Studies on the morphology of colonies of bacilli of BCG-Poland substrain. I. The influence of iron on the type of BCG-Poland colonies.

The influence of iron concentration in Sauton's medium solidified with agar on the type of colonies of BCG-Poland substrains, BCG-Rio de Janeiro, BCG-France, BCG-Denmark and BCG-Japan substrains has been examined. Of all the studied BCG substrains only the BCG-Poland substrain formed rough (R) and smooth (S) colonies. In the investigated substrains rough colonies became smaller with the decrease of iron concentration but they retained their characteristic surface roughness. The smooth colonies which in the same conditions appeared only in BCG-Poland substrain did not display such dependency on iron concentration. When the incubation period was prolonged secondary rough colonies appeared among the smooth ones, regardless of the iron concentration in the medium.     

Relative persistence capacity of BCG substrains in mouse spleen. Computerized statistical analysis. Multiple comparison.

The relative persistence capacity in mouse spleen of 10 and 9 BCG substrains from liquid and dried vaccines, respectively, was evaluated in two studies. Recoverable BCG colony counts from mouse spleen were determined at given days on solid medium in the two studies during a period of 1-360 and 1-345 days, respectively, after the intravenous BCG vaccination, performed with two different viable units. From 36,000 (study 1) and 21,600 (study 2) recoverable BCG colony counts, 180 and 108 mean relative persistence capacity values were estimated to test the residual virulence during the follow-up time, using computerized statistical analysis. The early and late trends of mean relative persistence capacity of the BCG substrains in mouse spleen were tested by linear regression analysis and analysis of variance and covariance; then with ranked adjusted group mean relative persistence capacity, Gabriel's simultaneous test procedure was performed for multiple comparison to diminish type 1 error in statistical inference and in objective interpretation of the experimental results. The associations of the ranked mean relative persistence capacity of the BCG substrains at the different sacrifice days of mice were also analyzed by Kendall's test of concordance. The early, late, and overall relative persistence capacity reflects the residual virulence of the BCG substrains and provides information on the required protective efficacy (immunogenicity) and adverse reactions (reactogenicity), allowing the appropriate vaccination dose, expressed in viable units of the substrain used, to be determined.     

Studies on the morphology of colonies of bacilli of BCG-Poland substrain. II. Dissociation of BCG-Poland substrain on L?wenstein-Jensen egg medium.

The dissociation of colonies of the substrain BCG-Poland on L?wenstein-Jensen medium has been studied. It had been stated previously (Lipińska and Rzucid?o, 1974) that BCG-Poland substrain undergoes a process of dissociation which manifests itself in the appearance of smooth colonies alongside typical rough colonies. Attempts have been made to obtain pure culture of both colony types. Pure cultures composed of smooth or rough colonies respectively were obtained. The morphology and diameter of the smooth colonies depended on the size of the seed inoculum spread on the medium.     

Lipid phenotype of two distinct subpopulations of Mycobacterium bovis Bacillus Calmette-Guerin Tokyo 172 substrain

Bacillus Calmette-Guérin (BCG) Tokyo 172 is a predominant World Health Organization Reference Reagent for the BCG vaccine. Recently, the BCG Tokyo 172 substrain was reported to consist of two subpopulations with different colony morphologies, smooth and rough. Smooth colonies had a characteristic 22-bp deletion in Rv3405c of the region of difference (RD) 16 (type I), and rough colonies were complete in this region (type II). We hypothesized that the morphological difference is related to lipid phenotype and affects their antigenicity. We determined the lipid compositions and biosynthesis of types I and II. Scanning electron microscopy showed that type I was 1.5 times longer than type II. Phenolic glycolipid (PGL) and phthiocerol dimycocerosate (PDIM) were found only in type I. Although it has been reported that the RD16 is involved in the expression of PGL, type II did not possess PGL/PDIM. We examined the ppsA-E gene responsible for PGL/PDIM biosynthesis and found that the existence of PGL/PDIM in types I and II is caused by a ppsA gene mutation not regulated by the RD16. PGL suppressed the host recognition of total lipids via Toll-like receptor 2, and this suggests that PGL is antigenic and involved in host responses, acting as a cell wall component. This is the first report to show the difference between lipid phenotypes of types I and II. It is important to clarify the heterogeneity of BCG vaccine substrains to discuss and evaluate the quality, safety, and efficacy of the BCG vaccine.     

