RESUMO
The costimulatory signal provided to T cells through CTLA-4-ligand interactions is required for T cell activation resulting in increased interleukin 2 (IL-2) production in vitro, but its role in the production of IL-4 and other cytokines is unclear and few in vivo studies have been performed to confirm results of in vitro experiments. We have examined the in vivo effects of blocking CTLA-4 ligands on the T helper cell 2 (Th2)-associated mucosal immune response that follows oral infection of mice with the nematode parasite, Heligmosomoides polygyrus. CTLA-4Ig administration inhibited H. polygyrus-induced increases in mesenteric lymph node (MLN) B cell major histocompatibility complex class II expression and size and T cell-derived IL-4 gene expression. In addition, CTLA-4 immunoglobulin (Ig) partially blocked increased IL-3, IL-5, and IL-9 cytokine gene expression in Peyer's patch (PP) and MLN 8 d after primary inoculation of mice with the parasite. Increases in the number of IL-4- but not IL-5-secreting cells were also inhibited by CTLA-4Ig. H. polygyrus-induced elevations in serum IgE levels but not blood eosinophils, were markedly inhibited by CTLA-4Ig. These results suggest that stimulation of CD28 and/or CTLA-4 is required for T cell priming leading to IL-4 cytokine production, B cell activation, and IgE secretion during a Th2-like, mucosal immune response to a nematode parasite.
Assuntos
Antígenos de Diferenciação/imunologia , Heligmosomatoidea/imunologia , Imunoconjugados , Interleucina-4/biossíntese , Enteropatias Parasitárias/imunologia , Infecções por Strongylida/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Abatacepte , Animais , Antígenos CD , Antígenos de Diferenciação/genética , Linfócitos B/imunologia , Antígeno CTLA-4 , Feminino , Interleucina-4/antagonistas & inibidores , Interleucina-4/genética , Interleucina-4/imunologia , Enteropatias Parasitárias/parasitologia , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais , Infecções por Strongylida/parasitologiaRESUMO
The cytokine interleukin (IL) 12 stimulates T cell and natural killer cell production of interferon (IFN) gamma and inhibits T cell production of IL-4. We investigated the effects of IL-12 on cytokine gene expression, immunoglobulin (Ig)E, mucosal mast cell, and eosinophil responses, and the course of infection in mice inoculated with the nematode parasite Nippostrongylus brasiliensis, as well as the IFN-gamma dependence of these effects. IL-12 stimulated IFN-gamma and IL-10 gene expression during primary and secondary N. brasiliensis infections and inhibited IL-3, IL-4, IL-5, and IL-9 gene expression during primary infections but had little inhibitory effect during secondary infections. IL-12 inhibited IgE, mucosal mast cell, and blood and tissue eosinophil responses during primary infections, but only eosinophil responses during secondary infections. IL-12 enhanced adult worm survival and egg production during primary, but not secondary infections. IL-12 needed to be administered by day 4 of a primary infection to inhibit IgE and mucosal mast cell responses, and by day 6 to strongly inhibit eosinophil responses and to enhance worm survival and fecundity. Anti-IFN-gamma mAb inhibited the effects of IL-12 on IgE secretion, intestinal mucosal mastocytosis, and parasite survival and fecundity, but did not affect IL-12 inhibition of eosinophilia. These observations indicate that IL-12, if administered during the initiation of eosinophilia. These observations indicate that IL-12, if administered during the initiation of an immune response, can change the response from one that is characterized by the production of T helper (Th)2-associated cytokines to one characterized by the production of Th-1 associated cytokines. However, IL-12 treatment has less of an effect once the production of Th2-associated cytokines has become established. In addition, our results provide evidence that Th2-associated responses protect against, and/or Th1-associated responses exacerbate, nematode infections.
