Group A (vaccine 1): Toxoid phospholipase D (PLD) – a preparation of culture filtrate from isolated Corynebacterium pseudotuberculosis biovar 1. 1 ml of the filtrate was mixed with 1 ml of oil adjuvant vaccine, dose (2 ml) contained 23 μg PLD, and the vaccine was inoculated subcutaneously, in the middle third of the neck.

Group B (vaccine 2): PLD – bacterin composed of formalin-killed C. pseudotuberculosis whole cells mixed with the toxoid PLD vaccine. 164 killed bacteria cells and 23 μg PLD/1 ml, and the dose (2 ml) was inoculated subcutaneously, in the middle third of the neck.

Group C (vaccine 3): Toxoid PLD with covaccine 8 – covaccine 8 is an imported vaccine formulated from a mixture of clostridial toxins, obtained from Schering Plough Animal Health. The mixture contained 23 μg PLD in 40 ml of covaccine 8, 2 ml inoculated subcutaneously, in the middle third of the neck.

Group D (vaccine 4): Toxoid PLD vaccine combined with the poly­valent clostridial vaccine – Clostridium perfringens, Clostridium tetani, Clostridium septicum, Clostridium chauvoei, and Clostridium novyi. 40 ml of polyvalent clostridial vaccine mixed with 6 g lyophilised powder formulated culture filtrate PLD. The polyvalent is a local vaccine prepared by Veterinary Serum and Vaccine Research Institute (Abbasia, Cairo, Egypt). The 2 ml dose of the vaccine was inoculated subcutaneously in the middle third of the neck.

Group E (control): Unvaccinated animals.

All the vaccinated animals were revaccinated after 3 weeks of the first vaccination.

After 3 weeks of the last vaccination, all five groups were challenged with virulent biovar 1 sheep origin isolate with 2 ml suspension containing 4×106 colony forming unit (CFU), 1 ml vaccine was inoculated subcutaneously in the middle third of the each side of the neck. The non-specific cellular immune response represented by stimulated macrophages was measured at 2 weeks after the challenge while the specific excited lymphocytes response was assessed at 4 weeks.

Statistical analysis was done using model Generalised Linear Model (GLM) of Statistical Analysis System (SAS) and software version 6.12.

The specific immune response was evaluated through lymphocyte proliferation assay using ELISA BrdU kit, while the non-specific response was estimated by superoxide anion production and lysozyme activity assays.

Group I: Vaccinated with 0.1 ml of Bacillus Calmette–Guérin (BCG) (supplied by Veterinary Serum and Vaccine Research Institute, used for vaccination of sheep dose of 0.1 ml), simultaneously injected intradermally with the first dose of 2.0 ml of the prepared CLA vaccine injected subcutaneously, then booster dose injected 21 days apart.

Group II: Vaccinated with 2.0 ml of prepared CLA vaccine then booster dose injected 21 days apart (injected subcutaneously).

Group III: Control group.

They were evaluated as cellular and humoral immune responses, through evaluation by phagocytic activity and index, and differential count of leukocytes and ELISA, respectively.

Group I: Immune response mediated by phagocytic activity and phagocytic index between 0 and 44 days average of 62.25%;

Group II: Immune response mediated by phagocytic activity and phagocytic index between 0 and 44 days average of 61%;

Group III: Immune response mediated by phagocytic activity and phagocytic index between 0 and 44 days average of 55.5%.

Average humoral immune response (by optical density of colored reaction):

Group I: day 0: 0.136; day 21: 0.397; day 44: 0.394; day 60: 0.371;

Group II: day 0: 0.149; day 21: 0.372; day 44: 0.382; day 60: 0.332;

Group II: day 0: 0.143; day 21: 0.133; day 44: 0.145; day 60: 0.145.

All lambs were treated by ivermectin 200 μg per kg of body weight subcutaneous at the rate of 1 ml per 50 kg as anthelminthic treatment for internal and external parasites before starting the experiment.

Lambs divided into three groups each group consisted of five lambs.

Group I: five lambs as negative control.

Group II: five lambs vaccinated (a commercial vaccine prepared by Colorado Serum Company (U.S.A.), detoxified and purified the whole culture of C. pseudotuberculosis contains thimerosal as preservative used in healthy sheep as a killed bacterin-toxoid vaccine. The vaccine injected subcutaneously in a dose of 2 ml in axillary space and repeated in four weeks in opposite axillary space).

Group III: five lambs as positive control.

Humoral and cell mediated immune response was detected during the period of experiments, the temperature, pulse rate and respiration rate were determined weekly.

Cellular immune responses of the vaccinated and control groups were evaluated by delayed type of hypersensitivity (DTH) skin test.

