Slov Vet Res 2020: 57 (4):169–78 Original Research Article DOI 10.26873/SVR-1000-2020 UDC 615.371:579.84:616.932:636.5(620) THE EFFICACY OF VACCINATION OF LAYER CHICKENS WITH INACTIVATED FOWL CHOLERA BACTERIN PREPARED FROM LOCAL EGYPTIAN STRAINS OF Pasteurella multocida Wafaa Abd El-Ghany Abd El-Ghany1*, Hanan Ali Ahmed2, Ali Zaher Qandoos1, Mohamed Abd El-Rahman Bosila3 1Faculty of Veterinary Medicine, Cairo University, Cairo, 2Central Laboratory for Evaluation of Veterinary Biologics, Cairo, 3National Research Center, Cairo, Egypt *Corresponding author, E-mail: wafaa.ghany@yahoo.com Abstract: This study was carried out to evaluate the efficacy of vaccination of layer chickens with inactivated FC bacterin prepared from local Egyptian strains of Pasteurella multocida (P. multocida). A total of 200 layer chickens were divided into 5 equal groups, 40 for each. At the age of 6 weeks, chickens in groups (A) and (B) were vaccinated with P. multocida serotypes A:1 and A:3, respec- tively, booster doses were given after 3 weeks (9 weeks old) and challenge was done with virulent serotypes A:1 and A:3 at 2 weeks later (11 weeks old). Chickens in groups (C) and (D) were not vaccinated, only challenged with P. multocida serotype A:1 and A:3, re- spectively. Birds in group (E) were kept as non-vaccinated and non-challenged. Blood samples were collected weekly from all groups for humoral immune response. All the birds were kept under observation for signs, mortalities, lesions and re-isolation of challenging organism and for histopathological examination. Results of the mean Enzyme Linked Immuno-Sorbent Assay (ELISA) revealed that the highest level was at 5 weeks post vaccination as the titers reached to 3970 in group (A) and 3905 in group (B). The clinical signs, mortality rate and lesions were mild in the vaccinated birds while severe lesions were in non-vaccinated and challenged birds. The protection rates were 85 % and 80 % in groups (A) and (B); respectively, while 10 % and 20 % in groups (C) and (D); respectively. The re-isolation rates of P. multocida after challenge were 95 % and 90 % in non-vaccinated-challenged birds with P. multocida serotypes A:1 and A:3; respectively, while they were 25 % and 15 % in vaccinated-challenged groups with P. multocida serotypes A:1 and A:3; respectively. Histopathological examination of P. multocida vaccinated-challenged birds revealed mild to no microscopic lesions when compared with non-vaccinated challenged chickens. In conclusion, the prepared FC inactivated bacterin from the local Egyp- tian predominant P. multocida serovars proved efficacy and protection of layer chickens. Key words: Pasteurella multocida; chickens; immunization; protection; Egypt Received: 26 November 2019 Accepted for publication: 25 March 2020 Introduction Fowl cholera (FC) is a bacterial disease of domestic and wild birds that causes high economic losses including; deaths, weight losses and condemnations (1-3). Mortalities related to FC infection in layer chickens may range from few percentages up to 100 % (4). The incidence of FC is more common in mature layer chickens than young broilers because of age factors (5, 6). Infection with FC is caused by Pasteurella multocida (P. multocida) micro-organism (7). P. multocida is considered as Gram-negative coccobacilli, non-motile and non-spore former and capsulated organism (8). Vaccination against FC is considered as one of the most important worldwide strategy to decrease the incidence of infection (9, 10). Globally, living and inactivated (bacterin) vaccines are being used to immunize birds against FC (4, 11). Living attenuated vaccines have advantages regarding good protection with long immunity as well as cross-protection against P. multocida of different serotypes or surface lipopolysaccharide (LPS) structures (12). However, living attenuated FC vaccines may lack of maintainable and sustainable attenuation methods and/or instability which may 170 W. A. Abd