Genotypic analysis of Mycobacterium tuberculosis in Bangladesh and prevalence of the Beijing strain

Genotypic analysis was performed on 48 Mycobacterium tuberculosis complex strains collected from a hospital in Dhaka city. Deletion analysis showed that the isolates were all M. tuberculosis; 13 of them were found to be of the "ancestral" type, while 35 were of the "modern" type, indicating that both endemic (ancestral type) and epidemic (modern type) strains cause tuberculosis in Bangladesh. Genotyping based on the spoligotype and variable-number tandem repeats (VNTR) of mycobacterial interspersed repetitive units (MIRU) was also done. A total of 34 strains (71%) were grouped by spoligotyping into nine different clusters; the largest comprised 15 isolates of the Beijing genotype, whereas the remaining eight clusters consisted of two to five isolates. MIRU-VNTR typing detected 32 different patterns among 44 tested strains, and the 15 Beijing strains were further discriminated by MIRU-VNTR typing (7 distinct patterns for the 15 isolates). These results indicate that MIRU-VNTR typing, along with spoligotyping and deletion analysis, can be used effectively for molecular epidemiological studies to determine ongoing transmission clusters; to our knowledge, this is the first report about the type of strains prevailing in Bangladesh.     

Cord formation in BACTEC 7H12 medium for rapid, presumptive identification of Mycobacterium tuberculosis complex.

We evaluated cord formation in BACTEC 7H12 medium as a criterion for rapid identification of Mycobacterium tuberculosis complex. Kinyoun-stained smears, prepared from 270 radiometrically positive BACTEC 7H12 bottles, were examined independently by three observers. Smears from 93.2, 88.6, and 83.0% of the M. tuberculosis complex cultures were read as cord positive, and smears from 97.3, 97.8, and 99.5% of the mycobacteria other than M. tuberculosis cultures were read as cord negative by the three observers, respectively. There was 93.3% agreement between the observers. The presence of cords in BACTEC 7H12 medium can be a reliable criterion for rapid, presumptive identification of M. tuberculosis complex.     

The immunoprotective Anaplasma marginale major surface protein 2 is encoded by a polymorphic multigene family.

An Anaplasma marginale Florida msp-2 gene was cloned and expressed in Escherichia coli. Pulsed-field gel electrophoresis and Southern blot analysis revealed the presence of multiple msp-2 gene copies that were widely distributed throughout the chromosomes of all three strains examined. Genomic polymorphism among copies was greatest in the 5' end of msp-2 but also occurred in 3' regions. The presence of gene-copy-specific epitopes was indicated by the reactivity of the cloned msp-2 copy with some, but not all, monoclonal antibodies that bound native MSP-2. Multiple antigenically distinct MSP-2 molecules were expressed within strains and were coexpressed by individual A. marginale organisms. These results suggest that expression of polymorphic msp-2 gene copies is responsible for the significant percentages of A. marginale organisms within strains that do not react with individual anti-MSP-2 monoclonal antibodies. Sequence analysis revealed highly significant MSP-2 homology with two rickettsial surface proteins, A. marginale MSP-4 and Cowdria ruminantium MAP-1. Immunization with MSP-4 has been shown to induce protective immunity in a manner similar to that of immunization with MSP-2. These findings support the hypothesis that A. marginale surface proteins are targets of protective immune responses but are antigenically polymorphic.     

Detection of Anaplasma ovis infection in goats by major surface protein 5 competitive inhibition enzyme-linked immunosorbent assay.

