preparation and evaluation of a combined bivalent vaccine … 2020. 8. 1. · my mother, my father,...

Prof. Dr. Jakeen Kamal El Jakee Professor of Microbiology and Vice dean for

graduate studies and research affairs

Faculty of Veterinary Medicine

Cairo University

Dr. Eman Mohammed El Rawy Chief Researcher and Head of Aerobic

Bacterial Vaccines Department

Veterinary Serum and Vaccine Research

Institute-Abbasia - Cairo

Prof. Dr. Wafaa Abd El Ghany Abd

El Ghany Professor of Poultry Diseases


Cairo University

Dr. Mona Mohammed Shaker Chief Researcher

Mycoplasma Department

Animal Health Research Institute-Dokki-Giza

Cairo University


Department of Microbiology

Preparation and Evaluation of a Combined Bivalent

Vaccine Against Avian Pasteurella and Mycoplasma

Infections in Chickens

A thesis presented by

Fatma Fathy Ibrahim Hassan B.V.Sc., Fac. Vet. Med., Cairo University (2004)

M.V.Sc., Veterinary Medical Sciences

Fac. Vet. Med., Cairo University (2012)

For the Degree of Ph. D. in Veterinary Medical

Sciences (Bacteriology, Immunology, Mycology)

Under the supervision of

Prof. Dr. Mahmoud Essam Hatem Ahmed (late) Professor of Microbiology


Cairo University

2018

Scanned by CamScanner

Supervision sheet

This thesis is under supervision of:

Prof. Dr. Mahmoud Essam Hatem Ahmed (late)

Professor of Microbiology, Faculty of Veterinary

Medicine, Cairo University

Prof. Dr. Jakeen Kamal Abd El-Haleem El Jakee

Professor of Microbiology and Vice dean for graduate

studies and research affairs, Faculty of Veterinary


Prof. Dr. Wafaa Abd El Ghany Abd El Ghany

Professor of Poultry Diseases, Faculty of Veterinary


Dr. Eman Mohammed El Rawy

Chief Researcher and Head of Aerobic Bacterial Vaccines

Department, Veterinary Serum and Vaccine Research Institute,

Abbasia, Cairo

Dr. Mona Mohammed Shaker

Chief Researcher in Mycoplasma Department, Animal Health

Research Institute, Dokki, Giza

Cairo University


Department of Microbiology

Name: Fatma Fathy Ibrahim Hassan

Nationality: Egyptian.

Date and place of birth: 1/10/1982, Giza.

Degree: Ph.D. in Veterinary Medical Sciences (Bacteriology–Immunology–

Mycology), 2018.

Thesis Title: Preparation and evaluation of a combined bivalent vaccine against

avian Pasteurella and Mycoplasma infections in chickens.

Under Supervision of:

Prof. Dr. Mahmoud Essam Hatem Ahmed (late). Professor of Microbiology,

Faculty of Veterinary Medicine, Cairo University.

Prof. Dr. Jakeen Kamal Abd El-Haleem El Jakee. Professor of

Microbiology and Vice dean for graduate studies and research affairs, Faculty

of Veterinary Medicine, Cairo University.

Prof. Dr. Wafaa Abd El Ghany Abd El Ghany. Professor of Poultry

Diseases, Faculty of Veterinary Medicine, Cairo University.

Dr. Eman Mohammed El Rawy. Chief Researcher and Head of Aerobic

Bacterial Vaccines Department, Veterinary Serum and Vaccine Research

Institute, Abbasia, Cairo.

Dr. Mona Mohammed Shaker. Chief Researcher in Mycoplasma Department,

Animal Health Research Institute, Dokki, Giza.

Abstract

The present work was planned to study the immune response of chickens vaccinated

with locally prepared combined inactivated vaccine of M. gallisepticum and P. multocida

adjuvanted with Montanide ISA70. 150, 4 weeks old specific pathogen free chickens were

divided into five groups, the 1st group was vaccinated with P. multocida vaccine, the 2

nd

group was vaccinated with M. gallisepticum vaccine, the 3rd

group was vaccinated with

combined M. gallisepticum and P. multocida vaccine, the 4th

group was vaccinated with

imported M. gallisepticum vaccine and the 5th

group was kept unvaccinated as a control

group. The prepared vaccines were evaluated by determination of the cellular immunity by

heterophils/lymphocytes ratio and measurement of nitric oxide in the supernatant of

macrophage, and evaluation of the humoral immunity by indirect haemagglutination,

haemagglutination inhibition and ELISA techniques. The potency of the vaccines were

evaluated by the challenge and passive mouse protection tests against the challenge with the

virulent strain of M. gallisepticum (Eis3-10 strain) and P. multocida (serotypes A and D).

The results showed that combined inactivated vaccine of M. gallisepticum and P. multocida

adjuvanted with Montanide ISA70 induced high and long duration of antibody response and

significant protection against the challenge with virulent strain of M. gallisepticum (Eis3-10

strain).

Keywords: M. gallisepticum, P. multocida, inactivated vaccine, chickens, Montanide

ISA70.

My sincere special dedication to:

My Mother,

My Father,

My Sisters

Acknowledgement

First of all, I wish to thank ALLAH for helping me to complete this work and supported

me with his blessing and unlimited care.

I am deeply grateful to Late Mahmoud Essam Hatem, Professor of Microbiology,

Faculty of Veterinary medicine, Cairo University, for his sincere advice and help.

I would like to express my heartfelt thanks and appreciation to Prof. Dr. Jakeen Kamal

Abd El-Haleem El-Jakee, Professor of Microbiology and Vice dean for graduate studies and

research affairs, Faculty of Veterinary Medicine, Cairo University, for her kind supervision,

interest, valuable advice and giving almost help to accomplish this work.

I would like to express my sincere gratitude to Prof. Dr. Wafaa Abd El Ghany Abd El

Ghany, Professor of Poultry Diseases, Faculty of Veterinary Medicine, Cairo University, for her

kind supervision, interest, valuable advice and giving almost help to accomplish this work.

Words cannot express my deepest thanks and gratitude to Dr. Eman Mohammed El-

Rawy, Chief Researcher and Head of Aerobic Bacterial Vaccines Department, Veterinary Serum

and Vaccine Research Institute, Abbasia, Cairo for her valuable help, advices and faithful

supervision till the end of the present work.

My deep gratitude to Dr. Mona Mohammed Shaker, Chief Researcher in Mycoplasma

Department, Animal Health Research Institute, Dokki, Giza for her great help during the

practical part and valuable advice to accomplish this work.

Great thanks to staff members of the Aerobic Bacterial Vaccines Department, Veterinary

Serum and Vaccine Research Institute, Abbasia, Cairo for their continuous help and

encouragement.

Great thanks to staff members of the Mycoplasma Department, Animal Health Research

Institute, Dokki, Giza for their great help during the practical part.

List of contents

PAGE

Introduction……………………………………………………... 1

Review of literature…………………………………………….. 5

2.1. M. gallisepticum …………………………………………... 5

2.1.1. History ………………………………………………….. 5

2.1.2. Etiology …………………………………………………. 7

2.1.3. Pathogenesis and immunity ……………………………... 10

2.1.4. Isolation and identification ……………………………… 15

2.1.5. Serological identification ………………………………... 18

2.1.6. Economic importance……………………………………. 20

2.1.7. Vaccination………………………………………………. 21

2.2. P. multocida……………………………………………………… 29

2.2.1. History…………………………………………………… 29

2.2.2. Etiology………………………………………………….. 31

2.2.3. Pathogenesis and immunity……………………………… 34

2.2.4. Isolation and identification………………………………. 36

2.2.5. Serological identification ………………………………… 38

2.2.6. Economic importance…………………………………….. 39

2.2.7. Vaccination……………………………………………….. 39

Materials and methods…………………………………………. 43

3.1. Materials…………………………………………………………. 43

3.1.1. Strains used……………………………………………… 43

3.1.2. Imported M. gallisepticum vaccine…………………….... 43

3.1.3. Laboratory animals and birds …………………………... 44

3.1.4. Culture media…………………………………………..... 45

3.1.5. Supplements……………………………………………... 46

3.1.6. Stains used………………………………………………. 47

3.1.7. Materials used for vaccines preparation…………………. 47

3.1.8. Materials used for measurement of NO concentration in

the supernatant of macrophage…………………………………

48

3.1.9. Materials used for IHA test……………………………… 48

3.1.10. Materials used for HI test………………………………. 49

3.1.11. Materials used for ELISA test………………………….. 49

3.1.12. Equipments and apparatus ……………………………... 50

3.2. Methods…………………………………………………………...

51

3.2.1. Preparation of inactivated oil emulsion M. gallisepticum 51

II

vaccine………………………………………………………….

3.2.2. Preparation of inactivated oil emulsion P. multocida

vaccine………………………………………………………….

52

3.2.3. Preparation of combined inactivated oil emulsion vaccine

of M. gallisepticum and P. multocida…………………………...

53

3.2.4. Evaluation and quality control of the prepared vaccines.... 53

3.2.5. Experimental design…………………………………….... 54

3.2.6. Evaluation of the cellular immunity ……………………... 57

3.2.6.1. Determination of H/L ratio…………………………….. 57

3.2.6.2. Measurement of NO concentration in the supernatant of

macrophage………………………………………………………

57

3.2.7. Evaluation of the humoral immunity ……………………. 58

3.2.7.1. IHA test……………………………………………….. . 58

3.2.7.2. HI test ……………………………………………….… 60

3.2.7.3. ELISA test……………………………………………... 64

3.2.7.3.1. M. gallisepticum ……………………………………. 64

3.2.7.3.2. P. multocida……………………………………………….. 66

3.2.8. Evaluation of the potency of the vaccines………………... 69

3.2.8.1. Passive mouse protection test ………………………….. 69

3.2.8.2. Challenge test…………………………………………. 70

3.2.8.2.1. M. gallisepticum…………………………………………. 70

3.2.8.2.2. P. multocida………………………………………………. 70

Results…………………………………………………………….. 71

4.1. Results of sterility, purity and safety tests of the prepared

vaccines…………………………………………………………

71

4.2. Evaluation of the cellular immune response of chickens that

vaccinated with different vaccines ……........................................

71

4.2.1. Determination of H/L ratio ………………………………. 71

4.2.2. Estimation of NO concentration in the supernatant of

macrophage………………………………………………………

76

4.3. Evaluation of the humoral immune response of chickens

that vaccinated with different vaccines…….................................

80

4.3.1. IHA test………………………………………………….. 80

4.3.2. HI test ……………………………………………………. 86

4.3.3. ELISA test ……………………………………………….. 90

4.4. Evaluation of the potency of the vaccines………………….. 97

4.4.1. Passive mouse protection test ……………………………. 97

4.4.2. Challenge test ……………………………………………. 100

III

Discussion………………………………………………………… 103

Summary…………………………………………………………. 115

References………………………………………………………… 120

Arabic summary

IV

List of tables

Number Title Page

1 Experimental design 56

2 Evaluation of H/L ratio post vaccination with

different vaccines in chickens

73

3 Statistical analysis of H/L ratio between vaccinated

groups and control group

74

4 Statistical analysis of H/L ratio between groups of

mycoplasma

75

5 Statistical analysis of H/L ratio between combined

vaccine and P. multocida vaccine

75

6 Estimation of NO concentration in the supernatant of

macrophage post vaccination with different vaccines

in chickens

77

7 Statistical analysis of NO concentration between

vaccinated groups and control group

78


groups of mycoplasma

79


combined vaccine and P. multocida vaccine

79

10 Level of antibody titers against P. multocida type

“A” in chickens vaccinated with combined vaccine

of M. gallisepticum and P. multocida and P.

multocida vaccine by IHA

82

11 Level of antibody titers against P. multocida type

“D” in chickens vaccinated with combined vaccine

of M. gallisepticum and P. multocida and P.

multocida vaccine by IHA

83

V

12 Statistical analysis of IHA antibody titers against P.

multocida type “A” between vaccinated groups and

control group

84


multocida type “A” between combined vaccine and

P. multocida vaccine

84


multocida type “D” between vaccinated groups and

control group

85


multocida type “D” between combined vaccine and

P. multocida vaccine

85

16 Level of antibody titers against M. gallisepticum in

chickens vaccinated with different M. gallisepticum

vaccines by HI

87

17 Statistical analysis of HI antibody titers against M.

gallisepticum between vaccinated groups and control

group

88

18 Statistical analysis of HI antibody titers against M.

gallisepticum between groups of mycoplasma

89

19 Level of antibody titers against M. gallisepticum in

chickens vaccinated with different M. gallisepticum

vaccines by ELISA

92

20 Level of antibody titers against P. multocida in

chickens vaccinated with combined vaccine of M.

gallisepticum and P. multocida and P. multocida

vaccine by ELISA

93

21 Statistical analysis of ELISA antibody titers against

M. gallisepticum between vaccinated groups and

control group

94


M. gallisepticum between groups of mycoplasma

95

VI


P. multocida between vaccinated groups and control

group

96


P. multocida between combined vaccine and P.

multocida vaccine

96

25 Passive mouse protection test against the challenge

with P. multocida type ‘‘A’’ in chickens vaccinated

with combined vaccine of M. gallisepticum and P.

multocida and P. multocida vaccine

98

26

Passive mouse protection test against the challenge

with P. multocida type ‘‘D’’ in chickens vaccinated

with combined vaccine of M. gallisepticum and P.

multocida and P. multocida vaccine

99

27 Challenge test against M. gallisepticum (Eis3-10

strain) in chickens vaccinated with different M.

gallisepticum vaccines

100

28 Challenge test against P. multocida type ‘‘A’’ in



vaccine

101

29 Challenge test against P. multocida type ‘‘D’’ in



vaccine

102

VII

List of abbreviations

APC Antigen presenting cells

APHIS Animal and Plant Health Inspection Service

BCPP Bovine contagious pleuropneumonia

BSA Bovine serum albumin

CFU Colony forming unit

CRD Chronic respiratory disease

CrmA Cytadherence-related molecule A

E. coli Escherichia coli

EDTA Ethylene diamine tetra-acetic acid

ELISA Enzyme linked immunosorbent assay

GA-SRBC Glutaraldehyde-fixed sheep erythrocytes

HA Haemagglutination

HI Haemagglutination inhibition

H/L ratio Heterophils/lymphocytes ratio

HSOAV Haemorrhagic septicaemia oil adjuvant

vaccine

i.b Intrabursal

i.c Intracoelomic

VIII

iCGN Iota carrageenan

Ig Immunoglobulin

IHA Indirect haemagglutination

IL Interleukin

LPS Lipopolysaccharide

LPs Lipoproteins

M. gallisepticum Mycoplasma gallisepticum

M. haemolytica Mannheimia haemolytica

M. iowae Mycoplasma iowae

M. meleagridis Mycoplasma meleagridis

MPC Multilamellar positively charged

M. synoviae Mycoplasma synoviae

NCS Normal control serum

NK Natural killer cell

NO Nitric oxide

NPIP National Poultry Improvement Plan

O.D Optical density

OIE Office International des Epizooties

Omps Outer membrane proteins

P% Protection percentage

IX

PBS Phosphate buffer saline

PCR Polymerase chain reaction

PCV Packed cell volume

P. multocida Pasteurella multocida

PMT P. multocida toxin

PPLO Pleuropneumonia-like organisms

RBCs Red blood cells

RSA Rapid serum agglutination

RSPA Rapid serum plate agglutination

S/C Subcutaneously

SDS Sodium dodecyl sulfate

SPA Slide plate agglutination

SPF Specific pathogen free

SRBC Sheep erythrocytes

TNF Tumor necrosing factor

USA United States of America

1. Introduction

Mycoplasmosis is one of the most important poultry diseases

causing significant economic losses in many countries. Most of these

losses are related directly or indirectly to Mycoplasma gallicepticum

(M. gallisepticum) infection, with or without complicating factors

(Levisohn and Kleven, 2000).

M. gallisepticum is a bacterial pathogen of poultry that is

estimated to cause annual losses exceeding $780 million. The National

Poultry Improvement Plan (NPIP) guidelines recommend regular

surveillance and intervention strategies to contain M. gallisepticum

infections and ensure mycoplasma - free avian stocks (Hennigan et al.,

2012).

M. gallisepticum is a significant poultry pathogen involved in

severe economic losses of the poultry industry due to a reduction in egg

production, hatchability and downgrading of carcasses. Both horizontal

and vertical disease transmission leads to rapid spreading of this

pathogen in flocks. M. gallisepticum can cause severe chronic

respiratory disease (CRD) when present in concert with other poultry

pathogens including Newcastle disease virus, infectious bronchitis

virus and Escherichia coli (E. coli) (Stipkovits et al., 2012). Infections

with Avibacterium paragallinarum and Pasteurella multocida (P.

multocida) should also be ruled out (OIE, 2012).

Control of pathogenic avian mycoplasmas can consist of one of

three general approaches; maintaining flocks free of infection,

medication, and vaccination. Medication can be very useful in

Introduction

2

preventing clinical signs and lesions, as well as economic losses, but

cannot be used to eliminate infection from a flock and is therefore not a

satisfactory long-term solution. Vaccination against M. gallisepticum

can be a useful long-term solution in situations where maintaining

flocks free of infection is not feasible, especially on multi-age

commercial egg production sites (Kleven, 2008).

The effective method to prevent this infection is vaccination by

inactivated vaccines (Ferguson-Noel et al., 2012). The major

advantage of bacterins is their safety. Live attenuated vaccines may

have residual pathogenicity or may revert to the status before

attenuation (El Gazzar et al., 2011). Otherwise, Ley (2008) stated that

bacterins are considered to be of minimal value in the long-term control

of M. gallisepticum infection in multiple-age commercial layer

production sites. Also, Faruque and Christensen (2007) concluded

that inoculation of inactivated M. gallisepticum vaccine is not justified

and is too expensive at farm levels.

P. multocida is a major animal pathogen that causes a range of

diseases including fowl cholera. P. multocida infections result in

considerable losses to layer and breeder flocks in poultry industries

worldwide. P. multocida lipopolysaccharide (LPS) is a primary

stimulator of the host immune response and a critical determinant of

bacterin protective efficacy (Harper et al., 2016).

So, the goal of this study was preparation and evaluation of

combined inactivated vaccine of M. gallisepticum and P. multocida.

Introduction

3

So, the following steps were carried out:

1. Preparation of inactivated M. gallisepticum vaccine

adjuvanted with Montanide ISA70.

2. Preparation of inactivated P. multocida vaccine adjuvanted

with Montanide ISA70.

3. Preparation of combined inactivated vaccine of M.

gallisepticum and P. multocida adjuvanted with Montanide

ISA70.

4. Evaluation of the locally prepared inactivated vaccines for

their safety, sterility and purity.

5. Evaluation of the cellular immune response of the vaccinated

chickens by heterophils / lymphocytes (H/L) ratio and

measurement of nitric oxide (NO) in the supernatant of

macrophage.

6. Evaluation of the humoral immune response of the

vaccinated chickens by:

a. Indirect haemagglutination test (IHA).

b. Haemagglutination inhibition test (HI).

c. Enzyme linked immunosorbent assay (ELISA).

7. Evaluation of the potency of the prepared vaccines by:

Introduction

4

a. Passive mouse protection test.

b. Challenge test

8. Comparison of the efficacy of the combined inactivated

vaccine of M. gallisepticum and P. multocida with the

imported M. gallisepticum vaccine.

