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  3. INTRODUCTION • The genus Brucella consists of very small, non motile, aerobic gram negative, coccobacilli that grow poorly on ordinary media and have little or no fermentative powers. • They are strict parasites of animals and may also infect humans. • Cause a zoonotic disease called Brucellosis. • Infectious diseases transmitted from animals to human beings are called ZOONOSES.

  4. history Dr. David Bruce(1886): He was a British army physician who established the etiology of what was known as Malta fever or Melitosis. He insisted on studying the diseases he found in the exotic sites where he was stationed.He isolated a small miroorganism from the spleen of fatal cases in Malta & transmitted the disease to monkeys experimentally.

  5. Contd…. • Bruce called these organisms as Micrococcus melitensis. The British army did not appreciate Bruce’s meddling with indigenous diseases or his research efforts, so they shipped him to Tanganyika. As soon as Bruce arrived in eastern Africa, he started working on a new disease called SLEEPING SICKNESS and found the etiologic agent, which is now called Trypanosoma brucei. Later the etiologic agent of Malta fever was named Brucella melitensis (Brucella after Bruce & melitensis from Melita, the Roman name for Malta).

  6. Contd…. • Bang(1897):Described Brucella abortus, the cause of contagious abortion in cattle. • Zammit(1904):Showed that Brucella melitensis was transmitted to humans by goat’s milk. • Traum(1914): Isolated Brucella suis from pigs in USA.

  7. TAXONOMIC CLASSIFICATION • KINGDOM: Bacteria • PHYLUM: Proteobacteria • CLASS: Alphaproteobacteria • ORDER: Rhizobiales • FAMILY: Brucellaceae • GENUS: Brucella

  8. Morphology • Brucella are gram negative, non-motile coccobacilli. In older cultures, irregular forms appear. • SIZE: 0.5 - 0.7 x 0.6-1.5µm. • They are arranged singly, in pairs,short chains or small clusters. • They are nonsporing & noncapsulated. • Fimbriae have not been described. • These are weakly acid fast. • Modified ZN staining: Primary stain:1 in 10 diluted carbolfuchsin for 15 minutes without heating; Decolourizer:0.5% acetic acid solution for 15 seconds; Counterstain:Loeffler’smethylene blue. • Bipolar staining is not uncommon.

  9. CULTURAL CHARACTERISTICS • Brucellae are strict aerobes. • Br.abortus& Br.ovisare capnophilic(5-10% Co2). • Optimum pH – 6.6 – 7.4. • Optimum temp - 370c ( range 20-400c ). • They may grow on ordinary media though growth is slow & scanty . Growth is improved by addition of serum or liver extract.

  10. CONTD…. MEDIA EMPLOYED FOR CULTIVATING Brucellae: • Ordinary media like blood agar and chocolate agar. SPECIAL MEDIA: • Serum dextrose agar/broth. • Serum potato infusion agar/broth. • Trypicase soy agar/broth. • Tryptose agar/broth. • Brucella agar( enzymatic digest of casein, enzymatic extract of animal tissue, yeast extract NaCl, sodium bisulfite, dextrose, agar ; ph: 7.0+/- 0.2). It supports the growth of Brucellae, Strep.pneumoniae, Strep.viridans, Campylobacter species &N.meningitidis.

  11. CONTD…. SELECTIVE MEDIA: • Farrell’s medium: Serum dextrose agar containing bacitracin, cycloheximide, nalidixic acid, nystatin, polymyxin B & vancomycin. • Brodie and Sinton’s medium: Tryptone soya broth containing amphotericin B & cycloserine + above mentioned selective agents + 5% horse serum. • Mair’s medium: Contains gentian violet as selective agent.

  12. CONTD…. • On solid media, colonies take 2-3 days to develop; they are small, smooth, translucent, low convex, glistening with an entire edge. • On selective media, colonies appear after 4-5 days & are 1-2mm in size; on further incubation they increase in size to 4-6mm. • On serum dextrose agar, colonies are pale yellow and translucent. • On Mair’s medium, the colonies are blue to violet in colour with an almost black centre. • In liquid media, growth is uniform and a powdery or viscous deposit is formed in old cultures. • AST can be carried out on trypticase soy agar or MHA.

