brucellosis

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2. 2 Introduction & History Brucellosis: Disease of domestic and wild animals (zoonosis): Transmittable to humans. It has different non-specific symptoms and signs “No disease, not excepting tuberculosis and syphilis, is more protean in its manifestations”..(Simpson). Marston – Surgeon serving with Royal Artillery during Crimean war-reported first accurate description of human brucellosis. 1886, Bruce isolated Micrococcus (later Brucella) Melitensis from spleens of malta fever victims.

3. 3 Introduction and History (Cont’d.) Zamit – maltese physician, identified native goats as infection reservoir, during his work with Mediterranean fever commission 1904-1907. He also identified fresh goat’s milk as vehicle of transmitting disease from animals to humans. 1895, Bang (Danish Veterinarian) identified Bacillus (later Brucella) abortus as cause of contagious abortions in cattle. Evans (American bacteriologist) recognized relation of Malta fever agent and Bang’s disease

4. 4 Introduction and History (Cont’d.) Genus – Bacillus, micrococcus, causing Malta fever, renamed Brucella, to honor Bruce. Traum, 1914, isolated Brucella Canis, from aborted Kennel-bred dogs. Carmichael, 1966, isolated Brucella Canis, from aborted Kennel-bred dogs. Brucella ovis from sheep and Brucella neotomae from desert wood rats, added to Brucella species, but to date these two not shown to cause human infections.

5. 5 Introduction and History (cont’d.) 1994, British and American workers, independ- ently, isolated previuously unknown Brucella organism from carcasses of marine mammals and cetaceans on Scotland coast, and from dolphin in California. These isolates-homogenous with distinctive metabolic, sensitivities to dye, phage sensitivity-tentatively named Brucella Maris.

6. 6 THE PATHOGEN Brucellae: Small, GN cocobacilli, non-motile, non-spore forming. Brucellae grow aerobically. Some spp. Require supplemental carbon dioxide for primary isolation. Any high-quality peptone-based media enriched with blood or serum serve for in vitro cultivation. Isolation form clinical specimens require prolonged (= 30 days) incubation. Brucella strains always catalse-positive; but oxidase and urease and H2S production vary.

7. 7 The Pathogen (cont’d.) Major Brucellae species and their biovars differentiated by selective inhibition of growth on media containing dyes-e.g. Thionin and basic fuchsin. Genus Brucella divided into six (possibly seven) nomen spp. on basis of preferred hosts and cultural, metabolic and antigenic characteristics. DNA-DNA hybridization studies shown remarkable( >95%) homology between strains, suggesting mono-specific gems with subspecies corresponding evolutionary lineage adapted to specific hosts.

8. 8 The Pathogen (cont’d.) Wilson and Miles, first described major cell wall antigen and virulence factor of brucellae to be S-Lps containing A and M antigens. Presence of 4-amino, 4,6 dideoxymannose in Lps is responsible for antigenic cross-reaction with other G.N.B. e.g. - vibrio cholerae 01 and Yersinia 09. Numerous protein antigens maybe important in inducing protective immunity.

9. 9 Epidemiology Brucellosis – zoonosis – all infections, derive directly or indirectly from animals exposure. Disease exists world-wide, esp. Mediterranean, Arabic Peninsula, Indian subcontinent, parts of Mexico and Central South America. B. abortus found mainly in cattle, but others spp. like buffalo, camels, tales can be affected. B. Melitensis primary affects goats and sheep. Camels can be important source in some countries. B. Suis biovars 1-3 in domestic and feral swine, cause abattoir-assoc. human disease.

10. 10 Epidemiology (Cont’d.) B. Canis: Least common cause of human disease. Animals: Brucellosis, Ch. Infection, persisting for life. Brucellae localization in reproductive organs, accounts for major manifestations – abortion and sterility. Brucellae shed in large numbers in: Milk, urine, cyetic products of infected animals.

11. 11 Epidemiology (Cont’d.) Thus, Brucellosis constitutes occupational risk for: farmers, veterinarians, abattoirs and Laboratory personnel. Routes of transmission to human include: - Director contact with animals or their secretions, through cuts and skin abrasions. - Infected aerosols inhaled or inoculated into eye conjunctival sac. - Ingestion of unpasteurized dairy products.

