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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
Faculty of Veterinary Medicine
Cairo University
Dr. Mona Mohammed Shaker Chief Researcher
Mycoplasma Department
Animal Health Research Institute-Dokki-Giza
Cairo University
Faculty of Veterinary Medicine
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
Faculty of Veterinary Medicine
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
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
Cairo University
Faculty of Veterinary Medicine
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
8 Statistical analysis of NO concentration between
groups of mycoplasma
79
9 Statistical analysis of NO concentration between
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
13 Statistical analysis of IHA antibody titers against P.
multocida type “A” between combined vaccine and
P. multocida vaccine
84
14 Statistical analysis of IHA antibody titers against P.
multocida type “D” between vaccinated groups and
control group
85
15 Statistical analysis of IHA antibody titers against P.
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
22 Statistical analysis of ELISA antibody titers against
M. gallisepticum between groups of mycoplasma
95
VI
23 Statistical analysis of ELISA antibody titers against
P. multocida between vaccinated groups and control
group
96
24 Statistical analysis of ELISA antibody titers against
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
chickens vaccinated with combined vaccine of M.
gallisepticum and P. multocida and P. multocida
vaccine
101
29 Challenge test against P. multocida type ‘‘D’’ in
chickens vaccinated with combined vaccine of M.
gallisepticum and P. multocida and P. multocida
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).
Review of literatures
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).
Review of literatures
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).
Review of literatures
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,
Review of literatures
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
Review of literatures
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.
Review of literatures
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).
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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).
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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).
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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
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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
Review of literatures
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
Review of literatures
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).
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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
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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).
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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
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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
Review of literatures
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).
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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
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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).
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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
Review of literatures
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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.
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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
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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.
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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
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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).
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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
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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
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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
Review of literatures
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
Review of literatures
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.
Review of literatures
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).
Review of literatures
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
Review of literatures
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
Review of literatures
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
Review of literatures
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.
Review of literatures
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.
Review of literatures
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.
Materials and methods
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
Materials and methods
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.
Materials and methods
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.
Materials and methods
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
Materials and methods
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.
Materials and methods
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
Materials and methods
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
Materials and methods
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.
Materials and methods
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%.
Materials and methods
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
Materials and methods
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
Materials and methods
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).
Materials and methods
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
Materials and methods
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
Materials and methods
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:
Materials and methods
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.
Materials and methods
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.
Materials and methods
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
Materials and methods
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
Materials and methods
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.
Materials and methods
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
Materials and methods
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
Materials and methods
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.
Materials and methods
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.
Materials and methods
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
Materials and methods
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.
Materials and methods
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
G3: Combined M. gallisepticum and P. multocida 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
G3: Combined M. gallisepticum and P. multocida 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
Ftab: F tabulated Fcal: F calculated
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:
G1: P. multocida vaccine G2: M. gallisepticum vaccine
G3: Combined M. gallisepticum and P. multocida vaccine
G4: Imported M. gallisepticum vaccine G5: Control
Booster vaccination: at 8 weeks of age
Challenge: at 11 weeks of age
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
G1: P. multocida vaccine G2: M. gallisepticum vaccine
G3: Combined M. gallisepticum and P. multocida vaccine
G4: Imported M. gallisepticum vaccine G5: Control
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
Ftab: F tabulated Fcal: F calculated
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
G2: M. gallisepticum vaccine G4: Imported M. gallisepticum vaccine
G3: Combined M. gallisepticum and P. multocida vaccine
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
SS: Sum of squares MS: Mean of squares Df: Degree of freedom
Ftab: F tabulated Fcal: F calculated
Table (9): Statistical analysis of NO concentration between combined vaccine and P. multocida vaccine:
G1 G3 Mean 46.15857 52.41429 Variance 567.3665 861.6881 Observations 7 7 Pearson Correlation 0.862192
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
G3: Combined M. gallisepticum and 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).
ANOVA test was used to show difference of the antibody titers
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).
Paired t test was used to show difference of antibody titers against
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
4 weeks post challenge 128 512 2
6 weeks post challenge 512 1024 2
8 weeks post challenge 256 512 0
10 weeks post challenge 256 512 0
12 weeks post challenge 256 256 0
14 weeks post challenge 128 128 0
G1: P. multocida vaccine G5: Control
G3: Combined M. gallisepticum and P. multocida vaccine
1st vaccination: at 4 weeks of age
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:
Interval times of serum collection
Types of vaccines
G1 G3 G5
Prevaccination 2 2 0
1st vaccination
2 weeks post 1st vaccination 32 64 2
Booster vaccination
2 weeks post 2nd vaccination 64 128 2
Challenge
2 weeks post challenge 128 128 2
4 weeks post challenge 256 512 4
6 weeks post challenge 512 512 2
8 weeks post challenge 128 256 2
10 weeks post challenge 64 64 0
12 weeks post challenge 64 64 0
14 weeks post challenge 32 32 0
G1: P. multocida vaccine G5: Control
G3: Combined M. gallisepticum and P. multocida vaccine
1st vaccination: at 4 weeks of age
Booster vaccination: at 8 weeks of age Challenge: at 11 weeks of age
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
G1: P. multocida vaccine G5: Control
G3: Combined M. gallisepticum and P. multocida vaccine
Source of Variation SS Df MS F cal P-value F tab Between Groups 617059.5556 2 308529.8 7.964122 0.002224 3.402826 Within Groups 929759.1111 24 38739.96
Total 1546818.667 26
SS: Sum of squares MS: Mean of squares Df: Degree of freedom
Ftab: F tabulated Fcal: F calculated
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
G3: Combined M. gallisepticum and 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
G1: P. multocida vaccine G5: Control
G3: Combined M. gallisepticum and P. multocida vaccine
Source of Variation SS Df MS F cal P-value F tab Between Groups 163124.2667 2 81562.13 4.109341 0.027668 3.354131 Within Groups 535895.6 27 19847.99
Total 699019.8667 29 SS: Sum of squares MS: Mean of squares Df: Degree of freedom
Ftab: F tabulated Fcal: F calculated
Table (15): Statistical analysis of IHA antibody titers against P. multocida type “D” between combined vaccine and P. multocida vaccine:
G1 G3 Mean 128.2 176.2 Variance 23381.73333 36160.4 Observations 10 10 Pearson Correlation 0.901578123
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
G3: Combined M. gallisepticum and 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.
