Chicken egg antibodies for prophylaxis and therapy of infectious intestinal diseases. V. In vivo studies on protective effects against Escherichia coli diarrhea in pigs

A field study and a controlled infection trial showed the protective effect of egg yolk lyophilisate and whole egg lyophilisate against enterotoxic E. coli germs. The lyophilisates were gained from eggs of hens immunized against pilus antigen of porcine-enterotoxic E. coli. In a first field study using egg yolk antibodies, 92% of 299 diarrhea affected piglets were cured. In a further field study diarrhea affected piglets were cured after 3 days by application of egg yolk lyophilisate from immunized hens. Piglets treated only with egg yolk of not immunized hens showed no signs of recovery. The infection trial showed, that whole egg lyophilisate of immunized hens was as successful as a common antibiotic therapy in curing piglets, orally infected with 5 x 10(10) E. coli/feeding and animal. The present data show that chicken egg antibodies can be used for treatment of infectious diarrheal diseases in young animals. So far they represent a good alternative to the common used antibiotic therapy.     

Chicken egg antibodies for prophylaxis and therapy of infectious intestinal diseases. II. In vitro studies on gastric and enteric digestion of egg yolk antibodies specific against pathogenic Escherichia coli strains

Specific antibodies directed against enterotoxic E. coli strains from the yolk of immunized hens were exposed in vitro to the influence of varying digestive phenomena like reduction of pH and proteolytic digestion. The remaining antibody activity was tested in a specific ELISA system. It could be shown that already within the stomach a considerable loss in antibody activity caused by a lowering of pH and peptic cleavage can be expected. A further loss in antibody activity is due to the proteolytic effect of the pancreas proteases trypsin and chymotrypsin. It was found that antibodies in protein rich solutions like egg or yolk suspensions were more resistant than mere globulin fractions or antibodies isolated by affinity chromatography. Prospects for further in vivo tests are discussed.     

Characterization of specific egg yolk immunoglobulin (IgY) against mastitis-causing Escherichia coli

The objective of this study was to estimate the in vitro activity of egg yolk immunoglobulin (IgY) against mastitis-causing Escherichia coli. Specific IgY was produced by hens immunized with formaldehyde killed E. coli O111 in long-standing immunization response (titer > or =6400 for 100 days) and was isolated from yolks with a purity of 86% by water dilution, salt precipitations and ultrafiltration. Enzyme-linked immunosorbent assay (ELISA) indicated the produced IgY specifically targeted E. coli O111 and five other E. coli strains which were isolated from mastitic cows. The growth inhibition activity of the specific IgY to bacteria was dose-dependent with an effective concentration of 20mg purified IgY per milliliter. The phagocytic activity of E. coli either by milk macrophages (MPhi) or by polymorphonuclear neutrophil leukocytes (PMN) in the presence of specific IgY was significantly higher than that with nonspecific IgY or without IgY (p<0.05), suggesting that it enhanced phagocytic activity. The current work suggests that this specific IgY has potential as a therapeutic treatment for mastitis in dairy cows.     

Chicken egg antibodies for prophylaxis and therapy of infectious intestinal diseases. III. In vivo tenacity test in piglets with artificial jejunal fistula

Large quantities of specific egg antibodies can be produced with little effort by immunization of laying hens. Several antibody containing egg preparations were subjected to a tenacity test against digestive activity taking piglets with artificial jejunum fistulas as a model. Even with high doses of orally administered yolk lyophilisate no antibody activity could be found in the distal jejunum. The additional feeding of egg white, however, showed a significant protective effect on the yolks antibodies during the digestive act. No difference between egg white of immunized hens and egg white of unimmunized hens could be detected. Buffering the acidic gastric environment with sodium bicarbonate increased the antibody activity in the intestine by an average of 40%. Eggs administered in hot (70 degrees C) beef stock had higher antibody titers than eggs that were boiled for 4 minutes. Tenacity in antibodies fed in pellets was significantly lower than in powdered feed.     

Studies of the pathogenesis of enteric E. coli infections in weaned pigs. The significance of the milk of the dam in preventing the disease.

Milk from sows whose progeny developed post weaning E. coli diarrhoea (PWD milk) and from sows which were immunized by intramuscular vaccination using a homologous strain of E. coli (immune milk) were tested in ligated segments of pig intestine. The results showed that PWD milk neutralized the enterotoxigenic, fluid accumulating capacity of the lysate of the disease-causing E. coli pathogen. A similar effect was seen by using immune milk (Table I). Neither PWD milk nor immune milk contained sufficient antibacterial activity to neutralize the fluid accumulating capacity of live cultures of E. coli O149:K91, while such activity was contained in immune serum. It is concluded that milk from sows whose progeny developed PWD contains antibodies capable of neutralizing the enterotoxigenic effects of the homologous E. coli organisms. It is suggested that the presence in milk from these sows of antibody-mediated activity against enteropathogenic E. coli organisms may be instrumental in preventing the disease in the progeny during the suckling period and consequently, when this protective milk supply stops at weaning, the disease may develop in susceptible animals, mainly because their own production of specific E. coli antibodies is insufficient to prevent PWD.     