Detection of natural infection with Mycobacterium intracellulare in healthy wild-caught Chacma baboons (Papio ursinus) by ESAT-6 and CFP-10 IFN-γ ELISPOT tests following a tuberculosis outbreak

Background

The current tuberculosis vaccine is a live vaccine derived from Mycobacterium bovis and attenuated by serial in vitro passaging. All vaccine substrains in use stem from one source, strain Bacille Calmette-Guérin. However, they differ in regions of genomic deletions, antigen expression levels, immunogenicity, and protective efficacy.

Results

As a RecA phenotype increases genetic stability and may contribute restricting the ongoing evolution of the various BCG substrains while maintaining their protective efficacy, we aimed to inactivate recA by allelic replacement in BCG vaccine strains representing different phylogenetic lineages (Pasteur, Frappier, Denmark, Russia). Homologous gene replacement was achieved successfully in three out of four strains. However, only illegitimate recombination was observed in BCG substrain Russia. Sequence analyses of recA revealed that a single nucleotide insertion in the 5' part of recA led to a translational frameshift with an early stop codon making BCG Russia a natural recA mutant. At the protein level BCG Russia failed to express RecA.

Conclusion

According to phylogenetic analyses BCG Russia is an ancient vaccine strain most closely related to the parental M. bovis. We hypothesize that recA inactivation in BCG Russia occurred early and is in part responsible for its high degree of genomic stability, resulting in a substrain that has less genetic alterations than other vaccine substrains with respect to M. bovis AF2122/97 wild-type.     

An attempt at the use of immunological methods for the detection of enzymes of BCG substrains.

Immunological methods have been used to visualize peroxidase and dehydrogenase activities of substrains of BCG mycobacteria. Immunodiffusion (ID) and immunoelectrophoresis (IE) in agar gel and two-dimensional immunoelectrophoresis (2D-IE) in agarose gel were employed. In all these tests part of the ID, IE, and 2D-IE preparations were stained with Amido Black 10B (AB 10B) to visualize the total number of precipitin lines and part were stained at the same time with histochemical methods for vizualization of peroxidase and dehydrogenase activities.     

Antigenic Analysis of Virulent and Avirulent Strains of Trichomonas gallinae by Gel Diffusion Methods*

