Incidence of Japanese Encephalitis and Acute Encephalitis Syndrome Hospitalizations in the Medium-Endemic Region in Central India.

BACKGROUND: We estimated the incidence of Japanese encephalitis (JE) and acute encephalitis syndrome (AES) following routine immunization with the live-attenuated SA 14-14-2 JE vaccine. METHODS: We implemented enhanced surveillance of AES and JE hospitalizations in endemic districts in Maharashtra and Telangana States during 2015-2016 and 2018-2020. We estimated incidence and compared differences in the incidence of JE and AES between two states, and vaccinated and unvaccinated districts during two study periods. We also considered secondary data from public health services to understand long-term trends from 2007 to 2020. RESULTS: The annual AES incidence rate of 2.25 cases per 100,000 children in Maharashtra during 2018-2020 was significantly lower than 3.36 cases per 100,000 children during 2015-2016. The six JE-vaccinated districts in Maharashtra had significantly lower incidence rates during 2018-2020 (2.03, 95% CI 1.73-2.37) than in 2015-16 (3.26, 2.86-3.70). In addition, the incidence of both JE and AES in two unvaccinated districts was higher than in the vaccinated districts in Maharashtra. Telangana had a lower incidence of both JE and AES than Maharashtra. The AES incidence rate of 0.95 (0.77-1.17) during 2018-2020 in Telangana was significantly lower than 1.67 (1.41-1.97) during 2015-2016. CONCLUSIONS: The annual incidence rate of Japanese encephalitis was < 1 case per 100,000 children. It indicated accelerated control of Japanese encephalitis after routine immunization. However, the annual incidence of acute encephalitis syndrome was still > 1 case per 100,000 children. It highlights the need for improving surveillance and evaluating the impacts of vaccination.

Vanillic acid mitigates hyperinsulinemia induced ER stress mediated altered calcium homeostasis, MAMs distortion and surplus lipogenesis in HepG2 cells.

Hyperinsulinemia (HI) induced insulin resistance (IR) and associated pathologies are the burning and unsolvable issues in diabetes treatment. The cellular, molecular and biochemical events associated with HI are not yet elucidated. Similarly, no focused research on designing therapeutic strategies with natural products for attenuation of HI are seen in literature. Keeping this in mind we planned the present study to evaluate the alterations occurring at ER/Ca2+ homeostasis/mitochondria associated endoplasmic reticulum membranes (MAMs) in HepG2 cells during HI and to evaluate the possible beneficial effect of vanillic acid (VA) to mitigate the complications. An in vitro model of HI was established by treating HepG2 cells with human insulin (1 µM) for 24 h. Then, ER stress, Ca2+ homeostasis, MAMs, IR and hepatic lipogenesis were studied at protein level. Various proteins critical to ER, Ca2+ homeostasis and MAMs such as p-IRE-1α, ATF6, p-PERK, p-eIF2α, CHOP, XBP1, p-CAMKII, InsP3R, SERCA, JNK, GRP78, VDAC, Cyp D, GRP75, MFN2, PTEN and mTORC were studied and found altered significantly causing ER stress, defect in Ca2+ movements and distortion of MAMs. The decreased expression of IRS2 and an unaltered expression of IRS1 confirmed the development of selective insulin resistance in hepatocytes during HI and this was the crucial factor for the progression of the hepatic lipid accumulation. We found simultaneous treatment of VA is beneficial up to a certain extent to protect HepG2 cells from the adverse effect of HI via its antioxidant, antilipogenic, mitochondrial and ER protection properties.

Effectiveness of Japanese encephalitis vaccination among children in central India.