ANOVA test was used to show difference of the antibody titers
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).
ANOVA test was used to show difference of the antibody titers
against M. gallisepticum between G2, G3 and G4. This test revealed no
significant difference at p≤0.05 with Fcal= 3.082234 and Ftab=
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:
Interval times of serum collection
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
4 weeks post challenge 128 512 256 2
6 weeks post challenge 128 512 256 2
8 weeks post challenge 64 256 128 0
10 weeks post challenge 64 128 128 0
12 weeks post challenge 32 64 64 0
14 weeks post challenge 16 64 32 0
G2: M. gallisepticum vaccine G4: Imported M. gallisepticum vaccine
G3: Combined M. gallisepticum and P. multocida vaccine
G5: Control 1st vaccination: at 4 weeks of age
Booster vaccination: at 8 weeks of age Challenge: at 11 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
G3: Combined M. gallisepticum and P. multocida vaccine
G4: Imported 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
SS: Sum of squares MS: Mean of squares
Df: Degree of freedom
Ftab: F tabulated Fcal: F calculated
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
G2: M. gallisepticum vaccine G4: Imported M. gallisepticum vaccine
G3: Combined M. gallisepticum and P. multocida vaccine
Source of Variation SS Df MS F cal P-value F tab Between Groups 89412.26667 2 44706.13 3.082234 0.062277 3.354131 Within Groups 391620.4 27 14504.46
Total 481032.6667 29
SS: Sum of squares MS: Mean of squares
Df: Degree of freedom
Ftab: F tabulated Fcal: F calculated
Results
90
4.3.3. ELISA test:
The data illustrated in Table (19) revealed 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 data illustrated in Table (20) revealed that 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 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.
ANOVA test was used to show difference of the antibody titers
against M. gallisepticum between vaccinated groups and control group.
Results
91
This test revealed significant difference at p≤0.05 with Fcal= 6.495703
and Ftab= 2.866266 (Table 21).
ANOVA test was used to show difference of the antibody titers
against M. gallisepticum between G2, G3 and G4. This test revealed no
significant difference at p≤0.05 with Fcal= 1.166651 and Ftab=
3.354131 (Table 22).
ANOVA test was used to show difference of the antibody titers
against P. multocida between vaccinated groups and control group.
This test revealed significant difference at p≤0.05 with Fcal= 8.786982
and Ftab= 3.354131 (Table 23).
Paired t test was used to show difference of antibody titers against
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:
Interval times of serum collection
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
2 weeks post challenge 1039 1902 1636 0
4 weeks post challenge 2423 4166 3665 0
6 weeks post challenge 2541 4958 3927 0
8 weeks post challenge 2106 3551 3229 0
10 weeks post challenge 1624 2768 2199 0
12 weeks post challenge 1010 1860 1487 0
14 weeks post challenge 743 969 892 0
G2: M. gallisepticum vaccine G4: Imported M. gallisepticum vaccine
G3: Combined M. gallisepticum and P. multocida vaccine
G5: Control 1st vaccination: at 4 weeks of age
Booster vaccination: at 8 weeks of age Challenge: at 11 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:
Interval times of serum collection
Types of vaccines
G1 G3 G5
Prevaccination 0 0 0
1st vaccination
2 weeks post 1st vaccination 206 227 0
Booster vaccination
2 weeks post 2nd vaccination 2391 2487 0
Challenge
2 weeks post challenge 1714 1876 0
4 weeks post challenge 2517 2610 0
6 weeks post challenge 3164 4327 0
8 weeks post challenge 2199 2475 0
10 weeks post challenge 1279 1599 0
12 weeks post challenge 850 948 0
14 weeks post challenge 512 535 0
G1: P. multocida vaccine G5: Control
G3: Combined M. gallisepticum and P. multocida vaccine
1st vaccination: at 4 weeks of age Booster vaccination: at 8 weeks of age
Challenge: at 11 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
G3: Combined M. gallisepticum and P. multocida vaccine
G4: Imported M. gallisepticum vaccine
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
SS: Sum of squares MS: Mean of squares
Df: Degree of freedom
Ftab: F tabulated Fcal: F calculated
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
G2: M. gallisepticum vaccine G4: Imported M. gallisepticum vaccine
G3: Combined M. gallisepticum and P. multocida vaccine
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
SS: Sum of squares MS: Mean of squares
Df: Degree of freedom
Ftab: F tabulated Fcal: F calculated
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
G1: P. multocida vaccine G5: Control
G3: Combined M. gallisepticum and P. multocida vaccine
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
SS: Sum of squares MS: Mean of squares Df: Degree of freedom
Ftab: F tabulated Fcal: F calculated
Table (24): Statistical analysis of ELISA antibody titers against P. multocida between combined vaccine and P. multocida vaccine:
G1 G3 Mean 1483.2 1708.4 Variance 1166729.067 1774683.6 Observations 10 10 Pearson Correlation 0.980423598
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
G3: Combined M. gallisepticum and 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:
Interval times of serum collection
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
4 weeks post challenge 5 0 5 100 0 5 100 5 0 0
6 weeks post challenge 5 0 5 100 0 5 100 5 0 0
8 weeks post challenge 5 0 5 100 0 5 100 5 0 0
P % = No. of survived mice Ⅹ 100
Total No. of mice
S= Survived mice D=Dead mice
G1: P. multocida vaccine G5: Control
G3: Combined M. gallisepticum and P. multocida vaccine
1st vaccination: at 4 weeks of age Challenge: at 11 weeks of age
Booster vaccination: at 8 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:
Interval times of serum collection
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
2 weeks post challenge 5 0 5 100 0 5 100 5 0 0
4 weeks post challenge 5 0 5 100 0 5 100 5 0 0
6 weeks post challenge 5 0 5 100 0 5 100 5 0 0
8 weeks post challenge 5 0 5 100 0 5 100 5 0 0
P % = No. of survived mice Ⅹ 100
Total No. of mice
S= Survived mice D=Dead mice
G1: P. multocida vaccine G5: Control
G3: Combined M. gallisepticum and P. multocida vaccine
1st vaccination: at 4 weeks of age Challenge: at 11 weeks of age
Booster vaccination: at 8 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
G2: M. gallisepticum vaccine G4: Imported M. gallisepticum vaccine
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
G1: P. multocida vaccine
G3: Combined M. gallisepticum and P. multocida vaccine G5: Control
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
G1: P. multocida vaccine
G3: Combined M. gallisepticum and P. multocida vaccine G5: Control
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
combined M. gallisepticum and P. multocida vaccine and P. multocida
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.
