Complete Genome Sequence of Pseudomonas aeruginosa Mucoid Strain FRD1, Isolated from a Cystic Fibrosis Patient.

We announce here the complete genome sequence of the Pseudomonas aeruginosa mucoid strain FRD1, isolated from the sputum of a cystic fibrosis patient. The complete genome of P. aeruginosa FRD1 is 6,712,339 bp. This genome will allow comparative genomics to be used to identify genes associated with virulence, especially those involved in chronic pulmonary infections.

Complete Genome Sequence of a Virulent Strain, Streptococcus iniae ISET0901, Isolated from Diseased Tilapia.

Streptococcus iniae ISET0901 is a virulent strain isolated in 2007 from diseased tilapia. Its full genome is 2,070,856 bp. The availability of this genome will allow comparative genomics to identify virulence genes important for the pathogenesis of streptococcosis caused by S. iniae, as well as possible immunogens for vaccine development.

Complete Genome Sequence of a Moderately Virulent Aeromonas hydrophila Strain, pc104A, Isolated from Soil of a Catfish Pond in West Alabama.

Aeromonas hydrophila pc104A is a moderately virulent strain isolated from the soil of a catfish pond in west Alabama in 2010. Its full genome is 5,023,829 bp. The availability of this genome will allow comparative genomics to identify the virulence genes that are important for pathogenesis or immunogens for the purpose of vaccine development.

Transcriptional analysis of four family 4 P450s in a Puerto Rico strain of Aedes aegypti (Diptera: Culicidae) compared with an Orlando strain and their possible functional roles in permethrin resistance.

A field strain of Aedes aegypti (L.) was collected from Puerto Rico in October 2008. Based on LD50 values by topical application, the Puerto Rico strain was 73-fold resistant to permethrin compared with a susceptible Orlando strain. In the presence of piperonyl butoxide, the resistance of Puerto Rico strain of Ae. aegypti was reduced to 15-fold, suggesting that cytochrome P450-mediated detoxification is involved in the resistance of the Puerto Rico strain to permethrin. To determine the cytochrome P450s that might play a role in the resistance to permethrin, the transcriptional levels of 164 cytochrome P450 genes in the Puerto Rico strain were compared with that in the Orlando strain. Of the 164 cytochrome P450s, 33 were significantly (P < 0.05) up-regulated, including cytochrome P450s in families four, six, and nine. Multiple studies have investigated the functionality of family six and nine cytochrome P450s, therefore, we focused on the up-regulated family 4 cytochrome P450s. To determine whether up-regulation of the four cytochrome P450s had any functional role in permethrin resistance, transgenic Drosophila melanogaster Meigen lines overexpressing the four family 4 P450 genes were generated, and their ability to survive exposure to permethrin was evaluated. When exposed to 5 microg per vial permethrin, transgenic D. melanogaster expressing CYP4D24, CYP4H29, CYP4J15v1, and CYP4H33 had a survival rate of 60.0 +/- 6.7, 29.0 +/- 4.4, 64.4 +/- 9.7, and 11.0 +/- 4.4%, respectively. However, none of the control flies survived the permethrin exposure at the same concentration. Similarly, none of the transgenic D. melanogaster expressing CYP4J15v1 or CYP4H33 ?5 survived when they were exposed to permethrin at 10 microg per vial. However, transgenic D. melanogaster expressing CYP4D24 and CYP4H29 had a survival rate of 37.8 +/- 4.4 and 2.2 +/- 2.2%, respectively. Taken together, our results suggest that CYP4D24 might play an important role in cytochrome P450-mediated resistance to permethrin.

Complete Genome of the Attenuated Sparfloxacin-Resistant Streptococcus agalactiae Strain 138spar.

Through the selection of resistance to sparfloxacin, an attenuated Streptococcus agalactiae strain, 138spar, was obtained from its virulent parent strain, S. agalactiae 138P. The full genome of S. agalactiae 138spar is 1,838,126 bp. This genome will allow comparative genomics to identify genes associated with virulence, antibiotic resistance, or other characteristics.

Complete Genome Sequence of the Attenuated Novobiocin-Resistant Streptococcus iniae Vaccine Strain ISNO.

Streptococcus iniae ISNO is an attenuated novobiocin-resistant vaccine strain. Its full genome is 2,070,182 bp in length. The availability of this genome will allow comparative genomics to identify potential virulence genes important for pathogenesis of S. iniae and potential mechanisms associated with novobiocin resistance in this strain.

Complete Genome Sequence of the Highly Virulent Aeromonas hydrophila AL09-71 Isolated from Diseased Channel Catfish in West Alabama.

