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Evidence of Japanese encephalitis virus infections in swine populations in 8 provinces of Cambodia: Implications for national Japanese encephalitis vaccination policy
Acta Tropica 120 (2011) 146–150
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Acta Tropica journal homepage: www.elsevier.com/locate/actatropica
Evidence of Japanese encephalitis virus infections in swine populations in 8 provinces of Cambodia: Implications for national Japanese encephalitis vaccination policy Veasna Duong a , San Sorn b , Davun Holl b , Manju Rani c , Vincent Deubel a , Philippe Buchy a,∗ a
Virology Unit, Institut Pasteur in Cambodia, # 5, Monivong boulevard, Phnom Penh, Cambodia Department of Animal Health and Production, National Veterinary Research Institute, Ministry of Agriculture, Forestry and Fisheries, Cambodia, Phnom Penh, Cambodia c World Health Organization, Regional Office of the Western Pacific, United Nations Avenue, PO Box 2932, 1000 Manila, Philippines b
a r t i c l e
i n f o
Article history: Received 26 November 2010 Received in revised form 14 July 2011 Accepted 15 July 2011 Available online 22 July 2011 Keywords: Japanese encephalitis virus Serology Swine Vaccination Cambodia
a b s t r a c t Although Cambodia, a Southeast Asian country, is suspected to be highly endemic for Japanese encephalitis virus (JEV), there are no nationally representative data on JEV transmission. Most of the existing data on human disease comes from few sentinel hospitals, and there have been no previous studies or surveillance for JEV transmission among pigs—the amplifying hosts in the natural cycle of JEV transmission. In preparation to develop a nationwide vaccination policy, data are required to show transmission of JEV in all the geographical regions of Cambodia. Analysis of JEV transmission among pigs will provide additional data on geographical scope and intensity of JEV transmission in Cambodia and will help to inform human vaccination policies in Cambodia. In this study, 505 sera obtained from swine bred in familial settings from 8 different provinces in Cambodia were tested by hemagglutination inhibition (HI) and ELISA tests to assess the presence of an immunological response to a JEV infection. Three hundred and thirty two sera (65.7%) were tested positives by HI assay and 321 (63.5%) by ELISA. Our results indicate that pigs particularly older than 6 months (95.2%) were highly infected with JEV in the 8 provinces. The high prevalence of HI antibodies and the high HI titer (>160 in 65.2% of cases and ≥1280 in 24.6% of cases) found in this age group suggest the important role of pigs in the transmission cycle of JEV in nature as they become probably rapidly infected and repeatedly re-exposed to the virus. Since the current pig rearing practices (within the backyard of home) are the same all over Cambodia, the results suggest that the human disease is also likely to be highly prevalent in the other provinces and warrant comprehensive policies for human vaccination and strengthened surveillance for acute meningo-encephalitis. © 2011 Elsevier B.V. All rights reserved.
1. Introduction The Japanese encephalitis virus (JEV) is a zoonotic arbovirus belonging to the genus of flavivirus in the Flaviviridae family (ICTV, 2008). JEV is a member of the JEV serological complex which consists of 8 species and 2 strains or subtypes: Japanese encephalitis virus (JEV), Murray Valley encephalitis virus (MVEV), St. Louis encephalitis virus (SLEV), West Nile/Kunjin virus (WNV/KUNV), Alfuy virus, Cacipacore virus, Yaounde virus, Koutango virus, and Ustusu virus (Mackenzie et al., 2002; van den Hurk et al., 2009). JEV is one of the important viral etiologies of encephalitis in Asia estimated to cause 35,000–50,000 human cases and 10,000 deaths annually (Solomon et al., 2000; WHO, 2006). Though there is no
∗ Corresponding author. Tel.: +855 12 802982; fax: +855 23 725606. E-mail address: [email protected] (P. Buchy). 0001-706X/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.actatropica.2011.07.008
cure for the disease and almost half of the survivors are left with life-long disabilities, there are safe and effective vaccines available for human use which have been used effectively in China, Japan and Republic of Korea to control the human disease (Halstead and Thomas, 2010). Culex tritaeniorhynchus is considered as principal vector of zoonotic and human JEV transmission. Others established culex vectors like C. vishnui, C. gelidus, C. fuscocephala, C. bitaeniorhynchus, C. infula, C. whitmori and C. annulus are also important secondary or regional vectors (Halstead and Jacobson, 2003; Rosen, 1986; van den Hurk et al., 2009). Other mosquito species possibly implicated in the transmission cycles of JEV are Aedes togoi, Ae. vexans nipponii, Ae. esoensis and Ae. curtipes (Halstead and Jacobson, 2003; Rosen, 1986). Although humans and equine are the only vertebrate hosts in which JEV is known to induce fatal disease under natural conditions, many other wild and domestic vertebrates and avian hosts
V. Duong et al. / Acta Tropica 120 (2011) 146–150
