Brucella Growth

[Adam Makin]

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Minimal nutritional requirements and temperature limits of growth were studied in Brucella suis and, comparatively, in a few other Brucella species. In a saline basic medium including thiosulphate, ammonium sulphate and glucose with addition of 2 or 4 vitamins (nicotinic acid, thiamin and panthotenic acid, biotin), 24 out of 25 B. suis, 4/6 B. melitensis and 1/6 B. abortus strains were able to grow. Some strains, however, needed to be initially induced to grow by other ingredients, CO2, other vitamins, or amino acids, or by a prolonged incubation. In the saline basic medium without ammonium, glutamic acid and/or alanine and arginine, with or without glucose, supported the growth of all the B. suis and B. melitensis strains, except 2 which required a sulphur amino acid. Five out of 6 B. abortus strains did not grow in either medium without addition of one or several aromatic amino acids or, for one strain, aspartic acid, or valine. One strain could also be induced to grow in ammonium medium by other amino acids. In a rich medium with yeast extract, all Brucella species grew at 18 degrees C and 42.5 degrees (except one) while most B. suis (14/17) grew also at 15 degrees C and 44 degrees C, in contrast to other brucellae of which a few strains only grew at these temperatures. In saline ammonium glucose medium, yeast extract at 0.1 g/l provided all the required vitamins and amino acids for all brucellae and at 1 g/l, it even provided enough nitrogen to support growth without ammonium. Such basic saline medium with yeast extract may be advantageously used in routine Brucella culture, instead of the classic undefined peptone mediums. B. suis biovar 1 strains did not differ significantly in their minimal nutritional requirements, precluding the use of these requirements to differentiate the strains, in particular the Chinese vaccine strain S2 from the reference strain 1330 or from other strains from different parts of the world. Finally, B. suis which is endowed with a nearly complete synthetic potential may represent the parental Brucella species from which the melitensis and abortus species may have evolved.

Brucellosis is a severe acute febrile disease caused by bacteria of the genus Brucella. Relapses are not uncommon; focal lesions may occur in bones, joints, genitourinary tract, and other sites. Hypersensitivity reactions can follow occupational exposure. Infection may be subclinical. Chronic infections may occur.

Three species (B melitensis, B abortus, B suis)are important human pathogens; B canis is of lesser importance. Species are differentiated by production of urease and H2S, dye sensitivity, cell wall antigens and phage sensitivity. The major species are divided into multiple biovars.

Portals of entry are the mouth, conjunctivae, respiratory tract and abraded skin. Organisms spread, possibly in mononuclear pha gocytes, to reticuloendothelial sites. Small granulomas reveal a mononuclear response; hypersensitivity is a major factor.

Brucellosis is a zoonosis, acquired from handling of infected animals or consuming contaminated milk or milk products. Exposure is frequently occupational. The disease is now uncommon in the United States and Britain but common in the Mediterranean and Arabian Gulf regions, Latin America, Africa, and parts of Asia.

Diagnosis can be made clinically if there is a history of exposure. Blood cultures may be positive in early disease but serology is mainstay of diagnosis. Interpretation is complicated by subclinical infections and persistent levels of antibody.

Brucellosis is prevented by pasteurizing milk, eradicating infection from herds and flocks, and observing safety precautions (protective clothing and laboratory containment). The disease is treated with doxycycline, streptomycin and rifampin.

Bacteria of the genus Brucella cause disease primarily in domestic, feral and some wild animals and most are also pathogenic for humans. In animals, brucellae typically affect the reproductive organs, and abortion is often the only sign of the disorder. Human brucellosis is either an acute febrile disease or a persistent disease with a wide variety of symptoms. It is a true zoonosis in that virtually all human infections are acquired from animals. The disease is controlled by the routine practice of pasteurizing milk and milk products, as well as by comprehensive campaigns to eradicate the disease by destroying domestic animals which exhibit positive serologic reactions to brucellae. Vaccines providing some protection to cattle, sheep and goats are available.

The presentation of brucellosis is characteristically variable. The incubation period is often difficult to determine but is usually from 2 to 4 weeks. The onset may be insidious or abrupt. Subclinical infection is common.

