Charles McL. Press

Distribution of prion protein in the ileal Peyer's patch of scrapie-free lambs and lambs naturally and experimentally exposed to the scrapie agent

A sensitive immunohistochemical procedure was used to investigate the presence of prion protein (PrP) in the ileal Peyers patch of PrP-genotyped lambs, including scrapie-free lambs and lambs naturally and experimentally exposed to the scrapie agent. The tyramide signal amplification system was used to enhance the sensitivity of conventional immunohistochemical procedures to show that PrP was widely distributed in the enteric nervous plexus supplying the gut wall. In scrapie-free lambs, PrP was also detected in scattered cells in the lamina propria and in the dome and interfollicular areas of the Peyers patch. In the follicles, staining for PrP was mainly confined to the capsule and cells associated with vascular structures in the light central zone. In lambs naturally exposed to the scrapie agent, staining was prominent in the dome and neck region of the follicles and was also found to be associated with the follicle-associated epithelium. Similar observations were made in lambs that had received a single oral dose of scrapie-infected brain material from sheep with a homologous and heterologous PrP genotype 1 and 5 weeks previously. These studies show that the ileal Peyers patch in young sheep may be an important site of uptake of the scrapie agent and that the biology of this major gut-associated lymphoid tissue may influence the susceptibility to oral infection in sheep. Furthermore, these studies suggest that homology or heterology between PrP genotypes or the presence of PrP genotypes seldom associated with disease does not impede uptake of PrP.

In situ localisation of major histocompatibility complex class I and class II and CD8 positive cells in infectious salmon anaemia virus (ISAV)-infected Atlantic salmon.

It is assumed that the mobilisation of a strong cellular immune response is important for the survival of Atlantic salmon infected with infectious salmon anaemia virus (ISAV). In this study, the characterisation of immune cell populations in tissues of non-ISAV infected Atlantic salmon and during the early viraemia of ISAV was undertaken. Immunohistochemical investigations of spleen, head kidney and gills using monoclonal antibodies against recombinant proteins from MHC I, II and CD8 were performed on tissues from Atlantic salmon collected day 17 post-challenge in a cohabitant infection model. The localisations of MHC I and II in control salmon were consistent with previous reports but this study presents novel observations on the distribution of CD8 labelled cell populations in Atlantic salmon including the description of significant mucosal populations in the gills. The distribution of MHC I, MHC II and CD8 positive cell populations differed between control salmon and cohabitant salmon in the early stages of ISAV infection. The changes in MHC I labelled cells differed between organs in ISAV cohabitants but all investigated organs showed a decreased presence of MHC II labelled cells. Together with a clustering of CD8 labelled cells in the head kidney and a reduced presence of CD8 labelled cells in the gills, these observations support the early mobilisation of cellular immunity in the response of Atlantic salmon to ISAV infection. However, differences between the present study and the findings from studies investigating immune gene mRNA expression during ISAV infection suggest that viral strategies to interfere with protein expression and circumvent the host immune response could be operative in the early response to ISAV infection.

Ontogeny of leukocyte populations in the ileal Peyer's patch of sheep

Enzyme- and immunohistochemical methods were used to characterize the leukocyte populations present in the ileal Peyers patches of sheep foetuses between 68 and 135 d of gestation and particularly in the period around 100 d of gestation, when active lymphopoiesis begins. A wide variety of leukocytes including IgM+, CD5+, CD4+, CD8+ cells, and MgATPase+ dendritic cells were present at an early stage. Groups of IgM+ cells were seen immediately beneath the epithelium as early as 70 d of gestation. Conventional morphometric and computer-assisted morphometric techniques were used to confirm the significant expansion of these cell populations from 90 d of gestation. IgM+ and CD5+ cells were responsible for the vast majority of the increase in cell numbers. It was concluded that a diverse leukocyte population was present at the initiation of active lymphopoiesis in the ileal PP of the sheep foetus and that all members of this population were associated with the emergence of the dome/follicle primordia from which the B-cell follicle develops.

