Research Article

 

Efficacy Assessment of Avian Pasteurella multocida and Mycoplasma gallisepticum Local Vaccines

 

Fatma F. Ibrahim1, Wafaa A. Abd El-Ghany2*, Eman M. El Rawy1, Mona M. Shaker3, El-Jakee J2

1Veterinary Serum and Vaccine Research Institute, Abbasia, Cairo, Egypt; 2Faculty of Veterinary Medicine, Cairo University, Cairo, Egypt; 3Animal Health Research Institute, Dokki, Giza, Egypt.

 

Received | January 12, 2021; Accepted | May 09, 2021; Published | June 25, 2021

*Correspondence | Wafaa A Abd El-Ghany, Faculty of Veterinary Medicine, Cairo University, Cairo, Egypt; Email: [email protected]

Citation | Ibrahim FF, Abd El-Ghany WA, El-Rawy EM, Shaker MM, El-Jakee J (2021). Efficacy assessment of avian pasteurella multocida and mycoplasma gallisepticum local vaccines. J. Anim. Health Prod. 9(3): 213-221.

DOI | http://dx.doi.org/10.17582/journal.jahp/2021/9.3.213.221

ISSN | 2308-2801

Copyright © 2021 Abd El-Ghany et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

 

INTRODUCTION

 

Fowl cholera (FC) is a highly contagious disease caused by Pasteurella multocida (P. multocida) and has been recognized as an important disease in poultry for more than 20 years (Furian et al., 2016). The disease causes devastating economic losses in poultry industry through death, weight loss and condemnation of carcasses (Xiao et al., 2015). Vaccination against FC is considered as one of the most world-wide preventive measures to reduce the prevalence of the disease condition (Kardos and Kiss, 2005; Parvin et al., 2011). Inactivated P. multocida vaccines are widely used with successful results (Akhtar et al., 2016; Salama et al., 2019). Local vaccines of P. multocida that prepared mainly from inactivated whole bacterial cells induced protective immune response and good protection of birds against challenge (Glisson et al., 2008; Qandoos, 2018).

 

Mycoplasma gallisepticum (M. gallisepticum) infection is a complex, complicating and multifactorial disease posing a serious economic challenge to the prosperity of poultry enterprise in many parts of the world. M. gallisepticum is considered as an economically important respiratory disease problem for commercial and backyard poultry production systems (Talha, 2003). Infection with M. gallisepticum could resulting in high morbidity, poor feed conversion, decreased production, medication costs and high mortality when complicated with other infections (Mallinath and Hari Babu, 2013). The sero-prevalence of M. gallisepticum antibodies in broiler breeder flocks was 52.92% using Enzyme linked immuno sorbent assay (EI-Jakee et al., 2019). Prevention and control programs of avian mycoplasmosis are based on strict biosecurity, surveillance and eradication of infected breeder flocks (Raviv et al., 2008). Vaccination against M. gallisepticum can be a useful long term solution in situation where maintaining flocks free of infection is not feasible, especially in multiage commercial egg production sites (Kleven, 2008; Jacob et al., 2014).

 

Although development of different types of live and inactivated vaccines against FC and mycoplasmosis, both diseases are still circulating in commercial poultry farms and this situation necessitates the development of local vaccines from the predominant circulating field strains.

 

So, this study was planned to prepare inactivated vaccines from local strains of P. multocida and M. gallisepticum either alone or in combinations, evaluate these vaccines through measuring the immune response as well as detect the protection rates against the challenge with virulent P. multocida and M. gallisepticum strains.

 

MATERIALS AND METHODS

 

Preparation of Local Bacterins

Inactivated oil emulsion P. multocida and M. gallisepticum bacterins were prepared as Mukkur et al. (1982) and Yoder (1979); respectively. Equal parts of P. multocida serotypes (A and D) (Serotypes A and D were kindly obtained from Aerobic Bacterial Vaccines Department, Veterinary Serum and Vaccine Research Institute, Abbasia, Cairo) and M. gallisepticum (Field isolate of M. gallisepticum (Eis3-10) was kindly obtained from Mycoplasma Department, Animal Health Research Institute, Dokki, Giza, Egypt) were mixed. Equal amount of above culture was thoroughly mixed with Montanide ISA70 oil adjuvant and finally thiomersal was added at a final concentration of 0.01%. The prepared bacterin was tested for sterility from any other bacterial or fungal contaminants, purity and safety after inoculation in chickens and mice.

