Development of a new monoclonal antibody to ovine chimeric IgE and its detection of systemic and local IgE antibody responses to the intestinal nematode Trichostrongylus colubriformis

The J558L cell line, previously transfected with the ovine Cepsilon gene, was induced to secrete a chimeric IgE protein composed of the ovine heavy chain and a mouse light chain with MW of approximately 80 and 26 kDa, respectively. After purification, the chimeric protein was used to immunise BALB-c mice and monoclonal antibodies (mAbs) were generated. The mAb 2F1, which had greatest anti-IgE activity in preliminary screens, was chosen for further characterisation and an examination of systemic and local IgE responses to the intestinal nematode, Trichostrongylus colubriformis. The chimeric IgE protein was not recognised in enzyme linked immunosorbent assay (ELISA) by mAbs raised against ovine IgG1, IgG2, IgA or IgM. However, 2F1 was highly specific to the chimeric IgE protein, and did not cross-react with ovine IgG1, IgG2 or IgA. Western blot analysis also showed that 2F1 and secretory IgA (sIgA) did not cross-react, and that 2F1 and the anti-IgA mAb identified different MW bands from colostrum (approximately 200 and 400 kDa, respectively). 2F1 bound to mucosal mast cells (MMC) isolated from the intestines of lambs infected with T. colubriformis, but cultured bone marrow-derived mast cells (BMMC) required prior incubation with the chimeric IgE protein for this binding to occur. Distinctive staining of plasma cells and putative mast cells were observed using 2F1 on immunohistological sections of mesenteric lymph node and jejunum.ELISA incorporating 2F1 was able to detect >0.4 ng chimeric protein. Total IgE in ovine colostrum and intestinal homogenates was quantified using a capture ELISA, with known amounts of chimeric protein used to produce a standard curve. Colostrum from outbred Merino ewes had 0.55-11.05 ng ml(-1) total IgE, and their lambs, at necropsy after infection with a total of 18,000 T. colubriformis infective larvae over a 9-week period, had 45-620 ng g(-1) total IgE in intestinal tissue. Compared to genetically susceptible lambs, antigen-specific levels of IgE were significantly higher in genetically resistant lambs after infection with 4500 T. colubriformis infective larvae (TcL3) per week for 9 weeks (161.4 versus 44.8 geometric mean titres; P=0.043). In western blots, distinctive bands (19-21 and 27 kDa) from T. colubriformis larval antigen were differentially recognised by IgE, as identified by 2F1, in intestinal homogenates from genetically resistant animals.These results have demonstrated the value of 2F1 for quantification of IgE responses in samples derived from ovine fluids and tissues using ELISA, western blots and immunohistology. In this respect, it recognises native ovine IgE and does not require pre-treatment of the sample with denaturing agents or ammonium sulphate.     

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.     

Stimulation of mucosal and systemic antibody responses against recombinant transferrin-binding protein B of Actinobacillus pleuropneumoniae with chitosan after tracheal administration in piglets

This study evaluated the suitability of using a chitosan formulation as an adjuvant to enhance both the mucosal and systemic immune responses against recombinant transferrin-binding protein B (rTbp B) of Actinobacillus pleuropneumoniae via direct tracheal administration. The chitosan formulation was found to enhance mucosal immune response, as measured by the secretory IgA level in lung lavage fluid and lung homogenate extracts, and systemic immune response, as measured by the serum IgG level.     

Immunogenicity of recombinant Omp31 from Brucella melitensis in rams and serum bactericidal activity against B. ovis