Spleen and kidney of Atlantic salmon (Salmo salar L.) show histochemical changes early in the course of experimentally induced infectious salmon anaemia (ISA)

Infectious salmon anaemia (ISA) is a disease of farmed Atlantic salmon (Salmo salar L.) in Norway that affects both erythrocytic and leucocytic cells. Both cell types are possible target cells for the aetiological ISA agent, which is probably a virus. In the present study the distribution and phenotype of leucocyte populations in the spleen and head kidney of Atlantic salmon that were developing ISA have been examined. Frozen tissues were collected from fish at various times after inoculation with ISA-infective material. Immune and enzyme histochemical techniques were used to characterise the response of leucocyte populations. Acid phosphatase positive macrophages predominantly in the red pulp of the spleen appeared to have engulfed erythrocytes at day 4 after infection. Evidence of degradation products of phagocytosed erythrocytes was present in macrophages in red pulp of the spleen at day 7 after infection, in addition to the usual site of erythrophagocytosis in melanomacrophage accumulations. Signs of erythrophagocytosis were not found in the head or body portions of the kidney. The activation of macrophages in the spleen at day 7 was suggested by decreased reactivity for the enzyme 5 nucleotidase. From day 7, clusters of immunoglobulin positive (Ig +) cells were present in the head kidney, while from day 11, the ellipsoids of the spleen showed reactivity for Ig and complement factor C3. These observations are discussed in relation to early immunoglobulin production and possible immune complex trapping. The present results suggest that the leucocyte populations in Atlantic salmon respond to ISA infection through macrophage activation and the initiation of an immune response.

Investigation of the structural and functional features of splenic ellipsoids in rainbow trout (Oncorhynchus mykiss)

The ultrastructure of ellipsoids in the spleen of rainbow trout (Oncorhynchus mykiss) is described. The endothelium of terminations of arterioles bulged into the lumen, and gaps between the endothelial cells were evident. A continuous basal lamina was not present, and there were extensive interdigitations between the endothelial cells and surrounding reticular cells. The interdigitating processes were rich in microfilaments. Intravenously injected colloidal carbon, approximately 0.03 μm in diameter, was held in the reticular matrix of the ellipsoidal wall and taken up by macrophages that extended cellular processes among the reticular and endothelial cells. The intravenous injection of fluorescent polystyrene microspheres of known diameter showed that microspheres with a diameter of 0.5 or 1.0 μm were localised in the red pulp, whereas microspheres with a diameter of 0.15 μm were retained in ellipsoidal walls. Thus, the terminations of splenic arterioles in rainbow trout were found to be consistent with descriptions of ellipsoids in other vertebrates in that they possessed a speciallised cuboidal endothelium, lacked a continuous basal lamina, were surrounded by a sheath of macrophages and reticular cells, and had a sheath of macrophages and reticular cells, and had a role in the selective filtration and retention of bloodborne particles.

Depletion of CD8 alpha cells from tissues of Atlantic salmon during the early stages of infection with high or low virulent strains of infectious salmon anaemia virus (ISAV).

The virulence of an infectious salmon anaemia virus (ISAV) isolate is influenced by the response of the hosts immune system to virus infection. Here we report the fate of immune responsive cells in head kidney, spleen and gills of Atlantic salmon during infection with high and low virulent strains of ISAV. A comparison of real-time PCR detection of virus and immunohistochemical detection of immune responsive cells revealed that peak viral load was coincident with both an elevated presence of MHC class I cells and a marked depletion of CD8 alpha cells. There was a larger CD8 alpha population in tissues from salmon infected with the low virulent strain compared with tissues from salmon infected with the high virulent strain at early stages of infection. These findings suggest a protective role for the CD8 alpha cell population in immune defences against ISAV.

Phenotypic characterisation of intestinal dendritic cells in sheep

The present study was undertaken to identify dendritic cells (DCs) in the ileum and rectum of lambs and adult sheep. The distribution of these cells in four different intestinal compartments, i.e. lamina propria, lymphoid follicles, domes and interfollicular areas was assessed, and the presence of these cells in lambs and adult sheep was compared. Specimens were examined by using a number of potential DC markers (CD11c, CD205, MHC class II (MHCII), CD1b and CD209) in immunohistochemical and multicolour immunofluorescent procedures. The ovine ileal and rectal mucosa contain many CD11c+/CD205+ cells with a dendritic morphology, and the majority of these cells co-expressed MHCII. These double-positive cells were also labelled with the CD209 antibody in the lamina propria and interfollicular regions. Only very few cells expressed CD1b. In conclusion, a major DC population in ileum and rectum of sheep co-expressed the CD11c, CD205 and MHCII molecules. The CD209 antibody appeared to be a novel marker for a subpopulation of ovine intestinal DCs.

