ACID FAST BACILLI

INFECTIONS
Veterinary Medicine Program
Brawijaya University

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Mycobacteria
 Unique bacteria
 Cell wall contain a lot of waxy material (mycolic acid)
 inhibits the uptake of nutrients into the bacterial cell

 causes the cell to clump

these factors contribute to the slow growth rate

 Mycobacteria do not grow outside of a host except in
cultured media
 Slow growth rate

 Growth slowly at artificial medium

 Multiply approximately once every 20 hours

Mycobacteria
 Mycobacteria: slim rod, aerobic, non sporulated.
 As a acid fast bacteria (bakteri tahan asam). Cell wall contains
wax difficult to stain, but once stained, very difficult to
decolorized acid alcohol decololizer “acid-fast/acid
alcohol-fast” bacilli.
 Some are human & animal pathogens granulomatous
lesions necrotic caseous calcified
 M. tuberculosis: human tuberculosis, bovine, murine, cold
blooded
 M. paratuberculosis: Johne's disease cattle, sheep
 M. leprae: human leprosy
Mycobacteria pathogenic can be differentiated
(Runyon Groups) by:
 speed of growth (all are slower than most
other pathogens),
 production of chromogenic pigments (in
light, in dark, or none)
Mycobacterium Runyon Classification

Complex TB M. tuberculosis M. bovis

M. africanum M. canetti, M. microti
Photochromogen M. asiaticum M. marinum
M. kansasii M. simiae
Scotochromogen M. flavescens M. scrofulaceum
M. gordonae M. szulgai
Nonchromogen M. avium complex M. shimoidei
M. celatum M. terrae
M. haemophilum M. trivale
M. gastri M. ulcerans
M. genavense M. xenopi
M. malmoense M. nonchomogenicum
Rapid grower M. abscessus M. phlei
Group M. fortuitum M. smegmatis
Group M. chelonae M. vaccae
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Atypical mycobacteria
 Rapid grower mycobacteria
 Non pathogenic
 Divide into 4 groups:
1. non chromogenic
2. photochromogenic
3. scotochromogenic
4. catalase.
 M. phlei, M. smegmatis, M. kansasii, M.
fortuitum, M. ulcerans

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 Pathogenic Mycobacterium spp.

BCG

AIDS
patients
Mycobacterium tuberculosis
 Distributed around the world
 Human type (M. tuberculosis) tuberculosis.
Especially pathogen to human
Pathogen also to animal. Guinea pigs is susceptible
 Bovine type (M. bovis) : cattle, pig, dog, cat, horse,
rabbit, human
 Avian type (M. avium) : especially to poultries, human
 Murine type (M. murium): rodents
 Cold blooded type (M. piscium, M. marinum,
M. platypoecilis): fish, etc
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There are three types of TB - human, avian, and bovine:
1. Human TB is rarely transmitted to non-humans
2. Avian TB is typically restricted to birds (pigs and occasionally
other animals have been found to be susceptible)
3. Bovine TB - or cattle TB - is the most infectious, capable of
infecting most mammals. Bovine TB is caused by the
bacterium Mycobacterium bovis (M. bovis) which is part of
the Mycobacterium tuberculosis complex.
High Virulence?
1. Mycobacteria produces "cord factors” (glycolipid:
6,6’dimycolates of trehalose) serpentine cords.
Nature lipid in the cell wall resistant to drying,
acid/alkaline
Used as adjuvant for enhancing of humoral and CMI
responses.
2. N-acetyl-muramyl-L-alanyl-D-isoglutamine (MDP)
inhibits macrophage migration