SYNOPSIS. Antigenic constitution of 5 Trichomonas gallinae strains and substrains was analyzed by gel diffusion technics. Fresh isolates of the very virulent JB and of an avirulent SG strain as well as avirulent substrains JBC and SGC, derived from JB and SG respectively by prolonged in vitro cultivation, were used in the experiments. An originally avirulent AG strain that was attenuated still further and lost its infectivity for pigeons during many years of serial transfers in nonliving media also was analyzed. Two major groups of antigens, A and B, were differentiated on the basis of precipitin line patterns formed in gel diffusion reactions involving the 5 strains and substrains and antisera prepared in rabbits against each of these trichomonad stocks. Group A was subdivided further into subgroups [A] and (A). JB, JBC, AG, and SGC trichomonads appeared to share all or nearly all antigens of both these subgroups, but AG strain contained some unique [A] and (A) antigens in addition to those which it had in common with the remaining 4 strains and substrains. Group B antigens were divided into 5 subgroups, B₁ to B₅. The complete B₁ antigenic complex was found in JB and JBC trichomonads and part of this complex was present also in SG strain and SGC substrain. In all instances, subgroup B₁ antigens stimulated production of specific antibodies in rabbits and combined with these antibodies present in immune sera. The complete B₂ antigenic complex was found only in JBC substrain. Some subgroup B₂ antigens were present also in JB trichomonads. Very few of these, however, were capable of stimulating antibody production in rabbits. The more numerous B₂ elements of JB strain that did not stimulate immunologic responses in rabbits, might be in the form of incomplete hapten-like antigens. All subgroup B₂ antigens found in JB strain represented only a portion of the B₂ complex associated with JBC substrain. Subgroup B₂ was characteristic of SG and SGC trichomonads, the latter substrain differing from the parental SG strain in the levels of both B₂ and B₁ antigens; these differences, however, were purely quantitative. JB strain reacted with some of subgroup B₃ antibodies present in SG and SGC antisera, but failed to stimulate antibody formation against any of these antigens in rabbits. The B₃ elements of JB trichomonads might be incomplete antigens. AG strain was characterized by having B₄ and B₅ antigenic complexes. The very small part of subgroup B₄, represented by a weak precipitin line in reactions between JB strain or JBC substrain and anti-AG serum, suggested the presence of some incomplete B₄ antigens in these trichomonads. Irrespective of whether freshly isolated avirulent strains or substrains attenuated by prolonged in vitro cultivation are examined by gel diffusion, such organisms are found richer in subgroup B antigens than the fully virulent JB trichomonads. All the results suggest that there may be a direct relationship between antigenic constitution and virulence of T. gallinae strains.     

An elevation of plasma ISH concentration in spontaneously hypertensive rats (SHR).

Thyroid activity was tested in two substrains of SHR. Plasma level and pituitary content of TSH increased significantly in both substrains of SHR. As a result, the thyroid weight and thyroidal radioiodine uptake increased significantly. Plasma T3 concentration was decreased in Kyoto substrain but was normal in NN substrain, while plasma T4 concentration decreased significantly in both substrains. Since the pituitary content and plasma level of TSH were significantly higher in spite of the normal concentration of plasma T3, it is concluded that the pituitary "hormostat" is set at a higher level at least in the NN substrain of SHR.     

Revisiting the structure of the anti-neoplastic glucans of Mycobacterium bovis Bacille Calmette-Guerin. Structural analysis of the extracellular and boiling water extract-derived glucans of the vaccine substrains

The attenuated strain of Mycobacterium bovis Bacille Calmette-Guérin (BCG), used worldwide to prevent tuberculosis and leprosy, is also clinically used as an immunotherapeutic agent against superficial bladder cancer. An anti-tumor polysaccharide has been isolated from the boiling water extract of the Tice substrain of BCG and tentatively characterized as consisting primarily of repeating units of 6-linked-glucosyl residues. Mycobacterium tuberculosis and other mycobacterial species produce a glycogen-like alpha-glucan composed of repeating units of 4-linked glucosyl residues substituted at some 6 positions by short oligoglucosyl units that also exhibits an anti-tumor activity. Therefore, the impression prevails that mycobacteria synthesize different types of anti-neoplastic glucans or, alternatively, the BCG substrains are singular in producing a unique type of glucan that may confer to them their immunotherapeutic property. The present study addresses this question through the comparative analysis of alpha-glucans purified from the extracellular materials and boiling water extracts of three vaccine substrains. The polysaccharides were purified, and their structural features were established by mono- and two-dimensional NMR spectroscopy and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of the enzymatic and chemical degradation products of the purified compounds. The glucans isolated by the two methods from the three substrains of BCG were shown to exhibit identical structural features shared with the glycogen-like alpha-glucan of M. tuberculosis and other mycobacteria. Incidentally, we observed an occasional release of dextrans from Sephadex columns that may explain the reported occurrence of 6-substituted alpha-glucans in mycobacteria.     

Morphometric comparison of the rat mandible in f344 substrains, f344/du and f344/n.

