A cloned DNA probe identifies Cowdria ruminantium in Amblyomma variegatum ticks.

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.     

An immunoblotting diagnostic assay for heartwater based on the immunodominant 32-kilodalton protein of Cowdria ruminantium detects false positives in field sera.

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.     

Protective immunity to heartwater (Cowdria ruminantium infection) is acquired after vaccination with in vitro-attenuated rickettsiae.

A Senegalese (S) stock of Cowdria ruminantium was passaged on bovine umbilical endothelial cells with an average interval of 13.9 days (range, 8 to 34 days) between passages. The virulence of infected bovine umbilical endothelial cultures was tested in susceptible goats and sheep by intravenous inoculation of culture supernatant from passages 2 (51 days in vitro), 3 (69 days), 11 (229 days), 14 (264 days), and 16 (291 days). Both animals inoculated with passages 2 and 3 died of heartwater. However, clinical reactions were completely absent in goats and sheep that were inoculated with C. ruminantium from passages 11, 14, and 16. High antibody titers were detected, with immunofluorescence in all vaccinated animals, and a strong signal was found against a 32-kDa Cowdria protein in Western blots (immunoblots). Moreover, the vaccinated animals proved solidly immune when challenged with virulent Cowdria sp.-infected blood stabilate (S strain), whereas all control goats died. No attenuation of a second Cowdria stock (W) was achieved after 226 days in culture, at which time passage 17 was tested in a recipient goat which died of typical heartwater. This is the first report of vaccination with live attenuated C. ruminantium. These attenuated organisms may replace vaccination with virulent blood currently in use in areas where heartwater is endemic.     

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.     

Development and evaluation of PCR assay for detection of low levels of Cowdria ruminantium infection in Amblyomma ticks not detected by DNA probe.

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.     

A DNA probe cloned in Escherichia coli for the identification of Brugia malayi

This report describes a specific and sensitive DNA probe for the identification of Brugia malayi. A genomic DNA library produced from subperiodic B. malayi microfilariae was screened to detect clones containing DNA sequences which are highly repeated within the parasite genome. Several clones were further analyzed to identify those which hybridize specifically with B. malayi DNA but not with DNA from B. pahangi and Dirofilaria immitis. From these, clone pBm15 was selected because it hybridized with high sensitivity to B. malayi DNA as detected by autoradiography. Clone pBm15 was sensitive enough to detect two infective larvae or five microfilariae or 300 pg of purified B. malayi microfilarial DNA. This study forms the basis for the development of a specific and sensitive DNA probe for the identification of B. malayi in field specimens.     

Dot blot hybridization assay for chicken anemia agent using a cloned DNA probe.

A dot blot hybridization assay capable of detecting chicken anemia agent (CAA)-specific DNA in tissues from infected birds has been developed. The assay uses a 32P-labeled DNA probe prepared from cloned CAA-specific fragments representing the entire virus genome and has a sensitivity limit of between and 1 and 10 pg. DNAs from CAA isolates originating in the Federal Republic of Germany, Japan, the United States, the United Kingdom, and Australia were detected. Investigation of specimens from experimentally infected chicks indicated that virus-specific DNA was detected in the tissues of birds from 5 through 42 days after infection and that greater amounts were usually detected in the thymus than in the spleen, liver, feces, or blood. Tissues from specific-pathogen-free and broiler chicks which had become infected at an older age through contact with experimentally infected anemic chicks also contained CAA-specific DNA detectable by the assay. Thymuses from 1- to 2-week-old chicks from eight commercial broiler flocks which had been showing clinical signs characteristic of anemia-dermatitis syndrome were found positive by the hybridization technique, but thymuses from chicks obtained from broiler flocks which did not show such signs were found negative. Of the 35 positive samples (from 46 samples tested), 19 (54%) contained virus-specific DNA in sufficiently great amounts to permit 4-h autoradiography exposures and sample throughput times of 2 days. When compared with virus isolation, the CAA dot blot hybridization assay is time- and labor-saving.     

Development of a DNA probe for Streptococcus bovis by using a cloned amylase gene.

