Dorota Wadowska

Infectious salmon anaemia virus replication and induction of alpha interferon in Atlantic salmon erythrocytes

BackgroundInfectious salmon anaemia (ISA) virus (ISAV), which causes ISA in marine-farmed Atlantic salmon, is an orthomyxovirus belonging to the genus Isavirus, family Orthomyxoviridae. ISAV agglutinates erythrocytes of several fish species and it is generally accepted that the ISAV receptor destroying enzyme dissolves this haemagglutination except for Atlantic salmon erythrocytes. Recent work indicates that ISAV isolates that are able to elute from Atlantic salmon erythrocytes cause low mortality in challenge experiments using Atlantic salmon. Previous work on ISAV-induced haemagglutination using the highly pathogenic ISAV strain NBISA01 and the low pathogenic ISAV strain RPC/NB-04-0851, showed endocytosis of NBISA01 but not RPC/NB-04-0851. Real-time RT-PCR was used to assess the viral RNA levels in the ISAV-induced haemagglutination reaction samples, and we observed a slight increase in viral RNA transcripts by 36 hours in the haemagglutination reaction with NBISA01 virus when the experiment was terminated. However, a longer sampling interval was considered necessary to confirm ISAV replication in fish erythrocytes and to determine if the infected cells mounted any innate immune response. This study examined the possible ISAV replication and Type I interferon (IFN) system gene induction in Atlantic salmon erythrocytes following ISAV haemagglutination.ResultsHaemagglutination assays were performed using Atlantic salmon erythrocytes and one haemagglutination unit of the two ISAV strains, NBISA01 and RPC/NB-04-0851, of differing genotypes and pathogenicities. Haemagglutination induced by the highly pathogenic NBISA01 but not the low pathogenic RPC/NB-04-0851 resulted in productive infection as evidenced by increased ISAV segment 8 transcripts and increase in the median tissue culture infectious dose (TCID50) by 5 days of incubation. Moreover, reverse transcription (RT) quantitative PCR used to compare mRNA levels of key Type I IFN system genes in erythrocyte lysates of haemagglutination reactions with the two ISAV strains showed a higher relative fold increase of IFN-α in NBISA01 haemagglutinations compared to RPC/NB-04-085-1 haemagglutinations (33.0 – 44.26 relative fold increase compared to 11.29). Erythrocytes exposed to heat-inactivated virus or to polyinosinic:polycytidylic acid (polyI:C) or to L-15 medium alone (negative control assays) had minimal late induction (<3.5 relative fold increase) of STAT1 and/or ISG15 and Mx genes, whereas erythrocytes exposed to UV-inactivated virus lacked any cytokine induction.ConclusionISAV-induced haemagglutination by a highly pathogenic virus strain results in virus uptake and productive infection of Atlantic salmon erythrocytes accompanied by significant induction of IFN-α. This study also highlights the critical role of ISAV strain variation in the initial stages of the virus-cell interaction during haemagglutination, and possibly in the pathogenesis of ISA. Moreover, the study shows for the first time that fish erythrocytes immunologically respond to ISAV infection.

Ultrastructural and secretory heterogeneity of fa/fa (Zucker) rat islets.

Many previous studies of obese rodents documented biochemical changes in pancreatic islets that contribute to hyperinsulinemia in vivo. Those studies used heterogeneous populations of islets, although the size of islets from obese rats ranges from < 100 to > 500 microm. Here, functional and morphological changes in size-sorted (< 125 and > 250 microm diameter) islets from obese Zucker (fa/fa) rats were correlated. Ultrastructural examination revealed that > 250 microm cultured islets had an increased number of immature secretory granules in the beta cells. The number of degranulated beta cells in > 250 and < 125 microm cultured islets from fa/fa rats was higher than in lean rat islets (33 vs 25%). The glucose EC50 values for cultured islets were 4.64 +/- 0.43, 7.9 +/- 0.70 and 7.29 +/- 1.64 mmol.l(-1) for > 250 microm, < 125 microm, and lean groups, respectively. Inhibition of insulin secretion by 10 mmol.l(-1) mannoheptulose was reduced by 50% in > 250 microm islets compared with small islets. Studies of individual beta cells by reverse hemolytic plaque assay revealed 3-fold more cells from > 250 microm islets were stimulated by 1.4 mmol.l(-1) glucose than cells from < 125 microm islets. We conclude that functional defects in mixed size populations of islets from fa/fa rats are mainly due to alterations in the large islets, whereas smaller islets have relatively normal function. Exposure to high glucose exacerbates morphological and functional differences of large islets, which could have important implications in the transition to noninsulin-dependent diabetes when beta cell insulin production is unable to compensate for hyperglycemia.

Mutated ATP synthase induces oxidative stress and impaired insulin secretion in β-cells of female BHE/cdb rats

Adenosine triphosphate (ATP) is a critical determinant of β‐cell insulin secretion in response to glucose. BHE/cdb rats have a mutation in ATP synthase that limits ATP production, yet develop mild diabetes only with ageing. We investigated the cellular basis for reduced insulin secretion and compensatory mechanisms that mitigate the effects of the ATP synthase mutation.

