Antigenic relatedness and partial amino acid sequences of pili of Escherichia coli serotypes O1, O2, and O78 pathogenic to poultry

Pilus proteins from Escherichia coli serotypes O1, O2, and O78 pathogenic to poultry were compared with regard to their antigenic relatedness and partial amino acid sequences. Agglutination, immunodiffusion, and immunoblot assays with polyclonal antibodies to these pili showed that these pili not only share some common antigens but also contain antigens unique to each pilus. The partial amino-terminal amino acid sequences support our earlier findings that the pili are different but contain some structural homologies.     

Purification, characterization, and serologic characteristics of Bacteroides nodosus pili and use of a purified pili vaccine in sheep

Hair-like appendages (pili) were isolated and purified from Bacteroides nodosus, the etiologic agent of foot rot disease in sheep. Microscopic and biochemical analyses indicated that pili from organisms isolated in Australia, New Zealand, and the United States are morphologically and structurally similar. Pili are filamentous assemblies of identical protein subunits. Using specific antisera raised in rabbits against pili, 7 antigenic types were identified. A geographic pattern in the distribution of the pilus serotypes was not evident. In a preliminary vaccine trial, sheep vaccinated with purified pili developed resistance to challenge exposure to B nodosus. Protection was correlated positively with the serum anti-pilus antibody titers.     

Monoclonal antibodies against pili of serologically distinct Bacteroides nodosus.

Several monoclonal antibodies (McAbs) against pili of Bacteroides nodosus were examined to determine their reactivity with 11 different serotypes. One McAb was identified by enzyme-linked immunosorbent assay (ELISA) analysis that bound to nine of the 11 serotypes and another that bound to the remaining two serotypes tested. In addition, some McAbs demonstrated specificity for a single serotype, while others displayed specificities for up to five other serotypes. Comparison of immunoblot analysis with the ELISA revealed that the former method was not as sensitive in that all McAbs positive by the ELISA, were not positive by immunoblot. Possible explanations of these findings are discussed. There appear to be several antigenic determinants on B. nodosus pili and considerable sharing of these determinants between pili types. The 11 serotypes analyzed by the McAbs in this report are representative of all 20 US serotypes as well as the A-set and D-set categories of Australia. Therefore, the two epitopes recognized by two of the McAbs reported herein encompass all of the currently characterized B. nodosus serotypes and may provide a basis for bivalent vaccines efficacious for all types of B. nodosus induced footrot in sheep.     

动物性食品源大肠杆菌O血清型鉴定及其K88菌毛基因检测

本研究对河北省冀东地区农贸市场和超市采集的生猪肉、生鸡蛋和生羊肉等分离得到的20株大肠杆菌进行大肠杆菌血清型鉴定;并检测不同血清型大肠杆菌的K88菌毛基因。采用常规方法进行大肠杆菌的O血清型鉴定,用PCR方法检测K88菌毛基因。分离鉴定的20株大肠杆菌有7种血清型,包括O38、O78、O88、O11、O107、O91、O9,其中O38、O78为优势血清型菌,均占分离菌株的25%(5/20)。在分离的动物性食品源大肠杆菌中有30%(6/20)的菌株K88菌毛基因扩增呈阳性。结果表明,O78、O38为冀东地区动物性食品源大肠杆菌常见血清型,30%(6/20)的菌株K88菌毛基因扩增阳性。     

Laboratory and field trials with formalin-inactivated Escherichia coli (O78)-Pasteurella anatipestifer bacterin in white pekin ducks

A combination Escherichia coli serotype O78 and Pasteurella anatipestifer bacterin was developed and tested in white pekin ducks in laboratory and field trials. Inoculations with bacterin at 2 and 3 weeks of age provided significant protection against challenge with virulent E. coli O78 and Pasteurella anatipestifer serotypes 1, 2, and 5. No significant cross-protection was observed against heterologous E. coli serotypes, although there was a slight reduction in mortality in ducklings challenged with E. coli serotypes O2a and O119. In field trials, the E. coli-P. anatipestifer bacterin produced significant reduction of mortality in commercial white pekin ducks compared with P. anatipestifer bacterin.     

Antigenic structure and relationship between serotypes 1 and 2 of Haemophilus paragallinarum.

