Brucella

Brucellosis - a kind of infectious disease of humans and animals, caused by bacteria of the genus Brucella. About 500 primary diseases of people with brucellosis are registered annually in Russia.

The person is 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 was described under the names determined by its geographical distribution (Maltese, Neapolitan, Gibraltar, Mediterranean fever).

The causative agent was discovered in 1886 by D. Bruce, who discovered it in a preparation from the spleen of a soldier who died from Maltese fever, and called the Maltese micrococcus Micrococcus melitensis. It was found that the main carrier is goats and sheep, and infection occurs when raw milk is consumed from them. In 1897, B. Bang and B. Stribolt found the causative agent of an infectious abortion of cows - Bacterium abortus bovis, and in 1914 J. Traum discovered the causative agent of an infectious abortion of pigs - Brucella abortus suis. Conducted in 1916-1918 years. A.Evens comparative study of the properties of Brucella melitensis and Brucella abortus bovis showed that they almost do not differ from each other in many properties. In this regard, it was proposed to combine them in one group, named after Bruce - Brucella. In 1929, I. Heddleson included Brucella abortus suis in this group and proposed the Brucella genus to be divided into 3 species: Brucella melitensis (Micrococcus melitensis), Brucella abortus (Brucella abortus bovis) and Brucella suis (Brucella abortus suis).

The disease of people and animals caused by brucella, it was decided to call brucellosis. In the following, the Brucella genus was replenished with three more species: Brucella ovis was isolated from sheep suffering from epididymitis (1953), Brucella neotomae from shrubby rats (1957) and Brucella canis from beagle dogs (1966). According to Berdzhi classification (2001), brucellas belong to the Alphaproteobacteria class.

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Morphology of brucella

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

Biochemical properties of brucella

Brucella are aerobes or microaerophiles, do not grow under anaerobic conditions. The temperature optimum for growth is 36-37 ° C; pH 7.0-7.2; grow well on normal nutrient media, but better - with the addition of serum or blood. Recommended media: nutrient agar with the addition of serum (5%) and glucose; Agar, cooked on potato infusion, with the addition of 5% whey; blood agar; meat-peptone broth. A feature of Brucella abortus is its need for an elevated C02 content (5-10%) in a growth atmosphere. It is very typical for brucellae slow growth, especially in the first generations: when sowing from humans and animals, growth sometimes appears after 2-4 weeks. Brucella colonies are colorless, convex, round - S-shaped, or rough - R-form, tender and transparent at first, cloudy with age.

Colonies of Brucella canis, Brucella ovis and the 5th biotype of Brucella suis always have an R-form. The growth of brucella in broth medium is accompanied by uniform turbidity. For the growth of brucellas, thiamine, biotin, niacin are needed. Brucella ferment glucose and arabinose to form an acid without gas, do not form an indole, reduce nitrates to nitrites. The formation of hydrogen sulphide is most pronounced in Brucella suis.

A total of 10-14 antigenic fractions were detected in brucella by immunoelectrophoresis of extracts prepared from ultrasound-destroyed cells. Brucella have a common rhodospecific 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 found in other species (biovars) of brucella, but in different ratios, which must be taken into account when identifying them. Antigens common to Francisella tularensis, Bordetella bronchiseptica and Y. Enterocolitica (serotype 09) were found. Due to the fact that some of the brucella symptoms vary, the Brucella melitensis species are divided into 3 biovars, the Brucella abortus species by 9 and B. Suis by 5 biovars. For their differentiation into species and biotypes and identification, a complex of features is used that includes, in addition to morphological and tinctorial properties, also the need for C02 for growth, the ability to grow on media in the presence of certain dyes (basic fuchsin, thionine, safranin), to isolate H2S, to form urease, phosphatase, catalase (the activity of these enzymes is most pronounced in Brucella suis, they do not grow on medium with safranin), sensitivity to Tbilisian bacteriophage, agglutination with monospecific sera. If necessary, use additional metabolic tests: 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 the pigs, but the northern deer, and, taking into account its other characteristics, it is advisable to isolate Brucella rangiferis as an independent species.

