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• The genus Brucella consists of six species,
  four of which cause human brucellosis 布
  鲁菌病: Brucella melitensis 羊布鲁菌,
  Brucella suis 猪布鲁菌, Brucella abortus
  牛布鲁菌, and Brucella canis 犬布鲁菌
• Are all intracellular organisms
• B. neotomae; B. ovis
• Brucella are small (0.4~0.8
  ×0.5~1.5μm), non-motile,
  non-capasulate, gram-
  negative coccobacilli.
• The organism is aerobic,
  and their nutritional
  requirements are complex.
• All strains grow best in a
  medium enrich with animal
  serum and glucose
• 5-10% carbon dioxide
 Antigenic Structure and
• Two main antigen: A and M
• The three main Brucella differ from one another
  in the amount or the two main antigen they have
  in common :
 B.abortus : A:M=20:1
 B.melitensis: A:M=1:20
 B.suis:    A:M=2:1
                  B. abortus
• Bacteria is excreted in genital secretions
  (including semen), milk, colostrum.
• Survival time:
    Cheese at 4oC: 180 days !!!
    Water at 25oC: 50 days
    Meat and salted meat: 65 days
    Manure at 12oC: 250 days !!!!
• Widespread:       Cattle, Bison, Elk, Deer, Moose, Horse,
  Sheep, Goat, Swine, Donkey, Dogs, Birds, Hares, Fox, Rats,
  mice, Camels and Human.
                  B. abortus
                                                                                    B. abortus
• Bacteria is excreted in genital secretions
  (including semen), milk, colostrum.
• Survival time:                                                  • Sources of Human Infection:
                                                                        Raw milk and products /Direct contact
    Cheese at 4oC: 180 days !!!
    Water at 25oC: 50 days                                        • Portal of entry: oral mucosa, nasopharynx and
                                                                    conjunctivae, genital then X in regional lymph
    Meat and salted meat: 65 days                                   node and spread to RES (nodes of udder, uterus,
    Manure at 12oC: 250 days !!!!                                   erythritol...). Placentitis with endometritis. Fetus
                                                                    die with edema /congestion of lung, dissimenated
• Widespread:       Cattle, Bison, Elk, Deer, Moose, Horse,
                                                                    hemorrhages of epicardium and splenic capsule.
  Sheep, Goat, Swine, Donkey, Dogs, Birds, Hares, Fox, Rats,
                                                                    Bacteria in lung and digestive tract of the fetus.
  mice, Camels and Human.

                                                                                        B. suis
                                                                • Wild pigs, Rats, Swine.
              B. melitensis                                     • Abortion,metritis,
                                                                  bursitis, spondylitis
                                                                  (Lumbar and sacral),
• Goat (1886), Sheep,                                             arthritis, orchitis,
  Cow (1905 in                                                    paralysis.
  Malta), Swine,                                                                 Brucella canis
  Hares, Camels,
  Buffalo, Impala.                                             • Brucella canis was first described as a cause of
                                                                 abortion in beagles in the USA
                                                               • It was subsequently shown to infect dogs in many
                                                                 other countries, irrespective of breed
                                                               • An occasional cause of brucellosis in humans
Spread of Brucella in the body
         Incubation period
• Acute or subacute disease follows an incubation
  period which can vary from 1 week to 6 or more
• In most patients for whom the time of exposure
  can be identified, the incubation period is between
  2 and 6 weeks
• The length of the incubation period may be
  influenced by many factors
   – virulence of the infecting strain
   – size of the inoculum
   – route of infection
   – resistance of the host
             Portals of entry
• Oral entry - most common route
  – Ingestion of contaminated animal products
    (often raw milk or its derivatives)
  – contact with contaminated fingers
• Aerosols
  – Inhalation of bacteria
  – Contamination of the conjunctivae
• Percutaneous infection through skin
  abrasions or by accidental inoculation
       Clinical Manifestations
• The presentation of brucellosis is characteristically
• The onset may be insidious or abrupt
• Influenza-like with fever reaching 38 to 40oC
   – Limb and back pains are unusually severe,
     night sweating and fatigue are marked.
   – Anorexia, weakness, severe fatigue and loss of
     weight, depression
   – Headache
• The leukocyte count tends to be normal or reduced,
  with a relative lymphocytosis
   – Relative leukopenia
• On physical examination, splenomegaly may be the
  only finding.
• If the disease is not treated, the symptoms
  may continue for 2 to 4 weeks
  – Many patients will then recover spontaneously
  – Others may suffer a series of exacerbations
• May produce an undulant fever in
  which the intensity of fever and
  symptoms recur and recede at about 10
  day intervals.
   • Cyprus fever/Gibraltar fever/Malta
     fever/Rock fever/Undulant fever

