Craig A. Shoemaker

Genetic fingerprinting of Flavobacterium columnare isolates from cultured fish

Aims:  To evaluate the intraspecific diversity of the fish pathogen Flavobacterium columnare

Efficacy of single and combined Streptococcus iniae isolate vaccine administered by intraperitoneal and intramuscular routes in tilapia /Oreochromis niloticus

We evaluated the effectiveness of Streptococcus iniae vaccines prepared from formalin-killed .

Density and dose: factors affecting mortality of Streptococcus iniae infected tilapia /Oreochromis niloticus

Fish density and infectious dose have been suspected to affect the mortality rate of cultured fish exposed to Streptococcusiniae. We determined the effects of S. iniae dose and tilapia (Oreochromisniloticus) density on streptococcal disease mortality. Tilapia with a mean weight of 12.7 g were used and maintained at 25±1°C in aquaria supplied with flow-through water at 0.5 l/min with a 12 h light:12 h dark cycle. Density and dose were evaluated by stocking tilapia at low (5.6 g/l), medium (11.2 g/l) and high (22.4 g/l) density and administering 2.5×107, 5×107 and 1×108 colony-forming units (CFU)/ml of S. iniae by immersion (5 tanks per density and dose, 45 total tanks). Mortality was monitored for 28 days post challenge. A significant difference (P<0.05) was seen in mortality when comparing low (4.8%) and medium (28.4%) and low and high (25.6%) density treatments. No significant difference was observed when comparing medium- and high-density treatments. Two-way analysis of variance demonstrated density had a significant effect on S. iniae mortality (P=0.0001). Doses had little effect on mortality, except at high density by dose which did show a significant interaction (P=0.001). We have demonstrated density has a significant effect on streptococcal disease mortality in tilapia exposed to S. iniae by immersion. We also evaluated infection of susceptible tilapia using dead/moribund S. iniae infected fish (i.e., cohabitation by placing five dead/moribund fish into tanks for 48 h). No significant difference in mortality pattern was observed between immersion in 8.6×107 CFU/ml S. iniae (37.6% and 34.6%) and cohabitation with S. iniae infected tilapia (24.0%). Although, densities used were less than in most water-reuse production systems (30–290 g/l), tilapia density of 11.2 g/l and above was an important factor in mortality of tilapia infected with S. iniae. A health-management strategy would be to reduce fish density thus lowering streptococcal disease mortality.

Development and Use of Modified Live Edwardsiella ictaluri Vaccine against Enteric Septicemia of Catfish

The present study showed that E. ictaluri RE-33 vaccine does not cause ESC but does stimulate protective immunity. The RE-33 vaccinates were protected against ESC for at least 4 months following a single bath immersion in a low number of E. ictaluri RE-33 without booster vaccination. Antibody responses are weak after RE-33 vaccination. Edwardsiella ictaluri RE-33 vaccine presents no risk or hazard to catfish. RE-33 vaccine will prevent ESC caused by most isolates of E. ictaluri in catfish. We recently obtained from USDA, Animal Plant Health Inspection Service (APHIS), and the state veterinarians of Alabama and Mississippi, approval to field test the RE-33 vaccine in young catfish. About 2-3 million 10- to 30-day-old channel catfish in Alabama and Mississippi have been vaccinated since June 1997 with no adverse effects of vaccination.

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 anothers pathogenic lineages.

Rapid detection of columnaris disease in channel catfish (Ictalurus punctatus) with a new species-specific 16-S rRNA gene-based PCR primer for Flavobacterium columnare

A 16-S rRNA gene from the chromosomal DNA of the fish-pathogenic bacterium Flavobacterium columnare (formerly Flexibacter columnaris), strain ARS-I, was cloned, sequenced and used to design a polymerase chain reaction (PCR) primer set. The primer set amplified a specific 1193-bp DNA fragment from F. columnare strains but not from related bacteria, F. psychrophilum, F. aquatile, F. branchiophilum, or other bacterial pathogens of fish, Flexibacter maritimus, Cytophaga johnsonae, Edwardsiella ictaluri, E. tarda, Aeromonas hydrophila, and Streptococcus iniae or from the non-fish pathogen Escherichia coli. The PCR reaction conditions were optimized to permit detection of the organism from agar plates, broth culture, frozen samples, dead fish tissue, and live fish in less than 5 h (8 h, if the more sensitive nested PCR is used). DNA was extracted by a boiled-extraction method or by commercial column purification. The PCR product was detected at DNA concentrations below 0.1 ng and from as few as 100 bacterial cells. Nested PCR using universal eubacterial primers increased the sensitivity five-fold, allowing detection of F. columnare strains at DNA concentrations below 0.05 ng and from as few as 10 bacterial cells in apparently healthy, asymptomatic fish. The efficiency of this primer set was compared to the 16-S rRNA gene primer sets of Toyama et al. [Fish Pathol. 29 (1994) 271.] and that of Bader and Shotts [J. Aquat. Anim. Health 10 (1998) 311.]. The new primer set is as good or better than the previously published primer sets for detecting F. columnare in all samples and under all conditions tested.

