Molecular Characterization of an Endozoicomonas-Like Organism Causing Infection in the King Scallop (Pecten maximus L.).

One of the fastest growing fisheries in the UK is the king scallop (Pecten maximus L.), also currently rated as the second most valuable fishery. Mass mortality events in scallops have been reported worldwide, often with the causative agent(s) remaining uncharacterized. In May 2013 and 2014, two mass mortality events affecting king scallops were recorded in the Lyme Bay marine protected area (MPA) in Southwest England. Histopathological examination showed gill epithelial tissues infected with intracellular microcolonies (IMCs) of bacteria resembling Rickettsia-like organisms (RLOs), often with bacteria released in vascular spaces. Large colonies were associated with cellular and tissue disruption of the gills. Ultrastructural examination confirmed the intracellular location of these organisms in affected epithelial cells. The 16S rRNA gene sequences of the putative IMCs obtained from infected king scallop gill samples, collected from both mortality events, were identical and had a 99.4% identity to 16S rRNA gene sequences obtained from "Candidatus Endonucleobacter bathymodioli" and 95% with Endozoicomonas species. In situ hybridization assays using 16S rRNA gene probes confirmed the presence of the sequenced IMC gene in the gill tissues. Additional DNA sequences of the bacterium were obtained using high-throughput (Illumina) sequencing, and bioinformatic analysis identified over 1,000 genes with high similarity to protein sequences from Endozoicomonas spp. (ranging from 77 to 87% identity). Specific PCR assays were developed and applied to screen for the presence of IMC 16S rRNA gene sequences in king scallop gill tissues collected at the Lyme Bay MPA during 2015 and 2016. There was 100% prevalence of the IMCs in these gill tissues, and the 16S rRNA gene sequences identified were identical to the sequence found during the previous mortality event.IMPORTANCE Molluscan mass mortalities associated with IMCs have been reported worldwide for many years; however, apart from histological and ultrastructural characterization, characterization of the etiological agents is limited. In the present work, we provide detailed molecular characterization of an Endozoicomonas-like organism (ELO) associated with an important commercial scallop species.

The effectiveness of fishery net dips: Advice for the improvement of biosecurity measures.

Disease poses a major threat to aquaculture and commercial and recreational fisheries globally. Biosecurity measures have been implemented; however, empirical evidence of their efficacy in situ is lacking. Here, we present the results from a study conducted to examine the effectiveness of disinfectant net dips. Samples were collected from disinfectant net dips at 25 recreational fisheries in south-west England and assessed to determine (a) the level of bacterial contamination and (b) the reduction in titre of a target virus (infectious pancreatic necrosis virus, IPNV) following a contact time of 2 and 5 min. In addition, the study examined the reduction in target virus titre following exposure to laboratory prepared Virkon® , representing "clean," "dirty" and "diluted and dirty" conditions, for 2 and 5 min. Bacterial contamination was high in 64% of disinfectant samples, and, 76% of disinfectant samples did not effectively reduce the target virus titre in 2 or 5 min. Virus titre was successfully reduced following exposure to laboratory prepared Virkon® for 2 or 5 min, although dilution and contamination reduced the effectiveness. These results suggest that disinfectant net dips may not be working effectively on a high proportion of fishery sites. We provide recommendations for improving biosecurity.

Gene expression comparison of resistant and susceptible Atlantic salmon fry challenged with Infectious Pancreatic Necrosis virus reveals a marked contrast in immune response.

