S4 Strain Research Articles

Cloning, nucleotide sequence, and expression of the Brucella melitensis bp26 gene coding for a protein immunogenic in infected sheep

We have previously identified a Brucella melitensis 28 kDa cytosoluble protein (CP28) which was highly immunogenic in infected sheep and which in addition made possible the serological differentiation between infected and B. melitensis Rev.l vaccinated sheep. Monoclonal antibodies against CP28 were used to screen a B. melitensis 16M genomic library and to clone the corresponding gene. DNA sequencing of the gene encoding CP28 of B. melitensis 16M revealed that it was nearly identical to that of the recently published bp26 gene of Brucella abortus vaccine strain S19 coding for a periplasmic protein. The differences between the B. melitensis 16M gene and that of B. abortus S19 consisted of single nucleotide substitutions, one or two codon deletions, one codon addition, and most importantly a 21-bp deletion. The corresponding region of B. abortus S19 contains two 10-bp direct repeats which could have been involved in the genesis of the deletion. Expression of the B. melitensis 16M bp26 gene in Escherichia coli studied by the use of the monoclonal antibodies showed the same characteristics as reported for the B. abortus S19 bp26 gene, i.e. the presence of a higher molecular mass preprotein and a lower molecular mass band which probably corresponds to the mature protein exported to the periplasm. Immunoblotting performed with sera from either naturally infected or B. melitensis H38 experimentally infected sheep confirmed the importance of the B. melitensis CP28/BP26 protein as diagnostic antigen.

Tumor necrosis factor alpha activity in genital tract secretions of guinea pigs infected with chlamydiae.

Previous studies using the guinea pig model of chlamydial genital infection demonstrated that primary infection is associated with a marked acute inflammatory response early on, while chronic inflammation appears later, at a time when the level of infection is reduced. Challenge infections result primarily in a chronic inflammatory response. The stimuli that initiate inflammation and lead to tissue damage have not been defined. We investigated the possibility that tumor necrosis factors (TNFs) play a role in the inflammatory response to chlamydial genital tract infection. Cytotoxicity assays for TNF were performed on genital tract secretions collected from female guinea pigs during infection with the Chlamydia psittaci agent of guinea pig inclusion conjunctivitis. During the early days of primary infection, high levels of TNF-alpha were detected in genital tract secretions from inbred S2 strain and outbred Hartley strain guinea pigs. Significantly lower levels of TNF-alpha were detected in secretions from both strains during challenge infection. In general, the intensity of the TNF-alpha response was proportional to the intensity of infection. High TNF-alpha levels were present during primary infection at a time of marked neutrophil influx. Thus, TNF-alpha may play an important role in the response to primary chlamydial genital tract infection.

Differentiation of Two Strains of Mycoplasma gallisepticum with Monoclonal Antibodies and Flow Cytometry

Identification of infecting Mycoplasma spp. is difficult and not routine for strain. This paper describes a procedure for the rapid identification of the strain of M. gallisepticum. Monoclonal antibodies were prepared against M. gallisepticum F and M. gallisepticum S6. Aliquots of 24-hour broth cultures of these organisms were incubated briefly with either of the monoclonal antibodies. A second incubation was made with anti-mouse immunoglobulin conjugated to fluorescein isothiocyanate. Fluorescent intensity associated with the organisms was measured with a flow cytometer. The criterion for identification was a comparative increase in fluorescent intensity when the strain and monoclonal antibody were homologous. The procedure correctly differentiated the F and S6 strains of M. gallisepticum in a blind study.

Demonstration of the Genetic Stability of a Mycoplasma gallisepticum Strain Following in vivo Passage

A Mycoplasma gallisepticum strain designated 6/85 (MGI) exhibiting reduced virulence for both chickens and turkeys was sequentially passaged 10 times in each species. DNA extracted from organisms before passage and those isolated after the third, sixth, and 10th passages was studied by restriction endonuclease DNA analysis using BamHI, BglII, EcoRI, HindIII, and PstI endonucleases. The virulent-type strain designated S6 was used as a comparison. Comparison of DNA fragment patterns of MGI and S6 strains showed distinct differences, although some similarities were evident. Passage of the strain in vivo did not affect DNA fragment patterns of the MGI strain. Electrophoretic protein patterns produced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed very similar band patterns in both the MGI and S6 strains. The most notable differences were seen in bands located in the molecular-mass regions of approximately 46.5, 50-54, 58-64, and 105-140 kilodaltons. Alteration of band pattern profiles following in vivo passage of the MGI strain was apparent in a single band at approximately 86 kilodaltons that appeared to stain more intensely following passage.

Improvement of catalase activity and formaldehyde production in a mutant of a methanol yeast, Candida boidinii S2, grown on hydrogen peroxide-containing medium.

