Specific serum antibody responses in channel catfish (Ictalurus punctatus) provide limited protection against Streptococcus ictaluri challenge.

Passive immunization studies were conducted to determine the role of specific antibodies in immunity to Streptococcus ictaluri. Adult channel catfish (Ictalurus punctatus) were injected i.p. with tryptic soy broth as control or with 1.5 × 10(7)colony-forming units (cfu) S. ictaluri/fish at 0, 30, and 60 d, and serum was collected 90 d after the original challenge. Fish were passively immunized by i.p. injection with serum from the tryptic soy broth (TSB) control group, anti-S. ictaluri serum from fish immunized three times and sampled at 90 d (SSI), or heat-inactivated anti-S. ictaluri serum from fish immunized three times and sampled at 90 d (HISSI). These passively immunized fish were then challenged 72 h later with 1.5 × 10(8)cfu S. ictaluri/fish. Over 21 d, the mean cumulative percent survival was 43.3 (TSB), 63.3 (SSI), and 50.0 (HISSI). A significant difference in cumulative percent survival was noted between the TSB and the HISSI groups, and significant differences were noted between these groups and the SSI group. Serum obtained from immunized fish 72 h after passive immunization exhibited increased anti-S. ictaluri antibody levels. Twenty-one days after the challenge, the HISSI and SSI group antibody levels significantly increased above their corresponding pre-challenge levels. No significant (r(2)=0.0806; P<0.5985) correlation between increased pre-challenge specific serum antibody levels and survival after challenge was demonstrated when analyzing the control and passive immunization groups. The results indicate that both specific anti-S. ictaluri antibodies and non-specific immune responses are important for protection against S. ictaluri.

Efficacy of a modified live Flavobacterium columnare vaccine in fish.

Flavobacterium columnare is an aquatic bacterium that is responsible for columnaris disease. This aquatic pathogen has a worldwide distribution and is highly infectious to both warm and cold water fish. A modified live F. columnare vaccine was developed by repeated passage of a virulent strain on increasing concentrations of rifampicin that resulted in attenuation. Here we report vaccination/challenge trials to evaluate efficacy and safety. In separate laboratory trials, immersion vaccination of channel catfish (Ictalurus punctatus) fry between 10 to 48 days post hatch (DPH) with experimental vaccine or licensed product resulted in relative percent survival (RPS) between 57-94% following challenge. Similarly, a vaccination/challenge trial using largemouth bass (Micropterus salmoides) fry at 10 DPH was performed using various doses of licensed product under laboratory conditions. Results demonstrated safety of the vaccine and significant protection following challenge with RPS values between 74-94%, depending on vaccine dose. Together, these trials demonstrate the vaccine administered to early life-stage channel catfish and largemouth bass is safe and reduces mortality following challenge with F. columnare.

A commercial rapid optical immunoassay detects Streptococcus agalactiae from aquatic cultures and clinical specimens.

The BioStar STREP B Optical ImmunoAssay (STREP B OIA) (BioStar OIA Strep B Assay Kit; BioStar Incorporation, Louisville, CO, USA), commonly used for diagnosis of human maternal group B streptococcus (GBS) colonization, was evaluated for its diagnostic and analytical sensitivity and specificity to aquatic animal GBS isolates, cross-reactivity, and diagnosis and recovery of GBS directly from clinically- infected fish swabs. STREP B OIA identified 25 known fish and dolphin GBS isolates. Thirteen non-GBS negative control isolates from fish and other animals were negative, giving 100% analytical specificity and no cross-reactivity. Three groups of 6 Nile tilapia (Oreochromis niloticus) (mean weight of 40.60+/-1.70 g) each were inoculated intraperitoneally with either 10(6) colony-forming units (cfu) GBS/fish, 10(6) cfu Streptococcus iniae/fish or 100 microL of tryptic soy broth (TSB) and observed for mortality for 7 days. The nare and brain of all fish were swabbed and subjected to the STREP B OIA for detection of GBS antigen immediately after swabbing (0 h) or 24, 48 and 72 h post-swabbing and compared to conventional culture on trypticase soy agar with 5% sheep blood. The STREP B OIA method demonstrated a diagnostic sensitivity of 75.0% and a diagnostic specificity of 69.2% compared to direct TSA. The percent agreement between OIA and culture was 100%. GBS antigen could be retrieved by OIA following 72-h storage of swabs. These results demonstrate the utility of the STREP B OIA to identify GBS from culture and directly from swabs of clinically- infected fish.

