1

Clicker Data

• 19 clicker questions to date

• Best students got 18 (94.7%) correct

• Class averages – steady improvement– 1st lecture = 65%

– 2nd lecture = 85%

– 3rd lecture = 91%

• Note: no response = incorrect response

Clickers must be returned at the end of Thursday’s 1:30 lecture

no clicker – no grade for VPM 201

Mycobacteria: Orientation

Slow-growing Mycobacteria

M Avium Complex (MAC)

M Tuberculosis Complex (MTBC)

Leprosy

Mycobacterium tuberculosis

A highly successful pathogen

A Global Problem

Most common infectious disease of humans in the world.M. tuberculosis infects one new person every second.

2.1 billion people are infected2 million deaths / year

2

Magnitude of the Problem

TB is #1 killer of women, > all causes of maternal mortality. TB creates more orphans than any other infectious disease. TB is the leading cause of death among HIV-positive individuals. TB is not on the decline. TB infects 7-8 million people every year. TB >25% of all preventable adult deaths in the developing world.

Clinical Signs

Cough for more than three weeks

Blood in the sputum

Chest pain for more than one month

Increasing weakness and loss of weight

Clinical Signs

Consumption

Coughing & Cachexia

Pathology

Mediastinal lymph node

Pathology

Lung: milliary tuberculosis

3

Pathogenesis Details

Novel cell wall structure & chemistry.Responsible for mycobacteria being:

Acid-fastAntibiotic resistant

Resistant to environmentResistant to intracellular killing

Mycolates

Lipoarabinomannan

Arabinogalactan

Slow Growth Strategy

Growth rate is linked with virulence.Rapid growers, e.g., M. phlei = non-pathogenicModerate growers, e.g., M. avium = low virulence.Slow growers, e.g., M. tuberculosis = most virulent.

TB Controls Host Cell

• Regulates phagosome environment– Sustains pH at growth optimum– Selectively allows delivery of molecules like

transferrin to phagosome by fusion with vesicles in early endosomal network.

• Suppresses immune response– Sequesters itself away from antigen-

processing machinery of host cell.– Suppress ability of infected macrophage to

stimulate CMI.– Over-produce cell wall lipids (pthiocerol

dimycocerasate) that leave infected cell by exocytosis to suppress neighboring macrophages.

Control

• Education

• Screening general public: skin test

• Confirmatory testing: X-ray & culture or PCR

• Treatment:– Observed to assure compliance

• BCG vaccination (in high-prevalence countries)

Human TB Diagnostics

Screen by skin test Confirm by X-ray

Delayed-type hypersensitivity response to PPD: Purified Protein Derivative of M. tuberculosis. PPD is a Mtb culture (synthetic medium) filtrate from which proteins are precipitated by trichloroacetic acid or ammonium sulfate and concentrated by ultrafiltration.

4

Diagnostic Microbiology

Acid-fast stain of sputum

Anti-Mycobacterial Drugs

BCG Vaccine

“…the only thing worse than BCG is nothing.”D. Russell, Nature Reviews, August 2001.

New Challenges

Resurgence in USA due to AIDS, IV drug use, and drug-resistant strains.

“…the incidence of TB will invariably increase in most areas of the world if the rate of HIV infection in the adult population is high (≥5%).”

“…in the presence of a significant proportion of untreated HIV infection among adults, TB incidence cannot be reduced with current technologies.”

T.R. Frieden, Int.J.Epidemiol., 2002.

Our Worst Fear

MDR-TB = Resistant to:Isoniazid and rifamycin

XDR-TB = Resistant to:Isoniazid and rifamycin +Any Fluoroquinolone +Any one of 3 injectable 2nd line TB drugs

(capreomycin, kanamycin, amikacin)

Our Worst Fear

MDR-TB = Resistant to:Isoniazid and rifamycin

XDR-TB = Resistant to:Isoniazid and rifamycin +Any Fluoroquinolone +Any one of 3 injectable 2nd line TB drugs

(capreomycin, kanamycin, amikacin)

XDR-TB is raising the specter of something we have been worried might happen for a decade – the possibility of a virtually untreatable TB.

Paul Nunn, World Health Organization

5

First Case He Doc! Glad we ran into you. Take a look at the inside of this deer and tell us if it is

OK to eat the meat.

Pleural surface

Acid-fast Stained Smear What is your diagnosis?

Tuberculosis

What’s the probable cause?

A. Rhodococcus equi

B. Mycobacterium bovis

C. Salmonella typhimurium

D. Mycobacterium paratuberculosis

What characteristic(s) of M. bovis are most responsible for its pathogenicity?

A. Thick waxy cell wall

B. Potent exotoxins

C. Spore formation

D. Intracellular survival

E. Two of the above

6

Which antibiotic(s) are best for treatment of tuberculosis in cattle?

A. Isoniazid

B. Fluoroquinolones

C. Penicillin

D. None of the above TB is not treated

Which of the following states are currently classified as free of bovine TB?

A. Minnesota

B. Wisconsin

C. Michigan

D. All of the above

In which Canadian provinces is bovine TB most likely to be discovered?

