Basrah Journal of Veterinary Research,Vol.15, No.3,2016
Proceeding of 5th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
532
CASEOUS LYMPHADENITIS IN IRAQI SHEEP AND
ASSESSMENT OF VACCINATION WITH COMMERCIAL
VACCINE
Taha Yassin Ghani* , Faisal Ghazi Habasha*
* Department of Veterinary Internal and Preventive Medicine, College of Veterinary
Medicine, University Of Baghdad,Baghdad,Iraq
Keywords; lambs, C. pseudotuberculosis, challenge dose.
ABSTRACT
Experimental study included 15 lambs which were divided into 3 groups; each
group consisted of 5 lambs. A commercial Case- Bac vaccine was used to vaccinate
group II, while others groups served as positive and negative controls. All lambs in group
II were received two doses of vaccine at four weeks interval. The lambs in groups II and
III were challenged S/C with 9 x 108 CFU virulent isolated strain of C.
pseudotuberculosis.
Humoral and cell mediated immune response were detected during the period of
experiments, the temperature, pulse rate and respiration rate were determined weekly.
The protection in vaccinated group was 100% against challenge dose, while in the
group III one lamb died on fifth day post challenge. Pus was appeared in the rest of lambs
at the site of injection.
INTRODUCTION
Caseous lymphadenitis (CLA) is a chronic infectious disease of small ruminant
(sheep and goats) caused by the bacterium Corynebacterium pseudotuberculosis was
formerly known as Corynebacterium ovis (1).
Basrah Journal of Veterinary Research,Vol.15, No.3,2016
Proceeding of 5th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
533
The disease found in the major sheep and goat production all over the worlds
which was cause significant economic losses due to culling of affected animals, decrease
in reproductive efficiency in meat, wool and milk production, carcass and skin
condemnation due to abscessation (2).
The best strategy for control and prevention of CLA disease is immunization
especially in countries with high prevalence of infection (3). To make the control of CLA
successful, the identification of infected animals are necessary. Bacteriological culture is
mandatory to done to exclude other bacterial pathogens capable to produce suppurative
lesions (4).
This study was conducted to know the efficacy of commercial vaccine (Case-Bac
Vaccine) by immunization trials against the caseous lymphadenitis disease in sheep and
study cellular and humoral immunity.
MATERIALS AND METHODS
Experimental Animals:
Fifteen male lambs Awassi breed their ages ranged between 5-7 months old were
used in this study. The animals farmed in college of veterinary medicine university of
Baghdad. All lambs were treated by Ivermectin 200 μg per kg of body weight
subcutaneously at the rate of 1 ml per 50 kg as anthelminthic treatment for internal and
external parasite before starting the experiment and remained one month for adaptation.
Lambs divided into 3 equal groups (I, II, and III) each group consisted of 5 lambs.
All lambs were clinically healthy and immunological response to infection was
negative as determined by measurement of serum anti PLD (phospholipase D) IgG by
Enzyme Linked Immunosorbent assay (ELISA) test.
CASE-BAC vaccine:
A commercial vaccine prepared by Colorado Serum Company (U.S.A.),
detoxified and purified the whole culture of Corynebacterium pseudotuberculosis
contains thimerosal as preservative used in healthy sheep as a killed bacterin-toxoid
Basrah Journal of Veterinary Research,Vol.15, No.3,2016
Proceeding of 5th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
534
vaccine. The vaccine injected subcutaneously in a dose of 2 ml in axillary space and
repeated in four weeks in opposite axillary space.
ELITEST CLA:
An Enzyme Immuno-Assay for the detection of IgG antibodies specific for the
Corynebacterium pseudotuberculosis in sheep sera. The (ELISA) utilized a recombinant
form of phospholipase D (PLD) from C. pseudotuberculosis to detected anti-PLD IgG
antibodies in sera of sheep and goats infected with caseous lymphadenitis. The
commercial kit ELITEST CLA product of Hyphen BioMed, France was used for
detection of IgG antibodies specific for the causative agent of caseous lymphadenitis
(CLA) in sheep and goat sera. This kit was performed according to the manufactures
instruction, recombinant phospholipase D (rPLD) antigen used for determined antibodies
in the sera of animals.
Cell Mediated Immune Response:
Cellular immune responses of the vaccinated and control groups were evaluated
by Delayed Type of hypersensitivity (DTH) skin test.