Assuntos
Interleucinas/imunologia , Enteropatias Parasitárias/imunologia , Nippostrongylus/imunologia , Infecções por Strongylida/imunologia , Animais , Citocinas/imunologia , Eosinófilos/imunologia , Feminino , Imunoglobulina E/imunologia , Interferon gama/imunologia , Interleucina-12 , Enteropatias Parasitárias/parasitologia , Enteropatias Parasitárias/prevenção & controle , Intestinos/parasitologia , Células Matadoras Naturais/imunologia , Mastócitos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Infecções por Strongylida/parasitologia , Infecções por Strongylida/prevenção & controleRESUMO
North American genotypes of Trichinella spiralis (T-1), Trichinella nativa (T-2), Trichinella pseudospiralis (T-4), Trichinella murrelli (T-5), and Trichinella T-6 were examined for susceptibility to freezing in pork using time-temperature combinations that have been proven to inactivate T. spiralis. Infections were established in 3-month-old pigs of mixed sex and breed by oral inoculation of 10,000 muscle larvae (ML) (all genotypes, rodent-derived ML), 20,000 ML (T-1, T-4, and T-5; cat-derived ML), or 30,000 ML (T-2 and T-6; cat-derived ML). Pigs were euthanized 60 days postinoculation. Muscles from the tongue, masseter muscles, diaphragm, triceps, hams, neck, rump, and loins were ground, pooled, and mixed to ensure even distribution of larvae. Samples (20 g) containing each Trichinella species, genotype, and source combination were placed in heat-sealable pouches, transferred to a constant temperature refrigerant bath, and maintained according to defined time and temperature combinations. Larvae recovered from cold-treated pork samples were inoculated into mice to determine infectivity. Results indicated that the time-temperature combinations known to render pork safe for T. spiralis are sufficient to inactivate T. nativa and T-6 (the freeze-resistant isolates), T. murrelli (the most common sylvatic species in the United States excluding Alaska), and T. pseudospiralis (a species that lacks a muscle nurse cell). These data close a gap in knowledge about the effectiveness of freezing for inactivating these parasites in pork and should alleviate concern about the safety of frozen pork products from the United States.
Assuntos
Congelamento , Genótipo , Carne/parasitologia , Trichinella/classificação , Trichinella/genética , Animais , Doenças do Gato/parasitologia , Gatos , Conservação de Alimentos , Camundongos , América do Norte , Suínos , Doenças dos Suínos/parasitologia , Triquinelose/parasitologia , Triquinelose/veterináriaRESUMO
Among the meat sources of Toxoplasma gondii, pork is considered important in the epidemiology of toxoplasmosis in the USA. How soon after infection T. gondii forms tissue cysts in pork is unknown. In the present study, eight serologically negative Ë3 months old pigs were fed mouse tissues infected with VEG (Type III) strain of T. gondii and euthanized 7 (4 pigs) and 14 days (4 pigs) post-inoculation (p.i.). Meat from the right shoulder of each pig was bioassayed in mice for T. gondii tissue cysts by peptic digestion. From each pig, the shoulder muscle was cut at random spots into 5 g, 10 g and 50 g portions. Extreme care was taken to use different scalpels and forceps to minimize cross contamination among 17 samples (6 replicates of each 5 g and 10 g portions and 5 replicates of 50 g). From the four pigs euthanized at 7 days p.i., a composite of Ë200 g of leftover meat from each shoulder was bioassayed in cats and their feces were tested for oocyst excretion. All eight pigs developed T. gondii antibodies (modified agglutination test, MAT, 1: 80 or higher) and viable T. gondii was isolated from shoulder meat of each pig. All four cats fed pork from excreted T. gondii oocysts. The density of T. gondii, based on mouse infectivity, varied within 5-50 g samples each pig, and between pigs within the same group, day 7 versus day 14 p.i. There were no significant differences in mouse bioassay results obtained with day 7 versus day 14 infected pigs. Overall, the rate of isolation of T. gondii increased with sample size of meat bioassayed. Results demonstrate that tissue cysts are formed early in infection and they are unevenly distributed.
