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Objective: To test if patients recovering from COVID-19 are at increased risk of mental morbidities and to what extent such risk is exacerbated by illness severity., Design: Population-based cross-sectional study., Setting: Iceland., Participants: A total of 22 861 individuals were recruited through invitations to existing nationwide cohorts and a social media campaign from 24 April to 22 July 2020, of which 373 were patients recovering from COVID-19., Main Outcome Measures: Symptoms of depression (Patient Health Questionnaire), anxiety (General Anxiety Disorder Scale) and posttraumatic stress disorder (PTSD; modified Primary Care PTSD Screen for DSM-5) above screening thresholds. Adjusting for multiple covariates and comorbidities, multivariable Poisson regression was used to assess the association between COVID-19 severity and mental morbidities., Results: Compared with individuals without a diagnosis of COVID-19, patients recovering from COVID-19 had increased risk of depression (22.1% vs 16.2%; adjusted relative risk (aRR) 1.48, 95% CI 1.20 to 1.82) and PTSD (19.5% vs 15.6%; aRR 1.38, 95% CI 1.09 to 1.75) but not anxiety (13.1% vs 11.3%; aRR 1.24, 95% CI 0.93 to 1.64). Elevated relative risks were limited to patients recovering from COVID-19 that were 40 years or older and were particularly high among individuals with university education. Among patients recovering from COVID-19, symptoms of depression were particularly common among those in the highest, compared with the lowest tertile of influenza-like symptom burden (47.1% vs 5.8%; aRR 6.42, 95% CI 2.77 to 14.87), among patients confined to bed for 7 days or longer compared with those never confined to bed (33.3% vs 10.9%; aRR 3.67, 95% CI 1.97 to 6.86) and among patients hospitalised for COVID-19 compared with those never admitted to hospital (48.1% vs 19.9%; aRR 2.72, 95% CI 1.67 to 4.44)., Conclusions: Severe disease course is associated with increased risk of depression and PTSD among patients recovering from COVID-19., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY. Published by BMJ.)

The purpose of the present study was to study specific and natural antibody levels in individual cod juveniles before and after being vaccinated against Vibrio anguillarum. Different vaccine preparations and vaccination regimes, i.e. bathing, dipping, i.p. injection or combination of treatments were employed and the performance of different groups to bath challenge by the bacterium tested. Antibody responses to V. anguillarum antigens in groups vaccinated by bathing and/or dipping were negligible, while responses were observed in i.p. injected fish. Fish receiving i.p. injection in addition to bathing, showed significant antibody response. Both groups showed increased levels of natural antibodies while levels were low in other groups. Fish bathed or dipped showed higher mortality when challenged than untreated fish, while fish that received a second vaccination showed the best protection. It was not ascertained whether there is a long term difference between the effects of immersion versus i.p. injection as a booster method. Levels of antibodies against V. anguillarum antigens or natural antibodies in groups with the lowest mortalities show that neither could have been used to predict protection given by the vaccines tested.

In this study experimental challenges with Moritella viscosa, the causative agent of winter ulcers in salmonids, were performed on juvenile Atlantic cod and Atlantic halibut. The challenges involved both intramuscular (i.m.) and intraperitoneal (i.p.) injections and bath with a strain originally isolated from Atlantic salmon. Cod was found to be significantly more sensitive than halibut to the infection. Both fish species were found to be more sensitive to i.m. than i.p. challenges. Both challenges induced a systemic disease in cod and halibut, but only cod was infected with an experimental bath challenge. Pathognomonic signs were found to be comparable with those described in M. viscosa-infected salmon and turbot. The main distinguishing pathological sign was that the cod showed host response to M. viscosa infection resulting in granuloma formation in infected tissues, which is a known response of cod to a infection with another Gram-negative bacterium, Aeromonas salmonicida. Re-isolation of M. viscosa from kidneys of cod and halibut with clear disease signs was problematic and optimization of isolation measures is needed. The results of this study indicate that M. viscosa infection may be a risk factor in cod farming, but that halibut is more resistant.

