Brucellosis vaccines: past,present and future

The first effective Brucella vaccine was based on live Brucella abortus strain 19, a laboratory-derived strain attenuated by an unknown process during subculture. This induces reasonable protection against B. abortus, but at the expense of persistent serological responses. A similar problem occurs with the B. melitensis Rev.1 strain that is still the most effective vaccine against caprine and ovine brucellosis. Vaccines based on killed cells of virulent strains administered with adjuvant induced significant protection but also unacceptable levels of antibodies interfering with diagnostic tests. Attempts were made to circumvent this problem by using a live rough strain B. abortus 45/20, but this reverted to virulence in vivo. Use of killed cells of this strain in adjuvant met with moderate success but batch to batch variation in reactogenicity and agglutinogenicity limited application. This problem has been overcome by the development of the rifampicin-resistant mutant B. abortus RB51 strain. This strain has proved safe and effective in the field against bovine brucellosis and exhibits negligible interference with diagnostic serology. Attempts are being made to develop defined rough mutant vaccine strains that would be more effective against B. melitensis and B. suis. Various studies have examined cell-free native and recombinant proteins as candidate protective antigens, with or without adjuvants. Limited success has been obtained with these or with DNA vaccines encoding known protective antigens in experimental models and further work is indicated.     

Effects of challenge dose on the clinical and immune responses of cattle vaccinated with reduced doses of Brucella abortus strain 19

Fifty-seven pregnant beef heifers that were unvaccinated or previously vaccinated with Brucella abortus S19, at a dose of either 10⁹ or 10¹⁰ colony-forming units (CFU), were challenge-exposed intraconjunctivally with virulent B. abortus S2308 at a dose of 9.4 × 10⁶ CFU (Experiment 1) or 5.2 × 10⁷ CFU (Experiment 2). In Experiment 1, S19 afforded significant protection (P < 0.01) against challenge exposure in that 8 of 9 unvaccinated heifers, 1 of 11 vaccinated with 10⁹ CFU, and 3 of 10 vaccinated with 10¹⁰ CFU aborted or delivered weak, non-viable calves. In Experiment 2, vaccination did not afford significant protection (P> 0.05) in that 9 of 9 unvaccinated heifers, 8 of 10 vaccinated with 10⁹ CFU, and 8 of 8 vaccinated with 10¹⁰ CFU aborted. Serologic responses to B. abortus were determined by three standard tests, as well as a quantitative fluorometric immunoassay (FIAX) and an enzyme-linked immunosorbent assay. In Experiment 1, the early serologic response, 0–8 weeks after challenge, appeared greater for controls than for vaccinates, but in Experiment 2, the early response, 0–6 weeks after challenge exposure, appeared greater for vaccinates than for controls. The lymphocyte blast transformation assay, using heat-killed B. abortus as an antigen, was performed sequentially after challenge exposure. In general, mean responses were significantly higher (P < 0.05) for vaccinated than for non-vaccinated heifers. For individual heifers, an association could not be established between the lymphocyte blast transformation assay and the clinical response to challenge exposure.     

Paradigm shifts in vaccine development: lessons learned about antigenicity,pathogenicity and virulence of Brucellae

As part of a program to support the USDA Animal Plant Health Inspection Service Bovine Brucellosis Eradication Program, the Brucellosis Research Unit of the National Animal Disease Center (NADC) sought to develop a bovine brucellosis vaccine that would allow vaccinated animals to be distinguished from virulent field infected animals. In order to meet that goal, several avenues of research were undertaken to construct and test candidate vaccines, including Brucella abortus RB51. In early vaccine development studies, a subunit preparation obtained by extracting B. abortus with salts was studied as a candidate subunit vaccine. Later, molecular biological techniques were used both to clone genes encoding products found in the salt extract (BCSP31 and Cu–Zn SOD) and genes encoding proteins of B. abortus that were antigenic (HtrA) or possibly essential (two-component systems) for full virulence of B. abortus. In vitro systems using mammalian cells lines such as HeLa and macrophage-related were used along with the mouse model and host animal models. Results obtained at NADC and in other Brucellosis research laboratories, using survival in mammalian cell lines and the mouse model to access pathogenicity and virulence of genetically engineered strains, do not necessarily identify loci that are essential for full virulence or pathogenicity in the natural host, the bovine. Studies at NADC and other brucellosis laboratories showed that antigenicity was not a predictor of the effectiveness of a protein as a subunit vaccine.     

