Original Article
Assessment
of mycoplasma vaccine efficacy in reducing infection with Newcastle disease
virus
Evaluación de la eficacia de la vacuna contra
micoplasma para reducir la infección por el virus de la enfermedad de Newcastle
Hala Mahmoud* ORCID: https://orcid.org/0000-0003-2462-956X
Marwa Fathy Elsayed ORCID: https://orcid.org/0000-0002-3794-9071
Reem A. Soliman ORCID: https://orcid.org/0000-0003-4074-4584
Mounir El Safty ORCID: https://orcid.org/0000-0003-1199-1375
Moustafa A. Zaghloul
ORCID: https://orcid.org/0000-0002-8201-7417
Central Laboratory for Evaluation of Veterinary
Biologics, Agriculture Research Centre, Cairo, Egypt.
Corresponding author: haloldodo123@yahoo.com
ABSTRACT
The present work recorded
the impact of using Mycoplasma gallisepticum vaccines
on post-vaccinal response and protection against challenge with Newcastle
disease virus. Specific pathogen-free chickens were divided into eight groups
of forty chickens each. Group G1 was vaccinated with Mycoplasma gallisepticum live attenuated and Mycoplasma gallisepticum inactivated vaccines. Group G2 was
vaccinated with Mycoplasma gallisepticum live
attenuated, Mycoplasma gallisepticum
inactivated and Newcastle disease inactivated vaccines. Group G3 was vaccinated
with Mycoplasma gallisepticum live attenuated
vaccine. Group G4 was vaccinated with Mycoplasma gallisepticum
live attenuated and Newcastle disease inactivated vaccines. Group G5 was
vaccinated with Mycoplasma gallisepticum
inactivated vaccine. Group G6 was vaccinated with Mycoplasma gallisepticum inactivated and Newcastle disease
inactivated vaccines. Group G7 was vaccinated with Newcastle disease
inactivated vaccine. Group G8 was kept as non-vaccinated control. The
Newcastle disease hemagglutination inhibition antibodies and mortality
percentages were measured. Group G7 recorded the best protective Newcastle disease
hemagglutination inhibition antibody titer (7 log2). Group G2
recorded a marginal satisfactory antibody titer (6 log2) after
vaccination by the three tested vaccines. The remaining groups revealed
unsatisfactory titers ranged from 0-5. The protection levels for G2, G4, G6 and G7
ranged from 70% to 100%, but only G2 and G7 were considered protected. G1, G3, G5
and G8 showed typical clinical signs of Newcastle disease. The Mycoplasma
gallisepticum vaccines couldn’t improve the
response to Newcastle disease inactivated vaccine. The results suggest that Mycoplasma
gallisepticum
vaccination is immunosuppressive rather than immunomodulatory in Newcastle disease
vaccination.
Keywords: Mycoplasma gallisepticum; Newcastle disease virus; vaccines;
mortality; virus shedding.
RESUMEN
En el presente trabajo se registró el impacto de la
utilización de vacunas contra Mycoplasma gallisepticum sobre la respuesta posvacunal
y la protección frente al reto con el virus de la enfermedad de Newcastle.
Pollos libres de patógenos específicos se distribuyeron en ocho grupos de
cuarenta pollos cada uno. El grupo G1 se vacunó con vacunas vivas atenuadas e
inactivadas contra Mycoplasma gallisepticum. Al grupo G2 se le aplicaron las vacunas:
viva atenuada contra Mycoplasma gallisepticum, inactivada contra Mycoplasma
gallisepticum e inactivada contra la enfermedad
de Newcastle. El grupo G3 se inmunizó con la vacuna viva atenuada contra Mycoplasma gallisepticum;
el G4, con las vivas atenuadas contra Mycoplasma
gallisepticum e inactivada contra la enfermedad
de Newcastle; el G5, con la vacuna inactivada contra Mycoplasma
gallisepticum; el G6 con las vacunas inactivadas
contra Mycoplasma gallisepticum
y la enfermedad de Newcastle; el G7, con la vacuna inactivada contra la
enfermedad de Newcastle y el G8 se mantuvo como control no vacunado. Se
midieron los anticuerpos de inhibición de la hemaglutinación contra el virus de
la enfermedad de Newcastle y los porcentajes de mortalidad. El grupo G7
registró el mejor título de anticuerpos inhibidores de la hemaglutinación
contra la enfermedad de Newcastle (7 log2). El grupo G2 registró un
título de anticuerpos marginalmente satisfactorio (6 log2) tras la
vacunación con las tres vacunas ensayadas. Los demás grupos revelaron títulos
insatisfactorios que oscilaban entre 0 y 5. Los niveles de protección de los
grupos G2, G4, G6 y G7 oscilaron entre el 70% y el 100%, pero sólo G2 y G7 se
consideraron protegidos. Los grupos G1, G3, G5 y G8 mostraron signos clínicos
típicos de la enfermedad de Newcastle. Las vacunas contra Mycoplasma
gallisepticum no pudieron mejorar la respuesta a
la vacuna inactivada contra la enfermedad de Newcastle. Los resultados revelan
que la vacunación con Mycoplasma gallisepticum es más inmunosupresora que inmunomoduladora en la vacunación contra la enfermedad de
Newcastle.
