Significance of Freund's adjuvant/antigen injection granuloma in the maintenance of serum antibody response.

An experimental system involving injections of ovalbumin (OVA) and ferritin (FER) in Freund's incomplete adjuvant (FIA) into the right and left flank skin folds of sheep was used to study the influence of the FIA/antigen depot and the draining lymph node in maintaining an antibody response. Excision of the injection granuloma and draining lymph node from one side 2-3 months after injections resulted in a profound decrease in serum antibody titres. This response was observed in all eight sheep in the experimental group. In five of eight animals in another experiment, excision of the injection sites had no appreciable effect on antigen-specific antibody titres when compared with antibody specific for antigen on the intact side of the sheep. In the remaining three animals, excision of the injection site did cause some fall in titre. Radiotracer studies revealed that about one-third of the original [125I]OVA/FIA injected was present in the granuloma 20 weeks after injection. Lymphatic cannulation approaches were used to study the responsiveness of the lymph node draining an FIA/antigen granuloma established 12 weeks earlier and showed that increments of 1-2 mg OVA in saline administered adjacent to the granuloma at 6-7 day intervals gave rise to strong anti-OVA containing cell (AOCC) responses in lymph. There were 2-6-fold increases in serum antibody titre in response to 3-5 doses of OVA or FER (1-2 mg) in saline injected adjacent to the FIA/antigen injection site (which had been administered 14-16 weeks previously). It is concluded that the release rate of antigen from a FIA/antigen depot is insufficient to sustain maximal antibody levels in blood serum.

Lymphocyte phenotypes in the intestinal mucosa of sheep infected with Trichostrongylus colubriformis.

Monoclonal antibodies to ovine lymphocyte surface antigens were used in an immunohistochemical study of the intestine of sheep. In the epithelium CD8+ cells predominated whereas the majority of lamina propria T lymphocytes were CD4+. Infection of sheep with the parasitic nematode Trichostrongylus colubriformis including sufficiently large numbers of parasites to induce protective immunity did not alter the number of CD4+ or CD8+ lymphocytes in the intestinal mucosa. In contrast, exposure of naive sheep to a single large infection of T. colubriformis resulted in a substantial decrease in number of CD8+ cells and moderate decreases in number of CD4+ cells in the duodenal but not the jejunal mucosa. MHC class II antigens were not detected either in or on epithelial cells of the sheep small intestine.

Immune response of sheep to oral and subcutaneous administration of live aromatic-dependent mutant of Salmonella typhimurium (SL1479).

The major objective of the present study was to determine whether oral immunization with a live aromatic-dependent strain of Salmonella typhimurium (SL1479) was capable of stimulating an intestinal immune response in sheep similar to that induced by combined intraperitoneal injection followed by oral boosting. The results showed that repeated oral immunization was incapable of stimulating an anti-flagella antibody containing cell (ACC) response in the lamina propria of the intestine even though primary oral administration of 5 x 10(9) live SL1479 gave rise to an ACC response in intestinal lymph which was predominantly of the IgM isotype. ACC reached a peak 9-10 days after oral administration when ACC comprised 0.5-1% of total lymphocytes in lymph. An ACC response of similar isotope specificity also occurred in popliteal prefemoral lymph of unprimed sheep following regional subcutaneous injection of SL1479. Oral administration of SL1479 to orally primed sheep did not reinvoke an ACC response in lymph although IgG1-ACC were observed in medullary cords of mesenteric lymph nodes of sheep 6-8 days after the booster dose of SL1479. The results suggest that the protective immunity elicited by oral administration of SL1479 cannot be attributed to induction of a local intestinal antibody production.

Protection against experimental salmonellosis in mice and sheep by immunisation with aromatic-dependent Salmonella typhimurium.

Mice immunised by the oral or intraperitoneal route with a live aromatic-dependent strain of Salmonella typhimurium exhibited significantly less protection against oral challenge with 50 LD50 of an ovine isolate of S. typhimurium (12313) than when a bovine isolate with the same O antigens and phage-type as strain 12313 was used as the challenge organism. When challenged with 10 LD50, however, protection against both strains was significantly better than that obtained when mice were vaccinated with killed vaccines (heat-killed, acetone-killed or irradiated) even when the antigenic mass of the killed vaccine was increased by up to 500-fold in an attempt to compensate for the expected limited multiplication of the mutant organism. Sheep immunised with the live mutant strain by either the intramuscular or oral route were protected against oral challenge with the virulent ovine isolate of S. typhimurium; unimmunised sheep died of acute enteritis within 7 days, although there was no evidence of systemic invasion by the challenge organism. After challenge, immunised animals ate more food than the unimmunised controls and suffered only transient, mild diarrhoea. Serum antibody titres against O and H antigens measured by direct or antiglobulin tests were significantly higher in sheep immunised by the intramuscular route than in those immunised orally. Sheep in both immunised groups developed skin swellings within 30 min after intradermal inoculation with purified homologous lipopolysaccharide indicating development of immediate-type hypersensitivity, but only those immunised by the intramuscular route showed significant indurated skin swellings characteristic of delayed-type hypersensitivity 48 and 72 h post-inoculation.(ABSTRACT TRUNCATED AT 250 WORDS)

