Brucella Gram____? Shape?
Gram negative, small, coccobacilli (more coccoid) Pairs or clusters
Brucella Name the dominant surface antigens in LPS in rough versus smooth colonies
Rough: "R" antigen Smooth: "A" abortus or "M" melitensis antigens
Brucella What is significant about the cell wall?
Peptidoglycan layer = thicker than other Gram negative (modified acid fast stains for diagnosis)
Brucella Stable in environment?
No, obligate pathogen Survives 4 months in milk, urine, water, damp soil but NEEDS MAMMALIAN HOST
Brucella Often chronic or acute infections?
Mainly chronic infections
Brucella Target site
Reticuloendothelial system & genital tract Abortion & epididymitis/orchitis
Brucella Pathogenic species
*B. melitensis, B. abortus, B. suis* B. canis, B neotomae, B. ovis B. cetaceae, B. pinnipediae
Brucella Virulence factors
Cell Wall Erythritol Outer membrane proteins Superoxide dismutase & catalase T4SS
Brucella Erythritol role
4 carbon sugar alcohol *Allantoic fluid factors* (stimulates growth) Preferential location for reproduction & mammary glands
Brucella Omp (Outer membrane protein)
Stimulate delayed type hypersensitivity
Brucella Superoxide dismutase and catalase function
Defense against oxidative killing
Brucella T4SS function
Brucella Resistance factors
Survives freezing & thawing Survives in milk & other fluids Pasteurization kills it
Genome very stable Different species vary in host preference & degree in virulence
Brucella What type of colonies are more virulent? Smooth or rough?
Smooth more virulent than rough Depends on polysaccharide side chain in LPS
What factors change Brucella host preference and virulence?
CO2 requirements H2S production Urease production Susceptible to different concentrations of certain dyes Susceptibility to bacteriophage (Tbilisi phage)
How does Brucella abortus differ from other strains?
B. abortus is susceptible to Tbilisi phage, others are resistant
Brucella Zoologic & Geographic reservoirs
Obligate parasite Host preference (some broad range) Survival time in environment depends on temperature & moisture
Cattle Smooth colonies, virulent Bison, camels, yaks
Swine Smooth colonies, virulent Hares, reindeer
Goat & Sheep Smooth colonies, virulent Camelids
Wood Rat Smooth colonies, virulent
Brucella Modes of transmission
Ingest cow/goat milk In utero (direct) Venereal Urine (dogs) Insects (maybe, minor)
Describe Brucella's venereal transmission
Male sex gland infections Dissemination occurs without lesions Common in swine, sheep, dogs
Penetrates intact mucosa--> Macrophage--> survives --> Regional lymph node --> hematogenous dissemination & localization in reticulo-endothelial system & repro tract --> Abortions
How do Brucella species survive inside macrophages?
T4SS, Superoxide dismutase, catalase
How do Brucella species cause abortions?
Interference with fetal circulation / placentitis Endotoxin Fetal stress / Inflammation
What type of inflammation is shown in the picture?
What can cause Brucella infections to move extra-genitally? (Disseminate)
Grossly visible lesions in placenta Intercotyledonary thickening with yellow gelatinous fluid Necrotic cotyledon with thick brown exudate Aborted edematous fetus Male genitalia
Describe Brucellosis in males
Palpable enlargement of epididymus Increase neutrophils in semen (acute cases) Orchitis Scrotal dermatitis in dogs Results: decrease fertility, sometimes sterility
Describe orchitis from Brucellosis in males
Necrosis in testicular parenchyma with pus, prostatitis, fibrinopurulent, seminal vasculitis
What is shown in the picture? What species caused it?
Necrotic cotyledons in aborted material. B. melitensis
Brucella Disease Pattern Female reproductive tract
Abortion (most commonly) Retained placenta Mastitis (goats)
Brucella When will it cause cattle to abort?
Brucella When will it cause swine to abort?
At any time
Brucella When will it cause dogs to abort?
~50 days gestation
Brucella Repeat abortions or lasting immunity?
Females usually abort once Acquired immunity
What does this picture show?
Brucella melitensis Stillbirth fetus with necrotic placenta
What do these pictures show?
