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Background & objectives: Dengue (DEN) is a rapidly spreading arboviral disease transmitted by Aedes mosquitoes. Although it is endemic in India, dengue virus (DENV) infection has not been reported from tribal areas of Madhya Pradesh. Investigations were conducted to establish the aetiology of sudden upsurge of cases with febrile illness in June 2013 from tribal villages of Mandla district of Madhya Pradesh, India. Methods: The rapid response team of the National Institute for Research in Tribal Health, Jabalpur, conducted clinical investigations and field surveys to collect the samples from suspected cases. Samples were tested using molecular and serological tools. Collected mosquitoes were identified and tested for the presence of virus using semi nested reverse transcriptase-polymerase chain reaction (nRT-PCR). The sequences were analysed to identify serotype and genotype of the virus. Results: Of the 648 samples collected from 18 villages of Mandla, 321 (49.53%) were found to be positive for dengue. The nRT-PCR and sequencing confirmed the aetiology as dengue virus type 2. Eighteen per cent of patients needed hospitalization and five deaths were attributed to dengue. The virus was also detected from Aedes aegypti mosquito, which was incriminated as a vector. Phylogenetic analysis revealed that the dengue virus 2 detected belonged to cosmopolitan genotype of the virus. Interpretation & conclusions: Dengue virus serotype 2 was detected as the aetiological agent in the outbreak in tribal villages of Mandla district of Madhya Pradesh. Conducive man-made environment favouring mosquitogenic conditions and seeding of virus could be the probable reasons for this outbreak. Urgent attention is needed to control this new threat to tribal population, which is already overburdened with other vector borne diseases.

This work was done to study the bacterial diversity of mithun (Bos frontalis) fed on mixed tree leaves and rice straw based diet. Genomic DNA was extracted from the rumen liquor of mithun, 16S rDNA sequences were amplified, cloned and randomly selected for sequencing. The nearest neighbors were retrieved from the NCBI through a BLAST search and a phylogenetic tree was constructed. In our findings, 12% of clones showed similarity with known bacterial species (Prevotella ruminicola, Butyrivibrio fibrisolvens, Pseudobutyrivibrio ruminis, Succinivibrio dextrinosolvens and Ruminococcus flavefaciens) and 6% of clones showed similarity with known bacterial genus (Butyrivibrio species, Streptococcus species) of 97-100% similarities. Twenty-two percent of clones showed similarity with known bacteria (P. ruminicola, Prevotella species, Sporanaerobacter acetigenes, Clostridiales bacterium, Bacteroidetes bacterium) of 90-97% similarities. Sequences of all the clones were also classified by using taxonomic classifier software available at Ribosomal Database Project and classification showed that, all the clones were under four phyla, namely Bacteroidetes (54%), Firmicutes (36%), Proteobacteria (4%) and Tenericutes (2%). The experiment showed that, bacterial population in the rumen of mithun fed on mixed tree leaves and rice straw based diet harbor diversified species of bacteria responsible for lignocellulosic feedstuffs. Key words: Tree leaves, bacteria, clones, sequences.