Turbot was shown to be sensitive to injection challenges by Aeromonas salmonicida subsp. achromogenes (Asa). A systemic disease was induced and the bacterium was isolated from various internal organs. Histopathological changes involved haemorrhages, necrosis and degeneration in skin and muscle, haemorrhages and necrosis in kidney, degeneration in the heart muscle, and fusion of the secondary gill lamellae. A polyvalent commercial salmon vaccine, containing A. salmonicida subsp. salmonicida as one of five antigens, did not confer protection in turbot against an experimental Asa infection 13 weeks post-vaccination. Vaccination induced a significant antibody response against Asa cells but not against extracellular products of the bacterium. The results of the study indicate that Asa may be a potential threat to turbot farming and that the development of new turbot vaccines is needed.

Moritella viscosa is the causative agent of winter ulcers in farmed salmonids and Atlantic cod in countries around the North Atlantic. The bacterium has also been isolated from various marine fish species. Bacterial diseases have been a limiting factor in farming of turbot, but M. viscosa has not so far been isolated. In this study, turbot was shown to be sensitive to M. viscosa infection in experimental challenges. Pathological changes in infected turbot were comparable with those previously described for winter ulcers in salmon. A multivalent commercial salmon vaccine, containing M. viscosa as one of five antigens and a mineral oil adjuvant, did not protect turbot against challenge 13 weeks post-vaccination. Weight gain of vaccinated turbot compared with controls was not reduced 7 weeks post-vaccination. Vaccination did not induce a specific anti-M. viscosa response, while elevated anti-M. viscosa antibody levels were detected both in vaccinated and unvaccinated fish 5 weeks post-challenge. The vaccine did, however, induce an antibody response against Aeromonas salmonicida, another vaccine component. Minor intra-abdominal adhesions were detected in vaccinated fish and fish injected with a mineral oil adjuvant. The measurement of various innate humoral immune parameters did not reveal significant differences between vaccinated and control groups.

This open-label, nonrandomized study compared changes in hemostatic variables during NuvaRing and oral levonorgestrel 150 microg/ethinylestradiol 30 microg (LNG/EE) use for six cycles. Eighty-seven women started the study, 44 with NuvaRing and 43 with the LNG/EE oral contraceptive. For most procoagulation variables, there was no difference between NuvaRing and oral LNG/EE; only Factor VII levels increased in the NuvaRing group and decreased in the LNG/EE group. The majority of assessed variables show that anticoagulation and fibrinolytic activity was comparable between the NuvaRing and oral LNG/EE groups. Antithrombin activity and protein C activity both tended to be higher with NuvaRing. Levels of tissue plasminogen activator decreased in both groups but the reduction was smaller with NuvaRing. There were no significant differences in fibrin turnover between the treatment groups. The data show that both NuvaRing and oral LNG/EE are associated with a minimal effect on hemostatic variables., (Copyright 2004 Elsevier Inc.)

Atlantic halibut, Hippoglossus hippoglossus (L.), was shown to be sensitive to infection by three different isolates of Aeromonas salmonicida ssp. achromogenes in pre-challenge tests using intraperitoneal (i.p.) and intramuscular (i.m.) injections as well as bath challenges. A commercial furunculosis vaccine, Alphaject 1200, and an autogenous vaccine, AAS, based on the challenge strain, induced immune protection as shown in challenge tests 8 weeks post-immunization. The survival rate of vaccinated fish after i.p. challenge was 100%, whereas mortality of control fish was 61%. Employing i.m. challenge, relative percentage survival induced by the furunculosis vaccine and the AAS vaccine was 47 and 44, respectively. Mortality of i.m. injected controls was 68%. Vaccinated fish behaved normally following vaccination but the weight gain was significantly reduced in vaccinated fish 8 weeks post-vaccination compared with control fish receiving phosphate-buffered saline. At the same time, intra-abdominal adhesions were observed in fish injected with either of the two vaccines or adjuvant alone. Antibody response against A. salmonicida ssp. achromogenes was detected in sera from fish receiving either vaccine.