Japanese encephalitis (JE) disease among children continues in central India despite vaccination implemented in the routine immunization program. Therefore, we planned to estimate the JE vaccination effectiveness among children by undertaking a 1:2 individually-matched population-based case-control study from August 2018 to October 2020. The laboratory-confirmed JE cases aged 1-15 years were enrolled along with neighborhood controls without fever and encephalitis matched on the residence area, age and sex. The JE vaccination history was enquired from parents and verified independently from the vaccination cards available at home and records at health facilities. We enrolled 35 JE cases and 70 matched controls. The vaccination effectiveness of 86.7% (95% confidence interval [CI]: 30.8-94.7) was estimated on the per-protocol analysis of 31 case-control sets. The screening method provided an effectiveness of 89.5% (CI: 78.9-94.7) on using the population vaccination coverage of 90% reported earlier in the same area. In conclusion, JE vaccination offered a moderate level of protection among children in JE medium-endemic central India, similar to reports from high-endemic areas in India. The operational aspects of vaccination program implementation need to be evaluated to assess the impact of vaccination on the disease burden of JE in medium-endemic regions of India.

Electrocoagulation followed by sound agitation for removal of nitrogen and carbon-based pollutants from industrial wastewater.

The herculean imprecation of nitrogen-based pollutant like ammoniacal nitrogen (AN) and chemical oxygen demand (COD) on aquatic milieu is now a concern for the dye, pharma and fertiliser industries. Wastewater from these is characterised with high concentration of AN, COD and total dissolved solids (TDS), treatment of which is of utmost importance for a cleaner environment. In the current research work, an attempt was made to apply integrated electro-coagulation (EC) - sonication process for the removal of COD and AN from highly acidic dye intermediate wastewater containing high to very high concentration of COD and AN. Systematic laboratory experiments were conducted for the treatment of dye intermediate wastewater and influences of pH (5-11), applied voltage (0.5-4V) and electrolysis time (30-120 min) were investigated. A Response Surface Methodology (RSM) was used for optimization of major operating parameters for EC. The conditions for minimum fraction remaining (C/C0), was found to be same for both COD and AN, i.e. pH 7, time 90 min and applied voltage 2V. The C/Co value for COD and AN were 0.244 and 0.302, respectively. The C/Co value of COD and AN in combined EC-Sonication process with optimum operating conditions were 0.145 and 0.228 respectively with sonication time 60 min at a frequency of 33 kHz. Thus, EC - sonication process is an efficacious process for their removal from dye industrial wastewater.

A novel aureothin diepoxide derivative from Streptomyces sp. NIIST-D31 strain.

A novel vicinal diepoxide of alloaureothin was isolated from Streptomyces sp. NIIST-D31 strain along with three carboxamides, p-aminobenzoic acid and 1,6-dimethoxyphenazine. Exhaustive 2D NMR analysis and analysis of experimental, theoretical CD spectra aided in establishing the structure of compound 1. Compound 1 inhibits adipogenesis and accumulation of lipid droplets during the differentiation of 3T3-L1 cells.

Artificial germination of Enterocytozoon hepatopenaei (EHP) spores induced by ions under the scanning electron microscope.

Enterocytozoon hepatopenaei (EHP), is an emerging microsporidian pathogen responsible for hepatopancreatic microsporidiasis (HPM) in shrimps and is associated with severe growth retardation. The disease causes economic losses in shrimp aquaculture. In this study, EHP spore germination was induced and demonstrated with a scanning electron microscope (SEM). The ions (cations and anions) generated by high-energy electrons during frozen water radiolysis in the SEM specimen chamber induce EHP spore germination. This study is the first to demonstrate the induction of a microsporidian spore germination by ions generated under SEM. This study will enhance our understanding of EHP biology, life cycle and lead to the development of prophylactics and therapeutics for EHP control. Also, this method will help standardize the study of germination in other microsporidians.

Pretreatment of hydroethanolic extract of Dillenia indica L. attenuates oleic acid induced NAFLD in HepG2 cells via modulating SIRT-1/p-LKB-1/AMPK, HMGCR & PPAR-α signaling pathways.

