Boom‐bust like dynamics of invasive black bullhead (Ameiurus melas) in Lake Sava (Serbia)

The life‐history traits and population dynamics of the black bullhead Ameiurus melas (Rafinesque) were studied monthly from August 2009 to December 2012 in Lake Sava in Belgrade (Serbia). This period included a mass mortality event in May 2011 and subsequent population recovery during 2012. Available evidence suggests the mass mortality was caused by European catfish virus that only affected the black bullhead. Changes in key life‐history traits and the recovery potential (increase in catch‐per‐unit‐effort, doubling of young‐of‐the‐year to adult ratio, earlier maturation and increased fecundity) following the mass mortality event suggest population resilience typical of a recurring boom‐bust pattern and demonstrate the species’ capacity to recover rapidly and re‐establish following disturbance. Repeated systematic mass removals of both juveniles and adults (using fyke nets) will be required if future fishery management measures aimed at limiting the population size are to be effective.     

Amino acid changes in the capsid protein of a reassortant betanodavirus strain: Effect on viral replication in vitro and in vivo

Betanodavirus reassortant strains (RGNNV/SJNNV) isolated from Senegalese sole harbour an SJNNV capsid featuring several changes with respect to the SJNNV‐type strain, sharing three hallmark substitutions. Here, we have employed recombinant strains harbouring mutations in these positions (r20 and r20 + 247 + 270) and have demonstrated that the three substitutions affect different steps of the viral replication process. Adsorption ability and efficiency of viral attachment were only affected by substitutions in the C‐terminal side of the capsid. However, the concurrent mutation in the N‐terminal side seems to slightly decrease these properties, suggesting that this region could also be involved in viral binding. Differences in the intracellular and extracellular production of the mutant strains suggest that both the C‐terminal and N‐terminal regions of the capsid protein may be involved in the particle budding. Furthermore, viral replication in sole brain tissue of the mutant strains, and especially double‐ and triple‐mutant strains, is clearly delayed with respect to the wt strain. These data support previous findings indicating that the C‐terminal side plays a role in virulence because of a slower spread in the fish host brain and suggest that the concurrent participation of the N‐terminal side is also important for viral replication in vivo.     

Biocontrol of saprolegniosis in rainbow trout (Oncorhynchus mykiss Walbaum) using two bacterial isolates (LE89 and LE141) of Pseudomonas fluorescens

The probiotic activity of 15 bacterial isolates that inhibit Saprolegnia parasitica in vitro was tested for the biocontrol of saprolegniosis in rainbow trout (Oncorhynchus mykiss Walbaum), adding the bacteria to tank water for 14 days at a concentration of 10⁶ bacteria ml^?1 water. Pseudomonas fluorescens LE89 and Pseudomonas fluorescens LE141 were effective in controlling experimental infection with S. parasitica since of the fish treated with LE89, 24.5% ± 16.27% (p < 0.05) became infected, as did 42.8% ± 8.41% (p < 0.05) of those treated with LE141. Given their protective effect when administered in water, their effect was also studied when administered in feed before and after experimental infection. Both bacterial isolates survived low pH levels and the action of bile, grew in skin and intestinal mucus, were resistant to several antibiotics and survived in feed; however, neither of the two isolates prevented S. parasitica infection when administered in feed.     

Multilocus sequence typing detects new Piscirickettsia salmonis hybrid genogroup in Chilean fish farms: Evidence for genetic diversity and population structure

Piscirickettsia salmonisis the causative bacterial pathogen of piscirickettsiosis, a salmonid disease that causes notable mortalities in the worldwide aquaculture industry. Published research describes the phenotypic traits, virulence factors, pathogenicity and antibiotic‐resistance potential for various P. salmonisstrains. However, evolutionary and genetic information is scarce for P. salmonis. The present study used multilocus sequence typing (MLST) to gain insight into the population structure and evolution of P. salmonis. Forty‐two Chilean P. salmonisisolates, as well as the type strain LF‐89^T, were recovered from diseased Salmo salar, Oncorhynchus kisutchand Oncorhynchus mykissfrom two Chilean Regions. MLST assessed the loci sequences of dnaK, efp, fumC, glyA, murG, rpoD and trpB. Bioinformatics analyses established the genetic diversity among P. salmonis isolates (H = 0.5810). A total of 23 sequence types (ST) were identified, 53.48% of which were represented by ST1, ST5 and ST2. Population structure analysis through polymorphism patterns showed few polymorphic sites (218 nucleotides from 4,010 bp), while dN/dS ratio analysis indicated purifying selection for dnaK, epf, fumC, murG, and rpoD but neutral selection for the trpB loci. The standardized index of association indicated strong linkage disequilibrium, suggesting clonal population structure. However, recombination events were detected in a group of seven isolates. Findings included genogroups homologous to the LF‐89^T and EM‐90 strains, as well as a seven‐isolate hybrid genogroup recovered from both assessed regions (three O. mykiss and four S. salar isolates). The presented MLST scheme has comparative potential, with promising applications in studying distinct P. salmonis isolates (e.g., from different hosts, farms, geographical areas) and in understanding the epidemiology of this pathogen.     

