Anal immunisation of carp and rainbow trout with different fractions of aVibrio anguillarumbacterin

Carp and rainbow trout were anally intubated with different fractions of aVibrio anguillarumbacterin in order to determine which fraction of the bacterin results in optimal antigen transport and induced immunological memory following application to the gut. Antigen uptake in the hindgut was demonstrated in fish intubated with complete vaccine, supernatant of the vaccine and LPS extracted from the bacterin, while whole bacteria were not taken up. Fish received a secondary immunisation by intramuscular injection with complete vaccine 10 weeks after anal intubation. Primary antibody titres were significantly increased in carp 21 days after intubation with complete vaccine or supernatant of the vaccine. Secondary antibody titres were only significantly increased in carp which were intubated with supernatant of the bacterin. Furthermore, antibody titres in carp intubated with complete vaccine or LPS tended to be increased. In contrast to carp, trout required 10 times more supernatant to obtain similar results. In conclusion, the supernatant seemed to contain the most immunogenic part of the bacterin.     

Growth performance of two Indian major carps, catla (Catla catla) and rohu (Labeo rohita) fed diets containing different levels of Spirulina platensis

The influence of Spirulina platensis meal on the growth and carcass composition of two Indian major carps, catla, Catla catla and rohu, Labeo rohita was investigated in a 90-day culture trial. Four experimental diets were used and Spirulina replaced fish meal protein from the standard diet at 25%, 50%, 75% and 100% levels. There was no significant difference in the final weight attained by catla at all levels of Spirulina incorporation as compared to the fish-meal-based control diet. However, the replacement of fish meal by more than 25% Spirulina resulted in significantly superior growth of rohu. The specific growth rate and protein efficiency ratio recorded in rohu improved with higher levels of Spirulina inclusion, while in catla they did not differ significantly from the control treatment. In both the species, the digestibility of dry matter, protein and fat was found to improve marginally with increasing levels of Spirulina incorporation. The carcass composition showed an inverse relationship between protein and fat deposition. In general, fish fed with Spirulina diets had a significantly higher percentage of fat. The study demonstrated the usefulness of Spirulina for partial or complete replacement of fish meal in the diets of catla and rohu.     

Uptake and transport of intact macromolecules in the intestinal epithelium of carp (Cyprinus carpio L.) and the possible immunological implications

Summary Two protein antigens, horseradish peroxidase (HRP) and ferritin, have been administered to the digestive tract of carp. Electron-microscopical observations reveal considerable absorption of both antigens in the second segment of the gut (from 70 to 95% of the total length) and also, although to a lesser extent, in the first segment (from 0 to 70% of the total length). Even when administered physiologically with food, a large amount of ferritin is absorbed by enterocytes in the second gut segment.HRP and ferritin are processed by enterocytes in different ways. HRP seems to adhere to the apical cell membrane, probably by binding to receptors, and is transported in vesicles to branched endings of lamellar infoldings of the lateral and basal cell membrane. Consequently, most of the HRP is released in the intercellular space where it contacts intra-epithelial lymphoid cells. Only small amounts of HRP become localized in secondary lysosomes of enterocytes. Ferritin does not bind to the apical cell membrane; after uptake by pinocytosis, it is present in small vesicles or vacuoles that appear to fuse with lysosome-like-bodies. In the second segment, intact ferritin ends up in the large supranuclear vacuoles (after 8 h), where it is digested slowly. Although no ferritin is found in the intercellular space, ferritin-containing macrophages are present between the epithelial cells, in the lamina propria and also to a small extent in the spleen. The transport of antigens from the intestinal lumen, through enterocytes, to intra-epithelial lymphoid cells or macrophages may have immunological implications, such as induction of a local immune response and prospectives for oral vaccination.     

Widespread hybridization among species of Indian major carps in hatcheries, but not in the wild

Twenty‐one allozyme loci in samples of wild‐caught and hatchery‐reared Indian major carps from Bangladesh were analysed. Bayesian model‐based clustering analysis revealed the presence of four taxa, corresponding to the three known species along with a fourth unknown taxon present in two hatchery samples. Individual admixture coefficients showed that 24% of all hatchery‐reared fishes were hybrids, whereas a single hybrid was observed in the wild‐caught samples. Only catla Catla catla × rohu Labeo rohita and mrigal Cirrhinus cirrhosus × rohu hybrids were observed, the vast majority of which were F₁ hybrids, though five individuals represented putative backcrosses. Mitochondrial DNA analysis revealed that catla × rohu hybridization primarily involved catla males and rohu females, whereas mrigal × rohu hybrids primarily resulted from rohu males and mrigal females. Despite the high percentage of F₁‐hybrids in hatchery samples, reproductive barriers among species have so far precluded widespread introgression. Continued hybridization may eventually lead to a breakdown of species barriers, thereby compromising the genetic integrity of the species in the wild, and leading to production losses in aquaculture.     

