Granulomatous enteritis in rainbow trout (Oncorhynchus mykiss) associated with soya bean meal regardless of water dissolved oxygen level.

This study investigated morphological changes associated with soya bean meal-induced enteritis (SBMIE) in distal intestine (DI) of rainbow trout (Oncorhynchus mykiss) fed a soya bean meal (SBM)-based diet and exposed to normoxia or hypoxia created by optimal and low water flow rates, respectively. A 28-day adaption period was followed by a 42-day challenge period where 600 fish were subjected to dietary challenge and/or hypoxia. Twelve tanks each containing 50 juvenile trout were assigned randomly in triplicate to each treatment. Histopathological and immunohistochemical evaluation revealed pathological features that have not previously been described in association with SBMIE. Vacuolar degeneration of epithelial cells mainly at the base of mucosal folds, epithelial cysts, epithelial dysplasia, necrosis, shedding of necrotic cells, and granulomatous inflammation including infiltration of enlarged, sometimes finely vacuolated or "foamy" macrophages, multinucleated giant cells and increased proliferation of fibroblasts were observed. Acid-fast bacteria were not detected in enlarged macrophages; however, these cells contained AB-PAS- and sometimes cytokeratin-positive material, which was interpreted to be of epithelial/goblet cell origin. Hypoxia did not affect the morphological changes in DI. These results suggest that SBM was associated with a granulomatous form of enteritis in DI of rainbow trout regardless of water oxygen level.

Histological, digestive, metabolic, hormonal and some immune factor responses in Atlantic salmon, Salmo salar L., fed genetically modified soybeans.

The paper reports the second and final part of an experiment aiming to study physiological and health-related effects of genetically modified (GM) soybean meal (SBM) type Roundup Ready soybean (RRS) in diets for post-smolt Atlantic salmon. For 3 months salmon were fed diets containing 172 g kg(-1) full-fat SBM from RRS (GM-soy) or an unmodified, non-isogenic line (nGM-soy), or a reference diet with fishmeal as the sole protein source (FM). Slight differences in anti-nutrient levels were observed between the GM and nGM-soy. Histological changes were observed only in the distal intestine of the soy-fed fish. The incidence of moderate inflammation was higher in the GM-soy group (9 of 10 sampled fish) compared with the nGM-soy group (7 of 10). However, no differences in the concomitant decreases in activities of digestive enzymes located in the brush border (leucine aminopeptidase and maltase) and apical cytoplasm (acid phosphatase) of enterocytes or in the number of major histocompatibility complex class II+ cells, lysozyme activity, or total IgM of the distal intestine were observed. GM compared with nGM-soy fed fish had higher head kidney lysozyme (11,856 vs. 10,456 units g(-1) tissue) and a tendency towards higher acid phosphatase (0.45 vs. 0.39 micromol h(-1) kg(-1) body mass in whole tissue) activities, respectively. Plasma insulin and thyroxin levels, and hepatic fructose-1,6-bisphosphatase and ethoxyresorufin-O-deethylase activities were not significantly affected. It is not possible, however, to conclude whether the differences in responses to GM-soy were due to the genetic modification or to differences in soy cultivars in the soy-containing diets. Results from studies using non-modified, parental line soybeans as the control group are necessary to evaluate whether genetic modification of soybeans in diets poses any risk to farmed Atlantic salmon.

Response to soy: T-cell-like reactivity in the intestine of Atlantic salmon, Salmo salar L.

T-cell-mediated hypersensitivity could be central in soybean meal (SBM)-induced intestinal changes in salmon. However, tools for immunohistochemical detection of T cells have been lacking in teleosts, including Atlantic salmon. Application of a specific histochemical protocol allowed demonstration of T-cell-like reactivities in formalin-fixed, paraffin-embedded tissues using an antibody reacting to a conserved region of human CD3epsilon (Dako A0452). Characteristic staining was observed in cells of the thymus as well as distal intestine, skin, gills and spleen. These cells were negative for immunoglobulin M (IgM). Intestinal intraepithelial leucocytes were CD3epsilon positive. During the SBM-induced enteropathy, the mixed inflammatory infiltrate in the lamina propria of the distal intestine included many lymphocytes with a T-cell-like reactivity. Real-time polymerase chain reaction revealed significantly increased expression of a complex polypeptide (CD3pp), CD4 and CD8beta (P < 0.05) in the distal intestine of SBM-fed fish compared to fish meal-fed reference fish. Increased reactivity for extracellular IgM in the lamina propria and a positive material between the epithelial cells at the tips of the folds was observed, possibly due to leakage of IgM through an abrogated epithelial barrier. In conclusion, a T-cell-like response appears to be involved in this example of a food-sensitive enteropathy.

