Identification of gyrB and rpoB gene mutations and differentially expressed proteins between a novobiocin-resistant Aeromonas hydrophila catfish vaccine strain and its virulent parent strain.

A total of 10 and 13 missense mutations were found in the deduced gyrB and rpoB proteins, respectively, between avirulent AH11NOVO vaccine strain and its virulent parent strain AH11P. SDS-PAGE revealed that six proteins bands were significantly over-expressed in AH11NOVO whereas five bands were significantly over-expressed in AH11P. Mass spectrometry identified seven proteins from the over-expressed AH11NOVO gel bands and five proteins from the over-expressed AH11P gel bands. QPCR confirmed that all 12 genes corresponding to the proteins identified by mass spectrometry were significantly over-expressed in AH11NOVO or AH11P. When AH11NOVO proteins were subjected to Western blot analysis, 13 protein bands exhibited significantly stronger reactivity with hyper-immune catfish sera. Fifteen proteins were identified from immunogenic protein bands, including six (formate acetyltransferase, chaperone htpG, transketolase, ATP synthase subunit alpha, asparagine-tRNA ligase, and serine hydroxymethyltransferase) that were over-expressed in AH11NOVO proteins and three (elongation factor G, class II fructose-bisphosphate aldolase, and a putative uncharacterized 23 kDa protein) that were over-expressed in AH11P. In addition, the following six proteins were also identified from the immunogenic protein bands: pyruvate dehydrogenase E1 component, ATP synthase subunit beta, ribose-phosphate pyrophosphokinase, glyceraldehyde-3-phosphate dehydrogenase, 50S ribosomal L10, and 50S ribosomal L15. Our results might provide insights on how to develop novel efficacious vaccine against Aeromonas hydrophila infection.

Identification and virulence of Chryseobacterium indologenes isolated from diseased yellow perch (Perca flavescens).

AIM: To identify pathogen of diseased yellow perch and determine their virulence. METHODS AND RESULTS: Fifteen Gram-negative bacterial isolates were recovered from the skin lesions of diseased yellow perch (Perca flavescens). Based on API 20NE test, ten isolates were found to share 67.2-99.9% homologies with Chryseobactertium indologenes. Based on fatty acid methyl ester analysis, 13 isolates were found to share similarities with C. indologenes and other species of Chryseobacterium. Based on sequencing results of partial 16S rRNA gene, 13 isolates shared 99% identities (e value = 2e-50) with the 16S rRNA sequence of C. indologenes (GenBank HQ259684). Based on the 16S-23S rRNA intergenic spacer region (ISR) sequence, the 13 isolates shared 88% identity (e value = 1e-165) with the 16S-23S ISR sequence of C. indologenes (GenBank EU014570). T-coffee multiple sequence alignment revealed that the partial 16S rRNA or the 16S-23S ISR sequence of the 13 isolates shared 100% identity with each other. When healthy yellow perch were exposed to the 15 isolates by bath immersion (c. 6 × 10(7) CFU ml(-1) for 1 h), only C. indologenes isolates killed 10-20% of fish, whereas other isolates were avirulent. When yellow perch were exposed to C. indologenes by intraperitoneal injection, mortality was dose dependent, with LD(50) and LD(95) values of 1.5 × 10(8) and 3.2 × 10(8) CFU per fish, respectively. CONCLUSIONS: Chryseobactertium indologenes could be pathogenic to yellow perch. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report on the isolation of C. indologenes from diseased yellow perch. Virulence studies suggested that C. indologenes could become pathogenic to yellow perch.

Reproducible challenge model to investigate the virulence of Flavobacterium columnare genomovars in rainbow trout Oncorhynchus mykiss.

