MHC class II+ cells in the gills of Atlantic salmon (Salmo salar L.) affected by amoebic gill disease

Amoebic gill disease (AGD) is characterised by the association of Neoparamoeba sp. with hyperplastic gill tissue of affected fishes, however, the identity and role of host cells associated with AGD lesions are not known. Here, we investigated cells with an immunological role that were associated with AGD lesions by locating cellular MHC class II β chain. A tank housing Atlantic salmon (Salmo salar) was inoculated with Neoparamoeba sp., and MHC class II β chain expression in the gills was qualitatively assessed by immunohistochemistry. In AGD-naïve control fish, MHC class II⁺ cells were detected basolateral to the interlamellar epithelium as well as upon the interlamellar and secondary epithelium. In the gills of AGD affected fish MHC class II⁺ cells were observed in both affected and unaffected tissue. Within AGD lesions, numerous MHC class II⁺ cells were present and these cells exhibited variable levels of expression suggesting that like mammals, MHC class II expression is highly regulated. The presence of MHC class II⁺ cells within gill lesions is indicative of immune cell trafficking and these cells could contribute in an antigen presentation capacity to the development of an antibody response in fish chronically affected by AGD.     

Gene expression studies of host response to Salmonid alphavirus subtype 3 experimental infections in Atlantic salmon

Salmonid alphavirus subtype-3 (SAV-3) infection in Atlantic salmon is exclusively found in Norway. The salmonid alphaviruses have been well characterized at the genome level but there is limited information about the host-pathogen interaction phenomena. This study was undertaken to characterize the replication and spread of SAV-3 in internal organs of experimentally infected Atlantic salmon and the subsequent innate and adaptive immune responses. In addition, suitability of a cohabitation challenge model for this virus was also examined. Groups of fish were infected by intramuscular injection (IM), cohabited (CO) or kept uninfected in a separate tank. Samples of pancreas, kidney, spleen, heart and skeletal muscles were collected at 2, 4 and 8 weeks post infection (wpi). Pathological changes were assessed by histology concurrently with viral loads and mRNA expression of immune genes by real time RT-PCR. Pathological changes were only observed in the pancreas and heart (target organs) of both IM and CO groups, with changes appearing first in the pancreas (2 wpi) in the former. Lesions with increasing severity over time coincided with high viral loads despite significant induction of IFN-α, Mx and ISG15. IFN-γ and MHC-I were expressed in all tissues examined and their induction appeared in parallel with that of IL-10. Inflammatory genes TNF-α, IL-12 and IL-8 were only induced in the heart during pathology while T cell-related genes CD3ε, CD4, CD8, TCR-α and MHC-II were expressed in target organs at 8 wpi. These findings suggest that the onset of innate responses came too late to limit virus replication. Furthermore, SAV-3 infections in Atlantic salmon induce Th1/cytotoxic responses in common with other alphaviruses infecting higher vertebrates. Our findings demonstrate that SAV-3 can be transmitted via the water making it suitable for a cohabitation challenge model.     

Evaluation of bithionol as a bath treatment for amoebic gill disease caused by Neoparamoeba spp

This study examined the toxicity of bithionol to Atlantic salmon Salmo salar and rainbow trout Oncorhynchus mykiss in fresh- and seawater and the efficacy of bithionol as a 1h seawater bath treatment for amoebic gill disease (AGD). To examine toxicity, fish were bathed for 1, 3 and 6h in bithionol, an anti-protozoal at 0, 1, 5, 10, 25 and 35mgL(-1) with toxicity determined by time to morbidity. Efficacy was examined by bathing AGD-affected Atlantic salmon and rainbow trout for 1h at bithionol concentrations of 1-25mgL(-1). Efficacy was determined by examining gill amoeba counts and identifying percent lesioned gill filaments at 1 and 24h after bath exposure to bithionol. For both species, bithionol was determined to be toxic at 25 and 35mgL(-1) exhibiting median lethal times (LT50s) ranging from 21 to 84min. Morbidity occurred in the 5 and 10mgL(-1) treatments, however, due to sampling regime there were not enough fish available to calculate LT50s. Only bithionol at 1mgL(-1) was considered non-toxic with no signs of morbidity. Bithionol was more toxic in seawater than freshwater and had no acute effects on gill Na+/K+ ATPase and succinic dehydrogenase, or plasma osmolality and chloride concentration. Bithionol at 1mgL(-1) reduced percent lesioned gill filaments in Atlantic salmon and rainbow trout by 33 and 27 per cent, respectively, compared to the seawater control. Similarly, numbers of amoeba were reduced by 33 and 43 per cent for Atlantic salmon and rainbow trout, respectively, when compared to the seawater control. Furthermore, bithionol reduced percent lesioned gill filaments as much as did the current industry standard of freshwater. This study demonstrated that a 1h seawater bath containing 1mgL(-1) bithionol could be an improvement to the current method of treatment for AGD-affected Atlantic salmon and rainbow trout.     

