Japan.
J. Med. Sci. Biol., 31, 317-324, 1978
EPIDEMIOLOGICAL IN IV.
STUDIES ON JAPANESE ENCEPHALITIS KYOTO CITY AREA, JAPAN
NATURAL
INFECTION
IN
SENTINEL
OSAMUMAEDA, KUNIHACHITAKENOKUMA,
PIGS
YOSHIAKIKARO JI,
AKIO KURODA, OSAMUSASAKI, TOSHIROKARAKI and TAKASHIISHII* Kyoto City Research Institute of Public Health, Higashi-Takada-cho, Mibu, Nakagyo-ku, Kyoto 604, and *Department of Biology, College of General Education, University of Tokushima, Mishima, Nanjo, Tokushima 770, Japan (Received: April 27, 1977)
SUMMARY: Natural infection with Japanese encephalitis virus in three sentinel pigs held in separate experimental huts was examined daily by virus recovery from blood samples of the pigs and from mosquitoes after incubation for about 7 days from their blood feeding and by HI antibody titration of the blood samples. After a period of low infection rates, below 6%, for about two weeks in engorged Culex tritaeniorhynchus summorosus, high mosquito infections of over 30% from each viremic pig occurred for two to three days. The pigs may be probably have been bitten by many infected but not infective mosquitoes in a period of about 10 days before infection.
INTRODUCTION
Annual dissemination of Japanese encephalitis virus in the Kyoto City area was examined from two different aspects. First of all, JE virus dissemination all over the Kyoto City area was investigated by virus recovery from unfed Culex tritaeniorhynchus summorosus caught at some stations (Maeda et al., 1978a). JE virus infection was estimated also by virus recovery from engorged mosquitoes after incubation for seven to 10 days (Maeda et al., 1978b). More detailed knowledge seemed necessary to explain the dissemination as to how often each pig is bitten by infected mosquitoes for JE virus infection to occur and how many infected mosquitoes are produced from a viremic pig. Therefore, natural infection in three sentinel pigs each held in an experimental but was examined -continuously by various methods .
前 田 理 ・竹 之熊 国 八 ・唐 牛良 明 ・黒 田 晃 生 ・佐 々木 、 修 ・唐 木 利 朗(京 都 市衛 生 研 究 所 京 区壬 生 東 高 田 町1-2),石 井 孝(徳 島大 学 教 養 部 生 物 学 教 室 徳 島市 南常 三 島1) 317
京都市中
MAEDA
318
MATERIALS Design
of
2 •~ 2 •~ 2 m baited
traps
covered
devised wood
Location in
by
the
placed
of
dry
Fig. 1.
The of
wood
by
Kato
or
polyvinyl
shed:
summer
near ice
about
shed:
made
with
all
dig
and
trap 10
m
Experimental
An
et
every
a al.
but penthouse
(1966)
experimental
but
where as
reported
pig
shed
at
mosquito
collection
was
unfed
year a
were
for
collecting
of
corrugated
set
mosquitoes
was
vinyl.
on
the
in
1969
walls
Animal compactly
screen.
station •gM•h,
from
31
AND METHODS
experimental with
Vol.
et al.
built mosquitoes
(Maeda the
hut.
western
et border
and
have al.,
two been
1978a). of
a
in
They group
1972:
collected
of
were sheds
1978
JE
VIRUS
INFECTION
IN SENTINEL
PIGS
319
along the Kamo River. Mosquito collection: In 1969, two baited traps were set in the but facing inward for collecting escaping mosquitoes and another facing outward for collecting invading mosquitoes as shown in Fig. 1. In 1971, two baited traps were set in each but both facing outward for collecting invading mosquitoes. A pig of three months' old having no antibody to the virus was fed in each hut , and mosquitoes caught with these traps were collected every morning after the middle of July in 1969 and from late July in 1971. Most mosquitoes , easily entering the but through narrow crevices, did not enter the traps set facing outward and such mosquitoes were caught with aspirator tubes every morning . Virus recovery from mosquitoes: Mosquitoes caught by these methods were identified and counted. Unfed and engorged C. tritaeniorhynchus were separated each other and used for virus recovery mixing together all kind of collection . The former was used for determining the infection rate before feeding , and the latter for estimating the number of infected mosquitoes after biting the pig, or for determining the infection rate after feeding the viremic pig . The unfed TABLE I Natural infection with JE virus of the sentinel pig , P1, in 1969
MAEDA
320
Vol.
et al.
