J Vector Borne Dis 52, December 2015, pp. 321–328

Entomological and serological investigation of Japanese encephalitis in endemic area of eastern ,

Nikky Nyari1, Dharamveer Singh1, Kavita Kakkar1, Swati Sharma1, S.N. Pandey2 & T.N. Dhole1

1Department of Microbiology, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow; 2Department of Botany, Lucknow University, Lucknow, India

ABSTRACT

Background & objectives: Japanese encephalitis virus (JEV), a mosquito borne pathogen, is one of the major causes of viral encephalitis in eastern Uttar Pradesh, India. The objective of this work was to evaluate the entomological based virological surveillance of Japanese encephalitis (JE) in the highly endemic area of eastern Uttar Pradesh. Methods: The study was carried out during September 2010 to March 2013 in district of Uttar Pradesh. A total of 251 adult mosquito pools and 64 water samples containing larvae were collected from the District of Gorakhpur. Water pH, turbidity, and oxygen level were analyzed for vector breeding index (BI). In addition, 393 serum/cerebrospinal fluid (CSF) samples of acute encephalitis syndrome (AES) suspected cases were collected from the district hospital. Results: The various Culex species found included, Cx. quinquefasciatus (26.83%), Cx. vishnui (22.29%), Cx. pseudovishnui (20.73%), Cx. tritaeniorhynchus (12.71%), Cx. whitmorei (9.04%), and Cx. gelidus (8.25%). Highest minimum infection rate (MIR) was calculated for Cx. tritaeniorhynchus (2.32), followed by Cx. vishnui (1.98) and Cx. pseudovishnui (0.71). All the larvae samples were negative for JEV. The mean number larvae of Cx. tritaeniorhynchus and Cx. pseudovishnui was negatively correlated with pH (r = – 0.45 and r = – 0.63) and turbidity (r = – 0.30 and r = – 0.37). In contrast, positive correlation was observed in case of Cx. quinquefasciatus. A total of 41 clinical samples were found positive for JEV by IgM ELISA. The rainfall was significantly associated with Japanese encephalitis incidence and showed positive correlation to disease transmission (p = 0.02, r = 0. 66). Interpretation & conclusion: The findings showed the rapid dissemination of JEV within a population, facilitated by different species of Culex in the region. As JE is a vaccine-preventable disease, an immunization programme, an effective vector control strategy and application of standard hygiene practices in these endemic areas could result in a considerable reduction in morbidity and mortality due to JE. Key words Culex mosquitoes; ecology; Japanese encephalitis; surveillance; vector

