Production and characterization of two Streptococcus suis capsular type 2 mutants.

Two avirulent mutants of Streptococcus suis capsular type 2 (M2 and M42) were produced from a highly virulent strain. Mutant M2, obtained after serial subcultures of the parent strain in the presence of rabbit anti-capsular type 2 serum, no longer possessed the type-specific capsular antigen, as demonstrated by serotyping methods and immunoelectron microscopy. The Lancefield group D antigen could not be detected on the cell surface of this mutant using the immunogold labelling technique. SDS-PAGE of lysozyme treated cells demonstrated that a 44 kDa protein which was present in the parent strain, was absent in mutant M2. Immunoblotting using rabbit whole cell homologous anti-serum revealed that the protein was strongly immunogenic. Mutant M2 was totally avirulent in mice, and the homologous antiserum completely failed to protect mice against challenge with the parent strain. However, mutant M42, obtained after passages of the parent strain at 42 degrees C, remained capsulated but lacked the same 44 kDa protein as mutant M2. The quantity of sialic acid present in the capsule was similar to that of the parent strain. Despite the presence of antibodies against the capsule, antiserum prepared against M42 only partially protected mice against a challenge with the parent strain. The 44 kDa cell wall protein could act as a virulence factor as well as an important immunogen of S. suis capsular type 2.     

Attenuated live fowl cholera vaccine. I. Development of vaccine strain M3G of Pasteurella multocida

A live cholera vaccine was developed from a virulent avian septicemia strain of Pasteurella multocida serotype 1. The virulent parental strain was mutagenized with N-methyl-N'-nitro-N-nitroso guanidine. Mutants were selected that had either smaller colonies at 37 C or temperature sensitivity for growth at 41 C. Four small-colony mutants and 2 temperature-sensitive mutants were studied. All the mutants were avirulent for turkeys. Sixteen days after turkeys were vaccinated with each mutant, both the vaccinates and unvaccinated controls were challenge-exposed to virulent P. multocida of the homologous serotype and the heterologous serotype 3. Two of the small-colony mutant strains protected against both homologous and heterologous challenge. Suggested for a live cholera vaccine is P. multocida M3G, a small-colony-forming mutant, innocuous for both mice and turkeys and stable against reversion.     

Live attenuated vaccine-based control of necrotic enteritis of broiler chickens

A vaccine for necrotic enteritis (NE) of chickens would reduce the current need to prevent or treat the disease in broiler chickens with antimicrobial drugs. The objective of this study was to understand aspects of immunity to the disease. The first experiment examined the virulence of six strains of Clostridium perfringens isolated from cases of NE in broiler chickens. Using a 5-day experimental oral infection of 2-week-old broiler chickens, four of the six strains were found to be virulent. Pulsed-field gel electrophoresis and PCR showed that virulence was not associated with a plasmid encoding the beta2 toxin gene, cpb2, since this was present in virulent and one of the two avirulent strains. In the second experiment, two virulent and one avirulent strains were tested for their ability to immunize ("infection-immunization") chickens through the oral route. The procedure used experimental infection for 5 days followed by bacitracin treatment for 9 days, and then re-challenge 2 days later with a virulent strain, CP4. Infection-immunization with the virulent isolates protected chickens from subsequent virulent challenge, whereas the infection-immunization with the avirulent isolate did not. In a third experiment, two of four alpha-toxin-negative mutants of CP4 protected birds from experimental NE after oral immunization. These two mutants were also attenuated for virulence. We conclude that it is possible to immunize chickens successfully against NE and that immunogen(s) other than alpha-toxin are important in protective immunity against oral infection.     

Mechanism of protection induced in mice against Erysipelothrix rhusiopathiae infection by treatment with porcine antiserum to the culture filtrate of an attenuated strain

The mechanism of protection induced in mice against challenge with a virulent strain of Erysipelothrix rhusiopathiae by porcine antiserum to the culture filtrate (CF) of an attenuated strain was investigated. Death and bacterial growth in the spleens of mice challenged with the virulent strain were completely prevented by treatment with the antiserum. The protective effect of the serum was markedly decreased in mice in which polymorphonuclear leucocytes (PMN) were depleted by cyclophosphamide (CY) treatment but not in mice in which macrophages were blocked selectively by carrageenan (CG). The phagocytic rate of PMN and the number of bacteria ingested by PMN were significantly higher in mice treated with the antiserum than in mice treated with normal serum. These results indicate that anti-CF serum exerts its protective effect by opsonic activity and that opsonized E. rhusiopathiae are eliminated mainly by PMN.     