Antibody titer two weeks after first vaccination dose (mean optical density values):

Group I: 0.096 ± 0.013

Group II: 0.426 ± 0.26 – The maximum titre was reached in the eighth week after vaccination (1.984 ± 0.776) and then the antibody titers begun to decrease to 0.95 ± 0.446

Group III: 0.093 ± 0.01

Group I: was vaccinated with a vaccine prepared from C. pseudotuberculosis Pl 18 strain. The vaccine injected subcutaneously in a dose of 0.5 ml (two doses).

Group II: with a vaccine prepared from C. pseudotuberculosis Cy 5 strain. The vaccine injected subcutaneously in a dose of 0.5 ml (two doses).

Group III: was used as a control and was subcutaneously injected with 2.0 ml phosphate buffer solution (PBS).

Serum samples were collected prior to vaccination from all animals (mature stock and lambs). The microagglutination test was used to distinguish among vaccinated and unvaccinated animals.

Group I: control (no vaccine);

Group II: whole cells (WC) + 2-acetamido-2-

deoxy-3-O-D-2-propionyl-L-alanyl-

D-isoglutamine-D-glucopyranose-snglyceryl-

dipalmitoyl) (MDP-GDP), administered intramuscularly;

Group III: Glanvac, administered subcutaneously;

In 1993, lambs were randomly assigned to one of 3 groups:

Group I: control (no vaccine);

Group II:WC+MDP-GDP, administered intramuscularly;

Group III: Case-Vac, administered subcutaneously;

1994-1996, ewes and lambs were assigned to 2 groups:

Group I: control (saline), administered subcutaneously;

Group II: WC+MDP-GDP, administered intramuscularly;

It does not report the number of animals allocated to each group and the dose of each injection.

Identify major antigens of C. pseudotuberculosis isolated from different sheep flocks in Turkey and to evaluate the efficacy of the vaccine formulated as bacterin + toxoid + Freund’s complete adjuvant (FCA) against bacterial challenge in lambs. Cellular immune responses of the vaccinated and control groups were evaluated by DTH.

Serum samples were collected prior to vaccination from all animals (mature stock and lambs) and 1, 3, 6, 9, and 12 months after vaccination.

In all industry flocks, antibody titres for both lambs and mature stock were significantly higher than those of controls immediately prior to, and 1 month after, the booster vaccination.

In each group 50 lambs were artificially infected with C. pseudotuberculosis and 100 were uninfected, and were separated for 40 months. One lot of 50 infected lamb and 100 uninfected lambs were vaccinated against CLA.

Four weeks after the second vaccination, 50 lambs from each group were artificially infected with C. pseudotuberculosis and run separately for a further 4 weeks.

50 artificially infected lambs being run with 100 uninfected lambs.

Sorology was checked to verify soroconversion, by means of antibodies of C. pseudotuberculosis through ELISA.

Differences in CLA infection rates and seroconversion rates between groups of lambs were analysed using chi-square. Analysis of differences in numbers of lesions was done by Kruskal-Wallis one way analysis of variance.

In each group 50 lambs were vaccinated as indicated before artificial infection:

Group I: Vaccinated against CLA, enterotoxaemia and tetanus; percentage with lesions 4%.

Group II: Vaccinated against enterotoxaemia and tetanus; percentage with lesions 44%.

Group III: Vaccinated against enterotoxaemia and tetanus; percentage with lesions 48%.               

In each group 100 lambs were run with 50 artificially infected lambs:

Group I: Vaccinated against CLA, enterotoxaemia and tetanus; percentage with lesions 2%.

Group II: Vaccinated against CLA, enterotoxaemia and tetanus; percentage with lesions 20%.

Group III: Vaccinated against enterotoxaemia and tetanus; percentage with lesions 76%.

Evaluated percentage of lambs with lesion CLA and number of lesions, besides the presence of antibodies to C. pseudotuberculosis exotoxin and cell wall.

Lambs vaccinated against CLA had a 97% lower infection rate (groups I and II).

Unvaccinated lambs had a 76% infection rate (groups III).

Vaccinated lambs infected with CLA have 96% fewer lung abscesses compared with unvaccinated infected lambs and therefore less likely to spread this disease to other lambs.

Groups II: Unvaccinated; 44 lambs

Females and offspring were allocated to groups.

The animals were vaccinated between 2.5 and 3.5 months of age, the vaccination was repeated 1 month later and again 11 months later.

Each dose of vaccine consisted of 0.25 ml sterile saline, 0.25 ml mineral oil with 3% Arlacel A and either 5.0 mg dried whole C. pseudotuberculosis cells (initial vaccination and 1 month booster) or 1.0 mg dried whole cells (12 month booster). The total volume for each injection was 0.5 ml.

