Brucellae

Brucellosis is a unique infectious disease of humans and animals caused by bacteria of the genus Brucella. In Russia, about 500 primary cases of brucellosis in humans are registered annually.

People become infected with brucellosis mainly from domestic animals (sheep, goats, cows, pigs, reindeer - the main reservoir of the pathogen in nature). Brucellosis has existed for a long time and has been described under names determined by its geographical distribution (Maltese, Neapolitan, Gibraltar, Mediterranean fever).

The pathogen was discovered in 1886 by D. Bruce, who found it in a preparation from the spleen of a soldier who died of Malta fever, and called it the Maltese micrococcus - Micrococcus melitensis. It was established that its main carriers are goats and sheep, and infection occurs when consuming raw milk from them. In 1897, B. Bang and B. Stribolt discovered the pathogen of infectious abortion in cows - Bacterium abortus bovis, and in 1914, J. Traum discovered the pathogen of infectious abortion in pigs - Brucella abortus suis. A comparative study of the properties of Brucella melitensis and Brucella abortus bovis conducted in 1916-1918 by A. Ivens showed that they are almost indistinguishable from each other in many properties. In this regard, it was proposed to combine them into one group, named in honor of Bruce - Brucella. In 1929, I. Heddleson included Brucella abortus suis in this group and proposed dividing the genus Brucella into 3 species: Brucella melitensis (Micrococcus melitensis), Brucella abortus (Brucella abortus bovis) and Brucella suis (Brucella abortus suis).

The disease of humans and animals caused by brucellae was decided to be called brucellosis. Subsequently, the genus Brucella was supplemented with three more species: Brucella ovis was isolated from rams suffering from epididymitis (1953), Brucella neotomae - from bush rats (1957) and Brucella canis - from hound dogs (1966). According to the classification of Bergey (2001), brucellae belong to the class Alphaproteobacteria.

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Morphology of Brucella

Brucella have similar morphological, tinctorial and cultural properties. They are gram-negative small coccoid cells with a diameter of 0.5-0.7 μm and a length of 0.6-1.5 μm, located randomly, sometimes in pairs, do not have flagella, do not form spores and capsules. The G + C content in DNA is 56-58 mol %.

Biochemical properties of Brucella

Brucella are aerobes or microaerophiles, do not grow under anaerobic conditions. The optimum temperature for growth is 36-37 °C; pH 7.0-7.2; grows well on conventional nutrient media, but better with the addition of serum or blood. Recommended media: nutrient agar with the addition of serum (5%) and glucose; agar prepared on potato infusion, with the addition of 5% serum; blood agar; meat-peptone broth. A feature of Brucella abortus is its need for an increased content of CO2 (5-10%) in the growth atmosphere. Slow growth is very characteristic of brucellas, especially in the first generations: when sown from humans and animals, growth sometimes appears after 2-4 weeks. Brucella colonies are colorless, convex, round - S-shaped, or rough - R-shaped, delicate and transparent at first, becoming cloudy with age.

Colonies of Brucella canis, Brucella ovis and the 5th biotype Brucella suis always have an R-form. Brucella growth in broth media is accompanied by uniform turbidity. Thiamine, biotin, and niacin are necessary for the growth of Brucella. Brucella ferment glucose and arabinose with the formation of acid without gas, do not form indole, and reduce nitrates to nitrites. The formation of hydrogen sulfide is most pronounced in Brucella suis.

In total, 10-14 antigen fractions were detected in Brucella using immunoelectrophoresis of extracts prepared from cells destroyed by ultrasound. Brucella have a common genus-specific antigen, various other somatic antigens, including species-specific M (predominant in Brucella melitensis), A (predominant in Brucella abortus) and R (in rough forms). Antigens M and A are also detected in other species (biovars) of Brucella, but in different proportions, which must be taken into account when identifying them. Antigens common to Francisella tularensis, Bordetella bronchiseptica and Y. enterocolitica (serotype 09) were detected. Due to the fact that some features of brucellae vary, the species Brucella melitensis is divided into 3 biovars, the species Brucella abortus - into 9 and B. suis - into 5 biovars. For their differentiation into species and biotypes and identification, a complex of features is used, which includes, in addition to morphological and tinctorial properties, also the need for CO2 for growth, the ability to grow on media in the presence of certain dyes (basic fuchsin, thionine, safranin), to secrete H2S, to form urease, phosphatase, catalase (the activity of these enzymes is most strongly expressed in Brucella suis, they do not grow on a medium with safranin), sensitivity to the Tbilisi bacteriophage, agglutination with monospecific serums. If necessary, additional metabolic tests are used: the ability to oxidize certain amino acids (alanine, asparagine, glutamic acid, ornithine, citrulline, arginine, lysine) and carbohydrates (arabinose, galactose, ribose, D-glucose, D-erythritol, D-xylose).

