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.

Loop-mediated isothermal amplification for detection of the tomato and potato late blight pathogen, Phytophthora infestans.

AIMS: To design and validate a colorimetric loop-mediated isothermal amplification assay for rapid detection of Phytophthora infestans DNA. METHODS AND RESULTS: Two sets of loop-mediated isothermal amplification (LAMP) primers were designed and evaluated for their sensitivity and specificity for P. infestans. ITSII primers targeted a portion of the internal transcribed spacer region of ribosomal DNA. These primers had a limit of detection of 2 pg P. infestans DNA and cross-reacted with the closely related species Phytophthora nicotianae. Rgn86_2 primers, designed to improve assay specificity, targeted a portion of a conserved hypothetical protein. These primers had a limit of detection of 200 pg P. infestans DNA and did not cross-react with P. nicotianae. The specificity of the Rgn86_2 assay was tested further using the closely related species P. andina, P. ipomoeae, P. mirabilis and P. phaseoli. Cross-reactions occurred with P. andina and P. mirabilis, but neither species occurs on tomato or potato. Both primer sets were able to detect P. infestans DNA extracted from tomato late blight leaf lesions. CONCLUSIONS: Two colorimetric LAMP assays detected P. infestans DNA from pure cultures as well as infected leaf tissue. The ITSII primers had higher sensitivity, and the Rgn86_2 primers had higher specificity. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report of a LAMP assay for the detection of P. infestans, the causal organism of potato and tomato late blight. These assays have potential for immediate utility in plant disease research and diagnostic laboratories.

First Report of the European Lineage of Phytophthora ramorum on Viburnum and Osmanthus spp. in a California Nursery.

Phytophthora ramorum S. Werres & A.W.A.M. de Cock is the causal agent of sudden oak death in California and Oregon forests and ramorum blight on a broad range of host species in wildlands and nurseries. It is thought to be an introduced pathogen and only three clonal lineages are known (3). The North American lineage (lineage NA1, mating type A2) is responsible for infections in California and Oregon forests. The European lineage (lineage EU1, predominantly A1) is responsible for infections in Europe, but has also been found in nurseries in Oregon and Washington. A third lineage (NA2) has only been isolated in a few instances from nurseries in Washington and California. In June 2006, P. ramorum was isolated from diseased Viburnum tinus, Osmanthus heterophyllus, and O. fragrans cultivars from a Humboldt County retail nursery in northern California. We genotyped isolates and placed them into clonal lineages using microsatellite markers developed for P. ramorum (3,4). Genomic DNA was extracted from mycelia with the FastDNA SPIN kit (Q-Biogene, Morgan, Irvine, CA). Primers used were PrMS6, Pr9C3, PrMS39, PrMS43a, PrMS43b, and PrMS45 (3) and 18, 64, and 82 (4). We sized fluorescently labeled amplicons using capillary electrophoresis (3100 Avant Genetic Analyzer, Applied Biosystems, Foster City, CA). Isolate genotypes were compared with control isolates of known clonal lineage, including BBA9/95 (EU1), Pr102 (NA1), and WSDA3765 (NA2). Three of four isolates belonged to genotype EU1. The fourth isolate, obtained from O. fragrans, belonged to genotype NA1. We repeated genotyping on independent genomic DNA extractions and obtained identical results. Two EU1 isolates and the single NA1 isolate were tested for mating type (1) and found to be of A1, A1, and A2 mating type, respectively. The coexistence of A1 and A2 mating types in the same retail nursery suggests the potential for sexual reproduction, as is the case in P. infestans where clonal and sexual populations exist (2), although to date, sexual reproduction in nature has not been documented in P. ramorum. The California retail nursery infestation highlights the risks associated with the unintentional transport of host nursery stock infested with P. ramorum. References: (1) C. M. Brasier and S. Kirk. Mycol. Res. 108:823, 2004. (2) N. J. Grünwald and W. G. Flier. Ann. Rev. Phytopathol. 43:171, 2005. (3) K. Ivors et al. Mol. Ecol. 15:1493, 2006. (4) S. Prospero et al. Mol. Ecol. 16:2958, 2007.

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.

Subclinical paratuberculosis in goats following experimental infection. An immunological and microbiological study.

