Amphiphilic polyanhydride nanoparticles stabilize Bacillus anthracis protective antigen.

Advancements toward an improved vaccine against Bacillus anthracis, the causative agent of anthrax, have focused on formulations composed of the protective antigen (PA) adsorbed to aluminum hydroxide. However, due to the labile nature of PA, antigen stability is a primary concern for vaccine development. Thus, there is a need for a delivery system capable of preserving the immunogenicity of PA through all the steps of vaccine fabrication, storage, and administration. In this work, we demonstrate that biodegradable amphiphilic polyanhydride nanoparticles, which have previously been shown to provide controlled antigen delivery, antigen stability, immune modulation, and protection in a single dose against a pathogenic challenge, can stabilize and release functional PA. These nanoparticles demonstrated polymer hydrophobicity-dependent preservation of the biological function of PA upon encapsulation, storage (over extended times and elevated temperatures), and release. Specifically, fabrication of amphiphilic polyanhydride nanoparticles composed of 1,6-bis(p-carboxyphenoxy)hexane and 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane best preserved PA functionality. These studies demonstrate the versatility and superiority of amphiphilic nanoparticles as vaccine delivery vehicles suitable for long-term storage.

Lack of oral tolerance in C3H/HeJ mice.

Daily gastric intubation of lipopolysaccharide (LPS)-responsive C3H/HeN, BALB/c, and Swiss mice with SRBC for 2 wk resulted in oral tolerance, whereas similarly treated LPS-nonresponsive C3H/HeJ mice gave splenic anti-SRBC PFC responses, including the IgA isotype, after systemic challenge with antigen. Oral tolerance in LPS-responsive C3H/HeN mice was due to T suppressor (Ts) cells because significant Ts cell activity was demonstrated in both Peyer's patches (PP) and spleens of these animals. On the other hand, T cells from PP and spleens of identically treated C3H/HeJ mice exhibited mainly T helper cell activity. Prior treatment of PP or spleen cell preparations from tolerant C3H/HeN mice with anti-Lyt-2.1 resulted in good in vitro anti-SRBC PFC responses, especially IgA isotype responses in PP cell cultures. These results indicate that oral administration of a thymic-dependent antigen (SRBC) to LPS-responsive mice induced a Ts cell population in PP, which, after migration to peripheral lymphoid tissue (e.g., spleen), suppressed responses to systemically administered antigen. LPS-nonresponsive mice lack this Ts cell pathway and continually respond to oral administration of antigen.

Evaluation of CpG-ODN-adjuvanted polyanhydride-based intranasal influenza nanovaccine in pigs.

Influenza results in significant economic loss in the swine industry each year. A broadly protective swine influenza vaccine would have the dual benefit of protecting pigs from influenza A viruses (IAVs) and limiting their possible zoonotic transmission to humans. In this study, we developed polyanhydride nanoparticles-based swine influenza vaccine (KAg + CpG-nanovaccine) co-encapsulating inacticated/killed soluble antigen (KAg) and Toll-like receptor (TLR)-9 agonist (CpG-ODN). The immunogenicity and protective efficacy of KAg + CpG-nanovaccine was compared with KAg vaccine containing five-times greater quantity of antigens following heterologous virus challenge. Prime-boost intranasally delivered KAg + CpG-nanovaccine induced significantly higher levels of cross-reactive antigen-specific IgA antibody responses in the nasal cavity, greater lymphoproliferative response in peripheral blood mononuclear cells (PBMCs), and higher IFN-γ secretion during antigen-induced recall responses of PBMCs and tracheobronchial lymph nodes cells compared to those immunized with KAg alone. Importantly, KAg + CpG-nanovaccine provided better protective efficacy through a significant reduction in influenza-induced fever, 16-fold reduction of nasal virus shedding and 80-fold reduction in lung virus titers compared to those immunized with soluble KAg. Our results indicated that CpG-ODN-adjuvanted polyanhydride nanovaccine can induce higher mucosal antibody and cellular immune responses in pigs; and provide better protection as compared with intranasally delivered soluble KAg.

Mycobacterial ESAT-6 protein enhances mouse IFN-gamma responses to Mycoplasma hyopneumoniae P71 protein.

