Detection of bovine trichomoniasis with a specific DNA probe and PCR amplification system.

Trichomoniasis is a widespread, economically important venereal disease of cattle which causes infertility and abortion. Effective control of trichomoniasis has been impeded by the insensitivity of traditional diagnostic procedures, which require the isolation and cultivation of the parasite, Tritrichomonas foetus, from infected cattle. We developed a 0.85-kb T. foetus DNA probe by identifying conserved sequences in DNAs from T. foetus that were isolated from cattle in California, Idaho, Nevada, and Costa Rica. The probe hybridized specifically to DNAs of T. foetus isolates from different geographic areas but not to DNA preparations of Trichomonas vaginalis, bovine cells, or a variety of bacteria from cattle. The probe detected DNA from a minimum of 10(5) T. foetus organisms. To improve sensitivity, a partial sequence of the probe was used to identify oligonucleotide primers (TF1 and TF2) which could be used to amplify a 162-bp product from T. foetus DNAs by PCR. A chemiluminescent internal T. foetus sequence probe was hybridized to Southern blots of the amplification product. This system detected as few as one T. foetus organism in culture media or 10 parasites in samples containing bovine preputial smegma. Analysis of 52 clinical samples showed that 47 (90.4%) of the 52 samples were correctly identified, with no false-positive reactions. In comparison, the traditional cultivation method detected 44 (84.6%) of the 52 samples from T. foetus-infected and uninfected bulls. These results indicate that the PCR-based amplification system could be a useful alternative method for the diagnosis of bovine trichomoniasis.     

A putative RNA virus in Babesia bovis

Babesia bovis is an intraerythrocytic protozoan that causes bovine babesiosis. Agarose gel electrophoresis of nucleic acids extracted from two isolates of B. bovis reveals, besides bulk DNA, an ethidium bromide-stainable band at about 5.5 kb. Further characterization of the latter with DNase I, RNase and mung bean nuclease suggested it to be a double-stranded RNA. Sonicated parasites were fractionated in a CsCl buoyant density gradient. A sample containing the 5.5-kb RNA was analysed under an electron microscope and a virus-like particle was observed.     

Comparative studies on Marek's disease virus and herpesvirus of turkey DNAs.

DNA of Marek's disease virus (MDV) was compared to that of herpes virus of turkey (HVT). Centrifugation of the two virus DNAs in neutral glycerol and CsCl density gradients showed that the MDV genome was slightly larger than that of HVT and that the buoyant density (1.705 g/ml) of MDV DNA in CsCl gradients was slightly lower than that (1.707 g/ml) of HVT DNA. MDV and HVT DNAs were digested with either EcoRI or HindIII restriction endonuclease and analysed by 0.5% agarose gel electrophoresis. The cleavage patterns of HindIII or EcoRI DNA digests of two strains of these two viruses showed general similarities between the strains, but not between MDV and HVT. However, a few fragments of EcoRI or HindIII digests of MDV DNA co-migrated with those of HVT DNA. DNA-DNA reassociation kinetics and DNA-RNA hybridization between the two viruses indicated that MDV and HVT DNAs share detectable homology, although it is less than 5%. The DNA of a HVT variant, which has lost the ability to protect chickens from Marek's disease, appeared similar to DNA of the vaccine strain in the size buoyant density and in its restriction endonuclease cleavage pattern.     

Novel identification of differences in the kinetoplast DNA of Leishmania isolates by recombinant DNA techniques and in situ hybridisation

Kinetoplast DNA (kDNA) was extracted from marker isolates of three 'Old World' cutaneous leishmania, L. tropica, L. aethiopica and L. major. Restriction endonuclease digestion followed by electrophoresis showed that each isolate produced a unique pattern of DNA fragments. The kDNA of each isolate was hybridised to Southern blots of digests of all three kDNAs. This showed that the kDNA of each isolate contained sequences unique to that isolate as well as sequences common to all three isolates. The kDNA sequences of L. tropica and L. aethiopica were more closely related than either were to those of L. major. kDNA of each isolate was cloned and plasmids selected which contained a fragment of DNA unique to the isolate from which the kDNA originated. Whole kDNA was hybridised to organisms fixed on a microscope slide. In each case the kDNA hybridised strongly to the kinetoplast of the organism from which the DNA had been extracted. There was a small amount of cross-hybridisation between L. tropica and L. aethiopica confirming the result of the Southern blot hybridisation. The feasibility of a method of isolate identification using recombinant DNA probes containing sequences unique to a particular isolate or group of isolates is discussed.     