Profiling pro-inflammatory cytokine and chemokine mRNA expression levels as a novel method for selection of increased innate immune responsiveness

Previous studies using F1 reciprocal crosses and two parental lines of broilers show the sire is instrumental in determining the in vitro leukocyte function and cytokine/chemokine profile. Since the innate immune response is the primary means young chickens have to protect themselves, we hypothesize utilizing a novel genomics approach to select sires based on an elevated pro-inflammatory cytokine and chemokine profile. By identifying sires with increased pro-inflammatory cytokine (interleukin [IL]-1beta and IL-6) and chemokine (CXCLi2 and CCLi2) mRNA expression levels, we expect the progeny will also have elevated profiles. We characterized the pro-inflammatory cytokine and chemokine profile of 119 sires using quantitative real-time RT-PCR (qRT-PCR) and identified two populations with inherently high and low mRNA expression levels of IL-1beta, IL-6, CXCLi2, and CCLi2. Select high and low sires were then used to produce progeny for the second phase of the trial. Blood samples were collected from 214 progeny and the cytokine and chemokine mRNA expression levels determined. Progeny from high sires had significantly (P相似文献   

Cytokine mRNA expression in experimental porcine pneumonia

A porcine Pasteurella multocida (P. m.) infection model was established to study the spatial distribution of cytokine mRNA-expressing cells in lung tissue during acute pneumonia. The mRNA detection was performed by non-radioactive, formamide-free in situ hybridization (ISH) using oligonucleotides against the porcine interleukins (IL): IL-1 beta, IL-2, IL-4, IL-6, IL-8, TNF alpha and TGF beta. Cytokine mRNA-expressing macrophages were demonstrated by a double staining procedure combining immunohistochemistry (IH) using the primary antibody 2G6 with IL-1 beta, IL-6 and TGF beta ISH. With the exception of some stained TNF alpha-expressing cells, no IL mRNA was detectable in the lung of unaffected animals. The experimental P. m. pneumonia was characterized by a predominant, exudative and an additional proliferative interstitial component as well as abscess formation in the lung. Many cells of the region between the abscess membrane and the affected lung area showed high IL-6, IL-1 beta, IL-4 as well as TGF beta and few cells low IL-8 mRNA expression with characteristic distribution patterns. The ISH/IH double staining procedure revealed that at least some of the IL-6 or TGF beta-producing cells belonged to the 2G6-positive macrophages.     

Quantitative analysis of inflammatory and immune responses in dogs with gastritis and their relationship to Helicobacter spp. infection

The present study sought to quantitatively examine mucosal inflammatory and immune responses in dogs with gastritis and the relationship of these responses to infection with Helicobacter. Gastric biopsies from 30 dogs were evaluated for B- and T-lymphocytes, neutrophils, eosinophils, macrophages, and mast cells. Mucosal atrophy, fibrosis, cellularity, and severity of gastritis were graded qualitatively. Messenger-RNA (mRNA) for actin, interleukin-1beta (IL-1beta), IL-4, IL-8, and IL-10, transforming growth factor beta (TGF-beta), and interferon gamma (IFN-gamma) was quantified by polymerase chain reaction (PCR). The presence of Helicobacter spp. was determined by urease activity, histology, PCR, and enzyme-linked immunosorbent assay. mRNA for IL-1beta, IL-8, IL-10, TGF-beta, and IFN-gamma was detected in most dogs. IL-4 mRNA was detected in only 1 dog. Correlations were observed for IL-1beta versus IL-8 and IL-10; IL-8 versus IL-10, IFN-gamma, and TGF-beta; and IL-10 versus IFN-y. Mucosal pathology was related to cytokine mRNA expression (neutrophils to IL-8 and IFN-gamma, macrophages and lymphocytes to IFN-gamma, and fibrosis to IL-1beta). Gastritis was categorized as lymphoplasmacytic in all dogs, and its histologic severity correlated with atrophy, infiltration with lymphocytes and macrophages, and expression of IL-10 and IFN-gamma. Of the dogs examined, 76.7% were infected with Helicobacter spp. Infection was associated with increased expression of TGF-beta and fibrosis. Circulating anti-Helicobacter immunoglobulin G titers were higher in uninfected than infected dogs. We conclude that lymphoplasmacytic gastritis in dogs is characterized by concurrent activation of proinflammatory and immunomodulatory cytokines, with increased mRNA expression related to mucosal pathology. No significant associations between Helicobacter infection and proinflammatory cytokine expression, severity of gastritis, or differences in the pathogenicity of different Helicobacter spp. were found.     

