Pathogenic brucellae(causative agents of brucellosis): An overview

▶The History of Discovery

In 1886 on the Island of Malta the English bacteriologist D. Bruce demonstrated the presence of the causative agent of Malta fever in the spleen of a dead patient. Later in 1887 he isolated these bacteria in pure culture.

In 1896 the Danish scientist B. Bang established the etiology of contagious abortion of cattle. In 1914 the American investigator G. Traum isolated from pigs the bacteria responsible for contagious abortion of these animals. Other brucellae species were discovered in 1953, 1957, and 1966.

▶Classification of Brucellae

Brucellae belong to the family Brucellaceae, genus Brucella. For a long time they were classified into numerous species, depending on primarily affected host. Among them are brucellae of goats and sheep – Brucella melitensis; brucellae of cattle – Brucella abortus; brucellae of pigs – Brucella suis; brucellae of forest rats – Brucella neotomae; the causative agents of abortion in sheep – Brucella ovis; and B. canis of dogs.

However, DNA-DNA hybridization studies elaborated in 1980s suggested that Brucella is a monospecific genus. These data were confirmed in further studies, and the latest edition of Bergey’s Manual of Determinative Bacteriology consolidated all brucellae of medical importance within the only species Brucella melitensis.

Nevertheless, isolates from human infection are still classified into groups using the former species names. This correlates with the animal species the strains of brucelllae are predominantly isolated from (cattle, goats, pigs, and dogs). These groups are differentiated on the basis of their special phenotypical traits.

Human pathology is predominantly associated with B. melitensis, B. abortus, and B. suis.

▶Structure and Properties of Brucellae

  • Morphology

Isolates of Brucellae form small gram-negative ovoid-shaped coccobacteria. They have no spores or capsules (in some strains of Brucella melitensis the capsule is present).

Brucellae have unusual genome structure composed of two non-identical circular closed chromosomes without plasmids.

  • Cultivation

When recovered from patients, brucellae propagate slowly, being cultured for 1-2 weeks. In laboratory subcultures the growth may appear in 1-2 days. The optimal temperature for culture is 37°C, pH 6.8-7.2.

Brucellae are cultured on special media, e.g. liver-extract agar and liver-extract broth. They produce small, convex, smooth colonies with a white or pearly hue. Brucella abortus prefers to grow in atmosphere of 5-10% of carbon dioxide. Selective media containing certain dyes and antibiotics are used for isolating of bacteria. Blood agar culture renders nonhemolytic glistening colonies.

All brucella actively propagate in the yolk sac of chicken embryos.

  • Biochemical properties

Brucellae are aerobic bacteria. They produce catalase and oxidase.

The bacteria display weak carbohydrate fermentation (sometimes metabolize glucose).

Some strains hydrolyze urea and asparagin, reduce nitrates to nitrites, and metabolize proteins, peptones and amino acids with release of ammonia and hydrogen sulfide. The bacteria don’t liquefy gelatin.

Also they produce enzymes lipase, phosphatase, and hyaluronidase.

  • Antigenic structure

Brucellae are defined to contain two lipopolysaccharide antigens, A and M. Brucella melitensis carries predominantly M fraction, whereas Brucella abortus group – A fraction. Superficial L antigen has been demonstrated; it resembles the capsular Vi antigen.

  • Virulence factors

Despite evident virulence of brucellae strains, their genome doesn’t harbor genetic pathogenicity islands. So the virulence factors of brucellae remain not well-determined.

Brucellae don’t produce soluble toxins.

An endotoxin is released as a result of disintegration of the bacterial cell, but it poorly activates the innate immune response. It can’t be excluded that it maintains microbial survival inside phagocytes.

In addition, low-molecular weight components of microbial body block phagosome-lysosome fusion.

Microbial capsule protects brucellae from phagocytosis.

Also the bacteria express invasive enzymes, e.g. hyaluronidase and lipase.

  • Resistance

Brucellae are characterized by marked resistance and viability. They survive for a long time at low temperatures (up to 4 months in ice). Bacteria live for about 4 months in urine and animal feces, from 3 to 4 months in sheep’s wool and sheep’s cheese, for 1 month in dust, about 20 days in meat, and for 7 days in milk.

Nevertheless, brucellae are sensitive to high temperatures and disinfectants. At 60°C they are destroyed in 30 min, at 80-95° in 5 min, boiling kills them almost instantly.

They are easily inactivated by all conventional disinfectants.