Bacteria Associated with the Gut Tract of Larval Stages of the Aquatic Cranefly Tipula abdominalis (Diptera; Tipulidae)

Scanning electron microscopy, light microscopy, and direct isolations were used to examine the distribution and diversity of bacteria in the gut tracts of larval stages of Tipula abdominalis. The animal had an enlarged hindgut which housed a diverse bacterial community in the lumen and directly attached to the gut wall. Distinct localization was noted, with the most dense and most diverse community anterior to the rectum. A distinct architecture of bacteria occurred in this region, characterized by a layering or a “weblike” array of filamentous bacteria overlying mats of bacteria closely associated with the gut wall. Although morphological diversity was high in the hindgut, filamentous bacteria were the dominant morphology observed. The attached microbiota, sloughed during ecdysis, recolonized to the same density and diversity observed before the molt. The majority of the isolatable bacterial types were facultatively anaerobic. The distinct localization and attached nature of the hindgut bacteria and the recolonization after each molt suggest they are indigenous to this region of the gut tract.     

Nitrite toxicity in Indian major carps: sublethal effect on selected enzymes in fingerlings of Catla catla, Labeo rohita and Cirrhinus mrigala

The effects of 96-h sublethal exposure of nitrite (1, 2, 4, 8 and 10.4 mg l(-1)) on selected enzymatic activities in serum and tissues of fingerlings of catla (Catla catla), rohu (Labeo rohita) and mrigal (Cirrhinus mrigala) were studied for the first time in these species. All three species responded almost identically to nitrite exposure. With increasing nitrite concentration, reduction in activities was observed in acetylcholinesterase (AChE) in brain and liver; alkaline phosphatase (ALP) in serum, brain and gill; and acid phosphatase (ACP) in gill, while progressive increase in alanine aminotransferase (ALAT) and aspartate aminotransferase (ASAT) activities in brain, gill and serum, and ACP activity in serum and brain was observed. Lactate dehydrogenase (LDH) activity increased in gill, liver, kidney, brain and serum of all three species with increasing nitrite concentration up to 8 mg l(-1) followed by reduction at 10.4 mg l(-1). The study revealed nitrite stress causing alteration in activities of all measured tissue and serum enzymes in the fingerlings, and so stresses the need for proper management of this particular nutrient in water during carp culture.     

Ingestion of bacteria in suspension Indian major carps (Catla catla,Labeo rohita) and Chinese carps (Hypophthalmichthys molitrix,Aristichthys nobilis)

Single-species groups of fry of the planktivorous, filter-feeding carps (silver carp Hypophthalmichthys molitrix, bighead carp Aristichthys nobilis, rohu Labeo rohita and catla Catla catla) were exposed to the bacterium Chromobacterium violaceum to assess if they could detect or ingest unattached bacteria. The results indicate that planktivorous carp fry are able to ingest unattached bacteria and that the numbers ingested increase with time and with bacterial concentration in the media, although probably by passive means. It is also suggested that unattached bacteria are of little nutritional significance to carps.     

Isolation of tannase‐producing microbiota from the gastrointestinal tracts of some freshwater fish