Human Streptococcus agalactiae isolate in Nile tilapia (Oreochromis niloticus).

Streptococcus agalactiae, the Lancefield group B streptococcus (GBS) long recognized as a mammalian pathogen, is an emerging concern with regard to fish. We show that a GBS serotype Ia multilocus sequence type ST-7 isolate from a clinical case of human neonatal meningitis caused disease and death in Nile tilapia (Oreochromis niloticus).

Pathogenicity of Streptococcus ictaluri to channel catfish.

The pathogenicity of a Streptococcus ictaluri isolate in channel catfish Ictalurus punctatus at the fry (0.5 g), fingerling (15 g), and juvenile (55 g) stages was determined by experimental bath immersion and injection experiments. Channel catfish were exposed in 1-L immersion baths containing 10(8), 10(9), 10(10), 10(11) or 10(12) colony-forming units (cfu) of S. ictaluri. Fish were also injected intraperitoneally with 0.1 mL of bacterial solution for final doses of 10(4), 10(5), 10(6), 10(7), or 10(8) cfu of S. ictaluri per fish. Streptococcus ictaluri caused mortality in fry, fingerling, and juvenile channel catfish within 21 d postinfection. When mortalities were calculated based on size and challenge route, the cumulative percent mortalities were 11% for fry and 0% for fingerlings by the bath immersion route and 14% for fingerlings and 6% for juveniles by the injection route. Isolation of S. ictaluri from moribund and dead catfish was confirmed by the newly established BIOLOG profile (MicroLog3 system). The results indicate that channel catfish were only susceptible to high concentrations of S. ictaluri and that juvenile channel catfish were less susceptible, possibly explaining why little mortality has been attributed to S. ictaluri infection in catfish aquaculture.

Phylogenetic relationships among Streptococcus agalactiae isolated from piscine, dolphin, bovine and human sources: a dolphin and piscine lineage associated with a fish epidemic in Kuwait is also associated with human neonatal infections in Japan.

Streptococcus agalactiae, commonly known as group B streptococcus (GBS), is a cause of infectious disease in numerous animal species. This study examined the genetic relatedness of piscine, dolphin and human GBS isolates and bovine GBS reference strains from different geographical regions using serological and molecular serotyping and multilocus sequence typing (MLST) techniques. Piscine isolates originating from Kuwait, Brazil, Israel and the USA were capsular serotype Ia, a serotype previously unreported in GBS isolated from fish. Sequence typing of piscine isolates produced six sequence types (ST-7, ST-257, ST-258, ST-259, ST-260 and ST-261), the latter five representing allelic designations and allelic combinations not previously reported in the S. agalactiae MLST database. Genomic diversity existed between dolphin and piscine GBS isolates from Kuwait and other geographical areas. Piscine GBS isolates from Brazil, Israel, Honduras and the USA appeared to represent a distinct genetic population of strains that were largely unrelated to human and bovine GBS. The Kuwait dolphin and piscine lineage (ST-7, Ia) was also associated with human neonatal infections in Japan. Comparative genomics of piscine, human and bovine GBS could help clarify those genes important for host tropism, the emergence of unique pathogenic clones and whether these hosts act as reservoirs of one another's pathogenic lineages.

Flavobacterium columnare chemotaxis to channel catfish mucus.