are known to be susceptible to JEV infection (Halstead and Jacobson, 2003; Rosen, 1986; van den Hurk et al., 2009). Anti-JEV neutralizing, hemagglutination-inhibiting (HI) or IgG antibodies have been previously detected in cattle, goats, wild boars, rodents, chickens, ducks, pigeons, sparrows, herons, and egrets (Halstead and Jacobson, 2003; Lim et al., 2007; Pant, 2006; Rodrigues et al., 1981; Rosen, 1986; See et al., 2002; van den Hurk et al., 2009; Yang et al., 2007). Neutralizing antibody has been also detected in frugivorous bats in China (Cui et al., 2008) and might be implicated in the life cycle of the virus through overwintering mechanisms allowing the persistence of the viremia (Cui et al., 2008; Rosen, 1986; van den Hurk et al., 2009). In swine, JEV infection can cause fetal abortion and stillbirth in infected sows and aspermia in boars. In Cambodia, JEV was first isolated from mosquitoes in 1965 (Chastel and Rageeu, 1966). In 1995, suspected cases acute Japanese encephalitis in human were reported in 2 pediatric hospitals in Phnom Penh, though the cases were not laboratory confirmed (Sunnara, 1995). From 1996 to 1998, hospital based surveillance was conducted at National Pediatric Hospital in Phnom Penh for hemorrhagic fever, encephalitis and hepatitis syndromes. Nine cases (18%) among 50 children with clinical encephalitis tested positive for anti-JEV IgM antibodies (Chhour et al., 2002). In another Cambodian study, 16 out of 99 patients with encephalitis had serological evidence of JEV infection but the virus was neither detected by PCR nor recovered after inoculation into cell cultures (Srey et al., 2002). A hospital-based sero-epidemiological surveillance among children with meningoencephalitis was established in 2006 in five sentinel hospitals and JEV was identified as the etiology in 19% of all children admitted with clinical diagnosis of meningoencephalitis (Touch et al., 2009). Despite the above evidence from few hospitals on transmission of JEV in those geographical areas, due to limited number of hospitals from where these data were collected, the empirical basis to implement a nationwide vaccination policy still remains rather weak. It just introduced JE vaccine for infants on pilot basis in 3 provinces with sentinel surveillance in 2009 and may need data on transmission of JEV in other areas before extending vaccination to other provinces. Considering the logistic difficulty in establishing nationwide surveillance for acute meningo-encephalitis with laboratory confirmation, an alternative approach may be to examine the JEV transmission among pigs, the intermediate amplifying host, from wider geographical areas. Data on JEV infection in animal have never been reported in Cambodia, even in the swine reservoir which has been recognized as a good candidate to show the global epidemiological pattern of the virus (Arai et al., 2008; Geevarghese et al., 1991; Wu et al., 1999). In this study, we have collected swine blood sample to determine the prevalence of HI and IgG antibodies against JEV in swine population in Cambodia. Presence of JEV transmission among pigs will provide an indirect evidence on geographical spread of human JE disease and will help to make a stronger case for nationwide vaccination policy in Cambodia.
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2.2. Hemagglutination inhibition (HI) test HI test modified from the original method of Clarke was performed (Clarke and Casals, 1958). Sera were inactivated at 56 ◦ C for 30 min, treated with 15% kaolin and washed with goose red blood cells (GRBC) to remove any nonspecific inhibitors and agglutinins. Two-fold serial dilutions were conducted in 96-wells U-form microplate from an initial volume of 50 l sera diluted at 1:10 in borate buffered saline. Then, diluted sera were mixed with 25 l of JEV antigen. Crude JEV antigen was prepared from suckling mice brain infected with the JEV Nakayama strain using sucrose–acetone extraction method and diluted in order to obtain a titer at 8 HI units. After overnight incubation at 4 ◦ C, 50 l of GRBC (diluted 1:24 from a mother solution containing 10% of GRBC in a Dextrose-GelatinVeronal with phosphate buffer) was added to the wells. The plate was incubated at room temperature for 30 min. The HI titer was given by the reciprocal highest dilution of serum that inhibited the agglutination of GRBC. Several negative and positive swine sera were included in each microplate. To ensure a better specificity, only sera with titers greater than 20 HI units were considered as positive. 2.3. Indirect ELISA test The ELISA used to detect IgG antibody against JEV was adopted from the technique developed by Dong-Kun Yang (Yang et al., 2006). Briefly, the 96-well microplate (Maxisorp; Nunc-ImmunoTM Plates, Roskilde, Denmark) was coated with JEV antigen (identical to the one used in HI test) diluted 1:80 in carbonate buffer (pH 9.6) and incubated overnight at 4 ◦ C. After 1 h of incubation at 37 ◦ C with blocking buffer (PBS + 5% skim milk), 50 l serum samples (diluted 1:200 in PBS + 0.1% Tween 20 and 1% skim milk) were added and incubated at 37 ◦ C for 1 h. After 3 series of wash with PBS + 0.05% Tween 20, 50 l of anti-swine IgG HRP conjugate (KPL, Gaithersburg, MD, USA) diluted 1:2000 in PBS was added. After the 1 h at 37 ◦ C incubation, the same volume of ABTS (2,2 -Azinobis [3-ethylbenzothiazoline-6-sulfonic acid]diammonium salt) substrate (KPL) was added and the reaction was kept at room temperature for 15 min before adding 50 l of stopping solution (1% sodium dodecyl sulfate solution). The optical density was measured using the Ultramark Microplate Imaging System (Bio-Rad, Hercules, CA, USA) and the cut off was defined as 3 times the average OD value measured in the 3 negative controls included in each microplate. 2.4. Statistical test The data analysis was done using Stata/SE version 9.0 (StataCorp, TX, USA). The comparison between percentage variables was tested by Chi2 and Fisher’s exact test, between two numeric variables by Spearman test and between numeric and categorical variables by Kruskal–Wallis test. A p value inferior to 0.05 was considered significant. 3. Results
2. Materials and methods 2.1. Samples collection Three hundred and ninety three swine sera collected in Kampong Cham in February 2006 and Kampong Speu province in July 2006 were provided by National Veterinary Research Institute of Cambodia (NaVRI). In December 2007, we added 112 blood samples collected in a slaughter house in Phnom Penh from swine originated from 8 different provinces in Cambodia (Fig. 1).