Type 1 immune responses play an important role in the resolution of diseases with infectious or oncogenic etiologies. Vaccines for production animals frequently target humoral immune responses and are often ineffective in protecting against disease. In order to shift the immune response more toward cellular immunity (i.e., type 1 response), we tested the ability of a mycobacterial protein, early secretory antigenic target (ESAT-6), to enhance interferon-gamma (IFN-gamma) secretion during the recall response with a second antigen. The Mycoplasma hyopneumoniae membrane protein P71 was used as a test antigen in murine vaccination studies. The ESAT-6 open reading frame (ORF) was fused to DNA encoding P71 to produce a recombinant protein that was used to immunize BALB/c mice. Control mice immunized with P71 alone demonstrated a splenic response characterized by release of interleukin-10 (IL-10) and a balanced antigen-specific IgG1/IgG2a antibody response. The presence of ESAT-6 as a fusion partner with P71 during immunization, however, resulted in an enhanced P71-specific IFN-gamma response, decreased release of IL-10, and significantly greater (p < 0.05) IgG2a antibody levels in comparison to immunizing with P71 alone. These results demonstrate that ESAT-6 can modify the profile of an immunologic response to an accompanying immunogen.

Detection of tumor necrosis factor alpha from porcine alveolar macrophages using an L929 fibroblast bioassay.

Tumor necrosis factor plays a central role in the mediation of the pathophysiological sequelae of infection and inflammation in animals and humans. The elucidation of its role in respiratory disease of swine has not been investigated, due in part to the lack of a sensitive and specific quantitative assay for its presence in tissue and fluid samples. Here we describe the detection of porcine tumor necrosis factor utilizing L929 murine fibroblast cells and characterize various parameters affecting assay sensitivity. Plating cell density and length of exposure time to test supernatants were the most critical factors. Using standard assay conditions as described here, porcine tumor necrosis factor was detected in alveolar macrophage conditioned media diluted more than 400-fold. Specificity of the assay for porcine tumor necrosis factor was shown by inhibition of cytotoxicity with neutralizing polyclonal antibodies for human recombinant tumor necrosis factor. Furthermore, comparisons of bioactivity with tumor necrosis factor mRNA levels from lipopolysaccharide-stimulated porcine alveolar macrophages indicated that the L929 bioassay was specific for porcine tumor necrosis factor.

The IgA response: inductive aspects, regulatory cells, and effector functions.

In this review, we have emphasized our current studies on the inductive aspects of the IgA immune response and homeostatic mechanisms involved in the induction of oral tolerance. By use of unique inbred mouse strains in restricted microbial environments, we have provided evidence for a central role of LPS in systemic unresponsiveness to orally encountered antigens. We have continued studies on characterization of GALT lymphoreticular cell types, including accessory cells, regulatory T-cells, and precursor IgA B-cells. We have placed recent emphasis on characterization of antigen-specific Th-cell clones derived from murine PP, which preferentially support IgA isotype responses. Relevant areas for continued research have been emphasized in this review.

Morphologic and temporal characterisation of lesions in an enhanced murine model of Serpulina hyodysenteriae infection.

This laboratory has previously reported a murine model of Serpulina hyodysenteriae infection in which mice fed a defined diet, Teklad 85420 (TD), developed caecal lesions more consistently than mice fed a conventional rodent chow (CRC). The objectives of the current studies were to characterise and compare the time of onset of lesions, the morphological nature and severity of lesions and the extent of colonisation by S. hyodysenteriae in mice fed the two diets. In the first of two experiments, 50 C3H/HeJ and 50 C3H/HeOuJ mice were fed either TD or CRC and then half of each group was infected with S. hyodysenteriae. Mice (n = 5) from each group were killed and examined on days, 1, 2, 4, 9 or 17 after infection. Each mouse was examined grossly and microscopically and assigned lesion scores based on lesion severity. The second experiment was designed in an identical way to the first, but had slightly smaller group sizes (n=20). Mice (n=4) were killed for necropsy at the same five time points after infection and their caeca were homogenised and examined by quantitative bacteriology with media selective for S. hyodysenteriae. There were no differences in any finding due to mouse strain. Group lesion scores over the entire experimental period were significantly higher in mice fed TD (mean total lesion index = 13) than in mice fed CRC (mean total lesion index = 8.8). Lesions were also temporally distributed in a significantly different manner in that they appeared earlier (day 1) and persisted longer in the TD-fed mice in comparison to CRC-fed mice. Furthermore, lesions of equivalent severity from each treatment group presented identical microscopic features. Finally, quantitative bacteriological results indicated that there was no significant difference in the number of cfu of S. hyodysenteriae isolated from mice fed TD and those fed CRC. These results demonstrate that the characteristic severe lesions associated with S. hyodysenteriae infection in mice can occur 1 day after oral challenge in mice fed Teklad diet 85420. Bacteriological results further indicate that the enhancement of lesion formation in this model is not due to any significant effect of the diet on numbers of spirochaetes in the caeca of infected mice.