Review of literatures

5

2. Review of literatures

2.1. M. gallisepticum:

2.1.1. History:

The first accurate description of the avian mycoplasmosis was in

1905 by Dodd in England and termed Epizootic pneumoenteritis

(Dodd, 1905). In 1938, Dickinson and Hinshaw named the disease

(infectious sinusitis) of turkeys (Dickinson and Hinshaw, 1938). In

1943, Delaplane and Stuart cultivated an agent in embryos isolated

from chickens with CRD and later from turkeys with sinusitis

(Delaplane and Stuart, 1943). In the early 1950, Markham, Wong,

and Van reported that the organism was a member of the

Pleuropneumonia group (Brown, 2002).

The microorganisms of the class Mollicutes (Mycoplasma) were

first identified in 1898 as the etiological agent of the bovine contagious

pleuropneumonia (BCPP) and thereafter, all similar agents were named

pleuropneumonia-like (PPLO-like) organisms (Davis et al., 1973).

Avian mycoplasmosis was primarily described in turkeys in 1926 and

in chickens in 1936 (Charlton et al., 1996). Delaplane and Stuart

(1943) referred to it as CRD of poultry. Markham and Wong (1952)

associated the etiologic agent of CRD to the pathogen responsible for

the infectious sinusitis of turkeys. It was then considered as a member

of the PPLO group and later named as M. gallisepticum (Yoder, 1991).


6

A survey of commercial egg laying poultry in United States of

America (USA) revealed that 37% of laying flocks (262.6 million

layers) were infected with M. gallisepticum and causing an annual

losses of 97 million US $ (Johnson, 1983).

M. gallisepticum infection has been produced experimentally in

captive-reared wild turkeys (Rocke et al., 1988), house sparrows and

budgerigars (Lin et al., 1996). M. gallisepticum has been confirmed by

culture or polymerase chain reaction (PCR) in purple finches

(Charlton et al., 1996). M. gallisepticum was also isolated from a blue

jay that contracted conjunctivitis and from free-ranging American

goldfinches (Ley et al., 1997).

M. gallisepticum is an avian pathogen within the genus

Mycoplasma (class Mollicutes) which includes other species infecting

animals, humans, insects and plants (Razin, 1992).

M. gallisepticum is the most important pathogen in poultry

(Bradbury, 2001). M. gallisepticum infection occur mostly in chickens

and turkeys. However, they have been frequently isolated from quails,

and from several avian species (Lobão et al., 2003).

Mollicutes (mycoplasmas) are pathogenic in a wide range of

mammals (including humans), reptiles, fish, arthropods, and plants. Of

the medically important mollicutes, M. gallisepticum is of particular

relevance to Avian Agriculture and Veterinary science, causing CRD in

poultry and turkey (Dennard, 2011).


7

2.1.2. Etiology:

M. gallisepticum is a fastidious organism requiring a protein

based media enriched with 10-15% heat inactivated horse or swine

serum (or serum factors) and glucose and yeast factors. Since M.

gallisepticum is relatively resistant to penicillin (no cell wall) and

thallium, these antimicrobial agents are usually added to the media to

suppress bacterial and fungal growth (Kleven, 1998).

M. gallisepticum is minute in size with minimal genetic

information and with a total lack of bacterial cell wall. These properties

are reflected in a high degree of interdependence between M.

gallisepticum and the host animal, and in the fastidious nature of the

organism in vitro (Levisohn and Kleven, 2000).

Avian mycoplasmosis can be caused by several species of

Mycoplasma. Generally, Mollicutes (soft skin) are small prokaryotic

organisms being devoid of cell wall and lacking the genetic capacity to

synthesize one; but they have a single trilaminar membrane composed

of protein, glycoprotein, glycolipid, and phospholipid. M. gallisepticum

infections vary from asymptomatic to severe, depending on the

infecting strain and other factors. Uncomplicated infections frequently

cause no clinical signs or mortality in chickens. M. gallisepticum can

be introduced into a flock by live birds or hatching eggs, as well as the

movement of people and fomites. Sub-clinically infected small

backyard flocks can be a source of infection for commercial poultry

(Bradbury, 2001).


8

Their genome size is small; the base composition is poor in

guanine and cytosine with mol% G + C of DNA ranging from 23% to

40%, since they lack a cell wall, they are extremely pleomorphic; cell

shape being spherical, pear shaped, spiral, and filamentous forms

(Quinn et al., 2002).

M. gallisepticum is a highly infectious respiratory pathogen

affecting poultry. When present in concert with other respiratory

pathogens such as infectious bronchitis virus, Newcastle disease virus,

E. coli, or Haemophilus paragallinarum, a condition known as CRD

results. Mycoplasmosis can spread by both lateral and vertical routes,

so it is imperative that any control programme is based on maintaining

a disease free breeding flock. It is particularly difficult to keep multi-

age sites M. gallisepticum free. Lateral transmission occurs through

direct contact and indirectly through mechanical means by way of

fomites and mechanical vectors (Ley, 2003).

The clinical signs of M. gallisepticum in chicken include

coughing, sneezing, rales, ocular and nasal discharge, decrease in feed

consumption and egg production, increased mortality, poor hatchability

and lose weight. The gross lesions in birds with M. gallisepticum

include catarrhal inflammation of sinuses, trachea and bronchi. Air sacs

are often thickened and opaque, and may contain mucous or caseous

exudates, besides hyperplastic lymphoid follicles on the walls. At

slaughter, carcass condemnation may result from the presence of

airsacculitis, fibrinous perihepatitis and adhesive pericarditis,


9

interstitial pneumonia and salpingitis, which are often seen in chickens

(Charlton et al., 2005).

M. gallisepticum infection can cause primary disease in young

chickens typified by coughing, rales, airsacculitis and poor growth.

Adult birds seldom show clinical signs of infection but mild change in

egg quality and drops in egg production may be noticed (Collett,

2005).

Mycoplasmas are bacteria that lack cell wall and belong to the

class Mollicutes. Although they have been considered extracellular

agents, scientists admit nowadays that some of them are obligatory

intracellular microorganisms, whereas all other mycoplasmas are

considered facultative intracellular organisms (Nascimento et al.,

2005).

M. gallisepticum and Mycoplasma synoviae (M. synoviae)

belong to the class Mollicutes, order Mycoplasmatales, family

Mycoplasmataceae. It should be noted, however, that Mycoplasma

meleagridis (M. meleagridis) and Mycoplasma iowae (M. iowae) can

also cause disease in poultry, but M. gallisepticum and M. synoviae are

considered to be the most important of the pathogenic mycoplasmas,

and both occur world-wide. The transmission of M. gallisepticum can

be both vertical and horizontal. Vertical transmission of M.

gallisepticum has been known to occur in eggs laid by infected hens.

Horizontal transmission of M. gallisepticum occurs through direct

contact between infected and susceptible chickens, especially in flocks


10

with high population density. M. gallisepticum strains vary in

infectivity and virulence, and infections may sometimes be unapparent.

Lesions of the respiratory tract take the form initially of excess mucous

exudate followed by catarrhal and caseous exudate, which may form

amorphous masses in the air sacs. In turkeys and game birds, the

swollen infraorbital sinuses contain mucoid to caseous exudates was

seen (OIE, 2012).

Wild birds have been identified as important carriers, so direct

contact with such birds should be avoided (Dhondt et al., 2014).

2.1.3. Pathogenesis and immunity:

Cell-mediated immunity is thought to play a role but has not

been extensively investigated. Birds which lacked a fully functional

immune system due to either neonatal bursectomy and/or thymectomy,

showed an inability to clear M. gallisepticum from the trachea

(Mukherjee et al., 1990).

Serum nitric oxide is one of the end products produced by

macrophages as a result of their exposure to microbial products or

chemotactic agents, the presence of nitric oxide in appropriate

concentration during inflammation leads to immunomodulatory

functions of host defense (Florquin et al., 1994). Aderem and

Underhill (1999) reported that internalization by macrophages occurs

by a restricted number of phagocytic receptors present on their surface.


11

Stafford et al. (2002) reported that macrophages perform a variety of

functions other than phagocytosis; they act as secretory cells, produce

nitric oxide that kills intracellular microorganisms and also secrete

many different proteins such as lysosomal enzymes and cytokines that

play a key role in regulating immunity.

The primary habitats of mycoplasmas are the mucosal

membranes of the respiratory tract, and/or the urogenital tract, eyes,

mammary glands and joints. Most mycoplasmas are considered surface

parasites, rarely invading tissues, although spread to other organs

strongly suggests a transitory systemic infection, at the least. Adhesion

of mycoplasmas to host cells is a prerequisite for successful

colonization, and ensuing pathogenesis (Krause, 1996).

Mycoplasmas have oval, filamentous or flask shapes, and

several pathogenic species display a prominent polar tip organelle or

bleb structure that mediates attachment to the host target cells. This tip

structure is hemispherical, around 800x1250 A in circumferences and

composed of surface-exposed proteins, called adhesins or cytadhesions

proteins. These adhesins promote the attachment of mycoplasma

allowing the colonization of epithelial cell surfaces. The percentage of

proteins in mycoplasma membranes is much higher than other

prokaryotes. These proteins are considered to be the most dominant

antigens and are responsible for antigenic variation (Razin et al.,

1998).


12

Mycoplasmas, including M. gallisepticum, have recently been

demonstrated to have the ability to vary the expression of major surface

antigens, thus expressing a continually changing "antigenic profile" to

the immune system (Razin et al., 1998). Continual variability in the

expression of such surface antigens also occurs in vivo (Levisohn et

al., 1995b) and may be a major factor in the development of clinical

disease in addition to having a significant impact on the development

of serological responses (Levisohn et al., 1995a). The marked

heterogeneity with respect to presentation of the major surface antigens

provides a likely explanation of how mycoplasma infections are able to

persist in birds despite a strong immune response (Levisohn and

Kleven, 2000).

The critical event for M. gallisepticum pathogenesis is

attachment and colonization of host respiratory epithelium (Razin,

1999). Upon attachment they can cause release of mucus from goblet

cells leading to obstruction of tracheal lumen, and rounding and

exfoliation of epithelial cells as well as ciliostasis, squamous

metaplasia and sometimes lysis of epithelial cells (Dykstra et al.,

1985).

Mycoplasma infection has been shown to have a direct effect on

both B and T lymphocyte proliferation, cytokine release and antibody

production, which indicates combined antibody and cell mediated

response (Gaunson et al., 2000).


13

The chronic nature of mycoplasma infections demonstrates a

failure of the host immune system to deal effectively with these

organisms. Antigenic variation of surface proteins allows M.

gallisepticum to evade the host’s immune response through the

generation of escape variants (Glew et al., 2000).

Intracellular invasion and survival within eukaryotic cells by M.

gallisepticum may contribute to this organism’s resistance to the host’s

immune response and antimicrobial therapy (Winner et al., 2000).

Cytadherence to the epithelial surfaces of the host tissues is a

requirement for successful colonization. Research into the molecular

mechanisms of M. gallisepticum cytadherence has identified a

coordinate action between the primary cytadhesin, GapA, and at least

one cytadherence-related molecule, CrmA (Papazisi et al., 2002).

It is presumed that M. gallisepticum enters the respiratory tract

by inhalation, aerosol or via the conjunctiva (Bradbury, 2001). The

respiratory tract and lungs are frequent sites of infection. Mycoplasmas

are capable of destroying the cilia of cells in the respiratory tract thus

predisposing to secondary bacterial infection (Quinn et al., 2002).

M. gallisepticum appears to have the capacity to alter the

expression of surface antigens to evade the host immune response

(Kleven, 2002). This variable surface antigen expression could explain

the phenomenon of chronic infection and carrier state despite the

initiation of a strong immune response (Ley, 2003).


14

The organisms are extracellular and produce haemolysin,

proteases, nucleases and other toxic factors that can lead to the death of

host cells or to a chronic infection. Latency can occur and various

stresses predispose to mycoplasmal diseases (Quinn et al., 2002).

The pathogenic mechanism for disease includes adherence to

host target cells, mediation of apoptosis, innocent bystander damage to

host cell due to intimate membrane contact, molecular (antigen)

mimicry that may lead to tolerance, and mitotic effect for B and/or T

lymphocytes, which could lead to suppressed T-cell function and/or

production of cytotoxic T cell, besides mycoplasma by-products, such

as hydrogen peroxide and superoxide radicals. Moreover, mycoplasma

ability to stimulate macrophages, monocytes, T-helper cells and NK

(natural killer) cells, results in the production of substances, such as

tumor necrosing factor (TNF-α), interleukin (IL-1, 2, 6) and interferon

particularly interferon gamma (Nascimento et al., 2005). These

mechanisms may explain the transient suppression of humoral and

cellular immune responses during mycoplasma infection in birds, the

immune tolerance and auto immune diseases, as well as the massive

lymphoid cell infiltration in the respiratory tract and joint tissues of

infected fowls (Razin et al., 1998).

Lipoproteins (LPs) reside on the surfaces of the cell wall-less

mycoplasmas and are important factors in pathogenesis

(Noormohammadi, 2007). The importance of antibodies produced in


15

response to M. gallisepticum infection inhibited attachment of the

organism to epithelial cells (Grodio et al., 2009).

M. gallisepticum infection in chickens is associated with severe

inflammation of the trachea, air sacs and lungs. M. gallisepticum

cytadheres to the tracheal epithelium and mediates infiltration of

macrophages, heterophils and lymphocytes to the tracheal submucosa

(Majumder, 2014).

The Pathogenic mechanisms of M. gallisepticum include

adherence to host target cells, release of toxins, mediation of apoptosis

and immune evasion leading to obstruction of the tracheal lumen,

exfoliation of epithelial cells as well as ciliostasis. In addition,

mycoplasma by-products, such as hydrogen peroxide and superoxide

radicals, along with inflammatory cytokines can exacerbate the disease

conditions (Umar et al., 2017).

2.1.4. Isolation and identification (Diagnosis):

Diagnosis is based on a flock basis, and the presence of one or

more infected birds in the flock sample that constitute an infected flock.

General guidelines of NPIP require testing of 10% of the flock (a

minimum of 300 birds) before the onset of egg production and every

sixty to ninety days thereafter (Animal and Plant Health Inspection

Service (APHIS), 1997).

Mycoplasma can be detected in tissue fragments of affected

organs like trachea, air sacs and lungs. Besides synovial, ocular and


16

infraorbital sinus exudates, good sources are swabs from trachea and

air sacs, and pipped embryos (Ley and Yoder, 1997). Samples

collected for culture or PCR can be placed in a 50% solution of Frey’s

medium or phosphate buffered saline (PBS) (pH 7.8) in glycerol and

kept in freezer before being processed (Polo et al., 2002).

Mycoplasmas are fastidious organisms and require specific

growth factors, an isotonic medium and the absence of inhibitory

substances for growth. They require a protein based medium enriched

(supplemented) with serum or serum factors, yeast extracts, glucose

and bacterial and/or fungal inhibitors. Horse or swine serum

(inactivated at 56°C

for 1 hour) should be used in media for the growth

of M. gallisepticum (Quinn et al., 2002).

Diagnosis of M. gallisepticum infection can be made by various

methods, but the gold standard test for confirmation of diagnosis is

isolation and identification of the organism, such examinations

typically require 2–3 weeks to complete (Ley, 2008).

Recently, Office International des Epizooties (OIE) and NPIP

recommended PCR as a reliable test for the detection of M.

gallisepticum infections. Real-time PCR, which has distinct advantages

over conventional PCR, such as higher reliability, rapidity and

prevention of environmental contamination, has been used for the

detection of M. gallisepticum in poultry (Kahya et al., 2010).

Inoculated plates are incubated at 37°C in sealed containers.

Increased humidity and CO2 tension in the atmosphere have been


17

reported to enhance growth; these conditions may be obtained by the

inclusion of damp paper or cotton wool, and by flushing the container

with 5–10% CO2 in nitrogen, by placing a lighted candle in the

container, or by using a CO2 incubator or suitable gas-generating

system. Yeast extracts, the other growth factor, may be beneficial and

is usually supplied by commercial yeast autolysate or by fresh yeast

extract. Glucose is fermented by M. gallisepticum and is a common

supplement. Mycoplasmas are resistant to penicillin (as they lack cell

wall), which is added in the medium to inhibit the growth of Gram

positive bacteria; and the component thallium acetate, for which

mycoplasmas are relatively resistant, helps to inhibit Gram negative

bacterial and fungal contamination. M. gallisepticum can be identified

by immunological methods after isolation in mycoplasma media or by

detection of their DNA in field samples or cultures. DNA detection

methods based on the PCR are used in specialized laboratories. Once

validated, they can be used on swab material or cultures. Several

serological tests are used to detect M. gallisepticum antibodies. The

most commonly used are the rapid serum agglutination (RSA) test,

ELISA and HI tests. Several commercial M. gallisepticum antibody

ELISA kits are available (OIE, 2012).


18

2.1.5. Serological identification:

2.1.5.1. HI test:

The HI test is used to confirm various rapid serum plate

agglutination (RSPA) reactions. It is considered to be highly specific

but less sensitive than the slide plate agglutination (SPA) test. Infected

birds may not be test positive until three weeks or longer after infection

as it detects IgG. In addition, there is antigenic variation among M.

gallisepticum strains as measured by HI. Antigen prepared from one M.

gallisepticum strain may not adequately detect HI antibodies in

chickens infected with a different strain (Charles and Graham, 1989).

HI being derived from the Greek word hamia for blood and

agglutination meaning rapid clumping, haemagglutination (HA) by

definition means the rapid clumping of red blood cells (RBCs). HA

occurs when M. gallisepticum surface antigens form cross linkages

between red blood cells causing them to adhere to each other and

clump. The principle of the HI test is that immune serum contains

immunoglobulins that are able to specifically inhibit homologous M.

gallisepticum haemagglutinin from causing erythrocytes to clump

(Collett, 2005).

M. gallisepticum is capable of haemagglutinating avian RBCs,

and specific antibodies in sera cause inhibition. The HI test requires a

satisfactory haemagglutinating M. gallisepticum antigen, washed fresh

chicken or turkey RBCs, as appropriate, and the tested sera. The

antigen can be either a fresh broth culture or a concentrated washed


19

suspension of the mycoplasma cells in PBS. It may be difficult to

sustain a supply of high-titred broth culture antigen; however, the use

of concentrated antigen (usually containing 25–50% glycerol and

stored at -70°C), increases the likelihood of nonspecific reactions. The

HI test should be performed using 4HA units. There is no official

definition of positive and negative results for international trade but

NPIP of the USA states that titers of 1/80 or above are considered to be

positive and titers of 1/40 are strongly suspicious (OIE, 2012).

Asif et al. (2015) concluded that the RSA test is highly sensitive

for the detection of M. gallisepticum in poultry but HI based assay is

more specific and reliable than RSA and conventional diagnostic

techniques.

2.1.5.2. ELISA test:

The recommended ELISA kits have excellent sensitivity and

specificity, but transitory non-specific reactions may still occur, for

similar reasons to those occurring in the SPA test (Avakian and

Kleven, 1990).

In ELISA assay, the plates are coated with whole cell M.

gallisepticum antigen and the test samples are added, but the reaction is

assessed by the extent of blocking that occurs when the conjugated

monoclonal antibody is added (Czifra et al., 1993). Commercial

ELISA kits are widely available and are increasingly used for

serological confirmation (Kempf et al., 1994).


20

2.1.6. Economic importance:

M. gallisepticum is believed to cost the worldwide poultry

industry over $780 million every year. In the United States, it is

believed to cost over $120 million on egg production alone. Infection

can lead to the culling of an entire flock to prevent further spread (Ley

and Yoder, 1997).

