Colonisation studies using Campylobacter jejuni in chicks

White Leghorn chicks used in this study were hatched from specific pathogen-free eggs. The colonizing capability of Campylobacter (C.) jejuni strains was investigated in 6 experiments. The formation of specific antibodies associated to colonization was also detected. In each experiment, day of hatch chicks were randomly separated into three groups of 24 birds each: two groups colonized experimentally and one control group. Chicks were reared on the floor in three separated, adjacent rooms with sterilized wood shavings as litter. At 2 or 8 days of age, respectively, the chicks in the experimentally colonized groups received between 3.3 x 10(7) and 2.0 x 10(8) colony-forming units (CFU) of C. jejuni via oesophageal gavage. Furthermore, 7, 14, 21, 28, 42 and 56 days after inoculation, 4 chicks of each group were sacrificed by cervical dislocation, at which time blood, liver and faeces were collected for processing. Serum was centrifuged and Campylobacter-specific IgG, IgA and IgM antibodies were measured by an indirect enzyme-linked immunosorbent assay (ELISA). Altogether, the colonizing capability of 11 C. jejuni strains was examined. Surprisingly, there were large differences between the C. jejuni isolates. After these experiments, we could divide the isolates into three groups. 4 out of 11 isolates could not be reisolated, 2 isolates caused weak or delayed colonization and 5 C. jejuni produced strong, long-lasting colonization. In the first days of life (9 days), the C. jejuni-free SPF chicks (control animals) had high IgG titres in sera, which decreased markedly up to the age of 15 days. During the experiments the IgM and IgA titres remained nearly at the same level, i.e., the amounts of maternal antibodies were low and there was no evidence for antibody formation in the chicks themselves. Two- and 8-day-old chicks were inoculated with C. jejuni strain Penner 1. Two-day-old chicks were colonized 3 weeks after inoculation. In comparison with these animals, 8-day-old chicks were colonized already 2 weeks after inoculation. There is the assumption, that the higher maternal antibodies in 2-day-old chicks could be responsible for this delay. In chicks the C. jejuni colonization resulted in a marked IgG (but not IgM and IgA) increase. Apparently, there is a positive relationship between the counts of this pathogen in caeca and the IgG increase.     

Infection and reinfection of chickens with Salmonella typhimurium: bacteriology and immune responses.

Four-day-old chickens infected orally with a spectinomycin-resistant (Spcr) mutant of a highly invasive avian Salmonella typhimurium strain excreted salmonellae in the feces for at least 10 weeks. When these chickens were reinfected at this time with a nalidixic acid-resistant (Nalr) mutant of the same strain, they excreted this mutant in significantly smaller numbers (P less than 0.01) than did a previously uninfected control group. The Nalr mutant had a shorter survival rate in the tissues of the immunized chickens than in tissues of the control birds. The Spcr mutant stimulated strong IgG, IgA, and IgM responses in serum, small-intestinal contents, and bile. These were detected by enzyme-linked immunosorbent assay (ELISA) against antigens of crude whole bacterial cell protein sonicate, lipopolysaccharide, flagella, and outer-membrane proteins. There was some evidence of an anamnestic response with IgA in bile following reinfection with the Salmonella. The peak response of antibody-producing cells from the spleens of infected chickens, assayed by solid-phase ELISA, occurred at 3 weeks postinoculation. A strong delayed hypersensitivity reaction, detected by foot-pad swelling after inoculation with either whole-cell or outer-membrane proteins, was observed between 2 and 5 weeks after infection with the Spcr mutant. The data indicate that outer-membrane proteins are major immunogens for both humoral and cell-mediated arms of the immune system.     

