rifampin and Fish-Diseases

Streptococcus iniae is a re-emerging bacterial pathogen in freshwater and marine aquaculture worldwide. There are no commercial vaccines available for S. iniae in the United States, and autogenous vaccines are restricted to inactivated whole-cell preparations with limited protection against heterogenous strains. Live-attenuated vaccines (LAV) represent an advantageous alternative to these bacterins, as they induce robust cellular and humoral immunity, and may provide longer lasting protection through less stressful routes of administration. We investigated whether accumulation of mutations in S. iniae by serial passage in the presence of rifampin can generate immunogenic LAV conferring protection against challenge with heterologous wild-type (WT) S. iniae strains in Nile tilapia (Oreochromis niloticus). Three lineages of rifampin-resistant S. iniae strains were generated from three genetically distinct parent strains (n = 9) by multiple passages in increments of Rifamycin SV sodium salt. Growth in liquid media, extent of capsulation, antimicrobial susceptibility, survival in Nile tilapia whole blood, and cytotoxicity in an O. mossambicus endothelial cell line were compared between the passaged and WT strains. Nile tilapia challenges were used to assess strain virulence, generation of anti-S. iniae IgM, and the protection conferred by LAV candidates against virulent S. iniae. Rifampin-resistant strains demonstrated changes in growth rate and cytotoxicity in endothelial cells, as well as significant reductions in whole blood survival (p < 0.05). Selected strains also showed attenuated virulence in the Nile tilapia challenge model, and anti-S. iniae IgM generated against these strains demonstrated cross-reactivity against heterologous bacteria. Immunization by intracoelomic injection induced protection against a virulent WT strain of S. iniae, with relative percent survival up to 95.05%.

Topics: Animals; Bacterial Vaccines; Cell Line; Cichlids; Endothelial Cells; Fish Diseases; Immunoglobulin M; Rifampin; Streptococcal Infections; Streptococcus iniae; Vaccines, Attenuated

We tested the efficiency of 2 different antibiotics, rifampicin and oxolinic acid, against an established infection caused by fish pathogen Francisella noatunensis ssp. orientalis (F.n.o.) in zebrafish. The drugs were tested in the free form as well as encapsulated into biodegradable nanoparticles, either polylactic-co-glycolic acid (PLGA) nanoparticles or nanostructured lipid carriers. The most promising therapies were PLGA-rifampicin nanoparticles and free oxolinic acid; the PLGA nanoparticles significantly delayed embryo mortality while free oxolinic acid prevented it. Encapsulation of rifampicin in both PLGA and nanostructured lipid carriers enhanced its efficiency against F.n.o. infection relative to the free drug. We propose that the zebrafish model is a robust, rapid system for initial testing of different treatments of bacterial diseases important for aquaculture.

Topics: Animals; Anti-Bacterial Agents; Fish Diseases; Francisella; Gram-Negative Bacterial Infections; Lactic Acid; Lipids; Nanoparticles; Oxolinic Acid; Polyglycolic Acid; Polylactic Acid-Polyglycolic Acid Copolymer; Rifampin; Zebrafish

Aeromonas hydrophila, as a strong Gram-negative bacterium, can infect a wide range of freshwater fish, including common carp Cyprinus carpio, and cause the huge economic loss. To create the effective vaccine is the best way to control the outbreak of the disease caused by A. hydrophila. In this study, a live attenuated A. hydrophila strain, XX1LA, was screened from the pathogenic A. hydrophila strain XX1 cultured on medium containing the antibiotic rifampicin, which was used as a live attenuated vaccine candidate. The immune protection of XX1LA against the pathogen A. hydrophila in common carp was evaluated by the relative percent survival (RPS), the specific IgM antibody titers, serum lysozyme activity and the expression profiles of multiple immune-related genes at the different time points following immunization. The results showed that the variable up-regulations of the immune-related genes, such as the pro-inflammatory cytokine IL-1β, the chemokine IL-10 and IgM, were observed in spleen and liver of common carp injected in the vaccines with the formalin-killed A. hydrophila (FKA) and the live attenuated XX1LA. Specific antibody to A. hydrophila was found to gradually increase during 28 days post-vaccination (dpv), and the RPS (83.7%) in fish vaccinated with XX1LA, was significant higher than that (37.2%) in fish vaccinated with FKA (P<0.05) on Day 28 after challenged by pathogen. It was demonstrated that the remarkable immune protection presented in the group vaccinated with XX1LA. During the late stage of 4-week immunization phase, compared with FKA and the control, specific IgM antibody titers significantly increased (P<0.05) in the XX1LA group. The activity of the lysozyme in serum indicated no significant change among three groups. In summary, the live attenuated bacterial vaccine XX1LA, screened in this study, indicates the better protect effect on common carp against A. hydrophila, which can be applied in aquaculture of common carp to prevent from the disease outbreak in the future.

