Transcriptional Profiling of the Chicken Tracheal Response to Virulent Mycoplasma gallisepticum Strain Rlow.

Mycoplasma gallisepticum, the primary etiologic agent of chronic respiratory disease (CRD) in poultry, leads to prolonged recruitment and activation of inflammatory cells in the respiratory mucosa. This is consistent with the current model of immune dysregulation that ostensibly allows the organism to evade clearance mechanisms and establish chronic infection. To date, studies using quantitative reverse transcription-PCR (qRT-PCR) and microarrays have shown a significant transient upregulation of cytokines and chemokines from tracheal epithelial cells (TECs) in vitro and tracheal tissue ex vivo in response to virulent strain Rlow that contributes to the infiltration of inflammatory cells into the tracheal mucosa. To expand upon these experiments, RNA was isolated from tracheas of 20 chickens infected with M. gallisepticum Rlow and 20 mock-infected animals at days 1, 3, 5, and 7 postinoculation, and samples were analyzed for differential gene expression using Illumina RNA sequencing. A rapid host response was observed 24 h postinfection, with over 2,500 significantly differentially expressed genes on day 3, the peak of infection. Many of these genes have immune-related functions involved in signaling pathways, including Toll-like receptor (TLR), mitogen-activated protein kinase, Jak-STAT, and the nucleotide oligomerization domain-like receptor pathways. Of interest was the increased expression of numerous cell surface receptors, including TLR4 and TLR15, which may contribute to the production of cytokines. Metabolic pathways were also activated on days 1 and 3 postinfection, ostensibly due to epithelial cell distress that occurs upon infection. Early perturbations in tissue-wide gene expression, as observed here, may underpin a profound immune dysregulation, setting the stage for disease manifestations characteristic of M. gallisepticum infection.

Vaccination of BALB/c mice with an avirulent Mycoplasma pneumoniae P30 mutant results in disease exacerbation upon challenge with a virulent strain.

Mycoplasma pneumoniae is a significant human respiratory pathogen that causes high morbidity worldwide. No vaccine to prevent M. pneumoniae infection currently exists, since the mechanisms of pathogenesis are poorly understood. To this end, we constructed a P30 cytadhesin mutant (P-130) with a drastically reduced capacity for binding to erythrocytes and an inability to glide on glass substrates. This mutant was determined to be avirulent and cannot survive in the lungs of BALB/c mice. We also ascertained that the previously identified P30 gliding motility mutant II-3R is avirulent and also cannot be recovered from the lungs of mice after infection. Mutant P130 was then assessed for its efficacy as a live attenuated vaccine candidate in mice after challenge with wild-type M. pneumoniae. After vaccination with the P-130 P30 mutant, mice showed evidence of exacerbated disease upon subsequent challenge with the wild-type strain PI1428, which appears to be driven by a Th17 response and corresponding eosinophilia. Our results are in accordance with other reports of vaccine-induced disease exacerbation in rodents and emphasize the need to better understand the basic mechanisms of M. pneumoniae pathogenesis.

Pulmonary eosinophilia correlates with allergen deposition to the lower respiratory tract in a mouse model of asthma.

BACKGROUND: Eosinophilic infiltration into the airways is frequently associated with allergic asthma; however, the role of antigen deposition in mediating this phenomenon has not been studied in detail. OBJECTIVE: Using a murine model of ovalbumin (OVA) allergy, we examined how differential deposition of OVA during antigen challenge affects pulmonary eosinophilia, immune response and airway hyper-reactivity (AHR). METHODS: Differential allergen deposition to the upper respiratory tract (URT) alone or combined upper and lower respiratory tract (ULRT) was accomplished by administering OVA intranasally to either anaesthetized or unanaesthetized mice, respectively. BALB/c mice (6-7 weeks old) were sensitized with OVA-alum via the intraperitoneal route, and then challenged intranasally using OVA, with or without anaesthesia. AHR, enumeration of inflammatory cells and quantitative measurement of inflammatory cytokines and chemokines in bronchoalveolar lavage fluid (BALF), lung histopathology and immune responses were subsequently assessed. RESULTS: In sensitized animals challenged via the ULRT route, a profound eosinophilia and goblet cell hyperplasia was observed in lung tissue. Conversely, sensitized mice receiving an identical challenge dose via the URT route alone exhibited only negligible levels of inflammation. Interestingly, AHR and OVA-specific IgG(1) and IgE systemic responses were comparable between the two groups. CONCLUSION: This study indicates that direct exposure of allergen in the deep lung is highly correlated with airway eosinophilia and lung inflammation, but does not correlate with AHR or immune response.

