Rev. sci. tech. Off. int. Epiz., 1995,14 (3), 539-546
Serological comparison of type Asia 1 foot
and mouth disease virus isolates from
Thailand
W.J. DOUGHTY *, L.J. GLEESON *, K. PRASATSUWAN ** and
A. KONGTHON **
Summary: Antigenic variation of type Asia 1 foot and mouth disease (FMD)
virus in Thailand was examined using a total of 50 field viruses isolated between
1986 and 1992. A two-dimensional serum neutralisation test was used to
calculate r values for comparison of these isolates with a reference vaccine
strain, Asia 1 Bangkok 1960 (BKK/60). Viruses were also compared to two
field isolates, Asia 1 36-2/88 and Asia 1 45/88, and some were compared to
another vaccine strain, Asia 1 Nakhon Pathom 1984 (NPT/84). In 80% of cases,
field isolates were deemed to be significantly different from the vaccine virus
Asia 1 BKK/60, and the distribution of r values indicated that there would be
some advantage in selecting a new vaccine strain more closely related to
contemporary field strains.
K E Y W O R D S : Aphthovirus - Foot and mouth disease virus type Asia 1 Serological comparison - Thailand.
INTRODUCTION
Foot and mouth disease (FMD) is endemic throughout Thailand, with the exception
of the southern peninsula (administrative regions 8 and 9), which is a disease control
area and experiences only sporadic outbreaks. The endemic serotypes are types O, A
and Asia 1. Disease control strategies include animal movement control, animal health
measures and vaccination. At the time of this study, three monovalent vaccines formed
the basis of t h e vaccination p r o g r a m m e . T h e Asia 1 vaccine strain was an isolate
characterised by t h e World R e f e r e n c e L a b o r a t o r y for F o o t and M o u t h Disease in
Pirbright ( U n i t e d K i n g d o m ) in 1960 and locally designated Asia 1 Bangkok/1960
(BKK/60). O u t b r e a k s caused by s e r o t y p e Asia 1 viruses h a v e generally b e e n less
common in Thailand than those caused by the other two serotypes. In 1991, however,
Asia 1 was the most prevalent serotype causing outbreaks of F M D in the northern part
of the country (unpublished findings).
A study was u n d e r t a k e n to assess t h e degree of antigenic variation a m o n g field
strains of F M D virus type Asia 1 which caused outbreaks of F M D in Thailand over a
five-year period, and to investigate t h e suitability of t h e current vaccine strain for
* Commonwealth Scientific and Industrial Research Organisation, Australian Animal Health
Laboratory, P.O. Bag 24, Geelong, Victoria 3220, Australia.
** Department of Livestock Development, Foot and Mouth Disease Centre, Pak Chong, Nakhon
Ratchasima, 30130 Thailand.
540
continued use. All viruses were compared to the Asia 1 BKK/60 vaccine strain, and
most to two recent field isolates, Asia 1 36-2/88 and Asia 1 45/88, using the twodimensional serum microneutralisation test (SNT). T h e viruses Asia 1 36-2/88 and
Asia 1 45/88 were selected from widely-separated provinces, as examples of the field
strains prevalent at the time, for the production of antisera for the FMD-typing enzymelinked immunosorbent assay ( E L I S A ) . Some of the viruses were also c o m p a r e d to
another strain, Asia 1 Nakhon Pathom 1984 (NPT/84), which was developed for vaccine
production to control outbreaks of FMD in pigs in central Thailand.
MATERIALS AND METHODS
Viruses
T h e reference vaccine viruses Asia 1 BKK/60 and Asia 1 NPT/84 were vaccine
potency test challenge viruses, provided by the Foot and Mouth Disease Centre in Pak
Chong (Thailand) and adapted to growth in baby hamster kidney (BHK) cells.
The field strains Asia 1 36-2/88 and Asia 1 45/88 were isolated in B H K 21 cells
and passaged a total of six and five times, respectively, prior to use in the twodimensional SNT.
