Original Article
Using guinea pig as a model for evaluation of equine
influenza vaccine
Uso del cobayo como
modelo para la evaluación de la vacuna contra la gripe equina
Heba MG. Abdel-Aziz1 ORCID: https://orcid.org/0000-0001-9763-9123
Mohamed A. Abdrabo1 ORCID:
https://orcid.org/0000-0003-0702-2934
Dalia M. Omar1 ORCID: https://orcid.org/0000-0002-7282-4346
Nermin M. Monir1 ORCID:
https://orcid.org/0009-0008-4283-3250
Nermeen A. Marden1* ORCID:
https://orcid.org/0009-0008-6957-0446
Lamiaa M. Omar1 ORCID: https://orcid.org/0000-0002-8393-1788
1 Central Laboratory for Evaluation of Veterinary
Biologics (CLEVB), Agricultural Research Center (ARC), Cairo, Egypt.
Corresponding author: nermeen.marden@gmail.com.
ABSTRACT
Equine influenza is a highly
contagious viral disease, specially among 1-5 years old naive horses.
Vaccination is considered the best way to control the disease spread and
outbreaks. Although foals are the main animal used for evaluation of equine influenza
vaccines, guinea pigs were chosen
as an alternative model in the present work, as they have a negligible antibody
titer against equine influenza virus and are cheaper and easier to handle than
foals. Five equine influenza vaccine batches were evaluated in two animal
models, foals and guinea pigs, by injection of two doses/animal with 4 weeks
apart using 2 mL/animal/dose and evaluation of immune responses by
hemagglutination inhibition test and enzyme-linked immunosorbent assay. On the 7th
week post vaccination, equine influenza antibodies titers reached maximum
values of 9-10.2 and 8.7-10 hemagglutination inhibition units for foals and
guinea pigs, respectively; sample/negative ratios were 0.126-0.464 and
0.128-0.445 for both animals, respectively. The use of guinea pigs as an animal
model for the evaluation of equine influenza vaccines could be recommended
instead of foals.
Keywords: equine influenza virus; H3N8 subtype; hemagglutination inhibition tests;
ELISA.
RESUMEN
La
gripe equina es una enfermedad viral muy contagiosa, especialmente entre los
caballos jóvenes de 1 a 5 ańos de edad. La vacunación se considera la mejor
forma de controlar la propagación y los brotes de la enfermedad. Aunque los
potros son el principal animal utilizado para la evaluación de vacunas contra
la gripe equina, en el presente trabajo se eligieron cobayos como modelo
alternativo, ya que tienen un título insignificante de anticuerpos contra el
virus de la gripe equina y son más baratos y fáciles de manejar que los potros.
Se evaluaron cinco lotes de vacunas contra la gripe equina en dos modelos
animales, potros y cobayos, mediante la inyección de dos dosis/animal con 4
semanas de intervalo utilizando 2 mL/animal/dosis y
la evaluación de las respuestas inmunitarias mediante la prueba de inhibición
de la hemaglutinación y el ensayo inmunoenzimático.
En la 7Ş semana posvacunación, los títulos de
anticuerpos contra la gripe equina alcanzaron valores máximos de 9-10,2 y
8,7-10 unidades de inhibición de la hemaglutinación para potros y cobayos,
respectivamente; las relaciones muestras/negativos fueron de 0,126-0,464 y
0,128-0,445 para ambos animales, respectivamente. Podría recomendarse el uso de
cobayos como modelo animal para la evaluación de vacunas contra la gripe
equina, en lugar de potros.
Palabras clave: virus de la influenza equina subtipo H3N8; pruebas de inhibición de hemaglutinación;
ELISA.
