Experimental infection trials with European North Atlantic ranavirus (Iridoviridae) isolated from lumpfish (Cyclopterus lumpus, L.).

European North Atlantic ranavirus (ENARV, Iridoviridae), is a ranavirus species recently isolated from lumpfish (Cyclopterus lumpus, L.), which are used as cleaner fish in Atlantic salmon (Salmo salar) farming in Northern Europe. This study aimed to investigate (1) the virulence of ENARV isolates from Ireland, Iceland and the Faroe Islands to lumpfish; (2) horizontal transmission between lumpfish; and (3) virulence to Atlantic salmon parr. Lumpfish were challenged in a cohabitation model using intraperitoneally (IP) injected shedders, and naïve cohabitants. IP challenge with isolates from Iceland (1.9 × 107 TCID50  ml-1 ) and the Faroe Islands (5.9 × 107 TCID50  ml-1 ) reduced survival in lumpfish, associated with consistent pathological changes. IP challenge with the Irish strain (8.6 × 105 TCID50  ml-1 ) did not significantly reduce survival in lumpfish, but the lower challenge titre complicated interpretation. Horizontal transmission occurred in all strains tested, but no clinical impact was demonstrated in cohabitants. Salmon parr were challenged by IP injection with the Irish isolate, no virulence or virus replication were demonstrated. A ranavirus qPCR assay, previously validated for fish ranaviruses, was first used to detect ENARV in tissues of both in lumpfish and Atlantic salmon. This study provides the first data on the assessment of virulence of ENARV isolates to lumpfish and salmon, guidelines for the diagnosis of ENARV infection, and poses a basis for further investigations into virulence markers.

Multilocus sequence analysis reveals different lineages of Pseudomonas anguilliseptica associated with disease in farmed lumpfish (Cyclopterus lumpus L.).

The bacterium Pseudomonas anguilliseptica has in recent years emerged as a serious threat to production of lumpfish in Norway. Little is known about the population structure of this bacterium despite its association with disease in a wide range of different fish species throughout the world. The phylogenetic relationships between 53 isolates, primarily derived from diseased lumpfish, but including a number of reference strains from diverse geographical origins and fish species, were reconstructed by Multi-Locus Sequence Analysis (MLSA) using nine housekeeping genes (rpoB, atpD, gyrB, rpoD, ileS, aroE, carA, glnS and recA). MLSA revealed a high degree of relatedness between the studied isolates, altough the seven genotypes identified formed three main phylogenetic lineages. While four genotypes were identified amongst Norwegian lumpfish isolates, a single genotype dominated, irrespective of geographic origin. This suggests the existence of a dominant genotype associated with disease in production of lumpfish in Norwegian aquaculture. Elucidation of the population structure of the bacterium has provided valuable information for potential future vaccine development.

Mortality and skin pathology of farmed Atlantic salmon (Salmo salar) caused by exposure to the jellyfish Physalia physalis in Ireland.

In October 2019, during Tropical Storm Lorenzo, an unusual bloom of Physalia physalis (commonly known as the Portuguese man o' war) appeared on the west coast of Ireland. The jellyfish were observed aggregating in high numbers surrounding salmon pens on two Atlantic salmon farms with tentacles extending underwater through the nets into the pens. Acute skin lesions and mortality were reported as a consequence of exposure to P. physalis on both sites. The impact of noxious zooplankton species such as P. physalis may pose a significant and more frequent threat in future if increased adverse weather events continue to transport these and other jellyfish outside their normal range. This is the first published record of Physalia physalis causing mortality in cultured fish.

Lumpfish (Cyclopterus lumpus, Linnaeus) is susceptible to viral nervous necrosis: Result of an experimental infection with different genotypes of Betanodavirus.

In recent years, the use of cleaner fish for biological control of sea lice has increased considerably. Along with this, a number of infectious diseases have emerged. The aim of this study was to investigate the susceptibility of lumpfish (Cyclopterus lumpus) to Betanodavirus since it was detected in asymptomatic wild wrasses in Norway and Sweden. Three betanodaviruses were used to challenge lumpfish: one RGNNV genotype and two BFNNV genotypes. Fish were injected and monitored for 4 weeks. Brain samples from clinically affected specimens, from weekly randomly selected fish and survivors were subjected to molecular testing, viral isolation, histopathology and immunohistochemistry. Reduced survival was observed but was attributed to tail-biting behaviour, since no nervous signs were observed throughout the study. Betanodavirus RNA was detected in all samples, additionally suggesting an active replication of the virus in the brain. Viral isolation confirmed molecular biology results and revealed a high viral titre in BFNNV-infected groups associated with typical lesions in brains and eyes of survivor fish. We concluded that lumpfish are susceptible to Betanodavirus, as proven by the high viral titre and brain lesions detected, but further studies are necessary to understand if Betanodavirus can cause clinical disease in this species.

