金黄色葡萄球菌毒力及其免疫原性评价方法研究

为建立评价金黄色葡萄球菌毒力和用于筛选免疫保护性菌株的技术方法,选取小鼠腹腔攻毒方法确定的强毒力和弱毒力菌株各3株,经小鼠后腿肌肉注射不同剂量,比较20 d的临床病变差异,确定小鼠后腿内侧肌肉注射0.25 mL、OD600=0.6的剂量可以评价不同金黄色葡萄球菌的毒力。利用该方法比较了6株强毒力菌株的毒力差异,并用于2株免疫保护性菌株的筛选。结果证明建立的金黄色葡萄球菌毒力评价方法可以精确、客观比较不同菌株毒力差异,并可用于免疫保护性菌株的筛选。     

Analysis of Korean strains of infectious laryngotracheitis virus by nucleotide sequences and restriction fragment length polymorphism

Korean field strains of infectious laryngotracheitis virus (ILTV) were analyzed by comparison of nucleotide sequences of thymidine kinase (TK) and glycoprotein G (gG) genes and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) patterns. Main differences among TK gene sequence were found in both amino acid at 252 and mRNA polyadenylation signals. In virulent strains, amino acid 252 of TK gene was methionine but was threonine in low virulence and vaccine strains. The mRNA polyadenylation signals of TK gene were identified at 24bp downstream from the stop codon in virulent strains, but not in low virulence and vaccine strains. The gG gene of all virulent strains showed the same nucleotide sequence except for N87278 which had a gG gene sequence identical to that of vaccine strains. The virulent ILTV strains differed from low virulence and vaccine strains in PCR-RFLP patterns of TK and gG genes. The RFLP patterns of TK and gG genes of low virulence ILTV strains were identical to those of vaccine strains. In the case of N87278, the PCR-RFLP patterns of TK and gG genes were identical to those of virulent and vaccine strains of ILTV, respectively. From these results, ILTV field strains were classified into three groups according to sequences of TK and gG genes and PCR-RFLP, and the virulent ILTV strains could be discriminated from low virulence and vaccine strains by PCR-RFLP of TK gene. And it was suspected that N87278 might be produced by in vivo recombination between virulent and vaccine strains of ILTV.     

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.     

多重聚合酶链反应快速检测鉴别鸡毒支原体强毒株和弱毒疫苗株的研究

根据鸡毒支原体强毒株和弱毒疫苗株基因组的结构特点，设计合成了二对引物XZ1，XZ2和XZ45、XZ46，建立了一种同时检测鉴别MG野毒株和弱毒疫苗株的多重PCR技术。试验结果表明，用这两对引物对MG强毒株和弱毒疫苗侏进行多重PCR，强毒株只扩增出732bp一条带，而弱毒疫苗株则可同时扩增出732bp、524bp二条带，而对其他种类鸡支原体和其它禽病病原的扩增不出现任何条带，结果均为阴性；敏感性测定结果表明，该多重PCR最低能检出1Pg的MG强毒株和弱毒疫苗株的DNA模板。     

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     

Second-generation pseudorabies virus vaccine with deletions in thymidine kinase and glycoprotein genes

