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1.  In vivo gene expression and the adaptive response: from pathogenesis to vaccines and antimicrobials. 
Microbial pathogens possess a repertoire of virulence determinants that each make unique contributions to fitness during infection. Analysis of these in vivo-expressed functions reveals the biology of the infection process, encompassing the bacterial infection strategies and the host ecological and environmental retaliatory strategies designed to combat them (e.g. thermal, osmotic, oxygen, nutrient and acid stress). Many of the bacterial virulence functions that contribute to a successful infection are normally only expressed during infection. A genetic approach was used to isolate mutants that ectopically expressed many of these functions in a laboratory setting. Lack of DNA adenine methylase (Dam) in Salmonella typhimurium abolishes the preferential expression of many bacterial virulence genes in host tissues. Dam- Salmonella were proficient in colonization of mucosal sites but were defective in colonization of deeper tissue sites. Additionally, Dam- mutants were totally avirulent and effective as live vaccines against murine typhoid fever. Since dam is highly conserved in many pathogenic bacteria that cause significant morbidity and mortality worldwide, Dams are potentially excellent targets for both vaccines and antimicrobials.
PMCID: PMC1692776  PMID: 10874736
2.  Salmonella DNA Adenine Methylase Mutants Elicit Protective Immune Responses to Homologous and Heterologous Serovars in Chickens 
Infection and Immunity  2001;69(12):7950-7954.
Salmonella DNA adenine methylase (Dam) mutants that lack or overproduce Dam are highly attenuated for virulence in mice and confer protection against murine typhoid fever. To determine whether vaccines based on Dam are efficacious in poultry, a Salmonella Dam− vaccine was evaluated in the protection of chicken broilers against oral challenge with homologous and heterologous Salmonella serovars. A Salmonella enterica serovar Typhimurium Dam− vaccine strain was attenuated for virulence in day-of-hatch chicks more than 100,000-fold. Vaccination of chicks elicited cross-protective immune responses, as evidenced by reduced colonization (10- to 10,000-fold) of the gastrointestinal tract (ileum, cecum, and feces) and visceral organs (bursa and spleen) after challenge with homologous (Typhimurium F98) and heterologous (Enteritidis 4973 and S. enterica O6,14,24: e,h-monophasic) Salmonella serovars that are implicated in Salmonella infection of poultry. The protection conferred was observed for the organ or the maximum CFU/tissue/bird as a unit of analysis, suggesting that Dam mutant strains may serve as the basis for the development of efficacious poultry vaccines for the containment of Salmonella.
PMCID: PMC98898  PMID: 11705984
3.  Antibody Responses to MAP 1B and Other Cowdria ruminantium Antigens Are Down Regulated in Cattle Challenged with Tick-Transmitted Heartwater 
Serological diagnosis of heartwater or Cowdria ruminantium infection has been hampered by severe cross-reactions with antibody responses to related ehrlichial agents. A MAP 1B indirect enzyme-linked immunosorbent assay that has an improved specificity and sensitivity for detection of immunoglobulin G (IgG) antibodies has been developed to overcome this constraint (A. H. M. van Vliet, B. A. M. Van der Zeijst, E. Camus, S. M. Mahan, D. Martinez, and F. Jongejan, J. Clin. Microbiol. 33:2405–2410, 1995). When sera were tested from cattle in areas of endemic heartwater infection in Zimbabwe, only 33% of the samples tested positive in this assay despite a high infection pressure (S. M. Mahan, S. M. Samu, T. F. Peter, and F. Jongejan, Ann. N.Y. Acad. Sci 849:85–87, 1998). To determine underlying causes for this observation, the kinetics of MAP 1B-specific IgG antibodies in cattle after tick-transmitted C. ruminantium infection and following recovery were investigated. Sera collected weekly over a period of 52 weeks from 37 cattle, which were naturally or experimentally infected with C. ruminantium via Amblyomma hebraeum ticks, were analyzed. MAP 1B-specific IgG antibody responses developed with similar kinetics in both field- and laboratory-infected cattle. IgG levels peaked at 4 to 9 weeks after tick infestation and declined to baseline levels between 14 and 33 weeks, despite repeated exposure to infected ticks and the establishment of a carrier state as demonstrated by PCR and xenodiagnosis. Some of the serum samples from laboratory, and field-infected cattle were also analyzed by immunoblotting and an indirect fluorescent-antibody test (IFAT) to determine whether this observed seroreversion was specific to the MAP 1B antigen. Reciprocal IFAT and immunoblot MAP 1-specific antibody titres peaked at 5 to 9 weeks after tick infestation but also declined between 30 and 45 weeks. This suggests that MAP 1B-specific IgG antibody responses and antibody responses to other C. ruminantium antigens are down regulated in cattle despite repeated exposure to C. ruminantium via ticks. Significantly, serological responses to the MAP 1B antigen may not be a reliable indicator of C. ruminantium exposure in cattle in areas of endemic heartwater infection.