Field viruses were obtained from the Northern Veterinary Research and Diagnostic
C e n t r e in Hang Chat, Lampang, or the F M D C e n t r e in Pak Chong. Samples w e r e
regarded as being from distinct outbreaks if they originated from separate provinces or
separate districts within a province. Samples were submitted as tongue epithelium and
typed using the FMD-typing E L I S A (7) or the c o m p l e m e n t fixation test (4). T h e
40 virus samples obtained between 1986 and 1988 w e r e isolated in primary bovine
thyroid cells (19 samples), secondary bovine kidney (1), goat kidney (2) or B H K
cells (18). The majority (35 samples) were passaged a further three to six times in B H K
cells prior to use in the SNT. One isolate required 12 passages in B H K cells before it
could be used in the SNT test. The ten viruses from 1991 were all isolated in Instituto
Biológico-rim suino-2 (IB-RS-2) cells and used after two or three passages in these cells.
Antisera
Bovine antiserum to Asia 1 BKK/60 and pig antiserum to Asia 1 NPT/84 were
provided by the assay section of the F M D Centre in Pak Chong. The sera were obtained
after homologous challenge of animals vaccinated with monovalent Asia 1 vaccines
during vaccine potency testing.
R a b b i t antisera were p r o d u c e d to viruses Asia 1 36-2/88 and Asia 1 45/88 as
described by Have et al. (2), but using higher antigen levels and live virus preparations.
Briefly, rabbits received 40 ug of 146s antigen in Freund's complete adjuvant, and were
re-inoculated 28 days later with 20 ug of the same purified virus in either 5 0 %
incomplete Freund's adjuvant or phosphate-buffered saline with 0.025 mg/ml saponin.
Determination of serological relationships
All isolates were assessed for their serological relationships to the reference viruses,
in a two-dimensional SNT (5) with the modifications described below. Virus/serum
mixtures were incubated for 1 h at 37°C. B H K 21 or IB-RS-2 cells were added at a
seeding rate of 50,000 cells per microtitre well, and the plates were fixed and stained
541
with 0.05% methylene blue in 10% formal saline after incubation for three days at 37°C.
The titre of the serum was determined at 100 T C I D (50% tissue culture infective dose)
of virus, using a linear regression computer program. Duplicate tests were performed on
separate days for all isolates, to d e t e r m i n e the mean serum titre to the virus. If the
replicate titres varied by more than 0.25 log , the test was conducted a third time and
the mean used.
50
10
The relationship of any virus to a reference virus (V) was expressed as an r value
calculated using the following formula:
titre of reference antiserum against heterologous virus
r(V) =
titre of reference antiserum against homologous virus
Criteria for the i n t e r p r e t a t i o n of r values w e r e p r o p o s e d by Samuel et al. (6), as
follows :
- r = 0-0.19: a highly significant serological variation indicating a requirement for a
vaccine strain with a closer relationship to the field virus.
- r = 0.2-0.39: a significant difference from the reference strain, but protection may
be satisfactory if a sufficiently potent vaccine is used.
- r = 0.4-1.0: not significantly different from the reference strain as measured by the
test system used.
RESULTS AND DISCUSSION
The study was conducted in two phases. In the initial phase, tests were performed on
forty field viruses isolated from separate outbreaks in regions 1 , 3 , 4 , 5 , 6 and 7 between
June 1986 and N o v e m b e r 1988. In the second phase, ten field viruses isolated from
outbreaks in regions 5 and 6 between January 1991 and January 1992 were tested.
The origins of the field isolates are shown in Figure 1. No isolates were available
from region 2. Forty-four of the isolates were from cattle, three from buffalo and two
from pigs. The species of origin of one isolate was not known.
The fifty viruses were tested for their serological relationship to the vaccine virus
Asia 1 BKK/60. Many isolates gave low r values to Asia 1 BKK/60, and these were then
tested against antisera to two recent field isolates, Asia 1 36-2/88 and Asia 1 45/88.