ETHNOPHARMACOLOGICAL RELEVANCE: Dillenia indica L. is an edible plant from the Dilleniaceae family present in the forest of India and other Asian countries. Different parts of this plant are being used in the traditional system of medicines for various diseases like diabetes, indigestion, asthma, jaundice, and rheumatic pain by various rural communities. This plant is very common among Khamptis traditional healers, the rural community of the Dhemaji district of Assam, ethnic communities of Dibru-Saikhowa Biosphere Reserve of Northeast, India for various medicinal uses. It is observed as a 'vat' suppressant and 'pitta' boosting medicine in Ayurveda. AIM OF THE STUDY: The aim of this research was to evaluate the effect of hydroethanolic extract of Dillenia indica leaf (DI-HET) against non-alcoholic fatty liver disease (NAFLD) as it is reported effective against jaundice in traditional medicine. We are also planning to see the various molecular mechanisms responsible for its effect if it is efficacious. STUDY DESIGN/METHOD: An in vitro model for NAFLD was employed in this study. For this HepG2 cells were incubated with 100 µM of oleic acid (OA) for 24 h. For evaluation of the effect of DI-HET, the extracts (5 or 10 µg/mL) were pretreated to the OA group. Fenofibrate was the positive control. Various parameters relevant to lipogenesis and ß-oxidation of fatty acids like intracellular lipid accumulation, reactive oxygen species (ROS), mitochondrial stress, and key proteins were studied. RESULTS: DI-HET significantly reduced the intracellular lipid accumulation in OA treated cells. And also substantially decreased the expression of lipogenic proteins and increased ß-oxidation in the OA group. OA induced ROS generation was found to reduce with DI-HET treatment. Western blot analysis showed that the expression of LXR-α, SREBP-1C, SREBP-2, HMGCR, FAS, CD-36, and ACOX-1 were downregulated while that of SIRT-1, p-LKB-, p-AMPK, p-ACC, CPT-1, and PPAR-α upregulated in DI-HET treatment. LCMS/MS analysis showed the presence of polyphenols like naringenin, catechin, epicatechin, shikimic acid, syringic acid, vanillic acid, and kaempferol. CONCLUSION: These results suggest that DI-HET is effective against NAFLD by activation of the SIRT-1/p-LKB-1/AMPK signaling pathway via polyphenols present in the extract.

Effects of conventional and herbal irrigants on microhardness and flexural strength of root canal dentin: An in vitro study.

AIM: The aim of this in vitro study was to compare the effects of herbal irrigants with conventional irrigants on microhardness and flexural strength of root dentin. MATERIALS AND METHODS: Sixty extracted permanent maxillary canines were selected. Decoronated roots were sectioned longitudinally into buccal and lingual segments to get 120 specimens. These were embedded in auto polymerizing acrylic resin and further grounded with fine emery papers under distilled water. Of these, 100 root segments without any defects were selected, further divided into four test groups and a control group according to the irrigants used (n = 20). Group 1: 2.5% Sodium hypochlorite, Group 2: Miswak stick extract, Group 3: Cashew leaves extract. Group 4: Mango leaves extract and Group 5: Normal saline (control). All specimens were treated with 5 ml of each irrigant for 10 minutes and rinsed immediately. Dentin microhardness was measured with a Vickers indenter, and the flexural strength test was done using a universal testing machine. The data were analyzed using one-way ANOVA and the intergroup comparison by student t-test. RESULTS: The experimental groups showed a significant reduction in microhardness values when compared with the control group. Intragroup comparison among experimental groups, herbal irrigants showed the least reduction in microhardness values at cervical, middle, and apical thirds. When compared to the control group, the flexural strength values decreased significantly with experimental groups. CONCLUSION: Within the limitation of this study, it was concluded that herbal irrigants were least detrimental to root dentin microhardness when compared with conventional irrigant. But the flexural strength was equally reduced by both conventional and herbal irrigants.

Cost of Tuberculosis Care in Programmatic Settings from Karnataka, India: Is It Catastrophic for the Patients?

