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RESEARCH ARTICLE
Year : 2022 | Volume
: 59
| Issue : 4 | Page : 356-362
Molecular epidemiology of Japanese encephalitis in pigs and risk factors associated with causing Japanese encephalitis in pigs of Lakhimpur, the first case reported in the district of North East India
U Rajkhowa1, AG Barua1, D Malakar2
1 Department of Veterinary Public Health, College of Veterinary Science, Assam Agricultural University, Khanapara, Guwahati, Assam, India
2 Department of Biotechnology, Mizoram University, Aizawl, Mizoram, India
Correspondence Address:
U Rajkhowa
Department of Veterinary Public Health, College of Veterinary Science, Assam Agricultural University, Khanapara, Guwahati-781022, Assam
India
Source of Support: None, Conflict of Interest: None
CheckDOI: 10.4103/0972-9062.355966
Background & objectives: Japanese encephalitis (JE) is a major public health problem in India. The first outbreaks of JE have been reported from the North-eastern regions of Assam, particularly from the Lakhimpur district of Assam between July–August 1989. In Assam every year many people died due to JE. This study was performed to investigate the molecular epidemiology of JE in pigs in Lakhimpur district of Assam and the risk factors associated with causing Japanese encephalitis in pigs. This study will help to map out the endemic regions and to know where and when to apply the most control strategies towards the prevention and control of the disease.
Methods: A total of 342 serum samples from pigs were collected from 10 organized and 20 unorganized farms from 9 blocks and recorded to age, sex and breed and tested by RT-PCR. Pig farms and the surrounding environment were studied for assessment of farm-level risk factors responsible for JEV infection in pigs.
Results: Out of 342 samples tested for detection of the E gene of JEV, 14 samples were found to be positive with a prevalence rate of 4.09%. Age, sex and breed-wise higher cases were found in at the age group above 12 months, sex wise female and breed-wise local pigs. Pig farms less than 500 meters from risk factors like rice field, stagnant water source, wild bird exposure to farm and mosquito exposure at farm/ bite to pigs, found to be more numbers of JE cases.
Interpretation & conclusion: Molecular epidemiology of JE in pigs, and humans; positive at Lakhimpur recommend the need for uninterrupted surveillance of this virus in pigs specially those areas where pig population is more and all risk factors are present.
Keywords: Molecular epidemiology; Japanese encephalitis; Pigs; Assam
Japanese encephalitis (JE) is mosquito-borne viral disease-causing encephalitis in humans as well as in horses and abortion in pigs[1]. The virus is small, enveloped with a single strand positive-sense RNA genome belonging to the genus Flavivirus of the family Flaviviridae. Two distinguished immunotypes of JEV are the Nakayama strain representing the prototype strain and the JaGAr01 strain; was isolated in 1959 from the Culex mosquito in Japan[2]. JE causes considerable illness and mortality in India, more than one billion people are living at risk and they are prone to get the disease, sharing the majority of cases in the World Health Organization (WHO) South-East Asia Region[3],[4].
JEV is the most common cause of epidemic encephalitis and childhood mortality, globally with an estimated 35,000 to 50,000 cases and 10,000 deaths annually[5]. Pig-mosquito-pig and bird-mosquito-bird are two basic transmission cycles of JEV in nature. Human and horses are accidental hosts for the JE virus but infection can lead to illness and death[6]. The first clinical case of JE in India was found in 1955 at Vellore; Tamil Nadu[7]. After 1955, several outbreaks have been reported from the different states of the country, predominantly in the rural areas. In 1973, the first major outbreak occurred in Burdwan and Bankura, the two districts of West Bengal with about 700 cases and 300 deaths[8]. Since then, the virus is active in almost all parts of India and outbreaks have been reported from the states of Bihar, Uttar Pradesh, Assam, Manipur, Andhra Pradesh, Karnataka, Madhya Pradesh, Maharashtra, Tamil Nadu, Haryana, Kerala, West Bengal, Odisha, Union territories of Goa and Puducherry[9]. Genotype III (GIII) of JEV was found as the dominant genotype in a study carried out in Odisha, Manipur and Assam[10]. Every year people died of JE in Assam. During the year 2022 a total of 30 people died due to JE. Japanese encephalitis cases are seen more during the month of June to August. A hospital-based retrospective study conducted from January 2014 to December 2017 showed a total of 3531 acute encephalitis syndrome cases were included out of which JE positivity rate was from 32.9% in 2014 to 13.3% in 2017[11].
