1. Abd El-Hamid HS1; Ellakany HF 1;
Okeila MA2 & Abd El-Halim BA
Isolation and Molecular Detection of
M. gallisepticum F vaccine Like Strains from
Chicken Flocks Suffering from Respiratory
Problems in Egypt
1 Fac. Vet. Med., Damnhour Univ., Poultry and Fish Diseases Dept.
2 Microbiology Dept, Fac. Vet. Med., Alex. Univ., Egypt
2. There are continuous changes in the genetic
make up and antigenicity of MG field strains
leading to emergence of variant strains.
Variant strains of MG are source of lab. problem
in isolation, serologic diagnosis by ELISA as well
as PCR detection.
Restriction Fragment Length Polymorphism
(RFLP of pvpA gene) is a valuable molecular tool
for diagnosis of MG and for differentiation of F
vaccine like strain from field isolates.
Introduction
3. Aims of the present study
(1) To survey the incidence of MG in broiler, layer and breeder
flocks in 3 governorates ( البحيرة–الغربية–الشيخ كفر )
(2) To detect the genetic relatedness between classic MG field
isolates and
MG-F-vaccine like strains.
Method
1- Semi nested PCR on 2 steps using 4 types of primers of pvpA (a
phase-variable putative adhesin protein gene)
2- Restriction fragment length polymorphism (RFLP) assay of pvpA
Aim
(3) To study the pathogenicity and transmissibility of 3 selected
MG field isolates and also the transmissibility of MG-F
vaccine strain between an infected and contact chickens.
Method
1- Assessment of degree of clinical signs, air sac lesions after
an experimental infection.
2- PCR and serological testing at 14 and 28 days PI with 3
isolates and MG-F vaccine at 1 day old.
4. Material & Methods
All swabs were from respiratory diseased
chickens.
Source of samples:
33 broiler flocks,
12 layer flocks,
4 broiler breeder flocks and.
Samples: 49 tracheal swabs (4 tracheal swabs
/flock)
Swabs were suspended in Frey’s broth media and
stored at –20 C for PCR and bacteriological
cultivation.
5. Principles of The Semi Nested PCR
Two step amplifications were performed targeting
pvpA gene,
then amplification of C-terminus-encoding region of
pvpA gene
to increase specificity
Primer selection: (Boguslavsky et al., 2000).
Initial primers were selected from the conserved sequences of
pvpA gene of MG- R strain
forward primers: pvpA1F, located at nucleotide positions 415 - 437
(5'GCC AMT CCA ACT CAA CAA GCT GA3')
reverse primers: pvpA2R, located at nucleotide positions 1059 -
1081
(5'GGA CGT SGT CCT GGC TGG TTA GC3').
Secondary primers flanked the direct repeat sequence within the
C-terminus-encoding region of the pvpA gene
forward primers: pvpA3F, located at nucleotide positions 583 - 604
(5'GGT AGT CCT AAG TTA TTA GGT C3')
reverse primers: pvpA2R, located at nucleotide positions 1059 -
1081
6. Amplifications
All primers were obtained from AccuOligo®, BIONEER.
The PCR reactions were performed with 10 pmole per
reaction.
The amplification were done in 50 µl volumes.
25 µl Master mix (Fermentas # K1081), 0.5 µl from both
outer forward primers and reverse primers, 1 µl template
DNA and 23 µl Water nuclease free.
In the second amplification, 1 µl from the product of the
first amplification was used as template.
7. MG PCR + ve flocks were 37 (out of 49)
(Total incidence = 75.5%)
The individual incidence was:
66.6% in layers
75.75% in broilers
100% in broiler breeder
Results
9. RFLP testing was performed for PCR-positive MG and
MG-F vaccine to detect the genetic relatedness
among them.
The restriction enzyme digestion was performed in 30
µl reaction mixtures (10 µl of PCR product, 1 µl of
FastDigest® PvuII enzyme, 2 µl of 10× FastDigest®
green buffer, 17 µl of nuclease free water #R0581,).
Then mixtures were incubated at 37 C for 10 minutes.
The digested PCR products were run on 1.5% agarose
gel electrophoresis using 1X TBE buffer at a constant
80 Volt for 100 min.
Gels were visualized by UV light.
Restriction fragment length polymorphism
(RFLP)
10. Restriction fragment length polymorphism
(RFLP)
Data analysis:
RFLP data were analyzed through Total Lab. Software analysis
(version 1.1)
Also Cluster Analysis was performed via BLAST program (version 1).
Results
11. Phylogenic analysis of
the C-terminus-encoding portion of pvpA gene
based on RFLP results.
The isolates sampled in the same month
and from the same geographic area,
showed similar RFLP patterns and
located very closely in the phylogenic
tree with genetic similarity of more than
95%.
6 MG isolates (no. 1,3,4,5,6 & 19) were
more related to MG-F vaccine strain with
more than 92% genetic similarity.
2 MG isolates (no. 9 & 28) were both
from one layer flock, one isolate obtained
at 6 days of age while the other was
recovered at 25 wks of age,
But they showed 38% relatedness.
12. Experimental design:
A total number of 220-one-day-old grand parent
male chicks were divided into 6 groups:
Groups 1, 2, 3; contained 120 chicks (40 in each,
where 20 were infected with 3 individual isolates no.
23, 36 and 37 respectively, + 20 x 3 chicks in each
group were left in contact to the infected mates in the
same battery section).
Group 4; contained 40 chicks (20 chicks were
vaccinated with single dose of F strain vaccine + 20
chicks were left in contact in the same battery
section).
Group 5; included 40 chicks (20 were vaccinated
with double dose of F strain vaccine + 20 chicks
were left in contact in the same pen).
Group 6; contained 20 chicks kept as non
vaccinated, non infected control group in a separate
13. MG ELISA immune response to field isolates and MG-F strain
vaccine:
One dose- vaccinated group only one bird out of 5 showed
very low titers after 5 weeks PI.
Double dose vaccinated group; 1 bird with high titer; 2 birds
with low titers.
Contact – to vaccinated birds were negative along the trial for
5 weeks.
Contact – to infected birds were negative along the
experiment for 5 weeks.
Results
PCR from experimental chicks showed that contact non birds
were positive starting from 14th day PI indicating shedding and
lateral transmission.