BACKGROUND: TB diagnostic and treatment services in India are provided free of cost in the programmatic context across the country. There are different costs incurred during health care utilization, and this study was conducted to estimate such costs. Methodology. A longitudinal study was conducted among patients of three urban tuberculosis units (TUs) of Davangere, Belagavi, and Bengaluru, Karnataka. Trained data collectors administered a validated questionnaire and recorded monthly costs incurred by the patients which are expressed in median Indian National Rupees (INR). The analysis was done using SPSS version 23.0. A p value of <0.05 was taken as statistically significant. RESULTS: Among 214 patients, about 37%, 42%, and 21% belonged to Davangere, Belagavi, and Bengaluru, respectively. Median total pre- and postdiagnostic costs incurred across the three TUs were 3800 and 4000 INR, respectively. The direct nonmedical cost was higher for accommodation (median cost of 800 INR) and direct medical cost for non-TB drugs (median cost of 2000 INR). However, maximum direct medical and nonmedical costs were attributed to hospital admissions (1200 INR) and accommodation costs (700 INR) in the postdiagnostic period, respectively. The median indirect cost incurred was 300 INR overall, and the maximum total indirect cost was 40000 INR in the postdiagnostic period. About one-third of patients faced loss of income and 19.6% faced coping costs. Patients spent about 6.7% (0.97%-52.3%) of their income on TB treatment. About 12.3% patients faced catastrophic expenditure. Median cost was significantly higher among those seeking private health care facilities (12100 INR in private vs. 6800 INR in public; p < 0.05) during the prediagnostic period. Prediagnostic and diagnostic out-of-pocket expenditures (OPE) were significantly higher across all the three centres (p < 0.05). CONCLUSION: The TB patients experienced untoward expenditure under programmatic settings. The costs encountered by one in eight patients were catastrophic by nature.

Development of antiviral gene therapy for Monodon baculovirus using dsRNA loaded chitosan-dextran sulfate nanocapsule delivery system in Penaeus monodon post-larvae.

In the present study, a suitable carrier system was developed for the delivery of dsRNA into Penaeus monodon (P. monodon) post larvae to silence the Monodon baculovirus (MBV) structural gene of p74. The carrier system was developed by layer by layer adsorption of oppositely charged chitosan-dextran sulfate, on charged silica nanoparticles. The silica template was removedto produce multilayered hollow nanocapsules (CS-DS) that were utilized for dsRNA loading at an alkaline pH. The capsule's surface was modified by conjugating with shrimp feed for enhanced cellular uptake. In vivo cellular uptake of CS-DS/FITC loaded nanocapsules conjugated with feed was studied after oral administration into post-larvae. The results revealed that the encapsulated FITC was effectively delivered and exhibited a sustained release into the cytoplasm of shrimp post-larvae. The MBV challenge study for structural gene p74was conducted after 3-25 days of post infection (dpi) with respective CS-DS/dsRNA coated with feed. The results showed a significant survival rate of 86.63% and effective gene silencing in P. monodon. Our findings indicated that the delivery of dsRNA using shrimp feed coatedCS-DSnanocapsules could be a novel approach to prevent viral infections in shrimp.

Development of SYBR Green based real time PCR assay for detection of monodon baculovirus in Penaeus monodon.

