Intramuscular vaccination of Atlantic lumpfish (Cyclopterus lumpus L.) induces inflammatory reactions and local immunoglobulin M production at the vaccine administration site.

Atlantic lumpfish were vaccinated by intramuscular (im) or intraperitoneal (ip) injection with a multivalent oil-based vaccine, while control fish were injected with phosphate-buffered saline. Four lumpfish per group were sampled for skin/muscle and head kidney tissue at 0, 2, 7, 21 and 42 days post-immunization (dpi) for histopathology and immunohistochemistry (IHC). Gene expressions of secretory IgM, membrane-bound IgM, IgD, TCRα, CD3ε and MHC class IIß were studied in tissues by using qPCR. Im. vaccinated fish showed vaccine-induced inflammation with formation of granulomas and increasing number of eosinophilic granulocyte-like cells over time. On IHC sections, we observed diffuse intercellular staining of secretory IgM at the injection site at 2 dpi, while IgM + cells appeared in small numbers at 21 and 42 dpi. Skin/muscle samples from im. vaccinated fish demonstrated an increase in gene expression of IgM mRNA (secretory and membrane-bound) at 21 and 42 dpi and small changes for other genes. Our results indicated that im. vaccination of lumpfish induced local IgM production at the vaccine injection site, with no apparent proliferation of IgM + cells. Eosinophilic granulocyte-like cells appeared shortly after im. injection and increased in numbers as the inflammation progressed.

Vibrio tapetis from wrasse used for ectoparasite bio-control in salmon farming: phylogenetic analysis and serotyping.

So-called 'cleaner fish', including various wrasse (Labridae) species, have become increasingly popular in Norwegian salmon farming in recent years for biocontrol of the salmon louse Lepeophtheirus salmonis. Cleaner fish mortalities in salmon farms are, however, often high. Various bacterial agents are frequently associated with episodes of increased cleaner fish mortality, and Vibrio tapetis is regularly cultured from diseased wrasse. In the present study, we investigated the genetic relationships among 54 V. tapetis isolates (34 from wrasse species) by multilocus sequence analysis (MLSA; rpoD, ftsZ, pyrH, rpoA and atpA). In the resulting phylogenetic tree, all wrasse isolates belonged to sub-clusters within V. tapetis subsp. tapetis. Slide agglutination testing further confirmed the complete dominance amongst these isolates of 4 O-antigen serotypes, designated here as V. tapetis subsp. tapetis serotypes O1, O3, O4 and O5, respectively. A pilot challenge trial using serotypes O3, O4 and O5 did not indicate high pathogenicity towards ballan wrasse Labrus bergylta, thus questioning the role of V. tapetis as a primary pathogen of this fish species.

Phylogenetic analysis and serotyping of Vibrio splendidus-related bacteria isolated from salmon farm cleaner fish.

Cleaner fish, i.e. various wrasse (Labridae) species and lumpsucker Cyclopterus lumpus, are to an increasing extent used for biocontrol of the salmon louse Lepeophtheirus salmonis in European salmon farming. Although efficient de-licers, cleaner fish mortality levels in salmon farms are often high. Bacterial infections are common, and Vibrio splendidus-related strains are frequently identified during diagnostic investigations. The population structure of 112 V. splendidus-related isolates, derived primarily from wrasse species, was investigated by means of multilocus sequence analysis using 5 housekeeping genes (rpoD, ftsZ, pyrH, rpoA and atpA). Most isolates were found to be closely related to the V. splendidus type strain, yet displayed extensive genetic microdiversity. Slide agglutination testing using polyclonal rabbit antisera further indicated O-antigen variability. Intra-outbreak genetic and antigenic diversity suggests direct infection from seawater, rather than fish-to-fish transmission, as the main route of infection. The variable nature of isolates involved complicates qualified selection of representative candidate strains, e.g. for infection and vaccine trials.