A competitive inhibition enzyme-linked immunosorbent assay (ELISA) based on a major surface protein 5 (MSP5) B-cell epitope conserved among Anaplasma species was used to detect goats infected with Anaplasma ovis. We examined strains of A. ovis isolated from goats in Kenya and demonstrated that MSP5 and the target B-cell epitope, bound by monoclonal antibody ANAF16C1, were conserved. Sera from 149 goats in four regions of Kenya and from 302 goats in six U.S. states were tested for the presence of epitope-specific antibodies with the MSP5 competitive inhibition ELISA. Evidence that the assay can be used to detect A. ovis-infected goats includes the following: (i) 53 goats raised in confinement with arthropod control were all seronegative; (ii) six goats experimentally infected with A. ovis seroconverted at the same time that they developed detectable rickettsemia; (iii) seroconverted goats remained seropositive, consistent with the persistence of A. ovis in goats and the presence of anti-MSP5 antibody in cattle persistently infected with Anaplasma marginale; and (iv) 119 of 127 known A. ovis-infected goats in Kenya were seropositive. A. ovis infection, as determined serologically and by demonstration of infected erythrocytes, in goats from the four regions in Kenya was highly prevalent. In contrast, despite the presence of A. ovis and competent arthropod vectors in the United States, the prevalence of infection appeared to be very low. The high prevalence in Kenya and the occurrence of anemia in persistently infected goats may be impediments to current efforts to increase milk yields on small farms.     

RHD maternal-fetal genotype incompatibility and schizophrenia: extending the MFG test to include multiple siblings and birth order

Rh incompatibility disease (ie Rh hemolytic disease of the fetus and newborn) has been implicated as a risk factor for schizophrenia. Here, we extend the maternal-fetal genotype incompatibility (MFG) test used in an earlier case-parent trio study that found significant evidence for an increased risk of schizophrenia in RHD MFG-incompatible children. We modify the MFG test for case-parent trios to include any number of siblings. This modified test enables us to use more of the available data from the earlier study. The increased sample size not only gives us greater power to test for MFG incompatibility but it also enables us to model the impact of previous RHD MFG-incompatible pregnancies on the relative risk of RHD MFG incompatibility in later-born siblings. This modeling is important, because RHD MFG incompatibility is a proxy for Rh incompatibility disease, and the risk of Rh incompatibility disease increases with the number of previous RHD MFG-incompatible pregnancies. The best-fitting models are consistent with the hypothesized effect that previous incompatible pregnancies increase the risk of schizophrenia due to RHD MFG incompatibility. There was significant evidence that the relative risk of schizophrenia in the second- and later-born RHD MFG-incompatible children is 1.7, consistent with earlier estimates. Our extension of the MFG test has general application to family-based studies of maternal-genotype and MFG interaction effects.     

Babesia bovis merozoite surface antigen 1 and rhoptry-associated protein 1 are expressed in sporozoites, and specific antibodies inhibit sporozoite attachment to erythrocytes

We examined Babesia bovis sporozoites for the expression of two molecules, merozoite surface antigen 1 (MSA-1) and rhoptry-associated protein 1 (RAP-1), that are postulated to be involved in the invasion of host erythrocytes. Both MSA-1 and RAP-1 were transcribed and expressed in infectious sporozoites. Importantly, monospecific MSA-1 and RAP-1 antisera each inhibited sporozoite invasion of erythrocytes in vitro. This is the first identification of antigens expressed in Babesia sp. sporozoites and establishes that, at least in part, sporozoites and merozoites share common targets of antibody mediated inhibition of erythrocyte invasion.     

Immune and pathologic responses in mice infected with Brucella abortus 19, RB51, or 2308.

Immune and pathologic responses were measured for 20 weeks after infection of mice with Brucella abortus 19, RB51, or 2308. Live bacteria and bacterial antigens of 19 and RB51 persisted in spleens for 10 and 4 weeks after infection, respectively, whereas 2308 bacteria and bacterial antigens persisted for at least 20 weeks. Small germinal centers and profound lymphoid depletion occurred in spleens of mice during the first 4 weeks of infection with strain 19 or 2308; however, mice infected with strain RB51 had much larger germinal centers but no lymphoid depletion. At 4 weeks, only spleen cells from RB51-infected mice proliferated when incubated with 2308 bacteria. Large germinal centers in the spleen and spleen cell proliferative responses to 2308 did not appear in strain 19-infected mice until 6 weeks or in strain 2308-infected mice until 10 weeks. Similar proliferative responses to 2308 occurred in mice infected with strain 19 or RB51 at 6 weeks and in mice infected with strain 19, RB51, or 2308 at 10 weeks. However, at 20 weeks, spleen cell proliferative responses to 2308 occurred in mice infected with strain 19 or 2308 but not in mice infected with strain RB51. Mice infected with strain RB51 had lower and less persistent antibody titers to 2308 than did mice infected with strain 19 or 2308. Collectively, these results indicate that RB51-infected mice have less persistent immune responses to 2308 than do mice infected with 19 or 2308. The shorter duration of the responses probably resulted because RB51 is considerably less pathogenic and is cleared more rapidly from mice than are 19 and 2308.     