31
mosquitoes were stored at -70 C after identification until inoculation into baby mice and the engorged ones at the same temperature after incubation for about a week at 27 C. The detailed technique for virus recovery has been described (Maeda et al., 1978a). Virus recovery from and HI antibody measurement of pig blood samples: Blood samples were taken from each pig every morning, cleaned up by centrifuging and inoculated into baby mice for direct detection of viremia. In 1971, the blood samples were titrated in 3-day old mice by injecting intracerebrally 0.01 ml of each of serial fivefold dilutions. HI antibody in the sample was measured by the same method as described (Maeda et al., 1978a).
TABLE II Natural infection with JE virus of the sentinel pig, P2, in 1971
* The
fluorescent
antibody
test was positive
in one out
of eight
inoculated
mice.
1978
JE
VIRUS
INFECTION
IN SENTINEL
PIGS
321
RESULTS Mosquito
Collection
Most of the mosquitoes caught with the baited traps and aspirator tubes were C. tritaeniorhynchus. Over 90% of this species remained in each but every morning without entering the baited traps set facing outside. The mosquitoes of over 95% were engorged with the blood of each sentinel pig. Daily fluctuations in the number of C. tritaeniorhynchus were remarkable especially TABLE III Natural infection with JE virus of the sentinel pig, P3, in 1971
* The eight
fluorecent inoculated
antibody mice.
test was positive
with
brain
tiss
ues from
two out
of
MAEDA
322
Vol.
et al.
31
in that of the remainders in 1969 and 1971 and in that of mosquitoes caught with traps 2 and 3 in 1969. Detailed discussions about number of these mosquitoes were made by Ishii and Karoji (1976). Natural
Infection
o f Each Sentinel
Pig with JE Virus
Table I presents natural infection of pig Pl in 1969, including the results of virus recovery from the mosquitoes engorged on the pig and from the blood samples and of the titration of the blood samples for HI antibody. Tables II and III present the same information for 1971 in pigs P2 and P3, respectively, and of the titration for viremi a. In 1969, JE virus was recovered from the mosquitoes gorging on the sentinel pig at low infection rates below 2% in the period from July 23 to 31. After this period, high rates of infection occurred during August 1 to 5, the maximum rate being 33.3% on August 3. Viremia of the pig was first detected in the morning of August 5. These results indicate that the heavy mosquito infection resulted probably from viremia of the pig (Table I). In 1971, the virus was recovered from the mosquitoes gorging on each pig at a lower rate below 6% in the period from August 8 till the abrupt increase in the rate on August 29 in P2 and on September 2 in P3. The highest rates TABLE IV Dissemination of JE virus in the area shown by the virus recoveries from un fed C. tritaeniorhynchus
* Each
date
represents
collection
during
the
period
from the night of denoted date to the next morning. ** Virus recovery was made with each lump of unfed mosquitoes
caught
in both
huts.
