Preliminary evaluation of the use of the sefA fimbrial gene to elicit immune response against Salmonella enterica serotype Enteritidis in chickens

In the last 2 decades, the prevalence of Salmonella enterica serotype Enteritidis (Salmonella Enteritidis) has dramatically increased worldwide, becoming the leading cause of food-borne illnesses and an important public health issue. Many studies have suggested the role of the SEF14 fimbrial protein in the adhesion of Salmonella Enteritidis to the host. In the present study, the sefA gene, which encodes the main subunit of the SEF14 fimbrial protein, was cloned into a temperature-sensitive expression vector and transformed into a nonpathogenic, avirulent strain of Escherichia coli. The recombinant strain was used as a vaccine to elicit specific immune response against the SefA protein of Salmonella Enteritidis in 1-day-old chickens. The recombinant strain was reisolated from the intestines of treated birds for up to 21 days posttreatment, demonstrating its ability to colonize the intestinal tracts of 1-day-old chickens. In addition, immunoglobulin A (IgA) against the SefA protein was detected in intestinal secretions from treated birds at 7 days posttreatment and in bile samples from 14 to 21 days posttreatment by enzyme-linked immunosorbent assay. Nontreated birds did not show any evidence of intestinal colonization by the recombinant strain or anti-SefA IgA response in their bile or intestinal secretions. Preliminary evaluation of the recombinant strain showed a potential use of this strain to elicit protection against Salmonella Enteritidis infection in chickens. Further experiments are needed to study the ability of the recombinant strain to protect birds against Salmonella Enteritidis colonization.     

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.     

Quantitation of sheep IgG1, IgG2, IgA, IgM and albumin by radioimmunoassay

A solid phase radioimmunoassay procedure for the measurement of immunoglobulins (IgG1, IgG2, IgA and IgM) and albumin in sheep body fluids (serum, intestinal lymph, caudal mediastinal lymph, bile, mammary secretions and intestinal secretions) is described. This method was found to be easy to perform, rapid, sensitive and reproducible. Results obtained were consistent with those previously reported using radial immunodiffusion and nephelometric techniques.     

Quantitation of the immunoglobulins in reproductive tract secretions of the mare

IgG, IgA, IgM and albumin concentrations were measured in serum, follicular fluid and oviductal, uterine and intestinal secretions of the horse. Follicular protein concentrations were found to be dependent on serum concentration and molecular size. Of the immunoglobulins only IgG was detectable in oviductal secretions, but IgG:albumin ratios did not differ significantly from those in serum. IgG, IgA and IgM were measured in uterine secretions, with IgG predominant. Serum transudation into uterine secretions was minimal. In intestinal secretions, IgA levels were slightly higher than IgG, with albumin and IgM at low levels. In five mares with histories of chronic metritis, IgG, IgA and albumin concentrations were significantly elevated in uterine secretions.     

Immunisation of chickens to reduce intestinal colonisation with Campylobacter jejuni

1. Systemic and intestinal antibody titres were measured in chickens following subcutaneous, intraperitoneal (IP), oral (po) and combined IP/po administration of antigen, in soluble, emulsified or microparticulate form. Antigens tested included keyhole limpet haemocyanin (KLH), killed Campylobacter jejuni whole cells and purified campylobacter flagellin protein.

2. The effect of immunisation with purified flagellin protein or with killed C. jejuni whole cells in reducing intestinal colonisation was assessed. The ability of newly‐hatched chicks to respond to immunisation was limited, possibly because of the immaturity of the immune system rather than maternal suppression of an immune response. Only 5 of 13 birds that were first immunised when 1‐d‐old with KLH showed a systemic response, even after 4 immunisations, whereas 10 of 11 birds that were first immunised at 24 d‐old responded systemically.

3. In an immunisation and challenge experiment, birds that were immunised twice intraperitoneally, at 16 and 29 d‐old, with killed C. jejuni whole cells, had fewer C. jejuni, in the caecal contents than unimmunised control birds. This reduction in intestinal colonisation, to less than 2% of bacterial numbers in control birds, was associated with an increase in specific IgG in intestinal secretions. There was no significant increase in specific IgA or IgM in intestinal secretions following immunisation and challenge.

4. These results indicate that immunisation can reduce the level of intestinal infection with C. jejuni. The protection may be enhanced by developing improved methods of immunisation that stimulate production of increased titres of specific antibody in intestinal secretions, particularly specific IgA antibody.     

Eye-drop DNA can induce IgA in the tears and bile of chickens

DNA vaccines protect chickens against lethal virus infections but whether they induce local antibody which is associated with preventing viral entry, is unknown. We were able to show how avian DNA vaccines can induce local IgA. 65 μg plasmid DNA encoding the reporter protein beta-galactosidase induced antigen-specific IgA in the tears of 6/10 birds, IgA in the bile of 4/10 birds and IgG in the serum of 2/10 birds. Giving the DNA by the intramuscular route, as is more usual, induced lacrimal IgA in 2/8 birds, biliary IgA in no birds and serum IgG in 4/8 birds. Eye-drop DNA therefore favoured local IgA whereas intramuscular DNA favoured serum IgG. Further to this preliminary work eye-drop DNA should be improved by adjuvants and cytokines as a way of inducing protective IgA at the mucosal surfaces of the alimentary and respiratory tracts.     