 

Experimental Design

A total of 135, 4 weeks old specific pathogen free chickens were divided into 4 groups. The 1st group was 30 birds and vaccinated with P. multocida bacterin (G1), the 2nd group was 15 birds and vaccinated with M. gallisepticum bacterin (G2), the 3rd was 45 birds vaccinated with combined P. multocida and M. gallisepticum bacterin (G3) and the 4th group was 45 birds and kept as control non vaccinated chickens (G4). Each chickens in the vaccinated group received 0.5 ml of the tested vaccines subcutaneously (S/C) in a double doses with one month interval. The 1st and 2nd vaccine doses were at 4 and 8 weeks old; respectively. The study was done in accordance with the National Regulations on Animal Welfare and Institutional Animal Ethical Committee Recommendations and Approval.

 

Determination of Cellular Immunity

Relative proportion of heterophils to lymphocytes (H/L ratio) was determined in the staining blood films collected from chickens groups (Cotter, 2015). Blood samples were collected at the 3rd, 7th and 15th days after the first and second vaccinations and after the challenge. Blood films were examined to obtain counts of lymphocytes and granulocytes per 100 leukocytes. Obtained cell counts were used for calculation of H/L ratio.

 

Evaluation of Humoral Immunity

Serum samples were collected every 2 weeks till 25 weeks old for determination of antibody titers in vaccinated P. multocida types A and D (Sawada et al., 1982) and M. gallisepticum (Senterifit, 1983) chickens groups using indirect haemagglutination (IHA) and haemagglutination inhibition (HI) tests.

 

Evaluation of Bacterin Potency

Passive mouse protection test was done to evaluate the protection rate of sera collected from chickens groups vaccinated with either P. multocida types (A and D) or combined P. multocida and M. gallisepticum bacterins (Tabatabaei et al., 2007). About 0.2 ml of the sera of vaccinated above mentioned groups was S/C inoculated in 120 mice while 60 mice were kept as control. After 24 hours, each of vaccinated mice was challenged separately and S/C with 0.1 ml virulent P. multocida types (A and D) containing 100 lethal dose 50 (LD50). As well, challenge test for 11 weeks old chickens (3 weeks post-2nd vaccination) was done as OIE (2012), where each chicken in vaccinated groups was challenged with the same route and dose as mice. Both mice and chickens were kept under observation for 7 days later.

 

Statistical Analysis

Paired t-test was used for comparison of H/L ratio, antibody titers between G1 and G3 and between G2 and G3. ANOVA test was conducted for comparison of H/L ratio, antibody titers between vaccinated groups and control group. The level of significance for all statistical tests was set at (p ≤ 0.05). All statistical tests were performed through the statistical package for social studies (SPSS) version 19 for windows (IBM SPSS, Chicago, IL, USA).

 

Table 1: Evaluation of H/L ratio post vaccination with different vaccines in chickens

 

Interval times of blood collection	Groups
	G1	G2	G3	G4
Pre-vaccination	1.3	1.4	1.1	1.5
1st vaccination
At 3rd day	0.7	0.8	0.5	1.4
At 7th day	0.4	0.6	0.2	1.0
At 15th day	0.5	0.7	0.3	1.3
Booster vaccination
At 3rd day	0.3	0.5	0.2	1.2
At 7th day	0.1	0.4	0.1	0.9
At 15th day	0.2	0.5	0.2	1.3
Challenge
At 3rd day	0.2	0.4	0.2	1.2
At 7th day	0.1	0.3	0.1	1.0
At 15th day	0.4	0.6	0.3	1.5

G1= P. multocida bacterin, G2= M. gallisepticum bacterin, G3= Combined P. multocida and M. gallisepticum bacterin, G4= Control

1st vaccination at 4 weeks old, Booster vaccination at 8 weeks old, Challenge at 11 weeks old

 

Table 2: The level of antibody titers against P. multocida type (A) in chickens vaccinated with combined P. multocida and M. gallisepticum bacterin using IHA

 