A modified-live pseudorabies virus (PRV) vaccine, designated PRV(dlg92/d1tk), with deletions in the thymidine kinase (tk) and glycoprotein-gIII (g92) genes, was derived from the PRV (Bucharest [BUK]-d13) vaccine strain. The vaccine virus also contained a deletion in glycoprotein gI. Despite 3 deletions, PRV(dlg92/d1tk) replicated to high titers in cell culture from 30 C to 39.1 C. Enzyme assays and autoradiography revealed that PRV(dlg92/d1tk) did not induce a functional tk activity in infected tk- RAB(BU) cells (rabbit skin). Rabbit skin cells were infected with PRV(dlg92/d1tk), with vaccine strains derived from BUK or Bartha K strains of PRV or with the virulent Illinois (ILL), Indiana-Funkhauser (IND-F), and Aujeszky (Auj) strains of PRV and were labeled with [3H]mannose from 4 or 5 to 24 hours after infection to investigate whether these viruses induced the synthesis of glycoprotein gIII. Nonionic detergent extracts were prepared and immunoprecipitated with antisera from pigs vaccinated with tk(-)-PRV(BUK-d13) or tk+-Bartha K, pigs vaccinated with tk+-PRV(BUK) strains and then challenge exposed to tk+-PRV(IND-F), naturally infected domestic or feral pigs, and pigs vaccinated with tk-)-PRV(dlg92/d1tk). Mouse monoclonal antibodies against PRV glycoproteins gIII, gp50, and gII were also studied. After immunoprecipitation, labeled PRV-specific proteins were analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis and autoradiography. The PRV glycoprotein-gII complex, but not glycoprotein gIII, was synthesized in PRV(dlg92/d1tk)-infected cells. Glycoprotein gII and gIII were made in cells infected with PRV vaccine strains BUK, Bartha K, and BUK-d13 and with virulent PRV strains ILL, IND-F, and Auj. Cells infected with PRV(dlg92/d1tk) and with PRV strains ILL, IND-F, Auj, Bartha K, BUK, and BUK-d13, excreted into the cell culture medium a highly sulfated glycoprotein gX of about 90 kilodaltons. Antibodies to glycoprotein gIII were not detected in the sera of pigs inoculated with PRV(dlg92/d1tk), but were found in all other swine sera.     

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.     

Virulence and toxigenicity of capsular serogroup D Pasteurella multocida strains isolated from avian hosts

Five capsular serogroup D strains of Pasteurella multocida isolated from avian hosts were examined for virulence and toxigenicity. Virulence was based on development of lethal infections or lesions following intramuscular exposure of turkey poults. The four strains isolated from turkeys varied from slightly to moderately virulent; the strain isolated from a chicken was avirulent. Poults exposed by intra-airsac inoculation with relatively few organisms of the more virulent of the strains had a high mortality rate; however, intranasal exposure of poults with this strain did not cause clinical disease or establish infections. All strains from turkeys were toxigenic, producing heat-labile toxins that killed poults when administered intraperitoneally and caused focal dermal lesions when administered intradermally. Using these criteria, the strain from a chicken was not toxigenic. The demonstration of virulence, particularly the high mortality in poults exposed via air sacs, indicates avian capsular serogroup D strains are a potential cause of fowl cholera.     

Vaccine genotype and route of administration affect pseudorabies field virus latency load after challenge

The influence of vaccine genotype and route of administration on the efficacy of pseudorabies virus (PRV) vaccines against virulent PRV challenge was evaluated in a controlled experiment using five genotypically distinct modified live vaccines (MLVs) for PRV. Several of these MLVs share deletions in specific genes, however, each has its deletion in a different locus within that gene. Pigs were vaccinated with each vaccine, either via the intramuscular or intranasal route, and subsequently challenged with a highly virulent PRV field strain. During a 2-week period following challenge with virulent PRV, each of the vaccine strains used in this study was evaluated for its effectiveness in the reduction of clinical signs, prevention of growth retardation and virulent virus shedding. One month after challenge, tissues were collected and analyzed for virulent PRV latency load by a recently developed method for the electrochemiluminescent quantitation of latent herpesvirus DNA in animal tissues after PCR amplification. It was determined that all vaccination protocols provided protection against clinical signs resulting from field virus challenge and reduced both field virus shedding and latency load after field virus challenge. Our results indicated that vaccine efficacy was significantly influenced by the modified live vaccine strain and route of administration. Compared to unvaccinated pigs, vaccination reduced field virus latency load in trigeminal ganglia, but significant differences were found between vaccines and routes of administration. We conclude that vaccine genotype plays a role in the effectiveness of PRV MLVs.     