A total of 505 swine sera were tested by HI and ELISA tests. The average swine age was 4.3 months (standard deviation [SD]: 3.3, range: 20 days to 12 months). HI titer was >20 units in 332 (65.7%) samples and ELISA was positive in 321 (63.5%) samples (Table 1). Comparative analysis shows a significant correlation between HI and ELISA tests (r = 0.77, p < 0.001). In order to simplify the data reporting, only results from HI test (being more sensitive than ELISA in our hands) will be used thereafter.
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Fig. 1. Map showing the percentage of swine tested positive by HI test in 8 provinces.
The differences in HI test positive rates based on results obtained from swine originated from 8 different provinces are illustrated in Fig. 1. The positive rate was found very high in Prey Veng 100%, Svay Reang 100%, Kampot 100%, Pursat 96.3% and Kandal 96% and a bit lower in Kampong Speu 81.7%, Takeo 60% and Kampong Cham 52.5%. When age of swine from the 8 provinces was stratified into 4 groups (less or equal to 2 months, greater than 2–4 months, greater than 4–6 months and greater than 6), the HI test positivity rate in older swine was significantly higher than in younger one (p < 0.001) (Table 2). The mean HI titer in swine older than 6 months was significantly higher than in other 3 groups (p < 0.001) (Table 2). Moreover, among HI positive swine older than 6 months (118), 69.5% had a titer greater than 160 and 24.6% greater or equal to 1280. 4. Discussion Wild and domestic animals, and particularly pigs, which constitute the main reservoir/amplifying host in the natural cycle of JEV,
play a key role in the virus epidemiology and give an interesting and large picture of the epidemic situation (Halstead and Jacobson, 2003; van den Hurk et al., 2009). In Cambodia, human cases with encephalitis and highly suspected of JEV infection, based on the detection of IgM antibodies, have been reported based on studies done in few tertiary level hospitals (Chhour et al., 2002; Srey et al., 2002; Sunnara, 1995; Touch et al., 2009). However, case to develop a nationwide vaccination policy will be strengthened by availability of data on JEV transmission in broad geographical areas. The intensity of JEV transmission among pigs may shed some light on the geographical risk for the disease, but no routine pig surveillance or special studies have been carried out in Cambodia to document the disease in swine population. Routine pig surveillance has been carried out in Japan and South Korea, both of which have experienced high levels of JE disease in the past but controlled the human disease with JE vaccination, to assess the intensity of JE transmission and to predict any potential human outbreaks. In our study, 505 sera obtained from swine bred in familial settings in 8 different Table 2 HI result and mean titer by age group (n = 505).
Table 1 Comparison between results obtained by ELISA and HI test (n = 505).
Swine age groups
Swine positive by HI test (%), p < 0.001
Mean HI titer (SEa ) p < 0.001
≤2 months (n = 198) 2–4 months (n = 128) 4–6 months (n = 55) >6 months (n = 124)
94 (47.5) 72 (56.2) 48 (87.3) 118 (95.2)
246.6 (45.9) 219.5 (35.6) 428.4 (101.4) 698.31 (107.32)
Total
332 (65.7)
No. of samples tested by HI test
ELISA Positive Negative Total
Positive
Negative
Total
313 19
8 165
321 184
332
173
a
Standard error.