Brucellosis in Argentina

Brucellosis has been recognized in Argentina since the 19th century. Several studies demonstrated the presence of the disease in most of the domestic species. Actually, the estimate of prevalence is that between 10 and 13% of the farm animals are infected with bovine brucellosis with an individual rate of 4–5%. The annual economical losses have been estimated at US$ 60,000,000. The control of bovine brucellosis began in 1932 and successive resolutions have been issued since then. The current resolution indicates that B. abortus S19 is mandatory in female calves between 3 and 8 months of age. The vaccine strain B. abortus RB51 was provisionally approved but only for cattle older than 10 months of age. The brucellosis control program consists principally of test and slaughter. This methodology has been successful mainly in the dairy farms that have the incentive due to increased pricing because of obtaining a low prevalence of the disease. Brucellosis has been found in porcine, caprine, ovine and canine species. All Brucella species have been found in the country. Human brucellosis is an important disease and a national coordinated diagnostic net has been formed to better control the disease in man.     

In vitro stimulation of bovine milk lymphocytes standardization of the assay for bovine brucellosis

Lymphocytes of bovine milk origin were investigated by immunostimulation in vitro to standardize the assay for measuring the immune responses of the cells which might be useful in further understanding the immunopathology and diagnosis of bovine brucellosis. The lymphocytes were separated from whole freshly collected milk by centrifugation. The pellet of lymphocytes was washed in RPMI-1640 medium, cultured at different concentrations for different days and with Brucella abortus soluble antigen strain 1119-3 and Concanavalin A. Each culture was labelled with 1.0 μCi of methyl-[³H]thymidine 16–18 hours prior to termination of incubation at 37 C. Termination was done by cooling to 4 C. The cells were harvested for liquid scintillation counting spectrometry. In the groups of calfhood vaccinated cows and nonexposed milkers, a milk lymphocyte concentration of 2.0 × 10⁶/ml of medium yielded a statistically significant blastogenesis. The Brucella abortus soluble antigen concentration of 4.4 μg of protein/well was found optimal to induce significant immunostimulation. A period of 4 days of incubation of the milk lymphocyte in the test was found optimal in inducing statistically significant blastogenesis in this system.     

Discrimination between sheep antibodies to Brucella melitensis and to Brucella ovis

When preparations containing smooth Brucella abortus lipopolysaccharide (LPS) were used as antigens in an ELISA, strong positive reactions were obtained with sera from sheep infected with Brucella melitensis or with Brucella ovis. Oxidation of the LPS with sodium metaperiodate greatly reduced the extent of the cross-reactions with antisera to B. ovis, with little effect on the reactions with antisera to smooth B. melitensis. Periodate oxidation of hot saline extract (HSX) antigen of B. ovis markedly reduced its reactivity in ELISA with anti-B. ovis sera and eliminated cross-reactivity with anti-B. melitensis sera. The reactivity of HSX was maintained after treatment with proteinase K.

A simple ELISA system, in which replicate samples from a single serum dilution were tested in parallel against both B. ovis HSX antigen and periodate-oxidised smooth phase B. abortus LPS, was evaluated. It was found to discriminate well between antibodies induced by vaccination or virulent infection with B. melitensis strains and those induced by infection with B. ovis.     

How to substantiate eradication of bovine brucellosis when aspecific serological reactions occur in the course of brucellosis testing