Palabras clave: Mycoplasma gallisepticum; virus de la enfermedad de newcastle;
vacunas; mortalidad; esparcimiento de virus.
Recibido: 12 de enero de 2023
Aceptado: 14 de junio de 2023
Introduction
Newcastle
disease (ND) is a viral disease of poultry caused by Newcastle disease virus
(NDV), a single-stranded RNA avian paramyxovirus type 1. The disease is present
worldwide and infects most bird species, causing huge losses in the poultry
sector. In developing countries, outbreaks of ND have occurred in many areas,
resulting in severe economic and commercial losses. It is an endemic disease in
Egypt.(1)
The primary NDV
control strategies depend mainly on vaccination with live attenuated or
inactivated vaccines.(2) In
United States, during the outbreak of California in 2002–2003, about 2,500
premises (4 million birds) were depopulated. Losses were estimated at $162 US
million. In 2008, the World Organization for Animal Health (OIE) considered ND
with certain virulence criteria, a notifiable disease.(3)
Mycoplasma gallisepticum (MG) is a bacterial
pathogen which causes chronic respiratory disease (CRD) with economic losses.(4)
It was listed by the OIE as a primary cause of CRD of poultry.(3)
The infection usually produces mild symptoms with the ability to synergize with
other avian respiratory agents such as infectious bronchitis virus, NDV and Escherichia
coli, therefore mycoplasmosis is considered an
economically important disease.(5) Poultry producers depend mainly
on vaccination and biosecurity to manage mycoplasmosis.(6)
MG induces
immunosuppressive effects by damaging the immune system and affecting B and T
cells development, leading to an impairment of chicken immune system. In turn,
it leads to down-regulation of the post-vaccination immune response and
consequently results in limited development of protection. Earlier reports
demonstrated the immunosuppressive effect of MG in chickens vaccinated with NDV
vaccine. This was confirmed by the reduction of hemagglutination inhibition
(HI) titer and IgG antibody titers against NDV,(7)
as well as delayed cytokine response initiation. Additionally, there is a
lack of information concerning the immune response to mixed MG and NDV vaccines
in Egypt. Studies are needed to evidence the adverse effects of MG vaccination
on the immune system at the time of ND vaccination.
The present study was
conducted to evaluate the most protective and effective vaccination program
against ND and GM vaccines, in order to contribute to the optimization of
vaccination programs to achieve the best flock immune status.
Material and Methods
Vaccines
Three commercial vaccines
were used in the current study: a live attenuated MG vaccine, an inactivated MG
vaccine and an inactivated ND vaccine.
Virus
A virulent NDV local
isolate type (genotype 7): 7 NDV-B7-RLQP-CH-EG-12, accession No KM288609 was
supplied by the Central Laboratory for Evaluation of Veterinary Biologics
(CLEVB), Abbasia, Cairo, Egypt which has been
routinely used at CLEVB for challenge testing.
Specific pathogen free
chickens and eggs
Three hundred and twenty,
5-weeks old specific pathogen free (SPF) chickens and 9 days old embryonated chicken
eggs (ECE) were obtained from the SPF Egg Production Farm, Koum
Osheim, El-Fayoum, Egypt.
Blood samples were
collected from all groups prior to vaccination to assure its freedom of MG
and NDV, to be used as negative control for further investigations.
Experimental design
Eight groups of SPF
chickens, 40 chickens per each, were vaccinated and challenged at the following
time intervals (Table 1).
Table 1. Vaccination-challenge
groups design.
|
|
Age of Vaccination |
|
|
Group |
5 weeks |
8 weeks |
12 weeks |
NDV challenge age |
G1 |
MG live attenuated |
MG inactivated |
|
12 weeks |
G2 |
MG live attenuated |
MG inactivated |
ND inactivated |
16 weeks |
G3 |
MG live attenuated |
|
|
9 weeks |
G4 |
MG live attenuated |
ND inactivated |
|
12 weeks |
G5 |
|
MG inactivated |
|
12 weeks |
G6 |
|
MG inactivated |
ND inactivated |
16 weeks |
G7 |
ND inactivated |
|
|
9 weeks |
G8 |
Control |
|
|
16 weeks |
Vaccination and boosting
As shown in Table 1, at 5
weeks old, SPF chicks in G1, G2, G3 and G4 were immunized with live attenuated
MG vaccines and G7, with inactivated ND. Three weeks later (8 weeks old), G1
and G2 were boosted with inactivated MG; G4, with inactivated ND and G5 and G6
were vaccinated with inactivated MG. At 12 weeks old, G2 and G6 were vaccinated
with inactivated ND. All vaccines were administered by the recommended route
(subcutaneous) and dose (0.5mL).