Flavobacterium columnare is a Gram-negative bacterium that causes columnaris disease and has significant economic impacts on aquaculture production worldwide. Molecular analyses have demonstrated that there is genetic diversity among F. columnare isolates. A review of the published literature that used restriction fragment length polymorphism analysis of the 16S rRNA gene revealed that all isolates typed from salmonids were Genomovar I. Our objective was to develop a laboratory challenge model for F. columnare in rainbow trout Oncorhynchus mykiss (Walbaum) and use the model to determine the virulence of Genomovar I and II isolates. Six F. columnare isolates were obtained from rainbow trout experiencing losses due to columnaris disease and were determined to be Genomovar I. Three of these were chosen for a preliminary assessment of virulence, and isolate 051-10-S5 was chosen for additional experiments to determine the reproducibility of the waterborne challenge model. In 2 independent experiments, cumulative percent mortalities (CPM) were 49 ± 10% and 50 ± 19%. Challenge of rainbow trout with Genomovar I and II isolates demonstrated a difference in the CPM, with the Genomovar II isolates inducing significantly higher CPM. This reproducible waterborne challenge model for columnaris disease in rainbow trout will be useful to investigate host-pathogen interactions, vaccine development, and other potential control strategies. This research also provides a basis for further defining the molecular diversity and virulence associated with F. columnare genomovars in rainbow trout and other salmonid species.

Susceptibility of three strains of blue catfish, Ictalurus furcatus (Valenciennes), to Ichthyophthirius multifiliis.

This study compared the susceptibility of three blue catfish strains (D&B, USDA 101 and USDA 102) to the parasite Ichthyophthirius multifiliis (Ich). In Trial I, a cohabitation study (all strains stocked communally) was conducted and fish were exposed to theronts at 0, 200, 1000, 5000 or 25 000 theronts fish(-1), respectively. All fish died when exposed to theronts at 5000 or 25 000 theronts fish(-1). When exposed to 1000 theronts fish(-1), USDA 102 strain of blue catfish showed significantly lower mortality (78.5%) compared to USDA 101 and D&B strains (92.7% and 100%). In Trial II, the same three strains of blue fish were evaluated for their susceptibility to Ich with strains challenged in separate tanks by adding Ich theronts at 0, 200 and 1000 theronts fish(-1), respectively. All D&B and USDA 101 blue catfish died; however, 42.3% of USDA 102 strain survived the infection when exposed to 1000 theronts per fish. The results indicate that there are differences among strains of blue catfish for susceptibility to Ich, and these differences will be useful in the development of improved catfish germplasm for commercial aquaculture.

Attenuation of a virulent Aeromonas hydrophila with novobiocin and pathogenic characterization of the novobiocin-resistant strain.

AIM: To determine whether novobiocin resistance strategy could be used to attenuate a virulent Aeromonas hydrophila AH11P strain and to characterize the growth and pathogenic differences between the novobiocin-resistant strain and its virulent parent strain AH11P. METHODS AND RESULTS: A novobiocin-resistant strain AH11NOVO was obtained from a virulent Aer. hydrophila strain AH11P through selection of resistance to novobiocin. AH11NOVO was found to be avirulent to channel catfish (Ictalurus punctatus), whereas AH11P was virulent. When AH11NOVO vaccinated channel catfish were challenged with AH11P at 14 days postvaccination, relative per cent of survival of vaccinated fish was 100%. The cell proliferation rate of AH11NOVO was found to be significantly (P < 0.05) less than that of AH11P. In vitro motility assay revealed that AH11NOVO was nonmotile, whereas AH11P was motile. AH11NOVO had significantly (P < 0.05) lower in vitro chemotactic response to catfish mucus than that of AH11P. Although the ability of AH11NOVO to attach catfish gill cells was similar to that of AH11P, the ability of AH11NOVO to invade catfish gill cells was significantly (P < 0.05) lower than that of AH11P. CONCLUSIONS: The novobiocin-resistant AH11NOVO is attenuated and different from its parent AH11P in pathogenicity. SIGNIFICANCE AND IMPACT OF THE STUDY: The significantly lower chemotactic response and invasion ability of AH11NOVO compared with that of its virulent parent strain AH11P might shed light on the pathogenesis of Aer. hydrophila.

Expression profiles of seven channel catfish antimicrobial peptides in response to Edwardsiella ictaluri infection.