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provinces of Cambodia were tested by HI and ELISA tests to assess the existence of an immunological response to a JEV infection. A total of 332 sera (65.7%) were determined to be positive by HI assay and 321 (63.5%) by ELISA. Since the antibodies detected by either ELISA or HI test might correspond to those of other viruses in the same serological complex with JEV, we randomly selected 38 sera with different HI titer levels and tested them for plaque reduction neutralization assay (Russell et al., 1967). The results showed a good consistency between HI titer and 50% plaque reduction titer except for some sera with the lowest HI titer (≤160) (data not shown). Piglet acquires passively maternal antibodies which could be detected by neutralization test and HI assay. The neutralizing antibody against the JEV remains detectable in the majority of pigs until the age of 3–6 months and after this period, pigs become susceptible to virus (Geevarghese et al., 1987; Scherer et al., 1959). Almost 2/3 (60.7%) of pigs are slaughtered at the age greater than or equal to 6 months (Institut Pasteur in Cambodia, unpublished data). In the current study, the HI test was positive in 95.2% of pigs over 6 months and the high HI titers (>160 in 65.2% of cases and ≥1280 in 24.6% of cases) in this age group suggest probably recent and/or repeated exposures to JEV (Chang, 2002; Lim et al., 2007). These results highlight the potential impact of the JEV infection in swine, particularly at the age of reproduction, on the economy of those who basically rely upon pig rearing. They also reinforce the important role of over 6 month-old pigs in the maintenance of virus in the nature as they become probably rapidly infected and repeatedly re-exposed to the virus. In Cambodia, pigs are traditionally bred domestically, cohabit with human under free-range conditions and are mainly raised to be sold for meat after relatively short periods (10–12 months) (Samkol, 2008). According to the Ministry of Agriculture, Forestry and Fishery in Cambodia, there was a significant increase (11.9%) in pig production from 2000 to 2004 (MAFF, 2007) and a study conducted by the Center for Livestock and Agriculture Development in Cambodia found that each family in the 8 communities in Pursat province owned an average of 0.87 pig (CelAgrid, 2006). Annual farrowing or introduction of new piglets together with the removal of immune animals ensures the presence of non-immune pig being exposed to JEV every year. The finding in this study indicated that the high prevalence of HI antibodies in swine in 8 provinces (among top 15/24 most populated provinces in Cambodia) posed a potential risk of JEV transmission to human. This hypothesis could be supported by reports of highly suspected human cases of JEV infection previously reported among the 8 provinces where pigs were found positive (Chhour et al., 2002; Srey et al., 2002; Sunnara, 1995; Touch et al., 2009). Although seroprevalence rates have been reported in many mammal species, pig is the only mammal that is important in the JEV transmission cycle (van den Hurk et al., 2009). Therefore, in some areas, sentinel pigs have been used to monitor the activity of JEV and predict the possible occurrence of JEV infection in human (Arai et al., 2008; Chang, 2002; Geevarghese et al., 1991) because they fulfill the following criteria: (1) high natural infection rate, 98–100% (Scherer et al., 1959; van den Hurk et al., 2009), (2) high viremia titer lasting up to 4 days (Johnsen et al., 1974; Scherer et al., 1959), (3) ability to acquire blood samples easily, e.g. at slaughter house, (4) seroconversion in pigs preceding by 2–4 weeks the outbreak of JEV in human (Gingrich et al., 1987; Peiris et al., 1992; Wu et al., 1999), (5) easier interpretation of HI tests result in swine than in human in region where dengue and JEV co-circulate, (6) longer persistence of HI titer in swine (as long as 3 years) than in human (50% undetectable after 6–8 months) (Gajanana et al., 1995; Geevarghese et al., 1994), and (7) difficulty in detection of JEV infection in human: mostly unapparent (1:25–1:1000 symptomatic and asymptomatic ratio) (van den Hurk et al., 2009; WHO, 2007) and brief and low viremia titers (WHO, 2007).
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Despite the importance of pigs in JEV transmission cycle, there are few limitations in JEV surveillance in pigs. Pigs raised in Cambodia are rapidly slaughtered in a period of 10–12 months (MAFF, 2007). Moreover, pigs are not the only animals that are implicated in JEV cycle. Antibodies against JEV in other mammals and birds have been reported and these animals act as amplifying hosts (Nidaira et al., 2007; Ting et al., 2004; van-den-Hurk et al., 2008). Others domestic animals like chicken and goat were suggested as sentinels for JEV surveillance (Ting et al., 2004; Yang et al., 2007). Nonetheless, birds and poultry are not easy to bleed and goats are not widely bred in Cambodia. In addition, removing pig from areas of human habitation would not be an effective measure to reduce contact between amplifying hosts, vectors and humans (van-den-Hurk et al., 2008). Several studies have reported the presence of JEV transmission in regions where there is a relatively small number of pig population or where pigs are not raised (Konishi et al., 2009; See et al., 2002; Ting et al., 2004). Besides, in the present economical context in Cambodia, it would be difficult to eliminate peridomestic animal rearing. Therefore, our result underscores the implementation of good surveillance system of JEV in swine population and to a larger extent other domestic animals, and inclusion of human JEV vaccine in the National Vaccination Program. 5. Conclusion In this study, high positive rate of HI antibody against JEV in swine was reported and suggests: (1) a significant circulation of the virus in pig population in 8 provinces of Cambodia, (2) the potential impact on economy caused by the JEV infection on pigs reproduction, and (3) a risk of transmission of JEV to humans through close contact with pigs, and hence warranting a comprehensive human vaccination policy. A safe and effective live-attenuated vaccine (SA14-14-2) is available now for more than two decades and should be introduced into the routine immunization program for young with a catch-up campaign for older children, if resources allow. Regular serological surveillance of JEV infection in pigs may be very useful to better understand the epidemiology and seasonality of the virus and to be well prepared for outbreak in human. Competing interests The authors have declared that no competing interests exist. Acknowledgments We would like to thank the staff of the National Veterinary Research Institute of Cambodia for providing us the pig samples and the World Health Organization Regional Office of the Western Pacific for financial support. We would like to thank Professor Tomohiko Takasaki, National Institute of Health, Ministry of Health, Labour and Welfare, Japan and Prof. Cho Haewol, South Korea, Laboratory of the Microbiology and Immunology, College of Medicine, Eulji University for providing us positive and negative controls for ELISA and HI tests. We also express our sincere thanks to the staff members of Virology Unit, Institut Pasteur in Cambodia for their assistance. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. References Arai, S., Matsunaga, Y., Takasaki, T., Tanaka-Taya, K., Taniguchi, K., Okabe, N., Kurane, I., 2008. Japanese encephalitis: surveillance and elimination effort in Japan from 1982 to 2004. Jpn. J. Infect. Dis. 61, 333–338.
