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As a result of the European ban of in-feed growth-promoting antibiotics, new strategies are being developed to increase the resistance to disease in farm animals. In pig production, this is of particular importance during the weaning transition when piglets are subjected to major stressful events, making them highly sensitive to digestive disorders. At this time, the development of both innate and adaptive immunity at the mucosal surface is critical in preventing the potential harmful effects of intestinal pathogenic agents. Strategies aiming at stimulating natural host defences through the use of substances able to modulate immune functions have gained increasing interest in animal research, and different bioactive components a priori sharing those properties have been the subject of in vivo nutritional investigations in pig. Among these, yeast derivates (β-glucans and mannans) are able to interact with immune cells, particularly phagocytic cells. However, studies where they have been fed to pigs have shown inconsistent results, suggesting that their ability to target the sensitive immune cells through the oral route is questionable. The plant extracts, which would benefit from a positive image in the public opinion, have also been tested. However, due to a lack of data on the bioactive components of particular plants and the large diversity of species, it has proved difficult to prepare extracts of equivalent potency and thus, the literature on their influence on pig immunity remains inconclusive. In considering piglet immunity and health benefits, the most promising results to date have been obtained with spray-dried animal plasma, whose positive effects would be provided by specific antibodies and non-specific competition of some plasma components with bacteria for intestinal receptors. The major positive effect of spray-dried animal plasma is in reducing the infiltration of gut-associated lymphoid tissue by immune cells, which is likely to be the result of a decreased colonisation by potentially harmful bacteria. This review also highlights the limitations of some of the published in vivo studies on the immunomodulatory activity of certain feed additives. Among those, the lack of standardisation of extracts and the heterogeneity of piglet-rearing conditions (e.g. exposure to pathogens) are likely the most limiting.

Intestinal tissue samples were examined from 32 cats in which a histopathological diagnosis of alimentary lymphoma or multicentric lymphoma affecting the gastrointestinal tract had been made. These samples were re-evaluated histopathologically and serial sections were examined immunohistochemically with antisera specific for the lymphoid markers CD3, CD79a and BLA-36 and for class II molecules of the major histocompatability complex. The cats ranged in age from 4-16 years (median 10.5 years). The main presenting clinical signs were vomiting, diarrhoea and weight loss. The majority of alimentary lymphomas were of the B-cell type (n=15), whereas cases of T-cell lymphoma were fewer in number (n=8). Four cats had lymphoma of a mixed T-and B-cell phenotype. In five of the cats, immunohistochemistry suggested an inflammatory process, in contradiction to the original histopathological diagnosis of lymphoma. Immunolabelling would appear to be a useful adjunct to histopathology in classifying cases of feline alimentary lymphoma, and may help in distinguishing lymphoma from severe intestinal inflammation.

Developments in immunohistology allow the routine simultaneous use on tissue sections of three monoclonal antibodies, tagged with different fluorochromes. Such staining can identify seven different cell populations and the limiting factor is rapid, reliable and reproducible analysis. Future reliance on computer-assisted analysis of digitised images depends on validation against manual counting, often viewed as the 'gold standard'. In this study images were digitised from sections of normal porcine skin, inflamed skin and tonsil, simultaneously stained with three monoclonal antibodies. Combinations of staining were quantified by four manual counts and by pixel-based area measurement. On individual images, the correlation between automated and manual measurements was poor. Despite this, the concordance between manual and automated measurements in the means and variances of tissues was good, and both techniques identified the same changes in inflamed versus normal tissues. In addition, pixel-based counting permitted statistical analysis of co-localisation of cell types in tissue sections. We conclude that automated counting is acceptable for the assessment of tissues, is faster and provides less opportunity for observer variation than manual counting. We also demonstrate that the technique is applicable where more than three fluorochromes are used such that manual counting becomes essentially impossible.

