Distribution of Brucella abortus organisms in calves after conjunctival exposure

Thirty calves (3 to 4 months old) were exposed conjunctivally to a pathogenic strain of Brucella abortus. Calves were euthanatized and necropsied at postexposure hours 2 and 4, and at postexposure days (PED) 1, 4, 7, 14, 21, 42, and 49. Selected ocular, pharyngeal, and lymphoid tissues were cultured bacteriologically for brucellae to determine organism distribution. Brucella abortus organisms initially localized in the third eyelids, bulbar conjunctivae, and parotid lymph nodes and were detected in these structures until PED 42, 21, and 49 respectively. In calves euthanatized at PED 7, organisms were in other cranial lymph nodes (mandibular and retropharyngeal), and in calves euthanatized at PED 21, organisms were isolated from peripheral lymphoid tissues. Brucellae were not isolated from mesenteric and bronchial lymph nodes and from the spleen until PED 21. The pattern of isolation indicated that conjunctival exposure probably resulted in entrance of brucellae into the host via ocular tissues.     

Biosafety of Brucella abortus strain RB51 for vaccination of mature bulls and pregnant heifers.

OBJECTIVE: To determine shedding and colonization profiles in mature sexually intact bulls and pregnant heifers after vaccination with a standard calfhood dose of Brucella abortus strain RB51 (SRB51). ANIMALS: 6 sexually mature 3-year-old Jersey bulls and 7 mixed-breed heifers in midgestation. PROCEDURE: Bulls and pregnant heifers were vaccinated IM with the standard calfhood dose of 3x10(10) colony-forming units of SRB51. After vaccination, selected body fluids were monitored weekly for vaccine organism shedding. Pathogenesis was monitored in bulls by weekly breeding soundness examination and, in heifers, by delivery status of the calf. Vaccine organism colonization was assessed by obtaining select tissues at necropsy for bacterial culture. Serologic analysis was performed by use of numerous tests, including complement fixation, an SRB51-based ELISA, and immunoblot analysis. RESULTS: After vaccination, none of the vaccinated bulls or heifers shed SRB51 in their secretions. Results of breeding soundness examination for bulls were normal as was delivery status of the pregnant heifers (6 live births, 1 dystocia). At necropsy, SRB51 was not recovered from any of the selected tissues obtained from bulls, heifers, or calves; however, serologic analysis did detect SRB51-specific antibodies in all cattle. CONCLUSIONS AND CLINICAL RELEVANCE: Vaccination with the standard calfhood dose of SRB51 administered IM was not associated with shedding or colonization in sexually mature bulls or pregnant heifers. Also, under conditions of this study with small numbers of animals, IM vaccination with SRB51 does not appear to cause any reproductive problems when administered to sexually mature cattle.     

Selective media for isolation of Brucella abortus strain RB51

Brucella abortus strain RB51 (SRB51) is the standard vaccine used to protect cattle against brucellosis and is currently being used to vaccinate bison in the United States (US). Currently available media for culture of Brucella have not been evaluated for their ability to support growth of SRB51. In this study, five selective media for isolating brucellae, four commercially available media for gram-negative bacteria, and tryptose agar with 5% bovine serum (TSA) were compared to two SRB51 selective media developed in this study (rifampin brucellae medium (RBM), and malachite green brucellae medium (MGB)), for their ability to support growth and enhance recovery of SRB51. Four of the five media currently used for isolation of brucellae and two of the four media used for other Gram-negative bacteria did not support growth of SRB51. Modified Kuzdas and Morse (MKM), Brilliant Green, Skirrow's, RBM, and MGB supported growth of SRB51 in a manner similar to TSA. Recovery of SRB51 from tissues of SRB51-vaccinated bison was attempted on TSA, MKM, RBM, and MGB. From a total of 436 samples, SRB51 was isolated from 9.6, 4.3, 5.5, and 9.0% on TSA, MKM, RBM, or MGB media, respectively. Strain RB51 was recovered on only one medium (nine on TSA; three on RBM; and 9 on MGB) from 21 samples. Overgrowth of contaminating bacteria prevented potential detection of SRB51 from 9. 4, 5.5, 0.07, and 5.9% of samples on TSA, MKM, RBM, or MGB, respectively. These data suggest that the use of RBM and MGB, in combination with TSA, enhances the ability to recover SRB51 from tissue samples.     

Humoral and cell-mediated immune responses in non-pregnant heifers following infection and vaccination with Brucella abortus

Humoral and cell-mediated-immune responses to Brucella abortus were observed in non-pregnant heifers following infection alone; infection followed by vaccination; vaccination followed by infection; and vaccination alone. The humoral responses, as measured by the Rose Bengal test (RBT), complement fixation test (CFT), indirect haemolysis test (IHLT) and the enzyme-linked immunosorbent assay (ELISA) tended to be immediate and transient following infection alone, infection following vaccination and vaccination alone. However, when vaccination was superimposed on infection, reactions were maintained for at least 2 years. The cell-mediated-immune (CMI) responses were assessed by the lymphocyte stimulation test. The responses occurred after the humoral responses had peaked and were present for periods of 6-22 weeks. However, the level of stimulation was greater following infection than following vaccination, and the response when vaccination was superimposed on infection was present for less than 6 months.     

