禽大肠杆菌病免疫保护机理的研究

以禽病原性大肠杆菌O18、O78分离株制成超声波裂解铝佐剂灭活苗免疫14日龄鸡，以相同或不同外膜蛋白型（Outer membrane protein pattern,OMP型）的O18、O78分离株攻毒。结果表明：O78血清相同和不同OMP型分离株间能获得最大保护；O18血清型相同OMP型分离株间获得最大保护，而不同OMP型分离株间不能保护；上述两个血清型的分离株间不论OMP型是否相同，均缺乏保护。以间接ELISA试验、间接血凝试验分别测定了试验鸡临攻毒前针对大肠杆菌OMPs和脂多糖(Lipopolysaccharide,LPS）的抗体。结果表明：免疫组鸡血清上述两种抗体明显高于攻毒对照组；在免疫组，存活鸡临攻毒前血清中上述两种抗体滴度恒高于死亡鸡，但除3个组外，多数组差异不显著。攻毒对照组这一关系不稳定。结果说明：禽大肠杆菌疫苗的免疫保护，主要与O血清型有关，部分与OMP型有关，如O18分离株，免疫保护性抗原含OMPs,LPS等多个抗原表位。     

Quantitation by ELISA of pili and sheep antibodies to the pili of Bacteroides nodosus

ELISA systems have been developed to quantitate the isotypic antibody response of sheep naturally infected with B. nodosus isolate 198 or injected with pili from isolate 198 in oil emulsion vaccines. The predominant humoral antibody detected following vaccination was IgG1, with substantially lower amounts of IgG2 and IgM. The antibody response was relatively specific for the pilus antigen from isolate 198. Although weak cross reactivities were detected with antiserums to some other isolates, ELISA IgG antibody titres in excess of 200 offer a tentative identification of the isolates of B. nodosus involved in natural outbreaks of footrot. A related ELISA was also developed to quantitate the amount of pili in cell suspensions and crude preparations of pili used in vaccines.     

Comparison of alum-absorbed or non-alum-absorbed oil emulsion vaccines containing either pilate or non-pilate Bacteroides nodosus cells in inducing and maintaining resistance of sheep to experimental foot rot

Highly pilate (P) or non-pilate (NP) cells of Bacteroides nodosus were compounded into oil emulsion (O) either with or without prior absorption onto alum (A). The abilities of these four preparations (referred to as PAO, NPAO, PO and NPO vaccines) to stimulate antibody production and to protect sheep from foot rot were compared. Two injections of PAO vaccine protected sheep against homologous challenge 12 weeks after the second dose by PO, NPO and NPAO vaccines were less effective. Sheep were protected against homologous challenge for 14 weeks after a single dose of PAO vaccine and for 22 weeks after three doses; an ameliorative effect was still evident 40 weeks after the third dose. Protection against challenge with two heterologous strains was demonstrated at six weeks after three doses of vaccine. A numerical system of scoring the lesions also confirmed that foot rot in vaccinated sheep challenged outside the 'protective' period of the vaccine was somewhat less severe than in controls. PAO vaccine induced much higher and more persistent titres of agglutinins than the other vaccines tested. There was a relationship between agglutinin titres and resistance to homologous challenge.     

Studies on the use of Yersinia enterocolitica 09 antigens in rapid quantitative plate tests for the differentiation of Brucella and Yersinia infections in cattle and the diagnosis of bovine brucellosis

The use of the rapid quantitative plate agglutination test (QPAT) utilizing triphenyltetrazolium chloride (TTC) stained antigen for the differentiation of bovine brucellosis and yersiniosis is described. It was found with experimental laboratory animals and cattle, that titres against homologous antigen tended to exceed titres against heterologous antigen and that this reaction together with measurement of the Yersinia OH agglutinin titre could be used for purposes of differentiation. It was, however, not possible to differentiate these infections in serum from cattle with naturally-occurring antibodies. It was also shown that Brucella 0 agglutination in the QPAT was very weak in some sera with high titred anti-Brucella antibodies by serum agglutination, while Yersinia 0 agglutination was not. The QPAT, using Yersinia 0 antigen, therefore correlated well with the results obtained using Brucella 0 antigen in serum agglutination tests. It is suggested that Yersinia 0 antigen may be a suitable antigen for use in rapid agglutination tests because of this relative insusceptibility to false negative results.     