▶Pathogenesis and Clinical Findings in Brucellosis

Brucellosis is a zoonotic infection contracted by humans via direct or indirect contact with animals (sources of infection), which were infected (usually chronically) with Brucella.

The disease usually affects veterinary and zootechnical personnel, herdsmen, livestock handlers, etc.

Infection can be established via cutaneous (contact with infected tissues of animals), respiratory, or alimentary routes. Cheese made from unpasteurized goats’ milk is a particularly common vehicle.

Symptoms of brucellosis are generally non-specific, and the onset of illness may be acute or insidious. As a result of the systemic nature of brucellosis, almost any organ of the body might be infected.

The incubation period lasts about 1-6 weeks.

The pathogens spread from the portal of entry via lymphatic vessels and regional lymph nodes to the bloodstream and then to parenchymatous organs. Due to the marked resistance to phagocytosis the bacteria disseminate throughout the body. They stay long within phagosomes without inactivation. The protracted clinical course of brucellosis maintained by viable bacteria is generally regarded as chronic systemic infection (sepsis).

Granulomatous nodules and abscesses emerge in lymphatic tissue, liver, spleen, and bone marrow. The lesions contain viable brucellae, located within the infected cells. The invaded pathogen stimulates the reactions of delayed (cell-mediated) hypersensitivity. Overproduction of cytotoxic molecules enhances tissue damage.

Specific granulomas consist of mononuclear cells, epithelioid histiocytes and giant cells; active tissue inflammation results in focal necrosis with subsequent gradual fibrosis.

Clinical manifestations of brucellosis are highly variable – from faint febrile ilness (“fever of unknown origin”) to evident respiratory infection and joint involvement. Various complications (e.g., osteomyelitis or meningitis) occasionally occur.

Usually В. abortus causes mild disease without suppurative complications; В. suis infection tends to be chronic with suppurative lesions. В melitensis infection is more acute and severe.

A long-lasting immunity both cellular and humoral is acquired following brucellosis, and the patient usually becomes resistant to recurrent infection. Cell-mediated reactions (T-lymphocyte activation and phagocytosis) play the major role in pathogen elimination.

▶Laboratory Diagnosis of Brucellosis

The specimens taken from patient’s blood and urine (for isolation of the pathogen), serum (for detection of antibodies), milk and dairy products (for detection of brucellae) are examined. Any suspected Brucella isolates determined in the clinical laboratory should be handled in a biological safety cabinet.

PCR with primers specific to various brucella species is used for detection of pathogens directly in clinical specimens.

Blood or tissues samples are inoculated for culture into liver-extract or ascitic-fluid broth, or trypticase-soy broth. At intervals of several days, subcultures are made on solid media of similar composition. All cultures are incubated in 10% CO2 and should be observed and subcultured for at least 3 weeks prior to negative conclusion.

The brucellae are aerobic small gram-negative coccobacilli, which are nonhemolytic and oxidase-positive; they do not ferment lactose or glucose.

Most of strains are urease-positive. Bacteria matching the criteria are further tested for agglutination with specific anti-brucella serum.

Suspected Brucella isolates should be sent to a reference laboratory for final identification.

Brucella cultures may be isolated by the biological method. To aim this, guinea pigs or mice are injected with test materials. A month later the pure culture is isolated.

Serological tests are the most practically relevant in laboratory diagnosis of brucellosis. They are performed from the 2nd week after the disease onset.

Huddleson reaction or tentative slide agglutination test is usually applied for primary examination of specific antibodies in brucellosis.

Extended tube agglutionation test or Wright reaction validates the primary positive result of afflutination and confirms the diagnosis of brucellosis. Wright’s reaction is valued positive in a titer of specific antibodies 1:200 and more.

Allergic skin test (or Burne brucellin test) is used to determine delayed hypersensitivity in brucellosis. Burne test is conducted with specific infectious allergen brucellin derived from brucella cells. The analysis becomes positive from the 2nd-3rd week of the disease.

▶Treatment and Prophylaxis of Brucellosis

Because of their intracellular location, brucellae are not readily eradicated by antimicrobial agents. For the best results, the treatment must be prolonged. Administration of aminoglycosides (e.g., gentamycin), fluoroquinolones, and tetracyclines accelerates the recovery.

Chronic cases can be additionally treated with killed vaccine that activates antimicrobial immunity.

For specific prophylaxis of brucellosis various live and inactivated vaccines were introduced into clinical practice. They can be administered to protect the contact persons as well as the personnel with occupational risk of brucellosis. However, vaccination is not able to confer the long lasting high-grade immunity.