Using quantitative polymerase chain reaction (QPCR), the relative transcriptional levels of seven channel catfish antimicrobial peptide (AMP) genes (NK-lysin type 1, NK-lysin type 2, NK-lysin type 3, bactericidal permeability-increasing protein, cathepsin D, hepcidin and liver-expressed AMP 2) in response to Edwardsiella ictaluri infection were determined. None of the AMP genes tested was significantly upregulated at 2 h post-infection. Hepcidin was the only one that was significantly (P<0.05) upregulated at 4, 6 and 12 h post-infection. At 24 and 48 h post-infection, four AMPs (hepcidin, NK-lysin type 1, NK-lysin type 3 and cathepsin D) were significantly (P<0.05) upregulated. Among all the AMPs that were significantly upregulated at different time points, hepcidin at 4, 6 and 12 h post-infection was upregulated the most. When catfish were injected with different doses of E. ictaluri, all lethal doses were able to induce significant (P <0.05) upregulation of hepcidin in the posterior kidney, whereas sublethal doses failed to induce any significant upregulation of hepcidin. In vitro growth studies revealed that the presence of synthetic hepcidin peptide at a concentration of 16 µm or higher significantly inhibited the cell proliferation of E. ictaluri. Taken together, our results suggest that hepcidin might play an important role in the channel catfish defence against E. ictaluri infection.

Effect of short-term feeding duration of diets containing commercial whole-cell yeast or yeast subcomponents on immune function and disease resistance in channel catfish, Ictalurus punctatus.

Juvenile channel catfish, Ictalurus punctatus, were fed diets supplemented with yeast or yeast subcomponents (YYS) as commercial preparations of ß-glucan (MacroGard(®) and Betagard A(®)), mannan oligosaccharide (Bio-Mos(®) Aqua Grade), or whole-cell Saccharomyces cerevisiae (Levucell SB20(®)) at the manufacturers' recommended levels. Fish were fed experimental diets for 1 or 2 weeks prior to disease challenge (pre-challenge feeding periods) and sampled at the end of each feeding period to measure haematological and immune parameters and to determine the effects of dietary YYS on resistance to Edwardsiella ictaluri, the causative agent of enteric septicaemia disease (ESC). Feeding of experimental diets continued for 3 weeks post-challenge. In channel catfish fed diets supplemented with MacroGard(®), Betagard A(®), or Levucell SB20(®), survival in the 1 week pre-challenge feeding group and antibody titres in the 2 week feeding group were significantly higher post-E. ictaluri challenge in relation to catfish fed with the control diet. In fish fed these same three diets, survival to ESC was significantly higher after 1 week vs. 2 weeks feeding, while the antibody response was significantly higher after 2 weeks vs. 1 week. Lysozyme activity was also higher in the 1 week feeding group, but the increased activity was unrelated to diet. Feeding YYS-supplemented diets for a shorter duration of 1 week prior to challenge may prove beneficial in increasing resistance to ESC in channel catfish. However, we cannot discount that feeding YYS diets during the recovery period may have contributed to 'glucan overload' and reduced survival in the 2 week feeding group.

Evaluation of an in vitro cell assay to select attenuated bacterial mutants of Aeromonas hydrophila and Edwardsiella tarda to channel catfish.

AIMS: To evaluate the feasibility of using an in vitro cell assay to select attenuated bacterial mutants. METHODS AND RESULTS: Using catfish gill cells G1B, the feasibility of using an in vitro assay instead of in vivo virulence assay using live fish to select attenuated bacterial mutants was evaluated in this study. Pearson correlation analysis between in vitro virulence to G1B cells and in vivo virulence of Aeromonas hydrophila and Edwardsiella tarda revealed that there was a significant correlation between the two (r = -0.768, P value = 3.7 × 10(-16)). CONCLUSIONS: The in vitro cell assay might be initially used to screen large quantities of bacteria to select attenuated mutants of catfish pathogens. SIGNIFICANCE AND IMPACT OF THE STUDY: The in vitro cell assay using catfish gill cells to identify attenuated mutants of catfish pathogens will reduce cost involved in the in vivo virulence assay that requires many fish and aquariums.

Temperature effects on immune response and hematological parameters of channel catfish Ictalurus punctatus vaccinated with live theronts of Ichthyophthirius multifiliis.