BACKGROUND: Infectious Pancreatic Necrosis (IPN) is a highly contagious birnavirus disease of farmed salmonid fish, which often causes high levels of morbidity and mortality. A large host genetic component to resistance has been previously described for Atlantic salmon (Salmo salar L.), which mediates high mortality rates in some families and zero mortality in others. However, the molecular and immunological basis for this resistance is not yet fully known. This manuscript describes a global comparison of the gene expression profiles of resistant and susceptible Atlantic salmon fry following challenge with the IPN virus. RESULTS: Salmon fry from two IPNV-resistant and two IPNV-susceptible full sibling families were challenged with the virus and sampled at 1 day, 7 days and 20 days post-challenge. Significant viral titre was observed in both resistant and susceptible fish at all timepoints, although generally at higher levels in susceptible fish. Gene expression profiles combined with gene ontology and pathway analyses demonstrated that while a clear immune response was observed in both resistant and susceptible fish, there were striking differences between the two phenotypes. The susceptible fish showed marked up-regulation of genes related to cytokine activity and inflammatory response that evidently failed to protect against the virus. In contrast, the resistant fish demonstrated a less pronounced immune response including up-regulation of genes relating to the M2 macrophage system. CONCLUSIONS: While only the susceptible phenotype shows appreciable mortality levels, both resistant and susceptible fish can become infected with IPNV. Susceptible fish are characterized by a much larger, yet ineffective, immune response, largely related to cytokine and inflammatory systems. Resistant fish demonstrate a more moderate, putative macrophage-mediated inflammatory response, which may contribute to their survival.

Survival of viral haemorrhagic septicaemia virus and infectious haematopoietic necrosis virus in the environment and dried on stainless steel.

Viral haemorrhagic septicaemia virus (VHSV) and infectious haematopoietic necrosis virus (IHNV) are important viral pathogens posing a serious threat to salmonid fish. Survival of two isolates of IHNV and one of VHSV was assessed at temperatures ranging from 4 to 25°C: (a) after drying on stainless steel, (b) in cell culture medium, (c) in filtered river water, (d) in unfiltered river water, and (e) survival, adsorption and desorption in river sediment and five typical soil types. The viruses survived 1 hr to > 84 days depending on the conditions. Survival was inversely related to temperature and organic and inorganic content. Both viruses remained infectious after being dried on stainless steel for several weeks highlighting the risk of mechanical transmission and persistence in a dry environment. Both adsorbed to the soils from the river water inoculum, with titres between 5.56x104 and 2.58x108 TCID50 /ml after 1 hr. Clay soils adsorbed the least virus but had the greatest decrease in the river water inoculum (undetectable in ≤ 1 hr), and there was no desorption. Virus desorbed from the other soils into the surrounding water at different rates dependant on soil type (longest desorption was from chalk loam and sandy soil-detected at 28 days). When desorption was no longer detectable, virus persisted, adsorbed to the soil and remained infectious (the longest adsorption was detected in clay loam for ≥ 49 days, but all the viruses adsorbed to soils were likely to have survived longer than that detected, based on their rate of decay). The long survival of the viruses, particularly at cooler temperatures, highlights the risk of survival in the environment and waterborne spread. The data presented here are highly relevant for assessing risk of pathogen introduction via fomites (stainless steel) and for deciding on best control measures in the context of disease outbreaks.

Effects of lipoic acid and dihydrolipoic acid on total erythrocytic thiols under conditions of restricted glucose in vitro.

The effects of lipoic acid and dihydrolipoic acid were explored on total thiol maintenance in diabetic and non-diabetic human erythrocytes in vitro over 22 hr in a 37 degrees C incubation system with no added glucose. Over 18-22.5 hr after treatment in both non-diabetic and diabetic cells, lipoic acid (1 mM) was associated with greater loss of cellular thiols than dihydrolipoic acid (1 mM), compared to respective control values. At 0.1 mM, in non-diabetic cells, although lipoic acid-treated cells' thiol levels were significantly lower than control, there was no significant difference between dihydrolipoic acid-treated cells and control cells regarding thiol levels. In addition, at 0.1 mM, dihydrolipoic acid-treated diabetic cells showed a reduction in thiol levels compared to control. At 0.01 mM, lipoic acid-treated cells had significantly lower measured thiol levels compared with diabetic cells exposed to dihydrolipoic acid, whereas in non-diabetic cells, dihydrolipoic acid-treated erythrocytic thiol levels were significantly greater than those treated with lipoic acid, although there were no other significant differences between the groups. At 22.5 hr, control values of methaemoglobin rose to 6.4 +/- 1.1% in diabetic cells and 3.6 +/- 2.1% in non-diabetic cells. Lipoic acid (1 mM) showed greater methaemoglobin formation in diabetic rather than non-diabetic cells (13.6 +/- 1.5% versus 11.6 +/- 1.5%), whereas dihydrolipoic acid-treated diabetic and non-diabetic cells were less potent in methaemoglobin generation (8.5 +/- 2.4% and 8.4 +/- 1.4%, respectively). These studies suggest that in certain circumstances such as hypoglycaemia, lipoic acid administration may actually be detrimental to cellular oxidant protection status.

