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Vaccines stimulate both innate and adaptive immunity. Innate immune responses promote the response of the adaptive immune system to vaccine antigens. Injected antigens are captured and processed by dendritic cells. This antigen is processed and presented to helper T cells. These, in turn, trigger either a B cell response to make antibodies or a T cell response to generate cell-mediated immunity. Inactivated vaccine antigens are treated as exogenous antigens and trigger a response that is predominantly antibody mediated. Modified live vaccine antigens are treated as endogenous antigens and trigger cell-mediated responses. Both types of vaccine trigger a long-lasting memory cell response that results in prolonged protection against infection.

Psittacine proventricular dilatation disease (PDD) is a neurological disease caused by parrot bornaviruses. A competing theory suggests that intestinal colonization by Campylobacter species may also be a potential cause of PDD or that their presence may be required for disease development. This theory proposes that PDD results from the activities of antiganglioside antibodies on enteric neurons in a manner similar to the pathogenesis of Guillain-Barre syndrome in humans. We therefore cultured feces from domestic chickens as well as from multiple parrot species to determine whether Campylobacter spp. could be detected in the latter. We failed to detect Campylobacter in a flock of cockatiels known to be highly susceptible to experimental parrot bornavirus-induced PDD. Even in naturally infected psittacines suffering from clinical PDD, no Campylobacter species were detected. Conversely, Campylobacter was readily cultured from domestic poultry samples and confirmed by using matrix-associated laser desorption ionization mass spectroscopy/real-time polymerase chain reaction. We conclude that not only are Campylobacter infections of psittacines uncommon, but also that infection by Campylobacter species is not related to the etiology of PDD.

Paulina Escandon,1,2 J Jill Heatley,1,3 Luc R Berghman,2,4 Ian Tizard,1,2 Jeffrey MB Musser1,2 1Schubot Exotic Bird Health Center, College of Veterinary Medicine, Texas A&M University, College Station, TX 77843, USA; 2Department of Veterinary Pathobiology, College of Veterinary Medicine, Texas A&M University, College Station, TX 77843, USA; 3Department of Small Animal Clinical Sciences, College of Veterinary Medicine, Texas A&M University, College Station, TX 77843, USA; 4Department of Poultry Science, College of Agriculture & Life Sciences, Texas A&M University, College Station, TX 77843, USACorrespondence: Jeffrey MB Musser4467 TAMU, VTPB, College of Veterinary Medicine, College Station, TX 77843-4467, USATel +1 979 458 9946Fax +1 979 458 0321Email Jmusser@cvm.tamu.eduPurpose: This study evaluated the specificity of different avian secondary antibodies used in Western blot and dot-blot ELISA to detect avian bornavirus antibodies in bird plasma.Methods: Plasma samples were collected from: two Blue and gold macaws, one positive and one negative for avian bornavirus by RT-PCR; a Cockatiel and a Monk parakeet prior to and following experimental infection; and, two Mallards, one positive and one negative for avian bornavirus by RT-PCR Samples were analyzed by Western blot and dot-blot ELISA that incorporated recombinant avian bornavirus nucleoprotein as the target analyte. Four species-specific anti-IgY secondary antibodies were used in the assays: goat anti-macaw IgY, goat anti-bird IgY, goat anti-duck IgY, and rabbit anti-chicken IgY.Results: In the Western blot, anti-macaw IgY secondary antibody produced strong signals with Blue and gold macaw and Cockatiel positive plasma, but no signal with Mallard positive plasma. Anti-bird IgY secondary antibody produced strong signals with Blue and gold macaw, Cockatiel, and Mallard positive plasma. Anti-duck and anti-chicken IgY secondary antibody produced a strong and moderate signal, respectively, only with Mallard positive plasma. In the dot-blot ELISA, there was a distinct and significant difference (P

Current inactivated influenza vaccines are strain-specific and poorly effective against variant or mismatched viruses. They are standardized based on their hemagglutinin (HA) or ability to induce strain-specific hemagglutination inhibition (HAI) antibodies. The HA is known to undergo major conformational changes when exposed to the low pH environment of endosomes (pH 5.0 and 37°C), which are required for membrane fusion during virus cell entry. In an effort to improve these vaccines, influenza antigens treated under various low pH conditions were evaluated for increased cross-reactive antibody response and cross protection. It was found that a full range of structural and antigenic changes in HA could be induced by varying low pH treatment conditions from the mild (low pH at ≤25°C) to the strong (low pH at ≥37°C) as determined by analysis of potency, HA morphology, protease sensitivity, and reactivity with an anti-HA2 domain (CD) antibody. Inactivated antigens of both H1N1 and H3N2 strains treated at mild low pH conditions (0-25°C) exhibited only moderate HA structural and antigenic changes and markedly increased antibody response against HA2, the highly conserved part of HA, and cross protection against heterologous challenge in mice by up to 30% in survival. By contrast, antigen treated with low pH at 37°C showed more extensive structural and antigenic changes, and induced much less of an increase in antibody response against HA2, but a greater increase with response against HA1, and did not provide any increased cross protection. These results suggest that the increased response against HA2 obtained with the mild low pH treatment is associated with the increased cross protection. These antigens treated at the mild low pH conditions remained capable of inducing a high level of strain-specific HAI antibodies. Thus, they could readily be formulated as an inactivated influenza vaccine which not only provides the same strain-specific protection but also an increased cross protection against heterologous viruses. Such a vaccine could be particularly beneficial in cases of vaccine mismatch.

