Production, characterization and evaluation of virulence of an adhesion defective mutant of Flavobacterium columnare produced by beta-lactam selection.

AIMS: The aim of this study was to develop and evaluate mutants of Flavobacterium columnare, the causative agent of columnaris disease in fish. METHODS AND RESULTS: Serial passage on ampicillin (beta-lactam) enriched modified Hsu-Shotts medium resulted in a F. columnare mutant that differed from the parent strain in colony morphology, whole cell proteins, adhesion and virulence. The mutant differed from its parent in virulence during immersion challenge, but not during injection challenge or generation of antibodies. CONCLUSION: Flavobacterium columnare exposure to ampicillin produces both resistance to that antibiotic and produces mutants that lack or have reduced adhesion characteristics and modified ability to adhere to fish tissue. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first description of an adhesion-defective mutant of F. columnare and the effects of altered adhesion on columnaris disease. This mutant has considerable potential as a tool to study the role of adhesion in columnaris disease.

Comparative challenge model of Flavobacterium columnare using abraded and unabraded channel catfish, Ictalurus punctatus (Rafinesque).

The early entry of the fish pathogen Flavobacterium columnare and enhancement by abrasion was studied in channel catfish, Ictalurus punctatus (Rafinesque), using the polymerase chain reaction and a species-specific primer set for a bacterial 16S rRNA gene product. Evaluations were conducted following an abrasion bath immersion challenge with F. columnare. Abrasion, a practice which has historically been used prior to bacterial challenge, had significant effects on the early entry of the pathogen and on cumulative percent survival (CPS). The FvpF1-FvpR1 primer set was useful in detecting the early entry of F. columnare in mucus, skin, gill, blood, liver and trunk kidney tissues in both abraded and unabraded fish following immersion challenge at 29 +/- 2 degrees C. Bacteria were detected earlier in all tissues in abraded fish, except in the trunk kidney. These differences were not significant, except in the case of blood. Mucus, skin and gill tissues were positive for F. columnare earliest regardless of treatment (after 5 min in abraded fish and after 15 min in unabraded fish). CPS following challenge with F. columnare was significantly affected by abrasion, which supports the use of abrasion for the F. columnare challenge model for channel catfish.

Diethylstilbestrol regulates trophoblast stem cell differentiation as a ligand of orphan nuclear receptor ERR beta.

The orphan nuclear receptor ERR beta is expressed in undifferentiated trophoblast stem cell lines and extraembryonic ectoderm, and genetic ablation of ERR beta results in abnormal trophoblast proliferation and precocious differentiation toward the giant cell lineage. Here, we show that the synthetic estrogen diethylstilbestrol (DES) promotes coactivator release from ERR beta and inhibits its transcriptional activity. Strikingly, treatment of trophoblast stem cells with DES led to their differentiation toward the polyploid giant cell lineage. In addition, DES-treated pregnant mice exhibited abnormal early placenta development associated with an overabundance of trophoblast giant cells and an absence of diploid trophoblast. These results define a novel pathway for DES action and provide evidence for steroidlike control of trophoblast development.

Orphan nuclear receptor ROR alpha-deficient mice display the cerebellar defects of staggerer.

It has recently been shown that the neurological mutant mouse staggerer (sg) harbors a deletion within the Rora gene that encodes the orphan nuclear receptor ROR alpha. This deletion removes an exon encoding part of the ligand binding domain of the putative receptor, generating an ROR alpha truncated protein (ROR alpha(sg)). It is unknown whether sg acts as a null or highly hypomorphic allele. To address this question, we have generated a null mutation of Rora by targeted disruption of its DNA binding domain in ES cells. The Rora-/- mice are viable but display tremor, body imbalance, small size and die between 3-4 weeks, similar to the sg mouse. Histological examination of the cerebellum of Rora-/- and sg mice showed similar defects, including small size and fewer ectopically localized Purkinje cells. Northern blot analysis of cerebellar RNA showed that ROR alpha transcripts are still expressed in the Rora-/- and sg mutants, although with altered mobilities. However, the cerebellum of the Rora-/- mutant does not express the ROR alpha protein. Attempts to complement the defect of the Rora-/- with sg failed, demonstrating conclusively that the sg defects are caused by the absence of functional ROR alpha.

Occurrence of Loma cf. salmonae in brook, brown and rainbow trout from Buford trout hatchery, Georgia, USA.