M. gallisepticum is an infectious respiratory pathogen of

chickens and turkeys. It is the most pathogenic and economically

significant mycoplasma pathogen of poultry. Economic losses from

condemnation or downgrading of carcasses, reduced feed and egg

production efficiency, and increased medication costs are factors that

make this one of the costliest disease problems confronting commercial

poultry production worldwide (Ley, 2003).

Losses attributed to mycoplasmosis, mainly M. gallisepticum

infection, result from decreased egg production and egg quality, poor

hatchability, poor feed efficiency, increase in mortality and carcass

condemnations, besides medication costs (Nascimento et al., 2005).

M. gallisepticum economically affects the poultry industry

through increased mortality, decreased egg production and reduced

feed efficiency (Almanama, 2011).

M. gallisepticum is a bacterial pathogen of poultry that is

estimated to cause annual losses exceeding $780 million. The NPIP

guidelines recommend regular surveillance and intervention strategies

to contain M. gallisepticum infections and ensure mycoplasma-free


21

avian stocks, but several factors make detection of M. gallisepticum

and diagnosis of M. gallisepticum infection a major challenge. Current

techniques are laborious, require special expertise, and are typically

plagued by false results (Hennigan et al., 2012).

Avian mycoplasmosis is a complex, complicated and

multifactorial disease posing a serious economic challenge to the

prosperity of poultry enterprise in many parts of the world directly or

indirectly resulting from high morbidity, poor feed conversion,

decreased production, medication cost and high mortality when

complicated with other infections (Mallinath and Hari Babu, 2013).

2.1.7. Vaccination:

Vaccination is an option for controlling M. gallisepticum when

biosecurity measures fail to prevent the infection of poultry flocks with

these mycoplasmas. Both killed vaccines (bacterins) and living

vaccines are currently in commercial use (Whithear, 1996).

The control strategy of many countries is based on maintaining

M. gallisepticum free breeding flocks. M. gallisepticum–negative

breeding stock can be identified and maintained by serologic testing.

Heat treatment or tylosin can eliminate egg transmission from valuable

breeding animals. Biosecurity measures are important in preventing

transmission on fomites. Wild or pet birds can also carry M.

gallisepticum, and should be excluded from poultry operations.

Currently, inactivated and live attenuated vaccines are available to


22

poultry farmers. Although inactivated vaccines were not well accepted

in the past, they are often preferred today, mainly because there is no

risk of infection and because they do not affect M. gallisepticum

detection (Ley and Yoder, 1997).

Control of pathogenic avian mycoplasmas can consist of one of

three general approaches; maintaining flocks free of infection,

medication, or vaccination. Maintaining flocks free of pathogenic

mycoplasmas consists of maintaining replacements from mycoplasma-

free sources in a single-age and all-in all-out management system.

Good biosecurity and an effective monitoring system are necessary

aspects of this program. Medication can be very useful in preventing

clinical signs and lesions, as well as economic losses, but cannot be

used to eliminate infection from a flock and is therefore not a

satisfactory long-term solution. Vaccination against M. gallisepticum

can be a useful long-term solution in situations where maintaining

flocks free of infection is not feasible, especially on multi-age

commercial egg production sites (Kleven, 2008).

Prevention and control programs based on strict biosecurity,

surveillance (serology, culture, and molecular identification), and

eradication of infected breeder flocks are preferable. Nevertheless, the

rapid expansion of poultry production in restricted geographical areas

and the consequent recurring M. gallisepticum outbreaks necessitated

the implementation of additional measurements (Raviv et al., 2008).


23

Treatment of mycoplasma diseases is difficult since

Mycoplasma species lack a cell wall, which differentiates them from

bacteria and is thus resistant to some commonly used antibiotics.

Despite the seriousness of mycoplasma diseases, there are few effective

vaccines to combat them today. Indeed, those that are available are

whole-cell vaccines, some of which are semi virulent, provide only

transient or partial immunity and often induce unpleasant side effects

(Nicholas et al., 2009).

M. gallisepticum causes severe economic losses to the poultry

industry. Considering that eradication through elimination of positive

flocks is expensive, available vaccines do not protect against infection,

and the disease is difficult to effectively treated, new alternatives are

needed to control the disease (Moura et al., 2012).

Over 20 serotypes of M. gallisepticum have been discovered and

the one to which CRD is attributable is known as S-6 serotype. It is

found in chickens, turkeys and ducks. The R-strain is widely used for

the production of bacterins (inactivated vaccines) and it is the highly

pathogenic (virulent) strain; whereas the F, ts-11 and 6/85 strains are

widely used for live vaccine production and have relatively poor

pathogenicity (OIE, 2012).

2.1.7.1. Living vaccines:

Living M. gallisepticum vaccines include the F strain and

attenuated strains ts-11 and 6/85. The F strain reduces the decline in


24

egg production and has been used to displace endemic strains in

multiple-age flocks. The major disadvantage is the inherent virulence

of F strain. Strain ts-11 is less virulent and less infectious than F strain

and provides a somewhat weaker, but usually effective, long-term

protective immunity, which is vaccine-dose dependent. Strain 6/85 also

stimulates a weaker protective immune response than F strain and is of

low virulence and infectivity (Whithear, 1996).

Three vaccines are currently approved for mixed-age flocks; the

F strain, ts-11 and 6/85. The F strain retains some virulence but confers

lifetime immunity. Both ts-11 and 6/85 are less virulent than the F

strain but also less effective (Kleven, 2008).

2.1.7.2. Inactivated vaccines:

Vaccination with M. gallisepticum organisms adjuvanted to

multilamellar positively charged (MPC) liposomes or oil-emulsion

resulted in higher egg production, during the first month following

challenge, in comparison to the unvaccinated-challenged birds; A

significant immunoglobulin (Ig) response specific to M. gallisepticum

was observed in sera of chickens collected 3 weeks after the first and

second vaccination with M. gallisepticum adjuvanted with MPC

liposomes or oil-emulsion. Both groups had highly significant

protection against M. gallisepticum transmission in eggs laid during the

first month post challenge (Barbour and Newman, 1990).


25

Chickens immunized by sequential intracoelomic (analogous to

intraperitoneal route in mammals) and intrabursal (i.c./i.b.) routes with

inactivated M. gallisepticum bacterin mixed with 0.2% iota carrageenan

(iCGN) as an adjuvant were resistant to airsacculitis induced by a

subsequent aerosol challenge with virulent R strain M. gallisepticum.

Chickens immunized by the i.c./i.b. routes with the adjuvanted bacterin

had increased levels of circulating and local anti-M. gallisepticum IgG

but not IgM or IgA. Tracheal populations of M. gallisepticum were

reduced when compared with unimmunized controls (Elfaki et al.,

1992).

Inactivated M. gallisepticum vaccines appear to protect against

loss of egg production in layers (Evans et al., 1992). Inactivated M.

gallisepticum oil based vaccines stimulate a humoral antibody

response, which is indistinguishable from field exposure based on the

RSPA, HI and ELISA titers (Abd El-Motelib and Kleven, 1993).

Shafay (1995) concluded that the locally prepared combined

inactivated vaccine of M. gallisepticum and P. multocida gave

acceptable protection level in comparison with the monovalent M.

gallisepticum vaccine in vaccinated chickens.

M. gallisepticum bacterins contain an oil emulsion adjuvant can

reduce the decline in egg production associated with M. gallisepticum,

although they do not prevent infection. Newer adjuvants, such as

immune stimulating complexes, may provide effective immunity


26

without the tissue lesions caused by oil emulsion adjuvants (Whithear,

1996).

Vaccination with M. gallisepticum bacterin has been shown to

reduce, but not eliminate colonization by M. gallisepticum following

challenge. Generally, it is felt that bacterins are of minimal value in

long-term control of infection on commercial layer multiple-age

production sites (Ley, 2003).

The safety and efficacy of an oily bacterin as a vaccine

candidate against M. gallisepticum in heavy commercial broilers was

evaluated. It was observed that the candidate vaccine was able to

produce 73.7% immunity to the birds when compared with the control

commercial vaccine. The safety of the candidate vaccine was

confirmed since there were no adverse effects or mortality among the

birds when the vaccine was used (Rosado et al., 2004).

Inactivated M. gallisepticum vaccine became popular in the

early 1980s. Although originally used in commercial layer flocks to

prevent egg production loss, these bacterins were later used in broiler

breeder flocks to reduce the vertical transmission rate (Collett, 2005).

Feberwee et al. (2006) stated that vaccination with an

inactivated M. gallisepticum vaccine at 16 and 20 weeks of age does

not reduce the horizontal transmission of M. gallisepticum between

laying hens and also there was an effect of vaccination with a bacterin

(an inactivated vaccine) on the levels of potential shedding of M.

gallisepticum.


27

M. gallisepticum bacterin do protect chickens against respiratory

signs, airsaculitis, egg production losses and reducing egg transmission

(Kleven, 2008). The major advantage of bacterin is their safety. Live

attenuated vaccines may have residual pathogenicity or may revert to

the status before attenuation (El Gazzar et al., 2011).

M. gallisepticum bacterins are considered to be of minimal value

in the long-term control of M. gallisepticum infection in multiple-age

commercial layer production sites (Ley, 2008).

Ferguson-Noel et al. (2012) reported that the M. gallisepticum

bacterin and live F-strain vaccinations were both protective and

resulted in significant differences in air sac lesions, tracheal lesions,

and ovarian regression compared to the non vaccinated controls and the

recombinant fowl pox- M. gallisepticum vaccine in laying hens.

M. gallisepticum bacterins are used to reduce the level of egg

transmission in breeder pullets. Using of bacterins in broilers is limited

by the fact that birds vaccinated before 1–2 weeks of age are not

protected. Although bacterins may provide protection against

respiratory signs, airsacculitis, and egg production losses, vaccinated

flocks are readily infected. The duration of immunity is not known, but

most flocks are exposed within 1–2 months after vaccination (OIE,

2012).

Gondal et al. (2013) concluded that the formaldehyde

inactivated Montanide ISA70 based M. gallisepticum vaccine induced

protective level of anti-M. gallisepticum ELISA antibodies in broilers


28

that persist for more than 45 days post priming. The oil based vaccine

didn’t interfere with the maternal antibody titer of the birds.

Jacob et al. (2014) reported that the M. gallisepticum bacterin

given alone during the prelay period was not effective in protecting

against egg production losses, particularly during the late periods of

lay. In addition, when both M. gallisepticum bacterin and ts11 M.

gallisepticum vaccines were administered together as prelay vaccines,

M. gallisepticum bacterin did not provide any additional benefit over

that of ts11 M. gallisepticum for the various performance and egg

quality parameters investigated.

Bekele (2015) concluded that the oil based M. gallisepticum

vaccine induced protective level of anti M. gallisepticum antibodies in

chickens (protect infection from M. gallisepticum). In this study,

formaldehyde inactivated Montanide ISA70 based M. gallisepticum

vaccine from the PCR confirmed positive from Samuel local isolate of

National Veterinary Institute was prepared and evaluated in chickens.

The amount of immune antigen per 0.5 ml of the dose was 107 Colony

forming units (CFU) of the bacteria.

Gadallah (2015) reported that the locally prepared inactivated

combined M. gallisepticum and E. coli vaccine could help in protection

against the CRD and potentiate the humoral immune response in broiler

chicks.

Jacob et al. (2015) stated that the vaccination with M.

gallisepticum bacterin alone or in combination with ts11 M.


29

gallisepticum at 10 weeks of age with or without an F M. gallisepticum

vaccine overlay at 45 weeks of age didn’t adversely affect the internal

egg or eggshell quality of commercial layers as well as the

functionality of their reproductive systems throughout lay.

Obukhovska et al. (2015) concluded that the level of

macrophages in chickens increased rapidly during the first 10 days after

the second injection of inactivated vaccines against avian

mycoplasmosis [vaccines contained 30% of antigenic substrate (3×107

CFU) and 70% adjuvant (Mantanide ISA 70 VG)]. The highest value of

this indicator was recorded in the spleen and lungs of birds treated by

M. gallisepticum bacterin (24.125 % and 22.280 %, respectively). It

was shown that injection of inactivated vaccines against avian

mycoplasmosis in chickens promoted stimulation for primary link of

cellular immunity (macrophage).

Sarfaraz et al. (2017) reported that oil based combined M.

gallisepticum and avian influenza (H9N2) vaccine adjuvanted with

Montanide ISA-70 induced effective antibody response in the

vaccinated birds measured by ELISA and HI tests.

2.2. P. multocida:

2.2.1. History:

It has been over 125 years since Louis Pasteur first identified

that a bacterium was the causative agent of fowl cholera. In seminal

experiments, he also showed that repeated passage of the bacteria


30

produced an attenuated strain incapable of causing disease, but the

inoculation of birds with this strain could elicit a protective immune

response (Pasteur, 1880 and Pasteur, 1881).

Outbreaks of fowl cholera mostly occur in chickens, turkeys,

ducks, geese, quails and Japanese green pheasants. However, the

disease affects other types of poultry also, such as game birds reared in

captivity, companion birds, zoo birds and wild birds (Sawada et al.,

1999).

P. multocida was first shown to be the causative agent of fowl

cholera by Louis Pasteur in 1881. Since then, this Gram-negative

bacterium has been identified as the causative agent of many other

economically important diseases in a wide range of hosts (Harper et

al., 2006).

Fowl cholera is commonly found in mature chickens over 16

weeks of age but rarely occurs in young chickens of less than 8 weeks

of age (Petersen et al., 2001 and Glisson et al., 2008).

P. multocida was first discovered by Perroncito in 1878 and

named after Louis Pasteur who first isolated and described this Gram-

negative bacterium as the cause of fowl disease in 1880. Subsequently,

P. multocida was also found to cause atrophic rhinitis in pigs,

haemorrhagic septicaemia in cattle and respiratory diseases in many

other animals (Kubatzky, 2012). P. multocida is an animal pathogen of

worldwide economic significance that causes fowl cholera in poultry

and wild birds (Xiao et al., 2016).


31

2.2.2. Etiology:

The family Pasteurellaceae contains Gram-negative,

facultatively anaerobic and fermentative bacteria of the

genera Pasteurella, Haemophilus, and Actinobacillus. Approximately

20 different species of the genus Pasteurella have been identified using

phenotypic and genetic analyses. Of these species, P. multocida and P.

haemolytica are the most prominent pathogens in domestic animals

causing severe diseases and major economic losses in the cattle, swine,

sheep, and poultry industries. Fowl cholera in chickens and turkeys is

caused by various serotypes of P. multocida serogroup A and

characterized by acute septicemia and fibrinous pneumonia or chronic

fibrinopurulent inflammation of various tissues (Confer, 1993).

Fowl cholera, caused by P. multocida can result in either an

acute septicemia or chronic localized infections in domestic and wild

birds (Sander et al., 1998).

P. multocida is the causative agent of fowl cholera and other

diseases of production animals. Isolates are classified into five groups

based on capsular antigens and into 16 serotypes based on LPS

antigens. Strains causing fowl cholera are most frequently designated

A: 1, A: 3 or A: 4 (Adler et al., 1999).

The outcome of infections may range from peracute /acute

infections to chronic infections. In the former type of infections, few

clinical signs are observed before death and the lesions will be

dominated by general septicaemic lesions. In chronic forms of


32

P. multocida infections, suppurative lesions may be widely distributed,

often involving the respiratory tract, the conjunctiva and adjacent

tissues of the head (Christensen and Bisgaard, 2000).

P. multocida is a Gram-negative encapsulated bacterium that is

the causative agent of a range of animal pasteurellosis diseases,

including fowl cholera in poultry and wild birds, haemorrhagic

septicaemia in cattle and buffalo, atrophic rhinitis in swine, and

snuffles in rabbits (Harper et al., 2006).

Signs of infection in acute Fowl cholera are often present for

only a few hours before death that includes fever, anorexia, ruffled

feathers, mucous discharge from the mouth, nose and ears, cyanosis of

comb and wattles, general depression, diarrhea and increased

respiratory rate. Under natural conditions, mortality may range from

only a few percent to nearly 100% (Glisson et al., 2008). It is important

to note that recovered birds may remain as carriers even after 9 weeks

after infection (Kasten et al., 1997 and Glisson et al., 2008).

Fowl cholera is a contagious bacterial disease of domesticated

and wild avian species caused by infection with P. multocida. All avian

species are susceptible to P. multocida, although turkeys may be the

most severely affected. Often the first sign of disease is dead birds.

Other signs include fever, anorexia, depression, mucus discharge from

the mouth, diarrhoea, ruffled feathers, drop in egg production coupled

with smaller eggs, increased respiratory rate, and cyanosis at the time

of death. Lesions that are often observed include congested organs with


33

serosal haemorrhages, enlarged liver and spleen, multiple small

necrotic areas in the liver and/or spleen, pneumonia, and mild ascites

and pericardial oedema. Birds that survive the acute septicaemic stage

or those infected with organisms of low virulence may develop chronic

fowl cholera, characterized by localized infections. These infections

often involve joints, foot pads, tendon sheaths, sternal bursa,

conjunctivae, wattles, pharynx, lungs, air sacs, middle ears, bone

marrow, and meninges. Lesions resulting from these infections are

usually characterized by bacterial colonization with necrosis,

fibrinosuppurative exudate, and degrees of fibroplasias (OIE, 2012).

The species P. multocida comprises a heterogeneous set of

organisms that are common commensals of the oropharyngeal tract of

many vertebrate species. P. multocida strains are also the primary

causative agent of a wide range of animal diseases, including

haemorrhagic septicaemia in ungulates, fowl cholera in avian species,

atrophic rhinitis in pigs, and snuffles in rabbits (Wilkie et al., 2012). As

well as being primary pathogens, P. multocida strains may also be

involved as opportunistic pathogens associated with agents of other

diseases, including lower respiratory tract infections, such as bovine

respiratory disease complex in cattle, and enzootic pneumonia in cattle

and pigs (Talan et al., 1999).

It is likely that the initial infection with P. multocida occurs via

the respiratory tract, and, as with haemorrhagic septicaemia, may

rapidly progress to disseminated disease. Acute and peracute disease


34

involves rapid bacterial multiplication in the liver and/or spleen, and

often results in fatal septicaemia. Chronic forms of the disease include

localized infections in joints, wattles or nasal sinuses (Boyce et al.,

2010 and Wilson and Ho, 2013).

Pasteurella species are highly prevalent among animal

populations, where they are often found as part of the normal microbial

of the oral, nasopharyngeal, and upper respiratory tracts. Many

Pasteurella species are opportunistic pathogens that can cause endemic

disease and are associated increasingly with epizootic outbreaks

(Wilson and Ho, 2013).

2.2.3. Pathogenesis and immunity:

P. multocida is the most common cause of fowl cholera; the

possible virulence factors include the capsule,

endotoxin, outer membrane proteins (Omps), iron binding systems,

heat shock proteins, neuraminidase production and antibody cleaving

enzymes. The wild birds may be a source of infection to commercial

poultry. Carrier birds seem to play a major role in the transmission of

cholera. The site of infection for P. multocida is generally believed to

be the respiratory tract (Christensen and Bisgaard, 2000).