Antigenic structure and relationship between serotypes 1 and 2 of Haemophilus paragallinarum were analyzed by the rapid plate agglutination and cross-absorption tests. Encapsulated strain forming iridescent colony type of both serotypes 1 and 2 had at least three antigens: heat-labile and trypsin-sensitive (L), heat-labile and trypsin-resistant (HL), and heat-stable and trypsin-resistant (HS). The L was a major antigen located in a surface and divided serologically into three parts: L1, L2, and L3. The L1 was specifici to serotype 1, the L2 was specific to serotype 2, and the L3 was a common antigen shared by serotypes 1 and 2. The HL and HS were common antigens between serotypes. By trypsinization or heating at 121 C, L antigen lost its agglutinability and agglutinin-producing ability. Nonencapsulated organisms forming noniridescent colony type lacked the L antigen. These results suggested that antigenic structure of H paragallinarum serotypes 1 was L1, L3, HL and HS, while serotype 2 was L2, L3, HL, and HS.     

Purification and characterization of a protease from Actinobacillus pleuropneumoniae serotype 1, an antigen common to all the serotypes.

A high molecular-mass proteolytic enzyme of Actinobacillus pleuropneumoniae serotype 1, was purified from culture supernatants (CSN) by using DEAE-cellulose and sepharose-4B-gelatin chromatography. In 10% SDS-polyacrylamide gels copolymerized with porcine gelatin, the protease showed a single band of activity of > 200 kDa. However, minor molecular-mass proteolytic bands were observed when the protease was electrophoresed in the presence of either 5% beta-mercaptoethanol, 50 mM dithiothreitol, or 0.25 M urea. Furthermore, when the > 200-kDa purified protein was passed through a sucrose gradient, several bands with proteolytic activity were found: 62, 90, 190, and 540 kDa. The proteolytic activity was increased in the presence of calcium or zinc and was not affected after being heated at 90 degrees C for 5 min. Proteolytic activities were also observed in CSN from all A. pleuropneumoniae serotypes and biotypes. The purified protease hydrolyzed porcine IgA and IgG in vitro. In addition, by immunoblot the protease was recognized by serum of naturally infected pigs with serotypes 1 and 5, and by serum of pigs experimentally infected with serotypes 1, 2, 8, or 9. Serum of a pig vaccinated with CSN of a serotype 3 strain also recognized the protease, but not sera of pigs vaccinated with a bacterin (serotype 1). Proteins from CSN of all the serotypes, which were precipitated with 70% (NH4)2SO4, were recognized by a polyclonal antibody raised against the purified protease. Taken together these results indicate that an antigenic protease is produced in vivo by all the serotypes of A. pleuropneumoniae. The results indicate that proteases could have a role in the disease and in the immune response of pigs infected with A. pleuropneumoniae.     

Expression of Escherichia coli antigens in Salmonella typhimurium as a vaccine to prevent airsacculitis in chickens

Avian pathogenic Escherichia coli strains are associated with a variety of extraintestinal poultry diseases, including airsacculitis, colisepticemia, and cellulitis. A number of E. coli serotypes are associated with these diseases, although the most prevalent serotype is O78. Fimbrial proteins expressed by these strains appear to be important virulence factors, including type 1 fimbriae, P fimbriae, and curli. We have been working to develop an effective vaccine to protect chickens against these diseases. We have previously shown that an attenuated Salmonella typhimurium strain expressing O78 lipopolysaccharide provides protection against challenge with an O78 avian pathogenic E. coli strain. In this work, we have constructed an attenuated S. typhimurium that expresses both the O78 lipopolysaccharide and E. coli-derived type 1 fimbriae. In these studies, chickens were vaccinated at day of hatch and again at 2 wk of age. Birds were challenged at 4 wk of age. We found that the vaccine candidate provided significant protection against airsacculitis as compared to untreated controls or birds vaccinated with an attenuated S. typhimurium that did not express any E. coli antigens. In a separate experiment, challenged vaccinates showed significant weight gain compared to challenged nonvaccinates. We were not able to demonstrate protection against E. coli O1 or O2 serotype challenge, nor against challenge with wild-type S. typhimurium.     

Characterization of Escherichia coli isolated from cases of avian colibacillosis.