The shape of the mandible was compared by morphometric methods to ascertain the genetic differences between two substrains of F344 rats, F344/DuCrlCrlj and F344/NSlc. Since these two substrains are clearly different in survival and the incidence of age associated disorders; thus, some genetic differences are suggested to be present between them. Although previous microsatellite analysis did not detect any differences between the two F344 substrains, the present study clearly detected interesting differences in the mandible morphology. At 2 months of age, the F344/Du mandible was characterized by a larger size, especially in length, than the F344/N mandible. The shape of the mandible seemed to be more variable in F344/N. This clear substrain difference suggests the importance of the substrain recognition in F344 rats, especially in experimental usage.     

Paracoccidioides brasiliensis antigen batches from the same isolate show immunological and biochemical differences

We investigated the occurrence of antigenic and biochemical variability among Paracoccidioides brasiliensis antigen batches prepared according to the same protocol. Initially (experiment #1), we analyzed two antigen lots of two human isolates (Bt1 & Bt2), cultured in two media (PYG: bactopeptone, yeast extract, glucose; MMM: McVeigh & Morton medium) in SDS-PAGE and in two immunological tests (imunodiffusion-ID and footpad swelling test-FPT). Afterwards (experiment #2), we compared the antigenic profile of three antigen batches from three human isolates (Bt1, Bt2 & Bt3) by two-dimensional immunoelectrophoresis (2 D-IEP) against a reference system for P. brasiliensis antigens. In experiment #1, there were important intra- and inter-strain antigenic differences between batches of the fungal isolates cultured on both media. The block titration of the antigen batches for the immunological tests revealed correlation between protein concentration and biological activity in ID and no correlation in FPT. In experiment #2, the reference system for P. brasiliensis showed 26 antigen peaks. There were important differences between batches prepared from the same isolate and between batches from different isolates. Our data suggested the occurrence of instability in the synthesis of antigenic components by a same P. brasiliensis isolate, under controlled incubation conditions.     

Prophage type of recombinants produced by cell fusion between various combinations of lysogenic or non-lysogenic substrains from two Staphylococcus aureus L-forms

Streptomycin (SM)- or erythromycin (EM)-resistant lysogenic and non-lysogenic substrains were produced from two Staphylococcus aureus L-form strains lysogenic for different prophages, namely, EMT-L (prophage alpha) and 209P (prophage beta). Cells of these L-form substrains were fused in various combinations using polyethylene glycol (PEG), and the frequency of recombinants selected as double resistance to both SM and EM and the prophage types of these recombinants were examined. In all the combinations, the frequency of recombinants was greater when the cells were treated with PEG than when they were not, and the difference was statistically significant (p less than 0.01) in 13 combinations. Combination between the lysogenic SM-resistant EMT-L substrain [EMT(Smr-alpha)] and lysogenic EM-resistant 209P-L substrain [209P(Emr-beta)] and the reverse combination, between 209P(Smr-beta) and EMT(Emr-alpha), resulted in a majority of recombinants harboring prophage beta. The former combination yielded recombinants that all held both prophage alpha and beta.     

Substitution mapping in dahl rats identifies two distinct blood pressure quantitative trait loci within 1.12- and 1.25-mb intervals on chromosome 3

Substitution mapping was used to refine the localization of blood pressure (BP) quantitative trait loci (QTL) within the congenic region of S.R-Edn3 rats located at the q terminus of rat chromosome 3 (RNO3). An F2(SxS.R-Edn3) population (n=173) was screened to identify rats having crossovers within the congenic region of RNO3 and six congenic substrains were developed that carry shorter segments of R-rat-derived RNO3. Five of the six congenic substrains had significantly lower BP compared to the parental S rat. The lack of BP lowering effect demonstrated by the S.R(ET3x5) substrain and the BP lowering effect retained by the S.R(ET3x2) substrain together define the RNO3 BP QTL-containing region as approximately 4.64 Mb. Two nonoverlapping substrains, S.R(ET3x1) and S.R(ET3x6), had significantly lower BP compared to the S strain, indicating the presence of two distinct BP QTL in the RNO3 q terminus. The RNO3 q terminus was fine mapped with newly developed polymorphic markers to characterize the extent of the congenic regions. The two RNO3 BP QTL regions were thus defined as within intervals of 0.05-1.12 and 0.72-1.25 Mb, respectively. Also important was our difficulty in fine mapping and marker placement in this portion of the rat genome (and thus candidate gene identification) using the available genomic data, including the rat genome sequence.     