Feline immunodeficiency virus (FIV) is a lentivirus that causes feline acquired immunodeficiency syndrome. Infection can be transmitted experimentally via the vagina and rectum, making the cat a useful model for human immunodeficiency virus (HIV) infection. Some strains of FIV use the CXCR4 chemokine receptor in vitro to gain entry to feline cell lines, thymocytes and peripheral blood leucocytes (PBLs). In this study, the tissue expression of messenger ribonucleic acid (mRNA) encoding the CCR3, CXCR4 and CCR5 receptors was examined by reverse transcriptase polymerase chain reaction (RT-PCR). mRNA encoding each receptor was expressed by two feline T-cell lines (Mya-1 and FeTJ), a feline kidney fibroblast cell line (FKCU) and PBLs. Mesenteric lymph node, colon, rectum, uterus, cervix and vagina all expressed mRNA for CXCR4 and CCR5 whilst only lymph node expressed CCR3 mRNA. In order to locate this receptor mRNA expression, in-situ hybridization studies were performed with DNA probes specific for the chemokine receptor mRNAs. CCR5 and CXCR4 receptor mRNA was expressed by epithelial cells and some lamina propria cells of the colon and rectum. Epithelial cell expression of chemokine receptor mRNA was reduced in intensity towards the base of the crypts. Expression of CXCR4 receptor was also demonstrated immunohistochemically on some lamina propria and intraepithelial cells. The expression of these receptor molecules may be important in mucosal infection with FIV., (Copyright Elsevier Science Ltd. All rights reserved.)

The objective of this study was to examine measurable parameters that could be used to describe the gross and microscopical anatomy of the feline colon, which has not been previously characterized. Post-mortem data were collected from 35 specific pathogen-free cats. Gross morphometric data (total colonic length, wet weight, number of lymphoid aggregates) were collected together with microscopical measurement of crypt depth and numbers, intra-epithelial lymphocyte numbers and the number of proliferating cell nuclear antigen (PCNA)-positive cells and their relative position within the crypt. Colonic length as a percentage of intestinal length was remarkably constant (20.9+/-2.0%). Crypt depth, however, showed considerable inter-cat variation (149-688 microm); the crypts became deeper with distance from the anus. Cellular proliferation was predominantly in the lower part of the crypt, and the numbers of PCNA-positive cells increased with distance from the anus. The number of lymphoid aggregates varied with distance from the anorectum and appeared to reflect the bacterial load. Intra-epithelial lymphocytes were relatively sparse (3.9+/-2.7/100 epithelial cells); they showed considerable inter-cat variation but did not vary with distance from the anus. These data suggest that to improve the interpretation of morphometric parameters by reducing inter-cat variation, samples should be taken at a consistent distance from the anus, broad reference ranges having now been established.

The aim of this study was to investigate the distribution of leucocyte subsets in the small intestine of healthy adult cats (n=16). Immunohistochemical methods were used to identify leucocyte subsets within the mucosa of the duodenum, jejunum and ileum. Computer-aided morphometry was used to enumerate cells within the epithelial compartment, villous lamina propria and lamina propria adjacent to upper and lower crypt. Throughout the small intestine, IgA+ and IgM+ plasma cells were more prominent in the lamina propria adjacent to the lower crypt than in the villus, whereas IgG+ plasma cells were present in equal numbers in the crypt and villous regions. Overall, IgA+ plasma cells predominated and IgM+ plasma cells were higher in number than IgG+ plasma cells at each of the three anatomical locations. By contrast, T cells (CD3+) and T-cell subsets (CD4+ and CD8+) were present in greater numbers in the villous lamina propria than in the lamina adjacent to the crypts. Intraepithelial lymphocytes (IELs) were also characterized phenotypically, the majority being CD8+ T lymphocytes. Lamina propria macrophages and dendritic cells were characterized by expression of L1 and major histocompatibility complex (MHC) class II, and MHC class II expression by enterocytes overlying Peyer's patches, although rare, was also shown. The qualitative and quantitative data from this study provide a basis for comparison with cats with inflammatory enteropathies., (Copyright Harcourt Publishers Ltd.)

Ultrastructural, histochemical and immunohistochemical features of porcine intestinal lamina propria macrophages (LPMs), peripheral blood fibronectin-adherent cells (FACs) and splenic-adherent cells (SPACs) were compared. Freshly isolated FACs and SPACs were small and showed small cytoplasmic processes, little evidence of endocytic vacuoles, few lysosomes and sparse rough endoplasmic reticulum (RER). Fresh FACs were negative for acid phosphatase, non-specific esterase (NSE) and beta-galactosidase activity. Of the SPACs, 20-40% were positive for acid phosphatase, < 5% for NSE and 5-10% for beta-galactosidase. Pre-cultured FACs and SPACs were large and showed an abundance of endocytic vacuoles; they possessed dilated and prominent RER and > 95% were positive for the three enzyme activities. LPMs exhibited abundant endocytic vacuoles or vesicles and lysosomes but sparse RER, and > 85% were positive for the three enzymes. LPMs (24%), FACs (49%) and SPACs (40%) expressed MHC (major histocompatibility complex) class II glycoproteins. Macrophage-granulocyte antigens were detected in LPMs (14%), FACs (50%) and SPACs (33%). The results thus suggest that freshly isolated FACs differ from LPMs morphologically and in enzymic features, and the differences may represent part of the cell maturation process.

