Richard A. Shelby

Effects of Probiotic Diet Supplements on Disease Resistance and Immune Response of Young Nile Tilapia, Oreochromis niloticus

Abstract Probiotic microbial feed supplements are gaining wide acceptance in livestock production, and may be applicable to aquaculture production systems. In two experiments, fry of Nile tilapia, Oreochromis niloticuswere fed a basal diet supplemented with commercially-available probiotic microorganisms. Survival, growth, disease resistance, and immunological parameters were monitored for a maximum of 94 days. Bacterial populations were higher in the digestive tracts of tilapia receiving probiotic diets, and colony morphology suggested that viable probiotic bacteria persisted up to 48 hours after feeding. Survival was higher after 39–63 days in fish receiving the Bioplus probiotic, Bacillus spp. There was no difference in growth, lysozyme, total serum immunoglobulin, complement, specific anti-streptococcal antibody levels, or mortality due to Streptococcus iniae infection either before or after disease challenge. The results suggest that these commercially-available probiotic microorganisms do not provide beneficial effects in young Nile tilapia.

Effects of Yeast Oligosaccharide Diet Supplements on Growth and Disease Resistance in Juvenile Nile Tilapia, Oreochromis niloticus

Commercially available yeast and yeast subcomponents consisting mainly of β-glucan or oligosaccharide feed additives were added to diets of juvenile (12–18g) Nile tilapia (Oreochromis niloticus) at rates recommended by suppliers. Three experiments were conducted following a basic protocol with varied rates of supplementation, duration of feeding, and stocking densities. Experimental diets were fed twice daily to apparent satiation for a period of two or four weeks, at the end of which feed consumption and weight gain were measured. Following the experimental feeding period, serum components, including protein and immunoglobulin concentrations, as well as lysozyme and complement activities, were measured. A disease challenge was conducted with pathogenic isolates of Streptococcus iniae or Edwardsiella tarda. Weight gains were not significantly different in fish fed the supplemented diets when compared to the control diet. There were significant differences in fed intake within individual experiments; however, this effect was not consistent in all three experiments. Overall feed efficiency was not significantly affected by diet. There were no differences in serum components of fish sampled at two or four weeks. Fish fed the experimental diets did not have lower mortality or morbidity after disease challenge compared to fish fed the control diets. Specific antibody against S. iniae or E. tarda measured by ELISA did not reveal differences in the fish surviving the challenge. We conclude that the incorporation of these commercial yeast component products into the diet of juvenile Nile tilapia at these rates and for these feeding periods had no effect on growth, serum components, antibody responses, or survival following S. iniae or E. tarda infection.

Effects of Probiotic Bacteria as Dietary Supplements on Growth and Disease Resistance in Young Channel Catfish, Ictalurus punctatus (Rafinesque)

ABSTRACT Three separate experiments were conducted to determine the beneficial effects of probiotic bacteria added commercial catfish diets. These commercial probiotics, singly and in combination, were incorporated into a commercial diet fed to juvenile channel catfish, Ictalurus punctatus (Rafinesque), in aquaria. Microbial analyses were conducted to confirm viability of the bacteria under storage conditions. Fish were fed twice daily to apparent satiation and weighed biweekly for a period of 5 to 8 weeks. After that time, representative fish were bled for serological analysis of protein, immunoglobulin, complement, and lysozyme. Disease resistance was additionally determined in the remaining fish by observing mortality after immersion challenge with pathogenic Edwardsiella ictaluri, the causative agent of enteric septicemia (ESC). Specific anti-ESC antibody was measured by ELISA after 15 days in the fish which survived challenge. The viability of the bacteria did not decline in storage at 5°C and could be recovered between 106 and 107 colony forming units per gram (CFU/g) of diet after storage for 4 weeks. No significant differences in levels of protein, immunoglobulin, complement, or lysozyme were noted between fish consuming probiotic diets or the control diet. In one feeding trial, weight gain per fish was significantly less in the fish receiving a combination of Pediococcus and Enterococcus probiotic bacteria when compared to those consuming either bacterial supplement diet alone or the control diet. We conclude that these particular commercial dietary supplements lack specific growth promoting or immune stimulating effects in juvenile channel catfish.