In this study, exotoxins produced by 62 Aeromonas salmonicida strains and the bacterium Haemophilus piscium were analysed. Enzymatic assays, zymograms and serological detection were used to monitor secretion by bacterial strains of the previously described exotoxins P1, GCAT and AsaP1 and also the extracellular P2 metallo-gelatinase and a serine caseinase, which is different from the P1 protease and has not yet been characterized. Based on the results, the strains were divided into five groups. One comprised the type strains for A. salmonicida ssp. masoucida, H. piscium and 36% of the atypical isolates, and another, a type strain for A. salmonicida ssp. smithia together with 14% of the atypical isolates. A second type strain of A. salmonicida ssp. smithia was grouped with 8% of the atypical isolates. The largest group contained the type strains for A. salmonicida ssp. achromogenes and 38% of the atypical isolates. The type strains for A. salmonicida ssp. salmonicida were in the last group with all the four typical strains and 4% of the atypical isolates. The combination of zymogram and serological detection used is recommended as the most reliable method for characterizing A. salmonicida strains according to their exotoxin secretion.

An antigen-capture enzyme-linked immunosorbent assay (cELISA) based on monoclonal antibodies (mAb) was set up and evaluated for selective detection of salmonid antibody responses to the antigen P1, which is a weakly immunogenic exoprotease of typical Aeromonas salmonicida. This new assay permits a specific determination of anti-protease-antibodies, without antigen purification. Serum antibodies induced by the strongly immunogenic lipopolysaccharide could reliably be discriminated from anti-P1-antibodies. Antibody titres of 45 experimental antisera recorded by cELISA were moderately correlated with titres determined by routinely used indirect ELISA (iELISA) by detecting partially different antibody populations (r=0.753). Substitutions of immunoreactants and confirmatory immunoblotting strongly suggest that the mAb-based assay selectively recognises antibodies directed to epitopes of native protease. A conjugate of inhibited protease and cationized bovine serum albumin (cBSA) was found to engender a significant anti-protease-response in three salmonid species (P<0.05), whereas the unconjugated antigen and Apoject 1-Fural were proved to be ineffective. Recorded specific antibody titres were as high as 1:381,400, indicating a considerable enhanced immunogenicity of cBSA-conjugated P1 and high assay sensitivity. The established cELISA offers a promising approach to further improvement of monitoring fish humoral immune response to surface accessible epitopes of the immunosuppressive exoprotease, P1, and to scrutinize its protective significance.

Balb/c mice, injected i.p. with extracellular products (ECP) of Aeromonas salmonicida subsp. achromogenes (Asa), displayed symptoms similar to toxic shock syndrome. The LD(50) observed was between 1.5 and 2.0 microg g(-1) and the mice died within 19 h. Four inflammatory cytokines were measured in mice receiving sublethal ECP doses. TNF-alpha and IL-6 showed a sharp peak in the serum while IL-1 beta and IL-2 were not detected. When peritoneal macrophages were cultivated in the presence of ECP, AsaP1 (a toxic caseinolytic metallo-protease purified from ECP) or LPS, all cultures produced TNF-alpha, IL-6 and IL-1 beta. The same antigens were mitogenic in spleen cell cultures. Furthermore, IL-2 production, which is a normal T-cell response to ConA stimulation, was downregulated in spleen cell cultures from mice injected with ECP.

Two monoclonal antibodies (Mabs) binding to a toxic extracellular metallo-proteinase of Aeromonas salmonicida subsp. achromogenes, AsaP1, were produced. Both reacted with common epitopes of the native enzyme and recognized this 20 kDa antigen on Western blots. One of these Mabs had an inhibitory effect on the caseinase activity of the exotoxin. A Mab-based ELISA was set up and evaluated for serological detection of AsaP1 in bacterial culture filtrates. The exotoxin was identified serologically in the extracellular products of 11 of 26 atypical Aer. salmonicida isolates, including the type strain for subsp. achromogenes NCIMB 1110. The ELISA was approximately 100-fold more sensitive in detecting AsaP1 compared with an azocasein assay. The established serological test enables AsaP1 to be quantified reliably with a lower detection limit of about 0.12 ng ml-1 and has a potential use for the phenotypic differentiation of atypical Aer. salmonicida isolates.