Development and application of an ELISA for detecting antibodies to Salmonella enteritidis in chicken flocks.

Enzyme-linked immunosorbent assays (ELISAs) were developed for the detection of IgG antibody to Salmonella enteritidis in poultry flocks. A lipopolysaccharide (LPS) and heat-extracted (HE) antigen for use in the ELISA were evaluated together with the rapid slide test (RST), microagglutination test (MT) and the microantiglobulin (MAG) test. In experimentally infected specific pathogen free chickens, good correlation was seen between all tests although, generally, the MT and MAG detected antibody earlier and titres peaked earlier than the ELISAs. The LPS antigen detected antibody earlier than the HE antigen but the latter gave higher titres in the later stages of infection. Cross reactions were seen between S enteritidis and S typhimurium in the ELISAs although homologous reactions were always much higher. Antisera to S montevideo or S senftenberg gave weak positive reactions in both S enteritidis ELISAs. Serological and bacteriological examinations of representative samples from two commercial chicken flocks were carried out. In flock A the HE-ELISA and MAG test detected antibody in nearly all birds. The LPS-ELISA detected antibody in over 60 per cent of birds, while the MT and RST detected few seropositive birds. The whole blood test using the stained S pullorum antigen on the farm detected antibody in just under 25 per cent of the birds. S enteritidis was isolated from the organs of 25 per cent of the birds. All birds in flock B were seronegative by all tests; no salmonellae were isolated from the organs of these birds.     

Surface antigens of virulent strains of Aeromonas hydrophila

Antiserum was raised in rabbits to whole cells of a representative strain from a group of A. hydrophila strains exhibiting enhanced virulence for fish. The major surface antigens of the strain were identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. The lipopolysaccharide (LPS) was examined using SDS-PAGE and silver staining. It was found to possess O polysaccharide chains of homogeneous length that were highly immunogenic. The LPS was conserved both morphologically and antigenically throughout the high virulence group. Heat-labile protein antigens were detected after absorption of the antiserum with boiled cells of the homologous strain. Only one major protein antigen, with a molecular weight of approximately 52,000, was present in outer membrane preparations or in whole cell lysates. A representative strain from the high virulence group, strain TF7, was shown by electron microscopy to be covered by a regular surface protein array (S-layer) which was found to be composed primarily of the 52 KD protein antigen. All the other members of the A. hydrophila high virulence group were shown to possess similar S-layers.     

Cross-protection from Bacteroides nodosus vaccines and the interaction of pili and adjuvants

The effects of vaccination of Merino sheep with the purified pili or the whole cells of Bacteroides nodosus strain 198, either in oil or alum-oil adjuvant, on the severity of foot-rot induced with the homologous strain (198) and a heterologous strain (217) were determined in a field experiment, on flood irrigated pasture. The efficacy of the whole cell vaccines was comparable to that of purified pili vaccines, against homologous challenge, when both had a similar content of pilus antigen although the purified pili vaccines induced significantly greater homologous pilus agglutinating antibody titres than the whole cell vaccines. However, against heterologous challenge, the whole cell vaccines in oil (CO) or alum-oil (CAO) provided significantly greater protection than a purified pili-in-oil (PPO) vaccine, the number of severely affected feet in sheep vaccinated with PPO being similar to that of the unvaccinated group. The group vaccinated with purified pili in alum-oil (PPAO) was intermediate between these two extremes. The superior performance of the PPAO in comparison to the PPO vaccine, against heterologous challenge, was associated with significantly higher mean ELISA titres to the outer membrane complex. Western blot analyses implicated a role in cross-protection for outer membrane proteins, in particular a protein Mr 78,000. The PPO vaccine produced fewer, smaller and less persistent vaccination reactions at the inoculation sites than did the other vaccines. Bodyweight gains in the period prior to challenge were much lower for the groups vaccinated with CO and CAO than for the controls and those vaccinated with purified pili, due presumably to the larger vaccination reactions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Construction and evaluation of a delta cya delta crp Salmonella typhimurium strain expressing avian pathogenic Escherichia coli O78 LPS as a vaccine to prevent airsacculitis in chickens.