Akhtar et al. (2016) concluded that the formalin killed fowl
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
combined inactivated vaccine of M. gallisepticum and P. multocida
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.
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
combined M. gallisepticum and P. multocida vaccine and P. multocida
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.
Akhtar et al. (2016) concluded that the formalin killed fowl
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
combined inactivated vaccine of M. gallisepticum and P. multocida
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,
respectively in comparison with 1.0 for G5.
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,
respectively in comparison with 2.0 for G5.
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
7. References
Abbas, A. K.; Lichtman, A.H. and Pillai, S. (2007): Cells and tissues
of the adaptive immune system. In: Cellular and Molecular
Immunology, 6th
ed., Saunders Elsevier, Philadelphia, pp. 56.
Abdel-Aziz H.M.G.; El-Enbaawy, M.I.H.; Afifi, M.; Ibrahim, S.I.;
Omar, L. and Koudier, M.H. (2015): Efficacy of Montanide
ISA-70-VG as adjuvant to fowl cholera vaccine. J. Vet. Adv.,
5(3): 848-852.
Abd El-Motelib, T.Y. and Kleven, S.H. (1993): A comparative study
of Mycoplasma gallisepticum vaccines in young chickens. Avian
Dis., 37: 981–987.
Aderem, A. and Underhill, D.M. (1999): Mechanisms of phagocytosis
in macrophages. Ann. Rev. Immunol., 17: 593-623.
Adler, B.; Bulach, D.; Chung, J.; Doughty, S.; Hunt,
M.; Rajakumar, K.; Serrano, M.; van Zanden, A.; Zhang,
Y.and Ruffolo, C. (1999): Candidate vaccine antigens and genes
in Pasteurella multocida. J. Biotechnol., 73(2-3): 83-90.
Adler, H.E.; Fabricant, J.; Yamamoto, R. and Berg, J. (1958): Symposium on chronic respiratory disease of poultry, isolation
and identification of pleuropneumonia like organisms of avian
origin. Am. J. Vet. Res., 19: 440-447.
Ahmed, E.S.; Mahmoud, M.S. and Ghoniemy, W.A. (2010):
Immunological studies on a modified adjuvanted fowl
cholera vaccine. Minufiya Vet. J., 7(2): 325-330.
Akhtar, M.; Rahman, M.T.; Ara, M.S.; Rahman, M.; Nazir,
K.H.M.N.H.; Ahmed, S.; Hossen, M.L. and Rahman, M.B.
(2016): Isolation of Pasteurella multocida from chickens,
preparation of formalin killed fowl cholera vaccine, and
determination of efficacy in experimental chickens. J. Adv. Vet.
Anim. Res., 3(1): 45-50.
References
121
Almanama, M. A. (2011): Prevalence of Mycoplasma gallisepticum in
the ten licensed hatcheries in Gaza strip, Palestine. M.Sc. Thesis,
Faculty of Science, Islamic University,Gaza.
Animal and Plant Health Inspection Service (APHIS) (1997):
National Poultry Improvement Plan Guidelines and Regulations,
Vol. APHIS-91-55-038, United States Department of Agriculture,
Beltsville, Maryland, pp. 105.
Asif, M.; Khalid, S.; Sarfaraz, I.; Rahman, S.Ur. and Abdul Rehman
(2015): Comparative efficacy of rapid serum agglutination and
haemagglutination inhibition tests in the seroprevalence study of
Mycoplasma gallisepticum in poultry birds. Sci. Int. (Lahore),
27(5): 4317-4319.
Atlas, R.M. (2004): Hand book of Microbiological media, 3rd
ed., Boca
Raton, London, New York, Washington, D.C., CRC Press.
Avakian, A.P. and Kleven, S.H. (1990): The humoral immune response
of chickens to Mycoplasma gallisepticum and Mycoplasma
synoviae studied by immunoblotting. Vet. Microbiol., 24 (2): 155-
169.
Avakian, A.P.; Dick, J.W. and Derieux, W.T. (1989):
Fowl cholera immunity induced by various vaccines in broiler
minibreeder chickens determined by enzyme-linked
immunosorbent assay. Avian Dis., 33(1): 97-102.
Aye, P.P.; Angrick, E.J.; Morishita, T.Y. and Harr, B.S. (2001):
Prevalence and characteristics of Pasteurella multocida in
commercial turkeys. Avian Dis., 45: 182-190.
Barbour, E.K. and Newman, J.A. (1990): Preliminary data of efficacy
of Mycoplasma gallisepticum vaccines containing different
adjuvants in laying hens. Vet. Immunol. and Immunopathol.,
26(2): 115-123.
Bekele, L. (2015): Isolation, molecular identification and vaccine trial of
Mycoplasma gallisepticum in Ethiopia. M.Sc. Thesis, College of
Veterinary Medicine and Agriculture, Addis Ababa University,
Bishoftu.
References
122
Boyce, J.D.; Harper, M.; Wilkie, I.; Adler, B. (2010): Pasteurella. In:
Pathogenesis of Bacterial Infections in Animals (Eds. Gyles, C.L.;
Prescott, J.F.; Songer, J.G. and Thoen, C.O.), Wiley-Blackwell:
Ames, IA, USA, pp. 325–346.
Boyle, R.C. and Finlay, B. (2003): Bacterial pathogenesis: exploiting
cellular adherence. Curr. Opin. Cell. Biol., 15: 633-639.
Bradbury, J.M. (2001): Avian mycoplasmas. In: Poultry Diseases, 5th
ed. (Eds. Jordan, F.; Pattison, M.; Alexander, D. and Faragher,
T.), W.B. Saunders, London, UK, pp. 178–193.
Brown, D. (2002): Mycoplasmosis and immunity of fish and reptiles.
Frontiers in Biosci., 7: 1338-1346.