Flavobacterium columnare is a Gram-negative pathogen of many species of wild and cultured fish. Isolates from diseased channel catfish belong to either genomovar I or II. Genomovar II isolates were found to be more virulent than genomovar I isolates. The objective of the present study was to determine whether differences exist in the chemotactic response of these genomovars to mucus obtained from the skin, gills and intestines of healthy channel catfish using the capillary chemotaxis assay. Mucus from the skin and gill induced a greater chemotactic response by F. columnare than mucus from the intestine. Sixty percent of mucus from the skin of individual catfish yielded a positive chemotactic response from F. columnare. Finally, skin mucus induced a greater chemotactic response in genomovar II F. columnare than in genomovar I F. columnare isolates. The data indicate that mucus from channel catfish results in a chemotactic response by F. columnare. This positive chemotactic response may be an important first step for F. columnare colonization of channel catfish skin or gills. Although the role that chemotaxis plays in the virulence of F. columnare is not fully defined, the chemotactic response of genomovar ll isolates suggests that chemotaxis is associated with virulence.

The macrophage chemotactic activity of Streptococcus agalactiae and Streptococcus iniae extracellular products (ECP).

The ability of Streptococcus agalactiae and Streptococcus iniae to attract macrophages of Nile tilapia (Oreochromis niloticus) was investigated. The extracellular products (ECP) from S. agalactiae and S. iniae were tested in vitro for macrophage chemotaxis using blind-well chambers. The macrophages were obtained from the peritoneal cavity 4-5 days after intraperitoneal injection of squalene. Both macrophage chemotactic and chemokinetic activities were demonstrated using the S. agalactiae ECP. However, only chemotactic activity was shown for S. iniae ECP. High-pressure liquid chromatography fractionation revealed that semi-purified S. agalactiae and S. iniae ECPs had estimated molecular weights of 7.54 and 19.2kDa, respectively. The prominent chemotactic activities of ECP from S. agalactiae and S. iniae are likely to be involved in the proinflammatory responses of macrophages to S. agalactiae and S. iniae infections.

Immunization of eyed channel catfish, Ictalurus punctatus, eggs with monovalent Flavobacterium columnare vaccine and bivalent F. columnare and Edwardsiella ictaluri vaccine.

The efficacy of a modified live monovalent Flavobacterium columnare vaccine and bivalent F. columnare and Edwardsiella ictaluri vaccines were evaluated following immersion vaccination of eyed channel catfish (Ictalurus punctatus) eggs. The modified live F. columnare vaccine was grown in modified Shieh broth and administered at 1.35 x 10(7) CFU/ml for 15 min exposure (1l water). Booster immunization was conducted at day 34 with 2.17 x 10(7) CFU/ml for 15 min. Bivalent vaccines consisted of a 1:1 ratio of the modified live F. columnare and AQUAVAC-ESC vaccine for the 15 min exposure (1l immersion bath). Non-vaccinated controls were held in 1l water without vaccine for 15 min. Fish were challenged with F. columnare (ALG-00-530) by immersion at days 109, 116, and 137 post-primary immunization or E. ictaluri (AL-93-75) by immersion at day 116 (bivalent vaccine group). Efficacy of monovalent modified live F. columnare vaccine administered singly or with a booster vaccination was shown to be protective with relative percent survival (RPS) values ranging from 50.0 to 76.8. Some variation was seen in RPS values following bivalent immunization, ranging from 33.0 to 59.7 in the fish challenged with F. columnare and 44.5 to 66.7 in fish challenged with E. ictaluri. However, the RPS values were not statistically different. The results suggest that administration of live bivalent vaccine at the eyed-egg stage is safe and elicits protection upon single pathogen challenge.

First report of Streptococcus agalactiae and Lactococcus garvieae from a wild bottlenose dolphin (Tursiops truncatus).