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Acta Tropica journal homepage: www.elsevier.com/locate/actatropica
Evidence of Japanese encephalitis virus infections in swine populations in 8 provinces of Cambodia: Implications for national Japanese encephalitis vaccination policy Veasna Duong a , San Sorn b , Davun Holl b , Manju Rani c , Vincent Deubel a , Philippe Buchy a,∗ a
Virology Unit, Institut Pasteur in Cambodia, # 5, Monivong boulevard, Phnom Penh, Cambodia Department of Animal Health and Production, National Veterinary Research Institute, Ministry of Agriculture, Forestry and Fisheries, Cambodia, Phnom Penh, Cambodia c World Health Organization, Regional Office of the Western Pacific, United Nations Avenue, PO Box 2932, 1000 Manila, Philippines b
a r t i c l e
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Article history: Received 26 November 2010 Received in revised form 14 July 2011 Accepted 15 July 2011 Available online 22 July 2011 Keywords: Japanese encephalitis virus Serology Swine Vaccination Cambodia
a b s t r a c t Although Cambodia, a Southeast Asian country, is suspected to be highly endemic for Japanese encephalitis virus (JEV), there are no nationally representative data on JEV transmission. Most of the existing data on human disease comes from few sentinel hospitals, and there have been no previous studies or surveillance for JEV transmission among pigs—the amplifying hosts in the natural cycle of JEV transmission. In preparation to develop a nationwide vaccination policy, data are required to show transmission of JEV in all the geographical regions of Cambodia. Analysis of JEV transmission among pigs will provide additional data on geographical scope and intensity of JEV transmission in Cambodia and will help to inform human vaccination policies in Cambodia. In this study, 505 sera obtained from swine bred in familial settings from 8 different provinces in Cambodia were tested by hemagglutination inhibition (HI) and ELISA tests to assess the presence of an immunological response to a JEV infection. Three hundred and thirty two sera (65.7%) were tested positives by HI assay and 321 (63.5%) by ELISA. Our results indicate that pigs particularly older than 6 months (95.2%) were highly infected with JEV in the 8 provinces. The high prevalence of HI antibodies and the high HI titer (>160 in 65.2% of cases and ≥1280 in 24.6% of cases) found in this age group suggest the important role of pigs in the transmission cycle of JEV in nature as they become probably rapidly infected and repeatedly re-exposed to the virus. Since the current pig rearing practices (within the backyard of home) are the same all over Cambodia, the results suggest that the human disease is also likely to be highly prevalent in the other provinces and warrant comprehensive policies for human vaccination and strengthened surveillance for acute meningo-encephalitis. © 2011 Elsevier B.V. All rights reserved.
1. Introduction The Japanese encephalitis virus (JEV) is a zoonotic arbovirus belonging to the genus of flavivirus in the Flaviviridae family (ICTV, 2008). JEV is a member of the JEV serological complex which consists of 8 species and 2 strains or subtypes: Japanese encephalitis virus (JEV), Murray Valley encephalitis virus (MVEV), St. Louis encephalitis virus (SLEV), West Nile/Kunjin virus (WNV/KUNV), Alfuy virus, Cacipacore virus, Yaounde virus, Koutango virus, and Ustusu virus (Mackenzie et al., 2002; van den Hurk et al., 2009). JEV is one of the important viral etiologies of encephalitis in Asia estimated to cause 35,000–50,000 human cases and 10,000 deaths annually (Solomon et al., 2000; WHO, 2006). Though there is no
∗ Corresponding author. Tel.: +855 12 802982; fax: +855 23 725606. E-mail address: [email protected] (P. Buchy). 0001-706X/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.actatropica.2011.07.008
cure for the disease and almost half of the survivors are left with life-long disabilities, there are safe and effective vaccines available for human use which have been used effectively in China, Japan and Republic of Korea to control the human disease (Halstead and Thomas, 2010). Culex tritaeniorhynchus is considered as principal vector of zoonotic and human JEV transmission. Others established culex vectors like C. vishnui, C. gelidus, C. fuscocephala, C. bitaeniorhynchus, C. infula, C. whitmori and C. annulus are also important secondary or regional vectors (Halstead and Jacobson, 2003; Rosen, 1986; van den Hurk et al., 2009). Other mosquito species possibly implicated in the transmission cycles of JEV are Aedes togoi, Ae. vexans nipponii, Ae. esoensis and Ae. curtipes (Halstead and Jacobson, 2003; Rosen, 1986). Although humans and equine are the only vertebrate hosts in which JEV is known to induce fatal disease under natural conditions, many other wild and domestic vertebrates and avian hosts
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are known to be susceptible to JEV infection (Halstead and Jacobson, 2003; Rosen, 1986; van den Hurk et al., 2009). Anti-JEV neutralizing, hemagglutination-inhibiting (HI) or IgG antibodies have been previously detected in cattle, goats, wild boars, rodents, chickens, ducks, pigeons, sparrows, herons, and egrets (Halstead and Jacobson, 2003; Lim et al., 2007; Pant, 2006; Rodrigues et al., 1981; Rosen, 1986; See et al., 2002; van den Hurk et al., 2009; Yang et al., 2007). Neutralizing antibody has been also detected in frugivorous bats in China (Cui et al., 2008) and might be implicated in the life cycle of the virus through overwintering mechanisms allowing the persistence of the viremia (Cui et al., 2008; Rosen, 1986; van den Hurk et al., 2009). In swine, JEV infection can cause fetal abortion and stillbirth in