Microsatellite in the beta-tubulin gene of Toxoplasma gondii as a new genetic marker for use in direct screening of amniotic fluids

To examine the correlation between Toxoplasma gondii genotype and congenital human toxoplasmosis, the polymorphism of the microsatellite consisting of a dinucleotide (TG) repeat in the intron of the beta-tubulin gene was investigated by PCR. Thirty-four reference strains were studied, including 7 strains virulent in mice and 27 strains avirulent in mice. The seven virulent strains had a (TG)8 microsatellite, and the avirulent strains had a (TG)7 microsatellite. This confirms the dichotomy already observed for virulent and avirulent strains. Additionally, 37 samples of amniotic fluid from infected fetuses were tested. All of them had the (TG)7 microsatellite marker. This result confirms that most of the human cases of congenital toxoplasmosis are due to strains avirulent in mice. Nevertheless, their virulence in human fetuses was obvious, as numerous abnormalities were observed on ultrasonic examination. The new genetic marker is the first one directly used for typing T. gondii isolates without any bias due to cultivation of the parasite. This microsatellite marker is not sufficient to type the strains which are avirulent in mice; however, seeking more polymorphic microsatellites should be worthwhile to obtain new genetic markers for direct screening of biological samples.     

Heat shock proteins of Toxoplasma gondii

We have investigated heat shock protein (HSP) expression in mouse-virulent and -avirulent strains of Toxoplasma gondii by performing Western blot analysis using a monoclonal antibody against HSP65 of Mycobacterium bovis and a polyclonal antiserum against HSP70 of Plasmodium falciparum as primary antibodies. We initially observed that murine macrophages express HSP65 when infected with either virulent or avirulent strains, a result which contradicts previous reports. Differential HSP expression consistent which virulence was observed between strains, with high levels of a 70kDa HSP (HSP70) only detected in virulent strains in vivo. This protein was not observed in virulent strains in the immunocompromised mouse or in vitro, suggesting induction by immunological stress. This protein was only poorly expressed in avirulent strains. A 65kDa protein was observed in all strains in vivo and in vitro, suggesting a shared epitope with HSP70. These results are consistent with the hypothesis that the induced expression of HSP70 in virulent strains of T. gondii by immunological stresses may provide protection for these strains against cell damage associated with invasion of the host, allowing the virulent strains to persist as tachyzoites without the requirement for the encystation observed in avirulent strains.     

Nucleic acid amplification for rapid detection of Rhodococcus equi in equine blood and tracheal wash fluids

OBJECTIVE: To evaluate the ability of nucleic acid amplification techniques to detect Rhodococcus equi in equine buffy coat, blood, and tracheal wash fluid and to differentiate between virulent and avirulent strains of the bacteria. SAMPLE POPULATION: Blood anticoagulated with EDTA and tracheal wash fluid from healthy horses. PROCEDURE: Logarithmic dilutions of virulent and avirulent strains of R equi were added to equine buffy coat and tracheal wash fluid samples. The DNA was extracted and amplified by polymerase chain reaction (PCR), using primers specific for the 16S ribosomal subunit gene and the virulence plasmid of R equi. RESULTS: PCR with 16S ribosomal subunit primers amplified a 441-bp segment of DNA from virulent and avirulent strains of R equi, but not from samples containing other species of bacteria. The virulence plasmid primers amplified an 875-bp segment of DNA from virulent strains of R equi, but not from avirulent R equi, or from other species of bacteria. Virulent strains of R equi could be identified by PCR and differentiated from avirulent strains within 12 to 24 hours after sample collection, with as few as 10 to 100 organisms present. CONCLUSIONS: PCR can be used to rapidly and accurately identify R equi in equine blood and tracheal wash fluid samples and can differentiate between virulent and avirulent strains of the organism. CLINICAL RELEVANCE: Because PCR can confirm a diagnosis of R equi infection in horses more rapidly and specifically than use of standard culture techniques, extrapolation of this assay to soil and fecal samples could be useful in epidemiologic studies and studies of environmental disinfection or decontamination.     