12. 12 Epidemiology (Cont’d.) - Meat products: rare source of infection because: Meat is rarely eaten raw and organisms are present in low number of muscle tissue. - Blood and bone marrow may transmit disease when ingested in some cultures. - Human-to-human transmission: Unusual, but rare cases suspected to be sexually transmitted.

13. 13 Epidemiology (cont’d.) - One case reported recently, presumptively due to intra-uterine transmission. Aids patient, prone to infections by zoonoses, but Brucellosis occurred in very few of these patients. If CD4 not severely depressed, course of brucellosis in Aids pts. not different from disease in immunocompetent pts. Brucellosis not rare in children as previously believed. Brucellosis manifests similarly in: Neonates, children and adults.

14. 14 Pathogenesis B. melitensis and B. suis, more virulent than B. abortus and B. canis. Infection with any B. species, including attenuated vaccine strains can cause serious human disease. Disease determined by: - Host nutritional and immune status. - Size of infectious inoculum. - Route of transmission. Ex: -Low gastric juice PH, more effective in preventing B. abortus than B. melitensis infection when administered by oral route.

15. 15 Pathogenesis (cont’d.) -Therefore, drugs that decrease gastric acidity were implicated in food borne brucellosis. Once brucellae gain entry to body: PMN – Leukocytes attracted to inoculation site by chemotaxis. Normal human serum has limited bactericidal activity against brucellae, but it effectively opsonizes bacteria for phagocytosis by PMN – Leukocytes.

16. 16 Pathogenesis (cont’d.) Brucellae: Facultative intracellular, slowly dividing pathogens with capacity to survive and multiply within host phagocytic cells. Mechanism by which brucellae evade intracellular killing by PMN-Leukocytes incompletely under-stood, it is possible that bacteria property enable them to escape detection. Factors contributing to intracellular survival: - Production of adenine and guanine monophos- phate.

17. 17 Pathogenesis (cont’d.) -These suppress myeloperoxide – H2O2-halide system and cu-zn superoxide dismutase, which eliminates reactive oxygen intermediates. Spink compared brucellosis with typhoid fever because bacteria enter lymphatic and replicate within regional lymph nodes. Hematogenous dissemination then followed by localization of bacteria within organs rich in reticuloendothelial system, e.g. liver, spleen and B. marrow.

18. 18 Pathogenesis (Cont’d.) Brucellae ingested by mononuclear phagocytes survive and replicate initially. Intracellular survival within macrophages facilitated by inhibition of phagoxome-Lysosome fusion by soluble products of brucellae, and production of stress-induced proteins. Eventual elimination of virulent brucellae depends on activation of macrophages through develop-ment of Th-1type cell-mediated immunity. Cytokines contributing to anti-brucella activity of activated macrophages include: TNF-alpha, TNF-gamma, IL-1, IL-12.

19. 19 Pathogenesis (cont’d.) Major determinant of virulence and immuno-dominant antigen of Brucella is S-Lps. Growth of B. abortus in cattle placental tissue apparently enhanced by eruthritol, this may explain localization of brucellae in genital tracts of ungulates.

20. 20 HOST IMMUNITY Genetic studies in various animals showed that resistance to intracellular pathogens is polygenic; but single genes recognized to have major effect on immune-mediated resistance. Selected breeding of cattle yielded evidence for genetic determination of resistance to bovine brucellosis. Data suggested that resistance reflected in immunoglobulin allotypes and in difference in ability of macrophages to control B. abortus replication in vitro.

21. 21 Host Immunity (Cont’d.) S-Lps is major deter of virulence of brucellae and dominates antibody response. Humoral anti-bodies to S-Lps confer short-term protection as shown by passive transfer experiments using monoclonal and polyclonal antibodies. Antibodies to S-Lps used for diagnosis when bacterial isolation is unsuccessful. Variety of serologic tests used to measure anti-bodies to brucellae. Earliest was serum agglutina-tion test (SAT), devised by Wright and Smith 1897. SAT. Measures total quantity of agglutinating anti-bodies, but does not distinguish between immuno-globulin isotypes.