Biogeography of the fish pathogen Aeromonas salmonicida inferred by vapA genotyping.

A recently described typing system based on sequence variation in the virulence array protein (vapA) gene, encoding the A-layer surface protein array, allows unambiguous subtyping of Aeromonas salmonicida. In the present study, we compile A-layer typing results from a total of 675 A. salmonicida isolates, recovered over a 59-year period from 50 different fish species in 26 countries. Nine novel A-layer types (15-23) are identified, several of which display a strong predilection towards certain fish hosts, including e.g. Cyprinidae and Pleuronectidae species. Moreover, we find indications that anthropogenic transport of live fish may have aided the near global dissemination of two cyprinid-associated A-layer types. Comparison of whole genome phylogeny and A-layer typing for a subset of strains further resulted in compatible tree topologies, indicating the utility of vapA as a phylogenetic as well as an epizootiological marker in A. salmonicida. A Microreact project (microreact.org/project/r1pcOAx9m) has been created, allowing public access to the vapA analyses and relevant metadata. In sum, the results generated provide valuable insights into the global population structure of A. salmonicida, particularly in relation to its piscine host spectrum and the geographic distribution of these hosts.

Langerhans Cells Suppress CD49a+ NK Cell-Mediated Skin Inflammation.

Recruitment of innate immune effector cells into sites of infection is a critical component of resistance to pathogen infection. Using a model of intradermal footpad injection of Candida albicans, we observed that inflammation as measured by footpad thickness and neutrophil recruitment occurred independent of adoptive immunity but was significantly reduced in MyD88(-/-) and IL-6(-/-) mice. Unexpectedly, huLangerin-DTA mice (ΔLC) that lack Langerhans cells (LC) developed increased skin inflammation and expressed higher amounts of IL-6, suggesting a suppressive role for LC. Increased inflammation also occurred in Rag1(-/-) ΔLC mice but was reversed by Ab-mediated ablation of NK cells. CXCR6(+)CD49a(+) NK cells are a liver-resident subset that can mediate inflammatory skin responses. We found that exaggerated skin inflammation was absent in ΔLC × CXCR6(-/-) mice. Moreover, the exaggerated response in ΔLC mice could be adoptively transferred with liver CD49a(+) NK cells. Finally, CD49a(+) NK cells in ΔLC but not control mice were recruited to the skin, and inhibition of their recruitment prevented the exaggerated response. Thus, in the absence of LC, CD49a(+) liver NK cells display an inappropriately proinflammatory phenotype that results in increased local skin inflammation. These data reveal a novel function for LC in the regulation of this recently described subset of skin tropic NK cells.

Immune mediated shaping of microflora community composition depends on barrier site.

Barrier surfaces, such as the intestinal lining and the skin, are colonized by a diverse community of commensal microorganisms. Although commensal microorganisms clearly impact the host immune system, whether the immune system also shapes the commensal community is poorly understood. We used 16S rDNA deep sequencing to test whether mice with specific immune defects have an altered commensal microflora. Initially, skin swabs were obtained from wild-type and Langerhans Cell (LC) deficient mice. Despite the intimate contacts that LC make with the upper epidermis, no significant differences were observed in microbial community composition. Similarly, the skin of MyD88/TRIF(-/-), Rag1(-/-) and heterozygous littermate controls showed no alteration in their commensal communities. Next we examined mouth swabs and feces. We did not find a difference in the MyD88/TRIF(-/-) mice. However, we did observe a significant shift in the microbial composition in the feces and mouths of Rag1(-/-) mice. Thus, we conclude that the adaptive immune system modulates the microbial composition at mucosal surfaces in the steady-state but LC, adaptive immunity, and MyD88-dependent innate responses do not affect the skin microbiome revealing a major distinction between barrier sites.

Noncovalent assembly of anti-dendritic cell antibodies and antigens for evoking immune responses in vitro and in vivo.

Targeting of Ags directly to dendritic cells (DCs) through anti-DC receptor Ab fused to Ag proteins is a promising approach to vaccine development. However, not all Ags can be expressed as a rAb directly fused to a protein Ag. In this study, we show that noncovalent assembly of Ab-Ag complexes, mediated by interaction between dockerin and cohesin domains from cellulose-degrading bacteria, can greatly expand the range of Ags for this DC-targeting vaccine technology. rAbs with a dockerin domain fused to the rAb H chain C terminus are efficiently secreted by mammalian cells, and many Ags not secreted as rAb fusion proteins are readily expressed as cohesin directly fused to Ag either via secretion from mammalian cells or as soluble cytoplasmic Escherichia coli products. These form very stable and homogeneous complexes with rAb fused to dockerin. In vitro, these complexes can efficiently bind to human DC receptors followed by presentation to Ag-specific CD4⁺ and CD8⁺ T cells. Low doses of the HA1 subunit of influenza hemagglutinin conjugated through this means to anti-Langerin rAbs elicited Flu HA1-specific Ab and T cell responses in mice. Thus, the noncovalent assembly of rAb and Ag through dockerin and cohesin interaction provides a useful modular strategy for development and testing of prototype vaccines for elicitation of Ag-specific T and B cell responses, particularly when direct rAb fusions to Ag cannot be expressed.