Purification and characterization of goblet-cell mucin of high Mr from the small intestine of sheep.

Crude soluble mucus from sheep small intestine was freed of nearly all the nucleic acid contaminants by precipitation with protamine sulphate and treatment with nucleases. After removal of non-covalently bound proteins by equilibrium density-gradient centrifugation in CsCl, a high-Mr glycoprotein was isolated by repeated h.p.l.c. from the partially purified mucin. The high degree of purity of the high-Mr mucin was borne out by (a) the observation of a single boundary on analytical ultracentrifugation in the presence of 5M-guanidinium chloride and (b) the observation of apparent monodispersity on sedimentation-equilibrium analysis. The Mr of the highly purified mucin, determined by sedimentation equilibrium, was 5.0 (+/- 0.1) X 10(6) and was concentration-independent. Finally, only goblet cells and the mucus blanket lining the intestinal epithelial cells were immunofluorescent when guinea-pig anti-(highly purified mucin) serum was used in an indirect immunofluorescence assay. The above antiserum reacted with apparently equal strength with goblet cells and with free mucin in abomasum, caecum and colon. The chemical composition of the glycoprotein was 66% carbohydrate and 34% protein, 45% of the latter being composed of valine and threonine. The glycoprotein migrated anodally on immunoelectrophoresis and contained 7.1% (w/w) sulphate. Neutral hexoses accounted for nearly half of the total carbohydrate content, followed by galactosamine and glucosamine. Whereas fucose and sialic acid were present in only small amounts, uronic acid was not detectable in the highly purified mucus glycoprotein.

The effect of adjuvants and prior immunization on the rate and mode of uptake of antigen into afferent popliteal lymph from sheep.

Experiments were carried out to determine the effect of a range of adjuvants on the antibody response of sheep to a subcutaneous injection of ovalbumin. Incomplete Freund's adjuvant (IFA) was the best of the repository type adjuvants. From a range of soluble immunopotentiating substances tested, the polyion DEAE-dextran, or dextran sulphate, produced the greatest enhancement of the response. These results were confirmed in a factorial experiment, but no interactions were found between various classes of adjuvants. Further experiments were carried out to determine the effect of superior adjuvants, prior immunization or intradermal injection on the rate and mode of uptake of antigen from the injection site to afferent lymph. These experiments showed that, with IFA, most antigen is retained at the injection site, less than 15% entering lymph by 3 days. There was some delay in antigen uptake with adjuvant 65, while for aluminium phosphate precipitated antigen, the rate of uptake was the same as following a saline injection. There was some delay in uptake of antigen following injection with DEAE-dextran or in primed sheep but, following intradermal injection, the rate of uptake into lymph was the same as aqueous antigen injected subcutaneously. Experiments in which the distribution of radioactive antigen between cells and plasma in lymph was examined showed that, in unprimed sheep, virtually all antigen is unassociated with cells. In primed sheep, however, about 0.5% of antigen in lymph was found to be cell-associated. Gel filtration of lymph plasma from primed sheep demonstrated that nearly all the antigen in lymph is carried to the node as high molecular weight material, presumably antigen-antibody complexes.

The antibody-containing cell response in hepatic and intestinal lymph following intraperitoneal and intravenous administration of antigen in different adjuvants.

A comparison was made of the antibody-containing cell (ACC) response in hepatic and intestinal lymph of sheep following intraperitoneal injection of ovalbumin in Freund's complete adjuvant (FCA), Freund's incomplete adjuvant (FIA) or the bland vegetable oil preparation, adjuvant 65. There was a substantial ACC response in intestinal lymph following intraperitoneal injection of ovalbumin in either FCA or FIA. Responses were essentially similar in magnitude and for both adjuvants ACC were distributed mainly among the IgGl, IgA and IgM immunoglobulin classes. In contrast, there was a negligible ACC response in intestinal lymph following injection of antigen in adjuvant 65. The output of ACC in hepatic lymph and the immunoglobulin class distribution of the ACC following intraperitoneal injection of antigen in either FCA or FIA were similar and results were specificity comprising 32% of ACC at the peak of the response. In contrast to results for intestinal lymph, injection of antigen in IgGl- and IgM-specific cells in hepatic lymph. The ACC response was much smaller in magnitude than with the Freund's adjuvants. Intravenous injection of ovalbumin in FIA or adjuvant 65 gave rise to substantial ACC responses in hepatic lymph which contrasted with the barely detectable response in intestinal lymph. Following intravenous administration the great majority of ACC in hepatic lymph were of the IgM class irrespective of adjuvant used although ACC of the IgA class made a transitory appearance.