Evaluation in swine of a subunit vaccine against pseudorabies

A subunit vaccine against pseudorabies virus (PRV) was prepared by treating a mixture of pelleted virions and infected cells with the nonionic detergent Nonidet P-40 and emulsifying the extracted proteins incomplete Freund's adjuvant. Three 7-week-old pigs without antibodies against PRV were given 2 IM doses of this vaccine 3 weeks apart. Thirty days after the 2nd vaccination, 10(6) median tissue culture infective doses (TCID50) of a virulent strain of PRV were administered intranasally. Tonsillar and nasal swabs were collected daily between 2 and 10 days after challenge exposure. The pigs vaccinated with the subunit vaccine were not found to shed virulent PRV. Two groups of five 7-week-old pigs vaccinated with commercially available vaccines, either live-modified or inactivated virus, and subsequently exposed to 10(6) TCID50 of virulent PRV, shed virulent virus for up to 8 days. The subunit vaccine induced significantly higher virus-neutralizing antibody titers than either the live-modified or inactivated virus vaccine.     

Vaccination against bovine babesiosis with drug-controlled live parasites

Live Babesia divergens derived from gerbils were used to vaccinate cattle that had previously been treated with imidocarb dipropionate. Drug doses ranged from 1 to 2 mg/kg and animals were infected subcutaneously three to seven days later. After a further 35 days, vaccinated and control animals were given a heavy heterologous intravenous challenge. This regimen was effective for both avirulent and virulent strains in 12- to 18-month-old cattle. However, at low drug doses some animals reacted to the virulent vaccine strain and at high doses animals infected with the avirulent strain failed to seroconvert although they were still resistant to challenge. The variable infectivity of vaccine strains was a minor problem which can be overcome by strain selection and optimisation of infectivity using gerbils as experimental animals. Gerbils would also be useful as a source of parasites for the further development of in vitro cultures which could ultimately produce the vaccine.     

Immune response of pigs inoculated with virulent pseudorabies virus and pigs inoculated with attenuated or inactivated pseudorabies virus vaccine before and after challenge exposure

Pseudorabies virus (PRV) antibodies, detectable by indirect radioimmunoassay (IRIA), serum-virus neutralization test (NT), or microimmunodiffusion test (MIDT) were developed within 8 days after pigs were inoculated with virulent PRV or attenuated PRV vaccine. Indirect radioimmunoassay and NT titers in pigs inoculated with virulent PRV were developed at the same rate, with IRIA titers being higher than NT titers. Pigs inoculated with attenuated or inactivated PRV vaccine developed peak mean prechallenge NT antibody titers of 4 and 1 (reciprocals of serum dilutions), respectively. Pigs inoculated with attenuated PRV vaccine had peak mean prechallenge IRIA antibody titers of 6, whereas pigs inoculated with inactivated PRV vaccine had mean IRIA antibody titers of 64. Challenge exposure of swine inoculated with attenuated or inactivated PRV vaccine elicited quantitatively equivalent responses, as measured by IRIA or NT, which were higher than prechallenge titers. There were no false-positive IRIA, NT, or MIDT results obtained when sera from nonvaccinated, nonchallenge-exposed pigs were tested. It appears that the PRV infection status of a seropositive swine herd could be ascertained by serologically monitoring several representative animals from a herd, using the NT. If 2 or more tests of representative animals at 14-day intervals were done and the mean NT titer was 4 or less, it could be concluded that the herd was vaccinated against, but not infected with, virulent virus.     

Iscom of viral envelope proteins protects against Aujeszky's disease

An immunostimulating complex (iscom) containing the envelope proteins of pseudorabies virus (PRV) was prepared and its efficacy was evaluated in two experiments on sheep. In the first experiment, sheep were intramuscularly (i.m.) or intradermally (i.d.) vaccinated with PRV iscom doses varying between 1 and 81 micrograms. The vaccination was repeated on Day 21 and the animals were exposed to challenge infection by subcutaneous inoculation of 1000 TCID50 of the virulent Phylaxia strain on Day 35 after first vaccination. In the second experiment, sheep were i.m. vaccinated with single doses of iscom varying between 1 and 27 micrograms and challenge-infected on Day 14. It was found that: (1) the i.d. administration of PRV iscom has no advantage over i.m. administration (2); a single dose of greater than or equal to 3 micrograms of PRV iscom provided protection against the disease. In immunoblots, viral proteins of molecular masses 120, 109, 55, 53 and 32 kDa were detected with the sera obtained from iscom-vaccinated and subsequently challenge-infected sheep, but not with sera from sheep which were iscom-vaccinated only. The above findings indicated that: (1) by using iscom technology, potent subunit vaccines can be prepared to prevent Aujeszky's disease; (2) the selective incorporation of viral envelope proteins into iscoms gives the opportunity to discriminate between iscom-vaccinated and naturally infected animals.     