The mechanisms by which these bacteria can invade the mucosa,

evade innate immunity and cause systemic disease are slowly being

elucidated. Key virulence factors identified to date include capsule and

LPS. The capsule is clearly involved in bacterial avoidance of


35

phagocytosis and resistance to complement, while complete LPS is

critical for bacterial survival in the host. A number of other virulence

factors have been identified by both directed and random mutagenesis,

including P. multocida toxin (PMT), putative surface adhesins and iron

acquisition proteins. However, it is likely that many key virulence

factors are yet to be identified, including those required for initial

attachment and invasion of host cells and for persistence in a relatively

nutrient poor and hostile environment. Omps of P. multocida are some

of the virulent factors that play an important role in pathogenesis

(Harper et al., 2006).

P. multocida is a ubiquitous pathogen which causes a range of

diseases in diverse animal species. Components of the bacterial outer

membrane, such as trans membrane proteins and LPs, play key roles in

the interaction of the pathogen with the host environment and in the

host immune response to infection (Hatfaludi et al., 2010).

P. multocida is a capsulated, gram-negative cocco-bacillus that

can cause serious disease in a wide range of mammals and birds.

P. multocida strains are classified into 16 serovars based on LPS

antigens. LPS is an essential virulence factor of P. multocida. LPS is

also a major immunogen of P. multocida and protection against

infections caused by P. multocida is generally considered to be serovar

specific (Harper et al., 2011).

Primary infection with respiratory viruses or with Mycoplasma

species also predisposes animals to secondary infection with P.

mhtml:file://D:/fatma%20search/Fatma%20search2/Outer%20membrane%20proteins%20of%20Pasteurella%20multocida%20-%20ScienceDirect.mht!https://www.sciencedirect.com/science/article/pii/S0378113510000635#!


36

multocida and/or Mannheimia haemolytica (M. haemolytica) (Fulton,

2009 and Pardon et al., 2011).

The capsule and LPS of P. multocida constitute the major

components of the bacterial cell surface. They play key roles in a range

of interactions between the bacteria and the hosts they colonize or

infect. Both polysaccharides are involved in the avoidance of host

innate immune mechanisms, such as resistance to phagocytosis,

complement-mediated killing, and the bactericidal activity of

antimicrobial peptides; they are therefore essential for virulence. In

addition, LPS is a major antigen in the stimulation of adaptive immune

responses to infection (Harper et al., 2012).

P. multocida is a heterogeneous species that is a primary

pathogen of many different vertebrates. This Gram-negative bacterium

can cause a range of diseases, including fowl cholera in birds,

haemorrhagic septicaemia in ungulates, atrophic rhinitis in swine, and

lower respiratory tract infections in cattle and pigs. One of the primary

virulence factors of P. multocida is LPS. P. multocida LPS is a strong

stimulator of host immune responses (Harper and Boyce, 2017).

2.2.4. Isolation and identification (Diagnosis):

Diagnosis is always dependent upon isolation of the organism.

For the detection of subclinical infections, mouse passage of relevant

samples is recommended, but PCR and isolation attempts on selective

media may represent alternatives (Christensen and Bisgaard, 2000).


37

Detection of P. multocida from clinical specimen by isolation

and identification, PCR, specific hybridization probes, serological tests

and other alternative methods is critically evaluated. These detection

systems provide a wide spectrum of options for rapid diagnosis and for

detecting and understanding of latent infections in herd/flock health

control programmes, though PCR methods for detecting P. multocida

in clinical specimen appear increasingly preferred. Although P.

multocida infections can be rapidly diagnosed with molecular and

serological tests, isolation and accurate species identification are

central to epidemiological tracing of outbreak strains (Dziva et al.,

2008).

Diagnosis depends on isolation and identification of the

causative bacterium, P. multocida. Presumptive diagnosis may be

based on the occurrence of typical signs and lesions and/or on the

microscopic demonstration of myriad bacteria in blood smears, or

impression smears of tissues such as liver or spleen. P. multocida is

readily isolated, often in pure culture, from visceral organs such as

lung, liver and spleen, bone marrow, gonads or heart blood of birds that

succumb to the acute bacteraemic form of the disease, or from the

caseous exudates characteristic of chronic fowl cholera lesions. It is a

facultative anaerobic bacterium that grows best at 37°C. Primary

isolation is usually accomplished using media such as dextrose starch

agar, blood agar, and trypticase–soy agar. Isolation may be improved

by the addition of 5% heat-inactivated serum. Colonies range from 1 to


38

3 mm in diameter after 18–24 hours of incubation and are discrete,

circular, convex, translucent, and butyraceous. The cells are

coccobacillary or short rod-shaped, 0.2–0.4 × 0.6–2.5 μm in size, stain

Gram negative, and generally occur singly or in pairs. Bipolar staining

is evident with Wright or Giemsa stains or methylene blue, and are

usually encapsulated. Identification of P. multocida is based on the

results of biochemical tests, which include carbohydrate fermentation,

enzyme production, and selected metabolite production (OIE, 2012).

2.2.5. Serological identification:

Antigenic characterization of P. multocida is accomplished by

capsular serogrouping. Capsular serogroups, determined by a passive

haemaglutination test, are A, B, D, E, and F. All but serogroup E have

been isolated from avian hosts (Rimler, 1994).

Serological characterization of strains of P. multocida includes

capsular serogrouping and somatic serotyping. DNA fingerprinting can

differentiate among P. multocida having the same capsular serogroup

and somatic serotype. These characterizations require a specialized

laboratory with appropriate diagnostic reagents. Serological tests are

rarely used for diagnosis of fowl cholera. Serological tests, such as

agglutination, and passive haemagglutination, have been used

experimentally to demonstrate antibody against P. multocida in serum

from avian hosts; none were highly sensitive. Determinations of

antibody titers using ELISA assays have been used with varying


39

degrees of success in attempts to monitor seroconversion in vaccinated

poultry, but not for diagnosis (OIE, 2012).

2.2.6. Economic importance:

Fowl cholera is a highly contagious disease which is caused by

P. multocida and has been recognized as an important disease in

poultry for more than 200 years (Kwon and Kang, 2003 and Glisson

et al., 2008). It causes devastating economic losses to the poultry

industry through death, weight loss and condemnation of carcasses and

is associated with high morbidity in poultry especially chicken and

ducks and occurs sporadically or enzootically all over the world (Aye

et al., 2001 and Glisson et al., 2008).

Fowl cholera is a severe systemic disease that occurs in

domestic poultry and wild birds and results in significant economic

losses to poultry industries worldwide (Chrzastek et al., 2012). Fowl

cholera can manifest as a chronic, acute or peracute disease in most

avian species. It causes significant economic impact to poultry

industries worldwide, and outbreaks with high mortality are also seen

in wild birds, especially waterfowl (Descamps et al., 2012 and Wilkie

et al., 2012).

2.2.7. Vaccination:

The currently available P. multocida vaccines

are live P. multocida vaccines and bacterins. Potency tests for avian


40

P. multocida vaccines are a bacterial colony count for vaccines and

vaccination and challenge of birds for bacterins. Somatic antigens,

particularly LPS, appear to be of major importance in immunity

(Confer, 1993). Inactivated vaccines are widely used as the organisms

do not have any chance to be reverted to virulence to cause the disease

(Hopkins and Olson, 1997).

El-Bayomy and Daoud (2004) reported that there was an

elevation in protective values of fowl cholera adjuvanted vaccines

against challenge with virulent strains of P. multocida types A and D

for the serum of the vaccinated chicken groups.

Jabbri and Moazeni Jula (2005) concluded that the inactivated

trivalent fowl cholera vaccine consisted of serotypes 1, 3 and 4 P.

multocida strains provided 70-100% protection against challenge with

homologous strains in chickens and induced a considerable increase in

antibody titer after twice vaccination of 8 weeks chickens.

Vaccination is considered as one of the common preventive

measures worldwide to reduce the prevalence and incidence of fowl

cholera (Kardos and Kiss, 2005). Both live and inactivated (bacterins)

vaccines have been attempted to control the disease (Glisson et al.,

2008).

Ahmed et al. (2010) concluded that the inactivated fowl cholera

vaccine adjuvanted with Montanide ISA70 induced high and protective

antibody titers and gave 100 % protection in chickens against challenge

with virulent strains of P. multocida types A and D.


41

Fowl cholera is a highly contagious and economically important

disease of poultry worldwide. Control of fowl cholera depends mainly

on vaccination throughout the world (Parvin et al., 2011). The P.

multocida vaccines in general use are bacterins, containing aluminium

hydroxide or oil adjuvant, prepared from inactivated cells of serotypes

selected on the basis of epidemiological information. Commercial

bacterins are usually composed of serotypes 1, 3, and 4. Live vaccines

containing modified P. multocida are not generally used except in

North America (OIE, 2012).

Ievy et al. (2013) concluded that the oil adjuvanted fowl cholera

vaccine with 0.5ml dose induced high immune response and high

protection in chickens against challenge with virulent strain of P.

multocida and stated that the vaccination is practiced as preventive

measures in many countries of the world to reduce the incidence of the

disease and various scientists suggested that a local strain of higher

immunogenic value should be selected as vaccine strain for preparation

of bacterin with a view to control fowl cholera.

Abdel-Aziz et al. (2015) reported that the inactivated fowl

cholera vaccine adjuvanted with Montanide ISA-70-VG induced high

protection rates in chickens against challenge with virulent serotypes 5:

A and D: 2 (95 and 90%, respectively), and induced earlier and higher

immune response than that induced by the mineral oil formulated

vaccine.


42

Akhtar et al. (2016) concluded that the formalin killed fowl

cholera vaccine prepared from the isolated bacteria induced protective

immune response and conferred protection against challenge infection

caused by the virulent strain of P. multocida.

Materials and methods

43

3. Materials and methods

3.1. Materials:

3.1.1. Strains used:

3.1.1.1. M. gallisepticum:

Field isolate of M. gallisepticum (Eis3-10) was kindly obtained

from Mycoplasma Department, Animal Health Research Institute,

Dokki, Giza, Egypt. The strain was propagated in PPLO broth and agar

and used for the preparation of vaccine, and antigen and for challenge

test.

3.1.1.2. P. multocida:

P. multocida types A and D were kindly obtained from Aerobic

Bacterial Vaccines Department, Veterinary Serum and Vaccine

Research Institute, Abbasia, Cairo. The strains were used in vaccine

and antigen preparation as well as challenge test.

3.1.2. Imported M. gallisepticum vaccine:

It is a commercial vaccine containing M. gallisepticum (S6 strain)

inactivated concentrated culture which incorporated in oil emulsion.


44

3.1.3. Laboratory animals and birds:

3.1.3.1. Chickens:

A total of 150 specific pathogen free (SPF) chickens, 4 weeks old

were obtained from Kom Osheem farm in Fayoum, Egypt and reared

under complete hygienic measures in special isolators. These birds

were examined to ensure that they are free from bacterial pathogens

and they had neither a history of mycoplasmosis (M. gallisepticum) nor

fowl cholera (P. multocida) infections. Also, these chickens have no

history of vaccination with these strains.

Another 20 chickens from the same source were used in safety

test of the prepared vaccines (M. gallisepticum, P. multocida and

combined M. gallisepticum and P. multocida vaccines).

3.1.3.2. Rabbits:

Eight native rabbits, their body weight ranged between 1-1.5 Kg

were used for the passage of local isolates of P. multocida types A and

D.

3.1.3.3. Mice:

A total of 235 Swiss white mice about 18-20 g body weights (25

mice were used for evaluation of safety of P. multocida vaccine and

210 mice were used for evaluation of potency of P. multocida vaccine

and combined vaccine of M. gallisepticum and P. multocida). These


45

mice were obtained from the Laboratory Animals Department,

Veterinary Serum and Vaccine Research Institute, Abbasia, Cairo.

3.1.4. Culture media:

3.1.4.1. Media used for M. gallisepticum:

1. Frey’s medium (Frey et al., 1968):

The pH was adjusted to 7.8 with 20 % NaOH and the medium

was sterilized by filtration.

2. PPLO medium (Adler et al., 1958):

The pH was adjusted to 7.8.

3.1.4.2. Media used for P. multocida (Atlas, 2004):

1. Tryptose phosphate broth (Oxoid).

2. Nutrient broth and agar.

3.1.4.3. Media used for sterility tests of the prepared vaccines

(Atlas, 2004):

1. Nutrient agar medium (Oxoid):

It was used for the detection of aerobic bacterial contamination.

2. Sabouraud’s dextrose agar (Difco):

It was used for detection of fungal contamination.


46

3. Thioglycollate broth (Oxoid):

It was used for the detection of anaerobic bacterial contamination.

3.1.5. Supplements:

3.1.5.1. Enrichment:

1. Horse serum:

It was obtained in sterile form from the General Egyptian

Organization for Biological Products and Vaccines, Agouza, Egypt and

stored frozen at -20°C till used.

2. Fresh yeast extract (Difco):

100 g of dehydrated yeast extract were dissolved in 1000 ml

distilled water to form 10% solution and sterilized by Seitz-filter then

distributed in 100 ml bottles and stored at -20°C until used.

3.1.5.2. Inhibitors:

1. Thallium acetate (B.D.H.) stock solution:

It was prepared as 2% stock solution by dissolving 2 g of thallium

acetate in 100 ml distilled water, sterilized by autoclaving (121°C for

15 minutes) and stored at 4°C till used.


47

2. Penicillin G solution:

The bottle contains 1000,000 I.U.; the content was reconstituted

in sterile broth to give final concentration of 200,000 I.U. /ml then

stored in a freezer.

3.1.6. Stains used:

3.1.6.1. Gram’s stain (OIE, 2012):

It was used for studying the morphology of P. multocida.

3.1.6.2. Giemsa stain (Cotter, 2015):

It was used for determination of H / L ratio in blood films.

3.1.7. Materials used for vaccines preparation:

3.1.7.1. Formalin 37% (Analar):

It was used as inactivator and was added at a final concentration

of 0.5% for inactivation of M. gallisepticum and P. multocida.

3.1.7.2. Thiomersal: (Elanco products Co., USA):

It was used as preservative and was added at a final concentration

of 0.01%.

3.1.7.3. Montanide ISA70:

It is a mineral oil based adjuvant from complex water in oil

emulsion and mixed with the corresponding culture according to the


48

manufacture’s instructions. It was obtained from SEPPIC, France. It

was used by the ratio 50/50, (culture/oil).

3.1.7.4. PBS:

The pH was adjusted to 7.2.

3.1.8. Materials used for measurement of NO concentration in the

supernatant of macrophage:

3.1.8.1. Zymosan:

5mg/ml of PBS, obtained from Sigma Chemical Company.

3.1.8.2. Griess reagent:

It used for colorimetric measurement of NO production. It

consists of:

Sulphonamide 1 g

Naphthyl ethylene di-amine di-hydrochloride 0.1 g

H3PO4 2.5 ml

Distilled water up to 100 ml

3.1.9. Materials used for IHA test:

Capsular antigens of P. multocida (types A and D) and

glutaraldehyde-fixed sheep erythrocytes (GA-SRBC) were used.


49

3.1.10. Materials used for HI test:

0.5% homologous RBCs in PBS, pH 7.2.

3.1.11. Materials used for ELISA test:

3.1.11.1. M. gallisepticum:

M. gallisepticum antibody test kit (Proflok®, Synbiotics® Corporation,

No. 96-6533) was used for determination of antibody titers of M.

gallisepticum in chickens serum samples.

Reagents required to perform the test:

1- M. gallisepticum antigen coated plate

2- M. gallisepticum positive control serum

3- Normal control serum

4- Goat anti-chicken IgG (H+L) peroxidase conjugate solution

5- Dilution buffer

6- ABTS- Hydrogen peroxide substrate solution

7- Stop solution

8- Washing solution


50

3.1.11.2. P. multocida:

P. multocida antibody test kit (Proflok®, Synbiotics® Corporation, No.

96-6527) was used for determination of antibody titers of P. multocida in

chickens serum samples.

Reagents required to perform the test:

1- P. multocida antigen coated plate

2- P. multocida positive control serum

3- Normal control serum

4- Goat anti-chicken IgG (H+L) peroxidase conjugate solution

5- Dilution buffer

6- ABTS- Hydrogen peroxide substrate solution

7- Stop solution, 5% Sodium dodecyl sulfate (SDS)

8- Washing solution

3.1.12. Equipments and apparatus:

1- Carbon dioxide incubator

2- Stereoscope

3- Shaking water bath

4- Magnetic stirrer


51

5- Shaker

6- Spectrophotometer

7- ELISA reader: Dynatech laboratories

3.2. Methods:

3.2.1. Preparation of inactivated oil emulsion M. gallisepticum

vaccine (Yoder, 1979):

The selected seed culture of M. gallisepticum (Eis3-10 strain) was

inoculated into a starter culture flask (250 ml of Frey’s medium, pH

7.8). Fresh medium was inoculated with 24 hours broth culture

equivalent to 10% of the volume of medium used. The mycoplasma

cells were harvested and washed using PBS (pH 7.2) by centrifugation

at 12,000 r.p.m. for 30 minutes after 48 hours incubation at 37°C in

carbon dioxide incubator. After repeated three washings, a final

suspension of antigen was prepared to contain 1% packed cell volume

(PCV) in PBS in final product.

The antigen batch was inactivated with 0.5% formalin with

frequent agitation during 24 hours incubation at 37°C. The inactivated

broth was then cultured for the detection of viable mycoplasma by

inoculated of 0.1 ml into 5 ml of mycoplasma medium, then incubated

at 37°C for 14 days in carbon dioxide incubator.


52

The Montanide ISA70 oil adjuvant was added to batch of

inactivated mycoplasma vaccine after centrifuged and resuspended in

PBS to contain 5% PCV (the final product contained 1% PCV after

added the adjuvant). Equal amounts of aforementioned culture and

Montanide ISA70 oil (SEPPIC, France) were mixed thoroughly in a

ratio of 50/50 using a magnetic stirrer at approximately 300 r.p.m. for

15 minutes (water-in-oil emulsion). Finally, the thiomersal was added

at a final concentration of 0.01%.

3.2.2. Preparation of inactivated oil emulsion P. multocida vaccine

(Mukkur et al,. 1982):

Each serotype of P. multocida (A and D) was isolated from heart

blood of inoculated rabbits then propagated separately in tryptose

phosphate broth at 37°C aerobically for 24 hours to obtain a dense

culture containing approximately 3.25 x 1010 CFU/ml of each strain.

The culture was inactivated by addition of 0.5% formalin and incubated

at 37°C for 24 hours. The inactivated culture was then cultured for the

detection of viable pasteurella by streaked onto nutrient agar medium,

then incubated at 37°C for 24 hours. Equal amounts of culture of each

strain were mixed together. Equal amounts of aforementioned culture

and Montanide ISA70 oil (SEPPIC, France) were mixed thoroughly in

a ratio of 50/50 using a magnetic stirrer at approximately 300 r.p.m. for

15 minutes (water-in-oil emulsions). Finally, the thiomersal was added

at a final concentration of 0.01%.


53

3.2.3. Preparation of combined inactivated oil emulsion vaccine of

M. gallisepticum and P. multocida:

Equal parts (V/V) of the inactivated broth of M. gallisepticum

(Eis3-10 strain) and P. multocida strains (serotypes A and D) were

mixed using a magnetic stirrer. Aforementioned suspension was

adjusted its concentration to contain 3x1010 CFU per dose (5% PCV) of

M. gallisepticum according to Yoder (1979) and 3.25 x 1010 CFU/ml

of each strain of P. multocida according to Mukkur et al. (1982).

Equal amounts of aforementioned culture and Montanide ISA70 oil

(SEPPIC, France) were mixed thoroughly in a ratio of 50/50 using a

magnetic stirrer at approximately 300 r.p.m. for 15 minutes (water-in-

oil emulsions). Finally, the thiomersal was added at a final

concentration of 0.01%.