An experimental oral infection of goats with a caprine isolate of Mycobacterium a. subsp. paratuberculosis was used to investigate immunological and bacteriological events during the subclinical phase of infection. Seven goats at 5-8 weeks of age were given a bacterial suspension in milk-replacement three times weekly for 9 weeks. Six animals were kept as controls. Cellular recall responses against M. a. paratuberculosis were analysed by means of a lymphocyte proliferation test, an IFN-gamma assay and an IL-2 receptor assay. All inoculated animals had detectable CMI responses from 9 weeks post-inoculation and through the 2 years of study, although the responses were highest during the first year. Antibodies against M. a. paratuberculosis could be detected from weeks 15-20 in four of the seven animals, and one additional animal became antibody positive at week 35, while two inoculated animals did not produce significant antibody titres during the experiment. At about 1-year post-inoculation, two animals became faecal shedders, while two others started to excrete bacteria into faeces about 2 years post-inoculation. The appearance of M. a. paratuberculosis in faeces was not associated with a decline in cellular responses as far as could be assessed using the current methods for measuring CMI. Pathological lesions due to M. a. paratuberculosis infection and presence of bacteria were recorded in the intestine and/or mesenteric lymph nodes of five animals while lymph node changes suggestive of paratuberculosis were observed in one animal. Only the two animals with no signs of an active infection at necropsy showed a considerable decline in the cellular parameters during the last year of the study, particularly in the IFN-gamma assay. The two animals with the highest levels of M. a. paratuberculosis responsive CD8+ lymphocytes in the circulation about 1-year post-inoculation had no detectable lesions in the distal ileum and colon at necropsy, while high numbers of gammadelta T-cells responsive to M. a. paratuberculosis in the circulation were associated with disseminated lesions in the distal ileum and colon.

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.

Uptake of Mycobacterium avium subsp. paratuberculosis through the distal small intestinal mucosa in goats: an ultrastructural study.

Various pathogens gain access to the intestinal wall via specialized cells, the M cells, found among the follicle-associated epithelial cells overlying the domes of the Peyer's patches. The present study was undertaken to examine the uptake of live Mycobacterium avium subsp. paratuberculosis in the distal small intestine of goat kids. Following laparotomy, distal small intestinal segments of five goats were ligated and injected with bacterial suspension. After 1 hour, the intestinal segments were excised and fixed for light and electron microscopic studies. M. a. paratuberculosis organisms were observed by transmission electron microscopy at locations in the intestinal wall, suggesting transcellular transportation through the M cells. The organisms were present both in the cytoplasm of the M cells and in the cytoplasm of intraepithelial leukocytes found in M-cell pockets. Intercellular bacteria between M cells were occasionally seen. Bacteria were not observed in association with the absorptive epithelium. This study indicates that in goat kids, M. a. paratuberculosis enters the intestinal wall primarily through the M cells in the follicle-associated epithelium of the Peyer's patches.

Role of iron in rhizobacteria-mediated induced systemic resistance of cucumber.

ABSTRACT Seed treatment with the rhizosphere bacterium Serratia marcescens strain 90-166 suppressed anthracnose of cucumber, caused by Colleto-trichum orbiculare, through induced systemic resistance (ISR). When the iron concentration of a planting mix was decreased by addition of an iron chelator, suppression of cucumber anthracnose by strain 90-166 was significantly improved. Strain 90-166 produced 465 +/- 70 mg/liter of catechol siderophore, as determined by the Rioux assay in deferrated King's medium B. The hypothesis that a catechol siderophore produced by strain 90-166 may be responsible for induction of systemic resistance by this strain was tested by evaluating disease suppression by a mini-Tn5-phoA mutant deficient in siderophore production. Sequence analysis of genomic DNA flanking the mini-Tn5-phoA insertion identified the target gene as entA, which encodes an enzyme in the catechol siderophore biosynthetic pathways of several bacteria. Severity of anthracnose of cucumbers treated with the entA mutant was not significantly different (P = 0.05) from the control, whereas plants treated with wild-type 90-166 had significantly less disease (P = 0.05) than the control. Total (internal and external) population sizes of 90-166 and the entA mutant on roots did not differ significantly (P = 0.05) at any sample time, whereas internal population sizes of the entA mutant were significantly lower (P = 0.05) than those of the wild-type strain at two sampling times. These data suggest that catechol siderophore biosynthesis genes in Serratia marcescens 90-166 are associated with ISR but that this role may be indirect via a reduction in internal root populations.