Assuntos
Anticorpos Antiprotozoários/sangue , Doenças dos Suínos/patologia , Toxoplasma/fisiologia , Toxoplasmose Animal/patologia , Animais , Gatos , Fezes/parasitologia , Feminino , Masculino , Camundongos , Músculo Esquelético/parasitologia , Oocistos , Carne Vermelha/parasitologia , Ombro/parasitologia , Suínos , Doenças dos Suínos/parasitologia , Toxoplasmose Animal/parasitologiaRESUMO
The production of safe and healthy food products represents one of the main objectives of the food industry. The presence of microorganisms in meat and products containing meat can result in a range of human health problems, as well as economic losses to producers of these products. However, contaminated meat products continue to initiate serious and large-scale outbreaks of disease in consumers. In addition to outbreaks of diseases caused by bacteria and viruses, parasitic organisms, such as Toxoplasma gondii, are responsible for foodborne infections worldwide, and in the case of T. gondii, is considered the 2nd leading cause of death from foodborne illness in the U.S. Transmission of Toxoplasma gondii has historically been linked to the consumption of raw or undercooked meat products, including pork. Specific concerns with respect to pork products are ready-to-eat (RTE) pork meals. These are pork or products containing pork that are prepared by curing or drying, and are not intended to be cooked before being consumed. Previous studies have demonstrated that T. gondii is inactivated during dry cured sausage preparation, apparently in the batter during fermentation. In this study, we have analyzed timing of inactivation of T. gondii in freshly prepared pepperoni batter to confirm our previous findings, to determine how quickly inactivation occurs during fermentation, and to confirm what parameters of the sausage preparation are involved in inactivation of the parasite. Results from the current and previous study indicate that rapid inactivation of T. gondii bradyzoites occurs in low salt batter for dry cured sausage within 4â¯h of initiation of fermentation.
RESUMO
Curing processes for pork meat in the U.S. currently require individual validation of methods to demonstrate inactivation of Trichinella spiralis, a nematode parasite historically associated with pork. However, for protozoan parasites, no such strictures exist. It has been assumed, with little evidence, that curing processes required to inactivate Trichinella also inactivate Toxoplasma gondii. Currently no model of meat chemistry exists that can be correlated with inactivation of T. gondii. Given the possibility of the presence of T. gondii in pork meat, and the frequent use of pork for ready-to-eat (RTE) products not intended to be cooked, curing methods which inactivate T. gondii early in the curing process would be of great value to producers. In this study, we tested the effect of five variables - salt/brine concentration, water activity (aw), pH, temperature, and time on inactivation of T. gondii bradyzoites in tissue cysts using low and high endpoints for common curing treatments during preparation of dry cured pork sausage. Survival of T. gondii bradyzoites at each stage of preparation was assessed using a mouse bioassay. Results indicated that encysted T. gondii bradyzoites do not survive the early stages of the dry curing process within the endpoint parameters tested here, even at levels of NaCl that are lower than typically used for dry curing (1.3%). Exposure of T. gondii encysted bradyzoites to curing components in the formulated batter resulted in rapid inactivation of bradyzoites. These data suggest that the use of dry curing components may be effective for controlling T. gondii potentially transmitted through RTE meats, rendering them safe from risk with respect to T. gondii transmission to human consumers.
RESUMO
Helminth infections induce the production of type 2 cytokines, which contribute both to expulsion of the worms and inflammatory responses that can either protect or damage the host. Although IL-4 has been considered the most critical cytokine for both inflammation and protective immunity, recent observations indicate that IL-13 - a related cytokine - can have equal or even greater importance than IL-4 in inflammatory responses and host protection against infection.
Assuntos
Inflamação/etiologia , Interleucina-13/fisiologia , Infecções por Nematoides/imunologia , Animais , Sistema Digestório/parasitologia , Humanos , Interleucina-4/fisiologia , Linfócitos T/imunologiaRESUMO
Signaling through the T cell receptor must be accompanied by costimulatory signals for the differentiation of naive T cells to cytokine-producing effector T helper cells. The costimulatory signal through CD28 is required for T cell activation resulting in increased interleukin (IL)-2 production in vitro, but its role in the production of IL-4 and in the in vivo response is still unclear. We have examined the effects of blocking CTLA-4 (the CD28 homologue) ligand interactions on the in vivo development of IL-4-producing T helper effector cells during a primary mucosal immune response to the nematode parasite Heligmosomoides polygyrus and during a primary systemic immune response to immunogenic anti-IgD antibodies. Our results demonstrate that CD28 and/or CTLA-4 signaling is required for T cell priming leading to IL-4 cytokine production, B cell activation, and IgE secretion during both immune responses, suggesting that other signaling molecules do not substitute for these molecules in either of these two different immune responses. Furthermore, the CD28 ligands, B7-1 and B7-2, can substitute for each other in providing the required T cell costimulatory ligand interactions during the primary immune response to H. polygyrus. In contrast, memory T cells during the challenge immune response do not require CD28/CTLA-4 ligand interactions for IL-4 production and T helper effector function.
