Tuberculin elicited cellular immune response in the lactating bovine mammary gland vaccinated intramammarily with Mycobacterium bovis

Veterinary Immunology and Immunopathology, 13 (1986) 39--50 Elsevier Science Publishers B.V., Amsterdam -- Printed in The Netherlands

TUBERCULIN

ELICITED

GLAND VACCINATED

CELLULAR

IMMUNE

INTRAMAMMARILY

RESPONSE

39

IN THE LACTATING

WITH MYCOBACTERIUM

BOVINE MAMMARY

BOVIS

S. C. NICKERSON 1 and B. J. NONNECKE 2 ILouisiana

State

Experiment 2National

Animal

Department (Accepted

University

Agricultural

Center,

Louisiana

Station, Hill Farm Research Station, Homer, LA Disease

of Agriculture, l October

Center,

Agriculture

Research

P.O. Box 70, Ames, IA

Agricultural

71040 (U.S.A.)

Service,

United

States

50010 (U.S.A.)

1985)

ABSTRACT Nickerson, S. C. and Nonnecke, B. J., 1986. Tuberculin elicited cellular immune response in the lactating bovine mammary gland xaccinated intramammarily with Mycobacterium bovis. Vet. Immunol. Immunopathol., 13: 39-50. The development of a local antigen-specific sensitivity was monitored histologically and in secretions of the bovine mammary gland. Three cows in mid-lactation were immunized by injecting 50 ~i of a killed Mycobacterium boris vaccine into the dorsal secretory tissue of the rear marmmary glands; two cows served as unvaccinated controls. Ten weeks after vaccination, all cows were challenged by Intramammary infusion of 1.0 ~g tuberculin in 5 ml phosphate-buffered saline (PBS). Three quarters of each cow received tuberculin at 72, 48, and 24 hours before slaughter; a control quarter received PBS at 72 hours. Vaccinated cows exhibited an intense, local cellular reaction to tuberculin in teat-end tissues at all times post infusion; PBS-infused glands were normal. A moderate leukocyte response in parenchymal tissues adjacent to the gland cistern of tuberculin-lnfused quarters was observed, but deep parenchymal tissues were normal; no effect on milk yield was found. Tuberculin-infused quarters exhibited histological responses in teat cisternal tissues similar to those in delayed-type hypersensitivity reactions. Leukocytic accumulation was primarily macrophages and lymphocytes with few neutrophils. Erythema was observed in the distal half of the cistern, and fibrin deposits were found in subepithelial connective tissues. The epithelium, although distended with leukocytes, was intact and numerous mitotic figures were present. Unvaccinated cows showed no response to challenge. Results demonstrated a marked and specific cellular response in sensitized cows to challenge with tuberculin. INTRODUCTION Vaccines

against

mastitis-causing

bacteria

evaluated by specific antibody concentrations subsequent

susceptibility

al.,

Adlam

local

1975;

cellular

penetration infiltration

et

al.,

immune

of neutrophils

to

1981). response

Little to

dairy

cows

have

been

in blood and milk, as well as by

infection attention antigenic

(Stark, has

been

1970;

Brock

focused

stimulation.

on

et the

Following

into infected tissue sites, monocyte and lymphocyte

is a fundamental

0165-2427/86/$03.50

intramammary

in

manifestation

of inflammation

© 1986 Elsevier Science Publishers B.V.

and may indicate

40 the

development

Targowski

of

and

cell-mediated

cows

in

was

first in

cows.

accumulated

et al.,

i) if a specific

CMI

local

response

antigenic provide

recently,

challenge.

evidence

gland

aureus

and

could

tissues

with

the

of this study was

be elicited

stimulation,

to

demonstrated

challenged

The purpose

that

response

~t was

in distal teat cisternal

1985).

antigenic

to to

the mammary

More

Staphylococcus

(Nickerson

following

response

the

involved

dairy

against

organism

glands

immune

were

cell populations

vaccinated

to determine mammary

(CMI)

antigens

that mononuclear

homologous

cellular (1975)

immunity

staphylococcal

of

a

Berman

and

in vaccinated

2) tissue

location

of the response.