Aeromonas hydrophila AL09-71 was isolated from diseased channel catfish in west Alabama during a 2009 disease outbreak. The full genome of A. hydrophila AL09-71 is 5,023,861 bp. The availability of this genome will allow comparative genomics to identify genes involved in pathogenesis or immunogens for the purpose of vaccine development.

Complete Genome Sequence of a Virulent Streptococcus agalactiae Strain, 138P, Isolated from Diseased Nile Tilapia.

Streptococcus agalactiae strain 138P was isolated from the kidney of diseased Nile tilapia in Idaho during a 2007 streptococcal disease outbreak. The full genome sequence of S. agalactiae 138P is 1,838,701 bp. The availability of this genome will allow comparative genomics analysis to identify genes for antigen discovery and vaccine development.

Vaccination of channel catfish with extracellular products of Aeromonas hydrophila provides protection against infection by the pathogen.

Aeromonas hydrophila, a Gram-negative bacterium, is one of the economically-important pathogens in modern aquaculture. Among various traits, extracellular products (ECP) secreted by the bacterium are considered to be essential factors for virulence. Whether vaccination with the ECP could produce immune protection in catfish against the pathogen was determined in this study. The results showed that fish vaccinated with ECP had 100% of relative percent survival (RPS) when challenged with the pathogen two weeks post vaccination. The anti-ECP serum from vaccinated fish could aggregate cells of homogeneous bacteria as well as other virulent strains (isolates) of A. hydrophila but not an A. veronii isolate and a low virulent field isolate. The agglutination titers increased from two weeks to four weeks post immunization and sustained a high level at week seven when the RPS remained at 100%. The anti-ECP serum could also provide naïve fish with immediate protection against A. hydrophila as evidenced by passive immunization. Immunoblotting analysis showed that the anti-ECP serum contained antibodies that bound to specific targets, including protein and lipopolysaccharide-like molecules, in the ECP. Mass spectrometric analysis identified following putative proteins that may serve as important immunogens: chitinase, chitodextrinase, outer membrane protein85, putative metalloprotease, extracellular lipase, hemolysin and elastase. Findings revealed in this study suggest that, while ECP prepared in a conventional and convenient way could be a vaccine candidate, further characterization of antibody-mediated targets in the ECP would uncover quintessential antigens for the future development of highly efficacious vaccines.

Comparative transcriptional analysis reveals distinct expression patterns of channel catfish genes after the first infection and re-infection with Aeromonas hydrophila.

To determine whether transcriptional levels of channel catfish (Ictalurus punctatus) genes are differentially regulated between a first infection with Aeromonas hydrophila and a re-infection, suppression subtractive hybridization (SSH) was performed in this study using anterior kidney cDNA after the re-infection as tester. Of the 96 clones isolated from the SSH library, 28 unique expressed sequence tags (ESTs) were obtained, of which eight were confirmed to be slightly but significantly (P < 0.05) more up-regulated by the re-infection at 6 h post infection (hpi). Expression kinetics studies at 3, 6, 12, 24, and 48 hpi revealed that the eight ESTs were significantly (P = 0.016) more up-regulated by the first infection, with a major peak at 3 hpi. A total of 96 genes reported in literature to be up-regulated by bacterial infections were selected and subjected to expression analysis at 3 hpi. Of the 96 selected genes, 19 were found to be significantly (P < 0.05) induced by A. hydrophila after the first infection and the re-infection. The 19 genes belonged to the following five main categories: 1) toll-like receptor (TLR2, TLR3, TLR5, TLR21); 2) antimicrobial peptide (NK-lysin type 1, NK-lysin type 2, NK-lysin type 3, cathepsin D, transferrin, hepcidin); 3) cytokine or chemokine (interleukin-1ß, interleukin-10, tumor necrosis factor α, chemokine CXCL-10); 4) signaling proteins (cadherin EGF LAG seven-pass G-type receptor 1, very large inducible GTPase 1, arginine deiminase type 2, lymphokine-activated killer T-cell originated protein kinase); 5) lysozyme (lysozyme c). Overall, the total 27 genes (8 ESTs plus the 19 selected genes) were significantly (P < 0.001) more induced by the first infection. Peaked expression of lysozyme c and serum lysozyme activity after the first infection were seen at 24 hpi, whereas that after the re-infection were seen at 12 hpi, suggesting that both innate and adaptive immunity were involved in the defense against the re-infection of A. hydrophila.

Recombinant goose-type lysozyme in channel catfish: lysozyme activity and efficacy as plasmid DNA immunostimulant against Aeromonas hydrophila infection.