Mast cells in common wolffish Anarhichas lupus L.: ontogeny, distribution and association with lymphatic vessels.

The morphology, ontogeny and tissue distribution of mast cells were studied in common wolffish(Anarhichas lupus L.) at the larval, juvenile and adult life stages using light and electron-microscopy and immunohistochemistry. Fish were sampled at 1 day, 1, 2, 3, 4, 8 and 12 weeks post-hatching in addition to 6 and 9 months and 2 years and older. From 8 weeks post-hatching, mast cells in common wolffish mainly appeared as oval or rounded cells 8-15 mm in diameter with an eccentrically placed, ovoid nucleus and filled with cytoplasmic granules up to 1.2 mm in diameter. Granules were refractile and eosinophilic to slightly basophilic in H&E and stained bright red with Martius-scarlet-blue and purple with pinacyanol erythrosinate in formalin-fixed tissues. Mast cells stained positive for piscidin 4 and Fc ε RI by immunohistochemistry. From 1 day to 4 weeks post-hatching, immature mast cell containing only a few irregularly sized cytoplasmic granules were observed by light and electron-microscopy in loose connective tissue of cranial areas. From 1 day post-hatching, these cells stained positive for piscidin 4 and Fc ε RI by immunohistochemistry. From 12 weeks post-hatching, mast cells showed a primarily perivascular distribution and were particularly closely associated with lymphatic vessels and sinuses. Mast cells were mainly located at the peripheral border of the adventitia of arteries and veins, while they were in intimate contact with the endothelium of the lymphatic vessels. Numerous mast cells were observed in the intestine. A stratum compactum, as described in salmonids, was not observed in wolffish intestine,nor were mast cells confined to a separate layer, a stratum granulosum. Lymphatic vessels consisting of endothelium, intimal connective tissue and a poorly developed basal lamina were observed in the intestine. Scanning electron microscopy was used to compare the structure and localization of intestinal mast cells of common wolffish and rainbow trout. Scanning electron microscopy also revealed endothelial surface features and confirmed the existence of three distinctly different types of vessels in the wolffish intestine. Rainbow trout mast cell granules appeared as intact globular structures while empty vacuoles were observed in common wolffish. Mast cells were closely associated with lymphatic vessels in common wolffish, but not in rainbow trout.

Sex-dependent effects of mechanical delousing on the skin microbiome of broodstock Atlantic salmon (Salmo salar L.).

Delousing strategies, including mechanical delousing, are typically used to treat Atlantic salmon (Salmo salar) sea lice infestations. In this study, we evaluate the impact of mechanical delousing (Hydrolicer) on the skin bacterial microbiome of broodstock female and male Atlantic salmon. 16S rDNA sequencing of salmon skin microbial communities was performed immediately before delousing, right after delousing and 2 and 13 days post-delousing (dpd). The skin bacterial community of female salmon was more diverse than that of males at the start of the experiment. Overall, hydrolycer caused losses in alpha diversity in females and increases in alpha diversity in males. Hydrolicer also caused rapid shifts in the skin microbial community composition immediately after delicing in a sex-specific manner. There was a decrease in abundance of Proteobacteria and Bacteriodetes in both female and male salmon, whereas Firmicutes and Tenericutes abundances increased. Interestingly, the female community recovered faster, while the male community remained dysbiotic 13 dpd due to expansions in Bacteroidetes (Pseudomonadaceae) and Firmicutes. Our data suggest that female broodstock are more resilient to Hydrolicer treatment due to their more diverse skin microbiota community, and that sex influences the skin microbial community and therefore host health outcomes during common farming manipulations.

Flavobacterium psychrophilum associated with septicaemia and necrotic myositis in Atlantic salmon Salmo salar: a case report.

