Transfection of non-susceptible cells with Ovis aries recombinant lymphocyte function-associated antigen 1 renders susceptibility to Mannheimia haemolytica leukotoxin

Mannheimia haemolytica is an important etiological agent of pneumonia in domestic sheep (DS, Ovis aries). Leukotoxin (Lkt) produced by this organism is the principal virulence factor responsible for the acute inflammation and lung injury characteristic of M. haemolytica caused disease. Previously, we have shown that the leukocyte-specific integrins, beta(2) integrins, serve as the receptor for Lkt. Although it is certain that CD18, the beta subunit of beta(2) integrins, mediates Lkt-induced cytolysis of leukocytes, it is not clear whether CD18 of all three beta(2) integrins, LFA-1, Mac-1 and CR4, mediates Lkt-induced cytolysis of DS leukocytes. Since polymorphonuclear leukocytes, which express all three beta(2) integrins, are the leukocyte subset that is most susceptible to Lkt, we hypothesized that all three beta(2) integrins serve as the receptor for Lkt. The objective of this study was to determine whether DS LFA-1 serves as a receptor for M. haemolytica Lkt. We cloned the cDNA for DS CD11a, the alpha subunit of LFA-1, and co-transfected it along with the previously cloned cDNA for DS CD18, into a Lkt-non-suceptible cell line. Transfectants stably expressing DS LFA-1 were bound by Lkt. More importantly, Lkt lysed the DS LFA-1 transfectants in a concentration-dependent manner. Pre-incubation of Lkt with a Lkt-neutralizing monoclonal antibody (MAb), or pre-incubation of transfectants with MAbs specific for DS CD11a or CD18, inhibited Lkt-induced cytolysis of the transfectants. Exposure of LFA-1 transfectants to low concentrations of Lkt resulted in elevation of intracellular [Ca(2+)](i). Taken together, these results indicate that DS LFA-1 serves as a receptor for M. haemolytica Lkt.     

Biotransformation of tuna waste by co-fermentation into an aquafeed ingredient

Dried skipjack tuna ( Katsuwonus pelamis) waste (red meat, gills, viscera, fins, etc.) were mixed with 25% wheat flour and inoculated with a starter culture of Lactobacillus plantarum National Collection of Industrial Microorganisms (NCIM) 2912 (10⁸–10⁹ cells mL⁻¹) and Bacillus licheniformis MTCC 6824 (10⁷–10⁸ cells mL⁻¹). Changes in the nutritional quality (crude protein, crude fat, crude ash, crude fibre and nitrogen-free extract and aminoacids) were monitored during a fermentation period of 14 days. The proximate analysis showed significant changes in the composition of L. plantarum -fermented tuna (LPFT) and B. licheniformis -fermented tuna (BLFT) from the unfermented raw materials. Fermentation of tuna waste has resulted in a significant ( P <0.05) increase in the protein content of tuna waste between days 6 and 12. All the amino acid contents in BLFT increased during fermentation, whereas, in LPFT the levels of serine, histidine, tyrosine, methionine, cystine and phenylalanine contents were decreased. A marginal increase in calcium and phosphorus levels was recorded in the fermented products. The results of the study suggest that LPFT or BLFT can be used as a novel aquafeed ingredient for different fish species.     

Ovarian histology of stunted pond-reared Macrobrachium rosenbergii females

The present study describes the ovarian histology of stunted freshwater prawn Macrobrachium rosenbergii. The ovarian maturation of stunted animals was examined and compared with similar‐aged normal females. Ten animals of the stunted group and each maturation stage of the normal group were sampled from the same pond and had their ovaries removed for histological analysis. Body weight, body length, ovarian weight and gonadosomatic index (GSI) were recorded for each female. The diameters of the different oocyte types were compared among groups through histological assessments. The ovarian histology of stunted M. rosenbergii females indicated that although the somatic growth is severely affected (7.6 g), some energy has been placed on the vitellogensis. Stunted females showed the simultaneous occurrence of previtellogenic, vitellogenic and mature oocytes in their ovarian tissue, but overall oocyte diameter and GSI (1.02%) were significantly affected when compared with normal females.     