In the simplest case, the onset is influenzalike with fever reaching 38 to 40C. Limb and back pains are unusually severe, however, and sweating and fatigue are marked. The leukocyte count tends to be normal or reduced, with a relative lymphocytosis. On physical examination, splenomegaly may be the only finding. If the disease is not treated, the symptoms may continue for 2 to 4 weeks. Many patients will then recover spontaneously but others may suffer a series of exacerbations. These may produce an undulant fever in which the intensity of fever and symptoms recur and recede at about 10 day intervals. Anemia is often a feature. True relapses may occur months after the initial episode, even after apparently successful treatment.

Most affected persons recover entirely within 3 to 12 months but some will develop complications marked by involvement of various organs, and a few may enter an ill-defined chronic syndrome. Complications include arthritis, often sacroiliitis, and spondylitis (in about 10 percent of cases), central nervous system effects including meningitis (in about 5%), uveitis and, occasionally, epididymoorchitis. In contrast to animals, abortion is not a feature of brucellosis in pregnant women. Hypersensitivity reactions, which may mimic the symptoms of an infection, may occur in individuals who are exposed to infective material after previous, even subclinical, infection.

Brucellae are Gram-negative coccobacilli (short rods) measuring about 0.6 to 1.5 μm by 0.5-0.7 μm. They are non-sporing and lack capsules or flagella and, therefore, are non-motile. The outer cell membrane closely resembles that of other Gram-negative bacilli with a dominant lipopolysaccharide (LPS) component and three main groups of proteins. The guanine-plus-cytosine content of the DNA is 55-58 moles/cm. No Brucella species has been found to harbor plasmids naturally although they readily accept broad-host-range plasmids.

The metabolism of the brucellae is mainly oxidative and they show little action on carbohydrates in conventional media. They are aerobes but some species require an atmosphere with added CO2(5-10 percent). Multiplication is slow at the optimum temperature of 37C and enriched medium is needed to support adequate growth.

Brucella colonies become visible on suitable solid media in 2-3 days. The colonies of smooth strains are small, round and convex but dissociation, with loss of the O chains of the LPS, occurs readily to form rough or mucoid variants. These latter forms are natural in B canis and B ovis as the LPS of these lack O chains.

A culture can be identified as belonging to the genus Brucella on the basis of colonial morphology, staining and slide agglutination with anti-Brucella serum, smooth or rough. Further classification is best done in a specialized laboratory. Identification to species level may be done by the procedures shown in Table 28-1. Further differentiation to biovars may be useful and is illustrated in Table 28-2. As a further refinement, tests for the oxidative metabolism of certain aminoacids and carbohydrates have been devised. Modern DNA hybridization tests, however, show that the currently named species show a high degree of homology and suggest that the genus could be appropriately reclassified as having a single species.

The application of techniques of molecular biology have allowed the cloning and characterization of several genes coding for outer membrane proteins, the use of PCR to identify the presence of brucellar DNA at genus and species level and the demonstration of species specific patterns of restriction fragment length polymorphism. It is predictable that this work will be extended to improve diagnostic tests and even vaccine development.

The organisms may gain entry into the body through a variety of portals (Fig. 28-1). Because the infection is systemic it is often not possible to determine which portal was involved in a particular case. Oral entry, by ingestion of contaminated animal products (often raw milk or its derivatives) or by contact with contaminated fingers, probably represents the most common route of infection even though this portal may not be the most vulnerable one. Inhalation of aerosols containing the bacteria, or aerosol contamination of the conjunctivae, is another route. Inhalation probably underlies some industrial outbreaks. Percutaneous infection through skin abrasions or by accidental inoculation has frequently been demonstrated.

Brucella species differ markedly in their capacity to cause invasive human disease. Brucella melitensis is the most pathogenic; B abortus is associated with less frequent infection and a greater proportion of subclinical cases. The virulence of B suis strains for humans varies but is generally intermediate.

In humans, the tissue lesions produced by Brucella species consist of minute granulomas that are composed of epithelioid cells, polymorphonuclear leukocytes, lymphocytes and some giant cells. In cases of infection with B melitensis these granulomas are particularly small although the toxemia associated with this organism is great. Necrosis is not common, and abscesses do not form, except in B suis infection. The fact that humans rapidly develop hypersensitivity to brucellar antigens suggests that many of the symptoms of human brucellosis result from the reaction of the host defenses.

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