ABSTRACT
The fish pathogen Aeromonas salmonicida was chromosomally marked with genes encoding bacterial luciferase, luxAB, isolated from Vibrio fischeri, resulting in constitutive luciferase production. During exponential growth in liquid batch culture, luminescence was directly proportional to biomass concentration, and luminometry provided a lower detection limit of approximately 10(sup3) cells ml(sup-1), 1 order of magnitude more sensitive than enzyme-linked immunosorbent assay detection. In sterile seawater at 4(deg)C, lux-marked A. salmonicida entered a dormant, nonculturable state and population activity decreased rapidly. The activity per viable cell, however, increased by day 4, indicating that a proportion of the population remained active and culturable. Putative dormant cells were not resuscitated after the addition of a range of substrates.
ABSTRACT
The small subunit ribosomal RNA (srRNA) gene was amplified from Gyrodactylus salaris using the polymerase chain reaction (PCR), cloned, and the complete gene sequence of 1966 bp determined. The V4 region of the srRNA gene was identified and amplified from single specimens of G. salaris, G. derjavini and G. truttae. Comparison of the V4 sequences from these three species revealed sequence differences from which restriction fragment length polymorphisms (RFLPs) were predicted and an oligonucleotide probe (GsV4) specific to G. salaris designed. Digestion of the amplified V4 region of the srRNA gene with Hae III and either Alw I, BstY I, Dde I or Mbo I provided a means of discriminating between G. salaris, G. derjavini and G. truttae. The GsV4 probe was used to detect the srRNA gene from G. salaris in Southern and dot blots of the amplified V4 region.
Subject(s)
Cestoda/genetics , Genes, Helminth/genetics , Polymorphism, Restriction Fragment Length , RNA, Ribosomal/genetics , Animals , Base Sequence , Cestoda/classification , Cloning, Molecular , DNA Probes , DNA, Helminth/analysis , Molecular Sequence Data , Polymerase Chain Reaction/methods , Salmon/parasitology , Sequence Analysis, DNA , Species SpecificityABSTRACT
Hybridisation with cosmid pRMB2, containing the vir locus of bordetella pertussis, showed that the Tn5 insertion in B. pertussis BP347 (Vir-) was located with 1 2.7 kB EcoRI fragment. When subcloned, this fragment alone was unable to complement BP347, but a larger 8.0 kb region which included the 2.7 kb EcoRI fragment, did restore expression of virulence associated properties to BP347, and to avirulent phase variants of both B. pertussis and B. bronchiseptica. EcoRI-digested DNA from stains of all four species of Bordetella showed homology to the cosmid genomic insert and to the 2.7 kb subclone, but B. avium showed a markedly different pattern of homology to the other species.