The objectives of this study were: 1) to investigate whether recombinant channel catfish lysozyme-g (CC-Lys-g) produced in Escherichia coli expression system possesses any lysozyme activity; and 2) to evaluate whether channel catfish lysozyme-g plasmid DNA could be used as an immunostimulant to protect channel catfish against Aeromonas hydrophila infection. Recombinant CC-Lys-g produced in E. coli expression system exhibited significant (P < 0.05) lytic activity against Gram-positive Micrococcus lysodeikticus and Gram-negative A. hydrophila. When pcDNA3.2-vectored recombinant channel catfish lysozyme-g (pcDNA-Lys-g) was transfected in channel catfish gill cells G1B, the over-expression of pcDNA-Lys-g offered significant (P < 0.05) protection to G1B cells against A. hydrophila infection. When channel catfish were intraperitoneally injected with pcDNA-Lys-g along with an adjuvant QCDCR, the transcriptional level of Lys-g was significantly (P < 0.05) increased. When pcDNA-Lys-g injected fish was challenged with a highly virulent A. hydrophila strain AL-09-71, pcDNA-Lys-g offered 100% protection to channel catfish at two days post DNA injection. Macrophages of fish injected with pcDNA-Lys-g produced significantly (P < 0.05) higher amounts of reactive oxygen species and nitric oxide than that of fish injected with pcDNA vector alone at two days post DNA injection. Taken together, our results suggest that pcDNA-Lys-g could be used as a novel immunostimulant to offer immediate protection to channel catfish against A. hydrophila infection.

Apolipoprotein A1 in channel catfish: transcriptional analysis, antimicrobial activity, and efficacy as plasmid DNA immunostimulant against Aeromonas hydrophila infection.

The objectives of this study were to: 1) determine transcriptional profiles of apolipoprotein A1 (ApoA1) in collected channel catfish tissues after infection with Aeromonas hydrophila by bath immersion; 2) investigate whether recombinant channel catfish apolipoprotein A1 produced in Escherichia coli expression system possesses any antimicrobial activity against A. hydrophila; 3) evaulate whether recombinant channel catfish apolipoprotein A1 plasmid DNA could be used as immunostimulant to protect fish against A. hydrophila infection. Quantitative PCR revealed that the transcription levels of ApoA1 in infected catfish were significantly (P < 0.05) more induced in the anterior kidney. Recombinant apoA1 produced in E. coli expression system exhibited lytic activity against Gram-positive Micrococcus lysodeikticus and Gram-negative A. hydrophila. When pcDNA3.2-vectored recombinant apoA1 was transfected in channel catfish gill cells G1B, the over-expression of pcDNA-ApoA1 offered significant (P < 0.05) protection to G1B cells against A. hydrophila infection. When channel catfish were intraperitoneally injected with QCDCR adjuvant formulated pcDNA-ApoA1 and challenged with a highly virulent A. hydrophila strain AL-09-71 at two days post injection, pcDNA-ApoA1 injection offered 100% protection to channel catfish. Macrophages of fish injected with pcDNA-ApoA1 produced significantly (P < 0.05) higher amounts of reactive oxygen species and nitric oxide than that of fish injected with pcDNA vector alone. Our results suggest that pcDNA-ApoA1 could be used as a novel immunostimulant to offer immediate protection to catfish against A. hydrophila infection.

G-protein coupled receptor 18 (GPR18) in channel catfish: expression analysis and efficacy as immunostimulant against Aeromonas hydrophila infection.

The objectives of this study were: 1) to determine the transcriptional profiles of G-protein coupled receptor 18 (GPR18) in channel catfish after infection with Aeromonas hydrophila compared to that in healthy catfish; 2) to determine whether over-expression of GPR18 in catfish gill cells will offer protection against infection of A. hydrophila; 3) to determine whether recombinant pcDNA-GPR18 could be used as an immunostimulant to protect channel catfish against A. hydrophila infection. Quantitative PCR revealed that the transcription levels of GPR18 in all tissues of infected catfish were significantly (P < 0.05) induced except in the intestine. When pcDNA3.2-vectored recombinant GPR18 was transfected in catfish gill cells G1B, the over-expression of pcDNA-GPR18 offered significant (P < 0.05) protection to G1B cells against A. hydrophila infection. When channel catfish were intraperitoneally injected with QCDCR adjuvant formulated pcDNA-GPR18 and challenged with a highly virulent A. hydrophila strain at 1-, 2-, 14-, and 28-days post treatment, pcDNA-GPR18 offered 50%, 100%, 57%, and 55% protection to channel catfish, respectively. Macrophages of fish treated with pcDNA-GPR18 produced significantly (P < 0.05) higher amounts of reactive oxygen species and nitric oxide than that of fish treated with pcDNA vector alone. In addition, serum lysozyme activity of catfish injected with pcDNA-GPR18 was significantly (P < 0.08) increased. Taken together, our results suggest that pcDNA-GPR18 could be used as a novel immunostimulant to provide immediate protection to channel catfish against A. hydrophila infection.

