Additional evidence that juvenile oyster disease is caused by a member of the Roseobacter group and colonization of nonaffected animals by Stappia stellulata-like strains.

Juvenile oyster disease (JOD) causes significant annual mortalities of hatchery-produced Eastern oysters, Crassostrea virginica, cultured in the Northeast. We have reported that a novel species of the alpha-proteobacteria Roseobacter group (designated CVSP) was numerically dominant in JOD-affected animals sampled during the 1997 epizootic on the Damariscotta River, Maine. In this study we report the isolation of CVSP bacteria from JOD-affected oysters during three separate epizootics in 1998. These bacteria were not detected in nonaffected oysters at the enzootic site, nor in animals raised at a JOD-free site. Animals raised at the JOD enzootic site that were unaffected by JOD were stably and persistently colonized by Stappia stellulata-like strains. These isolates (designated M1) inhibited the growth of CVSP bacteria in a disk-diffusion assay and thus may have prevented colonization of these animals by CVSP bacteria in situ. Laboratory-maintained C. virginica injected with CVSP bacteria experienced statistically significant elevated mortalities compared to controls, and CVSP bacteria were recovered from these animals during the mortality events. Together, these results provide additional evidence that CVSP bacteria are the etiological agent of JOD. Further, there are no other descriptions of specific marine alpha-proteobacteria that have been successfully cultivated from a defined animal host. Thus, this system presents an opportunity to investigate both bacterial and host factors involved in the establishment of such associations and the role of the invertebrate host in the ecology of these marine alpha-proteobacteria.

Multiplex reverse transcriptase PCR assay for simultaneous detection of three fish viruses.

A multiplex reverse transcriptase (RT)-PCR assay was developed for the simultaneous detection of three different fish viruses: infectious pancreatic necrosis virus (IPNV), infectious hematopoietic necrosis virus (IHNV), and viral hemorrhagic septicemia virus (VHSV). The sensitivity levels of the multiplex RT-PCR assay were 100, 1, and 32 50% tissue culture infective doses/ml for IPNV, IHNV, and VHSV, respectively.

Use of antibacterial agents To elucidate the etiology of juvenile oyster disease (JOD) in Crassostrea virginica and numerical dominance of an alpha-proteobacterium in JOD-affected animals.

Since 1988, juvenile oyster disease (JOD) has resulted in high seasonal losses of cultured Eastern oysters (Crassostrea virginica) in the Northeast. Although the cause of JOD remains unknown, most evidence is consistent with either a bacterial or a protistan etiology. For the purpose of discerning between these hypotheses, the antibacterial antibiotics norfloxacin and sulfadimethoxine-ormetoprim (Romet-B) were tested for the ability to delay the onset of JOD mortality and/or reduce the JOD mortality of cultured juvenile C. virginica. Hatchery-produced C. virginica seed were exposed in triplicate groups of 3,000 animals each to either norfloxacin, sulfadimethoxine-ormetoprim, or filter-sterilized seawater (FSSW) and deployed in floating trays on the Damariscotta River of Maine on 17 July 1997. Each week thereafter, a subset of animals from each group was reexposed to the assigned treatment. Repeated immersion in either a sulfadimethoxine-ormetoprim or a norfloxacin solution resulted in a delay in the onset of JOD mortality in treated animals and reduced weekly mortality rates. Weekly treatments with either norfloxacin or sulfadimethoxine-ormetoprim also resulted in a statistically significant reduction in cumulative mortality (55 and 67% respectively) compared to animals treated weekly with FSSW (81%) or those that had received only a single treatment with either norfloxacin, sulfadimethoxine-ormetoprim, or FSSW (77, 84, and 82%, respectively). Bacteriological analyses revealed a numerically dominant bacterium in those animals with obvious signs of JOD. Sequence analysis of the 16S rRNA gene from these bacteria indicates that they are a previously undescribed species of marine alpha-proteobacteria.

Bilateral ischemic tolerance of rat hippocampus induced by prior unilateral transient focal ischemia: relationship to c-fos mRNA expression.