Growth of Salmonella typhimurium and Salmonella enteritidis in egg yolks from highly immunized hens.

This experiment was conducted to ascertain whether the growth of Salmonella Enteritidis (SE) or Salmonella Typhimurium (ST) would be suppressed in the presence of antibodies contained in egg yolks. Specific pathogen-free chickens (102 days of age) were subcutaneously immunized with oil-adjuvanted bacterin of SE or ST, twice within a four-week interval. During 160 to 170 days of age, eggs were collected, the yolks were removed and mixed with an equal volume of physiological buffered saline, inoculated with ten colony forming units (CFU) of SE or ST, and incubated at 37 degrees C or 20 degrees C for 23 hr. The growth of organisms in each yolk solution was examined. The egg yolk derived from non-immunized hens was examined in the same manner as the controls. There was no difference in the growth titer between the antibody-positive yolk and the negative yolk. The result suggests that the antibodies in the yolk do not influence the growth of each organism, even if the hens are highly immunized.     

Resistance of broiler chickens to Escherichia coli respiratory tract infection induced by passively transferred egg-yolk antibodies

Egg-yolk antibodies induced by immunizing hens with selected Escherichia coli antigens were evaluated for their ability to protect broiler chickens against respiratory/septicemic disease caused by avian pathogenic E. coli (APEC). Seven groups of broiler breeder hens were vaccinated three times, 1 week apart with live E. coli, killed E. coli, E. coli antigens [lipopolysaccharide (LPS), type 1 pilus adhesin (FimH), P pilus adhesin (PapG), aerobactin outer membrane receptor (IutA)] or phosphate buffered saline (PBS). An O78 APEC strain was used for preparation of all the antigens. Egg yolk immunoglobulins (IgY) were purified from eggs of each group and antibody activity in serum and purified IgY was determined by enzyme-linked immunosorbent assay (ELISA). IgY (100mg) was injected intramuscularly into 11-day-old broiler chickens, which were challenged 3 days later with homologous (O78) or heterologous (O1 or O2) E. coli by the intra-air sac route. Mortality was recorded and surviving chickens were euthanized 1 week after the challenge and examined for macroscopic lesions. Passive antibodies against all antigens except FimH were protective (90-100%) against the homologous challenge, but only anti-PapG and anti-IutA were effective against heterologous challenge. Anti-PapG IgY provided the greatest protection against the three serogroups of E. coli used for challenge. Hence vaccination of broiler breeders to induce anti-PapG and anti-IutA antibodies may provide passive protection of progeny chicks against respiratory/septicemic disease caused by APEC.     

Immune responses in chickens against Salmonella typhimurium monitored with egg antibodies

Three mature hens were immunized with an Aro- mutant of Salmonella typhimurium beginning with a subcutaneous dose in adjuvant followed by two oral boosters. Isotype-specific antibodies were measured in the white and yolk eggs collected weekly over a period of 230 days. Two hens showed a memory response to the first oral booster, with large increases in egg yolk IgG and smaller increases in IgA and IgM antibodies in egg whites. Smaller amounts of IgA and IgM antibodies were found in egg yolks, and a slight increase in IgG occurred in the whites. One hen showed an increase in serum titers of all isotypes against S. typhimurium. The second hen had high serum titers before immunization was started which did not change. The third hen had a high level of IgM in the white of eggs before immunization was started. This hen showed erratic responses in egg white antibodies following immunization, no increase in IgA or IgM in yolks and only a slight increase in IgG, no increase in serum IgG, and was the only hen with a high level of IgM antibody against S. typhimurium in the bile, conditions reflecting a state of oral tolerance. With the exception of this hen, the results showed that IgA and IgM antibodies were aroused in hens by immunization with an avirulent mutant of S. typhimurium, and that these antibodies were present in the white of eggs from immunized hens.     