1978
JE
VIRUS
INFECTION
IN SENTINEL
PIGS
323
were 83.3% in P2 in the night of August 31 and 54.2% in P3 on September 3. Viremia was detected in the morning from August 29 to September 1 in P2 and from September 1 to 5 in P3. The high titers above 1.0 in log LD50in 3 days' old mice continued for two days in P2 and for 4 days in P3. High antibody titers were detected in blood samples after September 1 in P2 and after September 4 in P3 (Tables II and III). JE Virus Dissemination
at the Study Site
Table IV shows virus recoveries from lumps of unfed mosquitoes collected with the traps and aspirators in the huts in both years. The highest infection rates were 0.56 and 2.64% in respective years and were not significantly different from the rates in engorged mosquitoes except during the period of the occurrence of viremia in each pig as in Tables I to III. These results were not significantly different either from the rates in unfed mosquitoes caught by dry ice collection at station "M" as described before (Maeda et al., 1978a). DISCUSSION
The highest infection rate in C. tritaeniorhynchus gorging on each viremic pig did not reach 100%. This rate may depend on the viremic titer in each pig. Hulburt (1964) reported that viremia in pigs had viremic titers between 2.1 and 3.0 in 3-day old mice, and that the effective viremia over 30% infection of C. tritaeniorhynchus usually did not last longer than for two days before 2 days after infection, nor later than 4 days. In our laboratory experiments (Sasaki et al., unpublished data), four non-vaccinated pigs without JE antibody were challenged with JE virus; three with infective mosquitoes and the other with a virus solution. Mosquito infection by feeding on the viremic pigs was investigated with detection of viremia with blood samples . We obtained results similar to those described by Hulburt (1964), except for a case of mosquito infection on the first and second days after infection, although viremia was detected in all pigs in the period from the first to the fourth days after infection. From these results, it is very likely that the sentinel pigs were probably infected at night not so long before the occurrence of viremia in any pig . The number of the infected mosquitoes gorging on each sentinel pig was estimated daily by calculating the number of positive engorged mosquitoes and the infection rate from these mosquitoes as shown in Tables I to III . The sentinel pigs might be bitten by many infected mosquitoes during a period of about 10 days before the occurrence of viremia. If the highest viremia :occurred a few days after infection, the pigs might have been infected at the end of the period by biting by infective mosquitoes. This fact suggests that there are only a few infective mosquitoes in a given infected mosquito population in an early period every year. Cumulative infection rates in the period of viremia calculated for each pig
324
MAEDA
et al.
Vol.
31
were 72.1, 169.8, and 139.0%, respectively. The variation in the value amongg pigs is large and may be an important factor in production of infected mosquitoes and of human epidemic. The infection rates were investigated also with several other pig populations as described before (1978b). Cumulative rates, being somewhat high in the highest epidemic year of 1967, also differed among pig populations. Yamamoto (1971) investigated annual variation in the infection rate of unfed mosquitoes at some localities and reported that the rate did not correlate with the scale of the epidemic. However, the infection rate of unfed mosquitoes may more easily be affected by many factors than of engorged ones. The quantitative relation in the infection rate between engorged and unfed mosquitoes will be analyzed in a separate paper. REFERENCES
HURLBURT,H. S. (1964): The pig mosquito cycle of Japanese encephalitis in Taiwan. J. Med. Entomol., 1, 301-307. ISHII, T. ANDKAROJI,Y. (1976): Analysis of mosquito trap collection. 2. A comparison of varioust sample populations collected simultaneously at one station. J. Sci. Univ. Tokushima, 9, 15-46. KATO, M., ISHII, T., WATANABE,T. AND YOSHIDA,S. (1966): A newly dry ice baited trap for collecting mosquitoes. Japan. J. Sanit. Zool., 17, 83-88. MAEDA, O.,KARAKI,T., KURODA,A., KAROJI, Y., SASAKI, O.ANDTAKENOKUMA, K. (1978a): Epidemiological studies on Japanese encephalitis in Kyoto City area, Japan. II. Annual prevalence of virus dissemination based on virus recoveries from unfed Culex tritaeniorhynchus summorosus. Japan. J. Med. Sci. Biol., 31, 39-51. MAEDA, O., KARAKI,T., KURODA,A., SASAKI, O.,KAROJI, Y. ANDTEKENOKUMA, K. (1978b): Epidemiological studies on Japanese encephalitis in Kyoto City area, Japan. III. Seasonal prevalence of the virus infections in several pig populations shown by the virus recovery from engorged Culex tritaeniorhynchus summorosus. Japan. J. Med. Sci. Biol., 31, 277-290. SASAKI, O., KAROJI, Y., KURODA,A., KARAKI,T., TAKENOKUMA, K., MAEDA, O., KODAMA,K. AND SASAKI,M. (1978): Experimental challenge of the infected mosquitoes to the pigs immunized with the live attenuated Japanese encephalitis vaccine. Acta Virologica, in press. YAMAMOTO,H. (1971): (Seasonal prevalence of natural infection of Japanese encephalitis virus in vector mosquitoes from the epidemiological standpoint). Advan. Med. Zool., I, 77-103, Keigaku-Shuppan Co., Tokyo.