MATERIALS

AND METHODS

Animals Lactating tuberculin milk

multiparous

were

used.

production

Holstein-Freisian Animals

recorded were

were machine

before

and

evaluated

during

cattle, milked

skin

tested

twice

daily,

the vaccination

foremilk

samples

bacteriologically

at

determine

infection status using standard microbiological

negative and

quarter

trial.

weekly

to

Quarter

intervals

to

procedures.

Vaccination The vaccine

consisted

suspended

in

Inspection

Service,

vaccine

9

ml

to

the

paraffin

APHIS).

into secretory

proximity

of 1 gm (wet weight)

sterile

Three

tissue

oil

of killed

(USDA,

cows were

Mycobacterium

Animal

i~munized

and

by injecting

of right and left rear mammary

supramammary

lymph

nodes.

Two

Plant

control

bovis Health

50 ~l of

glands

in close

cows were

injected

with paraffin oil only.

In vivo tuberculin tests Ten weeks

after

vaccination,

the 5 cows were challenged APHIS)

in 5 ml pyrogen-free

duct into the teat cistern. prior

to slaughter

(0 hour).

ml PBS 72 hours prior

three

randomly

by infusing

phosphate-buffered Challenge

saline

was administered

Remaining

to slaughter.

selected

quarters

1.0 ~g of tuberculin

control

Quarter

(PBS)

of each

(PPD-bovis, through

of

USDA,

the teat

72, 48, and 24 hours,

quarters

were

infused with

samples were collected

5

prior to,

and at 6, 24, 30, 48, 54, and 72 hours after infusion to monitor total somatic and differential

cell counts.

Leukocyte enumeration Leukocyte

and characterization

counts were

determined

by the direct microscopic

method

using

4

41 replicate

milk

(Volu-Sol

Medical

observed

smears

defatted

Industries,

with

xylene

Inc.).

at 250 X to estimate

One

and

stained

hundred

with

fields

the mean number of leukocytes

Differential

cell counts were prepared with a cytocentrifuge

Stat-Stain.

Two hundred

cells per smear prepared

Stat-Staln

per

slide

per ml of milk. and stained with

in duplicate were

at 900 X and identified as lymphocytes, macrophages,

were

examined

or neutrophils.

Collection and processing of tissues for microscopy Immediately

after exsanguination,

from each quarter. zones.

parenchymal

Parenchyma was excised

and teat

tissues were

from a 5 cm-deep

incision

taken from 2

Zone I (deep parenchymal zone) was on the upper lateral aspect of the

quarter;

zone

adjacent

to

2

the

(cisternal gland

also collected.

parenchyma)

cistern.

was

Granulomas

toward

the

at vaccine

ventral

injection

surface,

sites were

Teats were severed at the point of attachment to the ventral

surface of the udder. Tissue fixed

specimens

in

0.i

M

of

cacodylate-buffered Samples oxide,

were

parenchyma

and

cacodylate-buffered

internal 2.5%

1.0% osmium tetroxide,

dehydrated

in a graded

and

I ~m-thick

for light microscopy.

of

ethanol,

washed

stained with

Based on light microscopic

acetate

the .teat were

followed

by

0.i

M

in propylene

Tissues were sectioned using an MT-5000

sections were

tissues were selected for electron microscopy, stained with uranyl

lining

and placed in 0.5% uranyl acetate.

series

and embedded in epoxy resins.

ultramicrotome,

tissues

glutaraldehyde

I% toluidine

examination,

blue

representative

and 60 to 90 nm-thick sections

and lead citrate and observed using a Philips EM

300 operated at 60 kV.

Quantitative morphology Teat-end underlying rosette Table

tissues.

area were

III were

minimum

those

from

each

tissue

examined.

quantified

quantity

characteristic and

For

connective

or

of

teat, the

cross-sections middle

Parameters

on a scale of

baseline

level

in PBS-infused quarters. the

rosette

area were

teat

for

the

1 to 5. based

of

the

cistern teat-end

the

and

Furstenberg's

tissues

A score of

on

epithelium

and

listed

in

i indicated the

prevalence

of

each

Tissues from the middle teat cistern quantitated

separately;

however,

data

were combined for each teat and included in Table III as teat-end tissues. Parenchymal tissues. examined.