Genetic diversity at alkB locus in Brucella abortus

DNA polymorphism of the alkB gene, a DNA repair gene, was assessed by PCR on Brucella abortus biovars 1 (strains 99, S19, 45/20, RB51 and 2308), 3 (Tulya strain), 5 (B3196 strain) and 6 (870 strain). A DNA repetitive element, named IS711, was detected in all studied biovars 1 and its complete nucleotide sequence was determined. We found that the element in alkB gene, bounded by 14 bp imperfect inverted repeats (IRs), is 840 bp long and appears to duplicate a consensus target site, CTAG. Analysing its nucleotide sequence of both forward and reverse strands, more than 10 open reading frames (ORFs) were found. Two potential transposase coding regions were chosen comparing all possible ORFs with the database. Comparing IS711 elements isolated from Brucella species, including both those characterized in our work and the published ones, differences in length and in nucleotide composition were observed among Brucella species, members of the same species and within the same strain. Our results confirm the heterogeneity of IS711 elements in Brucella genus and suggest the possibility to use this element to assess gene and genome diversity and to identify new molecular markers for Brucella species.     

Brucella abortus-specific immunoglobulin isotypes in serum and vaginal mucus from heifers vaccinated with Brucella abortus salt-extractable proteins and challenge exposed with virulent Brucella organisms

Serum and vaginal Brucella-specific immunoglobulin isotypes (IgG1, IgG2, IgM, and IgA), obtained from 62 crossbred beef heifers vaccinated with Brucella abortus salt-extractable proteins and subsequently challenge exposed with B abortus S2308, were studied. Brucella-specific IgG antibodies and Brucella-specific immunoglobulin isotypes were quantitated by a fluorometric immunoassay. Serum and vaginal immunoglobulin responses were evaluated as a method of distinguishing infected from noninfected heifers. Rivanol precipitation, complement-fixation, buffered-antigen brucellosis tests and an ELISA were performed on sera. For immunoglobulin isotypes, vaccinated heifers had mean antibody responses higher than baseline mean antibody responses for at least 31 weeks after vaccination. After challenge exposure, significant differences (P greater than 0.05) were not detected between mean antibody responses of vaccinated and nonvaccinated heifers. Vaginal Brucella-specific antibody responses did not correlate with protection from disease. Vaginal Brucella-specific IgM was detected only at the time of abortion. Vaginal IgA appeared specific for identification of virulent B abortus infection. All serotests appeared adequate in distinguishing baseline titers from titers of heifers that had aborted and were considered bacteriologic culture-positive. Results of serotests neither consistently distinguished vaccinates from challenge-exposed cattle nor distinguished heifers that were challenge exposed, had aborted, and were considered bacteriologic culture-positive adequately from heifers that were challenge-exposed, had not aborted, and were considered bacteriologic culture-negative. Brucella-specific IgA appeared to be the most effective in distinguishing vaccinated heifers from challenge- exposed heifers and heifers that were challenge exposed and had aborted, from heifers that were challenge exposed and had not aborted. Brucella-specific serum IgA was detected up to 13 weeks after abortion.     

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.     

Protection levels in vaccinated heifers with experimental vaccines Brucella abortus M1-luc and INTA 2

Brucella abortus M1-luc is a mutant strain derived from S19 vaccine strain in which most of bp26 sequence has been replaced by the luciferase coding gene. Strain I2 is a double mutant derived from M1-luc in which most of omp19 has been deleted without introduction of any genetic markers. In BALB/c mice, M1-luc presented equivalent performance to S19 regarding persistence, splenomegaly and protection against challenge. Interestingly, I2 was more attenuated than S19, with no reduction of protection against challenge. In order to evaluate the potential for vaccine use of these strains in the natural host, four groups of 15 heifers, 6-month old, were either non-vaccinated or vaccinated with S19, M1-luc or I2. To at reached 17-month old, heifers were synchronized with two doses of PGF2alpha and received natural service during 60 days with two bulls. Pregnant heifers were challenged at approximately six gestation months with virulent B. abortus S2308. Blood samples post-challenge of heifers were collected for serologic test as well as specimens of aborted fetuses and premature calves for bacterial isolation and histopathological analyses. Protection levels against abortion were 78.6% for S19, 81.8% for M1-luc and 45.5% for I2, compared to the 25% that did not abort from the non-vaccinated group. These results indicate that in bovines BP26 had no influence in protective capacity of S19, correlating with the results obtained in mice. However, contrarily to what was previously observed in mice, lack of expression of Omp19 rendered in less protection capacity of S19 in the natural host.     