The fourth biovar is Brucella suis, since its main carrier is not pigs, but reindeer, and taking into account its other features, it is advisable to distinguish it as an independent species, Brucella rangiferis.

The fifth biovar B. suis includes cultures isolated from aborted cows and sheep and containing a stable R-form of Brucella.

Brucella to Tbilisi phage: in the usual working dilution the phage lyses only B. abortus. However, in a dose equal to ten working dilutions the phage lyses, although weakly, strains of Brucella suis and Brucella neotomae.

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Brucella resistance

Brucella are relatively resistant in the environment. They survive in moist soil and water for up to 2-3 months, and at a temperature of 11-13 °C - up to 4.5 months; in stagnant waters - up to 3 months; in milk - up to 273 days; in butter - up to 142 days; in cheese - up to 1 year; in feta cheese - up to 72 days; in sour milk - up to 30 days; in kefir - up to 11 days. However, they are very sensitive to high temperatures - at 70 °C they die after 10 minutes, and when boiled - in a few seconds. Pasteurization of milk at 80-90 °C causes their death after 5 minutes. Brucella are also sensitive to various chemical disinfectants.

Brucella pathogenicity factors

Brucella do not form an exotoxin. Their pathogenicity is due to endotoxin and the ability to suppress phagocytosis, prevent "oxidative burst". Specific factors that suppress phagocytosis have not been studied sufficiently. The pathogenicity of brucella is also associated with hyaluronidase and other enzymes. It is essential that brucella have a very strong allergenic property, which largely determines the pathogenesis and clinical picture of brucellosis.

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Post-infectious immunity

Post-infectious immunity is long-lasting and strong, but repeated diseases are possible. Immunity is cross-linked (against all types of Brucella) and is caused by T-lymphocytes and macrophages. In immune people and animals, phagocytosis is complete. The role of antibodies in immunity is to stimulate phagocytic activity. A positive allergic reaction indicates not only sensitization of the body, but also the presence of immunity. A breakthrough in immunity can occur with infection by large doses of the pathogen or with its high virulence.

Epidemiology of brucellosis

The main carriers of brucellosis are sheep, goats (Brucella melitensis), cattle (Brucella abortus), pigs (Brucella suis) and reindeer (Brucella rangiferis). However, they can also spread to many other animal species (yaks, camels, buffalo, wolves, foxes, rodents, llamas, saigas, bison, horses, hares, hedgehogs, chickens, etc.). The transition of Brucella melitensis to cattle is especially dangerous, since in this case it retains its high pathogenicity for humans. Of all the species, the most pathogenic for humans in our country is Brucella melitensis. It causes human disease in more than 95-97% of all cases of brucellosis. Brucella abortus, as a rule, causes a latent form of the disease, and clinical manifestations are observed only in 1-3%. Brucella suis causes the disease even less frequently (less than 1%). The pathogenicity of brucellosis varies depending not only on the species, but also on the biovar. In particular, biovars 3, 6, 7, 9 of Brucella abortus are not inferior in virulence to Brucella melitensis. American variants of Brucella suis are also highly virulent, therefore, the etiologic role of individual species of brucellosis manifests itself differently in different countries. For example, in Mexico, the main role in the epidemiology of brucellosis is played by Brucella melitensis, in the USA - Brucella suis, and in Canada and some European countries - Brucella abortus. It is possible that biovars 3, 6, 7, 9 of Brucella abortus arose as a result of the migration of Brucella melitensis to cattle and its transformation.

In animals, brucellosis occurs as a general disease, the picture of which can be different. For large and small cattle, the most typical manifestations of the disease are infectious abortions, especially if they are mass. In pigs, abortions are less common, the disease occurs as chronic sepsis with damage to the joints, testicles and other organs. From sick animals, the pathogen is excreted with milk, urine, feces, pus and especially abundantly - during the period of miscarriage with the fetus, amniotic membranes and discharge from the birth canal, which are the most infectious material. Abundant reproduction of brucellae in the membranes of the fetus is associated with the presence of a polyhydric alcohol - erythritol, which serves as an important growth factor for Brunella of all species, except Brucella ovis.

