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.     

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.     

Incidence of brucellosis in Sri Lanka: an overview

Infection by Brucella abortus seems to be a major cause of abortions among cattle and buffaloes in Sri Lanka. The incidence of this disease is more prominent among the animals in the Dry zone of the country raised under extensive management systems. The present low incidence of this disease and the small size of the country may facilitate launching of an effective disease control scheme. The milk ring test (MRT) has proven to be usable in testing milk for the infection at farm level. An ELISA technique could be employed to test the seroprevalence of infection among MRT-positive animals. A program to purchase the diseased animals by the state for slaughter, and a countrywide vaccination program with B. abortus strain RB51 would enable the country’s livestock industry to eventually eradicate this disease.     

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.     

Killing of Brucella antigen-sensitized macrophages by T lymphocytes in bovine brucellosis

The present study was an investigation into the role of T lymphocytes in the killing of antigen-sensitized macrophages (MΦ) in bovine brucellosis. Following confirmation of bovine T lymphocyte cell lines derived from Brucella abortus Strain 19 vaccinated steers as antigen-specific in proliferation studies using various antigens, we adapted an apoptosis assay for evaluation of cytotoxicity by these bovine T cells against autologous monocyte-derived macrophages (MDMΦ) as target cells. Various B. abortus antigen preparations were tested including whole γ-irradiated B. abortus bacteria (γBA), a soluble cytosolic protein fraction and a membrane-associated protein fraction. Both polyclonal and cloned T lymphocyte cell lines exhibited cytotoxicity against MDMΦ targets in an antigen-specific fashion. Polyclonal and cloned T lymphocyte cell lines demonstrated cytotoxic responses to varying degrees against B. abortus antigens regardless of whether the antigen used was whole nonviable bacteria, a soluble protein extract or a membrane-associated fraction of extracted bacteria. To further develop correlation of these responses to an in vivo host defense mechanism, cytotoxicity was evaluated using target cells that had been infected with live B. abortus S19 or B. abortus Strain 2308. Cytotoxic responses were also demonstrated consistently against infected targets with either strain of B. abortus although in most cases, cytotoxicity was higher against target cells sensitized with γBA compared to those infected with live bacteria. Cloned T lymphocyte cell lines were all CD4⁺, CD8⁻ cells indicating that the observed cytotoxic responses were most likely due to an inflammatory T_h1 response and may represent an important host defense mechanism induced by vaccination with live attenuated strains of B. abortus in cattle.     

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.     

Epidemiology,zoonotic aspects,vaccination and control/eradication of brucellosis in India

In India, brucellosis was first recognised in 1942 and is now endemic throughout the country. The disease is reported in cattle, buffalo, sheep, goats, pigs, dogs and humans. B. abortus biotype-1 in cattle and buffaloes and B. melitensis biotype-1 in sheep, goats and man are the predominant infective biotypes. The long-term serological studies have indicated that 5% of cattle and 3% of buffaloes are infected with brucellosis. Economic losses due to brucellosis in livestock are considerable in an agrarian country like India. There is no organised and effective brucellosis control programme in the country. With the indigenous development of serum and milk based ELISA kits, the population survey of the disease has been undertaken on a large scale in several states and plans for the control of the disease through calf-hood vaccination are being worked out. An innovative approach—Bovine Brucellosis Progressive Control Programme (BBPCP) is targeted to overcome the basic problems of ban on cow slaughter, distress sale of animals following the positive serological diagnosis of brucellosis and absence of a disease control strategy. The work plan for the implementation of BBPCP is presented.     

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.     

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.     

The effects of Brucella strain 19 vaccination on titers in the tube agglutination test: a case control study.

The degree of agglutination in the tube agglutination test of the sera of 373 "reactor" cattle was compared to that of 800 "non-reactor" cattle from multiple reactor herds in southwestern Ontario. Vaccinated cattle had lower titers than did nonvaccinated cattle. Thus, vaccinated cattle were less likely to be classified as positive than nonvaccinated cattle irrespective of the method of interpreting the results of the tube test. When the full allowance for vaccination was used, vaccinated cattle were less likely to be classified as suspicious than nonvaccinated cattle. The extent of herd vaccination did not appear to markedly influence the effects of vaccination or the prevalence of reactors. Thus, it appeared that vaccination with strain 19 produced a small protective effect (inferred from the lower titers in vaccinated animals), but that the magnitude of this effect was markedly dependent on the appropriateness of the vaccine allowance for classifying cattle. Further work is needed on this aspect of vaccination with strain 19.     