Influence of reduced feed ration on Lepeophtheirus salmonis infestation and inflammatory gene expression in juvenile pink salmon

The effect of reduced feed ration on infestation levels with the sea louse Lepeophtheirus salmonis and gene expression in juvenile pink salmon Oncorhynchus gorbuscha was tested in three laboratory trials. Body weight was significantly lower among fish on the reduced ration for 27, 34, or 65 d than fish on the full ration. Neither the prevalence nor the abundance of L. salmonis differed between fish on full and reduced rations at any time in any trial. In trial 2, sea louse rejection was delayed among fish on reduced rations; however, the parasite was ultimately rejected from all fish in this trial regardless of ration. Proinflammatory gene expression in salmon exposed to L. salmonis was modulated by reduced rations. There was a reduction in the expression of interleukin-8 in pink salmon on reduced rations 7 d after exposure but not 14 d after exposure. In contrast, the 7-d expression of interleukin-1 beta (IL-1beta) was reduced in exposed pink salmon regardless of ration. By day 14, however, expression of IL-1beta was increased in association with reduced rations among exposed salmon. Similarly, the expression of tumor necrosis factor alpha (TNFalpha) was increased 14 d after exposure among salmon on a reduced ration. There was no evidence that short-duration exposure of otherwise healthy juvenile pink salmon to a reduced ration affected susceptibility to L. salmonis. The expression data do not suggest an obvious mechanism of louse rejection; rather, they indicate that a more comprehensive suite of inflammatory pathways should be surveyed to better understand the early pink salmon response to L. salmonis.     

Quantification of mRNA encoding cytokines and chemokines in nasal biopsies from dogs with sino-nasal aspergillosis

Canine sino-nasal aspergillosis is usually caused by Aspergillus fumigatus and is similar to human chronic erosive non-invasive fungal sinusitis. The pathogenesis of the disease is poorly understood. We investigated the nature of the local immune response mounted in canine sino-nasal aspergillosis. Quantitative RT-PCR was carried out on RNA isolated from nasal biopsies from diseased and control dogs, using specific assays designed to amplify mRNA encoding a panel of cytokines and chemokines. Canine sino-nasal aspergillosis was associated with significantly increased expression of mRNA encoding MCP-1, -2, -3 and -4, IL-8, IL-10, IL-18 and TNF-alpha relative to controls (P<0.01) but there was no difference between groups with respect to IL-4, IL-5, IL-6, IL-12, TGF-beta, and eotaxin-2 and -3. The up-regulation of proinflammatory cytokines and chemokines related to the influx of phagocytic cells might account for the localisation of this infection to the upper respiratory tract. The up-regulation of the expression of the immunomodulatory cytokine IL-10 in nasal tissue from affected dogs might be important in limiting the extent of local tissue destruction, but might also account for the fact that infected dogs are generally unable to clear this infection spontaneously.     

The Gill Pathogen Dermocystidium salmonis in Oregon Salmonids

Abstract

Intense infections of the gill pathogen Dermocystidium salmonis were associated with mortality of prespawning chinook salmon Oncorhynchus tshawytscha in several Oregon rivers in 1988. The occurrence of the pathogen in returning adult chinook salmon was monitored in several coastal Oregon stocks from 1989 to 1993. Although the prevalence of the pathogen was high in these fish (up to 66.6%), infection intensities were generally low, and no mortality attributable to D. salmonis was observed. In 1988, the pathogen was associated with a lethal epizootic among juvenile chinook salmon smolts at the Trask State Fish Hatchery near Tillamook, Oregon. Histological examination of gills from heavily infected fish revealed hyperplasia of gill epithelium and fusion of gill lamellae. When naturally infected smolts were transferred from fresh to salt water, the most heavily infected fish died within 10 d, and the number of D. salmonis cysts declined and disappeared from previously infected salmon after 21–42 d.     