By the fifth biovar, suis includes cultures isolated from aborted cows and sheep and having a stable R-form of brucella.

The ratio of brucella to the Tbilis phage: in normal working dilution phage only B. Abortus. However, in a dose equal to ten workers, strains of Brucella suis and Brucella neotomae, although weakly, are phaged.

[6], [7], [8], [9], [10], [11], [12]

Resistance of brucella

Brucella have relatively high stability in the external environment. They persist in moist soil and water up to 2-3, and at a temperature of 11-13 "C - up to 4.5 months, in non-renewable reservoirs - up to 3 months, in milk - up to 273 days, in oil - up to 142 days; in cheese - up to 1 year, in cheese - up to 72 days, in sour milk - up to 30 days, in yogurt - up to 11 days.However, they are very sensitive to high temperatures - at 70 ° C die in 10 minutes, and when boiled - for a few seconds.The pasteurization of milk at 80-90 ° C causes their death in 5 minutes Brucella are also sensitive to various chemical disinfectants.

Factors of pathogenicity of brucellae

Brucella do not form exotoxin. Their pathogenicity is due to endotoxin and the ability to suppress phagocytosis, to prevent "oxidative explosion". Specific factors that inhibit phagocytosis have not been adequately studied. The pathogenicity of brucellas is also associated with hyaluronidase and other enzymes. Essentially important is that brucella have the strongest allergenic property, which largely determines the pathogenesis and clinic of brucellosis.

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Postinfectious immunity

Postinfectious immunity is long lasting, but repeated diseases are possible. Immunity is cross-linked (against all Brucella species) and is caused by T-lymphocytes and macrophages. In immune individuals and animals phagocytosis is complete. The role of antibodies in immunity is to stimulate phagocytic activity. A positive allergic reaction testifies not only to the sensibilization of the body, but also about the presence of immunity. Breakthrough immunity can occur when infected with large doses of the pathogen or with its high virulence.

Epidemiology of brucellosis

The main carriers of brucella are sheep, goats (Brucella melitensis), cattle (Brucella abortus), pigs (Brucella suis) and reindeer (Brucella rangiferis). However, they can pass to many other species of animals (yaks, camels, buffaloes, wolves, foxes, rodents, llamas, saigas, bison, horses, rabbits, hedgehogs, chickens, etc.). Especially great epidemiological danger is the transition of Brucella melitensis to cattle, 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 the disease of people in more than 95-97% of all cases of brucellosis. Brucella abortus, as a rule, causes a latent form of the disease, and only in 1-3% clinical manifestations are noted. Even more rarely, the disease causes Brucella suis (less than 1%). The pathogenicity of brucella varies depending not only on the species, but also on the biowar. In particular, the biovars 3, 6, 7, 9 of Brucella abortus are not inferior to Brucella melitensis by virulence. American variants of Brucella suis are also highly virulent, so in different countries the etiological role of individual brucella species manifests itself in different ways. For example, in Mexico, Brucella melitensis plays a major role in the epidemiology of brucellosis, Brucella suis in the United States, and Brucella abortus in Canada and some European countries. It is not excluded that the biovar 3, 6, 7, 9 Brucella abortus arose as a result of migration to the cattle Brucella melitensis and its transformation.

In animals, brucellosis proceeds in the form of a common disease, the pattern of which varies. For large and small cattle, the most characteristic manifestations of the disease are infectious abortions, especially if they are of a massive nature. In pigs, abortions are less common, the disease proceeds as chronic sepsis with lesions of joints, testicles and other organs. From diseased animals, the pathogen is excreted with milk, urine, feces, pus and especially abundantly - during a miscarriage with the fetus, amniotic membranes and outflows from the birth canal that represent the most contagious material. The abundant reproduction of brucella in the fetal membranes is associated with the presence of a polyhydric alcohol in them, erythritol, which serves as an important growth factor for all Brunella species except Brucella ovis.