Collaborative work was financed by the EU to develop and assess new diagnostic tools that can differentiate between bovine brucellosis and bovine infections due to Yersinia enterocolitica O:9 either in conjunction with, or as an alternative to, the classical serological, bacteriological or allergic skin tests. Sixteen heifers were experimentally infected with Brucella abortus biovar 1 (five heifers), Brucella suis biovar 2 (two heifers), Y. enterocolitica O:9 (six heifers) and Y. enterocolitica O:3 (three heifers). Four heifers, naturally infected with Y. enterocolitica O:9 that presented aspecific brucellosis serological reactions were also included in the experiment. A self-limited infection was induced in cattle by B. suis biovar 2. All the brucellosis serological tests used, i.e. the slow agglutination test (SAW), the Rose Bengal test (RB), the complement fixation test (CFT), indirect and competitive ELISA’s, lacked specificity when used to analyze sera from Y. enterocolitica O:9 infected animals. A Yersinia outer membrane proteins (YOPs)-ELISA was also used and although the test is able to detect a Yersinia group infection, it provided no evidence of whether or not there is a possible brucellosis infection when dual infections are present. The brucellergen IFN-γ test showed a lack of specificity also. The only test that was proven to be specific is the brucellergen skin test. All brucellosis serological tests, except the indirect ELISA, were limited in their ability to detect B. abortus persistently infected animals.

Based on these experimental studies, a strategy was implemented as part of the year 2001 Belgian Brucellosis Eradication Program to substantiate the eradication of bovine brucellosis. Epidemiological inquiries have identified risk factors associated with aspecific serological reactions, possible transmission and infection of cattle by B. suis biovar 2 from infected wild boars; and both legal and administrative measures taken by the veterinary services. No cases of bovine brucellosis have been confirmed in Belgium since March 2000.     

Influence of the probiotic Bacillus cereus var. toyoi on the intestinal immunity of piglets

In a feeding trial, sows and piglets were fed with the probiotic bacterium Bacillus cereus var. toyoi as a feed additive, and the effects on immune cell populations were examined. The development of the gut immune system was determined for piglets at the ages of 14, 28, 35 and 56 days post partum. Tissue samples of the Jejunum and the continuous Peyer's patch were used for enumeration of intraepithelial lymphocyte populations by fluorescence activated flow cytometry and fluorescence microscopy. Both independent methods of investigation led to similar results: the population of intraepithelial CD8+ T cells was significantly enhanced in the probiotic group piglets (p ≤ 0.05), and the numbers of γδ T cells tended to be higher in the intestinal epithelium (p < 0.1) at the time of weaning (day 28). Lamina propria lymphocytes were also influenced by the treatment. Application of B. cereus var. toyoi resulted in significantly more CD25+ lymphocytes and γδ T cells in the probiotic group post-weaning. The occurrence of pathogenic Escherichia coli serogroups was also less frequent in the feces of piglets from the probiotic group. The finding that the CD8+ T cell population in the intestinal mucosa showed changes on day 28 indicated that the influence of B. cereus var. toyoi supplementation on the intestinal immune system started before weaning, an observation supported by changes in the intestinal microflora observed during the suckling-period. The results suggest that feeding of B. cereus var. toyoi to sows may result in beneficial effects on piglet health status independent of their feed supplementation.     

Incidence and control of brucellosis in the Near East region

In countries of the Near East region, brucellosis was reported in almost all domestic animals, particularly cattle, sheep and goats. Brucellosis in camels has been reported in Saudi Arabia, Kuwait, Oman, Iraq, Iran, Sudan, Egypt, Libya and Somalia. It has been reported even in racing camels in the United Arab Emirates. In Egypt, brucellosis has been reported also in buffaloes, equines and swine. Brucella melitensis biovar 3 is the most commonly isolated species from animals in Egypt, Jordan, Israel, Tunisia and Turkey. B. melitensis biovar 2 was reported in Turkey and Saudi Arabia, and B. melitensis biovar 1 in Libya, Oman and Israel. B. abortus biovar 1 was reported in Egypt, biovar 2 in Iran, biovar 3 in Iran and Turkey, and biovar 6 in Sudan. The countries with the highest incidence of human brucellosis are Saudi Arabia, Iran, Palestinian Authority, Syria, Jordan and Oman. Bahrain is reported to have zero incidence. Most human cases are caused by B. melitensis, particularly biovar 3. However, B. abortus has been responsible for an increasing number of cases in recent years, e.g. in Yemen, where B. abortus was identified in 45 cases and B. melitensis in 7 cases out of 330 cultures performed in 1995. Concerning control of brucellosis in animals, there is a controversy on the choice of policy. In some countries, the test and slaughter policy together with the vaccination of young females is adopted, in others, particularly with regard to sheep and goats; mass vaccination has been recently started. The most commonly used vaccines are B. abortus S19 and B. melitensis Rev.1 vaccines. B. abortus RB51 vaccine is used in some countries on small scale. Vaccination is limited to cattle and small ruminants.     