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.

Isolation in cell culture of nodavirus from farmed Atlantic halibut Hippoglossus hippoglossus in Norway.

Isolation in cell culture of nodavirus from Atlantic halibut Hippoglossus hippoglossus suffering from viral encephalopathy and retinopathy (VER) is described. The cell line SSN-1 was inoculated with tissue material from affected juveniles (60 d after first feeding). Extensive cytopathic effects (CPE) developed approximately 5 d after inoculation, and were also observed after several passages in the same cell line. Cells from infected cultures showed reactivity with an antiserum against sea bass Dicentrarchus labrax nodavirus in an indirect immunofluorescence test. Analysis of infected cells with reverse transcriptase-polymerase chain reaction (RT-PCR) resulted in a product of the predicted size using primers specific for striped jack Pseudocaranx dentex nodavirus. Electron micrographs of infected SSN-1 cells demonstrated virus particles that were approximately less than 30 nm. Challenge of Atlantic halibut larvae (4 d post-hatching) with supernatants from infected SSN-1 cells resulted in development of VER as verified by immunohistochemistry performed on larvae sampled from Day 9 after challenge. The present results show that a nodavirus from Atlantic halibut has been isolated using the SSN-1 cell line and that virus propagated in cell culture retained virulence.

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.

Bacterial isolation, immunological response, and histopathological lesions during the early subclinical phase of experimental infection of goat kids with Mycobacterium avium subsp. paratuberculosis.

The diagnosis of Mycobacterium avium subsp. paratuberculosis infection is difficult, especially in the early stages of disease. This is due to the long incubation period, the variable lag phase associated with bacterial proliferation, and the multifocal distribution of slowly developing lesions. There are few previous studies of the early stages of experimental paratuberculosis in goats. In the present study, the ability of conventional diagnostic methods to detect M. a. paratuberculosis infection during the early stages of infection was assessed. Eight goat kids were experimentally infected with M. a. paratuberculosis and subjected to a series of immunological and bacteriological tests before being euthanatized at various times postinfection. At postmortem examination, the ages of the kids ranged from 1 1/2 to 12 months. Of the eight goats infected, three had histopathological evidence of paratuberculosis. Two of these goats were positive with bacteriology, but only one was also positive with all immunological tests. One animal had a positive immunological response, but infection could not be demonstrated by bacteriologic or histopathologic examination. Histopathologic lesions were found in the jejunum, in the ileum, and in one mesenteric lymph node, but only the mesenteric lymph nodes and one retropharyngeal lymph node gave positive results following bacteriologic culture. The disparity between the localization of histopathologic lesions and bacteriologic results emphasizes the need for exhaustive sampling to confirm a diagnosis during the early phase of an infection. It also highlights the need for a better understanding of the biology of M. a. paratuberculosis and its interaction with the immune system of the host.

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.

Detection of viral antigen in placenta and fetus of cattle acutely infected with bovine viral diarrhea virus.

The reproductive organs and fetuses of seven Norwegian Red heifers were investigated for the presence of bovine viral diarrhea virus (BVDV) antigen during the time of initial transplacental transmission of the virus. The heifers were inoculated with a noncytopathogenic BVDV at day 85/86 of gestation and were slaughtered at day 7, 10, 14, 18, or 22 postinoculation (pi). Cryostat sections of uterus, ovaries, placentomes, intercotyledonary fetal membranes, and fetal organs were examined using immunohistochemical techniques. A double immunofluorescence technique was used to identify cells that showed staining with antibodies against the leukocyte common antigen CD45 or the intermediate filament vimentin and BVDV antigens. The earliest stage of infection at which BVDV antigen could be detected in the fetuses was 14 days pi. At this stage, BVDV antigen was detected in cells of mesenchymal origin in the lungs and in large cells that morphologically resembled immature megakaryocytes in the liver. In the intercotyledonary fetal membranes and in the placentomes, BVDV antigen was not detected until 18 and 22 days pi, respectively. BVDV antigen was not detected in maternal tissue from any of the heifers. The present results indicate that fetal infection with BVDV can take place without preceding or simultaneous high concentrations of BVDV in uterus or placenta of acutely infected heifers.