Comparative assessment of a metabolically attenuated Mycoplasma gallisepticum mutant as a live vaccine for the prevention of avian respiratory mycoplasmosis.

In a previous study, signature sequence mutagenesis (SSM) was used to identify a mutant with a disruption of the gene encoding the metabolic factor, dihydrolipoamide dehydrogenase, and that mutant was designated Mg 7. The current study assessed the safety, immunogenicity and efficacy of Mg 7 in comparison to two commercially available vaccines (ts-11 and F) as well as a laboratory vaccine strain, GT5. Intratracheal vaccination of chickens with all four attenuated mutants induced varying levels of protection against intratracheal challenge with virulent Mycoplasma gallisepticum strain R(low). Mg 7 vaccinated chickens rapidly cleared the challenge strain, had lower histopathologic tracheal lesion scores when compared to unvaccinated chickens, and mounted a strong humoral anti-M. gallisepticum-specific IgG response. The IgG levels increased 2- to 3-fold upon R(low) challenge. Mg 7 induced a greater level of protection against intratracheal R(low) challenge than that observed with the other three attenuated strains, as evidenced by a lower recovery of R(low) from tracheas and lower histopathologic lesion scores in tracheas and air sacs. Based on these findings, Mg 7 appears to have good potential as a safe and effective vaccine for the prevention of avian mycoplasmosis.

Energy status, lipid-soluble vitamins, and acute phase proteins in periparturient Holstein and Jersey dairy cows with or without subclinical mastitis.

In a retrospective, case-controlled, observational study, associations among indices of negative energy balance, plasma lipid and lipid-soluble vitamin concentrations, plasma acute phase protein status, and occurrence of a new subclinical intramammary infection (IMI) during the periparturient period were determined. Cows were paired based on breed and expected parturition date (EPD) and monitored from the cessation of lactation through wk 8 of the subsequent lactation. A cow was identified as developing a new IMI if the intramammary pathogen isolated postpartum differed from that isolated in wk -9 (relative to EPD). Mean body condition score (BCS) of cows at wk -9 was 3.71 +/- 0.12. Fifteen Holstein and 15 Jersey dairy cows met the study selection criteria. Cows with a new IMI had greater body condition score, body weight, and body weight loss compared with cows that did not develop a new IMI. Prepartum plasma concentrations of beta-carotene were greater for Jersey cows with a new IMI compared with Jersey cows without a new IMI and Holstein cows, regardless of IMI status. However, there was a significant delay in recovery of plasma concentrations of beta-carotene postpartum for Jersey cows with a new IMI compared with Jersey cows without a new IMI. Plasma alpha-tocopherol, albumin, and retinol binding protein concentrations were greater during the periparturient period for cows without a new IMI. Plasma haptoglobin was increased at wk 1 postpartum for cows without a new IMI. Milk protein and lactose percentages and milk urea N were decreased and somatic cell counts were increased in cows identified with a new IMI compared with cows that did not develop a new IMI. Dairy cows with greater tissue energy stores prepartum and reduced plasma proteins, beta-carotene, and alpha-tocopherol had a greater risk for developing a new IMI during the periparturient period.

Immune response to a mucosally administered aflatoxin B1 vaccine.