Serum resistance and virulence of Escherichia coli isolated from turkeys

Twenty-five strains of Escherichia coli isolated from turkeys were characterized for their serum resistance and virulence. An in vitro bactericidal assay was used to determine the serum resistance of E coli. Virulence was determined by survival time after IV inoculation of each strain into 3-week-old turkeys. Serum-resistant E coli strains were generally found to be virulent for turkeys, whereas serum-sensitive E coli strains were avirulent. Of the 25 strains, 18 strains were placed in the 2 categories of serum-resistant/virulent and serum-sensitive/avirulent. Five strains were serum-resistant and avirulent, and 2 strains were serum-sensitive and virulent. Serum resistance appears to be an important determinant of virulence for E coli in turkeys; however, the requirement for other virulence factors, in addition to serum resistance, was suggested by the finding that 5 serum-resistant strains were avirulent in turkeys.     

Avirulent ts and thymidine kinase-deficient mutant of Aujeszky's disease virus.

The temperature-sensitive (ts), thymidine kinase-deficient (TK-) mutant designated ZHtsTK- strain, of Aujeszky's disease virus (ADV) was isolated from a virulent strain with the treatments using 5-bromodeoxyuridine and arabinosylthymine. The ZHtsTK- strain was easily distinguished from the other virulent ADV strains by plaque size on HmLu-1 and chicken embryo fibroblast cells and by restriction endonuclease analyses using Bam HI, Sal I and Kpn I. The ZHtsTK- strain was avirulent for mice, guinea pigs and rabbits, and produced neutralizing antibodies to ADV in these animals. The rabbits inoculated with the ZHtsTK- strain did not shed detectable amounts of virus after dexamethasone treatment. The ZHtsTK- strain was also avirulent for 5-day-old piglets and did not cause disease. No virus was detected from the piglets inoculated intramuscularly in the nasal swabs or the tissues examined on postinoculation day 9. These findings presented here suggested that there is a significant correlation between pathogenicity and properties such as ts and TK-, and the combination of ts and TK- properties plays a much larger role in reducing virulence for animals.     

TonB is essential for virulence in avian pathogenic Escherichia coli

Avian pathogenic Escherichia coli (APEC) strains have multiple iron-uptake systems that facilitate adaptation to iron-restricted environments and are believed to assist in colonisation of the host. These systems include several TonB-dependent transporters of ferri-siderophores encoded by the chromosome and the large virulence plasmid common to APECs. The tonB gene of the virulent APEC strain E956 was replaced with a selectable antibiotic resistance marker using Lambda Red recombinase mutagenesis. The phenotype of the ΔtonB E956 mutant was compared to the parent strain under various culture conditions and in chickens experimentally infected via the respiratory route. The mutant was resistant to streptonigrin, impaired in its ability to adapt to growth in iron-depleted medium and had greater tolerance of oxidative stress than the parental strain. The mutant was avirulent in chickens, did not affect the growth of chicks and colonisation was mostly limited to the trachea. This study has demonstrated that TonB is essential for virulence in APEC.     

猪多杀性巴氏杆菌对HeLa细胞附着能力的研究

本研究通过猪肺疫的活菌疫苗和死菌疫苗多杀性巴氏杆菌菌株(Pasteurella multocida,Pm)对小鼠的毒力试验测定它们的毒力性。结果表明,死菌疫苗Pm的毒力性比活菌疫苗Pm的强,即死亡率分别为10 0 %和0 %。通过两菌株对He L a细胞的附着试验测定它们的附着能力,结果证明强毒菌的附着能力明显地比弱毒菌强(P<0 .0 1) ,平均附着数分别为11.96和2 .4 4 ;从上述菌株细胞荚膜中分别提取荚膜蛋白,用SDS- PAGE分离测定两菌株荚膜蛋白质结构,结果表明39k Da荚膜蛋白是强毒菌的特异性蛋白。以上研究结果证明Pm的毒力与He L a细胞的附着能力是密切相关的,同时暗示本菌39k Da荚膜蛋白可能与它们的毒力和He L a细胞的附着能力有关     