Orquitis - Swelling & irregular conformation of tail of epididymis of ram infected with Brucella ovis.
Brucella Extragenital manifestations in Swine
Arthritis Lumbar spondylitis Tissue necrosis Posterior paralysis
Brucella Extragenital manifestations in Dogs
Meningoencephalitis Osteomyelitis Discospondylitis Anterior uveitis Ocular manifestations
Brucella Extragenital manifestations in Cattle
Hygromas - Chronic (false bursa, on joint sacs)
May be secondary to abortion
Brucella Extragenital manifestation in Horses
Poll evil or fistulous withers
Brucella Extragenital manifestation in Humans
Undulant Fever Malta Fever
What body system does undulant fever affect?
Common signs of Undulant Fever
Mild lymphadenopathy, splenomegaly, hepatomegaly 2-3 weeks post-exposure Fever, chills, night sweats, fatigue, muscle & joint pain, backaches, depression, insomnia, arthritis, osteomyelitis, endocarditis
How do humans get Undulant fever?
Handling infected tissues Often: aborted fetuses, placentas, post-abortion uterine fluieds, milk, milk products Human-human rare
What human professions are at risk for Brucellosis?
Veterinarians, ranchers, slaughterhouse workers
What Brucella species can affect humans?
B. melitensis** B. suis B. abortus B. canis
Which Brucella species do not affect humans, generally?
B. ovis B. neotomae (always questionable)
What predisposes an animal to a Brucellosis infection?
Younger more resistant Sexually mature animals MORE susceptible, (infected for life possible) Higher herd size & density
What helps SPREAD Brucellosis in swine herds?
New boars Confine breeding swine in common pens Feral swine
What helps CONTROL Brucellosis in swine herds?
Separate the breeding pairs Quarantine boars Limit exposure to feral population Eliminate infected boars Minimize exposure to aborted tissue
How is Brucellosis often spread in sheep herds?
Dissemination during breeding season Older rams more likely infected Homosexual rams spread it
What are common differentials to Brucellosis in older rams?
Actinobacillus seminis Histophilus somni
How is Brucellosis spread in dogs?
Close confinement/ Kennels Urine!
Brucella Immune mechanisms in pathogenesis
Antibodies = protective & detrimental Phagocytes disseminate the organism
Describe the role of antibodies in a Brucella infection
Low levels= complement High levels = opsonization IgA autoantibodies in dogs
What class of immunoglobulin are used in low level antibody production?
Mainly initially IgM and low IgG (Cause complement-mediated lysis)
What class of immunoglobulin are used in high-level antibody production?
High levels of IgG Survive inside phagocytes Interfere with complement and opsonize bacteria
Brucella Immunologic aspects Mechanisms of Resistance & recovery
Effective immunity = **cell mediated** Macrophages & NK cells
What cytokines are released in cell mediated immunity against Brucella infections?
IL-2, -12, -18 IFN-g TNF
When will an animal develop the most effective immune response to Brucella?
**Better response if infected prior to sexual maturity**
Brucella Vaccinations in cattle
B. abortus 45/20 (killed, 2 doses) B. abortus 19 & RB51 (live-attenuated, 1 dose, 4-8mo old)
What can happen if you vaccinate a full grown bull for B. abortus?
What are some problems with the B. abortus vaccines in cows?
Abortions Serological false positives
Brucella vaccines for Goats and Sheep
B. melitensis Rev 1 (Live attenuated) B. melitensis H38 (killed) -Sometimes protects against B. ovis
Are there vaccines for B. suis or B. canis?
Can vaccines for B. melitensis be used in eradicated countries?
Cannot be used in countries free of B. melitensis because of serological test interference
What is important about submitting suspected Brucella specimens?
Biosafety level 3 practices (prevent aerosolization) Submit to reference labs!
What samples should be collected for Brucella lab diagnosis?
Abscess material, semen, vaginal fluids, milk samples, blood (dogs, bacteremia of B. canis) Necropsy & Abortion material
What necropsy material is collected for Brucella lab diagnosis?
Spleen, liver, udder, lymph nodes, epididymis, testicle, accessory male sex organs
What abortion material is collected for Brucella lab diagnosis
Abomasal fluids Lungs of aborted fetus Placenta
What direct exam methods are used for Brucella Lab diagnosis?