Received: 14 de junio de 2023
Accepted: 13 de diciembre de 2023
Introduction
Equine influenza (EI) is still one of the most
common highly contagious respiratory diseases affecting equines caused by two
equine influenza A virus (EIV) subtypes, H7N7 and H3N8 (formerly known as
equi-1 and equi-2 respectively), members of genus Alfainfluenzavirus
within the family Orthomyxoviridae. EIV causes high morbidity
(outbreaks) but low mortality; H3N8 is the parent subtype of all present EIV strains.(1)
In susceptible equines, clinical signs of EI include pyrexia, nasal
discharge and a harsh dry cough, while pneumonia in young foals and donkeys,
and encephalitis in horses, have been described as rare events.(2)
In Egypt, three outbreaks
of EIV H3N8 were recorded in horses, mules and donkeys. The first one occurred
in October 1989 in Monufia Governorate in the Nile
Delta;(3) it is believed to be caused by EIV
H7N7 or H7N7 mixed with H3N8.(4).The second outbreak in Egypt was
reported in 2000 in the Nile Delta and Upper Egypt,(5,6) while the
most recent and severe EIV H3N8 outbreak was in 2008.(7) The
infections spread to several provinces in a short time, and the virus had 98%
genetic identity with H3N8 viruses from USA and Japan.(4)
Vaccination is still the best method to overcome and prevent the EIV
outbreaks. Currently, there are three different types of EI vaccines: whole
inactivated, live attenuated and vaccines based on viral vectors. The whole
inactivated EIV vaccine is still the most widely used.(8,9,10)
In addition, it is well accepted and recognized that emergency vaccination has
contributed to reduction of EIV transmission in many countries, both in terms
of speed and distance of transmission.(11)
In Egypt, EI vaccines must
contain at least one of both EIV subtypes, H7N7 and H3N8, especially the
current circulating H3N8 strain to obtain a potent vaccine.(8)
Furthermore, trials for the preparation of EI inactivated vaccines using
different adjuvants have been conducted at the Veterinary Serum and Vaccine
Research Institute (VSVRI); these vaccines have been evaluated by the Central
Laboratory for the Evaluation of Veterinary Biological Products (CLVEB).(12,13)
Foals are the main animals used for the evaluation of EI vaccines. Other
animal models have been used to study influenza viruses including mice, guinea
pigs and ferrets which have many advantages like negligible antibody
titers to influenza viruses, relative low cost, availability, small size, and easy handling and housing.(14)
The main disadvantage of the mouse model is the need to use
mouse-adapted virus to achieve productive infection and clinically apparent
signs of disease, while guinea pigs model has the advantage of efficiently
transmitting influenza viruses to others of its species without the need to use
guinea pig-adapted virus.(14) Ferrets have the disadvantage of
relatively limited commercial availability, more complex husbandry requirements
and greater expense than mouse and guinea pig models, which can make it
difficult to perform experiments with adequate power.(14)
The present work was
carried out to provide an alternative animal model (guinea pigs) to horses for
the evaluation of EIV vaccines, in order to save cost, effort and time, in
addition to the easier handling of guinea pigs and their negligible EI antibody
titers.(15)
Materials and
Methods
Antigen
Inactivated lyophilized EIV
H3N8 antigen with hemagglutination (HA) titer 7 log2 was supplied by
VSVRI, Abbasia, Cairo to be used in the Hemagglutination
Inhibition (HI) test for evaluation of EIV antibody titer in collected sera of
vaccinated animals.
Vaccine batches
Five monovalent inactivated
EI vaccine batches were used in this study. Three batches were provided
by VSVRI, having (A/equine/Egypt(H3N8)/6066-NAMRU3-VSVRI/2008) strain, while
the other two batches were imported by Zoeits, having
(A/equine-2/Kentucky/97(H3N8)-American Lineage) strain.