• Most affected persons
  recover entirely within                      Undulant fever

  3 to 12 months
• Some will develop                     39.5

  – involvement of various           37.0

  – a few may enter an ill-defined
    chronic syndrome.
• Arthritis, often sacroiliitis, and spondylitis
  (in about 10 percent of cases)
• central nervous system involvement including
  meningitis (in about 5%)
• Uveitis, epididymo-orchitis
• Endocarditis very rare
• In contrast to animals, abortion is not a
  feature of brucellosis in pregnant women.
Chronic Brucellosis- Depression
             Population risk
• The main source of infection for the general
  population is dairy produce prepared from
  infected milk.
• B. melitensis presents the greatest hazard.
• The milk of infected sheep and goats may contain
  large numbers of viable organisms, which become
  concentrated in products such as soft cheeses.
• Indeed, soft cheese has been recognized as a
  major vehicle of infection in the Mediterranean
  region, the Middle East and Latin America
          Occupational hazard
• Infection arises from occupational or domestic
  contact with infected animals or with an
  environment contaminated by their discharges
• Farmers and their families, abattoir workers,
  butchers and veterinarians are particularly at
• Infected animals that have recently aborted or
  given birth present the greatest hazard
Extending spectrum of zoonosis
• The recent isolation of distinctive Brucella strains,
  tentatively named Brucella maris, from marine
  animals in the United Kingdom and the United
  States extends the ecologic range of the genus and,
  potentially, its scope as a zoonosis
   – seals, sea otters, dolphins and porpoises
• An incident of laboratory-acquired infection
  suggests that this type is pathogenic for humans
• Infection could result from occupational contact
  with infected seals or cetaceans.
• Pasteurization of dairy products and use of
  protective clothing prevent human infection.
  More importantly, systematic identification
  and elimination of infected animals and
  vaccination of animals reduces the reservoir.
• Eradication of brucellosis in cattle can be attempted by
  test and slaughter,active immunization of heifers with
  avirulent live strain 19,combined testing,segregation,
  and immunization.Cattle are examined by means of
  agglutination tests
• Active immunization of humans against brucella
  infection is experimental.Control rests on limitation of
  spread and possible eradication of animal
  infection,pasteurization of milk and milk products, and
  reduction of occupational hazards wherever possible.
• Brucella may be susceptible to tetracyclines or
• Symptomatic relief may occur within a few days
  after treatment with these drugs is begun.
• However ,because of their intracellular location,the
  organisms are not readily eradicated completely from
  the host.
• For best results,treatment must be
  prolonged.Combined treatment with streptomycin
  and a tetracycline may be considered
     Genus Escherichia

     Genus Yersinia
              • Y. pestis
              • Y. enterocolitica
              • Y. pseudotuberculosis
         Biological Features

– Small, 0.5-0.8 μm in width
  1.0-2.0 μm in length.
– Gram-negative rods.
– Sometimes appearing as
  cocco bacilli.
– Bipolar Staining:Retaining
  stain at the ends of cells.
         Biological Features
• Cultural Features
  – Facultative anaerobes.
  – Optimal growth temperature range form 28˚C
    to 30˚C.
  – Optimal growth pH: 6.9~7.2.
  – Growth is more rapid in media containing
    blood or tissue fluids.
  – Nonmotile when grow at temperatures above
    30 ˚C.