Bacterial immunogens and protective immunity in swine.

This review provides a limited discussion of antibody-mediated immune responses to bacterial pathogens which cause disease in swine. Serum antibody titers or responses have been used to correlate immunization or convalescence with protection from a given disease or infectious agent. Though much effort has been devoted to the elucidation of the host's antibody response to bacterial antigens, there are limited examples where an antibody response to a singular antigen has induced protection from disease. Antibody responses have been shown to be very effective in neutralizing bacterial exotoxins, e.g. Escherichia, Pasteurella, Actinobacillus, and inhibiting the ability of bacterial pathogens to colonize mucosal surfaces, e.g. Escherichia, Salmonella. The protective role of monospecific antibody responses to other bacterial components are less clear; however, antibody responses are generally polyclonal in nature and are an important component of the host immune response following the onset of disease. Anticapsular antibodies have been shown to enhance phagocytosis of numerous pathogens, e.g. Actinobacillus, Streptococcus, Pasteurella. Humoral immune responses directed against the lipopolysaccharide (LPS) of many Gram-negative organisms have been shown to enhance phagocytosis and the activation of complement, e.g. Salmonella. Anti-LPS antibodies have also aided in the identification of the serotypic diversity of Gram-negative organisms, e.g. Serpulina, Salmonella, Pasteurella. Antibody responses to the outer membrane proteins of Gram-negative organisms enhance phagocytosis, activation of complement, or inhibit bacterial adhesion to host cell. Adhesion of Gram-positive microorganisms, e.g. Staphylococcus, Streptococcus, Clostridium, to eukaryotic cells can be inhibited by antibody against the peptidoglycan and these peptidoglycan-specific antibodies may also facilitate opsonization of these organisms. Mycoplasma species have been shown to be metabolically inhibited by antibody directed against membrane proteins. In addition to the protective aspects of humoral immunity, the host's antibody response has been used as a diagnostic and epidemiological tool to identify or determine the prevalence of infectious agents.

Characterization of mitogen-stimulated porcine lymphocytes using a stable fluorescent dye (PKH2) and multicolor flow cytometry.

Stimulation of lymphocyte proliferation using mitogens or specific antigens is a method that is used frequently to assess immune responsiveness. While useful, lymphocyte blastogenesis, or [3H]-thymidine incorporation, provides little information regarding the response of specific subsets to the stimulant. Here, we report that the fluorescent cell membrane probe, PKH2, is a useful tool for measuring the proliferation of porcine lymphocyte subpopulations by utilizing multicolor flow cytometry. For this study, mitogen-induced proliferation of porcine peripheral blood mononuclear cells (PBMCs) was measured using [3H]-thymidine incorporation as well as a flow cytometric-based proliferation assay. From the [3H]-thymidine incorporation data alone, it was observed that PBMC stimulated with either concanavalin A (Con A), phytohemagglutinin (PHA) or pokeweed mitogen (PWM) demonstrated greater proliferation on day 3 than on day 5 of culture. Using the PKH dye and flow cytometric analysis, the responsiveness of specific lymphocyte subsets to mitogen stimulation was detected. The predominant subsets of porcine lymphocytes responding to Con A or PHA stimulation were CD4(+)CD8(+), CD4(-)CD8alpha(hi), CD4(-)CD8alpha(lo) and gammadelta TCR(+) cells. PWM stimulation induced responses by CD4(+)CD8(+), CD4CD8alpha(hi) but not by CD4(-)CD8alpha(lo) or gammadelta TCR(+) cells. Con A stimulation resulted in a sustained proliferation of CD8alpha(hi) cells over the 5-day period while PHA stimulation resulted in proliferation that peaked within the first 3 days. Little or no proliferative responses were detected within the IgM(+) population (e.g. B cells). This is the first study to define the contribution of individual lymphocyte subsets to mitogen-induced proliferation of porcine PBMCs.

