Brucellosis to be made in 2 hours, compared to 6 days by the usual cultural procedures.

Heifers injected with 10(8) (n = 40), 10(9) (n = 39), or 10(10) (n = 39) colony-forming units of Brucella abortus strain 19 were conjunctivally exposed to 10(7) colony-forming units of strain 2308 during gestation. At parturition, milk from each quarter of the udder, a piece of placenta, and 2 swab specimens of the uterus from the dam plus a swab specimen of the rectum from each calf were cultured for Brucella. If the calf was dead or died, additional specimens of lung, stomach contents, and a mediastinal lymph node also were cultured. Days in gestation was determined for each heifer, using data from rectal palpation after breeding and crown-rump length and weight of calf at parturition, with the median value used for data analysis. In each vaccine dosage group, the proportion (%) of heifers developing brucellosis increased as days in gestation at exposure increased. Strain 2308 was isolated from 3 (11%) of 26, 16 (25%) of 64, and 18 (64%) of 28 heifers that were grouped as less than 121, 121 to 150, and greater than 150 days in gestation at time of exposure, respectively. Thirty-two (86%) of the 37 infected heifers were less than 260 days in gestation at parturition, and calves were premature. Heifers with premature calves were more likely to be infected, and tissues were more likely to yield multiple isolations of strain 2308, regardless of days in gestation at exposure or of days after exposure to parturition. Days after exposure to premature parturition of infected heifers ranged from 35 to 110.

Over a 3 year period specimens were collected from single reacting cattle to the complement fixation test for brucellosis from large herds in north Queensland. Twenty-four such reactors were destroyed for culture, and Brucella abortus was isolated on 3 occasions. The existence of such low levels of Br. abortus in large herds should not be overlooked in the eradication campaign. Explanations were found for 17 of the single reactors, and included introduction of cattle to the herd, exposure to adjoining infected herds or vaccination with Strain 19.

Five calves from Brucella abortus reactor cows bought after they had been on infected farms for two to six weeks, were reared in isolation and examined serologically for approximately six months prior to cultural and biological examination post mertem. One calf which had been serologically negative to the Rose-Bengal, serum agglutination and complement fixation tests for the last eight weeks of its life yielded a culture of Br abortus biotype 1. This serotype had also been isolated from its dam at parturition.

Pregnant goats in midgestation (7 to 16 weeks) were conjunctivally exposed to Brucella abortus strain 2308 to evaluate their applicability as an animal model for bovine brucellosis. Brucellae were isolated from uterine fluid and/or placental specimens of 10 of 12 does at parturition. Six of the 10 infected does delivered dead fetuses and 1 of the 10 delivered live, premature twins. Dead fetuses typically contained brucellae in multiple tissues, whereas brucellae generally were not isolated at birth from live kids. After parturition, B abortus was excreted in the milk and uterine fluids of the infected does. At necropsy (6 weeks after parturition), organisms in the doe were primarily in the uterus and in the lymph nodes that drained the mammary glands, uterus, and head. Brucella abortus was most often isolated from the cranial lymph nodes of neonates that had remained with their dam for 6 weeks after parturition. Serum anti-Brucella antibody concentrations were determined by use of standard tube agglutination, mercaptoethanol agglutination, Rivanol plate tests, card tests, complement fixation, hemolysis-in-gel tests, and an enzyme-linked immunosorbent assay. Serologic responses were detected 2 to 3 weeks after exposure and remained detectable until parturition. Antibody titers increased after parturition in does shedding B abortus at parturition. Anti-Brucella antibody was not detected in neonates before colostrum intake. The neonate's postcolostral titers were similar to those in the dam at the time of parturition. Milk anti-Brucella antibody was detected in milk (milk ring test) from infected and noninfected mammary glands.(ABSTRACT TRUNCATED AT 250 WORDS)