           Flea   Bite
                         Respiratory Tract
         Antigenic Structure
• F1 Antigen:
• V,W Antigen:
• Yersinia Outer membrane Protein (Yop)
• Murine Toxin (MT)
     0.3%-0.4% formaldehyde Toxoid
• Endotoxin (LPS)
 Ca2+ Dependent
                V-W Gene        F1 Gene         F1 Antigen

                Plasmid                     Plasmid

W Antigen
             V Antigen
            Y. Pestis Virulence factors schematic diagram
                                         Invade        Lymph Nodes
                                                       In Groin and
    Y. pestis            Phagocyte

  Enter                Pneumonic Plague               Bubonic Plague
  Respiratory System

                                     Invade Blood Stream
Septicemic Plague                                            meningitis
• Clinical Forms :
  – Bubonic Plague: High fever, Swelling,
    Bleeding, Necrosis of lymph nodes
  – Pneumonic Plague: chills, cough, respiratory
    failure, circulatory collapse ——Black Death
  – Septicemic Plague: Fever (39-40 ˚C) , Shock ,
   Y.Enterocolitica & Y.Pseudotuberculosis
• Gram negative, No capsule, No spore, Facultative
• V-W antigen
• More than 50 serotypes of Y.Enterocolitica
  6 serotypes of Y.Pseudotuberculosis
• Diseases:
   –   Gastroenteritis
   –   terminal ileitis, appendicitis, mesenteric lymphadenitis,
   –   dermatitis contusiformia, arthritis
   –   Septicemia
• Sanitary precautions, Antibiotic
• Plague

  – Probably originated in Asia or central Africa.
  – One of the earliest record pandemics occurred
    in 542 B.C.
  – Three pandemics in the history.
  – 1989-1998:5440 cases, 681 dead.

         Humoral                  Cellular
         Immunity                 Immunity

       Antibody To:
    1) F1 Ag     2) V,W Ag      Phagocytose

Promote phagocytose , agglutinate and kill bacteria
• A. Specimens:
  –    Aspirates of lymph nodes
  –    Cerebrospinal fluid
  –    Blood
  –    Sputum
 B.   Smears:
   Giemsa’sstain
   immunofluorescent stain
• C. Culture:
   – All materials Cultured on blood agar and
     MacConkey’s agar and in infusion broth
   – Positive in 24 hours
   – Tentatively identified by biochemical reations
     Definite identified by immunofluorescence

CAUTION: All cultures are highly infectious and must be
         handled with extreme caution
• D. Serology:
      In patients who have not been previously
  vaccinated, a convalescent serum antibody
  titer of 1:16 or greater is presumptive
  evidence of Y.pestis infection.A titer rise in
  two sequential specimens confirms the
  serologic diagnosis.
• Streptomycin
• Tetracycline:
      alternative drug
      combination with streptomycin
      essential for control early in disease
• Sulfonamides
Summary of Yersinia infections
Spore-Forming Gram-Positive Bacilli:
                     Bacillus Species
• At least 48 species are known but only
• B. anthracis and B. cereus cause defined diseases in
 B. anthracis is responsible for the disease anthrax.
               This is a disease primarily of animals (zoonosis) but humans can
                acquire via handling, inhaling or ingesting contaminated animal
 B. cereus is predominantly responsible for food
  poisoning in humans.
 Bacitracin and polymyxin are two well-known
  antibiotics obtained from Bacillus species.
   Spores of many Bacillus species are resistant to heat, radiation, disinfectants
    and desiccation
• It was from studies on anthrax that Koch
  established his famous postulates in 1876
• Pasteur (1881) developed a vaccine against
B. anthracis Gram stain
 demonstrating spores
 B. anthracis,
Colony on SBA
 “STICKY” Consistency of
B. anthracis’ Colony on SBA
Anthrax infections are classified
       by route of entry

