novobiocin and Fish-Diseases

In an attempt to develop attenuated bacteria as potential live vaccines, four chemicals (gossypol, proflavine hemisulfate, novobiocin, and ciprofloxacin) were used to modify the following four genera of bacteria through chemical-resistance strategy: (1) Aeromonas hydrophila (9 isolates); (2) Edwardsiella tarda (9 isolates); (3) Streptococcus iniae (9 isolates); and (4) S. agalactiae (11 isolates). All bacteria used in this study were able to develop high resistance to gossypol. However, only some bacteria were able to develop resistance to proflavine hemisulfate, novobiocin, or ciprofloxacin. When the virulence of resistant bacteria was tested in tilapia or catfish, none of the gossypol-resistant isolate was attenuated, whereas majority of the proflavine hemisulfate-resistant isolates were attenuated. However, all proflavine hemisulfate-attenuated bacteria failed to provide significant protection to fish. Eight novobiocin- or ciprofloxacin-resistant Gram-positive bacteria (S. agalactiae and S. inaie) were found to be attenuated. However, none of them offered protection higher than 70%. Of seven attenuated novobiocin- or ciprofloxacin-resistant Gram-negative isolates (A. hydrophila and E. tarda), only one (novobiocin-resistant E. tarda 30305) was found to safe and highly efficacious. When E. tarda 30305-novo vaccinated Nile tilapia were challenged by its virulent E. tarda 30305, relative percent of survival of vaccinated fish at 14- and 28-days post vaccination (dpv) was 100% and 92%, respectively. Similarly, E. tarda 30305-novo offered 100% protection to channel catfish against challenges with virulent parent isolate E. tarda 30305 at both 14- and 28-dpv. Our results suggest that the development of live attenuated bacterial vaccines that are safe and efficacious is challenging, although it is feasible.

Topics: Aeromonas hydrophila; Animals; Bacterial Vaccines; Ciprofloxacin; Drug Resistance, Bacterial; Edwardsiella tarda; Fish Diseases; Gossypol; Ictaluridae; Novobiocin; Proflavine; Streptococcus; Tilapia; Vaccines, Attenuated; Virulence

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.

Topics: Aeromonas hydrophila; Animals; Anti-Bacterial Agents; Bacterial Proteins; Bacterial Vaccines; Blotting, Western; Catfishes; DNA Gyrase; DNA-Directed RNA Polymerases; Drug Resistance, Bacterial; Fish Diseases; Fructose-Bisphosphate Aldolase; Gram-Negative Bacterial Infections; Mutation; Novobiocin

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.. 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.. The novobiocin-resistant AH11NOVO is attenuated and different from its parent AH11P in pathogenicity.. 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.

Topics: Aeromonas hydrophila; Animals; Anti-Bacterial Agents; Bacterial Vaccines; Cells, Cultured; Chemotaxis; Fish Diseases; Gills; Ictaluridae; Novobiocin; Vaccination; Vaccines, Attenuated; Virulence

The efficacy of a novel attenuated Edwardsiella ictaluri vaccine (B-50348) was determined in channel catfish (Ictalurus punctatus) by bath immersion and intraperitoneal (IP) injection. The vaccine was developed from a virulent strain of E. ictaluri (AL93-58) through selection for novobiocin resistance. When channel catfish (average weight 10 g) were IP injected with 4.2 × 10⁶ colony-forming units (CFU) of the attenuated vaccine B-50348, no fish died. However, when the same age and size matched group of the catfish were IP injected with a lesser amount (2.4 × 10⁶ CFU/fish) of modified live RE-33 vaccine or the AL93-58 virulent strain (2.5 × 10⁶ CFU/fish) of E. ictaluri, 65% and 95% fish died, respectively. When channel catfish were challenged with AL93-58, relative percent survival values of vaccinated fish were all greater than 90% at 22, 32, and 63 days post B-50348 vaccination through intraperitoneal injection. By bath immersion, at 37 and 57 days post vaccination of B-50348, relative percent survival values were both 100% when fish were challenged by virulent E. ictaluri AL93-58. Our results suggest that B-50348 could be used as a novel safe and efficacious vaccine against ESC in channel catfish.