INTRODUCTION states in India and the western regions of (1540 cases, 259 deaths)12. Peak number of JE cases are reported Japanese encephalitis (JE) virus is the most impor- from May to October in northern India, during and just tant mosquito-borne flavivirus causing morbidity and after the rainy season that coincides with heavy rains and mortality in humans1. In addition to JEV in India, there flooding13. Several ecological, biological and social fac- are several other endemic arboviruses like dengue, yel- tors influence larval and adult vector population and trans- low fever, tick-borne encephalitis, West Nile and Rift mission of JE14. JE has been isolated from 16 species of Valley fever virus that cause acute encephalitis syndrome mosquitoes belonging to the genera of Culex (10), Anoph- (AES)2. The JE case-fatality rate varies across geographi- eles (3) and Mansonia (3)15. JE is a zoonotic disease where cal regions ranging from 10 to 30% among infected pa- large water birds act as carriers, mosquitoes as vectors tients3-5. JE has been the leading cause of AES in and domestic pigs are amplifying host, while humans are Asia with 50,000 cases and 10,000 deaths reported incidental hosts16. Climate is a major environmental fac- annually6-8. In 1940, JE was first identified in China, and tor influencing vector-borne disease epidemiology17. Out- in 1949, it was identified in Korea and subsequently re- breaks of JE in India are facilitated by several factors like ported from India, in 19549-10. The following year, JEV temperature pattern, distribution of vector population, strain was identified at Vellore, India in 195511. More agriculture practices (wetland rice crop) swine rearing and recent outbreaks have been reported from Uttar Pradesh their proximity to human populations1, 18-20. (3551 cases, 764 deaths) and Bihar (238 cases, 58 deaths) JE is a vaccine preventable disease. Due to the wide- 322 J Vector Borne Dis 52, December 2015 spread use of JE vaccine, JE cases have been reduced in (Fig. 1). According to the 2011 census, the Gorakhpur China, Korea, and Japan21. JEV vaccination (live attenu- district has a population of 4,440,895 with a population ated Chinese vaccine SA-14-14-2) was started in Uttar density of 1337 inhabitants/km2, sex ratio of 950 females Pradesh on 27 May 2007 by the Govt. of India. The vac- for every 1000 males and a literacy rate of 70.83%. cination programme covered 97% of targeted 10 million children between 1 and 15 yr-old, in the endemic Dis- Entomological survey tricts of Uttar Pradesh. JE vaccination has significantly contributed to the decline of JE positive cases from 35.88% Surveillance of adult mosquitoes: Adult mosquitoes were as reported during 2005–06 to 7.28% during 2008–09 in collected from September 2010 to March 2013 from dif- the State of Uttar Pradesh, which contributes >80% of ferent areas of Gorakhpur, such as paddy crops, wheat, cases and deaths respectively in the country22. Entomo- millet, mustard, fodder plants, and other crops near houses logical surveillance was performed in association with using a battery operated, back packed aspirator23. The serological findings of JE incidence in the highly endemic selection of these sampling sites was based on the history area of eastern Uttar Pradesh, India, to correlate the influ- of high JE incidences from different blocks, viz. ence of environmental factors with vector breeding ecol- Barhalganj, , Bhathat, Charganwa, Gagha, Gola ogy and their involvement in disease transmission. Bazaar, Jungle Kaudia, Khajni and . The time period for mosquito collection was between 0700 and MATERIAL & METHODS 0900 hrs. Dead mosquitoes were discarded, and live ones were labeled and transported to the Sanjay Gandhi Post Study site Graduate Institute of Medical Sciences (SGPGIMS) labo- The study site, is well connected ratory, Lucknow, where they were identified according with all seven endemic districts of eastern Uttar Pradesh to species identification keys developed by Reuben in India. In 2009, a total of 686 cases of AES were re- et al 24 and stored at –20°C until tested. ported; out of which 76 cases and 19 deaths were con- firmed due to JEV. Gorakhpur district lies between lati- Collection of larvae: A total of 11 larval breeding sites tude 26° 46' N and longitude 83° 22' E. The district covers were surveyed in each nine blocks. The breeding habitats an area of 3483.8 km2. It is surrounded by Maharajganj varied from human habitat water containing low lying district to the north, Kushinagar and Deoria districts in flooded areas to animal hoof prints, ponds, puddle/bor- the east, Ambedkar Nagar, Azamgarh, and Mau districts row pits, paddy fields and stagnant water bodies. Water in the south, and Sant Kabir Nagar district in the west samples were collected by using 350 ml standard larval

