Prevalence of antibodies to Japanese encephalitis virus among pigs in Bali and East Java, Indonesia, 2008.
ABSTRACT Japanese encephalitis virus (JEV) is a fatal disease in Asia. Pigs are considered to be the effective amplifying host for JEV in the peridomestic environment. Bali Island and Java Island in Indonesia provide a model to assess the effect of pigs on JEV transmission, since the pig density is nearly 100-fold higher in Bali than Java, while the geographic and climatologic environments are equivalent in these areas. We surveyed antibodies to JEV among 123 pigs in Mengwi (Bali) and 96 pigs in Tulungagung (East Java) in 2008 by the hemagglutination-inhibition (HAI) test. Overall prevalences were 49% in Bali and 6% in Java, with a significant difference between them (P < 0.001). Monthly infection rates estimated from age-dependent antibody prevalences were 11% in Bali and 2% in Java. In addition, 2-mercaptoethanol-sensitive antibodies were found only from Bali samples. Further, the average HAI antibody titer obtained from positive samples was significantly higher in Bali (1:52) than Java (1:10; P < 0.001). These results indicated that JEV transmission in nature is more active in Bali than East Java.
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Jpn. J. Infect. Dis., 63, 58-60, 2010
*Corresponding author: Mailing address: Department of Interna-
tional Health, Kobe University Graduate School of Health Sci-
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Prevalence of Antibodies to Japanese Encephalitis Virus among Pigs
in Bali and East Java, Indonesia, 2008
Atsushi Yamanaka1,2, Kris Cahyo Mulyatno1, Helen Susilowati1, Eryk Hendrianto1, Takako Utsumi1,2,
Mochamad Amin1, Maria Inge Lusida1, Soegeng Soegijanto1, and Eiji Konishi2,3*
1Indonesia-Japan Collaborative Research Center for Emerging and Re-Emerging Infectious Diseases,
Institute of Tropical Disease, Airlangga University, Surabaya 60115, Indonesia; 2Center for Infectious Diseases,
Kobe University Graduate School of Medicine, Kobe 650-0017; and 3Department of International Health,
Kobe University Graduate School of Health Sciences, Kobe 654-0142, Japan
(Received September 4, 2009. Accepted November 19, 2009)
SUMMARY: Japanese encephalitis virus (JEV) is a fatal disease in Asia. Pigs are considered to be the effective
amplifying host for JEV in the peridomestic environment. Bali Island and Java Island in Indonesia provide a
model to assess the effect of pigs on JEV transmission, since the pig density is nearly 100-fold higher in Bali than
Java, while the geographic and climatologic environments are equivalent in these areas. We surveyed antibodies
to JEV among 123 pigs in Mengwi (Bali) and 96 pigs in Tulungagung (East Java) in 2008 by the hemagglutination-
inhibition (HAI) test. Overall prevalences were 49% in Bali and 6% in Java, with a significant difference between
them (P < 0.001). Monthly infection rates estimated from age-dependent antibody prevalences were 11% in Bali
and 2% in Java. In addition, 2-mercaptoethanol-sensitive antibodies were found only from Bali samples. Further,
the average HAI antibody titer obtained from positive samples was significantly higher in Bali (1:52) than Java
(1:10; P < 0.001). These results indicated that JEV transmission in nature is more active in Bali than East Java.
Japanese encephalitis virus (JEV) is a mosquito-borne
flavivirus distributed throughout Asia. It causes Japanese
encephalitis (JE), with an estimated 30,000 to 50,000 cases
and 10,000 deaths reported every year (1). JEV exists in a
transmission cycle between Culex mosquitoes and birds in
nature. In a peridomestic environment, pigs are considered
to be an effective amplifying host.
Bali Island is adjacent to Java Island in the Indonesian
archipelago. Based on statistics of the pig population in In-
donesia, 2008 (2), a large number of pigs (899,582 heads)
existed in Bali Island (5,633 km2), whereas only a small pig
population (227,953 heads) was raised in Java Island (127,499
km2). The majority of Balinese and Javanese are Hindu and
Muslim, respectively, which probably affects the number of
pigs raised in the respective islands. The densities of pigs are
nearly 100-fold different at 160 and 1.79 heads/km2 in Bali
and Java, respectively. Rural areas containing rice fields and
pig farms provide an almost complete environment to main-
tain and amplify JEV in the presence of vector mosquitoes in
both Bali (3) and Java (4).
Reflecting the difference in swine populations, confirmed
JE cases have been reported mainly from Bali (5,6) and only
recently from Java (7,8). Therefore, pigs may act as an impor-
tant amplifier in these islands. However, no antibody surveys
among pigs have been published from Bali or East Java, to
the best of our knowledge. The present small survey of JEV
antibodies was carried out using pig sera collected in Bali
and East Java.
Serum samples were collected from 123 pigs at a farm in
Mengwi of Bali and 96 pigs at a farm in Tulungagung (East
Java province) of Java in 2008. Samples in Bali were col-
lected in the dry season (August), while samples in Java were
collected in the rainy season (March through April). Since
pigs are considered to have frequent natural exposures, the
ages of subjects were limited to 1–6 months, and approxi-
mately 20 individuals were used in each age group (Table 1),
except for Java samples aged 6 months (unavailable) and 1
month (the number was half that of other groups). The pigs
were housed in these farms under similar environments where
the farms were 3,000–5,000 m2 in area and adjacent to rice
fields. These two study sites were located in a single area
designated the East Java/Bali region from agricultural and
climatologic aspects (9), providing equivalent environments
involved in transmission of JEV by vector mosquitoes.