22. 22 HOST IMMUNITY (Cont’d.) In time, IgM antibodies titers decline, and with treatment IgG antibodies titers fall consistent with recovery. Failure of IgG titer to decline is prognostic of relapse or chronic infection. Currently, there is interest in cytoplasmic protein antigens in both smooth and rough strains, which could be used for diagnostic purposes. Reddin and colleagues modified SAT, by adding 2-mercapto-ethanol(2ME) to differentiate agglutina-tion by IgG-antibodies, and showed that IG antibodies correlated well with active infection.

23. 23 Host Immunity (cont’d.) Combination of SAT and 2 ME test shown to be useful to monitor Brucellosis course and response to therapy. Application of enzyme-linked immunoabsorbent assay (ELISA) measures immune response to therapy. IgM antibodies appear within 1st week of infection and followed by switch to IgG synthesis after 2nd week. In time, IgM antibodies titers decline and with treat- ment IgG antibodies, titers fall consistent with recovery.

24. 24 Host Immunity (cont’d.) Failure of IgG titer to decline is prognostic of relapse or chronic infection. Currently, there is interest in cytoplasmic protein antigens in both smooth and rough strains, which could be used for diagnostic purposes.

25. 25 Clinical Manifestation Symptoms of Brucellosis: non-specific, e.g. fever, sweats, malaise, anorexia, headache, backpain. Onset: acute or insidious, beginning within 2 to 4 weeks after inoculation. An “Undulant” fever pattern observed if patients go untreated for long periods. Some patients c/o malodorous sweat and peculiar mouth taste. Depression common and often out of proportion to severity of symptoms. In comparison to plethora of somatic complaints, physical abnormalities are few. Mild lymphadenopathy reported in 10 to 20% of cases.

26. 26 Clinical Manifestation Splenomegaly or hepatomegaly in 20 to 30% of cases. Brucellosis: systemic infection in which any organ or system of the body can be involved. Attempts to categorize the disease into acute, subacute and chronic, according to symptoms, length and severity are purely arbitrary. Disease referred to focal or localized when involvement of specific organ predominates. However, there is little evidence that such complication represent distinct subset of patients.

27. 27 Clinical Manifestation (cont’d.) When CNS or heart involved, such cases: difficult to treat and outcome can be affected. Problem in interpreting literature on brucellosis is differentiating acute and chronic forms of brucellosis. Because of necessity to treat patients for prolonged periods, relapse not uncommon, especially if therapy discontinued prematurely. Most relapses occur within 3 to 6 months of discontinuing therapy. Disease considered chronic if infection persists more than 12 months (arbitrary definition).

28. 28 Clinical Manifestation (cont’d.) Chronic Brucellosis usually caused by persisting deep foci of infection, e.g. suppurative lesions in bone, joints, liver, spleen or kidneys. In contrast, some patients experience delyaed convalescence after treatment, with persisting non-specific complaints of ill health, notably fatigue. Such disease distinguished from true chronic brucellosis by absence of objective signs of disease, as fever, in addition, chronic brucellosis characterized by persistently high IgG antibodies titres in serum, but:-

29. 29 Clinical Manifestation (cont’d.) Patients with delayed convalescence: poorly understood, some authorities attribute that to pre-existing psychoneurosis exacerbated by brucellosis. Such patients present diagnostic dilemma, because their complaints resemble brucellosis, but further antimicrobial therapy, ineffective, and patients often believe they suffer from incurable brucellosis.

30. 30 Clinical Manifestation (cont’d.) Patients with delayed convalescence: poorly understood, some authorities attribute that to pre-existing psychoneurosis exacerbated by brucellosis. Such patients present diagnostic dilemma, because their complaints resemble brucellosis, but further antimicrobial therapy, ineffective, and patients often believe they suffer from incurable brucellosis. Relapse not usually caused by antibiotic resistance, because strains of brucellae isolated during relapse have antibiogram identical to original infecting strains.

31. 31 Complications Nervous System Direct CNS invasion by brucellosis less than 5% although depression and mental inattention occur commonly in brucellosis. CNS Complications:- - Meningitis - Encephalitis - Myelitis – radiculoneuritis

32. 32 Complications (cont’d.) - Brain abscess - Epidural abscess - Demyelinating syndromes - Meningovascular syndromes - Acute and chronic meningitis: Most frequent CNS complication, and can be presenting finding, or occur late in disease course. - Brucella meningitis difficult to distinguish from other causes of meningitis.