Immunologic Analysis by Quantitative Fluorescent Antibody Methods of the Effects of Prolonged Cultivation on Trichomonas gallinae*

SYNOPSIS. Antisera were developed in rabbits against homogenized antigens prepared from 3 axenic lines of Trichomonas gallinae: JB(VI), the 6th isolate of the very virulent Jones' Barn strain which was kept frozen in liquid nitrogen and was fully pathogenic for pigeons at the time at which it was employed for immunization; JB (VI)C, a substrain derived from JB(VI), but attenuated during continuous in vitro cultivation for 1 year; and JB(V)C, a substrain of the 5th isolate of the Jones' Barn strain attenuated during continuous in vitro cultivation for over 3 years. Globulin fractions of the antisera were conjugated with fluorescein isothiocyanate (FITC), and fractionated on DEAE-cellulose columns to yield highly specific fractions. Intact, formalin-fixed flagellates of each line were stained on slides with each labelled antiserum under standardized conditions. Fluorescence emitted by individual organisms was measured with the aid of a special microfluorimetric apparatus. Experiments involving direct staining, one-step inhibition, and cross-absorption methods indicated the presence of unique and cross-reacting antigenic systems in each of the 3 lines of trichomonads. The 2 substrains attenuated in the course of prolonged in vitro cultivation, altho antigenically similar, could be readily differentiated from each other. Further, both of them appeared to contain more antigenic systems than the still fully virulent JB(VI) parasites.     

Trypanoplasma salmositica: experimental infections in rainbow trout, Salmo gairdneri.

Trypanoplasma salmositica was successfully cultured in Hanks' medium with 10% heated fetal calf serum. The culture forms were morphologically similar to blood forms and were infective to rainbow trout by inoculation. T. salmositica produced a disease in experimentally infected rainbow trout (Salmo gairdneri). The clinical signs were anemia, exophthalmia, abdominal distension with ascites, and splenomegaly. These clinical signs were observed in fish that were infected with three substrains (field substrain, cultured substrain, and cloned substrain) thus satisfying Koch's postulates. The anemia was microcytic and hypochromic and was coincident with increasing parasite number in the blood. The hemoglobin in the infected fish dropped from a normal of about 6 g% to about 1 g% in the first 10 weeks postinfection. Similarly, the hematocrit and red cell count declined as the infection progressed. Abdominal distension and exophthalmia was obvious 10 weeks postinfection. Up to 5 ml ascites fluid were recovered from each of three fish. The fluid contained large numbers of Trypanoplasma and macrophages. Some of the macrophages were engulfing the Trypanoplasma. At about this time the spleen in the infected fish was enlarged 5 to 10 times over that of control fish. The hematocrit centrifuge technique was less sensitive than wet mount examinations for the detection of the organism in blood. Fluctuations in parasite number during the course of infection may be due to antigenic change by the parasite to evade the host immune system.     

Genealogy of the 129 inbred strains: 129/SvJ is a contaminated inbred strain

The 129 mouse is the most widely used strain in gene targeting experiments. However, numerous substrains exist with demonstrable physiological differences. In this study a set of simple sequence length polymorphisms (SSLPs) was used to determine the relatedness of selected 129 substrains. 129/SvJ was significantly different from the other 129 substrains and is more accurately classified as a recombinant congenic strain (129cX/Sv), being derived from 129/Sv and an unknown strain. This mixed genetic background could complicate gene targeting experiments by reducing homologous recombination efficiency when constructs and ES cells are not derived from the same 129 substrain. Additionally, discrepancies due to different genetic backgrounds may arise when comparing phenotypes of genes targeted in different 129-derived ES cell lines. Received: 2 December 1996 / Accepted: 10 February 1997     

