Cellular and humoran immune responses to corynebacterium presudotuberculosis infection in goats
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Date
1986
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Department of microbiology, College of veterinary and animal sciences, Mannuthy
Abstract
Caseous lymphadenitis was experimentally produced in cross-bred
malabari goats aged 8 to 12 months by bilateral inoculation of 1 x 106 viable
C. pseudotuberculosis (ATCC 19410) through intradermal, subcutaneous and
submucosal routes. The clinical picture, immune response and pathological
features were studied up to a period of 13 weeks. The development of immune
response in experimentally infected goats was assessed, by comparing the data
with those of the controls, with respect to total serum protein, serum protein
fractions, antibody activity of the serum, leukocyte count, counts of
lymphocyte subpopulations, leukocyte migration inhibition index and skin
hypersensitivity reaction. Pathological features in the lymphnodes and other
tissues of infected goats necropsied at 15 days interval were also studied.
Initial febrile reaction which lasted for 72 to 96 h, local inflammatory
changes caused at the site of inoculations during the first two to three weeks
of infection and the development of lesions typical of caseous lymphadenitis in
local/regional lymphnodes within 21 days post-inoculation were the main
features of clinical manifestations of the disease. aS a result of infection,
neutrophilic leukocytosis was maximum during the 2nd week of infection. No
appreciable was maximum during the 2nd week of infection. No appreciable
change in counts of other cells in terms of their absolute numbers was noted
during the entire period of observation.
The humoral immune response in infected goats was indicated by the
rise in serum protein, antitoxic antibody and B-lymphocyte count in the
peripheral blood. The serum protein concentration increased to significant
levels from the 5th week onwards and it reached the peak value (11.346 g%)
by the 8th week. From the 3rd week onwards haemolysis inhibition test, which
detected goats and persisted till the end of the observation period. The peak
antibody level was recorded by the 5th week of infection and thereafter there
was gradual reduction in the titre. Significantly high percentage of B-
lymphocyte was recorded in infected animals from the 2nd to 10th week, except
at the 5th week. The percentage of B-cells in infected goats ranged between
12.3 + 0.85 – 17.63 + 1.2 while it was 8.56 + 0.75-12.3 + 1.09 in control goats.
This was considered as an indication of stimulation of humoral immune
response.
The cell-mediated immune response was evidenced by the increased T-
lymphocyte count in the peripheral blood, inhibition of leukocyte migration
and the development of delayed skin hypersensitivity. The mean percentage of
T-lymphocytes in the peripheral blood of infected goats by E-rosette assay
recorded an initial reduction at the first week (18.44 + 1.4) followed by an
increase which was significant during the 5th, 6th, 7th, 8th, 12th and 13th week of
infection. The maximum value was recorded (35.24 + 1.58) at the 13th week. In
the case of control goats the percentage values ranged from 24.55 + 3.66 to
26.74 + 1.34. The T-lymphocyte count in the peripheral blood enumerated the
ANAE method did not show any significant change even after infection. In the
experimentally infected goats, leukocyte migration inhibition index was less
than 0.8 during post-infection period while the control goats had the index
value above 0.8. Significant reduction in the migration index was noted by
45th day of infection and the maximum reduction was on the 60th day.
Intradermal injection of the toxic supernatant of the culture elicited
characteristic delayed skin hypersensitivity reaction in all the experimentally
infected goats while there was no reaction in the controls. The positive
reaction was found to be maximum by the 48th hour post-injection.
The pathological changes were characterized by an initial stimulatory
hyperplastic reaction in the lymphnodes and this was accompanied by
necrobiotic changes typical of caseous lymphadenitis. The hyperplastic
stimulatory reactions were characterized by the presence of several active
follicles with well developed germinal centres in the cortex, distinct medullary
cords densely lined with plasma cells and sinus histiocytosis indicating the
early elicitation of humoral immune response to the bacterium or to its in vivo
products.
The results obtained from the present study revealed the operation of
both cell-mediated and humoral immune responses in goats against C.
pseudotuberculosis infection. Of the various methods employed to monitor the
immune response, leukocyte migration inhibition and delayed skin
hypersensitivity tests were found to be of value in assessing the cell-mediated
immune response and haemolysis inhibition test for humoral immune
response. Leukocyte migration inhibition test and haemolysis inhibition test
could be employed for early diagnosis of C. pseudotuberculosis infection in
goats.
FINDINGS :
The immune responses and pathological features in Corynebacterium
pseudotuberculosis infection were studied by experimental infection of cross-
bred Malabari goats of S-12 months of age. Single cell bacterial suspension in
chilled sodium chloride bile salt solution was used for this purpose. Goats
were inoculated at both sides of the body by three routes viz., intradermal,
subcutaneous and submucosal, with 2 x 106 bacteria per site of injection. The
experimentally infected and control goats were observed for clinical
manifestations of caseous lymphadenitis for a period of 13 weeks.