The purpose of this study was to determine whether transient unilateral (2 h) middle cerebral artery occlusion (MCAo) is capable of inducing bilateral ischemic tolerance in hippocampal CA1 neurons when temporary bilateral forebrain ischemia by two-vessel occlusion (2VO) is carried out 3 days later, and to explore the relationship of this tolerance to the regional expression of c-fos and hsp-70 mRNA. Rats were sacrificed 4 days after 2VO, and normal-appearing neurons in CA1 subregions were counted. Rats subjected to MCAo and 2VO showed significant protection of CA1 neurons in both hippocampi, whereas rats which underwent sham MCAo and 2VO typically had severe bilateral destruction of CA1 neurons (normal neuron counts, ipsilateral medial CA1: 59.8 +/- 7.2 vs 16.6 +/- 7.8 (mean +/- s.e.m.); middle CA1: 50.0 +/- 4.7 vs 16.0 +/- 8.8; lateral CA1: 43.5 +/- 5.7 vs 13.8 +/- 6.3; contralateral, medial CA1: 52.3 +/- 6.3 vs 17.0 +/- 6.4; middle CA1: 43.3 +/- 4.7 vs 19.8 +/- 8.1; lateral CA1: 45.5 +/- 4.6 vs 26.0 +/- 10.3, respectively). This neuronal tolerance was preceded by the early bilateral induction of c-fos mRNA, which may in turn lead to expression of critical target genes that promote cell recovery.

Computer-assisted image-averaging strategies for the topographic analysis of in situ hybridization autoradiographs.

We report the application of a computer-based image-averaging strategy to the quantitative topographic analysis of in situ hybridization autoradiographs, based upon a disparity-analysis algorithm. We illustrate this approach for a representative antisense riboprobe-the astrocytic marker, glial fibrillary acid protein (GFAP)-in the setting of fluid-percussion brain injury in rats. Sequential coronal autoradiographs in individual animals are first digitized and aligned by disparity analysis. Next, coronal sections of all brains of a given experimental group are placed in register with one another, using a common anatomic reference level. One brain of the series serves as a template, and corresponding sections of other brains are mapped into its contour at each level. In this manner, average and standard deviation image data sets may be generated. With thresholding techniques, individual data sets can be dichotomized with respect to a chosen threshold, and frequency maps can be generated at each coronal level, displaying numbers of brains showing supra-threshold levels of mRNA at each pixel location. Pixel-by-pixel statistical comparison of data sets obtained under two different conditions (e.g., 30 min vs. 24 h following brain trauma) is then feasible. A digitized functional-anatomic brain atlas may be fitted to the images to assist analysis. Computer-based image analysis of in situ hybridization autoradiographs greatly extends the utility and applicability of this technique.

Overcoming a defect in generalized recombination in the marine fish pathogen Vibrio anguillarum 775: construction of a recA mutant by marker exchange.

A defect in generalized recombination has prevented the use of marker exchange for the construction of specific chromosomal mutations in the marine fish pathogen Vibrio anguillarum 775. Through the use of large segments of homologous DNA, we were successful in overcoming this defect and used marker exchange to construct a recA mutant of V. anguillarum H775-3. A recombinant cosmid carrying the recA gene of V. anguillarum 775 in the center of a 25-kb cloned DNA insert was isolated by complementation of methyl methanesulfonate (MMS) sensitivity in Escherichia coli HB101. The recA gene was inactivated by inserting a kanamycin resistance gene into recA, and the mutant gene was subsequently introduced into V. anguillarum H775-3 by conjugal mobilization. Isolation of recombinants between cosmid-borne recA::kan sequences and chromosomal DNA was facilitated by the introduction of an incompatible plasmid, and Southern hybridization was used to verify the presence of recA::kan in the chromosomal DNA of the recA mutant. V. anguillarum carrying recA::kan was considerably more sensitive to UV radiation and to MMS than was its parent, and near wild-type levels of resistance to MMS and UV light were restored by introduction of cloned recA genes from both E. coli and V. anguillarum. These results indicate that recA is required for DNA repair in V. anguillarum and demonstrate the utility of this modified marker exchange technique for the construction of mutations in this economically important fish pathogen.

Genomic variation of aquatic birnaviruses analyzed with restriction fragment length polymorphisms.