The present study was carried out to investigate the molecular epidemiology of JE in pigs in Lakhimpur district of North East India; Assam and the risk factors associated with causing Japanese encephalitis in pigs.
Material & MethodsStudy area
The present investigation was carried out from the period October 2019 to April 2021 from 10 organized and 20 unorganized farms in the Lakhimpur district of Assam, India. Lakhimpur is in the northern part of Assam of situated between the 27.2064°N, latitude and 94.1514° E. JE endemic areas were identified based on the previous year’s medical cases. Samples were collected from all 9 blocks of Lakhimpur district namely - North Lakhimpur, Telahi, Nowboicha, Bihpuria, Narayanpur, Karunabari, Dhokuakhana and Ghilamara.
Sample collection
Age-, sex-, and breed-wise details of collected samples
A total of 342 samples were collected from 9 blocks and recorded age, sex and breed. Lakhimpur district is divided into nine blocks - North Lakhimpur, Telahi, Nowboicha, Bihpuria, Narayanpur, Karunabari, Dhokuakhana and Ghilamara. Human JE cases were reported from all blocks of this district and pig population is also present in every block. Based on the previous year’s medical cases and the locations where pig populations were more, only that places were selected for this study for sampling. From each block three locations were selected and from each location one farm was selected for this study. For molecular study, inclusion criteria were made, if total pig number in farm was two then only one pig was sampled and if more than two pigs in a farm then one per two pigs were sampled. Age-wise three groups were selected viz., 3-6 months, 7-12 months, and above 12 months.
RNA extraction and complementary DNA synthesis
The RNA was extracted from serum samples collected from endemic zone suspected for JEV and live attenuated vaccine using QIAamp viral RNA kit (Qiagen, Germany) as per the standard protocol. The cDNA was synthesized for extracted RNA with the help of RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific, USA). In a 0.2 ml RNase free PCR tube, 11 μl of template RNA and 1 μl random primer were added which placed on ice and the cDNA was synthesized for extracted RNA with the help of RevertAid First Strand cDNA Synthesis Kit in a final volume of 20 μl. The tube was vortex gently and centrifuged briefly and further incubated at 65°C for 5 min in a thermocycler. Immediately it was chilled on ice, spins downed and then the vial was placed back on ice. The vial was spin briefly and added 4 μl of 5X Reaction Buffer, 1μl RiboLockRNase Inhibitor (20 U/μL), 2 μl 10 mMdNTP Mix and 1 μl RevertAid M-MuLV RT (200 U/μL). The tube was mixed gently and centrifuged briefly then incubated for 5 min at 25°C followed by 60 min at 42°C. The reaction was terminated by heating at 70°C for 5 min. The reverse transcription reaction product was used directly in PCR applications or stored at -20°C for less than one week. For longer storage, -80°C was used.
Reverse transcription-polymerase chain reaction (RT-PCR)
RNA envelope (E) gene of JEV was targeted anticipated product size was 400 bp[12]. Standardization of RT-PCR was done from live attenuated JEV vaccine (strain SA-14-14-2) using E gene-specific primer and used as a positive control during the present study.
PCR amplification
PCR was carried out in a final volume of 20 μl using 2 μl cDNA, 2 μl specific primer amplifying 1 μl for each Forward & Reverse, 6 μl of nuclease free water, and 10 μl DreamTaq Green PCR Master Mix (2X, Thermo Scientific).
Confirmation of PCR amplicons
The amplified PCR products were confirmed by agarose gel electrophoresis using 2% percent agarose. The gel was then visualized in a gel documentation system.
