The mutagenesis of a type IV secretion system locus of Piscirickettsia salmonis leads to the attenuation of the pathogen in Atlantic salmon, Salmo salar.

Piscirickettsiosis is a threatening infectious disease for the salmon industry, due to it being responsible for significant economic losses. The control of outbreaks also poses considerable environmental challenges. Despite Piscirickettsia salmonis having been discovered as the aetiological agent of the disease more than 25 years ago, its pathogenicity remains poorly understood. Among virulence factors identified so far, type four secretion systems (T4SS) seem to play a key role during the infection caused by the bacterium. We report here the genetic manipulation of P. salmonis by means of the transference of plasmid DNA in mating assays. An insertion cassette was engineered for targeting the icmB gene, which encodes a putative T4SS-ATPase and is carried by one of the chromosomal T4SS clusters found within the genome of P. salmonis PM15972A1, a virulent representative of the EM-90-like strain. The molecular characterization of the resulting mutant strain demonstrated that the insertion interrupted the target gene. Further in vitro testing of the icmB mutant showed a dramatic drop in infectivity as tested in CHSE-214 cells, which is in agreement with its attenuated behaviour observed in vivo. Altogether, our results demonstrate that, similar to other facultative intracellular pathogens, P. salmonis' virulence relies on an intact T4SS.

Prevalence, geographic distribution and phenotypic differences of Piscirickettsia salmonis EM-90-like isolates.

Early reports accounted for two main genotypes of Piscirickettsia salmonis, a fish pathogen and causative agent of piscirickettsiosis, placing the single isolate EM-90 apart from the prototypic LF-89 and related isolates. In this study, we provide evidence that, contrary to what has been supposed, the EM-90-like isolates are highly prevalent and disseminated across Chilean marine farms. Molecular analysis of 507 P. salmonis field isolates derived from main rearing areas, diverse hosts and collected over 6 years, revealed that nearly 50% of the entire collection were indeed typed as EM-90-like. Interestingly, these isolates showed a marked host preference, being recovered exclusively from Atlantic salmon (Salmo salar) samples. Although both strains produce undistinguishable pathological outcomes, differences regarding growth kinetics and susceptibility to the antibiotics and bactericidal action of serum could be identified. In sum, our results allow to conclude that the EM-90-like isolates represent an epidemiologically relevant group in the current situation of piscirickettsiosis. Based on the consistency between genotype and phenotype exhibited by this strain, we point out the need for genotypic studies that may be as important for the Chilean salmon industry as the continuous surveillance of antimicrobial susceptibility patterns.

Seasonal modulation of growth hormone mRNA and protein levels in carp pituitary: evidence for two expressed genes.

Adaptation of eurythermal fish to naturally varying environmental conditions involves modulation of expressions of various factors in the hypothalamo-hypophyseal axis. Here we used three complementary approaches to assess the seasonal variation of growth hormone (GH) protein and mRNA levels in pituitary glands of acclimatized carp fish. First, a polyclonal antibody raised against an oligopeptide derived from the carp GH sequence was used for immunohistochemistry; second, oligonucleotides specific for GH transcripts were used for in situ hybridization. Specific immunodetection of GH coincides with visualization of GH mRNA in the proximal pars distalis, the specific location of somatotroph cells in carp pituitary gland. Finally, competitive RT-PCR analyses confirmed that GH expression exhibits seasonal cyclical reprogramming with higher levels in summer- than in winter-adapted fish. The expression pattern suggests an important role for GH in the molecular mechanisms underlying the acclimatization process. In parallel, amplification of sequences from the fourth intron and adjacent sites from exons IV and V demonstrates the existence of a new GH gene previously undescribed. The detection of transcripts corresponding to each gene suggests that both GH gene copies are active in the duplicated carp genome and that they are similarly affected by seasonal adaptation.