The serums were analysed by microagglutination assay for the detection of antibodies to C. pseudotuberculosis. Animals were monitored for the development of abscesses by the technician at each serum collection visit and by routine observation by the farm managers.

The level of significance of the microagglutination was determined by the Student's t-test and was used the logrank test for difference in proportions of new cases in each group over time.

The vaccine appeared to produce a persistently elevated serum antibody titre to C. pseudotuberculosis as measured by the microagglutination assay. There was also a statistically significant degree of protection in sheep against the development of clinical CLA.

The vaccine could have a significant role in the control of CLA in infected sheep.

Group 1 – immunised with monocomponent vaccine (vaccine prepared from C. pseudotuberculosis); 38 lambs; administered subcutaneously.

Group 2 – immunised with the combined vaccine (vaccine prepared from C. pseudotuberculosis and Clostridium perfrigens D, Clostridium novyi B, Clostridium tetani, Clostridium septicum and Clostridium chavouei); 38 lambs; administered subcutaneously.

Group 3 – immunised with the combined vaccine (vaccine prepared from C. pseudotuberculosis, Clostridium perfrigens D, Clostridium novyi B, Clostridium tetani, Clostridium septicum and Clostridium chavouei and selenium); 24 lambs; administered subcutaneously.

Group 4 – control; 33 lambs

Serology and necropsy were performed (to verify lesions caused by CLA).

All three vaccines afforded an equal and high level of protection.

91% (91/100) of vaccinated sheep exhibiting no lesions of caseous lymphadenitis CLA, compared with 51.5% (17/33) affected sheep in the control group).

Average lesion counts were 1.2 per affected vaccinated sheep and 4.5 per affected control sheep.

Antitoxin responses to the clostridial toxoids incorporated in the combined vaccines were not affected by inclusion of the C. pseudotuberculosis toxoid or the sodium selenate.

The results indicate the overall efficacy of vaccines in controlling CLA infection.

Vaccine was prepared from C. pseudotuberculosis toxoid combined with aluminum hydroxide as adjuvant.

Each sheep received two doses of 2 ml of the appropriate vaccine administered subcutaneously in the neck. The interval between doses was 34 days.

Control group (unvaccinated): 17 lambs of which 15 were affected by CLA.

Group 1 (vaccine high toxine-toxoid: cell-free toxoid): 14 lambs of which three were affected by CLA.

Group 2 (vaccine high toxine-toxoid-cells: toxoid with formalin-killed cells): 15 lambs of which four were affected by CLA.

Group 3 (vaccine low toxine-toxoid: cell-free toxoid):18 lambs of which four were affected by CLA.

Group 4 (vaccine low toxine-toxoid-cells: toxoid with formalin-killed cells): 20 lambs of which five were affected by CLA.

Necropsy results:

Control group (unvaccinated): 15 (15/17) were affected. Ten sheep had lesions in both lung and carcase, 4 had lung lesions only and one had carcase lesions only. The average number of lesions per affected sheep was 27.1

Group 1 – 3 (3/14) were affected the lesions being found in the lungs only. The average number of lesions per affected sheep was 2.0.

Group 2 – 4 (4/15) were affected, all with carcase lesions only. The average number of lesions per affected sheep was 1.5

Group 3 – 4 (4/18) were affected, 3 with lung lesions only and one with kidney lesions only. The average number of lesions per affected sheep was 3.0.

Group 4 – 5 (5/20) were affected, one with carcase and lung lesions, 2 with carcase lesions only and 2 with lung lesions only. The average number of lesions per affected sheep was 3.6.

The protective potency of the vaccines was not improve by the inclusion of cells of C. pseudotuberculosis.

Group 1: was vaccinated of using 2 ml a C. pseudotuberculosis bacterin given subcutaneously in the right axillary region at the beginning of the study.

Group 2: was vaccinated twice, being the first vaccination done on the same day as the group 1 and the second 4 weeks after

Group 3 (unvaccinated): control group was given saline solution (2 ml subcutaneously).

Blood samples were collected immediately prior to the first vaccination and then weekly until week 11. From week 11 to the end of the study (week 25), samples were collected approximately every 2 weeks.

ELISA was used to detect antibodies against C. pseudotuberculosis.

13 weeks after inoculation all lambs were necropsied without knowledge of treatment groups to verify the presence of abscesses.

The average number of abscesses per lamb was seven for group 1, four for group 2 and 32 for group 3.

The results of the study indicated that the vaccine provided immunological protection of lamb against challenge exposure of C. pseudotuberculosis.