Anisakid larvae in Atlantic salmon (Salmo salar L.) grilse and post-smolts: molecular identification and histopathology.

The molecular identification and histopathology are described for the parasitic larvae of a nematode species present in the abdominal cavity of Atlantic salmon ( Salmo salar ) grilse caught in fish traps on their natal river in the west of Ireland and post-smolts collected during experimental trawls on the continental shelf edge of the northeast Atlantic Ocean. Larvae in the adult and juvenile salmon were identified as Anisakis simplex sensu stricto by PCR amplification and RFLP and sequencing of the ITS gene and PCR amplification and sequencing of the cox2 gene. Parasitic nematode larvae in the grilse were either encapsulated in the abdominal mesentery associated with the pyloric ceca or on the serosal surface of the liver and in the vent region. In some fish, larvae were found in the parenchyma of the liver and muscularis circularis of the intestine. In general, the larvae induced a limited cellular response apart from the occurrence of focal melanin macrophage aggregates and individual eosinophilic granular cells in the connective tissue capsule. Melanin macrophage aggregates were also present among the hepatocytes adjacent to encapsulated larvae in the liver. The reaction to the parasites was more severe in the wall of the intestine. Encapsulated nematode larvae caused displacement, vacuolation, and necrosis of the circular muscle fibers. The stratum compactum was also disrupted with focal areas of degeneration. Overall, the intestinal wall had a hypercellular appearance with extensive cellular infiltration comprising eosinophilic granular cells, macrophages, lymphocytes, and fibrocytes. The post-smolts were caught in May during the early oceanic phase of their life cycle. In these fish, A. simplex sensu stricto larvae were found lying free on the serosal surface of the intestine and liver without any apparent histologic changes. This is the earliest in the marine migration of Atlantic salmon that A. simplex sensu stricto infection has been recorded.

Mapping studies and genetic analysis of transfer genes of the multi-resistant IncHI2 plasmid, R478.

A bank of transfer-defective Tn7 insertion mutants of the multi-resistant IncHI2 megaplasmid, R478, was generated. Complementation analysis of these mutations identified a large 144-kb transfer-associated region of R478. A 6.8-kb segment from the transfer region was sequenced. The precise locations of Tn7 insertion within four distinct R478::Tn7 transfer-defective mutants were mapped and each insertion was found to disrupt a specific open reading frame. These transfer-associated determinants of R478 were designated htdB (H transfer determinant), htdD, htdT and htdC. Both htdB and htdC encoded amino acid sequences that showed a low homology with pilus biosynthetic proteins encoded by the F plasmid.

Spatial and temporal variation in the infestation of sea trout (Salmo trutta L.) by the caligid copepod Lepeophtheirus salmonis (Krøyer) in relation to sources of infection in Ireland.

Infestations of post-smolt sea trout (Salmo trutta L.) by the salmon louse (Lepeophtheirus salmonis Krøyer) were characterized in 42 estuaries over a 5 year period in Ireland. Spatial variation in infestation was more significant than temporal trends and existed at 3 levels; between regions (regions > 100 km of coastline), between bays within regions (bays < 50 km in length) and between estuaries within bays (distance between estuaries < 10 km). The observed spatial structure in infestations inferred that production of the infective larvae varied between regions and bays and that there was limited movement of fish and infective larvae between regions and bays. In addition the different levels of infestation recorded between estuaries in the same bay indicated short spatial scale variability in parasite transmission. Significantly higher infestations occurred in bays that contained lice-infested farmed salmon. Lice-infested wild spring salmon, which were present in estuaries of some systems, did not have a significant positive impact on infestations.

Displacement of OmpF loop 3 is not required for the membrane translocation of colicins N and A in vivo.

The pore-forming colicins N and A require the porin, OmpF, in order to translocate across the outer membrane of Escherichia coli. We investigated the hypothesis that in vivo, colicins N and A may traverse the outer membrane through the OmpF channel. In order to accommodate a polypeptide in the pore, the mid-channel constriction loop of OmpF, L3, would need to undergo a conformational change. We used five OmpF cystine mutants, which fix L3 in the conformation determined by X-ray crystallography, to investigate L3 movement during colicin activity in vivo. Sensitivity to colicins N and A of E. coli cells expressing these OmpF cystine mutants was determined using cell survival and in vivo potassium efflux and fluorescence assays. Results indicate that gross movement of L3 is not required for colicin N or A activity and that neither of these colicins crosses the outer membrane of E. coli through the lumen of the OmpF pore.

Characterization of a region of the IncHI2 plasmid R478 which protects Escherichia coli from toxic effects specified by components of the tellurite, phage, and colicin resistance cluster.