The effect of route of administration of antigen on the antibody-containing cell response in lymph of sheep.

The output and immunoglobulin class of antibody-containing cells (ACC) in lymph from different regions of adult sheep has been compared following intraperitoneal or subcutaneous injection of ovalbumin in Freund's complete adjuvant (FCA). Intraperitoneal injection of antigen in FCA elicited no response at all in popliteal lymph, but there were substantial responses in lymph from the hepatic, coeliac (which carries lymph mainly from the abomasum and anterior duodenum) and intestinal lymphatic ducts. A significant increase in the number of blast cells accompanied the ACC response. ACC reached a peak 7- days after injection when ACC comprised 3.7% +/- 0.7%, 2.3% +/- 0.7% and 3.3% +/- 0.6% of total lymphocytes in lymph from the hepatic, coeliac and intestinal ducts respectively. At this time ACC were distributed among three immunoglobulin classes IgM, IgG1 and IgA with the latter constituting 25%--40% of ACC in lymph from the three regions. ACC did not appear in intestinal lymph following subcutaneous injection of ovalbumin in FCA in the leg immediately below the tarsus. This contrasted with the vigorous response in popliteal lymph where 6 days after the injection ACC constituted 3.9% of cells in lymph. Most (83%) of the ACC were IgG1-specific at the peak of the response. The remaining ACC were distributed among the IgM, IgG2 and IgA classes with the latter comprising 3% of total ACC.

IgA-containing cells in the ruminant intestine following intraperitoneal and local immunization.

Experiments are described which demonstrate that a single intraperitoneal (i.p.) injection of antigen in Freund's complete adjuvant results in the appearance of IgA-specific antibody-containing cells (ACC) in the intestinal lamina propria of sheep and that these cells reach the intestine via the intestinal lymph and blood circulation. Following intraintestinal administration of antigen to sheep immunized i.p. 2 weeks previously, an enhanced ACC response occurs in the intestine but cannulation and drainage of the intestinal duct does not interfere with this enhancement. Evidence is presented which suggests that the enhanced ACC response may be accounted for by antigen-induced local proliferation of ACC in the lamina propria of the intestine.

Intestinal response of sheep to intraperitoneal immunization.

The aim of the present study was to develop an immunization procedure which centrally stimulated the IgA system of sheep with the release of antibody-containing cells (ACC) of the IgA class into intestinal lymph. It was found that intraperitoneal injection of ovalbumin resulted in a substantial output of ACC in intestinal lymph. ACC of the IgA class reached a peak 8--9 days after intraperitoneal injection when they comprised 1.4% of cells in lymph. ACC of the IgM and IgG1 classes comprised 3.5 and 2.9% of cells in lymph at this time. The output of ACC of the IgA, IgM and IgG1 classes in lymph at the peak of the response was respectively, 3.7 X 10(6), 9.8 X 10(6) and 8 X 10(6) cells/h. In marked contrast to rats, virtually no ACC appeared in intestinal lymph of sheep following intraduodenal infusion of ovalbumin in animals primed 2 weeks earlier by intraperitoneal injection of antigen in Freund's complete adjuvant (CFA).

The kinetic behaviour of radiolabelled IgG1 derived from different sources in adult sheep and neonatal lambs.

The turnover rate and pool volume of radiolabelled autologous IgG1 was similar to homologous IgG1 in adult sheep. In neonatal lambs there was no significant difference for either of these parameters between homologous serum IgG1 and maternal colostrum IgG1. However, the pool volume for IgG1 expanded by approx. 30% over the experimental period in the lambs. When the turnover rates were corrected for this increase they were found to be similar to those observed for IgG1 in adult animals.

The immune system on the ruminant mammary gland and its role in the control of mastitis.