Fig. 1: Map showing positive JEV cases and mosquito pools at different sample collection site of Gorakhpur district, Uttar Pradesh during 2010–13. Nyari et al: Surveillance of Japanese encephalitis in endemic area 323 dipper from the same25. Dips were taken gently with a in Culex species. The MIR was calculated as number of 2–3-min pause, to allow the mosquito larvae to move viruses detected in mosquitoes by species/total freely in the air–water interface and henceforth a mini- number of mosquitoes of that species tested multiplied mum of 5–10 dips were made from small and large water by 100027. sources respectively. Larvae were identified after micro- scopic observation24. Only Culex larvae were placed in ELISA glass/plastic vials containing water and stored at 4°C. Anti-JEV IgM and anti-dengue IgM antibodies in CSF Water analysis was performed using the Water Analyzer and serum samples were tested by IgM capture ELISA system (Type- 371, Systronics India Ltd.) for pH, turbid- (Panbio, Brisbane, Australia). The diluted sera and CSF ity and dissolved oxygen level. were added to the assay plate, which contained anti- human IgM antibodies coated on the surface of the wells Collection of clinical specimens and incubated at 37°C for 60 min. Concurrently horse- Patients with clinical symptoms of high grade fever radish peroxidase (HRP)-Conjugated anti-JEV/dengue (≥39 °C) for <10 days with any two of the following symp- monoclonal antibody was added to diluted JEV/dengue toms: headache, vomiting, unconsciousness, convulsions, virus type 1-4 antigens as per the kit manufacturer’s in- abnormal movements, stupor, delirium, altered sensorium, structions. The absorbance was measured at 450 nm us- neck rigidity, presence of kernig’s sign; admitted to dis- ing an ELISA reader (Finstruments, Multiskan Model, trict hospital of Gorakhpur, were enrolled for the study. Lab systems Finland, Type-347). The kit includes nega- The 217 blood and 176 CSF samples were collected from tive, positive and cut-off controls and results are based suspected AES patients. Serum samples were separated on a sample cut-off optical density ratio. from whole blood and aliquots stored at –80°C for fur- ther investigation. Meteorological data Meteorological data including temperature (°C), rain- Ethical approval fall (mm), and relative humidity (%) were obtained from Ethical approval was obtained for human samples by the meteorological department, Government of India on the Ethical committee bioethics cell, SGPGIMS, regular basis during the study period. Lucknow. Data surveillance Processing of specimens Data recorded from collected samples included each Adult and larval mosquitoes were pooled (20–25 patient’s name, age, residential address along with their mosquitoes/pool) by species, location and date of collec- clinical diagnosis and environmental condition. All ento- tion. Each pool was homogenized using a motor driven mological, epidemiological and laboratory data were en- tissue grinder with 3 to 5 ml of Eagle’s minimal essential tered and analyzed using SPSS, version 20 and prism, medium (MEM) media1. The homogenate was subjected version 5.01. Age, gender and environmental factor for to centrifugation and supernatant was used for further JE distributions in endemic areas of Uttar Pradesh were screening. determined.

RT-PCR Statistical analysis Viral RNA from adult mosquitoes and larvae were Data were analysed using SPSS Version 20 (SPSS extracted using QIAamp viral RNA mini kit according to Inc., USA). The significant difference between number the manufacturer’s instructions (Qiagen). Quantitative real of larvae and area were assessed by Kruskal–Wallis test. time polymerase chain reaction (qRT-PCR) for JEV (Ro- Chi-square test and z test were performed to test the sig- tor Gene 6000, Corbett Research, Australia) was done nificant difference in MIR with the different areas and using Geno Sen’s JEV real time PCR kit (Genome diag- between species respectively. Spearman’s coefficient of nostic, India).The qRT-PCR were carried out by reverse co-relation was applied to check the correlation between transcription at 50°C for 15 min, activation at 95°C for number of dips and breeding index (BI). Bivariate corre- 10 min, followed by 45 cycles at 95 °C for 15 sec, 50°C lation analysis was applied to check if the environmental for 30 sec and 72°C for 15 sec26. factors play role in disease transmission. For breeding index analysis, a linear regression model was performed Minimum infection rate (MIR) to determine if the rate of breeding index affects the rate The MIR was used to compare virus infection rates of positive cases in JE endemic areas. 324 J Vector Borne Dis 52, December 2015

RESULTS Larval and water analysis A total of 350 dips were taken from 11 different sam- A total of 6784 mosquitoes were collected which com- pling sites representing rural areas of study block. Out of prised 1820 Cx. quinquefasciatus (26.83%), 1512 Cx. 350, 117 were found positive for Culex larvae. Gola vishnui (22.29%), 1406 Cx. pseudovishnui (20.73%), 862 Bazaar (40.67%) showed maximum breeding index fol- Cx. tritaeniorhynchus (12.71%), 624 Cx. whitmorei lowed by Khajni (21.98%) and Bansgaon (21.75%) (Table (9.20%) and 560 Cx. gelidus (8.25%). Out of these Cx. 2). Each place differed significantly for the presence of quinquefasciatus, Cx. vishnui and Cx. pseudovishnui Culex larvae (p = 0.01). Percentage of positive dips was were the most commonly collected mosquitoes from found to be positively correlated with breeding index the surveyed area. The maximum number of Cx. (p = 0.93) indicating increase in number of Culex species tritaeniorhynchus was found in Charganwa (15.43%), after every dip. A total of 64 larval pools were made from Barhalganj (12.18%), and Gola Bazaar (11.95%). A total the most frequently collected species such as Culex vishnui of six out of 251 pools were positive for JEV by real-time (21), Cx. pseudovishnui (17) Cx. quinquefasciatus (14) PCR which included three pools of Cx. vishnui, two of Cx. and Cx. tritaeniorhynchus (12). The range of physico- tritaeniorhynchus and one of Cx. pseudovishnui. Although, chemical parameters of the collected water was: pH 6 to Cx. gelidus, Cx. whitmorei and Cx. quinquefasciatus did 8.13, turbidity 5.03–17.81 and 3.96 to 10.71 oxygen not show any positivity against JEV. Highest MIR was levels (Table 3). The mean number larvae of found in Cx. tritaeniorhynchus (2.32) and Cx. vishnui (1.98) Cx. tritaeniorhynchus and Cx. pseudovishnui was nega- as shown in Table 1. Distribution of positive mosquito pools tively correlated with pH (r = – 0.45 and r = – 0.63) and in different areas was estimated. Two positive mosquito turbidity (r = – 0.30 and r = – 0.37). The mean pools were found in Gola Bazaar. On the other hand, number larvae of Cx. quinquefasciatus was positively Bansgaon, Khajni, Charganwa and Pipraich accounted only correlated with pH (r = 0.47) and turbidity (r = 0.22). for one positive mosquito pool in each site (Fig. 1). No sig- All larvae samples were negative for JE virus by RT- nificant difference was observed in MIR among different PCR indicating that there is no transovarial infection of species of Culex (p = 0.89). JE virus.