The isolation and characterization of two bacterial species, Streptococcus agalactiae and Lactococcus garvieae, previously unreported in wild marine mammals are described from a freshly dead bottlenose dolphin, Tursiops truncatus, from Kuwait Bay, Kuwait, in September 2001. Conventional and rapid identification systems were used to determine that isolates from muscle and kidney were S. agalactiae and L. garvieae, respectively. The isolates were gram-positive, catalase-negative, oxidase-negative, nonhemolytic cocci. The S. agalactiae was serotyped to group antigen B, whereas the L. garvieae could not be assigned to any serogroup. These Kuwait isolates displayed considerable homogeneity with corresponding American Type Culture Collection (ATCC) type isolates. Although the dolphin S. agalactiae isolate was nonhemolytic, it was biochemically similar to S. agalactiae isolated from mullet sampled in the concurrent Kuwait Bay fish kill. Some biochemical heterogeneity was observed between the dolphin isolates and corresponding mammalian ATCC type isolates, especially with Voges Proskauer, alanine-phenylanaline-proline arylamidase, and alpha-galactosidase tests. Nile tilapia, Oreochromis niloticus, experimentally infected with the dolphin S. agalactiae and L. garvieae isolates experienced 90% and 0% mortalities, respectively. This is the first isolation of S. agalactiae and L. garvieae from a wild marine mammal, and the microbial characteristics established here provide pertinent information for the future isolation of these bacteria.

Passive immunization of Nile tilapia (Oreochromis niloticus) provides significant protection against Streptococcus agalactiae.

A study was conducted to determine the role of specific antibodies in immunity to Streptococcus agalactiae. Adult Nile tilapia (Oreochromis niloticus) were injected i.p. with tryptic soy broth as control or with S. agalactiae vaccine. Ninety days later, fish were challenged with 1.5x10(4)CFUS. agalactiae fish(-1). Blood was drawn from all fish 90d after vaccination and 25d after challenge, and the acquired serum was injected i.p. in fingerling Nile tilapia. These passively immunized fish were subsequently challenged 72h later with 1.5x10(4)CFUS. agalactiae fish(-1), and significantly less (P<0.0001) mortalities were noted among fish administered serum containing specific anti-S. agalactiae antibodies (0.0-10.0% mortalities) than in control groups (63.3-72.7% mortalities). Heat-inactivation of serum produced no significant differences in mortalities than non-heat-treated serum in groups administered serum containing specific antibodies from vaccinated fish (P<0.9455) or vaccinated-challenged fish (P<0.0781). Pre-challenge serum samples indicate that the passively immunized fish had significantly increased (P<0.0001) specific antibody levels over control fish. A highly significant (r(2)=0.5892; P<0.0001) correlation between increased pre-challenge specific serum antibody OD levels and survival after challenge was demonstrated when analyzing the control and passive immunization groups. The results of this study indicate that specific anti-S. agalactiae antibodies play a primary role in immunity to S. agalactiae in fish.

A vaccination and challenge model using calcein marked fish.

A vaccination and challenge cohabitation model was established and evaluated using Nile tilapia (Oreochromis niloticus), the fluorescent chromophore calcein, and a Streptococcus iniae vaccine. Tilapia were non-invasively calcein marked, sham-vaccinated (CMSV) and cohabited with non-marked sham-vaccinated (NMSV) or non-marked S. iniae vaccinates (NMV) as a single unit. After 30 d, the cohabitants were challenged with a virulent isolate of S. iniae by intraperitoneal (ip) injection and the cumulative mortality was measured over a period of 15 d. Calcein marking did not have a significant effect on S. iniae susceptibility as mortality of CMSV and NMSV was not significantly different (P=0.6756). Nor did calcein marking have an effect on the vaccination and challenge cohabitation model. The results showed that the cumulative mortality of CMSV (N=160) was significantly greater (P<0.0003) than those of NMV (N=160). The results of the calcein marking trials indicate that the most suitable calcein concentration and exposure time to produce detectable fluorescent marking of tilapia was 500 mg L(-1) for 4 h. Furthermore, the calcein marks were readily visible in the calcified skeletal structures of head and fins using a portable handheld UV lamp set at 365 nm wavelength. Calcein appears to be a valuable tool for non-invasive, non-lethal, non-stressful, mass marking of fish to differentiate between sham- and pathogen-vaccinated fish in this cohabitation model. The vaccination and challenge cohabitation model also offers the statistical advantage of using individual fish as the experimental unit maintained in the same aquarium.