Forty-four western Canadian isolates of Escherichia coli associated with colibacillosis of turkeys and chickens were examined for serotype, antibiotic resistance, and production of aerobactin. The isolates belonged to fourteen O serogroups, with 39% of the strains being non-typeable. A high frequency of resistance to tetracycline, kanamycin, neomycin, cephalothin, streptomycin and erythromycin was observed. Most isolates produced aerobactin. Ten E. coli belonging to serogroups O1, O2 and O78 were also examined for pili production, hemagglutination, serum sensitivity, production of iron-regulated outer membrane proteins (IROMPS), and virulence. All isolates examined produced pili, exhibited mannose-sensitive hemagglutination of avian red blood cells and produced IROMPS under iron-restricted growth conditions. The five isolates of serogroup O1 and O2 were resistant to killing by turkey serum and were highly virulent. Only two of the five isolates of serogroup O78 were serum resistant. No correlation between serum resistance and virulence was observed in serogroup O78.     

Adhesin-receptor interactions mediating the attachment of pathogenic Escherichia coli to chicken tracheal epithelium

Specific adherence of pathogenic Escherichia coli (serotypes O1, O2, and O78) to chicken tracheal epithelium was investigated using adherence-inhibition procedures. The role of pilus as adhesin was studied by blocking the pilus with antipilus antibodies. The nature of the host cell receptor was determined by blocking bacterial adhesion with specific carbohydrates or lectins and destroying the receptor with sodium metaperiodate. Antipilus antibodies to all three serotypes significantly (P less than or equal to 0.05) inhibited their adherence. Sodium metaperiodate considerably inhibited the adhesion of all three serotypes, indicating a role for monosaccharides in the host cell receptor. D-Mannose and its derivative methyl-alpha-D-mannopyranoside inhibited the adhesion of serotypes O1 and O78, indicating a role for these sugars in the host cell receptor; this was further supported by the inhibition of both serotypes after treatment of tracheal epithelium with concanavalin A. None of the sugars or lectins used inhibited adhesion of serotype O2.     

An enzyme-linked immunosorbent assay for detection of antibodies against Escherichia coli: association between indirect hemagglutination test and survival

An enzyme-linked immunosorbent assay (ELISA) was modified for detection of antibodies against the two main pathogenic serotypes of Escherichia coli: serotypes O78:K80 and O2:K1. The ELISA was a more sensitive and repeatable test than the indirect hemagglutination test (IHT), which is a common method for detecting antibodies against E. coli. Cross-reactivity between the two strains was measured by reacting antisera of each serotype against homologous and heterologous antigens. The results suggest that aside from similar determinants expressed by the two serotypes, serotype O2:K1 expresses more strain-specific determinants than does O78:K80. Comparison of mean antibody titers of immunized chicks by IHT and ELISA along the primary response revealed that during the first 15 days after immunization with inactivated E. coli, the titers in both tests were parallel. After 15 days post-immunization, antibody titers measured by IHT decreased rapidly, whereas titers measured by ELISA decreased only slightly. In addition, a higher correlation was found between titers detected by ELISA and survival through challenge with E. coli than between titers detected with IHT and survival through challenge. The results suggest that the ELISA is a better test for detection of antibody in flocks suspected of being infected with E. coli.     

Antigenic relationship of Ibaraki,bluetongue, and epizootic Hemorrhagic disease viruses

Ibaraki virus, which causes a bluetongue-like disease of cattle in Japan, was compared antigenically with the four serotypes of bluetongue virus (BTV) found in the U.S. and with the two serotypes of epizootic hemorrhagic disease virus (EHDV). No antigenic relationship was found between Ibaraki virus and BTV serotypes 10, 11, 13, and 17 in tests for group or serotype-specific antigens. However, Ibaraki virus and EHDV were related antigenically. The agar gel precipitin and indirect fluorescent antibody tests for group antigens showed two-way cross relationships between Ibaraki virus and EHDV serotypes 1 and 2. The more restrictive serotype-specific neutralization test revealed that antigenic relatedness was stronger between Ibaraki virus and the serotype 2 (Alberta strain) of EHDV than between Ibaraki virus and the serotype 1 (New Jersey strain) of EHDV.     