Identification of significant variation in the composition of lipophosphoglycan-like molecules of E. histolytica and E. dispar

The lipophosphoglycan-like (LPG-like) molecules of E. histolytica virulent strains are clearly distinct from those of the avirulent E. histolytica and E. dispar strains. Abundant 'LPG' levels are apparently limited to virulent strains, while lipophosphopeptidoglycans ('LPPG's) are common to both virulent and avirulent strains of E. histolytica and E. dispar. It is therefore conceivable that 'LPPG' performs a function that is essential to survival within the host, while the 'LPG' performs a more specific function related to virulence.     

Pathogenicity of K. ozaenae for mice and chick embryos

The authors tested the virulence of K. ozaenae--its old museum strains, the freshly isolated ones and those passaged on meat-peptone agar; experiments were carried out on mice and chick embryos. To mice the culture was administered intraperitoneally, subcutaneously, intranasally, and to embryos--into the allantoic cavity and on the chorioallantoic membrane. Irrespective of the method of administration, the freshly isolated strains were highly virulent both for mice and for chick embryos. The virulence of such strains decreased in the process of passaging on the nutrient medium. Old museum strains were of low virulence for albino mice and avirulent for chick embryos. In comparing the virulent and avirulent strains there were found no differences in the antigenic structure and toxicity of the Boiven complex, cytoplasm, membrane, capsular polysaccharide or whole virulent and avirulent bacteria killed by heating.     

Chromosome-mediated resistance of Yersinia enterocolitica serotype O9 to intracellular killing by mouse peritoneal macrophages

The survival of Yersinia enterocolitica serotype O9 within mouse peritoneal macrophages was investigated. To evaluate the role of the virulence plasmid in the resistance to intracellular killing, an isogenic pair of virulent (plasmid-bearing) and avirulent (plasmid-less) O9 strains was used. The virulent strain was able to express plasmid-encoded outer membrane proteins and to colonize the Peyer's patches of orally infected mice. When mice were infected intraperitoneally, both strains were recovered at similar rates and over the same time from the peritoneal cavity. When in vitro assays were performed, both strains showed similar resistance to intracellular killing by monolayers of resident and inflammatory peritoneal macrophages. Previous opsonization of bacteria did not modify their survival within macrophage monolayers. We concluded that serotype O9 strains display a chromosome-mediated resistance to intracellular killing by mouse peritoneal macrophages. Moreover, macrophage resistance does not seem to be of importance for virulence of serotype O9 strains in mice.     

Lung resistance protein (LRP) gene expression in adult acute myeloid leukemia: a critical evaluation by three techniques

Restriction fragment length polymorphism analysis of chromosomal DNA from 22 strains of Toxoplasma gondii were characterised using SalI and PstI restriction endonucleases and the TGR1E specific repetitive sequence as a probe. Two virulent strains, RH and P-CZ, had previously been isolated from humans, the remaining 20 strains were isolated from animals in the Czech Republic in 1994 and 1995. Among the 20 recently isolated strains, 19 belonged to an avirulent lineage and only one strain from the wild cat Felis silvestris belonged to a virulent lineage.     

Differential study of two strains of Toxoplasma gondii

This study aimed to differentiate between the virulent and avirulent strains of T. gondii by studying morphometric measurement using C.I.A.S., determination of the relative DNA content and extract, SDS poly acrylamide gel electrophoresis and isoenzyme on cellulose acetate gel. Identifying these differences may be useful in studying different disease types and in pathogenic mechanisms. Besides, the presence of common proteins between them may be of value for diagnostic purposes and for formulation of vaccines.     