Suppression of IgA synthesis in chickens

Four different protocols were tested for the induction of IgA deficiency in chickens: (I) inoculation of anti-alpha intra-peritoneally (i.p.) on alternate days after hatching up to a period of three weeks; (II) bursectomy within 6 h and at 24 h after hatching; (III) in-ovo injection of anti-alpha on the 17th day of embryonation followed by bursectomy at 24 h of hatch and a single injection of anti-alpha i.p. on the day of hatching; (IV) as in III above but bursectomy within 6 h of hatch, followed by three further injections of anti-alpha on days 3, 10 and 34 after hatching. Treatment (I) produced temporary dysgammaglobulinemia during the period of treatment. Bursectomy at 24 h of hatch rendered 75% of the chicks IgA deficient up to four weeks of age. Early bursectomy within 6 h of hatch resulted in substantial improvement of IgA suppression. Such chicks, when tested at 4, 6 and 10 weeks of age, were found to be 81, 72 and 58.3% IgA deficient, respectively. With treatment (III) all the treated birds were IgA deficient at four weeks of age. However, as the birds grew older, IgA appeared in the serum so that at the age of 12 weeks only 27.3% were deficient. Treatment (IV) completely suppressed the IgA system of 13 out of 14 chickens. These chickens lacked both serum and secretory IgA as well as IgA-containing cells in their intestinal mucosa. Both IgG and IgM continued to be produced.     

Monoclonal antibodies against ovine IgA purified from lung lavage fluid

Ovine secretory IgA (sIgA) has been purified to relative homogeneity by ammonium sulphate precipitation (to 40 per cent w/v) of lung lavage fluid from 3- or 12-month-old lambs, followed by molecular sieve chromatography on a Sephacryl S300 matrix. Three peaks A, B and C with molecular sizes corresponding to 550,000, 400,000 and 165,000 respectively were eluted from the column. Immunoelectrophoresis, radial immunodiffusion and enzyme-linked immunosorbent assays (ELISAs) with class specific antiserum confirmed that peak B contained only IgA. Polyacrylamide gel electrophoresis of peak B under reducing conditions resolved three subunits corresponding to secretory component, heavy and light chains. Hybridomas generated by fusing spleen cells from IgA-primed Balb/C mice with murine myeloma (Sp2/0) were screened for IgA-specific monoclonal antibodies against a panel of ovine IgG2, IgG1, IgA and IgM. One particular clone, F3-4B4, identified as murine IgG1, was monospecific against ovine IgA with no cross reactivity against bovine immunoglobulins. This hybridoma was successfully tested as a serological probe by ELISA profiling to locate IgA containing fractions in the course of immunoglobulin purification from biological fluids.     

枣多糖对免疫抑制蛋雏鸡生长性能和免疫指标的影响

为探究金丝小枣多糖（ZJP）对免疫抑制蛋雏鸡生长性能和免疫指标的影响，本试验将900只健康且体重相近的1日龄京红1号蛋鸡随机分为6组，每组6个重复，每个重复25只鸡。Ⅰ组为空白对照组，饲喂基础饲粮；Ⅱ~Ⅵ组在7~9日龄时注射100 mg/kg体重的环磷酰胺，每天1次，建立免疫抑制模型，并分别在基础饲粮中加入600 mg/kg黄芪多糖（APS）及0、400、800和1 600 mg/kg ZJP。试验期为6周。结果显示，ZJP可在不同程度上显著缓解环磷酰胺导致的42日龄体重、22~42和1~42日龄的平均日增重、胸腺和法氏囊指数、聚合免疫球蛋白受体、分泌型免疫球蛋白A （sIgA）及血清中免疫球蛋白A （IgA）和IgG的降低及22~42日龄料重比（F/G）的升高，并且料重比、IgA和IgG达到了APS组的水平。由此得出，在本试验条件下，在基础饲粮中添加适宜水平的ZJP可不同程度改善环磷酰胺所致的生长缓慢和免疫功能低下。     

Mucosal humoral immunity to experimental Salmonella enteritidis infection in the chicken crop