Charles, H.D. and Graham, H. P. (1989): Mycoplasmosis. In: A
Laboratory Manual for the Isolation and Identification of Avian
Pathogens, 3rd
ed., Kendall/Hunt publishing com, University of
Pennsylvania, pp.57-62.
Charlton, B.R.; Bermudez, A.J.; Boulianne, M.; Eckroade, R.J.;
Jeffrey, J.S.; Newman, L.J.; Sander, J.E. and Wakenell, P.S.
(1996): Avian Disease Manual (Ed. Charlton, B.R.), Kennett
Square, Pennsylvania, USA, American Association of Avian
Pathologists, pp. 115-125.
Charlton, B.R.; Bermudez, A.; Boulianne, M.; Eckroade, R.; Jeffrey,
J.; Newman, U.; Sander, J. and Wakenell, P. (2005): Avian
mycoplasmosis update. Rev. Bras. Cien. Avic. J., 1: 115-125.
Christensen, J.P. and Bisgaard, M. (2000): Fowl cholera. Rev. Sci.
Tech., 19(2): 626-637.
Chrzastek, K.; Kuczkowski, M.; Wieliczko, A.K.; Bednarek, K.J.;
Wieliczko, A. (2012): Molecular epidemiologic investigation of
polish avian Pasteurella multocida strains isolated from fowl
cholera outbreaks showing restricted geographical and host-
specific distribution. Avian Dis., 56: 529–536.
References
123
Collett, S.R. (2005): Monitoring broiler breeder flocks for Mycoplasma
gallisepticum infection after vaccination with ts-11. M.Sc. Thesis,
Faculty of Veterinary Science, University of Pretoria.
Confer, A.W. (1993): Immunogens of Pasteurella. Vet.
Microbiol., 37(3-4): 353-368.
Cotter, P.F. (2015): An examination of the utility of heterophil-
lymphocyte ratios in assessing stress of caged hens. Poultry
Science, 94: 512–517.
Czifra, G.; Sundquist, B.; Tuboly, T. and Stipkovits, L. (1993):
Evaluation of a monoclonal blocking enzyme linked
immunosorbent assay for the detection of Mycoplasma
gallisepticum-specific antibodies. Avian Dis., 37: 680-688.
Davis, B.D.; Dulbecco, R.; Eisen, H.N.; Ginsberg, H.S. and Wood,
W.B. (1973): Infecções bacterianas e micóticas. São Paulo (SP):
EDART.
Delaplane, J.P. and Stuart, H.O. (1943): The propagation of a virus in
embryonated chicken eggs causing a chronic respiratory disease
of chickens. Amer. J. Vet. Res., 4:325-332.
Dennard, R. (2011): Proteomic variations between a Mycoplasma
gallisepticum vaccine strain and a virulent field isolate. Ph. D.
Thesis, Collage of Arts and Sciences,Georgia State University,
http://scholarworks.gsu.edu/biology_diss/99.
Descamps, S.; Jenouvrier, S.; Gilchrist, H.G. and Forbes, M.R.
(2012): Avian cholera, a threat to the viability of an Arctic seabird
colony. Plos One, 7(2): e29659.
Dhondt, A.A.; DeCoste, J.C.; Ley, D.H.; Hochachka, W.M. (2014):
Diverse wild bird host range of Mycoplasma gallisepticum in
Eastern North America. Plos One 9(7), e103553.
Dickinson, E. and Hinshaw, W. (1938): Treatment of infectious
sinusitis of turkeys with argyrol and silver nitrate. Am. Vet. Med.
Assoc. J., 93:151-156.
References
124
Dodd, S. (1905): Epizootic pneumo-enteritis of the turkey. J.Comp.
Pathol. Ther., 18: 239-245.
Dykstra, M.J.; Levisohn S.; Fletcher O.J. and Kleven S.H. (1985):
Evaluation of cytopathologic changes induced in chicken tracheal
epithelium by Mycoplasma gallisepticum in vivo and in vitro. Am.
J. Vet. Res., 46(1): 116-122.
Dziva, F.; Muhairwa, A.P.; Bisgaard, M. and Christensen, H. (2008):
Diagnostic and typing options for investigating diseases
associated with Pasteurella multocida. Vet. Microbiol., 128(1-2):
1-22.
El-Bayomy, A.A. and Daoud, A.M. (2004): Efficacy of double
adjuvant system on the immune response of fowl cholera
vaccinated chickens. J. Egypt. Vet. Med. Assoc., 64(2): 215-329.
Elfaki, M.G.; Kleven, S.H.; Jin, L.H. and Ragland, W.L. (1992):
Sequential intracoelomic and intrabursal immunization of
chickens with inactivated Mycoplasma gallisepticum bacterin and
iota carrageenan adjuvant. Vaccine, 10: 655-662.
El Gazzar, M.; Laibinis, V.A. and Ferguson-Noel, N. (2011):
Characterization of a ts-11-like Mycoplasma gallisepticum isolate
from commercial broiler chickens. Avian Dis., 55: 569–574.
Evans, R.D.; Hafez, Y.S. and Orthel, F.W. (1992): Mycoplasma
gallisepticum vaccination-challenge: an egg-production model.
Avian Dis., 36: 956-963.
Faruque, M.R. and Christensen, J.P. (2007): Impacts of Mycoplasma
gallisepticum vaccine on Newcastle disease vaccination and
protection in parent stock flocks. Bangladesh J. Microbiol., 24(1):
62-64.
Feberwee, A.; Banniseht-Wysmuller, Th. von; Vernooij, J.C.M.;
Gielkens, A.L.J. and Stegeman, J.A. (2006): The effect of
vaccination with a bacterin on the horizontal transmission of
Mycoplasma gallisepticum. Avian Pathol., 35(1): 35-37.
References
125
Ferguson-Noel, N.; Cookson, K.; Laibinis, V.A. and Kleven, S.H.
(2012): The efficacy of three commercial Mycoplasma
gallisepticum vaccines in laying hens. Avian Dis., 56(2): 272–
275.
Florquin, S.; Amraoui, Z.; Duboi, C.; Decuyper, J. and Goldman, M.
(1994): The protective role of endogenously synthesized nitric
oxide in Staphylococcal enterotoxin-B induced shock in mice. J.
Exp. Med., 180: 1153-1158.