Topics: Aeromonas hydrophila; Animals; Antibodies, Bacterial; Bacterial Vaccines; Carps; Fish Diseases; Gram-Negative Bacterial Infections; Immunoglobulin M; Interleukin-10; Interleukin-1beta; Rifampin; Vaccines, Attenuated

Flavobacterium psychrophilum is the etiologic agent of bacterial coldwater disease in salmonids. Earlier research showed that a rifampicin-passaged strain of F. psychrophilum (CSF 259-93B.17) caused no disease in rainbow trout (Oncorhynchus mykiss, Walbaum) while inducing a protective immune response against challenge with the virulent CSF 259-93 strain. We hypothesized that rifampicin passage leads to an accumulation of genomic mutations that, by chance, reduce virulence. To assess the pattern of phenotypic and genotypic changes associated with passage, we examined proteomic, LPS and single-nucleotide polymorphism (SNP) differences for two F. psychrophilum strains (CSF 259-93 and THC 02-90) that were passaged with and without rifampicin selection.. Rifampicin resistance was conveyed by expected mutations in rpoB, although affecting different DNA bases depending on the strain. One rifampicin-passaged CSF 259-93 strain (CR) was attenuated (4 % mortality) in challenged fish, but only accumulated eight nonsynonymous SNPs compared to the parent strain. A CSF 259-93 strain passaged without rifampicin (CN) accumulated five nonsynonymous SNPs and was partially attenuated (28 % mortality) compared to the parent strain (54.5 % mortality). In contrast, there were no significant change in fish mortalities among THC 02-90 wild-type and passaged strains, despite numerous SNPs accumulated during passage with (n = 174) and without rifampicin (n = 126). While only three missense SNPs were associated with attenuation, a Ser492Phe rpoB mutation in the CR strain may contribute to further attenuation. All strains except CR retained a gliding motility phenotype. Few proteomic differences were observed by 2D SDS-PAGE and there were no apparent changes in LPS between strains. Comparative methylome analysis of two strains (CR and TR) identified no shared methylation motifs for these two strains.. Multiple genomic changes arose during passage experiments with rifampicin selection pressure. Consistent with our hypothesis, unique strain-specific mutations were detected for the fully attenuated (CR), partially attenuated (CN) and another fully attenuated strain (B17).

Topics: Animals; Anti-Bacterial Agents; DNA-Directed RNA Polymerases; Drug Resistance, Bacterial; Fish Diseases; Flavobacteriaceae Infections; Flavobacterium; Lipopolysaccharides; Molecular Sequence Data; Oncorhynchus mykiss; Polymorphism, Single Nucleotide; Proteome; Rifampin; Selection, Genetic; Sequence Analysis, DNA; Serial Passage; Survival Analysis; Virulence

Flavobacterium psychrophilum is the aetiologic agent of bacterial coldwater disease and rainbow trout fry syndrome. In this study, we compared a wild-type strain (CSF 259-93) with a rifampicin-resistant strain and virulence-attenuated strain of F. psychrophilum (CSF 259-93B.17). The attenuated strain harboured a mutation in the rpoB gene consistent with resistance to rifampicin. Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and mass spectrometry demonstrated an altered proteome with eight proteins characteristic for the parent strain and six that were unique to the attenuated strain. Immunoblotting with a diagnostic monoclonal antibody (FL-43) identified a putative antigen (FP1493) that was subsequently cloned, expressed as a recombinant protein and confirmed as recognized by FL-43. 2D-PAGE, immunoblotting with rainbow trout, Oncorhynchus mykiss (Walbaum), convalescent antisera and mass spectrometry of bacterial whole-cell lysates revealed several uniquely expressed immunoreactive proteins including FP1493. An FP1493 recombinant subunit vaccine was tested, but did not provide protection against challenge with the CSF259-93 strain. While the exact mechanism responsible for altered protein synthesis and attenuation of CSF 259-93B.17 is still unknown, the differentially expressed immunoreactive proteins are a valuable resource to develop subunit vaccines and to identify proteins that are potentially involved in disease.

Topics: Animals; Antibodies, Bacterial; Bacterial Proteins; Bacterial Vaccines; Drug Resistance, Bacterial; Fish Diseases; Flavobacteriaceae Infections; Flavobacterium; Gene Expression Regulation, Bacterial; Immunization; Proteome; Rifampin; 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