Background The hepatitis B surface antigen (HBsAg) has been administered over the last 20 years as a parenteral vaccine against the hepatitis B virus (HBV). Despite high seroconversion rates, chronic infection rates are still high worldwide. Orally delivered vaccines provide a practical alternative to injected vaccines, potentially helping poorly responding populations and providing a viable alternative for populations in remote locations. Anamnestic responses are vital to establishing the efficacy of a given vaccine and have been assessed in this study using a plant-based oral delivery platform expressing HBsAg. Methods Long-term immunological memory was assessed in mice injected with a primary dose of Recombivax ® and boosted with orally-delivered HBsAg wafers, control wafers, or parenterally-delivered commercial vaccine (Recombivax ® ). Results Mice boosted with HBsAg orally-administered wafers displayed sharp increases in mucosal IgA titers in fecal material and steep increases in serum IgA, whereas mice boosted with Recombivax ® showed no detectable levels of IgA in either fecal or serum samples following four boosting treatments. Long-term memory in the orally-treated mice was evidenced by sustained fecal IgA, and serum IgA, IgG, and mIU/mL over one year, while Recombivax ® -treated mice displayed sustained serum IgG and mIU/mL. Furthermore, sharp increases in these same antibodies were induced after re-boosting at 47 and 50 weeks post-primary injection. Conclusions Orally-delivered vaccines can provide long-term immune responses mucosally and systemically. For sexually-transmitted diseases that can be acquired at mucosal surfaces, such as HBV, an oral delivery platform may provide added protection over a conventional parenterally administered vaccine.

Typhoid fever remains a serious public health problem with a high impact on toddlers and young children. Vaccines against the Vi capsular polysaccharide are efficacious against typhoid fever demonstrating that antibodies against Vi confer protection. The currently licensed Vi typhoid vaccines have however limited efficacy and are manufactured by a complex process from wild-type bacteria. Due to these inherent issues with the current vaccines, an alternative vaccine based on an O-acetylated high molecular weight (HMW) polygalacturonic acid (GelSite-OAc™) was generated. The HMW polygalacturonic acid shares the same backbone as the Vi polysaccharide of Salmonella Typhi. The GelSite-OAc™ has a high molecular weight (>1 × 106 Da) and a high degree of O-acetylation (DOAc) (>5 μmole/mg), both exceeding the potency specifications of the current Vi vaccine. Studies in Balb/c mice demonstrated that GelSite-OAc™ was highly immunogenic, inducing a strong antigen-specific antibody response in a DOAc- and dose-dependent manner which was comparable to or higher than those induced by the licensed Vi vaccine. Importantly, the GelSite-OAc™ was shown to be fully protective in mice against lethal challenge with Salmonella Typhi. Furthermore, the GelSite-OAc™ demonstrated a boosting effect or memory response, exhibiting a >2-fold increase in antibody levels upon the second immunization with either GelSite-OAc™ or the Vi vaccine. This novel boosting effect is unique among polysaccharide antigens and potentially makes GelSite-OAc™ effective in people under 2 years old. Together these results suggest that the GelSite-OAc™ could be a highly effective vaccine against Salmonella Typhi.

Many studies address the influence of the gut microbiome on the immune system, but few dissect the effect of T cells on gut microbiota and mucosal responses. We have employed larval thymectomy in Xenopus to study the gut microbiota with and without the influence of T lymphocytes. Pyrosequencing of 16S ribosomal RNA genes was used to assess the relative abundance of bacterial groups present in the stomach, small and large intestine. Clostridiaceae was the most abundant family throughout the gut, while Bacteroidaceae, Enterobacteriaceae, and Flavobacteriaceae also were well represented. Unifrac analysis revealed no differences in microbiota distribution between thymectomized and unoperated frogs. This is consistent with immunization data showing that levels of the mucosal immunoglobulin IgX are not altered significantly by thymectomy. This study in Xenopus represents the oldest organisms that exhibit class switch to a mucosal isotype and is relevant to mammalian immunology, as IgA appears to have evolved from IgX based upon phylogeny, genomic synteny, and function.

