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Intestinal lymph-duct cannulae were established in normal and mesenteric lymphadenectomized (MLNx) pigs for the 6-day duration of a local intestinal immune response to a protein antigen (ovalbumin). The daily output of anti-ovalbumin-containing cells (AOCC) in intestinal lymph and the numbers of AOCC in the intestinal lamina propria at the end of the experiment were recorded. Very few AOCC were recovered in the intestinal lymph of normal pigs whereas in MLNx pigs large numbers were recovered reaching a peak output on day 4. However, there were significantly more AOCC detected in the jejunal lamina propria of normal pigs than MLNx pigs despite continuous drainage of intestinal lymph throughout the response. The absence of AOCC from efferent intestinal lymph of normal pigs, the failure of chronic intestinal-lymph drainage to abrogate the AOCC response in the intestine of these pigs and the reversal of these findings in MLNx pigs indicate that, in contrast to other species, lymphoblasts are diverted from porcine intestinal lymph, and probably enter the blood circulation at the level of the mesenteric lymph node (MLN).

Levamisole injection resulted in an elevation in the T helper:T suppressor (H:S) subset ratio in rats at 24 h after injection due to a selective depression in the cytotoxic/suppressor subset. This response was shown to be conditionable and could be reenlisted 14 days later by re-exposure to the conditioned stimulus. Rats were conditioned using a taste aversion paradigm by pairing levamisole injection with the novel taste of saccharin. Fourteen days later, after a second exposure to saccharin without levamisole injection, H:S ratios were elevated in the blood of these rats compared to control rats injected with levamisole but fed normal water or rats fed saccharin without levamisole injection.

Previous studies in sheep indicate that a combination of intraperitoneal and intramammary immunisation with ovalbumin results in a population of anti-ovalbumin-containing plasma cells of the IgG1 isotype in the immunised mammary gland. These cells are not present in the unimmunised contralateral gland. The studies reported here were undertaken to determine the source of these cells. The failure of chronic intestinal lymphatic drainage or either mesenteric or supramammary lymphadenectomy to abrogate this response was interpreted as evidence against their origin from gut-associated or mammary-associated lymphoid tissue. It is concluded that the success of prior intraperitoneal immunisation in stimulating an enhanced IgG1-specific response to local antigen in the mammary gland probably results from recruitment of cells from systemic lymphoid tissue primed by intraperitoneal immunisation.

Procedures for the diagnosis of Br. ovis infection in rams were evaluated by examining 10 rams artificially infected by preputial inoculation. Observations were undertaken at weekly intervals for 1 year to follow changes in clinical, bacteriological and serological findings. Clinical lesions were detected in 1 ram 3 weeks after inoculation and in all rams by 8 weeks; lesions were undetectable in 3 rams at the completion of the trial. The presence of inflammatory cells in semen samples was the earliest indication of infection being demonstrated in 2 rams at 2 weeks and in all rams by 8 weeks; subsequently 86% of samples were positive. Br. ovis was detected in semen smears from 3 rams at 4 weeks but only one in all rams (at 27 weeks); overall 52% of semen smears were positive from 4 weeks onwards. Br. ovis was cultured from semen of 5 rams after 4 weeks and from all rams at 5 weeks; therafter 97% of samples were positive. All rams developed significant titres to the CFT between 2 and 9 weeks; therafter the CFT was a reliable indication of infection in 6 rams, highly suggestive in 3 and unreliable in one. By 8-10 weeks all rams developed significant titres to the IHA which were then maintained in all rams for the remainder of the trial.

Previous studies have shown that cyclophosphamide, a drug with a broad spectrum of cytotoxic activity and one that produces noxious gastrointestinal side effects, can elicit taste aversion conditioning when paired with a non-immunosuppressive oral stimulus (saccharin) resulting in immunosuppression after subsequent exposure to the paired stimulus (1). The study reported here indicates that rabbit anti-rat lymphocyte serum (ALS) which is selectively cytotoxic for lymphocytes and does not produce sensory side effects can similarly induce taste aversion conditioning of the immune response. Rats were exposed to oral saccharin paired with ALS injection. Upon subsequent reexposure to saccharin alone the immunosuppressive effects of ALS were reenlisted and the primary mixed lymphocyte culture response of conditioned rats to allogeneic lymphocytes was suppressed by 35% compared to controls. The demonstrated influence of psychologic factors on the immune response has far reaching implications, especially to human medicine, and their role in the course of disease and recovery in man demands further investigation.

The oral cavity is relatively deficient in lymphoid tissue capable of directing the inductive and effector events required for effective mucosal IgA responses to local antigen. Recent evidence regarding a common mucosal immune system has raised new possibilities in immunization of mucosal surfaces. This review describes the role of the intestine in contributing to responses at distant mucosal sites and in particular emphasizes the immune potential of the intestine in determining the success of immunization strategies to control dental caries.