Immunization with E. coli produced recombinant T. gondii SAG1 with alum as adjuvant protect mice against lethal infection with Toxoplasma gondii

Polyclonal rabbit antibodies against recombinant Toxoplasma gondii SAG1 antigen expressed in E.coli recognize T. gondii and the antibodies significantly reduced T.gondii adherence and/or invasion into the host cell as did a monoclonal antibody against a conformational epitope of the SAG1 antigen. Groups of outbread NMRI mice were immunized with recombinant T. gondii SAG1 antigen in alum. The antibody response to immunizations was dominated by a Th2 response with production of T.gondii specific IgG1 antibodies. Challenge with tachyzoites from the virulent RH-strain produced a Th1 response dominated by the production of specific IgG2a antibodies and moderately boosted the IgG1 response, and challenge with bradyzoites from the avirulent SSI119-strain showed the same pattern. Immunization with rSAG1 resulted in a significant increased survival after challenge with tachyzoites of the RH-strain. Immunization with E.coli expressed recombinant SAG1 in alum induce partial protective immunity against lethal infection with T. gondii in mice.     

Basis for substrate specificity of the Toxoplasma gondii nucleoside triphosphate hydrolase

The Toxoplasma gondii nucleoside triphosphate hydrolase is the most active E-type ATPase yet identified, and was the first member of this new gene family to be cloned (Bermudes D, Peck KR, Afifi-Afifi M, Beckers CJM, Joiner KA. J Biol Chem 1994;269:29252-29260. Previous work also identified two isoforms of the enzyme in the virulent RH strain, and demonstrated that internal fragments of the genes encoding these isoforms were found differentially in virulent versus avirulent organisms (Asai T, Miura S, Sibley D, Okabayashi H, Tsutomu T, J Biol Chem 1995;270:11391-11397). We now show that the NTPase 1 isoform is expressed in avirulent strains, whereas virulent strains express both the NTPase 1 and NTPase 3 isoforms. The avirulent PLK strain lacks the gene for NTPase 3, explaining the absence of expression. Despite the fact that NTPase 1 and NTPase 3 are 97% identical at the amino acid level, recombinant NTPase 1 is a true apyrase, whereas recombinant NTPase 3 cleaves predominantly nucleotide triphosphates. Furthermore, native and recombinant NTPase 3 but neither native nor recombinant NTPase 1 bind to ATP-agarose, further distinguishing the two isoforms. Using chimeras between the NTP1 and NTP3 genes, we show that a block of twelve residues at the C-terminus dictates substrate specificity. These residues lie outside the regions conserved among other E-ATPases, and therefore provide new insight into substrate recognition by this class of enzymes.     

Cloned genes of the aerobactin system of virulent avian Escherichia coli do not confer virulence to recombinant strains

1. We cloned the aerobactin region and its receptor from pMV14, a large nonconjugative plasmid isolated from the virulent strain UEL14, to assess the importance of the aerobactin iron uptake system as a virulence determinant in septicemic avian Escherichia coli. 2. The physical map of the region of the recombinant plasmid (pGMV1) containing the genes for synthesis of aerobactin and its receptor was very similar to the corresponding region in pABN1 containing the genetic determinants for the aerobactin system of pColV-K30. 3. The 74-kDa outer-membrane protein encoded by pGMV1 cross-reacted immunologically with the 74-kDa aerobactin receptor protein encoded by pABN1. 4. Various avirulent E. coli strains carrying the recombinant plasmid, which contains only the aerobactin system, were assayed for virulence and were found to be avirulent for chickens. Only the wild-type aerobactin-producing strain was virulent in a pathogenicity test for chickens. 5. These results show that the aerobactin system by itself does not confer virulence, and that other factors are necessary for virulence of avian strains of E. coli.     

