BRUCELLA General Features • • • • Gram-negative coccobacillus Obligate aerobic, fastidious Nonsporing, nonmotile, noncapsulated Human infection is usually associated with occupational or domestic exposure to infected animals or their products History • 1886- David Bruce - British army physician isolated the first recognized species, Brucella melitensis • Melita is Roman name for Malta from Malta Island (Europe)  Malta fever • 1897- Danish physician Bernhard Bang isolated Brucella abortus from cattle (Bang’s disease) • 1914 - Jacob Traum - Brucella suis was isolated from aborted swine in USA. Nomen System of Classification Antigenic Structure • Two major types of lipopolysaccharide (LPS) antigens - M & A • P resent in varying proportion in three major species of Brucella • B. Melitensis - M antigen is predominant B. Abortus - A antigen is predominant • • B. Suis - either M or A antigens • Virulent colonies on primary isolation– smooth and possess LPS antigen. On repeat subcultures, strains may lose LPS  rough strains which may not agglutinate with anti-M or anti-A antisera Pathogenesis • Transmission - From infected animals to man. No man-to-man transmission • Direct contact: MC route - contact with infected animal tissue, blood, urine, vaginal discharge or placenta with abraded skin or mucosa of men, who are involved in animal handling • Food-borne: - Ingestion of raw milk or dairy products • Air-borne: Inhalation of dust or aerosols in infected cowshed or slaughter houses. Pathogenesis • Spread • Initial site of infection → local lymph glands → spillover to bloodstream  bacteremia → disseminate to various organs. • Organs Involved - Facultative intracellular pathogens, primarily infecting reticuloendothelial system - Special predilection for placenta (erythritol) • Musculoskeletal tissues & genitourinary systems Pathogenesis • Intracellular Survival and Virulence Factors • cell-wall LPS - Endotoxin activity is relatively low, it plays a key role in: • Rresistance to phagocytosis and serum complement-mediated killing of bacteria • Suppressing phagosome-lysosome fusion • Diverting the internalized bacteria into vacuoles located in endoplasmic reticulum, where intracellular replication takes place • D evelopment of pyrogenicity. Pathogenesis • Other virulence factors • Type IV secretion system (VirB): Secretory system that regulates intracellular survival in phagosomes & trafficking • Cu-Zn superoxide dismutase: Inhibits reactive oxygen radicals (B.abortus) • Nucleotide-like substances - Inhibit phagolysosome fusion & reactive oxygen radicals produced from neutrophils (B.abortus) Host’s Immune Response • Cell-mediated immunity (CMI) - key to control infection • Activation of T helper-1 (TH1) interferon γ  macrophage activation & killing of intracellular Brucella • TH2 activation IL-4, IL-6 and IL-10 downregulates protective TH1 immune response & stimulates humoral immunity • Antibodies play only a minor role - ineffective om intracellular orgamisms Clinical Manifestations • Incubation period - 1 week to several months • Classic triad: Fever with profuse night sweats, arthralgia/arthritis & hepatosplenomegaly • Typhoid-like illness: Except that it is less acute, less severe with undulating pattern of fever and more musculoskeletal symptoms • Undulating fever: Typical remittent course, Clinical Manifestations • Musculoskeletal symptoms – in one-half of patients, mimic skeletal tuberculosis • Vertebral osteomyelitis - lumbar and low thoracic • Septic arthritis: Knee, hip, sacroiliac & shoulder joints • Other nonspecific symptoms: abdominal pain, headache, diarrhea, rash, weakness/fatigue, weight loss, vomiting, cough, pharyngitis, & refusal to eat (children) Clinical Manifestations • CNS: Depression and lethargy with meningitis or lymphocytic • CVS: Endocarditis rarely • Genitourinary manifestations: Acute epididymo-orchitis, prostatitis, salpingitis and pyelonephritis. • All nomen species produce similar manifestations with slight variations - B. melitensis - more acute and aggressive - B. suis - focal abscess - B. abortus - insidious and chronic - B. canis - acute gastrointestinal symptoms EPIDEMIOLOGY • Zoonotic disease, worldwide • Human infection closely related to the prevalence in various domestic animals • Endemic area: Areas where animals are raised in large numbers - Mediterranean zone, Eastern Europe, Central Asia, Mexico and South America • Rare in most European countries, Australia and North America • Prevalence: True prevalence of human brucellosis is difficult to estimate. Under-reported - inapparent or difficulty in diagnosis EPIDEMIOLOGY • Sources of infection - Infected animals excreting the organisms in urine, milk, placenta or vaginal discharge - Contaminated animal food products – dairy products, especially soft cheeses, milk, icecream and rarely raw meats and bone marrows. • People at