Frequent exposure of mice to crude Brucella abortus proteins down-regulates immune response

Mice repeatedly immunized via the intraperitoneal route with a Brucella abortus antigen lost their ability to develop a strong in vitro lymphoproliferative response. This result correlates with a decreased tendency of the lymphoid population to produce interferon-gamma when stimulated in culture with the immunizing antigen. With respect to the humoral response, as the number of immunizations increased, the animals produced more specific immunoglobulin M and immunoglobulin G1 antibodies. It is postulated that the long-term exposure of an animal to Brucella antigen changes the nature of the immune response from a T-cell-mediated response to a humoral response favouring the establishment of the disease.     

Effect of dose of Brucella abortus strain 19 in yearling heifers on the relative risk of developing brucellosis from challenge exposure with strain 2308

Yearling heifers were given SC injections of 10(8) (n = 40), 10(9) (n = 44), or 10(10) (n = 44) colony-forming units of Brucella abortus strain 19 (S19). The proportion of heifers with positive serologic test results at 1 month following vaccination increased as the dose of S19 increased. These proportions decreased with time, and all heifers had negative card, rivanol, and complement fixation test results within 4 months. Positive ELISA results persisted beyond 4 months in all three S19 dose groups; however, all heifers were ELISA-negative within 9 months after vaccination. Comparable lymphocyte transformation activity was stimulated by S19 dose of 10(9) or 10(10) and approximately half of the heifers in both groups had a positive stimulation index at 9 months. Immunity of the pregnant heifers was challenged 9 months after vaccination with 10(7) B abortus strain 2308 as follows: diluent controls (n = 69); 10(8) B abortus S19 (n = 40); 10(9) B abortus S19 (n = 39); and 10(10) B abortus S19 (n = 39). Tissue specimens from heifers were obtained at parturition and necropsy for culturing of B abortus. The proportion of heifers that developed brucellosis, ie, had positive culture results, increased as gestation days at challenge exposure increased. The effect of gestational age was controlled in the analysis using logistic regression. The relative risk of brucellosis was reduced to 0.38, 0.15, and 0.06 for B abortus S19 doses of 10(8), 10(9), and 10(10), respectively, compared with diluent controls at 1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of growth rate and exposure to bulls on age at puberty in beef heifers

Two experiments were conducted to test the following hypotheses: 1) exposure of beef heifers to sterile bulls increases the proportion of heifers attaining puberty by 14 mo of age and 2) rate of growth interacts with bull exposure to influence age at puberty in beef heifers. In Exp. I, heifers were assigned to one of two treatments: 1) heifers were exposed to bulls (BE; approximately 70-d period of exposure) or 2) heifers were isolated from bulls (NE) and served as controls. In Exp. II, heifers were assigned to either BE or NE treatments (175-d period of exposure to bulls) and were fed to gain at a moderate (MG; .6 kg/d) or high (HG; .8 kg/d) growth rate. Blood samples were collected twice weekly to determine concentrations of progesterone indicative of onset of corpus luteum function and puberty. In Exp. I a greater (P less than .05) proportion of heifers receiving the BE treatment than of heifers receiving the NE treatment initiated corpus luteum function by 14 mo of age. In Exp. II, there was a bull exposure x growth rate interaction (P less than .05). The effect of bull exposure was greater within the HG groups than within the MG groups. However, heifers fed to attain a moderate or high growth rate and exposed to bulls attained puberty at younger ages than heifers not exposed to bulls and fed to attain a moderate or high growth rate. Mean ages at puberty were 375, 422, 428, and 449 (pooled SEM = 8.6) d for heifers in the BE-HG, BE-MG, NE-HG, and NE-MG groups, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparative behaviour of Brucella abortus strains 19 and RB51 in the pregnant mouse.

Pregnant BALB/c mice received various doses of either Brucella abortus strain 19, a smooth vaccine strain, or B abortus strain RB51, a stable rough organism, intraperitoneally on day 9 of gestation to compare the relative pathogenicity of the two attenuated strains. Nine days after inoculation, spleens and placentas were collected for bacteriological and histopathological examination. A dose of 10(7.5) and strain 19 organisms produced a severe necrosuppurative placentitis occasionally accompanied by fetal death. This dose resulted in a 10-fold higher level of splenic infection than did a dose of 10(9.5) strain RB51 organisms, which produced only mild to minimal placentitis not associated with fetal death. Strain 19 infected mice showed seroconversion in the standard tube agglutination test in contrast to the seronegative titre of strain RB51 infected mice. The results of this study corroborate previous investigations on the relative pathogenicity and the serological response of the non-pregnant mouse to strain RB51.     