Chicken-type lysozyme in channel catfish: expression analysis, lysozyme activity, and efficacy as immunostimulant against Aeromonas hydrophila infection.

To understand whether chicken-type lysozyme (Lys-c) in channel catfish was induced by infection of Aeromonas hydrophila, the transcriptional levels of Lys-c in skin, gut, liver, spleen, posterior kidney, and blood cells in healthy channel catfish was compared to that in channel catfish infected with A. hydrophila by bath immersion. Quantitative PCR revealed that the transcription levels of Lys-c in infected catfish were significantly (P < 0.05) induced in all five tissues tested as well as in blood cells. Recombinant CC-Lys-c produced in Escherichia coli expression system (R-CC-Lys-c) exhibited significant (P < 0.05) lytic activity to Gram-positive Micrococcus lysodeikticus and Gram-negative A. hydrophila. When pcDNA3.2-vectored recombinant channel catfish lysozyme-c (pcDNA-Lys-c) was transfected in channel catfish gill cells G1B, the over-expression of pcDNA-Lys-c offered significant (P < 0.05) protection to G1B against A. hydrophila infection. When channel catfish were intraperitoneally injected with QCDCR adjuvant formulated pcDNA-Lys-c and challenged with a highly virulent A. hydrophila strain AL-09-71 at 1-, 2-, 14-, and 28-days post treatment, pcDNA-Lys-c offered 75%, 100%, 60%, and 77% protection to channel catfish, respectively. Macrophages of fish treated with pcDNA-Lys-c produced significantly (P < 0.05) higher amounts of reactive oxygen species and nitric oxide than that of fish treated with pcDNA vector alone. Taken together, our results suggest that pcDNA-Lys-c could be used as a novel immunostimulant to protect channel catfish against A. hydrophila infection.

Expression and activity of recombinant proaerolysin derived from Aeromonas hydrophila cultured from diseased channel catfish.

Aerolysin is one of the putative toxins in extracellular products (ECP) produced by Aeromonas hydrophila, an important pathogen of catfish. To better understand the molecular mechanism and mode of action of this toxin, proaerolysin-coding gene was cloned from the genomic DNA of an A. hydrophila strain, cultured from diseased channel catfish, and heterologously expressed in E. coli. Functional recombinant proaerolysin was obtained, revealing some unique properties. The purified recombinant proaerolysin was inactive but could be activated by treatment with furin, trypsin, and ECP although different treatments produced different cleavage profiles and resulted in differential hemolytic and cytotoxic activities. The highest activity was observed from aerolysin processed by furin while treatment of proaerolysin with trypsin and ECP resulted in reduced activities. The unprocessed proaerolysin, though not hemolytic in vitro, had the same cytopathic effect on cultured walking catfish gill cells as the furin-processed had. In in vivo assay, the recombinant proaerolysin was found to be lethal to catfish when injected via intraperitoneal (IP) route. The lethal toxicity was acute and dose-dependent, as observed in IP injection of live A. hydrophila. This is the first recombinant proaerolysin confirmed to be a virulence factor; the recombinant protein could be used to further evaluate virulence, pathogenicity and antigenicity associated with A. hydrophila infection.

Development of live attenuated Streptococcus agalactiae as potential vaccines by selecting for resistance to sparfloxacin.

To develop attenuated bacteria as potential live vaccines, sparfloxacin was used in this study to modify 40 isolates of Streptococcus agalactiae. Majority of S. agalactiae used in this study were able to develop at least 80-fold resistance to sparfloxacin. When the virulence of the sparfloxacin-resistant S. agalactiae isolates were tested in 10-12g Nile tilapia by intraperitoneal injection at dose of 2×10(7)CFU/fish, 31 were found to be avirulent to fish. Of the 31 avirulent sparfloxacin-resistant S. agalactiae isolates, 30 provided 75-100% protection to 10-12g Nile tilapia against challenges with a virulent S. agalactiae isolate Sag 50. When the virulence of the 30 sparfloxacin-resistant S. agalactiae isolates was tested in 3-5g Nile tilapia by intraperitoneal injection at dose of 2×10(7)CFU/fish, six were found to be avirulent to 3-5g Nile tilapia. Of the six avirulent sparfloxacin-resistant S. agalactiae isolates, four provided 3-5g Nile tilapia 100% protection against challenges with homologous isolates, including Sag 97-spar isolate that was non-hemolytic. However, Sag 97-spar failed to provide broad cross-protection against challenges with heterologous isolates. When Nile tilapia was vaccinated with a polyvalent vaccine consisting of 30 sparfloxacin-resistant S. agalactiae isolates at dose of 2×10(6)CFU/fish, the polyvalent vaccine provided significant (P<0.001) protection to both 3-5g and 15-20g Nile tilapia against challenges with 30 parent isolates of S. agalactiae. Taken together, our results suggest that a polyvalent vaccine consisting of various strains of S. agalactiae might be essential to provide broader protection to Nile tilapia against infections caused by S. agalactiae.