Assuntos
Antígenos CD/imunologia , Antígeno B7-1/imunologia , Linfócitos T CD4-Positivos/imunologia , Heligmosomatoidea/imunologia , Imunoconjugados , Interleucina-4/biossíntese , Ativação Linfocitária , Glicoproteínas de Membrana/imunologia , Abatacepte , Animais , Antígenos de Diferenciação/imunologia , Antígeno B7-2 , Antígenos CD28/metabolismo , Antígeno CTLA-4 , Diferenciação Celular , Imunoglobulina D/imunologia , Interleucina-4/imunologia , Camundongos , Modelos Imunológicos , Transdução de SinaisRESUMO
Multiple pathways may be involved in the development of interleukin 4 (IL-4) producing T helper (Th) cells and the associated type 2 immune response. Increasing evidence suggests that the strength of signals delivered to the T cell may favor the development of the type 2 response. In contrast, antigen-presenting cell- (APC) derived stimuli produced following pattern recognition receptor binding during the innate response promotes the development of interferon-gamma (IFN-gamma) producing cells and the associated type 1 immune response. In many cases, the balance between increased signaling strength and the innate response may determine whether the type 2 response develops. T cell receptor (TCR), CD4, and costimulatory molecule interactions may all contribute to signal strength, but the type 2 immune response may be particularly dependent on the availability of coreceptor and costimulatory molecule interactions. B7 ligand interactions are required for the development of the type 2 immune response and interaction of CD28 with either B7-1 or B7-2 can provide sufficient signals for its initiation. In B7-2-deficient mice, the initial type 2 immune response is intact, but the response is not sustained, suggesting that B7-2 is important at later stages of the type 2 immune response. The roles of CD28 and CTLA-4 during the type 2 response remain unclear. The type 2 response to infectious pathogens is pronounced in CD28-/- mice, suggesting that other costimulatory molecule interactions can substitute for CD28 for the development of IL-4 producing T cells and the associated type 2 immune response.
Assuntos
Antígeno B7-1/metabolismo , Antígenos CD28/metabolismo , Imunoconjugados , Interleucina-4/biossíntese , Células Th2/imunologia , Abatacepte , Animais , Antígenos CD , Antígenos de Diferenciação/metabolismo , Antígeno B7-1/genética , Antígenos CD28/genética , Antígeno CTLA-4 , Camundongos , Camundongos Mutantes , Transdução de Sinais , Infecções por Strongylida/imunologiaRESUMO
Sulfo-NHS-biotin (aqueous soluble) and NHS-biotin (organic soluble) labeled similar SDS-2ME (sodium dodecyl sulfate/beta-mercaptoethanol) soluble cuticular proteins of second stage larvae (L2) and third stage Ascaris suum larvae (L3). Comparable analysis of biotin-labeled fourth stage larvae (L4), young adults, and mature adult Ascaris suum revealed strong labeling of several SDS-2ME soluble cuticular proteins with NHS-biotin, while sulfo-NHS-biotin appeared to strongly label a single SDS-2ME soluble cuticular protein. Both biotin probes labeled only cuticular proteins, since no evidence of internal labeling was observed in any developmental stage examined by either electroblot analysis or by electron microscopy. Our data suggest a greater cuticular permeability to the organic soluble biotin reagent in the later developmental stages (greater than L3) of A. suum than to the aqueous soluble biotin reagent, and may indicate the presence of a hydrophobic barrier in the cuticle of the later stages of the parasite.
Assuntos
Ascaris/crescimento & desenvolvimento , Biotina/análogos & derivados , Proteínas de Helminto/análise , Succinimidas , Animais , Ascaris/análise , Ascaris/ultraestrutura , Eletroforese em Gel de Poliacrilamida , Feminino , Microscopia Eletrônica , SuínosRESUMO
The cuticles from distinct developmental stages of Ascaris suum were isolated by a combination of mechanical disruption and detergent treatment of larvae or by surgical removal of cuticle from adults. Proteins from the isolated cuticles were solubilized with SDS and 2-mercaptoethanol (2ME) and analyzed by PAGE. Cuticular proteins from the third and fourth larval stages (L3 and L4) were comparable to adult, but differences in the number of bands were observed. The soluble proteins from the adult, L3 and L4 were readily degraded by bacterial collagenase, suggesting that these proteins are collagen-like structural elements of the cuticle. The soluble proteins from the L2 differed from the adult and other larval stages in both the number and molecular weight of protein bands and their lack of collagenase sensitivity. Antibodies made against the soluble cuticular proteins reacted with the medial and basal layers of the cuticle but not the external cortical or epicuticular regions. A significant amount of the cuticle was not solubilized by 2ME and was not digested by bacterial collagenase. These insoluble cuticular proteins were probably derived from the epicuticular and external cortical regions of the cuticle. Different developmental stages of A. suum were biotinylated and examined by electron microscopy. An organic soluble biotin reagent labeled all stages in a transcuticular pattern, while an aqueous soluble biotin labeled only the external cortical and epicuticular regions of the L4 and adult cuticle. These data indicate the presence of a hydrophobic barrier in the cuticle of later stages of the parasite.