MHC class II-restricted, CD4(+) T-cell proliferative responses of peripheral blood mononuclear cells from Mycobacterium bovis-infected white-tailed deer.

White-tailed deer are significant wildlife reservoirs of Mycobacterium bovis for cattle, predators, and, potentially, humans. Infection of cattle with M. bovis stimulates an antigen-specific T-cell response, with both CD4(+) and CD8(+) cells implicated in protective immunity. Few studies, however, have examined lymphocyte subset responses to experimental M. bovis infection of white-tailed deer. In this study, a flow cytometric proliferation assay was used to determine the relative contribution of individual peripheral blood mononuclear cell subsets of M. bovis-infected white-tailed deer in the recall response to M. bovis antigen. Naive deer were challenged with M. bovis by cohabitation with infected deer. These M. bovis-challenged deer developed significant in vivo (delayed-type hypersensitivity) and in vitro (proliferative) responses to M. bovis purified protein derivative (PPD). At necropsy, typical tuberculous lesions containing M. bovis were detected within lungs and lung-associated lymph nodes of infected deer. The predominant subset of lymphocytes that proliferated in response to in vitro stimulation with PPD was the CD4(+) subset. Minimal proliferative responses were detected from CD8(+), gamma delta TCR(+), and B-cells. Addition of monoclonal antibodies specific for MHC II antigens, but not MHC I or CD1 antigens, abrogated the proliferative response. Together, these findings indicate that while CD4(+) cells from infected deer proliferate in the recall response to M. bovis antigens, this response is not sufficient to clear M. bovis and immunologic intervention may require stimulation of alternate subsets of lymphocytes.

Lymphocyte subset proliferative responses of Mycobacterium bovis-infected cattle to purified protein derivative.

Despite highly successful eradication efforts in several countries, Mycobacterium bovis infection of cattle remains a significant health concern worldwide. Immune mechanisms of resistance to and/or clearance of M. bovis infection of cattle, however, are unclear. Recent studies have provided evidence supporting a role for CD4(+), CD8(+), and gammadelta TCR(+) T cells in the response of cattle to M. bovis. In the present study, we utilized a flow cytometric-based proliferation assay to determine the relative contribution of individual lymphocyte subsets in the response to M. bovis infection and/or sensitization with mycobacterial purified protein derivative (PPD). Peripheral blood mononuclear cells (PBMC) from M. bovis-infected cattle proliferated in response to in vitro stimulation with M. bovis PPD. CD4(+) T cells and gammadelta TCR(+) cells were the predominate subsets of lymphocytes responding to PPD. gammadelta TCR(+) cells also proliferated in non-stimulated cultures; however, the gammadelta TCR(+) cell proliferative response of infected cattle was significantly (p<0.05) greater in PPD-stimulated cultures as compared to non-stimulated cultures. Intradermal injection of PPD for comparative cervical testing (CCT) induced a boost in the in vitro proliferative response of CD4(+) but not gammadelta TCR(+) cells of infected cattle. Administration of PPD for CCT also boosted interferon-gamma (IFN-gamma) production by PBMC of infected cattle following in vitro stimulation with M. bovis PPD. Injection of PPD for CCT did not, however, elicit a proliferative or IFN-gamma response in cells isolated from non-infected cattle. These data indicate that CD4(+) and gammadelta TCR(+) cells of M. bovis-infected cattle proliferate in a recall response to M. bovis PPD and that the CD4(+) cell response is boosted by intradermal injection with PPD for CCT.

Systemic and mucosal immune responses of pigs to parenteral immunization with a pepsin-digested Serpulina hyodysenteriae bacterin.