Shrimp farming is one of the most important aquaculture activities. Expansion and intensification of shrimp farming has been accompanied with the outbreak of diseases, which threaten the development and sustainability of the industry. Viral diseases are the major challenges faced by shrimp farming industries. The prevention/control of such diseases have become critical in determining the viability of the shrimp farming. The disease caused by monodon baculovirus (MBV) is the major limiting factor especially in shrimp hatchery. There are no therapeutic measures to control the viral diseases. So the detection of the disease is crucial in the prevention and spread of the disease. Hence, in this study, SYBR Green based real time polymerase chain reaction (PCR) assay was developed for the detection of MBV. The sensitivity of the real time PCR was determined using 10-fold dilutions of purified plasmid DNA with the concentration in the range of 10(1)-10(5) copies per reaction. The assay could detect as low as 12 copies indicating that the assay was sensitive and could be effectively used for the quantification of MBV. The real time PCR assay was found to be specific and did not show any amplification with P. monodon infected with infectious hypodermal and hematopoietic necrosis virus (IHHNV), white spot syndrome virus (WSSV) and hepatopancreatic parvo-like virus (HPV). The novelty of this assay is that it could be employed for diagnosis of low level MBV infection in broodstock using fecal matter of shrimp, a non-invasive diagnostic tool.

Enrichment and identification of large filamentous sulfur bacteria related to Beggiatoa species from brackishwater ecosystems of Tamil Nadu along the southeast coast of India.

Beggiatoa species are filamentous sulfide-oxidizing bacteria belonging to the family Beggiatoaceae that contains several largest bacteria known today. These large sulfur bacteria occur in diverse ecosystems and play an important role in the global sulfur, nitrogen and phosphorus cycle. In this study, sediment samples from brackishwater shrimp culture ponds and other brackishwater ecosystems from Tamil Nadu, southeast coast of India, were enriched for Beggiatoa species. Extracted hay medium supplemented with catalase was used and were incubated for two weeks at 28°C. Out of seven set-ups, four yielded positive growth of filamentous sulfide-oxidizing bacteria. The filaments were several millimeters long, ranged in width between 2 and 15 µm and exhibited typical gliding motility. The 16S rRNA gene of four single filaments representing the four positive enrichments was subjected to PCR-DGGE followed by sequencing. All four filaments were affiliated to the Beggiatoaceae, but showed less than 89% identity with the Beggiatoa type strain Beggiatoa alba and less than 93% identity with any other sequence of the family. One of the four filaments revealed a nearly full-length 16S rDNA sequence (1411bp) and it formed a monophyletic cluster with two of the partial DGGE-16S rRNA gene sequences (99-100% identity) within the Beggiatoa species cluster. These organisms could possibly represent a novel genus within the family Beggiatoaceae. The fourth partial sequence affiliated with less than 93% sequence identity to the genera Parabeggiatoa, Thioploca and Thiopilula, and was likewise strongly delineated from any sequence published in the family.

Total synthesis and antifungal activity of (2S,3R)-2-aminododecan-3-ol.

We report the total synthesis of (2S,3R)-2-aminododecan-3-ol has been achieved starting from commercially available 10-undecenoic acid. The key steps involved are Sharpless asymmetric epoxidation, Miyashita's boron-directed C-2 regioselective azidolysis, generated the asymmetric centers and in situ detosylation and reduction of azido tosylate. The antifungal activity of the synthesized (2S,3R)-2-aminododecan-3-ol was evaluated on several Candida strains and was comparable to miconazole, a standard drug.

Comparative efficacy of double-stranded RNAs targeting WSSV structural and nonstructural genes in controlling viral multiplication in Penaeus monodon.

RNA interference (RNAi) is a potential strategy to control shrimp viral diseases, including the white spot disease caused by White Spot Syndrome Virus (WSSV). Selection of genes for targeting is an important criterion. We have compared the efficacy of dsRNAs targeting structural (vp28 and vp281) and nonstructural genes (rr1 and dnapol) of WSSV in controlling viral multiplication in Penaeus monodon. Targeting the rr1 and vp28 genes provided better protection (93.3% and 90% survival respectively) compared to vp281 and dnapol in experimentally infected shrimp. Temporal transcriptional analysis of the corresponding genes and PCR-based diagnosis of WSSV in samples collected at different time points in the experiment supported this observation, thereby indicating that targeting a combination of rr1 and vp28 would be effective in limiting WSSV multiplication.

Viral metagenomics: a tool for virus discovery and diversity in aquaculture.