Interval time of serum collection	Groups
	G1	G3	G4
Pre-vaccination	2	2	0
1st vaccination
2 weeks post 1st vaccination	64	128	2
Booster vaccination
2 weeks post 2nd vaccination	256	256	2
Challenge
2 weeks post-challenge	128	128	4
4 weeks post-challenge	128	512	2
6 weeks post-challenge	512	1024	2
8 weeks post-challenge	256	512	0
10 weeks post-challenge	256	512	0
12 weeks post-challenge	256	256	0
14 weeks post-challenge	128	128	0

G1= P. multocida bacterin, G3= Combined P. multocida and M. gallisepticum bacterin, G4= Control

1st vaccination at 4 weeks old, Booster vaccination at 8 weeks old, Challenge at 11 weeks old

 

Table 3: The level of antibody titers against P. multocida type (D) in chickens vaccinated with combined P. multocida and M. gallisepticum bacterin using IHA

 

Interval time of serum collection	Groups
	G1	G3		G4
Pre-vaccination	2	2		0
1st vaccination
2 weeks post 1st vaccination	32	64		2
Booster vaccination
2 weeks post 2nd vaccination	64	128		2
Challenge
2 weeks post-challenge	128		128	2
4 weeks post-challenge	256		512	4
6 weeks post-challenge	512		512	2
8 weeks post-challenge	128		256	2
10 weeks post-challenge	64		64	0
12 weeks post-challenge	64		64	0
14 weeks post-challenge	32		32	0

G1= P. multocida bacterin, G3= Combined P. multocida and M. gallisepticum bacterin, G4= Control

1st vaccination at 4 weeks old, Booster vaccination at 8 weeks old, Challenge at 11 weeks old

 

Table 4: The level of antibody titers against M. gallisepticum in chickens vaccinated with combined P. multocida and M. gallisepticum bacterin using HI

 

Interval time of serum collection	Groups
	G2	G3	G4
Pre-vaccination	2	2	0
1st vaccination
2 weeks post 1st vaccination	32	64	2
Booster vaccination
2 weeks post 2nd vaccination	64	128	2
Challenge
2 weeks post-challenge	128	256	4
4 weeks post-challenge	128	512	2
6 weeks post-challenge	128	512	2
8 weeks post-challenge	64	256	0
10 weeks post-challenge	64	128	0
12 weeks post-challenge	32	64	0
14 weeks post-challenge	16	64	0

G2= M. gallisepticum bacterin, G3= Combined P. multocida and M. gallisepticum bacterin, G4= Control

1st vaccination at 4 weeks old, Booster vaccination at 8 weeks old Challenge at 11 weeks old

 

Table 5: Passive mouth protection test against challenge with P. multocida type (A) in chickens vaccinated with combined P. multocida and M. gallisepticum bacterin

 

Interval times of serum collection	Total number of mice	Groups
		G1			G3			G4
		D	S	P%	D	S	P%	D	S	P%
Pre-vaccination	5	5	0	0	5	0	0	5	0	0
1st vaccination
2 weeks post 1st vaccination	5	1	4	80	0	5	100	5	0	0
Booster vaccination
2 weeks post 2nd vaccination	5	0	5	100	0	5	100	5	0	0
Challenge
2 weeks post challenge	5	0	5	100	0	5	100	5	0	0
4 weeks post challenge	5	0	5	100	0	5	100	5	0	0
6 weeks post challenge	5	0	5	100	0	5	100	5	0	0
8 weeks post challenge	5	0	5	100	0	5	100	5	0	0

P%= Number of survived mice/Total number of mice X 100

S= Survived mice, D= Dead mice, G1= P. multocida bacterin, G3= Combined P. multocidaand M. gallisepticum bacterin

G4= Control 1st vaccination at 4 weeks old, Booster vaccination at 8 weeks old, Challenge at 11 weeks old

 

Table 6: Passive mouth protection test against challenge with P. multocida type (D) in chickens vaccinated with combined P. multocida and M. gallisepticum bacterin

 

Interval times of serum collection	Total number of mice	Groups
		G1			G3			G4
		D	S	P%	D	S	P%	D	S	P%
Pre-vaccination	5	5	0	0	5	0	0	5	0	0
1st vaccination
2 weeks post 1st vaccination	5	0	5	100	0	5	100	5	0	0
Booster vaccination
2 weeks post 2nd vaccination	5	0	5	100	0	5	100	5	0	0
Challenge
2 weeks post challenge	5	0	5	100	0	5	100	5	0	0
4 weeks post challenge	5	0	5	100	0	5	100	5	0	0
6 weeks post challenge	5	0	5	100	0	5	100	5	0	0
8 weeks post challenge	5	0	5	100	0	5	100	5	0	0

