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.

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.

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.

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.

Identification of mariner elements from house flies (Musca domestica) and German cockroaches (Blattella germanica).

Full-length mariner elements were isolated and sequenced from house flies (Musca domestica) and German cockroaches (Blattella germanica). The amino acid sequence of the house fly mariner element (accession number: AF373028) showed 99.5% identity with Mos1 and peach elements, whereas the German cockroach mariner element (accession number: AF355143) showed 98.8% and 99.8% identity, respectively. Sequence analysis revealed that the mariner elements in house flies and German cockroaches differed from the active Mos1 mariner element by seven and 15 nucleotides, respectively. Four essential nucleotide substitutions at positions 64, 154, 305, and 1203, which have been proposed to contribute to the loss of activity of the inactive elements, were detected in the German cockroach mariner element. In contrast, although the mariner element in house flies contained substitutions at positions 64, 154, and 305, it retained T at position 1203, identical to active mariner elements. Mariner is present in approximately eight copies in the German cockroach genome.

Ultrasound scanning may reduce the need for barium radiology in the assessment of small-bowel Crohn's disease.

AIM: Bowel wall thickening on transabdominal ultrasound scanning (USS) correlates well with Crohn's disease (CD) activity. However, the role of USS in the assessment of CD has not been fully defined. This study compared USS and barium follow-through (BaFT) in the assessment of CD. METHODS: A retrospective case series was undertaken of 24 children with CD who underwent USS and BaFT during the same period of clinical disease activity. RESULTS: For terminal ileum abnormality, all 13 cases with abnormal USS had abnormality on BaFT. USS did not detect five cases with affected terminal ileum and two cases with isolated proximal small-bowel CD. CONCLUSION: BaFT is a more sensitive indicator of small-bowel CD than USS. USS has good positive predictive value of distal small-bowel CD, but BaFT should be performed in the presence of a normal USS where there is strong clinical evidence of CD.

Extracorporeal life support for children with meningococcal septicaemia.

OBJECTIVE: To describe the short-term outcome of children with meningococcal sepsis treated with extracorporeal membrane oxygenation (ECMO) in a single centre. DESIGN: Retrospective analysis of case notes. SETTING: The Heartlink ECMO Centre, Glenfield Hospital, Leicester. PATIENTS: Eleven children (8 boys) out of a total caseload of 800 patients were treated for meningococcal sepsis with ECMO. INTERVENTIONS: Extracorporeal membrane oxygenation. RESULTS: All children with meningococcal sepsis treated with ECMO had a Glasgow Meningococcal Septicaemia Prognostic Score (GMSPS) > or = 12 (positive predictive risk of death of approximately 90%). Five children had adult respiratory distress syndrome (ARDS) and six had refractory shock with multi-organ dysfunction syndrome (MODS) at presentation for ECMO. All five children in the ARDS group survived, four of five receiving veno-venous (VV-) ECMO therapy. In contrast, only one of six children with refractory shock with MODS survived, all of whom required veno-arterial (VA-) ECMO therapy. CONCLUSIONS: Most children with meningococcal sepsis and severe ARDS can be successfully treated with VV-ECMO. In contrast, children with refractory shock and MODS die despite treatment with VA-ECMO. This report does not resolve whether ECMO therapy offers any advantage over conventional therapy in treating severe meningococcal sepsis.