PMCID: PMC96068  PMID: 11238227
4.  Molecular characterization of the oafA locus responsible for acetylation of Salmonella typhimurium O-antigen: oafA is a member of a family of integral membrane trans-acylases. 
Journal of Bacteriology  1996;178(20):5904-5909.
Lipopolysaccharide (LPS) coats the surface of gram-negative bacteria and serves to protect the cell from its environment. The O-antigen is the outermost part of LPS and is highly variable among gram-negative bacteria. Strains of Salmonella are partly distinguished by serotypic differences in their O-antigen. In Salmonella typhimurium, the O-antigen is acetylated, conferring the 05 serotype. We have previously provided evidence that this modification significantly alters the structure of the O-antigen and creates or destroys a series of conformational epitopes. Here we report the detailed mapping, cloning, and DNA sequence of the oafA gene. The locus contains one open reading frame that is predicted to encode an inner membrane protein, consistent with its role in modification of the O-antigen subunit. The OafA protein shows homology to proteins in a number of prokaryotic and one eukaryotic species, and this defines a family of membrane proteins involved in the acylation of exported carbohydrate moieties. In many of these instances, acylation defines serotype or host range and thus has a profound effect on microbe-host interaction.
PMCID: PMC178445  PMID: 8830685
5.  Laboratory reared Amblyomma hebraeum and Amblyomma variegatum ticks differ in their susceptibility to infection with Cowdria ruminantium. 
Epidemiology and Infection  1995;115(2):345-353.
The susceptibility of laboratory reared Zimbabwean Amblyomma hebraeum and A. variegatum ticks to infection with geographically distinct Cowdria ruminantium strains was investigated by feeding both species simultaneously on individual sheep infected with one of the four strains (Crystal Springs [Zimbabwe], Ball 3 [South Africa], Gardel [Guadeloupe] and Nigeria [Nigeria]). A. hebraeum ticks demonstrated a high susceptibility to infection with all four C. ruminantium strains. In comparison, A. variegatum were less susceptible to infection with the Crystal Springs and Ball 3 strains (P < 0.001), but showed a similar susceptibility to the Gardel and Nigeria strains. The differences in susceptibility of A. variegatum to infection with the four strains of C. ruminantium correlated with the origin of these strains. The consistently higher susceptibility of A. hebraeum ticks to infection with geographically different C. ruminantium strains may be one explanation for the observation that heartwater is a more serious problem where A. hebraeum is the vector of the disease.
PMCID: PMC2271405  PMID: 7589273
6.  Sequence heterogeneity of the major antigenic protein 1 genes from Cowdria ruminantium isolates from different geographical areas. 