基因A型和C型鸭甲型肝炎病毒卵黄抗体的研制及应用

旨在研制基因A型和C型鸭甲型肝炎病毒(DHAV)单价及二价高免卵黄抗体,以满足实际生产中基因A型和C型DHAV单独或混合感染防控的需要。采用基因A型和C型DHAV强毒株作为抗原制备单价和二价油乳剂灭活疫苗免疫接种高产蛋鸡,制备了高效价的基因A型和C型DHAV单价及二价高免卵黄抗体,卵黄抗体鸭胚中和效价在1∶204与1∶281之间,单价及二价卵黄抗体防治试验证明,所制备的卵黄抗体防治效果良好。     

鸡抗猪大肠杆菌高免卵黄抗体的研制与应用

应用仔猪大肠杆菌标准菌株和地方分离菌株免疫商品蛋鸡，收获高免蛋制备多价卵黄抗体。经预防、攻毒试验及临床治疗效果观察，对仔猪免疫保护率可达90％以上，攻毒保护率98％以上，临床治愈率95％以上。     

Bovine monoclonal antibodies to the F5 (K99) pilus antigen of E. coli, produced by murine/bovine hybridomas

Lymph node cells from calves immunized with purified pilus antigen of K99+ enterotoxigenic E. coli (ETEC) were fused with mouse myeloma (NSO) cells, and with non-Ig producing mouse/calf hybridomas or with a bovine Ig-producing mouse/calf/calf secondary hybridoma. Lines secreting bovine monoclonal IgG1 specific for K99 pilus antigen in an ELISA were obtained in each case. The two lines derived from xenohybridoma fusion partners have been secreting anti-K99 bovine monoclonal antibody for over one year in continual passage. None of the antibodies cross-reacted with other pilus types including K88, CFAI, CFAII, 987P or CP; they all inhibited agglutination of horse RBC (which have a K99 receptor) in the presence of K99 antigen; they showed positive fluorescence in an indirect binding assay on K99+ ETEC and inhibited K99+ ETEC adhesion to piglet enterocytes. These antibodies have potential prophylactic and therapeutic use in control and treatment of diarrhoea.     

抗奶牛乳腺炎多价卵黄抗体的制备及含量测定

本研究的目的是制备抗奶牛乳腺炎主要致病菌的多价卵黄抗体并检测特异性抗体的含量。采用金黄色葡萄球菌、无乳链球菌和大肠杆菌混合抗原免疫产蛋母鸡。通过水稀释法分离卵黄抗体。采用ELISA法检测抗体效价和特异性抗体含量。多价抗体与大肠杆菌的结合效价最高可达25600,与金黄色葡萄球菌和无乳链球菌的结合效价为12800。每毫升卵黄液中大肠杆菌、金黄色葡萄球菌和无乳链球菌特异性卵黄抗体（egg yolk immunoglobulin,IgY）的最高含量分别为2.13、2.01和1.92 mg。免疫后特异性抗体含量随时间变化趋势与抗体效价变化趋势一致。     

抗猪流行性腹泻病毒卵黄抗体的制备及应用

本试验利用猪流行性腹泻病毒(porcine epidemic diarrhea virus,PEDV)SC株免疫产蛋鸡,收集高免卵黄,采用水稀释法和水稀释-盐析法获得鸡源抗PEDV卵黄抗体,采用已建立的ELISA方法对卵黄抗体效价进行测定。以3日龄无母源抗体的易感仔猪为试验动物,对抗PEDV卵黄抗体的治疗效果及安全性进行测定,并进一步利用自然发病猪场的治疗效果进行验证。结果表明,经PEDV SC株细胞毒免疫的鸡可在2次加强免疫后15 d产生高水平的特异性抗体。在实验室治疗试验中,攻毒治疗组仔猪的存活率达60%,攻毒对照组存活率为20%;用于自然发病猪场时,饲喂抗PEDV卵黄抗体饲料的仔猪存活率亦为60%,而自然发病对照组的存活率为10%。以上结果表明抗PEDV卵黄抗体对感染PEDV的仔猪有一定治疗作用。     

Inhibition of adhesion of F18+ Escherichia coli to piglet intestinal villous enterocytes by monoclonal antibody against blood group H-2 antigen

Enterotoxigenic and verotoxigenic F18⁺ Escherichia coli colonising the pig small intestine, adhere to receptors on intestinal villous enterocytes by F18 fimbriae. The aim of the present study was to define the F18R nature. The knowledge on the nature of this receptor could be important for the development of receptor-based treatments against F18⁺ E. coli-induced disease. The adhesion of F18⁺ E. coli to pig intestinal villous enterocytes was analysed in an in vitro assay. The adhesion of F18⁺ E. coli but not of F4ac⁺ E. coli was strongly inhibited by monoclonal antibodies (mAb) with blood group H-2 specificity. Conversely, blood group H-1 specific mAb could not inhibit the adhesion of F18⁺ E. coli nor F4ac⁺ E. coli. Moreover, the blood group H-2 trisaccharide strongly inhibited the adhesion of F18⁺ E. coli, but only partially the adhesion of F4ac⁺ E. coli. These data demonstrate that the F18 receptor contains the blood group antigen H-2 (-fuc-(1-2)-β-Gal-(1-4)-GlcNAc) as major carbohydrate.     