Viruses are abundant biological entities on earth and the emergence of viral pathogens has become a serious threat to aquaculture and fisheries worldwide. However, our response to viral pathogens has been largely reactive, in the sense that a new pathogen is usually not discovered until it has already reached epidemic proportions. Current diagnostic methods such as PCR, immunological assays and pan-viral microarrays are limited in their ability to identify novel viruses. In this context, the knowledge on the diversity of viruses in healthy and disease situations becomes important for understanding their role on the health of animals in aquaculture species. Viral metagenomics, which involves viral purification and shotgun sequencing, has proven to be useful for understanding viral diversity and describing novel viruses in new diseases and has been recognized as an important tool for discovering novel viruses in human and veterinary medicine. With the advancements in sequencing technology and development of bioinformatics tools for nucleic acid sequence assembly and annotation, information on novel viruses and diversity of viruses in marine ecosystems has been rapidly expanding through viral metagenomics. Novel circoviruses and RNA viruses in Tampa bay pink shrimp, annelovirus in sea lion, picornavirus in ringed seals and several new viruses of marine animals have been recently described using viral metagenomics and this tool has been also recently used in describing viral diversity in aquaculture ponds. Further, a large amount of information has been generated on the diversity of viruses in the marine environment using viral metagenomics during the last decade. There exists a great potential with viral metagenomics for discovering novel viruses in asymptomatic marine candidate animals of aquaculture/mariculture, some of which may assume pathogenic status under high density culture and stress. Additionally, viral metagenomics can help our understanding of viruses present in aquaculture/mariculture settings and routine pathogen surveillance programmes.

Laem-Singh Virus: A Probable Etiological Agent Associated with Monodon Slow Growth Syndrome in Farmed Black Tiger Shrimp (Penaeus monodon).

Among the emerging diseases in shrimp aquaculture, monodon slow growth syndrome (MSGS) is a major concern in South and Southeast Asia. Shrimp farming in Thailand was severely affected during 2000-2002 due to MSGS, which caused an economic loss, of about US$ 300 million. MSGS is characterized by abnormally slow growth with coefficients of size variation of >35 %, that has impacted P. monodon production in Thailand. A new shrimp virus, Laem-Singh virus (LSNV) was identified to be associated in MSGS affected shrimp. LSNV a RNA virus of about 25 nm diameter is phylogenetically related to the insect-borne viruses in the families Barnaviridae, Tymoviridae and Sobemoviridae an important histopathological observation is exclusively noticed in growth-retarded shrimp. The LSNV infections have been confirmed in various organs of infected shrimp such as lymphoid organ, gills and nervous tissues by various diagnostic techniques such as reverse transcription polymerase chain reaction (RT-PCR), in situ hybridization, quantitative real-time RT-PCR and reverse transcription loop-mediated isothermal amplification combined with a lateral flow dipstick (RT-LAMP-LFD) and these tools are available for the diagnosis of LSNV. Recently, an integrase containing element has been identified in absolute association with LSNV in stunted growth shrimp. The transmission of LSNV through horizontal and vertical routes has been experimentally demonstrated. The known natural host-range of LSNV includes P. monodon and other penaeid shrimp. The putative RdRp gene involved in replication of LSNV was targeted for dsRNA-mediated gene silencing and appeared to be effective in a dose-dependent manner. Since the discovery of LSNV in 2006 in Thailand, it has been added to the list of viruses to be excluded from domesticated specific pathogen-free stocks of P. monodon and it has been recommended that shrimp farmers avoid stocking post larvae positive for LSNV to prevent MSGS in their farms.

Supra dietary levels of vitamins C and E enhance antibody production and immune memory in juvenile milkfish, Chanos chanos (Forsskal) to formalin-killed Vibrio vulnificus.