Mutants exhibiting high catalase activity were derived from Candida boidinii S2 strain AOU-1, from among mutants resistant to H2O2, NaN3 or 3-amino-1, 2, 4-triazole (ATA). The catalase activity of an ATA-resistant strain was improved by means of a methanol-limited chemostat culture with H2O2 supplementation. The catalase activity increased with increasing H2O2 concentration in the feed medium in the range where methanol did not remain. Alcohol oxidase activity increased after adaptation of the cells to H2O2. Cells of mutant strain SA051 grown under the optimal culture conditions produced 1200 mM formaldehyde in the reaction mixture.

Evaluation of Attenuated Strains of Mycoplasma gallisepticum as Vaccines in Young Chickens

Five trials were conducted to evaluate the virulence and the vaccination efficacy of the F, R, S6, and A5969 strains of Mycoplasma gallisepticum (MG) at different in vitro passage levels. Vaccination was done by eye-drop or aerosol, and efficacy was evaluated in terms of air-sac lesions after aerosol challenge with the R strain of MG. Continuing medium passage of these strains of MG resulted in gradual attenuation. Aerosol vaccination with highly attenuated MG at 21 days of age was more effective in stimulating the development of immunity than was eye-drop vaccination at 7 days, but aerosol vaccination induced more air-sac lesions. Aerosol vaccination with high-passaged F or S6 strain provided good immunity against MG challenge. Back-passage in chickens of the F strain of over 300 medium passages did not substantially increase the virulence, but back-passage of the S6 strain at the 258th passage and F strain at the 11th and 252nd passage did result in increased virulence. Both low- and high-passaged F and S6 strains were virulent for turkey poults, but only the high-passage F strain did not significantly (P less than 0.05) affect body weight.

The response of chickens to experimental infection with strains of M. gallisept1cumof different virulence andM. gallinarum

Three strains of M. gallisepticum comprising S6 of low passage (virulent), S6 of high passage and A514 of high passage in artificial media and one M. gallinarum strain of high passage, were inoculated into 18-day-old chick embryos. Four groups of infected chicks hatched, one corresponding to each mycoplasma strain; these were investigated during the first 28 days of life and again for a period in lay. During the first 28 days of life, the virulent S6 strain was recovered more frequently from a wider variety of tissues for a longer time than were the other two strains of M. gallisepticum; this strain also produced more severe clinical disease including nervous signs, respiratory lesions and swollen hocks. Although nervous signs were confined to birds infected with the virulent S6, both S6 strains were isolated from brains and hocks, suggesting this strain's proclivity for these sites. One A514 infected chick developed unilateral eye enlargement from which mycoplasma were isolated. All strains of mycoplasma were isolated from respiratory tissues, indicating a proclivity common to all. Recoveries of mycoplasma were often associated with signs and/or lesions but not always, and recoveries also occurred in the absence of signs and lesions. M. gallinarum caused no clinical signs or lesions in young chicks and was rarely isolated. In mature birds, mycoplasma were isolated from the respiratory tissues of some infected with either of the two S6 strains or with M. gallinarum, but not from A514 infected chickens. In both younger and mature chickens, mycoplasma were most likely to be recovered from respiratory tissues and the infra-orbital sinus. Most chicks infected with the virulent S6, showed positive haemagglutination inhibition (HAI) reactions from 4 days of age which preceded the rapid serum agglutination (RSA) reactions. In contrast, chicks infected with other strains of M. gallisepticum either showed no positive HAI reactions, or developed them after 21 days. In either case, positive RSA preceded HAI reactions. The numbers of mature birds examined were small, but, comparing isolation of M. gallisepticum with serological results, it appears that false positives occur with the RSA and both false positives and negatives with the HAI test. No egg transmission of mycoplasma was observed.

Immunochemistry of Capsular Glucans Isolated from Streptococcus Bovis

Abstract Dextran-like capsular materials were isolated from the culture filtrates of Streptococcus bovis strains C3, C36, C48, C106, and S19 by DEAE-cellulose chromatography. Quantitative precipitin analysis revealed a marked immunologic relationship among the capsular glucans of S. bovis. Inhibition studies, employing diglucose inhibitors, indicated that isomaltose was the most effective inhibitor of the reaction between the glucans and antiserum. Isomaltose, for example, inhibited 90% of the reaction between C36 glucan and its homologous antiserum. On the other hand, maltose at a concentration of 8 µmol/mg was almost as effective as isomaltose in inhibiting the reaction between C106 glucan and its homologous antiserum. These results indicate that isomaltose is a common feature of the capsular glucans isolated from a few selected strains of S. bovis. Further, the immunologic relationship which exists among the various glucans is, in part, dependent upon the proportion of the α 1–6 linkages as well as α 1–4 linkages in the carbohydrate chains. Evidence has been presented which indicates a chemical analogy between S. bovis glucans and clinical dextran.

