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Lecture 2 ....Brucellae ................................................................. .

THE BRUCELLAE
The brucellae are obligate parasites of animals and humans and are characteristically located intracellularly. They are relatively inactive metabolically. Brucella melitensis typically infects goats; Brucella suis , swine; Brucella abortus, cattle; and Brucella canis, dogs. Other species are found only in animals.. The disease in humans, brucellosis (undulant fever, Malta fever), is characterized by an acute bacteremic phase followed by a chronic stage that may extend over many years and may involve many tissues.

Morphology & Identification


A. TYPICAL ORGANISMS The appearance in young cultures varies from cocci to rods 1.2 m in length, with short coccobacillary forms predominating. They are gram-negative but often stain irregularly, and they are aerobic, nonmotile, and non-spore-forming. B. CULTURE Small, convex, smooth colonies appear on enriched media in 25 days. C. GROWTH CHARACTERISTICS Brucellae are adapted to an intracellular habitat, and their nutritional requirements are complex. Some strains have been cultivated on defined media containing amino acids, vitamins, salts, and glucose. Fresh specimens from animal or human sources are usually inoculated on trypticase-soy agar or blood culture media. B abortus requires 510% CO2 for growth, whereas the other three species grow in air. Brucellae utilize carbohydrates but produce neither acid nor gas in amounts sufficient for classification. Catalase and oxidase are produced by the four species that infect humans. Hydrogen sulfide is produced by many strains, and nitrates are reduced to nitrites. Brucellae are moderately sensitive to heat and acidity. They are killed in milk by pasteurization. D. VARIATION The typical virulent organism forms a smooth, transparent colony; upon culture, it tends to change to the rough form, which is avirulent. The serum of susceptible animals contains a globulin and a lipoprotein that suppress growth of nonsmooth, avirulent types and favor the growth of virulent types. Resistant animal species lack these factors, so that rapid mutation to avirulence can occur. D-Alanine has a similar effect in vitro.

Antigenic Structure
Differentiation among brucella species or biovars is made possible by their characteristic sensitivity to dyes and their production of H2S. Few laboratories have maintained the procedures for these tests, and the brucellae are seldom placed into the traditional species. Because brucellae are hazardous in the
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Lecture 2 ....Brucellae ................................................................. .

laboratory, tests to classify them should be performed only in reference public health laboratories using biosafety precautions.

Pathogenesis & Pathology


Although each species of brucella has a preferred host, all can infect a wide range of animals, including humans. The common routes of infection in humans are the intestinal tract (ingestion of infected milk), mucous membranes (droplets), and skin (contact with infected tissues of animals). Cheese made from unpasteurized goats' milk is a particularly common vehicle. The organisms progress from the portal of entry, via lymphatic channels and regional lymph nodes, to the thoracic duct and the bloodstream, which distributes them to the parenchymatous organs. Granulomatous nodules that may develop into abscesses form in lymphatic tissue, liver, spleen, bone marrow, and other parts of the reticuloendothelial system. In such lesions, the brucellae are principally intracellular. Osteomyelitis, meningitis, or cholecystitis also occasionally occurs. The main histologic reaction in brucellosis consists of proliferation of mononuclear cells, exudation of fibrin, coagulation necrosis, and fibrosis. The granulomas consist of epithelioid and giant cells, with central necrosis and peripheral fibrosis. The brucellae that infect humans have apparent differences in pathogenicity. B abortus usually causes mild disease without suppurative complications; noncaseating granulomas of the reticuloendothelial system are found. B canis also causes mild disease. B suis infection tends to be chronic with suppurative lesions; caseating granulomas may be present. B melitensis infection is more acute and severe. Persons with active brucellosis react more markedly (fever, myalgia) than normal persons to injected brucella endotoxin. Sensitivity to endotoxin thus may play a role in pathogenesis. Placentas and fetal membranes of cattle, swine, sheep, and goats contain erythritol, a growth factor for brucellae. The proliferation of organisms in pregnant animals leads to placentitis and abortion in these species. There is no erythritol in human placentas, and abortion is not part of brucella infection of humans.

Clinical Findings
The incubation period is 16 weeks. The onset is insidious, with malaise, fever, weakness, aches, and sweats. The fever usually rises in the afternoon; its fall during the night is accompanied by drenching sweat. There may be gastrointestinal and nervous symptoms. Lymph nodes enlarge, and the spleen becomes palpable. Hepatitis may be accompanied by jaundice. Deep pain and disturbances of motion, particularly in vertebral bodies, suggest osteomyelitis. These symptoms of generalized brucella

Lecture 2 ....Brucellae ................................................................. .

infection generally subside in weeks or months, although localized lesions and symptoms may continue. Following the initial infection, a chronic stage may develop, characterized by weakness, aches and pains, low-grade fever, nervousness, and other nonspecific manifestations compatible with psychoneurotic symptoms. Brucellae cannot be isolated from the patient at this stage, but the agglutinin titer may be high. The diagnosis of chronic brucellosis is difficult to establis h with certainty unless local lesions are present.