During a 6 mo study of moribund trout from Buford hatchery, Buford, Georgia, USA, a Loma cf. salmonae microsporidian parasite was studied in the gills of brook trout Salvelinus fontinalis, brown trout Salmo trutta, and rainbow trout Oncorhynchus mykiss. This parasite was morphologically similar to L. salmonae and L. fontinalis but differed in spore size. Scanning and transmission electron microscopy demonstrated that xenomas were embedded in gill filaments. Transmission electron micrographs prepared from fresh tissue showed mature spores with 12 to 15 turns of their polar tube. Spore diameters for the Georgia strain from formalin-fixed gill tissues measured 3.5 (SD +/- 0.1) by 1.8 (SD +/- 0.1) microns. Electron micrographs of formalin-fixed, deparaffinized tissues of rainbow trout from Pennsylvania and West Virginia show spores with a diameter of 3.5 (+/- 0.2) by 1.7 (+/- 0.1) microns and 3.4 (+/- 0.2) by 1.8 (+/- 0.1) microns, respectively. Transmission electron micrographs of spores from Pennsylvania and West Virginia show that mature spores from both states had 13 to 15 turns of their polar tubes. Measurements from transmission electron micrographs prepared from alcohol-fixed tissues from Virginia fish contained spores with a diameter of 3.0 (+/- 0.3) by 1.1 (+/- 0.3) microns and 12 to 15 turns of their polar tubes. These measurements are consistent with L. salmonae and therefore suggest that the parasite is present on the east coast of the United States. During the height of the Georgia epizootic, the percentage of fish with observed xenomas reached 62.2% (N = 87), and the highest number of xenomas counted per 10 gill filaments was 133 (N = 87). The microsporidian epizootic occurred either during the autumn months or when intake river water quality reached combined iron-manganese concentrations as high as 1.01 (mean 0.44, SD +/- 0.42) mg-1.

Comparison of whole-cell antigens of pressure- and formalin-killed Flexibacter columnaris from channel catfish (Ictalurus punctatus).

OBJECTIVE: To identify and compare immunodominant antigens in whole-cell lysates of pressure- and formalin-killed Flexibacter columnaris. ANIMALS: Sera from naturally infected and vaccinated channel catfish. PROCEDURES: Whole-cell lysates of pressure- and formalin-killed F columnaris were compared, and antigens were isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The antigens were identified by staining, western blotting, and specific monoclonal antibodies to glycoproteins. Western blotting was performed, using sera from channel catfish (Ictalurus punctatus) with naturally acquired F columnaris infection and sera from channel catfish vaccinated with an experimental prototype F columnaris vaccine. RESULTS: Whole-cell lysates of pressure and formalin-killed F columnaris shared 4 proteins: 100, 80, 66, and 60 kd. The 60-kd antigen was a glycoprotein. Western blotting, using sera from naturally infected channel catfish, revealed the same proteins for pressure- and formalin-killed F columnaris. Sera from vaccinated fish reacted only to pressure-killed lysate antigens. CONCLUSIONS: Pressure- and formalin-killed F columnaris whole-cell lysates share 100-, 80-, 66-, and 60-kd proteins and are recognized by antibodies from naturally infected catfish and those vaccinated with formalin-killed F columnaris. Formalin treatment modifies or inactivates the 60-kd protein antigens, rendering them unrecognizable to antibodies from channel catfish naturally infected with F columnaris, suggesting that formalin-killed F columnaris may not be suitable for use as a bacterin against columnaris disease.

The orphan nuclear receptor estrogen-related receptor alpha is a transcriptional regulator of the human medium-chain acyl coenzyme A dehydrogenase gene.

