Changing paradigm in the epidemiology of Japanese encephalitis in a non-endemic region.

BACKGROUND & OBJECTIVES: Japanese encephalitis (JE) is a mosquito-borne zoonotic disease. The JE virus (JEV) does not cause any disease among its natural hosts and transmission continues through mosquitoes belonging to Culex vishnui subgroup. This study was aimed to investigate the prevalence of JEV in mosquitoes and humans in the Thanjavur district, a non-endemic region for JE, in Tamil Nadu, by using standard available assays. METHODS: : A sero-surveillance study was conducted in Thanjavur district among the normal rural school children in the 5-12 yr age group, during the JE season (October) and post-JE season (February) from 2011 to 2013 for the detection of JEV infection. Vector abundance studies were carried out from 2011 to 2014. JE seropositivity and its association between the seasons were analysed statistically. RESULTS: : The occurrence of JE infection among children aged 5-12 yr was very high in the study area. The infection rates for JE in two consecutive seasons for 2011-12 and 2012-13 were 32.2 and 65.2%, respectively. The Cx. tritaeniorhynchus sp. dominated the catch, and was majorly responsible for the transmission. There was a significant difference in the human infection rate compared to the years 1991-92 and 1992-93; and a marked decrease in the cattle to pigs ratio (123 : 1) compared to the studies in 1991-93. INTERPRETATION & CONCLUSION: : The study unearthed the prevailing situation of JE among children, who are at higher risk of developing the disease during the transmission season. The decrease in the cattle to pigs ratio might be the one of the reasons for increase in the JEV infection among the children population compared to 20 years before. This trend requires urgent attention as it could be prevented with effective surveillance systems and vaccines.

Eimeria species occurrence varies between geographic regions and poultry production systems and may influence parasite genetic diversity.

Coccidiosis is one of the biggest challenges faced by the global poultry industry. Recent studies have highlighted the ubiquitous distribution of all Eimeria species which can cause this disease in chickens, but intriguingly revealed a regional divide in genetic diversity and population structure for at least one species, Eimeria tenella. The drivers associated with such distinct geographic variation are unclear, but may impact on the occurrence and extent of resistance to anticoccidial drugs and future subunit vaccines. India is one of the largest poultry producers in the world and includes a transition between E. tenella populations defined by high and low genetic diversity. The aim of this study was to identify risk factors associated with the prevalence of Eimeria species defined by high and low pathogenicity in northern and southern states of India, and seek to understand factors which vary between the regions as possible drivers for differential genetic variation. Faecal samples and data relating to farm characteristics and management were collected from 107 farms from northern India and 133 farms from southern India. Faecal samples were analysed using microscopy and PCR to identify Eimeria occurrence. Multiple correspondence analysis was applied to transform correlated putative risk factors into a smaller number of synthetic uncorrelated factors. Hierarchical cluster analysis was used to identify poultry farm typologies, revealing three distinct clusters in the studied regions. The association between clusters and presence of Eimeria species was assessed by logistic regression. The study found that large-scale broiler farms in the north were at greatest risk of harbouring any Eimeria species and a larger proportion of such farms were positive for E. necatrix, the most pathogenic species. Comparison revealed a more even distribution for E. tenella across production systems in south India, but with a lower overall occurrence. Such a polarised region- and system-specific distribution may contribute to the different levels of genetic diversity observed previously in India and may influence parasite population structure across much of Asia and Africa. The findings of the study can be used to prioritise target farms to launch and optimise appropriate anticoccidial strategies for long-term control.

Cryptic Eimeria genotypes are common across the southern but not northern hemisphere.

The phylum Apicomplexa includes parasites of medical, zoonotic and veterinary significance. Understanding the global distribution and genetic diversity of these protozoa is of fundamental importance for efficient, robust and long-lasting methods of control. Eimeria spp. cause intestinal coccidiosis in all major livestock animals and are the most important parasites of domestic chickens in terms of both economic impact and animal welfare. Despite having significant negative impacts on the efficiency of food production, many fundamental questions relating to the global distribution and genetic variation of Eimeria spp. remain largely unanswered. Here, we provide the broadest map yet of Eimeria occurrence for domestic chickens, confirming that all the known species (Eimeria acervulina, Eimeria brunetti, Eimeria maxima, Eimeria mitis, Eimeria necatrix, Eimeria praecox, Eimeria tenella) are present in all six continents where chickens are found (including 21 countries). Analysis of 248 internal transcribed spacer sequences derived from 17 countries provided evidence of possible allopatric diversity for species such as E. tenella (FST values ⩽0.34) but not E. acervulina and E. mitis, and highlighted a trend towards widespread genetic variance. We found that three genetic variants described previously only in Australia and southern Africa (operational taxonomic units x, y and z) have a wide distribution across the southern, but not the northern hemisphere. While the drivers for such a polarised distribution of these operational taxonomic unit genotypes remains unclear, the occurrence of genetically variant Eimeria may pose a risk to food security and animal welfare in Europe and North America should these parasites spread to the northern hemisphere.

