Effect of Vaccination with Brucella abortus Strain RB51 on Heifers and Pregnant Cattle

Thirteen cows, which had been vaccinated as calves with strain 19, were revaccinated twice or three times as adults with 1×10⁹ cfu of B. abortus strain RB51. Their serological responses following adult vaccination remained negative to conventional brucellosis surveillance tests. Vaccination with strain RB51 during the eighth month of pregnancy did not induce abortion, although strain RB51 was recovered from milk for up to 69 days after vaccination. In a parallel study, thirteen 8- to 10-month-old heifers were vaccinated as calves with 10⁹ cfu of strain RB51. The heifers remained seronegative to conventional brucellosis surveillance tests but antibody responses to RB51 could be demonstrated using an indirect ELISA. This study showed that multiple vaccination with strain RB51 did not induce seroconversion to brucellosis surveillance tests. In addition, this study suggests that 10⁹ cfu of strain RB51 is safe for use in pregnant cattle.     

Use of Brucella abortus vaccine strain RB51 in pregnant cows after calfhood vaccination with strain 19 in Argentina

One hundred and seven pregnant cows, which had been calfhood vaccinated with Brucella abortus strain 19 (S-19) were revaccinated with either S-19 or strain RB51 (S-RB51). All S-19-revaccinated animals seroconverted, while none of the RB51-revaccinated animals seroconverted. Two out of 25 (8%) S-19-revaccinated animals aborted, while none of the 57 RB51-revaccinated group aborted. Four of the S-19-revaccinated animals shed S-19 in the milk for at least 7 days, while only 1 cow shed S-RB51 for at least 3 days (but <7 days) post-parturition. Revaccination of strain 19 calfhood-vaccinated, pregnant cattle with S-RB51 appears to be a safe procedure with no diagnostically negative consequences.     

Use of Brucella abortus vaccine strain RB51 in pregnant cows after calfhood vaccination with strain 19 in Argentina

One hundred and seven pregnant cows, which had been calfhood vaccinated with Brucella abortus strain 19 (S-19) were revaccinated with either S-19 or strain RB51 (S-RB51). All S-19-revaccinated animals seroconverted, while none of the RB51-revaccinated animals seroconverted. Two out of 25 (8%) S-19-revaccinated animals aborted, while none of the 57 RB51-revaccinated group aborted. Four of the S-19-revaccinated animals shed S-19 in the milk for at least 7 days, while only 1 cow shed S-RB51 for at least 3 days (but <7 days) post-parturition. Revaccination of strain 19 calfhood-vaccinated, pregnant cattle with S-RB51 appears to be a safe procedure with no diagnostically negative consequences.     

Use of serology and real time PCR to control an outbreak of bovine brucellosis at a dairy cattle farm in the Nile Delta region,Egypt

Background

Bovine brucellosis remains one of the most prevalent zoonotic infections affecting dairy cattle in developing countries where the applied control programs often fail. We analyzed the epidemiologic pattern of bovine brucellosis in a dairy cattle herd that showed several cases of abortions after regular vaccination with RB51 (B. abortus vaccine). In 2013 thirty dairy cows, from a Holstein-Friesian dairy herd with a population of 600 cattle, aborted five months post vaccination by a regular RB51 vaccine. Blood samples were drawn from milking cows and growing heifers, as well as heifers and cows pregnant up to 6 months. These samples were collected in June 2013 (n?=?257) and May 2014 (n?=?263) and were tested by real time (rt)-PCR as well as serological tests, in particular Rose Bengal Test (RBT), Enzyme-Linked Immunosorbent Assays (ELISA) and Fluorescence Polarization Assay. Tissue specimens were also collected from an aborted fetus and cultured. Isolates were subjected to bacteriological typing tests at the genus and species levels.

Results

Five months post vaccination with RB51 vaccine, Brucella (B.) DNA was detected in blood samples of cows by rt-PCR. The serological tests also revealed the spread of Brucella field strains within the herd in 2013. Four Brucella isolates were recovered from specimens collected from the aborted fetus. These isolates were typed as follows: one B. abortus RB51 vaccine strain and three isolates of B. abortus field strain. The seropositive cows with positive rt-PCR might indicate an infection by the Brucella field strain; while the positive rt-PCR results from seronegative animals may either be due to circulating RB51 vaccine DNA in vaccinated animals or to circulating field strain in infected animals before seroconversion.

