Brucella

• 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 classification

• 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 (in

cluding 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

• Sources of Human Infection: Raw milk and products /Direct contact

• Portal of entry: oral mucosa, nasopharynx and conjunctivae, genital then X in regional lymph node and spread to RES (nodes of udder, uterus,erythritol...). Placentitis with endometritis. Fetus die with edema /congestion of lung, dissimenatedhemorrhages of epicardiumand splenic capsule. Bacteria in lung and digestive tract of the fetus.

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. melitensis

• Goat (1886), Sheep, Cow (1905 in Malta), Swine, Hares, Camels, Buffalo, Impala.

B. suis• Wild pigs, Rats, Swine.

• Abortion,metritis, bursitis, spondylitis(Lumbar and sacral), arthritis, orchitis, paralysis.

Brucella canis• 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 per

iod which can vary from 1 week to 6 or more months.

• 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 (ofte

n 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

variable• 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.

COURSE OF BRUCELLOSIS• 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.

Brucellosis• Cyprus fever/Gibraltar fever/Malta

fever/Rock fever/Undulant fever

Undulant fever

39.5

37.0

• Most affected persons recover entirely within 3 to 12 months

• Some will develop complications

– involvement of various organs,

– a few may enter an ill-defined chronic syndrome.

COMPLICATIONS

• 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.

LARGE JOINTS

SPONDYLITIS 脊椎炎

SACROILIITIS 骶髂关节炎

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 risk

• 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.

CLINICAL DIAGNOSIS

Sanitary

• 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.

Prevention• Eradication of brucellosis in cattle can be attempted by t

est 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.

Treatment• Brucella may be susceptible to tetracyclines or ampic

illin.• Symptomatic relief may occur within a few days afte

r treatment with these drugs is begun.• However ,because of their intracellular location,the o

rganisms 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

YERSINIA

Genus Yersinia• Y. pestis

• Y. enterocolitica

• Y. pseudotuberculosis

EnterobacteriaceaeGenus Escherichia

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.

Pathogenicity

Transmission:

FleaRespiratory Tract

Bite

Antigenic Structure

• F1 Antigen:

• V,W Antigen:

• Yersinia Outer membrane Protein (Yop)• Murine Toxin (MT)

0.3%-0.4% formaldehyde Toxoid

• Endotoxin (LPS)

Y. Pestis Virulence factors schematic diagram

F1 AntigenF1 Gene

PlasmidPlasmid

Ca2+ Dependent Gene V-W Gene

W Antigen

V AntigenLPS

Pathogenesis

Y. pestis Phagocyte

Lymph Nodes

In Groin and Axilla

Enter

Respiratory System

Invade

Pneumonic Plague Bubonic Plague

Invade Blood StreamSepticemic Plague meningitis

Pathogenicity

• 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 ,

DIC

Y.Enterocolitica & Y.Pseudotuberculosis

• Gram negative, No capsule, No spore, Facultative anaerobes

• 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

Epidemiology• Plague

– Probably originated in Asia or central Africa.– One of the earliest record pandemics occurred i

n 542 B.C.– Three pandemics in the history.– 1989 － 1998 ： 5440 cases, 681 dead.

Immunity

Cellular Immunity

Humoral Immunity

1) F1 Ag 2) V,W Ag Phagocytose

Antibody To:

Promote phagocytose , agglutinate and kill bacteria

Diagnosis• A. Specimens:

– Aspirates of lymph nodes– Cerebrospinal fluid– Blood– Sputum

B. Smears:Giemsa’s stain immunofluorescent stain

Diagnosis• 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

Diagnosis• 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.

Treatment

• Streptomycin

• Tetracycline:

alternative drug

combination with streptomycin

essential for control early in disease

• Sulfonamides

Summary of Yersinia infections

Bacillus

Spore-Forming Gram-Positive Bacilli:

Bacillus Species

• At least 48 species are known but only • B. anthracis and B. cereus cause defined

diseases in humans. 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 products.