We describe the first case from Norway of increased mortality in Atlantic salmon Salmo salar (L.), with septicaemia and necrotic myositis, associated with infection by Flavobacterium psychrophilum. The outbreak occurred in smolt of 60 to 100 g in fresh water on a land-based farm in Western Norway during winter 2008-2009. The water temperature was < 5 degrees C and the accumulated mortality was 7.0%. Necropsy of dead and moribund fish revealed a swollen dark spleen, pale liver, serohaemorrhagic ascites and haemorrhage in the abdominal fat and muscle. F. psychrophilum was isolated from the kidney and spleen of diseased fish. Muscle biopsy revealed the presence of long filamentous rods in necrotic areas of skeletal muscle. Immunohistochemistry was positive for F. psychrophilum. Identification of cultured isolates as F. psychrophilum was confirmed using phenotypic testing and sequencing of the 16S rRNA gene. Analysis by allele-specific polymerase chain reaction (allele-specific PCR) indicated that 2 different genotypes of the bacterium were present in the outbreak.

Short-term ANG II produces renal vasoconstriction independent of TP receptor activation and TxA2/isoprostane production.

The relative contributions of vasoconstrictor and of dilator systems are balanced in health. The balance is reset in disease, often favoring a predominant role of vasoconstrictors, perhaps due to positive interactions between constrictor systems. For example, in hypertension, chronic high levels of angiotensin II (ANG II) stimulate the production of thromboxane (TxA2/PGH2) and/or isoprostane that activate constrictor thromboxane prostanoid (TP) receptors in the vasculature. The present study evaluated a modest concentration of ANG II administered acutely into the renal artery on urinary excretion of TxB2 and isoprostane and possible renal TP receptor activation that might amplify ANG II-induced renal vasoconstriction. TP receptors were blocked with SQ29548 coinfused with ANG II. Results were compared with a time control group of continuous ANG II infusion (40 ng.min(-1).kg body wt(-1)) over 90 min. TP receptor antagonism during 30-60 min had no effect on the reduction in renal blood flow (RBF) produced by ANG II (15.8 +/- 2.8 vs. 13.2 +/- 4.9%) (P > 0.6). Likewise, there was no difference between groups during ANG II-induced renal vasoconstriction between 60-90 min in presence or absence of TP receptor antagonist (RBF -8.6 +/- 4.0 vs. -9.6 +/- 4.5%) (P > 0.8). Systemic arterial pressure was stable throughout, so RBF changes reflected localized changes in renal vascular resistance. Urinary excretion of TxB2 and isoprostane were nearly doubled by ANG II. The present data indicate that short-term intrarenal infusion of ANG II rapidly increases renal production of TxA2 but that the ANG II-induced renal vasoconstriction is independent of TP receptor activation during the initial 90 min of local challenge with ANG II.

Angiotensin II-induced calcium signaling in the afferent arteriole from rats with two-kidney, one-clip hypertension.

The aim of this study was to investigate ANG II-induced Ca2+ signaling in freshly isolated afferent arterioles (AA) from two-kidney, one-clip hypertensive (2K1C) rats, which have an elevated plasma and renal ANG II level, and different perfusion pressure and vascular tone in the clipped and nonclipped kidney. The Ca2+ responses in vessels from 2K1C and control rats were similar in all groups (P>0.1). The intracellular Ca2+ (Cai2+) response in the afferent arteriole after 10(-8) M ANG II stimulation was 0.57+/-0.10, 0.50+/-0.07, 0.48+/-0.04, and 0.36+/-0.05 in the control, sham, nonclipped, and clipped kidney, respectively. These data were consistent with the finding of unchanged AT(1a)R mRNA levels in AAs from all groups. Although the absolute values were similar, the dose-response curves to ANG II were different. In the control, sham, and nonclipped kidney from 2K1C, the dose-response curve leveled off between 10(-8) and 10(-6) M ANG II. In the clipped kidney, the dose-response curve was linear, with a significantly increased response at 10(-6) M compared with 10(-8) M ANG II (P<0.05). Inhibition of cyclooxygenase-1 (COX-1) with indomethacin enhanced the ANG II response in the nonclipped (Delta0.30+/-0.09) and clipped (Delta0.30+/-0.09) kidneys from 2K1C (P<0.005), but not in control rats (Delta-0.02+/-0.11, P>0.8). Conclusively, the ANG II-induced Cai2+ response was reduced by COX-1-derived prostaglandins in 2K1C, in contrast to control animals, where the COX-1 inhibition had no effect. COX-2 inhibition with NS-398 did not increase the ANG II-mediated Cai2+ response in any of the groups.