Conclusion

The results herein suggest that PCR can be a good supplementary tool in an outbreak situation, if an assay is available that can differentiate vaccine and field strains with a high analytical sensitivity. We recommend using RBT and ELISA in parallel in outbreak situations, to identify as many infected animals as possible during the initial screenings. This test procedure should be repeated for at least three successive negative tests, with one month interval.

     

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.     

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.     

Intracellular trafficking study of a RB51 B. abortus vaccinal strain isolated from cow milk

Brucella is responsible for one of the major worldwide zoonoses. Over the last century, several vaccines have been used against brucellosis. Among these, the rough vaccine Brucella abortus RB51 was introduced with the idea that it would not interfere with the diagnosis of brucellosis. Recently, RB51 has been isolated from milk and vaginal exudates from vaccinated cows, thus raising the possibility of extensive bacterial replication in these animals. We hypothesized that shedding of RB51 might be related to a change in its intracellular cell cycle. Therefore, we have compared the intracellular trafficking in CHO cells of the virulent B. abortus 2308 and two RB51 strains, the vaccinal strain and the one isolated from cow milk. Both RB51 strains were transiently observed in phagosomes characterized by the presence of the early endosomal marker EEA1 and then were found in cathepsin D-enriched lysosomal compartments, in which they eventually underwent degradation at later post-infection times. In contrast, the virulent 2308 strain replicated within the endoplasmic reticulum. These results suggest that a change in intracellular trafficking cannot account for Brucella shedding in adult vaccinated cows.     

Isolation of <Emphasis Type="Italic">Brucella melitensis</Emphasis> from a RB51-vaccinated seronegative goat

The purpose of this study is to determine the etiology of abortions presented in a goat herd declared as free of brucellosis and vaccinated with RB51 located in Mexico. The serological diagnosis of brucellosis in 33 animals was performed. The study included three goats that aborted in the last third of gestation and 15 goats that gave birth normally; samples of milk and vaginal exudate were subjected to bacteriological study. All animals were negative for serological diagnosis, and isolation of Brucella melitensis was achieved in a single goat from vaginal exudate. However, the particularity is that this goat was negative to the card, indirect ELISA, and radial immunodiffusion tests. Isolation of a field strain was confirmed by biochemical test resistance to rifampicin and PCR. It is concluded that a goat which aborted in the last third of gestation was found spreading B. melitensis through vaginal discharge despite being vaccinated with RB51 and seronegative for brucellosis.     

The protoxin Cry1Ac of Bacillus thuringiensis improves the protection conferred by intranasal immunization with Brucella abortus RB51 in a mouse model

Brucellosis is a zoonotic disease affecting many people and animals worldwide. Preventing this infection requires improving vaccination strategies. The protoxin Cry1Ac of Bacillus thuringiensis is an adjuvant that, in addition to increasing the immunogenicity of different antigens, has shown to be protective in different models of parasitic infections. The objective of the present study was to test whether the intranasal co-administration of pCry1Ac with the RB51 vaccine strain of Brucella abortus confers protection against an intranasal challenge with the virulent strain B. abortus 2308 in BALB/c mice. The results showed that co-administration of pCry1Ac and RB51, increased the immunoprotection conferred by the vaccine as evidenced by the following: (1) decrease of the splenic bacterial load when challenged intranasally with the virulent strain; (2) greater in vivo cytotoxic activity in response to the transference of previously infected cells; (3) further proliferation of cytotoxic TCD8+ cells in response to stimulation with heat-inactivated bacteria; (4) increased production of TNF-α and IFN-γ; and (5) significant IgG2a response. These results indicate that the use of the Cry1Ac protein as a mucosal adjuvant via the intranasal route can be a promising alternative for improving current RB51 vaccine against brucellosis.     