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3. Virulent strains Mycobacteria able to disrupt the
membrane of phagosomal alveolar macrophages
loss of the normal function
4. Production high NO (nitric oxide) at phagocytosis
& inducing apoptosis of immunologic cells
primarily T cells hyporesponsiveness
Inhibit apoptosis living intracellular inhibit
killing mechanism by phagocyte cells
3. Cord factors enhance function of mitochondrial
membrane the phagocyte cells survive &
multiply in the phagosome the need of O2
Cord factor swelling & disruption of liver host
mitochondrial the need of causing disease
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 Tuberculosis characterized by long productive
cough
 Therapy: Isoniazid, rifampin and ethambutol
 M. leprae has never been cultured in vitro but can be
grown on the footpads of armadillos
Characteristics properties of the
M. tuberculosis & M. bovis cultures
 Obligate aerobes
 Need an enriched medium
Serum, potatoes, egg: Dorset egg medium
Glycerin: Lowenstein-Jensen medium
Growth at 4-8 weeks. Generation time 18 hrs
Human strain is eugonic, yellow pigment
Bovine strain dysgonic
 In the broth medium: cluster growth at the surface
disperse by Tween 80 detergent

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 Acid Fast Staining
(Ziehl Nelssen)

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Resistance to physical & chemical agents

 Most strain M. tuberculosis very heat sensitive was

dead at 600C for 15’
 More resistant to lethal effect of antiseptics compare
to others strain
 Susceptible to common detergents and sunlight
 Survive longer in the dark places, stool, sputum,
spoiled material, in cold weather

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Antigen & Toxin

 All strains have closed relationship

differentiation is difficult
 The antigens too complex serologic
differentiation
 No toxin

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BOVINE TUBERCULOSIS

VETERINARY MEDICINE
UB.
Implications
 Zoonotic
 Bovine tuberculosis is a human health issue in many
foreign countries where the milk is not pasteurized and
there are high rates of human infection
 Tuberculosis (TB) is a contagious disease of both
animals and humans
 Can be transmitted from animals to humans and vice
versa
WHAT IS BOVINE TUBERCULOSIS
Bovine tuberculosis (BT) is a serious respiratory
disease, chronic disease of animals caused by a
bacterium called Mycobacterium bovis (M. bovis),
which is closely related to the bacteria that cause
human and avian tuberculosis.

This disease can affect practically all mammals,

causing a general state of illness, coughing and
eventually death
The name Tuberculosis comes from the nodules,
called tubercles
Until the 1920s when control measures began in
developed countries, it was one of the major
diseases of domestic animals throughout the world.
Today TB remains an important disease of cattle,
wild animals, and is a significant zoonosis.
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Epidemiology

 Human tbc, bovine tbc & Johne’s disease

Ubiquitous in some countries
Infected in all ages
 Transmission
 Common route:
inhalation respiratory lymph node & lung
ingestion (contaminated food & water) GIT
 Can be transmitted through tissue, skin, congenital, saliva,
other discharges

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Geographic Distribution

Center for Food Security and Public Health, Iowa

State University, 2012
The true host of M. bovis is cattle the disease
has been reported in many other domesticated and
non-domesticated animals.

Isolations have been made from: buffaloes, bison,

sheep, goats, equines, camels, pigs, wild boars,
deer, antelopes, dogs, cats, foxes, mink, badgers,
ferrets, rats, primates, llamas, kudus, elands, tapirs,
elks, elephants, sitatungas, oryxes, addaxes,
rhinoceroses, possums, ground squirrels, otters,
seals, hares, moles, raccoons, coyotes and several
predatory felines including lions, tigers, leopards
and lynx.
 How is the disease transmitted
and spread?
Contagious
Spread by contact with infected domestic and wild
animals by inhaling infected droplets which are
expelled from the lungs by coughing.
Calves and humans can also become infected by
ingesting raw milk from infected cows.
The disease is slow months or years to kill an
infected host animal spread to many other
before begins to manifest clinical signs
Movement of undetected infected domestic animals
and contact the major ways of spreading the
disease.
Signs

Depends on location of the infection & wide of the

lesion
 Cattle Pulmonary infection

Tuberculosis mastitis: non pathognomonic

udder inflammation, decrease the milk production
chronic: necrosis, fibrosis
 In mammalian the signs similar with cattle