Parameters

Four tissue samples from each of zones I and II were

characterized

in Table III were quantitated

teat-end tissues except for secretory activity. score of

3 if fully

characteristics

secretory,

I if nonsecretory,

of the other two scores.

as with the

This parameter was assigned a and 2 if tissue displayed

42 RESULTS Milk production, There

was

somatic cell counts, and leukocyte differentials

no

effect

of

vaccination

on

quarter milk yield in the three vaccinated lbs per day,

and 4.0 ibs for the first 5 weeks

cows produced injection,

milk

an average of 6.4

production.

Average

after immunization.

ibs per quarter per day before

Milk SCC from control cows during the challenge period averaged (Table

I).

Quarters

-48,

and -24 hours were

362,

and

177 X

103

Quarters

cells/ml

at

This

PPD-challenged at

to

increase

control

-48,

control values

respectively, cows

330

X

was

and

quarters,

cows challenged with

above PBS

103

cells/ml

probably

-24

cell

but within

infused with

quarters which averaged

-72,

PPD-challenged

cells/ml,

hours

of

elevated

of vaccinated

-72

cells/ml).

cells/ml

Control

paraffin

oil

and 5.9 ibs per quarter per day during the following 6 weeks.

cells/ml

milk.

daily

cows prior to immunization was 4.3

due

PBS at

to

and averaged

the

range

increased 0 hour

the

140 X 103 PPD at -72,

for normal

from 96 X 103

(X

elevated

237,

208

cell

X

103

counts

in

2780 X 103 , 2808 X 103 , and 1753 X 103

hours, counts

respectively. rose

In

initially

at

saline

(PBS)

each the

of

the

6 hour

three

sampling

then declined toward 0 hour.

TABLE I Effect

of

tuberculin

(PPD)

challenge

and

infusion

on

total

somatic cell counts (SCC) in quarters of control and vaccinated cows

Time of chalQuarter lense treatment

Tmt.

Time (hours) prior to slaushter -72 -66 -48 -42 -24 -18 0 .................... SCC/ml X 103 ................

Control -72 -72 -48 -24

PBS PPD PPD PPD

190 250

160 260

160 310 280

90 230 310

130 180 450 190

120 240 390 170

130 190 380 170

-72 -72 -48 -24

PBS PPD PPD PPD

96 210

160 7800

130 4940 3140

140 3270 4600

120 1560 3210 160

480 1120 2070 3300

330 560 1020 1800

Vaccinated

Examination after

initial

increased, (Table

of differential PPD

while

II).

challenge lymphocytes

cell counts of control

or

PBS

infusion,

and neutrophils

the

cows demonstrated

percentage

decreased

or

that

of macrophages

remained

the

same

Quarters of vaccinated cows infused with PBS showed an increase

4S TABLE II Effect of tuberculin leukocyte counts

(PPD) challenge

(Type:

L = lymphocytes,

quarters of control and vaccinated

Tmt.

Time of challense

Control

-72

PBS

-72

PPD

-48

PPD

-24

PPD

-72

PBS

-72

PPD

-48

PPD

-24

PPD

Vaccinated

in neutrophils and

Quarter treatment

from -72

macrophages.

All

6 to 24 hours

quarters

challenged

M = macrophages,

N = neutrophils)

in

Time (hours) prior to slaushter -72 -66 -48 -42 -24 -18 0 ........... Percentage of cells ..........

