4Brucella: Overview Primarily a disease of animals. Common where significant disease among domestic animals.Common names- Undulant fever, Malta fever, Mediterranean remittent fever.Brucella can go through intact skin.Facultative intracellular bacteria
5Morphology & Physiology Small gram-negative coccobacillusGrows slowly (7 days), at 370 C.On subculture, a minimum of 48 h growthAerobic growth on Chocolate agar and Sheep blood agarWill not grow on MacConkey or Eosin methylene blue (EMB) agar
6Morphology & Physiology Non-pigmented and non-hemolyticNon-motileOxidase: positiveCatalase: positiveUrease: strongly positive, less than 2 hours. Some species within 5 minutes.
7Microscopic Characteristics Brucella spp.poorly stainingsmall gram-negative coccobacilliseen mostly as single cellsappearing like “fine sand”
8Brucella melitensis colonies A. Grows slowly on most standard laboratory media. Usually not visible at 24h.B. Pinpoint, smooth, translucent, non-hemolytic at 48h.
9Public Health Aspects Brucella: Sources Brucellosis caused by 1 of 4 Brucella species:B. abortusSome strainsrequire 5% CO2on initialisolation.Brucella abortus is enzootic in elk and bison in the greater Yellowstone National Park area
132 patient populations Individuals who work with unvaccinated animals B. abortus and B. suisInfections result from:direct contactinhalationIndividuals who ingest unpasteurized dairy productsB. melitensis is the most common agentFarmers, veterinarians, slaughterhouse workers, hunters and laboratory workers.People who travel to or migrate from rural areas of Latin America and the Middle East. Endemic in dairy animals, particularly goats and camels.
14Host Animal - Brucellosis Asymptomatic or mild disease.Predilection for organs rich in erythritol (breast, uterus, placenta, epididymis).Causes sterility, abortions or carrier state in non-human animals.
15Human - BrucellosisSymptomsAcute PhaseAdvanced DiseaseChronic FormSymptoms may not develop for up to 2 months from time of exposureAcute phase is < 8 weeks from onset with non-specific “flu like” symptoms and intermittent feverAdvanced disease is the undulant form, <1 year from onset. Symptoms are undulant fevers, arthritis and epididymo-orchitis in males.Chronic disease (>1 year from onset) can mimic miliary tuberculosis with suppurative lesions in the liver, spleen, and bone. The patient may have recurrent fevers, arthritis, depression, and chronic fatigue syndrome. Brucellosis may lead to granulomatous hepatitis, peripheral arthritis, leucopenia, thrombocytopenia, meningitis, and endocarditis.
16Pathogenesis Brucella mucosal epithelium Following penetration of the mucosal epithelium (ingestion/inhalation routes, abraded skin, or the conjunctiva) the bacteria are transported via macrophages to the regional lymphTransported to lymph nodes, spleen, liver and bone marrow.
17Pathogenesis X Phagosome LysozomeXPhagosomeInside the macrophage the brucella causes an inhibition of phagosome-lysosome fusion.Survival in macrophages establishes chronic infections
18Pathogenesis No exotoxins LPS does not activate the alternative complement pathwayAcute lymphadenitisGranulocyte production in lymphatic tissue, spleen, liver, bone marrow, lymph nodes and kidneys.A potential bioterrorist agent
19Diagnosis Symptoms and history Serological agglutination tests Culture Blood and bone marrow culturesSpleen, liver, joint fluid or abscesses
20TreatmentTetracycline, doxycycline, or trimethoprimsulfamethoxazole in combination and rifampin or gentamicin for 6 weeks to prevent reoccurring infection.
22Tularemia: Overview Primary reservoir in US Insect vectors Rabbits and muskratsInsect vectorsTicksInfection viaInsect bitesHandling contaminated animal tissuesInhalation of aerosolsIngestion of contaminated food or waterExposure in a laboratory settingRabbit fever
23Tularemia: Overview Gram-negative coccobacilli. Low infectious dose Two subspecies of F. tularensis:subspecies tularensis (type A)subspecies holarctica (type B)10 to 50 organisms by aerosol or intradermal route.Subspecies tularensisHighly virulentMost common biovar in North AmericaTypically associated with rabbitsSubspecies holarcticaLess virulentMost common biovar in Europe and Asia
24Morphology & Physiology Tiny gram-negative coccobacillusNonmotile, encapsulatedAerobic slow growing (48 hours) CFastidious organism requires sulfhydryl (cysteine, IsoVitaleX) supplementation for growthGrows wells onChocolate agarBuffered charcoal yeast extract agarfastidious organism and requires cysteine supplementation for good growth on general laboratory media-Cysteine heart agarEnriched chocolate agarBuffered charcoal yeast extract agar
25Colony Characteristics After 48 hours incubationColoniesVery smallwhite to gray to bluish-grayWill not grow on MacConkey or EMB plates.F. tularensis may at first grow on SBA, but upon subsequent passage will fail to grow on standard SBA. On cysteine supplemented agar plates, it is a gray-white, opaque colony, usually too small to be seen at 24 h. After incubation for 48 h or more, colonies are very small, white to grey to bluish-grey. F. tularensis will not grow on MacConkey or EMB plates.F. tularensis on chocolate agar 48 hours growth.