 In dogs & cats: digestive

 In poultry beginning by digestive sign, pale of the

mucosa membrane, intermittent diarrhea
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CLINICAL SIGNS
Pulmonary
 usually has a prolonged course, and symptoms take
months or years to appear.
 The usual clinical signs include:
 weakness,
 loss of appetite,
 weight-loss,
 fluctuating fever,
 intermittent hacking cough, sometimes cough up
blood/ massive bleeding
 diarrhea,
 large prominent lymph nodes.
 the bacteria can lie dormant in the host without
causing disease.
Extra pulmonary

 Commonly in immunosuppressed animals

 Extra pulmonary infection sites include:
 The pleura (in tuberculous pleurisy),

 The central nervous system (in tuberculous meningitis),

 The lymphatic system (in scrofula of the neck),

 The genitourinary system (in urogenital tuberculosis),

and
 The bones and joints = "osseous tuberculosis”.
A form of osteomyelitis.
Sometimes, bursting of a tubercular abscess through
skin results in tuberculous ulcer.
An ulcer originating from nearby infected lymph nodes is
painless, slowly enlarging and has an appearance of
"wash leather”.
A potentially more serious, widespread form of TB is
called "disseminated" TB, commonly known as miliary
tuberculosis. Miliary TB makes up about 10% of extra
pulmonary cases
LESIONS
In approximately 90% of tuberculosis cattle, lesions
primarily involved the lymph nodes of the respiratory
system.
Lesions can also be found in thoracic cavity, and
mesenteric lymph nodes.
A granuloma or tubercle forms as the body tries to
wall off the infected macrophages with fibrous
tissue.
The granuloma is usually 1-3 cm in diameter, yellow or
gray, round and firm.

On cut section, the core of the granuloma consists of

dry yellow, caseous, or necrotic cellular debris.
The infection can spread hematogenously to lymph
nodes and other areas of the body and cause smaller,
2-3 mm in diameter, tubercles.
The formation of these smaller tubercles is known as
“miliary tuberculosis”. The histological lesions consist
of necrotic cells in the center of the tubercle surrounded
by epitheloid cells and multinucleated giant cells all
encapsulated by collagenous connective tissue.

The necrotic core of cells can often become calcified

as the tubercle matures

Tubercles are usually located in the respiratory lymph

nodes and the lungs.
Animal Immune Response
 Immune system recognizes bacteria
 Inflammatory cells (macrophages) are sent to dispose of
it
 Mycobacterium is resistant to destruction
 once ingested by the macrophages it may replicate
and kill the macrophage
 Immune system continues to send macrophages to help
destroy the bacteria
 Results in an accumulation of living and dead
macrophages at the site of the bacteria
 Accumulation is called a tubercle
 Thick capsule may form around the tubercle; called a
granuloma

Pericardial granuloma
Diagram of
a Granuloma
a fibrin layer
develops around
granuloma (fibrosis),
further “walling off”
the lesion.
Typical
progression in
pulmonary TB
involves
caseation,
calcification and
cavity formation.
 DIAGNOSIS
 The standard method for detection of BT is the
tuberculin test, where a small amount of antigen is
injected into the skin, and the immune reaction is
measured.
 Based on T-cell response to mycobacterial proteins. The
caudal-fold tuberculin test (CFT) is performed using 0.10
cc of a purified protein derivative (PPD).
Injected intradermally in the skin of the tail.

After CFT & CCT diagnostic laboratory:

 Gross examination

 Histopathological examination
Examined for a reaction 72 hours post inoculation.
If there is a reaction:
- Discolored raised area, the animal is classified as a
responder and undergoes a comparative cervical
tuberculin test (CCT).
- The test is more sensitive and helps to determine
whether the animal is infected with M.
bovis By injecting 0.10 cc of a more potent
PPD intradermally into the cervical skin.
CCT
 CCT test must be conducted within 10 days or after 60
days of the initial CFT test.
 The veterinarian clips two areas of the neck, measures
the skin thickness with calipers, and intradermally
injects bovine PPD and avian PPD. Responses at the
injection site are evaluated and measured 72 hours after
the injections. The differences in pre and post test skin
thickness determine the test result.
 The CCT test is used to classify cattle as negative,
suspect or reactors. CCT suspect animals either remain
under quarantine for retest in 60 days, or are examined
for lesions at postmortem. CCT test reactor animals are
examined for lesions at post mortem.
TB Testing Summary

1. Caudal Fold Tuberculin Test:

a. Screening test
b. One injection in the caudal fold
c. Read 72 +/– 6 hours later
d. If negative, no further action is required
e. If positive, the animal is classified as a responder
and requires further testing.