3.2.4. Evaluation and quality control of the prepared vaccines:

The prepared vaccines were tested for purity, sterility and safety

tests according to OIE (2012).

3.2.4.1. Purity and sterility tests:

In accordance with OIE (2012), the vaccines were tested for

confirmation that the vaccines must be free from any bacterial and

fungal contamination. Vaccines were inoculated on nutrient agar and

thioglycolate broth and incubated at 37°C for 48-72 hours and on

Sabouraud’s dextrose agar at 25°C for 14 days. Also, inoculation was


54

made on mycoplasma broth which was followed by cultivation on

mycoplasma agar and incubated at 37°C for 14 days on 10% CO2. The

pure vaccines showed no growth on these media.

3.2.4.2. Safety tests:

1. SPF chickens inoculation test:

A total of 15 SPF chickens, 4 weeks old were inoculated

subcutaneously (S/C) with 0.5 ml per bird of the M. gallisepticum

vaccine; P. multocida vaccine and combined vaccine of M.

gallisepticum and P. multocida (5 chickens for each vaccine) and

5 SPF chickens were kept as a control. The inoculated chickens

were kept under observation for 14 days.

2. Mice inoculation test:

This test was carried out by inoculation of Swiss white mice with

0.2 ml of P. multocida bacterin. The inoculated mice were kept under

observation for 3-7 days.

3.2.5. Experimental design:

A total of 150, 4 weeks old SPF chickens were divided into five

groups, the 1st group was vaccinated with P. multocida vaccine (G1),

the 2nd group was vaccinated with M. gallisepticum vaccine (G2), the

3rd group was vaccinated with combined M. gallisepticum and P.

multocida vaccine (G3), the 4th group was vaccinated with imported M.

gallisepticum vaccine (G4) and the 5th group was kept unvaccinated as


55

a control group (G5). The vaccinated chickens were received vaccines

in a dose of 0.5 ml in 2 doses with 1 month interval. Blood samples

were collected at 3rd, 7th and 15th days after first, second vaccination

and after challenge for the determination of the cellular immunity by

H/L ratio and estimation of NO concentration in the supernatant of

macrophage. Also, serum samples were collected every 2 weeks till 25

weeks of age for the determination of the humoral immune response of

the vaccinated chickens by IHA, HI and ELISA techniques. The

potency of the vaccines was evaluated by the challenge (at 11 weeks of

age) and passive mouse protection tests against the challenge with the

virulent strain of M. gallisepticum (Eis3-10 strain) and P. multocida

(serotypes A and D).


56

Table (1): Experimental design:

Groups 1 2 3 4 5

Types of

vaccines P.

multocida M. gallisepticum

Combined

vaccine

Imported M.

gallisepticum

vaccine

Control

No. of

chickens 30 15 45 15 45

Dose/

Route 0.5 ml S/C upper dorsal part of the neck

1st dose At 4 weeks of age

2nd dose At 8 weeks of age

Challenge At 11 weeks of age

Blood

samples

Used for H/L ratio and measurement of NO in the

supernatant of macrophage at 3rd, 7th and 15th days after first,

second vaccination and after challenge

Serum

samples

Used for IHA, HI, ELISA and Passive mouse protection

tests every 2 weeks till 25 weeks of age


57

3.2.6. Evaluation of the cellular immunity:

3.2.6.1. Determination of H/L ratio (Cotter, 2015):

Blood samples (1ml) were collected from wing veins into

ethylene diamine tetra-acetic acid (EDTA) tubes as an anticoagulant,

Monolayer films made by pushing approximately 3μL of blood across a

standard microscope slide were dried immediately by a hot air stream.

Slides were then immersed in 95% ethanol and post fixed for 10 to 15

min. Films were stained by Giemsa stain. Blood films were examined

to obtain counts of lymphocytes and granulocytes per 100 leukocytes.

Obtained cell counts were used for calculation of the relative

proportion of heterophils to lymphocytes (H/L ratio).

3.2.6.2. Measurement of NO concentration in the

supernatant of macrophage:

Monocytes were isolated from pooled buffy coats of vaccinated

chickens, incubated at 37°C for 2 hours then the non adhered cells were

discarded. Differentiation of monocytes into macrophages was carried

out by culture for 3-5 days with 10% fetal calf serum. Zymosan

(5mg/ml of PBS) from Sigma Chemical Company was washed with

sterilized PBS, then coating with complement through process of

opsonization by incubation with species serum for 1hour at 37°C then

centrifuged and resuspended in sterilized PBS. For the assay of

phagocytosis, cells were incubated with zymosan particles for 1 h and


58

overnight, at each time the supernatant over macrophage was collected

and nitric acid concentration was measured in it (Municio et al., 2013).

The measurement of NO in the supernatant of macrophage was

assessed according to the assay described by Rajaraman et al. (1998);

the test depends on that nitrite is a stable oxidation product of NO,

which correlates with the amount of NO present in the supernatant of

macrophage. The amount of stable nitrite was determined by mixing

the supernatant of macrophage with colorless Griess reagent which

results in formation of purple complex. The degree of the color

development was measured spectrophotometrically using ELISA reader

at 570 nm.

3.2.7. Evaluation of the humoral immunity:

3.2.7.1. IHA test:

It was carried out according to Sawada et al. (1982) for

measuring antibody titers against P. multocida types A and D in

vaccinated chickens using GA-SRBC and capsular antigens of P.

multocida types A and D. The vaccine is concluded effective if it

induce seroconversion in sera of vaccinated chickens. Antibody titers

against P. multocida in the vaccinated chickens were estimated as

follows:


59

1. Preparation of GA-SRBC:

A 100 ml suspension of fresh sheep erythrocytes (SRBC) in

Alsever solution was washed by centrifugation (650 x g for 20 min) six

times with five or six volumes of saline (0.85% NaCl). After the last

wash, the packed cells were suspended in PBS to yield a 10%

suspension (vol/vol) and chilled to 4°C in an ice bath. A 25% solution

of glutaraldehyde (Eastman Kodak Co.) was diluted to 1% (vol/vol)

with PBS and chilled to 4°C. A 10% suspension of washed SRBC was

mixed with an equal volume of the 1% solution of glutaraldehyde, and

the mixture was incubated at 4°C for 30 min with gentle stirring. The

mixture was then centrifuged at 650 x g for 10 min at 25°C. The

pelleted, fixed cells were suspended in PBS, washed three times with

PBS by centrifugation, and suspended in PBS containing 0.1% sodium

azide to yield a 10% suspension. The GA-SRBC was stored at 4°C.

2. Preparation of capsular antigens of P. multocida types A and

D:

P. multocida serotypes A and D were prepared by heating the

24 hours of tryptose phosphate broth culture at 56°C for 30 minutes.

The bacterial cells were separated by centrifugation at 1500 rpm for 10

minutes; the supernatant fluid contained the antigen. The thiomersal

was added at a final concentration of 0.01% to prevent bacterial

contamination. The antigen stored at 4°C.


60

3. Sensitization of the GA-SRBC with pasteurella antigens:

0.2 ml of GA-SRBC was added to 3 ml bacterial extract which

were then thoroughly mixed and incubated at 37°C for 2 hours. The

sensitized cells were separated by centrifugation at 1500 rpm for 15

minutes and washed once with 10 ml of saline after which sufficient

saline was added to give 1% final concentration.

4. IHA test:

The IHA test was performed with a microtiter system (Dynatech

Laboratories, Inc.). Serial two fold dilutions of antiserum were made in

BSA-PBS (bovine serum albumin-PBS), and 0.025 ml of the sensitized

SRBC was added to 0.025 ml of the antiserum dilution in U-bottom

plates. The plates were shaken and allowed to stand for 1 to 2 h at 25°C

before SRBC settling patterns were read. The IHA titer was expressed

as the reciprocal of the highest dilution of serum showing a definite

positive pattern (flat sediment), as compared with the pattern of the

negative control (smooth dot in the center of the well). Controls

consisted of unsensitized SRBC plus test serum and sensitized SRBC

plus diluent.

3.2.7.2. HI test:

It was carried out according to Senterifit (1983) for measuring

antibody titers against M. gallisepticum (Eis3-10 strain) in the

vaccinated chickens.


61

• Antigen HA titration:

1. Standard HA test was performed on mycoplasma antigen to

determine titer of antigen.

a) 50 µl of PBS was dispensed into each well of 3 rows of a 96

well microtiter plate.

b) 50 µl of stock antigen was dispensed into the first well of 2

rows.

c) A serial two fold dilutions (50 µl) was performed used a 12

channel pipettor. The dilution series would be from 1:2-

1:4096.

d) 50 µl of 0.5% homologous RBCs was added to each well of

all three rows. The row with no antigen served as a RBCs

control.

2. The plate was incubated at room temperature (approximately 30-

60 minutes) until the control RBCs gave tight buttons. The HA

titer was read as the last well to give a complete lawn

(haemagglutination).

3. The stock antigen was diluted to 4 HA units for the HI test. The

HI assay using the (dilution in saline technique) used half the

volume of antigen 25 µl rather than 50 µl. The dilution required


62

to give 4 HA units in 25µl was calculated by dividing the stock

antigen HA titer by 8.

• HI assay (Dilution in saline technique):

1. The one column (A-H) of a 96 well, U-bottom microtiter plate

was labeled for each sample, each positive and negative control

sera, antigen backtitration and RBCs control.

2. 40 µl of PBS was added to wells of the top row (row A) of the

plate.

3. 25 µl of PBS was added to all remaining wells of the plate.

4. 10 µl of each test sera was added to well A of each column

(made a 1:5 sera dilution).

5. 25 µl was serially diluted from well A through H used a 12

channel pipettor. The final 25 µl was discarded. Row A =

1:5.....row H = 1: 640.

6. With an Oxford doser, 25 µl of 4 HA unit antigen was added to

wells B through H. Wells A served as the serum control.

7. An antigen backtitration (antigen control) was prepared by

added 25 µl of PBS to each well of one column. 25 µl of diluted

antigen was added to well A and serially diluted 25 µl from well

A-D. This prepared 1:2, 1:4, 1:8 and1:16 dilutions (it is


63

recommended that the antigen control backtitration be

performed before the diluted antigen is used in the assay;

dilution problems can be detected and corrected before the

inappropriately diluted antigen is used and an invalid assay is

performed).

8. A column of wells blank was leaved for a RBCs control (PBS +

RBCs, no antigen).

9. The plate was agitated gently and incubated for 30 minutes at

room temperature.

10. 50 µl of 0.5% RBCs was added to all wells.

Note: Do not agitate after RBCs have been added (agitation may

result in false positive reactions by causing the RBCs to roll into the

wells, resulting in false buttons).

11. The plate was covered with sealing tape and incubated at room

temperature (approximately 60 minutes) until control RBCs

gave a tight button.

12. The reaction was read on mirrored plate reader.

13. The titer was reported as the reciprocal of the last dilution to

give a tight button of RBCs. The final dilution scheme included

the antigen in the dilution calculation as follows: B=1:10,

C=1:20, D=1:40, E=1:80, F=1:160, G=1:320, H=1:640.


64

3.2.7.3. ELISA test:

3.2.7.3.1. M. gallisepticum:

a) An M. gallisepticum antigen coated test plate was removed

from the protective bag and labeled according to dilution plate

identification.

b) 50µl dilution buffer was added to all wells on the test plate.

c) 50 µl diluted M. gallisepticum positive control serum was

added to wells A1, A3 and H11. Pipette tip was discarded.

d) 50 µl / well of each of the diluted serum samples and normal

control serum samples was transferred from the dilution plate to

the corresponding wells of the M. gallisepticum coated test plate

used an 8 or 12 channel pipette. The pipette tips were discarded

after each row of sample was transferred. Transfer of samples to

the ELISA plate should be done as quickly as possible.

e) The plate was incubated for 30 minutes at room temperature.

f) The liquid from each well was taped out into an appropriate

vessel contained bleach or other decontamination agent.

g) Each well was filled with 300 µl wash solution used an 8 or

12 channel pipette and was allowed to soak in wells for 3

minutes; then the contents were discarded into an appropriate


65

waste container. The inverted plate was taped to ensure that all

residual liquid was removed. The wash procedure was repeated

2 more times.

h) 100 µl diluted anti-chicken IgG peroxidase conjugate was

dispensed into each assay well used an 8 or 12 channel pipette.

The pipette tips were discarded.

i) The plate was incubated for 30 minutes at room temperature.

j) The plate was washed as in steps f and g above.

k) 100µl substrate solution was dispensed into each test well

used an 8 or 12 channel pipette. The pipette tips were discarded.

l) The plate was incubated 15 minutes at room temperature.

m) 100 µl diluted stop solution was added to each test well used

an 8 or 12 channel pipette.

n) The bubbles were allowed to dissipate before reading plate.

Manual processing of data:

a) The plate was read used an ELISA plate reader set at 405-

410 nm.

b) The average positive control serum absorbance (optical

density {O.D.}) was calculated used the absorbance values

of wells A1, A3 and H11. The average normal control serum


66

(NCS) absorbance was calculated used values obtained from

wells A2, H10 and H12. The both averages were recorded.

c) The average NCS absorbance was subtracted from the

average positive absorbance. The difference was the

corrected positive control.

d) SP = (Sample absorbance) – (Average NCS absorbance)

Corrected positive control absorbance

e) An M. gallisepticum ELISA titer could be calculated by the

following suggested equation:

Log 10 titer = (1.464 x log10 SP) + 3.197

Titer = antilog of log10 titer

3.2.7.3.2. P. multocida:

a) A P. multocida antigen test plate was removed from the

protective bag and labeled according to serum dilution plate

identification.

b) 50µl dilution buffer was added to all wells on the test plate.

c) 50 µl diluted P. multocida positive control serum was added

to wells A1, A3 and H11. Pipette tip was discarded.


67

d) 50 µl / well of each of the diluted serum samples and normal

control serum samples was transferred from the serum dilution

plate to the corresponding wells of the P. multocida coated test

plate used an 8 or 12 channel pipette (yielded a 1: 100 dilution).

The pipette tips were discarded after each row of sample was

transferred. Transfer of samples to the ELISA plate should be

done as quickly as possible.

e) The plate was incubated for 30 minutes at room temperature.

f) The liquid from each well was taped out into an appropriate

vessel contained bleach or other decontamination agent.

g) Each well was filled with 300 µl wash solution used an 8 or

12 channel pipette and was allowed to soak in wells for 3

minutes; then the contents were discarded into an appropriate

waste container. The inverted plate was taped to ensure that all

residual liquid was removed. The wash procedure was repeated

2 more times.

h) 100 µl diluted anti-chicken IgG peroxidase conjugate was

dispensed into each assay well used an 8 or 12 channel pipette.

The pipette tips were discarded.

i) The plate was incubated for 30 minutes at room temperature.

j) The plate was washed as in steps f and g above.


68

k) 100µl substrate solution was dispensed into each test well

used an 8 or 12 channel pipette. The pipette tips were discarded.

l) The plate was incubated 15 minutes at room temperature.

m) 100 µl diluted stop solution was added to each test well used

an 8 or 12 channel pipette.

n) The bubbles were allowed to dissipate before reading plate.

Manual processing of data:

a) The plate was read used an ELISA plate reader set at 405-

410 nm.

b) The average positive control serum absorbance (O.D.) was

calculated used the absorbance values of wells A1, A3 and

H11. The average NCS absorbance was calculated used

values obtained from wells A2, H10 and H12. The both

averages were recorded.

c) The average NCS absorbance was subtracted from the

average positive absorbance. The difference was the

corrected positive control.

d) SP = (Sample absorbance) – (Average NCS absorbance)

Corrected positive control absorbance


69

e) A P. multocida ELISA titer could be calculated by the

following suggested equation:

Log 10 titer = (1.464 x log10 SP) + 3.197

Titer = antilog of log10 titer

3.2.8. Evaluation of the potency of the vaccines:

3.2.8.1. Passive mouse protection test (Tabatabaei et al., 2007):

The test was used to evaluate the protection rate of the vaccinated

serum against challenge with the virulent strains of P. multocida

(serotypes A and D) allover the intervals of the blood collection. 0.2 ml

of the serum collected (after first and second vaccination and

throughout 8 weeks after challenge) from groups of chickens

vaccinated with P. multocida vaccine and combined M. gallisepticum

and P. multocida vaccine inoculated S/C in 120 mice and 60 mice were

kept as a control group. After 24 hours, the vaccinated mice with P.

multocida vaccine and combined vaccine were challenged with virulent

strains of P. multocida (serotypes A and D). The cell suspensions of P.

multocida (serotypes A and D) were prepared in a concentration

contained 109 CFU/ml undergoing serial dilution, 0.1 ml of the virulent

P. multocida cell suspension containing 100 LD50 of each strain

separately was inoculated S/C in each mice. These mice were

undergoing observation for 7 days and recorded the results.


70

3.2.8.2. Challenge test:

3.2.8.2.1. M. gallisepticum:

Every chicken from groups of M. gallisepticum vaccine,

combined M. gallisepticum and P. multocida vaccine and imported M.

gallisepticum vaccine was challenged by intranasal inoculation with 0.1

ml of a 24 hours broth culture of the virulent M. gallisepticum (Eis3-10

strain) containing 109 CFU /ml at 11 weeks of age (3 weeks post

second vaccination). These chickens were undergoing observation for 7

days and recorded the results (Whithear, 1996).

3.2.8.2.2. P. multocida:

Every chicken from groups of P. multocida vaccine and combined

M. gallisepticum and P. multocida vaccine was challenged by S/C

inoculation with 0.1 ml of cell suspensions of the virulent P. multocida

(serotyes A and D). The cell suspensions were prepared in a

concentration contained 109 CFU /ml undergoing serial dilution, 0.1 ml

of the virulent P. multocida cell suspension containing 100 LD50 of

each strain separately was inoculated S/C in each chicken at 11 weeks

of age (3 weeks post second vaccination). These chickens were

undergoing observation for 7 days and recorded the results (OIE,

2012).

Results

71

4. Results

4.1. Results of sterility, purity and safety tests of the prepared

vaccines:

The prepared vaccines were free from any bacterial and fungal

contamination and there was no growth on mycoplasma broth and agar.

The prepared vaccines were proved to be safe after inoculation of

chickens and Swiss white mice without any respiratory signs or

mortality.

4.2. Evaluation of the cellular immune response of chickens

that vaccinated with different vaccines:

4.2.1. Determination of H/L ratio:

The data illustrated in Table (2) revealed that the H/L ratio at 7th

day post 1st vaccination for G1, G2, G3 and G4 were 0.4, 0.6, 0.2 and

0.3, respectively in comparison with 1.0 for G5. While, at 7th day post

2nd vaccination the H/L ratio for G1, G2, G3 and G4 were 0.1, 0.4, 0.1

and 0.2, respectively in comparison with 0.9 for G5. The H/L ratio at

7th day post challenge for G1, G2, G3 and G4 were 0.1, 0.3, 0.1 and 0.1,

respectively in comparison with 1.0 for G5.

ANOVA test was used to show difference of H/L ratio between

vaccinated groups and control group. This test revealed significant

Results

72

difference at p≤0.05 with Fcal= 14.93316 and Ftab= 2.578739 (Table

3).

ANOVA test was used to show difference of H/L ratio between

G2, G3 and G4. This test revealed no significant difference at p≤0.05

with Fcal= 2.553292 and Ftab= 3.354131 (Table 4).