Pancreatitis associated with hyperlipoproteinaemia type I in mink (Mustela vison): earliest detectable changes occur in mitochondria of exocrine cells.

Pancreatic tissue from young mink homozygous for a mutation in the lipoprotein lipase gene was studied by light and electron microscopy, with the aim of describing the earliest detectable changes in a process which rapidly progresses into overt pancreatitis. The mutation leads to hyperlipoproteinaemia, corresponding to hyperlipoproteinaemia type I in man. Assessment of relevant hepatic and pancreatic enzymes were included in the investigation. The earliest detectable changes consisted of widespread swelling and vacuolation of exocrine cells, arising mainly from swollen mitochondria. To a lesser extent, vesiculation of endoplasmic reticulum occurred. Mitochondria exhibited various changes, including cavitation and dilution of the matrix, with shortened and disorganized cristae displaced towards the periphery. Lamellar figures that developed within mitochondria were numerous. Acinar lumina were somewhat dilated, while plasma membranes were relatively well preserved and secretory granules seemed unchanged. Exfoliative processes progressively occurred, resulting in total necrosis of groups of parenchymal cells, while intercalated ducts were spared. The necrosis was rapidly followed by inflammatory reactions. The activity of the mitochondrial enzyme carnitine O-palmitoyltransferase, essential for the transport of fatty acids into the mitochondria, was lower in the pancreas than in the liver. The activity of the peroxisomal fatty acid beta-oxidation was high in the liver and low in the pancreas of both lipoprotein lipase-deficient and control mink. It is concluded that pancreatic lesions associated with hyperlipoproteinaemia start in exocrine cells, and are most probably the result of a metabolic disturbance, possibly a toxic effect of an excess of free fatty acids.

Transportation of prion protein across the intestinal mucosa of scrapie-susceptible and scrapie-resistant sheep.

To determine the mechanisms of intestinal transport of infection, and early pathogenesis, of sheep scrapie, isolated gut-loops were inoculated to ensure that significant concentrations of scrapie agent would come into direct contact with the relevant ileal structures (epithelial, lymphoreticular, and nervous). Gut loops were inoculated with a scrapie brain pool homogenate or normal brain or sucrose solution. After surgery, animals were necropsied at time points ranging from 15 min to 1 month and at clinical end point. Inoculum-associated prion protein (PrP) was detected by immunohistochemistry in villous lacteals and in sub-mucosal lymphatics from 15 min to 3.5 h post-challenge. It was also detected in association with dendritic-like cells in the draining lymph nodes at up to 24 h post-challenge. Replication of infection, as demonstrated by the accumulation of disease-associated forms of PrP in Peyer's patches, was detected at 30 days and sheep developed clinical signs of scrapie at 18-22 months post-challenge. These results indicate discrepancies between the routes of transportation of PrP from the inoculum and sites of de novo-generated disease-associated PrP subsequent to scrapie agent replication. When samples of homogenized inoculum were incubated with alimentary tract fluids in vitro, only trace amounts of protease-resistant PrP could be detected by western blotting, suggesting that the majority of both normal and abnormal PrP within the inoculum is readily digested by alimentary fluids.

Detection of PrP(Sc) in rectal biopsy and necropsy samples from sheep with experimental scrapie.