Experimental animals
Eighteen native breed
foals, 6 months old, were reorganized into six
groups (three foals/group). In addition, a total number of 30 guinea pigs, Cavia porcelus, of average weight 700 g/wt
was divided into six groups (five animals/group). Both foals and guinea pigs
were checked to be seronegative (pre-vaccination) for EIV H3N8 by HI test.(2)
Ethical approval
All experimental animals in
this study were conducted in strict accordance and adherence to the relevant
policies regarding animal handling according to international, national, and/or
institutional guidelines for the care of animals and were approved by the
Research Ethical Committee at the National Research Center, Cairo, Egypt.
Experimental design
Each vaccine batch was injected intramuscularly into foals and guinea pigs of
each group using a dose of 2 mL for both animal species as follows: vaccine
batches No. 1, 2, 3, 4, 5 were injected in groups 1, 2, 3, 4, 5 of foals,
respectively and groups 1, 2, 3, 4, 5 of guinea pigs, respectively. Groups No.
6 of both foals and guinea pigs were kept as
control groups. Vaccinated groups (1-5) were injected with a booster dose of
vaccine, 4 weeks after the first dose.
Foal blood samples were
collected from the jugular vein and guinea pig blood samples, by heart
puncture, each week after vaccination until the 7th week to monitor
antibody titer. Collected blood samples were allowed to clot and sera were
separated by centrifugation. Serum samples from foals and guinea pigs were heat
inactivated at 56°C for 30 min to remove nonspecific hemagglutinin.(2)
Hemagglutination-inhibition
(HI) test
HI test(2,16)
was performed using U-bottom micro-titer plates; hemagglutination (HA) test(2)
was carried out firstly to appropriately determine 4 HA units of EI H3N8
antigen.
The presence of HI
antibodies in serum was tested by HI. Briefly, inactivated sera were twofold
serially diluted in Phosphate Buffered Saline (PBS), then 4 HA units of
reconstituted H3 antigen were added. Plates were shaken and incubated at room
temperature (27°C) for 30 min to allow the reaction to take place; then equal
volume of 1% washed chicken red blood cells (RBCs) was added to each well and
plates were incubated for 30 min at room temperature (27°C). The test was read
by tilting the micro-titer plate at an angle to observe RBCs streaming at the
bottom of the well. The HI titer was taken as the highest dilution of serum
with complete inhibition of agglutination in the serial dilution.
Commercial ELISA
The commercial ELISA kit
(IDEXX Influenza A, REF 99-53101/ Lot No. 8140) supplied by IDEXX was used to
determine Influenza A virus antibodies in serum samples. The ELISA test was
done according to manufacturer’s instructions.
The presence or absence of
antibody to influenza A was determined by the sample to negative (S/N) ratio
for each sample.
·
Samples with S/N
values < 0.60 were considered influenza A antibody positives.
·
Samples with an S/N ratio ≥ 0.60 were considered
negative for the presence of influenza A antibodies.
Results and
Discussion
The spread of EIV infection
and the severity of disease are mostly reduced by the use of potent inactivated
EI vaccines containing epidemiologically relevant virus strains.(8,9,10)
Inactivated EI vaccines are
adjuvanted vaccines containing either inactivated whole viruses or their
subunits and provide protection by inducing humoral antibody response to the
hemagglutinin protein; multiple doses are required to maintain protective
levels of antibody.(2)
For preparation of
inactivated EIV vaccines, vaccinal strains are propagated in specific pathogen
free embryonated chicken eggs, then the strains are concentrated and purified
before inactivation with agents such as formalin or beta-propiolactone.(2)
The efficacy requirements
for EI vaccines may vary according to the National Authority, but usually
include the assessment of the serological response in horses and virus
challenge studies in susceptible horses.(2)
Results obtained by HI test
using serum samples from five groups of foals vaccinated with five different
lots of EI vaccines revealed that vaccinated animals had EI-specific antibodies
recording their highest titers (9 to 10.2) at 7 weeks post vaccination (Table
1); ELISA S/N ratios (0.126 to 0.464) were similar to the HI results (Table 2).
