Glycosaminoglycans improves early development of zona-free 8-cell rat embryos to blastocysts in a chemically defined medium, but not the pregnancy rate following transfer of the blastocysts

The objective of the present study was to clarify the possible role of the zona pellucida (ZP) in early development of rat embryos and to determine the effect of glycosaminoglycans on the development of ZP-free 8-cell embryos before or after embryo transfer at the blastocyst stage. Eight-cell embryos were divided into three groups comprised of, 1) intact controls, 2) embryos with the ZP was removed with acidic solution and 3) pairs of ZP-free 8-cell embryos aggregated in a small hollow. These embryos were cultured in a chemically defined mR1ECM for 24 h. Developmental ability to the blastocyst stage and mean cell number in the blastocyst was lower in ZP-free embryos than in intact controls. When these blastocysts were transferred, the farrowing rate and efficiency of embryos developed to term were also lower in ZP-free embryos, but not in the aggregated ones. Supplementation with hyaluronan (HA; 63-250 μg/ml) or heparan sulfate proteoglycan (HS; 15 μg/ml) significantly improved blastocyst formation of ZP-free embryos and the cell number in the blastocyst by reducing the incidence of apoptosis. However, there were no beneficial effects of HA or HS on farrowing and newborn rates after transfer of the blastocysts. In conclusion, the ZP plays roles in maintaining successful development of early rat embryos at least from the 8-cell stage not only to the blastocyst stage but also to posttransfer stages. Glycosaminoglycans, such as HA or HS, appear to contribute to successful cleavage during early development to the blastocyst stage but may be insufficient to maintain the posttransfer survival of ZP-free embryos.     

Experimental infection of opossums with Brucella abortus

Thirteen opossums (Didelphis virginiana) trapped in east central Alabama were fed approximately 1.5 X 10(9) Brucella abortus colony forming units. Serologic responses to at least 1 of 3 tests developed in 8 of the 13 opossums. Brucella abortus was recovered from 18 of 159 blood samples from 4 of the 13 opossums and from 7 of 159 fecal samples from 6 of them. All culture-positive feces had been excreted within 4 days after exposure. Sixty-four urine, 123 saliva, and 78 vaginal samples were culture-negative. Eleven baby opossums, in their mothers' pouches at the time of capture, were culture-negative. Brucella abortus was isolated from 10 of 13 adult opossums. Ten additional opossums were trapped and tested for brucellosis. One had a tube agglutination titer of 1:25, and B abortus was isolated from the liver and spleen. Brucella abortus was isolated from lung and spleen of 8 seronegative opossums. The remaining 8 opossums were negative to all tests.     

Non-specific seroreactions against Brucella abortus in ruminants in New Zealand and the presence of Yersinia enterocolitica 0:9

The level of non-specific reactions found in the brucellosis serology of ruminants in New Zealand was very low until July 1992. This changed when, in an export consignment of 1071 deer, 35% reacted in the Brucella abortus tube agglutination test with titres varying from 50 to 200 IU. The reactors were also positive in the Rose-Bengal agglutination test and most of them reacted in the complement fixation test with titres varying from 10 to 80 IU. Yersinia enterocolitica 0:9 was later isolated from one deer of this consignment. It was the first isolate of this serotype recovered in New Zealand from an animal. Shortly after, false reactors occurred more frequently than before in sera from Brucella abortus accredited free cattle herds. As the involvement of Yersinia enterocolitica 0:9 was suspected in these cases, faecal samples from reactors and in-contact animals were cultured for this organism. Yersinia enterocolitica 0:9 was isolated from nine of 19 herds showing one or more false Brucella abortus seroreactions. Prior to 1990, Yersinia enterocolitica serotype 0:9 had not been isolated in New Zealand, despite the recovery of a number of other bio- or serotypes of the organism from humans and animals. From 1990 onward, serotype 0:9 began to be isolated from human faecal samples with increasing frequency. Since the first isolations from deer and cattle in 1992, it has now also been recovered from a cat and an alpaca and from cattle without any association with false positive Brucella abortus reactions. All serotype 0:9 isolates were of biotype 2.     

The use of immunochemically purified anti-brucella antibody in a direct fluorescent antibody test for Brucella abortus

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.     