Serum plate agglutination
Blood samples were
collected on 28th day after last immunization for serological
testing by serum plate agglutination (SPA) test to
assure formation of protective antibody against MG.(3)
Hemagglutination inhibition
test
The collected serum samples
of all experimental groups were also tested to determine the NDV antibody titer
of each vaccinated group by HI test as mentioned in OIE.(8) Two fold serial dilutions of serum samples were
applied from 1/2 to 1/2048 against 4 HA units of ND antigen 106 EID50/0.1
mL by HA test.(8) The geometric mean
of ND HI antibody titer was calculated.(8,9)
The mean HI titer has to be not less than 6 1og2.
Challenge test
Ten birds from each group
were challenged (at different weeks according to the group, Table 1) with 106
LD50 NDV(7 NDV-B7-RLQP-CH-EG-12, accession No KM 288609), 1 mL/
bird, and were inspected for further 6 days for clinical signs and mortalities.(8,10)
Virus shedding post challenge was shown by tracheal swabs collected on days 1,
3 and 5 from live birds.(11) All NDV swabs were titrated using 9 day
old SPF ECE;(12) virus shedding was calculated using Kärber method.(9) At the end of observation
days, live birds with moderate to severe signs were humanly euthanized for
detection of ND post-mortem gross lesions.(13) NDV
protection percentage has to be 90% or higher.
Ethical approval
All
animal related procedures were applied with relevant guidelines and regulations
of Veterinary Cairo University Institutional Animal Care and Use Committee (Vet.CU-IACUC),
according to local Egyptian laws. The study was approved ethically by CLEVB,
Cairo, Egypt.
Results
Serum
plate agglutination of Mycoplasma gallisepticum vaccinated groups
All
sera from the MG vaccinated groups showed positive results by serum plate
agglutination test.
Hemagglutination inhibition
titers of Newcastle disease vaccinated groups
The
mean HI titer was calculated for all experiment groups (G1-G8) after blood
samples were collected separately from each vaccinated group before the
challenge test. The G7 immunized with ND vaccine (at 5 weeks) recorded the best
protective ND antibody titer (7 log2). Also, G2 recorded a marginal
satisfactory antibody titer (6 log2) after vaccination with the
three tested vaccines. The remaining groups revealed unsatisfactory titers
ranging from 0-5 (Table 2).
Table
2. Newcastle disease virus hemagglutination inhibition titers of
vaccinated groups.
Group |
G1 |
G2 |
G3 |
G4 |
G5 |
G6 |
G7 |
G8 |
HI titer log2 |
0 |
6 |
0 |
5 |
0 |
4.1 |
7 |
0 |
Post challenge protection rates
The
protection level was evaluated according to the percentage of mortality,
morbidity and virus shedding after inoculation of tracheal swabs into 9-day-old
SPF ECE. The groups vaccinated with ND showed highest protection rates as
follow: 90%, 80%, 70% and 100% for groups G2, G4, G6 and G7, respectively. Only
G2 and G7 were considered protected (Table 3).
Table
3. Protection percentages against virulent NDV post challenge.
|
|
n= 10 |
|
Groups |
Positive |
Negative |
Protection (%) |
G1 |
8 |
2 |
20 |
G2 |
1 |
9 |
90 |
G3 |
8 |
2 |
20 |
G4 |
2 |
8 |
80 |
G5 |
10 |
0 |
0 |
G6 |
3 |
7 |
70 |
G7 |
0 |
10 |
100 |
G8 |
10 |
0 |
0 |
Groups
G1, G3, G5 and G8 showed typical clinical signs of NDV, mainly nervous
manifestations with nasal and ocular discharge. At post mortem (PM) inspection,
the same groups showed characteristics of NDV infection, such as punctate
hemorrhages in the proventriculus and cecal tonsils,
with congested trachea and catarrhal exudates (Fig. 1).
Fig.
1. Post challenge lesion, post mortem inspection,
showing pin pointed hemorrhages at the proventriculus.
The
results of NDV shedding post challenge were consistent with mortality and
morbidity rates (Fig. 2). Groups immunized with ND vaccines (G2, G4, G6 and G7)
showed low or no virus shedding post challenge.
Fig.