Tannins are the most abundant among the plant‐derived antinutrients that bind readily with protein and other macromolecules to form indigestible complexes, thereby reducing the nutritional value of the plant feedstuffs. Presence of tannase‐producing gut microbiota in herbivorous animals has been suggested to overcome the antinutritional effects of tannins. However, this topic has been less investigated in herbivorous/omnivorous fish species. The present study was undertaken to evaluate the presence of tannase‐producing autochthonous microbiota in the gastrointestinal (GI) tracts of some culturable freshwater teleosts and to identify most promising tannase‐producing strains by molecular methods. Isolation and enumeration of tannase‐producing autochthonous microbiota have been carried out in the gut of ten culturable freshwater teleosts, namely catla (Catla catla), silver carp (Hypophthalmichthys molitrix), rohu (Labeo rohita), grass carp (Ctenopharyngodon idella), mrigal (Cirrhinus mrigala), common carp (Cyprinus carpio), bata (Labeo bata), kalbasu (Labeo calbasu), tilapia (Oreochromis mossambicus), and Nile tilapia (Oreochromis niloticus). Culturable heterotrophic and tannase‐producing microbial populations evaluated on tryptone soya agar and selective tannic acid agar media, respectively, revealed the maximum in the hindguts of all fish species studied. Out of 72 tannase‐producing colonies, 18 randomly selected isolates were maintained as pure cultures and evaluated quantitatively for tannase production. Among these, four most promising tannase producers were identified by 16S/26S rDNA sequencing following nucleotide blast and deposited in the National Centre for Biotechnology Information (NCBI) GenBank. The strain LR01 isolated from rohu was a bacterium, Enterobacter asburae (GenBank Accession No. GU939631 ). However, the strains CM02, OM01 and LR03 isolated from mrigal, tilapia and rohu were yeasts and identified as Pichia kudriavzevii (GenBank Accession No. GU939629 ), Candida tropicalis (GenBank Accession No. GU911469 ) and Candida parapsilosis (GenBank Accession No. GU939630 ), respectively. To the authors' knowledge, the present study is the first to report tannase‐producing autochthonous microbiota in the gut of freshwater teleosts. Tannin‐degrading microbiota detected in the present study may endow the fish with some ecological advantages by enabling them to overcome the anti‐nutritional effects of plant tannins.     

Culture-based fisheries in minor perennial reservoirs in Sri Lanka: variability in production, stocked species and yield implications

Sri Lanka is blessed with a large number of irrigation reservoirs. Depending on their hydrological regimes they are broadly categorized into perennial and seasonal reservoirs, and are secondarily used for inland fisheries. The Ministry of Fisheries and Aquatic Resources in Sri Lanka initiated a programme to introduce culture‐based fisheries in minor perennial reservoirs (< 250 ha) whereby 15 minor perennial reservoirs situated in eight administrative districts were selected for introduction of culture‐based fisheries in its initial year of implementation. These reservoirs were stocked with fish fingerlings of catla Catla catla, rohu Labeo rohita, common carp Cyprinus carpio, Nile tilapia Oreochromis niloticus and post‐larvae of freshwater prawn Macrobrachium rosenbergii. Stocking commenced in 2004; stocking data from 2004, 2005 and 2006 are considered in this study. Stocking densities varied from 217 to 870 ha^?1 year^?1, with an average density of 425 ha^?1 year^?1. Fish catch statistics for 2005, 2006 and 2007 were considered; high variability in fish yield among reservoirs as well as in some reservoirs between the years were observed. Mean annual fish yield from all 15 reservoirs increased from 57.3 to 208.1 kg ha^?1 year^?1 after the introduction of culture‐based fisheries. Overall fish production in all 15 reservoirs increased by 263%, ranging from 42.8 to 1344%. Overall contributions of Nile tilapia, catla, rohu, common carp and freshwater prawn to fish harvest were 47.4, 27.2, 16.9, 4.3 and 0.7%, respectively. Biomass gain per fingerling stocked with respect to catla, rohu, common carp and freshwater prawn are 0.51, 0.42, 0.26 and 0.04 kg, respectively, indicating attractive monetary gains through stocking of these species. Results indicate the viability of culture‐based fisheries in minor perennial reservoirs. Possible reasons for high variability in fish yield and areas for follow‐up studies are discussed.     

Development of monoclonal antibodies to rohu [Labeo rohita] immunoglobulins for use in immunoassays