Fitness cost, gyrB mutation, and absence of phosphotransferase system fructose specific IIABC component in novobiocin-resistant Streptococcus iniae vaccine strain ISNO.

To understand the fitness cost of novobiocin-resistance in an attenuated Streptococcus iniae vaccine strain ISNO compared to its virulent parent strain ISET0901, cell proliferation rate of the two strains were compared to each other. Our results revealed that the cell proliferation rates of ISNO were significantly (P<0.05) smaller than that of ISET0901. To understand whether there was any mutation at the target site of novobiocin, DNA gyrase subunit B (gyrB) was sequenced from both strains. Sequencing results revealed a point mutation of AGA to AGC, resulting in a deduced amino acid substitution of R635S. To determine whether any unique DNA sequence was present in ISET0901 but absent in ISNO, PCR-select bacterial genome subtractive hybridization was performed. A phosphotransferase system fructose specific IIABC component sequence was confirmed to be present in ISET0901 but absent in ISNO. Using genomic DNAs from ten field-strains of S. iniae as templates, the phosphotransferase system fructose specific IIABC component sequence was found to be present in five highly virulent strains, but absent in five avirulent strains. Taken together, our results suggest that: (1) As fitness cost of novobicin resistance, ISNO had significantly smaller cell proliferation rate; (2) point mutation at target site gyrB resulting in R635S substitution was associated with novobiocin resistance in ISNO; and (3) phosphotransferase system fructose specific IIABC component was associated with virulence of S. iniae.

Biochemical and molecular characterization of the novobiocin and rifampicin resistant Aeromonas hydrophila vaccine strain AL09-71N+R compared to its virulent parent strain AL09-71.

To understand the fitness cost of novobiocin- and rifampicin-resistance in an attenuated Aeromonas hydrophiila vaccine strain AL09-71 N+R compared to its virulent parent strain AL09-71, colony size, cell size, cell proliferation rate, chemotactic response, and the ability to invade catfish gill cells of the two strains were compared. Our results revealed that: (1) the cell size and the colony size of AL09-71 N+R was significantly (P<0.05) smaller than that of AL09-71; (2) the proliferation rate of AL09-71 N+R was significantly (P<0.05) slower than that of AL09-71; (3) AL09-71 N+R had a significantly (P<0.05) lower chemotactic response to catfish mucus than that of AL09-71; 4) the ability of AL09-71 N+R to invade catfish gill cells was significantly (P<0.05) lower than that of AL09-71. To understand whether target site mutation might play a role in antibiotic resistance, novobiocin's target site DNA gyrase subunit B gyrB and rifampicin's target site RNA polymerase subunit B rpoB were sequenced from the two strains. Our results revealed the following five mutations: (1) two missense mutations (CGC to ATC resulting in arginine/R to serine/S; TAC to TGC resulting in tyrosine/Y to cysteine/C) between AL09-71 gyrB and AL09-71 N+R gyrB; (2) three missense mutations (GAC to AAC resulting in aspartic acid/D to asparagine/N; CTG to CCG resulting in leucine/L to proline/P; CTG to CCG resulting in leucine/L to proline/P) between AL09-71 rpoB and AL09-71 N+R rpoB. To determine whether any unique DNA sequences were present in AL09-71 but absent in AL09-71 N+R, PCR-select bacterial genome subtractive hybridization was performed. Of 96 clones selected from the subtractive genomic DNA library, 32 sequences were found. None of the 32 sequences was confirmed to be present in AL09-71 but absent in AL09-71 N+R. At the transcription level, 29 of the 32 genes were found to be expressed greater than 10-fold in AL09-71 N+R compared to that in AL09-71.

Attempt to develop live attenuated bacterial vaccines by selecting resistance to gossypol, proflavine hemisulfate, novobiocin, or ciprofloxacin.

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.