In this report, we show that chickens, infected with Salmonella enteritidis (SE) by oral gavage, produce secretory immunoglobulin As (sIgAs) that specifically bind to numerous SE antigens. Chickens infected with SE showed strong sIgA response against flagella in both bile and crop. The optical density values of enzyme-linked immunosorbent assay (ELISA) tests in positive bile and crop were 1.17 and 0.38, respectively, and were significantly different from those of negative samples. Western immunoblotting revealed that approximately 13.5-, approximately 56-, approximately 62-, approximately 80-, and approximately 143-kD polypeptides were immunodominant proteins in bile, whereas approximately 56-, approximately 62-, and approximately 80-kD polypeptides were found to be strong antigens in crop. These results indicate that the crop may function as another site for mucosal immunity, and the SE flagella-based ELISA of crop samples can provide a useful screening test of SE exposure in chickens.     

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.     

Immunoglobulin class distribution of systemic and mucosal antibody responses to Newcastle disease in chickens

In serum, tracheal wash fluid, and bile from chickens that were inoculated with live or inactivated Newcastle disease virus (NDV), the kinetics and immunoglobulin (Ig) class distribution of an antibody response were demonstrated. The Ig classes (IgM, IgG, and IgA) were captured using monoclonal antibodies (MAbs) in enzyme-linked immunosorbent assays (Ig-capture ELISA). The antibody specificity of the captured Ig was confirmed by binding of NDV. After inoculation with live virus, antibodies of the IgG and IgM classes were mainly found in serum. IgM was produced early from day 4 postexposure (PE) onward, IgG was detected later from day 7 PE onward, and in the tracheal wash fluid and bile, all three Ig classes were demonstrated. After inoculation of inactivated virus, a delayed response of all three classes was observed in serum, and only IgM and IgG were recognized in the tracheal fluid and bile. The type of vaccine and the mute of antigen entrance may have determined the immunoglobulin class produced. The Ig-capture ELISA assay developed in this study can be useful for evaluating various strategies to improve the efficacy of Newcastle disease vaccines and to study the evoked immune mechanisms.     

Colonization characteristics of Campylobacter jejuni in chick ceca

We report our findings on several parameters influencing cecal colonization of chickens by Campylobacter jejuni. Thirty-five colony-forming units (CFU) of a composite culture of C. jejuni colonized the ceca of one-half of the newly hatched chicks challenged by oral gavage. A challenge dose of 3500 CFU/chick consistently colonized the ceca of all chicks challenged. Challenge doses of approximately 10(5) CFU of C. jejuni per chick resulted in consistent cecal colonization, regardless of whether the birds were challenged 1, 2, or 3 days post-hatch. Four isolates showed consistently strong cecal colonization abilities, whereas two isolates colonized the ceca in only 20 of 122 chicks when given levels of 10(5) CFU per chick. One of these poorly colonizing isolates was repeatedly transferred by fecal-oral passage through chicks; subsequently, this isolate was able to consistently colonize chicks. Competitive exclusion (CE) microflora did not diminish the colonization rates for C. jejuni. Birds treated with five different CE cultures were colonized at a rate of 81 of 84 chicks; control chicks were similarly consistently colonized (45 of 46 chicks).     

大围山微型鸡与科宝肉鸡免疫球蛋白含量及CATH-2基因表达差异研究

试验以相同条件下饲养的4、8、12周龄的大围山微型鸡和科宝肉鸡为研究对象,分别测定其免疫球蛋白含量(IgA、IgG、IgM)及胸肌、肝脏、小肠中的CATH-2 mRNA表达量,比较二者之间的差异。结果显示:大围山微型鸡血液中免疫球蛋白含量总体上低于科宝肉鸡,两个鸡种随着日龄的增加IgA、IgG含量均呈现先下降后上升趋势,IgM含量呈现下降趋势,且两个试验鸡种在4周龄时IgA、IgM、IgG含量均不同程度的高于8、12周龄。CATH-2 mRNA表达量在两个鸡种间存在差异,大围山微型鸡胸肌和肝脏中CATH-2 mRNA表达量均低于科宝肉鸡,而小肠中CATH-2 mRNA表达量显著高于科宝肉鸡。结果表明:在相同饲养条件下,大围山微型鸡免疫机能强于科宝肉鸡,具有潜在的较强的抗感染能力。     

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.     