Streptococcus bovis is a normal inhabitant of the rumen but has been implicated as a causative agent for ruminal lactic acidosis and related problems. While rarely isolated from humans, S. bovis has been identified as a causative agent for endocarditis, meningitis, and septicemia. Recent reports have also suggested a correlation between human colonic carcinoma and increased levels of S. bovis. Identification of S. bovis strains of human origin has been problematic because of variations in results of biochemical tests compared with results for ruminal strains. We have tested a cloned amylase gene from the ruminal strain S. bovis JB1 as a potential DNA probe for rapid and accurate identification of S. bovis strains from all sources. DNAs from strains identified as S. bovis, of both human and ruminal origin, were found to hybridize with the probe under stringent conditions. The probe also hybridized with variants of S. bovis that did not grow on starch. The probe did not hybridize with DNA isolated from other bacteria of human colonic and ruminal origin, including Bacteroides thetaiotaomicron, Bacteroides ruminicola, Butyrivibrio fibrisolvens, and Enterococcus faecalis but did demonstrate hybridization with Streptococcus salivarius.     

Comparison of alkaline phosphatase-conjugated oligonucleotide DNA probe with the Sereny test for identification of Shigella strains.

We compared an alkaline phosphatase-conjugated oligonucleotide DNA probe with the Sereny test to determine the sensitivity and specificity of the probe in detecting virulent Shigella strains. The probe hybridized with all 52 Sereny-test-positive strains (sensitivity, 100%) and 4 of 21 Sereny-test-negative strains (specificity, 81%). The probe did not hybridize with any of the Sereny-test-negative S. dysenteriae type 1 strains. This nonradioactive, synthetic probe provides a simple, rapid way to test a large number of strains simultaneously in a field setting, which will contribute to an improved understanding of the epidemiologic patterns of shigellosis in developing countries.     

A species-specific DNA probe for the detection of Mycoplasma gallisepticum.

An 800-base-pair DNA fragment from a partial genomic library of Mycoplasma gallisepticum was selected and used as a probe for the selective detection of this avian pathogen. The specificity and sensitivity of this probe were demonstrated by using dot blot and Southern hybridizations.     

Comparison of protease-resistant prion protein inhibitors in cell cultures infected with two strains of mouse and sheep scrapie

The transmissible spongiform encephalopathies (TSEs) are fatal neurodegenerative diseases. A primary therapeutic target for TSE intervention has been a protease-resistant form of prion protein known as PrP(Sc) or PrP-res. In vitro testing of mouse scrapie-infected cell cultures has identified many PrP-res inhibitors that also have activity in vivo. Here we identify 32 new inhibitors of two strains of mouse scrapie PrP-res. Furthermore, to investigate the species-specificity of these and other PrP-res inhibitors, we have developed a high-throughput cell culture assay based on Rov9 cells chronically-infected with sheep scrapie. Of 32 inhibitors of murine PrP-res that were also tested in the Rov9 cells, only six showed inhibitory activity against sheep PrP-res. The three most potent inhibitors of both murine and ovine PrP-res formation (with 50% inhibition at < or =5 microM) were tannic acid, pentosan polysulfate and Fe(III) deuteroporphyrin 2,4-bisethyleneglycol. The latter two have anti-mouse scrapie activity in vivo. These results identify new inhibitors of murine and ovine PrP-res formation and reinforce the idea that compounds effective against PrP-res from one species or strain cannot be assumed to be active against others.     

Distinction of species and strains of mycoplasmas (mollicutes) by genomic DNA fingerprints with an rRNA gene probe.

Genomic fingerprints of Acholeplasma laidlawii, Mycoplasma hominis, and Mycoplasma pneumoniae strains were obtained by Southern blot hybridization of the digested mycoplasmal DNAs with an rRNA gene probe. The hybridization patterns revealed genotypic heterogeneity among A. laidlawii and M. hominis strains and a remarkable degree of homogeneity among M. pneumoniae strains isolated from pneumonia patients during a 10-year period. Genomic fingerprints with the rRNA gene probe can thus serve as indicators of intraspecies genetic homogeneity or heterogeneity and can provide a new, sensitive tool for strain identification with a potential for application in epidemiology.     

Detection of Neospora from tissues of experimentally infected rhesus macaques by PCR and specific DNA probe hybridization.

