DDT & deltamethrin resistance status of known Japanese encephalitis vectors in Assam, India.

Background & objective: Japanese encephalitis (JE) outbreaks are common in Assam, northeastern State of India. Information on resistance in known JE vectors in the affected area is important for effective control measures. This study was undertaken to determine the species abundance of JE vectors endemic to Sibsagar district of Assam, and their susceptibility against DDT and deltamethrin. Methods: Adult mosquitoes were collected using CDC light trap and aspirators from human dwellings from 13 endemic villages falling under three Primary Health Centres. Collected mosquitoes were identified and unfed female mosquitoes were used for DDT and deltamethrin sensitivity bioassay. The bioassay was performed following WHO protocol using standard susceptibility test kit. Knockdown time (KDT) was monitored at every 10 minutes intervals, whereas mortalities were recorded 24 h post-exposure. Vector density and resistance status were mapped using geographic information system (GIS) technique. Results: A total of 7655 mosquitoes were sampled under three genera, i.e. Anopheles, Culex and Mansonia, and nine species, the JE vector Cx. vishnui group (31.78%) was the most predominant species, followed by Ma. uniformis (16.81%) and Ma. indiana (16.45%). All vector species were suspected to be resistant to DDT and sensitive to deltamethrin, except Ma. indiana, which was suspected to deltamethrin resistant. The KDT50 and KDT95 values of vector mosquitoes for DDT were significantly higher as compared to deltamethrin. The probit model used to estimate KDT50 and KDT95 values did not display normal distribution of percentage knockdown with time for all the vectors tested for DDT and deltamethrin, except for Ma. indiana for deltamethrin assay and Cx. gelidus for the DDT assay. Interpretation & conclusion: Differences in insecticide resistance status were observed between insecticides and vector species. The results of this study provided baseline data on insecticide resistance in known JE vectors of Sibsagar, Assam. The maps generated may allow better communication in control operations and comparison of changes in susceptibility status of these vectors over time.

Development of ELISA based detection system for lethal toxin of Clostridium sordellii.

Background & objectives: Clostridium sordellii and its toxins are associated with diseases in animals as well as human. C. sordellii produces two protein toxins (lethal toxin and haemorrhagic toxin). Lethal toxin has gained more importance due its high toxicity. The present study was carried out to develop a sandwich ELISA for detection of lethal toxin of C. sordellii. Methods: The catalytic domain (1.6kb) of lethal toxin of C. sordellii was PCR amplified, cloned into pQE30 UA vector and transformed into Escherichia coli SG 13009. Expression conditions were optimized and the recombinant protein was purified under native condition using Ni-NTA affinity chromatography, confirmed by SDS-PAGE and Western blot. Antibody was generated against the purified recombinant protein using Freund’s complete and incomplete adjuvants (FCA and FIA) in BALB/c mice and rabbit. A sandwich ELISA was optimized for the detection of lethal toxin. Results: The maximum recombinant protein expression was achieved at 0.5 mM IPTG (isopropylthiogalactoside) induction 4.0 h of post-induction. The polyclonal antibody raised in mice and rabbit showed a titre up to 1:512000. The produced antibody was highly sensitive with the detection limit of 0.3 ng/ml of lethal toxin at 1:4000 dilutions of mice (capturing) and rabbit (revealing) antibody. Interpretation & conclusions: An ELISA based detection system was developed for the detection of lethal toxin of C. sordellii. The developed detection system was found to be specific as there was no cross-reactivity with any other clostridial toxins. It will be useful for the detection of lethal toxin of C. sordellii in clinical and environmental samples.

Oroxylum indicum– a medicinal plant of North East India: An overview of its nutritional, remedial, and prophylactic properties.

Oroxylum indicum (family: Bignoniaceae) or Broken bones tree, which is distributed throughout India and South East Asia. Oroxylum indicum is known by such regional names as Bhatghila, Tona, Bhut-vriksha, Shyonaka, and Hanyu pinyin. Over the past two decades, many reports have appeared in mainstream scientific journals describing its nutritional and medicinal properties. While much of this recent enthusiasm indeed appears to be justified, it is critical to separate rigorous scientific evidence from anecdote. The present review provides the complete information about literatures of Oroxylum indicum as botanical descriptions, vernacular names, biological activity of plant parts, ethanomedicinal uses and current status of research with scope of investigation of Oroxylum indicum for future research. The structures of twenty eight isolated compounds from different parts of Oroxylum indicum with IUPAC names, molecular formula, formula weight, melting points were also reported in this study.