Detergent-extracted recombinant Omp31 (rOmp31 extract) from Brucella melitensis produced in Escherichia coli was previously identified as a protective immunogen against B. ovis in mice. In this study, we evaluated the immunogenicity of rOmp31extract in rams. This immunogen was emulsified in an oil adjuvant and administered three times with 4 and 8 weeks intervals. Antibody response was measured in serum by whole B. ovis ELISA. Specific antibodies to purified rOmp31 (pET-Omp31) were detected by Western blotting and indirect ELISA. In addition, isotype specific antibodies were measured in tears. Serum bactericidal activity against B. ovis in the presence of complement was measured in vitro. Cellular immune response was explored by intradermal testing with purified rOmp31. Immunization with rOmp31 extract induced IgG specific antibodies in serum able to bind to whole B. ovis cells. Furthermore, strong inhibition in a competitive ELISA (with an Omp31-specific monoclonal antibody) suggested that a proportion of Omp31-specific antibodies were directed against a loop containing a protective epitope. Serum antibodies killed efficiently B. ovis in vitro in the presence of either guinea pig or ovine serum. Tears had both IgG and IgA antibodies to equivalent titers. Finally, immunized rams showed skin reactivity to Omp31. These data demonstrate that B. melitensis Omp31, a protective antigen identified in the mouse model, induces antibody and cellular immune mechanisms in sheep.     

Analysis of the humoral immune response to Oestrus ovis in ovine

Antibody responses (IgG, IgM and IgA) against Oestrus ovis were analyzed in sheep and in first year grazing lambs from Sardinia (Italy) by an indirect-enzyme-linked immunoassay test and L2 O. ovis excretory/secretory antigens. Serum samples from 208 sheep were obtained prior to be slaughtered, and then heads were removed and cut open along their longitudinal axis to collect the parasites from the nasal cavities, turbinates and sinus. Besides this, blood samples were monthly collected from the lambs of G-1 (maintained under field conditions) and the lambs of G-2 (kept housed since birth to avoid Oestrus infestations) throughout a year. In the sheep, a positive significant correlation was observed between the number of first instar O. ovis larvae and the values of IgM, and between the second instar larvae and the IgG optical densities. In the lambs, all classes of antibodies increased significantly from July in G-1. The highest values of IgG were reached in September (IgG) and decreased in November-December. The IgM response peaked in November, and very low values of IgA were observed during the study. Matching these data with chronobiology of O. ovis in this region, we conclude that the first infection occurs on May, stimulating the production of humoral antibodies. The reduction of the IgG antibody levels starting from October means the beginning of the diapause while the IgM response seems to be associated to the presence of L1 in the nasal cavities. The data obtained led us to forecast an early treatment of the ovine on June-July, which should keep away from the maturation of O. ovis L1 larvae, avoiding the development of clinical lesions and interrupting the life cycle of this parasite.     

Influence of Campylobacter jejuni cecal colonization on immunoglobulin response in chickens

The immunoglobulin response of chickens to colonization by Campylobacter jejuni isolates B-540 and Clin-1 was monitored. Chicken humoral IgG and biliary secretory IgA (sIgA) responses were assessed by enzyme-linked immunosorbent assay (ELISA). Samples were taken from 128 C. jejuni-colonized chickens and 104 uncolonized chickens housed in a controlled environment. An indirect ELISA was performed using the homologous isolate of C. jejuni as the capture antigen and was developed with the specific goat anti-chicken IgG or IgA alkaline phosphatase conjugates. The ELISA absorbance values of the test samples at 405 nm (serum diluted 1:32 and bile diluted 1:10) were normalized in direct proportion to standard sera and bile sample values. In the colonized chickens, humoral IgG activities were highest at hatch, dropped to their lowest level after 2 weeks, and increased by 8 weeks to levels similar to those detected at hatch. The sIgA activity was lowest at hatch and increased by 4 weeks in colonized chickens while remaining lower in the control chickens. Chickens colonized with isolate B-540 showed a primary sIgA response during the first 4 weeks and reached a plateau over the final 4 weeks. In spite of these limited humoral and secretory immunoglobulin responses, once the chicken ceca was colonized by C. jejuni, the organism persisted throughout the 8-week experiment.     