The conserved N-linked glycosylation site on the Fc domain of IgG1 antibodies is essential for maintaining a functionally active conformation of the antibody. Different glycoforms of the Fc exhibit widely different effector functions. Similarly, therapeutic antibodies, with engineered glycosylation, exhibit altered binding to cellular Fc receptors (FcRs). Here, X-ray crystallographic structures were obtained for biosynthetic intermediate glycoforms of human IgG1 Fc bearing: unprocessed oligomannose-type, intermediate hybrid-type, and mature complex-type glycans. The fully processed Fc protein crystallised in an “open” conformation with glycans forming canonical stabilising interactions on the protein surface. Analysis of the biosynthetic intermediates revealed that these stabilising hydrophobic protein-glycan interactions are formed only after processing by Golgi -mannosidase II. Mutagenesis of hydrophobic residues on Fc disrupted crucial protein-glycan interactions resulting in the selective destabilization of the 3-arm of the glycan chain with the 6-arm closely matching that seen for the native structure. However, carbohydrate analysis of released glycans shows increased processing on both arms indicating a more accessible and flexible glycan in the mutant structure suggesting that the crystallographic structure of these antibody glycans represents a minor low-energy conformation. The importance of Fc glycosylation is highlighted by endoglycosidases which eliminate Fc effector function. The crystallographic structure of enzymatically deglycosylated IgG Fc revealed a significant collapse of the of Cγ2 domains resulting in a ‘closed’ quaternary conformation, incompatible with Fc receptor binding. This provides a structural explanation for immune deactivating properties of endoglycosidases including those under preclinical development for the treatment of antibody-mediated immune pathology. One such bacterial endoglycosidase, Endo S, was studied further and revealed a specificity for complex-type glycans of the type found on IgG but no hydrolytic activity towards an engineered IgG Fc with oligomannose-type glycans. Introduction of both the engineered monoclonal IgG and endoglycosidase in serum led to a dramatic increase in FcR binding as the competitive binding of serum IgG for FcRs was selectively eliminated. This approach is a general technique for boosting the effector signal of therapeutic antibodies.

The effects of feeding Artemia nauplii enriched with or without poly-ß-hydroxybutyrate (PHB) and/or highly unsaturated fatty acids (HUFA) on Siberian sturgeon (Acipenser baerii) larvae were investigated. Feeding larvae with PHB-enriched nauplii (PHB, PHB + HUFA) decreased the growth performance of the larvae. PHB affected the body composition by increasing the lipid content of the whole body and decreasing total saturated, monoenoic, n3, n6 and decosahexanoeic acid (DHA) in the larvae, indicating that the PHB addition affected lipid metabolism. A high activity of pepsin was observed in the digestive extracts of PHB treatments (PHB, PHB + HUFA), while PHB suppressed amylase activity in the intestine of the larvae. Based on molecular analysis, PHB changed the microbial community in the distal intestine of the larvae. The highest counts of goblet cells were observed in the HUFA-containing treatments (HUFA, PHB + HUFA), indicating that HUFA addition may improve the mucosal barrier defence system. The overall quality of the larvae was evaluated by exposing them to different salinities and ammonia stress levels. PHB decreased survival rates in these challenges. Our results show that optimal PHB doses for bio-encapsulation into Artemia remain to be determined for further application at the earliest larval stages of sturgeon.

Bacteria belonging to the genus Bacillus are amongst the most intensively studied group of bacteria for use as probiotics in aquaculture. However, the exact mechanism of action of these bacteria is often not well described, and the microbiota that are naturally present in cultures of test organisms often compromise the interpretation of the results. The present study aimed to evaluate the putative probiotic effect of Bacillus sp. LT3 in a model system with gnotobiotic brine shrimp Artemia franciscana larvae. The strain significantly increased the survival of brine shrimp larvae challenged with Vibrio campbellii when administered 6 h before the challenge. Under these conditions, LT3 was able to colonize the brine shrimp gastrointestinal tract and to decrease the in vivo pathogen activity as indicated by the bioluminescence of the V. campbellii associated with brine shrimp larvae. In order to investigate the effect of the Bacillus strain on the innate immune system of the brine shrimp larvae, prophenoloxidase and transglutaminase mRNA levels were monitored, while heat shock protein 70 mRNA levels were measured as an indicator of physiological stress. Interestingly, 12 h after challenge, the prophenoloxidase mRNA level in the larvae pre-treated with LT3 and challenged with V. campbellii was approximately 8-fold higher than in the other treatments. Further, a decreased mRNA level of transglutaminase gene and heat shock protein 70 gene suggested that pretreatment with LT3 results in less stress and tissue damage in the brine shrimp larvae upon V. campbellii challenge. These results indicated that Bacillus sp. LT3 could improve the survival of brine shrimp larvae when challenged with pathogenic V. campbellii, both by decreasing the in vivo activity of the pathogen and by priming the innate immune response through activating the prophenoloxidase system.