Hepatic hyperplasia and damages induces by zearalenone Fusarium mycotoxins in BALB/c mice / Hiperplasia e danos hepáticos induzidos por micotoxinas do gênero Fusarium-zearalenone em camundongos BAB/c

CONTEXT: Zearalenone is a mycoestrogen and considered a mycotoxin. OBJECTIVE: To establish whether zearalenone produced hepatotoxicity via oral administration. METHODS: Zearalenone was orally administered at a dose of 50 mg, 100 mg and 200 mg ZEN/body weight/daily, respectively, for 14 days to three groups of BALB/c mice. Diagnostic modalities used to evaluate hepatic damage and impaired hepatic function pre- and post zearalenone administration included hepatic marker enzyme activity, pentobarbital sleeping time, cytochrome P-450 activities and histopathologic evaluation of liver. RESULTS: Significant histopathologic changes viz. sinusoidal congestion, cytoplasmic vacuolization, hepatocellular necrosis and neutrophil infiltration were observed after evaluating of liver section from each group after accumulated zearalenone exposure. Further, zearalenone exposure increased activities of alanine transaminase, aspartate transaminase and lipid peroxides whereas activities of tissue glutathione and cytochrome P450 were decreased as compared to control mice. Zearalenone also increased the sleeping time and decreased sleeping latency after pentobarbital through intraperitoneal route as compared to control mice which indicates that the impairment of hepatic metabolizing enzymes by zearalenone. CONCLUSION: Zearalenone is a potential hepatotoxin by oral route.

CONTEXTO: Zearalenone é um micoestrógeno e considerado como micotoxina. OBJETIVO: Avaliar se o Zearalenone produz hepatotoxicidade por administração via oral. MÉTODOS: Zearalenone foi administrada por via oral em doses de 50 µg, 100 µg e 200 µg/peso corporal/dia/14 dias, respectivamente, para três grupos de camundongos BAB/C. Modalidades diagnósticas usadas para avaliar o dano hepático e comprometimento da função hepática pré- e pós-administração de Zearalenone incluíram atividade enzimática de marcadores hepáticos, tempo de sono por pentobarbital, atividade do citocromo P-450 e avaliação histopatológica hepática. RESULTADOS: Alterações histopatológicas significantes como congestão sinusoidal, vacuolização citoplasmática, necrose hepatocelular e infiltração neutrofílica foram observadas após avaliação histológica de cada grupo após exposição acumulada de Zearalenone. Além disto, a exposição à Zearalenone incrementou a atividade das enzimas alanina transaminase e aspartato transaminase e peróxidos lipídicos, ao passo que as atividades teciduais de glutationa e citocromo P-450 diminuiram, quando comparadas com camundongos-controle. Zearalenone também aumentou o tempo de sono e diminuiu a latência do sono após a administração de pentobarbital por via intra-abdominal, quando comparados com camundongos-controle, o que indica o comprometimento das enzimas do metabolismo hepático por ela. CONCLUSÃO: Zearalenone é uma potente hepatotoxina quando administrada por via oral.

In-vitro Antifungal activity of Sapium sebiferum L. against Aspergillus niger and Aflatoxigenic Aspergillus flavus.

Plants have provided a source of inspiration for novel drugs compounds, as plant derived medicines have made large contributions to human health and well-being. In present study, the methanolic, ethanolic, chloroformic and petroleum ether extracts of Sapium sebiferum leaves were investigated for their antifungal activity against Aspergillus niger and Aflatoxigenic Aspergillus flavus. Results obtained showed that all the extracts reduced colony growth by 1-32%. It was found that among all the leaf extracts, methanolic and ethanolic extracts have maximum percentage growth inhibition (26%) against Aspergillus flavus, while methanolic extracts showed maximum percentage growth inhibition (32%) against Aspergillus niger.

Evaluation of In-vitro antibacterial and anticandidal activity of Sapium sebiferum L.

The Sapium sebiferum leaf extracts were determined for their antimicrobial activities against Staphylococcus aureus, Micrococcus luteus ,Pseudomonas aeruginosa, Salmonella typhi, Klabsiella oxytoca, Listeria monocytogenes, E.coli, Saccharomyeces cereviceae and Candida albicans. The antimicrobial activities of these strains were compared with standard antibiotics (amoxicillin). Results obtained showed that all the extracts except aqueous (hot and cold) were effective against all the microbial strains. The methanolic extract of Sapium sebiferum leaves was most effective against all the bacterial strains and thus displayed highest zone of 21.0 mm at concentration of 100 mg/ml against Listeria monocytogenes ( MIC value is 25 mg/ml). Salmonella typhi and Klabsiella oxytoca did not show any activity. The anticandidal activity of methanolic, ethanolic and petroleum extracts showed maximum inhibition zone of 24.0 mm (MIC value is 25mg/ml).

Malaria incidence among paramilitary personnel in an endemic area of Tripura.