In the 300 million years since the divergence of the avian and mammalian lines, numerous differences have evolved between the antibody responses of birds and mammals. The most significant of these differences results from the use of the bursa of Fabricius by birds. This unique organ serves as a site where B cells, the cells that will eventually produce antibodies, are first selected for their ability to produce antibodies against foreign antigens. This selection process is preceded by a relatively short period when the developing B cell generate a wide array of antibody receptor molecules by gene conversion. Thus, unlike mammals that can generate new antigen-binding specificities throughout their lives, birds can only do so during a short period within the bursa before hatching. A second key difference lies in the chemical structure of the major immunoglobulin (Ig) involved. Thus in mammals, this is IgG. In birds it is now recognized as a distinctly different immunoglobulin called IgY. In addition, some birds can produce a small-version of the IgY that lacks a full-sized Fc region. Its biological significance is unclear. Finally, birds lay eggs and thus must pass maternal immunoglobulins on to their offspring within the egg contents. For this reason, the egg yolk is full of IgY, whereas the albumin is rich in IgA. Pigeons are a notable exception to this situation when they secrete a "crop milk" rich in IgA. Notwithstanding these differences, it must be emphasized that the biological role of antibodies is identical in all species tested to free the body of extracellular invaders. For this reason, the basic kinetics and function of the antibody response is well conserved between the different vertebrate classes.

Immunity to carbohydrate antigens plays an important role in resistance to infectious agents (19, 24). Natural carbohydrate-specific antibodies are found in normal humans and animals. They are produced independently of immunization (3), and their presence is considered to be a result of the immune response to normal environmental antigens such as the bacterial flora in the gut. Examples include natural blood group alloagglutinins (33) and human anti-alpha-galactosyl antibodies (23). However, these natural anticarbohydrate antibodies might also be the result of subclinical or unrecognized infections. Studies with human anti-alpha-galactosyl antibodies have shown that these antibodies may play an important role in host defense (23). In addition to anticarbohydrate antibodies, lectins present in serum and other body fluids also play an important role in host defense. A major group of these lectins consists of collectins (5, 7). Collectins are C-type lectins whose binding to ligands is calcium dependent. So far, five collectins have been identified: conglutinin, collectin 43 (CL-43), mannose-binding protein (MBP), and pulmonary surfactant proteins A (SP-A) and D (SP-D). Three of them (conglutinin, CL-43, and MBP) are found in serum, and the other two (SP-A and SP-D) are found in the lung (7). Conglutinin and CL-43 are found only in cattle (1, 5, 14). Thus, cattle are unique in that they possess three serum collectins (conglutinin, CL-43, and MBP) whereas other animals, including humans, are known to have only one (MBP). The sugar-binding preferences of these three bovine lectins are similar, as demonstrated by inhibition assays with monosaccharides; both MBP and conglutinin preferentially bind mannose and N-acetylglucosamine (GlcNAc), while CL-43 binds mannose and N-acetylmannosamine (7, 14). These collectins are an important part of innate immunity (5). They bind to their targets through their C-terminal carbohydrate recognition domains and activate complement or interact with phagocytic cells through their N-terminal domains, leading to the destruction of the targeted microorganisms. Collectins are especially important in newborns and the young, whose immune systems are not fully capable of mounting an efficient specific immune response. A congenital deficiency of MBP is responsible for numerous cases of immunodeficiency in children, who as a result suffer from recurrent infections (28–30). Adult humans with MBP deficiency, however, do not suffer from repeated infections as children do. This suggests that the development of specific humoral responses may have compensated for the loss of MBP. Mannan or alpha-mannan is a common fungal antigen. It generally consists of an α(1-6)-linked mannose backbone to which short α(1-2)- and/or α(1-3)-linked mannose side chains are attached (11, 26). β(1-2)-linked mannose side chains may also be present (11, 25). The detailed structure may differ among species. The mannan antigens from the yeasts Candida spp. and Saccharomyces cerevisiae have been extensively studied (10, 11, 26). The S. cerevisiae mannan, which has structural and antigenic features similar to those of the Candida mannan, is commonly used as an affinity ligand for isolation or detection of collectins, especially MBP (7, 28). Mannans with similar structural features are also found in other fungi, including Trichophyton mentagrophytes (9) and Aspergillus oryzae (18), and in bacteria such as Mycobacterium tuberculosis and Mycobacterium bovis (8, 32). Some epitopes from yeast mannan cross-react with mannose-containing polysaccharides from other microorganisms, including Serratia marcescens (21) and Salmonella thompson (20). Many of these fungi, including Candida albicans, are important pathogens. C. albicans is a normal inhabitant of the digestive tract, oral cavity, and vagina. Infections usually occur endogenously; i.e., they are caused by yeasts already present in the body. In cows, C. albicans, along with several other Candida species, can cause mastitis (13, 27). Vaginal infection by Candida in humans is particularly widespread (4). Antimannan antibodies have been found in normal and infected humans (15, 31), and they have been shown to be protective against yeast infections in experimental animals (4, 35). However, their protective role has not been directly demonstrated in humans. Recently, the anti-S. cerevisiae mannan antibodies found in normal humans have been suggested to be associated with inflammatory bowel disease (22). Given the ubiquitous nature and the disease-causing potential of mannan-containing microorganisms, examination of antimannan antibodies could potentially yield important clues regarding immunity against these microorganisms. This is of particular interest with regard to cattle because they, unlike other mammals, possess three serum collectins, all of which are capable of binding to the mannan antigen. Studies of these antibodies in cattle may provide useful information on the role of antimannan antibodies and their relationship to lectin-mediated innate responses.