鸡大肠杆菌多价1型菌毛亚单位疫苗在鸡体内的免疫原性研究

用1日龄雏鸡免疫评价了鸡大肠杆菌多价1型菌毛油乳剂苗在鸡体内的免疫原性。用含1型菌毛的3个不同血清型（O1、O78及O88）菌株,大容量培养后提取菌毛制备多价菌毛油乳剂苗。接种多价菌毛油乳剂苗的鸡用同源菌株攻毒后出现10％～11．2％的死亡率,阳性对照组鸡攻毒后出现70％～80％的死亡率;用异源菌株（O36）攻毒后,免疫鸡出现33．3％的死亡率,而未免疫阳性对照组鸡死亡率达70％。免疫鸡在气囊,心包及肝脏的病变非常轻微,且攻毒后非免疫鸡能更有效地清除侵入气囊的大肠杆菌。     

Serotypes of Escherichia coli isolated from healthy infants in Berlin,Germany and Melbourne,Australia

The characterization of Escherichia coli strains isolated from healthy infants under one year of age with respect to O:H serotype, K1 and K5 antigens in two disparate parts of the developed world was the purpose of this investigation. A total of 450 strains were examined, 264 from Berlin and 186 from Melbourne. Of all the 220 different O:H serotypes found, 179 were only isolated once, 90 in Berlin and 89 in Melbourne. However, 30 of the 41 O:H serotypes (73.2%) found more than once were isolated in both centers. The most commonly identified serotypes were found in both centers and included O1:H-; O1:H7; O2:H2; O2:H4; O2:H7; O4:H5; O6:H-; O6:H1; O15:H1; O18:H7; O25:H1; and 075:H-. Potentially pathogenic serotypes were found in both cities. Enteropathogenic E. coli (EPEC)-associated serotypes (O18:H7; O26:H-; O44:H34; O86:H-; O128:H2) were present in 11 cases and enterohaemorrhagic E. coli (EHEC)-associated types including O26:H11; O128:H2) were present in four cases. The distributions of serotypes found were similar in the two cities, strongly suggesting the wider applicability of these results.     

The serological response of cattle immunized with Yersinia enterocolitica O:9 or O:16 to Yersinia and Brucella abortus antigens in enzyme immunoassays

Sera from calves immunized with Yersinia enterocolitica serotypes O:9 or O:16 were tested by indirect enzyme linked immunosorbent assay (ELISA) using lipopolysaccharide (LPS) preparations from Brucella abortus or Y. enterocolitica O:9 or O:16 for their antibody content of the IgG1 or IgG2 subclasses. High IgG1 responses were present with the three antigens in both groups although some individual variations between animals were noted. The IgG2 responses were modest and in some cases not above background 'noise'. Thus IgG2 antibody was not measurable in sera from serotype O:9 injected calves when using serotype O:16 LPS or in serotype O:16 injected calves when using B. abortus or serotype O:9 LPSs. A competitive ELISA using B. abortus O-polysaccharide and a monoclonal antibody to B. abortus LPS (initially designed to differentiate the antibody responses of cattle naturally infected with B. abortus from those vaccinated with strain 19) was used on sera from both groups of calves. Using this test, no antibody was detected in the group immunized with serotype O:16 and except for one animal in the serotype O:9 immunized group, only low levels of antibody were transiently in evidence. One animal in this group responded with quite high levels of competing antibody which, however, declined towards the end of the test period. The competitive ELISA may prove a useful serological tool for differentiating vaccinal and field infection titers to B. abortus and also to eliminate cross-reactions observed with Y. enterocolitica serotypes.     

Serotyping of Haemophilus pleuropneumoniae in the Netherlands: with emphasis on heterogeneity within serotype 1 and (proposed) serotype 9

Four hundred and forty-three Dutch field isolates of Haemophilus pleuropneumoniae were serotyped by rapid slide agglutination (RSA) using specific antisera against serotypes 1 to 5 and against the recently proposed types 6 to 9. The predominant serotypes were 9 (49%) and 2 (32%). Serotypes 1, 3, 5, 7 and 8 were isolated in small numbers: together they accounted for 3% of the total. Five percent of the isolates were not typable either due to autoagglutination or because they were not agglutinated by any of the available antisera. The remaining 49 strains (11%) agglutinated in more than one antiserum and could therefore not be properly classified. Forty-four of these 49 strains agglutinated in both anti type 1 and anti type 9 serum. Antigenic relationships between serotype 1, serotype 9 and isolates reacting with both antisera were studied using immunodiffusion and RSA with adsorbed sera. Serotype 9 strains appeared not to be a homogenous group. Isolates agglutinating exclusively in anti type 9 serum can be divided into two groups: one closely related and another hardly related to serotype 1. Serotype 9 reference strain 13261 belongs to the latter. Type 1 + 9 strains have antigens in common with serotypes 1 and 9, but they also have their own specific antigenic material. Such strains are proposed as a new serotype 10.     