P%= Number of survived mice/Total number of mice X 100; S= Survived mice; D= Dead mice

G1= P. multocida bacterin, G3= Combined P. multocida and M. gallisepticum bacterin, G4= Control

1st vaccination at 4 weeks old, Booster vaccination at 8 weeks old, Challenge at 11 weeks old

 

Table 7: Challenge test against P. multocida type (A) in chickens vaccinated with combined P. multocida and M. gallisepticum bacterin

 

Groups	G1	G3	G4
Total number of chickens	15	15	15
D	1	0	15
S	14	15	0
P%	93	100	0

P%= Number of survived chickens/Total number of chickens X 100

S= Survived chickens, D= Dead chickens

G1= P. multocida bacterin, G3= Combined P. multocida and M. gallisepticum bacterin, G4= Control

 

Table 8: Challenge test against P. multocida type (D) in chickens vaccinated with combined P. multocida and M. gallisepticum bacterin

 

Groups	G1	G3	G4
Total number of chickens	15	15	15
D	0	0	15
S	15	15	0
P%	100	100	0

P%= Number of survived chickens/Total number of mice X 100

S= Survived chickens, D= Dead chickens

G1= P. multocida bacterin, G3= Combined P. multocida and M. gallisepticum bacterin, G4= Control

 

Table 9: Challenge test against M. gallisepticum in chickens vaccinated with combined P. multocida and M. gallisepticum bacterin

 

Groups	G2	G3	G4
Total number of chickens	15	15	15
Birds with respiratory manifestations	3	1	15
P%	80	93	0

P%= Number of survived chickens/Total number of chickens X 100

G2= M. gallisepticum bacterin, G3= Combined P. multocida and M. gallisepticum bacterin, G4= Control

 

 

RESULTS

 

The prepared vaccines were free from any bacterial and fungal contaminations. They were pure and proved their safety after inoculation in chickens and mice without signs or mortalities.

 

The data illustrated in Table (1) reveals that the H/L ratio at 7th day post 1st vaccination for G1, G2 and G3 was 0.4, 0.6 and 0.2; respectively in comparison with 1.0 in G4. However, at 7th day post 2nd vaccination, the ratio for G1, G2 and G3 was 0.1, 0.4 and 0.1; respectively but 0.9 in G4. The H/L ratio for G1, G2 and G3 at the 7th day after challenge was 0.1, 0.3 and 0.1; respectively while 1.0 in G4. Using ANOVA, there were significant differences (p ≤ 0.05) in H/L ratio between vaccinated groups (G1, G2 and G3) and the control one (G4).

 

From the results of IHA in Table (2), it can noticed that the antibody titers against P. multocida type (A) at 2 weeks post 1st vaccination were 64 and 128 for G1 and G3; respectively compared with 2.0 in G4. The antibody titers at 2 weeks post 2nd vaccination were 256 for both G1 and G3 in comparison with 2.0 in G4. At 6 weeks post-challenge, the titers were 512 and 1024 for G1 and G3 while 2.0 for G4.

 

Table (3) shows that the IHA antibody titers against P. multocida type (D) at 2 weeks post 1st vaccination were 32 and 64 for G1 and G3; respectively but 2.0 for G4. The titer was 64 for G1, 128 for G3 and 2.0 for G4 at 2 weeks post 2nd vaccination. The antibody titers at 6 weeks post challenge were 512 for G1 and G3 and 2.0 for G4.

 

It was observed significant differences (p ≤ 0.05) in antibody titers against P. multocida type (A) and type (D) between vaccinated groups (G1 and G3) and the control one (G4).

 

The data demonstrated in Table (4) reveals that the levels of HI antibody titers against M. gallisepticum at 2 weeks post 1st vaccination were 32 for G2, 64 for G3 and 2.0 for G4. But at 2 weeks post 2nd vaccination, the titers were 46 and 128 for G2 and G3; respectively compared with 2.0 for G4. The HI titers at 6 weeks post challenge for G2, G3 and G4 were 128, 512 and 2.0; respectively.

 

There were significant differences (p ≤ 0.05) in antibody titers against M. gallisepticum between vaccinated groups (G2 and G3) and the control one (G4).