Killing of Edwardsiella ictaluri by macrophages from channel catfish immune and susceptible to enteric septicemia of catfish.

The role of peritoneal macrophages in immunity to enteric septicemia of catfish (ESC) after infection with live Edwardsiella ictaluri was investigated. Channel catfish macrophage-mediated bacteriocidal activity was dependent on the macrophage:bacteria ratio. Ratios of 1:1 to 1:12 exhibited significant differences (P < or = 0.05) in killing between macrophages from immune fish when compared to killing by macrophages from susceptible fish at 2.5 h. At 5 h, macrophages from immune fish were capable of effective killing (83.3%) at a 1:24 effector:target ratio, whereas macrophages from susceptible fish killed significantly (P < or = 0.05) less (56.9%). Macrophage bacteriocidal activity was significantly greater (P < or = 0.05) in macrophages from individual immune fish (93.4%) compared to macrophages from individual susceptible fish (85.4%). The kinetics of macrophage killing showed a linear increase in bacteriocidal activity from 1 to 3 h. Opsonization with immune serum enabled macrophages from immune fish to kill bacteria more effectively (93.8 vs. 75.9%) at 2.5 h. Opsonization of E. ictaluri with immune serum significantly suppressed the killing ability of macrophages from susceptible fish (46.2%) at 2.5 h. The results suggest that macrophages from fish immune to ESC had a greater capacity to kill E. ictaluri than macrophages from susceptible fish especially when E. ictaluri were opsonized with anti-E. ictaluri antibody.

Technique for Identifying Flavobacterium columnare Using Whole-Cell Fatty Acid Profiles

Abstract Isolates of Flavobacterium columnare (29 from diseased fish and three American Type Culture Collection cultures [ATCC 23463, 49512, 43622]) were identified by use of biochemical characteristics prior to generating whole-cell fatty acid profiles. The microbial identification system (MIS; Microbial ID, Newark, Delaware), a gas chromatography system, was used to generate the fatty acid profiles of F. columnare. The MIS contains databases of clinically and environmentally important bacteria that are represented by over 100 genera, including Flavobacterium spp. (F. aquatile [ATCC 11947] and F. mizutaii). Flavobacterium columnare is not included in the databases because it does not grow on standard media. Fatty acid profiles of F. columnare were generated with the CLIN40 protocol established by MIS after growth of the bacteria in modified Shieh broth. The fatty acid composition of F. columnare isolates determined by the CLIN40 method consisted of 10 major fatty acids (those present at levels > 1%): 11-...

Efficacy of a modified live Edwardsiella ictaluri vaccine in channel catfish as young as seven days post hatch

Channel catfish (Ictalurus punctatus) were vaccinated by immersion at 12, 14, 16 and 31 days post hatch with modified live Edwardsiella ictaluri RE-33 vaccine in 1997 and 7 and 10 days post hatch in 1998. At 20 to 21 days post vaccination, the groups of vaccinates and non-vaccinates were challenged with virulent E. ictaluri and monitored for mortality for at least 14 days following challenge. Results showed the vaccine to be efficacious in channel catfish as young as 7 days post hatch with relative percent survival ranging from 58.4 to 77.5.

First Report of Yersinia ruckeri Biotype 2 in the USA

A polyphasic characterization of atypical isolates of Yersinia ruckeri (causative agent of enteric redmouth disease in trout) obtained from hatchery-reared brown trout Salmo trutta in South Carolina was performed. The Y. ruckeri isolates were biochemically and genetically distinct from reference cultures, including the type strain, but were unequivocally ascribed to the species Y. ruckeri, based on API 20E, VITEK, fatty acid methyl ester profiles, and 16S rRNA gene sequencing analysis. These isolates were nonmotile and unable to hydrolyze Tween 20/80 and were therefore classified as Y. ruckeri biotype 2. Genetic fingerprint typing of the isolates via enterobacterial repetitive intergenic consensus (amplified by polymerase chain reaction) and fragment length polymorphism showed biotype 2 as a homogeneous group distinguishable from other Y. ruckeri isolates. This is the first report of Y. ruckeri biotype 2 in the USA.