Prevention of hypertension and organ damage in 2-kidney, 1-clip rats by tetradecylthioacetic acid.

Dietary lipids are reported to affect the blood pressure in both humans and experimental animal models with hypertension. In the present study, 2-kidney, 1-clip (2K1C) hypertensive rats were treated with the modified fatty acid tetradecylthioacetic acid (TTA) from the time of clipping or after hypertension was established. TTA treatment attenuated the development of hypertension and reduced established 2K1C hypertension. The mRNA level of renin in the clipped kidney and the plasma renin activity were markedly reduced, and the plasma angiotensin II level tended to decrease after TTA treatment. In addition, TTA reduced the mRNA level of angiotensinogen in white adipose tissue. Prevention of organ damage was demonstrated by normal urinary excretion of protein, maintained serum albumin, lower heart weight, and clearly reduced vascular, glomerular, and tubulointerstitial damage in the nonclipped kidney. Renal function was not affected as estimated by unchanged plasma creatinine. Furthermore, the serum levels of triacylglycerol and cholesterol were reduced by TTA. The serum fatty acid composition was changed, resulting in a favorable increase of oleic acid. However, the levels of all of the omega-3 fatty acids and of linoleic acid were reduced, and no change was seen in the level of arachidonic acid, but the urinary excretion of 8-iso-prostaglandin F2alpha was declined. In conclusion, TTA attenuated the development of hypertension, reduced established hypertension, and prevented the development of organ damage in 2K1C rats, possibly by reducing the amounts of the vasoconstrictors angiotensin II and 8-iso-prostaglandin F2alpha and by inducing a favorable increase of oleic acid in serum.

Enhanced response to AVP in the interlobular artery from the spontaneously hypertensive rat.

Arginine vasopressin (AVP) induces exaggerated intracellular free calcium (Cai2+) responses in preglomerular smooth muscle cells from young spontaneously hypertensive rats (SHR) due to increased density of the AVP V1a receptor. The intention of the present paper was to examine the relative contribution of afferent arterioles (AA) and interlobular artery (ILA) in AVP- and norepinephrine-induced calcium signaling. The kidneys were perfused with agar solution in vivo, and thin cortical slices were enzyme digested to produce isolated agar-filled vascular fragments. Calcium responses were recorded in fura 2-loaded cells by Ca2+ imaging. Diameter changes were measured after AVP stimulation and mRNA for V1a was measured on isolated vessel fragments. SHR had a significantly higher baseline calcium ratio and lower resting diameter compared with normotensive Wistar-Kyoto rats (WKY). Stimulation with AVP (10(-7) M) in ILA fragments from SHR induced a ratio increase of 0.49 +/- 0.09, significantly higher than the ratio increase in AA from SHR (0.20 +/- 0.03, P < 0.01) and in ILA from WKY (0.24 +/- 0.03, P < 0.01). Stimulation with norepinephrine (10(-7) M) induced responses homogeneously distributed between the segments and strains. Nifedipine treatment or removal of external calcium (Cao2+) reduced the norepinephrine-induced peak response. Both norepinephrine- and AVP-induced sustained responses were abolished after Cao2+ removal in SHR and WKY (P < 0.01). Measurements of V1a receptor mRNA on isolated segments showed a threefold increase in ILA from SHR. The present findings indicate that the exaggerated Ca2+ and contractile response to AVP in SHR is mainly mediated through ILA vasoconstriction.