 

As shown in Table (5), the protection percentage (P%) against challenge of mice with virulent strain of P. multocida type (A) at 2 weeks post 1st vaccination was 80 and 100% for G1 and G3; respectively compared with 0% in G4. At 2 weeks post 2nd vaccination and 8 weeks post challenge, the P% were 100% for both G1 and G3 while 0% in G4.

 

The results in Table (6) shows that the P% against challenge of mice with virulent strain of P. multocida type (D) at 2 weeks post 1st vaccination was 100% for both G1 and G3 but 0% for G4. Also, 2 weeks post 2nd vaccination and 8 weeks post challenge, the P% were 100% for both G1 and G3 while 0% in G4.

 

The results of challenge test of chickens with virulent strain of P. multocida type (A) revealed P% 100% for G3, 93% for G1 and 0% for G4 (Table 7). Moreover, challenge test of chickens with virulent strain of P. multocida type (D) revealed P% 100% for both G1 and G3 and 0% for G4 (Table 8). Chickens challenged with virulent strain of M. gallisepticum showed P% of 80% in G2 and 93% in G3 compared with 0% in G4 (Table 9).

 

DISCUSSION

 

The cellular immune response of chickens that vaccinated with different bacterins was evaluated by H/L ratio. The results indicated that there were significant differences (p ≤ 0.05) between the vaccinated groups (G1, G2 and G3) and the control one (G4), and also between G1 and G3 but no difference between G2 and G3. These data agreed with Gaunson et al. (2006) who reported that M. gallisepticum vaccine activated cellular immune responses in tracheal mucosa including natural killer and cytotoxic T cells responses that are important for the immunity. Also, Abbas et al. (2007) stated that M. gallisepticum vaccine induced specific immune responses in vaccinated birds in the form of production of specific antibodies and non-specific factors/cytokines particularly interferon Gamma that activate antigen stimulated B cells, macrophages, cytotoxic T- cells and NK cells. Moreover, Kreslavsky et al. (2012) and Suling et al. (2012) explained that the formaldehyde inactivated Montanide ISA70 based M. gallisepticum vaccine causes irritation at inoculation site and induces granuloma formation/development of lymphoid tissues, where the macrophages or antigen presenting cells in the granuloma ingest the microbial antigen from oily suspension and present the microbial protein on their surface in association with self MHC II. Also, the T helper cells of the vaccinated birds recognize their specific antigens on surface of antigen presenting cells and undergo the process of blast formation, proliferation and differentiation into effectors and memory T lymphocytes. Concerning P. multocida, Harper et al. (2016) reported that lipopolysaccharide is a primary stimulator of the host immune response and a critical determinant of bacterin protective efficacy.

 

The humoral immune response of chickens vaccinated with P. multocida bacterin as well as combined P. multocida and M. gallisepticum bacterin was evaluated by IHA. There were significant differences (p ≤ 0.05) in antibodies titers between the vaccinated groups (G1 and G3) and the control one (G4) regarding P. multocida types (A and D). However, significant difference (p ≤ 0.05) in antibodies titers between G1 and G3 was found in P. multocida type (A) not type (D). These findings were parallel with Ahmed et al. (2010) and Abdel-Aziz et al. (2015) who concluded that inactivated FC vaccine adjuvanted with Montanide ISA-70-VG induced early and high immune response with long duration measured by IHA test. It was estimated that prepared P. multocida vaccines containing oil adjuvant Mantonide ISA70, ISA774 and W/O emulsion based on tween/span produced high immune response in 4 and 8 weeks old chickens (Belloc et al., 2008).

 

The humoral immune response of vaccinated chickens with different M. gallisepticum bacterins was evaluated by HI. It was found that there were significant differences (p ≤ 0.05) in antibodies titers between the vaccinated groups (G2 and G3) and the control one (G4), while no differences between G2 and G3. These results are in the same manner with Barbour and Newman (1990) who recorded significant immunoglobulin response specific to M. gallisepticum in the sera of chickens collected 3 weeks after the 1st and 2nd vaccination with oil-emulsion vaccine. The potency of the prepared bacterins was evaluated by passive mouse protection test against challenge with virulent strains of P. multocida types (A and D) in chickens vaccinated with P. multocida bacterin and combined P. multocida and M. gallisepticum bacterin. The P% post the 2nd vaccination and 8 weeks post challenge was 100% in G3. Similarly, El-Bayomy and Daoud (2004) found an elevation in the protective values of FC adjuvanted vaccines against challenge with virulent strains of P. multocida types (A and D). Moreover, Youssef and Tawfik (2011) concluded that inactivated Pasteurella vaccine adjuvanted with Montanide ISA50 induced 3.85 and 3.69 log protection in mice against challenge with rabbit P. multocida types (A and D).