The genes for the immunodominant major antigenic protein 1 (MAP1) of Cowdria ruminantium from four African and two Caribbean isolates were cloned, restriction mapped, and sequenced to identify conserved epitopes for development of serodiagnostic tools for heartwater. Restriction length polymorphisms were observed among the respective MAP1 genes analyzed and were confirmed by sequencing. The sequence data generated for these isolates were compared with data for the previously reported Senegal isolate MAP1 gene. These sequences were found to differ from each other by 0.6 to 14.0%. These differences translate into a 0.8 to 10.0% variation in the predicted protein sequence. In the entire coding sequence, several amino acid substitutions were identified in addition to deletions or insertions at three regions of the gene. These variable regions are referred to as variable regions I, II, and III. From the sequence data, an evolutionary distance tree was constructed; this tree suggested that at least two genetically distinct C. ruminantium strains exist in the Caribbean: the isolate from Antigua is similar to that from Senegal, while the isolate from Guadeloupe is closely related to that from Sudan.
PMCID: PMC170360  PMID: 8807206
7.  Use of a specific immunogenic region on the Cowdria ruminantium MAP1 protein in a serological assay. 
Journal of Clinical Microbiology  1995;33(9):2405-2410.
Currently available serological tests for cowdriosis (Cowdria ruminantium infection) in domestic ruminants are hampered by their low specificities because of cross-reactivity with Ehrlichia spp. The use of recombinant major antigenic protein (MAP1) of C. ruminantium for serodiagnosis was investigated. Overlapping fragments of the MAP1 protein were expressed in Escherichia coli and were reacted with sera from sheep infected with either C. ruminantium or Ehrlichia ovina. Two immunogenic regions on the MAP1 protein, designated MAP1-A and MAP1-B, were identified. MAP1-A was reactive with C. ruminantium antisera, E. ovina antisera, and three MAP1-specific monoclonal antibodies, whereas MAP1-B reacted only with C. ruminantium antisera. An indirect enzyme-linked immunosorbent assay (ELISA) based on MAP1-B was further developed and validated with sera from animals experimentally infected with C. ruminantium or several Ehrlichia spp. Antibodies raised in sheep, cattle, and goats against nine isolates of C. ruminantium reacted with MAP1-B. Cross-reactivity with MAP1-B was limited to Ehrlichia canis and Ehrlichia chaffeensis, two rickettsias which do not infect ruminants. Antibodies to Ehrlichia spp. which do infect ruminants (E. bovis, E. ovina, and E. phagocytophila) did not react with MAP1-B. Antibody titers to C. ruminantium in sera from experimentally infected cattle, goats, and sheep were detectable for 50 to 200 days postinfection. Further validation of the recombinant MAP1-B-based ELISA was done with sera obtained from sheep raised in heartwater-free areas in Zimbabwe and from several Caribbean islands. A total of 159 of 169 samples which were considered to be false positive by immunoblotting or indirect ELISA did not react with MAP1-B.(ABSTRACT TRUNCATED AT 250 WORDS)
PMCID: PMC228424  PMID: 7494037
8.  Acetylation (O-factor 5) affects the structural and immunological properties of Salmonella typhimurium lipopolysaccharide O antigen. 
Infection and Immunity  1995;63(2):437-441.
The lipopolysaccharide (LPS) of gram-negative bacteria serves as a barrier between the cell and its environment. The LPS O antigen is the immunodominant portion of the molecule and thus has a significant effect on the interaction between a bacterial pathogen and the host organism. Antibodies directed against O antigen are vital to the immune response to infection. In this study, we have characterized the interaction between a series of monoclonal immunoglobulin A antibodies and the LPS of Salmonella typhimurium. Using one of these antibodies, we have previously shown that monoclonal immunoglobulin A is sufficient to protect against S. typhimurium infection, both in vivo and in vitro. Here, we show that recognition of LPS by the monoclonal antibodies is affected by acetylation of the O antigen on the abequose moiety, the determinant of the O5 epitope. Although recognition of LPS by several of the monoclonal antibodies is completely dependent on acetylation, the antibodies recognize clearly separable epitopes. This suggests that acetylation of O antigen affects the three-dimensional structure of the molecule and thus creates and destroys a series of conformational antigenic determinants. We have shown that a change in the acetylation state of LPS has no effect on virulence. However, acetylation has important consequences for the mucosal immune response and thus could potentially have profound implications for the ability of an immune host to respond to a subsequent infection.