Assessment of farm-level risk factors of Japanese encephalitis virus infection in pigs of Lakhimpur district
The endemic areas for JE cases in the Lakhimpur district were visited based on previous medical data and the pig farms and the surrounding environment was studied for assessment of farm-level risk factor responsible for JEV infection in pigs. The questionnaire for farmers was formulated to gather information regarding (i) age, sex, and breed of pig and (ii) Exposure of pigs in that farm to probable risk factors including closeness of farm to the rice field, the closeness of farm to standing water sources and wild bird exposure to farm and mosquito exposure at farm/bite to pigs, that can be a source of JEV transmission to farms. A distance of 500 m was taken from farms to all 4 risk factors to find out the percentage of farms infected with JEV located within 500 m or more and to find out the association between closeness to risk factors with JE infection in the farms.
Statistical analysis
All the data obtained from serological survey in pigs of Lakhimpur district were subjected to standard statistical procedures using SAS enterprise guide-4.3.
Ethical statement: Not applicable
ResultsIn the present study, a total of 342 serum samples from 10 organized and 20 unorganized farms from all the 9 blocks of the Lakhimpur district were subjected to RTPCR analysis for the detection of the E gene of JEV. Out of 342 samples, 14 (4.09%) samples were found positive in RT-PCR [Table 1] & [Figure 1]. Human JE cases were reported almost all the district of Assam. Positivity rate was found 12.5% at Lakhimpur district. Boginadi, Nowboicha, Dhakuakhan and North Lakhimpur block where human cases were more pig JE positive cases were also found to be more.
Table 1: Prevalence of Japanese encephalitis in pigs among 9 blocks of Lakhimpur district, Assam, India
Figure 1: Representative agarose gel electrophoresis image of E gene of JEV by RT-PCR. L1: Ladder, L2: Positive control, L3: Negative control, L5, L6, L8, L9, L10: Positive field isolates.Age, sex, and breed wise distribution of JE cases
Among the age group, the highest cases were seen in the age group of above 12 months (5.98%) followed by 7-12 (2.75%) months and 3-6 months (3.75%) [Table 2]. Regarding sex-wise cases were found more in females (4.34%) compared to males (4.81%) [Table 3]. Positivity rate was found more in crossbreed animals (4.39%) than local (3.75%) [Table 4].
Farm-level risk factors associated with Japanese encephalitis infection in pigs of Lakhimpur district The closeness of farms to rice field
Paddy fields are mostly in low land areas and during the summer season, most of the paddy fields are filled with flood and rainwater. Out of the total 30 farms included in the study, 20 farms were located within 500 meters from standing water sources of which 8 (40%) farms were found positive for JEV [Figure 1]. The rest 10 farms were at a distance of more than 500 meters from standing water sources out of which 2 (20%) farms were found positive for JEV infection [Table 5].
Table 5: Farm level JE infection and association with various associated risk factorsThe closeness of farms to standing water source
Pigs are reared mostly by the tribal community of Lakhimpur district. Tribal habitation is generally near the bank of the rivers. During the study, farms in the non-tribal and municipality areas were also investigated for the presence or absence of stagnant water sources near the pig farms [Figure 2]. Out of the total farms included in this study, a total of 7 (41.17%) farms out of 17 farms are located within 500 meters and 3 (23.07%) farms out of 13 farms located more than 500 meters from the rice field were found positive for JEV infection [Table 5] & [Figure 2]
Wild bird exposure to pig farms
Pigs are mostly reared by the open housing system in the Lakhimpur district, only a few farms were seen following organized form of pig husbandry. Most pigs are left loose to feed and in rice fields, they were seen closely interacting with wild birds. Large numbers of wild migratory birds are also visiting the wild bird sanctuary present in the district. Ardeid birds play an important role as a reservoir of Japanese encephalitis virus. Birds like cattle egrets and black night heron are reservoir of the virus. A total of 8 (42.10%) farms out of 19 farms encountered wild birds and only 2 (18.18%) farms out of 11 which do not encounter wild birds were found positive for JEV [Table 5] & [Figure 3].