Serpulina hyodysenteriae infection of pigs, swine dysentery, causes a mucohemorrhagic diarrhoea resulting in significant economic losses to swine producers. The pathogenesis of this disease is poorly understood. Regardless, commercial vaccines have been developed and are in use. Thus, the present study was designed to examine cellular immune responses induced by parenteral S. hyodysenteriae vaccination. Significant antigen-specific interferon-gamma (IFN-gamma) and blastogenic responses were detected from peripheral blood lymphocytes isolated from vaccinated pigs. However, poor IFN-gamma responses were detected from colonic lymph node lymphocytes from these same pigs despite significant antigen-specific blastogenic responses. In addition, peripheral blood IFN-gamma responses were diminished by either in vitro depletion of CD4 expressing cells or by in vitro treatment with porcine IL-10. Colonic lymph node IFN-gamma responses were not inhibited by treatment with porcine IL-10. Vaccination also resulted in increased percentages of both mucosal and peripheral blood CD8 single positive cells with concurrent decreases in percentages of CD4 single positive cells as compared to percentages of these same populations from non-vaccinated pigs. In conclusion, these studies show that parenteral S. hyodysenteriae vaccination results in cellular immune responses detectable both peripherally (systemic immunity) as well as at the site of infection (mucosal immunity). However, it appears that regulatory mechanisms affecting IFN-gamma production in response to S. hyodysenteriae antigen differ between peripheral and colonic compartments.

The search for Brachyspira outer membrane proteins that interact with the host.

Little is known about the outer membrane structure of Brachyspira hyodysenteriae and Brachyspira pilosicoli or the role of outer membrane proteins (OMPs) in host colonization and the development of disease. The isolation of outer membrane vesicles from B. hyodysenteriae has confirmed that cholesterol is a significant outer membrane constituent and that it may impart unique characteristics to the lipid bilayer structure, including a reduced density. Unique proteins that have been identified in the B. hyodysenteriae outer membrane include the variable surface proteins (Vsp) and lipoproteins such as SmpA and BmpB. While the function of these proteins remains to be determined, there is indirect evidence to suggest that they may be involved in immune evasion. These data may explain the ability of the organism to initiate chronic infection. OMPs may be responsible for the unique attachment of B. pilosicoli to colonic epithelial cells; however, the only B. pilosicoli OMPs that have been identified to date are involved in metabolism. In order to identify further B. pilosicoli OMPs we have isolated membrane vesicle fractions from porcine strain 95-1000 by osmotic lysis and isopycnic centrifugation. The fractions were free of contamination by cytoplasm and flagella and contained outer membrane. Inner membrane contamination was minimal but could not be completely excluded. An abundant 45-kDa, heat-modifiable protein was shown to have significant homology with B. hyodysenteriae Vsp, and monoclonal antibodies were produced that reacted with five B. pilosicoli-specific membrane protein epitopes. The first of these proteins to be characterized is a unique surface-exposed lipoprotein.

Treatment with agmatine inhibits Cryptosporidium parvum infection in infant mice.

Cryptosporidium parvum is an intracellular protozoan parasite that causes enteric infection and diarrhea in a wide range of mammalian hosts, including humans and economically important livestock species. There are no effective vaccines or drug treatments available for cryptosporidiosis. Cryptosporidium parvum utilizes a unique metabolic pathway for the synthesis of polyamines, forming agmatine as an intermediary metabolite. We treated infant mice with oral doses of agmatine for 2 days before, the day of, and 5 days following experimental infection with C. parvum. Mice treated with agmatine were significantly less infected with C. parvum than were control mice receiving phosphate-buffered saline. Mice treated with agmatine only on the day of experimental infection with C. parvum were also significantly less infected than were control mice. These data suggest that exogenous agmatine alters the metabolism of C. parvum sufficient to interfere with its ability to colonize the mammalian intestine.

B cells are required for the induction of intestinal inflammatory lesions in TCRalpha-deficient mice persistently infected with Cryptosporidium parvum.

Mice with targeted disruptions in the T-cell receptor alpha gene (TCRalpha-/-) spontaneously develop inflammatory intestinal lesions with extensive B-cell lamina propria infiltrates. Cryptosporidium parvum infection accelerates intestinal lesion formation in TCRalpha-/- mice. In the present study, TCRalpha-/- mice were crossed with JH-/- (B-cell-deficient) mice and challenged with C. parvum to determine if B cells are required for intestinal lesion development. TCRalpha-/- x JH-/- mice challenged with C. parvum, either as neonates or adults, became persistently infected, whereas TCRalpha-/+ x JH-/+ heterozygote control mice cleared the parasite. Cryptosporidium parvum colonization of TCRalpha-/- x JH-/- mice was heaviest in the distal ileum, with fewer parasites detected in the cecum and distal colon. Despite persistent infection, TCRalpha-/- x JH-/- mice did not develop inflammatory or hyperplastic intestinal lesions as detected in C. parvum-infected TCRalpha-/- mice. These findings demonstrate that B cells are a necessary component for the development of inflammatory intestinal lesions of C. parvum-infected TCRalpha-/- mice.

