Effect of transcutaneous immunization with co-administered antigen and cholera toxin on systemic and mucosal antibody responses in sheep

Direct application of antigens to skin together with an adjuvant, a procedure called transcutaneous immunization (TCI), can induce systemic immune responses in mice, humans, cats and dogs. In previous studies we found that cholera toxin (CT) applied topically on unbroken skin induces systemic antibody and lymphocyte proliferative responses in sheep. The current study examined whether concurrent administration of CT and tetanus toxoid (TT) delivered transcutaneously could induce specific antibody responses to both antigens in sheep. Antibodies to both TT and CT were induced by TCI although antibody titres in serum to TT were higher in sheep receiving TT plus alum by intramuscular injection (n=5) than TT plus CT by TCI (n=5). The ratio of IgG1/IgG2 antibody to TT in serum was near unity, and the route of immunization, TCI versus injection, did not influence this ratio. In contrast, the ratio of IgG1/IgG2 antibody differed significantly between the two antigens, TT and CT, delivered by TCI, with a higher proportion of IgG1 antibody in serum to CT than TT. Antibody to TT was detected in lung washes from TCI and injection groups, with IgG1 predominating over IgG2 in both groups. IgA antibodies to CT and TT were detected in sera of CT and TT-immunized groups respectively but in lung washes IgA antibody to TT was detected only in the injection group. Results show that TCI induced systemic antibody responses to CT and the co-administered antigen TT, whereas no evidence was obtained for mucosal IgA responses following TCI.     

日粮蛋白质、赖氨酸、蛋氨酸及苏氯酸水平对早期断奶仔猪免疫机能的影响

本研究分两次试验进行 ,试验 1研究两个蛋白质水平 ( 1 8% ,2 0 % )及三个赖氨酸水平 ( 1 .0 % ,1 .3%和 1 .6 % )对机体免疫机能的影响 ;试验 2研究两个蛋氨酸水平 ( 0 .2 9% ,0 .39% )和三个苏氨酸水平( 0 .6 0 % ,0 .6 8%和 0 .76 % )对机体免疫机能的影响。试验日粮均为简单型日粮 ,试验动物为早期断奶仔猪 ,其日龄体重分别为 2 5± 3日、6 .78± 0 .0 8kg与 2 6± 1日龄、6 .6 8± 0 .0 4kg。测定不同蛋白质和氨基酸水平下 ,免疫器官重量、淋巴细胞数量及组成、血液IgG效价、注射PHA皮褶厚度变化及SRBC(绵羊红细胞 )免疫后抗体效价。结果表明 ,日粮不同蛋白质和赖氨酸水平对血清IgG效价无影响 ,在皮褶厚度的变化的影响上 ,两者交互作用极显著 (P <0 .0 1 )。苏氨酸及蛋氨酸的不同水平显著影响血液中IgG效价及抗SRBC抗体效价 (P <0 .0 5) ,机体免疫机能最佳时适宜的蛋白质、赖氨酸、蛋氨酸及苏氨酸水平分别为 1 8% ,1 .3% ,0 .39%及 0 .6 8%。     

A reagin-like antibody in horse serum: 1. Occurrence and some biological properties

The demonstration of a reagin-like antibody againstCulicoides pulicaris extract in the serum of horses and ponies affected with recurrent seasonal dermatitis (sweet itch) is reported. This antibody can confer Prauznitz-Küstner (P–K) sensitivity on homologous skin for up to 5 days and, like human IgE, is thermolabile and susceptible to 2-mercaptoethanol reduction. It is eluted on diethylaminoethyl dextran-52 anion exchange chromatography independently of IgG, IgG(T) and IgM, and its elution characteristics indicate similarity in net molecular charge to human IgE.The P–K response observed in horse skin is biphasic, and is morphologically similar to the late cutaneous anaphylactic response in man. Both phases of the P–K response are dependent upon the reagin-like antibody, although other serum factors appear involved in the delayed phase of the response.     