The frequency distribution of r values to Asia 1 BKK/60 a n t i s e r u m is shown in
Figure 2, and to Asia 1 36-2/88 and Asia 1 45/88 antisera in Figure 3. One virus isolated
from cattle in region 7 in 1986 (Asia 1 163/86) was clearly distinguishable from all three
reference viruses (all r values < 0.06). This virus had an r value to the Asia 1 NPT/84 pig
vaccine virus of 0.22. T h e virus had b e e n re-isolated from original bovine t o n g u e
epithelium, purified by two limiting dilution passages and typed as Asia 1 by E L I S A ,
after initial testing had produced very low r values. Figures 2 and 3 show the majority of
r values at the lower end of the range to Asia 1 BKK/60, at a slightly higher range to
Asia 1 45/88 and distinctly higher for Asia 1 36-2/88. The mean r value to Asia 1 BKK/60
was 0.29, with 32% of values < 0.2 and 80% < 0.4. The mean r value to Asia 1 45/88 was
0.33, with 14% < 0.2 and 6 5 % < 0.4. The mean r value to Asia 1 36-2/88 was 0.83, with
only one value < 0.2 (2.3%) and none between 0.2 and 0.4.
542
Geographical distribution of selected foot and mouth disease virus type Asia 1 isolates
in Thailand between 1986 and 1992
The r value distribution showed a significant antigenic drift away from the vaccine
strain Asia 1 BKK/60, which was originally isolated in 1960. But the study also
demonstrated that, while 80% of field isolates examined were significantly different
from Asia 1 BKK/60, the pattern of r values to the three reference viruses showed little
antigenic variation across the Asia 1 field viruses causing F M D outbreaks. This pattern
was essentially similar for the viruses isolated between 1986 and 1988 and those isolated
in 1991, suggesting that no new strains had caused o u t b r e a k s in that time. N o
subsequent isolates were detected with r value patterns similar to the very low values
observed for the 1986 isolate Asia 1 163/86. A similar observation was made with Asia 1
isolates from India, where antigenic drift from the historical vaccine strain India 8/79
was demonstrated by two-dimensional SNT, but a close relationship was found between
field strains and one contemporary vaccine candidate strain (1).
The bovine antiserum to Asia 1 BKK/60 was the most a p p r o p r i a t e r e a g e n t for
assessing the ability of the vaccine strain to control disease outbreaks (3). The rabbit
antisera to the two field isolates were used to study further the serological relationships
of the outbreak viruses. Although it might have b e e n preferable also to use bovine
antiserum in these neutralisation tests, the rabbit antisera were useful for showing
relationships between the field isolates. A small study using ten of the Asia 1 viruses,
543
FIG.
2
Frequency distribution of r values to the Asia 1 vaccine strain BKK/60 of fifty Asia 1
viruses isolated from foot and mouth disease virus outbreaks in six regions of Thailand
( 1 , 3 , 4 , 5 , 6 and 7) between January 1986 and January 1992
and comparing r values obtained with bovine and rabbit antiserum to Asia 1 BKK/60,
gave mean r values of 0.3 (range 0.113-0.99) and 0.36 (range 0.06-0.776), respectively,
indicating similar results in both test systems.
This study demonstrated that, while there was little antigenic variation across the
population of Asia 1 field viruses causing F M D outbreaks in Thailand, as assessed by
serum neutralisation, the r values to the vaccine strain Asia 1 BKK/60 were generally
low, with 80% of the viruses regarded as significantly different from Asia 1 BKK/60 (6).
This vaccine strain could be expected, in an emergency, to give protection against the
field strains if a vaccine of good potency was administered m o r e t h a n once. But it
appears that the efficacy of any Asia 1 vaccine used in Thailand would be enhanced by
incorporating a vaccine strain m o r e closely related to the majority of current field
strains. While it must be acknowledged that no conclusions should be drawn with regard
to the likelihood of vaccine efficacy on the basis of data derived using a rabbit
antiserum, the r values suggested that a strain such as Asia 1 36-2/88 could confer
greater protection against the majority of field strains.