J. Med. Sci. Biol., 31, 317-324, 1978
EPIDEMIOLOGICAL IN IV.
STUDIES ON JAPANESE ENCEPHALITIS KYOTO CITY AREA, JAPAN
NATURAL
INFECTION
IN
SENTINEL
OSAMUMAEDA, KUNIHACHITAKENOKUMA,
PIGS
YOSHIAKIKARO JI,
AKIO KURODA, OSAMUSASAKI, TOSHIROKARAKI and TAKASHIISHII* Kyoto City Research Institute of Public Health, Higashi-Takada-cho, Mibu, Nakagyo-ku, Kyoto 604, and *Department of Biology, College of General Education, University of Tokushima, Mishima, Nanjo, Tokushima 770, Japan (Received: April 27, 1977)
SUMMARY: Natural infection with Japanese encephalitis virus in three sentinel pigs held in separate experimental huts was examined daily by virus recovery from blood samples of the pigs and from mosquitoes after incubation for about 7 days from their blood feeding and by HI antibody titration of the blood samples. After a period of low infection rates, below 6%, for about two weeks in engorged Culex tritaeniorhynchus summorosus, high mosquito infections of over 30% from each viremic pig occurred for two to three days. The pigs may be probably have been bitten by many infected but not infective mosquitoes in a period of about 10 days before infection.
INTRODUCTION
Annual dissemination of Japanese encephalitis virus in the Kyoto City area was examined from two different aspects. First of all, JE virus dissemination all over the Kyoto City area was investigated by virus recovery from unfed Culex tritaeniorhynchus summorosus caught at some stations (Maeda et al., 1978a). JE virus infection was estimated also by virus recovery from engorged mosquitoes after incubation for seven to 10 days (Maeda et al., 1978b). More detailed knowledge seemed necessary to explain the dissemination as to how often each pig is bitten by infected mosquitoes for JE virus infection to occur and how many infected mosquitoes are produced from a viremic pig. Therefore, natural infection in three sentinel pigs each held in an experimental but was examined -continuously by various methods .
前 田 理 ・竹 之熊 国 八 ・唐 牛良 明 ・黒 田 晃 生 ・佐 々木 、 修 ・唐 木 利 朗(京 都 市衛 生 研 究 所 京 区壬 生 東 高 田 町1-2),石 井 孝(徳 島大 学 教 養 部 生 物 学 教 室 徳 島市 南常 三 島1) 317
京都市中
MAEDA
318
MATERIALS Design
of
2 •~ 2 •~ 2 m baited
traps
covered
devised wood
Location in
by
the
placed
of
dry
Fig. 1.
The of
wood
by
Kato
or
polyvinyl
shed:
summer
near ice
about
shed:
made
with
all
dig
and
trap 10
m
Experimental
An
et
every
a al.
but penthouse
(1966)
experimental
but
where as
reported
pig
shed
at
mosquito
collection
was
unfed
year a
were
for
collecting
of
corrugated
set
mosquitoes
was
vinyl.
on
the
in
1969
walls
Animal compactly
screen.
station •gM•h,
from
31
AND METHODS
experimental with
Vol.
et al.
built mosquitoes
(Maeda the
hut.
western
et border
and
have al.,
two been
1978a). of
a
in
They group
1972:
collected
of
were sheds
1978
JE
VIRUS
INFECTION
IN SENTINEL
PIGS
319
along the Kamo River. Mosquito collection: In 1969, two baited traps were set in the but facing inward for collecting escaping mosquitoes and another facing outward for collecting invading mosquitoes as shown in Fig. 1. In 1971, two baited traps were set in each but both facing outward for collecting invading mosquitoes. A pig of three months' old having no antibody to the virus was fed in each hut , and mosquitoes caught with these traps were collected every morning after the middle of July in 1969 and from late July in 1971. Most mosquitoes , easily entering the but through narrow crevices, did not enter the traps set facing outward and such mosquitoes were caught with aspirator tubes every morning . Virus recovery from mosquitoes: Mosquitoes caught by these methods were identified and counted. Unfed and engorged C. tritaeniorhynchus were separated each other and used for virus recovery mixing together all kind of collection . The former was used for determining the infection rate before feeding , and the latter for estimating the number of infected mosquitoes after biting the pig, or for determining the infection rate after feeding the viremic pig . The unfed TABLE I Natural infection with JE virus of the sentinel pig , P1, in 1969
MAEDA
320
Vol.
et al.