Diagnosis of Chikungunya dominated co-infection with dengue during an outbreak in south India (2010 and 2012).

Following a report of dengue outbreak from January 2010 to 2012 in the Tirunelveli, Theni, Dharmapuri and Thiruvallur districts of Tamil Nadu state, India, an investigation was carried out. The study was to demonstrate the probable presence of Chikungunya viral antibodies in patients clinically suspected of dengue fever. Out of 331 samples analysed, dengue viral antibodies were observed in 14.8% (n = 49) of patients, while 16.6% (n = 55) were positive for Chikungunya viral specific IgM antibodies. In the four districts surveyed, patients found positive for Chikungunya were found to be higher than dengue. The clinician should consider Chikungunya in the differential diagnosis of dengue-like infection appearing in the community.

Seasonal abundance & role of predominant Japanese encephalitis vectors Culex tritaeniorhynchus & Cx. gelidus Theobald in Cuddalore district, Tamil Nadu.

BACKGROUND & OBJECTIVES: Japanese encephalitis (JE) is the leading cause of viral encephalitis in Asia. The first major JE outbreak occurred in 1978 and since 1981 several outbreaks had been reported in the Cuddalore district (erstwhile South Arcot), Tamil Nadu, India. Entomological monitoring was carried out during January 2010 - March 2013, to determine the seasonal abundance and transmission dynamics of the vectors of JE virus, with emphasis on the role of Culex tritaeniorhynchus and Cx. gelidus. METHODS: Mosquito collections were carried out fortnightly during dusk hours in three villages viz. Soundara Solapuram, Pennadam, Erappavur of Cuddalore district. Mosquitoes were collected during dusk for a period of one hour in and around the cattle sheds using oral aspirator and torch light. The collected mosquitoes were later identified and pooled to detect JE virus (JEV) infection by enzyme linked immunosorbent assay (ELISA). RESULTS: A total of 46,343 mosquitoes comprising of 25 species and six genera were collected. Species composition included viz, Cx. tritaeniorhynchus (46.26%), Cx. gelidus (43.12%) and other species (10.62%). A total of 17,678 specimens (403 pools) of Cx. gelidus and 14,358 specimens (309 pools) of Cx. tritaeniorhynchus were tested, of which 12 pools of Cx. gelidus and 14 pools of Cx. tritaeniorhynchus were positive for JE virus antigen. The climatic factors were negatively correlated with minimum infection rate (MIR) for both the species, except mean temperature (P<0.05) for Cx. gelidus. INTERPRETATION & CONCLUSIONS: High abundance of Cx. tritaeniorhynchus and Cx. gelidus was observed compared to other mosquito species in the study area. Detection of JEV antigen in the two species confirmed the maintenance of virus. Appropriate vector control measures need to be taken to reduce the vector abundance.

Dengue vectors prevalence and the related risk factors involved in the transmission of dengue in Thiruvananthapuram district, Kerala, South India.

BACKGROUND & OBJECTIVES: A longitudinal, entomological and virological study was conducted from 2007 to 2010 in four dengue fever affected areas of Thiruvananthapuram district, Kerala to understand the risk factors involved in the dengue transmission. METHODS: Aedes surveys were carried out seasonally in the selected localities both indoors and peridomestic sites. Water holding containers were sampled for the presence of immature. Outdoor and indoor resting/landing mosquitoes were collected. Blood meal identification was performed by gel diffusion test and viral assay using the ELISA test. RESULTS: The species found were Aedes (Stegomyia) aegypti (Linn.), Ae. (Stegomyia) albopictus (Skuse) and Ae. (Stegomyia) vittatus (Bigot). Aedes aegypti and Ae. albopictus immature stages were also found during the study period. Aedes aegypti was the only prevalent species in the water-starved Vizhinjam, a rural coastal area with breteau index (BI) ranging from 40 to 271. Aedes albopictus was recorded in rest of the three surveyed localities­two urban and one rural ghat areas of Thiruvananthapuram district. INTERPRETATION & CONCLUSION: The vector control measures should be focused mainly on source reduction of water storage containers present in both outdoor (Ae. albopictus and Ae. vittatus) and indoor (Ae. aegypti). To achieve effective vector management, a public health response beyond routine larviciding or focal spraying is essential throughout the year.