Antibody reactivity in mice and cats to feline enteroepithelial stages of Toxoplasma gondii.

The reactivity of antibodies in mice and cats to feline enteroepithelial stages of Toxoplasma gondii was examined by means of an indirect immunofluorescent antibody test. Mice immunized with feline enteroepithelial stage (FES) parasites produced antibodies not only against FES, but also against tachyzoites, sporozoites/oocysts, tissue cysts and one part of the infected feline enterocytes. After absorption with tachyzoites, the titer of antibodies reactive to enterocytes was significantly reduced. In contrast, the titer of antibodies reactive to FES remained unchanged. The antibodies from cats immunized with FES, reacted specifically to FES, but not to tachyzoites, tissue cysts or enterocytes. These results suggest that FES parasites may have stage-specific antigen(s).     

轮状病毒和大肠杆菌不同免疫方式免疫奶牛乳中抗体消长规律的研究

以引起婴儿腹泻的轮状病毒RV和大肠杆菌E.coli免疫妊娠后期的奶牛，使之产生抗这两种病原的抗体。免疫方式分为两种：一种是分别用E.coli和RV作免疫原免疫奶牛，另一种以两者同时免疫奶牛。然后定期采乳，分别以试管凝集反应和反应间接血凝抑制试验检测大肠杆菌抗体和轮状病毒抗体，并比较这两种不同免疫方式免疫奶牛乳中抗体的消长规律。试验结果表明：单独以大肠杆菌或轮状病毒作免疫原与这两者同时作免疫原，免疫奶牛乳中抗体消长规律相似，抗体效价也相近，且均可维持近两个月。     

抗猪繁殖与呼吸综合征病毒卵黄抗体的制备及临床应用

制备猪繁殖与呼吸综合征病毒高免卵黄抗体,研究其治疗效果。以猪繁殖与呼吸综合征灭活苗免疫产蛋鸡,采用ELISA方法检测卵黄抗体效价,收集高效价卵黄,采用氯仿抽提和硫酸铵盐析法纯化卵黄抗体。ELISA方法测定收集抗体效价为1∶8 000,测定纯化的抗体浓度为7.85mg/mL,微生物学检验及安全性试验表明制备的卵黄抗体安全可靠,人工感染治愈率和临床应用治愈率分别为100%和89.1%,表明制备的卵黄抗体对猪繁殖与呼吸综合征具有显著的治疗效果。     

猪圆环病毒2型高免卵黄抗体的研制与应用

为制备猪圆环病毒2型(porcine circovirus serotype2,PCV-2)高免卵黄抗体,并对其治疗效果进行研究,通过自制猪圆环病毒2型(PCV-2)灭活苗,免疫SPF产蛋鸡,收集高免蛋,制备PCV-2高免卵黄抗体,对高免卵黄抗体进行无菌检验、活体动物安全性检测,用琼脂扩散方法进行效价检验和人工感染保育猪治疗试验,并将其应用于临床实践,观察其治疗效果。结果抗体效价达到1∶64;人工感染治疗试验治愈率达100%;临床试验治愈率为96%,应用效果良好。     

抗小鹅瘟卵黄抗体脱脂方法研究

通过对产蛋母鸡免疫接种小鹅瘟JS1株油乳荆灭活苗,从而获得含高效价小鹅瘟病毒抗体的鸡蛋.采用聚乙二醇6000和泊洛沙姆进行脱脂试验,并优化脱脂条件.结果表明,聚乙二醇6000和泊洛沙姆对鸡卵黄具有良好的脱脂作用,其中聚乙二醇6000价格低廉,更适合大规模工业化生产.     

The chicken egg as a supply of polyclonal antibodies

Polyclonal antibodies can be isolated not only from the blood of immunized mammals but also from the egg yolk of immunized chickens. The advantages of this alternative method are: 1) Birds produce antibodies against highly conserved mammalian proteins. 2) The quantity of antigen needed for an efficient immune response is very low (20-30 micrograms). 3) The use of complete Freund's adjuvant leads to long lasting titers of yolk antibodies yielding a total amount of 65 mg specific antibodies per month. 4) The purification of antibodies is simple, inexpensive and quick. Polyethylene glycol precipitation is sufficient to obtain a purity of more than 90%. 5) Chicken antibodies are acid- and heat-resistant and might therefore be orally applied to prevent or to cure infectious intestinal diseases of young animals or humans. 6) Immunization with complete Freund's adjuvant is well tolerated and produces no inflammatory reactions and 7) collecting eggs is, in contrast to bleeding animals, non-invasive. In this review we present both, the method how to produce and to isolate yolk antibodies as well as their possible application in science, diagnosis, prophylaxis and therapy.