Assuntos
Ascaris/crescimento & desenvolvimento , Animais , Antígenos de Helmintos , Ascaris/análise , Ascaris/imunologia , Biotina , Colágeno/isolamento & purificação , Larva/análise , Sondas Moleculares , Proteínas/imunologia , Proteínas/isolamento & purificação , SolubilidadeRESUMO
Mucohemorrhagic enteritis syndrome in swine has a complex etiology with largely unknown pathogenesis. We have observed that inoculation of pigs with swine whipworm, Trichuris suis, initiates an interaction with resident bacterial flora to induce mucohemorrhagic enteritis. The role of bacteria in this mixed infection was demonstrated using 4 treatment groups. One group of pigs was inoculated with 2500 embryonated T. suis eggs alone, while a second group received T. suis eggs along with broad spectrum antibiotic treatment. Two other control groups of pigs were uninoculated and were either treated with antibiotic or untreated. Pigs inoculated with T. suis eggs exhibited diarrhea, mucosal edema, inflammatory cell infiltration, bacterial accumulation at the site of worm attachment in the proximal colon, and intestinal adenomatosis associated with the intracellular Ileal symbiont intracellularis bacteria. In addition, enlarged lymphoglandular complexes (LGCs) containing numerous extracellular bacteria, eosinophils, lymphocytes, macrophages, and neutrophils were observed in the distal colon. The other group of pigs that was inoculated with T. suis but treated with antibiotics had lesions localized to the site of worm attachment and histologically normal LGCs with no invasive bacteria in the distal colon. The groups of uninoculated pigs, with or without antibiotic treatment, exhibited no pathology or bacterial invasion. It appears that the complex pathogenesis of necrotic proliferative colitis in pigs may be linked to worm induced suppression of mucosal immunity to resident bacteria. Further, the association between bacteria,lymphocytes and macrophages in the LGCs of pigs infected with T. suis suggests an antigen-processing role for these structures in the colon. Further, the complex pathogenesis of necrotic proliferative colitis in pigs may be linked to worm induced suppression of mucosal immunity to resident bacteria.
Assuntos
Colite/veterinária , Doenças dos Suínos/etiologia , Tricuríase/veterinária , Trichuris/patogenicidade , Animais , Bactérias/imunologia , Colite/etiologia , Colite/imunologia , Colo/imunologia , Colo/microbiologia , Colo/patologia , Feminino , Tolerância Imunológica , Imunidade nas Mucosas , Infecções Oportunistas/etiologia , Infecções Oportunistas/imunologia , Infecções Oportunistas/veterinária , Suínos , Doenças dos Suínos/imunologia , Doenças dos Suínos/microbiologia , Tricuríase/etiologia , Tricuríase/imunologiaRESUMO
Pigs fed Ascaris suum eggs attenuated by short wave ultraviolet-radiation developed resistance to challenge infection. Per os inoculation of pigs on three successive weeks with 10,000 eggs irradiated to total exposures of 150,100 and 75 microW-min/cm2, respectively, resulted in an 88% reduction in the number of larvae recovered from the lungs 7 days after challenge with 10,000 infective eggs. Peripheral blood lymphocytes (PBL) from vaccinated pigs were specifically stimulated in vitro to incorporate tritiated thymidine by egg hatching fluid (Ea) and by excretory-secretory products obtained from cultures and from cultures of third-stage larvae developing to fourth-stage (L3-4). PBL were also specifically stimulated by living L2. Ea and L2 stimulated pig PBL significantly at 7 days after the first inoculation; responses to L2-3 and L3-4 developed 7 days after a second inoculation. The antigen-responsive cells in the PBL population were non-immunoglobulin-bearing lymphocytes. Antibodies to Ea and L2-3 were not detected in the serum of vaccinated pigs, and only 3 of 7 pigs had low concentrations of serum antibodies to L3-4.
