Transcript CHAPTER 8

Veterinary Immunology
Type IV
Hypersensitivity
Dr. Chi-Young Wang
The Tuberculin Reaction

A skin test for an identification of
animals suffering from tuberculosis

Purified protein derivative (PPD)
tuberculin: growing organisms in
synthetic medium, killing them with
steam, and filtering

PPD is precipitated with TCA
The Tuberculin Reaction

The major antigenic component is
the heat-shock protein HSP65

A normal animal-no apparent
response for tuberculin

Animals infected with TB, a delayed
hypersensitivity occurs (indurated
swelling)

The inflammation begins between
12 and 24 hours, reaches its
greatest intensity by 24 to 72 hours,
and may persist for several weeks
before fading gradually

The lesion is infiltrated with
lymphocytes and macrophages,
although neutrophils are present
in the early hours of the reaction

When tuberculin is injected
intradermally, it is taken up by
Langerhans cells (macrophages),
which then migrate to the draining
lymph node

They present antigen to memory T
cells that respond by generating
Th1 effector cells

The circulating Th1 cells
accumulate around the antigen
deposit; the injected site is
infiltrated with γ/δ+, WC1+ T cells;
no B cells

The recruited Th1 cells secrete IFN-
γ, IL-2, and IL-16; The first two upregulate adherence molecules; IL-2
stimulates CXCL8, CCL5, and XCL1
which activates Tcells; IL-16 attracts
CD4+ T cells

The macrophages release serotonin and
CXCL1 and CCL2 which attract basophils;
T cell-derived chemokines CCL2 and
CCL3 can induce mast cell degranulation

By 60-72 hours, the predominate
lymphocytes are α/β+, CD4+, and CD8+ ;
macrophages accumulate as a result of
the production of CXCL8 and IFN-γ
Tuberculin Reactions in Cattle

0.05 ml of PPD tuberculin derived
from M. tuberculosis or
Mycobacterium bovis is injected
into one anal fold and the injection
site is examined 72 to 96 hours later
(The single intradermal test; SID
test)-A firm lump
Tuberculin Reactions in Cattle

In USA, two shots (one in the vulva
and the other into an anal fold);
other countries, the side of neck
(neck site is more sensitive than the
anal fold)

But the cross-reaction such as
Mycobacterium avium or Norcardia
Tuberculin Reactions in Cattle

If animals exposed to
nonpathogenic mycobacteria, false
positive occurs

False-negative SID tests with
advanced tuberculosis, in animals
with very early infection, claved
within the preceding 4 to 6 weeks
Tuberculin Reactions in Cattle
, in very old cows, and in animals tested
during 1 to 10 weeks

The lack of reaction (anergy) seen in
Johne’s disease due to the blocking
factor

The comparative test involves
intradermal inoculation of avian and
bovine tuberculins; each tuberculin
Tuberculin Reactions in Cattle
is injected into the side of neck at
separate sites..(90% sensitive and
99% specific)

Short thermal test: a large volume
of tuberculin solution is given
subcutaneously and examined for a
rise in temperature between 4 and 8
hours later (due to IL-1 from
macrophage)
Tuberculin Reactions in Cattle

Stormont test: giving 2 doses of
tuberculin at the same injection site
7 days apart

Both used in postpartum cows and
heavily infected animals

Repeated tuberculin testing results
in a period of decreased reactivity
and the induction of antibodies antiHSP 70
Pathological Consequences

Mycobacterium tuberculosis is resistant
to intracellular destruction until M1
macrophages are activated by Th1 and
dead organisms are very slowly removed
because they contain large quantities of
metabolized waxes

Many macrophages fail to prevent
bacterial growth so they die
Pathological Consequences

Other macrophages fuse to form
multinucleated giant cells

After 4-5 weeks, microscopic
granulomas enlarge and coalesce. The
lesion is consisted of necrotic debris, a
layer of fibroblasts, lymphocytes, and
macrophages, which are called
epithelioid cells; The entire called
“tubercle”
Pathological Consequences
Pathological Consequences

The mycobacteria are unable to multipy
within the caseous tissue because of its
low pH and lack of oxygen

If the host mounts an Th1 responsessufficient to control; if Th2 responsesthe organisms spread to lung, and
results in liquefaction of the caseous
center of tubercle

