Today, scientists and practitioners involved in the prevention of infectious diseases consider brucellosis to be one of the most dangerous zoonoses. It should be borne in mind that the official data are far from exhaustive concerning the intensity of manifestations of this infection in individual livestock species. Over 500,000 new cases of human brucellosis are reported in more than 170 countries each year [1]. It is also recognized as one of the most common worldwide infections with a high probability of infection in laboratory and plant conditions. An example is the 2019 outbreak of the disease among people in Lanzhou, China which occurred at a brucellosis vaccine plant. A biological accident resulted in the release of an aerosol containing live Brucella into the external environment. After screening more than 55,000 people, 6,620 were found to be positive for brucellosis.

In the Russian Federation over the past 10 years, the epidemic situation with brucellosis has been characterized as unfavorable with a downward trend in the incidence rate. Between 2011 and 2020, 3,508 new-onset cases of brucellosis in humans were registered. Complications of an epidemiological situation with this infection are due to epizootic problems among large and small cattle. According to the data of the Russian Ministry of Agriculture, during the period from 2010 to 2020, there were 4,283 contamination zones in the Russian Federation with 95,979 sick large cattle and 398 contamination zones with brucellosis of small cattle where 15,880 sick sheep and goats were found. The analysis of disease in farm livestock indicates the persistence of the long-term upward trend of epizootic problems for large cattle brucellosis in Russia1.

The main causes of epizootic problems persistence for brucellosis in Russia include non-compliance with veterinary requirements for the livestock acquisition, sale, and housing including violations of veterinary rules for examining cattle for brucellosis and vaccinating against brucellosis, the unauthorized movement of sick cattle within the administrative territory of the country, lack of proper control by municipalities over the registration of livestock (especially in the private sector), untimely delivery of sick animals for slaughter, the presence of undetected epizootic foci and Brucella carriers.

Associated with long-term epizootic problems, the incidence of brucellosis in humans has stabilized in recent years. The largest number of cases is registered in the administrative regions like the North Caucasian Federal District, Southern Federal District, and Siberian Federal District which has the highest incidence of large cattle (over 80% of the total sick animals in the Russian Federation) and small cattle (over 90%). Individual owners of animals, persons professionally connected with animal husbandry, processing of products and raw materials from animals remain at high risk for brucellosis. The main source of the pathogen is brucellosis in large cattle and small cattle; the leading conjunctive routes are contact and dietary. Human infection is a result of close contact with sick animals (livestock rearing, veterinary care, slaughtering and dressing such animals, and eating meat and dairy products contaminated with Brucella without sufficient heating).

The main pathogenic species of the genus Brucella that may induce serious epizootic and epidemic complications are B. melitensis, B. abortus, and B. suis. Worldwide, B. melitensis is considered the species that causes the most severe and acute forms of infection. This species occurs in 80 to 90% of humans brucellosis cases.

The purpose of this review is to analyze the literature on the history of use, specific prevention of brucellosis, and the problem of creating new safe and effective vaccines against brucellosis to immunize humans and animals.

Over more than a century-long history of controlling brucellosis epizootics, the world has developed two of the most effective strategies for reducing and eradicating infection, such as forming specific immunity in susceptible herds using vaccines and eliminating the focus of infection, which is identified during routine and diagnostic studies for epizootological indications. The culling of brucellosis seropositive animals was (and still is) used in many developed European and American countries. This method of disease control is effective for high livestock levels only2. In regions with a poor zootechnical inventory of public and private livestock, especially when using free-range animal husbandry, with the intensive movement of animals between herds, an effective system for identifying infected livestock is almost impossible. Veterinary services are often faced with a situation when 2 to 3 days after blood sampling for laboratory examination, it is not possible to find seropositive animals diagnosed during the survey. Under such conditions, vaccination becomes the main means of controlling brucellosis [2].

Some authors maintain that for brucellosis-unfavorable regions, where eradication of the infection with total replacement of sick livestock with healthy cattle is impossible for socio-economic reasons, mass vaccination is the main measure to control brucellosis in animals and a key element in the system3 of infection prevention in humans [3–6].

Due to the significant economic losses and public health hazards caused by brucellosis, measures are being taken to prevent the spread of infection through animals vaccination, since the high human incidence rate is associated with the spread of infection among livestock and the consumption of contaminated dairy products and meat. In this regard, new veterinary regulations were developed, which came into force on March 1, 2021, and are valid until March 1, 2027. On September 1, 2021, new SanPiN 3.3686-21 "Sanitary and Epidemiological Requirements for the Prevention of Infectious Diseases" will come into force4.