The IncHI2 plasmid R478 specifies resistance to potassium tellurite (Te(r)), to some bacteriophages (Phi), and to pore-forming colicins (PacB). The genes encoding the three phenotypes are linked, and an 8.4-kb fragment of R478 DNA encoding them cannot be subcloned unless cocloned with a second section of the plasmid. Subclone pKFW4A contains a 5.9-kb BamHI-EcoRI fragment which caused some toxicity when present in Escherichia coli cells. Bacterial cells containing freshly transformed pKFW4A, examined by light microscopy and electron microscopy, had a filamentous morphology consistent with a block in septation. Insertion of transposon Tn1000 into terZ, -A, -B, and -C genes of pKFW4A resulted in the loss of the filamentation phenotype. Deletion of several regions of the clone confirmed that these latter components are involved in the filamentation phenotype. The region specifying protection from toxicity caused by the larger 8.4-kb fragment (encompassing this cluster and the entire 5.9-kb section of pKFW4A) was sequenced and analyzed by T7 polymerase expression and Tn1000 mutagenesis. Three open reading frames, terW, terY, and terX, were identified in a 2.6-kb region. Two polypeptides with approximate molecular masses of 18 and 28 kDa were expressed in CSRDE3 cells and were consistent with TerW (17.1 kDa; 155 amino acids [aa]) and TerY (26.9 kDa; 248 aa), whereas a protein of 213 aa deduced from terX was not observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The terX gene product shows strong identity with the previously identified TerE, TerD, and TerZ polypeptides, and there is a conserved motif of 13 residues, GDN(R/L)TG(E/A)GDGDDE, within this group of polypeptides. Complementation analysis indicated that terW, located approximately 6.0 kb upstream of terZ, brings about protection of cells from toxic effects of components of the Te(r), Phi, and PacB cluster.

Phage inhibition, colicin resistance, and tellurite resistance are encoded by a single cluster of genes on the IncHI2 plasmid R478.

A region of the IncHI2 plasmid R478, encoding the phenotypes of tellurite resistance (Ter), phage inhibition (Phi), and colicin resistance (PacB), was cloned and sequenced. Analysis indicated seven open reading frames (ORFs), whose genes were designated terZ, -A, -B, -C, -D, -E, and -F. Five of these predicted ORFs (A to E) had extensive amino acid homology with the previously reported ORFs of the IncHI2 Ter operon from plasmid pMER610. There were domains of highly conserved amino acid residues within the group TerA, -D, -E, and -F and within TerD, -E, and -Z, but no consensus could be found among all five putative polypeptides. There were also regions of good identity and similarity between individual pairs of ORFs which was not reflected in the multiple alignments. The three phenotypes were expressed in Escherichia coli DH5 alpha by an 8.4-kb EcoRI insert subcloned from a cosmid of R478. The latter insert was clonable only as a double insertion with a 4.5-kb fragment, and forced deletion of the smaller fragment was lethal to cells. This lethality was not dependent on the cloned orientation of either fragment, suggesting that there is a trans-acting element in the 4.5-kb fragment. Tn1000 mutagenesis of one of the double-insert clones, pDT2575, showed that the phenotypes, including multiple colicin resistance, were genetically linked. Transpositions into terD, terC, and terZ reduced or abolished all phenotypes, while inserts into terE and terF had no effect on the phenotypes. Insertions in terA reduced phage inhibition levels only. The presence of the terZ and terF ORFs in pMER610 was confirmed, and derivatives of this plasmid mediated Phi, PacB, and Ter.

Genetic and nucleotide sequence analysis of the gene htdA, which regulates conjugal transfer of IncHI plasmids.

IncHI plasmids are naturally repressed for conjugative transfer and do not allow efficient propagation of the IncH pilus-specific phage Hgal. Transposons Tn7, Tn5, and TnlacZ were inserted into IncHI plasmids R478, R477-1, and R27, respectively, leading to the isolation of several plasmid mutants which exhibited increased levels of transfer and also permitted good lysis with phage Hgal. A 4.3-kb HindIII fragment from R478 reversed both phenotypic effects of derepression for the R477-1::Tn5 and the R478::Tn7 derivatives, pKFW99 and pKFW100, respectively. Exonuclease III deletions of this fragment and nucleotide sequence analysis indicated that the gene responsible for transfer repression, named here htdA, encoded a polypeptide of 150 amino acids. Cloning and sequence analysis of pDT2454 (R27::TnlacZ) revealed that the transposon had inserted into an open reading frame (ORF) which had an 83% amino acid identity with the R478 htdA gene. Maxicell analysis showed both the R27 and R478 HtdA products had molecular masses of 19.9 kDa. Conjugation experiments showed that the cloned htdA determinants caused a significant reduction of the transfer frequencies of wild-type R478 and R27 plasmids. Examination of both R478 derepressed mutants, pKFW100 and pKFW101, indicated that both transposon insertions occurred upstream of the htdA ORF. The results suggest that HtdA is a regulatory component of IncH plasmid transfer and also show that the region upstream of the htdA ORF is involved in transfer repression. The locations of the htdA determinants were identified on the plasmid maps of R27 and R478.

Restriction endonuclease mapping of the HI2 incompatibility group plasmid R478.

A restriction map of the 272-kb IncHI2 plasmid R478 was constructed by using the enzymes ApaI, XbaI, SalI, and XhoI. The map was derived from cloned restriction fragments from R478 inserted into cosmid and plasmid vectors as well as from double-digestion analysis of R478 and R478 miniplasmids. All previously known resistance determinants were cloned from R478, and their positions were located on the restriction map. A region involved in incompatibility was cloned and mapped. The location of a previously unreported arsenite resistance gene was also determined. The genes encoding tellurite resistance, colicin B resistance, and phage inhibition were found to be associated with a 6.7-kb SalI fragment of R478.