Mosquito exposure at farm/biting to pigs
Mosquitoes act as a vector for JEV. A total of 17 numbers of pig farms, where farmers had reported that they had seen mosquitoes biting their pigs. Culex gelidus and Cx. tritaeniorhynchus are the two main vectors responsible for transmission of JE. Out of these 17 numbers of farms, 9 (52.94%) farms recorded antibodies against JEV, rest 13 farms where farmers reported that they used insecticide around the farms to control mosquitoes and regularly fogging around the farms by burning the locally available materials and they had not seen mosquitoes biting pigs but 1 (7.69%) farm among these 13 farms showed JE seropositivity [Table 5].
Mapping of an endemic zone of 9 blocks of Lakhimpur district based on disease causal factors
Based on the results, a map had been drawn to establish the occurrence of JE cases in all the 9 blocks of the Lakhimpur district and has been summarized in [Figure 4]. All the blocks have been divided into three categories i.e., highly endemic, moderately, and low or no endemic zone of Japanese encephalitis infection in pigs. Among 9 blocks higher prevalence was found in Boginadi, Nowboicha, North Lakhimpur and they fall under a highly endemic zone for JEV infection in pigs. All the 4 risk factors included in the present study are present in these blocks. The blocks which come under moderately endemic zone are Bihpuria, Dhakuakhana, and Karunabari. The blocks which fall under low or no endemic zone are Telahi, Narayanpur, and Ghilamara blocks.
Figure 4: Occurrence of JE cases in 9 blocks of Lakhimpur district, Assam, India based on molecular data. Discussion Prevalence of Japanese encephalitis
The present study revealed the highest prevalence in Boginadi (10.52%) block among the 9 blocks followed by North Lakhimpur (7.89%), Nowboicha (7.89%), Dhokuakhana (5.17%), Bihpuria (2.63%), and Karunabari (2.63%). It may be because these blocks have large numbers of pig population which are reared by loose housing system, farms are situated close to paddy fields, and a large number of stagnant water bodies formed by overflowing rivers are present in these blocks. The presence of rice filed, standing water sources near pig farms were highly susceptible to getting the risk of JEV infection. Water lodges in rice fields act as a source for mosquito breeding and spread the disease to large geographical areas leading to higher prevalence[13].
The blocks Telahi (0%), Narayanpur (0%), and Ghilamara (0%) showed no prevalence for JE infection in the pig populations. It was observed in the present study period that these two blocks had less pig population and the farmers of these blocks were taking some precautionary measures in the pig farms against mosquito bites by using insecticides around the farms to control mosquitoes and regularly fogging around the farms by burning the locally available things like hay, dry leaves, and papers. It was also noticed that a loose housing system of rearing pigs was uncommon in these blocks, thereby minimizing exposure to mosquitoes and wild birds[14].
Age-wise prevalence of Japanese encephalitis in pigs
The present study was conducted to find out the possible association among various age groups for the Japanese encephalitis virus. A slightly higher prevalence rate with higher age groups was observed in this study. It may be because the pigs with higher age groups had an increased level of exposure to mosquitoes due to their longer rearing time than the lower age groups. Piglet acquires passively maternal antibodies which could be detected by a serological test. 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 the virus[15].
Sex-wise seroprevalence of Japanese encephalitis in pigs
Male and females are equally susceptible to JEV infection. In the present study, cases were found slightly more in females. The reasons for the slightly higher prevalence in female pigs maybe because female pigs were reared for a longer duration for breeding purposes, leading to longer exposure time and higher risk for acquiring the infection. Similar findings were observed where the higher prevalence in females (22.22%) is more than in males (13.04%)[13].
Breed-wise seroprevalence of Japanese encephalitis in pigs
Breed-wise prevalence for JE in pigs was found higher in local pigs (4.39%) compared to the crossbreeds (3.75%). Similar findings were observed in China with higher prevalence in local pigs (12.10%) compared to exotic breeds (10.60%)[16]. The higher prevalence in local pigs may be attributed to their raising by the free-range system with the possibility of more exposure to mosquitoes as compared to crossbreeds which are generally raised in pig pens. In the present study, it was found that all pigs despite breed, age and sex are equally exposed and at the risk of JEV infection. There was no significant association among age, sex, and breed for JE in pigs[13].