Serological response produced in chickens by three strains of Mycoplasma gallisepticum.

SummaryThree strains of Mycoplasma gallisepticum, S6, F and WS7, were used to infect 3 groups of broiler chickens. Serum antibody response was investigated using the stained antigen plate agglutination (SA) test and the haemagglutination inhibition (HI) test. The HI test was more strain specific and was associated with low molecular weight (IgG) antibodies. Chickens infected with one strain did not always give an HI reaction with the heterotypic antigen. The SA test was associated with high molecular weight (IgM) antibodies. Chickens infected with the three strains always reacted serologically with the homotypic and heterotypic SA antigens. In chickens infected with the S6 and WS7 strains, the IgM and IgG antibody titres remained consistently high during the period of the experiment; in chickens infected with the F strain, the IgG antibodies had disappeared by 7 weeks after infection although the IgM antibodies persisted.

Immunochemical Studies on the Cross-Reactivity between Streptococcus Bovis, Strain S19, and Group A Streptococcal Carbohydrates

Summary Terminal β-N-acetylglucosaminide residues are the prominent antigenic determinant of the cell-wall carbohyrate of S. bovis, strain S19. This carbohydrate gave a precipitin reaction with antisera of rabbits immunized with either Group A streptococci or S. aureus. The antigenic cross-reactivity among S19 carbohydrate, Group A carbohydrate and S. aureus teichoic acid is due to the fact that the immunodominant determinants of each antigen are terminal β-N-acetylglucosaminide residues. The antigenic similarity of these three antigens was emphasized by patterns of identity in double-diffusion studies in agar.

Intracellular Production of Brucella L Forms I. Recovery of L Forms from Tissue Culture Cells Infected with Brucella abortus

Hatten, Betty A. (The University of Texas Southwestern Medical School, Dallas), and S. Edward Sulkin. Intracellular production of Brucella L forms. I. Recovery of L forms from tissue culture cells infected with Brucella abortus. J. Bacteriol. 91:285-296. 1966.-Infectivity of virulent Brucella abortus strain 3183 was less for hamster macrophages after a 2-hr adsorption period than for an attenuated strain (S19) and its tissue culture variant (30). Both strains S19 and 30 were very toxic for the cells, but 3183 was not toxic. Two types of L forms were recovered from a large percentage of hamster kidney cell cultures when disintegration of infected cells was accelerated by tissue culture medium of high pH. One type grew in finely granular microcolonies, was isolated from cells infected for short periods of time, and often reverted to the bacterial form. The other type occurred in small irregularly shaped forms which later developed into round bodies. Both stained specifically with fluorescein-conjugated B. abortus antiserum. Semisolid media containing 0.7% agar provided optimal subsurface L-form growth. L forms also grew well in Thioglycollate Medium but grew poorly in other liquid media. Surface L-form growth was supported by several agar media, but CO(2) was required for optimal growth. Monolayers infected with strain 3183 and examined immediately after adsorption contained occasional small, round bodies. Bizarre forms increased in number with time and, after 24 to 72 hr, large pink-staining inclusions were often present which persisted for several days. Also appearing at about the same time were smaller, dark-staining forms which were first seen in clusters but later dispersed and finally occurred in chainlike configurations. Direct fluorescent-antibody stains of infected cells established that the intracellular forms were related to the infecting strain of B. abortus.

The antigens of Trichomonas foetus isolated from cows and pigs.

The serological properties of the two strains of Trichomonas foetus (strain B and strain M) isolated from uterine infections in cows in Belfast and in northern England, respectively, and two strains isolated from the caecum of pigs were in vestigated with rabbit antisera to each of the four strains by direct agglutination and by precipitation of a saline extract of the dired organisms in tubes and in gel-diffusion plates.There was considerable cross-reaction between the four strains. By the use of predominantly polysaccharide fractions from strains B and M and the predominantly protein fraction TF 4 from stratin B, the polysaccharide complex of lines could be distinguished from the protein lines.The major protein antigens were common to all four strains, but the major polysaccharide antigens were only partially shared.The cow strains B and M were readily distinguished from each other but had a variable though slight serological relationship. The two pig strains were very closely related to each other but were not identical. Cross-reaction revealed a relation between the cow and the pig strains. The cow strain B was more closely related than the M strain to the pig strains.The serological distinctions do not justify the separation of the pig and the cow strains of T. foetus into two species.I wish to thank Prof. W. T. J. Morgan, F.R.S. for preparations of diethylene glycol extracts and kind advice and Dr Winifred Watkins for ethanol fractionation of antigen and for enzyme preparations. I am also grateful to Dr A. E. Pierce of the Institute of Animal Physiology at Babraham for the isolation of clones from the S2 strain.