Hemagglutination-inhibition (HAI) assay was performed
by a micro-modification of the method of Clarke and Casals
(10), with 4 hemagglutinin units of the JEV antigen
(Nakayama strain; Denka Seiken, Niigata, Japan). Sera with
an HAI antibody titer of 1:10 or higher were considered
positive, and those with 1:20 or higher were treated with 2-
mercaptoethanol (2-ME) to detect 2-ME-sensitive antibodies.
When the difference between HAI antibody titers before and
after treatment with 2-ME was 4-fold or greater, the sample
was determined to contain IgM antibodies to JEV.
Overall, 60 (49%) of 123 pigs in Bali and 6 (6%) of 96
pigs in Java were positive for HAI antibodies, showing a sig-
nificant difference between them (P < 0.001 by the chi-square
test with the Yates’ correction factor; Table 1). Comparisons
in each age group also detected significant differences between
Bali and Java, except for pigs aged 1 month. The antibody
prevalence increased with age, except for Bali subjects aged
2 months or less, which were probably affected by maternal
antibodies: the duration of maternal antibodies in most pig-
lets is 2 months (11,12). Average monthly infection rates
estimated from age-dependent antibody prevalences were
11% in Bali and 2% in Java, supposing that sterile immunity
due to maternal antibodies is negligible and that these pig
populations were infected at the same frequency during 6
months. Moreover, 5–24% of pigs aged over 3 months in Bali
with an average of 13% (11/83) possessed IgM antibodies,
and this percentage was comparable to the monthly infection
rate estimated as described above (11%).
HAI antibody titers were distributed from <1:10 to 1:640
in Bali samples, whereas the maximum antibody titer in Java
samples was 1:10. The average HAI antibody titer obtained
from positive samples was significantly higher in Bali (1:52)
than Java (1:10; P < 0.001 by the Student’s t test).
The significantly higher qualitative (antibody prevalence)
and quantitative (antibody titer) results obtained with Bali
samples compared to Java samples relate to the difference in
pig density between Bali and Java. One report available on a
JEV antibody survey among pigs in Indonesia indicated a
prevalence of as high as approximately 90%, but the survey
was done in West Java and Central Java in the early of 1970s
(or before) with pig subjects of older ages (6 to 24 months
old; 13). In addition, one report from Bali Island described
an antibody prevalence of approximately 70%, but this was
described as “unpublished data” without details (6).
The serodiagnostic method used in the present study (HAI
test) detects antibodies cross-reactive to dengue viruses, which
are also distributed in the present survey areas. However,
vector mosquitoes that can transmit dengue viruses (Aedes
aegypti and Aedes albopictus) are anthrophilic, and the rural
area has low human densities, particularly around pig farms,
with only low levels of dengue virus activity, if any. Thus, it
is highly probable that the antibodies detected by an HAI test
using JEV antigens were those against JEV, although the pos-
sibility of measuring cross-reactive dengue antibodies is not
completely ruled out.
In conclusion, natural JEV activities were significantly
more prevalent in Bali than Java. High percentages of pigs
were infected before age 6 months in Bali, which may provide
a large number of infected mosquitoes in nature. Although
less active in Java, JEV did circulate and produce relatively
high antibody prevalences among humans (14).
This work was supported in part by grants-in-aid through the Program of
Founding Research Centers for Emerging and Re-emerging Infectious Dis-
eases, the Ministry of Education, Culture, Sports, Science and Technology
(MEXT), Japan, and through Research on Emerging and Re-emerging In-
fectious Diseases, the Ministry of Health, Labour and Welfare of Japan (H20-
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Table 1. Prevalence of antibodies to JEV among pigs in Bali and East Java, Indonesia, 2008
No. of samples with HAI antibody
Total no.Monthly No. of samples% of samples
titers (reciprocal) of:1)
infection with IgMwith IgM
<10 10 20 40 80 160 320 640
Bali1 20 14 4 2 6 30– 0 0
2 2015 2111 525**– 315
3 21 16 2 3 524* 7 3 14
4 21 11 1 4 32 1048*11 1 5
5 21 4 6 7 3117 81*** 15 5 24
6 20 3 1 5 6311 178513 210
123 6314 18 17631160 49***11 1411
Java1 1010 0 0– 0 0
2 28 28 0 0**– 0 0
3 1919 0 0* 0 0 0
4 18 16 2 211* 3 0 0
5 2117 4 419*** 4 0 0
9690 6 6 6*** 2 0 0
1): When the number of samples was zero, the result is indicated as a blank.
2): Significant differences between Bali and Java in each age group are indicated by *(P < 0.05), **(P < 0.01), and ***(P < 0.001) as determined by the chi-
square test with the Yates’ correction factor. Comparison was done using pigs aged 1 to 5 months.
3): Calculated by dividing the “% positive” by the average survival period of pigs in each age group. The average survival period was supposed to be 0.5
+ the number of months used for representing the age of pigs: for instance, pigs aged 4 months were supposed to have survived 4.5 months in average.
Pigs aged 3 months or older were used for calculation, since pigs aged 1 or 2 months may have maternal antibodies: the “–” indicates “not calculated”.
4): Percentages on the “Total” line indicates averages of the results obtained in each of 1–6 months, unless otherwise specified.
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