A panel of four monoclonal antibodies produced in our laboratory, MIL1, MIL2, MIL3, MIL4, and the type-specific monocyte/granulocyte marker 74-22-15 were used to isolate and to discriminate between monocytes, macrophages and granulocytes derived from porcine peripheral blood, lung and gut lamina propria. Two-colour flow cytometry and cell sorting showed that while no monoclonal antibody was specific for just a single cell population, each cell type had a unique and characteristic combination of surface antigens. These differences could be used to identify and purify monocytes, macrophages, neutrophils, eosinophils and basophils from the three different sites. The study also demonstrated similarities and differences within cell types from the same site and from different sites: polymorphonuclear neutrophils (PMN) from peripheral blood were subdivided into two subpopulations by the presence or absence of the surface antigen recognized by MIL4, while PMN from alveolar lavage did not express this antigen. Peripheral blood eosinophils were also divided into subpopulations by the presence or absence of the same surface antigen. Lamina propria eosinophils strongly expressed the MIL4 marker and differed morphologically from blood eosinophils. Peripheral blood basophils and lamina propria mast cells were morphologically similar and expressed similar antigens. Monocytes and alveolar macrophages also expressed the same surface antigens.

Four fractions of IgG from normal dog serum have been successfully isolated by gel filtration followed by protein A and protein G affinity chromatography using the fast protein liquid chromatography (FPLC) system. Protein A chromatography produced three peaks: peak 1 was fallthrough material consisting of components which did not bind to protein A, peak 2 consisted of bound material eluting at pH 6, and peak 3 contained bound material eluting at pH 3.5. The three peaks were then subjected individually to protein G affinity chromatography. Peak 1 from protein A chromatography produced a fallthrough peak followed by a weakly binding component which eluted at pH 8, and was called peak w. Peak 2 from protein A chromatography bound to protein G and eluted as a single peak at pH 3.8, and was called peak x. Peak 3 from protein A chromatography emerged as two separate peaks (y and z) off the protein G column; peak y bound and eluted at pH 4.1, and peak z bound weakly to protein G and emerged as a broad band at pH 8. Peaks w, x, y and z have been named gamma w, gamma x, gamma y and gamma z, respectively, and there purified IgG fractions were used to immunize mice for the preparation of monoclonal antibodies (McAbs). To date, two sets of McAbs have been produced: one which recognizes an epitope present in both gamma w and gamma z fractions and another set of McAbs which recognizes an epitope in the gamma x and gamma y fractions.

PROBIT estimates biologic activity by weighted probit regression analysis and comparison with a known standard. The program is intended to speed up calculation of the concentration of cytokines in large numbers of cell supernatants. Data are input from either sequential or free-form text files created with a spreadsheet or text editor. This allows transfer of data from a beta-counter equipped with a suitable terminal without retyping. Results are displayed and/or printed as relative potency (% of standard) and 50% effective dose (ED50).

A monoclonal antibody is described which recognises an epitope associated with a receptor for interleukin-2 (IL-2) on pig lymphocytes. The monoclonal antibody inhibits high affinity binding of radiolabelled recombinant human IL-2 (rhIL-2) by pig lymphoblasts and also non-competitively inhibits both pig-TCGF and rhIL-2 maintained proliferation. By flow cytometry the antigen is apparently not present on freshly isolated blood lymphocytes but is detectable on small cells between 6 and 12 h after activation and on large cells by 24-h. These findings are comparable with those obtained using monoclonal antibodies recognising the 55 kDa alpha chain of the human and mouse IL-2 receptor (p55, TAC) expressed on activated cells in vivo and in vitro. However, the molecular weight of the porcine antigen is between 65 and 70 kDa.