Demonstration of infectious salmon anaemia virus (ISAV) endocytosis in erythrocytes of Atlantic salmon

Infectious salmon anaemia (ISA) virus (ISAV) is a fish orthomyxovirus that has recently been assigned to the new genus Isavirus within the family Orthomyxoviridae. It possesses the major functional characteristics of the virus family including haemagglutinating, receptor destroying enzyme (RDE), and fusion activities associated with the virion surface proteins. It is generally accepted that ISAV agglutinates erythrocytes of several fish species and that the ISAV RDE activity dissolves this haemagglutination reaction except for Atlantic salmon (Salmo salar) erythrocytes. We used electron microscopy to examine the physical interaction between ISAV and erythrocytes from Atlantic salmon and rainbow trout (Oncorhynchus mykiss) during haemagglutination. We present evidence that ISAV enters into Atlantic salmon erythrocytes. Atlantic salmon erythrocytes incubated with ISAV for 4 hours showed endocytosis of the virus particles, which is consistent with virus infection. These observations suggest that the lack of dissolution of ISAV-induced haemagglutination of Atlantic salmon erythrocytes favours virus infection of the erythrocytes. Moreover, such a haemagglutination-infection phenotype is fundamentally different from haemagglutination by avian and mammalian orthomyxoviruses, and is indicative of a different pathogenesis for the fish orthomyxovirus.

Histochemical and ultrastructural analysis of pathology and cell responses in gills of channel catfish affected with proliferative gill disease.

Pond-reared channel catfish Ictalurus punctatus with proliferative gill disease (PGD), caused by the myxozoan parasite Henneguya spp., were examined with light and transmission electron microscopy to better characterize the inflammatory response during infection. The early stages of disease are characterized by the destruction of collagen in the matrix of the gill filament cartilage causing weakness and breaks within the gill filaments. These early lesions lacked a notable inflammatory response around the disrupted cartilage, a chondrocyte response was not apparent, and the parasite was not present, suggesting that the cartilage breaks occur prior to inflammation and arrival of the parasite in the gill. In later lesions, a significant inflammatory response was generated in areas of disrupted cartilage, and the inflammatory infiltrate was composed of a mixed population of granulocytes including neutrophils and cells that resembled eosinophils. The majority of eosinophil-like cells demonstrated evidence of degranulation. Trophozoites of Henneguya spp. were surrounded by a uniform population of cells believed to be neutrophils. The granulocytes were infiltrated within the dense collagen layer of the gill filament cartilage and often appeared within chondrocyte lacunae in place of the chondrocyte. The gill lamellae adjacent to the lesions were fused and contained an inflammatory infiltrate containing granulocytes and cells with pericentriolar granules that resembled previous descriptions of Langerhans-like cells. These cells were abundant within damaged lamellar epithelium, but were only rarely found within the gill filament. Lesions that appeared to be recovering lacked the dense collagenous layer around the cartilage and contained hyperplastic and hypertrophic chondrocytes that formed a callus. Other chondrocytes in the lesions had ultrastructural features indicative of cell death.

Proliferative Pododermatitis Associated with Virus-like Particles in a Northern Gannet

Small multifocal lesions of proliferative pododermatitis were observed in an emaciated adult male northern gannet (Morus bassanus). Ultrastructurally, these lesions were associated with numerous virus-like particles with a size and morphology suggestive of Papovaviridae. DNA in situ hybridization with probes for avian polyomaviral and papillomaviral nucleic acid and an immunohistochemical test for the presence of papillomaviral antigen failed to identify this virus further. To our knowledge, papovavirus-like particles have not been recognized previously in this avian species.

The effects of temperature and body size on immunological development and responsiveness in juvenile shortnose sturgeon (Acipenser brevirostrum)

Sturgeon are an important evolutionary taxa of which little is known regarding their responses to environmental factors. Water temperature strongly influences growth in fish; however, its effect on sturgeon immune responses is unknown. The objective of this study was to assess how 2 different temperatures affect immune responses in shortnose sturgeon (Acipenser brevirostrum) relevant immune organs such as the meningeal myeloid tissue, spleen, thymus and skin. These responses were studied in 2 different sizes of same age juvenile sturgeon kept at either 11 °C or 20 °C (4 treatment groups), before and after exposure to an ectoparasitic copepod (Dichelesthium oblongum). Based on a differential cell count, temperature was found to strongly influence immune cell production in the meningeal myeloid tissue, regardless of the fish sizes considered. Morphometric analysis of splenic white pulp showed a transient response to temperature. There were no differences between the groups in the morphometric analysis of thymus size. Splenic IRF-1 and IRF-2 had similar expression profiles, significantly higher in fish kept at 20 °C for the first 6 weeks of the study but not by 14 weeks. In the skin, IRF-1 was significantly higher in the fish kept at 11 °C over the first 6 weeks of the study. IRF-2 had a similar profile but there were no differences between the groups by the end of the trial. In conclusion, higher water temperatures (up to 20 °C) may have beneficial effects in maximizing growth and improving immunological capacity, regardless of the fish sizes considered in this study.