Vaccination is recognized by the WHO as an effective method of preventing human infectious diseases. High efficiency, simplicity, and the possibility of a full-scale range of vaccinated persons to prevent disease on a mass scale have made active immunoprophylaxis in most countries of the world a state priority. Vaccination measures include the selection of persons to be vaccinated, the choice of vaccine and defining the scheme of its use, and (if necessary) the control of effectiveness, relief of possible pathological reactions and complications [7].

In Russia, a live vaccine prepared from the B. abortus 19-BA vaccine strain is used to immunize people against brucellosis. The formulation was developed in the 1950s at the Gamaleya Research Institute under the direction of Academician P.A. Vershilova5.

Scientists worldwide have been striving for many years to create effective vaccines to immunize humans against brucellosis since the existing brucellosis vaccines [2][8–10], including Russian-made ones based on the B. abortus 19-BA strain, can not be considered safe.

Nowhere in the world, except in the USSR, China, and Mongolia, people have been vaccinated against brucellosis [11][12]. The reasons most states have refused to vaccinate people with live brucellosis vaccines were the results of comparative clinical trials of harmlessness, reactogenicity, and immunological efficacy of brucellosis vaccines based on the B. abortus 19-BA and B. melitensis Rev-1 strains.

In the 1950s in the USSR, during the extensive epizootic of brucellosis, the human vaccine prophylaxis (vaccination since 1952, revaccination since 1956) played a significant role in the decrease of incidence. By 1964, the number of new cases decreased by six times. Afterwards, despite the increase in vaccinations, human morbidity did not decrease; therefore, the vaccination rate among the population in some regions was limited [11]. A great contribution to the study of brucellosis specific prevention was made by the staff of the Rostov-on-Don Anti-Plague Institute (G.A. Balandin, I.I. Polyakov, N.P. Prostetova, M.S. Drozhevkina, V.S. Uralyova, and others). Based on the brucellosis department of the Institute, the production of "Brucellin" was organized, which is widely used in practice in Bürne's allergic test. It has been shown that great harm is done by revaccination without prior serological examination. Reducing immunoprophylaxis, especially repeated revaccination, was dictated by the need to reduce the side effects of the vaccine. The results of these studies subsequently led to significant changes in the instructions for preventing brucellosis in humans using the B. abortus 19-BA vaccine strain [13].

In the 1990s, economic difficulties in Russia led to a 6-7-fold decrease in brucellosis immunoprophylaxis in humans. On average, 1,100-1,300 people were immunized against brucellosis per year. From 2003 to 2005, risk groups were vaccinated only in a few subjects of Russia (the Republics of Kalmykia, Altai, Buryatia; Rostov, Orenburg, and Irkutsk regions). In other brucellosis-unfavorable subjects, such as the Stavropol Territory, the Republic of Khakassia, and the Karachay-Cherkessia Republic, where a high percentage of "professional" brucellosis is observed, no immunization was performed6.

At present, vaccination against brucellosis in the Russian Federation is included in the vaccination schedule for epidemic indications and is carried out under the current regulatory documents in the case of a threat of small cattle infection with brucellosis in foci of goat-sheep type7. Specialists of territorial establishments of Rospotrebnadzor decide on specific prophylaxis among people within a focus of brucellosis of small cattle inclusive of the veterinary service data.

According to SP3.1.7.2613-10 "Brucellosis Prophylaxis" in humans, specific prophylaxis of this disease is carried out in cases of increased risk of infection with the most pathogenic for humans B. melitensis strain. Scheduled vaccination is carried out for persons over 18 years of age from occupational risk groups and persons performing work on procurement, storage, and processing of raw materials and livestock products obtained from farms where brucellosis disease is registered, on the slaughter of cattle with brucellosis, procurement and processing of meat and meat products obtained from them, and breeders, veterinary workers, zootechnicians in farms enzootic for brucellosis; employees of bacteriological laboratories working with live cultures8. Vaccination of permanent and temporary workers engaged in animal husbandry is carried out according to epidemic indications until no cases of ovine and caprine type brucellosis among animals (both small and large cattle) are registered in farms, and the personnel of enterprises processing raw materials and livestock products until no brucellosis are registered in farms where livestock, raw materials, and livestock products come from. In areas free of ovine and caprine type brucellosis, no immunization of personnel of farms unfavorable for brucellosis caused by B. abortus, B. suis, and B. canis was performed.

The tactics for specific prophylaxis of brucellosis in humans remained the same. However, in the last 30 years, the proportion of persons not professionally connected with animal husbandry has increased (70% of livestock owners, 30% of other contingents) [14], and their scheduled vaccination according to epidemiological indications is not provided for in the current documentation6.