infected sows and aspermia in boars. In Cambodia, JEV was first isolated from mosquitoes in 1965 (Chastel and Rageeu, 1966). In 1995, suspected cases acute Japanese encephalitis in human were reported in 2 pediatric hospitals in Phnom Penh, though the cases were not laboratory confirmed (Sunnara, 1995). From 1996 to 1998, hospital based surveillance was conducted at National Pediatric Hospital in Phnom Penh for hemorrhagic fever, encephalitis and hepatitis syndromes. Nine cases (18%) among 50 children with clinical encephalitis tested positive for anti-JEV IgM antibodies (Chhour et al., 2002). In another Cambodian study, 16 out of 99 patients with encephalitis had serological evidence of JEV infection but the virus was neither detected by PCR nor recovered after inoculation into cell cultures (Srey et al., 2002). A hospital-based sero-epidemiological surveillance among children with meningoencephalitis was established in 2006 in five sentinel hospitals and JEV was identified as the etiology in 19% of all children admitted with clinical diagnosis of meningoencephalitis (Touch et al., 2009). Despite the above evidence from few hospitals on transmission of JEV in those geographical areas, due to limited number of hospitals from where these data were collected, the empirical basis to implement a nationwide vaccination policy still remains rather weak. It just introduced JE vaccine for infants on pilot basis in 3 provinces with sentinel surveillance in 2009 and may need data on transmission of JEV in other areas before extending vaccination to other provinces. Considering the logistic difficulty in establishing nationwide surveillance for acute meningo-encephalitis with laboratory confirmation, an alternative approach may be to examine the JEV transmission among pigs, the intermediate amplifying host, from wider geographical areas. Data on JEV infection in animal have never been reported in Cambodia, even in the swine reservoir which has been recognized as a good candidate to show the global epidemiological pattern of the virus (Arai et al., 2008; Geevarghese et al., 1991; Wu et al., 1999). In this study, we have collected swine blood sample to determine the prevalence of HI and IgG antibodies against JEV in swine population in Cambodia. Presence of JEV transmission among pigs will provide an indirect evidence on geographical spread of human JE disease and will help to make a stronger case for nationwide vaccination policy in Cambodia.
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2.2. Hemagglutination inhibition (HI) test HI test modified from the original method of Clarke was performed (Clarke and Casals, 1958). Sera were inactivated at 56 ◦ C for 30 min, treated with 15% kaolin and washed with goose red blood cells (GRBC) to remove any nonspecific inhibitors and agglutinins. Two-fold serial dilutions were conducted in 96-wells U-form microplate from an initial volume of 50 l sera diluted at 1:10 in borate buffered saline. Then, diluted sera were mixed with 25 l of JEV antigen. Crude JEV antigen was prepared from suckling mice brain infected with the JEV Nakayama strain using sucrose–acetone extraction method and diluted in order to obtain a titer at 8 HI units. After overnight incubation at 4 ◦ C, 50 l of GRBC (diluted 1:24 from a mother solution containing 10% of GRBC in a Dextrose-GelatinVeronal with phosphate buffer) was added to the wells. The plate was incubated at room temperature for 30 min. The HI titer was given by the reciprocal highest dilution of serum that inhibited the agglutination of GRBC. Several negative and positive swine sera were included in each microplate. To ensure a better specificity, only sera with titers greater than 20 HI units were considered as positive. 2.3. Indirect ELISA test The ELISA used to detect IgG antibody against JEV was adopted from the technique developed by Dong-Kun Yang (Yang et al., 2006). Briefly, the 96-well microplate (Maxisorp; Nunc-ImmunoTM Plates, Roskilde, Denmark) was coated with JEV antigen (identical to the one used in HI test) diluted 1:80 in carbonate buffer (pH 9.6) and incubated overnight at 4 ◦ C. After 1 h of incubation at 37 ◦ C with blocking buffer (PBS + 5% skim milk), 50 l serum samples (diluted 1:200 in PBS + 0.1% Tween 20 and 1% skim milk) were added and incubated at 37 ◦ C for 1 h. After 3 series of wash with PBS + 0.05% Tween 20, 50 l of anti-swine IgG HRP conjugate (KPL, Gaithersburg, MD, USA) diluted 1:2000 in PBS was added. After the 1 h at 37 ◦ C incubation, the same volume of ABTS (2,2 -Azinobis [3-ethylbenzothiazoline-6-sulfonic acid]diammonium salt) substrate (KPL) was added and the reaction was kept at room temperature for 15 min before adding 50 l of stopping solution (1% sodium dodecyl sulfate solution). The optical density was measured using the Ultramark Microplate Imaging System (Bio-Rad, Hercules, CA, USA) and the cut off was defined as 3 times the average OD value measured in the 3 negative controls included in each microplate. 2.4. Statistical test The data analysis was done using Stata/SE version 9.0 (StataCorp, TX, USA). The comparison between percentage variables was tested by Chi2 and Fisher’s exact test, between two numeric variables by Spearman test and between numeric and categorical variables by Kruskal–Wallis test. A p value inferior to 0.05 was considered significant. 3. Results
2. Materials and methods 2.1. Samples collection Three hundred and ninety three swine sera collected in Kampong Cham in February 2006 and Kampong Speu province in July 2006 were provided by National Veterinary Research Institute of Cambodia (NaVRI). In December 2007, we added 112 blood samples collected in a slaughter house in Phnom Penh from swine originated from 8 different provinces in Cambodia (Fig. 1).