Local intestinal immune response to Escherichia Coli heat-labile (LT) enterotoxin: LT antitoxin levels in healthy, diseased and antigen-fed pigs

With purified LT toxin and IgA, specific anti-LT enterotoxin activity was demonstrated in small intestinal contents of 27 pigs. After 60 days of age, rise in intestinal LT antitoxin titer was observed. Feeding LT containing E. coli antigen increased LT antibody levels in the intestinal secretions, but decreased antibody titers in sera. In post-weaning E. coli diarrhea LT antibody levels in intestinal secretions and sera decreased significantly. This phenomenon can be related to the occurrence of the frequently observed post-weaning E. coli diarrhea.     

B-cell infiltration in the respiratory mucosa of turkeys exposed to subtype C avian metapneumovirus

Turkeys exposed to avian metapneumovirus (aMPV) subtype C showed extensive lymphoid cell infiltrations in the nasal turbinates of the upper respiratory tract. The cellular infiltration occurred after the first virus exposure but not after re-exposure. Quantitation of the relative proportions of mucosal immunoglobulin (Ig)A+, IgG+, and IgM+ cells in controls and virus-exposed turkeys revealed that at 7 days after the first virus exposure, when mucosal infiltration was well pronounced, there was a significant increase (P < 0.05) in the numbers of infiltrating IgA+ but not of IgG+ and IgM+ cells. After the second virus exposure, although the overall numbers of mucosal lymphoid cells were similar in the virus-exposed and control turkeys, the relative proportions of IgA+ and IgG+ cells were significantly higher in the virus-exposed turkeys (P < 0.05) than in controls. Furthermore, elevated levels of aMPV-specific IgA were detected in the nasal secretions and the bile of virus-exposed birds after the second but not after the first virus exposure. These results suggest, for the first time, the possible involvement of local mucosal immunoglobulins in the pathogenesis of aMPV in turkeys.     

In ovo oral vaccination with cmpylobacter jejuni establishes early development of intestinal immunity in chickens

1. Chick embryos were orally immunised at day 16 of incubation by injection of heat‐killed Campylobacter jejuni organisms into the amniotic fluid. The response to vaccination was observed at 5 d after hatching or, in some birds which received a postnatal oral booster vaccination, at 7 d after hatching, and the response was observed at 14 d of age.

2. The titres of antibody in serum, bile and intestinal scrapings, the distribution of immunoglobulin‐containing cells in the spleen, duodenum and ileum and the expression on peripheral blood leukocytes (PBL) of the T cell surface markers CD3, CD4 and CD8 were determined.

3. Whereas low titres of anti‐flagellin antibody were detected in serum, bile and intestinal scrapings of unimmunised birds, high titres were observed in immunised birds.

4. An increase in antibody of all isotypes was detectable in serum but the elevation in IgA antibody in intestinal scrapings and bile was particularly striking. This response was reflected in a dramatic increase in immunoglobulin‐containing cells, detected by fluorescent histology, particularly diose associated with IgA and IgM isotypes in the spleen and intestine of immunised birds.

5. Secondary oral boosting after hatching resulted in a depression in serum anti‐flagellin antibody in immunised birds compared to pre‐boosting titres (although still significandy higher than in non‐immunised controls) but an increase in IgA antibody in intestinal scrapings and bile. The number of immunoglobulin‐containing cells was also increased after boosting.

6. Neither immunisation regimen caused a significant change in the numbers of circulating CD3, CD4 or CD8 T cells.

7. These results indicate that in ovo oral immunisation with C. jejuni antigens stimulates the precocious development of immunity in chicks.     

Immunoglobulin concentrations in ovine body fluids.

Ovine IgG, IgM and IgA and antisera specific for these immunoglobulins were prepared. The specific antisera were used to estimate the immunoglobulin concentrations in certain sheep body fluids. IgA was shown to be the major immunoglobulin in saliva, lung and lachrymal fluid, tracheobronchial and nasal secretions while IgG was the predominant immunoglobulin in colostrum, milk, bile and serum.     