  13. BIOCHEMICAL REACTIONS • Catalase: Positive • Oxidase: Positive(except: Br.neotomae, Br.ovisand some strains of Br.abortus) • Indole: Negative(not produced) • MR: Negative • VP: Negative • Citrate: Not utilized • Urease: Positive(except Br.melitensiswhich gives variable results). • Nitrate reduction: Positive (except Br.ovis). • H2S production: Br.neotomae, many strains of Br.abortusand some strains of Br.suis.

  14. Resistance • Brucella are killed by heat at 600c in 10min,in milk they are rapidly destroyed by pasteurisation. • They are also killed by 1% phenol in 15min • They are also sensitive to direct sunlight & acid. • It survives for weeks in soil & manure. • They may also survive for many weeks in meat. • Sensitive to many disinfectants and antibiotics including ampicillin, co-amoxyclav, cephalosporins, aminoglycosides, tetracyclines, chloramphenicol, ciprofloxacin, sulphonamides and cotrimoxazole; relatively resistant to vancomycin, nalidixic acid and polymyxins.

  15. Antigenic structure • Primary isolates of Brucella species are said to exhibit a smooth(S) morphology, but subcultured isolates become rough(R). • Brucellae have no capsule. Instead, these forms reflect changes in outer membrane. • The transition from S to R form appears appears to be a response to D-alanine excreted by S forms and to the lowering of Po2 level of the medium during the growth of Brucellae. • Three surface antigens have been identified on brucellae, two of which are expressed by S forms and one of which is expressed by R forms.

  16. CONTD…. • The antigens of S forms are more specific than those of R forms and their presence varies among individual species. • Wilson and Miles, first described major cell wall antigen and virulence factor of Brucellae to be S-LPS containing A and M antigens • Antigen A is dominant(20 times more than M) in Br.abortus. • Antigen M is dominant(20 times more than A ) in Br.melitensis. • Antigenic pattern is intermediate in Br.suis. • Primary isolates of Br.canisand Br. ovis express R antigens.

  17. CONTD…. • Antigenic cross reactions exist between Brucellae and V.cholerae, Esch.coli O:116 & O:157, Salmonella serotypes group N(O:30 antigen Kauffman and White), Ps.maltophila, Y.enterocolitica and F.tularensis. • Presence of 4-amino 4,6 dideoxymannose in LPS is responsible for antigenic cross-reaction with other GNB. e.g. - Vibrio cholerae 01 and Yersinia species.

  18. classification CRITERIA FOR CLASSIFICATION: • Co2 requirement • H2S production • Tolerance of bacteriostatic dyes • Agglutination with monospecific antisera • Lysis by specific bacteriophage DNA analysis has shown that there is no significant difference between different species. But, the present classification has been retained because of it’s epidemiologic usefulness and convenience.

  19. Species of Brucella • Br.melitensis • Br.abortus • Br.suis • Br.canis • Br.ovis • Br.neotomae • Br. cetae • Br. pinnipedalis

  20. Biotypes • Br.abortus - 7 biotypes(1,2,3,4,5,6,9) • Br.melitensis - 3 biotypes(1,2,3) • Br.suis - 5 biotypes(1,2,3,4,5) • Br.canis - 1 biotype • Br.ovis - 1 biotype • Br.neotomae - 1 biotype

  21. EPIDEMIOLOGY • Brucellosis – zoonotic disease – all infections occur directly or indirectly from animal exposure. • Disease exists world-wide, especially Mediterranean region, Arabic Peninsula, Indian subcontinent, parts of Mexico and Central & South America. • Br.abortusfound mainly in cattle, but other species like buffaloes, camels, dogs, horses, deer, goats , sheep and man can be affected. • Br.melitensisprimarily affects goats and sheep. Camels can be important source in some countries.Also infects man. • Br.suisbiovars 1-3 infecct pigs, biovar 4 reindeers and biovar 5 small rodents.

  22. CONTD…. • Br.canis: Least common cause of human disease. • Br.ovis and Br.neotomae do not cause human infection. • Br.cetae and Br.pinnipedalis have been isolated from marine mammals. • Human infections are mainly caused by Br.melitensis in India, Br.abortus in Great Britain and Br.suis in USA. • Animals acquire infection through the gastrointestinal tract, skin and mucous membranes or through aerosols • Animals usually recover from infection spontaneously but continue to shed bacteria for varying intervals of time in in vaginal secretions, urine and milk.