Frey, M.L.; Hanson, R.P. and Anderson, D.P. (1968): A medium for
the isolation of avian Mycoplasmas. Am. J. Vet. Res., 29: 2163-
2171.
Fulton, R.W. (2009): Bovine respiratory disease research (1983-2009).
Anim. Health Res. Rev., 10:131–139.
Gadallah, F.M. (2015): Preparation and evaluation of bivalent
inactivated vaccine against chronic respiratory disease in broiler
chickens. Ph.D. Thesis, Fac. Vet. Med., Beni-Suef University,
Egypt.
Gaunson, J.E.; Philip, C.J.; Whithear, K.G. and Browning, G.F.
(2000): Lymphocytic infiltration in the chicken trachea in
response to Mycoplasma gallisepticum infection. Microbiol., 146
(5): 1223-1229.
Gaunson, J.E.; Philip, C.J.; Whithear, K.G. and Browning, G.F.
(2006): The cellular immune response in the tracheal mucosa to
Mycoplasma gallisepticum in vaccinated and unvaccinated
chickens in the acute and chronic stages of disease. Vaccine, 24:
2627-2633.
Glew, M.D.; Browning, G.F.; Markham, P.F. and Walker I.D.
(2000): pMGA phenotypic variation in Mycoplasma
gallisepticum occurs in vivo and is mediated by trinucleotide
repeat length variation. Infect. Immun., 68(10): 6027-6033.
References
126
Glisson, J.R.; Hofacre, C.L. and Christensen, J.P. (2008): Fowl
cholera. In: Diseases of Poultry (Eds. Saif, Y.M.; Barnes, H.J.;
Glisson, J.R.; Fadly, A.M.; McDougald, L.R. and Swayne, D.E.),
Blackwell Publishing, Ames, Iowa, USA, pp. 739-758.
Gondal, M.A.; Rabbani, M.; Muhammad, K.; Yaqub, T.; Babar,
M.E.; Sheikh, A.A.; Ahmad, A.; Shabbir, M.Z. and Khan,
M.I. (2013): Antibodies response of broilers to locally prepared
oil based Mycoplasma gallisepticum vaccine. J. Anim. Plant Sci.,
23(4): 1094-1098.
Gong, Q.; Qu, N.; Niu, M.; Qin, C.; Cheng, M.; Sun, X. and Zhang,
A. (2013): Immune responses and protective efficacy of a novel
DNA vaccine encoding outer membrane protein of avian
Pasteurella multocida. Vet. Immunol. Immunopathol., 152(3-4):
317-324.
Grodio, J.; Buckles, E. and Schat K. (2009): Production of house finch
(Carpodacus mexicanus) IgA specific anti-sera and its application
in immunohistochemistry and in ELISA for detection of
Mycoplasma gallisepticum-specific IgA. Vet. Immun. and
Immunopathol. J., 132: 288-294.
Harper, M. and Boyce, J.D. (2017): The myriad properties
of Pasteurella multocida Lipopolysaccharide.Toxins, 9(8): 254.
Harper, M.; Boyce, J.D. and Adler, B. (2006): Pasteurella multocida
pathogenesis: 125 years after Pasteur. FEMS. Microbiol. Lett.,
265: 1–10.
Harper, M.; Boyce, J.D. and Adler, B. (2012): The key surface
components of Pasteurella multocida: Capsule and
lipopolysaccharide. Curr. Top. Microbiol. Immunol., 361: 39–51.
Harper, M.; Cox, A.D.; Adler, B. and Boyce, J.D. (2011): Pasteurella
multocida lipopolysaccharide: the long and the short of it. Vet.
Microbiol., 153(1-2): 109-115.
References
127
Harper, M.; John, M.; Edmunds, M.; Wright, A.; Ford, M.; Turni,
C.; Blackall, P.J.; Cox, A.; Adler, B. and Boyce, J.D. (2016):
Protective efficacy afforded by live Pasteurella multocida
vaccines in chickens is independent of lipopolysaccharide outer
core structure. Vaccine, 34(14): 1696-1703.
Harper, M.; Michael, F.St.; John, M.; Vinogradov, E.; Steen, J.A.;
Dorsten, L.V.; Steen, J.A.; Turni, C.; Blackall, P.J.; Adler, B.;
Cox, A.D. and Boyce, J.D. (2013): Pasteurella multocida
Heddleston serovar 3 and 4 strains share a common
lipopolysaccharide biosynthesis locus but display both inter-and
intrastrain lipopolysaccharide heterogeneity. J. Bacteriol.,
195(21): 4854–4864.
Hatfaludi, T.; Al-Hasani, K.; Boyce, J.D. and Adler, B. (2010): Outer
membrane proteins of Pasteurella multocida. Vet. Microbiol., 144
(1–2): 1-17.
Hennigan, S.L.; Driskell, J.D.; Ferguson-Noel, N., Dluhy, R.A.,
Zhao, Y., Tripp, R.A. and Krausea, D.C. (2012): Detection and
differentiation of avian mycoplasmas by surface-enhanced raman
spectroscopy based on a silver nanorod array. Appl. Environ.
Microbiol., 78(6): 1930–1935.
Hopkins, B.A. and Olson, L.D. (1997): Comparison of live avirulent
PM-1 and CU fowl cholera vaccines in turkeys. Avian Dis., 41:
317-325.
Ievy, S; Khan, M.F.R.; Islam, M.A. and Rahman, M.B. (2013):
Isolation and identification of Pasteurella Multocida from
chicken for the preparation of oil adjuvanted vaccine. Microbes
Health, 2(1): 1-4.
Jabbri, A.R. and Moazeni Jula, G.R. (2005): Fowl cholera: Evaluation
of a trivalent Pasteurella multocida vaccine consisted of serotypes
1, 3 and 4. Arch. Razi Ins., 59: 103-111.
References
128
Jacob, R.; Branton, S.L.; Evans, J.D.; Leigh, S.A. and Peebles, E.D.
(2014): Effects of live and killed vaccines against Mycoplasma
gallisepticum on the performance characteristics of commercial
layer chickens. Poult. Sci., 93: 1403–1409.
Jacob, R.; Branton, S.L.; Evans, J.D.; Leigh, S.A. and Peebles, E.D.