Previous studies have demonstrated that passage of pathogenic bacteria on increasing concentrations of the antibiotic rifampicin leads to the attenuation of virulence and these resistant strains may serve as live attenuated vaccines. Two rifampicin resistant strains of Flavobacterium psychrophilum, 259-93A.16 and 259-93B.17, were generated by passage on TYES plates containing increasing concentrations of rifampicin. Electrophoretic analysis of whole-cell lysates prepared from the parent and resistant strains identified five differentially expressed proteins between the 259-93B.17 strain and parent strain, while there were no differences identified between the 259-93A.16 and parent strain. The LPS banding patterns were identical between all three strains. Bacterial challenges of rainbow trout (Oncorhynchus mykiss Walbaum) with the resistant strains demonstrated that the 259-93B.17 strain was highly attenuated and the 259-93A.16 strain was modestly attenuated at the challenge doses tested. Immunization of rainbow trout with the live attenuated 259-93B.17 strain by intraperitoneal injection resulted in significant protection against challenge with the virulent parent F. psychrophilum strain at 8 and 15 weeks post-immunization and fish exhibited elevated specific antibody titers. Importantly, immersion delivery of the 259-93B.17 strain stimulated protective immune responses in fish at 10 weeks post-immunization. The results demonstrate that the rifampicin resistant 259-93B.17 strain may serve as an effective live attenuated vaccine for the prevention of F. psychrophilum infections.

Topics: Animals; Anti-Bacterial Agents; Antibodies, Bacterial; Bacterial Vaccines; Drug Resistance, Bacterial; Fish Diseases; Flavobacteriaceae Infections; Flavobacterium; Immunization; Oncorhynchus mykiss; Rifampin; Vaccines, Attenuated; Virulence

The biological properties of Edwardsiella ictaluri RE-33 rifampicin-mutant and its parent strain EILO were analysed. RE-33 is an avirulent isolate used as a modified live vaccine against enteric septicaemia of catfish. Electrophoretic analysis of lipopolysaccharide (LPS) patterns showed high homology between both isolates. Further characterization of LPS by immunoblotting revealed the main differences in LPS composition. The RE-33 isolate lacks the high molecular weight bands of LPS (HMW-LPS). Outer membrane protein analysis also showed some immunological differences between RE-33 and the EILO parent strain. Only two fingerprinting techniques, fatty acid composition analysis and Biolog phenotypic profiles, were able to discriminate between both isolates.

Topics: Animals; Bacterial Outer Membrane Proteins; Drug Resistance, Bacterial; Edwardsiella ictaluri; Enterobacteriaceae Infections; Fatty Acids; Fish Diseases; Genome, Bacterial; Ictaluridae; Lipopolysaccharides; Mutation; Phenotype; Rifampin

A nested reverse transcriptase (RT) PCR assay detected mRNA of the salmonid pathogen Renibacterium salmoninarum in samples of RNA extracts of between 1 and 10 cells. Total RNA was extracted from cultured bacteria, Atlantic salmon (Salmo salar L.) kidney tissue and ovarian fluid seeded with the pathogen, and kidney tissue from both experimentally challenged and commercially raised fish. Following DNase treatment, extracted RNA was amplified by both RT PCR and PCR by using primers specific for the gene encoding the major protein antigen of R. salmoninarum. A 349-bp amplicon was detected by polyacrylamide gel electrophoresis and silver stain. Inactivation of cultured bacteria by rifampin or erythromycin produced a loss of nested RT PCR mRNA detection corresponding to a loss of bacterial cell viability determined from plate counts but no loss of DNA detection by PCR. In subclinically diseased fish, nested RT PCR identified similar levels of infected fish as determined by viable pathogen culture. Higher percentages of fish testing positive were generated by PCR, particularly in samples from fish previously subjected to antibiotic chemotherapy where 93% were PCR positive, but only 7% were nested RT PCR and culture positive. PCR can generate false-positive data from amplification of target DNA from nonviable pathogen cells. Therefore, nested RT PCR may prove useful for monitoring cultured Atlantic salmon for the presence of viable R. salmoninarum within a useful time frame, particularly samples from broodstock where antibiotic chemotherapy is used prior to spawning to reduce vertical pathogen transmission.

Topics: Animals; Anti-Bacterial Agents; Culture Media; DNA, Bacterial; Erythromycin; Female; Fish Diseases; Gram-Positive Bacterial Infections; Kidney; Micrococcaceae; Ovary; Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; Rifampin; RNA, Bacterial; RNA, Messenger; Salmo salar; Sensitivity and Specificity

In vitro studies have shown that rifampicin is an effective antibiotic for mycobacteria infections. Two species of tropical fish, the Firemouth Cichild Cichlasoma meeki and the Congo Tetra Phenacogrammus interruptus, were used to determine whether oral application of rifampicin might serve as an effective treatment for mycobacteriosis in tropical fish. Fish of the two species were infected with M. marinum under controlled conditions. Six or twelve weeks after infection, treatment was begun with medicated fish food containing rifampicin in combination with tetracyclin. Histological examination of epithelial cell granuloma in the anterior and posterior kidneys, as well as in liver and spleen, showed that antibiotic treatment could somewhat reduce the intensity of, but could not successfully eliminate infection. In addition, after treatment, acid-resistant rods could still be isolated from histological samples and M. marinum could be cultured from organ samples.

Topics: Administration, Oral; Animals; Anti-Bacterial Agents; Fish Diseases; Fishes; Mycobacterium; Mycobacterium Infections; Perches; Rifampin; Tropical Climate