Immunoglobulins in mammary secretion are derived from blood serum or are made locally by cells of the lymphocyte-plasma cell series situated close to the glandular epithelium. The major immunoglobulin in colostrum and milk of ruminants, IgG1, is derived from the blood and is transferred into secretion selectively relative to IgG2, probably by a mechanism requiring specific receptor sites on the basal of intercellular membrane of the glandular epithelium. Acute inflammation causes suppression of selective transfer of IgG1, but there is a marked increase in the transfer of proteins, such as IgG2 and serum albumin, which enter secretion nonselectively. Infusion of antigen into the mammary gland of ruminants some weeks before parturition induces a persisting local production of antibody, most of which is associated with IgA and IgM. IgA cells in the mammary gland probably originate in the intestine, and prior antigenic stimulation of the gut may be required for maximal IgA antibody responses in the gland. Local immunization with staphylococcal vaccines gives a measurable degree of protection against staphylococcal challenge. Systemic immunization with viable staphylococci leading to subcutaneous abscess formation elicits significant protection to subsequent mammary challenge which is attributable, at least in part, to specific antibody of the IgG2 class cytophilic to polymorphs.

The relationship between the transfer of immunoglobulins, sodium and potassium into mammary secretion of the parturient ewe.

The experiment comprised two sections. First, radiotracer techniques were used to study the metabolism of IgG1 and IgG2 in 5 non-pregnant and 4 pregnant ewes. In the pregnant ewes, the rates of synthesis for IgG1 and IgG2 were similar to the rates observed in non-pregnant animals. However, the irreversible loss of IgG1 was significantly greater than IgG2 in pregnant ewes and IgG1 in non-pregnant ewes. Additionally, it was found that most of the IgG1 and virtually all of the IgG2 in mammary secretion was serum derived. Secondly, the levels of sodium, potassium and lactose and the selective index for IgG1 in mammary secretions of 5 pregnant ewes were monitored over the parturient period. The values of all the measures remained relatively constant until one day before parturition. From one day pre-partum, the levels of potassium and lactose in mammary secretion began to increase and had risen 2-3 fold by 5 days post-partum. Over the same period, the selective index for IgG1 decreased 20 fold, wehreas the level of sodium fell from approximately 32 mmol/l to 18 mmol/l. The concentration of IgG1 in plasma slowly declined from approximately 23 g/l to 15 g/l over the last 10 days of pregnancy. During the parturient period, the decline in plasma IgG1 levels, in comparison with IgG2, without alteration in the rates of synthesis of either immunoglobulin, supports the hypothesis that selective transport of IgG1 into mammary secretion occurs without degradation. The results also indicate that the transport of sodium and potassium into mammary secretions are altered over the parturient period.

The origin of immunoglobulins in salivary secretion of sheep.

A comparative study was made on the secretion of IgG1 and IgA into parotid and submaxillary saliva of sheep. The purpose of the study was to examine the suggestion that in the external secretory organs of ruminants there is an inverse relationship between the capacity to selectively transfer IgG1 and the development of the IgA secretory system. Despite a marked difference in the secretion of IgA between the parotid and submaxillary salivary glands, the magnitude of selective transfer of IgG1 was similar. Thus, there appears to be no relationship between the selective transfer of IgG1 and the secretion of IgA into the saliva of sheep.

The effect of pre-partum milking on the transfer of immunoglobulin into mammary secretion of cows.

The significance of local effects associated with mammary involution on transfer of immunoglobulin and especially on the selective transfer of IgG1 into mammary secretion of cows approaching parturition has been determined. This was carried out by measuring the changes in the concentration of IgG1, IgG2, IgM and IgA in serum and mammary secretion of 5 cows in which two mammary glands were milked continuously (twice daily) during the period preceding parturition, while the other two glands were allowed to undergo normal involution. In the secretion of unmilked glands of all cows there was a substantial increase in the concentration of IgG1 as cows approached parturition. In contrast, the increases in the concentration of IgG1 and in the selective index for IgG1 of milked glands were either virtually non-existent (1 cow) or generally reduced in magnitude and delayed in time of onset (4 cows). It is clear from the results that continued milking of a mammary gland throughout pregnancy tends to maintain milk production in the milked gland and at the same time reduces the massive selective transfer of IgG1 into secretion of that gland.

The origin of the immunoglobulins in intestinal lymph of sheep.

Various immunoglobulins were labelled with radioactive iodine and their distribution between intestinal lymph and plasma followed in order to determine the origin of the immunoglobulins found in intestinal lymph. By comparing specific activities in plasma and lymph, it was computed that 25 percent of the IgG1 and IgG2 and 90 percent of the IgA in intestinal lymph were locally synthesised. The results suggest that virtually all of the IgA and a proportion of the IgG1 computed to be synthesised locally were derived from plasma cells of corresponding specificity in the lamina propria of the intestine.