higher risk - farmers, shepherds, goatherds, butchers and abattoir workers in endemic areas LABORATOR Y DIAGNOSIS...Culture • Sample • Blood, bone marrow, CSF, joint fluid or other tissues. • Blood - collected during the febrile period before starting antibiotics. Multiple blood cultures (5–10 mL, 2–3 times a day) over 3 consecutive days yield better result • Bone marrow culture remains positive even after starting antibiotics and gives a higher yield than blood culture. • Culture Conditions - Highly fastidious & growth is enhanced by addition of blood or serum - Obligate aerobes, but growth is promoted in the presence of 5–10% CO2 -P rimary isolation requires prolonged (several weeks) incubation at 37°C. LABORATORY DIAGNOSIS • Culture Media - Biphasic media (Castaneda’s) - Brain heart infusion (BHI) broth/agar - Serum dextrose broth/agar • Advantage - Less contamination rate • Incubated for 2-4 weeks or more LABORATORY DIAGNOSIS • A utomated systemsBACTEC and BacT/ALERT - Take less time for isolation (7–10 days) & have better recovery rates LABORATORY DIAGNOSIS • Culture Smear and Motility Testing • Blood agar & Chocolate agar - Small, smooth, transparent, low convex nonhemolytic colonies • Gram-staining - gramnegative coccobacilli • Nonmotile, non-capsulated, nonsporing Biochemical Tests • • • • Do not ferment carbohydrates Catalase positive Oxidase positive Urease test - variable but often rapidly positive, especially with strains of B. suis and B. canis • Nitrate is reduced to nitrite (except B. ovis) • Citrate, indole, MR and VP tests are negative. Nomen Species Identification • - Based on: Preference of animal host CO2 requirement H2S production Tolerance to bacteriostatic dyes, such as basic fuchsin and thionine - Agglutination with monospecific antisera: Predominant A or M antigen of LPS - Lysis by bacteriophage - Tbilisi phage Serological Tests (Antibody Detection) • Standard Agglutination Test (SAT) - Gold standard test against which other serological tests are compared - Principle - Tube agglutination test - Significant titer >1:160 in nonendemic areas - In endemic areas demonstration of rising titer by repeating the test after 2–4 weeks is more reliable Standard Agglutination Test • Interpretation: • SAT detects antibodies against antigens of smooth LPS • Use of B. abortus can detect antibodies against B. melitensis, B. abortus & B. suis but not B. Canis (as it lacks smooth LPS) • SAT measures total quantity of agglutinating antibodies but does not differentiate between immunoglobulin isotypes • In acute infection, IgM antibodies appear early and are followed by IgG and IgA antibodies. • As the disease progresses, IgM levels decline, and the avidity and subclass distribution of IgG and IgA change. Thus, SAT may become negative. Standard Agglutination Test  • • • False-negative SAT may occur due to: Prozone phenomenon – overcome by serial dilution of sera ‘Blocking' or nonagglutinating IgG or IgA : Can be removed by: Prior heating of serum at 55°C for 30 min 4% saline to dilute patient’s sera Coombs antiglobulin test (most reliable). False-positive SAT - due to antigenic cross reactions Escherichia coli O157, Francisella tularensis, Salmonella enterica group N, Stenotrophomonas maltophilia and Vibrio cholerae. Serological Tests to Detect IgG Antibody • • • • -mercaptoethanol (2ME) agglutination test: 2 Destroys agglutinability of IgM, not altering IgG SAT performed in 2ME treated serum detect only IgG ELISA detecting antibodies to LPS antigen – More sensitive Can detect IgM and IgG antibodies separately Molecular Method PCR - rrs-rrl gene, Omp2 gene (outermembrane protein), insertion sequence IS711 • Brucellin skin test & guinea pig inoculation - obsolete now. Diagnosis of Brucellosis in Animals  Isolation from milk and dairy products: Culture of cream of the cow’s milk  Antibody detection in milk: • Milk ring test: Pooled milk + drop of the stained Brucella antigen in a narrow test tube and incubated at 70°C for 40–50 minutes Positive - Blue ring at the top, with milk unstained • • Rose Bengal card test and whey agglutination test Treatment • G old-standard regimen in adults: Streptomycin for 14–21 days plus doxycycline for 6 weeks • WHO regimen in adults: Rifampin for 6 weeks plus doxycycline for 6 weeks • Relapse or treatment failure occurs in 5–10% of cases • For CNS involvement: Ceftriaxone is added to the regimen and treatment is prolonged for 3–6 months. Prevention  Prevention in Animals: • Test and slaughter: Active case finding by skin test or CFT • Vaccine: Live attenuated vaccine using B. abortus 19 strain for cattle and B. melitensis rev-1 strain for sheep and goat  Prevention in Humans: • General precautions - pasteurized milk or properly cooked food. Prevent direct contact with animals. • Vaccine: Live attenuated B. abortus 19–BA • Provides short term protection & high reactogenicity.