The development of immune response in experimentally infected goats
was assessed by comparing the data with those of the controls with respect to
total serum protein, serum protein fractions, antibody activity of the serum,
leukocyte counts, counts of lymphocyte sub-populations, leukocyte migration
inhibition index and skin hypersensitivity reaction.
Gross and histopathological changes in the lymphnodes and other
tissues of necropsied goats were studied at 15 days interval for a period of 90
days.
All experimentally infected goats exhibited rise in temperature, general
weakness, lethargy and impaired appetite which lasted for 72 to 96 h. The
sites of inoculations showed varying degree of inflammatory reaction during
the first two to three weeks of infection. All experimentally inoculated goats
except one developed lesions typical of caseous lymphadenitis in regional/local
lymphnodes within 21 days post-inoculation. Route of infection did not
influence the ability to set up lesions in lymphnodes. Although massive dose of
bacterial (1.2 x 107) was administered, none of the goats had fatal infection
indicating that goats are relatively resistant to this infection. Majority of goats
did not develop generalized form of caseous lymphadenitis as there was no
lesions in visceral/deep seated lymphnodes or organs.
The normal serum protein concentration of cross-bred Malabari goats
was estimated to range from 7.187 to 9.750 g %. Consequent to experimental
infection, serum protein concentration was increased and recorded significant
rise from the 5th week onwards reaching the peak value by the 8th week –
11.346 g%.
Estimation of quantitative distribution of serum protein fractions was
done
by
agar
gel
electrophoresis
and
densitometer
tracing
of
electrophoretogram. Though there was initial increase in globulin content in
infected animal followed by a decrease, no significant alteration in the
albumin-globulin ration (A:G ratio) was noted compared to the control
group.
C. psuedotuberculosis was cultivated in lemco proteose broth
containing sheep serum and incubated aerobically at 370C for 72 h.
Supernatant obtained from the above culture, having maximum haemolysin
titre and dermonecrotoxicity was used as the toxin of the bacterium in the
present studies.
The haemolysin content of the culture supernatant was estimated by the
haemolysis test using sheep red cells. A maximum titre of 1:256 was found in
the culture aged 72 h. The dermonecrotoxicity of the toxic culture
supernatant was tested by intradermal inoculation into the rabbit skin. The
inflammatory and necrotic reactions were maximum by 48 h post-injection.
Specific antibody activity against toxin of C. pseudotuberculosis in the
serum was monitored by haemolysis inhibition test and the test was adjudged
as a useful test for detecting humoral
immune
response
to
C.
pseudotuberculosis infection in goats. In infected goat from the 3rd week of
infection onwards MIT was positive while it was negative in control goats
during the period of 13 weeks of observation. The peak antibody level was
achieved by the 5th week of infection and thereafter the titre was found to
dwindle gradually till the 11th week. Towards the end of the observation
period (12th week) there was a marginal increase in the antibody titre, which
would be considered as secondary immune response against the toxin of the
multiplying bacteria.
Infected goats showed leukocytosis during the entire period of
observation and maximum leukocytosis was observed during the 2nd week of
infection. The periodical fluctuation in leukocytosis indicated the recurrent
flare up of bacterial invasion in the body.
The absolute lymphocyte count obtained both at pre and post-infection
periods with experimentally infected goats did not show any change which
indicated no deleterious effect on peripheral blood lymphocytes. Throughout
the period of observation infected animals showed numerically low
lymphocyte percentage in differential counts and with several samples the
percentage distribution was significantly low.
Absolute counts of neutrophils were consistently high in experimental
goats when compared to those of controls and the same was reflected in
differential count also. The other blood cells were absolutely without any
change in infected as well as control goats.
Density gradient centrifugation using Ficoll-paque (1.077 g/ml,
centrifuged at 720 x g) was found quite useful for separation of mononuclear
leukocytes from the whole blood of goats. Such separated mononuclear cells
were found to contain on an average 91.80% lymphocytes and 8.2%
monocytes with an average viability of 91.2%.
Peripheral blood B-lymphocytes of goats were successfully enumerated
by EAC rosette assay employing bovine red cells. The normal percentage of
B-cells was estimated to range 8.56 and 12.3. Significantly high percentage of
B-cells was recorded in infected animal from the 2nd to 10th week post-
infection except at the 5th week. B-cell percentage in infected goats ranged
between 12.3 + 0.85-17.63 + 1.2 while it was 8.56 + 0.75 to 12.3 + 1.09 in
control goats indicating the operation of humoral immune response to
concurrently boost the specific antibody activity in the serum.