Serum immunoglobulins [Ig] of rohu [Labeo rohita] were purified by affinity chromatography using bovine serum albumin as capture ligand. The purified rohu Ig [r-Ig] had a molecular weight [MW] of 880 kDa as determined with gel filtration chromatography. The heavy chain of r-Ig had an MW of 77.8 kDa and that of light chain was 26.4 kDa in SDS-PAGE. Purified r-Ig was used for the production of two anti-rohu Ig monoclonal antibodies [D7 and H4] that belonged to subclass IgG2b and IgG1, respectively. Both the MAbs were specific to heavy chain of r-Ig as seen in Western blotting. Anti-rohu Ig MAb was used as a diagnostic reagent in ELISA and immunocytochemical assays to demonstrate its application for sero-surveillance and for immunological studies in rohu. A competitive ELISA was used to demonstrate the antigenic relatedness of r-Ig with whole serum Ig of other fish species. Cross reactivity of anti-rohu Ig MAb was observed with serum Ig of Catla catla and Cirrihinus mrigala. No reactivity to serum Ig of Ophiocephalus striatus and Clarias gariepinus was seen. Anti-rohu Ig MAb was found to be suitable for the detection of pathogen specific [Edwardsiella tarda] antibodies in serum of immunized rohu by an indirect ELISA. In flow cytometry using D7 MAb, the mean percentage [+/-SE] of Ig positive cells in spleen and blood of rohu were found to be 64.85% [+/-2.34] and 51.84% [+/-2.55] of gated lymphocytes, respectively. Similarly, D7 MAb also stained 52.84% [+/-1.30] and 10.5% of gated lymphocytes in kidney and thymus, respectively. The anti-rohu Ig MAbs also showed specific staining of Ig bearing cells in spleen sections by the indirect immunoperoxidase test.     

Exploring the gut microbiota composition of Indian major carp,rohu (Labeo rohita), under diverse culture conditions

Gut microbiota of freshwater carps are often investigated for their roles in nutrient absorption, enzyme activities and probiotic properties. However, little is known about core microbiota, assembly pattern and the environmental influence on the gut microbiota of the Indian major carp, rohu. The gut microbial composition of rohu reared in different culture conditions was analysed by 16S rRNA amplicon sequencing. There was variation on gut microbial diversity and composition. A significant negative correlation between dissolved oxygen content (DO) and alpha diversity was observed, thus signifying DO content as one of the key environmental factors that regulated the diversity of rohu gut microbial community. A significant positive correlation was observed between phosphate concentration and abundance of Actinobacteria in different culture conditions. Two phyla, Proteobacteria and Actinobacteria along with OTU750868 (Streptomyces) showed significant (p < 0.05) differences in their abundance among all culture conditions. The Non-metric multidimensional scaling ordination (NMDS) analysis using Bray-Curtis distances, showed the presence of unique gut microbiota in rohu compared to other herbivorous fish. Based on niche breadth, 3 OTUs were identified as core generalists, persistent across all the culture conditions whereas the specialists dominated in the rohu gut microbiota assembly. Co-occurrence network analysis revealed positive interaction within core members while mutual exclusion between core and non-core members. Predicted microbiota function revealed that different culture conditions affected the metabolic capacity of gut microbiota of rohu. The results overall indicated the significant effect of different rearing environments on gut microbiota structure, assembly and inferred community function of rohu which might be useful for effective manipulation of gut microbial communities of rohu to promote better health and growth under different husbandry settings.     

The time-course of appearance and net accumulation of horseradish peroxidase (HRP) presented orally to juvenile carp Cyprinus carpio (L.)

A sensitive enzyme-linked immunosorbent assay (ELISA) technique was used in order to determine horseradish peroxidase (HRP) uptake from the carp gut. HRP was detected in blood plasma and various tissues within 15 min of oral intubation. The time-course of net accumulation (uptake-degradation) over a 2 hr period was recorded. The presence of HRP reached a maximum in the body tissues approximately 1 hr after intubation and on a microgram/g wet weight basis the order of accumulation within the tissues was spleen greater than kidney greater than liver. The total organ accumulation (net) was in the order liver greater than kidney greater than spleen.     

Utilization of fermented silkworm pupae silage in feed for carps

Fermented silkworm pupae (SWP) silage or untreated fresh SWP pastes were incorporated in carp feed formulations replacing fishmeal. The feed formulations were isonitrogenous (30.2-30.9% protein) and isocaloric (ME = 2905-2935 kcal/kg). Feeding under a polyculture system consisting of 30% each of catla (Catla catla), mrigal (Cirrhinus mrigala) and rohu (Labeo rohita) with 10% silver carps (Hypophthalmychthys molitrix) was carried out in ponds to evaluate the nutritive quality of SWP silage. Survival rate, feed conversion ratio and specific growth rate, respectively, were 84.2%, 2.10 and 2.39 for fermented SWP silage, 65.8%, 2.98 and 2.26 for untreated SWP and 67.5%, 3.16 and 2.20 for fishmeal indicating clearly that the fermented SWP silage was nutritionally superior to untreated SWP or fishmeal. The dietary influence on the proximate composition of whole fish was marginal.     