Background & objectives: Paramilitary operations along the Indo-Bangladesh border are adversely affected by malaria induced morbidity and mortality. Villages surrounding the paramilitary installations often serve as disease reservoirs. Malaria incidence in Tripura State Rifles (TSR) units in Dhalai District of Tripura was studied and the role of the village population in disease transmission was also assessed. Methods: Mass blood surveys were carried out among TSR personnel and villagers during 2007 to 2009. Malaria diagnosis through blood smear examination and rapid detection kits was done, and percentage parasitaemia was determined. Activity of malaria vectors was monitored using CDC light traps. Results: Slide positivity rates (SPR) in the neighbouring villages (51.4%) was significantly higher than that in TSR (27.7%) (P<0.0001). Malaria incidence in villages did not show seasonal variability while it was lowest during post-monsoon season in TSR (P<0.325; OR = 0.74). Per cent Pf parasitaemia was high in TSR (0.29) as compared to villagers (0.20) (P<0.0001). Anopheles minimus and An. dirus were the major malaria vectors observed. Interpretation & conclusions: Paramilitary and public health authorities should adopt targeted measures to reduce the malaria incidence in the villages surrounding the paramilitary installations as the village populations play a major role in disease transmission.

Heterologous expression of staphylococcal enterotoxin B (seb) gene for antibody production

Staphylococcal food poisoning (SFP) is caused by the members of superantigen family called staphylococcal enterotoxins (SEs). About 20 different types of SEs are produced by Staphylococcus aureus out of which type A (SEA), B (SEB), C (SEC) and D (SED) are commonly implicated in SFP. Among these, SEB is the most potent toxin and has also gained the status of biological warfare (BW) agent. Therefore, detection of SEB is of utmost importance. Any immunological detection system for SEB requires specific and sensitive antibodies which inturn depends on the purity of the SEB. In the present investigation, seb gene of S. aureus was cloned and expressed in E. coli along with biotin as fusion partner to facilitate the purification process. The yield of purified recombinant SEB was 13.1 mg/L of culture broth. Biotin tag from the biotinylated toxin was removed by protease cleavage, and both biotinylated and non-biotinylated toxin types were used for raising hyperimmune antiserum. Antisera were also specific for SEB amongst different kinds of food poisoning agents tested by indirect plate ELISA and western blot analysis. The quality of the antisera raised in this study was found superior to the commercially available antiserum. The investigation suggests that construction of recombinant staphylococcal enterotoxin B is a good alternative for production of pure enterotoxin to be used in antibody generation.

Development of an immunodetection test for a botulinum-like neurotoxin produced by Clostridium sp. RKD.

BACKGROUND & OBJECTIVES: Clostridial neurotoxins are among the most toxic substances known and cause severe illnesses in both humans and animals. A neurotoxigenic Clostridium sp. (strain RKD) isolated from intestine of decaying fish produced a novel, botulinum type B like neurotoxin as suggested by mouse bioassay, protection with anti-botulinum antibodies and PCR. The aim of the present investigation was to develop a laboratory based detection assay as an alternative to the mouse bioassay without compromising sensitivity and specificity. METHODS: Growth and toxin production were carried out in trypticase peptone yeast-extract glucose (TPYG) broth. Toxicity was estimated in terms of minimum lethal dose (MLD) by mouse bioassay. The toxin was partially purified by acid precipitation. It was used for toxoid preparation by formaldehyde treatment. This purified IgG was used for detection of neurotoxin using indirect ELISA. The culture supernatant was concentrated using a stirred cell with a 50 kDa cut-off membrane at 4 degrees C. Further purification was carried out using Prep cell. Fractions showing toxicity and sufficient purity were pooled, concentrated and analyzed on sodium dodecyl sulphatepolyacrylamide gel electrophoresis (SDS-PAGE). RESULTS: The toxin was purified with a recovery of 8.56 per cent. Polyclonal antiserum was raised in mice using partially purified toxin with a titre of 1: 80000. A detection assay with sensitivity of approximately 15 and 300 ng/ml for partially purified and crude toxins, respectively were achieved using an indirect ELISA method. INTERPRETATION & CONCLUSION: The Clostridium sp. RKD produced a potent neurotoxin earlier shown to have novelties. A specific detection assay for the neurotoxin has been developed that may be useful both from food safety and clinical point of view.

Detection of spores of Bacillus anthracis from environment using polymerase chain reaction.

A sensitive PCR based detection of Bacillus anthracis spores from environnment was standardized. Specific 1247bp amplicon could be detected with template concentration as low as 13 pg. Sensitivity was enhanced to 10 fold by nesting with second set of primers, forming 208bp amplicon. Extraction of DNA from spores purified from soil samples by aqueous polymer two-phase system followed by partial germination and freeze-thaw treatment yielded best results. Soil sample spiked with spores (8x10(2)/g of sample) could be detected with this method.