Scrapie diagnosis is based on the demonstration of disease-associated prion protein (PrP(Sc)) in brain or, in the live animal, in readily accessible peripheral lymphoid tissue. Lymphatic tissues present at the rectoanal line were readily obtained from sheep without the need for anaesthesia. The presence of PrP(Sc) in such tissue was investigated in sheep infected orally with scrapie-infected brain material. The methods used consisted of immunohistochemistry and histoblotting on biopsy and post-mortem material. PrP(Sc) was detected in animals with PrP genotypes associated with high susceptibility to scrapie from 10 months after infection, i.e., from about the time of appearance of early clinical signs. In the rectal mucosa, PrP(Sc) was found in lymphoid follicles and in cells scattered in the lamina propria, often near and sometimes in the crypt epithelium. By Western blotting, PrP(Sc) was detected in rectal biopsy samples of sheep with the PrP genotype VRQ/VRQ, after electrophoresis of material equivalent to 8 mg of tissue. This study indicated that rectal biopsy samples should prove useful for the diagnosis of scrapie in sheep.

Filter function and immune complex trapping in splenic ellipsoids.

The role of splenic ellipsoids in the trapping of particulate material and immune complexes was investigated in mink (Mustela vison). The ellipsoids were prominent, with typical features such as a permeable endothelium and a discontinuous basement membrane surrounded by a sheath of macrophages and reticular cells. Ellipsoidal trapping of circulating particles was demonstrated 10 min after intracardiac injection of colloidal carbon and fluorescent microspheres. Preformed peroxidase-antiperoxidase immune complexes were detected in ellipsoids 10 min and also 1 h after intracardiac injection. Erythrocytes were frequently observed in the ellipsoidal sheath, and many phagocytized fragments of erythrocytes were found in the ellipsoidal macrophages. It was concluded that mink ellipsoids are effective blood filters with a role in retention of circulating particulate material, and that mammalian splenic ellipsoids also have the ability to trap immune complexes.

Mapping PrPSc propagation in experimental and natural scrapie in sheep with different PrP genotypes.

Twenty-one orally inoculated and seven naturally infected sheep with scrapie were examined for PrP(Sc) in peripheral tissues and in the central nervous system (CNS), using immunohistochemistry. In the inoculated group, VRQ (valine at codon 136, arginine at codon 154 and glutamine at codon 171)/VRQ sheep generally had a greater accumulation of the pathologic form of prion protein (PrP(Sc)) in peripheral tissues, as compared with VRQ/ARQ (alanine at codon 136, arginine at codon 154, and glutamine at codon 171) animals at corresponding time points after inoculation. PrP(Sc) was not detected in the ileal Peyer's patch, the spleen, the superficial cervical lymph node, and peripheral nervous tissues of several inoculated VRQ/ARQ animals. All inoculated VRQ/VRQ sheep, but only one of eight inoculated VRQ/ARQ animals, were PrP(Sc)-positive in the CNS. Thus, the propagation of PrP(Sc) seemed slower and more limited in VRQ/ARQ animals. Tissue and cellular localization of PrP(Sc) suggested that PrP(Sc) was disseminated through three different routes. PrP(Sc)-positive cells in lymph node sinuses and in lymphatics indicated spreading by lymph. The sequential appearance of PrP(Sc) in the peripheral nervous system and the CNS, with satellite cells as early targets, suggested the periaxonal transportation of PrP(Sc) through supportive cells. Focal areas of vascular amyloid-like PrP(Sc) in the brain of five sheep, suggested the hematogenous dissemination of PrP(Sc). There was a poor correlation between the amount of PrP(Sc) in the CNS and clinical signs. One subclinically affected sheep showed widespread PrP(Sc) accumulation in the CNS, whereas three sheep had early clinical signs without detectable PrP(Sc) in the CNS. A VV(136) (homozygous for valine at codon 136) sheep inoculated with ARQ/ARR (alanine at codon 136, arginine at codon 154, and arginine at codon 171) tissue succumbed to disease, demonstrating successful heterologous transmission. Less susceptible sheep receiving VRQ/VRQ or ARQ/ARR material were PrP(Sc)-negative by immunohistochemistry, enzyme-linked immunosorbent assay, and western blot.

Differentiation of the follicle-associated epithelium in ileal Peyer's patch and production of 50-nm particles are maintained in B-cell-depleted fetal sheep.