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 anthrax

B. anthracis Gram staindemonstrating spores

B. anthracis, Colony on SBA

“STICKY” Consistency ofB. anthracis’ Colony on SBA

Anthrax infections are classified by route of entry

• Cutaneous

• Gastrointestinal

• Respiratory

Cutaneous Anthrax

• > 95% of naturally occurring cases> 95% of naturally occurring cases• Spores enter breaks in skin after contact with Spores enter breaks in skin after contact with

contaminated animal productscontaminated animal products• PapulePapule 丘疹 - Vesicle- Vesicle 水泡 - Ulcer - Eschar- Ulcer - Eschar 焦

痂• Up to 20% case fatality rate if untreatedUp to 20% case fatality rate if untreated• Mortality with treatment < 1%Mortality with treatment < 1%

• After a 2- to 3-day incubation period, a small pimple or papule appears at the inoculation site.

• 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 Rat-bite fever Ulceroglandular tularemia Plague Glanders Rickettsialpox Orf Staphylococcal lymphadenitis Cutaneous tuberculosis Leprosy Buruli ulcer

Pseudomonas aeruginosa Streptobacillus moniliformis, Spirillum minor Francisella tularensis Yersinia pestis Pseudomonas pseudomallei Rickettsia akari Parapoxvirus Staphylococcus aureus Myocbacterium tuberculosis Mycobacterium leprae Mycobacterium ulcerans

Cutaneous anthrax

For cutaneous and gastrointestinal anthrax, low-level germination occurs at the primary site, leading to local edema and necrosis

Inhalation

• 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

Gastrointestinal• Ingestion of contaminated meat produces

systemic symptoms which can lead to death • Mortality by gastrointestinal anthrax may

be 50%

Gastrointestinal and pulmonary anthrax are both more dangerous than the cutaneous form because they are usually identified too late for treatment to be effective

PATHOGENESIS• 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)

ANTHRAX TOXINSANTHRAX TOXINS

LF

EF

PA

PA

PA

LF

HostProtease

HOST CELL

20 kDa

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

acidification of the endosome

the LF or EF cross the membrane into the cytosol via PA-mediated ion-conductive channels

Effects of anthrax exotoxins on macrophages• Edema toxin is a

calmodulin 钙调节蛋白 -dependent adenylate cyclase that increases intracellular levels of cyclic AMP (cAMP) on entry into most types of cell

• This is believed to alter water homeostasis– resulting in massive

edema

Effects of anthrax exotoxins on macrophages• Lethal toxin is a zinc

metallo-protease that causes a hyperinflammatory condition in macrophages– activating the oxidative

burst pathway • release of reactive

oxygen intermediates– production of

proinflammatory cytokines• responsible for shock

and death.

• MAPKK denotes mitogen-activated protein kinase kinase

•Endospores are phagocytosed by macrophages and germinate•Macrophages containing bacilli detach and migrate to the regional lymph node.

•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 spread•In pulmonary anthrax, peribronchial hemorrhagic lymphadenitis blocks pulmonary lymphatic drainage

• leading to pulmonary edema•Death results from septicemia, toxemia, or pulmonary complications

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 protective.

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 isreservoir 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 products

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 Anthrax

• Nonindustrial anthrax

• usually affects people who work with animals or animal carcasses

– 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 anthrax

Injured skin or mucous membranes inoculated by spores from the soil or a contaminated animal or carcass

Skin infection begins as a raised itchy bump and within 1-2 days develops into a vesicle and then a painless ulcer, usually 1-3 cm in diameter, with a characteristic black necrotic area in the center. Lymph glands in the adjacent area may swell.

People in endemic areas in contact with infected animals of contaminated soils; people who work with animals materials (hides, fur, wool, hair) imported from endemic area, such as farmers, veterinarians, knackers, butchers and laboratorians.

Intestinal anthrax

The ingestion of poorly cooked meat or milk from infected animals

Initial signs of nausea, loss of appetite, vomiting, and fever are followed by abdominal pain,

vomiting of blood, and severe diarrhea.

Pulmonary anthrax

Inhalation of spore-containing dust where animal hair or hides are being handled

begins abruptly with high fever and chest pain, progresses rapidly to a systemic hemorrhagic pathology

Bacillus Cereus 蜡样芽胞杆菌• B. cereus food poisoning results from the ingest

ion 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 vegetables

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 factors

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 products

• 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.