In the present study, a mucosal vaccine was used in an effort to elicit serum IgG and intestinal secretory IgA against the mycotoxin aflatoxin B1 (AFB) in chickens. AFB was coupled to carrier proteins (BSA and porcine thyroglobulin) for use as a vaccine and ELISA coating antigen, respectively. Seven-day-old broiler chicks were divided into groups of 10 and immunized with one of four vaccine preparations: 1) AFB-BSA conjugate alone, 2) AFB-BSA linked to the B subunit of the recombinant heat-labile enterotoxin of Escherichia coli (rLT-B), 3) AFB-BSA admixed with rLT-B, or 4) AFB-BSA mixed with cholera toxin (CT). Each vaccine preparation was administered perorally, intrarectally, or intraperitoneally, with a booster immunization given 2 wk later. Sera and feces were collected weekly and assayed using isotype specific ELISA. All three routes of immunization elicited significant serum IgG responses; however, the intraperitoneal route was strongest for all vaccine preparations tested. The serum IgG immune response to the AFB-BSA conjugate was enhanced by co-administration of rLT-B but not by covalent coupling to rLT-B or coadministration with CT. Secretory IgA anti-CT and anti-rLT-B antibodies were detected in fecal supernatants, but no anti-AFB responses could be detected. As all 12 treatment groups produced significant levels of serum IgG anti-AFB, any of these approaches, including oral administration without adjuvant, may afford the chicken some level of protection through simple immuno-interception of free AFB.

Intranasal immunization of guinea pigs with an immunodominant foot-and-mouth disease virus peptide conjugate induces mucosal and humoral antibodies and protection against challenge.

Guinea pigs immunized intranasally with a keyhole limpet hemocyanin-linked peptide, corresponding to the prominent G-H loop of the VP1 protein of foot-and-mouth disease virus, raised substantial levels of antipeptide and virus-neutralizing antibodies in sera and of peptide-specific secretory immunoglobulin A in nasal secretions. In groups of animals immunized intranasally without adjuvant, 86 percent were fully protected upon challenge with homotypic virus. Surprisingly, animals given the peptide conjugates plus the mucosal adjuvant cholera toxin were afforded only partial protection in that primary lesions were observed in most animals, although spread to other feet was prevented. These results indicate that intranasal inoculation with the peptide offers a potential route of vaccination against foot-and-mouth disease and may be useful for eliciting protection in the upper respiratory tracts of susceptible animals.

A modified live Mycoplasma gallisepticum vaccine to protect chickens from respiratory disease.

The aim of this study was to assess the efficacy of a modified live Mycoplasma gallisepticum vaccine (GT5) for the protection of chickens against infection and respiratory disease. GT5 was constructed by the reconstitution of the avirulent high passage R (R(high)) strain with the gene encoding the major cytadhesin GapA. GT5 expressed GapA on its surface yet retained the phenotypic characteristics of the parental R(high) strain. Birds vaccinated with GT5 were protected upon challenge with the virulent low passage R (R(low)) strain as evidenced by a complete absence of tracheal lesions 2 and 4 weeks post-challenge, in contrast to sham immunized/challenged control birds. Modest amounts of IgG, and little, if any secretory IgA or IgM anti-M. gallisepticum were found in tracheal washings following vaccination. However, copious amounts of specific IgA were found following challenge, especially in sham immunized birds. This suggests that the tracheal IgG elicited by GT5 vaccination may have been responsible for blocking the initial colonization of R(low), thereby resulting in protection.

High affinity binding site-mediated prevention of chemical absorption across the gastrointestinal tract.

Preventing mucosal absorption of low-molecular weight compounds such as carcinogens, toxins and drugs could help prevent many diseases. To characterize the effects of dose and timing on high-affinity binding site mediated sequestration of specific chemical ligands in the gastrointestinal tract, avidin was perorally-administered to mice either prior to or mixed with 3H-biotin. Avidin enhanced fecal 3H-biotin excretion in a dose-dependent manner, consistent with the accepted mechanism of egg white-induced biotin deficiency syndrome. Avidin administration up to 4 h before 3H-biotin administration also enhanced fecal 3H-biotin excretion. Activated charcoal (AC) reduced 3H-biotin absorption when mixed with 3H-biotin before ingestion, but was ineffective when ingested prior to 3H-biotin. These studies suggest that ingestion of high-affinity protein binding sites can establish an absorptive barrier at the gastrointestinal mucosa to prevent the uptake of unwanted low molecular-weight chemicals.