Duration of protective antibodies and correlation with survival in Nile tilapia Oreochromis niloticus following Streptococcus agalactiae vaccination.

Streptococcus agalactiae is a major piscine pathogen that causes significant morbidity and mortality among numerous species of freshwater, estuarine and marine fishes. Considering the economic importance of fishes susceptible to S. agalactiae throughout the world, an efficacious S. agalactiae vaccine was developed using an extracellular product (ECP) fraction and formalin-killed whole cells of S. agalactiae. A vaccine study was conducted by intraperitoneal (i.p.) injection in Nile tilapia Oreochromis niloticus in order to determine the duration of protection and its correlation to antibodies specific for this pathogen. After 47, 90 or 180 d post-vaccination (DPV), the fish were i.p. challenged with approximately 2.0 x 10(4) S. agalactiae colony-forming units (CFU) fish(-1) to determine the duration of protective immunity. The percent survival in control fish i.p.-injected with sterile TSB was 16,16, and 4% on 47, 90 and 180 DPV, respectively, while the percent survival for the vaccinated fish was 67, 62 and 49%, respectively. The specific mean antibody concentration of the vaccinated fish was significantly higher than that of the control fish, with significant correlation between the ELISA optical density (OD) and protection. These results indicate that the specific antibody has a correlation with protection following immunization with the S. agalactiae vaccine and that the vaccine can confer protection against S. agalactiae up to 180 DPV.

Efficacy of Streptococcus agalactiae (group B) vaccine in tilapia (Oreochromis niloticus) by intraperitoneal and bath immersion administration.

We evaluated the effectiveness of a Streptococcus agalactiae vaccine in tilapia (Oreochromis niloticus) for prevention of streptococcal disease. The vaccine was prepared from formalin-killed cells and concentrated extracellular products (greater than 3 kDa) of a single isolate of S. agalactiae (ARS-KU-MU-11B). Intraperitoneal (IP) and bath immersion (BI) vaccine trials were conducted at two temperatures, 32 and 26 degrees C, and mean fish weights, 5 and 30 g. Control tilapia were injected with tryptic soy broth. Thirty gram tilapia vaccinated and challenged by IP injection with 1.5 x 10(4) colony-forming units (CFU)/fish of Streptococcus agalactiae at 30 days post-immunization had a relative percent survival (RPS) of 80. Smaller tilapia vaccinated and challenged under similar conditions had an RPS of 25. An RPS of zero was noted in 30 g fish IP vaccinated with Streptococcus iniae and IP challenged with S. agalactiae. The 5 and 30 g tilapia bath immunized with S. agalactiae and IP challenged with 3.6 x 10(5) and 1.7 x 10(6) CFU/fish of S. agalactiae had RPS values of 34. Intraperitoneal administration of the vaccine provided efficacious protection only in the 30 g tilapia regardless of whether the fish were immunized and challenged at 26 or 32 degrees C. Bath immunization of both 5 and 30 g tilapia resulted in RPS values that were two times lower than those achieved with IP vaccination. The results of this study suggest that there is a lack of cross-protection of S. iniae bacterins against S. agalactiae challenge. Protection against S. agalactiae infection is, however, provided through vaccination with a S. agalactiae modified bacterin vaccine.

Warmwater fish vaccinology in catfish production.

The ability of the fish industry to provide a continuous supply of fish protein depends on both proper biosecurity and strategies to significantly reduce the risk of infectious diseases. Vaccination is a safe and effective means to prevent disease and to increase the productivity and profitability of farmed fish. Vaccines are likely to be the prime prophylactic measure of the future because of rapidly developing advances in fish vaccine technology and producer acceptance. The efficacy of a vaccine is influenced by a variety of factors that must be considered in the development of vaccination strategies for fish. This review highlights some of these factors. The response to a modified live vaccine against Edwardsiella ictaluri is used for illustration.

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.