Inge Bjerkås

Redescription of Neospora caninum and its differentiation from related coccidia

Neospora caninum is a protozoan parasite of animals, which before 1984 was misidentified as Toxoplasma gondii. Infection by this parasite is a major cause of abortion in cattle and causes paralysis in dogs. Since the original description of N. caninum in 1988, considerable progress has been made in the understanding of its life cycle, biology, genetics and diagnosis. In this article, the authors redescribe the parasite, distinguish it from related coccidia, and provide accession numbers to its type specimens deposited in museums.

Identification and characterization of a novel intraepithelial lymphoid tissue in the gills of Atlantic salmon

In addition to being the respiratory organ in fish, the gills form a barrier against the external milieu. Innate and adaptive immune system components have been detected in the gills, but lymphoid cell accumulations similar to that seen in the mammalian mucosa have not been described. The present investigations revealed cell accumulations on the caudal edge of interbranchial septum at the base of the gill filaments in the Atlantic salmon. Cytokeratin immunohistochemical staining and identification of a basal membrane and desmosome cell junctions by electron microscopy showed that the cell accumulation was located intraepithelially. Major histocompatibility complex (MHC) class II+ cells were detected by immunohistochemistry, and laser capture micro‐dissection and subsequent RT‐PCR analysis revealed expression of T‐cell receptor transcripts in the investigated tissue, suggesting the presence of T cells. The intraepithelial tissue reported here may be a suitable location for immune surveillance of gill infections, as well as a target site for new vaccine approaches and investigations of epithelial immunity. This is the first description of a lymphocyte cell aggregation within a teleostian gill epithelium network, illustrating a phylogenetically early form of leukocyte accumulations in a respiratory organ.

Vaccination-Induced Systemic Autoimmunity in Farmed Atlantic Salmon

Over half of the salmon consumed globally are farm-raised. The introduction of oil-adjuvanted vaccines into salmon aquaculture made large-scale production feasible by preventing infections. The vaccines that are given i.p. contain oil adjuvant such as mineral oil. However, in rodents, a single i.p. injection of adjuvant hydrocarbon oil induces lupus-like systemic autoimmune syndrome, characterized by autoantibodies, immune complex glomerulonephritis, and arthritis. In the present study, whether the farmed salmon that received oil-adjuvanted vaccine have autoimmune syndrome similar to adjuvant oil-injected rodents was examined. Sera and tissues were collected from vaccinated or unvaccinated Atlantic salmon (experimental, seven farms) and wild salmon. Autoantibodies (immunofluorescence, ELISA, and immunoprecipitation) and IgM levels (ELISA) in sera were measured. Kidneys and livers were examined for pathology. Autoantibodies were common in vaccinated fish vs unvaccinated controls and they reacted with salmon cells/Ags in addition to their reactivity with mammalian Ags. Diffuse nuclear/cytoplasmic staining was common in immunofluorescence but some had more specific patterns. Serum total IgM levels were also increased in vaccinated fish; however, the fold increase of autoantibodies was much more than that of total IgM. Sera from vaccinated fish immunoprecipitated ferritin and ∼50% also reacted with other unique proteins. Thrombosis and granulomatous inflammation in liver, and immune-complex glomerulonephritis were common in vaccinated fish. Autoimmunity similar to the mouse model of adjuvant oil-induced lupus is common in vaccinated farmed Atlantic salmon. This may have a significant impact on production loss, disease of previously unknown etiology, and future strategies of vaccines and salmon farming.

Antigen-sampling cells in the salmonid intestinal epithelium

Antigen uptake has been shown to occur in the teleost intestine, but so far, limited information is available on the distribution and nature of cells involved in the process, and M cells, known for their antigen-sampling abilities in mammals, have not been identified. Here, different intestinal segments from salmonid fish were exposed to gold-BSA to identify antigen-sampling cells. Sections from exposed intestine were examined by light and electron microscopy. Uptake of gold-BSA was restricted to very few dendritic-like cells and to a limited number of epithelial cells located in the mucosal folds in the second segment of the mid-intestine. Gold-positive epithelial cells displayed diverging and electron-dense microvilli with channels intruding into the cytoplasm. A lectin binding experiment demonstrated the presence of cells with mammalian M-cell characteristics in the identical regions. As the identified epithelial cells shared some morphological similarities with immature mammalian M cells, this phenotype may represent evolutionary early antigen-sampling enterocytes.