L M N L M N L M N L M N L M N L M N L M N L M N

to 0 hour,

60 14 26 55 18 27

49 23 28 31 39 30

48 21 30 36 32 32 42 13 45

21 59 20 46 21 33 26 14 60

32 39 29 33 38 29

32 42 26 15 6 79

34 33 33 13 26 61 17 36 47

28 45 27 I0 30 60 18 12 70

and concomitant

PPD-challenged

later,

(PBS) infusion on differential

cows

Type

marked increase in the percentage peaked

and saline

quarters

of neutrophils

followed

37 44 19 46 26 28 32 35 33 46 16 38 31 37 32 16 36 48 22 35 43 27 48 25

decreases

of

40 39 21 46 25 29 36 30 34 34 29 37 27 26 47 21 43 36 32 31 37 13 32 55

51 25 24 51 22 27 38 24 38 30 31 39 27 28 45 32 42 26 37 32 31 19 25 56

in lymphocytes

vaccinated

cows

showed

a

after initial infusion, which

by a decline

at 0 hour

(in the case of

at -72 and -48 hours).

Mammary histology Teat-end infused

I0

tissues weeks

from later

tuberculin exhibited epithelial postmortum Histological

lining

72,

of 48,

cows

vaccinated

and

24

exhibited which

marked was

demonstrated

epithelium,

as

well

primarily

of

macrophages

as

erythema

limited a

marked

subepithelial and

to

before

M.

and

leukocytic

upon of

infiltration

tissues

cells,

half

with

(Fig. few

and with

The cistern

induration

distal

bovis

slaughter

(Table III).

the

connective

lymphoid

against

hours

a marked response to challenge

examination study

quarters at

i)

gross

the of

teat. the

composed

neutrophils.

44 TABLE III Quantitative control

analysis a of teat-end

cows infused w i t h saline

and

parenchymal

tissues

(PBS) or challenged

of vaccinated

with tuberculin

and

(PPD) at

-72, -48, and -24 hours prior to slaughter Vaccinated cows PPD PPD PPD -72 -48 -24

PBS -72 Teat end tissues M i t o t i c divisions Erythrocytes Neutrophils Macrophages Lymphocytes Plasma cells Circulating cells Fibrin Parenchymal zone I Neutrophils Macrophages Lymphoid cells Secretory activity P a r e n c h y m a l zone II Neutrophils Macrophages Lymphoid cells Secretory activity

1 1 i i I 2 i i

3 3 2 4 5 4 3 3

3 4 3 4 4 3 3 4

2 3 3 3 3 3 2 3

I 1 4 I i 2 1 I

1 I I i i i I i

1 i 2 I 2 2 i I

i I 3 i 2 2 i 1

i I i 3

1 i 1 3

I 1 i 3

I i 1 3

I i i 3

I i 1 3

i I I 3

i 1 I 3

i i i 3

3 1 2 3

4 3 4 2

2 2 2 2

i I 1 3

i I I 3

i I i 3

2 1 i 3

aparameters were quantitated baseline level, 5 = m a x i m u m 3 = fully secretory. Leukocytes

72 hours

after

mononuclear were

abundant (Fig.

cisternal

were

was

observed

small blood normal

vessels

(Fig.

in

subepithelial were

also

often

damaged 3).

mitotic

observed

although

(Fig.

and numbers

Numerous

were

but

neither

in cross-sections

cistern,

in the

2),

lining,

(Fig. 2),

teat

challenge.

leukocytes

most

together

on a scale of 1 to 5: 1 = m i n i m u m quantity or quantity. Secretory activity: i = nonsecretory,

were more prevalent

in those of the mid

N o n v a c c i n a t e d cows PPS PPD PPD -72 -48 -24

PBS -72

nor

tended

stroma

in

where

between

distended

with

necrosed,

and

cells

also

Teat-end

tissue

parenchymal

areas

rosette

to be greater

figures

subepithelial

found

Mononuclear 3).

of Furstenberg[s

cells

as

Erythrocytes

they were

often

packed

epithelia.

infiltrating numerous

tended

at 48 and

identified

areas.

cistern

than

The

macrophages

mitotic

figures

to accumulate

from PBS-infused

around

quarters

was

in appearance.