A person becomes infected from animals (very rarely from a sick person) mainly by contact or contact-household means (80-90% of all diseases). The alimentary method of infection is observed mainly when consuming unpasteurized milk from sick animals or dairy products prepared from it, as well as water. All persons who constantly or temporarily deal with animals or animal raw materials due to their profession (shepherds, cattlemen, milkmaids, veterinary workers and others) can become infected by contact or contact-household means. Brucella enters the human body through the skin or, much more often, through the mucous membranes of the mouth, nose, eyes (brought in by dirty hands).

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Symptoms of brucellosis

The incubation period of brucellosis varies from 1 week to several months.

The pathogen enters the lymph nodes via the lymphatic pathways; multiplying, it forms a "primary brucellosis complex", the localization of which depends on the site of the entry gate, but most often it is the lymphatic apparatus of the oral cavity, the retropharyngeal, cervical, submandibular glands and the lymphatic apparatus of the intestine. From the lymph nodes, the pathogen enters the blood and spreads throughout the body, selectively affecting the tissues of the lymphohematopoietic system. Bacteremia and generalization of the process lead to severe allergization of the body. Brucellosis occurs as chronic sepsis. This is due to the incomplete nature of phagocytosis. Being and multiplying inside cells, including phagocytes, brucellae are inaccessible to either antibodies or chemotherapy drugs. In addition, they can transform into L-forms inside cells and in this form persist in the body for a long time, and returning to the original form, cause a relapse of the disease.

The symptoms of brucellosis are very diverse and complex. They depend primarily on the allergy and intoxication of the body and on which organs and tissues are involved in the infectious process. Most often, the lymphatic, vascular, hepatosplenic, nervous, and especially the musculoskeletal systems are affected. Brucellosis is characterized by a long course (sometimes up to 10 months), in severe cases it can lead to long-term loss of ability to work and temporary disability, but still the disease, as a rule, ends in complete recovery.

Laboratory diagnostics of brucellosis

Brucellosis is diagnosed using a biological test, bacteriological method, serological reactions, Burnet allergic test and DNA-DNA hybridization method. The material for the study is blood, bone marrow, conjunctival secretion, urine, breast milk (in nursing mothers), less often - feces, periarticular fluid. Since the main place of residence of the pathogen in the body are cells of the hemo- or lymphopoietic systems, preference should be given to the isolation of hemo- or myeloculture. In bacteriological research, it is necessary to provide conditions for the growth of Brucella abortus (need for CO2). Identification of isolated brucella cultures is carried out on the basis of the signs specified in Table 30. A biological test (infection of guinea pigs) is resorted to in cases where the material is heavily contaminated with foreign microflora and it is difficult to obtain a pure culture of the pathogen directly from it. Serological reactions can be used either to detect antigens of the pathogen or to detect antibodies to it. To detect brucellosis antigens that can circulate in the blood either in free form or in the form of antigen + antibody complexes (CIC - circulating immune complexes), the following reactions are used: RPGA (especially using erythrocyte diagnostics with monoclonal antibodies to the genus-specific antigen of Brucella); aggregate-hemagglutination reaction (AGR); erythrocytes carry antibodies to brucellosis antigens; coagglutination, precipitation and IFM reactions. To detect antibodies in the patient's serum, the following are used: Wright agglutination reaction, Coombs reaction (to detect incomplete antibodies), indirect immunofluorescence reaction, RPGA, IFM, RSK, OFR, as well as accelerated reactions on glass: Heddleson, Rose Bengal, latex agglutination, indirect hemolysis reaction (erythrocytes sensitized with Brucella LPS are lysed in the presence of antibodies and complement).

How to prevent brucellosis?

Brucellosis vaccine is a specific prophylaxis of brucellosis. Vaccination is carried out using a live vaccine prepared from the B. abortus strain (live brucellosis vaccine - LBB), only in foci of goat and sheep brucellosis. The vaccine is applied cutaneously, once. Revaccination is carried out only for individuals with negative Burnet test and serological reactions. Since LBB has a strong allergenic effect, a chemical brucellosis vaccine (CBV) prepared from brucella cell wall antigens has been proposed instead. It has high immunogenicity, but is less allergenic. A suspension of killed brucellae (killed therapeutic vaccine) or LBB can be used to treat chronic brucellosis (stimulate the formation of post-infection immunity).