Aquatic birnaviruses are the most ubiquitous and diverse group of viruses in the family Birnaviridae. Several cause different diseases in a variety of fish species, such as infectious pancreatic necrosis virus in salmonids in North America, Europe, and Asia and European eel virus in eel in Asia. Most isolates are antigenically related and belong to a single serogroup (serogroup A) comprising nine serotypes. Previous studies with monoclonal antibodies have demonstrated considerable variation in epitope profiles even among strains within a single serotype. The few studies of genomic variation among these viruses, which have focused on the NS/VP3 coding region, demonstrated the existence of several genogroups that generally did not correlate with antigenic groups. In this study, PCR was used to amplify a 1,180-bp cDNA genomic fragment representing most of the VP2 (the major outer capsid protein) coding region from five serotype A type strains and 17 Asian isolates. The PCR products were digested with nine different restriction enzymes. Restriction fragment length polymorphism profiles demonstrated heterogeneity among the tested viruses; however, the isolates from Asia were closely related to each other. Cluster analysis of the restriction fragment length polymorphism patterns demonstrated that these viruses could be divided into four major genogroups. In contrast to previous studies of variation in the NS/VP3 coding region, these genogroups based on variation in the VP2 coding region correlated with a serological classification based on VP2-specific monoclonal antibody reaction patterns. Furthermore, all Asian isolates tested belonged to one genogroup typified by the serotype type strain Ab.

Long-term expression of a fluorescent reporter gene via direct injection of plasmid vector into mouse skeletal muscle: comparison of human creatine kinase and CMV promoter expression levels in vivo.

Expression of a fluorescent reporter gene has been studied using two alternate promoters to transcribe the green fluorescent protein (gfp) from Aequorea victoria. The human cytomegalovirus (CMV) enhancer/ promoter or the human muscle-specific creatine kinase promoter (CKM) were inserted along with the gfp cDNA into a plasmid expression vector based on a modified adeno-associated virus genome. Naked plasmid DNA was injected into the hamstring muscle of mdx mice and gfp gene expression determined from frozen muscle sections taken at 4, 14, and 42 days postinjection. Fluorescence patterns obtained by photomicroscopy and quantitative fluorescence measurements indicated a near-linear increase in the accumulation of the gfp in skeletal muscle during the length of the study, with gfp expression at 42 days being roughly four times the values obtained at 4 days. The levels of expression of gfp from the CKM construct were consistantly higher than for the CMV construct. The CKM promoter/expression vector combination demonstrates significant potential for simple, direct delivery and long-term, high-level expression of genes in skeletal muscle.

Mutation segregation and rapid carrier detection of X-linked muscular dystrophy in dogs.

OBJECTIVES: To use exon 7-specific genomic polymerase chain reaction (PCR) products to identify the genotypes of normal, affected, and carrier female dogs in pedigrees segregating Golden Retriever muscular dystrophy (GRMD), and to confirm the concordant segregation of the mutation in all carrier and affected dogs presently available. DESIGN: The GRMD mutation is found in the consensus splice acceptor site in intron 6 of the canine dystrophin gene. PCR cycle-sequencing and restriction fragment length polymorphism/PCR were used for determination of the pattern of segregation of the point mutation which causes GRMD. ANIMALS: Normal, clinically affected, and obligate carrier dogs in pedigrees of GRMD. PROCEDURE: DNA from blood was amplified, using PCR and primers that bracket all of exon 7 of the canine dystrophin gene as well as 100 base pairs of intron on either side. PCR products were either cycle-sequenced directly or submitted to a second round of PCR, using 1 of the original primers coupled with a mutagenic restriction fragment length polymorphism-primer, which thus creates an artificial restriction site. Digestion with Stu I detected the normal allele. To detect the affected allele, Sau96 I was used to digest the 310-base pair exon 7 genomic fragment directly. CONCLUSIONS: Simple, clear diagnosis of carrier status was possible using these methods. This mutation is passed through all carrier and affected dogs in both United States GRMD colonies and the colony in Australia. CLINICAL RELEVANCE: Rapid, accurate diagnosis of carrier and affected dogs will enhance study of this homologue of Duchenne muscular dystrophy.

Wood-drying condensate from Eastern white pine induced cytotoxicity and genotoxicity in vitro.