To evaluate the dependence of the differentiation of the follicle-associated epithelium (FAE) on the presence of follicular B-cells, the FAE of ileal Peyer's patch follicles was examined in B-cell-depleted fetal lambs. The FAE of these rudimentary follicles, which are devoid of lymphocytes, showed normal differentiation, including carbonic anhydrase reactivity and ultrastructural characteristics of transcytosis, extensive interdigitation of the lateral plasma membrane and the shedding of membrane-bounded particles, approximately 50 nm in size, resembling exosomes. These 50-nm membrane-bounded particles were abundant in the extracellular space of the epithelium and the dome but no particles were found in the rudimentary follicles. This study confirms that the rudimentary follicles consist of clusters of follicular dendritic cells. Our findings suggest that the differentiation of FAE of ileal Peyer's patch and the production of the 50-nm particles constitute features that appear to be independent of B-cells.

Kinetics of glycosaminoglycan deposition in splenic AA amyloidosis induced in mink.

The kinetics of splenic glycosaminoglycan (GAG) expression in mink has been investigated during the course of AA amyloid induction, i.e. at 3 to 6 weeks of lipopolysaccharide (LPS) treatment. Splenic amyloid was demonstrated by means of Congo red staining in five of 19 LPS-treated mink. Chondroitin/dermatan sulfate (CS/DS), as well as heparan sulfate proteoglycans (HSPG), was extracted from amyloid and control spleens. Independently of the presence of amyloid, the total amount of splenic GAGs increased with the duration of LPS treatment, and an HSPG population was found confined to the LPS-treated spleens. The differential expression of various PG and GAG epitopes in mink spleen was investigated with the help of immunohistochemistry. The amyloid deposits were shown to contain GAG chains of CS and HS, and the core proteins of DSPG decorin and the HSPGs perlecan and agrin. Decorin and perlecan were shown in normal spleens localized to the splenic ellipsoids, an early target for AA amyloid deposition. The constitutive expression of PGs at predilection sites for amyloid deposition and their increased expression in the tissues developing amyloidosis at these early stages show that PGs are available for the formation and deposition of AA amyloid.

Accumulation of CD25+ CD4+ T-cells in the draining lymph node during the elicitation phase of DNCB-induced contact hypersensitivity in lambs.

The elicitation phase of DNCB induced contact hypersensitivity in lambs was studied, and the presence of CD25+ cells in the lymph nodes draining the contact site was measured. Confocal laser scanning microscopy was used to capture images of two sets of triple immunofluorescence labellings. One set labelled CD25+, CD4+ and CD3+ cells, while the other labelled CD25+, VPM30+ and CD4+ cells. The CD25+ subpopulation labellings were assessed by area measurements in a morphometric protocol. The CD25+CD4+CD3+ cells were found to be increased in the DNCB treated group. This subpopulation of CD25+ cells comprised 75% of all CD25+ cells measured. The CD25+VPM30+CD4+ cells were also found to be increased in the DNCB group, but comprised only 17% of the total CD25+ cells measured. Since the VPM30 antibody detects an antigen found on activated T-cells, it was concluded that a substantial proportion of the triple CD25+CD4+CD3+ cells could represent a regulatory phenotype that may be active in suppressing the formation of effector immune cells in CHS of sheep.

The effect of dosage, gestational age and splenectomy on anti-IgM interception of prenatal B-cell development in sheep.

The administration of a single bolus of anti-IgM antibody to foetal lambs early in pregnancy produces prolonged B-cell depletion. The present study investigated this depletion by examining the effect, on B-cell development in the ileal Peyer's patches, of varying the timing and dosage of antibody administration and by supplementing anti-IgM with surgical splenectomy. The capacity of a 1 mg bolus of anti-IgM to deplete Peyer's patches of B cells was lost if its administration was deferred until two thirds of the way through pregnancy, but persisted beyond this time if weight-adjusted doses were used. Splenectomy of the foetus performed at an earlier age failed to extend the age at which a 1 mg dose of antibody remained effective. As the concentration of murine immunoglobulin in foetal serum was greatly reduced after 21 days, it is inferred that ongoing suppression of B-cell development is not dependent on the continued presence of murine immunoglobulin. The enduring nature of suppression could be attributable to a limited period during which differentiation of B cells from stem cells normally occurs, although further studies will be needed to investigate this and other possible explanations for the effect of anti-IgM treatment on prenatal B-cell development in sheep.