Quantitative Changes Associated with Calving in the Levels of Bovine Immunoglobulins in Selected Body Fluids I. Changes in the Levels of IgA, IgG1 and Total Protein

Levels of bovine IgA, IgG1 and total protein (TP) were determined in serum, saliva, tears and individual quarter lacteal secretions of six Holstein-Friesian cows sampled from six weeks before to four weeks after parturition. Hierarchal analyses of variance indicated significant variations among weeks, cows and quarters of the udder. A precipitous but non proportional drop in the levels of IgA and IgA1 in lacteal secretions occurred at calving. There was a concomitant increase in IgG1, and decrease in IgA, in serum. Correlation studies supported the concept of selective transport of IgG1 from serum to lacteal secretions in regulated amounts independent of serum IgG1 levels. Changes in the IgG1/TP ratio of serum and lacteal secretions supported the idea of a decrease in the selective transport mechanism. Correlation studies and estimations of secretory IgA (SIgA) in serum suggest that serum IgA is derived from IgA synthesized in secretory tissues. Highly significant correlations between IgA and IgG1 levels in all secretions postpartum suggest that local IgA synthesis and either IgG1 transport or local IgG1 synthesis are initiated by the same stimuli. Although some of the variation in the level reported for IgA and IgG1 in secretions resulted from protein dilution, much of the variation represents physiological differences between individual animals and tissues in the same animal. An IgG2/IgG1 ratio approaching that of serum occurred in a mastitic quarter of one cow. IgA was the principal immunoglobulin in saliva and tears, comprised a greater proportion of the immunoglobulin in milk whey than in prepartum lacteal secretions and was a minor immunoglobulin in bovine serum.     

Monoclonal anti-equine IgE antibodies with specificity for different epitopes on the immunoglobulin heavy chain of native IgE

In this study we describe the generation of monoclonal antibodies (mAbs), which recognize different epitopes of the equine IgE constant heavy chain. Equi-murine recombinant IgE (rIgE), composed of the murine V(H)186.2 heavy chain variable region, linked to the equine IgE constant heavy chain and expressed together with the murine lambda(1) chain in J558L cells was used to immunize BALB/C mice. A total of 17 different mAbs were obtained, which recognized the rIgE heavy chain constant region. None of the mAbs reacted with monoclonal equine isotypes IgM, IgG1 (IgGa), IgG3 (IgG(T)), IgG4 (IgGb) or isolated equine light chains, IgGc and IgA from horse serum, or the native mAb B1-8delta, expressing the same heavy chain variable regions and light chains. One of the mAbs (alphaIgE-132) recognized the recombinant equine IgE, but did not recognize any protein in equine serum, i.e. native IgE. A total of 16 mAbs detected a serum protein of approximately 210,000Da on Western blots, corresponding to the expected MW of native IgE. In addition, one of the mAbs (alphaIgE-176) detected a protein of 76,000Da under reducing conditions, most likely the equine IgE heavy chain. According to binding inhibition studies, the equine IgE specific mAbs recognize at least two different epitopes of the equine IgE. In an ELISA using two anti-IgE mAbs which recognized different epitopes, no significant differences in the concentration of total serum IgE could be detected between adult Icelandic horses with IgE-mediated type I allergy (summer eczema) and healthy control animals. In Icelandic horse foals, no serum IgE could be measured 6 months post partum. All anti-IgE mAbs recognized a small population (1.3+/-0.5%) of leukocytes from adult Icelandic horses by surface immunofluorescence, but no cells could be detected in foal blood. The stained leukocytes from adult horses could be enriched by magnetic cell sorting and contained 32% basophils, 53% monocytes and/or large lymphocytes, 13% small lymphocytes and 2% eosinophils.     

Protein profiles of dense-centered forms of five chlamydial strains of animal origin.

Purified dense-centered form of 1 bovine strain (LW613) and 3 ovine strains (B577, 034-EYE, and 047-EYE) of Chlamydia psittaci and 1 murine strain of Chlamydia trachomatis (MoPn) were dissociated in the presence of sodium dodecyl sulfate (SDS) and 2-mercaptoethanol. The number of polypeptides detected in the 5 strains varied between 17 and 20, with a molecular weight range of 29,000 to 120,000. Two polypeptides predominated and comprised approximately a third of the total protein in each of the 5 strains. The average molecular weights of the 2 polypeptides were 89,000 and 85,250 for 4 of the strains, and the polypeptide molecular weights were 100,000 and 98,000 for the ovine abortion strain (B577). Molecular weights and proportional composition of the polypeptides permitted differentiation of the chlamydial strains. The 3 strains from naturally occurring conjunctivitis or polyarthritis (LW613, 034-EYE, and 047-EYE) had similar polypeptide profile in the 75,000 to 100,000 molecular weight range. The polypeptides of the ovine abortion strain (B577) differed significantly from these 3 strains. Eight of the polypeptides of this strain had a molecular weight of 100,000 or greater, and 3 of the predominant polypeptides were in excess of 100,000. In contrast, some of the polypeptides of the murine strain had lower molecular weights than the 4 other strains. Three predominant polypeptides had molecular weights below 50,000.     