Eastern white pine is one of the most important commercial species of wood in the Northeast. Condensates extracted from this wood were tested to detect potential cytotoxicity and genotoxicity in Chinese Hamster Ovary (CHO) cells in the absence of S-9 activation. Cytotoxicity was measured by the Trypan blue exclusion assay, mitotic index (MI) and proliferative rate index (PRI). Genotoxicity was measured by the chromosome aberration (CA) assay and sister chromatid exchange (SCE) analysis. Both cytotoxic and genotoxic effects were observed. Laboratory-generated Eastern white pine condensate reduced the viability of CHO cells. The number of viable cells was roughly inversely proportional to dosage over a range of 91 percent to 58 percent survival in treated groups as compared to 2.4 x 10(5) viable cells (100 percent) in the control. The mitotic index (MI) data also showed an inverse correlation with dosage. The highest scorable dose limited by toxicity was determined to be 1 ml of Eastern white pine condensate in a total of 10 ml of medium. Lastly, a dose response curve was observed using the CA assay and also with the SCE analysis. The present findings support results obtained from Ames testing of Eastern white pine condensate and also corroborate results derived from human peripheral-blood lymphocytes.

Toxigenicity of culture filtrates of Salmonella enteritidis isolates on three mammalian cell lines.

Culture filtrates of 28 Salmonella enteritidis isolates were tested for toxicity on Vero-, CHO-, and human foreskin fibroblast (HFF) cells. Cytopathic effects on HFF cells were extensive, and were observed even with some filtrates diluted 1:256. Vero cells showed effects with filtrates diluted up to 1:16, and CHO cells gave weak or no reaction. All isolates produced iron-binding siderophores as determined by reactions on chrome-azurol-S medium.

Cytotoxicity and genotoxicity of wood drying condensate from Southern Yellow Pine: an in vitro study.

We tested condensates from Southern Yellow Pine for potential cytotoxicity and genotoxicity in CHO-WBL and human peripheral blood lymphocytes (PBL) in the absence of S-9 activation. Cytotoxicity was evaluated by the Trypan blue exclusion assay, mitotic index (MI) and proliferative rate index (PRI). Genotoxicity was measured by the chromosome aberration (CA) assay and sister chromatid exchange (SCE) analysis. Both cytotoxic and genotoxic effects were observed. Laboratory-generated Southern Yellow Pine condensate reduced the viability of CHO-WBL cells. The number of viable cells was roughly inversely proportional to dosage over a range of 100% to 31% in treated groups, in both experiments, as compared to 2.6 x 10(5) (100%) in the control. The MI data in both CHO cells and PBL also showed an inverse correlation. The highest scorable dose limited by toxicity was determined to be 1 ml of Southern Yellow Pine condensate in 10 ml total of medium. Lastly, a dose response curve was observed in CHO cells, as well as in PBL, using the CA assay and also with the SCE analysis. The present findings corroborate the results from Ames testing and represent the only information currently available on the genotoxic potential of these chemicals.

Detection and identification of aquatic birnaviruses by PCR assay.

A reverse transcriptase polymerase chain reaction (RT-PCR) assay was developed for the detection and identification of aquatic birnaviruses. The four sets of primers (PrA, PrB, PrC, and PrD) that we used are specific for regions of cDNA coded by genome segment A of aquatic birnaviruses. PrA identifies a large fragment (1,180 bp) within the pVP2-coding region, and PrB identifies a 524-bp fragment within the sequence amplified by PrA. Primer set PrC frames a genome fragment (339 bp) within the NS-VP3-coding region, and PrD identifies a 174-bp sequence within the fragment identified by PrC. PrB and PrD amplified cDNAs from all nine recognized serotypes of aquatic birnavirus serogroup A as well as the N1 isolate that may represent a 10th serotype. These results indicate that these three primer sequences are highly conserved and can be used in PCR assays for group identification of these viruses. PrA routinely produced amplification products from eight serotypes but exhibited variable results with one serotype, and primer PrC identified 6 of the 11 virus isolates tested. The qualitative sensitivity of the RT-PCR assay was evaluated by comparison of the results with those of cell culture isolation assays. With the exception of one sample, the RT-PCR assay with primer PrD was as accurate as cell culture isolation for detecting virus in kidney and spleen tissues from naturally infected, asymptomatic carrier fish. These results indicate that the RT-PCR assay can be a rapid and reliable substitute for cell culture methods for the detection of aquatic birnaviruses.

