Biomedical subjects

Craig A Shoemaker

Publications and source records attributed to Craig A Shoemaker.

11 recordsLinked to original sources

PubMed2007

Molecular typing of isolates of the fish pathogen, Flavobacterium columnare, by single-strand conformation polymorphism analysis.

Flavobacterium columnare intraspecies diversity was revealed by analyzing the 16S rRNA gene and the 16S-23S internal spacer region (ISR). Standard restriction fragment length polymorphism (RFLP) of these sequences was compared with single-strand conformation polymorphism (SSCP). Diversity indexes showed that both 16S-SSCP and ISR-SSCP improved resolution (D>or=0.9) when compared with standard RFLP. ISR-SSCP offered a simpler banding pattern than 16S-SSCP while providing high discrimination between isolates. SSCP analysis of rRNA genes proved to be a simple, rapid, and cost-effective method for routine fingerprinting of F. columnare.

Animals↗

PubMed2006

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.

Animals↗

PubMed2006

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.

Animals↗

PubMed2006

Immunofluorescent test for simultaneous detection of Edwardsiella ictaluri and Flavobacterium columnare.

Enteric septicemia of catfish (ESC) and columnaris disease are 2 bacterial diseases significantly affecting the aquaculture industry, and thus rapid diagnosis of disease is imperative for making judicious management decisions. A rapid indirect fluorescent antibody (IFA) test with antibody conjugated fluorochromes having 2 different spectral properties (Alexa Fluor 488-emitting green fluorescence, and Alexa Fluor 594-emitting red fluorescence) was compared with bacteriological culture (accepted standard) for simultaneous detection of Edwardsiella ictaluri (EI) and Flavobacterium columnare (FC) in 3 groups of experimentally infected channel catfish (Ictalurus punctatus Rafinesque), and a fourth group that acquired an aquarium-infection with F. columnare. A total of 303 samples (derived from kidney, brain and nares) from 101 fish were concurrently examined by both tests. Fish in the 3 experimentally infected groups (I to III) were culture positive for the bacteria with which they were infected, and fish in Group IV, (the spontaneously infected fish) revealed F. columnare only. The IFA test compared favorably in sensitivity (EI= 80.7 %; FC = 87.2%) and specificity (EI = 83.9%; FC = 88.9%) with the standard bacteriological culture. The positive predictive value (EI = 96.2% Group I, 90.8% Group II, 93.7% Groups I and II combined; FC = 95.2% Group II, 95.3% Groups II, III and IV combined) was high, while the negative predictive value (EI = 66.7% Group I, 31.3% Group II, 59.5% Groups I and II combined; FC = 73.7% Group II, 72.7% Groups II, III and IV combined) was relatively low. The IFA test will serve as an efficient tool for rapid simultaneous detection of E. ictaluri and F. columnare in outbreaks of disease.

Animals↗

PubMed2006

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.

Animals↗

PubMed2005

Evaluation of a loop-mediated isothermal amplification method for rapid detection of channel catfish Ictalurus punctatus important bacterial pathogen Edwardsiella ictaluri.

Channel catfish Ictalurus punctatus infected with Edwardsiella ictaluri results in 40--50 million dollars annual losses in profits to catfish producers. Early detection of this pathogen is necessary for disease control and reduction of economic loss. In this communication, the loop-mediated isothermal amplification method (LAMP) that amplifies DNA with high specificity and rapidity at an isothermal condition was evaluated for rapid detection of E. ictaluri. A set of four primers, two outer and two inner, was designed specifically to recognize the eip 18 gene of this pathogen. The LAMP reaction mix was optimized. Reaction temperature and time of the LAMP assay for the eip 18 gene were also optimized at 65 degrees C for 60 min, respectively. Our results show that the ladder-like pattern of bands sizes from 234 bp specifically to the E. ictaluri gene was amplified. The detection limit of this LAMP assay was about 20 colony forming units. In addition, this optimized LAMP assay was used to detect the E. ictaluri eip 18 gene in brains of experimentally challenged channel catfish. Thus, we concluded that the LAMP assay can potentially be used for rapid diagnosis in hatcheries and ponds.

Animals↗

PubMed2005

Transmission and detection of Flavobacterium columnare in channel catfish Ictalurus punctatus.

A specific and rapid PCR detection method for Flavobacterium columnare based on the 16S-23S rDNA intergenic spacer region (ISR) of the ribosomal RNA operon has been developed. The ISR of 30 F. columnare strains and other Flavobacterium species was amplified using universal primers and sequenced. Once F. columnare specific sequences within the ISR were recognized, specific PCR primers were designed against them (FCISRFL and FCISRR1). The primers were sensitive and able to detect as low as 7 colony forming units from pure culture by PCR. The new PCR detection method was applied to experimentally infected channel catfish. Two different experiments in which channel catfish fingerlings were infected by intramuscular injection or by immersion bath showed the advantage of the PCR method over standard culture techniques. F. columnare was detected by PCR in both tank water and catfish tissue samples with a higher frequency and in less time than standard microbiological methods. Furthermore, PCR detection confirmed that F. columnare can be transmitted horizontally indirectly through the water column without fish-to-fish contact. The newly developed PCR detection method for F. columnare was more sensitive and rapid than standard culture on bacteriological media for detection of F. columnare in channel catfish tissues and in tank water.

Analysis of Variance↗

PubMed2004

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.

Animals↗

PubMed2004

Immune response induced by N-lauroylsarcosine extracted outer-membrane proteins of an isolate of Edwardsiella ictaluri in channel catfish.

A virulent isolate of Edwardsiella ictaluri (AL-93-75), the causative agent of enteric septicaemia of catfish (ESC), was used to derive a lipopolysaccharide-reduced N-lauroylsarcosine outer-membrane protein (OMP) fraction vaccine. The OMP fraction was analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and compared to whole-cell lysate, purified lipopolysaccharide (LPS) and a crude cell-wall fraction. The OMP fraction contained less than 2% (W/V) LPS. SDS-PAGE showed that whole cell lysates contained 27 proteins from 107 to 14.3 kDa, whereas OMP contained nine proteins from 97 to 14.3 kDa, LPS contained two proteins at 45 and 37 kDa bands and a smear of bands below 14.3 kDa, and cell wall fraction contained 21 proteins from 97 to 8 kDa. Channel catfish, Ictalurus punctatus, were vaccinated with 12.5 microg/100 microl OMP and immunogenicity was confirmed by subsequent Western blots. Blots showed that 97, 80, and 19 kDa proteins were immunogenic. Rapid enzyme-linked immunosorbent assays (ELISA) demonstrated that OMP produced a weak, but observable antibody response by 21 days post injection. OMP concentrations of 3.13, 6.25, 12.5, 25, and 50 microg/100 microl total protein were tested for protective immunity. Marginal protection by relative percent survival (RPS) was only seen for fish injected with 12.5 microg/100 microl with RPSs between 55-67.5%. A booster dose of 12.5 microg/100 microl OMP did not significantly enhance protection.

Animals↗

PubMed2004

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.

Animals↗

PubMed2003

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.

Animals↗