(2015): Effects of different vaccine combinations against
Mycoplasma gallisepticum on the internal egg and egg shell
characteristics of commercial layer chickens. Poult. Sci., 94: 912–
917.
Jaffri, K.T.; Gill, Z.J.; Bhatti, A.R. and Raza, A. (2006): Immune
response of buffaloe calves to haemorrhagic septicemia oil
adjuvant and alum precipitated vaccine. Int. J. Agri. Biol., 8(5):
645–647.
Johnson, D. (1983): Role of management and sanitation in controlling
mycoplasma outbreaks. Avian Dis. J., 27: 342-343.
Kahya, S.; Temelli, S.; Eyigor, A. and Carli, T. (2010): Real-time
PCR culture and serology for the diagnosis of Mycoplasma
gallisepticum in chicken breeder flocks. Vet. Microbiol. J., 144:
319-324.
Kardos, G. and Kiss, I. (2005): Molecular epidemiology investigation
of outbreaks of fowl. Journal of Clinical Microbiology, 43: 2959-
2961.
Kasten, R.W.; Carpenter, T.E.; Snipes, K.P. and Hirsh, D.C. (1997):
Detection of Pasteurella multocida specific DNA in turkey flocks
by use of the polymerase chain reaction. Avian Dis., 41: 676-682.
Kempf, I.; Gesbert, F.; Guittet, M.; Bennejean, G. and Stipkovits, L.
(1994): Evaluation of two commercial enzyme-linked
immunosorbent assay kits for the detection of Mycoplasma
gallisepticum antibodies. Avian Pathol., 23: 329-338.
Kleven, S.H. (1998): Mycoplasmosis. In: The Merck Veterinary
Manual, 18th
ed. (Ed. Aiello, S.E.), Merck and Co., Inc.,
Whitehouse Station, NJ, USA, pp. 1926-1932.
References
129
Kleven, S.H. (2002): Recent developments in mycoplasma diagnosis
and control. Proceedings of the Western Poultry Disease
Conference, 51: 109-113.
Kleven, S.H. (2008): Control of avian mycoplasma infections in
commercial poultry. Avian Dis., 52: 367–374.
Krause, D.C. (1996): Mycoplasma pneumoniae cytadherence:
unravelling the tie that binds. Molec. Microbiol., 20 (2): 247-253.
Kreslavsky, T.; Gleimer, M.; Miyazaki, M.; Choi, Y.; Gagnon, E.;
Murre, C.; Sicinski, P. and Boehme, H.V. (2012): β-selection
induced proliferation is required for αβ T cell differentiation.
Immunity, 37(5): 840-853.
Kubatzky, K.F. (2012): Pasteurella multocida and immune cells. Curr.
Top. Microbiol. Immunol., 361: 53-72.
Kwon, Y.K. and Kang, M.I. (2003): Outbreak of Fowl cholera in
Baikal teals in Korea. Avian Dis., 47: 1491-1495.
Levisohn, S. and Kleven, S.H. (2000): Avian mycoplasmosis
(Mycoplasma gallisepticum). Rev. Scient. Tech., 19(2): 425-442.
Levisohn, S.; Menaker, D.; Rosengarten, R. and Yogev, D. (1995a):
The effect of surface antigenic variation on diagnosis of
Mycoplasma gallisepticum infection of poultry. In: Proc. 44th
Western Poultry Disease Conference, 5-7 March, Sacramento,
California, University of California, Davis, 126.
Levisohn, S.; Rosengarten, R. and Yogev, D. (1995b): In vivo
variation of Mycoplasma gallisepticum antigen expression in
experimentally infected chickens. Vet. Microbiol., 45: 219-231.
Ley, D.H. (2003): Mycoplasma gallisepticum infection. In: Diseases of
Poultry, 11th
ed. (Eds. Saif, Y.M.; Barnes, H.J.; Glisson, J.R.;
Fadly, A.M.; McDougald, L.R. and Swayne, D.E.), Iowa State
University Press, Ames, Iowa, USA, pp. 722-744.
References
130
Ley, D.H. (2008): Mycoplasma gallisepticum infection. In: Diseases of
Poultry, 12th
ed. (Eds. Saif, Y.M.; Fadly, A.M.; Glisson, J.R.;
McDougald, L.R.; Nolan, L.K. and Swayne, D.E.), Blackwell
Publishing, Ames, IA., pp. 807-834.
Ley, D. H. and Yoder, H.W.Jr. (1997): Mycoplasma gallisepticum
infection. In: Diseases of Poultry, 9th
ed. (Eds. Calnek, B.W.;
Barnes, H.J.; Beard, C.W.; McDougald, L.R. and Saif, Y.M.),
Iowa State University Press, Ames, IA., pp. 194–207.
Ley, D.H.; Berkhoff, E. and Levisohn, S. (1997): Molecular
epidemiologic investigations of Mycoplasma gallisepticum
conjunctivitis in songbirds by random amplified polymorphic
DNA analyses. Emerging Infect. Dis., 3: 375-380.
Lin, M.; Wu, Y.; Cheng, J.; Lin, G.; Tung, M.; Lan, Y.; Sung, H.
and Cheng, C. (1996): Isolation of avian Mycoplasmas and
Salmonella spp. and serological survey of Newcastle disease, egg
drop syndrome, pullorum disease and two avian mycoplasmas in
sparrows flying around chicken farms. Taiwan Vet. J., 66: 125-
131.
Liu, T.; Garcia, M.; Levisohn, S.; Yogev, D. and Kleven, S.H. (2001):
Molecular variability of the adhesin-encoding gene pvpA among
Mycoplasma gallisepticum strains and its application in diagnosis.
J. Clinic. Microbiol., 39: 1882-1888.
Lobão, M.L.; Lima, C.B.S.; Nascimento, E.R.; Santos, M.G.;
Medeiros, M.I.M.; Barreto, M.L.; Lemos, M.; Silva, R.C.F.
and Pereira, V.L.A. (2003): Micoplasmoses em Avestruzes
(Struthio camelus). In: 8ª Semana de Extensão da UFF, Niterói,
Rio de Janeiro, Brasil, pp.181-182.
Majumder, S. (2014): Role of Mycoplasma gallisepticum and host
airway epithelial cell interaction in inflammation. Ph.D. Thesis.
University of Connecticut, United States of America.