SEROLOGICAL CLASSIFICATION OF AUSTRALIAN STRAINS OF ERYSIPELOTHRIX RHUSIOPATHIAE ISOLATED FROM PIGS, SHEEP, TURKEYS AND MAN

154 strains of Erysipelothrix rhusiopathiae from pigs, sheep, turkeys and man were serotyped by using the double diffusion gel precipitation test. Ten of the 18 serotypes were detected in 151 of the strains. Three strains failed to react with any of the type specific antisera. It was found that serotype 1a shared an antigen(s) with serotype 1b, and that serotype 6 shared an antigen(s) with serotype 14. Serotype 2a and 2b were difficult to distinguish. Since serotypes 1 and 2 were isolated from cases of septicaemia in pigs, and since serotypes 1, 2, 4 and 7 were isolated from cases of arthritis, it was suggested that factors other than serotype were important in causing the various forms of swine erysipelas. The fact that the distribution of serotypes 1a, 1b and 2b between septicaemic and arthritic pigs was similar supported the conclusion that arthritis was consequent to bacteraemia. Serotypes 1a, 1b, 2b, 5, 12 and 15 were isolated from cases of arthritis in sheep, and serotypes 1a and 5 from cases of erysipelas in turkeys. Serotype 2b was isolated from a human specimen.     

Cross-protection of Salmonella abortusovis, S. choleraesuis, S. dublin and S. gallinarum in mice induced by S. abortusovis and S. gallinarum: bacteriology and humoral immune response

Cross-protection induced by primary infection with Abortusovis and Gallinarum was examined against challenge injection with these Salmonella serotypes as well as with Dublin and Choleraesuis, the other virulent serotypes. Abortusovis induced efficient protection against the other Salmonella. Gallinarum was ineffective against Choleraesuis. Even with low multiplication in mice, the Gallinarum J91 strain induced a weak but significant protection against Dublin (same O group serotype). The antibodies in the blood of mice were tested with ELISA specific for the Salmonella antigens used to prime or to challenge animals. The Gallinarum J91 strain was detected to be more antigenic in ELISA than the other Salmonella antigens. It is difficult to conclude on a correlation between IgM or IgG antibodies and induction of protection, because of the variability in immune response according to the different serotype used. Nevertheless, the negative linkage between a number of bacteria in the spleen of mice challenged with Gallinarum and Dublin, and the level of IgM and IgG antibodies specific for the challenging serotype, showed that humoral immune response could be one element of cross-protection, mainly by the immune response against the same O serotype.     

奶牛粪样中致病性大肠埃希菌的分离与鉴定

大肠埃希菌血清型众多,一些具有特殊血清型的大肠埃希菌对人和动物有致病性。本试验从包头市侯家营奶牛场77头外表健康成年泌乳荷斯坦奶牛采集新鲜粪样,进行细菌分离培养,共分离到疑似大肠埃希菌69株,经过形态观察、生化鉴定、动物致病性试验和血清型鉴定,其中12株为致病性大肠埃希菌,均为强毒株,占分离菌数的17.4%,另57株为非致病性大肠埃希菌,占分离菌数的82.6%。同时对12株致病性大肠埃希菌进行了"O"血清型鉴定,分属7种血清型O78、O6、O9、O98、O1、O8、O149,分别占定型菌株的25.0%(3/12)、8.3%(1/12)、16.7%(2/12)、8.3%(1/12)、8.3%(1/12)、8.3%(1/12)、25.0%(3/12)。     

Serological characterization of Haemophilus pleuropneumoniae (Actinobacillus pleuropneumoniae) strains and proposal of a new serotype: serotype 9

Ten strains of H. pleuropneumoniae isolated from 10 herd outbreaks of pleuropneumonia were studied by means of the slide agglutination test, the indirect haemaggluitiniation (IHA) test and by gel diffusion. The strains were antigenically homogeneous and serologically distinct from serotypes 1 through 8. It is therefore proposed to refer these strains to a new serotype: serotype 9, with strain CVJ 13261 as the type strain.In addition to the serotype-specific capsular antigens, capsular antigen of serotype 1 (strain 4074) could be demonstrated in the 10 strains by means of gel diffusion analyses.In cross protection studies it was shown that the antigenic determinants shared by serotypes 9 and 1 were unable to yield a sufficient protection against disease. Thus, parenteral immunization with a killed 6-h culture of serotype 9 did not afford an acceptable protection against challenge with serotype 1 since only 3 of the 5 vaccinates were protected. The reverse experiment showed that parenteral immunization with serotype 1 only protected 1 out of 4 vaccinates.