Paired t test was used to show difference of H/L ratio between G1

and G3. This test revealed significant difference at p≤0.05 with tcal=

3.354102 and ttab= 2.262157 (Table 5).

Results

73

Table (2): Evaluation of H/L ratio post vaccination with different vaccines in chickens:

Interval times of blood collection

Types of vaccines G1 G2 G3 G4 G5

Prevaccination 1.3 1.4 1.1 1.2 1.5 1st vaccination

At 3rd day 0.7 0.8 0.5 0.6 1.4 At 7th day 0.4 0.6 0.2 0.3 1.0 At 15th day 0.5 0.7 0.3 0.4 1.3

Booster vaccination At 3rd day 0.3 0.5 0.2 0.3 1.2 At 7th day 0.1 0.4 0.1 0.2 0.9 At 15th day 0.2 0.5 0.2 0.3 1.3

Challenge At 3rd day 0.2 0.4 0.2 0.2 1.2 At 7th day 0.1 0.3 0.1 0.1 1.0 At 15th day 0.4 0.6 0.3 0.4 1.5

G1: P. multocida vaccine

G3: Combined M. gallisepticum and P. multocida vaccine

G2: M. gallisepticum vaccine

G4: Imported M. gallisepticum vaccine

G5: Control

1st vaccination: at 4 weeks of age

Booster vaccination: at 8 weeks of age

Challenge: at 11 weeks of age

Results

74

Table (3): Statistical analysis of H/L ratio between vaccinated groups and control group:

Groups Count Sum Average Variance G1 10 4.2 0.42 0.130667 G2 10 6.2 0.62 0.097333 G3 10 3.2 0.32 0.088444 G4 10 4 0.4 0.097778 G5 10 12.3 1.23 0.044556

G1: P. multocida vaccine G2: M. gallisepticum vaccine


G4: Imported M. gallisepticum vaccine G5: Control

Source of Variation SS Df MS F cal P-value F tab

Between Groups 5.4808 4 1.3702 14.93316 7.7E-08 2.578739

Within Groups 4.129 45 0.091756 Total 9.6098 49

SS: Sum of squares MS: Mean of squares

Df: Degree of freedom

Ftab: F tabulated Fcal: F calculated

Results

75

Table (4): Statistical analysis of H/L ratio between groups of mycoplasma:

Groups Count Sum Average Variance G2 10 6.2 0.62 0.097333 G3 10 3.2 0.32 0.088444 G4 10 4 0.4 0.097778

G2: M. gallisepticum vaccine G4: Imported M. gallisepticum vaccine


Source of Variation SS Df MS F cal

P-value F tab

Between Groups 0.482666667 2 0.241333 2.553292 0.09647 3.354131 Within Groups 2.552 27 0.094519

Total 3.034666667 29 SS: Sum of squares MS: Mean of squares Df: Degree of freedom


Table (5): Statistical analysis of H/L ratio between combined vaccine and P. multocida vaccine:

G1 G3 Mean 0.42 0.32 Variance 0.130667 0.088444 Observations 10 10 Pearson Correlation 0.977757

Hypothesized Mean Difference 0 Df 9 t Stat (t calculated) 3.354102 P(T<=t) one-tail 0.004234 t Critical one-tail 1.833113 P(T<=t) two-tail 0.008468 t Critical two-tail (t tabulated) 2.262157

G1: P. multocida vaccine G3: Combined M. gallisepticum and P. multocida vaccine

Results

76

4.2.2. Estimation of NO concentration in the supernatant of

macrophage:

The data illustrated in Table (6) revealed that the NO

concentration in the supernatant of macrophage at 7th day post 2nd

vaccination for G1, G2, G3 and G4 were 67.08, 45.2, 53.9 and 46.3,

respectively in comparison with 16.3 for G5. While, at 7th day post

challenge the NO concentration for G1, G2, G3 and G4 were 80.8,

78.3, 102.6 and 94.1, respectively in comparison with 15.2 for G5.

ANOVA test was used to show difference of NO concentration

between vaccinated groups and control group. This test revealed

significant difference at p≤0.05 with Fcal= 3.359872 and Ftab=

2.689628 (Table 7).

ANOVA test was used to show difference of NO concentration

between G2, G3 and G4. This test revealed no significant difference at

p≤0.05 with Fcal= 0.721788 and Ftab= 3.554557 (Table 8).

Paired t test was used to show difference of NO concentration

between G1 and G3. This test revealed no significant difference at

p≤0.05 with tcal= 1.10747 and ttab= 2.446912 (Table 9).

Results

77

Table (6): Estimation of NO concentration in the supernatant of macrophage post vaccination with different vaccines in chickens:






Interval times of blood Collection

Types of vaccines G1 G2 G3 G4 G5

Prevaccination 9.83 10.9 15.4 15.1 8.12 Booster vaccination

At 3rd day 34.7 19.7 25.2 24.1 11.06 At 7th day 67.08 45.2 53.9 46.3 16.3 At 15th day 29.6 29.4 47.4 43.9 14.0

Challenge At 3rd day 49.4 23.3 47.8 41.5 11.7 At 7th day 80.8 78.3 102.6 94.1 15.2 At 15th day 51.7 44.7 74.6 62.7 10.8

Results

78

Table (7): Statistical analysis of NO concentration between vaccinated groups and control group:

Groups Count Sum Average Variance G1 7 323.11 46.15857 567.3665 G 2 7 251.5 35.92857 508.3557 G3 7 366.9 52.41429 861.6881 G 4 7 327.7 46.81429 674.5048 G 5 7 87.18 12.45429 8.125562




Source of Variation SS Df MS F cal P-value F tab Between Groups 7042.401154 4 1760.6 3.359872 0.021872 2.689628 Within Groups 15720.24409 30 524.0081

Total 22762.64524 34

SS: Sum of squares MS: Mean of squares Df: Degree of freedom


Results

79

Table (8): Statistical analysis of NO concentration between groups of mycoplasma:

Groups Count Sum Average Variance G2 7 251.5 35.92857 508.3557 G3 7 366.9 52.41429 861.6881 G4 7 327.7 46.81429 674.5048



Source of Variation SS Df MS F cal P-value F tab

Between Groups 983.8209524 2 491.9105 0.721788 0.499421 3.554557

Within Groups 12267.29143 18 681.5162 Total 13251.11238 20



Table (9): Statistical analysis of NO concentration between combined vaccine and P. multocida vaccine:


Hypothesized Mean Difference 0 Df 6 t Stat (t calculated) -1.10747 P(T<=t) one-tail 0.15525 t Critical one-tail 1.94318 P(T<=t) two-tail 0.310501 t Critical two-tail (t tabulated 2.446912 G1: P. multocida vaccine


Results

80

4.3. Evaluation of the humoral immune response of chickens

that vaccinated with different vaccines:

4.3.1. IHA test:

From Table (10), it can be noticed that the antibody titers against

P. multocida type “A” 2 weeks post 1st vaccination for G1 and G3 were

64 and 128, respectively in comparison with 2.0 for G5. While, 2

weeks post 2nd vaccination the antibody titers were 256 for both groups

(G1 and G3) in comparison with 2.0 for G5. The antibody titers 6

weeks post challenge for G1 and G3 were 512 and 1024, respectively

in comparison with 2.0 for G5.

From Table (11), it can be noticed that the antibody titers against

P. multocida type “D” 2 weeks post 1st vaccination for G1 and G3 were

32 and 64, respectively in comparison with 2.0 for G5. While, 2 weeks

post 2nd vaccination the antibody titers for G1 and G3 were 64 and 128,

respectively in comparison with 2.0 for G5. The antibody titers 6 weeks

post challenge were 512 for both groups (G1 and G3) in comparison

with 2.0 for G5.

ANOVA test was used to show difference of the antibody titers

against P. multocida type “A” between vaccinated groups and control

group. This test revealed significant difference at p≤0.05 with Fcal=

7.964122 and Ftab= 3.402826 (Table 12).

Results

81

Paired t test was used to show difference of antibody titers against

P. multocida type “A” between G1 and G3. This test revealed

significant difference at p≤0.05 with tcal= 2.34520788 and ttab=

2.306004133 (Table 13).


against P. multocida type “D” between vaccinated groups and control

group. This test revealed significant difference at p≤0.05 with Fcal=

4.109341 and Ftab= 3.354131 (Table 14).


P. multocida type “D” between G1 and G3. This test revealed no

significant difference at p≤0.05 with tcal= 1.8 and ttab= 2.262157158

(Table 15).

Results

82

Table (10): Level of antibody titers against P. multocida type “A” in chickens vaccinated with combined vaccine of M. gallisepticum and P. multocida and P. multocida vaccine by IHA:

Interval times of serum collection

Types of vaccines

G1 G3 G5

Prevaccination 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







G1: P. multocida vaccine G5: Control



Booster vaccination: at 8 weeks of age Challenge: at 11 weeks of age

Results

83

Table (11): Level of antibody titers aga inst P. multocida type “D” in chickens vaccinated with combined vaccine of M. gallisepticum and P. multocida and P. multocida vaccine by IHA:


Types of vaccines

G1 G3 G5


1st vaccination


Booster vaccination


Challenge












Results

84

Table (12): Statistical analysis of IHA antibody titers against P. multocida type “A” between vaccinated groups and control group:

Groups Count Sum Average Variance G1 9 1922 213.5556 20373.78 G3 9 3330 370 95844 G5 9 10 1.111111 2.111111




Total 1546818.667 26



Table (13): Statistical analysis of IHA antibody titers against P. multocida type “A” between combined vaccine and P. multocida vaccine:

G1 G3 Mean 213.5555556 370 Variance 20373.77778 95844 Observations 9 9 Pearson Correlation 0.86183536

Hypothesized Mean Difference 0 Df 8 t Stat (t calculated) -2.34520788 P(T<=t) one-tail 0.023515886 t Critical one-tail 1.859548033 P(T<=t) two-tail 0.047031773 t Critical two-tail (t tabulated) 2.306004133 G1: P. multocida vaccine


Results

85

Table (14): Statistical analysis of IHA antibody titers against P. multocida type “D” between vaccinated groups and control group:

Groups Count Sum Average Variance G1 10 1282 128.2 23381.73 G3 10 1762 176.2 36160.4 G5 10 14 1.4 1.822222




Total 699019.8667 29 SS: Sum of squares MS: Mean of squares Df: Degree of freedom


Table (15): Statistical analysis of IHA antibody titers against P. multocida type “D” between combined vaccine and P. multocida vaccine:


Hypothesized Mean Difference 0 Df 9 t Stat (t calculated) -1.8 P(T<=t) one-tail 0.052695335 t Critical one-tail 1.833112923 P(T<=t) two-tail 0.105390669 t Critical two-tail (t tabulated) 2.262157158 G1: P. multocida vaccine


Results

86

4.3.2. HI test:

The data illustrated in Table (16) revealed that the antibody titers

against M. gallisepticum 2 weeks post 1st vaccination for G2, G3 and

G4 were 32, 64 and 64, respectively in comparison with 2.0 for G5.

While, 2 weeks post 2nd vaccination the antibody titers for G2, G3 and

G4 were 64, 128 and128, respectively in comparison with 2.0 for G5.

The antibody titers 6 weeks post challenge for G2, G3 and G4 were

128, 512 and 256, respectively in comparison with 2.0 for G5.


against M. gallisepticum between vaccinated groups and control group.

This test revealed significant difference at p≤0.05 with Fcal= 6.415084

and Ftab= 2.866266 (Table 17).


against M. gallisepticum between G2, G3 and G4. This test revealed no


3.354131 (Table 18).

Results

87

Table (16): Level of antibody titers against M. gallisepticum in chickens vaccinated with different M. gallisepticum vaccines by HI:


Types of vaccines

G2 G3 G4 G5

Prevaccination 2 2 2 0

1st vaccination

2 weeks post 1st vaccination 32 64 64 2

Booster vaccination

2 weeks post 2nd vaccination 64 128 128 2

Challenge

2 weeks post challenge 128 256 128 4









G5: Control 1st vaccination: at 4 weeks of age


Results

88

Table (17): Statistical analysis of HI antibody titers against M. gallisepticum between vaccinated groups and control group:

Groups Count Sum Average Variance G2 10 658 65.8 2272.4 G3 10 1986 198.6 34000.04 G4 10 1186 118.6 7240.933 G5 10 12 1.2 1.955556

G2: M. gallisepticum vaccine



G5: Control

Source of Variation SS Df MS F cal P-value F tab Between Groups 209365.9 3 69788.63 6.415084 0.001356 2.866266 Within Groups 391638 36 10878.83

Total 601003.9 39




Results

89

Table (18): Statistical analysis of HI antibody titers against M. gallisepticum between groups of mycoplasma:

Groups Count Sum Average Variance G2 10 658 65.8 2272.4 G3 10 1986 198.6 34000.04 G4 10 1186 118.6 7240.933




Total 481032.6667 29




Results

90

4.3.3. ELISA test:


against M. gallisepticum 2 weeks post 1st vaccination for G2, G3 and

G4 were 157, 360 and 241, respectively in comparison with 0 for G5.

While, 2 weeks post 2nd vaccination the antibody titers for G2, G3 and

G4 were 729, 996 and 965, respectively in comparison with 0 for G5.

The antibody titers 6 weeks post challenge for G2, G3 and G4 were

2541, 4958 and 3927, respectively in comparison with 0 for G5.


against P. multocida 2 weeks post 1st vaccination for G1 and G3 were

206 and 227, respectively in comparison with 0 for G5. While, 2 weeks

post 2nd vaccination the antibody titers for G1 and G3 were 2391 and

2487, respectively in comparison with 0 for G5. The antibody titers 6

weeks post challenge for G1 and G3 were 3164 and 4327, respectively

in comparison with 0 for G5.

The humoral immune response of the vaccinated chickens firstly

detected 2 weeks post 1st vaccination and continued till 14 weeks post

challenge. The antibody titers reached peak levels at 6 weeks post

challenge.


against M. gallisepticum between vaccinated groups and control group.

Results

91




against M. gallisepticum between G2, G3 and G4. This test revealed no


3.354131 (Table 22).


against P. multocida between vaccinated groups and control group.




P. multocida between G1 and G3. This test revealed no significant

difference at p≤0.05 with tcal= 2.057045087 and ttab= 2.262157158

(Table 24).

Results

92

Table (19): Level of antibody titers against M. gallisepticum in chickens vaccinated with different M. gallisepticum vaccines by ELISA:


Types of vaccines

G2 G3 G4 G5

Prevaccination 0 0 0 0

1st vaccination

2 weeks post 1st vaccination 157 360 241 0

Booster vaccination

2 weeks post 2nd vaccination 729 996 965 0

Challenge










G5: Control 1st vaccination: at 4 weeks of age


Results

93

Table (20): Level of antibody titers against P. multocida in chickens vaccinated with combined vaccine of M. gallisepticum and P. multocida and P. multocida vaccine by ELISA:


Types of vaccines

G1 G3 G5


1st vaccination


Booster vaccination


Challenge










1st vaccination: at 4 weeks of age Booster vaccination: at 8 weeks of age


Results

94

Table (21): Statistical analysis of ELISA antibody titers against M. gallisepticum between vaccinated groups and control group:

Groups Count Sum Average Variance G2 10 12372 1237.2 811260.4 G3 10 21530 2153 2776937 G4 10 18241 1824.1 1946091 G5 10 0 0 0

G2: M. gallisepticum vaccine G5: Control



Source of Variation SS Df MS F cal P-value F tab Between Groups 26961825.28 3 8987275 6.495703 0.001258 2.866266 Within Groups 49808602.5 36 1383572

Total 76770427.78 39




Results

95

Table (22): Statistical analysis of ELISA antibody titers against M. gallisepticum between groups of mycoplasma:

Groups Count Sum Average Variance G2 10 12372 1237.2 811260.4 G3 10 21530 2153 2776937 G4 10 18241 1824.1 1946091



Source of Variation SS Df MS F cal P-value F tab Between Groups 4304388.2 2 2152194 1.166651 0.326616 3.354131 Within Groups 49808602.5 27 1844763

Total 54112990.7 29




Results

96

Table (23): Statistical analysis of ELISA antibody titers against P. multocida between vaccinated groups and control group:

Groups Count Sum Average Variance G1 10 14832 1483.2 1166729 G3 10 17084 1708.4 1774684 G5 10 0 0 0



Source of Variation SS Df MS F cal

P-value F tab

Between Groups 17230759.47 2 8615380 8.786982 0.00115 3.354131 Within Groups 26472714 27 980470.9

Total 43703473.47 29



Table (24): Statistical analysis of ELISA antibody titers against P. multocida between combined vaccine and P. multocida vaccine:


Hypothesized Mean Difference 0 Df 9 t Stat (t calculated) -.057045087 P(T<=t) one-tail 0.034906642 t Critical one-tail 1.833112923 P(T<=t) two-tail 0.069813285 t Critical two-tail (t tabulated) 2.262157158 G1: P. multocida vaccine


Results

97

4.4. Evaluation of the potency of the vaccines:

4.4.1. Passive mouse protection test:

The data illustrated in Table (25) revealed that the protection

percentage (P%) against the challenge with virulent strain of P.

multocida type “A” 2 weeks post 1st vaccination for G1 and G3 were

80% and 100%, respectively in comparison with 0% for G5. While, 2

weeks post 2nd vaccination and 8 weeks post challenge the P% were

100% for both groups (G1 and G3) in comparison with 0% for G5.

The data illustrated in Table (26) revealed that the P% against

the challenge with virulent strain of P. multocida type “D” 2 weeks

post 1st vaccination were 100% for both groups (G1 and G3) in

comparison with 0% for G5. Also, 2 weeks post 2nd vaccination and 8

weeks post challenge the P% were 100% for both groups in comparison

with 0% for G5.

Results

98

Table (25): Passive mouse protection test against the challenge with P. multocida type ‘‘A’’ in chickens vaccinated with combined vaccine of M. gallisepticum and P. multocida and P. multocida vaccine:


Total no. of

mice

Types of vaccines

G1 G3 G5

D S P% D S P% D S P%

Prevaccination 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




P % = No. of survived mice Ⅹ 100

Total No. of mice

S= Survived mice D=Dead mice



1st vaccination: at 4 weeks of age Challenge: at 11 weeks of age


Results

99

Table (26): Passive mouse protection test against the challenge with P. multocida type ‘‘D’’ in chickens vaccinated with combined vaccine of M. gallisepticum and P. multocida and P. multocida vaccine:


Total no. of

mice

Types of vaccines

G1 G3 G5

D S P% D S P% D S P%

Prevaccination 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





P % = No. of survived mice Ⅹ 100

Total No. of mice

S= Survived mice D=Dead mice



1st vaccination: at 4 weeks of age Challenge: at 11 weeks of age


Results

100

4.4.2. Challenge test:

The data illustrated in Table (27) showed that the P% against the

challenge with virulent strain of M. gallisepticum was 93% for G3 and

87% for G4 and the lowest P% was in G2 (80%) in comparison with

0% for G5.

The data illustrated in Table (28) revealed that the P% against the

challenge with virulent strain of P. multocida type ‘‘A’’ was 100% for

G3 and 93% for G1 in comparison with 0% for G5.

The data illustrated in Table (29) revealed that the P% against the

challenge with virulent strain of P. multocida type ‘‘D’’ was 100% for

G3 and G1 in comparison with 0% for G5.