The hepatitis B virus continues to be a major pathogen worldwide despite the availability of an effective parenteral vaccine for over 20 years. Orally-delivered subunit vaccines produced in maize may help to alleviate the disease burden by providing a low-cost, heat-stable alternative to the parenteral vaccine. Oral subunit vaccination has been an elusive goal due to the large amounts of antigen required to induce an immunologic response when administered through the digestive tract. Here we show that high levels of HBsAg were obtained in maize grain, the grain was formed into edible wafers, and wafers were fed to mice at a concentration of approximately 300 μg/g. When these wafers were made with supercritical fluid extraction (SFE)-treated maize material, robust IgG and IgA responses in sera were observed that were comparable to the injected commercial vaccine (Recombivax(®)). In addition, all mice administered SFE wafers showed high secretory IgA titers in fecal material whereas Recombivax(®) treated mice showed no detectable titer. Increased salivary IgA titers were also detected in SFE-fed mice but not in Recombivax(®) treated mice. Wafers made from hexane-treated or full fat maize material induced immunologic responses, but fecal titers were attenuated relative to those produced by SFE-treated wafers. These responses demonstrate the feasibility of using a two-dose oral vaccine booster in the absence of an adjuvant to induce immunologic responses in both sera and at mucosal surfaces, and highlight the potential limitations of using an exclusively parenteral dosing regime.

The immune system consists of various types of cells and molecules that specifically interact with each other to initiate the host defense mechanism. Recent studies have shown that carbohydrates and lectins (carbohydrate-binding proteins) play an essential role in mediating such interactions. Both lectins and carbohydrates are widely distributed in the mammalian tissues as well as in microorganisms. Carbohydrates, due to their chemical nature, can potentially form structures that are more variable than proteins and nucleic acids. Lectins can exist in either soluble or cell-associated form, and although overall structures vary, invariably possess carbohydrate-recognition domains (CRD) with various specificities. The interaction between lectins and carbohydrates have been shown to be involved in such activities as opsonization of microorganisms, phagocytosis, cell adhesion and migration, cell activation and differentiation, and apoptosis. The number of lectins identified in the immune system is increasing at a rapid pace. The development in this area has opened a new aspect in studying the immune system, and at the same time, provided new therapeutic routes for the treatment and prevention of disease.

Hepatitis B remains a major global health problem despite the availability of a safe and effective vaccine. Segments of the population lack access to or respond poorly to the parenteral vaccine, perpetuating the infection-transmission cycle. A low cost, orally delivered vaccine has the potential to alleviate many of these problems. Here we describe the expression of a bioencapsulated hepatitis B surface antigen (HBsAg) in maize and its immunogenicity, demonstrating for the first time a commercially feasible oral subunit vaccine production system for a major disease. This work surmounts previous barriers to plant-produced vaccines by expressing HBsAg at much higher levels and retaining antigen immunogenicity post-processing: factors which facilitated a robust immune response in mice without the need for an adjuvant. This method provides a practical solution to the delivery of a low-cost, stable oral vaccine.

The adjuvant properties of a polydispersed beta-(1,4)-linked acetylated mannan, acemannan (ACE-M), were evaluated. Day-old broiler chicks were randomly selected and allocated to four flocks (Vac 1-4). The Vac 1 flock was sham vaccinated with saline. The Vac 2 flock was vaccinated with an oil-based vaccine (Breedervac III; Newcastle disease virus (NDV), infectious bursal disease virus (IBDV) and infectious bronchitis virus). The Vac 3 flock was vaccinated with a vaccine-ACE-M mixture, and the Vac 4 flock was vaccinated with vaccine and ACE-M at separate anatomical sites. ELISA titres to NDV and IBDV were determined. The immune response to NDV at 21 days postvaccination (PV) was significantly enhanced (P less than or equal to 0.05) by the addition of ACE-M to the vaccine, compared with vaccination without ACE-M. Subsequently, the vaccine-ACE-M mixture appeared to suppress the immune response to NDV. However, at day 35 PV, 95% of the Vac 3 chicks compared with 90% of the Vac 2 and 89% of the Vac 4 chicks exhibited protective titres. The response to IBDV differed from that to NDV. At day 21 PV the immune response to IBDV was essentially the same for all flocks that received vaccine, i.e. addition of ACE-M to the vaccine did not significantly enhance the immune response; however, it did significantly (P less than or equal to 0.05) sustain the immune response at days 28 and 35. In addition to the observed effect on titres to NDV and IBDV, ACE-M also had an effect on flock immunity.(ABSTRACT TRUNCATED AT 250 WORDS)