Characterization of the caprine model for ruminant brucellosis

The relationship between man, the goat, and brucellosis is historical. Today Brucella melitensis and Brucella abortus pose a serious economic and public health threat in many countries throughout the world. Infection of pregnant goats and sheep with B. melitensis results in abortion during the third trimester of pregnancy. Although nearly eradicated in the US, bovine brucellosis is still a problem in many countries and the potential for re-infection of domestic stock from wildlife reservoirs in this country is a regulatory nightmare. Humans infected with this pathogen develop undulant fever, which is characterized by pyrexia, arthritis, osteomyelitis, and spondylitis. Although available for both organisms, currently available vaccines have problems ranging from false positive serological reactions to limited efficacy in different animal species. With the continued need for new and better vaccines, we have further developed a goat model system to test new genetically derived strains of B. melitensis and B. abortus for virulence as measured by colonization of maternal and fetal tissues, vaccine safety, and vaccine efficacy.     

Value of serologic reactions at 2 months following strain 19 vaccination of cattle herds with brucellosis

Non-reactor cows in a dairy herd and six beef herds quarantined because of brucellosis were vaccinated with Brucella abortus Strain 19 and tested by rivanol and complement-fixation (CF) tests. Cows with rivanol 100 and CF 80 test titers at 2 months post-vaccination (p.v.) were defined as test positives. In the dairy herd, 46 test positives were diagnosed as follows: 17 (37%) had field strain infection; 1 (2%) had a Strain 19 infection; an additional 18 (39%) were brucellosis reactors at 4 months p.v.; 10 (22%) had declining or negative serologic tests at 4 months p.v. In the beef herds, 58 test positives were diagnosed as follows: 19 (33%) had field strain infection; 5 (9%) had Strain 19 infection; an additional 21 (36%) were brucellosis reactors at 6 months p.v.; 13 (22%) had declining or negative serologic tests at 6 months p.v.

Since the majority of the test-positive cattle were diagnosed as either infected with B. abortus or brucellosis reactors, segregation of these cattle should reduce field strain exposure for the remaining cattle in the herd and therefore reduce the number of new cases of brucellosis.     

Serologic survey of wild cervids for potential disease agents in selected national parks in the United States

A total of 589 serum specimens were collected from mule deer (Odocoileus hemionus) (133) and wapiti (Cervus elaphus) (456) in eight national parks and/or adjacent lands in the western USA. Thirty two percent of the samples were collected from immobilized animals and 68% from hunter-killed animals in or near Glen Canyon National Recreation Area, Bryce Canyon National Park (NP), and Zion NP, Utah; Yosemite NP, California; Rocky Mountain NP, Colorado; Upper Yellowstone NP, Montana, and Grand Teton NP, Wyoming. Serum specimens were tested for the presence of antibodies against selected disease agents. Overall seroprevalences for mule deer were 77/133 (58%) for parainfluenza-3 virus (PI-3), 42/133 (32%) for bovine herpesvirus-1 (BHV-1), 79/133 (59%) for bovine virus diarrhea virus (BVD), 73/133 (55%) for respiratory syncytial virus (RSV), 14/133 (11%) for bluetongue virus (BT), 18/133 (14%) for epizootic hemorrhagic disease vims (EHD), 3/133 (2%) for Borrelia burgdorferi, and 1/133 (1%) for Francisella tularensis. None of the deer sera presented antibodies for Leptospira spp., Brucella abortus and Anaplasma marginale. For wapiti, overall prevalences were 262/456 (57%) for PI-3, 211/456 (46%) for BHV-1, 251/456 (55%) for BVD, 247/456 (54%) for RSV, 1/456 ( < 1%) for BT, 16/456 (4%) for Leptospira pomona, 13/456 (3%) for Leptospira hardjo, and 8/456 (2%) for B. abortus. No antibody titers were detected for EHD, A. marginale, and other Leptospira serotypes. This survey documents seroprevalence of selected park cervid populations to domestic livestock pathogens. Further research on the epidemiology of these potential pathogens in wild ungulates in national parks is recommended.     

