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)     

Response of Bali cattle (Bos javanicus) to vaccination with Brucella abortus strain 19 in West Timor

A trial was conducted in two villages (one containing cattle infected with brucellosis and one not containing infected cattle) in Timor, Indonesia to determine the serological response to vaccination with Brucella abortus strain 19 in Bali cattle (Bos javanicus) (n = 599). Mature female cattle were immunised with low-dose strain 19 (2x10(8)-6x10(8) colony forming units) and calves (6-12 months) with high-dose strain 19 (4x10(10)-12x10(10) colony forming units). Other mature females and calves were inoculated with sterile vaccine diluent and formed a non-vaccinated in-contact control group. The seroprevalence and mean titres were highest in the vaccinated cattle 3 months after vaccination. These then receded, however, 1% of vaccinated calves and 1.9% of vaccinated cows from the village without infected cattle were still seropositive on the complement-fixation test (CFT) 24 months after vaccination. Non-vaccinated seropositive animals were more likely to have aborted or had a stillbirth and were less likely to have produced a calf than were seronegative cows from the village containing infected animals. We concluded that strain 19 vaccine induced protection in Bali cattle and that this vaccine might play an important role in the control of bovine brucellosis in Timor.     

Effect of stage of gestation on efficacy of Brucella abortus strain-19 vaccination in cattle.

Seventy-nine cattle in all stages of gestation were inoculated with a low dose (2.5 x 10(8) colony-forming units) of Brucella abortus strain 19, then challenge exposed with pathogenic B abortus strain 2308 during the subsequent gestation. A brucellosis case was defined by isolation of strain 2308 from dam or calf samples. Cumulative incidence of brucellosis cases was 48, 33, 25, or 47% for cattle that were, respectively, not pregnant, or 19 to 87, 100 to 167, or 190 to 253 days in gestation at vaccination. The cumulative incidence was 56% in 27 nonvaccinated controls. The 95% confidence intervals for risk ratios included 1 in all cattle, except those that were 100 to 167 days in gestation at vaccination (ie, second trimester); the confidence interval for this group was 0.21 to 0.97. The prevented fraction (1-risk ratio) attributed to strain 19, in ascending order, was 0.14, 0.16, 0.4, or 0.55, respectively, for cattle that were not pregnant, or were 190 to 253, 19 to 87, or 100 to 167 days in gestation at vaccination. Potential confounders of breed, pen effect, and gestation days at challenge exposure did not significantly affect results. Results supported the hypothesis that stage of gestation at vaccination will affect the prevented fraction of brucellosis, or efficacy of strain 19, in cattle vaccinated with a low dose and, therefore, is one factor that may explain variation in strain 19-induced protection.     

Brucellosis in heifers weaned from seropositive dams

Fifty-six heifers were weaned from dams that were card-test positive for brucellosis. Forty-four dams were positive by rivanol and complement-fixation tests and Brucella abortus field strain was isolated from 14. Numbers of expected pregnancies following natural breeding and numbers of viable calves produced were not reduced in the heifers. Persistent B abortus infection was documented in 2 of 37 parturient heifers from reactor dams. The frequency of infection was 1 of 10 in strain 19-vaccinated heifers, and 1 of 27 in nonvaccinated heifers. The 2 persistently infected heifers had atypical serologic reaction patterns before normal parturitions.     

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.     

Relationship of fetal age at conjunctival exposure of pregnant heifers and Brucella abortus isolation

Pregnant heifers were exposed by a conjunctival inoculation with 1 X 10(7) colony-forming units of Brucella abortus strain 2308. At parturition, milk and uterine samples from dams plus samples from dead calves were cultured bacteriologically for Brucella. The logistic regression probability of B abortus isolation increased from 0.22 to 0.90, as fetal age at exposure of heifers increased from 60 to 150 gestation days. Strain 2308 was recovered at parturition from 14 (64%) of 22, 17 (71%) of 24, and all 28 (100%) heifers that were at gestation days less than 127, 127 to 157, and greater than 157, respectively, at time of exposure. The number of infected heifers and the number of samples positive for B abortus were significantly increased as fetal age at exposure of heifers increased from gestation days less than 127 to greater than 157 (chi 2 greater than 10, P less than 0.005).     

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.     