Avian Borna virus (ABV) has recently been shown to be the causal agent of proventricular dilatation disease (PDD) a lethal neurologic disease of captive psittacines and other birds. An immunoblot assay was used to detect the presence of antibodies against avian Borna virus in the serum of affected birds. A lysate from ABV-infected duck embryo fibroblasts served as a source of antigen. The assay was used to test for the presence of antibodies to ABV in 117 birds. Thirty of these birds had biopsy or necropsy-confirmed proventricular dilatation disease (PDD), while the remaining 87 birds were apparently healthy or were suffering from diseases other than PDD. Sera from 27 of the 30 PDD cases (90%) contained antibodies to ABV. Seventy-three (84%) of the apparently ''healthy'' birds were seronegative. Additionally, sera from seven macaws and one parrot trapped in the Peruvian Amazon were seronegative. Positive sera recognized the bornaviral nucleopro- tein (N-protein). While the presence of antibodies to ABV largely corresponded with the development of clinical PDD, 14 apparently healthy normal birds possessed detectable antibodies to ABV. The existence of a carrier state was confirmed when 13 of 15 apparently healthy cockatiels were shown by PCR to have detectable ABV RNA in their feces. Western blot assays may be of significant assistance in diagnosing proventricular dilatation disease. Many apparently healthy birds may however be seronegative while, at the same time, shedding ABV in their feces.

Apoptosis, or programmed cell death, is believed to be the mechanism for depletion of lymphocytes recognizing self-antigens following clonal expansion in the bursa of Fabricius. Although bursal apoptosis has previously been shown to increase following in vivo exposure to glucocorticoids, the microanatomical site of induced or normal apoptosis has not been unequivocally established. Presently, we adapted the existing terminal deoxynucleotidal transferase-mediated dUTP nick-end labeling (TUNEL) assay for use with neonatal bursae. Similar to previous reports, TUNEL revealed that normal apoptosis is preferentially, but not exclusively, ongoing in bursal follicular cortical cells. Administration of a single dose of a synthetic glucocorticoid (dexamethasone) or androgen (19-nortestosterone) did not significantly (P < 0.05) alter follicular lymphocyte numbers or apoptosis per unit of area at the time points evaluated post-administration (6 or 24 h). However, administration of 19-Nortestosterone increased the interfollicular epithelial thickness, a change usually associated with edema, within 6 h following treatment. Additionally, administration of the androgen 19-nortestosterone significantly decreased the number of proliferating cells as detected using mouse anti-proliferating cell nuclear antigen (PCNA) as a primary immunohistochemical antibody. In normal (control) bursal sections, occasional follicles consisting of predominantly apoptotic cells were observed (0.26% of follicles). Such follicles were consistently one-tenth the area of normal follicles. This incidental finding may suggest occasional occurrence of a common signal for deletion, such as a common integral or clonal mistake, viral infection, or an aberrant paracrine signal.

Immunity against the carbohydrate components of microorganisms mediated by antibodies is an important part of host defenses. Humans and closely related primates, but not other mammals, possess natural anti-Galα1-3Gal antibodies which also, although less avidly, react with melibiose (Galα1-6Glc). Using an enzyme-linked immunosorbent assay (ELISA) with melibiose-bovine serum albumin as an antigen, we analyzed bovine anti-alpha galactosyl antibodies with respect to specificity and distribution in individual animals. Inhibition assays showed that melibiose was the strongest inhibitor, followed equally by stachyose (Galα1-6Galα1-6Glcβ1-2Fru) and raffinose (Galα1-6Glcβ1-2Fru) and then by Galβ1-6Gal, Gal, and Galα1-2Gal. Others, including Galα1-3Gal and Galα1-4Gal, only exhibited minor inhibition. Thus, these bovine anti-alpha galactosyl antibodies appeared to preferentially react with Galα1-6Glc or Galα1-6Gal. The distinction of this specificity from that (Galα1-3Gal) of human antibodies was further demonstrated by the poor reaction of bovine serum to the Galα1-3Gal antigen in comparison to human serum. All 27 healthy bovine serum samples of the three age groups (newborn, calf, and adult) tested contained such antibodies with titers increasing with age. The antibodies purified by affinity chromatography using a melibiose-agarose column were mainly of the immunoglobulin G (IgG) isotype with a concentration of >23 μg/ml in most samples. IgG1 was found to be the primary antimelibiose IgG isotype in all age groups by isotype-specific ELISA, but a significant increase in IgG2, an isotype more related to innate immunity, was observed in calves and adults, compared to newborns. The purified antibodies reacted with the type II bovine strain ofStreptococcus agalactiae, a common pathogen of bovine mastitis. Thus, these anti-Galα1-6Glc or Galα1-6Gal antibodies in cattle might be involved in defense against microbes bearing this or the related epitopes.