It was done two weeks after booster dose of vaccination. Delayed Type of
hypersensitivity was assessed by the skin thickness that was measured before and after
the inoculation of C. pseudotuberculosis antigen (5)
The prepared soluble Ag was given intra dermally in the lower flank region. The
skin measurement was done by using metal Vernier caliper micrometer to estimate
double skin fold thickness before and after the I/D injection with 0.1 ml Ag contained 0.5
mg protein which was determined by means of (6 and 7).
Preparation of C. pseudotuberculosis inoculum:
The bacterium was cultured on blood agar plate and then growth was collected by
using sterile cotton swabs. Bacteria were suspended in phosphate buffered saline (PBS)
in sterile test tubes by rolling swab gently against the tube sides. The bacterial suspension
Basrah Journal of Veterinary Research,Vol.15, No.3,2016
Proceeding of 5th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
535
was adjusted to a final concentration of (9x108) CFU per ml for challenge dose.
MacFarland tubes were used to estimate colony forming unit (CFU) as mentioned by (8).
One ml inoculum was given subcutaneously in the flank region about 10 cm above the
pre femoral lymph node of each lamb.
Experimental design:
Basrah Journal of Veterinary Research,Vol.15, No.3,2016
Proceeding of 5th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
536
Statistical analysis:
All data were analyzed statistically using one way ANOVA test by Microsoft
program (SPSS). The level of statistical significance was set at (P< 0.05) as described by
(9). The chi-square test was achieved to compare between percentage of the study.
RESULTS
The clinical examination of group II (G II) moderate increase in rectal
temperature, pulse, and respiration (40.24 ± 0.45 0C), (98 ± 3.16/ minute), and (41.6 ±
3.85/ minute) respectively were recorded after vaccination as showed in Figures (1, 2,
and 3). All lambs returned to their normal activities within 3-4 days post vaccination.
Slight localize swelling was noticed at the sight injection and disappeared within one
week.
Basrah Journal of Veterinary Research,Vol.15, No.3,2016
Proceeding of 5th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
537
Basrah Journal of Veterinary Research,Vol.15, No.3,2016
Proceeding of 5th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
538
The Lambs in group III none vaccinated after challenged they revealed moderate
elevation in average in temperature, pulse rate and respiration rate as showing in Table
(1).
Table (1) Clinical Response (TPR) to Challenge in all Lambs Groups
Mean ± SD Groups
Respiration Pulse Temperature.
31.2 ± .5.215 80.5 ± 5.727 39.02 ± 0.497 GI
38.6 ± 3.975 94 ± 0.316 40.2 ± 0.316 GII
54.6 ± 5.702 112 ± 3.162 40.62 ± 0.130 GIII
The site of injection showed redness and induration and clinical sings persisted
for 5 – 7 days. On the 5th day post challenged the lamb number one died while the other
lambs showed inflammation and abscess formation at the site of injection.
Humoral Immune response
All lambs were negative on indirect ELISA and no antibody titer was recorded
pre vaccination. The level of anti PLD IgG antibodies of each animal were determined
and presented in Figures (4 and 5) as the absorbance values at 450 nm obtained using
indirect ELISA. Two weeks after the first dose of vaccination the antibody titer
developed in all vaccinated lambs (GII) with mean optical density (OD) values (0.426 ±
0.26) while the control groups (GI and GIII) OD values were (0.096 ± 0.013) and (0.093
± 0.01). The maximum titer was reached at the 8th week post vaccination (1.984 ± 0.776)
then the antibody titers begun to decrease to reach to (0.95 ± 0.446) as in Figure (4)
Basrah Journal of Veterinary Research,Vol.15, No.3,2016
Proceeding of 5th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
539
All lambs in GII showed no signs of infection during the experimental period. The
lambs in GIII appeared rising antibodies titer two weeks post challenged and reached the
maximum level within 5 weeks post challenged were the mean optical density was 1.691
± 0.89 and then declined to 1.508 ± 0.09 after 3 weeks as in Figure (5).
Delayed Type Hypersensitivity- Skin test
All vaccinated lambs had positive skin reaction after intradermal injection of
antigen which manifested by induration and thickening of the skin. Two weeks after the
second dose of the case-bac vaccine, skin reactivity was determined in two groups. While
negative control group GI did not show any skin reaction as in Figure (6) and Table (2).
