Development of methods for the genetic manipulation of Flavobacterium columnare

<p>Abstract</p> <p>Background</p> <p><it>Flavobacterium columnare </it>is the causative agent of columnaris disease, a disease affecting many freshwater fish species. Methods for the genetic manipulation for some of the species within the <it>Bacteroidetes</it>, including members of the genus <it>Flavobacterium</it>, have been described, but these methods were not adapted to work with <it>F. columnare</it>.</p> <p>Results</p> <p>As a first step toward developing a robust set of genetic tools for <it>F. columnare</it>, a protocol was developed to introduce the <it>E. coli </it>– <it>Flavobacterium </it>shuttle vector pCP29 into <it>F. columnare </it>strain C#2 by conjugal mating at an efficiency of 1.5 × 10^-3antibiotic-resistant transconjugants per recipient cell. Eight of eleven <it>F. columnare </it>strains tested were able to receive pCP29 using the protocol. pCP29 contains the <it>cfxA </it>and <it>ermF </it>genes, conferring both cefoxitin and erythromycin resistance to recipient cells. Selection for pCP29 introduction into <it>F. columnare </it>was dependent on <it>cfxA</it>, as <it>ermF </it>was found not to provide strong resistance to erythromycin. This is in contrast to other <it>Flavobacterium </it>species where <it>ermF</it>-based erythromycin resistance is strong. The green fluorescent protein gene (<it>gfp</it>) was introduced into <it>F. columnare </it>strains under the control of two different native <it>Flavobacterium </it>promoters, demonstrating the potential of this reporter system for the study of gene expression. The transposon Tn<it>4351 </it>was successfully introduced into <it>F. columnare</it>, but the method was dependent on selecting for erythromycin resistance. To work, low concentrations of antibiotic (1 μg ml^-1) were used, and high levels of background growth occurred. These results demonstrate that Tn<it>4351 </it>functions in <it>F. columnare </it>but that it is not an effective mutagenesis tool due to its dependence on erythromycin selection. Attempts to generate mutants via homologous recombination met with limited success, suggesting that RecA dependent homologous recombination is rare in <it>F. columnare</it>.</p> <p>Conclusion</p> <p>The conjugation protocol developed as part of this study represents a significant first step towards the development of a robust set of genetic tools for the manipulation of <it>F. columnare</it>. The availability of this protocol will facilitate studies aimed at developing a deeper understanding of the virulence mechanisms of this important pathogen.</p

Development of Methods for the Genetic Manipulation of \u3cem\u3eFlavobacterium columnare\u3c/em\u3e

Background: Flavobacterium columnare is the causative agent of columnaris disease, a disease affecting many freshwater fish species. Methods for the genetic manipulation for some of the species within the Bacteroidetes, including members of the genus Flavobacterium, have been described, but these methods were not adapted to work with F. columnare. Results: As a first step toward developing a robust set of genetic tools for F. columnare, a protocol was developed to introduce the E. coli – Flavobacterium shuttle vector pCP29 into F. columnare strain C#2 by conjugal mating at an efficiency of 1.5 × 10-3 antibiotic-resistant transconjugants per recipient cell. Eight of eleven F. columnare strains tested were able to receive pCP29 using the protocol. pCP29 contains the cfxA and ermF genes, conferring both cefoxitin and erythromycin resistance to recipient cells. Selection for pCP29 introduction into F. columnare was dependent on cfxA, as ermF was found not to provide strong resistance to erythromycin. This is in contrast to other Flavobacterium species where ermF-based erythromycin resistance is strong. The green fluorescent protein gene (gfp) was introduced into F. columnare strains under the control of two different native Flavobacterium promoters, demonstrating the potential of this reporter system for the study of gene expression. The transposon Tn4351 was successfully introduced into F. columnare, but the method was dependent on selecting for erythromycin resistance. To work, low concentrations of antibiotic (1 μg ml-1) were used, and high levels of background growth occurred. These results demonstrate that Tn4351 functions in F. columnare but that it is not an effective mutagenesis tool due to its dependence on erythromycin selection. Attempts to generate mutants via homologous recombination met with limited success, suggesting that RecA dependent homologous recombination is rare in F. columnare. Conclusion: The conjugation protocol developed as part of this study represents a significant first step towards the development of a robust set of genetic tools for the manipulation of F. columnare. The availability of this protocol will facilitate studies aimed at developing a deeper understanding of the virulence mechanisms of this important pathogen