The renal vascular response to ANG II injection is reduced in the nonclipped kidney of two-kidney, one-clip hypertension.

The ANG II receptor 1 (AT(1)R) level in the nonclipped kidney of two-kidney, one-clip hypertension (2K1C) has shown to be unchanged despite a high circulating angiotensin (ANG) II level. To examine the vasoreactive response to ANG II in this kidney, injections of ANG II into renal artery were performed 6 wk after clipping of the kidney and compared with normotensive controls. The renal blood flow (RBF) response to 2.5 ng ANG II was measured by a Transonic transit-time flowmeter, before and after indomethacin and candesartan treatment, and analyzed by a computer program. The RBF response to 5 ng arginine-vasopressin (AVP) was examined for comparison with ANG II. The mRNA for AT(1A) and AT(1B) as well as Western blotting for AT(1)R in renal resistance vessels were determined, and plasma renin activity (PRA) was measured. Systolic blood pressure was 183 +/- 4 mmHg in 2K1C rats compared with 113 +/- 1 mmHg in controls (P < 0.001). PRA was significantly increased in 2K1C animals (P < 0.05). Injection of ANG II reduced RBF with 10 +/- 2% in the nonclipped kidney and 24 +/- 3% in controls (P < 0.001). After indomethacin, the RBF response increased from 10 +/- 2 to 20 +/- 3% (P < 0.02) in 2K1C rats and from 24 +/- 3 to 34 +/- 6% in controls (P < 0.01). The doses of candesartan needed to completely inhibit RBF response to ANG II were 30 microg/kg in the nonclipped kidney and 100 microg/kg in controls (P < 0.001). Western blotting and mRNA for AT(1A) and AT(1B) in the nonclipped kidney were similar to the controls. The results indicate that despite no difference in total AT(1)R levels, functional AT(1)R is downregulated in the nonclipped kidney of 2K1C rats.

Enhanced Ca2+ response to AVP in preglomerular vessels from rats with genetic hypertension during different hydration states.

Exaggerated arginine vasopressin (AVP)-induced calcium signaling and renal vasoconstriction, characteristic in young spontaneously hypertensive rats (SHR) during euvolemia, are related to greater amounts of V1a receptor mRNA and V1a protein in preglomerular resistance arterioles. The present study determined whether V1a receptor density and calcium signal transduction in the renal vasculature of young SHR is regulated appropriately during physiological changes in hydration state. [3H]AVP ligand binding documented two- to threefold greater density of V1a receptors in euvolemic SHR vs. Wistar-Kyoto (WKY) rats. Parallel changes in V1a receptor density were observed in both strains during chronic water loading (plus approximately 50 fmol/mg) and during dehydration (minus approximately 50 fmol/mg). Affinity was unchanged. Real-time RT-PCR demonstrated that V1a mRNA in preglomerular arterioles was three times greater in euvolemic SHR. Dehydration decreased expression approximately 50% in renal vessels independent of rat strain; water loading increased V1a mRNA. Thus V1a receptor regulation correlated with changes in mRNA in a normal manner in response to chronic changes in AVP concentration, albeit set at a higher level in SHR. In dehydrated animals, AVP increased the cytosolic Ca2+ concentration ([Ca2+]i) by 60 +/- 5 and 112 +/- 13 nM cytosolic Ca2+ in WKY and SHR, respectively (P < 0.01), whereas in hydrated animals the [Ca2+]i increase was 168 +/- 10 and 220 +/- 18 nM, respectively (P < 0.05). In all hydration states, calcium signaling was greater in SHR compared with WKY (P < 0.05). Calcium signaling paralleled changes in the receptor density and mRNA. Mechanisms other than hydration state per se are likely to be responsible for the two- to threefold difference in the V1a receptor density between WKY and SHR in the renal vasculature at the critical age of 6 wk.