ADAM10-mediated E-cadherin release is regulated by proinflammatory cytokines and modulates keratinocyte cohesion in eczematous dermatitis.

Acute eczema is an inflammatory skin disease characterized by the formation of small intraepidermal blisters, reduction of the adhesion molecule E-cadherin from the keratinocyte surface, and impaired keratinocyte cohesion. Here, we reveal that the disintegrin and metalloprotease ADAM10 is critically involved in regulating E-cadherin cell-surface expression in cultured primary human keratinocytes and in diseased human skin. Proinflammatory cytokines, transforming growth factor-beta, and lipopolysaccharide led to increased release of soluble E-cadherin by activating mitogen-activated protein kinase signaling in cultured keratinocytes. Moreover, these stimuli decreased the amount of pro-ADAM10 and increased the level of the active protease, leading to loss of E-cadherin from the cell surface and decreased keratinocyte cohesion. In situ examination and immunoblot analyses of E-cadherin and ADAM10 expression in lesional skin of eczema revealed that the reduction of E-cadherin expression in areas of blister formation closely correlated with increased level of ADAM10 expression and elevated E-cadherin shedding. Our data suggest that ADAM10-mediated E-cadherin proteolysis leads to the impaired cohesion of keratinocytes observed in eczematous dermatitis and provide previously unreported insights into the understanding of the molecular mechanisms involved in inflammatory diseases with loss in epithelial integrity.

Constitutive expression and regulated release of the transmembrane chemokine CXCL16 in human and murine skin.

The CXC-chemokine ligand 16 (CXCL16) is expressed as a transmembrane adhesion molecule and can be released as a chemoattractant. Both functions are carried out by binding of CXCL16 to its receptor, CXC-chemokine receptor 6 (CXCR6). We here provide early evidence that CXCL16 is expressed in situ by epidermal keratinocytes of normal skin on messenger RNA and protein level and released into the wound exudate upon injury. Cultured human and murine keratinocyte cell lines (HaCaT and PAM212, respectively) as well as primary keratinocyte cultures constitutively express transmembrane CXCL16 on the cell surface. Soluble CXCL16 is released by its limited proteolytic cleavage involving the disintegrin-like metalloproteinase (ADAM)10 but not the closely related ADAM17, as shown by specific inhibitors and small-interfering RNA knockdown experiments. This shedding of CXCL16 is reduced by serum starvation but enhanced by cell stimulation with ionomycin or by UVB irradiation. Soluble CXCL16 from keratinocytes was shown to bind and activate CXCR6, and marked expression of this receptor was found on a subpopulation of T cells in the dermis. Thus, CXCL16 is constitutively expressed on the surface of human epidermal keratinocytes, released upon cell activation or photodamage and may then target CXCR6-expressing T cells in the dermis.

ADAM10 mediates E-cadherin shedding and regulates epithelial cell-cell adhesion, migration, and beta-catenin translocation.

E-cadherin controls a wide array of cellular behaviors, including cell-cell adhesion, differentiation, and tissue development. We show here that E-cadherin is cleaved specifically by ADAM (a disintegrin and metalloprotease) 10 in its ectodomain. Analysis of ADAM10-deficient fibroblasts, inhibitor studies, and RNA interference-mediated down-regulation of ADAM10 demonstrated that ADAM10 is responsible not only for the constitutive shedding but also for the regulated shedding of this adhesion molecule in fibroblasts and keratinocytes. ADAM10-mediated E-cadherin shedding affects epithelial cell-cell adhesion as well as cell migration. Furthermore, the shedding of E-cadherin by ADAM10 modulates the beta-catenin subcellular localization and downstream signaling. ADAM10 overexpression in epithelial cells increased the expression of the beta-catenin downstream gene cyclin D1 dose-dependently and enhanced cell proliferation. In ADAM10-deficient mouse embryos, the C-terminal E-cadherin fragment is not generated, and the full-length protein accumulates, highlighting the in vivo relevance for ADAM10 in E-cadherin shedding. Our data strongly suggest that this protease constitutes a major regulatory element for the multiple functions of E-cadherin under physiological as well as pathological conditions.