The extent to which sleep deprivation interferes with immunity in the respiratory tract to influenza virus has been assessed in mice. Mice were orally immunized with influenza virus on two occasions separated by a one week interval and challenged intranasally one week later. Some animals were deprived of sleep for a 7 h period immediately following challenge. Three days after challenge, virus clearance and virus specific antibody were determined in lungs of sleep deprived and normally sleeping mice and the results compared with unimmunized mice subjected to the same protocol. Whereas immunized, normal sleep mice achieved total virus clearance, sleep deprivation in immunized mice completely abrogated this effect such that sleep deprived animals behaved as though they had never been immunized. There was no difference in viral clearance in unimmunized mice whether sleep deprived or not, indicating that sleep deprivation did not itself have a direct effect on viral replication. The data reported here support the concept that sleep is a behavioral state which is essential for optimal immune function in the presence of a respiratory tract pathogen.

An immunisation procedure involving intraperitoneal followed by oral administration of a killed Escherichia coli vaccine was used to reduce losses associated with colibacillosis in young pigs reared under commercial conditions. Mortality was reduced in both the preweaning and postweaning period. The mortality rate among vaccinated piglets was half that of unvaccinated controls and by 3 weeks after weaning the total weight of pigs produced was 33% higher in the vaccinated group than in the control group.

An intestinal immunisation procedure involving systemic priming and oral boosting was investigated in lambs with a view to providing vaccination control of enteritis in young animals. The procedure stimulated the appearance of antibody-containing cells of IgA specificity in the intestine and lambs immunised in this way with a bacterial vaccine were protected against subsequent challenge with live enteropathogenic bacteria. This immunisation regime therefore provides a useful method for the stimulation of IgA immunity in the intestine of ruminants and it is anticipated that it will have general application to a variety of enteric diseases.

Antibody responses were measured in colostrum-fed (CF) and colostrum-deprived (CD) calves immunised at ages varying from birth to four months using a variety of antigens with and without adjuvant. In addition immunoglobulin production in CD calves over the same period was compared with that of CF calves. A marked unresponsiveness to antigens injected at birth was observed if maternal antibody specific for the antigen used was present in the circulation. However, significant responses to all antigens tested occurred in CD calves and also in CF calves immunised with an antigen (egg albumin) to which there was no maternal antibody. Since these responses were not as great as those in older calves immunised similarly it is clear that age was influencing the response. It was also found that subsequent responsiveness was not significantly enhanced or impaired by neonatal exposure to antigen. With respect to immunoglobulin production the results conclusively demonstrated that endogenous production occurred much earlier in CD calves than CF calves and even after 128 days the serum concentrations of IgG1 and IgA in CD calves exceeded those for CF calves. Thus it appears that the most important consideration in immunological responsiveness of neonates is not so much immaturity of the lymphoid system as the effects of maternal antibody on the cells in that system.

The levels of the immunoglobulins IgA, IgG1, IgG2, and IgM were measured in serum and fluid from various locations in the reproductive tract of normal rams. These fluids included semen, preputial washings, and fluid from the accessory sex glands (ASG), vasa deferens, rete testes, and tissue fluid from the seminal vesicles, bulbourethral glands, epididymal tails and efferent ducts. In addition, the prevalence of specific Ig-containing cells (ICC) was measured in sections of formalin fixed tissues stained by an indirect peroxidase-antiperoxidase labelling technique. Mean IgA levels in semen (1.23 mg/ml) and ASG fluid (0.46 mg/ml), were higher than in serum (0.19 mg/ml) and were at levels higher than IgG1 or IgG2 levels in semen, ASG fluid, and preputial washings, thus confirming the existence of a local immune system primarily in the ASG of ram genitalia. Relatively low concentrations of IgA and IgG in other genital fluids and IgG levels in these fluids were consistent with diffusion from serum. The relatively high prevalence of IgA-containing cells in bulbourethral (56% of all ICC) and prostate (49%) glands confirmed these tissues as major sites of local Ig production. ICC were also found in large numbers beneath pelvic urethral and preputial epithelia, but these were predominantly IgG-containing (88 and 72% respectively).

The lack of progress in development of vaccines to stimulate local protection in the urinary tract is attributable in part to the lack of information regarding mechanisms of local immunity and to the difficulty in antigen administration at this site. Experiments reported in this paper indicate that the urinary tract in rats forms part of an effector network linking mucosal organs (the common mucosal immune system). Concomitant immunization of the intestine and urinary tract with ovalbumin resulted in the appearance at both sites of antiovalbumin-containing cells (ACC) of IgA specificity. The gut origin of ACC in the urinary tract was confirmed by demonstrating in similarly immunized rats abrogation of the urinary tract response by chronic drainage of the thoracic lymphatic duct during the post-challenge period and by demonstrating the appearance of radiolabelled ACC in the urinary tract after injection of labelled autologous thoracic duct lymphocytes collected during the post-challenge period. These experiments indicate a role for oral immunization in enhancing the local antibody response in the urinary tract against invading pathogens.