Table (27): Challenge test against M. gallisepticum (Eis3-10 strain) in chickens vaccinated with different M. gallisepticum vaccines:

Type of vaccines G2 G3 G4 G5

Total no. of chickens 15 15 15 15

No. of chickens showing respiratory

signs

3 1 2 15

P % 80 93 87 0


G3: Combined M. gallisepticum and P. multocida vaccine G5: Control

Results

101

Table (28): Challenge test against P. multocida type ‘‘A’’ in chickens vaccinated with combined vaccine of M. gallisepticum and P. multocida and P. multocida vaccine:

Types of vaccines G1 G3 G5

Total no. of chickens 15 15 15

D 1 0 15

S 14 15 0

P % 93 100 0

P % = No. of survived chickens Ⅹ 100

Total No. of chickens

S= Survived chickens D=Dead chickens



Results

102

Table (29): Challenge test against P. multocida type ‘‘D’’ in chickens vaccinated with combined vaccine of M. gallisepticum and P. multocida and P. multocida vaccine:

Types of vaccines G1 G3 G5

Total no. of chickens 15 15 15

D 0 0 15

S 15 15 0

P % 100 100 0

P % = No. of survived chickens Ⅹ 100

Total No. of chickens

S= Survived chickens D=Dead chickens



Discussion

103

5. Discussion

For many years, the control of M. gallisepticum in most of the

world has been based on the maintenance of breeding stock that is free

of M. gallisepticum and on biosecurity (Ley, 2008). However, M.

gallisepticum vaccines may be employed in situations where this

approach is not feasible such as endemically infected multi-age

facilities and areas of dense poultry populations (Kleven, 2008). While

M. gallisepticum bacterins reduced the severity of lesions and egg

production losses but did not completely prevent M. gallisepticum

colonization of the chicken respiratory tract upon challenge (OIE,

2012).

M. gallisepticum is further complicated with other poultry

pathogens causing avian influenza, NewCastle disease, infectious

bronchitis, fowl cholera, coryza and E. coli (Liu et al., 2001). So this

study was conducted for preparation and evaluation of locally prepared

combined inactivated vaccine of M. gallisepticum and P. multocida

adjuvanted with Montanide ISA70, and for comparison of its efficacy

with the imported M. gallisepticum vaccine.

The cellular immune response of chickens that vaccinated with

different vaccines was evaluated by H/L ratio. The H/L ratio of G3 at

7th

day post 2

nd vaccination and post challenge was lower than that of

G2 and G4 (Table 2). There was a significant difference of H/L ratio

between vaccinated groups and control group (Table 3). Also, there

Discussion

104

was a significant difference of H/L ratio between G1 and G3 (Table 5).

On the other hand there was no significant difference of H/L ratio

between G2, G3 and G4 (Table 4). 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 cell responses that are important for the immunity. Also,

Abbas et al. (2007) stated that the M. gallisepticum vaccine induced

specific immune responses in the vaccinated birds in the form of

production of specific antibodies and production of nonspecific 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 tissue. The

macrophages or antigen presenting cells (APC) in the granuloma ingest

the microbial antigen from oily suspension and present the microbial

protein antigen on their surface in association with self MHC II. The T

helper cells of the vaccinated birds recognize their specific antigens on

surface of the APC and undergo the process of blast formation,

proliferation and differentiation into effector and memory T

lymphocytes.

Discussion

105

Among P. multocida Harper et al. (2016) reported that P.

multocida LPS is a primary stimulator of the host immune response and

a critical determinant of bacterin protective efficacy.

Also, the cellular immune response of the vaccinated chickens

was evaluated by estimation of NO concentration in the supernatant of

macrophage. The NO concentration in the supernatant of macrophage

of G3 at 7th

day post 2

nd vaccination and post challenge was higher than

that of G2 and G4 (Table 6). There was a significant difference of NO

concentration between vaccinated groups and control group (Table 7).

On the other hand there was no significant difference of NO

concentration between G2, G3 and G4 (Table 8). Also, there was no

significant difference of NO concentration between G1 and G3 (Table

9). These data were in the same manner with Florquin et al. (1994)

who explained that serum NO is one of the end products produced by

macrophages as a result of their exposure to microbial products, the

presence of NO in appropriate concentration during inflammation leads

to immunomodulatory functions of host defense.

Obukhovska et al. (2015) concluded that the level of

macrophages in chickens increased rapidly during the first 10 days after

the second injection of inactivated M. gallisepticum vaccines

adjuvanted with Mantanide ISA 70. It was shown that inoculation of

inactivated vaccines against avian mycoplasmosis in chickens

promoted stimulation for primary link of cellular immunity

(macrophage).

Discussion

106

Nascimento et al. (2005) stated that genus Mycoplasma has

ability to stimulate macrophages, monocytes, T-helper cells and NK

cells, results in the production of substances, such as TNF-α, IL-1, 2, 6

and interferon particularly interferon gamma. Moreover, Majumder

(2014) explained that M. gallisepticum cytadheres to the tracheal

epithelium and mediates infiltration of macrophages, heterophils and

lymphocytes to the tracheal submucosa.

Zhang et al. (2013) stated that the capsule is a major virulence

factor of P. multocida serotype A: 3 strain. Also, Harper et al. (2013)

reported that P. multocida is a Gram-negative pathogen and the

causative agent of fowl cholera and the major outer membrane

component LPS is both an important virulence factor and a major

immunogen.

The humoral immune response of chickens that vaccinated with

combined M. gallisepticum and P. multocida vaccine and P. multocida

vaccine was evaluated by IHA. The antibody titers against P. multocida

type “A” 2 weeks post 2nd

vaccination for G1 and G3 were 256, while

the antibody titer of G3 6 weeks post challenge was higher than that of

G1 (Table 10). There was a significant difference of antibody titers

between vaccinated groups and control group and between G1 and G3

(Tables 12 and 13). The antibody titer against P. multocida type “D” of

G3 2 weeks post 2nd

vaccination was higher than that of G1 and the

antibody titers 6 weeks post challenge for G1 and G3 were 512 (Table

11). There was a significant difference of antibody titers between

Discussion

107

vaccinated groups and control group (Table 14). On the other hand

there was no significant difference of antibody titers between G1 and

G3 (Table 15). These data agreed with Abdel-Aziz et al. (2015) who

concluded that the inactivated fowl cholera vaccine adjuvanted with

Montanide ISA-70-VG induced early and high immune response with

long duration measured by IHA test. Also, Ievy et al. (2013) concluded

that the oil adjuvanted fowl cholera vaccine induced high immune

response measured by IHA.

Ahmed et al. (2010) concluded that the inactivated fowl cholera

vaccine adjuvanted with Montanide ISA70 induced high and protective

antibody titers measured by IHA.

Also, Youssef and Tawfik (2011) reported that the inactivated

rabbit pasteurellosis vaccine adjuvanted with Montanide ISA50

induced protective antibody titer against P. multocida and gave high

and long duration of antibody level measured by IHA test. Moreover,

Jaffri et al. (2006) recorded that the haemorrhagic septicaemia oil

adjuvant (HSOAV) vaccine gave protective antibody titer (IHA titer

1:64) up to 300 days after booster shot and antibodies could be

detectable in serum of the vaccinated animals up to 420 days (IHA titer

1:4).

The humoral immune response of the vaccinated chickens with

different M. gallisepticum vaccines was evaluated by HI. The antibody

titers against M. gallisepticum of G3 and G4 2 weeks post 2nd

Discussion

108

vaccination were higher than that of G2. G3 induced the highest

antibody titer 6 weeks post challenge (Table 16). There was a

significant difference of antibody titers between vaccinated groups and

control group (Table 17). On the other hand there was no significant

difference of antibody titers between G2, G3 and G4 (Table 18). These

data were in the same manner with Barbour and Newman (1990) who

stated that a significant Ig response specific to M. gallisepticum was

observed in sera of chickens collected 3 weeks after the first and

second vaccination with oil-emulsion M. gallisepticum vaccine.

Asif et al. (2015) concluded that HI based assay is more specific

and reliable than RSA and conventional diagnostic techniques.

The humoral immune response of the vaccinated chickens with

different M. gallisepticum vaccines was evaluated by ELISA. G3

induced the highest antibody titer against M. gallisepticum 2 weeks

post 2nd

vaccination and 6 weeks post challenge (Table 19). There was

a significant difference of antibody titers between vaccinated groups

and control group (Table 21). On the other hand there was no

significant difference of antibody titers between G2, G3 and G4 (Table

22). These data agreed with Gondal et al. (2013) and Bekele (2015)

who reported that the formaldehyde inactivated Montanide ISA70

based M. gallisepticum vaccine induced protective level of anti M.

gallisepticum antibodies in chickens. Also, Sarfaraz et al. (2017)

reported that oil based combined M. gallisepticum and avian influenza

(H9N2) vaccine adjuvanted with Montanide ISA-70 induced effective

Discussion

109

antibody response in the vaccinated birds measured by ELISA and HI

tests.

These data were explained by Harper et al. (2012) who

reported that the capsule and LPS of P. multocida constitute the major

components of the bacterial cell surface. They play key roles in a range

of interactions between the bacteria and the hosts they colonize or

infect. Both polysaccharides are involved in the avoidance of host

innate immune mechanisms, such as resistance to phagocytosis,

complement-mediated killing, and the bactericidal activity of

antimicrobial peptides; they are therefore essential for virulence. In

addition, LPS is a major antigen in the stimulation of adaptive immune

responses to infection.

The humoral immune response of chickens vaccinated with


vaccine was evaluated by ELISA. G3 induced the highest antibody titer

against P. multocida 2 weeks post 2nd

vaccination and 6 weeks post

challenge (Table 20). There was a significant difference of antibody

titers between vaccinated groups and control group (Table 23). On the

other hand there was no significant difference of antibody titers

between G1 and G3 (Table 24). These data agreed with Jabbri and

Moazeni Jula (2005) who concluded that the inactivated trivalent fowl

cholera vaccine consisted of serotypes 1, 3 and 4 P. multocida strains

induced immunogenic response in vaccinated chickens. ELISA assay

Discussion

110

showed a considerable increase in antibody titer after twice vaccination

of 8 weeks chickens.


cholera vaccine prepared from the isolated bacteria induced protective

immune response and significant increase in the antibody titer

measured by ELISA.

Also, Avakian et al. (1989) reported that

polyvalent fowl cholera oil-based bacterin induced a high antibody titer

in broiler minibreeder hens measured by ELISA technique.

Moreover, Perelman et al. (1990) concluded that

commercial inactivated P. multocida bacterin induced high antibody

titers measured by ELISA in turkeys.

Youssef and Tawfik (2011) reported that the inactivated rabbit

pasteurellosis vaccine adjuvanted with Montanide ISA50 induced a

considerable immunity and gave high and long duration of antibody

level measured by ELISA.

The potency of the vaccines was evaluated by passive mouse

protection test against the challenge with the virulent strains of P.

multocida types A and D in chickens vaccinated with combined M.

gallisepticum and P. multocida vaccine and P. multocida vaccine. The

P% against the challenge with virulent strains of P. multocida types A

and D 2 weeks post 2nd

vaccination and 8 weeks post challenge was

Discussion

111

100% for G3 (Tables 25 and 26). These data were in the same manner

with those of El-Bayomy and Daoud (2004) who found that there was

an elevation in protective values of fowl cholera adjuvanted vaccines

against challenge with virulent strains of P. multocida types A and D

for the serum of the vaccinated chicken groups.

Also, Youssef and Tawfik (2011) concluded that the inactivated

rabbit pasteurellosis vaccine adjuvanted with Montanide ISA50

induced 3.85 and 3.69 log protection in mice against the challenge with

P. multocida serotypes A and D, respectively. Moreover, Jaffri et al.

(2006) reported that HSOAV vaccine gave prolonged and strong

protection against the challenge dose in passive mouse protection test.

The potency of the vaccines was evaluated by the challenge test

against M. gallisepticum (Eis3-10 strain) in chickens vaccinated with

different M. gallisepticum vaccines (Table 27). The highest P% against

the challenge with M. gallisepticum was in G3 (93%). These data were

in the same manner with those of Bekele (2015) who concluded that

the formaldehyde inactivated Montanide ISA70 based M. gallisepticum

vaccine induced 100% protection against M. gallisepticum. All

chickens did not show clinical signs or post mortem changes after

challenge test. Also, Ferguson-Noel et al. (2012) found that the M.

gallisepticum bacterin was protective and resulted in significant

differences in air sac lesions, tracheal lesions, and ovarian regression

compared to the non vaccinated controls.

Discussion

112

Moreover, Shafay (1995) concluded that the locally prepared


gave acceptable protection level in comparison with the monovalent M.

gallisepticum vaccine in vaccinated chickens. In addition, Gadallah

(2015) reported that the locally prepared inactivated combined M.

gallisepticum and E. coli vaccine induced protection against the CRD

and elicited the humoral immune response in broiler chickens.

Kleven (2008) concluded that M. gallisepticum bacterins

protected chickens against respiratory signs, airsaculitis, egg

production losses and reducing egg transmission. While OIE (2012)

reported that M. gallisepticum bacterins reduced the severity of lesions

and egg production losses but did not completely prevent M.

gallisepticum colonization of the chicken respiratory tract upon

challenge.

These data were explained by Gong et al. (2013) who stated

that the two Omps (OmpH and OmpA) are the major immunogenic

antigens of avian P. multocida, which play an important role in

inducing immune responses that confer resistance against infections.

Moreover, Boyle and Finlay (2003) found that the Omps promote

adherence to host cell surfaces and are therefore likely to be involved in

P. multocida virulence. Also, Noormohammadi (2007) found that LPs

reside on the surfaces of the cell wall-less mycoplasmas and are

important factors in pathogenesis.

https://www.ncbi.nlm.nih.gov/pubmed/?term=Gong%20Q%5BAuthor%5D&cauthor=true&cauthor_uid=23340446

Discussion

113

Also, the potency of the vaccines was evaluated by the challenge

test against P. multocida types A and D in chickens vaccinated with


vaccine. The P% against the challenge with P. multocida types A and D

was 100% for G3 (Tables 28 and 29). These data agreed with Ahmed

et al. (2010) who concluded that the inactivated fowl cholera vaccine

adjuvanted with Montanide ISA70 gave 100% protection in chickens

against challenge with virulent strains of P. multocida types A and D.

Also, Abdel-Aziz et al. (2015) reported that the inactivated fowl

cholera vaccine adjuvanted with Montanide ISA-70-VG induced high

protection rates in chickens against challenge with virulent serotypes 5:

A and D: 2 (95 and 90%, respectively). Moreover, Jabbri and

Moazeni Jula (2005) recorded that the inactivated trivalent fowl

cholera vaccine consists of serotypes 1, 3 and 4 P. multocida strains

provided 70-100% protection in chickens against challenge with

homologous strains.

Ievy et al. (2013) concluded that the oil adjuvanted fowl cholera

vaccine induced high protection in chickens against challenge with

virulent strain of P. multocida.


cholera vaccine prepared from the isolated bacteria induced protection

in chickens against challenge with the virulent strain of P. multocida.

Discussion

114

So, it could be concluded that the locally prepared combined

inactivated M. gallisepticum and P. multocida vaccine induced a

considerable immunity in chickens as it gave early, high and long

duration of antibody response. Also, it was efficient and safe in

protection of chickens against M. gallisepticum and P. multocida

infections. Depending on the obtained results, it could be suggested to

use this combined vaccine for control of M. gallisepticum in poultry

industry.

Summary

115

6. Summary

Mycoplasmosis is one of the important poultry diseases causing

significant economic losses in poultry industry. M. gallisepticum is the

most economically significant mycoplasma pathogen of poultry; M.

gallisepticum causes CRD in chickens, reduced egg production, high

mortality rates among young birds, and increased carcass

condemnations.

Vaccination has been suggested as a useful tool to control M.

gallisepticum in chickens. The present work was planned to study the

immune response of chickens vaccinated with locally prepared


adjuvanted with Montanide ISA70.

One hundred and fifty, 4 weeks old SPF chickens were divided

into five groups, the 1st

group was vaccinated with P. multocida vaccine

(G1), the 2nd

group was vaccinated with M. gallisepticum vaccine (G2),

the 3rd

group was vaccinated with combined vaccine of M.

gallisepticum and P. multocida vaccine (G3), the 4th

group was

vaccinated with imported M. gallisepticum vaccine (G4) and the 5th

group was kept unvaccinated as a control group (G5).

The cellular immune response of the vaccinated chickens was

evaluated by H/L ratio. The H/L ratio at 7th

day post 1

st vaccination for

G1, G2, G3 and G4 were 0.4, 0.6, 0.2 and 0.3, respectively in

Summary

116

comparison with 1.0 for G5. While, at 7th

day post 2nd

vaccination the

H/L ratio for G1, G2, G3 and G4 were 0.1, 0.4, 0.1 and 0.2,

respectively in comparison with 0.9 for G5. The H/L ratio at 7th

day

post challenge for G1, G2, G3 and G4 were 0.1, 0.3, 0.1 and 0.1,


Also, the cellular immune response was evaluated by estimation

of NO concentration in the supernatant of macrophage. The NO

concentration in the supernatant of macrophage at 7th

day post 2

nd

vaccination for G1, G2, G3 and G4 were 67.08, 45.2, 53.9 and 46.3,

respectively in comparison with 16.3 for G5. While, at 7th

day post

challenge the NO concentration for G1, G2, G3 and G4 were 80.8,

78.3, 102.6 and 94.1, respectively in comparison with 15.2 for G5.

The humoral immune response of the vaccinated chickens was

evaluated by IHA, HI and ELISA tests. The results of IHA test revealed

that the antibody titers against P. multocida type “A” 2 weeks post 1st

vaccination for G1 and G3 were 64 and 128, respectively in

comparison with 2.0 for G5. While, 2 weeks post 2nd

vaccination the

antibody titers were 256 for both groups (G1 and G3) in comparison

with 2.0 for G5. The antibody titers 6 weeks post challenge for G1 and

G3 were 512 and 1024, respectively in comparison with 2.0 for G5.

The antibody titers against P. multocida type “D” 2 weeks post 1st

vaccination for G1 and G3 were 32 and 64, respectively in comparison

with 2.0 for G5. While, 2 weeks post 2nd

vaccination the antibody titers

Summary

117

for G1 and G3 were 64 and 128, respectively in comparison with 2.0

for G5. The antibody titers 6 weeks post challenge were 512 for both

groups (G1 and G3) in comparison with 2.0 for G5.

The results of HI test revealed that the antibody titers against M.

gallisepticum 2 weeks post 1st

vaccination for G2, G3 and G4 were 32,

64 and 64, respectively in comparison with 2.0 for G5. While, 2 weeks

post 2nd

vaccination the antibody titers for G2, G3 and G4 were 64, 128

and128, respectively in comparison with 2.0 for G5. The antibody titers

6 weeks post challenge for G2, G3 and G4 were 128, 512 and 256,


The results of ELISA test showed that the antibody titers against

M. gallisepticum 2 weeks post 1st

vaccination for G2, G3 and G4 were

157, 360 and 241, respectively in comparison with 0 for G5. While, 2

weeks post 2nd

vaccination the antibody titers for G2, G3 and G4 were

729, 996 and 965, respectively in comparison with 0 for G5. The

antibody titers 6 weeks post challenge for G2, G3 and G4 were 2541,

4958 and 3927, respectively in comparison with 0 for G5.