Immunomodulation with killed Propionibacterium acnes in guinea pigs simultaneously vaccinated with Brucella abortus strain 19

Immunomodulation with killed Propionibacterium acnes was attempted in guinea pigs simultaneously vaccinated with Brucella abortus strain 19. Two groups, each comprised of 9 guinea pigs, were injected by different routes (s.c. and or i.v.) with 1.4 mg of P. acnes and 5 X 10(8) CFU of B. abortus, S-19, while 3 other groups each received either P. acnes, B. abortus S-19, or saline (s.c.). The antibody titers to B. abortus measured at 6, 10 and 14 weeks after vaccination indicated no significant (P less than 0.01) response in the 2 groups immunopotentiated with P. acnes concurrent with B. abortus S-19 vaccination. The delayed hypersensitivity response to 3 Brucella antigens conducted 8 weeks after immunization did not show a significant difference between the B. abortus S-19 vaccinated group compared with the 2 groups immunopotentiated and vaccinated. However, the proliferative response of lymphocytes to the B. abortus soluble antigen diluted 1:100 indicated significantly enhanced blastogenesis in the (s.c.) immunopotentiated and immunized guinea pigs compared with the B. abortus S-19 vaccinated group. A slightly enhanced response was also observed in the group immunopotentiated (i.v.) and vaccinated (s.c.). The guinea pigs were challenged with B. abortus strain 2308 and necropsied 4 weeks later. The mean splenic CFU of the Brucella in the group immunopotentiated (i.v.) and vaccinated (s.c.) was significantly decreased when compared with the guinea pigs vaccinated with B. abortus S-19 alone. These findings indicated that P. acnes administered simultaneously with B. abortus S-19 vaccine was able to augment the immune response in guinea pigs. Immunomodulation as evidenced by enhanced clearance of B. abortus from the spleens of immunopotentiated animals was presumably brought about by activated macrophages or a T-cell mediated cytolytic mechanism or both.     

Cleavage of bovine immunoglobulin G1 in whey by an extracellular material from Brucella abortus.

Culture extracts of in vitro grown Brucella abortus were demonstrated to cleave a part of the Fc portion of bovine immunoglobulin G1 in whey but not in serum or as a purified protein from serum. Supernates from Strains 19 and 2308 of B. abortus were both capable of this hydrolysis whereas living cells were not. The cleavage process was independent of antibody activity to B. abortus, appeared to require factor(s) found only in some whey samples and was ineffective with the other bovine immunoglobulins.     

Diagnostic characterization of a feral swine herd enzootically infected with Brucella.

Eighty feral swine were trapped from a herd that had been documented to be seropositive for Brucella and which had been used for Brucella abortus RB51 vaccine trials on a 7,100-hectare tract of land in South Carolina. The animals were euthanized and complete necropsies were performed. Samples were taken for histopathology, Brucella culture, and Brucella serology. Brucella was cultured from 62 (77.5%) animals. Brucella suis was isolated from 55 animals (68.8%), and all isolates were biovar 1. Brucella abortus was isolated from 28 animals (35.0%), and isolates included field strain biovar 1 (21 animals; 26.3%), vaccine strain Brucella abortus S19 (8 animals, 10.0%), and vaccine strain Brucella abortus RB51 (6 animals, 7.5%). Males were significantly more likely to be culture positive than females (92.9% vs. 60.6%). Thirty-nine animals (48.8%) were seropositive. Males also had a significantly higher seropositivity rate than females (61.9% vs. 34.2%). The relative sensitivity rates were significantly higher for the standard tube test (44.6%) and fluorescence polarization assay (42.6%) than the card agglutination test (13.1%). Lesions consistent with Brucella infection were commonly found in the animals surveyed and included inflammatory lesions of the lymph nodes, liver, kidney, and male reproductive organs, which ranged from lymphoplasmacytic to pyogranulomatous with necrosis. This is the first report of an apparent enzootic Brucella abortus infection in a feral swine herd suggesting that feral swine may serve as a reservoir of infection for Brucella abortus as well as Brucella suis for domestic livestock.     

Adherence of Haemophilus somnus to bovine embryos after in vitro exposure

Preimplantation bovine embryos were exposed in vitro to Haemophilus somnus to determine whether the bacteria would adhere to zona pellucida-intact embryos or would adhere to or infect zona pellucida-free embryos. The effect of H somnus on in vitro embryonic development also was investigated. After exposure to H somnus and before washing, some of the zona pellucida-intact embryos were held in antibiotic-containing medium. Haemophilus somnus was isolated from 10 to 42 zona pellucida-intact embryos and none of the zona pellucida-free embryos. Haemophilus somnus was not recovered from any of the 32 antibiotic-treated embryos. The coculture system was not compatible with normal embryonic development, and all embryos had begun to degenerate by the end of the 18-hour exposure period.     

A serological comparison of complement fixation reactions using Brucella abortus and B. melitensis antigens in B. abortus infected cattle

Brucella abortus and B. melitensis antigens were used in parallel on the National Standard Brucella abortus antiserum and on field sera coming from cattle where practically exclusively B. abortus biotypes 1 and 2 have been isolated over the last 11 years. With the National Standard serum the titres to B. melitensis were consistently lower than those to B. abortus antigen. Most were 1 dilution (twofold) lower. Although a similar trend was seen with the field sera, there were 7/346 sera which had twofold or higher titres to B. melitensis antigen. Although this may be due to the vagaries of the test it also warrants closer investigation of the animals concerned to see whether M-antigen predominant Brucella biotypes are possibly present. The use of the dual antigens could identify herds which are infected only with A-antigen predominant brucellae but would not be reliable for classifying individual animals.     