Immunologic Analysis by Gel Diffusion Technics of the Effects of Prolonged Cultivation on Trichomonas gallinae*

SYNOPSIS. Antisera were developed in rabbits against 3 axenic lines of Trichomonas gallinae: JB(VI), the 6th isolate of the very virulent Jones' Barn strain, which was kept frozen in liquid nitrogen and had its full pathogenicity for pigeons when it was employed for immunization; JB(VI)C, a substrain derived from JB(VI), but attenuated during continuous in vitro cultivation for 1 year; and JB(V)C, a substrain of the 5th isolate of the Jones' Barn strain attenuated during continuous in vitro cultivation for over 3 years. All antisera were reacted on gel diffusion slides with varying concentrations of homologous and heterologous antigens. Two groups of precipitin bands, arbitrarily labeled A and B, were seen on the slides. Analysis of these bands revealed the common genetic makeup of the 3 trichomonad lines with respect to the group A bands. However, the group B antigenic system was strong in the attenuated JB(VI)C and JB(V)C substrains, and very weak in the fully pathogenic JB(VI) strain. These differences are discussed in the light of their possible relationship to pathogenicity.     

Schistosoma mansoni: mice infected with different worm strains.

Infections with five geographical strains and substrains of Schistosoma mansoni were compared in mice. Two substrains (Lc-1 and Lt-1) were derived from the parent (L-1) St. Lucian strain on the basis of differing infectivity for various snail strains. The Puerto Rican strains (PR-1 and PR-2) were obtained with an interval of 25 years. Consistent differences among the lines were found in egg distribution and numbers of eggs in tissues and feces. One Puerto Rican strain (PR-2) and one St. Lucian substrain (Lc-1) had longer prepatent periods than the other strains. Mice infected with the PR-1 strain consistently had the highest egg accumulation in the tissues per worm pair. Relatively few eggs were passed in the feces of the Lt-1 strain. By Week 9 of infection, eggs were noted in the spleens of mice carrying each of the strains and substrains.     

Expression of retroviral vectors in transgenic mice obtained by embryo infection.

Pre-implantation embryos were infected with the retroviral vector MMCV-neo, which carries the neomycin resistance (neo) gene and the v-myc gene. Three transgenic substrains (M-TKneo 1-3) were derived which stably transmit a single intact copy of the vector. In all of the substrains, expression of the neo gene from the internal thymidine kinase (TK) promoter was detected, with two of the substrains expressing the gene in all tissues analysed. In the third substrain, the vector had integrated on the X chromosome and neo expression varied between different tissues. A second series of transgenic mice were obtained with the retroviral vector SAX, in which the human adenosine deaminase cDNA (ADA) is under the control of an internal SV40 promoter. Four substrains (M-SAX 1-4) were analysed; however, no expression of the ADA cDNA was detected. In all mice, no expression was found of the genes under the control of the viral 5' long terminal repeats (LTRs). In the M-TKneo substrains the vector was hypomethylated irrespective of its expression whereas in the M-SAX mice the vector was hypermethylated. These results demonstrate for the first time that the TK promoter can apparently express a gene in all tissues of adult mice and that retroviral vectors with internal promoters may provide an alternative to DNA injection for the efficient expression of genes in transgenic mice.     

Length growth of two Escherichia coli B/r substrains.

Length growth of synchronized Escherichia coli B/r substrain A (ATCC 12407) and B/r substrain F26 (Thy his) was followed with an electron microscope. Cells were grown with doubling times (tau) of 60 min (B/rA) and of 82 and 165 min (B/rF26). Different length growth patterns were found for the two substrains. In B/rF, the length growth rate increased about midway in the cell cycle. For tau = 165 min, the rate increase was preceded by a short period of slow growth. For B/r A (r = 60 min), this period seemed to occur at the beginning of the cell cycle. The possibility is raised that the different length growth patterns are related to different deoxyribonucleic acid replication patterns of the respective strains.