THE INDIRECT HAEMOLYSIS TEST (IHLT) FOR BOVINE BRUCELLOSIS—COMPARISONS WITH THE COMPLEMENT FIXATION TEST (CFT) IN VACCINATED AND EXPERIMENTALLY INFECTED CATTLE

SUMMARY Cattle were vaccinated with Brucella abortus strain 19 subcutaneously in doses ranging from the normal (2.0 ml) dose of standard vaccine down to 1/400 of the normal dose, and via the conjunctival sac with 1/2 or 1/20 of the normal dose. Under all vaccination regimes serum antibody titres in the complement fixation test (CFT) rose more rapidly, reached higher levels, declined more slowly and involved a greater proportion of animals, than titres in the indirect haemolysis test (IHLT). The interval between the first positive serological test and parturition was determined for 54 pregnant cows infected with a virulent field strain (VRI 3) of B. abortus. On average the CFT titre rose to 1/4 43 days, and 1/8 33 days, before parturition, while the IHLT reached a 2/8 reaction 31 days before parturition.     

Milk yield and reproductive performance of brucellosis-vaccinated but seropositive Holstein cows

The aim of this research was to study if seropositivity for brucellosis in vaccinated cows against this disease hampers reproductive performance and milk production in high-yielding Holstein cows. For this purpose 1,026 healthy cows and 372 cows seropositive for brucellosis were enrolled in this study. Cows positive to card test and subsequently to the rivanol test were further subjected to the radial immunodiffusion (RID) test. It was found that only 11 % of the presumably infected cows by brucellosis screening tests were really infected with this disease. The reproductive performance of the group of cows with 11 % Brucella-infected animals was not impaired; overall pregnancy rate did not differ between seropositive and healthy cows (30.9 vs. 29.6 %). The abortion rates were similar between seropositive cows (5.3 %) and seronegative animals (6.9 %). Cows in the herd with 11 % Brucella-infected animals produced significantly more milk than unaffected cows over a 305-day lactation (10,684?±?1,720 vs. 10,345?±?1,736; mean ± SD; P?<?0.05). It was concluded that in dairy herds vaccinated against brucellosis with both 19 and RB51 strains, supplemental tests such as RID need to be conducted on all reactors in order to maintain diagnostic accuracy. These results also indicate that 11 % animal prevalence of brucellosis did not exert a detrimental effect on 305-day milk yield and reproductive performance in high milk-yielding Holstein cows.     

VACCINATION OF HEIFERS WITH A REDUCED DOSE OF BRUCELLA ABORTUS STRAIN 19 VACCINE BEFORE FIRST MATING

Ten Jersey heifers aged 14 to 23 months were vaccinated with 2.25 times 10⁸ cells of living Brucella abortus strain 19 vaccine. They and 10 similar non-vaccinated heifers were subsequently mated and when about 6 months pregnant were challenged by the conjunctival application of a virulent culture of B. abortus. The serological response to vaccination was much less than is usually seen following vaccination with the normal dose of strain 19, especially when the indirect haemolysis test was used. A persistent vaccinal reaction was observed in one heifer. Significant resistance to infection was demonstrated which was greater than that previously observed in calfhood vaccinates given the full dose but less than that shown by cows given the smaller dose in early pregnancy. The effectiveness of strain 19 vaccination appears to be related to the age of the animal at vaccination.     

Rough vaccines in animal brucellosis: structural and genetic basis and present status

Brucellosis control and eradication requires serological tests and vaccines. Effective classical vaccines (S19 in cattle and Rev 1 in small ruminants), however, induce antibodies to the O-polysaccharide of the lipopolysaccharide which may be difficult to distinguish from those resulting from infection and may thus complicate diagnosis. Rough attenuated mutants lack the O-polysaccharide and would solve this problem if eliciting protective immunity; the empirically obtained rough mutants 45/20 and RB51 have been used as vaccines. Strain 45/20 is reportedly unstable and it is not presently used. RB51 is increasingly used instead of S19 in some countries but it is rifampicin resistant and its effectiveness is controversial. Some controlled experiments have found good or absolute protection in adult cattle vaccinated orally (full dose) or subcutaneously (reduced dose) and in one field experiment, RB51 was reported to afford absolute protection to calves and to perform better than S19. Controlled experiments in calves, however, have shown reduced doses of RB51 to be ineffective, full doses only partially effective, and RB51 less effective than S19 against severe challenges. Moreover, other observations suggest that RB51 is ineffective when prevalence is high. RB51 is not useful in sheep and evidence in goats is preliminary and contradictory. Rough mutants obtained by molecular biology methods on the knowledge of the genetics and structure of Brucella lipopolysaccharide may offer alternatives. The B. abortus manBcore (rfbK) mutant seems promising in cattle, and analyses in mice suggest that mutations affecting only the O-polysaccharide result in better vaccines than those affecting both core and O-polysaccharide. Possible uses of rough vaccines also include boosting immunity by revaccination but solid evidence on its effectiveness, safety and practicality is not available.     