The efficacy of bacteriological procedures for the isolation of Brucella abortus from abattoir material

A process of emulsifying and centrifuging abattoir specimens before plating out is described. Brucella abortus was isolated more successfully by this process than by conventional methods, especially in low grade infections. The 5 different media used were equally effective in our attempts at isolation, but growth did not necessarily occur on all 5 plates. In dairy cows, specimens from supra-mammary lymph nodes, udder and iliac lymph nodes accounted for a high percentage of positive isolations.     

Humoral immune response in pregnant heifers inoculated with Neospora caninum tachyzoites by conjunctival route

The aim of this study was to compare systemic humoral immune responses in pregnant heifers inoculated with Neospora caninum tachyzoites by conjunctival and intravenous routes. Twenty nine heifers separated in three experimental groups were studied: Group 1 (n=10 animals) and Group 2 (n=9 animals) were inoculated with 10(8) of N. caninum tachyzoites by conjunctival and intravenous routes at 5th month of gestation, respectively; Group 3 (n=10 animals) were non-inoculated control animals. An indirect fluorescent antibody test (IFAT) and western immunoblotting (IB) were used to analyze the humoral immune response. All animals from Group 1 developed N. caninum specific antibody responses after conjunctival inoculation recording the highest antibody titer (mean+/-SE: 160+/-49.9) at 6th month of gestation. There were statistical differences between humoral immune responses found in Group 1 and 2 being higher in the second one at 6.5th, 8.5th and 9th months of gestation (P<0.05). Interestingly, all heifers from Group 1 reverted to seronegative status at the end of gestation. No increase in antibody was detected in the uninfected control group. Same pattern of N. caninum antigens was recognized by sera from heifers inoculated by conjunctival route and heifers inoculated by intravenous route. Recognized antigens were 116, 92, 84, 77, 45, 40, 25-26 and 17-18 kDa. The conjunctival instillation of N. caninum tachyzoites in pregnant heifers induces specific systemic antibodies. Further work is needed in order to clarify the consequences of this novel experimental route of infection not only on the fetus but also on the dam.     

Immunogenicity of Brucella abortus salt-extractable proteins

The immunogenic properties of salt-extractable proteins and chromatographic fractions thereof from Brucella abortus were evaluated in lemmings (Dicrostonyx rubricatus). The efficacy of the Brucella proteins as immunogens was determined after challenge with virulent B. abortus strain 2308 and was based on protection against clinical signs and gross lesions of brucellosis, as well as on numbers of viable Brucella in the spleen. Vaccination of lemmings with as little as 0.1 microgram of salt-extractable proteins (CSP) suppressed splenic infection, resulting in reduced numbers of viable organisms per spleen of 5-6 logs compared to non-vaccinated controls. Protein fractions separated by column chromatography were generally effective in reducing splenic infection, and contained proteins with molecular weights of 30,000, 20,000 and 12,000. Vaccines containing chemically modified dodecanoyl-CSP offered no additional advantage over unmodified CSP vaccines.     

Photoperiod influences age at puberty of heifers

Two experiments were conducted to determine if exposure of prepubertal heifers to supplemental lighting hastens the onset of puberty. In Exp. 1, 16 heifers were paired according to birth date (April 21 to July 4) and assigned randomly to exposure to either 18 h light/d (L) or natural photoperiods (N) from 22 wk of age until puberty. Twenty-two heifers in Exp. 2, born between February 27 and March 31 and between May 3 and May 17, 1981, were exposed to L or N from 24 wk of age until March 23, 1982. In Exp. 2, animals were bred at all estrous periods until conception. Age at first ovulation and first estrus were less (P less than .01 for Exp. 1 and P less than .10 for Exp. 2) for L than N heifers. Average ages at first estrus were 318 (L) and 367 d (N) for Exp. 1 and 367 (L) and 394 d (N) for Exp. 2. Age at conception in Exp. 2 was similar for L (380 d) and N (396 d) groups. There were no significant differences between L and N heifers in changes in body weight for either experiment. There was a photoperiod X age interaction (P less than .06) for ovarian volume in Exp. 1 because the rate of ovarian growth was greater for L than N heifers. Concentrations of LH were not affected by photoperiod in Exp. 1 and not measured in Exp. 2. There were no significant changes in LH concentrations between 22 and 34 wk of age. When expressed relative to first ovulation, LH levels were highest at 7 and 2 wk before first ovulation. Concentrations of prolactin in Exp. 1 were not significantly affected by photoperiod. It was concluded that supplemental lighting after 22 or 24 wk of age reduced ages at first ovulation and first estrus in heifers born from February to July. These effects of photoperiod were accompanied by changes in ovarian development.     

The relationship between the isolation of Brucella abortus and serological status of infected, non-vaccinated cattle

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.