Studies of the migration of antigen-specific regulatory T cell subsets responding to gut immunization were undertaken to clarify their migratory potential and the role of antigen in their localization. In initial experiments, lymphocytes collected from the thoracic duct of rats after immunization of Peyer's patches (PP) with keyhole limpet hemocyanin (KLH), were enriched for T helper (Th) cells and labelled with the fluorochrome H33342. In other experiments, a higher frequency of antigen-specific T cells was achieved by short-term culture of the enriched Th cells in the presence of KLH and the blast cells labelled with 3H-thymidine. The distribution of both populations was determined after injection into immunized and unimmunized syngeneic recipients. Whereas the uncultured population (predominantly small Th cells) localized almost exclusively in follicular lymphoid tissues, the cells expanded by secondary culture (predominantly Th blasts) appeared in the gut lamina propria (LP) initially, then in PP and mesenteric lymph nodes. The Th blasts in the LP were almost always seen in close proximity to the gut epithelium. However, the migration of neither population appeared to be influenced significantly by antigen, in contrast to previous findings with regard to IgA-committed B cells. The initial subepithelial location of Th blasts in the gut LP and their subsequent appearance in PP may provide a mechanism by which antigen presented by epithelial cells could influence B cell differentiation in PP through modulation of signals expressed by these T cells.

Evidence is presented which indicates that recirculating lymphocytes originating from the intestine in pigs are returned to the blood circulation at the level of the mesenteric lymph nodes (MLN) and not via efferent intestinal lymph. This was demonstrated by three observations: (1) removal of all MLN resulted in a thirty-fold increase in lymphocyte numbers in efferent lymph of pigs, but not in rats; (2) there are about twenty-five times more lymphocytes in afferent intestinal lymph than efferent intestinal lymph in normal pigs; (3) 51Cr-labelled lymphocytes injected into afferent lymphatics are mostly recovered in the node tissue or efferent lymph of sheep, and very few in the venous drainage, whereas in pigs relatively few labelled cells are recovered in the node or in efferent lymph.

The contribution of gut-associated lymphoid tissue (GALT) to the local response in the mammary gland is well documented in laboratory animals and has been evaluated in this study in ruminants. Ewes were immunized intraperitoneally (IP) with antigen in Freund's complete adjuvant (FCA), a procedure which stimulates the production of antibodies of the IgA class in the intestine, and challenged intramammarily (IMam) either during colostrum formation or mammary gland involution. Despite a substantial IgA antibody-containing cell (ACC) response in the intestine in IP immunized sheep, there was no evidence to suggest a relocation of IgA-specific ACC to the mammary gland. There was, however, an IgA antibody response in mammary secretion of IP immunized animals, regardless of whether the mammary gland was locally immunized, but the origin of this antibody is unclear. IP/IMam immunized sheep did have an enhanced antigen-specific ACC response of the IgG1 isotype in locally immunized glands, but whether these cells were of GALT or systemic origin is also unclear.

A sleep-inducing substance Factor S (FS) has been identified as a member of the muramyl peptide family of molecules which constitute the cell wall of bacteria. FS-like substances are unable to be synthesized by mammals suggesting that the symbiotic bacteria of the gastrointestinal tract are a prime source of FS. The present paper considers the relationship between the ontogenesis of Slow Wave Sleep (SWS) and the colonization of the intestine with strictly anaerobic bacteria. The practice of coprophagy in rats and rabbits is considered as an efficient method of FS ingestion during the sleep period.

Foetal lambs were immunized orally 6-15 days before birth by introducing horse spleen ferritin into the amniotic fluid. Immunized and non-immunized lambs were killed at birth, usually before they had suckled, blood and intestinal contents were collected and single cell suspensions were prepared from spleen, mesenteric lymph nodes and jejunum. Specific antibody was detected in serum and intestinal contents of all immunized lambs which had not suckled. Specific antibody was usually not detected in samples from non-immunized lambs. In immunized lambs antibody activity in serum was associated with IgM and in intestinal contents with IgA and IgM. In agreement with these findings, the levels of IgM and IgA in serum and intestinal contents of immunized lambs were relatively high. Generally, immunoglobulins were not detected in samples from non-immunized lambs. Relatively high proportions of cells secreting specific antibody were present in the tissues of immunized but not non-immunized lambs. In the spleen most of the cells were secreting IgM antibody, in mesenteris lymph nodes IgM cells predominated and small numbers of IgA cells were detected, and in the jejunum approximately equal numbers of IgA and IgM cells were secreting specific antibody.