Manifestations of systemic autoimmunity in vaccinated salmon.

The development of systemic autoimmunity may result as an undesired side-effect following vaccination, and this condition was recently shown to occur in farmed salmon (Salmo salar). Several of previously reported side-effects following vaccination of fish should therefore be reviewed in the light of this condition. Here, organs and pathological changes in three separate groups of fish severely affected by vaccination were investigated by different morphological methods (n=84). Granulomas or microgranulomas were observed at the injection site and in several organs. Mott cells were observed in all tissues examined. Pannus-like changes with lymphocyte infiltrates were observed in spines. In conclusion, the reactions following vaccination were of a systemic nature that may be explained by a pathogenetic mechanism caused by systemic autoimmunity.

Intestinal morphology of the wild Atlantic salmon (Salmo salar).

The worldwide‐industrialized production of Atlantic salmon (Salmo salar) has increased dramatically during the last decades, followed by diseases related to the on‐going domestication process as a growing concern. Even though the gastrointestinal tract seems to be a target for different disorders in farmed fish, a description of the normal intestinal status in healthy, wild salmon is warranted. Here, we provide such information in addition to suggesting a referable anatomical standardization for the intestine. In this study, two groups of wild Atlantic salmon were investigated, consisting of post smolts on feed caught in the sea and of sexually mature, starved individuals sampled from a river. The two groups represent different stages in the anadromous salmon life cycle, which also are part of the production cycle of farmed salmon. Selected regions of gastrointestinal tract were subjected to morphological investigations including immunohistochemical, scanning electron microscopic, and morphometric analyses. A morphology‐based nomenclature was established, defining the cardiac part of the stomach and five different regions of the Atlantic salmon intestine, including pyloric caeca, first segment of the mid‐intestine with pyloric caeca, first segment of the mid‐intestine posterior to pyloric caeca, second segment of the mid‐intestine and posterior intestinal segment. In each of the above described regions, for both groups of fish, morphometrical measurements and regional histological investigations were performed with regards to magnitude and direction of mucosal folding as well as the composition of the intestinal wall. Additionally, immunohistochemistry showing cells positive for cytokeratins, α‐actin and proliferating cell nuclear antigen, in addition to alkaline phosphatase reactivity in the segments is presented. J. Morphol. 274:859–876, 2013.

Granulomatous Uveitis Associated with Vaccination in the Atlantic Salmon

This study addressed histologic and immunopathologic changes in ocular tissues and investigated the distribution of major histocompatibility class II (MHC class II)-positive cells in Atlantic salmon (Salmo salar) suffering from severe postvaccination disease. Twenty-nine fish with generalized inflammation, probably a result of vaccination, were investigated. One individual that had escaped vaccination was included in the study. Material was investigated by cultivation methods for fungi and bacteria. Histology using conventional staining procedures and immunohistochemistry with antisera against MHC class II β chain were performed. No growth was observed from the cultivation investigations. Histology revealed occlusion of the lumen in the larger choroid vessels and in the choriocapillaris, inflammatory infiltrations and loss of structure in the choroid rete, and, in some cases, aggregations of multinucleated giant cells (MGC) and Splendore-Hoeppli material. Immunohistochemistry demonstrated massive MHC class II+ cellular infiltrations in the uveal tract. Such infiltrations were also seen in the ventral ciliary cleft, a condition that is associated with glaucoma. Immunoreactive cells included dendritelike cells, epithelioid cells, and MGCs. The endothelia of smaller vessels were frequently MHC class II+, and immunoreactive infiltrations were seen in the optic nerve in several individuals. No pathologic changes were detected in the unvaccinated individual. In conclusion, generalized inflammatory reactions in fish may lead to severe ocular inflammation, occlusion of uveal vessels, and perivascular changes with MHC class II+ upregulation in cells in the uveal tract and optic nerve.

Vaccination Induces Major Histocompatibility Complex Class II Expression in the Atlantic Salmon Eye

The aim of the study was to investigate the presence, distribution and density of major histocompatibility complex (MHC) class II+ cells in the ocular tissues of the Atlantic salmon, Salmo salar, prior to and following vaccination. Eyes were collected 14 days prior to and at 4, 11, 25 and 39 days and 4 months subsequent to vaccination with a commercial fish vaccine. A quantitative analysis was performed in sections on the number of immunopositive cells in the retinal layers. In all groups, MHC class II+ cells were detected in the area of the limbus but not in the central parts of the cornea. In the uvea, immunopositive cells were present in unvaccinated and vaccinated fish. Abundant immunopositive cells were identified in the choroid rete (or choroid gland) in all groups as well as in the ventral ciliary cleft, where macrophage‐like MHC class II+ cells were seen. Quantitative histology of the retina revealed a significant increase in MHC class II+ cells in the outer plexiform layer (OPL) and the inner nuclear layer (INL) 4 days following vaccination. Positive cells were detected in all layers of the retina with the exception of the photoreceptor layer.