Diagnostic Laboratory Tests


A. SPECIMENS Blood should be taken for culture, biopsy material for culture (lymph nodes, bone, etc), and serum for serologic tests. B. CULTURE Brucella agar was specifically designed to culture brucella species bacteria. The medium is highly enriched andin reduced formis used primarily in cultures for anaerobic bacteria. In oxygenated form, the medium grows brucella species bacteria very well. However, infection with brucella species is often not suspected when cultures of a patient's specimens are set up, and brucella agar incubated aerobically is seldom used. The brucella species bacteria will grow on commonly used media, including trypticase soy medium with or without 5% sheep blood, brain heart infusion medium, and chocolate agar. Blood culture media (see below) readily grow brucella species bacteria. All cultures should be incubated in 810% CO2 at 3537 C and should be observed for 3 weeks before being discarded as negative; liquid media cultures should be blindly subcultured during this time. Bone marrow and blood are the specimens from which brucellae are most often isolated. The method of choice for bone marrow is to use pediatric Isolator tubes, which do not require centrifugation, with inoculation of the entire contents of the tube onto solid media. Media used in semiautomated and automated blood culture systems readily grow brucellae, usually within 1 week; however, holding the cultures for 3 weeks is recommended. Negative cultures for brucella do not exclude the disease because brucellae can be cultivated from patients only during the acute phase of the illness or during recurrence of activity. After a few days of incubation on agar media, the brucellae form colonies in the primary streak that are < 1 mm in diameter. They are nonhemolytic. The observation of tiny gram-negative coccobacilli that are catalase-positive and oxidase-positive suggests brucella species. All further work on such a culture should be done in a biologic safety cabinet. A Christensen urea slant should be inoculated and observed frequently. A positive urease test is characteristic of brucella species. B suis and some strains
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Lecture 2 ....Brucellae ................................................................. .

of B melitensis can yield a positive test less than 5 minutes after inoculating the slant; other strains will take a few hours to 24 hours. Bacteria that meet these criteria should be quickly submitted to a reference public health laboratory for presumptive identification. If organisms resembling brucellae are isolated, they are typed by H2S production, dye inhibition, and agglutination by absorbed sera. C. SEROLOGY IgM antibody levels rise during the first week of acute illness, peak at 3 months, and may persist during chronic disease. Even with appropriate antibiotic therapy, high IgM levels may persist for up to 2 years in a small percentage of patients. IgG antibody levels rise about 3 weeks after onset of acute disease, peak at 68 weeks, and remain high during chronic disease. IgA levels parallel the IgG levels. The usual serologic tests may fail to detect infection with B canis . 1. Agglutination test To be reliable, serum agglutination tests must be performed with standardized heat-killed, phenolized, smooth brucella antigens. IgG agglutinin titers above 1:80 indicate active infection. If the serum agglutination test is negative in patients with strong clinical evidence of brucella infection, tests must be made for the presence of blocking antibodies. These can be detected by adding antihuman globulin to the antigen-serum mixture. 2. Blocking antibodies these are IgA antibodies that interfere with agglutination by IgG and IgM and cause a serologic test to be negative in low serum dilutions (prozone) although positive in higher dilutions. These antibodies appear during the subacute stage of infection, tend to persist for many years independently of activity of infection, and are detected by the Coombs antiglobulin methods.

3. ELISA assays- IgG and IgM antibodies may be detected using ELISA assays, which use
cytoplasmic proteins as antigens. These assays tend to be more sensitive and specific than the agglutination assays.

Immunity
An antibody response occurs with infection, and it is probable that some resistance to subsequent attacks is produced. Immunogenic fractions from brucella cell walls have a high phospholipid content; lysine predominates among eight amino acids; and there is no heptose (thus distinguishing the fractions from endotoxin).

Treatment
Brucellae may be susceptible to tetracyclines or ampicillin. Symptomatic relief may occur within a few days after treatment with these drugs is begun. However, because of their intracellular location, the

Lecture 2 ....Brucellae ................................................................. .

organisms are not readily eradicated completely from the host. For best results, treatment must be prolonged. Combined treatment with streptomycin and a tetracycline is recommemded.

Epidemiology, Prevention, & Control


Brucellae are animal pathogens transmitted to humans by accidental contact with infected animal feces, urine, milk, and tissues. The common sources of infection for humans are unpasteurized milk, milk products and cheese and occupational contact (eg, farmers, veterinarians, slaughterhouse workers) with infected animals. Cheese made from unpasteurized goat's milk is a particularly common vehicle for transmission of brucellosis. Occasionally the airborne route may be important. Because of occupational contact, brucella infection is much more frequent in men. The majority of infections remain asymptomatic (latent). Infection rates vary greatly with different animals and in different countries. Outside the USA, infection is more prevalent. Eradication of brucellosis in cattle can be attempted by test and slaughter, active immunization of heifers with avirulent live strain 19, or combined testing, segregation, and immunization. Cattle are examined by means of agglutination tests. Active immunization of humans against brucella infection is experimental. Control rests on limitation of spread and possible eradication of animal infection, pasteurization of milk and milk products, and reduction of occupational hazards wherever possible.

Dr. Shama M.J Saadaldin Al-Shadidi 3rd grade / College of Medicine Mustansiryah University 1/4/2012 Updated 2012