Comparison between immune responses and resistance induced in BALB/c mice vaccinated with RB51 and Rev. 1 vaccines and challenged with Brucella melitensis bv. 3

BALB/c mice were immunized with live rough Brucella abortus RB51 or smooth Brucella melitensis Rev. 1 vaccines and challenged with a B. melitensis field strain. Protection was assessed by a variety of serological tests and recovery of vaccinal and challenge strains by culture. Mice vaccinated with RB51 gave negative results in the conventional serological tests prior to challenge, namely; standard tube agglutination test (SAT), Rose Bengal plate test (RBPT), buffered acidified plate antigen test (BAPAT), and mercaptoethanol test (MET). Sero-conversion took place to a whole-cell bacterial buffered RB51 antigen after vaccination and persisted for 7 weeks post-vaccination. Mice challenged with B. melitensis were assessed for bacterial load and immune response for 12 weeks after challenge. Protection units were showed that Rev. 1 vaccine was superior to RB51 vaccine in protection of mice against B. melitensis. However, RB51 vaccine has the advantage that it would not elicit antibodies to standard serological tests based on the LPS O antigen. RB51 vaccine could therefore be used for control of B. melitensis infection and avoid confusion in the use of standard sero-diagnostic tests.     

Enhanced efficacy of recombinant Brucella abortus RB51 vaccines against B. melitensis infection in mice

Brucella abortus strain RB51 is an attenuated rough strain, currently being used as the official live vaccine for bovine brucellosis in the USA and several other countries. In strain RB51, the wboA gene, encoding a glycosyltransferase required for the O-side chain synthesis, is disrupted by an IS711 element. Recently, we have demonstrated that strain RB51WboA, RB51 complemented with a functional wboA gene, remains rough but expresses low quantities of O-side chain in the cytoplasm. Mice vaccinated with strain RB51WboA develop greatly enhanced resistance against challenge with B. abortus virulent strain 2308. We have also demonstrated that overexpression of Cu/Zn superoxide dismutase (SOD) in strain RB51 (RB51SOD) significantly increases its vaccine efficacy against strain 2308 challenge. In this study, we constructed a new recombinant strain, RB51SOD/WboA, that over expresses SOD with simultaneous expression of O-side chain in the cytoplasm. We tested the vaccine potential of strains RB51SOD, RB51WboA, RB51SOD/WboA against challenge with virulent Brucella melitensis 16M and B. abortus 2308 in mice. In comparison with strain RB51, strain RB51SOD induced better protection against strain 2308, but not strain 16M, challenge. Similar to strain RB51WboA, vaccination with strain RB51SOD/WboA resulted in complete protection of the mice from infection with strain 2308. When challenged with strain 16M, mice vaccinated with either strain RB51WboA or strain RB51SOD/WboA were significantly better protected than those vaccinated with strain RB51 or RB51SOD. These results suggest that strains RB51WboA and RB51SOD/WboA are good vaccine candidates for inducing enhanced protection against B. melitensis infection.     