Comparison of oral and intraperitoneal toxicity of yessotoxin towards mice.

Currently, yessotoxin is regulated among the toxins in the diarrhetic shellfish poisoning (DSP) complex. Yessotoxin is equally acutely toxic towards mice upon intraperitoneal injections as those algal toxins giving diarrhea, but is not diarrheagenic. Its presence in mussels may therefore lead to overestimation of risk of DSP in consumers when the standard mouse bioassay is used. Arguments are presented for the use of analytical methods instead of the mouse bioassay for the diarrheagenic DSP toxins and yessotoxin. Yessotoxin was found to be more than ten times less toxic to mice via the oral route, compared with intraperitoneal injections. Even at 10mg/kg body weight, the highest dose ever tested orally, yessotoxin did not kill the mice. By means of light microscopy of several organs, moderate changes were only observed in the heart. Ultrastructural studies revealed swelling of heart muscle cells leading to separation of the organelles. Effects were most pronounced close to the capillaries. The pathological changes were clearly dose dependent, and the lowest oral dose where any effects were seen was 2.5mg yessotoxin per kg.

Effect of early fetal splenectomy on prenatal B-cell development in sheep.

The contribution of early splenic B-cell populations to the colonization of the ileal Peyer's patch was investigated following the surgical removal of the spleen in a series of 56-day-old fetal sheep. The fetuses were killed at 140 days of gestation and the ileal Peyer's patch, the distal jejunal lymph node which drains the Peyer's patch, and a peripheral lymph node, the superficial cervical lymph node, were examined. Enzyme and immunohistochemical evaluation concluded that the distribution of B cells, T cells and stromal cells in the ileal Peyer's patch was similar in splenectomized and normal fetal sheep. Thus, the presence of the fetal spleen was not essential for the colonization of the ileal Peyer's patch and other early sites of B-cell accumulation would appear capable of generating the necessary precursor populations. Investigation of B-cell populations in lymph nodes used a combination of terminal deoxynucleotidyl-transferase-mediated deoxyuridine-triphosphate nick-end-labelling (TUNEL) histochemistry and immunofluorescence to determine the average number of apoptotic B cells in the primary follicles of the outer cortex of splenectomized and normal lambs. A significantly increased number of apoptotic B cells was present in the distal jejunal lymph node but not in the superficial cervical lymph node of splenectomized lambs. This finding suggests that splenectomy affected prenatal B-cell development in fetal sheep and raises questions as to the regulation of B-cell lymphopoiesis in a species using a post-rearrangement organ of diversification.

Characterization of proteoglycans and glycosaminoglycans in splenic AA amyloid induced in mink.

Amyloidosis of the protein AA type is readily induced in mink using repeated injections of bacterial lipopolysaccharide (LPS). We have characterized splenic proteoglycans/glycosaminoglycans (PGs/GAGs) in mink during amyloidogenesis. Moderate to rich amounts of amyloid exhibiting green birefringence was demonstrated by polarization microscopy of the splenic section stained with Congo red in seven out of eight minks after 10 weeks of LPS-treatment, and a significant increase in the total amount of PGs and GAGs in AA amyloid spleens was observed (two to eight times that in unstimulated animals). Intact PGs as well as free GAGs were extracted, and heparan sulfate (HS) was the most abundant GAG in the amyloid as well as in the control spleens. The GAGs showing the most pronounced increase in the amyloid spleens was of the chondroitin sulfate/dermatan sulfate (CS/DS) type and these were extracted in the form of free GAG chains. We conclude that there is a selective enrichment of PGs/GAGs in extracted splenic amyloid in the mink, which confirms to previous observations in human amyloid as well as in other animal species, supporting their pathogenic significance in the formation of AA amyloid.

Accessory cell populations in draining lymph nodes of lambs in the elicitation phase of DNCB-induced contact hypersensitivity.