Entomological investigations into an epidemic of Japanese encephalitis (JE) in northern districts of West Bengal, India (2011-2012).

BACKGROUND & OBJECTIVES: Japanese encephalitis (JE) is one of the most important arboviral diseases of human beings with outbreaks in many parts of Southeast Asia including India. We present the entomological findings of an outbreak occurred in northern part of West Bengal during 2011-2012 with special emphasis on the role of JE vectors in different seasons. METHODS: Adult mosquito collections were made with the help of mouth aspirators, aided by flash lights during day time resting inside human and animal habitations as indoor, and resting outside field grasses, bushes, underneath of culverts and bridges as outdoor, and in and around the pig enclosures and cattle sheds during dusk period in JE affected villages from Cooch Behar, Dakshin Dinajpur, Darjeeling and Jalpaiguri districts in North West Bengal. In all study villages, a long handled with enamel bowl dipper was used to obtain immature stages of mosquitoes from various breeding habitats. RESULTS: A total of 19 different types of mosquito breeding habitats were examined for vectors of JE. From these habitats, 23.7 per cent were positive for breeding during the study period. Overall, nine different species were recorded through emergence, but none was positive for JE virus when subjected for detection of virus. Adult mosquitoes of more than 50 per cent of the potential JE vector species obtained through dusk and the rest through indoor and outdoor collections in all seasons. Altogether, 27 different species were recorded. Most of these were JE vectors. INTERPRETATION & CONCLUSIONS: Our results showed that in addition to Cx. vishnui subgroup, detection of JE virus antigen in Cx. quinquefasciatus indicated the possible maintenance of JE virus in nature through poor vector mosquitoes throughout the year. Since, all potential vector species reported elsewhere in India were also found in this region and fluctuated in density in different seasons, a proper integrated vector control programme needs to be implemented to control JE transmission.

Dengue NS1 and prM antibodies increase the sensitivity of acute dengue diagnosis test and differentiate from Japanese encephalitis infection.

Accurate and early diagnosis of dengue infection is essential for dengue case management. In outbreak conditions, it is essential to include two different tests to diagnose dengue and the choice depends on the number of days after the onset of illness in which the sample is collected. During the laboratory diagnosis of dengue in late acute and convalescent phase by MAC-ELISA, it is necessary to rule out possible cross reactions of closely related flavivirus, such as Japanese encephalitis virus which is commonly co-circulating. In the present investigation, the usefulness of dengue virus NS1 and prM antibodies in diagnosing and differentiating dengue from Japanese encephalitis infection was assessed using samples collected during out-breaks. It was shown here that, detection of antibodies against dengue NS1 and prM proteins increases the sensitivity of dengue diagnosis until 15days. Moreover, detection of antibodies against both proteins was able to differentiate dengue from Japanese encephalitis infection.

High-level expression of functionally active dengue-2 non-structural antigen 1 production in Escherichia coli.

Detection of nonstructural protein (NS1) is an important diagnostic marker during acute phase of dengue infection. Not only for diagnostic purpose, the protein had important role in vaccine design as well, as a candidate for studying virus assembly and maturation. Various researchers employed different expression systems and strategies for recombinant NS1 protein production. Attempts to express NS1 protein in prokaryotic and yeast expression system result in formation of insoluble protein which needs to undergo refolding to attain native structural and functional forms. Here, we report the production of soluble NS1 protein in E. coli by using appropriate vector and employing suitable culture conditions to maximize protein production. Proteins were purified using metal affinity chromatography. SDS-PAGE and western blot analysis reveal the native structure of NS1 protein. Solid phase ELISA using the recombinantly expressed antigen with positive and negative dengue samples showed that the expressed protein retains its antigenic and immunological properties. To our knowledge, this is the first report on the successful production of functionally active recombinant dengue-2 NS1 protein production without undergoing any in vitro posttranslational modification process.