33. 33 Complications (cont’d.) Nuchal rigidity occurs in <50% of cases. Brucella meningitis difficult to distinguish from other causes CSF: Lymphocytic pleocytosis, elevated protein, low to normal glucose. G. stain usually negative. Culture positive <25% of cases. Diagnosis made by finding specific antibodies in CSF

34. 34 Histologic findings: - Inflammation of leptomeninges - Adhesive arachnoiditis - Vasculitis - Leukoencephalitis GIT 70% of patients with brucellosis - Anorexia, abd. Pain, nausea, vomiting, diarrhea or constipation.

35. 35 GIT 70% of patients with brucellosis (cont’d) - Pathologic lesions: - Intestinal mucosa hyperemia with peyer’s patches inflammation. - Acute ileitis radiologically and histologically in patients with colitis B. melitensis. Hepatobiliary system (HPS) - Liver, largest reticuloendothelial organ, probably always involved in brucellosis. - LFT, usually slightly elevated.

36. 36 Hepatobiliary system (HPS) - Brucella hepatitis pathologic findings spectrum is variable. - B. abortus infection: Granulomas indistinguishable from sarcoidosis. - In contrast: B. melitensis: Lesions range from small, almost insignificant mononuclear cell aggregates surrounding foci of necrosis scattered throughout parenchyma, to diffuse non-specific inflammation resembling viral hepatitis.

37. 37 Hepatobiliary system (HPS) (cont’d.) - Occasionally: Collections of mononuclear cells forming loose granuloma may be found. - Suppurative abscess of liver and spleen common with B. suis infection, and occasionally with B. melitensis. - Hepatic lesions resolve with antimicrobial therapy, and in absence of other causes (HCVm HBV, alcohol abuse). No cirrhosis, despite inflammation severity.

38. 38 Hepatobiliary system (HPS) (cont’d.) - Brucella rarely causes acute cholecystitis, pancreatitis and spontaneous bacterial peritonitis (SBP). MSS - Osteoarticular complications reported in 20-60% of pts. infected with brucellosis. - Bone and joint lesions include:- ? Arthritis ? Spondylitis

39. 39 MSS - Osteoarticular complications reported in 20-60% of pts. infected with brucellosis. - Bone and joint lesions include: ? Arthritis ? Spondylitis ? Osteomyelitis ? Tenosynovitis ? Bursitis ? Sacroiliitis most commonly reported complication.

40. 40 MSS (cont’d.) - Synovial fluid analysis from brucella effusions reveal: ? Mononuclear cells predominance, and brucellae recovered in about 50% of cases. ? Reactive post infectious spondyloarthropathy described in some pts. This may be caused by circulating immune complexes, but no association found with specific HLA phenotype. ? Spondylitis, predominantly, involving lumbar spine more common among elderly pts. and rarely associated with paraspinal abscess.

41. 41 MSS (cont’d.) ? Radiographic abnormality generally occur late, but bone scans may detect inflammation early in disease. ? Bone scan useful in differentiating sacroiliitis from hip joint involvement. ? Earliest radiographic findings in spondylitis: straightening of spine and narrowing of disk space. ? CT scan useful for detecting: - Joint destruction - Vertebral osteomyelitis - Paraspinal abscess

42. 42 C.V.S. - Endocarditis occurs in less than 2% of cases, but accounts for majority of brucellosis-related deaths. - Before effective therapy, including valve replace- ment surgery, Brucella endocarditis is nearly always fatal. - Aortic valve is affected more often than mitral valve. - Both native and prosthetic valve infections reported.

43. 43 C.V.S. (cont’d.) - Mycotic aneurysms of brain, aorta and other vessels are secondary complications, especially in B. suis infections.

44. 44 Respiratory System - Airborne transmission of brucellosis common in abbatoirs. - Respiratory tract involvement ranges from flu- like illness with normal chest radiograph results to bronchitis, broncopneumonia, lung nodules, miliary lesions, hilar adenopathy and pleural effusions. - Rarely brucellae identified by stain or culture of expectorated sputum.