网状内皮组织增殖病病毒感染SPF雏鸡后局部体液免疫球蛋白含量变化

试验对ＳＰＦ雏鸡接种网状内皮组织增殖病病毒（ＲＥＶ）后,应用间接酶联免疫吸附分析法检测了泪液、气管液、肠液和胆汁的免疫球蛋白ＩｇＧ、ＩｇＭ、ＩｇＡ含量的动态变化。结果发现,感染ＳＰＦ雏鸡泪液、气管液、肠液和胆汁的ＩｇＧ、ＩｇＭ、ＩｇＡ含量于感染后７～４２ｄ明显降低,表明ＲＥＶ感染鸡的局部体液免疫能力降低     

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。     

高纯度鸡IgA的制备与鉴定

本试验应用乙醇（ＰＥＧ）粗提后，ＤＥＡＥ纤维素离子交换，Ｓｅｐｈａｒｏｓｅ－４Ｂ凝胶过滤层析方法，从鸡胆汁分离提取ＩｇＡ，ＳＤＳ－ＰＡＧＥ电泳表明可达电泳纯。     

An Unusual Necrotizing cholangiohepatitis in Broiler Chickens

A diagnosis of toxic necrotizing cholangiohepatitis was made in approximately 35% of a flock of 23,000 commercial broiler chickens. Affected chickens were small, and had discrete green foci throughout the liver. Seven to twelve days prior to shipping the flock to slaughter, premixes containing oxytetracycline, monensin, and 3-nitro-4-hydroxyphenylarsonic acid had been added to the ration because the birds had wet droppings and were growing slowly. The 3-nitro-4-hydroxyphenylarsonic acid had been inadvertently incorporated at ten times the recommended level. Liver and kidney of affected birds had elevated levels of total arsenic, and special strains demonstrated arsenic in necrotic foci in liver. Histologically there was marked dilation of intrahepatic bile ducts, and patchy necrosis and hyperplasia of the bile duct epithelium. Damage to bile ducts may have been a toxic manifestation of altered metabolism and excretion of the three drugs being fed to birds suffering from intercurrent diarrhea and/or undefined interactions among the drugs.     

A method for preparation of IgA from bovine mammary secretions.

A method for preparing purified IgA from bovine mammary secretions is described. Whey was initially fractionated by gel filtration and fractions containing IgA were pooled, concentrated and digested with pepsin. The digest was rechromatographed on the same type of gel twice and the resulting IgA preparation tested for purity by an enzyme immunoassay procedure. Five different preparations tested were found to contain no measureable IgM or IgG2 and 0.8% to 1.1% IgG1 on a weight basis. If colostral whey was digested with pepsin prior to chromatography, the IgA preparations contained 1.1% to 2% IgG1 and no measureable IgM and IgG2. The procedure provides a reasonably easy method of eliminating most of the contaminating IgG1 (dimeric) and allows preparation of quantities of IgA for immunochemical studies and standardization of serological techniques.     

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.     

Inhibition of adhesion of Escherichia coli K88 antigen by mammary secretions of susceptible and resistant sows

Anti-adhesive activities of colostrum and milk from genetically susceptible sows, which protected their susceptible offspring in an outbreak of neonatal diarrhoea caused by K88-positive Escherichia coli, were compared with the activities in mammary secretions of resistant dams that did not protect their susceptible progeny. There was significantly more anti-adhesive antibody in the secretions of susceptible sows than in resistant sows, both during the disease period, and 1 year later. Fractionation of colostrum by gel filtration and ion-exchange chromatography led to identification of the anti-adhesive antibodies as including both IgA and IgM.     

Coronaviral enteritis of young calves: virologic and pathologic findings in naturally occurring infections.

Coronavirus was detected in 113 (16.4%) of 689 calf enteric disease accessions received at the South Dakota Animal Disease Research and Diagnostic Laboratory from Mar 1, 1975 to Oct 31, 1976. During this period, modified live coronaviral vaccine was not in common use in this area. The virus was detected by negative stain electron microscopy of feces and intestinal contents and by fluorescent antibody examination of intestine and colon sections. Calves with coronaviral enteritis ranged in age from 1 day to 3 months, with most cases occurring between 1 and 7 days. At necropsy, a green, mucoid fecal cast was commonly seen in the colon. Histopathologic examination revealed necrotizing enterocolitis.     

Local immunity in the respiratory tract of the chicken. II. The secretory immune response to newcastle disease virus and the role of IgA

The nature, specificity and characteristics of the secretory immune response in the respiratory tract of the chicken were investigated in young specific-pathogen-free chickens after vaccination with live lentogenic and inactivated Newcastle disease virus (NDV). Virus-neutralizing (VN) activity considerably exceeding transudation levels from serum were detected in lachrymal fluid, saliva and tracheal washes following infection by both ocular and oral routes. Heat inactivated virus inoculated into the trachea evoked neither serum nor secretory VN activity, whereas a commercial inactivated virus vaccine in mineral oil adjuvant stimulated high titres of serum antibody and some VN activity in tracheal fluids.Antibody in secretions limited, but did not prevent, reinfection of the trachea when birds were challenged 2 weeks later. In contrast to an elevation of circulating antibody titre, challenge induced only a repeated primary response of secreted antibody.All secretions contained IgA which, at least in saliva, accounted for 85% of its activity, the remainder being due to IgG. Fluorescent localization of immunoglobulin producing cells (IPC) demonstrated large numbers containing IgA in association with the upper respiratory tract, particularly in the Harderian gland which contained dense aggregations of plasma cells, many of which were producing IgA.It is concluded that the respiratory tract of the chicken possesses an antibody mediated secretory immune system analogous to that of mammalian species.     

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.