Table 1. Mean
hemagglutination inhibition equine influenza antibody titers in sera of
vaccinated foals.
|
Foal groups |
Mean EI-HI antibody titers*/ Weeks post
vaccination |
||||||||
|
0 |
1 |
2 |
3 |
4 |
Booster dose |
5 |
6 |
7 |
|
|
1 |
0 |
3.5 |
4.2 |
6.3 |
6.8 |
7.5 |
8.3 |
9 |
|
|
2 |
0 |
3.8 |
5 |
6.4 |
6.9 |
8.3 |
9 |
9.5 |
|
|
3 |
2 |
3.6 |
4.7 |
6.2 |
7.5 |
8 |
9.6 |
10 |
|
|
4 |
2 |
3.5 |
4.5 |
6.5 |
7.7 |
8.7 |
9 |
10 |
|
|
5 |
2 |
4 |
5.1 |
6.8 |
7.4 |
8.7 |
9.8 |
10.2 |
|
|
6 |
2 |
2 |
2 |
2 |
2 |
2 |
2 |
2 |
|
*EI-HI antibody
titer: equine influenza hemagglutination inhibition antibody titer (calculated
as the highest dilution of serum with complete inhibition of agglutination in a
twofold serial dilution of the original sera).
Table 2. Sample/Negative
ratios of vaccinated foals’ sera
tested by equine influenza ELISA.
|
Foal groups |
EI-ELISA antibody S/N*/ Weeks post vaccination |
||||||||
|
0 |
1 |
2 |
3 |
4 |
Booster dose |
5 |
6 |
7 |
|
|
1 |
0.978 |
0.740 |
0.549 |
0.535 |
0.211 |
0.550 |
0.211 |
0.163 |
|
|
2 |
0.899 |
0.728 |
0.540 |
.470 |
0.290 |
0.464 |
0.456 |
0.260 |
|
|
3 |
0.987 |
0.665 |
0.540 |
0.470 |
0.290 |
0.464 |
0.456 |
0.260 |
|
|
4 |
0.720 |
0.650 |
0.432 |
0.343 |
0.175 |
0.544 |
0.432 |
0.464 |
|
|
5 |
0.734 |
0.633 |
0.422 |
0.229 |
0.253 |
0.163 |
0.261 |
0.126 |
|
|
6 |
0.955 |
0.920 |
0.703 |
0.852 |
0.729 |
0.876 |
0.987 |
0.795 |
|
**EI-ELISA antibody S/N: equine influenza enzyme linked immunosorbent assay antibody sample/negative ratio.
Evaluation by ELISA depends
on calculation of the signal-to-noise ratio (S/N), which quantifies and
compares the level of a desired signal to the level of background noise
(undesired signal). S/N was calculated by dividing the mean signal of the
clinical study sample by the mean signal of the negative control analyzed,
therefore a low S/N ratio indicates that there is a higher than optimal
background noise. For the used ELISA kit, S/N values < 0.60 should be
considered positive to influenza A antibodies.(17)
Vaccinated guinea pigs with
the same vaccine batches showed the same pattern of HI titers (8.7 – 10, Table
3) and ELISA S/N ratio (0.128 - 0.445, Table 4). These findings indicate the
possibility and validity of the use of guinea pigs instead of foals to evaluate
EI vaccines.
Table 3. Mean hemagglutination
inhibition equine influenza antibody titers in sera of vaccinated guinea pigs.
|
Guinea pig groups |
Mean EI-HI antibody titers*/ Weeks post
vaccination |
||||||||
|
0 |
1 |
2 |
3 |
4 |
Booster dose |
5 |
6 |
7 |
|
|
1 |
0 |
3 |
4 |
6 |
6.5 |
7.5 |
8.3 |
8.7 |
|
|
2 |
0 |
3.5 |
4.6 |
6 |
6.5 |
8 |
8.5 |
9 |
|
|
3 |
0 |
3.3 |
4.4 |
6 |
7 |
8 |
9 |
9.6 |
|
|
4 |
0 |
3 |
4.6 |
6 |
7 |
8.2 |
9.2 |
9.5 |
|
|
5 |
0 |
3.6 |
4.9 |
6.3 |
7 |
8.3 |
9.4 |
10 |
|
|
6 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
*EI-HI antibody
titer: equine influenza hemagglutination inhibition antibody titer (calculated as the highest
dilution of serum with complete inhibition of agglutination in a twofold serial
dilution of the original sera).