This study evaluated the influence of temperature on the immune responses and hematological parameters in channel catfish Ictalurus punctatus immunized via intraperitoneal injection with live theronts of Ichthyophthirius multifiliis. Fish were distributed in 18 aquaria and received 9 treatments: 4 groups of fish were vaccinated with live theronts and maintained at constant temperature 15 °C, 20 °C, 25 °C and 30 °C; 3 groups of fish vaccinated and subjected to cycling temperature regime from 15-25 °C, 20-25 °C and 20-30 °C, changed 5 °C each day; 2 groups of fish were not vaccinated and served as controls at 25 °C, one with Ich challenge and the other without challenge. Non vaccinated fish and those vaccinated at 15 °C or 15-25 °C did not show anti-Ich antibodies in the serum 14 and 21 days post-immunization. The antibody levels were significantly higher from fish vaccinated at 25 °C, 30 °C, 20-25 °C and 20-30 °C compared to fish at 15 °C, 20 °C and 15-25 °C both 14 and 21 days post-immunization. At constant water temperature, fish vaccinated at 15 °C showed significantly higher mortality rate (67.8%, P < 0.05) than those vaccinated at 20 °C, 25 °C, and 30 °C (0-10.7% mortalities). At cycling water temperature, fish vaccinated at 15-25 °C showed significantly higher mortality rate (67.8%) than those vaccinated at 20-25 °C and 20-30 °C (P < 0.05). Twenty days after immunization fish vaccinated at 30 °C and 20-30 °C showed significant increase in the red blood cells, white blood cells, thrombocytes and monocytes. Six days after challenge with I. multifiliis theronts the fish showed decreased white blood cells, thrombocytes and monocytes. This study suggests that vaccinated catfish were severely impacted by low temperature, either at 15 °C constant temperature or at 15-25 °C cycling temperature. The fish showed no anti-Ich antibodies and suffered high mortality similar to non vaccinated control fish.

An in vitro screening method to evaluate chemicals as potential chemotherapeutants to control Aeromonas hydrophila infection in channel catfish.

AIMS: To develop an in vitro screening method to be used for identifying potential effective chemotherapeutants to control Aeromonas hydrophila infections. METHODS AND RESULTS: Using catfish gill cells G1B and four chemicals (hydrogen peroxide, sodium chloride, potassium permanganate and D-mannose), the feasibility of using an in vitro screening method to identify potential effective chemotherapeutants was evaluated in this study. In vitro screening results revealed that, at concentration of 100 mg l⁻¹, H2O2 was the only chemical tested that was able to completely abolish the attachment and invasion of Aer. hydrophila to catfish gill cells. In vivo virulence studies using live channel catfish through bath immersion confirmed that H2O2 was the only chemical tested that was able to significantly (P < 0·001) reduce the mortality (from 90 or 100% to 0 or 20%) caused by Aer. hydrophila infections. CONCLUSIONS: The in vitro screening method using catfish gill cells G1B could be used to initially identify potential effective chemotherapeutants to control Aer. hydrophila. SIGNIFICANCE AND IMPACT OF THE STUDY: An in vitro screening method using catfish gill cells to identify potential effective chemotherapeutants described here will cut cost in research compared with the method of using live fish to screen lead compounds for fish disease control.

Expression profiles of toll-like receptors in anterior kidney of channel catfish, Ictalurus punctatus (Rafinesque), acutely infected by Edwardsiella ictaluri.

Using quantitative PCR (QPCR), the relative transcriptional levels of five toll-like receptors (TLR2, TLR3, TLR5, TLR20a and TLR21) were studied in the channel catfish, Ictalurus punctatus (Rafinesque), under uninfected and acutely infected conditions [1-, 2-, 4-, 6-, 12-, 24-, 36- and 48-h post-injection (hpi)]. Under uninfected conditions, the transcriptional levels of the five TLRs were significantly lower than that of 18S rRNA (P < 0.001). QPCR results also revealed that the transcriptional levels of TLR20a and TLR5 were higher than those of TLR2, TLR3 or TLR21. The transcriptional level of TLR3 was significantly lower than that of the other four TLRs (P < 0.001). However, when channel catfish were acutely infected by Edwardsiella ictaluri through intraperitoneal injection, the transcriptional levels of TLRs increased significantly (P < 0.005) at 6 hpi. Among the five TLRs studied, the transcriptional levels of TLR3, TLR5 and TLR21 were never significantly lower than under uninfected conditions (P = 0.16, 0.27 and 0.19, respectively), suggesting these three TLRs might play important roles in host defence against infection by E. ictaluri. The amount of E. ictaluri in the anterior kidney increased at 12 and 24 hpi but decreased at 36 and 48 hpi. Our results suggest that TLRs are important components in the immune system in the channel catfish, and their rapid transcriptional upregulation (within 6 hpi) in response to acute E. ictaluri infection might be important for survival from enteric septicaemia of catfish.