Vaccination p r o g r a m m e s are a key element in F M D control strategies, and
laboratory investigations of relationships between field viruses and vaccine strains
make a critical c o n t r i b u t i o n to the success of these p r o g r a m m e s . This study
demonstrates the need for continual monitoring of these relationships.
544
FIG.
3
Frequency distribution of r values to two Asia 1 field viruses isolated in 1988, of Asia 1
viruses isolated from foot and mouth disease virus outbreaks in six regions of Thailand
( 1 , 3 , 4 , 5 , 6 and 7) between January 1986 and January 1992
ACKNOWLEDGEMENTS
This work was supported by the Commonwealth Scientific and Industrial Research
Organisation, Australian A n i m a l H e a l t h Laboratory, the A u s t r a l i a n C e n t r e for
International Agricultural Research and the Department of Livestock Development of
the Royal Thai Government.
*
* *
545
COMPARAISON SÉROLOGIQUE D E SOUCHES D U VIRUS D E LA FIÈVRE
APHTEUSE (TYPE ASIA 1) EN THAÏLANDE. - W.J. Doughty, L.J. Gleeson,
K. Prasatsuwan et A. Kongthon.
Résumé : Les auteurs étudient la variation antigénique du virus de la fièvre
aphteuse (type Asia 1) en Thaïlande à partir de 50 souches sauvages isolées
entre 1986 et 1992. Une épreuve de séroneutralisation à deux dimensions a
permis de calculer les valeurs r et de comparer ces isolais avec une souche
vaccinale de référence, Asia 1 Bangkok 1960 (BKK/60). On a également
comparé les virus aux isolats de terrain, Asia 1 36-2/88 et Asia 1 45/88, et
certains d'entre eux à une autre souche vaccinale, Asia 1 Nakhon Pathom 1984
(NPT/84). Dans 80 % des cas, les isolats de terrain se sont révélés radicalement
différents de la souche vaccinale Asia 1 BKK/60. Quant à la distribution des
valeurs r, elle indiquait qu'il y aurait intérêt à sélectionner une nouvelle souche
vaccinale plus étroitement apparentée aux souches de terrain actuelles.
MOTS-CLÉS : Comparaison sérologique - Thaïlande - Virus de la fièvre
aphteuse - Virus de la fièvre aphteuse de type Asia 1.
*
COMPARACIÓN SEROLÓGICA D E LOS VIRUS DE LA FIEBRE AFTOSA D E
TIPO ASIA 1 AISLADOS EN TAILANDIA. - W.J. Doughty, L.J. Gleeson,
K. Prasatsuwan y A. Kongthon.
Resumen: Entre 1986 y 1992 se investigó la variación antigénica que exhibe en
Tailandia el tipo Asia 1 del virus de la fiebre aftosa. Para la realización de dicho
estudio se utilizó un total de 50 virus salvajes aislados durante aquel período.
La aplicación de una prueba de neutralización bidimensional en suero permitió
calcular los valores r de comparación entre los aislados y una cepa de referencia
para vacunas conocida como Asia 1 Bangkok 1960 (BKK/60). Los virus
fueron comparados además con otras dos cepas salvajes, Asia 1 36-2/88 y Asia
1 45/88, y algunos de ellos con otra cepa utilizada para la producción de
vacunas, Asia 1 Nakhon Pathom 1984 (NPT/84). Los resultados indicaron que
los aislados eran, en el 80% de los casos, significativamente distintos de la cepa
de vacuna Asia 1 BKK/60. A tenor de la distribución de los valores de r; puede
afirmarse que sería ventajoso seleccionar una nueva cepa para la producción de
vacunas, más afín a las cepas salvajes contemporáneas.
PALABRAS CLAVE: Aphthovirus - Comparación serológica - Tailandia Tipo Asia 1 del virus de la fiebre aftosa.
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