Three different culturing techniques were compared and evaluated to determine the most effective method for isolating Brucella abortus from bovine supramammary lymph nodes (SM's). In method I, the SM was sliced in half, and the inner surface was minced finely with a sterile scalpel. The minced surface was spread onto the agar surface of 4 selective media. In method II, the SM was cut into small pieces and placed in a bag with a volume of phosphate-buffered saline equal to the volume of the lymph node. The bag was placed in a laboratory blender and the SM was macerated for 5 min. The tissue suspension was spread with a sterile cotton swab onto the agar surface of 4 selective media. In method III, the SM was processed in the laboratory blender. One milliliter of the suspension was pipetted into a flask of biphasic medium, and 2 ml of the suspension was pipetted into another flask of biphasic medium. A total of 626 SM's from 285 cows were cultured. Brucella abortus was isolated from 149 (52.3%) cows by 1 or more methods. Brucella abortus was isolated from 136 cows by method I. 137 cows by method II, and 86 cows by method III. Nine (3.2%) cows were positive by method I only, 11 (3.9%) cows by method II only, and 2 (0.7%) cows by method III only. The isolation rate for method III was significantly lower than for method I or II. There was no significant difference between methods I and II.

With the aim of knowing more exactly what species and biotypes are responsible for brucellosis in France, 2,058 strains have been examined since 1972. Of these, 1,693 were isolated from cattle, 190 from sheep, 10 from hare, 9 from goats, 3 from swine and 3 from field mice (Apodemus sylvaticus); 150 were of cases of human infection. Of the 18 types presently recognized for the genus Brucella, 11 were identified within the sample. In animal strains, they were B. abortus biotypes 1, 2, 3, 4 and 9, B. suis biotypes 1 and 2, B. melitensis biotypes 1, 2 and 3, and B. ovis. All these types--except B. suis 2 and B. ovis--were identified within the human strains. Of these 11 types, 3 were widely predominant both in man and animals: B. abortus biotypes 1 and 3 and B. melitensis biotype 3, which together total 84.2% of the sample's strains. The results also emphasized the preference of B. abortus, B. melitensis and B. ovis for a decided animal host: 99.4% of the bovine strains were B. abortus, 98.1% of those isolated from ewes and goats were B. melitensis, and all the B ovis strains were from cases of ram epididymitis. Of the 150 human strains, 115 were B. melitensis, 33 B. abortus and 2 B. suis. These data were discussed from a taxonomical and epidemiological point of view.

Calvings in a herd infected by Brucella abortus were monitored by cultural examination of milk and mucus and by serological and cultural examination of 5 ml samples of the blood. Out of 129 samples from which culture of B abortus was attempted, only one yielded a positive result.

Results listing the identification of brucella isolates received by the National Brucellosis Reference Centre, National Biological Standards Laboratory, Canberra from 1981 to 1985 are presented. The distribution of brucella species and biotypes is shown on a host and state basis. Cultures isolated in Australia were identified as Brucella abortus biotypes 1, 2 and 4, and Strain 19; B. suis biotype 1, and B. ovis. B. melitensis biotype 3 was recovered from man infected in the Mediterranean area. B. abortus biotype 1 was the most frequent isolate. Atypical cultures isolated from cattle included B. suis biotype 1, and erythritol-utilising mutants of Strain 19. The epizootiological implications of these findings are discussed in relation to their impact on the national campaign to eradicate bovine brucellosis.

Ligated ileal loops of calves were inoculated with Brucella abortus and examined at 2, 4, 6, 10, and 24 hours post-inoculation. B. abortus was identified by light and electron microscopy using immunoperoxidase and antibody-coated colloidal gold techniques. B. abortus was detected in vesicles, phagolysosomes, and large vacuoles of lymphoepithelial cells. Numbers of intracellular bacteria decreased with time after inoculation. B. abortus was also seen between and below lymphoepithelial cells and free in the dome interstitium and intestinal lymph vessels. Neutrophils and macrophages in both epithelium and lamina propria contained intact or degraded bacteria within phagosomes, phagolysosomes, and multivesicular bodies. These studies showed that (1) transepithelial migration of B. abortus occurred principally by dome lymphoepithelial cell endocytosis and transport, and (2) B. abortus was degraded by macrophages and neutrophils of the gut-associated lymphoid tissue.