Neospora is a newly recognized Toxoplasma-like cyst-forming coccidian parasite that causes abortion or congenital infections in naturally or experimentally infected animals. In this study, pregnant rhesus macaques were inoculated with culture-derived tachyzoites of a bovine Neospora isolate, and tissue samples from various major organs were collected from dams and fetuses for the detection of parasite DNA by using oligonucleotide primers COC-1 and COC-2 for PCR amplification of a conserved coccidial nuclear small-subunit rRNA gene sequence, and amplification products were confirmed by hybridization with a Neospora-specific DNA probe. PCR products were amplified from DNAs of different fetal monkey tissues, including brain, heart, lung, liver, spleen, skeletal muscle, skin, and placenta. In addition, Neospora DNA was amplified from the brain, heart, and lung tissues of infected rhesus macaque dams. The PCR and probe hybridization system may provide an effective method for the detection of Neospora infection in fetuses and dams from nonhuman primates and may be useful in determining the zoonotic potential of Neospora.     

Prior enrichment of human immunodeficiency virus DNA with probe DNA particles for efficient PCR diagnosis.

We report here a novel method by which one can recover human immunodeficiency virus (HIV) DNA in a small volume (approximately 50 microliters) of solution from 8 ml of crude cell lysate which contains as few as several HIV DNA copies. The method uses the specific binding of HIV DNA to HIV probe DNA particles. The HIV DNA thus concentrated on the particles can be subjected to the PCR assay. The method also enables the examination of 100 individual blood specimens in a combined form.     

Macrophage activity in resistant and susceptible mouse strains infected with Mycobacterium lepraemurium.

The level of activation of peritoneal macrophages following subcutaneous inoculation of resistant (C57BL) and susceptible (BALB/c) mice was assessed by monitoring superoxide anion and hydrogen peroxide production and also tumour cell cytostasis. The level of systemic macrophage activation appeared to correlate with bacterial load, rather than resistance to infection. It was observed that the more susceptible (BALB/c) strain developed higher and more sustained levels of systemic macrophage activation, whereas the more resistant (C57BL) strain showed only low transient levels of macrophage activation. In contrast, in vivo challenge of subcutaneously infected C57BL mice, via the intra-peritoneal route, with heat-killed Mycobacterium lepraemurium and thioglycollate resulted in a high level of macrophage activation compared with similarly treated uninfected mice. Similar treatment of susceptible BALB/c mice, however, did not result in enhanced macrophage activation. It was also observed that high levels of macrophage activation occurred in T-cell deprived C57BL mice following infection with M. lepraemurium.     

Rapid screening for B19 parvovirus DNA in clinical specimens with a digoxigenin-labeled DNA hybridization probe.

A rapid dot blot hybridization assay for the detection of B19 parvovirus DNA in human sera was developed. Small portions of four serum samples were mixed, filtered onto a nylon membrane, and hybridized with a digoxigenin-labeled DNA probe; for each membrane, 380 serum samples could be tested. When a dot was positive by the hybridization assay, the four serum samples dotted together were separately tested to identify the sample positive for B19 DNA. A total of 10,150 serum samples submitted for viral serological and laboratory investigation with no specific requests for B19 testing were analyzed. Nine serum samples were positive for B19 DNA by dot blot hybridization assay, and the results were confirmed by electron microscopy. This method has proven to be reliable, economical in terms of time and costs, and useful for large-scale screening of clinical specimens, both for diagnostic work and for a source of antigen.     

A repetitive DNA probe specific for a North American sylvatic genotype of Trichinella.

A partial genomic DNA library constructed in pUC 13 using DNA from a sylvatic isolate of Trichinella spiralis (T. spiralis T5) was differentially screened with radiolabeled homologous genomic DNA and with DNA from T. spiralis T1. One clone was identified and designated pUPB-3.7 which, by slot blot and Southern blot analyses, reacted specifically with T. spiralis T5 DNA and did not cross-react with DNA from any other T. spiralis genotype. The 482-bp repetitive sequence which is 70% rich in A and T residues, comprises at least 2.7% of the parasite genome and can detect as little as 0.4 ng of DNA. When used to assess the prevalence of T. spiralis T5 in Indiana wildlife, DNA from 19 of 20 independently obtained sylvatic isolates reacted positively with the pUPB-3.7 probe indicating that within this geographical locality, T. spiralis T5 is the predominating genotype in wild mammals.