Table 1. Adult mosquito collection with minimum infection rate (MIR) at different block

Species Barhalganj Bansgaon Bhathat Charganwa Gagha Gola Jungle Khajni Pipraich Total Total Positive MIR Bazaar Kaudia no. of pools pools mosquito

Cx. vishnui 152 173 127 205 110 137 149 218 241 1512 56 3 1.98 Cx. tritaeniorhynchus 105 78 93 133 90 103 87 94 79 862 32 2 2.32 Cx. gelidus 64 54 97 59 78 42 71 25 70 560 22 0 0 Cx. whitmorei 79 76 73 57 73 70 71 61 64 624 24 0 0 Cx. pseudovishnui 195 176 153 112 177 141 157 159 136 1406 52 1 0.71 Cx. quinquefasciatus 179 213 198 225 231 169 171 251 183 1820 65 0 0

Table 2. Dipper index and breeding index of larvae in water sample by dip method

Block/Site name Total no. of Positive dips Dipper index Mean no. of Breeding index dips (%) larvae per dip

Barhalganj 57 14 24.5 10.00 ± 3.136 9.12 Bansgaon 32 15 46.8 7.73 ± 3.453 21.75 Bhathat 44 12 27.2 10.67 ± 4.376 11.60 Charganwa 46 18 39.1 6.28 ± 3.045 17.15 Gagha 42 8 19 8.88 ± 3.563 1.69 Gola Bazaar 27 15 55.5 10.47 ± 3.226 40.67 Jungle Kaudia 30 7 23.3 9.00 ± 2.828 2.1 Khajni 39 17 43.5 7.82 ± 3.127 21.98 Pipraich 33 11 33.3 8.64 ± 3.042 8.64 Nyari et al: Surveillance of Japanese encephalitis in endemic area 325

Table 3. Mean of larvae on different water variables in various blocks

Block/Site name Barhalganj Bansgaon Bhathat Charganwa Gagha Gola Bazaar Jungle Kaudia Khajni Pipraich