鸡贫血因子病局部粘膜免疫功能变化的研究

用鸡贫血病毒感染１日龄ＡＡ雏鸡,以未感染同龄ＡＡ雏鸡的对照,在感染后７,１４,２１,２８,３５,４２,４９日龄检测其哈德尔腺及盲肠扁桃体Ｔ细胞和ＩｇＧ＾＋、ＩｇＭ＾＋、ＩｇＡ＾＋抗体生成细胞数量,泪液,气管液、肠液中ＩｇＭ、ＩｇＭ、ＩｇＡ含量的动态变化。     

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.     

Local antibody responses in the bile and faeces of sheep infected with Fasciola hepatica

The effect of infection with the liver fluke Fasciola hepatica on serum, bile and faecal immunoglobulin and antibody levels was studied in Scottish Blackface sheep. In the serum the immunoglobulins showing the most marked increase were IgG1 and IgG2 and their maximal values were reached at 16 weeks after infection. In the bile IgG2 rose to peak values at two weeks and IgG1, IgA and IgM were maximal at four weeks after infection. The levels of faecal IgG and IgA were low after primary infection but after reinfection a rapid increase in IgA concentration was observed within one to two weeks. Haemagglutinating antibody levels against egg antigens, juvenile and adult excretory-secretory antigens and adult fluke somatic antigens were evaluated. In the sera high titres were observed starting from two to four weeks after infection and persisting until 14 to 16 weeks. Bile haemagglutinating antibodies against excretory-secretory antigens showed the highest level at two and four weeks after infection while antibodies against adult somatic antigens reached maximal titres between four and eight weeks. Faecal antibody levels after primary infection were low but increased rapidly within two weeks after reinfection, coinciding with the elevation in faecal IgA concentration. However, there was no reduction in the number of flukes established in reinfected animals.     

The absence of cecal colonization of chicks by a mutant of Campylobacter jejuni not expressing bacterial fibronectin-binding protein.

Campylobacter jejuni is a common cause of human gastrointestinal illness throughout the world. Infections with C. jejuni and Campylobacter coli are frequently acquired by eating undercooked chicken. The ability of C. jejuni to become established in the gastrointestinal tract of chickens is believed to involve binding of the bacterium to the gastrointestinal surface. A 37-kD outer membrane protein, termed CadF, has been described that facilitates the binding of Campylobacter to fibronectin. This study was conducted to determine whether the CadF protein is required for C. jejuni to colonize the cecum of newly hatched chicks. Day-of-hatch chicks were orally challenged with C. jejuni F38011, a human clinical isolate, or challenged with a mutant in which the cadF gene was disrupted via homologous recombination with a suicide vector. This method of mutagenesis targets a predetermined DNA sequence and does not produce random mutations in unrelated genes. The parental C. jejuni F38011 readily colonized the cecum of newly hatched chicks. In contrast, the cadF mutant was not recovered from any of 60 chicks challenged, indicating that disruption of the cadF gene renders C. jejuni incapable of colonizing the cecum. CadF protein appears to be required for the colonization of newly hatched leghorn chickens.     