26Microscopic Characteristics Gram Stain showsTiny Gram negative coccobacillusPleomorphicPale stainingSeen mostly as individual cellsDifficult to interpret due to minute size and poor staining.Tiny, faintly staining, pleomorphic gram-negative rods ( mcm X mcm) are noted; cells are smaller than those of Haemophilus species.
27Phenotypic Characteristics Grows slowly at COxidase-negativeWeakly catalase-positive (may be negative)Urea-negativeNitrate-negativeNon-motileBeta-lactamase-positiveSatellite or XV test-negative (unlike Haemophilus)
28Tularemia: Public Health Modes of humans infectionBite of infected flies, or ticksHandling contaminated animal tissues or fluidsDirect contact with or ingestion of contaminated water, food, or soilInhalation of infective aerosols (most likely BT route)Occupational exposure risk for laboratory workers, landscapers, farmers, veterinarians, hunters, trappers, cooks, and meat handlers
29Tularemia: Public Health Endemic in USMajority of cases occur May – September (tick exposure) or winter (hunters).Most in rural areas.Arkansas, Missouri and Oklahoma
30Symptoms Incubation period: 3-5 days (range 1-21 days) Clinical presentation can be divided into groupsUlceroglandular (45-85%) /glandular (10% to 25%)TyphoidalPneumonicOculoglandularOropharyngeal/GastrointestinalProminent lymphadenopathyRecovery followed by permanent immunityTularemia can range from a mild infection to a severe life-threatening illness.
31Tularemia Clinical Types Clinical presentation based on the route of infectionF. tularensis, able to pass through unbroken skin. Incubation period = 1-21 days
32Typhoidal tularemia Bacteremia- Sepsis Fever, chills, headache, myalgias, malaise, sore throat, and anorexia.Likely bioterrorism presentation.Typhoidal tularemia involves a systemic illness without anatomic localization of infection.Organisms enter the bloodstream through breaks in the skin or through mucous membranes and may affect the lungs and reticuloendothelial organsNecrotic foci can occur in any involved organ, and caseating granulomas may develop.Sepsis may occur, leading to shock, organ system failure, adult respiratory distress syndrome, and disseminated intravascular coagulation.
33Pneumonic tularemia Entry into lungs via Severe atypical pneumonia AerosolshematogenousSevere atypical pneumoniaLikely BT presentationOrganisms enter the lungs either through inhalation of infectious aerosols or through hematogenous spread. The infectious dose by the respiratory route is 10 to 50 organismsOnce in the lungs, the organisms rapidly enter pulmonary macrophages (within minutes) and begin replicating. An accumulation of neutrophils and macrophages, can be seen at sites of bacterial replication. The neutrophils appears to play more of a destructive than protective role in the host response.
34Pathogenesis Pathogenesis Virulence factors for F tularensis have not been well defined.Tularensis is a facultative intracellular pathogen that multiplies predominantly within macrophages.
35Facultative intracellular pathogen Capsule protects against complement killingMacrophage uptakebacterial surface polysaccharidesserum complementcomplement C3 receptorsLPS - O antigenprevents maturation of the phagosomemultiply to high levels in cytosolBacterial release via apoptosis
36Diagnosis Symptoms & History Direct staining of clinical specimens with a fluorescein-labeled antibodies.Serum antibody titers of 1:160 or greaterCulture on cysteine-rich mediaNotify Laboratory personnel if you suspect Francisella since it is HIGLY INFECTIOUS
37Treatment of Tularemia Prompt removal of ticks and insect repellent can prevent disease.AntibioticsStreptomycin is the drug of choice
39Overview: 3 species cause human disease Yersinia pestisYersinia enterocolyticaYersinia pseudotuberculosis
40Overview: Plague Yersinia pestis; a gram-negative bacterium. Three forms of clinical illness;BubonicSepticemicPneumonicPneumonic is the only one transmitted through aerosals.