The effect of experimentally induced contact hypersensitivity on accessory cell populations in draining lymph nodes of lambs was studied. Previous studies of draining lymph nodes of lambs during the elicitation phase of CHS have shown that there are significant changes in T-cell subpopulations, particularly CD4(+) cells and gamma delta T-cells, but the behaviour of accessory (antigen presenting) cell populations was not investigated. The immunohistochemical presence of accessory cell populations was determined using markers for CD68, Pan MHCII, MHCII DQ, MHCII DR, OvCD1w1 (putative human CD1a/c-like) and OvCD1w2 (human CD1b-like). Ten lambs were sensitised, and 14 days later re-challenged, by applying the hapten di-nitro-chloro-benzene (DNCB) together with an acetone and olive oil (AOO) vehicle, onto the skin. Cryosections of the draining lymph nodes were stained immunohistochemically for the accessory cell markers. Using an image analysis system, the areas of staining in the lymph nodes from the challenged animals were compared with measurements in control animals. A significant increase in staining for CD68(+) cells was detected in the cortex of the DNCB-treated group (p=0.003). A significant increase in staining for the Pan MHCII marker was also observed in the DNCB group (p=0. 013). These results show that MHCII(+) cells and CD68(+) cells constitute a prominent cell population in the cortex of the regional lymph nodes of lambs in the late elicitation phase of DNCB-induced contact hypersensitivity.

Distribution of MHC-II and CD1 molecules in the skin of lambs and changes during experimentally-induced contact hypersensitivity.

The presentation of antigen to specific T-cell populations is a crucial event during the elicitation phase of contact hypersensitivity (CHS). Significant changes in CD4(+) T-cell and gammadelta T-cell populations occur in the skin of sheep 48h after re-exposure to dinitrochlorobenzene but the expression of antigen presentation molecules such as MHC-II and CD1 at this stage of the hypersensitivity response has not been investigated. In the present study, a panel of monoclonal antibodies recognising CD1 and MHC-II subtypes was used in combination with computer assisted morphometric analysis to estimate the distribution of antigen presentation molecules in the superficial and deep dermis of the ears of lambs during the elicitation phase of CHS. The MHC-II molecules showed predominantly a perivascular and peri-appendageal distribution in the dermis and there were scattered MHC-II(+) cells in the basal and suprabasal layers of the epidermis. The CD1w2(+) (CD1b-like) molecules were present on distinct cells that were scattered evenly through the dermis, whereas CD1w3(+) (CD1c-like) molecules were almost exclusively detected on or in close association with the vascular endothelium. There was a significant increase in the presence of MHC-DQ(+) cells in the superficial dermis of dinitrochlorobenzene-treated animals compared with both an untreated control group and a vehicle-treated control group. However, MHC-DQ/DR(+) and CD1w3(+) cells only showed a significant increase compared with the vehicle-treated control group. The present study shows that the distribution of molecules involved in antigen presentation to CD4(+) T-cells and gammadelta T-cells changes during the elicitation phase of CHS in sheep, and suggests a role for MHC-DQ molecules on antigen presenting cells. However, the changes in distribution and expression of MHC-II and CD1 subtypes argue against a prominent role for a CD1-dependent pathway for T-cell recognition in the clinical cutaneous hypersensitivity response in sheep. Based on the expression of MHC-II molecules and CD1c molecules, we also suggest a potential role for endothelial cells in antigen presentation during the clinical dermatitis reaction.

Increased gammadelta T-cell populations in draining lymph nodes of lambs during the elicitation phase of dinitrochlorobenzene-induced contact hypersensitivity.

Ten lambs were sensitised with the hapten DNCB in an acetone/olive oil vehicle. The hapten/vehicle solution was applied onto the skin on the shaved ventral surface of the right ear. Two weeks later these lambs were rechallenged with the DNCB/vehicle solution. Simultaneously, ten non-sensitised lambs were treated with vehicle only, serving as vehicle controls. The 20 lambs were slaughtered 48 h after challenge/vehicle treatment, along with ten untreated animals serving as normal controls. Specimens of draining lymph nodes were collected from the 30 animals. All lambs were between 149 and 187 days old. Lymph node cryosections were stained for several leukocyte markers using monoclonal antibodies with the ABC immunohistochemical method. The stained sections were subsequently assessed in three different cortical compartments in each section, using an image analysis system. The resulting measurements from the three groups were compared. A marked increase of gammadelta T cells was detected in the DNCB group. The number of CD4+ T helper cells was decreased in the DNCB group compared with the normal control group, but not with the vehicle control group. No differences were revealed for CD8+ T cytotoxic cells or B cells. These findings were interpreted to be the consequences of possible downregulatory mechanisms protecting the lymphoid tissue from hypersensitivity. The prominence of gammadelta T-cells could indicate that these cells are involved in downregulation.