Macrophage function in mammary glands of Brucella abortus-infected cows and cows that resisted infection after inoculation of Brucella abortus

Nonvaccinated pregnant cows were segregated retrospectively into 2 groups following inoculation with Brucella abortus strain 2308. One group resisted infection (resistant cows) and the other group developed active infections (susceptible cows) and subsequently aborted. Mammary gland macrophages collected from the 2 groups of cows were compared, using in vitro functional assays. In a chemiluminescence assay, mammary gland macrophages from resistant cows produced significantly (P = 0.014) higher oxidative burst activity than did macrophages from susceptible cows. Macrophages from resistant cows had significantly (P = 0.038) greater bacteriostatic activity against B abortus than did macrophages from susceptible cows. Differences in lysosomal enzymatic activity or Fc receptor expression were not observed for macrophages from the 2 groups of cows. Differences in macrophage function may be one factor responsible for natural resistance to Brucella infection in cattle.     

Enzyme-linked immunosorbent assay for detecting antibodies to Brucella abortus in bovine milk and serum

An enzyme-linked immunosorbent assay (ELISA) method was evaluated for the detection of antibodies to Brucella abortus in cows milk. Milk samples from seropositive or -negative cows were sed to determine the distribution of absorbance values to classify milk as ELISA positive or ELISA negative. Brucella abortus was isolated from milk samples from 10 (45%) of the 22 cows whose milk and serum were ELISA positive. The ELISA was evaluated and determined to be an appropriate method for detecting antibodies to B abortus in bovine milk.     

Serologic and bacteriologic test results after adult vaccination with strain 19 in three dairy herds infected with brucellosis.

Milk culture data and serologic test results were evaluated after adult vaccination with Brucella abortus strain 19 in cattle of 3 large California dairy herds infected with brucellosis. Strain-19 organisms were isolated by culture of milk from 1.9% of the vaccinated cows. Isolation of field strain of B abortus varied directly with magnitude of complement-fixation (CF) and rivanol titers. At time of milk culture, 74% of cows from which field strain was isolated had CF titer greater than or equal to 160, compared with 58% of cows from which strain 19 was isolated. Cows with CF titer greater than or equal to 160 at 2 months or greater than or equal to 80 to 4 months after adult vaccination were more likely to be correctly classified as reactors (on the basis of subsequent milk culture results and/or persistently high serologic titer) than were cows with lower CF titer at these times. Cows from which B abortus strain 19 was isolated from milk were more likely to maintain persistent serologic titer than were cows from which neither strain of B abortus was isolated.     

Brucellosis of camels in Kuwait

This study investigated the presence of Brucella antibodies in serum and milk obtained from camels in Kuwait. Brucella strains were also isolated from the foetus using standard technique (Webridge Lab Techniques). Three serological tests for serum were adopted. These tests were Rose Bengal Plate Test (RBPT), the Serum Tube Agglutination Test (SAT) and the Complement Fixation Test (CFT). The RBPT was used for all sera samples, and both SAT and CET were used for the positive RBPT. Camels that showed a titer of 1:40 in SAT or 1:5 in CFT or greater were considered positive. Thirteen of the samples examined were found positive by CFT (at 1:5); by SAT, they showed a titer of 1:20. One serological test, the Milk Ring Test (MRT), was used for milk. Here 3 and 2 were considered positive reactors but 1+ was considered suspicious. We were unable to isolate the Brucella organism from Sedemine and Cream of milk, but we isolated them from Foetus Brucella abortus and it is confirmed by Webridge Laboratory, U.K. It is Brucella abortus (Biovar 1). The prevalence rate was 14.8% from serum by the CFT and RBPT methods and 10.8% by the SAT method. For milk, the prevalence rate was 8.0%. Two Brucella abortus were isolated from 5 foetuses.     

Isolation of Brucella abortus and Brucella abortus, strain 19, from cattle.

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.     

Brucellosis induced by RB51 vaccine in a pregnant heifer

Brucellosis developed in a 14.5-month-old Gelbvieh heifer after the animal was vaccinated with the calfhood dose of strain RB51 Brucella abortus vaccine s.c. during the fourth month of its first pregnancy. The heifer experienced dystocia and was euthanatized during cesarean section because of a large uterine tear. The fetus was dead at delivery. Suppurative placentitis and fetal pneumonia were evident at necropsy. Brucella abortus strain RB51 was isolated from the placenta and the fetus' lung.     

