Joyce J. Evans

Protection against heterologous Streptococcus iniae isolates using a modified bacterin vaccine in Nile tilapia, Oreochromis niloticus (L.)

Streptococcus iniae is a significant pathogen impacting aquaculture production worldwide. The objectives of this study were to determine whether a developed modified S. iniae (ARS-98-60) bacterin vaccine is efficacious in Nile tilapia, Oreochromis niloticus (L.), against challenge with heterologous isolates from diverse geographical locations and to evaluate protein and antigenic variability among the isolates tested. Two groups of tilapia (approximately 5 g) were intraperitoneally (IP) vaccinated with 100 microL of the vaccine or sham vaccinated with 100 microL of sterile tryptic soy broth and held for 28 days. Fish were challenged with each isolate by IP injection of 2-3 x 10(7) CFU per fish using calcein to mark fish prior to cohabitation for challenge. The results demonstrated significant protection against all challenge isolates, and relative percent survivals ranged from 79% to 100%. SDS-PAGE analysis of whole-cell lysate proteins from the S. iniae isolates demonstrated similar protein profiles between 10 and 31 kDa and variation in profiles between 35 and 100 kDa. Western blot analysis using antiserum from vaccinated fish (ARS-98-60) demonstrated shared immunogenic proteins among all isolates in the molecular mass range of 22-35 kDa and high molecular mass material >150 kDa. The results suggest that the developed S. iniae vaccine has broad ranging protection among isolates exhibiting different protein profiles.

Molecular typing of Streptococcus agalactiae isolates from fish

The genetic variability among Streptococcus agalactiae isolates recovered from fish was characterized using single-stranded conformation polymorphism (SSCP) analysis of the intergenic spacer region (ISR), and amplified fragment length polymorphism (AFLP) fingerprinting. A total of 46 S. agalactiae cultures isolated from different fish species and geographic origins as well as related reference strains were included in the study. ISR-SSCP divided the S. agalactiae isolates analysed into five distinct genotypes. Genotype 1 grouped all Kuwait isolates while genotype 4 clustered the majority of non-Kuwait isolates (USA, Brazil and Honduras). AFLP analysis offered a higher resolution level by dividing the isolates into 13 different genotypes. Two different AFLP profiles were identified within the Kuwait isolates. When data from both ISR-SSCP and AFLP were combined through a multidimensional analysis (MDS), a good correlation between geographical origin and genotypes was observed. Both AFLP and ISR-SSCP revealed genetic differences between S. agalactiae isolates from fish. While AFLP offered a higher resolution, ISR-SSCP also provided valid information being a simpler and faster method.

First isolation and characterization of Lactococcus garvieae from Brazilian Nile tilapia, Oreochromis niloticus (L.), and pintado, Pseudoplathystoma corruscans (Spix & Agassiz)

Lactococcus garvieae infection in cultured Nile tilapia, Oreochromis niloticus (L.), and pintado, Pseudoplathystoma corruscans (Spix & Agassiz), from Brazil is reported. The commercial bacterial identification system, Biolog Microlog, confirmed the identity of L. garvieae. Infectivity trials conducted in Nile tilapia using Brazilian Nile tilapia L. garvieae isolates resulted in a median lethal dose-50 of 1.4 x 10(5) colony-forming units (CFU)/fish. This is the first evidence of the presence of this pathogen from Brazilian fish. In addition, this is the first report of L. garvieae infection in either Nile tilapia or pintado. Collectively, this evidence expands the geographical range of fish hosts, number of fish hosts harbouring L. garvieae and carbon source utilization by L. garvieae fish isolates. Furthermore, the Biolog system may be an alternative technique to polymerase chain reaction for the identification of L. garvieae and discrimination between closely related bacterial species.

Effect of Iron Supplementation to Cottonseed Meal Diets on the Growth Performance of Channel Catfish, Ictalurus punctatus

Abstract Three basal diets containing 0, 27.5, and 55% solvent-extracted cottonseed meal (CSM) as replacements of 0, 50, and 100% of solvent-extracted soybean meal (SBM) on an equal nitrogen basis were each supplemented with three levels (40, 336, and 671 mg) of iron (Fe) from ferrous sulfate heptahydrate (3×3 factorial experiment). Each diet was fed to juvenile channel catfish, Ictalurus punctatus in triplicate aquaria twice daily to apparent satiation for 10 weeks. Dietary levels of CSM significantly affected weight gain (WG), feed intake (FI), feed efficiency ratio (FER), protein efficiency ratio (PER), and apparent protein utilization (APU), with diet containing 27.5% CSM provided the best performance. Total replacement of SBM by CSM decreased weight gain and feed consumption, possibly due to toxic effect of free gossypol. Supplementation of dietary iron from iron sulfate heptahydrate at a 1:1 weight ratio of iron to free gossypol had no effect on gossypol toxicity. Histological examinations of fish fed CSM containing (27.5% and 55%) diets had increased glycogen accumulation in liver as compared to that of fish fed the SBM-based diets. Fish fed 55% CSM and 0% SBM-based diets had increased liver necrosis and liver and anterior kidney pigment deposition. These parameters were not evident in fish fed diets containing 27.5% CSM. Dietary levels of iron and interactions between CSM and iron had no effect on the liver, spleen, or anterior head kidney histology. WG, FI, PER, and APU were significantly affected by the interaction between dietary levels of CSM and Fe. For SBM-based diets, these parameters linearly increased with increasing dietary level of iron. This effect was not observed for diets in which 50 and 100% of SBM were replaced by 27.5 and 55% CSM. Survival, and serum and liver iron content were not affected by dietary levels of CSM, iron, or their interaction. The effect of CSM on whole body composition was seen only for fish fed the 55% CSM diets, with fish fed these diets having lowest fat and highest moisture content. Dietary level of iron and the interaction between iron and CSM had no effect on whole body composition.