45. 45 G. U. T. - Renal involvement is rare, although brucella have been recovered from urine. - Interstitial nephritis, pyelonephritis, exudative glomerulonephritis, and IgA nephropathy have been reported. - Orchitis occurs in up 20% of men with brucellosis. - Testes or epididymis infiltrated with lympho- cytes and plasma cells and there is atrophy of seminiferous tubules.

46. 46 G.U.T. cont’d.) - In women, rare cases salpingitis, cervicitis and pelvic abscess reported. - Principal brucellosis in animals is spontaneous abortion and presence of erythritol in tissues of susceptible animals, thought to play role in localization of brucellae in genital tract. - Brucellosis can result in human abortions, but unclear whether it is more frequent than with other bacteremic infections.

47. 47 Hematologic Complications - Hematologic manifestations of brucellosis include: anemia, leukopenia, thrombo- cytopenia and clotting disorders. - Granulomas found in B. marrow in up to 75% of cases, but they are small and indistinct. - Severe thrombocytopenia with cutaneous purpura reported and maybe associated with antiplatelets antibodies, or hemophagocytic histiocytes in bone marrow.

48. 48 Cutaneous Complications - Cutaneous lesions occur in 5% of patients with brucellosis. - Many transient, nonspecific lesions described, including rashes, papules, ulcers, petechiae, purpura, and vasculitis.

49. 49 Ocular Complications - Many ocular complications reported in patients with brucellosis. - Uveitis, late manifestation, consisting of chronic iridocyclitis, mummular keratitis, multifocal choroiditis, and optic neuritis. - Brucella uveitis considered non-infectious immune response that response to topical and systemic corticosteroid therapy. - Rare cases of endophthalmitis reported, in which brucellae isolated from vitreous humor.

50. 50 Diagnosis - Because sx. of brucellosis nonspecific, it is important to: ? Obtain detailed history including: - Occupation, exposure to animals, travel to enzootic areas, and ingestion of high-risk foods, as unpasteurized dairy products. - WBC: normal or low, and may not suggest infectious process. - Anemia, leukopenia, and thrombocytopenia, common findings.

51. 51 Diagnosis (cont’d.) - E.S.R. variable and of little diagnostic value. - Diagnosis of brucellosis made with certainty, when brucellae recovered from blood, bone marrow, or other tissues. - Rate of isolation from blood ranges from 15- 70% depending on methods used and incubation period. - When brucellosis is suspected, Lab. should be informed to maintain cultures for minimum of 4 weeks.

52. 52 Diagnosis (cont;d.) - Bone marrow cultures have higher yield than blood. - Most Labs. Now use rapid isolation techniques, (e.g. BACTEC, Dupont Isolator…etc.), which are satisfactory for recovering brucellae. - Faster isolation time reported for lysis concentration method. - Preliminary studies using PCR, with random or selected primers, are promising, but require additional evaluation.

53. 53 Diagnosis (cont’d.) - Presumptive identification of brucella spp. Made on the basis of morphologic, cultural, and sero- logic features, but confirmation requires: - Oxidative metabolism - Phage-typing, or genotyping procedures. - Results of rapid bacterial identification system should be interpreted with caution, because some brucella isolates misidentified as moraxella phenylpyruvica.

54. 54 Diagnosis (cont’d.) - In the absence of bacteriologic confirmation, presumptive diagnosis made on the basis of high or rising titers of specific antibodies. - Variety of serologic tests applied to brucellosis, SAT, most widely used. - No single titer of brucella antibodies always “diagnostic”, but, most cases of active infection, have titer higher than 1:160. - Rose Bengal test, rapid screening method, but positive sera should always be confirmed by SAT.

55. 55 Diagnosis (cont’d.) - “Febrile agglutinin” tests insensitive, and should not be relied on for diagnosis. - False-negative reactions in SAT, result from prozone phenomenon, and false-positive results from cross-reaction with antibodies to yersinia, cholera, and tularemia. - False-negative and false-positive reactions, avoidable by routinely diluting serum beyond 1:320.

56. 56 Diagnosis (cont’d.) - V. rarely, presence of blocking antibodies causes negative reaction, but, blocking substances identifiable by coombs test or blocking assay. - Brucella “ELISA” can be definitive, when aggluti- nation tests equivocal.