Eradication of bovine brucellosis in the 10th Region de Los Lagos,Chile

The process of Bovine Brucellosis Eradication that began in 1996 in the 10th Region de Los Lagos of Chile will be reviewed. The region comprises the most important dairy area of the country and it has the largest concentration of brucellosis infected herds. Based on the information gathered by an epidemiological surveillance system, the results of the eradication process for the years 1996 till 2001 are presented as rates of Milk Ring Test (MRT) positive dairies, rates of brucellosis reactors (bovines) in livestock markets and slaughterhouses, and the annual incidence and prevalence of brucellosis infected herds.

During the period the rates of positive dairies, bovine reactors in livestock markets and slaughterhouses, and the annual incidence and prevalence of infected herds have experienced a decrease, while the rate of bovine reactors in slaughterhouses has remained stable. Data on the preventive measures taken, such as vaccination of female bovines and Certification of Brucellosis Free Herds, are also shown. The surveillance system has allowed the detection of infected herds, while the measures of prevention and cleaning of infected herds have allowed a reduction in the incidence and prevalence of the infection by Brucella abortus.     

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.     

An overview of the epidemiology and epizootology of brucellosis in selected countries of Central and Southeast Europe

The objective of this paper is to give an overview of the epidemiologic and epizootic status of brucellosis in selected countries of Central and Southeast Europe (Balkan region). Based on dimension of the disease problem, there is a need to establish collaboration in the eradication and prevention of brucellosis between all countries in the region. Although there were no readily accessible data concerning epidemiology and epizootology of brucellosis in these countries, the limited official and published data were analyzed.

The incidence of brucellosis caused by Brucella melitensis in sheep, goats and humans is a very significant problem in Macedonia and Greece. In Greece, cattle are also affected either by B. melitensis or B. abortus. The disease is an endemic problem in some regions of Yugoslavia and includes B. suis biovar 2 in pigs and in Croatia, B. melitensis in sheep, goats and human is found occasionally. No problem appears to exist with brucellosis in Bulgaria.

Financial well-supported brucellosis control programs of the European Union that will include all countries, regardless of the magnitude of brucellosis incidence, are needed for eradication and control of brucellosis.     

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.     

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.     

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.     

Brucellosis in Central America

Brucellosis is a disease of domestic animals and humans in Central America (CA). Bovine and swine brucellosis caused by Brucella abortus and Brucella suis, respectively, have been identified in all CA countries, while ovine and caprine brucellosis caused by Brucella melitensis has been detected in Guatemala. The prevalence of bovine brucellosis is estimated between 4 and 8%, with higher prevalence in dairy herds, with losses calculated at US$ 25 million per year. National Control Programs based in calf vaccination and removal of the reactors have had little impact in the control of brucellosis in CA. In a region where experimentation with new vaccines is not affordable, unrestricted adult vaccination by the conjunctival route with S19 is recommended. This strategy is expected to reduce the prevalence and density of the bacteria to numbers where “clean” vaccination would be possible. Thereafter, serological identification and elimination of the reactors could be initiated under more favorable conditions of herd infection.     

Clinical and serological evidence of bovine babesiosis and anaplasmosis in St. Lucia

One hundred fifty-nine Holstein calves were imported into St. Lucia from the U.S.A. An outbreak of babesiosis occurred 17 days post-arrival, and an outbreak of anaplasmosis occurred 5 months after importation. Sera obtained 3, 6 and 12 months post-importation revealed a high prevalence of IFA titres to Babesia bovis and B. bigemina 3 months after arrival and an increase in titres to Anaplasma marginale 6 months after arrival. Sera obtained arrives from native cattle from several places on the island indicated infection rates of 80, 65 and 64% with A. marginale, B. bigemina and B. bovis, respectively. The rapid card test only indicated a 25% prevalence of infection of native cattle by A. marginale. This low prevalence was probably due to deterioration of serological activity during shipment.     

The Brucella genome at the beginning of the post-genomic era

The year 2002 began with the publication of the first complete genome sequence for a Brucella species, that of the two replicons of B. melitensis 16M. Hopefully in 2002, the complete genome of B. suis 1330, and, perhaps, a B. abortus strain will be published. This is the culmination of over 30 years investigation of the composition, structure, organisation and evolution of the Brucella genome. Brucella research must now adapt to the new challenges of the post-genomic era.     

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.