T-lymphocytes of goats were identified and enumerated by E-rosette
assay and ANAE activity. Goat lymphocytes presented several receptors to
sheep red cells, as majority of rosettes presented erythrocytes at the entire
periphery of lymphocytes.
The mean percentage of E-rosette positive lymphocytes in the
peripheral blood of control goats ranged from 24.55 + 3.66 to 26.74 + 1.34
during 13 weeks of observation. The E-rosette technique employed in the
present study was assumed unaffected by unknown variables as the data
recorded in the control goats remained near normal throughout the
observation period.
During the first two weeks of infection E-rosette positive lymphocyte
count was found numerically decreased and the reduction was significant at
the first week (18.44 + 1.40). From the third week onwards an increase in the
E-rosette forming cells was observed and significant increase was noted
during the 5th, 6th, 7th, 8th, 12th and 13th week of infection, the maximum being
at the 13th week (35.24 + 1.58).
T-cells were also identified and enumerated based on the demonstration
on ANAE activity. Fixing of mononuclear cells in acetone-citric acid solution
enabled the fixed smears to be stored in dry state without any interference to
the enzymic activity for longer periods. T-lymphocytes presented one or two
localized red coloured reaction product in the cytoplasm adjacent to the cell
membrane.
Mean percentage of ANAE positive cells in experimental goats was
28.09 + 1.51 while it was 35.80 + 4.86 for control goats when estimated before
the start of the experiment. During infection, the count of ANAE positive cells
in the peripheral blood did not show any change as the mean percentage
ranged between 28.9 + 2.06-33.78 + 1.99 as against the corresponding values
(30.83 + 3.5-36.91 + 3.61) in controls.
In infected animals significant hike in E-rosette positive lymphocyte
counts was recorded while such a change could not be observed with ANAE
positive lymphocytes. Thus the results of T-cell estimation by E-rosette assay
and ANAE demonstration indicated that estimation of total rosette forming
cells could reflect better the T-cell competence.
Cell mediated immune response to C. pseudotuberculosis infection in
goats was demonstrated by leukocyte migration inhibition test under agarose.
A population density of 1.5 x 108 leukocytes/ml was found suitable for LMIT.
Toxic culture supernatant having haemolysin titre 1:16 whose pH adjusted to
7.2 could be successfully used as antigen in the test. In experimentally infected
goats leukocyte migration index was less than 0.8 during post-infection period
while with control goats it was above 0.8. Significant reduction in LMI index
was noted by 45th day of infection through 75 days showing maximum
reduction by the 60th day.
Intradermal injection of toxic culture supernatant elicited characteristic
delayed type skin hypersensitivity reaction in all experimentally infected
goats, while a negative reaction in controls. Skin hypersensitivity reaction was
found to be maximum by 48 h post-injection.
Histopathology of skin biopsy taken from the site of inoculation
revealed infiltration of lymphocytes, and macrophages at perifollicular and
periglandular areas, congestion of blood vessels with perivascular infiltration
of lymphocytes and macrophages and dermal oedema.
Tuberculin failed to produce a positive skin hypersensitive reaction in
C. pseudotuberculosis infected or control goats.
From 15th day onwards, experimentally infected goats which were
necropsied presented gross lesions typical of caseous lymphadenitis in
lymphnodes. The lesions were found to confine to superficial lymphnodes
adjacent to the site of inoculations.
The histological changes observed in lymphnodes were basically of two
types: hyper-plastic stimulatory reaction and degenerative changes. The
changes were hyper-plastic reactive follicles with well distinguished germinal
centre, accumulation of lymphocytes and varying degrees of sinus
histiocytosis in medullary region, dense lining of medullary cords with plasma
cells, depletion of lymphocytes from the cortical area; subcapsular and
cortical oedema, congestion of blood vessels, haemorrahage, infiltration of
mononuclear cells in lymphatics and blood vessels, accumulation of
macrophages and plasma cels in the medulla, dilatation of sinusoids, fibrous
tissue proliferation, degenerative and necrotic changes of lymphocytes in the
cortex and medulla, fibrous tissue encapsulated focal areas of caseation of
calcification surrounded by lymphocytes, macrophages and giant cells and
finally conversion of parenchyma to a caseated mass enclosed in fibrous tissue
capsule.
In brief, the results obtained from the present study revealed the
operation of both cell-mediated and humoral immune responses in goats
against C. pseudotuberculosis infection. Of the various methods employed to
monitor the immune response, leukocyte migration inhibition and delayed
skin hypersensitivity tests were suitable for ascertaining the cell-mediated
immune response and haemolysis inhibition test for humoral immune
response. Leukocyte migration inhibition test and haemolysis inhibition test
can be successfully employed for the early diagnosis of C. pseudotuberculosis
infection in goats.
Description
PhD
Keywords
Citation
170979