 

Vaccination challenge test for estimation of protective indices is the main for evaluation and quality control of any prepared vaccine (OIE, 2012). The potency of the prepared bacterins was evaluated by challenge of chickens with virulent strains of P. multocida types (A and D) and the results revealed that the P% was (100%) in G3. Jabbri and Moazeni Jula (2005), Ahmed et al. (2010), Ievy et al. (2013), Abdel-Aziz et al. (2015), Ali and Sultana (2015) and Akhtar et al. (2016) demonstrated high protection rate of inactivated FC vaccines in chickens.

 

The results of potency of the prepared bacterins against challenge of chickens with virulent strain of M. gallisepticum showed the highest P% (93%) in G3. The same of this result, Bekele (2015) concluded that formalin inactivated Montanide ISA70 based M. gallisepticum induced 100% protection manifested by absence of signs and lesions after challenge. Kleven (2008), Ferguson-Noel et al. (2012) and OIE (2012) found that M. gallisepticum bacterin was protective as there were significant differences in air sacs, tracheal and ovarian lesions of the vaccinated birds compared to the non-vaccinated controls. Bekele and Assefa (2018) demonstrated that 16 weeks old chickens vaccinated with inactivated oil-emulsion adjuvant (Montanide ISA 70) M. gallisepticum did not show clinical signs or post mortem changes (100% protection).

 

CONCLUSION

 

It could be concluded that the locally prepared combined inactivated P. multocida and M. gallisepticum vaccine elicited good cellular and humoral immune responses as well as high protection of chickens against both diseases.

 

acknowledgements

 

The authors are grateful to Faculty of Veterinary Medicine, Cairo University, Egypt for the supervision and support, as well as Aerobic Bacterial Vaccines Department, Veterinary Serum and Vaccine Research Institute, Abbasia, Egypt and Mycoplasma Department, Animal Health Research Institute, Dokki, Egypt for covering all the expenses of the experiment needed to conduct the work.

 

CONFLICT OF INTEREST

 

All authors have no conflict of interest.

 

authors contribution

 

Ibrahim FF performed the experiments and wrote the manuscript. El-Jakee J, Abd El-Ghany WA and El-Rawy EM designed the experiments and reviewed the manuscript. Shaker MM and Ibrahim FF designed the experiments and prepared the vaccine. All authors read and approved the final version.

 

REFERENCES

 

• Abbas AK, Lichtman AH, Pillai S (2007). Cells and tissues of the adaptive immune system. In: Cellular and Molecular Immunology, 6th ed., Saunders Elsevier, Philadelphia, pp, 56.

• Abdel-Aziz, HMG, El-Enbaawy MIH, Afifi M, Ibrahim SI, Omar L, Koudier MH (2015). Efficacy of Montanide ISA-70-VG as adjuvant to fowl cholera vaccine. J. Vet. Adv., 5(3): 848-852. https://doi.org/10.5455/jva.20150315015734

• Ahmed ES, Mahmoud MS, Ghoniemy WA (2010). Immunological studies on a modified adjuvanted fowl cholera vaccine. Minufiya Vet. J., 7(2): 325-330.

• Akhtar M, Rahman MT, Ara MS, Rahman M, Nazir K, Ahmed S, Hossen ML, Rahman MB (2016). Isolation of Pasteurella multocida from chickens, preparation of formalin killed fowl cholera vaccine, and determination of efficacy in experimental chickens. J. Adv. Vet. Anim. Res., 3(1): 45-50. http://dx.doi.org/10.5455/javar.2016.c130

• Ali MZ, Sultana S (2015). Determination of humoral immune response in chickens against formalin-inactivated alum-precipitated fowl cholera vaccine. Int. J. Anim. Biol., 1(4): 114-117.