A reagin-like antibody in horse serum II. Anti-human IgE induced reversed cutaneous anaphylaxis-like responses in horse skin

Fc specific anti-human IgE serum induced prolonged reversed cutaneous anaphylaxis (RCA)-like reactions in horse skin. Morphologically and histologically, these reactions resembled passively induced late cutaneous anaphylaxis responses in human skin, but differed from reversed passive Arthus responses induced in horse skin using anti-horse IgG serum. The induction of RCA-like responses in horse skin by anti-human IgE indicates shared Fc antigenic determinants on human IgE and a horse homocytotropic or reagin-like antibody.     

Hypersensitivity to dietary components in young farm animals: isolation and partial purification of bovine immunoglobulin E

An antibody of known specificity and active in long (72 hours) latent period passive cutaneous anaphylactic reactions, was isolated and partially purified from bovine serum. This antibody was not associated with immunoglobulins IgG, IgM or IgA. A rabbit antiserum raised against this antibody and used as an immunoabsorbent, successfully recovered skin sensitising antibody from bovine reaginic serum.     

Preliminary study using an indirect ELISA for the detection of serum antibodies to Alternaria in domestic cats

Alternaria is a saprophytic fungus that is widespread in the environment; it is an opportunistic pathogen and causes disease in human beings and domestic animals. Fungal spores gain entry to the host through skin lesions and cause slow-growing, soft to firm, subcutaneous swellings, either with or without ulcers. An indirect ELISA was developed for the detection of anti-Alternaria immunoglobulin G (IgG) antibodies in serum to determine the prevalence of Alternaria exposure in domestic cats. Fifty-two of 63 cats had detectable levels of anti-Alternaria IgG antibody. There were no correlations between the concentration of antibody and the sex, breed or living environment of the cats, but cats less than two years of age had significantly lower concentrations than older cats. The cats with disease caused by culture-confirmed Alternaria infections did not have significantly higher concentrations of antibody than the healthy cats or cats with other diseases.     

Prophylaxis of experimentally induced ovomucoid allergy in neonatal pigs using Lactococcus lactis

Probiotic Lactococcus lactis (LL) is immunomodulatory and may prevent allergy by biasing from type-2 to a type-1 immune response. We hypothesized that newborn pigs pre-treated orally with LL are protected against allergy to ovomucoid (Ovm). Pigs were assigned to two treatment groups. Piglets were pretreated orally on days of age 1-7, 10, 12, 14, 21, 28 and 35 with LL (n=30) or medium (control, n=32) and sensitized to Ovm by intraperitoneal injection together with cholera toxin on days 14, 21 and 35. Pigs were orally challenged with egg white (day 46) and assigned scores for allergic signs. Outcomes were measured as direct skin tests, serum antibody to Ovm [IgG (H+L); IgE; IgG(1) and IgG(2)] and cytokine production by mitogen-stimulated blood mononuclear cells (BMC). Clinical signs and skin test positivity were less frequent in the LL group (p ≤ 0.0001). Serum antibody associated with IgG (H and L), IgE, IgG(1) or IgG(2) was significantly increased on day 46 (post-sensitization) compared to day 14 (pre-sensitization) (p ≤ 0.0001). The LL-treated pigs had more IgE and IgG(2)-related antibody activity and lower IgG(1)/IgG(2) and IgE/IgG(2) ratios indicating a type-1 bias in immune response (p ≤ 0.05). Concentration of type-2 cytokines interleukin IL-4 and IL-10 were significantly lower in supernatants of stimulated BMC of LL-treated pigs (p ≤ 0.0001). Interferon-γ, TGF-β and IL-13 were not detected in control or treated animals. Thus, oral treatment of neonatal pigs with LL significantly reduced subsequent frequency of allergy to Ovm associated with reduced type-2 immune response correlates hence supporting the "hygiene hypothesis" and potential use of LL as a neonatal immunoregulator.     