Basrah Journal of Veterinary Research,Vol.15, No.3,2016
Proceeding of 5th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
540
Table (2) DTH-Skin Test Reaction in Vaccinated Lambs Groups
Soluble Ag
0.5 mg/ml
Skin thickness
(mm)
Time/hours
72 48 24 0
Control
negative
group (GI)
Range
Mean ±
SD
0.97-1.62
1.344 ±
0.369
1.15-2.04
1.62 ±
0.345
1.12-1.82
1.586±
0.317
1.07-1.80
1.518 ±
0.327
Vaccinated
group
(GII)
Range
Mean ±
SD
1.12-1.69
1.49 ±
0.223
2.15-3.39
2.81 ±
0.537
2.25-3.47
2.87 ±
0.467
2.04-2.95
2.47 ±
0.420
Positive
control
group (GIII)
Range
Mean ±
SD
1.22-1.45
1.36 ±
0.083
2.45-2.99
2.73 ±
0.673
2.94-3.35
3.07 ±
0.194
2.14-2.74
2.55 ±
0.278
Basrah Journal of Veterinary Research,Vol.15, No.3,2016
Proceeding of 5th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
541
DISCUSSION
The data of CLA incidence in sheep flocks in Iraq have not been recorded yet.
Caseous lymphadenitis in sheep in Iraq is a disease which was not studied until now; and
if it was done, with very limited and restricted works regarding isolation of C.
pseudotuberculosis as an isolate, or in a group of isolates that were surveyed with other
pathogens.
This study is the first one conducted using the commercial vaccine Case-Bac for
vaccination of sheep in Iraq. Antibody levels due to CLA vaccine were detected using
ELISA test as in many studies of serological tests (10; 11; and 12). Vaccination with
Case-Bac vaccine resulted in a complete protection against challenge and these results
coincide with full protection achieved by (8) and with (13). The study showed that there
was fluctuation in individual animal responses to vaccination in which antibody titer
differed from one lamb to another and the OD ranged from 0.409 to 2.409 on the 8th week
post vaccination. This fluctuation may be due to the individual differences of immune
status or may be due to persistent of other infection.
Vaccinated group (G II) showed increased in antibodies level (OD absorbance)
after 3 weeks of the first dose of vaccine and reache the maximum level at the seventh
weeks post vaccination. Similar finding has been reported by (13) while antibodies titer
decreased after 3 weeks of challenge.
In unvaccinated control group (G III) antibodies increased 2weeks after challenge
and declined after 3 weeks as this fact agreed also with (13). Vaccination subcutaneously
has been shown to induced humoral and cell mediated responses (14). Antibody titers of
vaccinated sheep were measured by ELISA to evaluate the humoral response which
induced by vaccine and play a role in protection against CLA (15 and 16). C.
pseudotuberculosis well known as a facultative intracellular microorganism, so cell
mediated immune response play a major role in protection and providing adequate
protection against CLA (8).
Basrah Journal of Veterinary Research,Vol.15, No.3,2016
Proceeding of 5th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
542
In conclusion, this vaccine appeared to offer an excellent protection against the
development of CLA in sheep and the vaccine could play an important role in the control
of the disease in infected sheep flocks.
مرض التھاب الغدد التجبنی فی الاغنام العراقیھ وتقییم التحصین باللقاح التجاری
طھ یاسین غنی و فیصل غازی حباشة
فرع الطب الباطنی والوقائی ،کلیة الطب البیطری، جامعة بغداد ،بغداد ،العراق
الخلاصھ
تضمنت الدراسة الحقلیة 15 حملا .قسمت الحملان الى 3 مجامیع متساویھ تحتوی کل مجموعة على خمسة
واعطیت جرعتین بینھما فترة اسبوعین بینما Case-Bac حملان. لقحت حملان المجموعھ الثانیة باللقاح التجاری
بقیت حملان المجموعة الاولى کمجموعة سیطره سالبة. تم تعریض حملان المجموعتین الثانیة والثالثة الى جرعة
9 تحتوی عتره ضاریة من جرثومة وتدیات التدرن الکاذب .وتم قیاس مستوى المناعھ x108 CFU تحدی تحت الجلد
الخلطیة فی جمیع المجامیع اسبوعیا،وکذلک تم قیاس مستوى المناعھ الخلویھ باستخدام اختبار الجلد کما تم قیاس درجة
الحرارة ومعدل النبض ومعدل التنفس اسبوعیا .وکانت نسبة الحمایھ من الاصابة التجریبة بجرعة التحدی فی
المجموعھ الملقحة 100 % بینما المجموعة الثالثة سجلت موت احد الحملان فی الیوم الخامس من الحقن مع اصابة
بقیة الحملان بخراجات فی منطقة الحقن.
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Proceeding of 5th International Scientific Conference,College of Veterinary Medicine
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University of Basrah,Iraq
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