Lymphoid follicles in the gastric mucosa of dogs. Distribution and lymphocyte phenotypes

The occurrence and distribution of lymphoid follicles within the stomachs of 36 dogs that did not have macroscopic gastric lesions are presented. The dogs ranged in age from less than 1 year to over 13 years. The number of follicles varied between the different regions of the stomach, being most numerous (15.6 follicles cm-2) and uniform in size (about 1 mm in diameter) in the fundus. The number and size of follicles in the antrum varied widely between dogs. Age-related changes in the distribution of follicles were not found following simple linear regression analysis. The phenotypes of lymphocytes in gastric lymphoid follicles of nine dogs aged from less than 1 year to 5 years were determined using monoclonal antibodies specific for canine leucocyte antigens and an indirect immunoperoxidase technique. The follicles had an organized distribution of lymphocytes subsets in that a predominantly B cell area contained some CD4+ cells and very few CD8+ cells and was adjacent to an area containing mostly T cells. Computer-assisted morphometric analysis was used to quantify the overall presence of the various lymphocyte subpopulations. Follicles in the fundus and body regions possessed similar percentages of lymphocytes averaging 42%, 22% and 3% of the area occupied by B cells, CD4+ and CD8+ cells, respectively. It is concluded that lymphoid follicles are a normal constituent of the canine gastric mucosa and possess a lymphocyte composition similar to that reported by others for solitary intestinal follicles.

Development and cell phenotypes in primary follicles of foetal sheep lymph nodes

Lymph nodes from sheep foetuses and postnatal lambs were examined to determine the participation of different leucocyte populations in primary follicle formation, with special emphasis on the emergence and subsequent development of follicular dendritic cells during late gestation and early postnatal life. A series of immune and enzyme histochemical markers was used. The first 5′-nucleotidase-positive primary follicles were found at 80 days gestational age (gestation in sheep is 150 days) in superficial cervical lymph nodes. In the last month of gestation the primary follicles possessed follicular dendritic cells, macrophages, dendritic cells, and CD5-positive lymphocytes, in addition to IgM-positive cells. Follicular dendritic cells in primary follicles were found to be ultrastructurally immature. These follicular dendritic cells were characterised by a few, coarse surface projections and many ribosomes attached to the endoplasmic reticulum. A final differentiation to mature follicular dendritic cells was coincident with the postnatal germinal centre reaction. Computer-assisted morphometric analysis demonstrated that the size of 5′-nucleotidase-positive primary follicles in the distal jejunal lymph node, but not in the superficial cervical lymph node, increased significantly during late gestation. It was concluded that stromal cells in primary follicles of foetal sheep lymph nodes were a continuously developing population but that ultrastructural maturity was only achieved in the germinal centres of postnatal lambs.

Compartments within the lymph node cortex of calves and adult cattle differ in the distribution of leukocyte populations: an immunohistochemical study using computer-assisted morphometric analysis.

The combination of an immunohistochemical technique and a panel of monoclonal antibodies was used to investigate the presence of leukocyte populations in the distal jejunal lymph node of 3-4 week old calves and adult cattle. The application of computer-assisted morphometric analysis enabled information to be obtained on the distribution of leukocyte populations in lymphoid compartments of the lymph node cortex. Semi-quantitative estimates of the areas of staining in histological sections showed that calves possessed significantly fewer B-cells and CD4+ cells in the outer cortex and significantly fewer T-cells (CD4+, CD8+ and gamma delta T-cells) in the deep cortex. These findings were interpreted to be a possible consequence of immunosuppression resulting from the passive transfer of maternal immunity in colostrum. The presence of some B-cell follicles in the region defined as the deep cortex suggested the on-going differentiation of this predominantly T-cell compartment. The larger presence of interdigitating cells (IDC) in the deep cortex of calves than adults was suggested by significantly larger CD1+ populations and it was argued that this could be the result of the confrontation with exogenous antigen faced by calves in early postnatal life. Antigen presenting populations, pan MHC II+ and MHC II DQ+ populations, were increased in all compartments of calf lymph nodes but were not significantly different from the populations in adult lymph nodes. Variance component analysis of the data generated in the present study showed that the image analysis technique was an effective and statistically powerful approach to investigate leukocyte populations within the specific microenvironments of the lymph node.