Juveniles of milkfish, Chanos chanos (Forsskal), were fed two independent supra dietary levels of vitamins C (500 and 1500 mg kg(-1) feed, T1 and T2) and E (50 and 150 mg kg(-1), T3 and T4). Milkfish fed diets with supra (in addition to the vitamins present in the control diet) and normal levels (T5 containing 90 and 1.2mg of vitamins C and E, respectively, kg(-1) of feed) of vitamins were immunized (ip) with formalin-killed Vibrio vulnificus (FKVV). Priming and booster antibody responses to the injected bacterin were significantly (P<0.05) better in the milkfish juveniles fed supra dietary levels. Survival response of the experimental fish fed supra dietary levels of vitamins (T1, T2 and T3) was significantly (P<0.01) better than that of the control set. Protective response against virulent bacterial challenge of the vaccinated fish fed vitamin-supplemented diets (T2 and T3) was better than the control (T5) and T1 and T4. Memory factor reflecting immunological memory was superior in the fish fed vitamin-supplemented diets. Diets supplemented with either 1500 mg of Vitamin C or 50mg of Vitamin E kg(-1) produced the best antibody responses, final survival and protective response upon challenge. No conclusive inferences could be drawn on the growth responses from the experiment.

Nodavirus infection causes mortalities in hatchery produced larvae of Lates calcarifer: first report from India.

Larvae (15 to 21 d post hatch, dph) of the Asian sea bass Lates calcarifer (Bloch) suffered heavy mortalities (60 to 90%) during the hatchery-rearing phase. Darkened and moribund larvae showed no evidence of bacterial or parasitic infections. Tissue sections of brain and spinal cord showed clear necrotic vacuolation. Electron microscopy revealed membrane-bound viral particles in the cytoplasm of the nerve cells. The viral particles measured 28 to 30 nm in diameter. Primer sets, designed for the amplification of the RNA2 segment of the piscine nodavirus coat protein gene, were used in the RT-PCR analysis of moribund larvae of 20 and 21 dph which produced the amplified product of 430 bp. The clinical manifestations, pathology and electron microscopy observations supported by the RT-PCR analysis suggest that the nerve necrosis was due to nodavirus infection in the larvae. This is the first report of piscine nodavirus infection from the Indian sub-continent.

Evaluation of Pseudomonas sp. PM 11 and Vibrio fluvialis PM 17 on immune indices of tiger shrimp, Penaeus monodon.

Occurrence of widespread epizootics among cultured stock of shrimp has put research programmes on preventive approaches such as application of probiotics on a high priority in aquaculture. In the present study two bacteria, Pseudomonas sp. PM 11 and Vibrio fluvialis PM 17 were selected as candidate probionts from a pool of bacteria isolated from gut of farm reared sub-adult shrimp and tested for their effect on the immunity indicators of tiger shrimp. Sub-adult shrimp, weighing 14 to 22 g were treated in separate experiments with Pseudomonas sp. PM 11 and V. fluvialis PM 17 at 10(3) bacterial cells ml(-1) in the experimental shrimp culture tanks. One set of experimental animals was treated every 3 days and another set of animals every 7 days with each of the candidate probionts. Estimation of immunological indicators such as haemocyte counts, phenol oxidase and antibacterial activity showed declining trends. The haemocyte counts dropped from 31 x 10(3) to 65 x 10(3) ml(-1) on the first day to 4-16 x 10(3) ml(-1) on the 45th day. Similarly, the phenol oxidase activity declined from 12-32 units on the first day to 11-14 units on 45th day of the experiment. Antibacterial activity of haemolymph reduced to 46-67 percent on the 45th day of the experiment. The results of the study suggest that, the criteria used for the selection of putative probiotic strains in the present study, such as predominant growth on primary isolation media, ability to produce extracellular enzymes and siderophores, did not bring about the desired effect in vivo and improve the immune system in shrimp. Hence, new protocols have to be evolved for selection of microbe(s) as putative probiotics and that, detailed understanding of proven probiotics, employed presently on empirical basis may provide a clue on the selection procedure.