PMCID: PMC173014  PMID: 7529745
9.  Development and evaluation of PCR assay for detection of low levels of Cowdria ruminantium infection in Amblyomma ticks not detected by DNA probe. 
Journal of Clinical Microbiology  1995;33(1):166-172.
The sensitivities of a PCR assay and a DNA probe assay were compared for the detection of Cowdria ruminantium in Amblyomma ticks that were fed on C. ruminantium-infected, clinically reacting, and recovered carrier animals. The PCR assay and DNA probe detected infection in 86.0 and 37.0%, respectively, of 100 ticks fed on a febrile animal. In 75 ticks fed on carrier animals, PCR and the DNA probe detected infection in 28.0 and 1.33% of ticks, respectively. This demonstrates that the DNA probe has poor sensitivity for the detection of low levels of infection in ticks and that PCR is necessary for this purpose. The PCR assay had a detection limit of between 1 and 10 C. ruminantium organisms and did not amplify DNA from Ehrlichia canis, which is phylogenetically closely related to C. ruminantium, Theileria parva, or uninfected Amblyomma hebraeum or A. variegatum. PCR detected infection in A. hebraeum and A. variegatum adult ticks infected with one of six geographically different C. ruminantium strains. Amplification was also possible from desiccated ticks and ticks fixed in 70% ethanol, 10% buffered formalin, or 2% glutaraldehyde. The PCR assay supersedes the DNA probe and older detection methods for the detection of C. ruminantium in ticks, particularly those fed on carrier animals, and is suitable for both prospective and retrospective studies which require accurate detection of C. ruminantium in individual ticks. Application of the PCR assay should significantly improve the understanding of heartwater epidemiology, particularly through the determination of field tick infection rates.
PMCID: PMC227901  PMID: 7699036
10.  Size variation of the major immunodominant protein of Cowdria ruminantium. 
An immunodominant response is made to a polypeptide of approximately 32 kDa in animals infected with the rickettsial pathogen Cowdria ruminantium. We show here using cultured strains of the rickettsia from different geographical areas that the apparent size of this polypeptide varies with strain origin. Changes in the primary structure between strains should be considered in the design of vaccines and diagnostic tests based on this antigen.
PMCID: PMC368408  PMID: 8556531
11.  Detection of Cowdria ruminantium by means of a DNA probe, pCS20 in infected bont ticks, Amblyomma hebraeum, the major vector of heartwater in southern Africa. 
Epidemiology and Infection  1993;110(1):95-104.
A DNA probe, pCS20, previously described for use in detection of Cowdria ruminantium infections in Amblyomma variegatum (the principal vector of heartwater) hybridized with C. ruminantium DNA in organs of laboratory-infected A. hebraeum adult ticks (the major southern African vector of heartwater). The probe hybridized with C. ruminantium DNA in 46/49 midguts from male ticks and 26/29 from females, thus indicating infection. Corresponding salivary glands were less heavily infected, but infections were more numerous in glands from males. Infection in ticks was confirmed by transmission of the disease to susceptible goats. The probe did not hybridize with DNA from uninfected ticks or with DNA from a spotted fever group rickettsia commonly associated with A. hebraeum in Zimbabwe. The C. ruminantium specific pCS20 DNA probe can be applied to determine accurately the infection rates in the two major vectors of heartwater and the risk of exposure of ruminants in endemic areas.
PMCID: PMC2271963  PMID: 8432329
12.  Conconavalin A-stimulated bovine T-cell supernatants inhibit growth of Cowdria ruminantium in bovine endothelial cells in vitro. 
Infection and Immunity  1994;62(2):747-750.