A total of 505 swine sera were tested by HI and ELISA tests. The average swine age was 4.3 months (standard deviation [SD]: 3.3, range: 20 days to 12 months). HI titer was >20 units in 332 (65.7%) samples and ELISA was positive in 321 (63.5%) samples (Table 1). Comparative analysis shows a significant correlation between HI and ELISA tests (r = 0.77, p < 0.001). In order to simplify the data reporting, only results from HI test (being more sensitive than ELISA in our hands) will be used thereafter.
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Fig. 1. Map showing the percentage of swine tested positive by HI test in 8 provinces.
The differences in HI test positive rates based on results obtained from swine originated from 8 different provinces are illustrated in Fig. 1. The positive rate was found very high in Prey Veng 100%, Svay Reang 100%, Kampot 100%, Pursat 96.3% and Kandal 96% and a bit lower in Kampong Speu 81.7%, Takeo 60% and Kampong Cham 52.5%. When age of swine from the 8 provinces was stratified into 4 groups (less or equal to 2 months, greater than 2–4 months, greater than 4–6 months and greater than 6), the HI test positivity rate in older swine was significantly higher than in younger one (p < 0.001) (Table 2). The mean HI titer in swine older than 6 months was significantly higher than in other 3 groups (p < 0.001) (Table 2). Moreover, among HI positive swine older than 6 months (118), 69.5% had a titer greater than 160 and 24.6% greater or equal to 1280. 4. Discussion Wild and domestic animals, and particularly pigs, which constitute the main reservoir/amplifying host in the natural cycle of JEV,
play a key role in the virus epidemiology and give an interesting and large picture of the epidemic situation (Halstead and Jacobson, 2003; van den Hurk et al., 2009). In Cambodia, human cases with encephalitis and highly suspected of JEV infection, based on the detection of IgM antibodies, have been reported based on studies done in few tertiary level hospitals (Chhour et al., 2002; Srey et al., 2002; Sunnara, 1995; Touch et al., 2009). However, case to develop a nationwide vaccination policy will be strengthened by availability of data on JEV transmission in broad geographical areas. The intensity of JEV transmission among pigs may shed some light on the geographical risk for the disease, but no routine pig surveillance or special studies have been carried out in Cambodia to document the disease in swine population. Routine pig surveillance has been carried out in Japan and South Korea, both of which have experienced high levels of JE disease in the past but controlled the human disease with JE vaccination, to assess the intensity of JE transmission and to predict any potential human outbreaks. In our study, 505 sera obtained from swine bred in familial settings in 8 different Table 2 HI result and mean titer by age group (n = 505).
Table 1 Comparison between results obtained by ELISA and HI test (n = 505).
Swine age groups
Swine positive by HI test (%), p < 0.001
Mean HI titer (SEa ) p < 0.001
≤2 months (n = 198) 2–4 months (n = 128) 4–6 months (n = 55) >6 months (n = 124)
94 (47.5) 72 (56.2) 48 (87.3) 118 (95.2)
246.6 (45.9) 219.5 (35.6) 428.4 (101.4) 698.31 (107.32)
Total
332 (65.7)
No. of samples tested by HI test
ELISA Positive Negative Total
Positive
Negative
Total
313 19
8 165
321 184
332
173
a
Standard error.