  23. CONTD…. • Brucellosis constitutes an occupational risk for: farmers, veterinarians, butchers, animal handlers and laboratory personnel. • Routes of transmission to humans include: • Direct contact with animal tissues. • Ingestion of contaminated meat, raw infected milk or milk products. • Inhalation of aerosolized organisms. • Accidental exposure in the laboratory (containment level 3 room and category I or III exhaust protective cabinet needed). • Person to person spread usually does not occur but, very rarely transmission has been reported through placenta(in utero), breastfeeding, sexual intercourse and transfer of tissues including blood and bone marrow.

  24. Contd…. • In AIDS patients if CD4 counts are not severely depressed,then course of brucellosis in AIDS patients is not different from disease in immunocompetent patients. • Brucellosis is not rare in children as previously believed. • Brucellosis manifests similarly in: Neonates, children and adults

  25. pathogenesis • Infection with any Brucella species, including attenuated vaccine strains can cause serious human disease. • Br.melitensis is the most virulent of all the species. • Once these organisms gain entry into the body: PMNs are attracted to inoculation site by chemotaxis.

  26. CONTD…. • Normal human serum has limited bactericidal activity against brucellae, but it effectively opsonizes bacteria for phagocytosis by PMNs. • Brucellae are facultative intracellular, slowly dividing pathogens with capacity to survive and multiply within host phagocytic cells.

  27. CONTD…. • Mechanism by which brucellae evade intracellular killing by PMNs is incompletely under-stood. • Factors contributing to intracellular survival: Production of adenosine and guanosinemonophosphate which suppress granulocyte degranulation and myeloperoxidase – H2O2-halide system and Cu-Zn superoxide dismutase, which eliminate reactive oxygen intermediates.

  28. CONTD…. • PMNs act as “taxicabs” and carry these organisms to various lymphatic tissues. • Hallmark of brucellosis is formation of granulomas within lymphatic tissues. • Multiplication of brucellae within macrophages fills and destroys these cells and patients develop bacteremia.

  29. CONTD…. • If the cell mediated immune response that develops during this stage is insufficient, the liver, spleen, bone marrow and lymph nodes may become extensively infected. • Caseation necrosis may be seen in granulomas caused by Br.suis. • In patients with chronic brucellosis, granulomas form in the kidneys, central nervous system, subcutaneous tissues, testes and ovaries.

  30. CONTD…. • Brucellae ingested by mononuclear phagocytes survive and replicate initially. Intracellular survival within macrophages is facilitated by inhibition of phagosome-lysosomefusion by soluble products of brucellae, and production of stress-induced proteins. • Eventual elimination of virulent brucellae depends on activation of macrophages through development of TH-1type cell-mediated immunity. • Cytokines contributing to anti-brucella activity of activated macrophages include: TNF-α, TNFgamma, IL-1, IL-12.

  31. CONTD…. • Major determinant of virulence and immuno-dominant antigen of Brucella speciesis S-LPS. • Growth of brucellaein placental fluid of ruminants is apparently enhanced by erythritol(a carbon and energy source that promotes rapid multiplication of brucellae). In contrast, the placental fluid of humans contains erythrose, which does not support the growth of brucellae. Therefore, septic abortions are commonly seen among ruminants.


  33. COMPLICATIONS • Arthritis, Sacroileitis, large joints involved in children(Most common) • Epididymo orchitis • Meningitis • Optic neuropathy • Pappiledema • Uveitis • Radiculopathy • IC hemorrhages & stroke • Endocarditis • Pneumonitis, pleural effusion, empyema • Granulomatous hepatitis • Abscesses of the liver , spleen, thyroid and epidural space. • Cholecystitis • Erythema nodosum • Prosthetic device infections • Chronic skin ulcers

  34. Laboratory Diagnosis • Brucellosis is a febrile disease with varied clinical manifestations . Laboratory methods include 1. Culture 2. Serology 3. PCR 4.Hypersensitivity testing (Brucellin test)

  35. Specimens • Blood culture is the most definitive method for diagnosis of acute brucellosis. • Brucellae can also be isolated from bone marrow, vaginal discharge , liver, lymph nodes, CSF, urine, sputum, breast milk & seminal fluid.