Conconavalin A-stimulated bovine T-cell supernatants inhibited the growth of Cowdria ruminantium in bovine endothelial cells in vitro but did not affect their entry. This finding represents one mechanism by which T cells may control C. ruminantium multiplication and hence affect the severity of disease.
PMCID: PMC186172  PMID: 8300237
13.  Bacteriophage P22 transduction of integrated plasmids: single-step cloning of Salmonella typhimurium gene fusions. 
Journal of Bacteriology  1993;175(21):7086-7091.
Transcriptional fusions to Salmonella typhimurium chromosomal genes were constructed by integration of a suicide fusion vector into the chromosome by homologous recombination with random cloned chromosomal fragments. We describe here a transductional method using the generalized transducing phage of S. typhimurium, P22, to clone these fusions directly from the bacterial chromosome, in a single step, without the use of restriction enzymes. In this transduction, the phage packages the chromosomal fragment containing the integrated plasmid. Once introduced into the recipient, the plasmid circularizes by homologous recombination between the duplicated region determined by the cloned fragment. Although RecA mediates the majority of these events, the plasmid can circularize in a recA recipient. However, in this case, the event occurs at a much lower frequency and only when the transduction is done at a high multiplicity of infection. In addition to integrated fusion constructs, we also show that autonomously replicating low-copy-number plasmids can be transduced. In this case, transduction is dependent on homologous recombination between the plasmid and the donor chromosome via cloned sequences, in which the transducing particle effectively traps the integrated plasmid.
PMCID: PMC206837  PMID: 8226650
14.  An immunoblotting diagnostic assay for heartwater based on the immunodominant 32-kilodalton protein of Cowdria ruminantium detects false positives in field sera. 
Journal of Clinical Microbiology  1993;31(10):2729-2737.
Heartwater, a major constraint to improved livestock production in Zimbabwe, threatens to invade areas which have been previously unaffected. To monitor its spread in Zimbabwe, an immunoblotting diagnostic assay based on the responses of animals to the immunodominant, conserved 32-kDa protein of Cowdria ruminantium was evaluated. In this assay, no false reactions were detected with sera known to be positive and negative, but sera from some cattle, sheep, and goats from heartwater-free areas of Zimbabwe reacted strongly with the 32-kDa protein, suggesting that either these animals had previous exposure to heartwater or they were false positives. To investigate the possibility of previous exposure to heartwater, 11 immunoblot-positive and 6 immunoblot-negative sheep from heartwater-free areas of Zimbabwe were compared regarding their susceptibilities to challenge with C. ruminantium. Prior to challenge, C. ruminantium could not be detected in any sheep by transmission to Amblyomma hebraeum ticks or by the polymerase chain reaction (PCR) conducted with plasma samples. All sheep were equally susceptible to the challenge, and infection was confirmed by brain biopsy, necropsy, PCR, and transmission of C. ruminantium to ticks. Our data suggest that the immunoblot-positive reactions of sera from heartwater-free areas were due not to previous C. ruminantium infection but rather to antigenic cross-reactivity between C. ruminantium and another agent(s) such as Ehrlichia species. In conclusion, the immunodominant 32-kDa protein is not antigenically specific to C. ruminantium and its use in serological diagnosis of heartwater requires reevaluation.
PMCID: PMC265996  PMID: 8253974
15.  Repressor binding to a regulatory site in the DNA coding sequence is sufficient to confer transcriptional regulation of the vir-repressed genes (vrg genes) in Bordetella pertussis. 
Journal of Bacteriology  1993;175(2):519-527.