A new method for identification of Trichomonas vaginalis by fluorescent DNA in situ hybridization.

The protozoan flagellate Trichomonas vaginalis is responsible for human trichomoniasis, one of the most widespread sexually transmitted diseases in the world. Several methods are currently used for laboratory diagnosis, including direct microscopic observation, cell culture, immunological techniques, and more recently, DNA probing and gene amplification. This report describes an in situ hybridization technique with specific DNA probes labeled with either biotin, rhodamine, or fluorescein for detection of T. vaginalis with fluorescence microscopy. Repetitive DNA sequences were evident in the nuclei of the protozoa as intensively fluorescent regions, giving a spotted pattern. No cross-reactivity between the probes and the DNAs of mammalian cells, yeasts, or bacteria was noted. This technique is potentially useful for the diagnosis of human trichomoniasis in clinical samples.     

Biophysical properties of virions of human adenovirus of the type 6 and its DNA

A study has been made of human adenovirus type 6 (Ad 6) which belongs to the C subgroup of the nononcogenic adenoviruses. The buoyant density of the Ad 6 virions in CsCl gradient was 1.3545 +/- 0.0010 g/cm3. The DNA content determined from the buoyant density values and direct phosphorus analysis was 11.2 +/- 0.5 and 11.5 +/- 0.5 per cent, respectively. The S20,w0 and D20,w0 values for the Ad 6 virions were 795 +/- 18S and (3.255 +/- 0.035) X 10-8 cm2/s. The hydrodynamic parameters of the viral particle and its volume as well as the specific partial volume, the content and the molecular weight of the DNA have yielded a molecular weight of the virions of (203.6 +/- 10.0) X 10(6). The sedimentation constant and the intrinsic viscosity of the DNA have been found to be 32.7 +/- 0.7S and 93.5 +/- 5.0 dl/g, respectively. The molecular weight of the DNA found from these results is (23.4 +/- 0.8) X 10(6). The buoyant density of the Ad 6 DNA measured in CsCl and Cs2SO4 density gradients was 1.7128 +/- 0.0010 and 1.427 +/- 0.001 g/cm3, respectively. The GC content found from the buoyant density and the melting temperature (90.5 +/- 0.1 degrees C in 1 SSC and 75.0 +/- 0.1 degrees C in 0.1 SSC) was 52.6 +/- 1.0 per cent, that is, it is considerably lower than the GC content of 56-60 per cent which, according to the "Green's rule", should be typical of the DNAs of all the human adenoviruses of the subgroup C (30).     

Comparison of the DNAs of varicella-zoster viruses isolated from clinical cases of varicella and herpes zoster

The DNA of varicella-zoster virus (VZV) has been characterized by sucrose gradient sedimentation and by isopycnic banding in CsCl. Comparisons of the DNAs from different clinical isolates have been made by mixing radiolabeled DNAs prior to centrifugation. VZV-DNA sedimented just behind T4 DNA on neutral sucrose gradients. Thus VZV-DNA has a molecular weight of about 100 x 10⁶. There were no distinguishable differences in the sedimentation behavior in neutral sucrose gradients of the DNAs from several clinical VZV isolates. The buoyant density in CsCl of the DNA of VZV isolated from varicella was reproducibly slightly lighter than the buoyant density of the DNA of VZV isolated from herpes zoster.     

Purification of murine alpha-herpesvirus and some properties of its DNA

Strain Sumava Af of murine alpha-herpesvirus (MHV) banded in density gradient at the boundary between 10-20% and 20-30% Ficoll solution. In electron microscope, the partially purified virus visualized by negative staining exhibited intact or disrupted envelope structures and capsid particles of about 110 nm in outer diameter. The DNA extracted from partially purified virus and further purified in CsCl density gradient had the Tm-value of 79.0 degrees C. The molecular weight of about 90 million daltons was calculated for viral DNA according to its migration in agarose gel.     