A. PEI

B. Manitoba

C. BC

D. Quebec

Animal Version of TB Story

M. bovisTechnically, Mycobacterium tuberculosis subsp. bovis

• Source: infected animal, cattle…or?• Contact or inhalation• Invasion through respiratory mucosa• Uptake by macrophages• Survives intracellularly

• Arrests phagosome development• Suppresses host immune response

• Disseminates to regional lymph nodes granuloma caseous necrosis escape from host in respiratory secretions possible bacteremia / dissemination

• Clinical signs: wasting (cachexia)• Diagnosis – culture or skin test• REPORTABLE DISEASE – notify State Veterinarian / CFIA

Key Elements of the Story

• If cattle found infected;• Quarantine herd• Test entire herd

• Slaughter test-positive cows,…or• Slaughter entire herd

• Trace forward and trace backward for other infected herds• Treatment: none• Prevention – biosecurity, interstate regulations; no vaccine• Transmissible to humans: Zoonotic

Key Elements of the Story

7

Pathogenesis Details

Novel cell wall structure & chemistry.Responsible for mycobacteria being:

Acid-fastAntibiotic resistant

Resistant to environmentResistant to intracellular killing

Mycolates

Lipoarabinomannan

Arabinogalactan

Key Concepts

Intracellular bacterial pathogensBacteriologist’s view:

T-cell-mediated host responseImmunologist’s view:

Granulomatous tissue responsePathologist’s view:

Tuberculosis (TB) – like lesionFood safety inspector’s view

Practitioner’s view: Vaccines do not work well &antibiotic therapy is difficult

Clinical signs - wasting

From 1923 book on TB First accurate illustration of pathology, 1838.

Pathology - granulomas

Caseous Necrosis

Mediastinal lymph node

Lung

Readily Disseminates Classical Lesions are Granulomas

Bovine Spleen

Bovine Liver

MonkeyKidney

Granuloma = Tubercle

Caseous necrosis

Mineralization

Mononuclear cell inflammation

Fibrinous wall

8

Diagnosis

Caudal fold skin test M. bovis PPDSwelling = positive

Measures CMI response

Comparative Cervical Test

0

2

4

6

8

10

12

0 2 4 6 8 10 12

M. bovis PPD reaction (mm)

M. a

viu

m P

PD

rea

ctio

n (

mm

)

M. avium PPD reactor

M. bovis PPD reactor

Diagnosis and RegulationFlow Chart

Comparative Cervical TestDTH reaction toM. avium PPD

andM. bovis PPD

Compared(skin thickness measured with a calipers)

Culture: Special media + special labs

M. bovis is Zoonotic1993 Reviews Article on M. bovis in Humans

Stomach of child that got M. bovis from drinking raw milk, 1923 book illustration.

Herd of TB-free cattleused by Boston Children’shospital 1914-1940.

In the 1920s, 20% of all human cases of TB in the U.S. were caused by M. bovis.

Close to Home & In The News

On July 12, 2005, the Minnesota Board of Animal Health in conjunction with the United States Department of Agriculture (USDA) announced the confirmation of a tuberculosis (TB) infected beef cattle herd in Roseau County. This was the first positive herd identified in Minnesota since 1971, when the state was declared free from tuberculosis.

October, 2006; 6th herd found. Minnesota lost accredited TB-free status. Every bovine leaving the state must be TB tested.

9

Biological & Political Challenges

http://www.bovinetb.com/

Outstanding website!

Deer feeding congregation M. bovis transmission

Since 1995, bovine TB has been diagnosed in 398 white-tailed deer out of more than 88,000 tested.

Canadian Conundrum

Manitoba

New Zealand

Brushtail Possum

The wild animal reservoir of TB in New Zealand.

United Kingdom

The wild animal reservoir of TB in the U.K.

Badger

Unique ChallengesAnthropozoonosis

TB lung Asian elephantTesting protocol

18 U.S. elephants were found infectedwith M. tuberculosis, originating from humans. These animals then pass the infection back to humans. All U.S. elephants must now be tested annually.Treatment costs for an elephant are roughly$50,000 – requires one full year.

10

Unique ChallengesWildlife Health

Kruger National Park

Avian Tuberculosis

Etiology:M. avium (17%)M. genavense (71%)

Enlarged liverWith granulomas

Common in psittacine birdsand zoos with aviaries.

Leprosy M. leprae Leprosy = Hansen’s Disease

Armauer Hansen1841-1912

First to discover the leprosy bacillus.

Global distribution

Hansen’s reputation was tarnished when it was learned that he inoculated infectious material from leprosy victims into nurses and patients in an attempt to study the disease.

M. leprae Attacks Nerves

Leprosy lesionwith abundant

acid-fast bacteria

Can not be cultured in vitro

Grows in cooler parts of the body

Classification of Leprosy

RESISTANCE

TuberculoidSkin-NerveNo or few bacilliTuberculoid granuloma

LepromatousSkin-Nerve-EyeInternal organs

Numerous bacilliFoam cell granuloma

NO RESISTANCE

INFECTION

INDETERMINATE

BORDERLINE

11

Leprosy Models

Mouse footpadand armadillo;only means of

cultivatingM. leprae.

Feline LeprosyM. lepraemurium

Erosive skin lesionsFig. 2-63 inSmall Animal DermatologyW.B. Saunders

Nicely described in Greene, 3rd ed. pp. 477-479.

Who’s Winning,The Host or the Pathogen?