Farm-level risk factors associated with Japanese encephalitis in pigs
The closeness of pig farms to the rice field
In the present investigation, it was observed that the major agricultural product of the Lakhimpur district is rice and most of the farmers were using their land for rice cultivation. Rice fields are preferred for breeding of Cx. tritaeniorhynchus mosquito and during the rainy seasons plenty of waterlogging in the paddy field and temperature facilitates larval growth resulting in increasing the density of the mosquitoes and ultimately transfer of JEV to pigs. Moreover, wild birds flock come searching for food in rice fields, thus rice fields are an important transmission site for infections, where susceptible birds and mosquitoes exist[17]. The practice of paddy cultivation, the proximity of houses to water bodies, and suitable climatic factors were the most important environmental factors associated with several outbreaks in North-East India as it provides mosquito breeding sites[18].
The closeness of farm to standing water source
Standing water is a good source for mosquito breeding. The river flows across Lakhimpur district and forms large number of standing water pockets/reservoirs. In this study it was found that farms located within 500 meters from standing water sources, were found more positive than farms located more than 500 meters distance from standing water sources. The irrigated rice system where water is lodged through a canal system act as a standing water source providing favorable conditions for mosquito breeding, increasing the mosquito densities, and ultimately spreading the JEV to a large area[19].
Exposure of wild birds to farms
This study showed that wild bird exposure to pig farms is significantly associated with JEV infection status. Some researchers indicated that along with other animals, chickens could also possibly act as amplifying hosts. Seroprevalance of JEV in chicken was found 11% prevalence and 12% in guinea fowl during a study[20]. The migratory birds play a major role in the importation of JEV into new territories. In the present study it was observed that in the open housing system, birds come to share the feed given to pigs like kitchen garbage. When infected birds come to visit those farms and are bitten by mosquitoes, healthy pigs run the risk of getting bitten by this infected mosquito thereby spreading the disease on a large scale. The farms which never encounter wild birds were also found positive for JE. It may be due to other risk factors responsible for causing disease in those farms like infected mosquitoes maintained in the area[21].
Pig farm exposure to mosquito
Mosquito acts as a carrier of disease and transfer the disease from one farm to another. Japanese encephalitis virus has been isolated from over 30 species of mosquitoes. Cx. tritaeniorhynchus and Cx. vishnui mainly breeds in the paddy field. In the present study it was found that most of the farmers didn’t use any preventive measures for the control of mosquitoes. It was also observed that farm exposure to mosquito bites found higher infection in pigs. The farms situated close by are at high risk of acquiring the infection[21].
The analogy between finding of porcine seroprevalence and human JE cases
Based on the human JE cases data collected from the Joint Director of the Health Services, North Lakhimpur District, and the result found in the present study, it was observed that among the nine blocks, four blocks where human cases were already reported, more pig JE cases were also found. It may be due to high pig population and also presence of all risk factors like paddy field and major rivers of the district flows across these blocks forming many reservoirs on the bank of the rivers. In Ghilamara block, despite low seroprevalence in pigs, human JE cases were found more. This may be because pigs are not the single reservoir of the virus, ardeid birds are also the reservoir of the JE virus, and a block like Ghilamara is nearby to a bird sanctuary[22].
ConclusionJapanese encephalitis is a major concern in Assam, India as it is reported from almost all districts every year. Besides the development of several interventions, epidemiological studies revealed that Assam has become endemic for JE and JE-associated acute encephalitis syndrome (AES) and increased pig farming is the main reason for the spread of this disease. In the case of human beings, certain investigation has been done till now in a different geographical region of Assam but only a few works have been done on pigs to date. Considering public health importance, it is very much needed to investigate the seroprevalence and molecular detection of JE in pigs and assessment of farm-level risk factors of JEV infection in pigs.
Conflict of interest: None
AcknowledgementsThis study was supported by the Department of Biotechnology, Govt. of India. The authors are thankful to the Department of Veterinary Public Health, College of Veterinary Science, Assam Agricultural University, Khanapara, Guwahati-781022, Assam, India.
References
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