Enrichment of eicosapentaenoic acid from sardine oil with Delta5-olefinic bond specific lipase from Bacillus licheniformis MTCC 6824

Lipase derived from Bacillus licheniformis MTCC 6824 was purified to homogeneity by anion exchange chromatography on Amberlite IRA 410 (Cl-) and gel filtration using Sephadex G-100 as judged by denaturing polyacrylamide gel electrophoresis. The purified lipase was used for hydrolysis of triacylglycerol in sardine oil to enrich Delta5-polyunsaturated fatty acids (Delta5-PUFAs) namely, arachidonic acid (5,8,11,14-eicosatetraenoic acid, ARA, 20:4n-6) and eicosapentaenoic acid (5,8,11,14,17-eicosapentaenoic acid, EPA, 20:5n-3). The individual fatty acids were determined as fatty acid methyl esters (FAMEs) by gas-liquid chromatography and gas chromatography-mass spectroscopy as FAMEs and N-acyl pyrrolidides. The enzyme exhibited hydrolytic resistance toward ester bonds of Delta5-PUFAs as compared to those of other fatty acids and was proved to be effective for increasing the concentration of EPA and ARA from sardine oil. Utilizing this fatty acid specificity, EPA and ARA from sardine oil were enriched by lipase-mediated hydrolysis followed by urea fractionation at 4 degrees C. The purified lipase produced the highest degree of hydrolysis for SFAs and MUFAs (81.5 and 72.3%, respectively, from their initial content in sardine oil) after 9 h. The profile of conversion by lipase catalysis showed a steady increase up to 6 h and thereafter plateaued down. Lipase-catalyzed hydrolysis of sardine oil followed by urea adduction with methanol provided free fatty acids containing 55.4% EPA and 5.8% ARA, respectively, after complexation of saturated and less unsaturated fatty acids. The combination of enzymatic hydrolysis and urea complexation proved to be a promising method to obtain highly concentrated EPA and ARA from sardine oil.     

Population structure of the rice sheath blight pathogen <Emphasis Type="Italic">Rhizoctonia solani</Emphasis> AG-1 IA from India

The population structure of Rhizoctonia solani AG-1 IA causing rice sheath blight from India was evaluated for 96 isolates using seven RFLP loci. Nineteen of the isolates did not hybridise to R. solani AG-1 IA RFLP probes and rDNA analyses subsequently confirmed that they were either Ceratobasidium oryzae-sativae isolates or another Rhizoctonia sp. The population structure of the remaining 77 R. solani AG-1 IA Indian isolates was similar to that of a previously characterized Texas population. Clonal dispersal of R. solani AG-1 IA in India was moderate within fields and no clones were shared among field populations. Low levels of population subdivision and small genetic distances among populations were consistent with high levels of gene flow. Frequent sexual reproduction was indicated by the fact that most populations were in Hardy–Weinberg equilibrium (HWE). The two loci (R68 and R111) that deviated significantly from HWE showed an excess of heterozygosity. Although Texas and Indian populations were geographically very distant, they exhibited only moderate population subdivision, with an F_ST value of 0.193.     

Co-expression of ovine LPS receptor CD14 with Mannheimia haemolytica leukotoxin receptor LFA-1 or Mac-1 does not enhance leukotoxin-induced cytotoxicity