Five TnphoA fusions to vir-repressed genes (vrg genes) have been identified in the respiratory pathogen Bordetella pertussis. A comparison of vrg DNA sequences suggests a consensus DNA element within the coding regions of four of five vrg genes. To determine the role of this DNA sequence in vrg regulation, a nucleotide substitution mutation in the conserved region of vrg-6 was isolated. This mutant showed constitutively high levels of expression in the absence of antigenic modulators, MgSO4 and nicotinic acid, suggesting that this DNA element may be a control site for vrg repression. Moreover, Northern (RNA) analysis and transcriptional fusion analysis suggest that vrg genes are regulated at the transcriptional level. To determine whether sequences in the coding region were sufficient to respond to antigenic modulation, a vrg-6::TnphoA promoter deletion plasmid that contained a heterologous promoter driving the expression of vrg-6 coding sequences from the vrg-6 translation start site to the TnphoA fusion junction was constructed. This heterologous construct responded to modulators in a vir-dependent fashion, indicating that sequences upstream of the coding sequence are not required for antigenic modulation. Southwestern (DNA-protein) analysis and mutational studies suggest that the vrg consensus DNA sequence is specifically recognized by a 34-kDa vir-activated gene (vag) product, whose binding results in down-regulation of vrg transcript levels. We conclude, at least for the vrg::TnphoA fusion strains, that a site on the DNA that corresponds to a consensus sequence located in the vrg coding region is sufficient to confer the transcriptional regulation (repression) of vrg genes when B. pertussis strains are grown under nonmodulating conditions.
PMCID: PMC196167  PMID: 8419298
16.  Host RecJ is required for growth of P22 erf bacteriophage. 
Journal of Bacteriology  1993;175(1):288-290.
Growth of bacteriophage P22 erf is known to require host RecA recombination function. We show that the RecA function is necessary but not sufficient to restore the plaque-forming ability of phage P22 erf; such mutant phage also requires host RecJ function. The residual efficiency of plaquing of P22 erf in a recJ background (0.03%) is completely abolished in recJ recB hosts (< 0.001%), suggesting that the RecBCD nuclease can provide an alternative function allowing phage growth. One tentative explanation is that circularization of P22 erf DNA mostly proceeds through the RecF pathway of recombination; however, less efficient circularization via the RecBCD pathway may also occur. In a recJ background, lysates obtained upon induction of an erf prophage show reduced yield (10%), suggesting that growth of P22 erf may require host RecJ in a step(s) other than circularization of phage DNA.
PMCID: PMC196124  PMID: 8416903
17.  Monoclonal secretory immunoglobulin A protects mice against oral challenge with the invasive pathogen Salmonella typhimurium. 
Infection and Immunity  1992;60(5):1786-1792.
Hybridomas producing monoclonal immunoglobulin A (IgA) antibodies against Salmonella typhimurium were generated by mucosal immunization of BALB/c mice with attenuated strains of S. typhimurium and subsequent fusion of Peyer's patch lymphoblasts with myeloma cells. To test the role of secretory IgA (sIgA) in protection against Salmonella sp., we analyzed in detail the protective capacity of a monoclonal IgA, Sal4, produced in polymeric as well as monomeric forms, that is directed against a carbohydrate epitope exposed on the surface of S. typhimurium. BALB/c mice bearing subcutaneous Sal4 hybridoma tumors and secreting monoclonal sIgA into their gastrointestinal tracts were protected against oral challenge with S. typhimurium. This protection was directly dependent on specific recognition by the monoclonal IgA, since mice secreting Sal4 IgA from hybridoma tumors were not protected against a fully virulent mutant that lacks the Sal4 epitope. Although monoclonal Sal4 IgA was present in the bloodstreams and tissues of tumor-bearing mice, it did not protect against intraperitoneal challenge and did not possess complement-fixing or bacteriocidal activity in vitro. Taken together, these results indicate that secretion of sIgA alone can prevent infection by an invasive enteric pathogen, presumably by immune exclusion at the mucosal surface.
PMCID: PMC257074  PMID: 1373399
18.  A cloned DNA probe for Cowdria ruminantium hybridizes with eight heartwater strains and detects infected sheep. 
Journal of Clinical Microbiology  1992;30(4):981-986.