Avian pathogenic strains of Escherichia coli cause a number of extraintestinal diseases in poultry, including airsacculitis and colisepticemia. Expression of O78 lipopolysaccharide (LPS) is frequently associated with pathogenic isolates. Salmonella, a common poultry contaminant, is a major public health concern. The purpose of this work was to develop an E. coli vaccine for poultry with the use of an attenuated Salmonella typhimurium carrier that would benefit both the bird and the consumer. Orally administered attenuated S. typhimurium delta cya delta crp strains have been shown to provide excellent protection against wild-type Salmonella challenge in chickens. This work describes the construction of a delta cya delta crp derivative of an avian pathogenic S. typhimurium that expresses both the homologous group B determinants (O1,4,5,12) and the heterologous E. coli O78 LPS O antigens. This was accomplished by inserting the E. coli rfb region, which encodes the genes required for O78 expression, into the chromosomal cya gene of S. typhimurium, creating a defined deletion/insertion mutation. A delta crp mutation was introduced in a subsequent step. Expression of both O antigens was stable in vitro and in vivo. Vaccination of white leghorn chicks at day of hatch and 14 days with the recombinant vaccine strain induced serum immune responses against both S. typhimurium and E. coli LPS and protected the birds against subsequent challenge with an avian pathogenic E. coli O78 strain. Introduction of a mutation in rfc, which encodes the O antigen polymerase, reduced the chain length of the S. typhimurium LPS without affecting the expression of O78. The rfc mutation further enhanced the ability of the vaccine strain to protect chickens against E. coli challenge.     

Evaluation of the indirect fluorescent antibody test for Babesia major and Theileria mutans in Britain.

Use of the indirect fluorescent antibody (IFA) tests is described to detect antibodies to Theileria mutans and Babesia major in the sera of infected cattle. When antisera against T mutans and B major were tested against homologous antigens high antibody titres were recorded: when they were tested against each other or against Babesia divergens antigen insignificant titres (1/40 or less) were recorded. Thus the test was found to be species specific. Animals recovered from T mutans and B major infections retained significant levels of IFA titres for 22 and 11 months respectively. It is suggested that the IFA test could be used for field survey of the piroplasms of cattle in Britain.     

Duration of resistance to experimental footrot infection in Romney and Merino sheep vaccinated with Bacteroides nodosus oil adjuvant vaccine

Groups of 10-12 Romney and Merino wethers were challenged simultaneously with homologous experimental footrot infection after having received the second of 2 doses of Bacteroides nodosus (strain 198) vaccine 0, 4, 8, 12, 16 and 20 weeks previously. Inoculations were carried out 28 days apart and unvaccinated sheep of both breeds were challenged as controls. Most Romneys that had been vaccinated up to 16 weeks prior to challenge were resistant to footrot whereas 8 of 10 controls were susceptible. This resistance was lost by about 20 weeks after vaccination. By contrast, protection against challenge in vaccinated Merinos lasted only about 4-5 weeks, although residual benefits of vaccination were apparent after longer intervals from the reduced number and severity of foot lesions among vaccinates compared with controls. Agglutinin titres, which did not differ markedly after similar intervals between Romneys and Merinos, reached maximum levels between 4 and 8 weeks after the second vaccine dose and subsequently declined. Although peak titres were generally recorded at the time of maximum protection in Merinos, the relationship between agglutinin levels and resistance in the Romneys was ill-defined.     