Leukotoxin (Lkt) and LPS are the major virulence determinants of Mannheimia haemolytica that contribute to the pathogenesis of bovine and ovine pneumonic pasteurellosis. We have previously identified bovine and ovine CD18 as the functional receptor for Lkt. LPS complexes with Lkt resulting in increased thermal stability and enhanced cytotoxic activity of Lkt. Cellular recognition of LPS involves several different molecules including CD14. We hypothesized that expression of ovine CD14 together with LFA-1 or Mac-1 would enhance Lkt-induced cytotoxicity. Ovine cDNA for CD14 was amplified by PCR and cloned into mammalian expression vectors. The 1122 bp cDNAs for bighorn sheep (BHS) and domestic sheep (DS) CD14 encode 373 amino acids which exhibit 99% identity with each other. Ovine CD14 plasmids were transfected either into HEK-293 cells, or previous HEK-293 transfectants stably expressing ovine LFA-1 or Mac-1. Flow cytometric analysis of transfectants confirmed the cell surface expression of CD14. The transfectants expressing LFA-1 or Mac-1 and the transfectants co-expressing CD14 with LFA-1 or Mac-1 did not show any significant difference in Lkt-induced cytotoxicity when incubated with LPS complexed Lkt. In contrast, incubation of the LFA-1 or Mac-1 and LFA-1/CD14 or Mac-1/CD14 transfectants with Lkt which lacks LPS, resulted in reduced cytotoxicity. None of the above transfectants showed any difference in [Ca2+](i) elevation when incubated with both types of Lkt preparations. Lkt did not induce any cytotoxicity or [Ca2+](i) elevation in ovine CD14 transfectants or parent HEK-293 cells. Based on these findings, we conclude that expression of CD14 together with LFA-1 or Mac-1 does not enhance Lkt-induced cytotoxicity, whereas LPS enhances cytotoxicity by complexing with Lkt.     

Effect of plant secondary metabolites on legume pod borer, Helicoverpa armigera

The effect of various flavonoids, lectins and phenyl β-d-glucoside on larval survival, weights and the activities of digestive (total serine protease and trypsin) and detoxifying [esterase and glutathione-S-transferase] enzymes of Helicoverpa armigera larvae at 5 and 10 days after treatment (DAT) was studied through diet incorporation assay. Flavonoids (quercetin, cinnamic acid, caffeic acid, chlorogenic acid, catechin, trihydroxyflavone, gentisic acid, ferulic acid, protocatechuic acid and umbelliferone) were incorporated in artificial diet at 100, 500 and 1000 ppm, lectins: groundnut leaf lectin (GLL), concavalin (ConA) and phenyl β-d-glucoside at 2.5 and 5 μg mL^?1. Flavonoids such as chlorogenic acid, caffeic acid and protocatechuic acid at 1,000 ppm were more toxic to H. armigera larvae at 10 DAT than quercetin, catechin, cinnamic acid, trihydroxyflavone, gentisic acid, ferulic acid and umbelliferone. Larval growth and development were significantly reduced in H. armigera larvae fed on a diet with GLL and ConA at 5 μg mL^?1 compared to the larvae fed at 2.5 and 1.25 μg mL^?1 concentrations. The enzyme activities of the larvae were significantly reduced in flavonoid-treated diets. The flavonoids such as chlorogenic acid, caffeic acid, gentisic acid, trihydroxyflavone, catechin and protocatechuic acid, and lectins, GLL and ConA can be utilized in insect control programs.     

Efficacy of various lipid supplements in formulated pellet diets for juvenile Scylla serrata

Efficacy of sunflower oil (diet SF) and soybean oil (diet SB) alone and in combination with cod liver oil (diets M1‐2.80:1.40:1.40, M2‐2.80:2.24:0.56 and M3‐2.80:0.56:2.24; cod liver oil:sunflower oil:soybean oil) as lipid supplements (5.6%) in formulated diets (crude fat ～9.79%) for juvenile Scylla serrata (weight=0.28±0.07 g, carapace width=9.7±0.1 mm) were compared with diet CL, containing cod liver oil alone as the lipid supplement (6 diets × 24 crabs stocked individually, randomized block design). Growth performance, nutrient (protein and lipid) intake and gain of crabs fed M1, M2 and M3 were higher (P≤0.05) than the crabs fed SF and SB, but were not significantly different (P≥0.05) from crabs fed CL. Dietary fatty acids (FAs) are found to influence the FA profile of test crabs. Higher tissue levels of 16:1n‐7, 18:1n‐9 and 18:1n‐7 reflected the essential FA deficiency in crabs fed diets supplemented only with vegetable oils. Results confirmed that S. serrata could utilize vegetable oil supplements in the formulated diets as a partial replacement (50%) of cod liver oil without compromising growth and survival. Partial substitution of marine fish oil with suitable vegetable oils can reduce the feed cost considerably, in the context of rising fish oil prices.