The DNA probe pCS20, which was cloned from the DNA of the Crystal Springs heartwater strain from Zimbabwe, cross-reacted with DNAs of heartwater strains from all endemic areas, including four heartwater strains from Zimbabwe, two strains from South Africa, one strain from Nigeria, and the Gardel strain from the Caribbean island of Guadeloupe. By nucleic acid hybridization, the pCS20 DNA probe detected Cowdria ruminantium DNA in all DNA preparations made from plasma samples from infected sheep before and during the febrile reaction. Synthetic oligonucleotides were prepared for amplification of specific C. ruminantium DNA sequences by the polymerase chain reaction (PCR). Amplification of two DNA products (181 and 279 bp) from pCS20 DNA and C. ruminantium genomic DNA of heartwater strains was demonstrated. In contrast, amplification of these products or any other products was not possible from genomic DNAs of Anaplasma marginale, Babesia bigemina, Trypanosoma brucei brucei, Escherichia coli, and bovine endothelial cells. The cross-reactivities of the 32P-labeled PCR products with genomic DNAs from several heartwater strains were similar to those with the pCS20 DNA probe. A nucleic acid-based test that uses hybridization assays and PCR provides a sensitive method for the detection of heartwater in both animals and ticks and has applications in epidemiological studies for the disease, which may allow for improved disease control.
PMCID: PMC265197  PMID: 1572987
19.  A cloned DNA probe identifies Cowdria ruminantium in Amblyomma variegatum ticks. 
Journal of Clinical Microbiology  1991;29(11):2571-2577.
Heartwater, caused by Cowdria ruminantium and transmitted by ticks of the genus Amblyomma, is a constraint to ruminant animal production in sub-Saharan Africa. This rickettsial disease could spread from endemically infected areas of sub-Saharan Africa and certain Caribbean islands to other countries, including the United States, in which Amblyomma ticks exist. To detect C. ruminantium in tick vectors and animals, we made DNA probes from C. ruminantium DNA isolated from endothelial cell cultures. Two clones were evaluated; pCS20 from Crystal Springs (Zimbabwe) strain DNA had a 1,306-bp insert, and pCR9 from Kiswani (Kenya) strain DNA had a 754-bp insert. Both DNA probes detected 1 ng of Crystal Springs DNA; however, the pCS20 probe had a 10-fold-greater ability to discriminate between C. ruminantium DNA and DNA from other organisms. Also, the pCS20 probe did not hybridize to 400 ng (highest amount tested) of DNA from bovine cells, 3 protozoa, 3 rickettsiae, and 12 bacteria. In all experiments, C. ruminantium DNA was detected in midguts from 99 of 160 Amblyomma variegatum nymphs infected as larvae and in midguts from 38 of 80 adult ticks infected as nymphs but not in midguts from control nymphs and adults. The presence of C. ruminantium in nymphs and adults was confirmed by transmission of heartwater to goats. The DNA sequences of both probes were determined; synthetic oligonucleotides from pCS20 are recommended as DNA probes for C. ruminantium.
PMCID: PMC270375  PMID: 1774264
20.  recB and recC genes of Salmonella typhimurium. 
Journal of Bacteriology  1989;171(1):612-615.
We have investigated the genetic organization of the recB (exonuclease V) and recC (exonuclease V) genes of Salmonella typhimurium. A detailed genetic map is constructed that includes the relative order in the chromosome, P22 cotransduction frequencies, and the orientation of transcription of the recB and recC genes. In addition, the isolation and characterization of Mu dJ insertion mutations in recB and recC are discussed.
PMCID: PMC209634  PMID: 2644211
21.  Control of trypanodestructive antibody responses and parasitemia in mice infected with Trypanosoma (Duttonella) vivax. 
Infection and Immunity  1986;54(1):213-221.