Measurement of canine IgE using the alpha chain of the human high affinity IgE receptor

In vitro assays for allergen specific immunoglobulin E (IgE) are a convenient and reproducible alternative to intradermal skin testing in dogs. Such tests may be used to support a diagnosis of atopic dermatitis and to define appropriate allergens for immunotherapy. Current in vitro assays rely upon monoclonal or polyclonal antibodies as IgE detection reagents. However, in sera where allergen-specific IgG occurs in great excess, any IgE:IgG cross-reactivity of the detection reagent may result in lowered assay specificity. Therefore, we have developed an assay for canine IgE which uses a recombinant form of the extracellular part of the alpha chain of the human high affinity IgE receptor (FcvarepsilonRIalpha). Biotinylated FcvarepsilonRIalpha shows no significant binding to purified canine IgG, and recognizes a heat labile antibody in serum, with a detection limit of 73-146pg/ml. Comparison of assay signals using the labeled FcvarepsilonRIalpha and a highly specific anti-canine IgE monoclonal antibody (MAb) shows good agreement. The FcvarepsilonRIalpha is therefore a sensitive and specific alternative to polyclonal or monoclonal antibodies for canine serum IgE measurement.     

Immunologic responses of calves to aerosolized antigen: humoral response to ovalbumin

The humoral response of cattle to ovalbumin (OA), a nonenvironmental well-defined antigen, was studied. During 9 weeks of aerosolization, weekly serum and nasal secretion concentrations of immunoglobulin (Ig)G1, IgG2, IgM, IgA, and IgE were determined by enzyme-linked immunosorbent assay (ELISA) for OA specific antibody. Data from 3 calves given aerosol OA were compared and contrasted with data from 3 calves given aerosol saline solution and 1 calf given parenteral OA. The presence of cytotropic (skin sensitizing) antibody was evaluated during weeks 6 and 9 by direct skin testing with OA. A humoral response was induced in all 3 calves given aerosol OA. Serum IgG1 and IgG2 titers reached a maximum of 64,000 and 2,000, respectively, in calves given aerosol OA compared with 521,000 and 16,000, respectively, in the calf given parenteral OA. The ELISA did not detect an OA-specific IgM response. In contrast, all 3 calves given aerosol OA had serum IgA concentrations that increased to a peak by week 9. The mean IgA absorbance value for the 3 calves given aerosol OA was slightly greater than 5 times that of the calf given parenteral OA. Similarly, nasal secretions from calves given aerosolized OA had absorbance values that were 15-fold greater than that from the calf given parenteral OA. Calves given aerosol OA had antigen-specific IgE responses during weeks 6 to 8. The ELISA results were compared with results of passive cutaneous anaphylaxis tests. The presence of skin-sensitizing antibody was indicated by positive skin tests in the calves given aerosol OA and the calf given parenteral OA by week 9.     

Change of antibody levels to ferritin in the sera of foals after birth: Possible passive transfer of maternal anti‐ferritin autoantibody via colostrum and age‐related anti‐ferritin autoantibody production

Antibody (immunoglobulin G (IgG), IgM or IgA) levels relative to ferritin in six foal sera (three male and three female) after birth (day 0 and 2, 6, 10, 20, 28, 36, 40, 52 and 56 weeks of age) were semi‐quantitatively measured with normalization with antibody activity to ferritin in one adult horse serum. After addition of horse spleen ferritin to the serum sample, the complex formed between antibodies to ferritin in the serum and ferritin was co‐immunoprecipitated using antibody to horse spleen ferritin. Antibody classes of the co‐immnoprecipitate were detected with antibodies specific for horse IgG, IgM or IgA heavy chain. Six adult horse serum samples were found to have ferritin‐binding activities in all immunoglobulin classes examined. Although ferritin antibody activities (IgG, IgM and IgA) were scant in the foal sera before sucking colostrum (day 0), their activities increased at 2 weeks of age. IgG antibodies showed a biphasic response and IgM antibody activity increased up to 40 weeks of age. Antibody (IgG, IgM and IgA) activities to ferritin in three colostrum samples were significantly higher than in adult horse serum samples. These results demonstrate that antibody to ferritin in foal serum is derived from colostrum after birth and is produced thereafter.     

The effect of parenteral immunisation on antibody production in the pig colon

Local and systemic antibody production was studied in pigs to compare responses to live and killed bacterial antigen and purified protein antigen, with and without prior mucosal stimulation. Recovery from challenge with live bacteria and intramuscular injection with killed bacteria gave rise to similar high levels of serum IgG antibody, but the ratio of specific IgA to IgG in the colon was significantly higher after infection than following vaccination with killed bacteria. Vaccination with a protein antigen gave rise to serum and local antibody production. Prior feeding of the antigen had a tolerising effect on the serum antibody response, but production of IgG and IgA antibody by the colon was not suppressed.     