Detection of brucella antibodies of different immunoglobulin classes in cow milk by enzyme-linked immunosorbent assay

Enzyme-linked immunosorbent assays were conducted on milk of cows from which Brucella abortus was isolated and that of noninfected controls. Horseradish peroxidase-labeled rabbit antibovine immunoglobulins IgG, IgG1, and IgA were used as conjugates. A heat-killed whole-cell suspension of B abortus strain 19 was used as the antigen. Differences in antibody profiles were observed in milk of cows from which B abortus was isolated and in milk of noninfected cows. Antibody profiles were similar in milk of cows infected with B abortus and that of cows from which B abortus strain 19 was isolated.     

Biotypes of Brucella abortus and their value in epidemiologic studies of infected cattle herds.

Four Texas cattle herds containing cows infected with either Brucella abortus biotype 1, 2, or 4 were studied to determine the probability of transmission of Brucella between adjacent cattle herds, the most probable means by which Brucella was introduced into the herds, and the relative frequency of strain 19 isolation from vaccinated cattle. A total of 1,935 cattle in the four herds were tested for brucellosis; 339 reactors were identified, and isolations of B abortus were made from 143. The biotype of B abortus was used to determine that purchased cattle or reentry of bred heifers into the herds was probably responsible for introducing B abortus and that the biotype was not readily transmitted to adjacent herds. Three (9%) of 32 B abortus isolations from adult-vaccinated cattle were strain 19. The data supported the hypothesis that biotypes can be useful in determining the source of B abortus for cattle and in differentiating field and vaccine strain infections in adult-vaccinated cattle.     

Effects of killed Brucella abortus strain 45/20 vaccine on reindeer later challenge exposed with Brucella suis type 4

Six seronegative pregnant reindeer (Rangifer tarandus L) were vaccinated with killed Brucella abortus strain 45/20 with added adjuvant. These were challenge exposed with B suis type 4 after 90 days; at the same time, 4 seronegative, nonvaccinated, pregnant reindeer (controls) were given the challenge inoculum. Humoral antibodies were detected in the vaccinated reindeer by postvaccination day 14. A marked increase in antibody levels also occurred after they were challenge exposed, but did not reach the levels observed in control reindeer which seroconverted within 8 days after they were given the challenge inoculum. One control reindeer aborted at 45 days after challenge exposure (at 165 days of a normal 225-day gestation period), and the fawn of another lived only a few days after delivery. Brucella suis type 4 was isolated from tissues of 3 of 4 control reindeer and of 2 of their fawns. All vaccinated reindeer gave birth to live fawns which were culture negative, although 1 fawn lived only a few days. Brucella suis type 4 was isolated from tissues of 1 vaccinated reindeer. Under the conditions of this experiment, killed B abortus 45/20 vaccine provided increased resistance to brucellosis.     

Preliminary evidence for a diagnostic immunoglobulin G1 antibody response among culture-positive cows vaccinated with Brucella abortus strain 19 and challenge exposed with strain 2308

The sera of cows inoculated with Brucella abortus have a characteristically high titer of immunoglobulin (Ig) G1 antibodies to a soluble brucella antigen compared with sera of noninoculated vaccinated cattle. Concentrations of antigen-specific IgG1 were greater than 10-fold higher than those for IgG2, even though total IgG2 concentrations were higher than total IgG1 concentrations. Increases in IgG1 antibodies to Brucella abortus soluble antigen were detected shortly after vaccination in those cows from which strain 19 was isolated and by 28 weeks in cows from which strain 2308 was isolated. Increases in specific antibodies were not paralleled by increases in either total IgG1 or total IgG2 concentrations. Rather, there was a 15-fold to greater than 200-fold increase in specific activity, with up to 16% of the IgG1 specific for the brucella antigen used in the assay. Thus, measurement of changes in total IgG1 concentrations is not a reliable method to identify brucellosis-associated anti-Brucella abortus soluble antigen activity. Only one cow in a panel of 10 selected for detailed study showed a false-positive IgG1 titer, whereas some serologic assays showed as many as 4 or 5 false-positives. Results of the complement-fixation test, among the battery of serologic tests used for detection of brucellosis, best agreed with the occurrence of increased IgG1 antibody levels.     

Response of the badger (Meles meles) to infection with Brucella abortus

Three badgers exposed to conjunctival instillation of Brucella abortus strain 544 developed a mild subclinical infection accompanied by the production of agglutinating and complement fixing antibodies. Six months after inoculation, the infection could not be detected in one badger and was confined to lymphoid tissue in the other two. No cell mediated immune response to B abortus antigens could be detected by in vitro lymphocyte transformation tests or intradermal tests for delayed hypersensitivity during the observation period.