Long-term lipid-based total parenteral nutrition activates mononuclear cells and modulates membrane lipid composition in pigs

This study aims to assess the possible strain-dependent variations in detection of Toxoplasma antigens and antibodies. The virulent RH strain or avirulent Beverley strain of T. gondii were injected into mice, intraperitoneally, and their antigens, antibodies and parasites were identified from the blood or tissues; liver, brain and spleen by ELISA, Western blot and PCR. In mice infected with RH strain, circulating antigens and parasitemia were first detected from 2 days after infection, and Toxoplasma DNA were found in the blood, liver, brain and spleen from 3 days after infection. It was impossible to detect specific IgM and IgG antibodies to T. gondii, and any specific band was not found by Western blot. In mice infected with Beverley strain, circulating antigens were detected between day 10 and day 35. The Toxoplasma DNA was found in the blood and liver from day 15 until day 60, and in the brain from day 20. But Toxoplasma DNA in the spleen were mainly detected between day 10 and day 30. The IgM antibodies were first appeared on day 10 post-infection, and were noted obviously increased between day 15 and 25. The IgG antibodies were first detected on day 15, and showed progressively increased titers. The antibody binding bands were specific according to infection period. Sera from mice infected with Beverley strain reacted mainly with the antigen of 27.5-kDa and 32.5-kDa. In conclusion, mice infected with RH strain revealed Toxoplasma antigens strongly, but not antibodies. However, mice infected with Beverley strain revealed both the Toxoplasma antigens and antibodies. The present results showed that immune responses are different between avirulent and virulent T. gondii.     

CAMDAS: an automated conformational analysis system using molecular dynamics. Conformational Analyzer with Molecular Dynamics And Sampling

In order to investigate the relationship among virulent and avirulent Bacteroides fragilis strains, SDS-PAGE of whole-cell proteins (WP) and periplasmic proteins (PP) were used to establish a protein profile of strains isolated from human infections, fecal flora and environmental water. Despite different sources of the strains, no significant differences were observed as determined by the WP SDS-PAGE analysis. In contrast, the proteins obtained from the bacterial periplasm showed differences in the electrophoretic protein profile. Two distinct PP profile patterns were obtained. Pattern A included 6 out of the 8 virulent strains and pattern B, 6 out of 8 avirulent strains. Interestingly, an environmental strain that was capable of inducing abscesses in mice, had a PP profile highly similar to that of the virulent strains from human infections. These data indicate that PP from B. fragilis may be useful to characterize differences among virulent and avirulent strains. Moreover, strains isolated from environmental water may also be a source of exogenous infections by B. fragilis.     

Virulence of Rhodococcus equi isolates from patients with and without AIDS

Rhodococcus equi is an emerging opportunistic pathogen of human immunodeficiency virus-infected patients. Thirty-nine isolates of R. equi from immunocompromised patients with and without AIDS were analyzed for the presence of virulence plasmid DNA, expression of 15- to 17-kDa antigens, and their pathogenicities in mice. Of the human isolates, eight contained an 85-kb virulence plasmid, expressed 15- to 17-kDa antigens, and were virulent in mice. Nineteen isolates carried cryptic plasmids of various sizes, and the remaining 12 isolates did not contain any plasmids. These 31 isolates did not express virulence-associated antigens and were not virulent in mice. The results suggested that opportunistic infections in immunocompromised patients could be caused by both virulent and avirulent R. equi strains and that the pathogenesis of R. equi infection in immunocompromised patients appears to be different from that which occurs in foals.     

Species identification and virulence attributes of Saccharomyces boulardii (nom. inval.)