Table 4. Sample/Negative
ratios of vaccinated
guinea pigs’ sera tested by equine influenza ELISA
|
Guinea pig groups |
EI-ELISA S/N*/
Weeks Post Vaccination |
||||||||
|
0 |
1 |
2 |
3 |
4 |
Booster dose |
5 |
6 |
7 |
|
|
1 |
0.778 |
0.945 |
0.565 |
0.543 |
0.233 |
0.523 |
0.211 |
0.156 |
|
|
2 |
0.869 |
0.759 |
0.525 |
0.510 |
0.326 |
0.489 |
0.169 |
0.320 |
|
|
3 |
0.856 |
0.899 |
0.567 |
0.475 |
0.296 |
0.498 |
0.432 |
0.278 |
|
|
4 |
0.734 |
0.659 |
0.443 |
0.346 |
0.178 |
0.530 |
0.456 |
0.445 |
|
|
5 |
0.787 |
0.667 |
0.467 |
0.243 |
0.250 |
0.157 |
0.261 |
0.128 |
|
|
6 |
0.854 |
0.940 |
0.862 |
0.950 |
0.740 |
0.775 |
0.985 |
0.975 |
|
**EI-ELISA antibody S/N: equine influenza enzyme linked immunosorbent assay antibody sample/negative ratio. Positive ELISA S/N < 0.6.
Comparing the HI test results
in foals (Table 1) and guinea pigs (Table 3), there was a great relation
between them, ranged from 0.985 to 0.999. Also, there was a strong relation
between ELISA results in foal (Table 2) and guinea pigs (Table 4), ranged from
0.875 to 0.999.
Regarding the
use of laboratory animals as alternative models to horses for the evaluation of
EI vaccines, intranasal administration of a single dose of eq/GA/81 ca
vaccine virus induced neutralizing antibodies and conferred complete
protection against homologous virus challenge in the upper respiratory tract in
mice and ferrets.(18) In addition, one dose of the eq/GA/81 ca vaccine
also induced neutralizing antibodies and conferred complete protection in mice
and nearly complete protection in ferrets upon heterologous challenge with the
H3N8 (eq/Newmarket/03) virus.(2)
The level of
EI-specific antibodies, measured by HI assays, is correlated to protection
against homologous EI strains. Reduced clinical signs of disease and resistance
to infection with an EI strain homologous to the vaccine strain have been recorded.(19,20)
Conclusion
The use of guinea pigs as
an alternative animal model to foals for the evaluation of EI vaccines is
possible, very useful and highly recommended, since it gave results that were
approximately similar to those of foals, saving also the required cost and
efforts.
Acknowledgement
The authors are
grateful and thankful to the CLEVB and ARC for its appreciated cooperation and
support.
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Conflict of interest
The authors declare that
there is no conflict of interest.
Author’s
contributions
Heba MG. Abdel-Aziz: the owner of main idea and experimental design, applied
the experiments and wrote the manuscript.
Mohamed A. Abdrabo: followed up the practical work.
Dalia M. Omar:
followed up the practical work.
Nermin M. Monir: followed up the practical work.
Nermeen A. Marden: follow up the work, and
critically reviewed the manuscript.
Lamiaa M. Omar: supervisor and
follow up the work.
All authors reviewed and approved the final version of
this manuscript for publication.
*Researcher at Central Laboratory for Evaluation of Veterinary
Biologics (CLEVB), Agricultural Research Center (ARC), Cairo, Egypt