Cx. vishnui pH 7.61 ± 0.13 7.42 ± 0.19 7.13 ± 0.03 7.80 ± 0.34 7.74 ± 0.05 7.43 ± 0.15 7.92 ± 0.10 6.84 ± 0.22 6.6 ± 0.19 Turbidity 5.03 ± 0.30 6 ± 0.91 9.06 ± 1.02 7 ± 1.3 12 ± 1.02 7 ± 0.91 8 ± 0.144 16 ± 2 8 ± 0.144 Dissolved DO 5.63 ± 0.1 4.23 ± 0.91 5.44 ± 0.40 7 ± 1.43 8 ± 0.33 4 ± 0.92 6.03 ± 0.33 7.45 ± 0.89 6.57 ± 1.14 Cx. tritaeniorhynchus pH 7.14 ± 0.13 7.90 ± 0.77 6.25 ± 0.39 6.45 ± 0.87 7.45 ± 0.64 6.20 ± 0.48 7.34 ± 0.63 6.80 ± 1 7.44 ± 0.99 Turbidity 9.32 ± 1.02 5.71 ± 0.90 6.32 ± 0.79 14.87 ± 1.62 10.72 ± 1.40 9.26 ± 0.90 5.32 ± 0.57 7.63 ± 1.22 12.40 ± 1.72 Dissolved DO 3.26 ± 0.47 5.78 ± 0.91 6.22 ± 0.85 4.92 ± 0.14 5.66 ± 0.63 7.45 ± 1 6.14 ± 0.63 7.44 ± 0.73 5.04 ± 0.44 Cx. quinquefasciatus pH 7.32 ± 0.05 6.31 ± 0.13 7.14 ± 0.41 6.83 ± 0.91 7.90 ± 0.47 8.13 ± 0.73 6.21 ± 0.80 6 ± 0.61 7.32 ± 0.37 Turbidity 5.14 ± 0.71 12 ± 1.21 9.41 ± 0.64 6.57 ± 0.83 7.41 ± 1.14 17.81 ± 1.37 9 ± 1.93 6.37 ± 0.63 14 ± 0.93 Dissolved DO 7.32 ± 0.40 5.09 ± 0.9310.71 ± 1.04 6.32 ± 0.65 7.14 ± 0.94 5.32 ± 0.41 6.03 ± 0.91 5.43 ± 0.71 7.41 ± 0.64 Cx. pseudovishnui pH 6.22 ± 0.14 7.43 ± 0.91 6.43 ± 0.71 7.90 ± 0.83 6.52 ± 0.32 6.59 ± 0.14 5.98 ± 0.37 6.47 ± 0.44 7.46 ± 0.56 Turbidity 5.46 ± 0.16 7.93 ± 0.4412.11 ± 1.56 13.46 ± 1.77 8.19 ± 1.61 7.32 ± 0.57 6.14 ± 1.37 9.57 ± 1.40 12 ± 1.32 Dissolved DO 6.45 ± 0.19 7.43 ± 0.63 8.46 ± 0.71 4.34 ± 0.14 7.46 ± 1 .32 5.43 ± 0.64 7 ± 0.37 5.83 ± 0.91 6.32 ± 0.47

Serological investigation Sera collected from 217 patients during the study period, showed positivity for both JE and dengue viruses by IgM capture ELISA. A total of 22 (10.1%) serum samples were reactive against IgM antibody for JE, 4 (1.8%) were cross reactive for JE/dengue and 191 (88%) were negative for both. Similarly, 19 in 176 (8.7 %) CSF specimens were positive for anti-JE-IgM antibodies, con- firming the involvement of JEV as an etiologic agent. However, only 41 (61% male and 39% female) cases were confirmed JEV positive. Although, JE cases were ob- served in all the age groups, the highest numbers of JE positive cases were observed in the age group of 0–10 yr. In total, 16 patients between age of >1–5 yr; and 10 from >5–10 yr were positive for JE (Fig. 2). Out of 41, the maximum number of JEV was reported from Charganwa Fig. 2: Age and gender-wise analysis of JE incidence in study area. (10), followed by Gola Bazaar (8), Bansgaon (6), Khajni (5), and Pipraich (4) (Fig. 1). temperature, relative humidity) tested, only rainfall was Correlation of environmental factor with JEV incidences significantly associated with JE incidences (p = 0.02, r = It was found that out of the three variables (rainfall, 0.66) as shown in Fig. 3.

(a) (b) (c)

Fig. 3: Correlation and association of different environmental conditions with disease transmission— (a) Rainfall vs JE incidence; (b) Temperature vs JE incidence; and (c) Humidity vs JE incidence. 326 J Vector Borne Dis 52, December 2015