Induction of heat shock proteins (e.g. Hsp70) has been considered as a potential disease control and health management strategies in aquaculture. Recently, the compound Tex-OE®, a patented extract from the skin of the prickly pear fruit, Opuntia ficus indica, was reported to enhance the production of Hsp70 in fish and shrimp tissues without any adverse effect, protecting the animals against various abiotic stressors. Most of the studies on this compound primarily focused on the positive effects at the level of the host while the effects on the microorganisms in the culture environment were often overlooked. In our study, we aimed at evaluating the effect of this compound on the interaction of the host brine shrimp Artemia franciscana with pathogenic Vibrio. Pretreatment of axenically-grown Artemia with Tex-OE® conferred significant protection against V. campbellii in a conventional rearing system. However, continuous exposure of axenniclly-grown Artemia to Tex-OE® in an open system negatively affected their survival. In vitro experiments provided unequivocal evidence that continuous exposure of Tex-OE® modulated the growth and virulence (such as hemolytic and caseinase activity) of V. campbellii. Pretreatment of Vibrio with the compound also increased their virulence in an Artemia challenge assay. Based on the overall results, it can be suggested that the compound Tex-OE® has the potential to be used as a disease control tool in (shrimp) aquaculture systems. However, the possibility that such kind of compounds also modulate the microbial activity cannot be ignored making the outcome dependent on the composition of the standing microbial community.

Biologically active conformations of the IgG1 Fc homodimer are maintained by multiple hydrophobic interactions between the protein surface and the N-glycan. The Fc glycan modulates biological effector functions, including antibody-dependent cellular cytotoxicity (ADCC) which is mediated in part through the activatory Fc receptor, FcγRIIIA. Consistent with previous reports, we found that site-directed mutations disrupting the protein-carbohydrate interface (F241A, F243A, V262E, and V264E) increased galactosylation and sialylation of the Fc and, concomitantly, reduced the affinity for FcγRIIIA. We rationalized this effect by crystallographic analysis of the IgG1 Fc F241A mutant, determined here to a resolution of 1.9 Å, which revealed localized destabilization of this glycan-protein interface. Given that sialylation of Fc glycans decreases ADCC, one explanation for the effect of these mutants on FcγRIIIA binding is their increased sialylation. However, a glycan-engineered IgG1 with hypergalactosylated and hypersialylated glycans exhibited unchanged binding affinity to FcγRIIIA. Moreover, when we expressed these mutants as a chemically uniform (Man5GlcNAc2) glycoform, the individual effect of each mutation on FcγRIIIA affinity was preserved. This effect was broadly recapitulated for other Fc receptors (FcγRI, FcγRIIA, FcγRIIB, and FcγRIIIB). These data indicate that destabilization of the glycan-protein interactions, rather than increased galactosylation and sialylation, modifies the Fc conformation(s) relevant for FcγR binding. Engineering of the protein-carbohydrate interface thus provides an independent parameter in the engineering of Fc effector functions and a route to the synthesis of new classes of Fc domain with novel combinations of affinities for activatory and inhibitory Fc receptors.

Like other invertebrates, the brine shrimp Artemia franciscana relies solely on innate immunity, which by definition lacks adaptive characteristics, to combat against invading pathogens. One of the innate mechanisms is melanisation of bacteria mediated by the activation of the prophenoloxidase (proPO) system. The 70 kDa heat shock proteins (Hsp70) derived from either prokaryote (Escherichia coli) or eukaryote (Artemia), well conserved and immune-dominant molecules, protect Artemia against Vibrio campbellii. However, the molecular mechanisms by which these proteins protect Artemia against Vibrio campbellii infection are unknown. Here we demonstrated that feeding gnotobiotically grown Artemia with either Artemia Hsp70 or the E. coli Hsp70 equivalent DnaK, each overproduced in E. coli, followed by V. campbellii challenge enhanced the proPO system, at both mRNA and protein activity levels. Additionally, the Artemia fed with these proteins survived well in a Vibrio challenge assay. These results indicated that Hsp70s derived from either prokaryotic or eukaryotic sources generate protective immunity in the crustacean Artemia against V. campbellii infection by priming the proPO system. This is apparently the first in vivo report on priming activity of Hsp70 in an invertebrate.