The origin of the immunoglobulins in the upper respiratory tract secretion of sheep was determined by measuring the distribution between plasma and secretion of radiolabelled purified immunoglobulins and albumin. By calculation of the ratio of specific activity for each immunoglobulin between plasma and secretion, it was estimated that about 81% of IgA in secretion was of local origin, whereas IgM, IgG1, IgG2 and albumin were wholly derived from plasma. Estimates of the selectivity of transport of IgA and IgM into both respiratory tract secretion and saliva were obtained by calculation of a selective index relative to IgG1 or IgG2, which do not bind secretory component (SC). This was based on radioactivity ratios after the simultaneous injection of immunoglobulin labelled with different isotopes (IgA or IgM injected with either IgG1 or IgG2). These calculations revealed that both IgA and IgM were selectively transported into respiratory tract secretion and saliva. This provides further support for the proposition that SC-binding immunoglobulins may be transported from serum into secretions at a variety of mucosal sites dependent on SC availability. Since the IgA in serum of sheep is predominantly of gut origin, this provides an opportunity, in addition to relocation of gut-derived plasma cell precursors, by which the gut may contribute to extraintestinal mucosal responses.

A single intraperitoneal injection of ovalbumin in oil adjuvant in young lambs has been shown to result in the appearance in the intestinal lamina propria of antibody-containing cells, most of which contained antibody of IgA specificity. Intraperitoneal immunisation of lambs with a Salmonella typhimurium vaccine during the suckling period provided protection against postweaning challenge with live organisms. This response was shown to be associated with specific IgA antibody in intestinal secretion.

The IgA-secreting cells in the lamina propria of the small intestine are derived from large lymphocytes which enter the blood by way of the thoracic duct and then migrate into the gut where they complete their differentiation into plasma cells. Three aspects of this cellular traffic have been examined in rats. 1. The cells in thoracic duct lymph which give rise to IgA-secreting cells in the lamina propria are among those which carry surface IgA. Blast cells lacking surface immunoglobulin migrate mainly into the Peyer's patches and do not contribute to the IgA response. 2. Studies on a secondary antibody response to cholera toxoid, in which the challenge was given into a Thiry-Vella loop, showed that the antibody-containing blast cells in thoracic duct lymph were derived from Peyer's patches. The mesenteric nodes contributed little, if anything, to the cellular response in the lymph. 3. The idea that secretory component is a signal for the emigration of large lymphocytes from the blood into the lamina propria lacks experimental support. Secretory component does not bind to the IgA on the surface of thoracic duct cells. On the other hand, antigen in the gut may play an important part in immobilizing large lymphocytes in the lamina propria once they have migrated.

The existence of a hepato-biliary pump for IgA has been studied in sheep by measuring appearance of radioactivity in bile after simultaneous i.v. injection of IgG2 and homologous dimeric IgA each labelled with either 125I or 131I. These experiments confirm that in sheep, as in rodents, IgA is the predominant immunoglobulin in bile and is selectively transported into bile from serum relative to IgG2. In addition, there is evidence for local production of IgA in bile presumably by plasma cells within the liver and biliary tree.

The distribution of keyhole limpet haemocyanin (KLH)-specific helper cells for antibody responses of IgA, IgM and IgG isotypes in Peyer's patch (PP), mesenteric lymph node (MLN) and peripheral lymph node (PLN) was examined following oral, intraduodenal (ID), intraperitoneal (IP), intra-Peyer's patch (IPP) or subcutaneous (SC) immunization with KLH. Oral or ID immunization gave little or no response in any tissue studied. IP immunization with or without a subsequent ID challenge gave rise to a modest IgA and IgM helper response in MLN but a small IgA and IgM helper response in PP and PLN. IP immunization alone did not stimulate IgG-specific help in any tissues studied, but a small IgG helper response occurred in MLN and PLN after subsequent ID challenge. IPP was the most effective route of immunization, giving rise to a large helper response for IgA, IgM and IgG isotypes in PP, a smaller response in MLN and no response in PLN. The helper response following IPP immunization was not augmented by subsequent ID challenge. SC immunization gave a small but significant helper response for all isotypes in PLN but no response in PP or MLN. The kinetics of the helper response were examined in PP, MLN, PLN and thoracic duct lymph (TDL) following IPP immunization. The helper response for all isotypes in PP was maximal at 2 weeks and then declined. Similar kinetics but of lower magnitude were observed in MLN and TDL. The presence of IgA-specific helper cells in TDL demonstrates that these cells migrate, presumably from GALT, and may constitute an important component of mucosal responses at extraintestinal sites.