Man gets infected from animals (from the sick person very rarely) mainly by contact or by contact-household way (80-90% of all diseases). Alimentary mode of infection is observed mainly when using unpasteurized milk from sick animals or dairy products cooked from it, as well as water. Any person permanently or temporarily dealing with animals or animal raw materials by virtue of their profession (shepherds, cattlemen, milkmaids, veterinary workers and others) can become infected by contact or contact-household methods. Brucella penetrate into the human body through the skin or, much more often, through the mucous membranes of the mouth, nose, eyes (covered with dirty hands).

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Symptoms of brucellosis

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

On the lymphatic pathways the pathogen enters the lymph nodes; multiplying, forms the "primary brucellosis complex," the localization of which depends on the site of the entrance gates, but most often it is the lymphatic apparatus of the oral cavity, the pharyngeal, cervical, submaxillary glands and the lymphatic apparatus of the intestine. From the lymph nodes the pathogen penetrates into the blood and spreads throughout the body, selectively affecting the tissues of the lympho- hemopoietic system. Bacteremia and generalization of the process lead to a strong allergic organism. Brucellosis proceeds like chroniosepsis. This is due to the incomplete nature of phagocytosis. Being and reproducing inside cells, including phagocytes, brucellae are not available either for antibodies or for chemotherapy drugs. In addition, they can be converted inside the cells into L-forms and in this form persistently persist in the body, and returning to the original form, cause a relapse of the disease.

Symptoms of brucellosis are very diverse and complex. They depend primarily on allergization and intoxication of the body and on what organs and tissues are involved in the infectious process. Most often suffer from lymphatic, vascular, hepatolien, nervous, and especially musculoskeletal systems. Brucellosis is characterized by a prolonged course (sometimes up to 10 months), in severe cases it can lead to long-term disability and temporary disability, but nevertheless the disease usually ends in complete recovery.

Laboratory diagnostics of brucellosis

Diagnosis of brucellosis is carried out with the help of biological test, bacteriological method, serological reactions, allergic Burne 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 - bowel movements, periarticular fluid. Since the main location of the pathogen in the body are the cells of the hemo- or lymphopoietic system, preference should be given to the release of hemo- or myeloculture. In a bacteriological study, it is necessary to provide conditions for the growth of Brucella abortus (requirement for C02). The identification of the isolated Brucella cultures is carried out on the basis of the data indicated in Table. 30 signs. The biological sample (infection of guinea pigs) is used when the material is heavily polluted by extraneous microflora and it is difficult to obtain a pure culture of the pathogen directly from it. Serological reactions can be used either to detect the 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: RPHA (especially using erythrocyte diagnosticums with monoclonal antibodies to the rhodospecific antigen of brucella); aggregate-hemagglutination reaction (RAGA); erythrocytes carry antibodies to brucellosis antigens; reaction of coagglutination, precipitation and IFM. To detect antibodies in the patient's serum, the following are used: Wright agglutination reaction, Coombs reaction (for the detection of incomplete antibodies), indirect immunosupplification reaction, RPGA, IFM, RSK, OBS, and accelerated reactions on glass: Heddleson, rosebengal, latex- agglutination, indirect hemolysis reaction (erythrocytes sensitized with LPS brucella, in the presence of antibodies and complement are lysed).

How to prevent brucellosis?

The vaccine against brucellosis is a specific prophylaxis of brucellosis. Vaccination is carried out using a live vaccine prepared from B. Abortus strain (live brucellosis vaccine - WBC), only in foci of goat-sheep brucellosis. The vaccine is applied ocularly, once. Revaccination is carried out only to persons in whom the Burne test and serological reactions are negative. Since WBC has a strong allergenic effect, a chemical brucellosis vaccine (HBV), prepared from antigens of the brucella cell wall, has been proposed instead. It has a high immunogenicity, but is less allergenic. A suspension of killed brucella (killed curative vaccine) or HBV can be used to treat chronic brucellosis (stimulate the formation of post-infection immunity).