Assuntos
Ascaris/imunologia , Óvulo/efeitos da radiação , Raios Ultravioleta , Animais , Antígenos/administração & dosagem , Feminino , Suínos/imunologia , Vacinação , Vacinas AtenuadasRESUMO
The radiosensitivity of in vitro proliferative responses of porcine peripheral blood lymphocytes (PBL) was assessed. PBL were stimulated by Con-A, PHA, culture supernates from mitogen-stimulated porcine lymphocytes, or in the case of antigen-primed swine, specific antigens. The resulting levels of proliferation were assessed by a determination of the level of incorporation of tritiated thymidine in vitro, and in some cases by the presence of blast cells in the cultures. Porcine PBL were found to be more radioresistant than either mouse PBL or mouse spleen cells. Irradiation levels of greater than 3000 rads were necessary to arrest Con-A or PHA-induced proliferative responses. Proliferation induced by lymphokines in the form of supernates from mitogen-stimulated lymphocyte cultures was arrested in PBL that had received 3000 rads prior to culture. Antigen-induced proliferative responses in primed porcine PBL populations were the most radiosensitive, in that a previous irradiation with 500 rads was sufficient to completely abolish a secondary in vitro proliferative response.
Assuntos
Ativação Linfocitária/efeitos da radiação , Linfócitos/imunologia , Suínos/imunologia , Animais , Antígenos/imunologia , Células Cultivadas , Concanavalina A/farmacologia , Relação Dose-Resposta a Droga , Relação Dose-Resposta à Radiação , Raios gama , Mitógenos/farmacologia , Fito-Hemaglutininas/farmacologiaRESUMO
Interleukin 2 (IL2) or T cell growth factor (TCGF) has been characterized in a number of species but not in porcines. Porcine IL2 was detected in supernates (SN) of cultures of pig lymphocytes by: 1) the stimulation of the IL2-sensitive murine T cell line, CT6; 2) a costimulator assay involving porcine thymocytes; and 3) by the in vitro maintenance of antigen or mitogen-induced porcine lymphoblastoid cells. Porcine IL2 production by pig lymphocytes was induced by the mitogens Concanavalin A (Con A) Phytohemagglutiniin (PHA), and Pokeweed mitogen (PWM), but not by lipopolysaccharide (LPS). IL2 activity was demonstrated in the SN of mitogen-stimulated lymphocyte cultures as early as 24 hr after initiation of culture, reached peak levels at 48 hr, and decreased by 72 hr. Mitogens induced IL2 secretion by pig peripheral blood mononuclear cells, lymph node cells, and spleen cells, but not thymus cells. The cells responsible for IL2 production are presumptive T cells because: 1) they are nylon wool non-adherent; and 2) are non-surface-Ig bearing. In contrast, SN from cultures of surface Ig-positive cells had minimal IL2 activity. Porcine IL2 resembles rat and human IL2 in that it has an apparent molecular weight of approximately 15,000, and does not bind to DEAE-cellulose (DE-52) ion exchange columns equilibrated in 0.05 M sodium phosphate buffer (pH 7.6).
Assuntos
Interleucina-2/biossíntese , Suínos/imunologia , Linfócitos T/metabolismo , Animais , Bovinos/imunologia , Linhagem Celular , Células Cultivadas , Humanos , Interleucina-2/isolamento & purificação , Cinética , Tecido Linfoide/efeitos dos fármacos , Camundongos , Mitógenos/farmacologia , Ratos/imunologia , Linfócitos T/efeitos dos fármacosRESUMO
The development of immunity to Ascaris suum was studied in pigs immunized with isolated cuticle fragments from A. suum second and third stage larvae (L2/L3) and adult worms, and compared with other methods that stimulate a strong protective response in pigs. A significant protective response was seen in animals immunized with isolated cuticle fragments from A. suum L2/L3 and adults, but it was less than that seen in animals inoculated with UV-irradiated eggs or naturally exposed to eggs on a dirt lot. Significant IgG responses to 2-mercaptoethanol (2ME)-soluble cuticle components were seen in all groups, but the level of the antibody response did not relate to protection. Group differences in antibody and lymphocyte blastogenic responses to cuticle proteins indicated quantitative and qualitative stage specific differences in 2ME-soluble and insoluble cuticular proteins. Intestinal immunity was notably absent from cuticle immunized pigs because a marked liver white spot response was observed following the challenge inoculation. Thus, cuticle fragments from larval and adult A. suum are capable of inducing a protective response to larval migration; however, the development of intestinal immunity is not a direct function of exposure to these antigens.