Moritella viscosa bypasses Atlantic salmon epidermal keratocyte clearing activity and might use skin surfaces as a port of infection

Moritella viscosa is considered the main causative agent of winter ulcer disease in salmonid fish. In order to obtain more details on route of infection, we challenged Atlantic salmon (Salmo salar) epidermal keratocytes with M. viscosa and performed an Atlantic salmon immersion challenge. Although keratocytes were able to remove M. viscosa from surfaces, their engulfment capability appeared inefficient with reduced ability to reepithelialise superficial wounds (scale less skin surfaces) challenged with the bacterium. The immersion challenge revealed a significant connection between exposure area and mortality. Enhanced invasion ability and mortality was observed by M. viscosa exposure of the head and gill region compared to exposure of: the right side of the body; the left side of the body; or the body from pectoral to caudal fin (p=0.04). Ulcer development corresponded to area exposed (p=0.002), suggesting skin ulcer formation to result primarily from direct skin surface colonization. Ulceration of surfaces exposed to M. viscosa in parallel with occurrence of septicaemia suggests that both skin and gills may act as possible initiation sites for M. viscosa infections.     

Escherichia coli inoculation of porcine mammary glands affects local mRNA expression of Toll-like receptors and regulatory cytokines

The expression of mRNAs for the Toll-like receptors (TLRs) TLR2 and TLR4, pro- and anti inflammatory cytokines and their receptors was evaluated in mammary gland biopsy material collected from sows intramammarily inoculated with Escherichia coli strain O127 at parturition. Quantitative real-time RT-PCR analysis showed increased mRNA levels for TLR2, the proinflammatory cytokines interleukin IL-1beta and tumor necrosis factor-alpha TNF-alpha, and the anti-inflammatory cytokine IL-10 in the inoculated mammary glands 24h after inoculation. Increased mRNA levels of the proinflammatory cytokine IL-6 were only observed in the inoculated mammary glands of sows that developed clinical signs of mastitis. In contrast, the expression of the anti-inflammatory cytokine, transforming growth factor-beta 1 (TGF-beta1) mRNA was unaltered, as was mRNA expression for the IL-1 receptor type I (IL-1R1). Furthermore, IL-1beta and IL-10 mRNA expression was higher in the inoculated mammary glands of sows that developed clinical signs of mastitis compared with sows that remained clinically healthy. Notably, sows that developed clinical signs of mastitis had significantly lower pre-inoculation levels of IL-1beta mRNA than sows that remained clinically healthy. These findings suggest that development of coliform mastitis is associated with the level of local expression of regulatory cytokines in response to intramammary E. coli inoculation and infection.     

Transmission of Loma salmonae (Microsporea) to chinook salmon in sea water.

Transmission studies were conducted to determine if Loma salmonae was transmissible in sea water. Transmission of L. salmonae to chinook salmon (Oncorhynchus tshawytscha) held in sea water was achieved by exposing fish to macerated, infected gill tissue. Fish were exposed in seawater in a flow-through aquarium, and the infection was detected as soon as 5 wk after exposure. Heavily infected fish exhibited numerous xenomas in the branchial arteries, central venous sinusoids, and within the blood channels of the lamellae. The pathological changes were similar to those seen in pen-reared salmon with L. salmonae infections. The infection was not observed in Atlantic salmon (Salmo salar), Pacific herring (Clupea pallasi, family Clupeidae), or shiner perch (Cymatogaster aggregata, family Embiotocidae), experimentally exposed using identical methods. This study suggests that L. salmonae is transmissible to chinook salmon in seawater netpens. Fish farmers and fish health specialists should consider this possibility when developing and implementing strategies to control the infection.     