Cryptosporidium parvum-induced inflammatory bowel disease of TCR-beta- x TCR-delta-deficient mice.

Experimental inoculation of neonatal immunocompetent strains of mice with Cryptosporidium parvum results in a transient, noninflammatory enteric infection. In the present study, we show that inoculation of mice deficient in alphabeta and gammadelta T cells (TCR-beta- x TCR-delta-deficient mice) with C. parvum results in persistent infection and severe inflammatory bowel disease-like lesions. The most severe lesions in these mice were in the cecum with similar yet less severe lesions in the ileum and proximal colon. The most notable aspect of the histopathology was glandular hyperplasia with abscess formation, extensive fibrosis of the lamina propria with infiltrates of predominately polymorphonuclear cells and macrophages, and a few small aggregates of B cells. Persistently infected mice also developed extensive hepatic periportal fibrosis in association with C. parvum colonization of bile ducts. Lesions observed in TCR-beta- x TCR-delta-deficient mice were markedly different than previously described lesions detected in C. parvum-infected TCR-alpha-deficient mice. Cryptosporidium parvum-infected TCR-alpha-deficient mice have extensive infiltrations of B cells, whereas TCR-beta- x TCR-delta-deficient mice had only a few small aggregates of B cells. These findings indicate that although gammadelta T cells are not necessary for induction of intestinal inflammation in C. parvum-infected alphabeta T-cell-deficient mice, their presence does alter the morphology of the ensuing lesion.

Effects of dietary conjugated linoleic acid in nursery pigs of dirty and clean environments on growth, empty body composition, and immune competence.

Early-weaned pigs (n = 64) averaging 5.3 +/- 0.3 kg and distributed into two environments (dirty and clean) were used to evaluate effects of conjugated linoleic acid (CLA) on growth performance, immune competence, and empty body composition. A factorial (2 x 4) arrangement within a split-plot design, with four littermate pigs as the experimental unit for the environment, pig within litter as the experimental unit for dietary treatment, and d-0 body weight used as covariate, were used in data analysis. Diets were formulated to contain CLA at 0, 0.67, 1.33, or 2% and to exceed the NRC (1988) nutrient needs of pigs. Animals were given ad libitum access to feed for 7 wk in three phases (I, 1 to 2; II, 3 to 5; and III, 6 to 7 wk). Within phases, diets were isocaloric and isonitrogenous. In Phase I, as dietary CLA concentration increased, ADG and ADFI decreased linearly (P < 0.05 and P < 0.02, respectively). In Phase II, upon adaptation to dietary CLA supplementation, ADG increased quadratically (603, 623, 622, and 548 g/d; P < 0.01), ADFI decreased linearly (873, 840, 867, and 717 g/d; P < 0.02) and gain:feed ratio tended to increase linearly (691, 742, 715, and 763; P < 0.07). In Phase III, no differences in growth performance were attributed to either dietary or environmental treatments. The poor health status associated with the dirty environment induced a growth suppression; pigs in the clean room had a greater cumulative ADG (P < 0.01) and ADFI (P < 0.01) than pigs in the dirty room. In Phase I, lower plasma urea nitrogen levels observed in pigs found in the dirty room (P < 0.03) indicated a lower protein intake caused by a lower ADFI. The effects of dietary CLA on peripheral phenotypic profiles of lymphoytes did not appear until d 42. However, as indicated by the growth suppression of pigs in the dirty room, the negative effects of the environmental challenge on pig health and growth had already appeared during phase I. On d 42, CLA induced a linear increase in percentages of CD8+ lymphocytes (21.7, 22.3, 28.0, and 32.7%; P < 0.001). These data suggest that a 42-d dietary CLA supplementation preceding a disease challenge could have prevented disease-associated growth suppression. Also, CLA-mediated amelioration of particular infectious diseases will depend on which CD8+ T cell subset (i.e., CD8alphaalpha-immunoregulatory or CD8alphabeta-cytotoxic) is most influenced by dietary CLA supplementation.