2. NDV shedding post challenge with virulent Newcastle disease virus.
Discussion
Over
the past years, studies have been conducted on immunosuppressive effect of
bacteria at the time of ND, which may render vaccine efficacy by affecting the
immune response against ND vaccine.(14) The current study evaluates
the impact of using MG vaccines for chicken immunization before vaccination
with ND vaccine, for this purpose, the humoral immune response by detection of
antibodies anti-hemagglutinin protein using HI test and the ND vaccine
protective efficacy after challenge test were evaluated.(15)
The
antibody titer of the ND vaccinated groups is directly proportional to the
immunogen retention time. The present study showed that the humoral immune
response against NDV decreased in all MG vaccination models. Low titers of
antibodies against NDV in birds vaccinated with either live attenuated or
inactivated MG confirmed its immunosuppressive effect.(16)
The
present work also studied the impact of using live attenuated and inactivated MG vaccination on NDV experimental
infection. The immune response of all vaccinated groups was evaluated; sera
from groups G2, G4, G6 and G7 were especially considered, since these chickens
were vaccinated with NDV inactivated vaccine, in addition to different MG
vaccines in several schedules.
Groups
vaccinated with inactivated or live attenuated MG vaccines and ND vaccine
revealed lower NDV antibody titers compared to the group immunized with ND
vaccine only. This finding may be explained by the ability of the ND vaccine to
induce specific and non-specific immune responses against NDV, which
corresponds to the development of protective NDV antibody titers when the ND
vaccine was used. Also, an adequate amount of non-specific factors, such as
cytokines like interferon gamma, which activate the production of
antigen-stimulated B lymphocytes, cytotoxic T lymphocytes, macrophages and
natural killer lymphocytes, could be produced.(17)
On
the other hand, groups vaccinated with inactivated or live attenuated MG vaccines
before ND vaccination resulted in a marginal NDV antibody titer. This could be
attributed to defective B-lymphocyte production in the live attenuated MG
vaccinated group.(18) In
addition, IgM to IgG switching could not have occurred when the inactivated MG
vaccine was administered, but could be possible when the live vaccine was used.
Furthermore, interferon gamma may not be efficiently produced, which in turn
leads to inadequate macrophage activation.(17)
Mycoplasma
vaccines may induce an immunosuppressive effect by affecting B and T cells
progress, leading to a drastic impairment of the immune system, accompanied by
down regulation of post-vaccinal immune response to ND and the development of a
very limited post-vaccination protection against ND. These findings were
evidenced by the low level of antibody titers under the effect of MG vaccination.(14)
The
current work revealed that the level of humoral immune response to NDV was
decreased with both MG vaccines. The low NDV antibody titers in groups vaccinated
with MG and ND vaccines confirmed to be immunosuppressed. This
immunosuppression led to a negative impact on ND vaccination. This information
can be a guidance when design an appropriate vaccination strategy for the
prevention and control of NDV.(14)
Both
live attenuated and inactivated MG vaccines produced an adverse effect on ND
vaccine. Unsatisfactory HI titers suggest its negative impact.(19)
Unfortunately, there is no evidence of immunomodulatory effect of MG
vaccination before NDV vaccination.
Additionally,
a challenge with virulent NDV was applied to measure the protective immunity
induced against NDV. It is evident that the mortality rates of birds vaccinated
only with ND vaccine were very low after challenge. Only birds from G2 and G7
were protected (90% and 100%, respectively) when protection percentages and
shedding titers were calculated. Groups vaccinated with MG
live attenuated/ND vaccines G4) or MG inactivated /ND vaccines (G6) showed
an impotent protection percentage, but still better than ND non-vaccinated
groups (G1, G3, G5). This finding supports previous reports of the negative
effect of MG vaccines on the inactivated ND vaccine, since vaccinating only
with the ND vaccine was the best variant of those studied in the experiment,
the birds were able to persist the challenge without mortality, PM lesions,
shedding or clinical signs.(19)
On
the other hand, the lower protection levels demonstrated insufficient immunity
to protect chickens against NDV, which can be attributed to the
immunosuppressive effect of using MG vaccines before inactivated ND vaccination.(14)
Conclusions
MG
vaccination did not improve the response to ND vaccination, suggesting an
immunosuppressive rather than immunomodulatory effect.
Acknowledgment
The authors are grateful
and thankful to the Central Laboratory for Evaluation of Veterinary Biologics,
Cairo University for their appreciated cooperation and support.
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Conflict of interest
The authors declare that
there is no conflict of interest.
Author’s
contributions
Hala Mahmoud: performed the
experiments and wrote the manuscript.
Marwa Fathy Elsayed: performed the experiments and wrote the manuscript.
Reem A. Soliman: performed
the experiments.
Mounir El Safty: designed the experiments.
Moustafa A. Zaghloul:
performed the experiments.
All authors reviewed and
approved the final version of this manuscript for publication.
*PhD, senior
researcher at Agriculture Research Center, Central Laboratory for Evaluation of
Veterinary Biologics, Cairo, Egypt.