An experimental model of proliferative glomerulonephritis (GN) in the cat, which closely resembles human proliferative forms of GN, has been used to study the role of antibody and antibody affinity in the development of immune complex-mediated renal disease. The serum IgG and IgM antibody response to antigen, average antibody affinity (avidity) and affinity heterogeneity of the IgG and IgM populations was assessed at varying times after commencement of chronic immunization with the antigen, human serum albumin (HSA), by enzyme immunoassay. Cats could be classified according to whether they were "low", "intermediate" or "high" IgG responders, by quantification of serum IgG values. Cats with the lowest serum IgG values failed to develop glomerulonephritis. However, there was no relationship between actual IgG values and the severity of the induced disease. In contrast to IgG, there was no division of cats into low or high IgM anti-HSA responders. Again, cats with the lowest IgM values failed to develop GN, but, more interestingly, a late, marked increase in serum IgM anti-HSA occurred only in cats that developed clinical signs of GN (anterior uveitis and nephrotic syndrome). Maturation of average, functional IgG affinity (avidity) for HSA following chronic immunization was clearly demonstrated for all cats. At the end of the experiment, all cats had IgG of high affinity for HSA and the average affinity heterogeneity of the IgG populations was less than in measurements taken earlier. Values of IgG affinity at the end of the experiment were very similar both in cats which developed GN and in those which remained clinically, biochemically and pathologically normal. In contrast to IgG antibody, some cats developed IgM of increased affinity, whilst others produced antibody of reduced affinity, following chronic immunization. There was no correlation between the development of disease and the production of either low or high affinity IgM antibody. Data indicated that an alteration in IgM affinity occurred at a late stage, as serum IgM levels increased, in cats which progressed to develop GN. These findings suggested that an increase in both serum IgG and IgM anti-HSA values, in particular IgM, was associated with the development of a more severe immune complex renal disease in these cats. Although there was no evidence that differences in the average affinity of either the IgG or IgM antibody populations for HSA, were associated with the development of disease, the increase in IgM values was also accompanied by a concomitant alteration in IgM affinity for antigen.(ABSTRACT TRUNCATED AT 400 WORDS)

A model of chronic serum sickness was used to induce immune-complex glomerulonephritis in seven experimental cats, by daily intravenous inoculation of an increasing dose (5 to 35 mg) of human serum albumin (HSA). At week four, two of the seven animals developed anterior uveitis. At week 23, two different animals developed the subcutaneous oedema characteristic of the nephrotic syndrome (NS), whilst the other five cats appeared clinically normal. The kidneys were examined at necropsy by light microscopy and by transmission electron microscopy. The glomeruli of four animals (three with both proteinuria and uraemia, and one with proteinuria only) showed morphological changes under light microscopy. The abnormalities suggested that a diffuse mesangial proliferative glomerulonephritis (GN) had been induced in three cats and diffuse membranoproliferative GN induced in another. Ultrastructural studies revealed electron-dense deposits (immune-complexes) in six of the seven cats. Two cats without glomerular abnormalities by light microscopy had mesangial deposits and three cats with mesangial proliferative GN had deposits at mesangial, subendothelial and/or subepithelial sites. The single cat with membranoproliferative GN had deposits at mesangial, subendothelial, subepithelial and intramembranous sites. Immunohistological examination (peroxidase-antiperoxidase technique) showed that HSA and immunoglobulin (IgG and IgM) were deposited in the glomeruli of these cats. Deposits were the most dense in cats with more severe renal lesions. Deposits of IgM were most abundant. An extensive cellular infiltrate, comprising macrophages, neutrophils and plasma cells, was observed only in the four animals which showed abnormalities in glomerular ultrastructure. The disease induced in these cats thus appears to differ from the membranous nephropathy previously described in the cat and bears a close resemblance to immune complex (IC) disease in man. In view of the relatively few specific animal models of IC-mediated proliferative GN, this model has potential for application to the study of human IC disease.

Biochemical changes in plasma and urine were monitored in six cats before and during the induction of immune complex-mediated glomerulonephritis (ICGN) by daily intravenous administration of human serum albumin (HSA). The earliest indication of renal dysfunction in the cats was hypoalbuminaemia, which occurred as early as 13 weeks before cats developed clinical signs of renal disease. Proteinuria occurred 2 to 3 weeks before clinical disease, but was sensitive in predicting renal pathology in two cats that did not develop clinical signs of disease. In addition, increased activities of several urinary enzymes were detected in affected cats, with measurement of N-acetyl-beta-D-glucosaminidase and gamma-glutamyl transferase providing the earliest and most sensitive indication of renal damage. These plasma and urine measurements correlated more closely with the renal pathology, observed at postmortem, than clinical assessment of disease. It was concluded that ICGN in the cat could be diagnosed earliest by measurement of plasma protein concentration, whilst disease progress could be effectively monitored by including assays to measure urine protein and urine enzymes.