Evaluation and use of NS1 IgM antibody detection for acute dengue virus diagnosis: report from an outbreak investigation.

The usefulness of detecting circulating non-structural protein 1 (NS1) IgM antibodies for diagnosing acute dengue virus infection was evaluated during an outbreak investigation along with other routinely used laboratory diagnostic methods. For the first time, the samples were also tested for NS1 antigen detection. NS1 IgM antibody detects all the serum samples that were positive for NS1 antigen detection within first 5 days of infection. The sensitivity of the NS1 IgM ELISA was higher when compared with RT-PCR and therefore, it could be used for early diagnosis.

Molecular identification of mosquito vectors using genomic DNA isolated from eggshells, larval and pupal exuvium.

Correct and precise identification of mosquito vectors is important in many respects including development of vector control strategies. Conventional identification methods have limitations for sibling and closely related species of mosquitoes, stage and quality of the specimen used and this could be overcome by DNA-based identification methods using molecular markers such as nuclear ribosomal internal transcribed spacer (ITS) which do not demand intact or undamaged specimen. Genomic DNA is usually isolated from whole mosquito, legs, wings etc. Alternate sources for genomic DNA isolation such as eggshells, larval and pupal exuviae were explored in this study by amplifying the ITS markers. Standardization of genomic DNA extraction and ITS amplification were carried out with laboratory specimens. The same was applied to specimens collected from the field. The results show that PCR amenable genomic DNA could be isolated from fresh exuviae collected in the laboratory and not from older and/or field specimens. But exuviae of larvae and/or pupae collected in the field reared to adulthood in the laboratory yielded PCR amenable genomic DNA. The results also revealed that the ITS2 marker could very well differentiate Aedes aegypti and Aedes albopictus by producing amplicons of ~330 bp and ~520 bp, respectively. The genomic DNA from these alternate sources also supported the species-specific PCR to distinguish the Culex vishnui subgroup mosquitoes.

Longitudinal studies of Japanese encephalitis virus infection in vector mosquitoes in Kurnool district, Andhra Pradesh, South India.

A 4-yr (2002-2006) entomological study was carried out in Kurnool district, Andhra Pradesh state, south India, to identify the mosquito vectors of Japanese encephalitis virus (family Flaviviridae, genus Flavivirus, JEV). In total, 37,139 female mosquitoes belonging five genera and 18 species resting on vegetation were collected in villages and periurban areas at dusk. Mosquito species composition and pattern of JEV infection in mosquitoes varied in periurban and rural areas. In periurban area, Culex gelidus Theobald was abundant, making up 49.7% of total catch followed by Culex tritaeniorhynchus Giles (44.5%). In rural area, Cx. tritaeniorhynchus was predominant, making up 78.9% of total catch followed by Culex quinquefasciatus Say (10.8%), Anopheles subpictus Grassi (7.1%), and Cx. gelidus (1.1%). In light trap collections, Cx. gelidus and Cx. tritaeniorhynchus predominated in periurban and rural areas, respectively. Of 50,145 mosquitoes screened JEV isolations were made only from Cx. gelidus and Cx. tritaeniorhynchus. Based on high abundance and frequent JEV isolation, Cx. tritaeniorhynchus was found to be the principal vector in both areas, whereas Cx. gelidus plays a secondary vector role in periurban areas only.

In-silico homology modeling of three isoforms of insect defensins from the dengue vector mosquito, Aedes aegypti (Linn., 1762).

Dengue is a serious public health problem in tropical and subtropical countries. It is caused by any of the four serologically distinct dengue viruses, namely DENV1-4. The viruses are transmitted by Aedes mosquitoes. Understanding various defence mechanisms of insects has become a prime area of research worldwide. In insects, the first line of defence against invading pathogens includes cellular mechanisms and a battery of antimicrobial peptides such as defensins, cecropins etc. Defensins--cationic, cysteine-rich peptides consisting of approximately 40 amino acids with broad-spectrum activity against Gram-positive bacteria--have been reported from a wide range of organisms. In the dengue vector mosquito, Aedes aegypti, three isoforms of defensins are reported to be expressed in a spatial and temporal fashion. This report presents the three-dimensional structures of the three isoforms of Ae. aegypti defensins predicted by comparative modeling. Prediction was done with Modeller 9v1 and the structures validated through a series of tests. The best results of the prediction study are presented, and may help lead to the discovery of new synthetic peptides or derivatives of defensins that could be useful in the control of vector-borne diseases.