Bovine herpesvirus-1 infection of cattle: kinetics of antibody formation after intranasal exposure and abortion induced by the virus

The kinetics of antibody formation in Holstein heifers after primary and secondary intranasal inoculation of bovine herpesvirus-1 (BHV-1) and after BHV-1-induced abortion was determined. Sera were fractionated by gel filtration and ion-exchange chromatography. The antibody activity within serum immunoglobulin (Ig) isotypes was assessed, using a plaque-reduction neutralization assay. The primary immune response to BHV-1 infection was characterized by the appearance of IgM and IgG antibodies in serum by postinoculation day (PID) 7. Maximal IgG antibody activity occurred at PID 35 in nonpregnant heifers and at PID 14 in pregnant heifers. Thereafter, IgG antibody activity declined slowly in both groups of heifers. Maximal IgM antibody activity occurred at PID 14 in both groups of heifers and declined rapidly thereafter. The IgG antibody activity during primary immune responses was restricted to the IgG1 subclass. Secondary responses were characterized by anamnestic IgG antibody responses. Antibody activity was present within the IgG1 and IgG2 subclasses during secondary immune responses, but the increase in antibody activity during this period was primarily in the IgG2 subclass. Secondary IgM antibody formation was elicited by abortion induced by the intra-amniotic inoculation of BHV-1, but not by reexposure by the intranasal route. Abortion occurred in 1 heifer 28 days after intranasal BHV-1 inoculation. Abortion in this heifer was not associated with a secondary antibody response. The nature of BHV-1 antigenic exposure in the bovine determined the relative distribution of anti-BHV-1 antibody activity in serum IgM, IgG1, and IgG2. The formation of IgM antibody, with the exception of secondary intranasal exposure, indicated recent BHV-1 antigenic exposure.(ABSTRACT TRUNCATED AT 250 WORDS)     

A comparison of intradermal testing and detection of allergen-specific immunoglobulin E in serum by enzyme-linked immunosorbent assay in horses affected with skin hypersensitivity

Skin hypersensitivities (allergies) in horses are often diagnosed using clinical signs only. Intradermal testing or serological assays are diagnostic options to confirm the allergic nature of the disease and to identify the allergen(s). Our objective was to develop an allergen-specific enzyme-linked immunosorbent assay (ELISA) using a monoclonal antibody specific for horse IgE and to examine its potential for allergen detection in serum in comparison to intradermal testing. Intradermal testing with 61 allergen extracts was performed on 10 horses affected with skin hypersensitivity. Their sera were analyzed by ELISA for IgE antibodies to the same allergens. The kappa test of concordance was used for comparison of the results of both tests. Out of 61 allergen extracts, only two (Timothy and Quack) had kappa values greater than 0.60, suggesting a substantial agreement between skin testing and IgE ELISA. The statistical comparison of the remaining 59 allergens showed little or no concordance between the tests beyond chance. To identify parameters that may influence the sensitivity of the ELISA, the assay was modified to detect allergen-specific IgGb and IgG(T) in serum, and the protein content in all allergen extracts was determined by SDS-PAGE. The commercial allergen extracts revealed a high variation in detectable protein. High concentrations of allergen-specific IgG in horse serum were found to compete with IgE for binding to the plates. In conclusion, an ELISA using whole serum and crude allergen preparations provides limited diagnostic information in horses. The reliable diagnosis of allergens in equine skin hypersensitivity is essential to improve allergen-specific treatments, such as hyposensitization, or the development of allergy vaccines.     