Endometrial biopsies were collected on two occasions from mares resistant to (n = 3) and once from mares susceptible to persistent endometritis (n = 6). The endometrial tissue was minced and cultured in vitro for 24 h. No hemolytic complement activity was detected in the endometrial culture supernatant. Endometrial culture supernatant from mares with persistent endometritis contained titers of antibodies to Streptococcus zooepidemicus similar to those from resistant mares. However, the culture supernatant of biopsies from mares with endometritis was less effective (P < 0.05) at opsonizing S. zooepidemicus in vitro.

Mucosal lymphoid nodules were identified within the equine respiratory tract by an acetic acid fixation technique. Nodules were identified in foetuses from nine months gestational age, and estimates of total and regional nodule populations were made in foetal, neonatal and adult horses. Nodules occurred at specific sites within the tract, which probably relate to areas where inhaled antigens accumulate. The largest populations of nodules occurred in the nasopharynx and larynx, with smaller numbers in the nasal cavity, trachea and bronchi. There was an age-related change in the size of these nodule populations, with an increase in number from late gestation to the neonatal period to early adulthood (up to 5 years of age), followed by a decrease in older adults.

Atopic systems were more common in children with steroid-responsive nephrotic syndrome (S.R.N.S.) than in matched controls, and HLA-B12 was more common in children with S.R.N.S. than in adult controls. Atopic symptoms (particularly hayfever), positive prick tests with grass pollen antigens, and a higher mean serum concentration of IgE antibody to timothy grass pollen were more common in nephrotic children with HLA-B12 than in those without HLA-B12. There was also an increased frequency of the haplotype HLA-A1 and HLA-B8, mainly among the non-atopic patients.

The distribution of mucosa-associated lymphoid tissue along the equine respiratory tract was surveyed in light microscopical sections. Intra-epithelial lymphocytes and similar cells scattered in the lamina propria were identified in all sites from the nasal vestibule to bronchioles of 2 to 4 mm diameter. Isolated lymphoid patches, occasionally with nodules, were common in bronchioles, but the density of this bronchiole-associated lymphoid tissue (BRALT) varied between individual horses. Bronchus-associated lymphoid tissue (BALT) was infrequently encountered. In the upper respiratory tract, nodules dispersed within the nasal cavity, nasopharynx and near the auditory tube merit the collective term, nasal-associated lymphoid tissue (NALT). Laryngeal- and tracheal-associated lymphoid tissue (LTALT) was also identified, but this was limited to the epiglottis, arytenoid and rostral trachea. At all sites, the discrete lymphoid masses comprise primary or secondary nodules, a parafollicular and a dome area. The airway lining above was frequently modified into a lympho-epithelium and also showed varied patterns of infolding which might enhance antigen trapping.

Endometritis was induced in four progesterone-treated ovariectomized mares and in two seasonally anestrous mares by intrauterine infusion of Streptococcus zooepidemicus. The bacteria were suspended in phosphate buffered saline (PBS), or in nonimmune or hyperimmune serum. Uterine lavage was performed after 24 h. Significantly fewer (P < 0.05) bacteria were recovered from mares which received hyperimmune serum than from mares which received nonimmune serum or PBS. There was no significant difference between treatment with nonimmune serum or PBS. It is therefore suggested that increasing the availability of specific antibody reduced the severity of intrauterine infection.

Two histochemical staining techniques, Alcian blue/PAS and high-iron-diamine/Alcian blue, which differentiated neutral from sulphated and sialylated acid mucins were applied to sections of duodenum, ileum, caecum, colon and rectum from pigs aged one day, 3 weeks and 10 weeks. In each age group, sulphated-acid mucins predominated at all sites, particularly in the large intestine. In both the small and the large intestine, non-sulphated mucins occurred mainly at the bases of the crypts. Neutral mucins occurred in a few goblet cells in crypts and villi, either alone or together with acid mucins; neutral mucins increased away from the bases of the crypts. No changes were noted in the caecum, colon or rectum of pigs one, two, 5 or 13 days after weaning onto a diet containing soya protein. In the small intestine, there was a transient increase in the numbers of goblet cells in the crypts and villi, a relative increase in sulphated and a decrease in non-sulphated acid mucins and a change in the distribution of sulphated mucins in the crypts. No change in proportions of neutral and acid mucins was detected.