A mycolyl transferase mutant of Rhodococcus equi lacking capsule integrity is fully virulent

Rhodococcus equi is a mucoid Gram-positive facultative intracellular pathogen which can cause severe bronchopneumonia in foals and AIDS patients. A polysaccharide capsule which gives R. equi a mucoid appearance has long been suspected to be a virulence factor. Here, we describe a transposome mutant in the gene fbpA of strain R. equi 103 causing absence of a capsular structure. FbpA is a chromosomal gene homologous to antigen 85 (Ag85) mycolyl chain transferase gene of Mycobacterium tuberculosis. The mutant multiplied normally in isolated macrophages, was able to establish the unusual R. equi-containing vacuole in macrophages, was cytotoxic for macrophages, and was virulent in a mouse model. Colonies had a dry appearance on nutrient agar and defective capsule structure. Surprisingly, fbpA mutants cured of the virulence-associated plasmid were found in a phagosome that was more alkaline than that of the corresponding wild-type bacteria, were more cytotoxic and even multiplied to some extent. This study suggests that the capsule is not an important virulence factor of R. equi and that it may even counteract virulence traits.     

Studies on Erysipelothrix insidiosa S. rhusiopathiae. 1. Morphology, cultural features, biochemical reactions and virulence

Sixty-two E. insidiosa strains isolated from joints or regional lymph nodes of pigs were examined from the point of view of morphology, cultural aspects, biochemical activity and virulence. All the strains consisted of gram-positive, short rods, which were similar on solid and fluid media. All strains formed H₂S. Otherwise the biochemical activity was rather low except in 1 strain (no. 18), which was very active. One strain (no. 36) was rather inactive, since it showed no other activity than H₂S formation. This latter strain was the only one that was avirulent for mice. The rest of the strains (61) were strongly virulent for mice (LD50 0.5 × 10^−4.17 to 0.5 × 10^−8.5).Of 7 strains examined for virulence for pigs by intracutaneous injection of 0.1 ml broth culture, 6 were virulent. The 7th, which was avirulent, was the one that was also avirulent for mice.     

Pathogenicity of field isolates of Erysipelothrix rhusiopathiae in mice, rats and pigs

Eight field isolates of Erysipelothrix rhusiopathiae serotypes 1 and 2, from different sources, were examined for their pathogenicities for mice and pigs. Arthritogenicity for pigs correlated with virulence for mice at the highest and lowest levels, but not with strains of intermediate virulence. The most virulent strain was also arthritogenic in rats. In pigs, after repeated intravenous challenge the number of affected joints ranged from 0 to 11 of 12 examined. For the 8 strains, the mean number of affected joints ranged from 1 to 7.7 per pig. Clinical course and pathological findings were correlated, but the onset, severity and duration of lameness was variable both within and between groups. Clinical lameness, joint swelling and urticariae were of limited use as indicators of joint changes. The more virulent strains caused lameness as early as 2 days, whereas strains of low virulence took up to 8 weeks.     

Avirulence and immunogenicity in mice of a bovine mastitis Staphylococcus aureus mutant.

An avirulent mutant, designated RC122, was derived from Staphylococcus aureus bovine mastitis strain RC108 after N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis. Mutant RC122, which was isolated on the basis of reduced colony size, showed diminished virulence in mice (LD50 of RC122: 3.1 x 10(10) cfu vs LD50 of RC108: 2.3 x 10(7) cfu). Mutant RC122 grew more slowly than its parental strain and showed decreased production of several exoproteins, such as alpha- and beta-hemolysin, DNAse and coagulase. The production of its capsule was induced only under in vivo growth conditions. Clearance studies performed in the mouse kidney revealed that the kinetics of disappearance of the mutant was similar to that of its parental strain. Protection experiments carried out by intraperitoneal administration in mice showed that mutant RC122 conferred a good degree of protection from challenge with homologous and heterologous strains.     

Production and characterization of streptomycin dependent mutants of Pasteurella multocida from bovine haemorrhagic septicaemia.