Distribution of viral antigen in uterus, placenta and foetus of cattle persistently infected with bovine virus diarrhoea virus.

The tissue distribution and cellular localisation of bovine virus diarrhoea virus (BVDV) was investigated in the uterus, placentomes, intercotyledonary foetal membranes and foetal organs of three persistently infected (PI) pregnant heifers. The uterus and ovaries of a non-pregnant PI heifer were also included in the study. Cryostat sections were examined using immunohistochemical techniques and monoclonal antibodies against BVDV. A double immunofluorescence technique was used to identify BVDV positive cells that also showed staining for either the leukocyte common antigen CD45 or the cytoskeletal filament vimentin. BVDV antigen was detected in all the organs examined, and was present in both epithelial and non-epithelial cells. In all organs many of the virus-positive cells also showed reactivity for vimentin. In the foetal liver and spleen a small, scattered population of virus-positive cells showed reactivity for CD45. A few cells showed reactivity both for BVDV antigen and for CD45 in the placentomes and intercotyledonary foetal membranes. In contrast to earlier reports, only scattered cells in the foetal part of the placentomes, the cotyledons, showed reactivity for BVDV antigen. However, in the chorion of the intercotyledonary foetal membranes, a larger proportion of the trophoblast cells showed reactivity for BVDV, especially the binuclear trophoblast cells. In the uterus, pregnancy appeared to favour virus replication, as the section from the pregnant heifers showed much stronger staining and a higher proportion of viral antigen-positive cells than sections from the non-pregnant PI heifer.

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.

Development of accessory cells in B-cell compartments is retarded in B-cell-depleted fetal sheep.

Accessory-cell populations in the lymphoid tissues of fetal sheep were investigated following depletion of B cells. An intraperitoneal injection of an anti-IgM antibody early in gestation resulted in a marked depletion of IgM+ cells in lymphoid tissues. Immune and enzyme histochemical techniques were used to identify accessory-cell populations in the ileal Peyer's patch, spleen, and lymph nodes of B-cell-depleted fetal sheep. The rudimentary follicles in the ileal Peyer's patch showed strong enzyme reactivity for 5'nucleotidase, indicating the presence of follicular dendritic cells (FDCs). Enzyme reactivities for FDCs in primary follicles of the spleen and lymph nodes were absent, as were reactivities for metallophilic macrophages in the marginal zone of the spleen. MgATPase reactivity associated with dendritic-cell populations in the gut-associated lymphoid tissues was detected. A monoclonal antibody against complement receptor-2 (CD21) reacted with FDCs in the rudimentary follicles of the ileal Peyer's patch and immature FDCs in lymph nodes. The results suggest that the development of accessory-cell populations in B-cell compartments of peripheral but not central lymphoid tissues is dependent on the presence of B cells.

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.

Apoptosis in phagocytotic cells of lymphoid tissues in rainbow trout (Oncorhynchus mykiss) following administration of clodronate liposomes.

Macrophage function has been studied in vivo by using liposomes that contain dichloromethylene-bisphosphonate (clodronate liposomes) to deplete macrophage subpopulations. In the present study, the effects of intravenously injected clodronate liposomes on the head kidney and spleen of the rainbow trout (Oncorhynchus mykiss) were studied from 1 h to 7 days after injection. The rapid trapping of liposomes in the splenic ellipsoids was followed by depletion of ellipsoidal sheath macrophages and accumulation of particulate material and IgM in the ellipsoidal wall, findings illustrating the importance of ellipsoidal macrophages in the clearance of filtered substances trapped in the reticular matrix of the ellipsoidal wall. A reduced reactivity for acid phosphatase in the spleen and ultrastructural evidence of cell death in phagocytotic cells of the head kidney and spleen supported the selective effect of clodronate liposomes on macrophages in rainbow trout. Apoptotic bodies were prominent ultrastructural features in tissues collected from clodronate-liposome-treated rainbow trout. The increased presence of apoptotic cells in clodronate-liposome-treated trout compared with trout given liposomes containing phosphate-buffered saline was confirmed by using terminal deoxynucleotidyl-transferase-mediated deoxyuridine-triphosphate nick-end-labelling of cells with extensive DNA fragmentation. The characterization of liposome-mediated macrophage depletion in fish provides a useful model for further investigation of piscine macrophage function.