Development of an Indirect ELISA to Detect Humoral Response to Flavobacterium columnare Infection of Channel Catfish, Ictalurus punctatus

ABSTRACT The humoral antibody response of channel catfish, Ictalurus punctatus, to experimental Flavobacterium columnare infection was measured in control (non-infected) and infected (intraperitoneal- and intramuscular-injected) fish by an indirect enzyme-linked immunoassay (ELISA). The antibody response of the experimentally infected fish was significantly higher (P<0.0001) than that of the non-infected channel catfish by both indirect ELISA and agglutination. The indirect ELISA utilized goat anti-channel catfish IgM and a commercially available rabbit anti-goat IgG enzyme conjugate. The antibody response was directed against a cell-free sonicated antigen preparation derived from live whole F. columnare cells. Indirect ELISA and agglutination results correlated (r2 = 0.60) for anti-F. columnare antibody response in experimentally and non-infected fish. We were also able to correlate ELISA and agglutination results of 60 fish naturally infected with F. columnare(r2 = 0.76). Indirect ELISA will allow for rapid monitoring of humoral antibody, following natural exposure or vaccination against F. columnare, with a small sample of serum.

Cortisol Response of Nile Tilapia, Oreochromis niloticus (L.), to Temperature Changes

Abstract Nile tilapia, Oreochromis niloticus (L.), is a commercially-valuable fish species with high nutritional value. As a result of the intensive aquaculture of this species, handling, transport, and environmental changes that causes stress on these fish are unavoidable. The objective of this study was to determine the effects of gradual and acute temperature changes on juvenile tilapia. No significant difference (P > 0.05) was found among serum cortisol levels in juvenile tilapia when the water temperature was gradually increased from 27°C to 32°C, or 40°C, and maintained for 1 hour, although the levels were five times pretreat-ment levels. When tilapia acclimated to 27°C were subjected to 18°, 27°, 30°, 32°, 34°, 36°, 38°, or 40°C water for 10 minutes in a water bath, followed by a recovery period of 10 minutes at 27°C in the original aquaria, no significant difference (P > 0.05) in cortisol levels was observed among treatments except for significantly elevated levels at 38°C and 40°C. When tilapia acclimated to 27°C were subjected to the same temperature exposures but given a recovery period of 60 minutes at 27°C in the original aquaria, there was no significant (P > 0.05) increase in cortisol levels in tilapia among treatments from 18° to 36°C; but there was a significant (P > 0.05) increase between values from those treatments at 38° and 40°C. Acute temperature changes initiated the cortisol response as early as 10 minutes in fish following exposure to 38°C or 40°C and resulted in significant increases in the 38°C and 40°C treatments following 1 hour of recovery at 27°C. These results have implications for the management of tilapia during bacterial challenge, vaccination, and handling and transport during aquacultural activities.

Detection of Humoral Response to Streptococcus iniae Infection of Nile Tilapia, Oreochromis niloticus, by a Monoclonal Antibody-Based ELISA

Abstract A monoclonal antibody (Mab) was developed that was specific for Nile tilapia, Oreochromis niloticus, immunoglobulin (Ig). This Mab was identified as isotype IgG1 and showed no cross-reactivity with other fish species tested by indirect ELISA. Further testing by Western blotting indicated the Mab to be specific for the heavy chain of Nile tilapia Ig. A specific immunoassay was developed to measure immune response to Streptococcus iniae in Nile tilapia utilizing this Mab. There was a high degree of correlation with this assay compared to agglutination assay and ELISA based on polyclonal antibodies developed in goat. The Mab offers the potential for increased accuracy in the detection of vaccine and disease response to S. iniae in this commercially important fish species.