Saccharomyces boulardii (nom. inval.) has been used for the treatment of several types of diarrhea. Recent studies have confirmed that S. boulardii is effective in the treatment of diarrhea, in particular chronic or recurrent diarrhea, and furthermore that it is a safe and well-tolerated treatment. The aim of the present study was to identify strains of S. boulardii to the species level and assess their virulence in established murine models. Three strains of S. boulardii were obtained from commercially available products in France and Italy. The three S. boulardii strains did not form spores upon repeated testing. Therefore, classical methods used for the identification of Saccharomyces spp. could not be undertaken. Typing by using the restriction fragment length polymorphisms (RFLPs) of the PCR-amplified intergenic transcribed spacer regions (including the 5. 8S ribosomal DNA) showed that the three isolates of S. boulardii were not separable from authentic isolates of Saccharomyces cerevisiae with any of the 10 restriction endonucleases assessed, whereas 9 of the 10 recognized species of Saccharomyces could be differentiated. RFLP analysis of cellular DNA with EcoRI showed that all three strains of S. boulardii had identical patterns and were similar to other authentic S. cerevisiae isolates tested. Therefore, the commercial strains of S. boulardii available to us cannot be genotypically distinguished from S. cerevisiae. Two S. boulardii strains were tested in CD-1 and DBA/2N mouse models of systemic disease and showed intermediate virulence compared with virulent and avirulent strains of S. cerevisiae. The results of the present study show that these S. boulardii strains are asporogenous strains of the species S. cerevisiae, not representatives of a distinct and separate species, and possess moderate virulence in murine models of systemic infection. Therefore, caution should be advised in the clinical use of these strains in immunocompromised patients until further study is undertaken.     

The pH of the host niche controls gene expression in and virulence of Candida albicans

Little is known of the biological attributes conferring pathogenicity on the opportunistic fungal pathogen Candida albicans. Infection by this pathogen, as for bacterial pathogens, may rely upon environmental signals within the host niche to regulate the expression of virulence determinants. To determine if C. albicans responds to the pH of the host niche, we tested the virulence of strains with mutations in either of two pH-regulated genes, PHR1 and PHR2. In vitro, PHR1 is expressed when the ambient pH is at 5.5 or higher and deletion of the gene results in growth and morphological defects at neutral to alkaline pHs. Conversely, PHR2 is expressed at an ambient pH below 5.5, and the growth and morphology of the null mutant is compromised below this pH. A PHR1 null mutant was avirulent in a mouse model of systemic infection but uncompromised in its ability to cause vaginal infection in rats. Since systemic pH is near neutrality and vaginal pH is around 4.5, the virulence phenotype paralleled the pH dependence of the in vitro phenotypes. The virulence phenotype of a PHR2 null mutant was the inverse. The mutant was virulent in a systemic-infection model but avirulent in a vaginal-infection model. Heterozygous mutants exhibited partial reductions in their pathogenic potential, suggesting a gene dosage effect. Unexpectedly, deletion of PHR2 did not prevent hyphal development in vaginal tissue, suggesting that it is not essential for hyphal development in this host niche. The results suggest that the pH of the infection site regulates the expression of genes essential to survival within that niche. This implies that the study of environmentally regulated genes may provide a rationale for understanding the pathobiology of C. albicans.     

Selection and characterization of Toxoplasma gondii mutants resistant to artemisinin

Toxoplasma gondii infection, like malaria, is sensitive to inhibition by artemisinin (ART). Mechanisms of action for ART in malaria treatment have been proposed, but little is known about its effects in T. gondii infection. To better understand its inhibitory effects on T. gondii, mutants resistant to ART were selected by progressive culture in permissive levels of the drug. Five clonal isolates were established and characterized. The isolates were approximately 65-fold less sensitive to ART than is the parental RH and showed cross-resistance to the ART derivatives dihydroartemisinin and artemether. In addition to ART resistance, 1 clone (C9) formed morphologically unusual parasitophorous vacuoles and another (A2) was avirulent for mice and protected mice from challenge with the wild type. These clonal T. gondii mutant isolates will be useful for the study of not only the mechanism of action of ART but also parasite vacuole biology and virulence factors.     