Phenotyping and genotyping of Haemonchus contortus isolates reveals a new putative candidate mutation for benzimidazole resistance in nematodes

In order to monitor and eventually control the spread of drug-resistant Haemonchus contortus, knowledge of the molecular mechanisms underlying resistance is essential. Here we phenotypically and genotypically characterize three multidrug-resistant H. contortus field isolates from Australia and South Africa. All were significantly less susceptible to thiabendazole than a sensitive reference strain in an in vitro egg-hatch assay. The sensitivity was further reduced in a surviving population after treatment of infected sheep with albendazole. The beta-tubulin genes were amplified from genomic DNA of the H. contortus isolates, cloned, and sequenced. There was a high degree of sequence variation. The known mutation phenylalanine-200 to tyrosine (F200Y) occurred in 60% of the sequences from resistant isolates, but not in the sensitive reference. Interestingly, 90% of the beta-tubulin sequences from resistant isolates lacking tyrosine-200 carried another mutation nearby, glutamate-198 to alanine (E198A). This mutation was not found in the sensitive isolate, nor in sequences from resistant isolates carrying the mutation F200Y. However, the mutation E198A is known from benomyl-resistant isolates of phytopathogenic fungi such as Monilinia fructicola. The finding that alanine-198 correlates with thiabendazole resistance in H. contortus isolates from South Africa and Australia suggests that also in nematodes, the mutation E198A plays a role in benzimidazole resistance. Alanine-198 alleles of beta-tubulin can be detected by PCR-RFLP and we suggest to include this test in future surveys of H. contortus field populations.     

Detection of fish antibody against protein antigen of Aeromonas salmonicida by enzyme-linked immunosorbent assay using biotin-avidin system

Antibody against Aeromonas salmonicida was detected in sera from immunised or experimentally infected rainbow trout by enzyme-linked immunosorbent assay (ELISA) using the biotin-avidin system. The ELISA titre correlated well with the agglutinin titres of the sera, but the ELISA was found to be more sensitive than the agglutination test. When the rainbow trout serum was separated by column chromatography, antibody activity (determined by ELISA and agglutination test) was detected in the IgM fractions. Minimum cross reaction was observed in the ELISA system between antigen prepared from A salmonicida and antibodies against Vibrio species and other species of Aeromonas. The specificity of the ELISA was also confirmed by inhibition test. Immunisation of rainbow trout with a virulent strain of A salmonicida provided good protection, though no correlation was observed between the protection and the ELISA titres of sera.     

A selected water-in-oil emulsion: Composition and usefulness as an immunological adjuvant

A selected water-in-oil (W/O) emulsion was evaluated in terms of stability and antibody-response and side-effects in experimental animals.The results from stability tests performed at 4°C and 37°C indicate a storability of the W/O emulsion of more than a year under normal storage conditions. The antibody-response of rabbits after injection of E.coli K99 antigen and bovine serum albumin (BSA) using the W/O emulsion as vehicle was generally significantly higher than the response to the same antigens injected within incomplete Freund's adjuvant (IFA): twenty days after the first antigen injection the rabbits injected with antigen in the W/O emulsion showed average anti-K99 and anti-BSA titres which were ca 5x as high as the titres in sera from rabbits injected with the antigens in IFA. Thirty-two and forty-two days after the first antigen injections the W/O group showed anti-K99 titres which were respectively ca 3.5x and 2x higher than titres in the IFA group, while anti-BSA titres in the W/O group were respectively ca 13x and 6.5x higher than in the IFA group.Intra-muscular and/or subcutaneous injection of the W/O emulsion in rabbits and pigs resulted in a slight degeneration of local tissue. When antigens were present in the emulsion granulomatas were often observed. The rise in temperature in pigs after injection of killed E.coli 0149:K91,K88 in the W/O emulsion was comparable with that induced by E.coli in physiological saline.From these results it is concluded that the W/O emulsion is likely to be a promising basis for veterinary vaccines and that the application of the W/O emulsion may contribute to the limitation of the number of animals used for raising antibodies for general immunological purposes.     