El-Ghany, H. A. Ahmed, A. Z. Qandoos, M. A. Bosila lead to risk of returning their virulence. Therefore, inactivated FC bacterins are widely used as there is no chance of the vaccines reverted to virulence and cause the disease (13). In this regard, inactivated FC bacterins have comparative advantages over the living vaccines and are thus favored to protect chickens against infection caused by homologous P. multocida strains (14). Bacterins used for prevention of FC provide homologous but not heterologous protection (15). Immunogenic local strains of P. multocida should be selected as the ideal strains to prepare effective bacterin (16). There are 16 different serovars of P. multocida and the most common types associated with FC outbreaks are serovars 1, 3 and 4. Evaluation and quality control of the efficacy of locally prepared FC bacterin are based mainly on inactivation of P. multocida serovars, followed by vaccination and challenge test by which the protective indices are measured. According to FAO report, Egyptian poultry production systems are varying from rural very small-scale, extensive poultry production to highly intensive systems with over 70,000 birds per house in industrial commercial systems. The meat production or broiler sector has a pyramid structure; with grandparents at the top of the pyramid, the broilers at the bottom and the broiler breeders in between the two. Egypt is 100 % self- sufficient from table eggs as it produces nearly 13 billion commercial table eggs annually. Some studies have been conducted in Egypt considering the situation of P. multocida infection in layer, breeder and broiler chicken flocks. Researches indicated that the most common circulating P. multocida serotypes that circulating in Egyptian flocks are types A:1 and A:3 causing severe economic losses in back yard as well as layer and breeder chicken flocks (17-20). There is available commercial inactivated oil adjuvant bacterin used for vaccination of Egyptian layer and breeder chicken flocks against FC. This bacterin was prepared from local P. multocida serotypes A:1 and A:3 strains and used at age of 8-10 weeks and boostered at 16- 17 weeks. However, the bacterin doesn’t confer complete protection of flocks against P. multocida infection. Therefore, from time to another, trials have been done to prepare and test the efficacy of using locally prepared FC bacterin from the predominant circulating P. multocida serovars in chickens (21-23). Accordingly, this work was designed to evaluate the efficacy of vaccination of layer chickens with inactivated FC bacterin prepared from local Egyptian strains of P. multocida. Materials and methods Experimental design: A total of 200, day-old layer chickens was obtained from local hatcheries and reared on thoroughly cleaned and disinfected semi closed houses for 13 weeks. Birds were vaccinated using standard protocol for vaccination. Feed and water was given ad libitum. At 6 weeks old chickens, birds were divided into 5 equal groups 40 for each. Groups (A) and (B) were vaccinated with 0.5 ml I/M with P. multocida serotypes A:1 and A:3; respectively, booster doses were given after 3 weeks (9 weeks old), and challenge was done orally with virulent serotypes A:1 and A:3 at 2 weeks later (11 weeks old). Chickens in groups (C) and (D) were not vaccinated and challenged with P. multocida serotype A:1 and A:3; respectively. Birds in group (E) were kept as non-vaccinated and non- challenged. The experiment was done according to the National Guidelines and Regulations on Animal Welfare and Institutional Animal Ethical Committee (IAEC) of Cairo University with approval number (CU II F 100 18). Preparation of inactivated bacterin from the predominant P. multocida local strains Local P. multocida strains serotypes (A:1 and A:3) that were isolated from different Egyptian governorates were used. The used P. multocida strains were previously serologically and molecularly identified (20). Inactivated bacterin was prepared according