• Barbour EK, Newman JA (1990). Preliminary data of efficacy of Mycoplasma gallisepticum vaccines containing different adjuvants in laying hens. Vet. Immunol. Immunopathol., 26(2): 115-123. https://doi.org/10.1016/0165-2427(90)90061-V

• Bekele L (2015). Isolation, molecular identification and vaccine trial of Mycoplasma gallisepticum in Ethiopia. M.Sc. Thesis, College of Veterinary Medicine and Agriculture, Addis Ababa University, Bishoftu.

• Bekele L, Assefa T (2018). Inactivated vaccine trial of Mycoplasma gallisepticum in Ethiopia. Open J. Vet. Med., 8: 75-85. https://doi.org/10.4236/ojvm.2018.86009

• Belloc C, Dupus L, Deville S, Aucouturier J, Laval A (2008). Evaluation of safety and immune response induced by several adjuvants included in Pasteurella multocida vaccines in chickens. Rev. Vet. Med., 159(7): 371-375.

• Cotter PF (2015). An examination of the utility of heterophil-lymphocyte ratios in assessing stress of caged hens. Poult. Sci., 94: 512-517. https://dx.doi.org/10.3382%2Fps%2Fpeu009

• El-Bayomy AA, Daoud AM (2004). Efficacy of double adjuvant system on the immune response of fowl cholera vaccinated chickens. J. Egypt. Vet. Med. Assoc., 64(2): 215-329.

• EI-Jakee J, Marouf SH, Amin BH, Hedia RH (2019). Characterization of Mycoplasmae isolated from chicken. Biosci. Res., 16(2): 1843-1853.

• Ferguson-Noel N, Cookson K, Laibinis VA, Kleven SH (2012). The efficacy of three commercial Mycoplasma gallisepticum vaccines in laying hens. Avian Dis., 56(2): 272-275. https://doi.org/10.1637/9952-092711-reg.1

• Furian TQ, Karen AB, Vanessa LS, Luis SR, Camila NA, Vladimir PN, Carlos TP, Hamilton LSM (2016). Virulence genes and antimicrobial resistance of P. multocida isolated from poultry and swine. Braz. J. Microbiol., 47: 210-216. https://dx.doi.org/10.1016%2Fj.bjm.2015.11.014

• Gaunson JE, Philip CJ, Whithear KG, Browning GF (2006). The cellular immune response in the tracheal mucosa to Mycoplasma gallisepticum in vaccinated and unvaccinated chickens in the acute and chronic stages of disease. Vaccine. 24: 2627-2633. https://doi.org/10.1016/j.vaccine.2005.12.008

• Glisson JR, Hofacre CL, Christensen JP (2008). Fowl cholera. In: Diseases of Poultry, Saif YM, Barnes HJ, Glisson JR, Fadly AM, McDougald LR, Swayne DE (Editors). Blackwell Publishing, Ames, Iowa, USA; pp. 739-758.

• Harper M, John M., Edmunds M, Wright A, Ford M, Turni C, Blackall PJ, Cox A, Adler B, Boyce JD (2016). Protective efficacy afforded by live Pasteurella multocida vaccines in chickens is independent of lipopolysaccharide outer core structure. Vaccine, 34(14): 1969-1703. https://doi.org/10.1016/j.vaccine.2016.02.017

• Ievy S, Khan MFR, Islam MA, Rahman MB (2013). Isolation and identification of Pasteurella multocida from chicken for the preparation of oil adjuvanted vaccine. Microbes Health, 2(1): 1-4. https://doi.org/10.3329/mh.v2i1.17253

• Jabbri AR, Moazeni Jula GR (2005). Fowl cholera: Evaluation of a trivalent Pasteurella multocida vaccine consisted of serotypes 1, 3 and 4. Arch. Razi. Inst., 59: 103-111.

• Jacob R, Branton SL, Evans JD, Leigh SA, Peebles ED (2014). Effects of live and killed vaccines against Mycoplasma gallisepticum on the performance characteristics of commercial layer chickens. Poult. Sci., 93: 1403-1409. https://doi.org/10.3382/ps.2013-03748

• Kardos G, Kiss I (2005). Molecular epidemiology investigation of outbreaks of fowl cholera in geographically related poultry flocks. J. Clin. Microbiol., 43(6): 2959-2961. https://dx.doi.org/10.1128%2FJCM.43.6.2959-2961.2005

• Kleven SH (2008). Control of avian mycoplasma infections in commercial poultry. Avian Dis., 52: 367-374. https://doi.org/10.1637/8323-041808-review.1

• Kreslavsky T, Gleimer M, Miyazaki M, Choi Y, Gagnon E, Murre C, Sicinski P, Boehme HV (2012). β-selection induced proliferation is required for αβ T cell differentiation. Immunity, 37(5): 840-853. https://dx.doi.org/10.1016%2Fj.immuni.2012.08.020

• Mallinath KC, Hari Babu YA (2013). Study on isolation of mycoplasma from cases of avian mycoplasmosis. Frontier J. Vet. Anim. Sci., 2(2): 96-99.