31
mosquitoes were stored at -70 C after identification until inoculation into baby mice and the engorged ones at the same temperature after incubation for about a week at 27 C. The detailed technique for virus recovery has been described (Maeda et al., 1978a). Virus recovery from and HI antibody measurement of pig blood samples: Blood samples were taken from each pig every morning, cleaned up by centrifuging and inoculated into baby mice for direct detection of viremia. In 1971, the blood samples were titrated in 3-day old mice by injecting intracerebrally 0.01 ml of each of serial fivefold dilutions. HI antibody in the sample was measured by the same method as described (Maeda et al., 1978a).
TABLE II Natural infection with JE virus of the sentinel pig, P2, in 1971
* The
fluorescent
antibody
test was positive
in one out
of eight
inoculated
mice.
1978
JE
VIRUS
INFECTION
IN SENTINEL
PIGS
321
RESULTS Mosquito
Collection
Most of the mosquitoes caught with the baited traps and aspirator tubes were C. tritaeniorhynchus. Over 90% of this species remained in each but every morning without entering the baited traps set facing outside. The mosquitoes of over 95% were engorged with the blood of each sentinel pig. Daily fluctuations in the number of C. tritaeniorhynchus were remarkable especially TABLE III Natural infection with JE virus of the sentinel pig, P3, in 1971
* The eight
fluorecent inoculated
antibody mice.
test was positive
with
brain
tiss
ues from
two out
of
MAEDA
322
Vol.
et al.
31
in that of the remainders in 1969 and 1971 and in that of mosquitoes caught with traps 2 and 3 in 1969. Detailed discussions about number of these mosquitoes were made by Ishii and Karoji (1976). Natural
Infection
o f Each Sentinel
Pig with JE Virus
Table I presents natural infection of pig Pl in 1969, including the results of virus recovery from the mosquitoes engorged on the pig and from the blood samples and of the titration of the blood samples for HI antibody. Tables II and III present the same information for 1971 in pigs P2 and P3, respectively, and of the titration for viremi a. In 1969, JE virus was recovered from the mosquitoes gorging on the sentinel pig at low infection rates below 2% in the period from July 23 to 31. After this period, high rates of infection occurred during August 1 to 5, the maximum rate being 33.3% on August 3. Viremia of the pig was first detected in the morning of August 5. These results indicate that the heavy mosquito infection resulted probably from viremia of the pig (Table I). In 1971, the virus was recovered from the mosquitoes gorging on each pig at a lower rate below 6% in the period from August 8 till the abrupt increase in the rate on August 29 in P2 and on September 2 in P3. The highest rates TABLE IV Dissemination of JE virus in the area shown by the virus recoveries from un fed C. tritaeniorhynchus
* Each
date
represents
collection
during
the
period
from the night of denoted date to the next morning. ** Virus recovery was made with each lump of unfed mosquitoes
caught
in both
huts.