to method described by Borkowska-Opacka et al. (24). Simply, P. multocida strains were grown in brain heart infusion broth at 37oC for 16-24 hr to obtain a dense culture containing approximately 108 CFU of each strain. Formalin was added to the culture in final concentration of 0.2 % and the formalized culture was re-incubated at 37oC for 24 hr to ensure complete bacterial inactivation. Two percent of aluminium hydroxide gel was added in concentration of 20 % and was mixed well with the culture. Cultures were equally mixed 171 W. A. Abd El-Ghany, H. A. Ahmed, A. Z. Qandoos, M. A. Bosila together. The bacterin was standardized to contain 108 CFU/0.5ml dose. Finally, the bacterin was preserved with 0.01 % of thiomersal and stored at 4 C° until use. The quality control parameters of the locally prepared bacterin were sterility, safety and potency test (25). Briefly, the prepared bacterin was inoculated on bacteriological and mycological media to prove its sterility or purity from any bacterial or fungal contaminations. In addition, 0.5 ml of the prepared bacterin was inoculated in 5 chickens and the birds were kept under observation for a weeks to ensure that the bacterin induced no adverse effects like signs, lesions or mortalities. The potency test was applied by collection of blood samples and measuring of immunological parameters. Vaccination scheme Primary vaccination was done at the age of 6 weeks, 0.5 ml of the prepared inactivated P. multocida bacterin containing 108 CFU/0.5 ml was inoculated intramuscular (I/M) into the thigh region in chickens of groups (A) and (B). Booster vaccination was done at 9 weeks of age (3 weeks after primary vaccination) (24). Challenge test with virulent P. multocida Serovars of P. multocida that were used for bacterin preparation were used for chickens challenge. Both vaccinated and non-vaccinated chickens (groups A, B, C and D) were challenged I/M with virulent P. multocida serotypes A:1 and A:3 separately at 2 weeks after booster vaccination (11 weeks old) in a dose of 0.1 ml of bacterial suspension/bird containing 107/ml that inoculated I/M (26). Parameters for evaluation of inactivated P. multocida bacterin in chickens Immunological parameters: Detection of humeral immune response of vaccinated and challenged birds was done using Enzyme Linked Immuno-Sorbent Assay (ELISA). Commercial ELISA kit (ID-VET) was used and the test was done as manufacturer’s instructions. Blood samples were collected from the wing vein of birds in each group pre-vaccination and weekly after vaccination till the end of the study to determine the serum antibody titer of chickens. The results of antibody titers were determined from S/P ratio (Sample mean – negative control) Log10 Titer= 1.09 (log10 S/P) + 3.36 Clinical parameters The clinical signs, mortality rate and post- mortem lesions specific for P. multocida were recorded in groups two weeks post challenge for measuring bacterin protection rate. Re-isolation rate Liver was collected from dead as well as sacrificed living birds at the end of the study in each group for P. multocida re-isolation. It was done by inoculation of the liver on blood agar media and then by morphological and biochemical identification (27-29). Histopathological examination Three chickens from each group were sacrificed by cervical dislocation at the end of study (13 weeks old) for histopathological examination and tissue specimens were collected from heart, liver and spleen and then fixed in 10% formol saline solution. After proper fixation, the specimens were dehydrated in ascending grades of ethanol from 70 % to 100 %, diluted in alcohol (methyl, ethyl and absolute ethyle), cleared in xylol and manually embedded in paraffin at 56oC in hot air oven for 24 hr. Thin tissue sections, 5 µm thick were cut from paraffin blocks and stained with Hematoxylin and Eosin (HE) for histopathological examination through the light microscope (30). Results and discussion Fowl cholera is one of the