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.     

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 the European Union and Norway at the turn of the twenty-first century

Control and eradication programs of brucellosis in cattle, sheep, goats and pigs have been more or less successfully implemented within the Member States (MS) of the European Union (EU) and Norway after Word War II. As a result, the epidemiological situation of animal brucellosis is extremely diverse among different MS or regions within a MS and among the different animal species. Some MS, mainly North European countries, and Norway are declared “officially bovine brucellosis free” and/or “officially ovine and caprine (Brucella melitensis) free”. The situation is less favorable in Southern European countries, particularly as far as sheep and goat brucellosis are concerned. This situation has important zoonotic consequences as reflected in the number of human brucellosis cases due to B. melitensis that are still encountered in those countries. Brucellosis in swine has re-emerged as a result of spillover from the wild boar brucellosis (Brucella suis biovar 2) reservoir, particularly in outdoor reared pigs. Besides the actual challenge to eradicate brucellosis, further issues have to be addressed: (1) the management of false positive serological results that occur in the course of brucellosis testing, particularly in cattle; (2) the impact of wildlife brucellosis, particularly wild boar brucellosis in domestic animals; and (3) the importance of B. melitensis infection in cattle that are in contact with infected sheep.     

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.     

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.     

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.     

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.     

An epidemiological and surveillance overview of brucellosis in Romania

This article reports epidemiological investigations on the occurrence of brucellosis in Romania. Like in other former communist countries, data concerning epidemiology of brucellosis and published articles are very few. The epidemiology and control of brucellosis in Romania was analyzed using data made available by the Office International des Epizooties and Veterinary Service of Romania. Romania, like many other developed countries, has eradicated Brucella abortus from cattle since 1969. Brucellosis caused by Brucella melitensis has never been reported. The incidence of brucellosis in swine and sheep is very rare but still there are a few outbreaks in some regions. In 2000, the number of cases was 47 in swine and 270 cases in sheep. Vaccination against brucellosis is prohibited in Romania.     

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.     

Control of small ruminant brucellosis by use of Brucella melitensis Rev.1 vaccine: laboratory aspects and field observations

Brucellosis vaccines are essential elements in control programs. Since first developed in the mid-1950s, the Brucella melitensis vaccine strain Rev.1 has been used worldwide and its significant value in protecting sheep and goats in endemic areas recognized. This review provides historical background on the development of the vaccine, its use and field complications arising in Israel following changes in the strain’s pathogenicity. The urgent need for resolving cases of vaccine strain excretion in the milk, horizontal transfer and a unique case of human infection has led to identification of an atypical B. melitensis biovar 1 strain that resembles strain Rev.1 in susceptibility to penicillin and dyes. An omp2 based PCR method has been developed that traced the lineage of Israeli B. melitensis biovar 1 strains. This locus serves as an epidemiological tag for the Rev.1 vaccine strain. Despite the rapid development of new approaches in the field of vaccination, it is anticipated that in the near future the Rev.1 vaccine would remain the only accepted vaccine in national control programs.     

Epidemiology and control of brucellosis in China

The paper describes the history and evolvement of brucellosis in China. It presents the variation of epidemic situation, epidemiological characteristics, application of vaccines and control in brief. Before 1980s, human and animal brucellosis was quite severe; during 1980s, the incidence of human and animal brucellosis was relatively low, and seemed to decrease during the decade. During 1990s, there were no obvious changes in the incidence of animal brucellosis, but the incidence of human brucellosis increased, especially from 1995 to 2001. There are not only some common characteristics but also some differences in brucellosis epidemiology relative to that reported in the rest of the world. For the entire country, B. melitensis was the predominant strain associated with outbreaks, and the epidemic peak is from February to June. Several Brucella vaccines have been used in China for prevention and control of brucellosis. such as B. abortus 104 M in humans, B. suis S2 in animals. The introduction of comprehensive measures has allowed great progress in the prevention and control of brucellosis in China. Surveillance points were set-up countrywide to estimate the epidemic situation. In addition, we discussed the new characteristics of brucellosis in China, the influence of the El Nino phenomenon on brucellosis epidemic situation, the phenomenon of antigenic interference between Brucella species and some disadvantages of live Brucella vaccines.     

A review of serological investigations of brucellosis among farm animals and humans in northern provinces of iraq (1974-2004)

Brucellosis is an endemic anthropozoonotic disease in most Middle East countries including Iraq. It causes considerable loss of economy and energy, particularly in the rural areas. Since 1966 when the prevalence of brucella agglutinin from slaughtered sheep and goat was reported in Baghdad, several trials have been conducted including serological studies of brucellosis in the northern provinces of Iraq (Nainava, Duhok, Erbil, Kirkuk and Sulaimani). Humans, sheep, goats, cattle and buffalo have been involved. The studies also included isolation and identification of Brucella species, including B. abortus, B. melitensis and B. ovis. Local veterinary authorities, international organizations, universities and some NGOs have been involved. Moreover, limited efforts have been reported concerning the control and eradication of the disease among goats and sheep in some restricted areas. A limited vaccination program for newborn lambs and kids also has been conducted by the FAO, which was involved in the veterinary emergency plan for brucellosis in 2000. Northern provinces share an extensive border with Iran, Turkey and Syria, in addition to the direct and intimate contacts with other provinces of Iraq, which share borders with Jordan, Saudi Arabia and Kuwait. Hence any strategic planning for the control or eradication of this devastating anthropozoonotic disease in the country should be implemented regionally and not locally.     

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.     

A Review of Serological Investigations of Brucellosis Among Farm Animals and Humans in Northern Provinces of Iraq (1974–2004)

Brucellosis is an endemic anthropozoonotic disease in most Middle East countries including Iraq. It causes considerable loss of economy and energy, particularly in the rural areas. Since 1966 when the prevalence of brucella agglutinin from slaughtered sheep and goat was reported in Baghdad, several trials have been conducted including serological studies of brucellosis in the northern provinces of Iraq (Nainava, Duhok, Erbil, Kirkuk and Sulaimani). Humans, sheep, goats, cattle and buffalo have been involved. The studies also included isolation and identification of Brucella species, including B. abortus, B. melitensis and B. ovis. Local veterinary authorities, international organizations, universities and some NGOs have been involved. Moreover, limited efforts have been reported concerning the control and eradication of the disease among goats and sheep in some restricted areas. A limited vaccination program for newborn lambs and kids also has been conducted by the FAO, which was involved in the veterinary emergency plan for brucellosis in 2000. Northern provinces share an extensive border with Iran, Turkey and Syria, in addition to the direct and intimate contacts with other provinces of Iraq, which share borders with Jordan, Saudi Arabia and Kuwait. Hence any strategic planning for the control or eradication of this devastating anthropozoonotic disease in the country should be implemented regionally and not locally.     

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.     

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.     

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.     

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.