Brucella

Clinical Significance: Brucella melitensis

(sheep/goats) most important sources of brucellosis in humans

Identification of Brucella spp.:

CO2/H2S/Dyes Fuschin, Thionin/Serological Agglutination A, M, R- B. meitensis FT M B. abortus CH F A B. suis T AM B. canis T R B. ovis C T R B. neotomae H A

Clinical Significance: Brucella suis

affects both swine sexes infertility arbortion orchitis lesions of bones, joints

Molecular Biology Techniques: primers, probes

amplify/detect species-specific targets: 16S rRNA 23S rRNA 5S rRNA omp2

Brucellosis: infection in humans

male infertility arthritis endocarditis meningitis

BurR/BurS

(2)-component regulatory system: sensor kinase & response regulator modulates host cell cytoskeleton regulates expression of outer membrane proteins required for full virulence autophosphorylation BurR binds to regulatory region of DNA to turn on mode of operation

Molecular Biology Techniques: IS711

(PCR specific Assay) insertion sequence found in all Brucella occurs in variable numbers, and positions but in constant manner within a spp.

Molecular Biology Techniques: Phage-typing

10+ brucellaphage (narrow host range easily identifies genus, species) phage is associated w/ Brucella spp Phages (commercially available): Tb (Tbilisi) Fi ( Firenze) Wb (Weybridge) Iz (Izatnagar)

Brucella genomes sequenced:

2 circular chromosomes except B. suis biovar 3 (1 circular) all genomes similar (Seq., org., struct.) 0 plasmids/intact lysogenic phages

O-polysaccharide of S-LPS

2 main antigens: A, M (speciation & biotyping) 3 phenotypes: A+M- (B. abortus) A-M+ (B. melitensis) A+M+ (B. suis)

Transmission: Brucella

3 routes: ingestion (contaminated meats/dairy) direct contact (infected animals/products) inhalation (aerosolized organisms)

Brucellosis: human pathogenicity scale

B. melitensis > B. suis > B. arbortus > B. canis

Pathogenesis: Brucella BCV

BCV Brucella containing vacuole: survives w/in phagocytic/nonphagocytic cells interacts w/ early endosomes, late endosomes, lysosomes BCV fuses w/ the ER replicated BCV (rBCV)

Pathogenesis: Brucella rBCV

BCV fuses w/ the ER replicated BCV (rBCV): ER-derived BCVs are replication-permissive organelles after replication, bacteria exits host cell to reinfect adjacent cells

General Properties: Brucella morphology

Gram- coccobacilli, coccoid, rod-shaped no capsules singly, pairs, short chains, groups non-motile no spores

Pathogenesis: Brucella virulence factors

HSP60 heat-shock protein 60 (mediates attachment) CBG cyclic beta-1,2 glucan (early biogenesis BCV) VirB Type IV secretion apparatus BurR/BurS (2)-component regulatory system

General Properties: Brucella LPS

LPS (unconventional structure): low endotoxicity hidden from immune system recognition 2 types (sugars)- smooth-phase S-LPS (long o chain)(high virulence) rough-phase R-LPS (short o chain)(low virulence)(susceptible to complement)

Clinical Significance: names of the disease caused by Brucella spp.:

Most common term- Brucellosis Microbiologists: Sir David Bruce Bernard Bang Clinical presentation of the disease: undulant fever(humans) Sites of outbreaks: Mediterranean/Malta fever

Presumptive differentiation of Brucella spp. from similiar Gram negative organisms:

Oxidase/Motility/X, V factor- Brucella spp. +-- (Ox-pos) Acinetobact spp. --- Bordetella spp. ++- (Ox-pos, Motility) Haemophilus influenzae +-+ (Ox-pos, non-motile, X, V required) X, V factors preformed growth factors found in blood

Brucella species: R/S-LPS form type

Rough LPS form: B. canis (dogs) B. ovis (sheep) Smooth LPS form: B. melitensis (goats) 1st gene sequenced B. abortus (cattle) B. suis ( pigs) B. inopinata (humans)

General Properties: Brucella metabolism

STRICT Aerobes: chemoorganoheterotrophic respiratory metabolism (O2 as TEA) Catalase positive oxidase negative no acid prod. from carbohydrates gelatinase- negative VP negative Methyl red negative Indole negative most strains urease positive

VirB Type IV secretion apparatus

VirB Type IV secretion apparatus: General VirB, Specific VirB required for late survival events alows bacterial effectors to inject in host cell's cytosol subverts signaling pathways favors bacterial growth & pathogenesis

Clinical Significance: Brucellosis, source of infection

Zoonosis: infected animals consumption of milk/milk products Oral entry: raw milk /derivatives Direct contact: fingers common route of entry Inhalation: aerosol contamination of conjuctivae

Treatment: human Brucellosis

antibiotic combo: rifampin (prevents transcription, blocks reverse transcriptase) + tetracycline (inhibits translation) (+ streptomycin) prolonged treatment course: 4-6 wks. (Brucella is intracellular) no antibiotic resistance demonstrated

Clinical Significance: Bovine Brucellosis

caused by B. arbortus (cattle)

Laboratory Diagnosis: cultivation Brucella spp.

colonies small, non-hemolytic, non-pigmented- Agar: Brucella agar (specific; yeast, casein, animal tissue) blood agar (35-37ºC) chocolate MAC MacConkey agar Gram stain: not well retained in Brucella (Gram-) difficult in blood sample

Prevention, Control: Brucellosis

control of infection in livestock controls human brucellosis vaccination: animals RV51 vaccine

Pathogenesis: Brucella

facultative intracellular no classic virulence factors defeats both innate & adaptive immunity: lowers its stimulatory activity & toxicity for cells BCV Brucella containing vacuole

Brucellosis: infection B. melitensis

granulomas small major toxemia

Natural Habitats: Brucella

infected mammals/products not commensals not free-living Brucellosis: occupational disease (cattle, pigs, goats) common lab infection

Brucellosis: onset/ initial episode

influenza-like (fever) limb/back pain severe sweating, fatigue normal/reduced leukocyte count w/ lymphocytosis (high WBC) splenomegaly (enlarged spleen) patients may recover after 2-4 weeks/ suffer series of exacerbations

General Properties: Brucella cell wall

meso-DAP LPS (unconventional structure)

Brucella species: general

named according to host preference Most pathogenic: B. melitensis (goats) 1st gene sequenced B. abortus (cattle) B. suis ( pigs) B. inopinata (humans) Least pathogenic: B. canis (dogs) B. ovis (sheep) B. neotomae (desert wood rat)

Brucellosis: infection B. suis

necrosis abscesses (pus w/ infection)

Brucellosis: general

occupational exposure incubation period: 2-4 wks onset insidious/abrupt subclinical infection (asymptomatic carrier)

Clinical Significance: B. canis infection

occurs in dog handler

Brucella spp. deadly in daylight:

respond to blue wavelength replicate quickly LOVs signaling proteins (signal replication)

smooth vs. rough phase LPS

rough is mutant of smooth

Brucellosis: post-onset 4-8 wks; series of exacerbations

undulant fever (10 day intervals) anemia relapses (months) tissue lesions/ granulomas (inflammation)

Molecular Biology Techniques: other methods

whole-cell protein profile analysis by SDS-PAGE- PFGE RAPD-PCR RFLP-PCR MLVA MLST IS711 (PCR specific Assay) Multiplex-PCR AMOS-PCR ( uses 5 primers) Brucella-ladder PCR (uses 7 primers) Phage-typing