Puffy Skin Disease Is an Emerging Transmissible Condition in Rainbow Trout Oncorhynchus mykiss Walbaum.

The transmission of puffy skin disease (PSD) to rainbow trout Oncorhynchus mykiss Walbaum was tested in the laboratory by conducting co-habitation challenges with puffy skin (PS)-affected fish (Trojans) collected from the field. Two separate challenges were conducted using Trojans sourced from two different sites and diploid (first trial) or triploid (second trial) naïve fish. PSD-specific clinical signs were observed in both groups of naïve fish, with 66% of the fish sampled during the challenges showing signs of varying severity. The first clinical features of PSD were presented as white oval skin patches on one or both flanks 15-21 days post-challenge (dpc). The extent of the lesions ranged from 10 to 90% of the body surface, depending on the severity of the lesion. Both the severity and number of affected fish increased during the challenge. Macroscopically, oedema of the skin and multifocal petechial haemorrhaging were observed towards the end of the trials. Abnormal fish behaviour consisting of "flashing" and excessive mucous production was noted from 15 dpc onwards. Fish with severe PSD lesions also displayed inappetence and associated emaciation. Rodlet cells were observed in 41% of the fresh skin scrapes analysed from the second trial. Histologically epidermal oedema was observed in 31% of the naive fish showing gross pathology, with additional 12% displaying epidermal hyperplasia, mostly observed at the end of the challenge. Other concomitant features of the PSD lesions in challenged fish were epithelial erosion and sloughing, and occasionally mild or focal inflammation. No consistent pathology of internal organs was observed. The parasites Ichthyophthirius multifiliis and Ichthyobodo necator were observed in skin samples of a proportion of naïve challenged fish and in Trojans but not in control fish. The presence of these and other known fish pathogens in the skin of PSD-fish was confirmed by high-throughput sequencing analysis. In summary, we have demonstrated that PSD is a transmissible condition. However, even though a number of known fish pathogens were identified in the skin tissues of PSD-fish, the actual causative infectious agent(s) remain(s) unknown.

High prevalence of multidrug-tolerant bacteria and associated antimicrobial resistance genes isolated from ornamental fish and their carriage water.

BACKGROUND: Antimicrobials are used to directly control bacterial infections in pet (ornamental) fish and are routinely added to the water these fish are shipped in to suppress the growth of potential pathogens during transport. METHODOLOGY/PRINCIPAL FINDINGS: To assess the potential effects of this sustained selection pressure, 127 Aeromonas spp. isolated from warm and cold water ornamental fish species were screened for tolerance to 34 antimicrobials. Representative isolates were also examined for the presence of 54 resistance genes by a combination of miniaturized microarray and conventional PCR. Forty-seven of 94 Aeromonas spp. isolates recovered from tropical ornamental fish and their carriage water were tolerant to > or =15 antibiotics, representing seven or more different classes of antimicrobial. The quinolone and fluoroquinolone resistance gene, qnrS2, was detected at high frequency (37% tested recent isolates were positive by PCR). Class 1 integrons, IncA/C broad host range plasmids and a range of other antibiotic resistance genes, including floR, bla(TEM-1), tet(A), tet(D), tet(E), qacE2, sul1, and a number of different dihydrofolate reductase and aminoglycoside transferase coding genes were also detected in carriage water samples and bacterial isolates. CONCLUSIONS: These data suggest that ornamental fish and their carriage water act as a reservoir for both multi-resistant bacteria and resistance genes.