VACCINATION OF PREGNANT COWS WITH LOW DOSES OF BRUCELLA ABORTUS STRAIN 19 VACCINE

Two groups, each of 9 Jersey cows, were vaccinated subcutaneously with reduced doses of Brucella abortus strain 19 vaccine (measured by the number of bacteria in the vaccine dose) early in their second pregnancy. Ten weeks later they were challenged, along with a similar group of non-vaccinated cows, by conjunctival instillation of a virulent strain of B. abortus biotype 1. Cows in Group 1 received 1/20th and those in Group 2 received 1/400th the recommended dose of strain 19. Marked reduction in the serological response to vaccination was seen only in Group 2. Four cows in Group 1 excreted strain 19 after parturition, one of them aborted and another calved prematurely with heavy infection of the placenta and foetus with strain 19 in both cases. Resistance to challenge was similar in both vaccinated groups, and higher than previously demonstrated after conventional calfhood vaccination with strain 19. It is concluded that pregnant cows can be effectively vaccinated by the subcutaneous administration of a dose of strain 19 vaccine containing approximately 3 x 10(8) organisms without undue interference with subsequent serological tests or inconvenience resulting from persistence of strain 19 infection.     

Experiences in the control of brucellosis in cattle herds in the government district of Hannover

Caused by imported beef cattle new outbreaks of Brucellosis (subtype III) were observed in the government district of Hannover, which was "Brucellosis free" over a long period. With the aim to interrupt the series of infections from herd to herd it seemed to be necessary to introduce a herd screening system including frequent tests. It was not possible to screen the herds by the usual way of blood serum testing because of reasons of practicability and economy. A practicable alternative was the ELISA supported tank investigation. Four of the last six outbreaks were detected by this milk sampling. The other two infected herds were detected by only clinical symptoms because the lactating cows were not infected and had no contacts to the infected separately held heifers. The required test frequency in the dairy cattle herds could only be realized applying an ELISA supported highly sensitive tank milk test method. This method offered the chance of discovering infected herds as soon as possible and prevented greater economical losses and animal health risks. The tank milk screening of the dairy cattle herds to detect antibodies against Brucellosis (and additional EBL and IBR) has now become a standard method to continue the official status "free from..." This is a safer and a more economic alternative to the previous blood sampling of cattle older than two years with a three years interval because this method guarantees a safe information about the serological status of each cow which is now tested at least once a year, depending on the time of dry standing status.(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.     

Infectivity virulence and immunogenicity of Anaplasma centrale live blood vaccine

Cross-bred Bos taurus calves, aged between 6 and 8 months, were inoculated with the Onderstepoort Anaplasma centrale live blood vaccine. One group of 15 calves were inoculated once only, while a 2nd group of 15 were revaccinated 6 months later. All the animals were challenged with approximately 1 X 10(10) Anaplasma marginale parasites of a known virulent strain 8 months after the first vaccination. The results of blood smear examination and the card agglutination test indicated that only 20 out of 30 animals vaccinated contracted A. centrale infections after the first attempt, and 3 out of 5 after the second. The vaccine conferred only partial immunity to challenge with a virulent A. marginale strain.     

Evaluation of brucellosis RB51 vaccine for domestic water buffalo (Bubalus bubalis) in Trinidad

Thirty-two young domestic water buffalo (Bubalus bubalis) were obtained from a brucellosis-free farm to determine effectiveness of RB51 vaccination for prevention of Brucella infection under natural-exposure conditions in Trinidad. Study animals (20 males and 12 females 5–20 months old) were assigned to vaccination or control groups, using a block randomization design ensuring equal sex distributions between groups. The vaccination group received commercially available RB51 at the recommended calfhood dose of (1.0–3.4)×10¹⁰ colony-forming units (CFU) and controls received 2 ml sterile saline. Vaccination did not result in positive serologic results as measured by four traditional agglutination tests: standard tube agglutination test (STAT), standard plate agglutination test (SPAT), buffered plate agglutination test (BPAT), and card agglutination. Study animals were maintained in a brucellosis-positive herd in southern Trinidad with an estimated 56% prevalence to allow for natural exposure to B. abortus, which was evaluated using STAT, SPAT, BPAT, and card tests. Animals were sampled seven times over 2 years and were classified as positive if they had persistent agglutination titers or had Brucella isolated from specimens collected at completion of the study. Five of the original 32 study animals were lost to follow-up during the field trial. Six of the 14 (43%) vaccinated animals completing the study were classified as positive for Brucella infection—as were two of the 13 (15%) control animals (P=0.21). Isolates from four vaccinates and one control were confirmed as B. abortus biovar 1.