2. Comparative Cervical Tuberculin Test or Gamma Interferon Test:

a. Confirmatory tests
b. CCT test - injections in the skin of the neck with
bovine PPD and avian PPD, and read 72 hours later.
Gamma Interferon Test is a blood test.
• IfIf negative – no further action
• reactor – euthanize, necropsy and post mortem testing
• If suspect – option of euthanize, necropsy and
post mortem testing or remain under herd quarantine for repeat
confirmatory testing.
 X-ray infiltrations of immunocompetence cells
 Definitive diagnosis is made by growing the
bacteria in the laboratory, a process that takes at
least eight weeks.
 PCR
 ELISA, detect IFN-γ produce by NK cells
 The guidelines for tuberculin manufacture and
culture of M. bovis are detailed in the OIE Manual
of Diagnostic Tests and Vaccines for Terrestrial
Animals
Gross Lesions
 Finding gross lesions is not conclusive evidence that the
animal is infected with the disease
 Further testing is required

Lesions in the lungs of a 2 yrs. old heifer

REPORTING BY IUAT (International Union Against
Tuberculosis)

SCALE NUMBER OF THE BACTERIA

Negative No Acid fast bacteria (AFB)

Doubt 1-9 AFB/ 100 field examinations
Positive + 10-99 AFB/ 100 field examinations
Positive ++ 1-10 AFB/ field examinations
Positive +++ > 10 AFB/ field examinations
REPORTING BY WHO
CODE OF THE BACTERIA NUMBER
1. 0 - No AFB
2. 1 - 1 AFB/slide
3. 2 - 2 AFB/slide
4. 3 - 3 AFB/slide
5. 4 - 4 AFB/slide
6. 5 - 5 AFB/slide
7. 6 - 6-24 AFB/slide
8. 7 - 25 or more AFB/slide
9. 8 - 25 or more/line of examination
10. 9 - abundant/field examination
Process
 Culturing is performed under specific conditions that
favor growth of the bacteria
 Culturing usually takes 8 to 16 weeks

Growing culture colonies

 PREVENTION
The standard control measure applied to BT is test and
slaughter.
- post-mortem meat inspection tubercles in
the lungs and lymph nodes
- intensive surveillance including on-farm visits,
- systematic individual testing of cattle and removal
of infected and in-contact animals as well as
movement controls have been very successful in
reducing or eliminating the disease
Pasteurization of milk of infected animals

A number of new candidate vaccines are currently

being tested.
Vaccination for the prevention of bovine tuberculosis is
another option that is being investigated.
A vaccine was created for M. bovis in the 1920s by
Chalmette and Guerin. The BCG vaccine has
undergone some scrutiny due to variable efficacy.
The vaccine reduces the severity of the disease, usually
allowing the bacteria to infect only a few lymph nodes,
but does not prevent infection.
The vaccine can also cause false-positive caudal
fold skin tests, which can cause confusion when
testing is performed.
There are studies researching the efficacy of
combining the BCG vaccine with mycobacterium
protein and DNA.
The goal is to develop an enhanced vaccine that
can provide protection against the disease.
The vaccine would be given to wildlife in an effort
to prevent the spread of the disease to cattle
Treatment
Difficult & requires administration of multiple
antibiotics over a long period of time
Social contacts are also screened and treated if
necessary.
Antibiotic resistance is a growing problem in multiple
drug-resistant tuberculosis (MDR-TB) infections
 INH
 Pyrazinamide
 Streptomycin
 Rifampin
 Easy resistance to PAS (para-amino salicylic
acid)
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