Mallinath, K.C. and Hari Babu, Y.A. (2013): Study on isolation of
mycoplasma from cases of avian mycoplasmosis. Frontier J. Vet.
Anim. Sci., 2(2): 96-99.
References
131
Markham, F.S. and Wong, S.C. (1952): Pleupneumonia-like organisms
in the etiology of turkey sinusitis and chronic respiratory disease
of chickens. Poult. Sci., 31:902-904.
Moura, L.; Dohms, J.; Almeida, J.M.; Ferreira, P.S.; Biffi, C.P. and
Backes, R.G. (2012): Development and evaluation of a novel
subunit vaccine for Mycoplasma gallisepticum. Arq. Bras. Med.
Vet. Zootec., 64(6): 1569-1576.
Mukherjee, F.; Tiwary, B.K.; Prasad, R.L.; Jha, G.J. and Roy, M.K.
(1990): Role of T and B cells in immune response to Mycoplasma
gallisepticum infection in normal and immunocompromised
chickens. Indian J. Anim. Sci., 60: 1027-1031.
Mukkur, T.K.S.; Pyliotis, N.A. and Bones, A. (1982): Possible
immunological synergism among the protective antigens of P.
multocida type A. J. Comp. Pathol., 92: 249-260.
Municio, C.; Alvarez, Y.; Montero, O.; Hugo, E.; Rodríguez, M.;
Domingo, E.; Alonso, S.; Fernández, N. and Crespo, M.S.
(2013): The response of human macrophages to β-Glucans
depends on the inflammatory Milieu. Plos One, 8(4).
Nascimento, E.R.; Pereira, V.L.A.; Nascimento, M.G.F. and Barreto,
M.L. (2005): Avian mycoplasmosis update. Brazilian J. Poult.
Sci., 7(1): 1-9.
Nicholas, R.A.; Ayling, R.D. and McAuliffe, L. (2009): Vaccines for
mycoplasma diseases in animals and man. J. Comp. Pathol.,
140(2-3): 85-96.
Noormohammadi, A. H. (2007): Role of phenotypic diversity in
pathogenesis of avian mycoplasmosis. Avian Pathol., 36: 439–
444.
Obukhovska, O.V.; Stegniy, B.T.; Glebova, K.V.; Shutchenko, P.O.
and Medved, K.O. (2015): The macrophages accumulation in
chickens vaccinated against avian mycoplasmosis. J. Vet. Med.,
Biotech. Bios., 1(1): 5-8.
References
132
OIE (2012): Avian mycoplasmosis (Mycoplasma gallisepticum, M.
synoviae), Ch. 2.3.5. In: Manual of Diagnostic Tests and Vaccines
for Terrestrial Animals, 7th
ed., Vol. 1, France, pp. 455- 469.
Papazisi, L.; Frasca, S.Jr.; Gladd, M.; Liao, X.; Yogev, D. and
Geary, S.J. (2002): GapA and CrmA coexpression is essential for
Mycoplasma gallisepticum cytadherence and virulence. Infect.
Immun., 70: 6839–6845.
Pardon, B.; De Bleecker, K.; Dewulf, J.; Callens, J.; Boyen, F.;
Catry, B. and Deprez, P. (2011): Prevalence of respiratory
pathogens in diseased, nonvaccinated, routinely medicated veal
calves. Vet. Rec., 169: 278.
Parvin, M.S.; Siddique, M.P. and Islam, M.T. (2011): Humoral
immune response to fowl cholera vaccine in different breeds of
commercial birds. Bangl. J. Vet. Med., 9 (2): 127 – 131.
Pasteur, L. (1880): De l’att´enuation du virus du chol´era des poules. C
R Acad Sci., 91: 673–680.
Pasteur, L. (1881): Sur les virus-vaccins du chol´era des poules et du
charbon. C R Trav Congr Int Dir Agronom session de Versailles:
151–162.
Perelman, B.; Hadash, D.; Meroz, M.; Gur-Lavie, A.; Abramson, M.
and Samberg, Y. (1990): Vaccination of young turkeys
against fowl cholera. Avian Pathol., 19(1): 131-137.
Petersen, K.D.; Christensen, J.P.; Permin, A. and Bisgaard, M.
(2001): Virulence of Pasteurella multocida subsp. multocida
isolated from outbreaks of fowl cholera in wild birds for domestic
poultry and game birds. Avian Pathol., 30: 27-31.
Polo, P.A.; Nascimento, E.R.; Pereira, V.L.A.; Barreto, M.L.;
Nascimento, M.G.F.; Zuanaze, M.A.F.; Scanavine-Neto, H.
and Silva, R.C.F. (2002): Perfil sorológico de galinhas SPF
Imunizadas com vacinas e cepa atenuada de Mycoplasma
gallisepticum Diferenciadas Por PCR-RAPD. Revista Brasileira
de Ciência Avícola, Supl., 4:118.
References
133
Quinn, P.J.; Carter, M.E.; Markey, B.; and Carter, G.R. (2002): The
Mycoplasmas. In: Clinical Veterinary Microbiology, Mosby,
Virginia Tech and Blacksburg, USA, pp.320-326.
Rajaraman, V.; Nonnecke, B.J.; Franklin, S.T.; Hamell, D.C. and
Horst R.L. (1998): Effect of vitamin A and E on nitric oxide
production by blood mononuclear leukocytes from neonatal
calves fed milk replacer. J. Dairy Sci., 81: 3278-3285.
Raviv, Z.; Callison, S.A.; Ferguson-Noel, N. and Kleven, S.H. (2008):
Strain differentiating real-time PCR for Mycoplasma
gallisepticum live vaccine evaluation studies. Vet. Microbiol.,
129: 179–187.
Razin, S. (1992): Mycoplasma taxonomy and ecology. In:
Mycoplasmas: molecular biology and pathogenesis (Eds.
Maniloff, J.; McElhaney, R.N.; Finch, L.R. and Baseman, J.),
Washington, DC: American Society for Microbiology, pp. 3-22.
Razin, S. (1999): Adherence of pathogenic mycoplasmas to host cells.
Biosci. Rep., 19(5): 367-372.
Razin, S.; Yogev, D. and Naot, Y. (1998): Molecular biology and
pathogenicity of mycoplasmas. Microbiol. Mol. Bio. Rev., 62:
1094-1156.