Serum containing ovine IgG2 antibody specific for maedi visna virus envelope glycoprotein mediates antibody dependent cellular cytotoxicity

Antibody-dependent cell-mediated cytotoxicity (ADCC) specific for maedi visna virus (MVV) has never been described. The IgG antibody response to MVV is restricted to an IgG1 response whilst MVV specific IgG2 is never seen in persistently infected sheep. To determine whether the isotypic restriction of the antibody response is responsible for the lack of ADCC, an ADCC assay was developed using polyclonal serum raised to recombinant MVV ENV protein. Sheep immunised with a recombinant GST:SUenv fusion protein in complete Freund's adjuvant produced an antibody response which contained IgG1 and IgG2 antibodies. The activity of this serum in an ADCC assay was compared to serum from persistently infected sheep. Serum from immunised sheep mediated ADCC reactions whilst no activity was ever seen in persistently infected sheep serum. IgG2 may therefore be the possible effector isotype for ADCC reactions against MVV. Failure of the IgG2 dependent ADCC system in vivo may contribute to the persistence of MVV-infected macrophages in vivo.     

新生动物免疫球蛋白Fc受体的研究概况

影响新生动物死亡的一个关键原因是初乳中IgG的含量不足,使新生动物免疫力低下易感疾病而死亡。新生动物免疫球蛋白Fc受体(FcRn)是IgG在乳腺、肠道运输过程中的唯一受体,对初生动物吸收初乳中的IgG含量起关键作用。本文综述了FcRn的特点与生物学功能。     

Evaluation of systemic and secretory IgA concentrations and immunohistochemical stains for IgA-containing B cells in mucosal tissues of an Irish setter with selective IgA deficiency

Immunoglobulin A is the predominant secretory antibody at mucosal surfaces. In the dog, immunoglobulin A deficiency (IgAD) is characterized by low to absent serum IgA and normal to elevated serum immunoglobulin G (IgG) and immunoglobulin M (IgM) concentrations. However, studies comparing serum and secretory IgA in dogs have often documented a poor correlation, suggesting that serum concentrations should not be used to estimate mucosal secretion of this antibody. This report demonstrates the concurrent use of serum IgA, IgG, and IgM; secretory IgA (from bronchoalveolar lavage fluid); and immunohistochemical stains on bronchial and duodenal mucosa for IgA-containing B cells in a young Irish setter with recurrent respiratory and gastrointestinal signs.     

Attempts to localize the defect in dysgammaglobulinemia of UM-B19 chickens by studying the effect of immunomodulating substances on immunoglobulin and antibody production

The extreme decreased levels of IgG in dysgammaglobulinemic UM-B19 chickens are linked with decreased antibody activity. Antibody activity to T-dependent (although diminished) and T-independent antigens is present but is restricted to IgM and IgA antibodies. Complete Freund's adjuvant enhances the existing isotype pattern of serum immunoglobulins and antibodies. The antibody response to a "T-independent" antigen (B. abortus) is greatly increased by CFA in dysgammaglobulinemic chickens. Beside the appearance of high levels of IgG in dysgammaglobulinemic chickens during the first weeks of life and in transitory dysgammaglobulinemia, remarkable IgG synthesis can be temporarily induced by the mitogenic activity of LPS and even more by the regulatory function of Levamisole. Furthermore, LPS and Levamisole induce IgG antibody synthesis to concomitantly administered antigen, the IgG antibodies appearing within the normal time. Contrary to a missing feedback inhibition from total IgG to IgM serum immunoglobulins, a feedback inhibition from IgG to IgM antibodies is found. No correlation can be found between Levamisole-induced IgG immunoglobulin concentrations, and IgG antibodies. Germfree rearing for one week or longer prevents the dysgammaglobulinemic defect. The following conclusions are drawn: Early antigenic stimulation seems to be the inducing factor for dysgammaglobulinemia in UM-B19 chickens. A BG cell pool is still present in adult dysgammaglobulinemic chickens. This BG cell pool is probably diminished to a varying extent. T cell helper functions seem to be present (albeit they may be disturbed) and can be stimulated. IgG specific T cell suppression is probable. From these conclusions the etiology of the dysgammaglobulinemia in UM-B19 chickens is hypothesized to be primarily due to delayed bursal development: Immature BG cells are eliminated by environmental antigens during the neonatal period in a process similar to tolerance induction. This event, in turn, induces suppressor mechanism(s) or disturbance in helper mechanism(s).     

A longitudinal study of rotavirus antibody titers in swine in a closed specific pathogen-free herd.