most important problem facing poultry industry, so vaccination against the disease is practiced as preventive measures in many countries of the world including Egypt (31). Both live and inactivated P. multocida vaccines had been attempted to prevent the disease (4). Inactivated P. multocida vaccines are widely S P (positive control-negative control)= The Protection rate (%) = (Number of living birds) (Total number of birds ) 172 W. A. Abd El-Ghany, H. A. Ahmed, A. Z. Qandoos, M. A. Bosila used as the organisms do not have chance to be reverted to virulence to cause the disease (13). In the present work, an inactivated local bacterin was prepared using P. multocida field strains and its efficacy was determined. The quality control assessment of the prepared P. multocida bacterin showed it was sterile and free from any bacterial or fungal contaminations and it was safe as it didn’t produce any local or systemic reactions as well as no mortalities in inoculated birds. Table (1) shows the potency of the locally prepared P. multocida bacterin through determining the humoral immune response using ELISA. In both groups (A) (vaccinated and challenged birds with P. multocida type A1) and (B) (vaccinated and challenged birds with P. multocida type A3), the mean ELISA antibody titers increased from (80) pre-vaccination level to reach (2260) and (2010) at the 3rd week after primary vaccination; respectively, however, two weeks after secondary vaccination, the antibody titers reach (4350) and (3980) respectively, then declined to (2998) and (2679) one week after challenge then increased to (3970) and (3905) at two weeks after challenge, respectively. Results of the mean ELISA antibody levels in controls groups (C) (non-vaccinated and challenged birds with P. multocida type A:1) and (D) (non- vaccinated and challenged birds with P. multocida type A:3) were 60-80 before challenge and then increased to (95) at two weeks after challenge. Group (E) (non-vaccinated-non challenged control) showed steady mean ELISA antibody levels (65-80). Solano et al. (32) developed ELISA assay to determine the humoral immune response of chicken to P. multocida and compared the results with indirect haemagglutination (IHA) test as they concluded that the antibody titers measured by ELISA was at least twice as sensitive as IHA. Furthermore, Avakian et al. (33), Perelman et al. (9) and Esmaily et al. (34) recorded that polyvalent FC oil-based bacterin induced a high antibody titer in broiler breeder hens measured by ELISA technique. In addition, Jabbri and Moazeni Jula (35) stated that inactivated trivalent FC vaccine consisted of serotypes 1, 3 and 4 P. multocida strains induced immunogenic response in vaccinated chickens, as ELISA assay showed a considerable increase in antibody titer after twice vaccination of 8 weeks old chickens. Birds vaccinated two or three times between the ages of 7 and 20 weeks became sufficiently immune tolerated to FC challenge, while those were not vaccinated or only vaccinated at the age of 7 weeks were not sufficiently immunized using ELISA (36). The results of humoral immune response obtained in this study were comparable Akhtar et al. (16) who tested a formalin killed FC in 15 weeks old chickens and found an increase in humoral antibody titers after booster vaccination. The bird’s immune response to P. multocida bacterin was previously explained (37, 38). The capsule and LPS of P. multocida cell surface are considered primary stimulators of immune response and critical determinants of bacterin Table 1: Mean ELISA antibody titres of sera in chicken groups pre-vaccination; one, two and three weeks after primary vaccination; one and two weeks after secondary vaccination and one and two weeks after challenge Group Treatment P. multocida strain P. multocida mean antibody titers/ weeks after vaccination Weeks after challenge (11th wks old) Weeks after primary vaccination (6th wks old) Weeks after secondary vaccination (9th wks old) Pre-V* 