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provinces of Cambodia were tested by HI and ELISA tests to assess the existence of an immunological response to a JEV infection. A total of 332 sera (65.7%) were determined to be positive by HI assay and 321 (63.5%) by ELISA. Since the antibodies detected by either ELISA or HI test might correspond to those of other viruses in the same serological complex with JEV, we randomly selected 38 sera with different HI titer levels and tested them for plaque reduction neutralization assay (Russell et al., 1967). The results showed a good consistency between HI titer and 50% plaque reduction titer except for some sera with the lowest HI titer (≤160) (data not shown). Piglet acquires passively maternal antibodies which could be detected by neutralization test and HI assay. The neutralizing antibody against the JEV remains detectable in the majority of pigs until the age of 3–6 months and after this period, pigs become susceptible to virus (Geevarghese et al., 1987; Scherer et al., 1959). Almost 2/3 (60.7%) of pigs are slaughtered at the age greater than or equal to 6 months (Institut Pasteur in Cambodia, unpublished data). In the current study, the HI test was positive in 95.2% of pigs over 6 months and the high HI titers (>160 in 65.2% of cases and ≥1280 in 24.6% of cases) in this age group suggest probably recent and/or repeated exposures to JEV (Chang, 2002; Lim et al., 2007). These results highlight the potential impact of the JEV infection in swine, particularly at the age of reproduction, on the economy of those who basically rely upon pig rearing. They also reinforce the important role of over 6 month-old pigs in the maintenance of virus in the nature as they become probably rapidly infected and repeatedly re-exposed to the virus. In Cambodia, pigs are traditionally bred domestically, cohabit with human under free-range conditions and are mainly raised to be sold for meat after relatively short periods (10–12 months) (Samkol, 2008). According to the Ministry of Agriculture, Forestry and Fishery in Cambodia, there was a significant increase (11.9%) in pig production from 2000 to 2004 (MAFF, 2007) and a study conducted by the Center for Livestock and Agriculture Development in Cambodia found that each family in the 8 communities in Pursat province owned an average of 0.87 pig (CelAgrid, 2006). Annual farrowing or introduction of new piglets together with the removal of immune animals ensures the presence of non-immune pig being exposed to JEV every year. The finding in this study indicated that the high prevalence of HI antibodies in swine in 8 provinces (among top 15/24 most populated provinces in Cambodia) posed a potential risk of JEV transmission to human. This hypothesis could be supported by reports of highly suspected human cases of JEV infection previously reported among the 8 provinces where pigs were found positive (Chhour et al., 2002; Srey et al., 2002; Sunnara, 1995; Touch et al., 2009). Although seroprevalence rates have been reported in many mammal species, pig is the only mammal that is important in the JEV transmission cycle (van den Hurk et al., 2009). Therefore, in some areas, sentinel pigs have been used to monitor the activity of JEV and predict the possible occurrence of JEV infection in human (Arai et al., 2008; Chang, 2002; Geevarghese et al., 1991) because they fulfill the following criteria: (1) high natural infection rate, 98–100% (Scherer et al., 1959; van den Hurk et al., 2009), (2) high viremia titer lasting up to 4 days (Johnsen et al., 1974; Scherer et al., 1959), (3) ability to acquire blood samples easily, e.g. at slaughter house, (4) seroconversion in pigs preceding by 2–4 weeks the outbreak of JEV in human (Gingrich et al., 1987; Peiris et al., 1992; Wu et al., 1999), (5) easier interpretation of HI tests result in swine than in human in region where dengue and JEV co-circulate, (6) longer persistence of HI titer in swine (as long as 3 years) than in human (50% undetectable after 6–8 months) (Gajanana et al., 1995; Geevarghese et al., 1994), and (7) difficulty in detection of JEV infection in human: mostly unapparent (1:25–1:1000 symptomatic and asymptomatic ratio) (van den Hurk et al., 2009; WHO, 2007) and brief and low viremia titers (WHO, 2007).
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Despite the importance of pigs in JEV transmission cycle, there are few limitations in JEV surveillance in pigs. Pigs raised in Cambodia are rapidly slaughtered in a period of 10–12 months (MAFF, 2007). Moreover, pigs are not the only animals that are implicated in JEV cycle. Antibodies against JEV in other mammals and birds have been reported and these animals act as amplifying hosts (Nidaira et al., 2007; Ting et al., 2004; van-den-Hurk et al., 2008). Others domestic animals like chicken and goat were suggested as sentinels for JEV surveillance (Ting et al., 2004; Yang et al., 2007). Nonetheless, birds and poultry are not easy to bleed and goats are not widely bred in Cambodia. In addition, removing pig from areas of human habitation would not be an effective measure to reduce contact between amplifying hosts, vectors and humans (van-den-Hurk et al., 2008). Several studies have reported the presence of JEV transmission in regions where there is a relatively small number of pig population or where pigs are not raised (Konishi et al., 2009; See et al., 2002; Ting et al., 2004). Besides, in the present economical context in Cambodia, it would be difficult to eliminate peridomestic animal rearing. Therefore, our result underscores the implementation of good surveillance system of JEV in swine population and to a larger extent other domestic animals, and inclusion of human JEV vaccine in the National Vaccination Program. 5. Conclusion In this study, high positive rate of HI antibody against JEV in swine was reported and suggests: (1) a significant circulation of the virus in pig population in 8 provinces of Cambodia, (2) the potential impact on economy caused by the JEV infection on pigs reproduction, and (3) a risk of transmission of JEV to humans through close contact with pigs, and hence warranting a comprehensive human vaccination policy. A safe and effective live-attenuated vaccine (SA14-14-2) is available now for more than two decades and should be introduced into the routine immunization program for young with a catch-up campaign for older children, if resources allow. Regular serological surveillance of JEV infection in pigs may be very useful to better understand the epidemiology and seasonality of the virus and to be well prepared for outbreak in human. Competing interests The authors have declared that no competing interests exist. Acknowledgments We would like to thank the staff of the National Veterinary Research Institute of Cambodia for providing us the pig samples and the World Health Organization Regional Office of the Western Pacific for financial support. We would like to thank Professor Tomohiko Takasaki, National Institute of Health, Ministry of Health, Labour and Welfare, Japan and Prof. Cho Haewol, South Korea, Laboratory of the Microbiology and Immunology, College of Medicine, Eulji University for providing us positive and negative controls for ELISA and HI tests. We also express our sincere thanks to the staff members of Virology Unit, Institut Pasteur in Cambodia for their assistance. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. References Arai, S., Matsunaga, Y., Takasaki, T., Tanaka-Taya, K., Taniguchi, K., Okabe, N., Kurane, I., 2008. Japanese encephalitis: surveillance and elimination effort in Japan from 1982 to 2004. Jpn. J. Infect. Dis. 61, 333–338.
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