The antibody titers against P. multocida 2 weeks post 1st

vaccination for G1 and G3 were 206 and 227, respectively in

comparison with 0 for G5. While, 2 weeks post 2nd

vaccination the

antibody titers for G1 and G3 were 2391 and 2487, respectively in

comparison with 0 for G5. The antibody titers 6 weeks post challenge

Summary

118

for G1 and G3 were 3164 and 4327, respectively in comparison with 0

for G5.

The humoral immune response of the vaccinated chickens firstly

detected 2 weeks post 1st vaccination and continued till 14 weeks post

challenge. The antibody titers reached peak levels at 6 weeks post

challenge.

The potency of the vaccines was evaluated by passive mouse

protection test and challenge test. The results of passive mouse

protection test revealed that the P% against the challenge with virulent

strain of P. multocida type “A” 2 weeks post 1st

vaccination for G1 and

G3 were 80% and 100%, respectively in comparison with 0% for G5.

While, 2 weeks post 2nd

vaccination and 8 weeks post challenge the P%

were 100% for both groups (G1 and G3) in comparison with 0% for

G5.

The P% against the challenge with virulent strain of P. multocida

type “D” 2 weeks post 1st

vaccination were 100% for both groups (G1

and G3) in comparison with 0% for G5. Also, 2 weeks post 2nd

vaccination and 8 weeks post challenge the P% were 100% for both

groups in comparison with 0% for G5.

The challenge test showed that the P% against the challenge with

virulent strain of M. gallisepticum (Eis3-10 strain) was 93% for G3 and

87% for G4 and the lowest P% was in G2 (80%) in comparison with

Summary

119

0% for G5. The P% against the challenge with virulent strain of P.

multocida type ‘‘A’’ was 100% for G3 and 93% for G1 in comparison

with 0% for G5. The P% against the challenge with virulent strain of P.

multocida type ‘‘D’’ was 100% for G3 and G1 in comparison with 0%

for G5.

The data in the present work showed that the combined

inactivated vaccine of M. gallisepticum and P. multocida adjuvanted

with Montanide ISA70 induced high and long duration of antibody

response and significant protection against the challenge with virulent

strain of M. gallisepticum (Eis3-10 strain).

References

120

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4

النوع دا منووجد أن نسبة الحمایة ضد العدوى بالعترة الضاریھ من الباستریال مالتوسی

"D" 1بالنسبھ للمجموعتان %100كانت الجرعة األولى أسبوعین منبعدG ،G3 ارنھ بالمق

ع منأسابی 8وبعد الجرعھ الثانیھ بعد أسبوعین من آیضوأ %0عطت التى أ G5بالمجموعھ

بالمقارنھ 1G ،G3بالنسبھ للمجموعتان %100 كانت نسبھ الحمایھ اختبار التحدى

. %0 التى أعطت G5بالمجموعھ

سبتیكمالیالمیكوبالزما جالضاریھ من ضد العدوى بالعترة جرى اختبار التحدىوقد أ

% 87و G3 ھمجموعلل %93 كانت ن نسبھ الحمایھوقد تبین أ) Eis3-10( من النوع

% حمایة للمجموعھ0مقارنھ %) G2 )80مجموعھ لاقل نسبھ حمایھ فى وأ G4 مجموعھلل

G5. وعلتوسیدا من الناالضاریھ من الباستریال م العترةب العدوى نسبة الحمایة ضد "A"

% حمایة0 مقارنھG1 مجموعھلل %93و G3 مجموعھلل% بالنسبة 100لى تصل إ

لنوعا من التوسیداالضاریھ من الباستریال م نسبة الحمایة ضد العدوى بالعترة .G5للمجموعھ

"D" مجموعتانلل% بالنسبة 100لى تصل إ G3 و G1 حمایة للمجموعھ 0 مقارنھ %G5.

سبتیكمیكوبالزما جالیالمن اللقاح المشترك المیت من وقد تبین من ھذه الدراسھ أ

ھ مناعیھ ى یعطى استجابكمحسن مناع ISA70لیھ مونتانیدوالمضاف إ والباستریال مالتوسیدا

یكمالیسبتجالمیكوبالزما الضاریھ من ضد العدوى بالعترة یلھ وحمایھ معتبرةطو عالیھ لمدة

.)Eis3-10( من النوع

3

بعد كم لیسبتید المیكوبالزما جاجسام المناعیھ ضوجد أن عیاریھ األاختبار االلیزا وفى

، 2G ،G3بالترتیب بالنسبھ للمجامیع 241، 360، 157كانت الجرعة األولى أسبوعین من

G4 لمقارنھ بالمجموعھباG5 كانت ھ الجرعھ الثانی بعد أسبوعین منبینما ،0عطت التى أ

لتى ا G5بالمقارنھ بالمجموعھ 2G ،G3 ،G4بالنسبھ للمجامیع بالترتیب 965، 996، 729

ھ بالنسببالترتیب 3927، 4958 ،2541كانتاختبار التحدى أسابیع من 6وبعد 0 أعطت

. 0 التى أعطت G5ارنھ بالمجموعھ بالمق 2G ،G3 ،G4للمجامیع

ة الجرع أسبوعین منبعد وجد أن عیاریھ األجسام المناعیھ ضد الباستریال مالتوسیدا

G5عھبالمقارنھ بالمجمو 1G ،G3بالترتیب بالنسبھ للمجموعتان 227، 206كانت األولى

النسبھ بترتیب بال 2487، 2391كانت الجرعھ الثانیھ بعد أسبوعین منبینما ،0عطت التى أ

بار اخت أسابیع من 6وبعد 0 التى أعطت G5بالمقارنھ بالمجموعھ 1G ،G3للمجموعتان

موعھ بالمقارنھ بالمج 1G ،G3بالترتیب بالنسبھ للمجموعتان 4327، 3164 كانت التحدى

G5 0 التى أعطت .

نموقد وجد أن المناعة المصلیة للدجاج المحصن تبدأ فى الظھور بعد أسبوعین

لى إ ناعیةاألجسام الم تصلو ،اختبار التحدىسبوع بعد أ 14لمدة ة األولى ثم تستمر الجرع

.اختبار التحدى أسابیع من 6بعد أعلى مستوى

اختبار و فئرانالحمایة الغیر مباشر فى ال اللقاحات عن طریق اختبار وقد تم تقییم كفاءة

یة ضدن نسبة الحماأ ووجد الفئران وقد أجري إختبار الحمایة الغیر مباشر فى .التحدى

الجرعة أسبوعین منبعد "A" لتوسیدا من النوعاالضاریھ من الباستریال م العترةب العدوى

بالمقارنھ 1G ،G3بالترتیب بالنسبھ للمجموعتان %100، %80كانت األولى

ع منیأساب 8وبعد الجرعھ الثانیھ بعد أسبوعین منبینما %0عطت التى أ G5بالمجموعھ

بالمقارنھ 1G ،G3بالنسبھ للمجموعتان %100 نسبھ الحمایھ كانتاختبار التحدى

. %0 التى أعطت G5بالمجموعھ

2

، 1Gالنسبھ للمجامیع بالترتیب ب 46.3، 53.9، 45.2، 67.08السابع بعد الجرعھ الثانیھ كان

2G ،G3 ،G4 بالمقارنھ بالمجموعھG5 بینما فى الیوم السابع بعد ، 16.3التى أعطت

1Gبالنسبھ للمجامیع بالترتیب 94.1، 102.6، 78.3، 80.8اختبار التحدى كانت النسبھ

،2G ،G3 ،G4 بالمقارنھ بالمجموعھG5 15.2 التى أعطت.

لغیر بواسطھ اختبارات تلزن الدم الدجاج المحصن فى ا تم قیاس المناعھ المصلیھ

جسام ألااریھ عیوجد أن فى اختبار تلزن الدم الغیر مباشر .االلیزاتثبیط تلزن الدم ومباشر و

، 46كانت ى الجرعة األول بعد أسبوعین من "A" نوعال من المناعیھ ضد الباستریال مالتوسیدا

بینما ،2عطت التى أ G5المقارنھ بالمجموعھ ب 1G ،G3 بالنسبھ للمجموعتان بالترتیب 128

بالمقارنھ 1G ،G3 بالنسبھ للمجموعتان 562 الجرعھ الثانیھ كانت بعد أسبوعین من

ترتیب بال 1024، 512 اختبار التحدى كانت أسابیع من 6وبعد 2 التى أعطت G5بالمجموعھ

. 2 التى أعطت G5بالمقارنھ بالمجموعھ 1G ،G3للمجموعتان بالنسبھ

عد ب "D" وجد أن عیاریھ األجسام المناعیھ ضد الباستریال مالتوسیدا من النوع

1G ،G3بالترتیب بالنسبھ للمجموعتان 32،64كانت الجرعة األولى أسبوعین من

، 64نت كا الجرعھ الثانیھ بعد أسبوعین منبینما ،2عطت التى أ G5بالمقارنھ بالمجموعھ

بعد و 2 التى أعطت G5بالمقارنھ بالمجموعھ 1G ،G3وعتان بالنسبھ للمجمبالترتیب 128

بالمقارنھ 1G ،G3بالنسبھ للمجموعتان 512 كانتاختبار التحدى أسابیع من 6

.2 التى أعطت G5بالمجموعھ

زما المیكوبالضد جسام المناعیھعیاریھ األوجد أن فى اختبار تثبیط تلزن الدم و

امیع لمجبالنسبھ ل بالترتیب 64، 32،64كانت ة األولىالجرع بعد أسبوعین منجالیسبتیكم

2G ،G3 ،G4 بالمقارنھ بالمجموعھG5 الجرعھ بعد أسبوعین منبینما ،2عطت التى أ

بالمقارنھ 2G ،G3 ،G4للمجامیع بالنسبھ بالترتیب 128، 128، 64كانت الثانیھ

256، 512 ،128 انتكاختبار التحدى أسابیع من 6وبعد 2 التى أعطت G5بالمجموعھ

. 2 التى أعطت G5بالمقارنھ بالمجموعھ 2G ،G3 ،G4للمجامیع بالنسبھ بالترتیب

الملخص العربى

یھدخسائر اقتصا مراض الدواجن التى تسببمن أھم أالمیكوبالزما مرض عتبری

نواع من أھم أ سبتیكمیالمیكوبالزما جال، ویعتبر میكروب فى صناعھ الدواجن فادحھ

فى CRD)( مرض الجھاز التنفسى المزمن وتسبب المیكوبالزما التى تصیب الدواجن

وزیادة نسبھ فى األعمار الصغیرة الوفیاتمعدالت ارتفاع یض،نتاج الب، نقص فى إالدواجن

.اإلعدام بالمجازر

. جالیسبتیكم فى الدواجن على المیكوبالزما لتحصین ھو الوسیلھ المثلى للسیطرةویعد ا

مشترك المیت المحلى القاح للاقامت ھذه الدراسھ على دراسھ الحالھ المناعیھ للدجاج المحصن ب

كمحسن ISA70مونتانید لیھإ جالیسبتیكم والباستریال مالتوسیدا مضاف من المیكوبالزما

5لى اسابیع) إ 4من الدجاج الخالى من األمراض (أعمارھم 150عدد تم تقسیم مناعى.

المجموعھ الثانیھ ، (G1)داالباستریال مالتوسیولى تم تحصینھا بلقاح المجموعھ األ ،مجموعات

المجموعھ الثالثھ تم تحصینھا بلقاح مشترك ، (G2)جالیسبتیكما المیكوبالزم تم تحصینھا بلقاح

المجموعھ الرابعھ تم تحصینھا ، (G3)المیكوبالزما جالیسبتیكم والباستریال مالتوسیدا من

ما المجموعھ الخامسھ فھى المجموعھ أ، (G4) جالیسبتیكمالمیكوبالزما بلقاح مستورد من

. (G5) الغیر محصنھ الضابطھ

بھعد نس بواسطة إختبار لدجاج المحصنفى ا المناعة الخلویة استم قی

heterophilsلىإ lymphocytes م السابع بعد الجرعھ األولى أن ھذه النسبھ فى الیوووجد

بالمقارنھ 1G، 2G ،G3 ،G4بالترتیب بالنسبھ للمجامیع 0.3، 0.2، 0.6، 4.0كانت

، 0.1م السابع بعد الجرعھ الثانیھ كانت النسبھ بینما فى الیو، 1.0التى أعطت G5بالمجموعھ

G5بالمقارنھ بالمجموعھ 1G، 2G ،G3 ،G4بالترتیب بالنسبھ للمجامیع 0.2، 0.1، 0.4

0.1، 0.1، 0.3، 0.1 بھاختبار التحدى كانت النس فى الیوم السابع بعد . 0.9التى أعطت

.1.0 التى أعطت G5لمجموعھ بالمقارنھ با 1G، 2G ،G3 ،G4بالنسبھ للمجامیع بالترتیب

فى السائل الطافى وكسید النیترك أقیاس تركیز بواسطة یضاأ المناعة الخلویةتم قیاس

الیوم فى ى السائل الطافى للبالعھ الكبیرةف وكسیدجد أن تركیز النیترك أوو للبالعھ الكبیرة

جامعة القاھرة

كلیة الطب البیطري

قسم المیكروبیولوجي

فاطمھ فتحى ابراھیم حسن : سمألا

مصریةالجنسیة :

لجیزةا، 1/10/2198 : تاریخ المیالد

2018،میكولوجیا ) –نولوجیاامی -الوجیتریو(بك دكتوراة فى العلوم الطبیة البیطریة : الدرجھ

شترك ثنائى ضد عدوى الباستریال والمیكوبالزما فى الدجاجتحضیر وتقییم لقاح م : عنوان الرسالة

كلیة الطب – ستاذ المیكروبیولوجيأ( (رحمھ هللا) أحمد محمود عصام حاتمستاذ الدكتور/ أل: ا شرافإتحت جامعة القاھرة) –البیطري

لشئون الدراسات العلیا ووكیل الكلیھ المیكروبیولوجي ستاذأ( جاكین كمال عبد الحلیم الجاكى ستاذ الدكتور/ألاجامعة القاھرة) –كلیة الطب البیطري – والبحوث

جامعة القاھرة) –كلیة الطب البیطري – مراض الدواجنأستاذ أ(وفاء عبد الغنى عبد الغنى ستاذ الدكتور/ألا

مصال األ معھد بحوث –قسم اللقاحات البكتیریھ الھوائیھورئیس (رئیس بحوثایمان محمد الراوى الدكتور/) القاھرة –العباسیھ –واللقاحات البیطریھ

)الجیزة –دقىال – صحھ الحیوانبحوث معھد – لمیكوبالزماقسم ا –ئیس بحوث(رمنى محمد شاكرالدكتور/

المستخلص

مشترك من المیت المحلى القاح للاقامت ھذه الدراسھ على دراسھ الحالھ المناعیھ للدجاج المحصن بتم تقسیم عدد .كمحسن مناعى ISA70مونتانید لیھإیكم والباستریال مالتوسیدا مضاف جالیسبت المیكوبالزما

ولى تم تحصینھا المجموعھ األمجموعات، 5لى إسابیع) أ 4(أعمارھم مراضالخالى من األ من الدجاج 150، المجموعھ الثالثھ تم المیكوبالزما جالیسبتیكم بلقاح ، المجموعھ الثانیھ تم تحصینھاالباستریال مالتوسیدابلقاح

، المجموعھ الرابعھ تم تحصینھا الیسبتیكم والباستریال مالتوسیداجالمیكوبالزما تحصینھا بلقاح مشترك من. الغیر محصنھ ما المجموعھ الخامسھ فھى المجموعھ الضابطھأ، المیكوبالزما جالیسبتیكم بلقاح مستورد منلى إheterophils بھسعد ن اللقاحات عن طریق قیاس المناعة الخلویة بواسطة إختبار وقد تم تقییم ھذهlymphocytes باستخدام وقیاس المناعھ المصلیھ السائل الطافى للبالعھ الكبیرة فىوقیاس النیترك اوكسید

باستخدام اختبار التحدى اتقاحاللتم تقییم فعالیة ،االلیزاو واختبار تثبیط تلزن الدماختبار تلزن الدم الغیر مباشرمن النوع المیكوبالزما جالیسبتیكم الضاریھ من وإختبار الحمایة الغیر مباشر فى الفئران ضد العدوى بالعترة

"Eis3-10" والباستریال ملتوسیدا من النوع " A ،D ". المشترك من المیت اح ن اللقأ وقد أظھرت النتائجیعطى إستجابھ كمحسن مناعى ISA70یدتانلیھ مونوالمضاف إ ریال مالتوسیداجالیسبتیكم والباستالمیكوبالزما

-Eis3" المیكوبالزما جالیسبتیكمالضاریھ من ضد العدوى بالعترة مناعیھ عالیة لمدة طویلھ وحمایھ معتبرة10".

.ISA70لقاح میت، دجاج، مونتانید ،باستریال مالتوسیدا ،جالیسبتیكممیكوبالزما : الكلمات الدالة

شرافإللجنة ا

:شراف منإلتتكون لجنة ا

(رحمه هللا) أحمد محمود عصام حاتم .د.ا

ةجامعة القاهر –كلية الطب البيطري –ستاذ الميكروبيولوجي أ

جاكين كمال عبد الحليم الجاكى . د.ا

كلية الطب البيطري – ووكيل الكليه لشئون الدراسات العليا والبحوث ستاذ الميكروبيولوجيأ

جامعة القاهرة –

وفاء عبد الغنى عبد الغنى .د.ا

جامعة القاهرة –كلية الطب البيطري –مراض الدواجن أستاذ أ

ايمان محمد الراوى .د

مصال واللقاحاتألمعهد بحوث ا –بكتيريه الهوائيه قسم اللقاحات ال ورئيس رئيس بحوث

ةالقاهر –العباسيه –البيطريه

منى محمد شاكر. د

ةالجيز –الدقى –بحوث صحه الحيوان معهد –قسم الميكوبالزما –رئيس بحوث

Scanned by CamScanner

جامعة القاهرة

الطب البيطرى كلية

قسم الميكروبيولوجى

تحضير وتقييم لقاح مشترك ثنائى ضد عدوى الباستريال

والميكوبالزما فى الدجاج

رسالة مقدمة من

براهيم حسنإفتحى ةفاطم ط.ب/ (2004) ةجامعة القاهر -كلية الطب البيطرى -البيطرية ةبكالوريوس العلوم الطبي

(2012) جامعه القاهرة -كليه الطب البيطرى -وم الطبيه البيطريهماجيستير العل

ةللحصول على درج

البيطرية فى العلوم الطبية دكتوراةال ميكولوجيا( -امينولوجيا -)بكتريولوجيا

شرافإ تحت

)رحمه هللا( أحمد محمود عصام حاتم /األستاذ الدكتور أستاذ الميكروبيولوجى

القاهرة ةجامع -كلية الطب البيطرى

2018

وفاء عبد الغنى عبد ستاذ الدكتور/ األ

الغنى مراض الدواجنأستاذ أ

جامعه القاهرة -الطب البيطرى كلية

منى محمد شاكر الدكتور/ قسم الميكوبالزما -رئيس بحوث

جيزةال -دقىال -صحه الحيوانمعهد بحوث

جاكين كمال الجاكى ستاذ الدكتور/األووكيل الكليه لشئون الدراسات كروبيولوجىالميأستاذ

العليا والبحوث

ةجامعه القاهر -كلية الطب البيطرى

الدكتور/ ايمان محمد الراوى

ئيهاقسم اللقاحات البكتيريه الهوورئيس رئيس بحوث

-العباسيه -مصال واللقاحات البيطريهمعهد بحوث األ

القاهرة

preparation and evaluation of a combined bivalent vaccine … 2020. 8. 1. · my mother, my father,...

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