Isolation, Purification, and Molecular Weight Determination of Serum Immunoglobulin from Gulf Menhaden: Development of an Enzyme-Linked Immunosorbent Assay to Assess Serum Immunoglobulin Concentrations from Atlantic Menhaden

An immunoglobulin M (IgM)-like immunoglobulin was isolated by polyethylene glycol precipitation from pooled serum collected from healthy gulf menhaden Brevoortia patronus. The immunoglobulin (Ig) was purified by Sephacryl-400 gel filtration chromatography. The molecular weight of unreduced, purified Ig was determined to be 850 kilodaltons (kD) by high-performance liquid chromatography. A goat antiserum against the purified Ig was produced and determined to react with the serum Ig of both gulf and Atlantic menhaden B. tyrannus by double gel diffusion. When reacted with sera from taxonomically unrelated species of fish, sheepshead minnow Cyprinodon variegatus, striped mullet Mugil cephalus, gulf flounder Paralichthys albigutta, and hybrid striped bass (white bass Morone chrysops × striped bass M. saxatilis), no precipitation bands developed. Furthermore, the specificity of the goat antiserum was shown by Western blot analysis to be for the 77,000-molecular-weight heavy chain of reduced and alkylated gulf and Atlantic menhaden Ig. An indirect enzyme-linked immunosorbent assay (ELISA) was developed for the assessment of Ig concentrations in Atlantic menhaden serum. To illustrate the applicability of the ELISA, we assessed the Ig concentration in the serum of 542 healthy Atlantic menhaden collected from inland bays of Delaware and Maryland in 2000 and 2001. The amount of Ig was estimated to be in the range 0.26-23.50 mg/mL, with a mean of 7.37 and a standard deviation of 5.12 mg/mL. The ELISA was reproducible, as determined by the inter- and intra-assay coefficients of variation (100·SD/mean) of 11.2% and 6.8%, respectively, and used very small amounts (1-2 μL) of serum to assess the Ig concentrations from menhaden.

Development of an immunoassay to measure the humoral immune response of hybrid striped bass Morone chrysops x M. saxatilis

Hybrid striped bass (HSB) were immunized with bovine serum albumin (BSA) and the specific anti-BSA immunoglobulin (Ig) was affinity purified from the resulting serum by means of an agarose gel-BSA column. The native Ig had an apparent molecular size of 893 KDD, by size exclusion chromatography, and when examined by polyacrylamide gel electrophoresis (PAGE) under denaturing conditions, resolved to heavy (H) and light (L) chains of 76 and 27 KDD, respectively. Affinity purified native HSB Ig was used to immunize a goat which produced specific anti-HSB Ig antibody (Ab). Purified native HSB Ig was also used to produce two murine monoclonal antibodies (mAbs) with specific affinities for H and L chain moieties of the HSB Ig molecule. Both polyclonal and monoclonal antibodies could be used individually in an indirect enzyme-linked immunosorbent assay (ELISA) to measure specific anti-BSA Ig in HSB serum. These antibodies could also be used in combination to measure total Ig in a capture ELISA format. Using both assays, the kinetics of the humoral immune response of HSB was measured for 98 days following two injections of BSA.

Relationship between Frequency of Skin Ulceration and Total Plasma Immunoglobulin Levels in Atlantic Menhaden from Delaware and Maryland Inland Bays

Abstract In the summer and fall of 2000, a high prevalence of ulcerative skin lesions in Atlantic menhaden Brevoortia tyrannus from inland bays of Delaware and Maryland caused public and governmental concern. The relationship between the frequency of skin ulceration and total plasma immunoglobulin (Ig) levels was investigated for Atlantic menhaden from these inland bays in 2000 and 2001. Total plasma Ig levels were determined using goat anti-menhaden Ig in an enzyme-linked immunosorbent assay. Total plasma Ig levels were higher in nonulcerated fish (7.4 ± 0.02 mg/mL [mean ± SE]; n = 543) than in skin-ulcerated ones (4.2 ± 0.42; n = 120). Skin ulcers were less frequent among small fish (67-80 mm fork length; 6%) than in medium (81-105 mm; 21%) or large fish (106-190 mm; 27%). Nonulcerated large fish had higher total plasma Ig levels (8.5 ± 0.44 mg/mL) than nonulcerated small fish (6.1 ± 0.28) but not medium fish (7.9 ± 0.42). The total plasma Ig levels were higher in all sizes of fish in 2001 (8.9 ± 0.26) ...