An enzyme-linked immunosorbent assay (ELISA) for the serodiagnosis of Toxoplasma gondii infections on single serum dilutions was developed. This test system is a standardized kit designed to detect circulating specific antibodies to Toxoplasma gondii in human sera. It consists of Toxoplasma gondii soluble antigen-coated microtitration multiwell plates, specific immunoglobulin-enzyme conjugate and other required reagents. In a clinical trial performed on sera from 1,035 clinically suspected toxoplasmosis cases, the Sabin Feldman Dye Test (SFDT) and this ELISA system agreed closely. Relative to the SFDT, the sensitivity and specificity of the latter was 98.0% and 97.6% respectively with a correlation coefficient of 0.97. In a further study of 121 sera, the Indirect Fluorescent Antibody Test (IFAT), the Indirect Haemagglutination Test (IHAT) and this ELISA procedure showed over 90% agreement, with correlation coefficient of 0.98 and 0.95 respectively. Within the working concentration of specific antibody to T. gondii in human serum, there was a linear relationship between the ELISA values and the WHO international standard for human anti-Toxoplasma serum.

Human and animal forms of African trypanosomiasis are characterised by sustained hypocomplementaemia, gross hypergammaglobulinaemia M, and profound immunosuppression. It is suggested that this hypocomplementaemia is probably due to the action of a trypanosome-derived complement-activating factor and that the elevated IgM levels may be the combined result of this decomplementation, together with a subsequent failure of the normal IgM-to-IgG antibody switch mechanism and polyclonal B-lymphocyte activation by a trypanosome-generated mitogen The immunosuppression in this disease may be a result of the collective immunosuppressive effects of trypanosome-derived immune-modulating free fatty acids, polyclonally stimulating B-cell mitogen, and complementactivating factors.

It was postulated that the anemia commonly seen in mammalian trypanosomiasis, and specifically in Trypanosoma congolense-infected calves, was of immunological origin. Specifically, we postulated that trypanosome antigen-antibody-complement complexes, deposited on the surface of erythrocytes of infected calves, resulted in their immune elimination leading to clinical anemia. This hypothesis was tested experimentally. Immunoglobulins bound to the erythrocytes of 13 infected calves were detected by a direct antiglobulin test from 7 to 10 days post infection. The reaction was strongest between 3 and 9 weeks and remained inconsistently positive until the calves were killed by euthanasia 15 to 18 weeks after infection. Erythrocytes reacting positively in this test were then lysed and immunoglobulins were eluted from the washed stromata by means of a low pH buffer. Sixteen out of 74 eluates prepared in this way and concentrated, contained IgM and IgG. Antibody activity of these eluates against T. congolense was demonstrated by means of the complement fixation test, the indirect hemagglutination test, and the indirect antiglobulin test. It is considered that the original hypothesis has been essentially proven.

Changes in antibody responses in adult mice infected with Trypanosoma congolense and subsequently challenged with unrelated antigens (sheep red blood cells and pneumococcal polysaccharide) were studied. Immune responses were significantly depressed within 1 week of infection, and complete suppression of both IgM and IgG responses to both types of antigen was established 15 days after immunization. Coincidentally with the development of high parasitaemias, background IgM plaque-forming cell responses to sheep red cell antigen significantly increased in non-immunized T. congolense-infected animals. Autolysates of T. congolense and chloroform-soluble extracts of the autolyzed trypanosome were found to be mitogenic in vitro for the spleen cells of normal mice. Fractionation of these extracts by thin-layer chromatography indicated that the mitogenic activity migrated with the free fatty acids. Substitution of the relevant saturated and unsaturated free fatty acids in the autolyzed trypanosome extracts with commercial pure fatty acids in the mouse spleen cultures indicated that the mitogenicity was due to palmitic and stearic acids. It is suggested that the general immunosuppressing effect of trypanosomes may be attributed, at least in part, to the polyclonal activation, and subsequent depletion and/or clonal exhaustion of B-cells as a result of blastogenic stimulus from the parasites. This may operate, at least in part, through the generation of B-cell mitogenic saturated fatty acids.

Yersinia enterocolitica O9 was shown to provoke O, OH and H antibody response in calves. Brucella abortus failed to generate Yersinia H antibody response and generated Brucella O antibodies that cross-reacted with Yersinia O and OH antigens. The presence of Yersinia H agglutinins along with a higher titre of Yersinia OH antibody than Brucella O antibody is suggestive of subclinical infection with yersiniosis rather than brucellosis. Cross-absorption of sera from calves with established Yersinia infections indicated that absorption of sera with Brucella O antigens, although completely removing Brucella O antibodies, failed to remove completely Yersinia O, OH and H antibodies, and thus provides an additional method of distinguishing between the two infections.