Preparation of porcine IgA and its detection using the fluorescent antibody technic]

The preparation of immunoglobulin A (IgA) from porcine colostrum, intestinal content and serum is described. The best results were achieved with colostrum, from which an antigen of satisfactory purity was prepared by purification on Sephadex G-200, on DEAE cellulose and subsequent filtration on Sephadex G-200. The serum to this antigen raised in rabbits was adsorbed to an immunoadsorbent from porcine serum (PS) or porcine IgG. The adsorbtion of the serum against secretory IgA (SIgA) to PS removed its undesirable heterologous and nonspecific reactivity. The anti-SIgA serum adsorbed in this way still reacted with IgA from porcine serum. In the direct and indirect immunofluorescent staining we detected the main antigenic determinants of the SIgA molecule, i. e. the heavy chains and the secretory component.     

Dynamics of the humoral immune response of calves infected and re-infected with Cooperia punctata

The dynamics of the humoral immune response of calves were analysed after primary infection and re-infection with the intestinal nematode Cooperia punctata. 12 male 5 month-old Holstein-Friesian calves were randomly divided into two groups A and B. At the beginning of the experiment Group A animals were each infected experimentally with a single oral dose of 130,000 infective third stage larvae (L3) of C. punctata. The animals of Group B were kept as non-infected controls. The two calves from Group A with the highest infections died of cooperiosis at 32 and 44 days after infection (DAI), respectively. On DAI 100 the calves were treated with the recommended dose of oxfendazole. On DAI 180 the remaining four calves of Group A and three animals of Group B (B1) were infected with 260,000 L3 of C. punctata, while the other three calves of Group B (B2) served as non-infected controls. Monitoring of the humoral immune response predominantly demonstrated an IgG1 response against both adult and L3 antigen of C. punctata. Moreover, re-infections increased the levels of these immunoglobulins. IgA levels were less increased than IgG1 and no significant increase was observed in IgG2 and IgM levels. Immunoblotting analysis showed that total IgG present in the serum of the primary infected animals mainly reacted against adult proteins of 12-14 and 17-20 kDa and against L3 proteins of 33 and 43 kDa. After re-infection total IgG reacted with the same adult proteins but also with an adult 29 kDa protein.     

Allergen-specific IgE levels against crude mould and storage mite extracts and recombinant mould allergens in sera from horses affected with chronic bronchitis

Immunoglobulin E antibody (IgE) levels against four recombinant (r) mould allergens (r-Aspergillus fumigatus [rAsp f] 7, 8 and 9; r-Alternaria alternata 1 [rAlta1]) and crude mould (Aspergillus fumigatus, Alternaria alternata, Penicillium notatum) and storage mite extracts were determined by ELISA in sera from 24 pulmonary sound control horses and 26 horses suffering from chronic bronchitis/bronchiolitis (CB), also called chronic obstructive pulmonary disease (COPD). Serum IgG and IgA titres were also determined against Aspergillus fumigatus extract and rAsp f 8.IgE against the crude extracts could be measured in all sera, but there was no significant difference between CB-affected and control horses. In contrast, only 8-30% of the horses, depending on the r-allergen tested, had detectable IgE levels in serum against the r-allergens. Horses with CB had significantly more often detectable IgE levels than controls against rAlt a 1 (10/26 and 3/24, respectively, p=0. 054), rAsp f 7 (13/26 and 2/24, respectively, p<0.01) and rAsp f 8 (11/26 and 1/24, respectively, p<0.01). Only four horses (three CB-affected and one healthy, p0.05) had detectable IgE levels against rAsp f 9. Furthermore, CB-affected horses were often sensitised against two or more r-allergens (13/26 of the CB-affected horses) while only one of the 24 healthy horses had positive IgE levels against more than one r-allergens. Similarly to IgE levels, no significant differences between CB-affected and healthy horses were found for IgG titres against the Aspergillus fumigatus extract. However, horses with CB had significantly higher serum IgG titres against rAsp f 8 than healthy controls (median=28 versus 10 relative ELISA units [REU], p<0.01). Additionally, horses with detectable IgE titres against rAsp f 8 had significantly higher IgG titres against this r-allergen than horses with undetectable IgE titres (median IgG titres=46 and 13 REU, respectively; p<0.01). For serum IgA titres, neither differences between healthy and CB-affected animals nor correlations between IgA and IgG or IgE titres could be found.These results show that horses suffering from CB are more often sensitised to some Aspergillus fumigatus and Alternaria alternata allergens than control horses and that they are partly sensitised to the same fungal proteins as mould-allergic human patients. Furthermore, this study shows that r-allergens allow a much more sensitive determination of specific serum antibody levels by ELISA than crude mould extracts.     