1 2 3 1 2 1 2 A Vaccinated- challenged A:1 80 687 1760 2260 3400 4350 2998 3970 B A:3 80 654 1590 2010 3085 3980 2676 3905 C Non vaccinated-challenged control A:1 80 70 70 65 70 80 95 95 D A:3 80 75 60 60 75 70 90 95 E Non vaccinated- non challenged control _ 70 70 80 75 65 80 75 65 Pre V*: Pre-vaccination 173 W. A. Abd El-Ghany, H. A. Ahmed, A. Z. Qandoos, M. A. Bosila protective efficacy (39). Both play key roles in a range of interaction between the bacteria and the hosts they colonize or infect. Gong et al. (40) demonstrated that outer membrane proteins (H and A) of P. multocida are the major immunogene antigens which play an important role in confer resistance against infections. The Omps promote adherence to host cell surfaces and are therefore likely involve in P. multocida virulence (41). Our results revealed that, the clinical signs of P. multocida vaccinated and challenged chicken groups were mild depression, off food, diarrhea, septicaemia and congested mucous membrane of conjunctiva and buccal cavity. Severe signs were observed in non-vaccinated challenged controls groups, while no signs appeared in the non- challenged control group during the observation period. These results were in agreement with Levy et al. (42) who recorded signs of depression dullness, anorexia, greenish diarrhea and labored breathing in P. multocida challenged non vaccinated chickens, while the vaccinated birds did not show clinical signs except dullness and depression. Also, signs of FC in commercial layer flocks were recorded as depression, anorexia with ruffled feathers, mucous discharge from mouth and nares and cyanotic comb and wattles (43). The mortality rates in different groups that recorded here were 15 % and 20 % in vaccinated and challenged birds with P. multocida type A:1 and A:3; respectively, however, in non-vaccinated and challenged controls, they were 90 % and 80 % for P. multocida type A:1 and A:3; respectively (Table 2). These results were in a partial agreement with others (44, 45). The results showed that vaccinated chickens were resistant to challenge with P. multocida A:1 and A:3 strains, where the protection rates were 85 % and 80 % respectively, while they were 10 % and 20 % in the non- vaccinated-controls, respectively (Table 2). Two doses of prepared bacterin induced good protection (80-90 %) against challenge with P. multocida of homologous immunogenic type but low protection (10-30 %) against heterologous challenge (46). An Egyptian study concluded that locally prepared polyvalent bacterins should be used in cases of FC outbreaks, and the capsular antigen plays a little role in immunization when compared with the somatic antigen (47). Inactivated trivalent FC vaccine consists of serotype 1, 3 and 4 P. multocida provided 70-80 % protection in chickens against challenge with homologous strains (35). Also, some other Egyptian trials revealed that adjuvented local FC vaccine gave 100 % protection in chickens against challenge with virulent strains of P. multocida types A and D (48) as well as 95 % and 90 % for types A:5 and D:2; respectively (49). Furthermore, the protection rate was 100 % in chickens vaccinated twice with alum-precipitated FC vaccine (50). Post-mortem examination of chickens revealed mild and severe lesions in P. multocida vaccinated -challenged and non-vaccinated-challenged con- trol groups, respectively. The lesions included sep- ticaemia, congestion of internal organs, enlarged liver with sub-capsular hemorrhage, pericarditis and enlarged and congested spleen. These lesions were in accordance with other study (45) where the lesions of birds vaccinated with double doses of P. multocida local bacterin were congested heart and Group Chicken groups Challenge P. multocida Strain No. of chickens No. of survived birds No. of dead birds Mortality rate (%) Protection Rate (%) A Vaccinated- challenged A:1 40 34 6 15 85 B A:3 40 32 8 20 80 C Non vaccinated- challenged control A:1 40 4 36 90 10 D A:3 40 8 32 80 