Correlation of breeding index with JEV incidence tritaeniorhynchus indicates, infestation rate of larvae de- Vector breeding index was found to be statistically creased with increasing pH and turbidity. In contrast, significant with the positivity of JE cases in endemic ar- positive correlation indicates increase in infestation rate eas (p = 0.035 r2 = 0.491). However, Gola Bazaar, Khajni, of Cx. quinquefasciatus larvae with increasing pH and Bansgaon and Charganwa represented higher breeding turbidity28. index indicating the high clinical positivity of JE cases in On the basis of serological investigation; out of 393 the same areas. In contrast, maximum number of Cx. cases 41 were found reactive against IgM antibody and tritaeniorhynchus and Cx. vishnui were found in those had the history of illness for <10 days, indicative of an areas where number of ELISA positive cases was high active immune response. Although, in some districts of (Fig. 1). eastern Uttar Pradesh like Gorakhpur, Kushinagar, Maharajganj, Basti, Deoria, and some adjoining areas of DISCUSSION Bihar, the vaccine was provided through the National Vector Borne Disease Control Programme (NVBDC), Based on the entomological surveys done in cases are still reported annually33-34. In our study more Gorakhpur region, species of Culex mosquitoes were males were affected than females with the predominant found to be the most abundant species in all surveyed age group between 1 and 10 yr of age. The highest num- areas. This is because human habitat provided suitable ber of cases was observed in this age group possibly due breeding sites for the same28. In the present study, Cx. to their low immunity35. quinquefasciatus was the most abundant species found Moreover, the male individuals between the age of in urban, semi urban and rural areas. A wide range of Cx. >15 and 20 yr usually take an active part in crop cultiva- quinquefasciatus has also been documented in India and tion activities. The vector usually breeds in standing wa- other countries29. Being the cosmopolitan mosquito this ter in the cultivated rice paddies and grassy pools of wa- species is found abundantly near households of urban, ter where the majority of this age group is exposed to semi-urban and rural areas. In the present study the col- vector populations. Maximum JE incidences were re- lection of Cx. tritaeniorhynchus, a primary vector for JEV, ported from Charganwa, Gola Bazaar, Bansgaon, Khajni was found infrequent30. These differences in vector popu- and Pipraich which might be due to abundance of spe- lation might be attributed to changes in annual agricul- cific vectors, Cx. tritaeniorhynchus and Cx. vishnui. The tural practices, improper usage of insecticides in the agri- overall report of low positivity of JEV was probably due culture fields and variation in optimum humidity to decline of primary vector in this region. A significant influencing the evaporation of water from breeding statistically positive correlation was noted in the present sites16, 31. In the study, highest MIR was recorded for Cx. study between vector indexing and JE incidence. Thus, tritaeniorhynchus. Similar to the study of Kanojia et al. vector breeding index might be associated with the posi- most of the JE cases were found between August and tivity of JE cases in this endemic region. November, following the decline of vector populations, Viral diagnosis is tedious and expensive, and may particularly Cx. tritaeniorhynchus. Few environmental not be possible for individual patients. Now, JE is con- factors including rainfall, temperature and relative humid- sidered the most common form of sporadic encephalitis ity, are also responsible for virus transmission32. in Uttar Pradesh. Lots of programmes were conducted by Result of our study represents that rainfall might be the Govt. of India for minimization of JE risk by control- the important risk factor for JEV transmission. Present ling vector density. In rainy season, the incidence of both study reflects that the larvae of Culex species were abun- JEV and AES circulation was found to be increased. It dantly found in Gola Bazaar, Khajani, Bansgaon and was observed that some of the environmental conditions Charganwa exhibiting the high BI values. It was suggested were statistically significant risk factors for transmission that the water condition, i.e. pH, turbidity and dissolved of JEV virus. A result of JEV negative in CSF and serum oxygen of the breeding sites of larval species were found samples for ELISA also indicate the possibility of other quite favourable for breeding of JE vectors in these ar- pathogens that cause encephalitis, e.g. enteroviruses, such eas. In the study, some study areas showed low BI value. as coxsackie virus and herpes simplex virus in this area. Although, we didn’t quantified the presence of aquatic species that may cause harm to the larvae, but taking CONCLUSION other studies into consideration, besides water conditions this may be one of the possible reasons for the low BI The current study has identified the potential or ac- values in the studied areas28. Negative correlation of Cx. tual larval habitats of mosquitoes in the region and has Nyari et al: Surveillance of Japanese encephalitis in endemic area 327 represented several correlations between vector density India, 2005. 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Correspondence to: Dr T.N. Dhole, Professor and Head, Department of Microbiology, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Rae Bareli Road, Lucknow–226 014, Uttar Pradesh, India. E-mail: [email protected]

Received: 23 July 2015 Accepted in revised form: 15 September 2015