Immunoglobulin concentrations in serum and nasal secretions of calves at the onset of pneumonia

Immunoglobulin (Ig) concentrations in serum and in nasal secretions were correlated with pneumonia and diarrhea during the first 12 weeks of life in 56 calves. The peak onset of pneumonia occurred between 2 and 4 weeks of age when the calves' serum IgG1, IgG2, and IgA concentrations were lowest. As IgG2 concentrations increased, fewer calves developed pneumonia. Peak onset of pneumonia was also correlated with the lowest IgG and IgA concentrations in the calves' nasal secretions. Most calves developed pneumonia when serum concentrations of IgG1 were less than 1.5 g/dl, IgG2 less than 0.3 g/dl, IgA less than 0.1 g/dl, and IgM less than 0.2 g/dl and when the combined IgG and IgA values in nasal secretions were less than 0.2 mg of Ig/mg of protein. In study A, diarrhea preceded pneumonia in 63% of 56 calves. In study B, 38% of 23 calves had diarrhea and/or hemorrhagic feces before pneumonia. Seemingly, there was a relationship between diarrhea and pneumonia. Furthermore, pneumonia occurred at or just after the time when IgG1, IgG2, and IgA concentrations in serum and the combined IgG and IgA concentrations in nasal secretions were lowest. Pneumonia is a common disease of calves between 1 and 5 months of age, a period coinciding with the usual low point in serum immunoglobulin (Ig) concentrations due to catabolism of passively acquired antibodies. Calves that absorb less than adequate amounts of Ig may be susceptible to pneumonia at approximately 2 months of age, when serum Ig concentrations would be lowest.(ABSTRACT TRUNCATED AT 250 WORDS)     

Helicobacter pylori-specific immunoglobulin synthesis in gnotobiotic piglets: evidence for the induction of mucosal immunity in the stomach

Short term tissue biopsy cultures and paired, sera, bile and gastric and intestinal contents from Helicobacter pylori-infected gnotobiotic piglets were tested for the synthesis of H. pylori-specific immunoglobulin (Ig) isotype production by antigen-specific ELISA from post-infection days (PIDs) 2-28. Serum antibody levels in all three Ig isotypes were elevated from baseline values by PID 14, serum IgM levels reached peak levels on PID 14 and by PID 28 bile was strongly positive for IgA and IgG.Intestinal, but not gastric contents from infected piglets, contained IgA-specific antibody from PID 14 onward. Gastric mucosal epithelia adjacent to areas of inflammation in infected but not uninfected control piglets produced readily detectable amounts of porcine secretory component (SC); IgA-positive plasma cells were identified in gastric submucosa and lamina propria in these areas. Culture fluid supernatants, collected from explanted gastric cardia and antra and intestinal ilea of H. pylori-infected piglets had trace amounts of IgA as early as PID 2 in some animals, and strong IgA reactivity in all by PID 28. Supernatants also contained H. pylori-specific IgG by PID 14. A strong gastric lymph node IgA response contrasted with moderate IgA production in mesenteric lymph nodes and spleen. Mucosal biopsy production of H. pylori-specific IgG was more evenly distributed throughout the lymphoid system. These data support the contention that the Ig response to H. pylori is initiated within the gastric compartment and matures over time to a generalized IgA-dominated mucosal and IgG-dominated nonmucosal humoral immune response.     