41Plague: Overview Natural disease of rodents Fleas that live on rodents transmit the bacteria to humans, in the bubonic form.This disease occurs in many areas of the world, including the United States.
42Plague: Overview U.S. averages 13 cases/yr (17 in 2006) Plague is endemic in the desert southwest.Most cases occur in summer.New Mexico and Colorado had the majority of plague cases in 2006
43Microscopic Characteristics Y. pestis appear as single cells or short chains of plump, gram-negative rods.
44Microscopic Characteristics Gram stain:In direct smears, bacterial cells may be inside or outside of leukocytes.The Gram smear morphology is suggestive but not specific for Y. pestis.Bipolar staining of a plague smearprepared from lymph aspiratedfrom a bubo of plague patient.
45Microscopic Characteristics Bipolar staining occurs when using Wayson, or Giemsa stain.CDC
46Colony Characteristics Grows well on most standard laboratory media.Sheep Blood AgarGray-white translucent coloniesPinpoint, gray-white, non-hemolytic at 24 hoursBlood agar plate of Yersinia pestis at 48 hours.CDC/Dr. Brodsky
47Y. pestis: Physiology Non-motile Pleomorphic gram-negative bacillus Urease, and oxidase negativeFacultative anaerobeOptimal growth at 28o CFacultative intracellular parasite
48Public Health Aspects of Plague Fleas carry Y. pestis in their intestinal tract.When feeding the fleas regurgitate uncapsulated organisms.Bacteria re-encapsulate and grow.Progeny are resistant to intracellular killing
51Bubonic Plague Regional lymphadenitis (Buboes) Cutaneous findings Inguinal, axillary, or cervical lymph nodes most common80% can become septic60% mortality if untreatedCutaneous findingsPossible papule, vesicle, or pustule at inoculation sitePurpuric lesions - late
52Bubo swollen inguinal lymph node or bubo. After the incubation period of 2-7 days, symptoms of the plague appear.
53Pneumonic Plague Pneumonic From aerosol or septicemic spread to lungs.Person-to-person transmission by respiratory droplet.100% mortality untreated.Pneumonia progresses rapidly to dyspnea, cyanosis.Death from respiratory collapse/sepsis.
54Septicemic Plague Primary or secondary Severe endotoxemia Secondary from bubonic or pneumonic forms100% mortality if untreatedSevere endotoxemiaSystemic inflammatory response syndromeShock, Disseminated intravascular coagulopathy (DIC)Adult Respiratory Distress Syndrome (ARDS)
55Y. pestis: Virulence Determinants 3 virulence encoded PlasmidsVirulence is up-regulated at 37°CCapsule (F1 antigen)
56Yersinia Outer Proteins (Yops) 11 different proteinsAntiphagocyticInhibit productionproinflammatory cytokinestumor necrosis factorCytotoxin2 are antiphagocyticOthers inhibit the proinflammatory cytokine, tumor necrosis factor production and induces macrophage apoptosisanother Yops is a cytotoxin that causes actin filament disruption
57Yops Targets dendritic cells macrophages Neutrophils does not target B and T lymphocytes
58F-1 Antigen Glycoprotein capsule expressed at 370 C Not expressed in flea hostAntiphagocyticAntibodies to F-1 are protective
59Plasminogen activator (fibrinolysin) and Coagulase Plasmid encoded proteinsPromote dissemination of organisms from the clot at the bite siteCoagulase is produced at 280 C but not at 320 C.
60Diagnosis Examination of Bubo aspirate, blood, sputum stained for bipolar stainingFluorescent-antibodyCulture (hazardous)
61Plague Treatment Y. pestis is susceptible to a variety of antibiotics. streptomycin, tetracycline, and doxycyclinePeumonic plague is contageous and isolation is recommended.
62Clinical Case30 year old man from Colorado, went to a hospital emergency department with a 3-day history of fever, nausea, vomiting, and right inguinal lymphadenopathy.Patient was not a hunter nor had he been in the woods recently but he did have dogs.He was discharged home without treatment.Lymphadenopath could be from Francisella tularensis (Ulceroglandular or Glandular) or Yersinia pestis (bubonic). What should you look for? Answer: Look for papules or pustules. No papules or pustules were seen on right leg.
63Cultures of blood and a lymph node aspirate. Three days later, the man returned and was hospitalized with sepsis and bilateral pulmonary infiltrates.One of the patient's dogs had serologic evidence of past Y. pestis infection.Cultures of blood and a lymph node aspirate.What should have been asked the first day? Answer: Any recent tick or flea bites.