The Hsp70 class of heat shock proteins (Hsps) has been implicated at multiple points in the immune response of both vertebrates and invertebrates. This class of chaperones is highly conserved in both sequence and structure, from prokaryotes to higher eukaryotes. In view of their high degree of homology, it was assumed that these Hsp70 proteins derived either from the prokaryotes or eukaryotes would have similar functions, especially in relation to their protective ability in a challenge assay. To verify this, we compared two evolutionary diverse Hsp70s, Artemia Hsp70 and Escherichia coli Hsp70 equivalent DnaK (each overproduced in E.coli), for their ability to protect Artemia against Vibrio challenge. Results showed that Artemia fed with E. coli producing Artemia Hsp70 or DnaK proteins, as assessed by immune-probing in western blots, survived better in a Vibrio challenge assay. The observed effects could be due to enhancement of the Artemia immune system as phenoloxidase activity was found to be increased by these proteins. These two Hsp70 proteins exhibit a high degree of homology, particularly in the peptide-binding domain (the putative innate immunity-activating portion) with 59.6% identity, indicating that the observed protective capacity of homologous or heterologous Hsp70 proteins might reside within this peptide-binding domain.

A feeding trial was conducted for 60 d to study the effect of dietary microbial phytase on growth performance and nutrient digestibility of rohu, Labeo rohita, fingerlings. One hundred and twenty fingerlings (average initial weight: 9.17 g/fish) were equally distributed into five experimental tanks, each with four replicates. Five isonitrogenous (35%) and isocaloric (16.79 kJ/g) diets were prepared from plant-based ingredients, supplemented with microbial phytase at the level of 0, 250, 500, 750, and 1000 U/kg diets and fed to T0, T1, T2, T3, and T4 groups, respectively. Weight gain %, food conversion ratio, protein efficiency ratio, and apparent net protein utilization were significantly (P < 0.05) improved in groups fed phytase-supplemented diets compared to control, the highest being observed in T 3 group. Maximum apparent digestibility of phosphorus and crude protein was recorded in T 3 group. Bone ash, phosphorus (P), and calcium (Ca) contents were also significantly (P < 0.05) increased in phytase-fed groups. However, maximum was recorded in T 3 group. Results from the present study indicated that addition of 750 U microbial phytase/kg diets effectively improved nutrient utilization, bone mineralization, and hence growth of L. rohita fingerlings.

Experiment was conducted to study the effect of dietary protein level on growth and nutrient utilization by angel fish (Pterophyllum scalare) juveniles. Fifty-four juveniles (average wt. 2-2.5 g) were equally divided in three treatments with each of three replicates. Three formulated diets with graded protein levels, T1 (35% CP), T2 (40% CP) and T3 (45% CP) were fed to juveniles for 45 days. A trend of higher weight gain %, SGR, FER and PER was found with the increased CP level in the feed. Feed intake was similar in all the groups. T3 group fed with 45% CP registered highest weight gain % (43.26 ± 2.07), SGR (0.78 ± 0.04), FER (0.29 ± 0.01), which were significantly higher (P

Global fishmeal production from wild-catch sources cannot continue to increase indefinitely; suitable alternatives have to be found for sustainable aquaculture. Plant-based aquafeed seems to be the ideal alternative to this, but has its own limitations. Plant ingredients are rich in phytic acid, which reduces the bioavailability of nutrients like minerals and protein to the fish, thereby causing aquaculture pollution. Dietary phytase treatment reduces the aquaculture pollution by improving the bioavailability of nutrients, and reduces the feed cost as evident from poultry and piggery. Phytase activity is highly dependent upon the pH of the gut. Unlike mammals, fish are either gastric or agastric, and hence, the action of dietary phytase varies from species to species. In this article, the authors attempt to summarise various effects of phytase on nutrient utilization, growth of fish and aquatic pollution.