Transient enhancement of the serum antibody response to Brucella abortus strain 19 in cattle treated with levamisole

Two experiments were done on 57 steers. These cattle were allotted to 8 groups (4 groups/experiment) and vaccinated with 1 to 3 X 10(9) colony-forming units of Brucella abortus strain 19. Cattle in 3 of the 4 groups/experiment were given 6 mg of levamisole/kg, subcutaneously, either at the time of vaccination (day 0), 7 days later, or at both times. Serum antibody titers to B abortus were measured sequentially for 28 days in experiment 1 and for 56 days in experiment 2, using the card test, Rivanol test, complement-fixation test, fluorometric immunoassay, and an enzyme-linked immunosorbent assay. In general, the highest mean antibody titers, as determined by all serologic tests, occurred in steers treated with levamisole at 7 days after vaccination or in those treated at the time of vaccination and 7 days later. By the card test on day 56, there was a significantly (P less than 0.05) greater number of seropositive cattle among those given levamisole 7 days after seropositive cattle among those given strain 19 alone. Simultaneous administration of strain 19 and levamisole did not alter antibody responses to B abortus.     

Protection by oral administration of brucella abortus strain 19 against an oral challenge exposure with a pathogenic strain of Brucella

Twenty heifers were vaccinated orally with Brucella abortus strain 19. These heifers and 21 control heifers were challenge exposed in midgestation with strain 2308 by the oral route. Ten of 19 pregnant control heifers aborted and 14 were culture positive. Two additional heifers were seropositive at slaughter. Strain 2308 was recovered from 4 vaccinates at slaughter; none of the vaccinates aborted. Titers after oral vaccination persisted for less than 82 days.     

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.     

Relationship of days in gestation at exposure and development of brucellosis in strain 19-vaccinated heifers

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.     

Safety of vaccination against brucellosis with the rough strain in pregnant cattle

Brucellosis is an infectious and contagious disease that profoundly impacts public health. However, in many countries, disease prevention is restricted to the vaccination of calves, and there is no prophylactic strategy for pregnant heifers and cows. The aim of this study was to evaluate the safety of the rough strain vaccine against brucellosis in pregnant cattle. Crossbred cows (N = 96) at three gestational periods (early, mid, or late pregnancy) were randomly allocated into the vaccine treatment group or to the control group. We then compared the percentage of pregnancies reaching full term, live calves 60 days after delivery, and seropositive calves. There was no effect of vaccination in any of the gestational periods on the evaluation endpoints. In conclusion, vaccination against brucellosis with the rough strain is safe for pregnant cattle at all gestational periods.

     

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.     

An evaluation of the anamnestic test for brucellosis in cattle of the northern pastoral area

SUMMARY An investigation of the anamnestic test for brucellosis using Brucella abortus 45/20 vaccine was carried out in 3 groups of weaner cattle on 2 farms in western Queensland. Each group originally consisted of about 500 cattle. They were bled before and at 6 or 10 weeks after vaccination and again in the following year. The serums were tested by the complement fixation (CFT), Rose Bengal (RBT) and indirect haemolysis tests (IHLT). Most of the cattle reacting to one or more of the tests were killed and selected tissues were subjected to bacteriological examination for B. abortus. B. abortus was isolated from 19 of 30 (63%) pre-vaccinal reactors, 23 (24%) of 96 cattle reacting at 6 or 10 weeks after vaccination (the anamnestic test) and 1 (2%) of 50 cattle reacting one year after vaccination. The reactor found to be infected the year after vaccination had high serological titres in each of the 3 serological tests: RBT of 3, CFT of 128 and IHLT of 256. A subsequent test showed the group to be brucellosis-free. The CFT was the most efficient test. In the pre-vaccination tests 17 of 19 infected animals were positive in the CFT compared with 11 positive in the IHLT and 17 in the RBT. In post vaccination tests 22 of 23 infected animals were positive in the CFT compared with 18 in the IHLT and 19 in the RBT. At the pre-vaccinal and anamnestic tests (6 or 10 weeks after vaccination) 19 of 57 (33%) cattle with CF titre of 4 or 8 yielded B. abortus on culture compared with none of 26 cattle with similar titres in the year after vaccination. The interpretation of CF titres in cattle following 45/20 vaccination needs to be re-examined.     

Brucella abortus-specific immunoglobulin in isotypes in serum and vaginal mucus from cattle vaccinated with strain 19 and challenge exposed with virulent strain 2308

The immunoglobulins (IgG1, IgG2, IgM, and IgA) of the Brucella-specific antibody response of 69 crossbred beef heifers were studied after Brucella abortus strain 19 vaccination and strain 2308 challenge exposure. The immunoglobulin isotype responses in serum and vaginal mucus were measured by use of fluorescent immunoassay. Serum antibody responses were detected also by 3 standard serologic tests (complement fixation [CF], Rivanol precipitation, and the CARD test] and 2 primary bindings assays that detect IgG antibodies. One month after vaccination, mean antibody titers for all immunoglobulin isotypes were higher for vaccinated cattle (n = 46) than for nonvaccinated controls (n = 23). After vaccination, IgA antibody responses in vaccinated cattle were only 2-fold higher than those for controls, whereas IgG1, IgG2, and IgM antibody responses were 3- to 90-fold greater than those for controls. Measurement of IgA antibody responses classified 21 of 39 vaccinates as seropositive after vaccination, whereas the other isotypes classified 28 or 34 cattle as seropositive. Three months after challenge exposure, the mean antibody responses for each isotype were higher in cattle that aborted or were culture positive than in cattle that did not abort and were culture negative. Although IgG1, IgG2, and IgM antibody titers were each of benefit in identifying B abortus-infected cattle, it did not appear that the magnitude of the antibody responses provided sufficient discrimination between S19-vaccinated cattle and S2308 challenge-exposed cattle. Serum IgA antibody responses were 10-fold higher after challenge exposure than after vaccination and may be a response to mucosal infection with the virulent organism.(ABSTRACT TRUNCATED AT 250 WORDS)     