The national literature is critical of the vaccine used for human immunization in Russia. There is an opinion on the low immunological and epidemiological effects of the vaccine preparation and the possible side effects and complications due to its use9 [11][15]. There has been described vaccine pathergy in people inoculated with live brucellosis vaccine in violation of the instructions for the drug administration [11][16]. It was noted that due to the low immunogenicity of the vaccine prepared from the B. abortus 19-BA vaccine strain and insufficient duration of post-vaccination immunity (up to 6 months), it was important to consider the timing of mass livestock works when planning immunization of the risk contingent6.

Vaccines based on the B. abortus 19, B. abortus 82, and B. abortus 75/79-AV strains (Shchelkovsky Biokombinat, Russia) and a vaccine against brucellosis of sheep and goats and infectious ram epididymitis from the B. melitensis Rev-1 strain (Agrovet LLC, Russia) are currently the most commonly used vaccines for animal immunization against brucellosis in the Russian Federation.

Live attenuated vaccines for immunization of animals are also widely used abroad [17–19]. S-strain vaccines based on B. abortus S 19, B. melitensis Rev-1, and R-forms of B. abortus RB-51 are mainly used for this purpose [20–22].

For a long time, bovine brucellosis vaccines from the B. abortus RB-51 nonagglutinogenic strain manufactured in the USA were used abroad, including in the Republic of Kazakhstan [23], for bovine brucellosis recovery. This strain was obtained from the B. abortus 2803 pathogenic strain by a series of passages on media with rifampicin [24]. More recently, data have appeared in the literature indicating the hazard of this vaccine. For example, between 2017 and 2018, human cases of brucellosis, directly related to the consumption of unpasteurized milk from cows immunized with a live vaccine based on the B. abortus RB-51 strain, were detected in the United States in Texas, New Jersey, and Pennsylvania [25].

Over the past decades, research by many scientists has focused on creating vaccines based on purified surface or intracellular proteins (peptides) of Brucella spp. [26][27]. Brucella spp. immunodominant proteins including outer membrane proteins (Omp16, Omp19, Omp25, Omp28, Omp31), ribosomal protein L7/L12 are considered as the basis for creating split vaccines [27–30]. An analysis of the worldwide development of new vaccines against brucellosis showed that the most effective preparations mainly used immunogenic membrane peptides of Omp31 and Omp22 Brucella, which also play a role in virulence, and the regulatory iron-containing protein Frp B [31].

Foreign researchers are developing vaccines for human and animal immunization based on vector (genetically engineered) preparations using Lactococcus lactis and influenza virus. The efficacy of recombinant L. lactis and B. abortus secreting superoxide dismutase was studied when administered alone or in combination with L. lactis producing IL-12. Oral vaccination of mice in different variations revealed the presence of specific antibodies to protect the animal from infection with the virulent B. abortus 2308 strain [32][33]. Scientists are working on candidate vaccines consisting of influenza A subtype H5N1 and H1N1 viruses expressing the ribosomal protein of Brucella L7/L12 and Omp16. This vaccine is recommended for cattle vaccination. In addition, research is underway to create an effective vector drug for human vaccination. A recombinant vector of influenza virus (rIVV) subtype H5N1 expressing amino acids of outer membrane proteins of Brucella Omp 16 and 19, ribosomal L7/L12, and proteins of Cu/Zn-superoxide dismutase was used. Studying 18 combinations of mono-, bi- and quadrivalent vaccines, the most effective combination was determined [34–36].

Chinese scientists propose a live vaccine based on the mutant B. melitensis M5-90 man B (M5-90ΔmanB) strain. Drug tests showed that the strain induced in the experiment the pronounced and sustained immune response, did not cause the production of agglutinins, and protected animals from brucellosis when infected with the B. melitensis 16 M highly virulent strain [37].

Other researchers have proposed a live attenuated vaccine based on the B. abortus 2308 (2308ΔgntR) mutant strain, which induces a high degree of protection in animals [38].

Russian researchers have experimented on the design and use of an inactivated split-conjugate vaccine for vaccinating small cattle [39]. These studies established the high immunogenicity of this vaccine for goats which will allows its effective use in brucellosis control in this species of animal. To enhance immunogenicity for sheep, the authors recommend the use of immunoprotectors capable of enhancing the humoral immune response of these animals to the injected antigen of the tested vaccine.