20 E Non vaccinated- non challenged control _ 40 40 0 0 100 Table 2: Results of protection rates in different chicken groups after vaccination with local P. multocida bacterin and challenge with the same serovars 174 W. A. Abd El-Ghany, H. A. Ahmed, A. Z. Qandoos, M. A. Bosila slight congestion of the liver and spleen, however the lesions in non-vaccinated-challenged birds were congestion of the subcutaneous blood ves- sels, dark red muscles, enlarged and congested liver and spleen and pericarditis. In other study, severe congestion, hemorrhages, pericardial and peritoneum exudations, enlargement of spleen and liver and white necrotic foci over liver were reported in chickens vaccinated with type A:1 of P. multocida at 14 day post-challenge (46). Table 3 reveals that P. multocida could be re-isolated from liver, heart blood and spleen of the chickens with high rate (90-95 %) and low rate (15-25 %) in non-vaccinated and vaccinated-chal- lenged birds; respectively. These findings sup- ported by Egyptian researcher Mahmoud (51) who found that the incidence of isolation of P. multocida was higher from non-vaccinated flocks than those from vaccinated ones. Partial agreement with ours revealed that P. multocida could not be recovered from immunized and challenged survived chick- ens, while it could be isolated from all dead or dis- eased birds (35). In this study, Table 4 and Figures 1-4 demonstrate the histopathological findings in different examined organs at the end of the experiment. Figure 1 shows the microscopic lesions of chickens vaccinated and challenged with P. multocida serotype A:1. Mild con- gestion of hepatic central vein and some inflamma- tory cells infiltrate (A), normal myocardium (B) and congested red pulp of spleen (C) were the observed lesions. In chickens vaccinated and challenged with P. multocida serotype A:3 (fig. 2) showed hydropic degeneration of hepatocytes (A), slight congestion of the myocardium (B) and hyperplasia of splenic lymphoid follicle (C). The previous results were in a partial agreement who noticed congestion with presence of some degenerative changes of liver, mild depletion of splenic lymphoid cells and slight myocarditis in P. multocida vaccinated and chal- lenged chickens (45, 52). Point of comparison Group A B C D Liver Congestion of central vein Inflammatory cells infiltrate Coagulative necrosis Hydropic degeneration + + - - - - - + - + + - + - - + Myocardium Congestion Inflammatory cells infiltrate Endocardial haemorrhages - - - + - - - + - - - + Spleen Red pulb congestion Lymphoid follicle hyperplasia Focal area of necrosis + - - - + - + - - - + + + = Present - = Absent Group Chicken groups Challenge P. multocida Strain No. of chickens Re-isolation % of P. multocida A Vaccinated- challenged A:1 40 6/40 (15%) B A:3 40 10/40 (25%) C Non vaccinated- challenged control A:1 40 38/40 (95%) D A:3 40 36/40 (90%) E Non vaccinated- non challenged control _ 40 0/40 (0%) Table 3: Results of re-isolation of P. multocida among chicken groups after vaccination with local P. multocida bacterin and challenge with the same serovars Table 4: Results of histopathological examination of chicken groups after vaccination with local P. multocida bac- terin and challenge with the same serovars 175 W. A. Abd El-Ghany, H. A. Ahmed, A. Z. Qandoos, M. A. Bosila Figure 1: Chickens vaccinated and challenged chickens with P. multocida serotype A:1 (group A) (H&E. X 200): A: Liver shows mild congestion of central vein and some inflammatory cells infiltrate, B: Heart showing normal myocardium, C: Spleen showing congested red pulp Figure 2: Chickens vaccinated and challenged chickens with P. multocida serotype A:3 (group B) (H&E. X 200): A: Liver showing hydropic degeneration of hepatocytes, B: Heart shows slight congestion of the myocardium, C: Spleen showing hyperplasia of lymphoid follicle Figure 3: Chickens experimentally challenged with P. multocida A:1 strain (group C) (H&E. X 200): A: Liver showing area of coagulative