Eastern white pine wood-drying condensate induced cytotoxicity and genotoxicity in human peripheral blood lymphocytes in vitro.

Condensate from eastern white pine, one of the commercially most important species of tree in the northeastern United States, was treated for potential cytotoxicity and genotoxicity in human peripheral blood lymphocytes in the absence of S-9 activation. Cytotoxicity was evaluated by mitotic index (MI) determination and proliferative rate index. Genotoxicity was measured by the chromosome aberration (CA) assay and sister chromatid exchange (SCE) analysis. Both cytotoxic and genotoxic effects were observed with laboratory-generated eastern white pine condensate. The MI data showed an inverse correlation between the MI and treatment dosage. A dose response curve was observed using the CA assay and also with the SCE analysis. The present findings thus corroborate the results from Ames testing and represent the only information currently available on the cytotoxic and genotoxic potential of these chemicals.

Virulence plasmid pJM1 prevents the conjugal entry of plasmid DNA into the marine fish pathogen Vibrio anguillarum 775.

Studies involving the introduction of cloned homologous genes into Vibrio anguillarum revealed that several plasmids could not be conjugally introduced into V. anguillarum 775(pJM1), but were transmissible to the pJM1-cured derivative H775-3. Recombinant pBR322 plasmids containing V. anguillarum genomic DNA inserts were mobilized from Escherichia coli donors, using pRK2013, into V. anguillarum H775-3 recipients at frequencies of 10(-6) to 10(-5) per recipient. When identical matings were performed with V. anguillarum 775(pJM1) recipients, the infrequent exconjugants recovered carried the pBR322-based plasmid but had lost the large virulence plasmid pJM1. Similar studies were carried out with plasmid RP4 and with recombinant derivatives of the closely related broad-host-range plasmid pRK290. While RP4 was transmissible from E. coli to V. anguillarum H775-3 at frequencies of 6.7 x 10(-2) per recipient, transmission to V. anguillarum 775(pJM1) recipients occurred at frequencies of only 2.5 x 10(-7). When pRK290 contained V. anguillarum DNA inserts, the only exconjugants recovered had lost pJM1, or contained pJM1 and a deletion derivative of the recombinant pRK290 plasmid where all of the DNA insert had been deleted. The use of Dam-, Dcm-, or EcoK- methylation-deficient E. coli donor strains failed to result in appreciable numbers of V. anguillarum 775(pJM1) exconjugants that contained the desired transferred plasmids. Following UV mutagenesis, a derivative of V. anguillarum 775(pJM1) was isolated that would accept conjugally transferred plasmid DNAs at frequencies similar to those observed when using V. anguillarum H775-3 recipients. These data suggest that virulence plasmid pJM1 mediates a restriction system that prevents conjugal transmission of plasmid DNA from E. coli donors into V. anguillarum 775(pJM1). This putative restriction system appears not to be directed towards Dam-, Dcm-, or EcoK-methylated DNA, and appears not to involve a Type II restriction endonuclease.

Molecular characterization of Salmonella enteritidis isolates from Maine poultry and poultry farm environments.

Eighty-six Salmonella enteritidis isolates obtained during a surveillance program of poultry farms in Maine were subjected to phage-typing, plasmid profiling and fingerprinting, outer-membrane polypeptide analysis, and antimicrobial sensitivity testing. Isolates were obtained from a variety of sources, including poultry-farm environmental samples, chicken organ samples, human stool samples, cat feces, and live-trapped rats and mice. These isolates were compared with 21 S. enteritidis isolates originating outside of Maine. Phage types isolated in Maine included 13a (60%); 14b (29%); 23 (5%); 8 (2%); and 2 (2%). All S. enteritidis isolates from Maine carried plasmid DNA, and 97% of these isolates carried a 40.3-megadalton plasmid alone (6%) or in conjunction with several smaller plasmids (91%). All 52 phage-type 13a isolates harbored 40.3- and 3.0-megadalton plasmids. All 25 phage-type 14b isolates carried 3.3- and 1.3-megadalton plasmids, and 22 isolates also carried the 40.3-megadalton plasmid. All isolates displayed highly similar outer-membrane polypeptide profiles and were sensitive to a variety of antimicrobials commonly used against gram-negative organisms. The above data suggest that phage type and plasmid content may be related in the cases of phage-type 13a and 14b isolates, and that traditional plasmid-borne antimicrobial resistance determinants were not present in Maine isolates. Results also indicate that phage-typing can be a valuable epizootiological tool for monitoring the potential spread of these strains throughout the Northeast.