In a newly established closed specific pathogen-free (SPF) swine herd, gilt/sow suckling and weaned pig rotavirus specific antibody titers were followed for three lactations by enzyme-linked immunosorbent assay (ELISA) to gain insight into the dynamics of herd antibody titers to group A rotavirus. Among gilts/sows, serum antirotavirus IgG titers increased during each lactation with a subsequent drop in titer between farrowings. Serum antirotavirus IgM titers declined during each lactation and with subsequent parity. Serum antirotavirus IgA titers remained constant during lactations and among parities. In colostrum and milk, antirotavirus IgA antibody was abundant. Differences in titer were not noticed between gilts and second litter sows but third litter sows had significantly higher titers than the first two groups. Antirotavirus IgG was high in colostrum but nearly nonexistent in milk. This titer did not vary significantly within or among parities. There was a linear regression in the titers of baby pig serum antirotavirus IgG from the post colostral sample through to seven weeks old, after which titer began to increase. No difference in baby pig serum antirotavirus IgG was noted among the three litters. Serum antirotavirus IgA and IgM were undetectable in baby pig sera after 2-3 weeks of age. Coproantibody to rotavirus was sporadically present in pig feces for 2-3 weeks after birth with highest titers in the IgA fraction. We conclude that although it is probable that age resistance of pigs to rotavirus diarrhea occurs, humoral immunity as measured by ELISA rotavirus antibody titers may not be intimately involved in virus clearance since in our studies baby pigs passively received large amounts of antibody but still excreted pathogenic virus. The finding of increasing levels of serum antirotavirus IgG in gilt/sow serum suggest that exposure to antigen of dams occur without significant increases in antirotavirus IgG titers in either colostrum, milk, or baby pig serum.     

Serum anti-trichostrongyle antibody responses of first and second season grazing calves

Serum anti-Ostertagia ostertagi and anti-Cooperia oncophora antibody responses were assessed in first season and second season calves grazing permanent paddocks. Calves without previous exposure to trichostrongyles were found to mount significant parasite-specific IgG1 antibody responses within two months of introduction to the pastures. A significant serum IgA response to O ostertagi and IgG2 responses to both O ostertagi and C oncophora antigens were also observed, but these responses were weaker. No consistent serum anti-trichostrongyle IgM responses were discernible in either age group. Second season grazing calves had significantly elevated IgG1, IgG2 and IgA antibody levels at turnout when compared to first season calves, but only IgA antibody levels against O ostertagi increased during the second grazing season. Comparison of serum antibody levels in first and second season calves grazed separately or together suggests that mixed grazing had no discernible effect on antigen priming.     

Development and validation of an ELISA to detect antibodies to Corynebacterium pseudotuberculosis in ovine sera

Several enzyme-linked immunosorbent assays (ELISAs) have been developed for the detection of antibodies to Corynebacterium pseudotuberculosis, the causative agent of caseous lymphadenitis (CLA). However, none are commercially available in the UK. It was therefore necessary to develop a new, economic ELISA for use in a research project studying the epidemiology of CLA in UK sheep. The ELISA with its diagnostic qualities is presented. The ELISA was developed using sonicated C. pseudotuberculosis and optimised to detect total antibody or IgG class antibody in serum. Receiver operating characteristic (ROC) curves were obtained and the area under the ROC curve was used to compare the sensitivity and specificity of the two ELISAs. Both versions of the ELISA were evaluated on a panel of 150 positive reference sera and 103 negative reference sera. Using the test at 100% specificity, the sensitivity of detection of total antibody was 71% (95% confidence interval 63-78%), and the sensitivity of detection of IgG antibody to C. pseudotuberculosis was 83% (76-89%), which compares favourably with other reported ELISA tests for CLA in sheep. The sensitivity of the IgG antibody assay may be higher because of the greater affinity of IgG class antibodies compared with the IgM antibodies also detected by the total antibody ELISA. The results of ROC analysis indicated that the IgG isotype ELISA was more accurate than the total antibody ELISA. The efficiency of the test was greatest when serum samples were run in a dilution series than when any single serum dilution was used. The ELISA is considered to be suitable for application in field studies of CLA in UK sheep.