Humoral immune responses of experimentally Eimeria ninakholyakimovae-infected goat kids

Although cellular immune reactions seem to be crucial for protective immune responses in Eimeria spp. infections, there are also evidences on an active involvement of the humoral counterpart. In the present study, we have analyzed the humoral response of goat kids subjected to primary and challenge infections with Eimeria ninakholyakimovae. Specific levels of IgG and IgM in serum samples and IgA in the ileal mucus were estimated. In infected kids, significantly increased levels of IgG were observed from 3 weeks post infection onwards in addition to an enhancement of specific IgM and secretory IgA levels. A wide range of peptides of sporulated oocyst antigen (SOA) was recognized by specific IgG as determined by immunoblotting. However, no correlations were found between immunoglobulin levels and OPG counts after challenge infection. Overall, these data indicate a significant specific humoral response of E. ninakohlyakimovae-infected goat kids that does not seem to convey immunoprotection. Further studies should be addressed to clarify if the lack of correlation might be associated to the type of antigen used for the immunoenzimatic assays, the age of the animals or other factors.     

感染鸡贫血病毒雏鸡接种新城疫疫苗后局部粘膜免疫功能的变化

雏鸡1日龄感染鸡贫血病毒（CAV），8日龄接种Lasota疫苗，以未感染免疫雏鸡为对照，于免疫后7、14、28d检测其哈德尔腺和盲肠扁桃体T细胞及IgG^ 、IgM^ 、IgA^ 抗体生成细胞数量，泪液、气管液、肠液、胆汁中IgG、IgM、IgA含量以及泪液、胆汁HI抗体滴度的动态变化。揭示了感染CAV雏鸡接种ND疫苗免疫后哈德尔腺、盲肠扁桃体的T细胞和IgG^ 、IgM^ 、IgA^ 抗体生成细胞数量，泪液、气管液、肠液、胆汁中免疫球蛋白IgG、IgM、IgA含量以及泪液、胆汁HI抗体滴度，均较未感染免疫雏鸡明显减少。表明眼部、呼吸道和消化道局部粘膜免疫防御能力减弱。     

Phagocytosis and intracellular killing of Campylobacter jejuni by elicited chicken peritoneal macrophages.

In vitro phagocytosis and intracellular survival of Campylobacter jejuni (strains B540 and Clin 1) in chicken peritoneal macrophages were studied. Macrophages were induced with Sephadex G-50 and harvested 48 hr later by peritoneal lavage. The extent of phagocytosis over time was determined by enumerating the intracellular C. jejuni after removal of extracellular C. jejuni with gentamicin. Pre-incubation of C. jejuni with antiserum generally enabled the macrophages to ingest greater numbers of cells than when the organism was pre-incubated in phosphate-buffered saline. C. jejuni were exposed to macrophage uptake for 30 minutes in a 5% CO2 incubator at 42 C. This suspension was then exposed to 12.5 micrograms gentamicin/ml to eliminate extracellular bacteria. Subsequently, the intracellular survival of C. jejuni was examined by monitoring its number within the macrophage at 30 minutes, 3 hr, and 6 hr after phagocytosis. Macrophages from C. jejuni-colonized chickens and from uncolonized control chickens were able to almost destroy the organism within the experimental period.     

Embryo vaccination of specific-pathogen-free chickens with infectious bursal disease virus: tissue distribution of the vaccine virus and protection of hatched chickens against disease

Vaccination of specific-pathogen-free chickens as 18-day embryos with the BVM isolate of infectious bursal disease virus (IBDV) resulted in extensive replication of the vaccine virus in the embryonic tissues. The virus was recovered from lung, thymus, proventriculus, liver, kidney, and spleen of embryos 1 day postvaccination, and recoverable virus persisted for at least 7 days. Replication and spread of the vaccine virus in chickens vaccinated as 18-day embryos was compared with that in chickens vaccinated at hatch. Distribution of the virus in tissues was more extensive, virus levels in tissues were generally higher, and detectable virus persisted longer in chickens vaccinated as 18-day embryos than in those vaccinated at hatch. Effective vaccine response could be initiated with 6.2 median embryo lethal doses, the lowest dose tested. Chickens immunized as embryos developed neutralizing antibody against IBDV and resisted challenge with pathogenic IBDV at 4, 6, 8, and 10 weeks of age.     

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.