• Cutaneous
• Gastrointestinal
• Respiratory
           Cutaneous Anthrax

•   > 95% of naturally occurring cases
•   Spores enter breaks in skin after contact with
    contaminated animal products
•   Papule丘疹- Vesicle水泡- Ulcer - Eschar焦痂
•   Up to 20% case fatality rate if untreated
•   Mortality with treatment < 1%
• After a 2- to 3-day incubation period, a small
  pimple or papule appears at the inoculation
• A surrounding ring of vesicles develops
• Over the next few days, the central
  papule ulcerates, dries, and blackens
  to form the eschar
Vesicles & Black Eschar
                 Painless & Edema
• The lesion is painless and is surrounded by marked edema that
  may extend for some distance
• Pus and pain appear only if the lesion becomes infected by a
  pyogenic organism
• Similarly, marked lymphangitis淋巴管炎and fever usually
  point to a secondary infection.
Evolution of an anthrax eschar in
        a 4-year-old boy
             DAY 6
DAY 10 - 15
Evolution of an anthrax eschar in a 4-year-old boy.
(A&B) the lesion when first seen (day 0).Note the arm swollen from the
characteristic edema.
(C) Day 6. (D) Day 10. (E) Day 15.
Although penicillin treatment was begun immediately and the lesion was sterile by
about 24 hours,
it continued to evolve and resolve as seen.
           Cutaneous anthrax
          Differential diagnosis
Ecthyma gangrenosum          Pseudomonas aeruginosa
Rat-bite fever               Streptobacillus moniliformis,
                             Spirillum minor
Ulceroglandular tularemia    Francisella tularensis
Plague                       Yersinia pestis
Glanders                     Pseudomonas pseudomallei
Rickettsialpox               Rickettsia akari
Orf                          Parapoxvirus
Staphylococcal lymphadenitis Staphylococcus aureus
Cutaneous tuberculosis       Myocbacterium tuberculosis
Leprosy                      Mycobacterium leprae
Buruli ulcer                 Mycobacterium ulcerans
          Cutaneous anthrax

For cutaneous and gastrointestinal
anthrax, low-level germination occurs at
the primary site, leading to local edema
and necrosis
• Bacillus spores are inhaled and ingested by
  alveolar macrophages肺泡巨噬细胞
• These cells carry the bacteria to the
  regional lymph nodes, causing necrotic
  hemorrhaging which leads to death
• Ingestion of contaminated meat produces
  systemic symptoms which can lead to death
• Mortality by gastrointestinal anthrax may be
Gastrointestinal and pulmonary
anthrax are both more dangerous
than the cutaneous form
because they are usually
identified too late for treatment
to be effective
• Anthrax infections result only if the bacteria
  produce a
    – i) capsule (poly-y-D-glutamic acid polypeptide)
    – ii) exotoxins
    – both encoded on plasmids
•   three proteins
•   protective antigen (PA) (82. 7 kDa)
•   lethal factor (LF) (90.2 kDa)
•   edema factor (EF) (88.9 kDa)

  PA               20 kDa

              PA                    EF

                             The complex (PA+LF or
                        PA   PA+EF) is internalized by

HOST CELL                    acidification of the endosome
                   LF        the LF or EF cross the
                             membrane into the cytosol via
                             PA-mediated ion-conductive
  Effects of anthrax exotoxins
       on macrophages
• Edema toxin is a
  dependent adenylate
  cyclase that increases
  intracellular levels of
  cyclic AMP (cAMP) on
  entry into most types of
• This is believed to alter
  water homeostasis
   – resulting in massive
    Effects of anthrax exotoxins
         on macrophages
• Lethal toxin is a zinc
  metallo-protease that causes
  a hyperinflammatory
  condition in macrophages
    – activating the oxidative burst
        • release of reactive
          oxygen intermediates
    – production of
      proinflammatory cytokines
        • responsible for shock
          and death.
•    MAPKK denotes mitogen-
    activated protein kinase
and germinate
bacilli detach
and migrate to
the regional
•Vegetative anthrax bacilli grow in the lymph node, creating
regional hemorrhagic lymphadenitis
•Bacteria spread through the blood and lymph and increase to
high numbers, causing severe septicemia
•High levels of exotoxins are produced that are responsible for
overt symptoms and death.
•In a small number of cases, systemic anthrax can lead to
meningeal involvement by means of lymphatic or hematogenous
•In pulmonary anthrax, peribronchial hemorrhagic lymphadenitis
blocks pulmonary lymphatic drainage
    • leading to pulmonary edema
Once they have been released from the
macrophages, there is no evidence that an
immune response is initiated against
vegetative bacilli
        Protective immunity
• Antibodies against protective antigen
• Both the noncellular human vaccines and
  live-spore animal vaccines confer protection
  by eliciting antibodies to protective antigen
• The poly-g-D-glutamic acid capsule of B
  anthracis is poorly immunogenic, and
  antibodies to the polysaccharide and other
  components of the cell wall are not
         Species differences
• Anthrax has been documented in a wide
  variety of warm-blooded animals
• Some species, such as rats, chickens, and
  dogs, are quite resistant to the disease
• Others (notably herbivores such as cattle,
  sheep, and horses) are very susceptible
• Humans have intermediate susceptibility.
     reservoir of B anthracis is
         contaminated soil
• Spores remain viable for long periods
• Herbivores, the primary hosts, become infected
  when foraging in a contaminated region
• Because the organism does not depend on an
  animal reservoir, it cannot readily be eradicated
  from a region
   – anthrax remains endemic in many countries
• Humans become infected almost exclusively
  through contact with infected animals or animal
         Cycle of infection in nature
• As a susceptible animal with anthrax approaches death, its
  blood contains as many as 109 bacilli/ml
• Necrosis of the walls of small blood vessels during the
  acute phase of the illness leads to hemorrhages and to
  characteristic bloody exudations from the mouth, nose,
  and anus, a highly diagnostic sign
• These exudates carry vast numbers of the bacilli
   – sporulate on exposure to air
   – produce a heavily contaminated environmental site
   – potentially capable of infecting other animals for
     many years
Handling of carcasses动物尸体
• Sporulation of B anthracis requires oxygen
  – therefore does not occur inside a closed carcass
  – regulations in most countries forbid
    postmortem examination of animals when
    anthrax is suspected
  – The vegetative cells in the carcass are killed
    in a few days by the process of putrefaction.
• In endemic areas, animals that die suddenly should
  be handled cautiously
• Livestock should be vaccinated annually.