A large number of streptomycin dependent mutants were produced from bovine haemorrhagic septicaemia strains of Pasteurella multocida. The mutants required a minimum concentration of 25-50 microgram/mL streptomycin for growth and tolerated a concentration of 200 mg/mL. These mutants were avirulent to mice, when inoculated alone, but some mutants killed mice when inoculated with streptomycin. Biochemically all mutants were uniform and similar to the wild type. Most mutants were stable, but a few produced streptomycin independent revertants. The rate of reversion varied with each mutant. Most revertants were highly virulent for mice, some totally avirulant and a few relatively avirulent.     

Bacterial adherence in mastitis caused by Escherichia coli.

The possible role of bacterial adherence in the pathogenesis of experimental mastitis in the mouse was examined with four strains of Escherichia coli. Two of these strains had a known adhesion antigen (K88) and two did not. The K88 antigen did not play a significant role in the virulence or infectivity of E. coli either in the murine or bovine mammary gland. Two E. coli strains, W1 (K88+) and J2 (K88-) were virulent in the mouse but did not adhere to epithelial cells. Both these strains produced clinical mastitis in the cow. A third strain, D282 (K88-), produced mild disease in the mouse but was avirulent in the cow. The fourth strain, 233/ID (K88+), was avirulent in both the mouse and the cow. Strains D282 and 233/1D were killed rapidly by bovine serum whilst J2 and W1 were more resistant. All strains were more sensitive than the control resistant strain E. coli P4, which is known to be highly virulent for the lactating udder.     

Brucella species lacking the major outer membrane protein Omp25 are attenuated in mice and protect against Brucella melitensis and Brucella ovis

To aid in the development of novel efficacious vaccines against brucellosis, Omp25 was examined as a potential candidate. To determine the role of Omp25 in virulence, mutants were created with Brucella abortus (BA25), Brucella melitensis (BM25), and Brucella ovis (BO25) which contain disruptions in the omp25 gene (Deltaomp25 mutants). Western immunoblot analysis and PCR verified that the Omp25 protein was not expressed and that the omp25 gene was disrupted in each strain. BALB/c mice infected with B. abortus BA25 or B. melitensis BM25 showed a significant decrease in mean CFU/spleen at 18 and 4 weeks post-infection, respectively, when compared to the virulent parental strain (P<0.05, n=5). Mice infected with B. ovis BO25 had significantly lower mean CFU/spleen counts from 1 to 8 weeks post-infection, at which point the mutant was cleared from the spleens (P<0.01, n=5). Murine vaccination with either BM25 or the current caprine vaccine B. melitensis strain Rev. 1 resulted in more than a 2log(10) reduction in bacterial load following challenge with virulent B. melitensis (P<0.01, n=5). Vaccination of mice with the B. ovis mutant resulted in clearance of the challenge strain and provided 2.5log(10) greater protection against virulent B. ovis than vaccine strain Rev. 1. Based on these data, the B. melitensis and B. ovis Deltaomp25 mutants are interesting vaccine candidates that are currently under study in our laboratory for their safety and efficacy in small ruminants.     

Susceptibility of vaccinated swine and mice to generalized infection with specific serotypes of Erysipelothrix rhusiopathiae

Swine were vaccinated with adsorbate bacterin made from Erysipelothrix rhusiopathiae of serotype 2 and were subsequently allotted to 4 exposure groups, each of which was exposed to 1 of the strains of E rhusiopathiae of serotypes 1, 2, 9, or 10. Mice were vaccinated with the same bacterin and were subsequently allotted to 60 exposure groups which were exposed to 60 strains of E rhusiopathiae, comprising 10 strains each of serotypes 1, 2, 4, 9, 10, and 11. Response to challenge of immunity in swine was determined by the presence of clinical signs of acute generalized erysipelas; response in mice was determined by the quantal (live-dead) method. Vaccinated swine were as susceptible to the strain of serotype 10 as were nonvaccinated control swine, whereas vaccinated swine were immune and control swine were susceptible to the strains of serotypes 1 and 2. The strain of serotype 9 was not sufficiently virulent to induce acute generalized erysipelas, even in control swine. Arthritis was not prevented by vaccination, but its frequency and severity were less in vaccinated swine exposed to strains of serotype 1 or 2 than in those exposed to strains of serotype 9 or 10. Vaccinated mice were significantly (P less than 0.05) more susceptible to the strains of serotype 10 than to those of any other serotype tested.     