Polyclonal hypergammaglobulinemia and autoantibody production induced by vaccination in farmed Atlantic salmon.

The introduction of oil-adjuvanted vaccines in salmon aquaculture made large-scale production feasible by reducing the impact of infections. Vaccines given intraperitoneally (ip) contain oil adjuvant such as mineral oil. However, in rodents, a single ip injection of adjuvant hydrocarbon oil induces lupus-like systemic autoimmune syndrome. We have recently reported that autoimmune disease in farmed salmon, characterized by production of various autoantibodies, immune complex glomerulonephritis, liver thrombosis, and spinal deformity, are previously unrecognized side effects of vaccination. In the present study, we examined whether vaccination-induced autoantibody production in farmed Atlantic salmon is a mere result of polyclonal B-cell activation. Sera were collected from 205 vaccinated and unvaccinated Atlantic salmon (experimental, 7 farms) and wild salmon. Total IgM levels and autoantibodies to salmon blood cell (SBC) extract in sera were measured by ELISA and the relationship between hypergammaglobulinemia and autoantibody production was analyzed. Comparison of endpoint titers vs levels/units using a single dilution of sera in detection of autoantibodies to SBC showed near perfect correlation, justifying the use of the latter for screening. Both total IgM and anti-SBC antibodies are increased in vaccinated salmon compared with unvaccinated controls, however, they do not always correlate well when compared between groups or between individuals, suggesting the involvement of antigen-specific mechanisms in the production of anti-SBC autoantibodies. The primary considerations of successful vaccine for aquaculture are cost-effectiveness and safety. Vaccination-induced autoimmunity in farmed Atlantic salmon may have consequences on future vaccine development and salmon farming strategy. Evaluation for polyclonal hypergamamglobulinemia and autoimmunity should be included as an important trait when vaccine efficacy and safety are evaluated in future.

A Gly98Val Mutation in the N-Myc Downstream Regulated Gene 1 (NDRG1) in Alaskan Malamutes with Polyneuropathy

The first cases of early-onset progressive polyneuropathy appeared in the Alaskan Malamute population in Norway in the late 1970s. Affected dogs were of both sexes and were ambulatory paraparetic, progressing to non-ambulatory tetraparesis. On neurologic examination, affected dogs displayed predominantly laryngeal paresis, decreased postural reactions, decreased spinal reflexes and muscle atrophy. The disease was considered eradicated through breeding programmes but recently new cases have occurred in the Nordic countries and the USA. The N-myc downstream-regulated gene (NDRG1) is implicated in neuropathies with comparable symptoms or clinical signs both in humans and in Greyhound dogs. This gene was therefore considered a candidate gene for the polyneuropathy in Alaskan Malamutes. The coding sequence of the NDRG1 gene derived from one healthy and one affected Alaskan Malamute revealed a non-synonymous G>T mutation in exon 4 in the affected dog that causes a Gly98Val amino acid substitution. This substitution was categorized to be “probably damaging” to the protein function by PolyPhen2 (score: 1.000). Subsequently, 102 Alaskan Malamutes from the Nordic countries and the USA known to be either affected (n = 22), obligate carriers (n = 7) or healthy (n = 73) were genotyped for the SNP using TaqMan. All affected dogs had the T/T genotype, the obligate carriers had the G/T genotype and the healthy dogs had the G/G genotype except for 13 who had the G/T genotype. A protein alignment showed that residue 98 is conserved in mammals and also that the entire NDRG1 protein is highly conserved (94.7%) in mammals. We conclude that the G>T substitution is most likely the mutation that causes polyneuropathy in Alaskan Malamutes. Our characterization of a novel candidate causative mutation for polyneuropathy offers a new canine model that can provide further insight into pathobiology and therapy of human polyneuropathy. Furthermore, selection against this mutation can now be used to eliminate the disease in Alaskan Malamutes.