Zone normal

II in

(gland

cistern)

appearance;

however,

tissues

were

frequently

lymphoid

cells

in PPD-challenged

peak at

48 hours post challenge.

alveolar

infiltrated

of

lumina with

glands. Secretory

PBS-infused

and

interalveolar

neutrophils,

Cellular activity

quarters

were

connective

macrophages,

infiltration

and

appeared

to

of zone II parenchyma

of

45

Figures 1 through 3 are tissue sections from teats of quarters of vaccinated cows challenged with PPD. Fig. I. Cross-section of the internal teat cistern lining at Furstenberg's rosette illustrating marked leukocyte infiltration into epithelia] and subepithelial tissue sites. Fibrin deposits are observed at arrows. Leukocytes tend to accumulate in areas adjacent to the mucosal surface, rather than in deeper tissues. X300.

PPD-challenged quarters at 24 and 48 hours tended to be lower than PBS-infused quarters Zone normal

(Table IIl). I

(deep)

parenchymal

in appearance with

activity.

Gross

tissues

no signs

examination

of

of

of

vaccine

administration revealed small granulomas, Microscopic

evaluation

lobuloalveolar (Fig.

4).

of

architecture

Tissues

PBS-

exhibited

injection

sites

demonstrated

still apparent, marked

PPD-infused

glands

or decrease

were

in secretory

10

weeks

after

approximately 2 to 3 cm in diameter.

granulomas was

and

inflammation

that

although

the parenchyma was

leukocytic

infiltration

the

involuted

with

dense

accumulation of lymphocytes and plasma cells (Fig. 5). Teat-end tissues from control cows exhibited no reaction to infusion of PBS or challenge with

PPD.

Parenchymal

zones

I 8nd II of

PBS- and PPD-infused

46

Fig. 2. Epithelial lining illustrating accumulation of vacuolated macrophages between apical and basal cell layers; note macrophage migrating into the basal epithelium (arrows). Erythrocytes are observed in the adjacent connective tissue. X800. Fig. 3. Cross section of the cisternal lining illustrating the accumulation of macrophages around blood vessel, and cells dividing in the teat epithelium (arrows). X800.

quarters

appeared

free

of

inflammation,

and

exhibited

normal

secretory

activity.

Electron microscopy of PPD-challensed quarters. confirmed

that

lymphocytes. which

infiltration

from

congested

Macrophages

in spaces between

capillaries,

frequently

and

epithelium.

and

of

also

accumulated

macrophages

numerous

lymphocytes

Frequently,

as dense,

were

leukocytes

also

contained

(Fig. 6).

follicle-like

incorporated

identified

and

erythrocytes remnants

in the cisternal

the apical and basal cell layers

lymphoid cells were often observed cells,

Ultrastructural evaluation

consisted

Macrophages were often observed with phagocytosed

exuded

neutrophilso

leukocytic

Subepithelial

accumulations

into

of

epithelium

the

of

cisternal

as immunoblasts were observed

in both epithelial (Fig. 7) and connective tissue sites, where they were found in various lymphocytes reticulum

phases

of

and plasma

mitosis.

Such

cells,

exhibited

and mitochondria,

cells

were

in

close

a few strands

association

with

of rough endoplasmic

and were filled with clusters

of polyribosomes.

47

Fig. 4. Section of a granuloma from the injection site of a vaccinated cow showing massive mononuclear cell infiltration and involution of parenchymal tissue. X200. Fig. 5. Dense accumulation of leukocytes in granuloma illustrating numerous lymphocytes and plasma cells. X800.

Discussion Results of this investigation demonstrated a marked and specific localized response in M. bovis-vaccinated cows to challenge with tuberculin.

Total SCC

in

after

glands

of

challenge, during

vaccinated

then

the

lymphocytes mammary

and

dramatically

toward

postchallenge possibly

specific

secretions

proliferative culture,

declined

early

chemoattractants,

cows

of

responses

0 hour. period

mediated antibody M.

over

increased

by

The

suggests

from

indicating prior sensitization

the

of

local

1985).

cows

unvaccinated

to antigen

of

controls

of

sensitized

Lymphocytes

showed

PPD

neutrophils

production

population

et al.,

bovls-vaccinated those

6 hours

predominance

a resident

(Nonnecke

by

in

significant to

(Nonnecke et al.,

PPD

in

1985).