Mucosal immunoadjuvant activity of the low toxic recombinant Escherichia coli heat-labile enterotoxin produced by Bacillus brevis for the bacterial subunit or component vaccine in pigs and cattle

A gene encoding the mature Escherichia coli heat-labile enterotoxin (LT) lacking the nick site in the A subunit by deleting tripeptides was introduced in a vector pNH301 and expressed extracellularly as mutant molecule of holotoxin at high levels in Bacilus brevis HPD31-S5 of the host bacterium. The mucosal adjuvant activities of the produced mutant LT (mLT) preparation were studied in pigs and cattle. Intranasal immunization of pigs with the recombinant subunit vaccine of Erysipelothrix rhusiopathiae or the component vaccine of Bordetella bronchiseptica mixed with the mLT resulted in a substantial enhancement of both mucosal and serum-specific antibody levels. The immunized pigs were also protected when challenge-exposed intradermally with a highly virulent E. rhusiopathiae strain or challenge-exposed intranasally with a highly virulent strain of B. bronchiseptica. The mLT intranasally administered with recombinant intimin (an outer membrane adhesin) of E. coli O157:H7 also induced an elevation of IgA-specific antibody in the nasal secretion and saliva of calves as well as an elevation of IgG1-specific antibody level against the intimin in the sera and colostrum of cows. The three kinds tested protein antigens were poorly immunogenic when antigen administered intranasally alone. The mLT intranasally administered at a higher effective dose did not induce local adverse reactions or diarrhea in pigs and cattle. The present study demonstrates that the recombinant mLT produced using the B. brevis expression system might represent promising immunoadjuvants for the potential application of intranasal vaccines directed against infectious diseases in pigs and cattle.     

猪链球菌2型Ⅲ型溶血素的研究

为了探讨猪链球菌2型(Streptococcus suis serotype 2,SS2)的Ⅲ型溶血素是否具有溶血活性以及Ⅲ型溶血素在SS2致病过程中的作用,本研究利用同源重组基因敲除法成功构建了SS205ZY的Ⅲ型溶血素(slyrp)基因缺失突变菌株△slyrp及双基因缺失突变菌株△sly/△slyrp,并比较了野生菌株和基因缺失突变菌株的溶血能力以及对小鼠的致病力.结果表明,slyrp基因敲除后可导致SS2裂解红细胞的能力有所下降,而双基因缺失突变菌株△sly/△slyrp的溶血能力完全丧失;slyrp基因敲除后对小鼠的致病力没有影响.结果提示猪链球菌2型Ⅲ型溶血素具有一定的溶血能力,该Ⅲ型溶血素在SS2感染过程中,对溶血素(sly)起协同作用,不是SS2主要的毒力相关基因.     

Cholesterol oxidase (ChoE) is not important in the virulence of Rhodococcus equi

To analyze further the role in virulence of the prominent cholesterol oxidase (ChoE) of Rhodococcus equi, an allelic exchange choE mutant from strain 103+ was constructed and assessed for virulence in macrophages, in mice, and in foals. There was no difference between the mutant and parent strain in cytotoxic activity for macrophages or in intra-macrophage multiplication. No evidence of attenuation was obtained in macrophages and in mice, but there was slight attenuation apparent in four intra-bronchially infected foals compared to infection of four foals with the virulent parent strain, based on a delayed rise in temperature of the choE-mutant infected foals. However, bacterial colony counts in the lung 2 weeks after infection were not significantly different, although there was a slight but non-significant (P=0.12) difference in lung:body weight ratio of the choE mutant versus virulent parent infected foals (mean 2.67+/-0.25% compared to 4.58+/-0.96%). We conclude that the cholesterol oxidase is not important for the virulence of R. equi.     

Immunization of mice against Streptococcus suis serotype 2 infections using a live avirulent strain.

In this study, the IgG response of mice injected with two virulent strains and one avirulent Streptococcus suis capsular type 2 strain was compared by Western blotting. The serum from mice immunized against the avirulent strain could recognize most proteins of the various strains tested and similar results were obtained with serum from mice injected with virulent strains. The live avirulent strain was injected twice (days 0 and 10) to groups of five mice, and four virulent strains from different geographical origins were used to challenge the animals. All mice, except one in one group, survived the challenge. These results suggest that a live avirulent strain could be used for immunization of swine, the natural host.