Antibody responses to a mucosally delivered HIV-1 gp120-derived C4/V3 peptide.

T1-SP10MN(A) is a synthetic peptide containing a T-helper (Th), cytotoxic T cell (CTL) and a B-cell epitope derived from the HIV-1 gp120 envelope protein. This peptide can elicit both systemic and mucosal antibody responses following nasal immunization in various species. In the present study, three different mucosal immunization strategies were performed in rabbits to determine which induced a more vigorous antibody response to T1-SP10MN(A). Nasal immunization followed by nasal boosting was found to be superior at inducing both serum IgG and vaginal secretory IgA (S-IgA) when compared to nasal followed by vaginal boosting. Conversely, vaginal priming followed by vaginal boosting elicited minimal serum IgG and vaginal S-IgA responses to T1-SP10MN(A), but moderate levels of vaginal IgG were detected. This study further demonstrates that vaginal immune responses can be elicited by immunization at distant and local mucosal sites.

Peroral administration of specific antibody enhances carcinogen excretion.

Ingested carcinogens may exert effects directly on the gastrointestinal epithelium or after absorption and transport to other tissues. To determine the effect of anti-carcinogen antibody ingestion on dietary carcinogen excretion, a mixture of specific IgA or IgG and the model carcinogen 125I-N-2-(4-hydroxyphenyl-acetamido) fluorene (125I-pHP-AAF) was perorally administered to mice. These mice excreted more total and antibody-bound radiotracer in feces compared with controls given a similar mixture containing nonspecific antibody. In addition, urinary radiotracer excretion was reduced by 96% in specific-antibody dosed mice, indicating reduced gastrointestinal absorption of 125I-pHP-AAF. Reduced radiotracer absorption was also reflected by a 56% reduction in radiotracer content in tissues from mice receiving specific antibody. Other mice received peroral IgA before i.p. injection of 125I-PH-AAF. Specific antibody treatment consistently increased intraluminal radiotracer sequestration, as indicated by the level of total and antibody-bound radiotracer partitioning to aqueous fecal extracts. Similarly, when a mixture of 125I-pHP-AAF and IgG were injected directly into the small intestine, more radioactivity appeared in the feces of mice given specific antibody. High-performance liquid chromatography analysis of aqueous fecal extracts indicated that the majority of fecal radiotracer from specific-antibody dosed mice was unmetabolized parent compound. Thus, peroral administration of AAF-specific antibodies mixed with 125I-pHP-AAF decreased gastrointestinal absorption and increased fecal excretion of the radiotracer, suggesting a novel mechanism for protection against environmental carcinogens.

Mucosal and systemic antibody responses to a C4/V3 construct following DNA vaccination of rabbits via the Peyer's patch.

A plasmid encoding T1-SP10MN(A), a peptide derived from immunodominant regions of human immunodeficiency virus type 1 gp120, was delivered to rabbit Peyer's patches using a helium-driven gene gun. Six weeks thereafter, 2 of 5 animals were given an intradermal booster immunization. Blood, feces, and vaginal washes were collected weekly and assayed by ELISA. High titer T1-SP10MN(A)-specific fecal and vaginal secretory IgA responses were observed, and the response appeared to be augmented following dermal booster immunizations. Specific serum IgG was also detected within 1 week of immunization and remained elevated through week 20 in the 2 animals receiving dermal boosts (titers > or = 6400). This study establishes the Peyer's patch as a promising target tissue for DNA vaccination and demonstrates the efficacy of gene gun-mediated delivery of foreign DNA to a mucosal tissue for the induction of an immune response.

Local and systemic tolerance to orally administered dinitrochlorobenzene is not broken by cholera toxin.