necrosis infiltrated with heterophils, B: Heart showing severe inflamma- tory cells infiltration of myocardium, C: Spleen shows congestion of red pulp and splenic artery Figure 4: Chickens experimentally challenged with P. multocida A:3 strain (group D) (H&E. X 200): A: Liver showing congestion of portal vein and hydropic degeneration of cytoplasm, B: Heart sub endocardial haemorrhage with heterophilic infiltration, C: Spleen shows severe congestion and focal area of necrosis (arrow) A B C A B C A B C A B C 176 W. A. Abd El-Ghany, H. A. Ahmed, A. Z. Qandoos, M. A. Bosila Histopathological lesions in chickens challenged with P. multocida A:1 strain (fig. 3) showed area of hepatic coagulative necrosis infiltrated with heterophils (A), severe inflammatory cells infiltration of myocardium (B) and congestion of splenic red pulp and artery (C). Moreover, in chickens experimentally challenged with P. multocida A:3 strain (fig. 4), the liver showed congestion of portal vein and hydropic degeneration of cytoplasm (A), the heart revealed sub endocardial haemorrhage with heterophilic infiltration (B) and the spleen demonstrated severe congestion and focal area of necrosis (C). 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Shilpa S, Verma PC. Pathology of P. multo- cida infection in chickens. Inter J Anim Res 2006; 40: 15–19. 54. Panna SN, Nazir KH, Rahman MB, Ahmed S, Saroare MG, Chakma S, Kamal T and Majumder UH. Isolation and molecular detection of Pasteurella multocida type A from naturally infected chickens, and their histopathological evaluation in artificially infected chickens in Bangladesh. J Adv Vet Anim Res 2015; 2 (3): 338–45. UČINKOVITOST CEPLJENJA KOKOŠI NESNIC Z INAKTIVIRANO BAKTERIJO KOLERE PERJADI, PRIPRAVLJENE IZ LOKALNIH EGIPTOVSKIH SEVOV BAKTERIJE Pasteurella multocida W. A. Abd El-Ghany, H. A. Ahmed, A. Z. Qandoos, M. A. Bosila Povzetek: Raziskava je bila izvedena z namenom ocenitve učinkovitosti cepljenja kokoši nesnic z inaktivirano bakterijo FC, pripravljeno iz lokalnih egiptovskih sevov bakterije Pasteurella multocida (P. multocida). Skupno 200 kokoši nesnic je bilo razdeljenih v 5 enakih skupin. V vsaki skupini je bilo 40 kokoši. Pri 6 tednih smo kokoši v skupinah A in B cepili s serotipoma P. multocida A:1 in A:3, po 3 tednih, ko so bile živali stare 9 tednov, so dobile poživitvene doze cepiva. Po dveh tednih (v starosti 11 tednov) so bile kokoši okužene z virulentnima serotipoma A:1 in A:3. Piščanci v skupinah C in D niso bili cepljeni temveč samo okuženi s serotipoma A:1 in A:3. Kokoši v skupini E niso bile niti cepljene, niti okužene. Vzorci krvi so bili odvzeti pri vseh skupi- nah tedensko za preverjanje humoralnega imunskega odziva. Vse kokoši smo stalno opazovali in beležili prisotnost bolezen- skih znakov, različnih ran in umiranje kokoši. Pri poginulih kokoših smo osamili bakterije ter opravili histopatološki pregled. Re- zultati encimsko-imunskega testa (ELISA) so pokazali da je bila najvišja stopnja zaščite dosežena 5 tednov po cepljenju, saj so titri dosegli 3970 v skupini A in 3905 v skupini B. Klinični znaki, stopnja umrljivosti in rane so bili pri cepljenih kokoših blagi, hude rane pa so bile vidne pri necepljenih in okuženih kokoših. Stopnja zaščite je bila v skupinah A in B 85- oziroma 80-odstotna, v skupinah C in D pa 10- oziroma 20-odstotna. Stopnje ponovne izolacije P. multocida po okužbi so bile 90 in 95 odstotkov pri kokoših, ki niso bile cepljene, medtem, ko so bile v skupinah, ki so bile okužene s P. multocida serotipa A:1 in A:3 15- in 25-ods- totkov. Histopatološki pregled cepljenih in okuženih kokoši je pokazal popolno odsotnost ali prisotnost blagih mikroskopskih poškodb, medtem ko so imele necepljene okužene kokoši bolj obsežne histopatološke poškodbe. Pripravljena inaktivirana bakterija FC iz lokalnih egiptovskih prevladujočih serovarov P. multocide se je izkazala za učinkovito zaščito kokoši nesnic. Ključne besede: Pasteurella multocida; kokoši; imunizacija; zaščita; Egipt