Analysis of classical swine fever virus replication kinetics allows differentiation of highly virulent from avirulent strains

To study the replication of classical swine fever virus (CSFV) in cell culture, kinetics of viral plus-strand RNA synthesis, of viral structural and non-structural protein expression as well as of secreted and cell-associated infectious virus were determined. Highly virulent, moderately virulent and avirulent strains that were tested in standardized animal experiments to confirm their virulence were used to search for in vitro parameters allowing the differentiation of strains according to their virulence. No significant qualitative or quantitative differences were found between the strains studied when either RNA replication or protein synthesis were investigated. However, the ratio of cell-associated virus versus secreted virus proved to be considerably lower for the highly virulent strains when compared to avirulent or moderately virulent strains. These data suggest that highly virulent strains of CSFV can be distinguished in cell culture from strains with reduced virulence.     

Relationship between virulence of Chlamydia psittaci strains and establishment of persistent infection of McCoy cells

The pathogenicity of chlamydial strains for their natural hosts and their ability to induce persistent infections in McCoy cells were compared. Both virulent and avirulent strains persistently infected McCoy cells, but the appearance of the cell culture varied between strains. Avirulent strains induced completely inapparent persistent infection (infection Type 1), while with invasive strains the culture alternated between periods of cell multiplication and periods of extensive cytopathic change (infection Type 2). The virulence of virulent strains was not attenuated, even after 6 months of culture, but after 2 or 3 months some avirulent strains produced infection Type 2 and became invasive for mice and abortive for ewes. This variation of virulence was accompanied by a modification of protein patterns.     

Intranasal vaccination of pigs against pseudorabies: absence of vaccinal virus latency and failure to prevent latency of virulent virus

The avirulent Bartha's K strain of pseudorabies virus (PRV) was used to vaccinate 8 pigs at 10 weeks of age by the intransal route (experiment 1). On postvaccination days (PVD) 63 and 91, pigs were treated with corticosteroids. Viral shedding could not be detected. Explant cultures of trigeminal ganglia and tonsils did not produce virus. Four pigs with maternal antibody were vaccinated intranasally with Bartha's (attenuated) K strain of PRV at 10 weeks of age and were challenge exposed with a virulent strain of PRV on PVD 63 (experiment 2). Corticosteroid treatment, starting on postchallenge exposure day 70 (PVD 133) resulted in viral shedding in 1 of 4 pigs. In another pig of these 4, a 2nd corticosteroid treatment was required to trigger reactivation. In both pigs, sufficient reactivated virus was excreted to infect susceptible sentinel pigs. Restriction endonuclease analysis indicated that viruses isolated from the 2 pigs after challenge exposure and corticosteroid treatment were indistinguishable from the virulent virus. Evidence was not obtained for simultaneous excretion of vaccinal and virulent virus. Of 4 pigs without maternal antibody vaccinated twice with 1 of 2 inactivated PRV vaccines, challenge exposed on PVD 84, and treated with corticosteroids on postchallenge exposure day 63 (PVD 147), 1 was latently infected, as evidenced by the shedding of PRV (experiment 3). However, its sentinel pig remained noninfected.(ABSTRACT TRUNCATED AT 250 WORDS)     

Histophilus somni IbpA Fic cytotoxin is conserved in disease strains and most carrier strains from cattle, sheep and bison