Evidence for the facultative intracellular behaviour of the fish pathogen Vibrio ordalii

Vibrio ordalii is an extracellular, Gram‐negative bacterium that produces vibriosis in salmonids. While pathogenesis is not fully understood, this bacterium has numerous likely genes for adhesion, colonization, invasion factors and, as recently suggested, intracellular behaviour. Therefore, this study aimed to clarify possible intracellular behaviour for V. ordalii Vo‐LM‐18 and ATCC 33509^T in the fish‐cell lines SHK‐1 and CHSE‐214. Confocal microscopy revealed Vo‐LM‐18 and ATCC 33509^T inside cytoplasm in both fish‐cell lines at 4 hr post‐inoculation (hpi). At 8 and 16 hpi, the proportion of fish cells invaded by both strains increased. Moreover, intracellular V. ordalii were observed after 8 hpi inside mouse embryonic fibroblasts (MEF), demonstrating that entry was not due to a cellular phagocytosis process. Flow cytometry confirmed immunocytochemistry results, with both V. ordalii evidencing statistically significant differences in the number of infected cells between 8 and 16 hpi. Interestingly, V. ordalii infection did not significantly damage fish cells, as determined by LDH liberation. Viable counts at 8 hpi detected, on average for both lines, 176 ± 47 CFU/ml of culturable intracellular Vo‐LM‐18 and ATCC 33509^T cells. These in vitro findings support the facultative intracellular behaviour of V. ordalii and may be of importance for understanding pathogenicity and survival in aquatic environments.     

Aeromonas salmonicida infects Atlantic salmon (Salmo salar) erythrocytes

Aeromonas salmonicida subsp. salmonicida (hereafter A. salmonicida) is the aetiological agent of furunculosis in marine and freshwater fish. Once A. salmonicida invade the fish host through skin, gut or gills, it spreads and colonizes the head kidney, liver, spleen and brain. A. salmonicida infects leucocytes and exhibits an extracellular phase in the blood of the host; however, it is unknown whether A. salmonicida have an intraerythrocytic phase. Here, we evaluate whether A. salmonicida infects Atlantic salmon (Salmo salar) erythrocytes in vitro and in vivo. A. salmonicida did not kill primary S. salar erythrocytes, even in the presence of high bacterial loads, but A. salmonicida invaded the S. salar erythrocytes in the absence of evident haemolysis. Naïve Atlantic salmon smolts intraperitoneally infected with A. salmonicida showed bacteraemia 5 days post‐infection and the presence of intraerythrocytic A. salmonicida. Our results reveal a novel intraerythrocytic phase during A. salmonicida infection.     

Fish photobacteriosis—The importance of rapid and accurate identification of Photobacterium damselae subsp. piscicida

MALDI‐TOF MS was tested for the identification of Photobacterium damselae subsp. piscicida on isolates grown on two media, cultured at three incubation times and applied on the target plate by the direct sample spotting (DS), by the on‐target extraction (OTE) and by the full extraction (FE) method, in triplicates. The identification of samples grown on blood agar (BA) outperformed identification on tryptic soya agar (TSA) by 0.64% for DS and OTE. The OTE gave the highest scores in both culture media, all incubation times and replicates. Reliable 24‐hr species identification was 61.54%, 84.61% and 53.85% for samples grown on TSA and identified by DS, OTE and FE, respectively. For isolates grown on BA, they were 76.92%, 96.15% and 30.77%, respectively. When identified by OTE, the 48‐hr identification was 93.58%, but for 72 hr declined to 71.79%. The reliable identification with the highest score from the first measurement was 100% only for OTE from BA (24 hr), whereas OTE from TSA gave 84.61% (24 hr), 76.92% (48 hr) and 84.61% (72 hr). The reliable MALDI‐TOF MS identification of Ph. damselae subsp. piscicida is incubation time, media, target plate preparation and replicate‐dependent.