  36. Blood culture • 10 ml of blood is withdrawn, 5ml being added to each of 2 bottles containing serum dextrose broth/trypticase soy broth & incubated at 37°c aerobically and in the presence of 5-10% Co2. • Subcultures are madeon solid media every 3-5 days starting on the fourth day for 8 weeks . • Blood is collected during the pyrexial phase, preferably when the temperature is rising. • Subcultures can be avoided by using Castaneda's method of blood culture. It contains both liquid (serum dextrose broth) & solid (serum dextrose agar) media in the same bottle. The bottle is incubated in upright position. For subculture, the bottle is tilted so that the broth flows over the surface of the agar slant. • BACTEC cultures may become positive in 5-6 days.

  37. Blood cultures are positive in 30 - 50% cases, even when repeated samples are tested. Bone marrow cultures yield a higher rate of isolation and remain positive long after the blood culture has become negative.

  38. Serology • As cultures are often unsuccessful, serological methods are important in diagnosis. • Antibodies (Ig G & Ig M ) appear within 7-10 days of onset of the disease. IgM antibodies appear first. They are rapidly followed by and superseded by IgG and to a lesser extent IgA antibodies. • The various serological tests are SAT, 2ME agglutination test, Coombs test, CFT, ELISA, RIA, indirect immunofluorescence test, IHA, Rose Bengal plate test, Castaneda strip test.

  39. Standard tube agglutination(SAT) • This is a tube agglutination test in which equal volumes of serial two fold dilutions (1:20 to 1:640) of patients serum in 0.4% phenol saline & standardized antigen ( killed suspension of standard strain of Brucella abortus) are mixed & incubated at 37°c for 24-48 hours. • 5 controls are used: 1ml antigen, 0.75 ml antigen+ 0.25 ml saline, 0.5 ml antigen and saline each, 0.25 ml antigen and 0.75 ml saline and 1 ml saline. 50% clearance is taken as positive(compared with 3rd tube).

  40. Contd…. • A single titre of more than 1:160 is presumptive evidence of brucella infection. Most patients with acute brucellosis develop agglutinin titres of 1:640 or more by the end of the 3rd or 4th week of illness. Thereafter the titres fall. • Demonstration of rising titres is important(4 fold rise). • Prozone effect and blocking effect of IgA and IgG antibodies. Blocking effect is taken care of by: Coombs test, 4% saline, high speed centrifugation and heating the test serum at 55°c for 30 minutes.

  41. Contd…. • Coombs test: It is the most reliable method for obviating the blocking effect and detecting ‘incomplete’ antibodies, • 2ME agglutination test: Helps to differentiate between 2ME sensitive(IgM) and 2ME resistant antibodies(IgG). Cholera, tularemia or yersinia induced agglutinins may be differentiated by the agglutinin absorption test and also as they are removed by treatment with 2ME.

  42. ELISA & RIA • These are very sensitive & helpful to distinguish IgA , IgG and IgM antibodies. • Complement fixation: It is more useful in chronic cases as it detects both Ig G & Ig M . Indirect immunofluorescence • It is a specific & sensitive method for detecting antibodies & may be positive even when agglutination tests are negative.

  43. Contd…. • IHA: Human ‘O’ group RBCs or sheep RBCs can be used. RBCs are sensitised with Br.abortus and Br.melitensis antigens. Test serum is inactivated at 56ºc for 30 minutes. Controls used are: test serum in a dilution of 1:50 with unsensitised RBCs, normal sera in dilutions 1:50, 1:100, 1:200 with sensitised RBCs, Brucella positive serum in dilutions ranging from 1:50 to 1:200,000 with sensitised RBCs and Brucella positive serum in a dilution of 1:50 with unsensitised RBCs.

  44. Rose Bengal Test (RBT) • Spot test for brucella diagnosis(humans and animals). • Detects specific antibodies (IgM and IgG types) but more effectively detect IgG-1 than IgM and IgG 2 • Low pH (3.6) of the antigen enhances the specificity of the test, the temperature of the antigen and the ambient temperature influence the sensitivity and specificity of the RBT.

  45. Principle The buffered acidic Rose Bengal antigen interacts with serum antibodies to produce agglutination, which is used for the early detection of Brucella specific antibodies. Antigen is coated with dyes(hematoxylin or eosin).

  46. Procedure • Place a drop (30 μl) of undiluted serum on a slide. • Add a drop of the reagent (Rose Bengal Brucella antigen) next to the drop of the serum. • Mix both drops by the disposable stirring stick, spreading them over the full surface of the circle. • Observe for the result

  47. Reading • Noagglutination= (- Ve) Negativeresult. • Agglutination= (+Ve) Positive results Positive test Negative test