Ontogeny of the immune system in Acipenserid juveniles

Sturgeon aquaculture has increased considerably worldwide but little is known about their immunological development and competence in early life stages. Culture of larvae is one of the most critical stages in intensive sturgeon farming, often associated with high mortality rates. The objective of this study was to characterize the developmental morphology (light and transmission electron microscopy, LM and TEM) of the meningeal myeloid tissue, spleen and thymus in Atlantic sturgeon (Acipenser oxyrinchus oxyrinchus) from hatching until 5 months old (2895°C·day (dd)). The spleen was first visible on 541 dd larvae LM sections and the other two immune organs in 768 dd samples (approximately 400 and 600 dd after onset of feeding). Generally, younger fish had significantly higher percentages of undifferentiated cells (meningeal myeloid tissue and spleen) and effective adaptive immune competence would not be expected in these fish on the onset of feeding, but further functional immune assessment is needed.

Lesions Associated with Postweaning Multisystemic Wasting Syndrome in Pigs from Prince Edward Island, Canada

Postweaning multisystemic wasting syndrome (PMWS) is a recently recognized swine disease originally reported in western Canada.2,9 Retrospective postmortem studies revealed that this condition first appeared in 1991, but it was not recognized as a specific disease until 1996.9 A similar syndrome has been described recently in pigs from the United States4,11 and Europe.1 The etiology of this syndrome is still under investigation, but porcine circovirus (PCV), a member of the family Circoviridae that includes chicken anemia virus, psittacine beak and feather disease virus, and a newly described pigeon circovirus,15 is thought to play an important role in the pathogenesis of this condition. The purpose of this paper is to describe and illustrate the most common microscopic findings associated with PMWS in pigs and to report the presence of PMWS in swine herds from Atlantic Canada. Six pigs, 5–12 weeks of age, from four swine herds located in Prince Edward Island were submitted to the Atlantic Veterinary College for postmortem examination. Five pigs were alive and one (pig no. 3) had been found dead in its pen. Pig nos. 2 and 3 and pig nos. 5 and 6 were herdmates. All pigs had a history of weight loss, dyspnea, and/or scouring, first noticed 1 or 2 weeks after weaning. Blood samples were obtained from live pigs prior to euthanasia. Complete postmortem examination was performed, and tissues were selected for histopathology, bacteriologic culture, and virologic analysis. Tissues for histopathology were fixed in 10% neutral buffered formalin, embedded in paraffin, cut at 5 mm, and stained with hematoxylin and eosin. In two cases, formalin-fixed tissue was fixed in 2% glutaraldehyde and postfixed in 1% osmium tetroxide, dehydrated in a graded series of ethanol, and infiltrated and embedded in epon/araldite for sectioning and examination by transmission electron microscopy. Thin sections were stained with a saturated solution of uranyl acetate and Sato lead stain. Tissues from five pigs (nos. 1, 2, 3, 5, and 6) were sent to the Veterinary Services Branch of Manitoba Agriculture for the detection of pathogenic PCV by polymerase chain reaction (PCR).8 Porcine reproductive and respiratory syndrome (PRRS) serology was done on serum of two pigs by an indirect fluorescent antibody test (IFAT) that was developed in house. Gradual dilutions of the test serum were made and reacted with PRRS virus (PRRSV)-infected MARC cells, washed, mounted, and evaluated by fluorescent microscopy. Lungs from three pigs and lymph nodes from two others were tested for the pres-

Aerococcus viridans var. homari: The presence of capsule and the relationship to virulence in American lobster (Homarus americanus).

The relationship between virulence and encapsulation of Aerococcus viridans var. homari was evaluated by growing virulent (Rabins) and avirulent (ATCC 10400) strains under varying culture conditions, and during challenge trials. Changes in capsule thickness were monitored using a modified lysine-ruthenium red (LRR) fixation method and transmission electron microscopy. The virulent Rabins strain possessed a prominent capsule of 0.252 μm±0.061 μm that was diminished by in vitro growth conditions to 0.206 μm±0.076 μm. The ATCC 10400 strain capsule thickness decreased from 0.157 μm±0.043 μm to 0.117 μm±0.043 μm after 10 in vitro passages. The virulent Rabins strain capsule was significantly thicker than the avirulent ATCC 10400 strain under all growth conditions. Rabins strain, regardless of pre-challenge growth conditions or dose (high dose 10(7) or low dose 10(2)), was able to kill lobsters in 7 days at 15°C. ATCC 10400 strain, regardless of pre-challenge growth conditions, killed lobster only at high doses (10(7)) with varying median time to death of ∼15 days, while at low doses (10(2)) all lobsters survived and no bacteria were present after 42 days. This work demonstrates the importance of the thickness of the A. viridans capsule to virulence in the American lobster.