Detection of African swine fever virus antigen and antibody by radioimmunoassay

Three groups of animals were infected with three different African swine fever virus isolates. Antibody responses were measured, using immuno electro-osmophoresis, reverse single radial immunodiffusion and radioimmunoassay (RIA). The RIA detected antibody at 3–4 days whereas the diffusion tests did not detect it until 10 days post inoculation. Virus survival in different transport media was compared, using haemadsorption assays and RIA. Glycerinated medium reduced infectivity titres, although it had no effect on the ability of the RIA to detect antigen.     

Relationship between protective activity and antigen structure of Haemophilus paragallinarum serotypes 1 and 2

The object in this investigation was to determine the relationship between protective activity and antigenic structure of Haemophilus paragallinarum, serotypes 1 and 2. A close relationship exists in both serotypes between protective activity and colonial phenotypic form (iridescent and noniridescent). Protective activities of both serotypes were related to a heat-labile, trypsin-sensitive (L) antigen of iridescent form that produced serotype-specific agglutinin to chickens. The chickens having the agglutinins were protected against challenge exposure with homologous strain, but not with heterologous strain. The chickens injected with unencapsulated organisms of noniridescent form that were derived from encapsulated organisms of iridescent form failed to produce both serotype-specific agglutinins and protection against challenge exposure with homologous strain. Most of the chickens injected with serotype 1 strain produced both hemagglutination-inhibition antibody and serotype 1-specific agglutinin, whereas those injected with serotype 2 produced serotype 2-specific agglutinin and protected against homologous challenge exposure. The protective activity was found in saline extract derived from encapsulated organisms of serotype 1, but was absent in those of serotype 2.     

Use of schizont and piroplasm antigens of Babesia equi in the indirect fluorescent antibody and complement fixation tests

Eight ponies infected with Babesia equi were investigated for their serological response to B. equi schizont and piroplasm antigen with the indirect fluorescent antibody test (IFAT) and complement fixation test (CFT). Piroplasm antigen was prepared from an infected splenectomized pony, while schizont antigen was produced from cultured lymphoid cells which contained B. equi macroschizonts. The IFAT detected a rise in antibody titres to schizont antigen as well as to piroplasm antigen, but differences were obtained in the duration of antibody detection. Significant antibody titres to piroplasm antigen were detectable for a longer period post infection than to schizont antigen. The complement fixation test was not effective in detecting specific antibodies to schizont antigen in contrast to piroplasm antigen. The schizont antibody titres were in general extremely low and not detectable in 3 horses. Neither test showed any serological cross-reaction with B. caballi and B. bigemina antiserum using schizont antigen.     

Comparison of the Antibody Response in Adult Cattle Against Different Epitopes of Brucella abortus Lipopolysaccharide

The comparison of serological responses in a sample of adult, vaccinated and field‐infected bovines with Brucella abortus is reported. Indirect enzyme immunoassay (EIA) titration curves and Western blotting tests for smooth‐type lipopolysaccharide (S‐LPS), rough‐type LPS (R‐LPS) and lipid A were performed. In the initial screening of sera, an overall prevalence of 20.5 % was found, which corresponds to a country with a high incidence of brucellosis. End‐point EIA titres against LPS antigens from vaccinated and field‐infected cows were not significantly different. However, the absorbance values in the titration curves were significantly higher for S‐LPS as compared with the other antigens. A high correlation coefficient (r=0.933) was obtained when the titres to R‐LPS versus lipid A were compared. Western blotting reactions of vaccinated and field‐infected animals were indistinguishable. S‐LPS, R‐LPS and lipid A epitopes were recognized in a heterogeneous manner. In general, the number of bovines that reacted against LPS was higher in the field‐infected group, with a stronger binding to S‐LPS. Based on our observations, the vaccinated and field‐infected bovines are capable of producing similar antibody responses to the Brucella main outer surface antigen, LPS. It should be emphasized that the humoral response of cattle to Brucella LPS contains significant amounts of antibodies to other antigenic moieties of this important surface molecule, which may contribute to the immunity to brucellosis.     