• Mukkur TKS, Pyliotis NA, Bones A (1982). Possible immunological synergism among the protective antigens of P. multocida type A. J. Comp. Pathol., 92: 249-260. https://doi.org/10.1016/0021-9975(82)90083-4

• OIE (2012). Avian mycoplasmosis (Mycoplasma gallisepticum, M. synoviae), Ch.2.3.5. In: Manual of Diagnostic Tests and Vaccines for Terrestrial Animals, 7th ed., Vol. 1, France, pp. 445-469.

• Parvin MS, Siddique MP, Islam MT (2011). Humoral immune response to fowl cholera vaccine in different breeds of commercial birds. Bangladesh J. Vet. Med., 9(2): 127-131.

• Qandoos AZ (2018). Recent studies on acute fowl cholera infection. MvSc, Thesis, (Poultry Diseases), Faculty of Veterinary Medicine, Cairo University, Egypt.

• Raviv Z, Callison SA, Ferguson-Noel N, Kleven SH (2008). Strain differentiating real-time PCR for Mycoplasma gallisepticum live vaccine evaluation studies. Vet. Microbiol., 129: 179-187. https://doi.org/10.1016/j.vetmic.2007.11.017

• Salama SS, Gadallah FM, Gamal FEZ, Abo-Elkhir, AAK, Ali MA (2019). Uses of single dose dependent and relative potency assays for evaluation of inactivated fowl cholera vaccine. J. World Poult. Res., 9(3): 133-138. https://dx.doi.org/10.36380/jwpr.2019.16

• Sawada T, Rimler RB, Rhoades KR (1982). Indirect hemagglutination test that uses glutaraldehyde-fixed sheep erythrocytes sensitized with extract antigens for detection of Pasteurella antibody. J. Clin. Microbiol., 15(5): 752-756. https://www.ncbi.nlm.nih.gov/pubmed/6808010

• Senterifit LB (1983). Preparation of antigens and antisera, Ch. F2. In: Methods in Mycoplasmology (Eds. Razin S. and Tully, J.G.), Vol.1, New York, Academic Press.

• Suling L, Miao T, Sebastian M, Bhullar P, Ghaffari E, Liu M, Symonds ALJ, Wang P (2012). The transcription factors Egr2 and Egr3 are essential for the control of inflammation and antigen induced proliferation of B and T cells. Immunity, 37: 685-696. https://dx.doi.org/10.1016%2Fj.immuni.2012.08.001

• Tabatabaei M, Moazeni Jula GR, Jabbri AR, Esmailzadeh M (2007). Vaccine efficacy in cattle against haemorrhagic septicemia with live attenuated aerA mutant of Pasteurella multocida B:2 strain. J. Cell Anim. Biol., 1(4): 062-065. http://www.academicjournals.org/JCAB

• Talha AFSM (2003). Investigation on the prevalence and significance of M. gallisepticum in village chickens and possibility of establishing M. gallisepticum free flocks and significance of M. gallisepticum on different production parameters in layer chickens in Bangladesh. MSc Thesis, The Royal Veterinary and Agricultural University, Denmark and Bangladesh Agricultural University, Mymensingh.

• Xiao K, Liu Q, Liu X, Hu Y, Zhao X, Kong Q (2015). Identification of the avian Pasteurella multocida phoP gene and evaluation of the effects of phoP deletion on virulence and immunogenicity. Inter. J. Mol. Sci., 217: pii: E12. https://dx.doi.org/10.3390%2Fijms17010012

• Yoder HWJr (1979). Serological response of chickens vaccinated with inactivated preparation of Mycoplasma gallisepticum. Avian Dis., 23(2): 493-506.

• Youssef EA, Tawfik HE (2011). Improvement of rabbit pasteurellosis vaccine using Montanide ISA50. Egypt. J. Agri. Res., 89(2): 697-708.