Impact of oxygen on metabolic fluxes and in situ rates of reductive acetogenesis in the hindgut of the wood-feeding termite Reticulitermes flavipes

The symbiotic digestion of lignocellulose in the hindgut of the wood-feeding termite Reticulitermes flavipes is characterized by two major metabolic pathways: (i) the oxidation of polysaccharides to acetate by anaerobic hydrogen-producing protozoa; and (ii) the reduction of CO2 by hydrogenotrophic acetogenic bacteria. Both reactions together would render the hindgut largely homoacetogenic. However, the results of this study show that the situation is more complex. By microinjection of radiolabelled metabolites into intact agarose-embedded hindguts, we showed that the in situ rates of reductive acetogenesis (3.3 nmol termite(-1) h(-1)) represent only 10% of the total carbon flux in the living termite, whereas 30% of the carbon flux proceeds via lactate. The rapid turnover of the lactate pool (7.2 nmol termite(-1) h(-1)) consolidates the previously reported presence of lactic acid bacteria in the R. flavipes hindgut and the low lactate concentrations in the hindgut fluid. However, the immediate precursor of lactate remains unknown; the low turnover rates of injected glucose (< 0.5 nmol termite(-1) h(-1)) indicate that free glucose is not an important intermediate under in situ conditions. The influence of the incubation atmosphere on the turnover rate and the product pattern of glucose and lactate confirmed that the influx of oxygen via the gut epithelium and its reduction in the hindgut periphery have a significant impact on carbon and electron flow within the hindgut microbial community. The in situ rates of reductive acetogenesis were not significantly affected by the presence of oxygen or exogenous H2, which is in agreement with a localization of homoacetogens in the anoxic gut lumen rather than in the oxic periphery. This adds strong support to the hypothesis that the co-existence of methanogens and homoacetogens in this termite is based on the spatial arrangement of the different populations of the gut microbiota. A refined model of metabolic fluxes in the hindgut of R. flavipes is presented.     

三种鲤科鱼类肠道内分泌细胞的初探

使用Grimelius嗜银染色法对草鱼,鲤,翘嘴红鳇3种不同食性的鲤科鱼的肠道内分泌细胞进行了研究,在3种鱼的整个肠道上均发现有内分泌细胞的分布。在前肠前段中,内分泌细胞分布最多,愈向后分布愈少,在肠褶各处均有内分泌细胞分布,以基部分布最密,内分泌细胞几乎都为开放型,位于上皮细胞和杯状细胞之间,将胞突伸向肠腔,有极少数内分泌细胞兼有开放型和封闭型细胞的特点,在它们顶端胞突伸向腔的同时,基部也伸出突起将分泌物送入邻近细胞或细胞间隙中,肠上皮中还发现一种与内分泌细胞具有同样嗜银特性的圆形颗粒。     

Bath immunisation of spawn,fry and fingerlings of Indian major carps using a particulate bacterial antigen

Larval mortality in Indian major carps is one of the major problems encountered in the pond culture system. The present investigation was carried out to investigate the proper age, duration of exposure, and optimum bacterin concentration for vaccinating rohu (Labeo rohita) and catla (Catla catla) at their early stages with a formalin killed Edwardsiella tarda bacterin suspension. The development of immunological competence was recorded with spawn of rohu and catla of 3 weeks of age exposed to a bacterin at a concentration 10(9) cfu ml(-1) for 15 min, where it persisted up to 4 weeks post vaccination. They showed significant resistance against challenge with virulent E. tarda bacteria. Significant antibody titre could be recorded in advanced fries and fingerlings exposed to 10(9) cfu/ml(-1) bacterin concentration for 45 and 60 min, respectively.     