Generation of immunity against Fusobacterium necrophorum in mice inoculated with extracts containing leucocidin

The capacity of extracts from toxigenic and non-toxigenic ruminant strains of Fusobacterium necrophorum to protect against challenge with homologous and heterologous bacteria was examined in mice. The numbers of F. necrophorum which were infective or lethal for mice increased 5- to 8-fold in animals which had been previously inoculated with complete Freund's adjuvant (FCA). Although preparations containing lipopolysaccharide (LPS) and outer membrane proteins (OMP) from several strains gave protection against a non-toxigenic strain (FnB-3), they did not significantly immunize mice against a challenge infection with a toxigenic bovine strain, FnB-1. Only material which had been prepared by gel filtration of 18-h liquid culture supernates of toxigenic F. necrophorum elicited significant immunity against homologous challenge with FnB-1. This preparation contained LPS and the majority of the leucotoxic activity. However, passive protection was not afforded to mice inoculated with bovine or rabbit sera which possessed high neutralization titres against the leucocidin.     

Occurrence of Escherichia coli O157 in Finnish cattle

Bovine faecal samples were collected during June-December 1997 at 14 major abattoirs slaughtering cattle in Finland. Escherichia coli O157 was isolated from 19 of the 1448 samples (1.31%) after enrichment and immunomagnetic separation (IMS). The positive faecal isolates originated from 16 farms and eight abattoirs. The occurrence of E. coli O157 was highest in July (8/204; 3.92%) and September (6/244; 2.46%). No E. coli O157 was detected in November and December, nor from the faecal samples from the northernmost region where cattle density is low. All of the isolates carried the eae gene and showed the enterohaemolytic phenotype. All except one were motile and had the flagella antigen H7. Seventeen of the isolates were positive for stx(2) gene and one carried both the stx(1) and stx(2) genes. Of the 17 isolates with stx genes, 16 were verocytotoxin-positive in a reversed passive latex agglutination test after polymyxin extraction but only eight without extraction. The isolates belonged to 10 different pulsed-field gel electrophoresis (PFGE) patterns. The most common PFGE pattern (1.42) was detected in eight isolates (42.1%). Four PFGE patterns (1.1; 1.6; 1.12; 1.14) were identical with those isolated from humans in Finland, suggesting that at least some human E. coli O157 infections may be of bovine origin.     

The effect of immaturity of the calf on immunological responses to strain 19 and killed 45/20 adjuvant vaccines.

Thirty brucellosis free calves with zero titres to the serum agglutination test (SAT), complement fixation test (CFT) and antiglobulin test (ABGT) were vaccinated with strain 19 at ages from seven hours to 198 days. Calves 75 days of age and older responded with normal serological patterns, developing high titres to all three tests. At 45 days and younger most calves responded with much reduced titres, some were negative to the SAT and CFT but all develped titres to the ABGT. Two of the younger group were subjected to an anamnestic test at about a year old and gave a positive response, indicating that the calf may be effectively primed with S19 as early as the first day of life. Three of the group were colostrumdeprived yet the patterns of their responses were similar to those of the colostrum-fed calves. Seventy-four zero titres calves were vaccinated with killed 45/20 adjuvant vaccine at ages from 60 to 320 days. Up to 200 days of age only seven of 33 calves gave positive response. From 200 to 280 days 18 of 29 responded and from 280 days of age all calves a positive response. The late development of competence to respond to this adjuvant vaccine is somewhat unusual and is discussed. It is suggested that the rough strain 45/20 may be a very weak antigen in cattle.