BCG感染巨噬细胞后内质网应激对细胞焦亡的调控作用

旨在探讨牛分枝杆菌疫苗株卡介苗（Bacillus Calmette-Guérin,BCG）感染人单核巨噬细胞THP-1细胞后内质网应激（endoplasmic reticulum stress,ERS）对细胞焦亡的调控作用及分子机制。在BCG单独感染THP-1细胞或与ERS抑制剂TUDCA共处理细胞后,采用qRT-PCR检测ERS标志性分子GRP78,细胞焦亡标志性分子GSDMD、Caspase1、NLRP3、IL-1β和IL-18在mRNA水平的表达,采用Western blot检测GRP78、Caspase12、GSDMD、Caspase1和NLRP3在蛋白水平的表达,采用ELISA检测IL-1β和IL-18的释放量,采用CCK-8检测细胞存活率。结果表明：在BCG单独感染THP-1细胞不同时间后,GRP78在mRNA水平和蛋白水平的表达均随感染时间延长而升高（P<0.01）,GSDMD蛋白表达在24 h达到最高,IL-1β和IL-18在mRNA水平的表达呈时间依赖性（P<0.01）。在BCG单独感染或与TUDCA共同作用细胞24 h后,与未感染对照组相比,BCG感染组GRP78、GSDMD、Caspase1、NLRP3、IL-1β和IL-18 mRNA水平表达上调（P<0.05）,GRP78、Caspase12、GSDMD、Caspase1和NLRP3蛋白水平表达上调（P<0.001）,IL-1β和IL-18的浓度增加（P<0.01）,细胞存活率下降（P<0.001）,而经TUDCA预处理的BCG感染组与BCG单独感染组相比,以上分子的表达下降,细胞存活率上升（P<0.01）。综上表明,在BCG感染THP-1细胞后,引起的ERS对细胞焦亡具有调控作用。     

Innate immune response in experimentally induced bovine intramammary infection with Staphylococcus simulans and S. epidermidis

ABSTRACT: Coagulase-negative staphylococci (CNS) are in several countries the most common bacteria isolated in subclinical mastitis. To investigate the innate immune response of cows to infections with two common mastitis-causing CNS species, Staphylococcus epidermidis and Staphylococcus simulans, experimental intramammary infection was induced in eight cows using a crossover design. The milk somatic cell count (SCC), N-acetyl-β-D-glucosaminidase (NAGase) activity, milk amyloid A (MAA), serum amyloid A (SAA) and proinflammatory cytokines interleukin (IL)-1β, IL-8, and tumor necrosis factor α (TNF-α) were determined at several time points before and after challenge. All cows became infected and showed mild to moderate clinical signs of mastitis. The spontaneous elimination rate of the 16 infections was 31.3%, with no difference between species. Infections triggered a local cytokine response in the experimental udder quarters, but cytokines were not detected in the uninfected control quarters or in systemic circulation. The innate local immune response for S. simulans was slightly stronger, with significantly higher concentrations of IL-1β and IL-8. The IL-8 response could be divided into early, delayed, or combined types of response. The CNS species or persistency of infection was not associated with the type of IL-8 response. No significant differences were seen between spontaneously eliminated or persistent infections.     

Cytokine and chemokine gene expression of IL-1beta stimulated equine articular chondrocytes

OBJECTIVE: To evaluate mRNA expression of several proinflammatory and anti-inflammatory cytokines and chemokines in equine unstimulated and interleukin-1beta (IL-1beta)-stimulated chondrocytes. STUDY DESIGN: In vitro experiment using equine chondrocyte cultures. SAMPLE POPULATION: Whole articular cartilage from metacarpophalangeal joints (n=5 horses; 10 fetlocks). METHODS: Chondrocyte monolayer cultures were established from digested adult equine articular cartilage and stimulated with 5 ng/mL of recombinant human IL-1beta. RNA was extracted from the cells 24 hours after stimulation. IL-1beta, IL-4, IL-6, IL-8, tumor necrosis factor-alpha (TNF-alpha), and ubiquitin (house keeping gene) mRNA expression were investigated by real-time RT-PCR. RESULTS: IL-1beta, IL-6, and IL-8 mRNA were expressed in unstimulated chondrocytes from macroscopically normal joints and were significantly up-regulated after stimulation (5/5 horses). IL-4 mRNA was not detected in any samples (0/5 horses). TNF-alpha mRNA, by comparison, was expressed in 2/5 unstimulated samples and in all stimulated samples but a considerable sample variation in response to IL-1beta stimulation was observed. CONCLUSIONS: Equine chondrocytes express mRNA for several proinflammatory cytokines and chemokines and IL-1beta modulates their expression. CLINICAL RELEVANCE: Chondrocytes express proinflammatory cytokines and chemokines capable of modulating a local inflammatory cascade in articular cartilage, which could potentially lead to focal degradation and osteoarthritis.