Research is also continuing to improve immunization schemes for animals with previously developed vaccines. The possibility of controlling brucellosis in acute foci using the B. abortus 82 vaccine strain has been worked out [3]. A scheme for large cattle vaccination and revaccination using the B. abortus 19 and 82 strains has been proposed [40]. The effectiveness of the two-stage method of cattle immunization against brucellosis in the sanitation of individual farms has been proven. The use of the brucellosis R-antigen in the complement-binding reaction makes it easy to separate sick animals from vaccinated animals [41]. The usefulness of the "Nitox-200" antibacterial drug before immunization in brucellosis-unfavorable herds of large cattle was shown, in response to which the recovery of animals occurred in four months already [42]. A concept was developed to optimize the use of the B. abortus 75/79-AB and 82 weakly agglutinogenic strains for immunization of European reindeer [43]. The efficiency of specific prevention of reindeer brucellosis was increased by determining the optimal doses of a vaccine from the B. abortus 19 and 82 strains [44]. In addition, a scheme has been developed for vaccination and recovery from brucellosis of an endangered herd of camels using the B. abortus 75/79-AB vaccine strain [45]. Researchers have compiled an algorithm of specific measures to ensure optimal protection against brucellosis, which includes the use of inactivated immunogen, highly agglutinogenic in young large cattle and low agglutinogenic in adult animals; specific anti-brucellosis immunization is performed not earlier than three months after immunosignificant events (antiparasitic treatment, immunization against other infections, etc.) [46].

In addition, Russian and foreign authors have shown that the vaccine efficacy is affected by these ways of administration; intraperitoneal and subcutaneous vaccination is much more effective for protection against aerosol brucellosis infection than intranasal vaccination [18]. Other researchers have established the effectiveness of the conjunctival method of vaccination; the presence of a developed network of lymphatic vessels and receptors in the conjunctival mucosa mobilizes and activates the functions of the mononuclear phagocytic system, the antigen penetrates the nasal cavity through the nasal lacrimal duct (increased absorption area) [42][47][48].

Improving the specific prevention of brucellosis is one of the most important tasks for both public health and veterinary medicine, given the fact that brucellosis is an infection common to humans and farm livestock. Brucellosis infection causes serious harm to the health of citizens professionally engaged in obtaining and processing livestock, as well as having livestock in subsidiary farms; the incidence of brucellosis in farm livestock leads to significant economic losses in animal husbandry.

Despite a half-century history of brucellosis vaccine development and the availability of promising research in recent decades, no commercially available drugs that are completely safe for human and animal immunization and that have long-term efficacy have been created to date. The existing system is imperfect; vaccines have residual virulence and require modifications, which determines the prospects for further developments in that direction [49].

At the same time, vaccination, which is the main factor of protection against brucellosis infection in animals, cannot provide complete control of the infection. One of the main tasks of post-vaccination monitoring of brucellosis and ensuring epizootic and epidemiological well-being of this infection remains the development of means and methods of differentiating the vaccinated from the infected. In this regard, the continuation of Russian scientists' work and experiments to develop systems of preventive and recreation activities against brucellosis in large and small cattle is required to improve the post-vaccination diagnosis of brucellosis [50–54].

1. https://www.snipchi.ru/updoc/2021/EPID_OBZOR_BRUZ_2020_2021.pdf accessed on May 5, 2021. 2. https://www.who.int/ru/news-room/fact-sheets/detail/brucellosis accessed on May 13, 2021. 3. Order of the Ministry of Agriculture of Russia of September 8, 2020 No. 533 "On approval of the Veterinary Rules for preventive, diagnostic, restrictive and other measures, the establishment and cancellation of quarantine and other restrictions aimed at preventing the spread and elimination of foci of brucellosis (including infectious ram epididymitis)" (Registered with the Ministry of Justice of Russia September 15, 2020 No. 59869). 4 Resolution of the Chief State Sanitary Doctor of the Russian Federation dated January 28, 2021 No. 4 "On the Approval of Sanitary Regulations and Standards SanPin 3.3686-21"Sanitary and Epidemiological Requirements for the Prevention of Infectious Diseases." 5. Vershilova P.A. Feder M.L., Polyakova A.M. Vaccinating people against brucellosis with a live vaccine// Voprosy infectsionnoy patologii i immunologii: Pr. AMNUSSR. – Vol. 2. – Moscow., 1954. – P. 231. 6. Zheludkov M.M. Brucellosis in Russia: Current Epidemiology and Laboratory Diagnostics: Author's abstract ... Dr of Medical Sciences / Zheludkov Mikhail Mikhailovich. – Moscow, 2009. – 52 p. 7. Order of the Ministry of Health of the Russian Federation of March 21, 2014, No. 125n "On Approval of the National Calendar of Prophylactic Immunizations and the Calendar of Prophylactic Immunizations for Epidemic Indications" (as amended on February 3, 2021). 8.Resolution of the Chief State Sanitary Doctor of the Russian Federation dated April 26, 2010 No. 39 "On Approval of SP 3.1.7.2613-10" (together with "SP 3.1.7.2613-10. Prevention of brucellosis. Sanitary and Epidemiological Rules"). 9. Tsirelson L.E. Clinical and immunological features of brucellosis associated with specific vaccination: Author's abstract ... Dr of Medical Sciences / Tsirelson Lyudmila Yekimovna. – Alma-Ata, 1992. – 38 p.