Rimler, R.B. (1994): Presumptive identification of Pasteurella
multocida serogroups A, D, and F by capsule depolymerisation
with mucopolysaccharidases. Vet. Rec., 134, 191–192.
Rocke, T.; Yuill, T. and Amundson, T. (1988): Experimental
Mycoplasma gallisepticum infections in captive-reared wild
turkeys. Wild life Dis. J., 24: 528- 532.
Rosado, I.; Ruedas, D.; Sanchez, L.; Chavez, Y. R.; Perez, T.; Lobo,
E.; Miranda, I.; Bulnes, C.; Martinez, S.(2004): Evaluation of a
bacterin against mycoplasma. Revista Cubana de Ciencia Avicola,
28(2): 33-39.
References
134
Sander, J.E.; Resurreccion, R.S.; Waltman, W.D. and McMurray,
B.L. (1998): Pasteurella challenge and ELISA serology
evaluation of broiler breeders vaccinated with live cholera
vaccine. Avian Dis., 42:190-193.
Sarfaraz, S.; Muhammad, K.; Yaqub, T.; Aslam, A.; Rabbani, M.;
Khalil, M. and Riaz, R. (2017): Antibody response of broilers to
oil based combined avian influenza (H9 N2) and Mycoplasma
gallisepticum vaccine. J. Anim. Plant Sci., 27(4): 1150-1154.
Sawada, T.; Borrathybay, E.; Kawamoto, E.; Koeda, T. and Ohta, S.
(1999): Fowl cholera in Japan: disease occurrence and
characteristics of Pasteurella multocida isolates. The bulletin of
the Nippon Veterinary and Animal Science University, 48: 21-32.
Sawada, T.; Rimler, R.B. and Rhoades, K.R. (1982): Indirect
hemagglutination test that uses glutaraldehyde- fixed sheep
erythrocytes sensitized with extract antigens for detection of
pasteurella antibody. J. Clin. Microbiol., 15(5): 752-756.
Senterifit, L.B. (1983): Preparation of antigens and antisera, Ch. F2. In:
Methods in Mycoplasmology (Eds. Razin S. and Tully J.G.), Vol.
1, New York, Academic press.
Shafay, S.M. (1995): Trials for combined vaccination against infectious
coryza, fowl cholera and mycoplasma infection. Ph.D. Thesis,
Fac. Vet. Med., Cairo University, Egypt.
Stafford, J.L.; Neumann, N.F. and Belosevic, M. (2002):
Macrophage-mediated innate host defense against protozoan
parasites. Crit. Rev. Microbiol., 28: 187-248.
Stipkovits, L.; Egyed, L.; Palfi, V.; Beres, A.; Pitlik, E.; Somogyi,
M.; Szathmary, S. and Denes, B. (2012): Effect of low-
pathogenicity influenza virus H3N8 infection on Mycoplasma
gallisepticum infection of chickens. Avian Pathol., 41(1): 51-57.
Suling, L.; Miao, T.; Sebastian, M.; Bhullar, P.; Ghaffari, E.; Liu,
M.; Symonds, A.L.J. and Wang, P. (2012): The transcription
factors Egr2 and Egr3 are essential for the control of
References
135
inflammation and antigen-induced proliferation of B and T cells.
Immunity, 37: 685-696.
Tabatabaei, M.; Moazeni Jula, G.R.; Jabbri, A.R. and Esmailzadeh,
M. (2007): Vaccine efficacy in cattle against haemorrhagic
septicemia with live attenuated aroA mutant of Pasteurella
multocida B:2 strain. Journal of Cell and Animal Biology, 1 (4):
062-065.
Talan, D.A.; Citron, D.M.; Abrahamian, F.M.; Moran, G.J.;
Goldstein, E.J. (1999): Bacteriologic analysis of infected dog
and cat bites. Emergency Medicine Animal Bite Infection Study
Group. N. Engl. J. Med., 340(2): 85–92.
Umar, S.; Munir, M.T.; Ur-Rehman, Z. and Subhan, S. (2017):
Mycoplasmosis in poultry: update on diagnosis and preventive
measures. World's Poultry Science Journal, 73(1): 17-28.
Whithear, K.G. (1996): Control of avian mycoplasmoses by
vaccination. Rev. Sci. tech. Off. int. Epiz., 15(4): 1527–1553.
Wilkie, I.W.; Harper, M.; Boyce, J.D. and Adler, B. (2012):
Pasteurella multocida: Diseases and pathogenesis. Curr.
Top.Microbiol. Immunol., 361: 1–22.
Wilson, B.A. and Ho, M. (2013): Pasteurella multocida: From zoonosis
to cellular microbiology. Clin. Microbiol. Rev., 26(3): 631–655.
Winner, F.; Rosengarten, R. and Citti, C. (2000): In vitro cell
invasion of Mycoplasma gallisepticum. Infect. Immun. J., 68:
4238 - 4244.
Xiao, K.; Liu, Q.; Liu, X.; Hu, Y.; Zhao, X. and Kong, Q. (2016):
Identification of the avian Pasteurella multocida phoP gene and
evaluation of the effects of phoP deletion on virulence and
immunogenicity. Int. J. Mol. Sci., 17(1): 12.
References
136
Yoder, H.W.Jr. (1979): Serological response of chickens vaccinated
with inactivated preparation of Mycoplasma gallisepticum. Avian
Dis., 23(2): 493-506.
Yoder, H.W.Jr. (1991): Mycoplasma gallisepticum infection. In:
Diseases of Poultry (Eds. Calnek, B.W.; Beard, C.W.; Barnes,
H.J.; Reid, W.M. and Yoder, H.W.Jr.), Iowa State University
Press, Ames, Iowa, USA, pp. 198- 212.
Youssef, E.A. and Tawfik, H.E. (2011): Improvement of rabbit
pasteurellosis vaccine using Montanide ISA50. Egypt. J. Agric.
Res., 89(2): 697-708.
Zhang, Y.F.; Wulumuhan, N.; Gong, F.J. and Entomack, B. (2013):
Construction and characterization of an acapsular mutant of
Pasteurella multocida strain P-1059 (A: 3). J. Vaccines Vaccin.,
4: 184.
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