Gram Stains Modified Gram Stains Modified Ziehl-Neelsen Macchiavello stains
Brucella Lab Dx Describe Gram Stains (fetal contents vs. semen)
Fetal contents reveal high # gram negative cocci Few numbers of bacteria in semen
Brucella Lab Dx Modified Gram Stain
Use carbol fuschin instead of safranin
Brucella Lab Dx Modified Ziehl-Neelsen (with good differentials)
0.5% Acetic acid to destain Stain Red DDx:: Coxiella burnetii and Chlamydophila abortus
Brucella Lab Dx Macchiavello stains
Mainly from fetal stomach content & placenta Stain red
Brucella Lab Dx Microscopy
Gram stain & immunofluorescence
Brucella Lab Dx Isolation - samples used?
Tissues Fluids Milk (cream & sediment)
Brucella Lab Dx Isolation - Agars used?
Serum dextrose Tryptose Columbia agar w/ 5% serum Brucella agars
Brucella Lab Dx Isolation - Mediums used?
Farrell's medium Thayer-martin modified medium
Brucella Lab Dx Isolation - Mediums - Conditions for culture
Aerobic environment, 37C, 10% CO2, 10-21 days Bluish color under obliquely transmitted light
What is the most sensitive method for Brucella laboratory diagnosis?
Describe animal inoculation in Brucella lab diagnosis
Necessary when low # are present Guinea pigs are most sensitive Test 3-6 weeks post inoculation
Describe the colonies on blood agar
Brucella Gram negative, coccobacilli, non-hemolytic Catalase & oxidase positive Urease positive (except B. ovis) No fermentation of glucose or lactose
Brucella species and biovar determinations?
Urease production CO2 requirements H2S production Oxidation of metabolic substrates Agglutination in mono-specific antisera Growth in presence of varying concentration of thionin Basic fuschin Phage typing
Molecular techniques in Brucella Lab diagnosis
PCR, real-time PCR, RLFP PCR Strain 19 - deletion in ery locus Strain RB51 - Insertion in wbo A gene
What makes strain 19 different from other Brucella strains.
Deletion in the ery locus
What makes strain RB51 different?
Insertion sequence in wbo A gene
What does the Brucellin Test test?
Type 4 Hypersensitivity
What tests for B. ovis best?
Agar gel immunodiffusion
Brucella Immuno Dx Rose-Bengal plate test
Antigen suspension, pH=3.6, allows IgG1 agglutination Positive results need confirmation
Brucella Immuno Dx Brucella milk ring test
Purple ring develops in top with cream (picture)
Brucella Immuno Dx Confirmatory tests
CFT - complement fixation test Indirect ELISA
Brucella Immuno Dx For canine brucellosis
Rapid slide agglutination test
Brucella Immuno Dx Which species share antigens?
Antigens shared with Y. enterocolitica O:9 and E. coli O:157
Brucella Control & Prevention
Depends on animal species Brucella Species Husbandry Availability/Efficacy of vaccines
Brucella Treatment Livestock
No treatment in livestock High failure Cost Problem maintaining infected during eradication program
Brucella Treatment Rams with B. ovis
Prior to palpable epididymal lesions, abscesses or fibrosis: Tetracycline or dihydrostreptomycin After these lesions - difficult to treat
Brucella Treatment Dogs
Neuter affected animals Antibiotics (tetracycline & dihydrostreptomycin) Cull males in breeding program Don't treat breeding colonies
Brucella Prevention Immunization alone
Reduces # abortions No eradication
Brucella Prevention Bovine Brucellosis Immunization THEN test & slaughter (Tests in dairy vs. beef)
Vaccinate young cattle, test @ sexual maturity Seropositives culled & slaughtered -Ring test in dairy -Blood test in beef cattle
Brucella Prevention Testing & removal without immunization
Depopulation of entire swine, sheep, goat population Re-stock
How often Rams palpated?
Rams tested twice per year by palpation
Brucella Prevention Dogs (# tests needed for "free" status)
Dogs tested before breeding Need 3+ seronegative test results for "free" status