Serum antibody response and Cryptosporidium parvum oocyst antigens recognized by sera from naturally infected sheep.

The response of specific serum immunoglobulins (IgG, IgM and IgA) and the major antigens of Cryptosporidium parvum recognized by these isotypes were investigated by using enzyme-linked immunosorbent assay and immunoblot techniques in lambs and ewes naturally infected throughout an outbreak of cryptosporidiosis. Serum samples were collected from 20 lambs the first day they showed diarrhoea (D1), and Days 11 and 22, in addition to single serum samples from 17 of their dams. Serum anti-C. parvum IgG, IgM and/or IgA antibodies were detected in lambs as early as Day 1. Levels of IgM antibodies remained steady from D1 to D11 and increased at D22, whereas the IgG response decreased from D1 to D11 and subsequently increased. In contrast, IgA antibodies rapidly fell from D1 and all lambs were seronegative at D11 and D22. The highest levels of specific antibodies were detected in sera from ewes. In fact, all ewes were seropositives for IgM and IgA isotypes and most (16/17) showed positive levels of IgG. Four protein fractions (37-39, 42-48, 51-57 and 60-69 kDa) were the most frequently recognized by IgG and IgM from lamb sera. A low molecular weight fraction (12-14 kDa) reacting with IgG and IgA in most lamb sera was scarcely recognized by IgM and three broad bands were frequently recognized by IgA antibodies (23-25, 51-57 and 90-95 kDa). The recognition pattern of 23-25 kDa peptides by IgA from lamb sera clearly increased with the age. Peptides of 42-48, 51-57, 60-69 and 71-78 kDa were most frequently recognized by IgG and IgM from ewe sera. In relation to IgA antibodies from ewe sera, a frequent immunoreactivity was found with proteins in the intervals between 12 and 22 kDa as well as between 32 and 34 kDa and practically all sera reacted with fractions from 42 to 95 kDa.     

Purification and identification of turkey immunoglobulin-A

Turkey immunoglobulin-A (IgA) was isolated from bile, intestinal secretions, and serum by affinity chromatography using monospecific anti-turkey IgA coupled to CNBr-activated Sepharose. The isolated immunoglobulin was antigenically distinct from IgM and IgG. The purity of IgA was demonstrated by immunodiffusion, immunoelectrophoresis, and analytical ultracentrifugation. The predominant forms of polymeric IgA in bile and intestinal secretions had respective So20w values of 16.1 and 15.2. Larger polymers (25-26S) were also present. Two molecular forms (8.5S and 17S) were found in serum. The 8.5S peak was higher than the 17S, indicating a greater concentration of 8.5 S.     

Adjuvant regulation of cytokine profile and antibody isotype of immune responses to Mycoplasma agalactiae in mice

Traditionally, adjuvants have been administered with antigens to enhance immunity. We studied the effect of several adjuvants such as Freund's complete adjuvant (FCA), Freund's incomplete adjuvant (FIA), lipopolysaccharide (LPS), homopolymers of polyinosinic-polycytidylic acid (poly I:C) and polyadenylic-polyuridylic acid (poly A:U), lithium chloride (LiCl), saponin Quil A and calcium phosphate gel (CaHPO(4)) on the immune response of mice to formalin-inactivated Mycoplasma agalactiae. The specific antibody or cytokine producing splenocytes were detected by ELISAspot and immunocytochemistry, respectively. Depending on the adjuvant given, the number of M. agalactiae-specific antibody producing cells was increased 2.5-6-fold. IgG was the major class of M. agalactiae-specific antibodies followed by IgM, IgA and IgE. Among IgG isotypes, FCA, FIA, Quil A and CaHPO(4) induced an IgG1 response with substantial increase of the IgG2a, IgG2b and IgG3 isotypes while poly I:C shifted the response toward an IgG2a/IgG3 production. Finally, poly A:U induced an IgG2b response while LPS and LiCl augmented the IgG3/IgG1/IgG2a secretion. FCA augmented IL-4, IL-5 and IL-10 production suggesting a strong Th2 response, while IFN-gamma and IL-12 remained low; poly I:C enhanced IFN-gamma, IL-12 and TNF-alpha eliciting a Th1 response; poly A:U resulted in a IL-10, IL-5, IL-6 and IL-12 secretion; and LPS enhanced the IL-10, IL-6 and TNF-alpha production. Our data show that adjuvants augment M. agalactiae-specific antibody production and lead to B cell isotype-switching via the appropriate cytokine milieu. Certain adjuvants, such as poly I:C, therefore, appear as promising immune enhancers for vaccination against M. agalactiae infections.     