After infection with a cloned population of Trypanosoma vivax, C57BL/6 mice controlled parasitemia during the exponential growth phase and survived, with intermittent parasitemia, for several weeks. In contrast, most mice of the C3H/He strain did not control the first wave of parasitemia and died within 9 to 13 days after infection. Control of parasitemia in C57BL/6 mice was mediated by the production of a variant surface glycoprotein-specific trypanodestructive antibody response which was accompanied by production of antibodies against antigens shared between procyclic and bloodstream T. vivax as well as antibodies against trinitrophenyl (TNP) and sheep erythrocytes. The infected C3H/He mice did not produce trypanodestructive antibodies or antibodies against procyclic antigens or TNP but did produce antibodies against sheep erythrocytes. Although infected C57BL/6 mice produced levels of serum immunoglobulin M four times higher than infected C3H/He mice, their parasite-induced B-cell DNA synthetic responses were similar, and both sets of mice developed similar numbers of spleen cells with cytoplasmic immunoglobulin M, a proportion of which could react with TNP. In vitro biosynthetic labeling studies accompanied by immunoglobulin precipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated that the immunoglobulin-containing cells of infected C3H/He mice synthesized and secreted less immunoglobulin than similar cells from infected C57BL/6 mice. We concluded that some parasite-induced antibody-forming cells in C3H/He mice, perhaps including parasite-specific and certainly including TNP-specific cells, had an impaired capacity to make and release immunoglobulin. Within 24 h after Berenil-mediated elimination of T. vivax from infected C3H/He mice, a population of cyclophosphamide-sensitive spleen cells produced large amounts of parasite-specific and TNP-specific antibody. We concluded that the defect in terminal B-cell function leading to suppressed parasite-specific and TNP-specific antibody responses was induced either by living trypanosomes or short-lived factors from degenerating trypanosomes or by short-lived parasite-induced host responses.
PMCID: PMC260139  PMID: 3489676
22.  Genetic analysis of the proBA genes of Salmonella typhimurium: physical and genetic analyses of the cloned proB+ A+ genes of Escherichia coli and of a mutant allele that confers proline overproduction and enhanced osmotolerance. 
Journal of Bacteriology  1983;156(3):1249-1262.
Because of the fact that proline overproduction relieves the inhibitory effects of high external osmotic strength in a number of procaryotes, we wished to clone a mutant allele, pro-74, that confers proline overproduction and enhanced osmotolerance on Salmonella typhimurium and Escherichia coli. Therefore, the pro-74 allele, originally located on an E. coli episome F'128, was cloned into pBR322. In a parallel experiment, the wild type proB+ A+ genes of E. coli were also cloned from F'128 into pBR322. Both the pro-74 and the proB+ A+ alleles were obtained on a 10.4-kilobase-pair fragment that also contained the unrelated phoE gene. Strains carrying either the wild-type proB+ A+ or the pro-74 alleles on pBR322 grew more slowly, both in minimal medium and media of elevated osmotic strength, than strains carrying the same alleles on the low-copy plasmid, F'128, indicating that some gene in the cloned region is deleterious in high copy. We constructed Tn5 insertion mutations in the proB and the proA genes of E. coli, carried on F'128 in S. typhimurium. Using P22 transduction in S. typhimurium, we transferred these proB and proA::Tn5 insertions from F'128 into the cloned proBA genes on pBR322. From the restriction maps of the plasmids thus generated, we determined the approximate locations of the proB and the proA genes. We also performed complementation tests of S. typhimurium and E. coli proB and proA mutants by using the F'128 proB and proA::Tn5 insertions. These tests revealed that the proBA genes of S. typhimurium form an operon, whose direction of transcription is from proB to proA. They also indicated that in S. typhimurium, as in E. coli, the proB+ gene encodes gamma-glutamyl kinase, and the proA+ gene encodes gamma-glutamyl phosphate reductase. Complementation tests also indicated that the pro-74 mutation is either in the proB structural gene, or its promoter-operator.
PMCID: PMC217975  PMID: 6315682

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