Estrogen-related receptor alpha (ERR alpha) is an orphan member of the superfamily of nuclear hormone receptors. ERR alpha was initially isolated based on its sequence homology to the estrogen receptor but is not activated by classic estrogens. To identify possible physiologic functions for this orphan receptor, we cloned the mouse ERR alpha cDNA and used it to characterize the expression of ERR alpha transcripts and to identify potential ERR alpha target genes. RNA in situ hybridization studies detect ERR alpha transcripts in an organ-specific manner through mid- to late embryonic development, with persistent high-level expression in brown adipose tissue and intestinal mucosa. In the adult mouse, ERR alpha is most highly expressed in kidney, heart, and brown adipocytes, tissues which preferentially metabolize fatty acids. Binding site selection experiments show that ERR alpha preferentially binds to an ERR alpha response element (ERRE) containing a single consensus half-site, TNAAGGTCA. An ERRE is present in the 5'-flanking region of the gene encoding medium-chain acyl coenzyme A dehydrogenase (MCAD), a key enzyme involved in the mitochondrial beta-oxidation of fat. The MCAD nuclear receptor response element 1 (NRRE-1) interacts in vitro with ERR alpha expressed in COS-7 cells. Supershift experiments show that endogenous ERR alpha present in nuclear extracts obtained from a brown fat tumor cell line (HIB) interacts with NRRE-1. In the absence of its putative ligand, ERR alpha does not activate the MCAD promoter in transient transfection studies; however, a VP16-ERR alpha chimera activates natural and synthetic promoters containing NRRE-1. In addition, ERR alpha efficiently represses retinoic acid induction mediated by NRRE-1. These results demonstrate that ERR alpha can control the expression of MCAD through the NRRE-1 and thus may play an important role in regulating cellular energy balance in vivo.

Placental abnormalities in mouse embryos lacking the orphan nuclear receptor ERR-beta.

Classical endocrine studies have shown that steroid hormones are required for the maintenance of pregnancy and placental viability. The oestrogen-receptor-related receptor beta (ERR-beta) is an orphan member of the superfamily of nuclear hormone receptors. Although ERR-beta is homologous to the oestrogen receptor and binds the oestrogen response element, it is not activated by oestrogens. Expression of ERR-beta during embryogenesis defines a subset of extra-embryonic ectoderm that subsequently forms the dome of the chorion, suggesting that ERR-beta may be involved in early placental development. Homozygous mutant embryos generated by targeted disruption of the Estrrb gene have severely impaired placental formation, and die at 10.5 days post-coitum. The mutants display abnormal chorion development associated with an overabundance of trophoblast giant cells and a severe deficiency of diploid trophoblast. The phenotype can be rescued by aggregation of Estrrb mutant embryos with tetraploid wild-type cells, which contribute exclusively to extra-embryonic tissues. Our results indicate that ERR-beta has an important role in early placentation, and suggest that an inductive signal originating from or modified by the chorion is required for normal trophoblast proliferation and differentiation.

Compound mutants for retinoic acid receptor (RAR) beta and RAR alpha 1 reveal developmental functions for multiple RAR beta isoforms.

Mice with targeted disruptions in retinoic acid receptor genes have been generated to assess the role of nuclear receptors as transducers of the retinoid signal during vertebrate development. Mice with mutations that disrupt all isoforms of the RAR alpha, RAR beta and RAR gamma genes as well as for the individual RAR alpha 1, RAR beta 2 and RAR gamma 2 have been described. By breeding the RAR alpha 1 and RAR beta strains together we have generated double mutants which have striking phenotypes not discernible in mice homozygous for the individual mutations. Mice lacking both RAR alpha 1 and RAR beta died shortly after birth because of hypoxia, although individual RAR alpha 1 and RAR beta mutants were phenotypically normal. As previously observed in RAR compound mutants, histological examination of 18.5 dpc fetuses of RAR alpha 1 -/-beta-/- double mutants revealed a number of congenital malformations which in many respects were similar to those observed in fetuses of vitamin A-deficient mothers. The regions of congenital defects in RAR alpha 1 -/-beta-/- double mutants included the eye, the skull, the respiratory tract, the heart, the aortic arch-derived great vessels, and urogenital system. The penetrance of malformations in RAR alpha 1 -/-beta-/- mutants was greater than that in the reported RAR alpha 1 -/-beta 2-/- double mutants. Moreover, RAR alpha 1 -/-beta-/- mutants exhibited hypoplastic lungs and ossified fusion between basioccipital and exoccipital bones that were not reported in the RAR alpha 1 -/-beta2-/- animals, and displayed ectopic thymus and an unique defect in testis suggesting specific roles for RAR beta 1, 3 and/or 4 isoforms in these structures. The RAR alpha 1 single mutant animals as well as RAR alpha 1-/- beta-/- double mutant mice were susceptible to the teratogenic effects of RA, demonstrating that RAR alpha 1 and RAR beta isoforms singly or in combination do not play a major role in RA-induced craniofacial malformation and limb deformities.