Effect of fowl adenovirus-1 (IBH isolate) on humoral and cellular immune competency of broiler chicks

Fowl adenovirus-1 (FAV-1), isolated from field outbreaks of inclusion body hepatitis (IBH), was administered orally to 3-week-old disease-free broiler chicks. Humoral immune competency was evaluated by determining the antibody response of infected chicks to sheep red blood cells (SRBC) and Brucella abortus. FAV-1 infection significantly decreased the antibody response of chicks to B. abortus (T-cell-independent antigen) by decreasing IgM responses, however, the decreased antibody response to SRBC (T-cell-dependent antigen) was statistically non-significant. Bursal index was also found lowered in infected chicks as compared to the control chicks. A significant decrease was seen in blastogenesis response of peripheral blood lymphocytes to phytohaemagglutinin (PHA-P) in FAV-1-infected chicks on 2 and 3 weeks post-infection (WPI). These results indicated that FAV-1 affects humoral as well as cellular immune competency of infected chicks.     

Major outer membrane proteins of Brucella spp.: past,present and future

The major outer membrane proteins (OMPs) of Brucella spp. were initially identified in the early 1980s and characterised as potential immunogenic and protective antigens. They were classified according to their apparent molecular mass as 36–38 kDa OMPs or group 2 porin proteins and 31–34 and 25–27 kDa OMPs which belong to the group 3 proteins. The genes encoding the group 2 porin proteins were identified in the late 1980s and consist of two genes, omp2a and omp2b, which are closely linked in the Brucella genome, and which share a great degree of identity (>85%). In the 1990s, two genes were identified coding for the group 3 proteins and were named omp25 and omp31. The predicted amino acid sequences of omp25 and omp31 share 34% identity. The recent release of the genome sequence of B. melitensis 16 M has revealed the presence of five additional gene products homologous to Omp25 and Omp31. The use of recombinant protein technology and monoclonal antibodies (MAbs) has shown that the major OMPs appear to be of little relevance as antigens in smooth (S) B. abortus or B. melitensis infections i.e. low or no protective activity in the mouse model of infection and low or no immunogenicity during host infection. However, group 3 proteins, in particular Omp31, appear as immunodominant antigen in the course of rough (R) B. ovis infection in rams and as important protective antigen in the B. ovis mouse model of infection. The major OMP genes display diversity and specific markers have been identified for Brucella species, biovars, and strains, including the recent marine mammal Brucella isolates for which new species names have been proposed. Recently, Omp25 has been shown to be involved in virulence of B. melitensis, B. abortus and B. ovis. Mutants lacking Omp25 are indeed attenuated in animal models of infection, and moreover provide levels of protection similar or better than currently used attenuated vaccine strain B. melitensis Rev.1. Therefore, these mutant strains appear interesting vaccine candidates for the future. The other group 3 proteins identified in the genome merit also further investigation related to the development of new vaccines.     

Antibody levels to Brucella abortus, toxoplasma gondii, and Leptospira serogroups, in sera collected from healthy people in Fiji

A collection of 300 sera from a predominantly rural community on the island of Viti Levu in Fiji were studied for the presence of antibodies to B. abortus, T. gondii and Leptospira serogroups. Significant levels of immunity were found to B. abortus and T. gondii and over half the population had diagnostic leptospiral antibody levels.     

An intradermal test for the diagnosis of brucellosis in extensively managed cattle herds

The application of a delayed hypersensitivity test for the diagnosis of bovine brucellosis was examined in a series of field experiments. The test is based on the intradermal injection of ‘Brucellin’, a lipopolysaccharide-free protein extract of Brucella abortus strain 45/20.

The Brucellin test was compared with the complement fixation (CF) test in 8656 cows of mixed age and known vaccination and herd status. An intradermal injection of 0.1 ml of the allergen was made in either the cervical region or the caudal fold. The injection site was examined 72 h after administration of Brucellin and any increase in skin thickness of 2 mm was regarded as positive.