Antibodies in calves on feed supplemented with chlortetracycline after vaccination with Brucella abortus strain 19

Twenty dairy heifers each consumed 350 mg of chlortetracycline/day in their feed. Four tests were performed on serum specimens from these and 20 control calves after vaccination with Brucella abortus strain 19. The numbers of positive test results on the card test and mean titers on the tube and rivanol agglutination and complement-fixation tests were compared in the 2 groups. Using the rivanol and complement-fixation tests, there were differences in the mean titers at weeks 5 and 6 after vaccination, but by week 10, differences were not found. The results suggest that addition of low concentration of chlortetracycline in feeds have minimal effects on postvaccinal serologic reactions determined after strain-19 inoculation.     

Early removal of cows with Brucellosis and the effect in strain 19 vaccinated cattle herds

The effect of early removal of cows excreting pathogenic Brucella abortus following Strain 19 vaccination of beef cattle herds was determined by comparing cumulative incidence (CI) of brucellosis reactors post-vaccination (p.v.). Adult female cattle in six herds were tested, reactors removed, and vaccinated with 3×10⁹ colony-forming units of B. abortus Strain 19. Cattle were tested at 2 months p.v. and culture-positive cattle were removed from a principal cohort of three herds at approximately 3 months p.v., and removal from a control cohort of three herds delayed until approximately 7 months p.v. Neither CI nor time to eliminate brucellosis was significantly different between cohorts.A review of the parturition data revealed that more of the infected cows in the principal cohort herds terminated gestation before removal. These data suggest that stage of gestation plus diagnostic and management alternatives to prevent parturition of infected cattle in the herd are more important factors in herd plans than early removal of postparturient infected cows following whole-herd Strain 19 vaccination.     

The first report in new zealand of Lymnaea columella say (Mollusca: gastropoda) an intermediate host of the liver fluke Fasciola hepatica L

Six herds of dairy cattle, in which infection had persisted after measures had been employed to eradicate brucellosis, were investigated in detail. One hundred and two animals out of 700 (14.6%) had evidence of the disease from cultural or serological tests. Only 4 infected animals aborted; the remaining 98 animals with brucellosis exhibited no clinical sign of the disease, and 52 were heifers calving for the first time. Sixty-five of the 700 animals (9.3%) produced brucella-infected vaginal discharges at the end of a normal period of gestation, and another 17 (2.4%) were detected only by the brucellosis (Brewer) card test (BCT) or complement fixation test (CFT). All the infected animals were removed from the herds immediately after detection.

About 2 weeks after all the cows had calved, 20 of the remain-ing 510 animals (3.9%) in 4 of the herds became positive to one or more tests and 8 excreted brucellae in their milk. The pregnancy of three 2-year-old primiparous heifers terminated normally and one aborted. All four discharged brucellae until they were slaughtered 20 to 35 days after parturition.

Twenty-five of the 102 infected animals were detected by bacteriological, and 29 by serological, means only. A compari-son was made of the results of the testsfrom 73 culturally positive animals; 4.1% of the sera were CFT positive but BCT negative, and 5.5% were BCT positive but CFT negative. An attempt has been made to explain the large numbers (34.2%) of infected animals that were serologically negative.     

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.     

Risk associated with animals moved from herds infected with brucellosis in Northern Ireland

The movement of cattle from herds infected with Brucella abortus was investigated in order to assess the control measures for eradication of brucellosis from the cattle population of Northern Ireland. Using recorded cattle movement data, a historical cohort study was designed and carried out to quantify the risk of seropositivity in bovine animals moved from herds infected with brucellosis. The study found that 3.1% of animals, moved in the 6-month period prior to disclosure of infection in the source herd and subsequently tested, were interpreted as seropositive in their destination herds. The odds of seropositivity were approximately 19 (95% confidence interval: 7.8-46.4) times higher in this cohort compared with animals from herds with no history of infection. A multivariate logistic regression model was constructed to examine factors influencing the risk of seropositivity in the exposed cohort of animals, identifying maternal status (whether the dam had been a brucellosis reactor) and age at leaving the infected herd as the main risk factors.