Histophilus somni causes bovine pneumonia, septicemia, myocarditis, thrombotic meningoencephalitis and arthritis, as well as a genital or upper respiratory carrier state in normal animals. However, differences in virulence factors among strains are not well studied. The surface and secreted immunoglobulin binding protein A (IbpA) Fic motif of H. somni causes bovine alveolar type 2 (BAT2) cells to retract, allowing virulent bacteria to cross the alveolar monolayer. Because H. somni IbpA is an important virulence factor, its presence was evaluated in different strains from cattle, sheep and bison to define whether there are syndrome specific markers and whether antigenic/molecular/functional conservation occurs. A few preputial carrier strains lacked IbpA by Western blotting but all other tested disease or carrier strains were IbpA positive. These positive strains had either both IbpA DR1/Fic and IbpA DR2/Fic or only IbpA DR2/Fic by PCR. IbpA Fic mediated cytotoxicity for BAT2 cells and sequence analysis of IbpA DR2/Fic from selected strains revealed conservation of sequence and function in disease and IbpA positive carrier strains. Passive protection of mice against H. somni septicemia with antibody to IbpA DR2/Fic, along with previous data, indicates that the IbpA DR1/Fic and/or DR2/Fic domains are candidate vaccine antigens for protection against many strains of H. somni. Since IbpA DR2/Fic is conserved in most carrier strains, they may be virulent if introduced to susceptible animals at susceptible sites. Conservation of the protective IbpA antigen in all disease isolates tested is encouraging for development of protective vaccines and diagnostic assays.     

Motility in relation to virulence of Bacteroides nodosus

Fourteen Bacteroides nodosus isolates from footrot lesions of sheep were examined microscopically and all were found to have twitching motility. The mean percentage of cells showing motility was 40% and 9% for virulent and benign strains, respectively. This corresponded with mean agar colony diameters of 17 mm and 7 mm, respectively, for these strains. Two strains of intermediate virulence had values of motility and colony diameter similar to the benign strains. However, the intermediate and the virulent strains produced relatively stable protease compared to the benign strains. All virulent, benign and intermediate strains produced abundant pili. Included for comparison in this study was an avirulent variant strain which was highly motile, formed large colonies and produced stable protease, but showed no pili on electron microscopy. It was concluded that the properties of motility and protease stability may be used to distinguish, in the laboratory, wild-type virulent, benign and intermediate strains of B. nodosus.     

强毒小肠结肠耶氏菌生物素标记基因探针的研究

在构建的小肠结肠耶氏菌毒性质粒DNA基因文库pYB1～8与pYP1～6的基础上,筛选出了pYB7和pYP6克隆株.用限制性内切酶Bam HI消化pYB7,Pst消化pYP6,可分离出3.8kb和6.4kb的插入性DNA片段.以这两个基因片段为目的基因,用生物素化dUTP和光敏生物素标记,获得了生物素标记的基因探针.该探针能检出10pg以上的强毒小肠结肠耶氏菌DNA,不与无毒小肠结肠耶氏菌及大肠杆菌、鼠伤寒沙门氏菌、金黄色葡萄球菌等18种对照菌反应,具有高度的特异性和敏感性.pYB7与pYP6探针对不同血清型及来源的小肠结肠耶氏菌检测,其结果与自凝性试验、依钙试验等结果相符;对小肠结肠耶氏菌强毒株与无毒株检定的准确率为100%.     

New serotype 2 and attenuated serotype 1 Marek's disease vaccine viruses: comparative efficacy

Attempts were made, through selection of optimum viral strains, to develop improved vaccines against Marek's disease (MD). Seven attenuated serotype 1 strains and 22 avirulent serotype 2 strains, both alone and in combination with the FC126 strain of serotype 3, were screened for protective efficacy against challenge with virulent and very virulent MD viral strains. The three viruses selected as most promising were evaluated alone and in various combinations and compared with commercially available vaccines, including FC126, bivalent (FC126 + SB-1), and CV1988/C, in 12 separate assays. Two of these new viruses--301B/1 (serotype 2) and Md11/75C/R2 (serotype 1)--were exceptionally protective compared with prototype vaccine strains. Four new monovalent and polyvalent vaccines based on these two isolates protected chickens better than FC126 alone or CV1988/C alone. Three of these new vaccines provided better protection than the bivalent (FC126 + SB-1) vaccine. Protective synergism was noted commonly between viruses of serotypes 2 and 3 but only sporadically between serotypes 1 and 2 or between serotypes 1 and 3. Strain CVI988/C was protective but was no better than FC126 alone, and it was less effective than bivalent (FC126 + SB-1) vaccine, even when used as a bivalent vaccine with FC126 or SB-1.