Surface rupture of multiple crustal faults in the 2016 Mw 7.8 Kaikōura, New Zealand, earthquake

Multiple (>20 >20 ) crustal faults ruptured to the ground surface and seafloor in the 14 November 2016 M w Mw 7.8 Kaikōura earthquake, and many have been documented in detail, providing an opportunity to understand the factors controlling multifault ruptures, including the role of the subduction interface. We present a summary of the surface ruptures, as well as previous knowledge including paleoseismic data, and use these data and a 3D geological model to calculate cumulative geological moment magnitudes (M G w MwG ) and seismic moments for comparison with those from geophysical datasets. The earthquake ruptured faults with a wide range of orientations, sense of movement, slip rates, and recurrence intervals, and crossed a tectonic domain boundary, the Hope fault. The maximum net surface displacement was ∼12  m ∼12  m on the Kekerengu and the Papatea faults, and average displacements for the major faults were 0.7–1.5 m south of the Hope fault, and 5.5–6.4 m to the north. M G w MwG using two different methods are M G w MwG 7.7 +0.3 −0.2 7.7−0.2+0.3 and the seismic moment is 33%–67% of geophysical datasets. However, these are minimum values and a best estimate M G w MwG incorporating probable larger slip at depth, a 20 km seismogenic depth, and likely listric geometry is M G w MwG 7.8±0.2 7.8±0.2 , suggests ≤32% ≤32% of the moment may be attributed to slip on the subduction interface and/or a midcrustal detachment. Likely factors contributing to multifault rupture in the Kaikōura earthquake include (1) the presence of the subduction interface, (2) physical linkages between faults, (3) rupture of geologically immature faults in the south, and (4) inherited geological structure. The estimated recurrence interval for the Kaikōura earthquake is ≥5,000–10,000  yrs ≥5,000–10,000  yrs , and so it is a relatively rare event. Nevertheless, these findings support the need for continued advances in seismic hazard modeling to ensure that they incorporate multifault ruptures that cross tectonic domain boundaries

The Use of Self-Inflating Hygroscopic Tissue Expanders to Facilitate Osteosarcoma Removal in a Massasauga Rattlesnake (Sistrurus catenatus)

A 0.34 kg adult female Massasauga rattlesnake (Sistrurus catenatus) was presented for evaluation of a subcutaneous mass affecting the ventral scales. The mass was diagnosed as a sarcoma via punch biopsy with no evidence of metastasis on diagnostic imaging. Surgical margins of 1-2 cm were planned to achieve complete excision of the neoplasm. A technique for tissue expansion was employed due to concerns regarding the ability to close the surgical site without excess tension or dehiscence. Two 27 mm diameter×5 mm hygroscopic self-inflating tissue expanders were placed subcutaneously under the lateral scales adjacent to the mass. Maximum skin expansion occurred over a four-week period, and no direct negative effects were noted. Excision of the primary mass was performed routinely five weeks after implant placement. Primary closure of the defect was achieved with minimal tension by incorporating the expanded skin. While the surgery was successful with no evidence of metastasis, the snake died of sepsis two weeks postoperatively. This is the first report of the use of self-inflating hygroscopic tissue expanders to help close a surgical defect in a reptile

Failure of translation of human adenovirus mRNA in murine cancer cells can be partially overcome by L4-100K expression in vitro and in vivo

Adaptive immune responses may be vital in the overall efficacy of oncolytic viruses in human malignancies. However, immune responses to oncolytic adenoviruses are poorly understood because these viruses lack activity in murine cells, which precludes evaluation in immunocompetent murine cancer models. We have evaluated human adenovirus activity in murine cells. We show that a panel of murine carcinoma cells, including CMT64, MOVCAR7, and MOSEC/ID8, can readily be infected with human adenovirus. These cells also support viral gene transcription, messenger RNA (mRNA) processing, and genome replication. However, there is a profound failure of adenovirus protein synthesis, especially late structural proteins, both in vitro and in vivo, with reduced loading of late mRNA onto ribosomes. Our data also show that in trans expression of the nonstructural late protein L4-100K increases both the amount of viral mRNA on ribosomes and the synthesis of late proteins, accompanied by reduced phosphorylation of eIF2α and improved anticancer efficacy. These results suggest that murine models that support human adenovirus replication could be generated, thus allowing evaluation of human adenoviruses in immunocompetent mice