Serum antibody isotype responses of Fasciola-infected sheep and cattle to excretory and secretory products of Fasciola species

This study investigated the immunoglobulin isotype responses of sheep and cattle chronically infected with Fasciola hepatica and Fasciola gigantica to adult F. hepatica excretory/secretory products (Fh-ES) or F. gigantica excretory/secretory products (Fg-ES), respectively. An antibody enzyme-linked immunosorbent assay (Ab-ELISA) was used to determine serum antibody (total Ig, IgG(1), IgM, IgG(2) and IgA) responses. At necropsy, the mean number of flukes recovered was lower in cattle than in sheep. All F. hepatica and F. gigantica infected sheep and cattle showed an increased total Ig levels from 3 to 4 weeks post-infection (wpi). Among isotypes IgG(1) was most dominant while IgM was the earliest (2 wpi) to be detected in both sheep and cattle infected with both F. hepatica and F. gigantica animals. IgG(2) response was early (2 wpi) in sheep infected by F. hepatica but there was no response in sheep infected with F. gigantica. There was a late and strong IgG(2) response in cattle infected with both flukes. The IgA isotype showed an early and a clear biphasic response in sheep with F. hepatica but was less pronounced in F. gigantica infected sheep. While IgA response to Fh-ES was noticed 5 wpi in F. hepatica infected cattle, it appeared much later (21 wpi) in those infected with F. gigantica. The dominance of IgG(1) isotype in infected sheep and cattle suggest an associated Th2 response. This early response to adult Fasciola spp. ES antigen suggests an early exposure to the antigen presumably through the cross-reacting ES products of juvenile flukes. There is clearly difference in IgG(2) isotype response in cattle (resistant) compared to sheep (susceptible). The late IgG(2) response in cattle may suggest late Th1 involvement in bovine cellular responses to adult Fh-ES/Fg-ES.     

Development of a protective immunity against Ostertagia leptospicularis in trickle-infected sheep and parallel changes of serum gastrin, pepsinogen and antibody levels

Nine lambs, approximately 9 months of age were allocated to three groups (A, B, C), with three animals in each. Sheep in Groups A and B were trickle-infected with doses of 1000 third-stage larvae (L3) of Ostertagia leptospicularis (five times per week) over periods of 7.5 and 10.5 weeks, respectively, and were subsequently treated with fenbendazole (7.5 mg/kg). Approximately 3 weeks after anthelmintic treatment, all sheep were challenged with a single dose of 100,000 L3, whereas sheep of Group C received the same dose as a primary infection. Sheep of Groups A and B were almost completely refractory against the challenge infection, as indicated by negative faecal egg counts and adult worm burdens. A relatively high infection level was present in the sheep of Group C. The results indicate that a comparatively short immunization period of 7.5 weeks is sufficient to protect lambs against subsequent larval challenge. During immunization, the pepsinogen-, gastrin- and IgA-responses were similar in the individual sheep. In contrast to parasite-specific IgG1 and IgG2 levels, IgA decreased rapidly after cessation of trickle infection and parallel anthelmintic treatment, and may therefore indicate current exposure to parasite antigen. After challenge, the majority of the immunized sheep exhibited immediate and short-term responses of pepsinogen, gastrin and IgA in the serum. The time course and the level of each of these responses were very similar in the individual sheep, suggesting that the release of pepsinogen, gastrin and IgA into the circulation was influenced by related mechanisms.