Topics: Animals; Bacterial Vaccines; Drug Resistance, Bacterial; Edwardsiella ictaluri; Enterobacteriaceae Infections; Enzyme-Linked Immunosorbent Assay; Fish Diseases; Ictaluridae; Novobiocin; Plasmids; Vaccines, Attenuated

A novel attenuated Streptococcus iniae vaccine was developed from a virulent strain of Streptococcus iniae (ISET0901) through selection for novobiocin resistance (named ISNO). The safety of ISNO was then evaluated in Nile tilapia (Oreochromis niloticus) through intraperitoneal (IP) injection. When male tilapia (average weight 10 g) were IP injected with 2×10(7) colony-forming units (CFU) of the attenuated S. iniae vaccine strain, no fish died. However, when the same age and size matched tilapia were IP injected with 2×10(7) and 1×10(5)CFU of the virulent parent strain of S. iniae, 100 and 90% fish died, respectively. Backpassage safety studies revealed that ISNO was unable to revert back to a virulent state. When IP vaccinated fish were challenged by the virulent ISET0901 strain of S. iniae, relative percent survival (RPS) values of vaccinated fish at 14, 28, 60, 90, and 180 days post ISNO vaccination (dpv) were 100, 100, 100, 89, and 75%, respectively, The RPS values of ISNO vaccinated fish (IP vaccination) against infections by five heterologous virulent strains of S. iniae (F3CB, 102 F1K, 405 F1K, IF6, and ARS60) at 60 dpv were 78, 90, 100, 100, and 100%, respectively. When tilapia were IP vaccinated by ISNO at dose of 1×10(2), 1×10(3), 1×10(4), 1×10(5), 1×10(6), and 1×10(7)CFU/fish, RPS values at 28 dpv were 81, 94, 100, 100, 100, and 100%, respectively. At 28 dpv, RPS of vaccinated fish by ISNO through bath immersion (1×10(7)CFU/ml) was 88%. ELISA results revealed that protection elicited by ISNO was due to antibody- as well as cell- mediated immunity. Our results suggest that ISNO could be used as a novel safe and efficacious vaccine to protect Nile tilapia from S. iniae infections.

Topics: Animals; Anti-Bacterial Agents; Bacterial Vaccines; Colony Count, Microbial; Drug Resistance, Bacterial; Fish Diseases; Male; Novobiocin; Streptococcal Infections; Streptococcus; Survival Analysis; Tilapia; Vaccination; Vaccines, Attenuated; Virulence

Three attenuated Aeromonas hydrophila vaccines were developed from the virulent 2009 West Alabama isolates through selection for resistance to both novobiocin and rifampicin. When channel catfish (Ictalurus punctatus) were IP injected with 4×105 colony-forming unit (CFU) of the mutants, no fish died. However, when the same age and size matched channel catfish were IP injected with similar amount of their virulent parents, 80-100% fish died. Similarly, when Nile tilapia (Oreochromis niloticus) were IP injected with 2×108 CFU of the mutants, no fish died. However, when Nile tilapia were IP injected with similar amount of the mutants, all fish died. Vaccination of channel catfish with the mutants at dose of 4×105 CFU/fish offered 86-100% protection against their virulent parents at 14 days post vaccination (dpv). Vaccination of Nile tilapia with the mutants at dose of 2×108 CFU/fish offered 100% protection against their virulent parents at 14, 28, and 56 dpv. Agglutination assay results suggested that protection elicited by the mutants was partially due to antibody-mediated immunity. Taken together, our results suggest that the three attenuated vaccines might be used to protect channel catfish and Nile tilapia against the highly virulent 2009 West Alabama isolates of A. hydrophila.

Topics: Aeromonas hydrophila; Alabama; Animals; Anti-Bacterial Agents; Antibodies, Bacterial; Bacterial Vaccines; Cichlids; Drug Resistance, Bacterial; Fish Diseases; Gram-Negative Bacterial Infections; Ictaluridae; Mutation; Novobiocin; Rifampin; Survival Analysis; Vaccines, Attenuated; Virulence

The susceptibility of six Vibrio harveyi strains to antibiotics was studied. Four strains originally isolated from diseased penaeids and two reference strains originally isolated from either sea water (ATCC 25919) or diseased Talorchestia sp. (ATCC 14126) were used in the present study. Results revealed that all three strains isolated in Taiwan exhibited resistance against nitrofurantoin, novobiocin and sulphonamide. The two reference strains and the strain isolated in Indonesia were susceptible to these three antibiotics.

Topics: Animals; Anti-Bacterial Agents; Chloramphenicol; Ciprofloxacin; Decapoda; Doxycycline; Drug Resistance, Microbial; Erythromycin; Fish Diseases; Fishes; Microbial Sensitivity Tests; Nalidixic Acid; Nitrofurantoin; Novobiocin; Oxolinic Acid; Oxytetracycline; Penicillins; Seawater; Streptomycin; Sulfonamides; Vancomycin; Vibrio; Vibrio Infections; Water Microbiology