Granuloma-a common chronic
inflammation
Allergic Contact Dermatitis

Reactive chemicals painted onto the
skin, they may bind to skin proteins
and resulting complexes are processed
by Langerhans cells

The Langerhans cells migatre to
draining lymph nodes and secret IL-12
and IL-18 , to which Th1 cells respond
Allergic Contact Dermatitis

These cells produce large amounts of IFNγand promote the activities of cytotoxic T
cells

Following exposure, macrophages and
lymphocytes infiltrate the dermis by 24 hours

Cytotoxic T cells destroy and remove the
altered cells leading to intraepithelial
vesicles
Allergic Contact Dermatitis

The inflammatory reaction as an intensely
pruritic skin disease called allergic contact
dermatitis and α/β T cells, γ/δT cells, B-1
cells, and natural killer (NK) Tcells are
involved

Formaldehyde, picric acid, aniline dyes, plant
resins, oils, organophosphates, neomycin,
and nickle and berylium
Allergic Contact Dermatitis
Allergic Contact Dermatitis
Allergic Contact Dermatitis

Ears of dogs treated with neomycin for otitis
externa

Carpet dyes exposed to the scrotum and
ventral abdomen of dogs

Neck exposed to dichlorvos in flea collars

Milking machine to dairy cow

Muzzle of dogs to component of plastic food
bowls
Allergic Contact Dermatitis

Closed patch tests –suspected allergens are
used to impregnate gauze swabs that are
then attached to the shaved skin with tape.
After 48 to 72 hours the dressing is removed
and the areas in contact with the swabs
examined

A positive-local erythema and vesiculation
Measurement of Cell-Mediated
Immunity

In vivo-Intradermal skin test is difficult
to quantitate

To measure the ability of an animal to
mount cell-mediated immune response
in general rather than response to one
specific antigen

To give a small skin graft or paint skin
Measurement of Cell-Mediated
Immunity
with a contact sensitizer such as
dinitrochlorobenzene to mount a cellmediated immune response
Measurement of Cell-Mediated
Immunity

In vivo technique-antigen is mixed with
peripheral blood lymphocytes and
cultured for 48 to 96 hours. Twelve
hours before harvesting, thymidine
labeled with tritium is added to the
culture

Stimulation index is the ratio of
radioactivity in the stimulated cultures
Measurement of Cell-Mediated
Immunity
to the radioactivity in the control

MTT assay is a pale yellow compound
that serves as a substrate for active
mitochondrial enzymes; it is sensitive
to quantify the increase in T cells
numbers triggered by antigen or
mitogens
Measurement of Cell-Mediated
Immunity

To measure T-cell mediated
cytotoxicity-the living cells take up and
retain chromium ions (51 Cr), but if the
cell dies, the chromium is released into
the extracellular fluid

Lymphocytes from an immune animals
mixed with 51Cr-labeled target cells; the
Measurement of Cell-Mediated
Immunity
mixture is incubated for 4 to 24 hours at
at 37 ℃;the amount of chromium
released is directly to the number of
target cells killed

Adding tuberculin PPD to heparinized
blood and incubating the mixture for 24
to 48 hours at 37 ℃;the plasma is
Measurement of Cell-Mediated
Immunity
assayed by ELISA; Mycobactrium bovis
and Mycobacterium avium PPD (falsepositive results) are used; ESAT-6
reduce the incidence of false-positive
results

The assay is at least as sensitive as the
single intradermal test
Measurement of Cell-Mediated
Immunity
assayed by ELISA; Mycobactrium bovis
and Mycobacterium avium PPD (falsepositive results) are used; ESAT-6
reduce the incidence of false-positive
results

The assay is at least as sensitive as the
single intradermal test
Measurement of Cell-Mediated
Immunity

ELISpot assay: a capture-antibody
directed against the cytokine of interest
is coated on the bottom of tissue culture
well

The cells to be tested are cultured on
this surface and exposed to the antigen
of interest
Measurement of Cell-Mediated
Immunity

The cells to be tested are cultured on
this surface and exposed to the antigen
of interest; Once the culture period is
completed, the presence of this bound
cytokine can be detected by a traditional
ELISA using specific detection antibody
and enzyme-labeled antiglobulin
Measurement of Cell-Mediated
Immunity
This results in the development of a
pattern of colored spots that each
correspond to the location of a cytokinesecreting cell