Such a resident population of sensitized lymphoid cells would be instrumental in

eliciting

a

response

to

subsequent

antigenic

challenge.

Contact

of

bacterial antigens with lymphocytes in the udder may result in sensitization, with subsequent exposure leading to CMI response (Targowski and Berman, 1975). Previous evaluations of local immunization techniques demonstrated limited success due to damage to secretory tissue and reduced milk yield (McDowell and

Figs. 6 and 7 are electron micrographs of teat-end tissues of quarters of vaccinated cows challenged with PPD. Fig. 6. Macrophage (M) in a space below the luminal epithelial cell layer. X3200. Fig. 7. Immunoblast with numerous cytoplasmic polysomes in the basal X5600. cisternal epithelium.

Watson,

1974;

minimal

damage

Watson,

1981).

to secretory

Results

parenchyma

of

the

present

and no effect

study

on milk

demonstrated

yield

following

local vaccination. Histological

examination

response

to

observed

in delayed-type

1975;

PPD

Wiener,

challenge.

1970)

which are manifested accumulation Localized

mitogenesis

Cytological

including

erythema,

at 24 to 72 hours.

of mononuclear

cells, tissue

with

lining.

antigen

tissue

and

(Outteridge

Resident subsequent

and Lee,

confirmed

features

hypersensitivity

in the connective

in the eplthelial contact

of teat-end

induration, The prominant

interaction

may with

identical

(Barrett, and

was

which

small blood function

cellular to

those

1983; David,

fibrin

feature

macrophages,

surrounding

macrophages

1981).

were

reactions

especially space

the specific

deposition the marked were

in the

lymphocytes,

found

vessels

and

initial

enhancing

49 Leukocytic

infiltration

was

greater

toward

the

distal

teat

cistern

at

Furstenberg's rosette in comparison with tissue from the middle portion of the cistern.

Likewise,

quantitation

quarters

demonstrated

uninfected

of a

teat-end greater

leukocyte

populations

concentration

at

from

Furstenberg's

rosette (Nickerson and Pankey, 1983), Mechanisms

involved

in the marked

concentration of protective

populations in teat-end tissues is unclear. the natural

route of antigen entry,

region of the gland is logical.

leukocyte

However, because the teat duct is

the recruitment of leukocytes to this

After breaching the teat duct,

the first

contact between microorganisms and the interior mucosal surfaces would involve the

epithelial

Mucosal

lining

surfaces

of

which

Furstenberg's

contact

the

rosette

external

and

resident

environment

leukocytes.

generally

contain

diffuse mononuclear cell infiltrates in close proximity to the epithelium, and are typical of intestinal and respiratory tracts as well as the mammary gland (Anderson and Anderson,

1981).

Thus,

specifically sensitized T lymphocytes

possibly homed to distal teat-end tissues near the route of antigen entry. Subsequent exposure would lead to a CMI response including lymphokine release, resulting in recruitment, activation, and immobilization of other leukocytes. B lymphocytes were probably present in teat-end tissues as evidenced by the frequent appearance of immunoblasts and dividing forms. morphologic

alterations

by

accumulating

rough

Such cells undergo

endoplasmic

reticulum

and

maturing into plasma cells upon contact with antigen (Wiener, 1970). In conclusion, results demonstrated that local vaccination of the lactating bovine

mammary

sacrificing tissue.

gland

milk

led

yield

to

or

sensitization

inflicting

to

a

specific

extensive

damage

to

antigen

without

milk-producing

Mammary tissue responses to tuberculin challenge were most dramatic

in distal teat-end tissue, exhibiting characteristics indistinguishable from delayed-type hypersensitivity reactions.

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responses

in

cows

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A.O.

lymphatic

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Anderson,

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In:

following

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with

staphylococcal

J. Comp. Pathol., 91: 105-113. N.D., D.L.

1981.

Structure

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and

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Res.

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B.A.

MI,

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Watson,

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