Polypeptides p40, pOM2, and pAngR are required for iron uptake and for virulence of the marine fish pathogen Vibrio anguillarum 775.

Insertions were created in three iron uptake genes in plasmid pJM1 of Vibrio anguillarum 775 to assess their in vivo effects on virulence in fish. Insertions that blocked p40, pOM2, and pAngR expression resulted in iron uptake-negative strains and in 4.2 x 10(5)-, 8.8 x 10(5)-, and 2.5 x 10(5)-fold attenuations in virulence, respectively. A strain with an insertion in the pAngR coding region still synthesized significant constitutive levels of the outer membrane protein pOM2 and persisted in fish for at least 14 days postinjection. The results demonstrate a direct relationship between virulence and three pJM1-encoded gene products and also the feasibility of constructing live attenuated strains of V. anguillarum that might be useful in future vaccines.

Molecular cloning of the recA analog from the marine fish pathogen Vibrio anguillarum 775.

The recA analog from Vibrio anguillarum 775 was isolated by complementation of recA mutations in Escherichia coli, and its protein product was identified. The recA analog promoted recombination between two partially deleted lactose operons, stimulated both spontaneous and mitomycin C-induced phage production in RecA- lambda lysogens, and restored near wild-type levels of resistance to UV radiation and methyl methanesulfonate.

Identification of polypeptides encoded by cloned pJM1 iron uptake DNA isolated from Vibrio anguillarum 775.

The XhoI fragment containing much of the iron uptake region of plasmid pJM1 was isolated from Vibrio anguillarum 775 and cloned into plasmid pBR322. Plasmid-encoded polypeptides were examined in maxicells of Escherichia coli, and transposon mutagenesis was used to map insertion mutations in the structural DNA encoding the OM2 polypeptide. Tn1000 insertions that mapped within OM2 and blocked maxicell expression of OM2 resulted in the loss of ferric iron-anguibactin receptor function when plasmids containing OM2:: Tn1000 insertions were introduced into V. anguillarum cells. Two iron-regulated polypeptides were identified in maxicell polypeptide profiles of E. coli SS201. A 20,000-dalton polypeptide was expressed in maxicells of SS201 grown under conditions of iron limitation but was barely detectable in profiles of SS201 cells that were grown under high-iron conditions. DNA encoding the 20,000-dalton polypeptide mapped downstream of and adjacent to the gene encoding OM2. DNA sequences required for production of a 46,000-dalton polypeptide mapped 4.5 kilobases downstream of the OM2 structural gene. The 46,000-dalton polypeptide was synthesized at high levels in E. coli SS201 maxicells grown under high-iron conditions, but synthesis of the protein was severely repressed under conditions of iron limitation. Iron-regulated expression of both proteins in maxicells of SS201 was relieved upon deletion of a 4.9-kilobase SalI-XhoI fragment of pJM1 DNA, which indicated that pJM1 DNA sequences present in the deleted fragment are required for regulated expression of both proteins in E. coli. Maxicells of SS201 harboring these deletion derivatives synthesized the 20,000-dalton polypeptide at very low constitutive levels and the 46,000-dalton polypeptide at high constitutive levels, regardless of the iron concentration of the growth medium. The observed regulation of the 20,000-dalton protein suggested that it might play a role either in siderophore biosynthesis or in the functional expression of OM2. The opposite regulatory pattern observed for the 46,000-dalton polypeptide suggested that it does not play a structural role in siderophore or OM2 biosynthesis, but the observed regulatory pattern might be expected if the 46,000-dalton protein played a negative regulatory role in siderophore biosynthesis.