Assuntos
Ascaríase/veterinária , Ascaris suum/imunologia , Proteínas de Helminto/imunologia , Imunização/veterinária , Pele/imunologia , Doenças dos Suínos/prevenção & controle , Animais , Anticorpos Anti-Helmínticos/análise , Ascaríase/prevenção & controle , Ensaio de Imunoadsorção Enzimática/veterinária , Imunidade , Imunoglobulina G/análise , Mucosa Intestinal/imunologia , Fígado/parasitologia , Fígado/patologia , Pulmão/patologia , Ativação Linfocitária/imunologia , Contagem de Ovos de Parasitas/veterinária , SuínosRESUMO
Swine parasitism exerts a significant economic impact worldwide. In the United States, the greatest losses are due directly or indirectly to the costs of zoonotic parasitisms. Three of the six most common foodborne parasitic diseases of humans in the United States are associated with pork consumption. These include toxoplasmosis, taeniasis or cysticercosis (caused by the pork tapeworm Taenia solium), and trichinellosis. Toxoplasmosis is of particular concern because of the fulminating disease that occurs in immunocompromised people. Generalizations and extrapolations of information derived from rodent and human studies, to swine parasitisms, are complicated by immunological differences between the hosts, and by the diverse biological characteristics of internal and external parasites studied. Swine studies thus far reported have demonstrated that protective immunity to helminth infection involves both cellular and humoral mechanisms, with antibodies and antibody-mediated responses playing important roles in preventing establishment of newly acquired larvae. Protection against protozoan parasites is primarily by cell-mediated strategies, whereas protective immunity to arthropod infestation is primarily through humoral mechanisms, principally those associated with type 1 hypersensitivity.
Assuntos
Doenças Parasitárias em Animais , Doenças dos Suínos/imunologia , Animais , Imunidade , Imunidade Celular/imunologia , Doenças Parasitárias/imunologia , SuínosRESUMO
A competitive PCR assay (cPCR) was used to quantify swine cytokine responses to parasite infection. Internal standards (deleted cDNA competitor molecules [DcDNA mimics]) were produced and tested for swine interleukin-12 (IL-12), interleukin-10 (IL-10) and hypoxanthine phosphoribosyltransferase (HPRT) from PCR generated cDNA cloned in plasmid vectors. Deletion clones for the cDNA competitor molecules (DcDNA mimics) were generated for IL-10, IL-12 and HPRT by PCR in a single step and verified by (1) amplification of the expected smaller PCR product with the original primers, (2) appropriate fragment size released by restriction digestion of the deleted clone, and (3) correct sequence of the new DcDNA insert. DcDNA mimics were used to quantitate cytokine gene mRNA production during experimental and natural infections of swine with the gastrointestinal nematode parasite Trichuris suis. Mesenteric lymph node cells were collected from control and infected pigs at the time of maximal pathogenicity (35 days after infection) and snap frozen. After RNA extraction, samples were reverse transcribed (RT) to cDNA. cPCR was performed using the housekeeping gene HPRT DcDNA mimic and HPRT specific primers to insure RNA integrity and concentration. Cytokine cDNA content in these samples was then quantitated using cytokine mimics and gene specific primers. IL-10 gene expression in MLN draining the colon of pigs experimentally infected with T. suis increased 10-20 fold at day 35 compared to control pigs. IL-12 gene expression was not detectable in MLN of these pigs, but was detectable in MLN of pigs exposed naturally to T. suis on a contaminated dirt lot that also exhibited signs of secondary bacterial invasion. Swine IL-10 and IL-12 gene expression can be quantitated in local mesenteric tissues. This cPCR assay will enable scientists to quantitate cytokine gene expression in swine and determine the nature of immune responses to important infectious diseases.