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.

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.

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.

The macrophage chemotactic activity of Edwardsiella tarda extracellular products.

The chemoattractant capabilities of Edwardsiella tarda extracellular products (ECP) were investigated from two isolates, the virulent FL6-60 parent and less virulent RET-04 mutant. Chemotaxis and chemokinesis were assayed in vitro using blind well chambers with peritoneal macrophages obtained from Nile tilapia, Oreochromis niloticus, 5 days following squalene injection. Non-purified ECP derived from both isolates stimulated predominantly chemokinetic migration of macrophages. Additionally, the ECP were semi-purified by high pressure liquid chromatography. The FL6-60 parent ECP yielded higher molecular weight components than did the ECP from the RET-04 mutant. The chemotactic activity of the macrophages for both the FL6-60 parent and RET-04 mutant semi-purified ECP was increased over the non-purified ECP and overall migration was primarily chemotactic. Exposure to ECP derived from virulent and less virulent E. tarda isolates promoted chemokinetic movement of macrophages that may be involved in inflammatory responses of Nile tilapia to E. tarda infection.

Flavobacterium columnare genomovar influences mortality in channel catfish (Ictalurus punctatus).

Flavobacterium columnare, causal agent of columnaris disease, is pathogenic to many species of freshwater fish throughout the world. The United States channel catfish (Ictalurus punctatus) aquaculture industry is severely impacted by columnaris disease. The majority of the F. columnare isolates recovered from diseased channel catfish belonged to either genomovars I or II. The objective of the present study was to determine if differences existed in the ability of these genomovars to induce mortality in channel catfish. Single strand conformation polymorphism analysis (SSCP) was used to ascribe the isolates used in this study to the appropriate genomovar. Immersion challenge experiments (15min immersion exposure to approximately 5x10(5) to 1x10(6) CFU/mL) were carried out to assess virulence of genomovar I and II isolates to channel catfish. The results demonstrated that genomovar II (n=4) isolates were significantly (P<0.05) more virulent to channel catfish fry (92-100% mortality) than genomovar I (n=3) isolates (0-46% mortality). In vivo adhesion of the genetically characterized F. columnare also correlated (r2=0.73) to increased mortality in the challenged fry. In fingerling channel catfish, significantly higher mortality (P<0.05) resulted with genomovar II isolates ALM-05-182 and ALG-00-530 as compared to all the genomovar I isolates (n=3). Mortality of genomovar II isolate BGFS-27 with similar to genomovar II isolate (ALG-00-530) and two genomovar I isolates (ALM-05-53 and 140). The results suggest that although both genomovars are present in the aquatic environment, genomovar II appears to be more pathogenic for channel catfish.

Passive immunization of channel catfish Ictalurus punctatus with anti-Flavobacterium columnare sera.

Passive immunization of channel catfish Ictalurus punctatus (Rafinesque) was conducted to determine if anti-Flavobacterium columnare serum was protective when injected intraperitoneally (i.p.) into channel catfish. The anti-F. columnare serum was produced by actively immunizing (i.p. injection) channel catfish with sonicated whole cells or purified lipopolysaccharide (LPS) of F. columnare in Freund's adjuvant. Serum anti-F. columnare activity was verified by Western blotting and ELISA of serum. Normal serum and sterile culture broth were used as controls. Complement was inactivated in all sera by heating. After 48 h, passively immunized fish were challenged with virulent F. columnare by i.p. injection. A group of unchallenged fish served as controls. The immune response of catfish to the antigenic fractions was different when examined by Western blotting. Antibody produced with whole-cell antigen responded to a broad range of molecular weight components, while LPS antigens were restricted to a pair of bands near 20 kDa. Control fish injected with culture medium experienced 100% mortality 14 d post-challenge. Relative percent survival was 77 and 73 for catfish passively immunized with anti-LPS and anti-whole-cell serum, respectively. Results suggest that antibodies in the serum are involved in the protective immune response against columnaris disease in channel catfish.