BACKGROUND: It is widely acknowledged that oral administration of many antigens induces antigen-specific systemic tolerance. In this study we tested the hypothesis that oral administration of a low dose of dinitrochlorobenzene (DNCB) could induce local tolerance in the absence of systemic tolerance. We also hypothesized that the mucosal adjuvant cholera toxin (CT), which prevents the induction of local and systemic tolerance to coadministered antigens, would be unable to break an established tolerance to an orally administered antigen. METHODS: Tolerance was induced in BALB/c mice by oral administration of either a high (5.0 mg) or a low (0.05 mg) oral dose of the contact-sensitizing agent DNCB. This pretreatment was followed by parenteral or oral administration of dinitrophenyl (DNP)-carrier protein conjugates. RESULTS: As anticipated, the high DNCB dose induced systemic tolerance, as evidenced by depressed delayed type hypersensitivity responses to DNCB and reduced serum IgG anti-DNP responses. Oral pretreatment with the high dose of DNCB also induced local tolerance, as indicated by reduced fecal IgA and IgG anti-DNP responses. Conversely, oral pretreatment with the low dose of DNCB induced only local, not systemic tolerance. In addition, CT was incapable of breaking the preexisting tolerance induced by oral DNCB pretreatment. CONCLUSION: This study and others support the notion that the mucosal arm of the immune system is more sensitive to the induction of tolerance than the systemic arm, and that CT may not be an effective adjuvant for antigens to which the mucosal immune system has previously been exposed.

Mucosal immune response to an HIV C4/V3 peptide following nasal or intestinal immunization of rabbits.

The HIV env-encoded synthetic peptide T1-SP10MN(A) contains immunodominant epitopes of the C4/V3 regions of gp120. The mucosal immunogenicity of this peptide in various vaccine preparations was first tested in rabbits using chronically isolated Thiry-Vella (T-V) ileal loops. Intestinal and serum samples collected from rabbits immunized via T-V loops demonstrated secretory IgA (S-IgA) and IgG anti-T1-SP10MN(A), respectively, when assayed by ELISA. Intranasal delivery of the peptide supplemented with cholera toxin (CT) resulted in serum IgG and S-IgA anti-T1-SP10MN(A) in vaginal and nasal secretions. This study further demonstrates the utility of rabbits as a convenient animal model for HIV vaccine research and the relationship between nasal immunization and vaginal immunity.

Mucosal unresponsiveness to aflatoxin B1 is not broken by cholera toxin.

Rabbits immunized via chronically isolated ileal loops with aflatoxin B1 (AFB) conjugated to porcine thyroglobulin (TG) mixed with the mucosal adjuvant cholera toxin (CT) produced very small mucosal antibody responses to AFB. Strong mucosal and systemic antibody responses to CT and TG were generated by this immunization protocol, suggesting that the observed unresponsiveness was specific to AFB. Parenteral immunization with AFB-TG produced strong serum IgG anti-AFB responses, indicating that the conjugate preparation was immunogenic and that the rabbits possess the requisite systemic B and T cell repertoires to recognize and respond to AFB. This mucosal unresponsiveness was distinct from oral tolerance, as animals immunized mucosally with AFB-TG mixed with CT produced vigorous serum IgG anti-AFB responses upon subsequent parenteral immunization with AFB-TG. In vitro mitogen stimulation of lymphocytes isolated from Peyer's patches and mesenteric lymph nodes of unimmunized rabbits revealed the presence of AFB-specific B cells at levels comparable with these found in the spleen. These observations indicate that unresponsiveness to AFB is hapten-specific, restricted to the mucosa, and refractory to the adjuvancy of CI.

Immunoprophylactic intervention in chemical toxicity and carcinogenicity.

Preventing or diminishing the effects of environmental carcinogens and toxicants using immunoprophylactic vaccines has been tested by various investigators for the last 60 y. This approach has not gained widespread interest in scientific or medical communities due to ambiguous results. However, advances in vaccine design and immunization protocols now offer a reliable mechanism for the induction of mucosal immunity which may afford protection from these agents at their site of absorption. Previous studies aimed at immunoprophylactic vaccination against carcinogens and toxicants are reviewed, and future directions for this area of research are discussed.

A simplified procedure for studies of intestinal immunity in rabbits.