A survey of serum samples from mammals trapped in Central Ontario showed that many contained antibodies to Toxoplasma gondii. The prevalence of infection as reflected by positive reactions in the Sabin-Feldman Dye Test appeared to be related to the type of diet of each species examined, and specifically, to the proportion of rodents in the diet. Of the fox blood samples tested, 84% were positive. The percentage of positive samples diminished through, coyote, mink, bear, fisher skunk, raccoon, marten and rabbit. Blood samples from squirrel, deer, hare and groundhog were negative.

A method of utilizing morphological changes in rat mast cells to determine reaginic antibody activity in bovine serum is described. This technique, which has been shown to be useful for the diagnosis of allergies in man, relies on the ability of antigen to degranulate mast cells sensitized with allergic serum. Experiments with radioactively-labelled allergic bovine globulin indicated the specificity of the binding of such proteins to rat mast cells. Cross-reaction between reaginic bovine antibody and human IgE was shown by a binding assay involving the uptake of 125I-labelled anti-human IgE globulin by mast cells incubated with bovine passive cutaneous anaphylaxis positive globulin.

SUMMARYBetween 1961 and 1974, 11934 samples of serum were tested by the Sabin- Feldman Dye test for the presence of antibodies to Toxoplasma gondii.Analysis of high-titred sera suggested that a 6-year cycle of high disease prevalence occurred across Canada. In addition, a decline in the percentage of positive reactions occurred each year in the Fall. The suggestion that this decline was due to dry conditions during the summer months was supported by the observation that differences in the prevalence of toxoplasma infection in ten Canadian cities were related to their average summer rainfall. The significance of these observations in relation to the epidemiology of toxoplasmosis in this country is discussed. The influence of patient age on the prevalence of infection was also investigated; the results obtained suggested that at least 75% of infants with high antibody titres against T. gondii had obtained these antibodies by placental transfer from their mothers.

The existence of enterobacterial common antigen in Yersinia enterocolitica and its absence in Brucella abortus were utilized in an attempt to provide a method to distinguish Brucella infections from infections with cross-reacting Yersinia. The indirect hemagglutination test was employed for this purpose. In experimental laboratory animals, the presence of anti-enterobacterial common antigen was found to be indicative of prior exposure to Y. enterocolitica rather than B. abortus. In cattle, however, low titers of anti-enterobacterial common antigen were present in all animals. It was observed that anti-enterobacterial common antigen titers either equaled or exceeded anti-Yersinia O titers in Yersinia-exposed animals, whereas in animals infected with B. abortus the anti-Yersinia O titer generally exceeded the anti-enterobacterial common antigen titer.

Injection of lipopolysaccharide from Escherichia coli intravenously into mice affected the ability of their peritoneal macrophages to adsorb antisheep erythrocyte cytophilic antibodies in vitro. After a single intravenous injection of lipopolysaccharide, the alteration in antibody uptake took the form of a triphasic response. An early evanescent increase in adsorptive capacity took place, which reached a maximum 5 min after injection and was followed immediately by a phase of depressed uptake of antibody. Enhancement of antibody uptake then increased to reach a maximum about 24 hr after injection, before declining slowly. The second enhancement phase was inhibited by prior immunization with E. coli or after multiple injections of lipopolysaccharide. Exposure of macrophages to lipopolysaccharide in vitro did not alter their capacity to take up cytophilic antibody. Changes in the capacity of macrophages and polymorphonuclear neutrophil leukocytes to adsorb opsonized erythrocytes were induced by injection of lipopolysaccharide and were similar to those induced with respect to the uptake of cytophilic antibodies by macrophages.

Summary Antigenic studies on variants of Trypanosoma brucei isolated from infected rabbits at different intervals were carried out by agglutination and by opsonic adherence tests with corresponding antisera isolated from the same rabbits. Organisms isolated from infected rabbits 2 and 5 wk after infection were relatively inagglutinable by sera collected at the same time and 3 wk later from the rabbit from which the trypanosomes were derived. Experiments on adherence of trypanosomes to macrophages in the presence of antibodies indicated the lack of opsonic adherence particularly in the presence of a prozone. These results suggest possible antigenic loss in the presence of antibodies.

SUMMARYThe changes in the titres of immunoconglutinin (IK), conglutinin and heterophile antibodies were measured in calves vaccinated with Br. abortus Strain 19. The IK titres rose rapidly following vaccination but returned to normal within ten weeks.A survey of the sera of over 300 cattle showed no significant correlation between the Brucella STA titre and of these ‘non-specific’ indicators. It is concluded that they are of little assistance in the serodiagnosis of brucellosis.

Triphenyltetrazolium chloride stained Brucella abortus 0 and Yersinia enterocolitica 09 0 and 0H antigens were used in the microplate agglutination test. It was shown that the results of these tests could be used to differentiate anti-brucella antibodies from antibodies induced by motile Y enterocolitica 09 in cattle sera.