Biological reactivity of Moraxella bovis lipopolysaccharide.

Lipopolysaccharide (LPS) was isolated from Moraxella bovis 118F and ATCC 10900, M ovis ATCC 33078, and M phenylpyruvica ATCC 23333 by hot phenol-water extraction. In silver-stained sodium dodecyl sulfate polyacrylamide electrophoresis gels, M bovis 118F LPS had a smooth profile, whereas the other Moraxella preparations appeared to be rough. The LPS preparations induced pyrogenicity and dermal Shwartzman reactions in rabbits, and induced production of tumor necrosis factor and interleukin-1 in vitro. Induction of tumor necrosis factor appeared to be among the most potent biological activities of M bovis LPS.

Flow cytometric analysis of canine colonic mucosal lymphocytes from endoscopically obtained biopsy specimens.

OBJECTIVE: To validate use of canine colonic biopsy specimens obtained via endoscopy as a source of mucosal lymphocytes (ML) for flow cytometric analysis. SAMPLE POPULATION: Mucosal biopsy specimens from 10 adult dogs. PROCEDURE: Mucosal lymphocyte subsets obtained from excised colon were compared with ML subsets obtained from biopsy specimens obtained by use of an endoscopic forceps (6 dogs). Endoscopic colonic biopsy specimens from 4 other dogs were used to define whether obtained ML were predominantly of intraepithelial or lamina propria origin. Mucosal lymphocytes were isolated and labeled, using commercially available monoclonal antibodies directed against canine cell surface antigens. Lymphocyte subsets (cytotoxic or helper T cells; B cells) were determined by use of flow cytometric analysis. RESULTS: A large number of viable ML was obtained after dissociation of the colonic epithelium from excised colon (45.5 + 21.5 X 10(6)) and endoscopic (7.2+/-3.4 X 10(6)) biopsy specimens. Lymphocyte subsets obtained with both methods were identical for each dog and consisted predominantly of intraepithelial lymphocytes, with some lymphocytes from the lamina propria. Collagenase digestion of excised colon also yielded a large number of viable lymphocytes from the lamina propria (56.7+/-20.4 X 10(6)), but collagenase digestion of endoscopic biopsy specimens was less rewarding. CONCLUSION AND CLINICAL RELEVANCE: A representative sample of viable intraepithelial ML is obtainable from endoscopic biopsy specimens. Flow cytometric analysis, a minimally invasive technique, can be used to study ML of client-owned animals.

Susceptibility of inbred mouse strains to infection with Serpula (Treponema) hyodysenteriae.

Several inbred strains of mice were inoculated with Serpula (Treponema) hyodysenteriae B204 to determine susceptibility to infection. Challenge doses of 10(7) or 10(8) spirochetes induced cecal lesions in C3H/HeJ mice and other C3H strains of mice. However, more than a 100-fold difference existed between the dose required to induce lesions in 50% of the infected C3H/HeJ mice (8.3 x 10(7)) and that required to induce them in 50% of the infected C3H/HeN mice (5 x 10(5)). C3H/HeJ mice lack a splenocyte mitogenic response to Escherichia coli lipopolysaccharide but exhibited a mitogenic response comparable to those of other C3H strains of mice when stimulated with S. hyodysenteriae endotoxin (butanol-water extract). Different inbred strains exhibited different susceptibilities to infection, with the strain C3H/HeN being the most susceptible on the basis of colonization and development of macroscopic cecal lesions. The ity gene had no apparent effect on susceptibility of mice challenged with S. hyodysenteriae. The involvement of the H-2 haplotype with susceptibility is unclear, but the mice bearing H-2k were more susceptible than mice with the H-2b, H-2d, or H-2q haplotype. These data support the hypothesis that the host's responsiveness to lipopolysaccharide influences the susceptibility to infection with S. hyodysenteriae. However, differences in susceptibility between inbred mice exist independent of the lps locus, suggesting that there are other inherent differences between mouse strains that affect susceptibility to infection by S. hyodysenteriae.