57. 57 Treatment - Antimicrobial therapy of brucellosis relieves symptoms, shortens illness duration, and reduces complication incidence, some of these complications be life-threatening. - Variety of agents active against brucellae, but in vitro susceptibility results do not always predict clinical efficacy, e.g. Beta-Lactam anti- biotics active in vitro, yet often clinically ineffective.

58. 58 Treatment (cont’d.) - Intracellular localization of brucellae, believed to offer some protection against antimicrobials, thus drugs with good intra-cellular penetration are necessary for cure. - Tetracyclines among most active drugs for treating brucellosis, but, relapse rate unaccept- ably high with single-drug therapy, thus combi- nation therapy is recommended. - Many studies showed tetracycline (500 mg PO x 4 times daily combined with streptomycin (1 g/day I.M.) for three weeks is the most effective treatment.

59. 59 Treatment (cont’d.) - Doxycycline, given (200 mg PO/day), has fever GIT side effects, thus becomes tetracyline of choice. - WHO, 1986, recommended use of Doxycycline 200 mg, once daily orally, combined with Rifampicin (600-900 mg once daily orally), both given for 6 weeks, as combination of choice. - Subsequent studies concluded that: - Doxycycline + Streptomycin is more effective than

60. 60 Treatment (cont’d.) - Doxycycline + Rifampicin, especially for pts. with complications such as spondylitis. - Rifamcpicin reported effective for treating brucellosis during pregnancy. - Inclusion of aminoglycosides in treatment regimen suggested, by studies, to be synergistic with other agents in vitro and clinical use.

61. 61 Treatment (cont’d.) - Streptomycin used as preferred aminoglycoside by most studies, reasons exist to use Gentamin instead, because Gentamicin is more active in vitro, less toxic, and can be given as single daily dose (SDD). However, more studies needed to establish optimal schedule and compare both agents (Strept. & Genta.) - Cotrimoxazole (TMP + SMX), gained initial enthusiasm, but subsequent comparative studies revealed unacceptable relapse rate.

62. 62 Treatment (cont’d.) - Cotrimoxazole + Aminoglycoside reported to be successful in treating children younger than 8 years of age, an age in which teeth staining contraindicates Tetracycline use. - Quinolones use found disappointing, despite in vitro activity and good cellular penetration. Thus, these agents are best reserved as adjunctive therapy. - Special problems arise in treating brucella complications, as meningitis, endocarditis, and no consensus of opinions regarding optimal regimen.

63. 63 Treatment (cont’d.) - Most authorities recommend: Doxycycline in combination with two or more other drugs with treatment continued for many months depending on response. - Doxycycline crosses BBB, better than generic Tetracycline, and use successfully with Cotrimoxazole (TMP/SMX) and Rifampicin for brucella meningitis and endocarditis.

64. 64 Treatment (cont’d.) - Third generation cephalosporins achieve high concentration in CSF, but brucella spp. susceptibility variable, thus in vitro sensitivity should be ensured. - Despite cure of brucella endocarditis, cases with antibiotic alone, most cases require combined medical-surgical approach. - Corticosteroids often recommended from neurobrucellosis, but, in the absence of controlled studies, their efficacy is unproved.

65. 65 * In K.S.A., because Rifampicin is one of the essential first line drugs for treating tuberculosis, its use in brucellosis, should be reserved for specific indications (e.g. neurobrucellosis, Brucella endocarditis and Brucellosis during pregnancy.

66. 66 Prevention - Prevention of human brucellosis depend on: Control and elimination of Brucellosis in domestic animals. - Effective attenuated live bacterial vaccines exist for: - B. abortus (Strain 19) - B. melitensis (Strain Rev-1) - No vaccines for B. suis and B. canis - On rare occasions, accidents with 19 and Rev-1 caused human brucellosis.

67. 67 Prevention (cont’d.) - B. abortus strain RB51 (stable rough mutant) largely replaced strain 19 as preferred bovine vaccine (USA). - Strain RB51, has advantage of protecting cattle without inducing antibody response, and RB51 appears to lack virulence for humans and despite accidents, no proven cases of human RB51 infection. - No licensed human vaccine again on brucellosis to deal.