Factor XIIIa positive dendritic cells are a major accessory cell population in the elicitation phase of DNCB-induced contact hypersensitivity.

The phenotypes and distribution of accessory cells in the ear skin of lambs during the elicitation phase of dinitrochlorobenzene (DNCB)-induced contact hypersensitivity (CHS) were examined using indirect immunoperoxidase histochemistry (ABC method), and a panel of antibodies. Thirty lambs, between 21 and 26 weeks of age, were divided into groups of 10. The shaved right ear of one group was treated with DNCB. Two weeks later this group was challenged with DNCB. One group was treated with the vehicle alone and the remaining group was left untreated. The lambs were slaughtered 48 h after challenge, and tissue specimens were collected from the ears of the three groups. Factor XIIIa+ (FXIIIa+) cells were prominent in the superficial dermis and showed predominantly a perivascular and subepidermal distribution. The other markers were less prominent, and whereas CD1+ cells and CD68+ cells showed a reaction pattern similar to the FXIIIa+ cells, CD14+ cells were found scattered predominantly in the deep dermis. There appeared to be an increase in FXIIIa+ cells, CD1+ cells, and CD68+ cells in the dermis of the DNCB-treated lambs 48 h after challenge. Only CD1+ cells were detected in epidermis of normal controls, and these cells appeared to be decreased in number in the two treated groups. Computer-assisted morphometric analysis was used to estimate the relative presence of the accessory cell subpopulations in the superficial and deep dermis and the entire dermis. A statistical analysis of the relative area of immunostaining showed a significantly increased presence of FXIIIa+ cells and CD68+ cells in the dermis of the DNCB-treated lambs 48 h after challenge. Interestingly, FXIIIa+ cells and CD68+ cells were also significantly increased in the vehicle treated group compared with untreated controls. We found no significant difference in the presence of CD1+ cells or CD14+ cells in the DNCB treated group compared with the controls. The study showed that FXIIIa+ DDC are the major accessory cell population in normal ear skin of lambs and the major responsive population during the elicitation phase of CHS. The lack of response in the CD1+ cell population suggests a less prominent role for the LC-related DC in the skin during the elicitation phase.

Prominence of gammadelta T cells in the elicitation phase of dinitrochlorobenzene-induced contact hypersensitivity in lambs.

Immunohistochemical techniques were used to identify T-cell subpopulations in the ear skin of lambs during the elicitation phase of dinitrochlorobenzene (DCNB)-induced contact hypersensitivity. Thirty lambs (21-26 weeks of age) were divided into groups of 10. The shaved right ear of one group was treated with DNCB. Two weeks later, this group was challenged with DNCB. One group was treated with the vehicle alone, and the remaining group was left untreated. The lambs were killed 48 hours after challenge, and tissue specimens were collected from the ears of the three groups. There was an increase in T-cell populations in the skin of the DNCB-treated lambs 48 hours after challenge. The majority of the T cells were CD8+ and associated predominantly with the blood vessels and adnexa of the superficial dermis. There was also an increased presence of CD4+ cells and gammadelta T cells in the superficial dermis. In the epidermis, clusters of gammadelta T cells and CD4+ cells were associated with microlesions. Computer-assisted morphometric analysis was used to estimate the relative presence of the T-cell subpopulations in the superficial and deep dermis and the entire dermis. Statistical analysis of the relative area of immunostaining showed that the significant increases in all T-cell subpopulations (CD4+, CD8+, and gammadelta T cells) in the entire dermis were accounted for by changes in the superficial dermis. The prominence of gammadelta T cells in delayed-type hypersensitivity in the skin of domestic ruminants has been the subject of conflicting reports. In the present study, CD4+ cell and gammadelta T-cell populations were of similar size in the normal and DNCB-treated lambs, suggesting an equal participation in the elicitation phase of contact hypersensitivity.