When administered into the caudal fold site the Brucellin test had a sensitivity relative to the CF test of between 52 ± 14% and 61 ± 6%. The relative specificity of the test exceeded 99%.

Calfhood vaccination with B. abortus strain 19 did not result in positive Brucellin test results. There was no evidence that the injection of Brucellin induced a serological response.

Despite the low relative sensitivity of the Brucellin test, it is a useful low-cost tool for identifying infected herds. It is a least as effective as slaughterhouse surveillance systems.     

Regulation of Brucella virulence by the two-component system BvrR/BvrS

The Brucella BvrR/BvrS two-component regulatory system is highly similar to the regulatory and sensory proteins of Sinorhizobium and Agrobacterium necessary for endosymbiosis and pathogenicity in plants, and very similar to a putative system present in the animal pathogen Bartonella. Mutations in the bvrR or bvrS genes hamper the penetration of B. abortus in non-phagocytic cells and impairs intracellular trafficking and virulence. In contrast to virulent Brucella, BvrR/BvrS mutants do not recruit small GTPases of the Rho subfamily required for actin polymerization and penetration to cells. Dysfunction of the BvrR/BvrS system alters the outer membrane permeability, the expression of several group 3 outer membrane proteins and the pattern of lipid A acylation. Constructs of virulent B. abortus chimeras containing heterologous LPS from the bvrS⁻ mutant demonstrated an altered permeability to cationic peptides similar to that of the BvrR/BvrS mutants. We hypothesize that the Brucella BvrR/BvrS is a system devoted to the homeostasis of the outer membrane and, therefore in the interface for cell invasion and mounting the required structures for intracellular parasitism.     

Brucella evolution and taxonomy

The genus Brucella contains alpha-Proteobacteria adapted to intracellular life within cells of a variety of mammals. Controversy has arisen concerning Brucella internal taxonomy, and it has been proposed that the DNA–DNA hybridization-based genomospecies concept be applied to the genus. According to this view, only one species, Brucella melitensis, should be recognized, and the classical species should be considered as biovars (B. melitensis biovar melitensis; B. melitensis biovar abortus; etc.). However, a critical reappraisal of the species concept, a review of the population structure of bacteria and the analysis of Brucella genetic diversity by methods other than DNA–DNA hybridization show that there are no scientific grounds to apply the genomospecies concept to this genus. On the other hand, an enlarged biological species concept allows the definition of Brucella species that are consistent with molecular analyses and support the taxonomical standing of most classical species. Both the host range as a long-recognized biological criterion and the presence of species-specific markers in outer membrane protein genes and in other genes show that B. melitensis, B. abortus, B. ovis, B. canis and B. neotomae are not mere pathovars (or nomenspecies) but biologically meaningful species. The status of B. suis is, however, less clear. These approaches should be useful to define species for the marine mammal Brucella isolates, as illustrated by the grouping of the isolates from pinnipeds or from cetaceans by omp2 gene analysis. It is shown that a correct Brucella species definition is important to understand the evolution of the genus.     

Brucellosis in Venezuela

Brucellosis is a public health problem in Venezuela and affects large numbers of animals. The most important biovar in the country is Brucella abortus. In cattle and buffalo it causes high rates of abortions in females and infertility in males; it is transmissible to occupationally exposed humans. In 1968, an official program was set up for the control and eradication of the disease and it is still in place. Amongst the control provisions, this program provides for the vaccination of female calves with strain 19 and the slaughtering of positive reactors following the official diagnosis (rapid agglutination in plate test). According to the official reports, the positive reactors ranged from 0.8 to 1.2% in the past few years. These values do not corroborate reports showing an average positive rate of 10.5% and even higher values in some areas of the country. The government is working to approve a new resolution that will replace the rapid agglutination in plate test with the Card Test, the use of 2-Mercaptoetanol, fixation of complement and competitive ELISA as confirmatory tests. In addition, there will be an obligatory vaccination with B. abortus strain 19 or B. abortus RB51 of all female calves between 3- and 8-month-old and a recommended revaccination at 10–15-month-old and adult cows in high prevalence areas. These measures should allow help to reduce the prevalence of the disease in cattle herds and thus minimize the risk for human populations.