Toxocara larval excretions and secretions collected from in vitro culture were used as antigen in passive haemagglutination and soluble antigen fluorescent antibody tests for the diagnosis of visceral larva migrans in experimental animals and man. Antibody to toxocaral secretions was detected in rabbits within 13 days of light Toxocara infection (ten larvae per kg) and within four days of heavy infection (10(4) larvae). Antibody was not detected following infection with 10(4) Ascaris suum larvae. In human sera, antibody was detected at low titre in 1% of 100 healthy adults and in 2% of 50 children. High titres were observed in one third of 170 patients with suspected visceral larva migrans and in 23 of 27 such patients presenting with an eosinophilia greater than 20%. In 25 patients with ocular lesions of an undiagnosed nature, four showed significant levels of anti-Toxocara antibody.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and Aroclor 1254 induced the cytochrome P-450 dependent monooxygenases, aryl hydrocarbon hydroxylase (AHH) and ethoxyresorufin O-deethylase (EROD) in rat hepatoma H-4-II E cells and C57BL/6J mice. It has been proposed that both Aroclor 1254 and 2,3,7,8-TCDD induce these enzymes via a common mechanism which features initial binding to the aryl hydrocarbon (Ah) cytosolic receptor protein. The major difference between these compounds was the relative potency (i.e. 2,3,7,8-TCDD much greater than Aroclor 1254). Cotreatment of rat hepatoma H-4-II E cells or C57BL/6J mice with a dose of 2,3,7,8-TCDD which submaximally induces AHH and EROD and a dose of Aroclor 1254 which exhibited little or no induction activity resulted in significant antagonism of the induction effects of 2,3,7,8-TCDD. For example, cotreatment of C57BL/6J mice with 2,3,7,8-TCDD (15 nmol/kg) and Aroclor 1254 (25, 75 and 150 mumol/kg) resulted in up to 23% antagonism of AHH induction by 2,3,7,8-TCDD. Moreover, cotreatment with a higher dose of the 2,3,7,8-TCDD agonist (30 or 50 nmol/kg) partially reversed some of the antagonism by Aroclor 1254. In vivo antagonism was observed only at Aroclor 1254/2,3,7,8-TCDD molar ratios of 1667:1, 5000:1 and 10,000:1. Administration of 2,3,7,8-TCDD (3.72 nmol/kg) to C57BL/6J mice resulted in a 76% decrease in the splenic plaque forming cell response to sheep red blood cells. This T-cell mediated immunotoxic effect of 2,3,7,8-TCDD segregates with the Ah locus. In contrast, administration of 5, 15, 75 and 150 mumol/kg of Aroclor 1254 resulted in impairment of the immune response only at the highest dose level. However, cotreatment of mice with 2,3,7,8-TCDD (3.72 nmol/kg) and Aroclor 1254 (5, 15 or 75 mumol/kg) resulted in no significant decrease in the plaque forming cell response and complete protection from the immunotoxicity of 2,3,7,8-TCDD. Cotreatment of the mice with Aroclor 1254 (75 mumol/kg) and a higher dose of the 2,3,7,8-TCDD agonist resulted in partial reversal of the protective effects of Aroclor 1254. The in vitro and in vivo data suggest that within specific antagonist/agonist dose ratios, Aroclor 1254 can antagonize at least 2 Ah receptor-mediated effects of 2,3,7,8-TCDD, namely AHH induction and immunotoxicity.

At least two complement-activating materials are present in Trypanosoma lewisi preparations. One, a relatively minor component, is trichloroacetic acid (TCA) precipitable and can be inactivated with phenyl methylsufonyl fluoride (PMSF), a broad specificity enzyme inactivator. This substance is therefore probably protin in nature and has enzyme activity, which can activate complement. The second complement-activating material was shown to be stable in TCA and not inhibited by PMSF. This component was shown to contain hexoses and probably some amino acids, although the ultraviolet absorbance was minimal at 280 and 260 nm. Terminal absorbance occurred at 214 nm. The amount of hexoses in different samples corresponded well with their complement-activating capacities.

summaryGrowth agglutination and growth inhibition tests were established for the diagnosis ofBrucella abortusinfection. The former involves the agglutination of living organisms while the latter is a bactericidal test. Using mouse, guinea-pig, rabbit and bovine serum it was shown that the growth agglutination test is approximately ten times, and the growth inhibition test one hundred times, more sensitive than the conventional tube agglutination test. It is suggested that these techniques may be of assistance in diagnosing bovine brucellosis in situations in which the tube agglutination test results are suspected of being falsely negative.

By using an in vitro test for phagocytosis, it was demonstrated that mouse antisheep erythrocyte macrophage-cytophilic antibodies did not significantly contribute to erythrocyte ingestion by mouse peritoneal macrophages.

The action of injected bacterial lipopolysaccharide upon the uptake of cytophilic antibodies by macrophages was found to be unrelated either to changes in surface charge or to the rate of anaerobic glycolysis of these cells. It is apparently mediated by alterations in the levels of material in the serum which enhances the uptake of cytophilic antibody by macrophages. This enhancing material does not appear to be an immunoglobulin nor can it be generated by the action of endotoxin on either whole blood or serum.