Stimulation of T-helper cell gamma interferon and immunoglobulin G responses specific for Babesia bovis rhoptry-associated protein 1 (RAP-1) or a RAP-1 protein lacking the carboxy-terminal repeat region is insufficient to provide protective immunity against virulent B. bovis challenge

Rhoptry-associated protein 1 (RAP-1) is a targeted vaccine antigen for Babesia bovis and Babesia bigemina infections of cattle. The 60-kDa B. bovis RAP-1 is recognized by antibodies and T lymphocytes from cattle that recovered from infection and were immune to subsequent challenge. Immunization with native or recombinant protein was reported to reduce parasitemias in challenged animals. We recently reported that the NT domain of B. bovis RAP-1 contained immunodominant T-cell epitopes, whereas the repeat-rich CT domain was less immunostimulatory for T lymphocytes from cattle immune to B. bovis. The present study was therefore designed to test the hypothesis that the NT region of RAP-1, used as a vaccine with interleukin-12 and RIBI (catalog no. R-730; RIBI Immunochem Research, Inc., Hamilton, Mont. [now Corixa, Seattle, Wash.]) adjuvant to induce a type 1 response, would prime calves for antibody and T-helper cell responses comparable to or greater than those induced by full-length RAP-1 containing the C-terminal repeats. Furthermore, a type 1 immune response to RAP-1 was hypothesized to induce protection against challenge. Following four inoculations of either recombinant full-length RAP-1 or RAP-1 NT protein, RAP-1-specific immunoglobulin G (IgG) titers, T-lymphocyte proliferation, and gamma interferon production were similar. Similar numbers of NT region peptides were recognized. However, in spite of the presence of strong RAP-1-specific IgG and CD4(+)-T-lymphocyte responses that were recalled upon challenge, neither antigen stimulated a protective immune response. We conclude that successful priming of calves with recombinant RAP-1 and adjuvants that elicit strong Th1 cell and IgG responses is insufficient to protect calves against virulent B. bovis challenge.     

Expression of L-Selectin (CD62L), CD44, and CD25 on activated bovine T cells

Mycobacterium bovis infection of cattle represents a natural host-pathogen interaction and, in addition to its economic and zoonotic impact, represents a model for human tuberculosis. Extravasation and trafficking of activated lymphocytes to inflammatory sites is modulated by differential expression of multiple surface adhesion molecules. However, effects of M. bovis infection on adhesion molecule expression have not been characterized. To determine these changes, peripheral blood mononuclear cells from M. bovis-infected cattle were stimulated with M. bovis purified protein derivative (PPD) or pokeweed mitogen (PWM) and evaluated concurrently for proliferation and activation marker expression. Stimulation with PPD or PWM increased CD25 and CD44 mean fluorescence intensity (MFI) and decreased CD62L MFI on CD4(+) cells from infected animals. CD62L MFI on PPD- and PWM-stimulated gammadelta T-cell receptor-positive (TCR(+)) and CD8(+) cells was also reduced compared to that of nonstimulated gammadelta TCR(+) and CD8(+) cells. Using a flow cytometry-based proliferation assay, it was determined that proliferating cells, regardless of lymphocyte subset, exhibited increased expression of CD25 and CD44 and decreased expression of CD62L compared to cells that had not proliferated. In contrast to proliferation, activation-induced apoptosis of CD4(+) cells resulted in a significant down regulation of CD44 expression. Lymphocytes obtained from lungs of M. bovis-infected cattle also had reduced expression of CD44 compared to lymphocytes from lungs of noninfected cattle. These alterations in surface molecule expression upon activation likely impact trafficking to sites of inflammation and the functional capacity of these cells within tuberculous granulomas.