Streptococcus iniae infection and tissue distribution in hybrid striped bass (Morone chrysops×Morone saxatilis) following inoculation of the gills

This study was designed to test the possibility that Streptococcus iniae enters through the gills and causes infection in hybrid striped bass. To determine the dose response, four groups of fish were inoculated with S. iniae via the gills with a dose of 5.010 5 , 2.610 6 , 5.010 6 , or 1.010 8 CFU/fish. One group of fish was inoculated with tryptic soy broth (TSB) via the gills to serve as controls. The cumulative percent mortality was 13%, 27%, 100% and 100% for 5.010 5 , 2.610 6 , 5.010 6 and 1.010 8 CFU/fish, respectively. We also examined the tissue dissemination of S. iniae at 0.5, 4, 8, 12, 24, 48 and 72 h after experimental gill inoculation. Fish were inoculated with 2.610 6 or 5.010 6 CFU/fish, which caused low and high mortality, respectively. Within 48 h, fish inoculated with the 2.610 6 dose were culture positive on the gill surface, blood of the first and second gill arches, blood of the third and fourth gill arches and the nares. However, for the dose of 5.010 6 CFU/fish, S. iniae was also isolated from the olfactory, optic and cerebellum regions of the brain, eye, head kidney, trunk kidney, spleen and liver at 48 h. For the 2.610 6 dose, S. iniae was not isolated until 48 h post-inoculation, but was isolated at 12 h for the 5.010 6 dose. The results of this study indicate that S. iniae can enter hybrid striped bass through the gills. However, mortality at similar S. iniae doses was lower than we previously observed by inoculation of the nares. D 2003 Elsevier Science B.V. All rights reserved.

Streptococcus agalactiae Vaccination and Infection Stress in Nile Tilapia, Oreochromis niloticus

Abstract The stress response following intraperitoneal (IP) injection of a non-adjuvant Streptococcus agalactiae vaccine in cultured warmwater Nile tilapia, Oreochromis niloticus, has not been investigated. Further, little or no information is available on stress following S. agalactiae infection and what effect, if any, vaccination has on susceptibility to infection. The objective of this study was to develop preliminary information on the associations between vaccination, stress, and infection. Blood glucose levels were used to evaluate stress in the fish at different time intervals following vaccination and challenge with S. agalactiae. Blood glucose levels were measured in vaccinates and controls at 0, 2, 6, 24 hours, and 28 days post-immunization (0 hours pre-challenge), and at 2,6, 24,48,72, and 312 hours following challenge with 1.5 × 104 colony forming units (CFU) of S. agalactiae/fish. Significant increases in blood glucose levels were observed only in association with the injection of the vaccine and at 2 hours after injection. After S. agalactiae challenge, both controls and vaccinates had significantly (P < 0.05) higher blood glucose values at 2, 24,48, and 72 hours than at 0 hours. However, blood glucose levels in vaccinates were significantly (P < 0.05) lower than the controls at 24, 48, and 312 hours. Blood glucose levels and mortality of the infected controls were significantly correlated (r2 = 0.9236, P = 0.0134). The cumulative mortality of the vaccinates and controls was 10% and 60% after 13 days post-challenge, respectively. The relative percent survival (RPS) was 83.4. Our results indicate that the vaccine was efficacious against S. agalactiae and induced short-term stress in tilapia. These preliminary results also suggested, for the first time, that vaccination may significantly reduce the infection stress associated with S. agalactiae infection in tilapia.

Development of an Indirect ELISA to Detect Humoral Response to Strep to coc cus iniae Infection of Nile Tilapia, Oreochromis niloticus

Abstract The humoral an ti body of Nile tilapia, Oreochromis niloticus, to Strep to coc cus iniae in fec tion was mea sured in two groups by an in di rect en zyme-linked immunoassay (ELISA). One treat ment group was ex per i men tally in oc u lated with S. iniae, and the other con sisted of a non-in oc u lated con trol. The an ti body re sponse of the ex per i men tal infected group was sig nif i cantly dif fer ent from the non-in fected tilapia. The in di rect ELISA uti lized goat anti-tilapia IgM and a com mer cially avail able rab bit anti-goat IgG en zyme con ju gate. The spec i fic ity of the an ti body re sponse was di rected against cell-free sonicated an ti gen prepa ra tion from whole S. iniae ARS-98-60 cells. The ELISA re sults were highly cor re lated with the ag glu ti na tion re sults for tilapia anti- S. iniae an ti body.