1978
JE
VIRUS
INFECTION
IN SENTINEL
PIGS
323
were 83.3% in P2 in the night of August 31 and 54.2% in P3 on September 3. Viremia was detected in the morning from August 29 to September 1 in P2 and from September 1 to 5 in P3. The high titers above 1.0 in log LD50in 3 days' old mice continued for two days in P2 and for 4 days in P3. High antibody titers were detected in blood samples after September 1 in P2 and after September 4 in P3 (Tables II and III). JE Virus Dissemination
at the Study Site
Table IV shows virus recoveries from lumps of unfed mosquitoes collected with the traps and aspirators in the huts in both years. The highest infection rates were 0.56 and 2.64% in respective years and were not significantly different from the rates in engorged mosquitoes except during the period of the occurrence of viremia in each pig as in Tables I to III. These results were not significantly different either from the rates in unfed mosquitoes caught by dry ice collection at station "M" as described before (Maeda et al., 1978a). DISCUSSION
The highest infection rate in C. tritaeniorhynchus gorging on each viremic pig did not reach 100%. This rate may depend on the viremic titer in each pig. Hulburt (1964) reported that viremia in pigs had viremic titers between 2.1 and 3.0 in 3-day old mice, and that the effective viremia over 30% infection of C. tritaeniorhynchus usually did not last longer than for two days before 2 days after infection, nor later than 4 days. In our laboratory experiments (Sasaki et al., unpublished data), four non-vaccinated pigs without JE antibody were challenged with JE virus; three with infective mosquitoes and the other with a virus solution. Mosquito infection by feeding on the viremic pigs was investigated with detection of viremia with blood samples . We obtained results similar to those described by Hulburt (1964), except for a case of mosquito infection on the first and second days after infection, although viremia was detected in all pigs in the period from the first to the fourth days after infection. From these results, it is very likely that the sentinel pigs were probably infected at night not so long before the occurrence of viremia in any pig . The number of the infected mosquitoes gorging on each sentinel pig was estimated daily by calculating the number of positive engorged mosquitoes and the infection rate from these mosquitoes as shown in Tables I to III . The sentinel pigs might be bitten by many infected mosquitoes during a period of about 10 days before the occurrence of viremia. If the highest viremia :occurred a few days after infection, the pigs might have been infected at the end of the period by biting by infective mosquitoes. This fact suggests that there are only a few infective mosquitoes in a given infected mosquito population in an early period every year. Cumulative infection rates in the period of viremia calculated for each pig
324
MAEDA
et al.
Vol.
31
were 72.1, 169.8, and 139.0%, respectively. The variation in the value amongg pigs is large and may be an important factor in production of infected mosquitoes and of human epidemic. The infection rates were investigated also with several other pig populations as described before (1978b). Cumulative rates, being somewhat high in the highest epidemic year of 1967, also differed among pig populations. Yamamoto (1971) investigated annual variation in the infection rate of unfed mosquitoes at some localities and reported that the rate did not correlate with the scale of the epidemic. However, the infection rate of unfed mosquitoes may more easily be affected by many factors than of engorged ones. The quantitative relation in the infection rate between engorged and unfed mosquitoes will be analyzed in a separate paper. REFERENCES
HURLBURT,H. S. (1964): The pig mosquito cycle of Japanese encephalitis in Taiwan. J. Med. Entomol., 1, 301-307. ISHII, T. ANDKAROJI,Y. (1976): Analysis of mosquito trap collection. 2. A comparison of varioust sample populations collected simultaneously at one station. J. Sci. Univ. Tokushima, 9, 15-46. KATO, M., ISHII, T., WATANABE,T. AND YOSHIDA,S. (1966): A newly dry ice baited trap for collecting mosquitoes. Japan. J. Sanit. Zool., 17, 83-88. MAEDA, O.,KARAKI,T., KURODA,A., KAROJI, Y., SASAKI, O.ANDTAKENOKUMA, K. (1978a): Epidemiological studies on Japanese encephalitis in Kyoto City area, Japan. II. Annual prevalence of virus dissemination based on virus recoveries from unfed Culex tritaeniorhynchus summorosus. Japan. J. Med. Sci. Biol., 31, 39-51. MAEDA, O., KARAKI,T., KURODA,A., SASAKI, O.,KAROJI, Y. ANDTEKENOKUMA, K. (1978b): Epidemiological studies on Japanese encephalitis in Kyoto City area, Japan. III. Seasonal prevalence of the virus infections in several pig populations shown by the virus recovery from engorged Culex tritaeniorhynchus summorosus. Japan. J. Med. Sci. Biol., 31, 277-290. SASAKI, O., KAROJI, Y., KURODA,A., KARAKI,T., TAKENOKUMA, K., MAEDA, O., KODAMA,K. AND SASAKI,M. (1978): Experimental challenge of the infected mosquitoes to the pigs immunized with the live attenuated Japanese encephalitis vaccine. Acta Virologica, in press. YAMAMOTO,H. (1971): (Seasonal prevalence of natural infection of Japanese encephalitis virus in vector mosquitoes from the epidemiological standpoint). Advan. Med. Zool., I, 77-103, Keigaku-Shuppan Co., Tokyo.