                                           Do I look
                                        that I am going
                                             to die?
   Non-Industrial vs Industrial
                   • Nonindustrial anthrax
• usually affects people who work with animals or animal
   – farmers, veterinarians, butchers
   – almost always cutaneous
                      • Industrial anthrax
• acquired from handling contaminated hair, hides, wool,
  bone meal, or other animal products
   – higher chance of being pulmonary as a result of the inhalation of
     spore-laden dust
             Transmission                Clinical syndrome               Who is at risk

Cutaneous    Injured skin or mucous      Skin infection begins as a      People in endemic areas
anthrax      membranes inoculated by     raised itchy bump and within    in contact with infected
             spores from the soil or a   1-2 days develops into a        animals of contaminated
             contaminated animal or      vesicle and then a painless     soils; people who work
             carcass                     ulcer, usually 1-3 cm in        with animals materials
                                         diameter, with a                (hides, fur, wool, hair)
                                         characteristic black necrotic   imported from endemic
                                         area in the center. Lymph       area, such as farmers,
                                         glands in the adjacent area     veterinarians, knackers,
                                         may swell.                      butchers and laboratorians.
Intestinal   The ingestion of poorly     Initial signs of nausea, loss
anthrax      cooked meat or milk         of appetite, vomiting, and
             from infected animals       fever are followed by
                                         abdominal pain,
                                         vomiting of blood, and
                                         severe diarrhea.
Pulmonary    Inhalation of spore-        begins abruptly with high
anthrax      containing dust where       fever and chest pain,
             animal hair or hides are    progresses rapidly to a
             being handled               systemic hemorrhagic
 Bacillus Cereus蜡样芽胞杆菌
• B. cereus food poisoning results from the
  ingestion of preformed enterotoxins, producing
  predominantly vomiting and diarrhea.
• The vomiting form is most often associated
  with ingestion of a heat stable toxin from
  contaminated rice, while the diarrheal form is
  most often associated with ingestion of a heat
  labile toxin from contaminated meat or
                 B cereus virulence factors

• A 38 to 46-kDa protein complex has been shown in animal models:
  – to cause necrosis of the skin or intestinal mucosa
  – to induce fluid accumulation in the intestine
  – a lethal toxin
• Responsible for the necrotic and toxemic nature of severe B cereus
  infections and for the diarrheal form of food poisoning
Bacillus cereus also produces two hemolysins
Phospholipases produced by B cereus may act as exacerbating
   by degrading host cell membranes following exposure of their
   phospholipid substrates in wounds or other infections
        Bacillus Food Poisoning
               Two Distinct Types
                    • Diarrheal type
• diarrhea and abdominal pain
• 8 to 16 hours after consumption of the contaminated food
• Associated with a variety of foods, including meat and
  vegetable dishes, sauces, pastas, desserts, and dairy

                 • Emetic呕吐 disease
• nausea and vomiting begin 1 to 5 hours after the
  contaminated food is eaten
• Boiled rice that is held for prolonged periods at ambient
  temperature and then quick-fried before serving is the
  usual offender, although dairy products or other foods are
  occasionally responsible.