Assuntos
DNA Complementar/química , Interleucina-10/genética , Interleucina-12/genética , Suínos/imunologia , Animais , Clonagem Molecular , Colo/imunologia , Colo/parasitologia , Primers do DNA/química , DNA Complementar/genética , Eletroforese em Gel de Ágar/veterinária , Regulação da Expressão Gênica , Hipoxantina Fosforribosiltransferase/química , Hipoxantina Fosforribosiltransferase/genética , Interleucina-10/análise , Interleucina-10/biossíntese , Interleucina-12/análise , Interleucina-12/biossíntese , Linfonodos/imunologia , Linfonodos/parasitologia , RNA Mensageiro/química , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Suínos/genética , Suínos/parasitologia , Doenças dos Suínos/imunologia , Tricuríase/imunologia , Tricuríase/veterinária , Trichuris/imunologiaRESUMO
Control of parasitic infections is dependent on the production of cytokines that activate mechanisms which limit invasion, reproduction or survival of the parasite. In contrast, conditions that induce inappropriate cytokine responses facilitate the spread of infection and ultimately exacerbate the level of disease. Measurement of local cytokine responses to different gastrointestinal parasites, such as the intracellular protozoan, Cryptosporidium parvum, and luminal dwelling nematodes like Nippostrongylus brasiliensis and Heligmosomoides polygyrus, reveal stereotype response patterns. In general, intracellular parasites stimulate type 1 responses where IFN-gamma is the predominant immune activator, while extracellular parasites stimulate type 2 responses where IL-4 plays a prominent role in elevating humoral immune mechanisms. Cytokines alter cellular function and the milieu of the intestinal lumen to affect the outcome of an infection. The importance of a particular response during the course of an infection can be studied by selective enhancement with an excess of exogenous recombinant cytokine or cytokine antagonists. For example, exogenous IL-12 enhances resistance to C.parvum, but suppresses the normally rapid cure of an infection with N. brasiliensis. Both mechanisms are dependent on expression of IFN-gamma. At the molecular level, exogenous IL-12 stimulates IFN-gamma production which elevates a protective type 1 response to C. parvum but converts the normally anti-worm type 2 response to a type 1 response that inappropriately regulates the infection. Alternatively, excess IL-4 plays a prominent role in modulating effector elements that change intestinal physiology to create a hostile environment for worm parasites. Exogenous IL-4 can cure chronic worm infection, while IL-4 antagonists interfere with protective responses to infection. These observations provide a paradigm for analysis of stereotype responses to different gastrointestinal parasites, and demonstrate how cytokine-induced immune system-dependent and independent effector mechanisms can limit parasitic infection, while inappropriate cytokine responses can exacerbate the state of disease.
Assuntos
Criptosporidiose/imunologia , Interferon gama/farmacologia , Interleucina-4/farmacologia , Intestinos/imunologia , Intestinos/parasitologia , Infecções por Strongylida/imunologia , Células Th1/efeitos dos fármacos , Células Th1/imunologia , Células Th2/efeitos dos fármacos , Células Th2/imunologia , Animais , Cryptosporidium parvum/imunologia , Intestinos/efeitos dos fármacos , Camundongos , Camundongos SCID , Nematospiroides dubius/imunologia , Nippostrongylus/imunologiaRESUMO
The cytokine interleukin-12 (IL-12) is a key molecule in the regulation of CD4 + T cell development and specifically potentiates T helper 1 responses in mouse and man. However, biological effects mediated by IL-12 have not been well defined in pigs. Herein, recombinant porcine IL-12 (rPoIL-12) was expressed in a swine poxvirus system as a biologically active heterodimer and used to stimulate bovine or swine lymphoblast cells. After 3 days of incubation, only bovine blasts were responsive to the rPoIL-12 treatment as monitored by cell proliferation in several independent trials. Similarly, i.m. administration of rPoIL-12 in the hind leg of 3-week-old pigs indicated a reduction in the number of interferon-gamma (IFN-gamma) producing lymphocytes isolated from inguinal lymph nodes. The porcine IL-12R beta2 (IL-12Rbeta2) sequence was cloned and results generated by reverse transcriptase polymerase chain reaction (RT-PCR) demonstrated that the expression of IL-12R on porcine blasts as measured by the relative levels of IL-12Rbeta2 mRNA was less than that in bovine blasts and are in agreement with the reduced proliferation response of swine blast cells to rPoIL-12 treatment. Real time PCR analysis demonstrated that after PBMC stimulation, bovine blasts had an 11-fold increase in IL-12Rbeta2 mRNA levels while porcine blasts had almost no change. These data support a mechanism for IL-12 stimulation in swine inconsistent with that observed in conventional models.