Macrophages activated by Listeria monocytogenes induce organ-specific autoimmunity

We have previously reported an experimental autoimmune model induced by the local infection of Listeria monocytogenes. The unilateral inoculation of virulent Listeria into a testis of a normal mouse induced a delayed-type hypersensitivity response against testicular antigen and caused autoimmune orchitis in the contralateral testis. The orchitis was transferred to naive mice by T cells from the intratesticularly infected mice. In this paper, we demonstrated that avirulent Listeria, which lacks the expression of listeriolysin O, failed to induce any anti-testicular responses or contralateral orchitis even when it was inoculated at a high dose into the testis. Furthermore, the intraperitoneal inoculation of virulent Listeria with testicular antigen induced the anti-testicular responses and orchitis although intraperitoneal inoculation of testicular antigen with avirulent Listeria failed to induce them. The difference between virulent and avirulent Listeria in the induction of anti-testicular responses was supposed to be dependent on the difference in macrophage activation by the two bacterial strains because, first, the anti-testicular responses were elicited in normal mice when macrophages from virulent Listeria-infected mice were intraperitoneally transferred with testicular antigen although no viable bacteria were detected from the macrophages, and secondly, in contrast, the intraperitoneal co-inoculation of macrophages from avirulent Listeria-infected mice and testicular antigen failed to elicit any anti-testicular responses. Finally, we found that the virulent Listeria-induced macrophages expressed a higher level of CD80 (B7-1) and CD86 (B7-2) molecules than did the avirulent Listeria-induced macrophages and naive peritoneal macrophages. These results thus suggest that virulent Listeria activates macrophages to induce autoreactive T cells while avirulent Listeria does not. The up-regulation of B7 molecules by virulent Listeria infection is a candidate of the mechanism for the activation of autoreactive T cells.     

Immune response and protection in mice inoculated with Leishmania amazonensis clones expressing different degrees of virulence

The induction of protective immunity to Leishmania amazonensis was investigated by injection of parasite clones of low and medium virulence into susceptible mice. To this end, L. amazonensis were cloned by limiting dilution and the clones' virulence was evaluated by the course of infection in susceptible mice. Clones originally derived from the spleen showed virulence variations in comparison with that of the parental population (PP) of parasites. Two low-virulence clones (SP 5 and SP 20) and one medium-virulence clone (SP 11), representative of the spectrum of derived clones, were compared with virulent parasites and with an avirulent strain (Josefa) as to their ability to induce T-cell immune responses and to protect BALB/c mice from infection with the virulent L. amazonensis PP. Clone SP 20 and clone SP 11 induced partial protection when injected by the intravenous and intradermal route, respectively. The avirulent Josefa strain induced neither T-cell responses nor protection. Low-virulence L. amazonensis clones can therefore be additional tools in vaccine investigation.     

Trypanosoma cruzi: maintenance in culture modify gene and antigenic expression of metacyclic trypomastigotes

In this study we examined whether the maintenance of Trypanosoma cruzi by long-time in axenic culture produces changes in gene expression and antigenic profiles. The studies were made with a Dm30L-clone from a low-virulent strain and a non-cloned virulent EP-strain of T. cruzi. Both parasites were maintained, for at least seven years, by successive alternate passage triatomine/mouse (triatomine condition), or by serial passage in axenic medium (culture condition). The comparison of the [35S]methionine metabolic labeling products of virulent and non-virulent parasites by 2D-SDS-PAGE, clearly indicates that the expression of metacyclic trypomastigotes (but not of epimastigotes) proteins have been altered by laboratory maintenance conditions. Western blot analysis of EP and Dm30L-epimastigotes using a serum anti-epimastigotes revealed that although most of antigens are conserved, four antigens are characteristics of triatomine condition parasites and three other are characteristics of culture condition parasites. Anti-metacyclics serum revealed significative differences in EP- and Dm30L-metacyclic trypomastigotes from triatomine condition. However, avirulent metacyclic forms were antigenically very similar. These results suggest that besides a possible selection of avirulent subpopulation from T. cruzi strains genetically heterogeneous when maintained by long time in axenic culture, changes in virulence might be due to post-translational modifications of the antigens induced by the absence of the natural alternability (vertebrate-invertebrate) in the life-cycle of T. cruzi.