The atypical isolates of Aeromonas salmonicida are becoming increasingly important as the frequency of isolation of bacteria belonging to this group continues to rise. The primary object of this study was to compare and evaluate the results obtained in various laboratories concerning the biochemical identification of atypical Aer. salmonicida before and after standardization of media and methods. Five laboratories examined 25 isolates of Aer. salmonicida from diverse fish species and geographical locations including the reference strains of Aer. salmonicida subsp. salmonicida (NCMB 1102) and Aer. salmonicida subsp. achromogenes (NCMB 1110). Without standardization of the methods, 100% agreement was obtained only for two tests: motility and ornithine decarboxylase. The main reason for the discrepancies found was the variation of the incubation time prior to reading the biochemical reactions. After standardization, improvement was obtained with the identification; however, disagreement was still observed between the different laboratories. These findings demonstrate the difficulties involved in a proper identification of atypical Aer. salmonicida and also that data presented in the literature on various strains of Aer. salmonicida are not readily comparable. This paper seems to be the first on standardization of microbiological tests for identification of fish pathogens and the results obtained show the need for standardization of methods both within and between laboratories.

Fifty two isolates of atypical Aeromonas salmonicida, recovered from a wide range of hosts and geographical locations, were heterogeneous in terms of molecular and phenotypic characteristics, and represented taxa which could not be accommodated by the current classification of four subspecies. Generally, there was incongruence between the molecular (PCR, RAPD and ribotyping) and phenotypic methods in terms of cluster membership. By PCR, 6 groups were described of which Group 1 encompassed 12 isolates including the type strain of A. salmonicida subsp. smithia. Group 2 accommodated 23 isolates including the reference cultures of subspecies achromogenes and masoucida. The named culture of Haemophilus piscium was recovered in Group 6. By ribotyping and RAPD, the reference cultures were recovered in separate groups. All methods pointed to the uniqueness of subspecies smithia. Most isolates contained 2-6 plasmids, of 2.3 to 150 kb in length. Nevertheless, all isolates possessed certain key characteristics, including Gram-negativity, and the absence of motility.

The pathogenicity of extracellular products (ECPs) from 24 atypical Aeromonas salmonicida strains was studied with respect to: lethality in Atlantic salmon, pathogenic effect in muscle, haemolytic activity, cytotoxicity in two fish cell lines and proteolytic activities. Furthermore, the relationship between lethality of ECPs and mortality caused by bacterial challenge was examined. Correlation was demonstrated between the pathogenic properties and proteolytic activities of the ECPs. Cytolytic (GCAT) activity comparable with that of the typical reference strain used (NCMB 1102) was not detected in ECPs of any of the atypical strain tested. An extracellular metallo-caseinase, AsaP1, was linked with lethal toxicity and a strong pathogenic effect. Furuncular-like lesions were produced by ECPs containing AsaP1 activity. One strain produced a lethal toxin which was neither caseinolytic nor with GCAT comparable activity. The examined atypical strains form at least three distinct groups based on different virulence mechanisms and extracellular proteases.

The humoral antibody response of healthy Atlantic salmon and of two groups of salmon, naturally infected with Aeromonas salmonicida ssp. achromogenes, was examined in some detail. One diseased group was chronically infected and the other recently infected. It was found that the humoral response of these two infected groups was quite different. The chronically infected fish showed poor specific response to the causative agent whereas the recently infected salmon produced strong specific antibody response. The chronically infected fish showed evidence of increased unspecific response including an elevated level of natural antibodies. The specific humoral response of the recently infected fish was primarily directed against two cell-associated antigens of the A. salmonicida ssp. achromogenes bacterium, the A-layer protein and the o-polysaccharide component of LPS. In the chronically infected fish the humoral response was primarily directed against the A-layer protein.