Improved uptake of plant-derived LTB-linked proteins in carp gut and induction of specific humoral immune responses upon infeed delivery

Oral vaccination of fish is an effortless and stress free immunisation method which can be used for almost any age. However, vaccination via the mucosal route does have disadvantages. For example, the vaccine may induce tolerance and has to be protected to escape digestion. Also the vaccine should be efficiently delivered to immune-competent cells in the gut or other lymphoid organs. In addition, it should be cost effective. Here we present a novel fish vaccination model using potato tubers as vaccine production and delivery system. The model vaccines discussed here include fusion proteins consisting of a gut adhesion molecule (LTB) and a viral peptide or green fluorescent protein (GFP) expressed in potato tubers. The adhesion molecule mediates binding to and uptake from the gut, whereas the viral peptide or GFP functions as model vaccine antigen provoking the induction of an immune response. We demonstrate that fusion to LTB facilitates an elevated uptake of the model vaccines in carp gut mucosa. The plant-derived fusion proteins also elicit a specific systemic humoral immune response upon oral application of crude tuber material incorporated into a standard dietary feed pellet. The data presented here show the promising potentials of the plant as a production system for oral vaccines in aquaculture and feed mediated immunisation of fish.     

Performance of different fish species for controlling weeds in rainfed lowland rice field

A field experiment was conducted during wet season to evaluate the performance of different fish species for biocontrol of weeds in rainfed waterlogged rice fields with fingerlings of three exotic carps – grass carp, Ctenopharyngodon idella (Val.) (Cyprinidae, Cypriniformes), silver barb, Puntius gonionotus (Bleeker) (Cyprinidae, Cypriniformes) and common carp, Cyprinus carpio (L.) (Cyprinidae, Cypriniformes) – and three Indian major carps – rohu, Labeo rohita (Ham.) (Cyprinidae, Cypriniformes), catla, Catla catla (Ham.) (Cyprinidae, Cypriniformes), mrigal, Cirrhinus mrigala (Ham.) (Cyprinidae, Cypriniformes). A total of 13 major weeds under the categories of grassy, sedges, broadleaf and aquatic weeds were observed in the rice fields. Grass carp reduced maximum weed biomass (weed control efficiency [WCE] 63% at 60 days after transplanting [DAT] and 62% at 100 DAT) followed by silver barb and common carp. Among the Indian carps, only rohu was effective in control of weeds (WCE, 23% at 60 DAT). The grain yield of rice (variety Varshadhan) slightly increased (4.2–4.5 t/ha), but straw yield was significantly higher (10.2–10.7 t/ha) under rice-fish farming. Fish yield was significantly higher in exotic carps (270–288 kg/ha/90days) due to higher specific growth rates (1.8–2% body weight/day). The study indicated that exotic carps (grass carp, silver barb and common carp in order) were more effective than Indian carps for control of weed in rainfed lowland rice fields and among the Indian carps, rohu showed potential for weed control.     

Augmentation of the Antibody Response of Atlantic Salmon by Oral Administration of Alginate-Encapsulated IPNV Antigens

The objective of the present study was to assess the effect of alginate-encapsulated infectious pancreatic necrosis virus antigens in inducing the immune response of Atlantic salmon as booster vaccines. One year after intraperitoneal injection with an oil-adjuvanted vaccine, post-smolts were orally boosted either by 1) alginate-encapsulated IPNV antigens (ENCAP); 2) soluble antigens (UNENCAP) or 3) untreated feed (control). This was done twice, seven weeks apart. Sampling was done twice, firstly at 7 weeks post 1^st oral boost and the 2^nd, at 4 weeks after the 2^nd oral boost. Samples included serum, head kidney, spleen and hindgut. Serum antibodies were analyzed by ELISA while tissues were used to assess the expression of IgM, IgT, CD4, GATA3, FOXP3, TGF-β and IL-10 genes by quantitative PCR. Compared to controls, fish fed with ENCAP had a significant increase (p<0.04) in serum antibodies following the 1^st boost but not after the 2^nd boost. This coincided with significant up-regulation of CD4 and GATA3 genes. In contrast, serum antibodies in the UNENCAP group decreased both after the 1^st and 2^nd oral boosts. This was associated with significant up-regulation of FOXP3, TGF-β and IL-10 genes. The expression of IgT was not induced in the hindgut after the 1^st oral boost but was significantly up-regulated following the 2^nd one. CD4 and GATA3 mRNA expressions exhibited a similar pattern to IgT in the hindgut. IgM mRNA expression on the other hand was not differentially regulated at any of the times examined. Our findings suggest that 1) Parenteral prime with oil-adjuvanted vaccines followed by oral boost with ENCAP results in augmentation of the systemic immune response; 2) Symmetrical prime and boost (mucosal) with ENCAP results in augmentation of mucosal immune response and 3) Symmetrical priming and boosting (mucosal) with soluble antigens results in the induction of systemic immune tolerance.