As interest in the development of oral vaccines continues to rise, alternative animal models for studies of mucosal immunity are needed. The present study examines a simplified procedure for delivering antigen to rabbit Peyer's patches via an indwelling cannula. The cannula was placed 3-4 cm proximal to the Peyer's patch, and was used to deliver four weekly doses of the potent mucosal immunogen, cholera toxin (CT). Anti-CT specific fecal secretory IgA (S-IgA), serum IgG and serum IgA were found in essentially equal amounts in rabbits with cannulas and in rabbits fitted with Thiry-Vella (T-V) isolated ileal loops. In contrast to animals with T-V loops, the intestinal flora of animals with cannulas contained less bacterial overgrowth with Pseudomonas sp. Further, the villus architecture remained histologically normal in appearance and there were fewer post-surgical complications associated with this technique than with T-V loops. This simplified technique should allow wider use of rabbits in studies of mucosal immunity.

Selective induction of mucosal immune responses to 2-acetylaminofluorene.

Mucosal vaccination with chemical carcinogens coupled to enterotoxins such as cholera toxin (CT) can elicit carcinogen-specific immunoglobulin secretion into the intestinal lumen. The present study examines the ability of several related bacterial enterotoxins and their subunits to act as adjuvants or carrier proteins in stimulating an intestinal secretory IgA (S-IgA) response to 2-acetylaminofluorene (AAF). Using Thiry-Vella loops in rabbits, CT, cholera toxin B subunit (CTB) and the recombinant B subunit of the heat labile enterotoxin from E. coli (rLTB) were all found to be effective carrier proteins and adjuvants for eliciting S-IgA anti-AAF. However, marked differences in the ratio of mucosal S-IgA to serum IgG production were observed. CT elicited the highest luminal S-IgA anti-AAF titers as well as the highest ratio of intestinal S-IgA/serum IgG when used as an adjuvant. Conversely, rLTB elicited a high serum IgG anti-AAF titer but only a modest intestinal S-IgA response. Dialysis studies using monoclonal IgA versus IgG anti-AAF on opposing sides of a semipermeable membrane demonstrated the potential importance of the intestinal S-IgA/serum IgG ratio. A high "intestinal" IgA/"serum" IgG ratio abolished carcinogen transfer to the "serum" side of the membrane, while a low ratio enhanced transfer. Thus, to generate an active mucosal immune response capable of blocking carcinogen absorption, the carrier protein or adjuvant should be selected to optimize the intestinal S-IgA/serum IgG ratio.

Characterization of the mucosal immune response to 2-acetylaminofluorene-protein conjugates.

Many environmental carcinogens gain access to the body only after traversing a mucosal surface. Due to their small size, most carcinogens are not recognized by the immune system and pass unhindered from the external to the internal environment. In previous studies, we demonstrated that secretory IgA directed against the carcinogen 2-acetylaminofluorene (AAF) can be elicited by covalently coupling AAF to the mucosal immunogen cholera toxin (CT). Rabbit intestines receiving secretions containing secretory IgA anti-AAF demonstrated a marked reduction in transmucosal absorption of carcinogen from the intestinal lumen to the mesenteric blood supply. In actively immune animals, however, recent data suggests that the disposition of luminal carcinogen may be influenced by the relative abundance of serum versus mucosally-based immunoglobulins. Our objective was to quantify the amount and isotype distribution of antibodies produced in response to AAF-carrier protein conjugates administered via different routes; using traditional parenteral carrier proteins and routes of administration, compared to mucosal carrier proteins and routes of administration. Administration of AAF-cholera toxin conjugates to isolated ileal (Thiry-Vella) loops in rabbits elicited a vigorous sIgA anti-AAF response in ileal secretions, with low levels of serum or intestinal IgG, or serum-based IgA produced concomitantly. All parenteral immunization protocols generated extremely high titers of serum IgG anti-AAF, with only moderate levels of sIgA produced concomitantly, even when mucosal boosting followed parenteral priming. When AAF-CT mucosal boosts were administered after intraperitoneal priming, a dramatic rise in serum, not secretory IgA was observed.