Purification of biologically active Epstein-Barr virus by affinity chromatography and non-ionic density gradient centrifugation

Epstein-Barr virus was purified by affinity chromatography on ricin agglutinin Sepharose followed by non-ionic density gradient centrifugation on Nycodenz. The purified virus was highly active in three biological assays: stimulation of immunoglobulin synthesis by B lymphocytes, transformation of B lymphocytes, and superinfection of Raji cells, as well as in an indirect immunofluorescent binding assay. Electron microscopy revealed intact viral particles free of contaminating membranous structures. CsCl density gradient analysis of nick-translated DNA showed material only at the expected viral density with no detectable material at the density of cellular DNA. SDS gel electrophoresis of radioiodinated virus revealed the characteristic viral polypeptides. This method produces highly purified, biologically active virions with an overall recovery of about 30%.     

Molecular probe for identification of Trichomonas vaginalis DNA.

Trichomoniasis is one of the most widespread sexually transmitted diseases in the world. Diagnosis can be achieved by several methods, such as direct microscopic observation of vaginal discharge, cell culture, and immunological techniques. A 2.3-kb Trichomonas vaginalis DNA fragment present in strains from diverse geographic areas was cloned and used as a probe to detect T. vaginalis DNA in vaginal discharge by a dot blot hybridization technique. This probe was specific for T. vaginalis DNA. It recognized strains from two regions in Italy (Sardinia, Piemonte) and from Mozambique (Africa). In addition, our probe did not cross-react with bacterial (Escherichia coli, Enterococcus spp., group B streptococci, Gardnerella vaginalis, Neisseria gonorrhoeae, Chlamydia trachomatis, and Lactobacillus spp.), viral (herpes simplex virus type 2), fungal (Candida albicans), protozoan (Entamoeba histolytica, Giardia lamblia, Plasmodium falciparum, Leishmania major, and Leishmania infantum), or human nucleic acids. The probe reacted with Pentatrichomonas hominis and Trichomonas foetus. The limit signal recognized by our probe corresponded to the DNA of 200 T. vaginalis isolates. The 2.3-kb probe was used in a clinical analysis of 98 samples. Of these, 20 samples were found to be positive both with the probe and by cell culture, and only 14 of these were positive by a standard wet mount method.     

Physicochemical properties of the DNAs of the fastidious adenovirus species 40 and 41

The DNAs of the two fastidious adenovirus species 40 and 41 (Ad40 and Ad41) have been studied by restriction endonuclease cleavage, CsCl density gradient centrifugation, and liquid-phase hybridization. For Ad40 DNA, cleavage maps were constructed for the restriction endonucleases EcoRI, SalI, ClaI, BstEII, BclII, and PvuI. The EcoRI, SalI, ClaI, and XhoI maps could be established for Ad41 DNA. The buoyant density in CsCl of DNAs of the fastidious species is 1.711 g/cm3 corresponding to a G + C content of 52%. The homology of the DNAs from Ad40 and Ad41 proved to be 62-69%. These results are consistent with previous reports on antigenic and DNA restriction enzyme analysis in which the classification into two species was proposed (J. C. De Jong, R. Wigand, A. H. Kidd, G. Wadell, J. G. Kapsenberg, C. J. Muzerie, A. G. Wermenbol, and R.-G. Fritzlaff, J. Med. Virol. 11, 215-231 (1983), and I. Uhnoo, G. Wadell, L. Svensson, and M. Johansson, Dev. Biol. Stand. 53, 311-318 (1983].     

Involvement of MAP kinases in apoptosis of macrophage treated with Trichomonas vaginalis

A primitive protozoan parasite Trichomonas vaginalis selectively activates the signal transduction pathways in macrophages (RAW264.7). This study evaluated the correlation of these signaling pathways and T. vaginalis-induced cell apoptosis. In macrophages infected with T. vaginalis, apoptosis was assessed on the basis of DNA fragmentation on agarose gel electrophoresis. Infection of macrophages with T. vaginalis induced tyrosine phosphorylation of several proteins. Infected cells with T. vaginalis were shown to associate with phosphorylation of the extracellular signal-regulated (ERK)1/2 kinase, p38, c-Jun N-terminal kinase (JNK) mitogen-activated protein (MAP) kinases on Western blot analysis. The present finding also demonstrated a link between the ERK1/2, JNK and p38 apoptotic pathways that was modulated by T. vaginalis infection.     

PCR-based study of conserved and variable DNA sequences of Tritrichomonas foetus isolates from Saskatchewan, Canada.

The protozoan parasite Tritrichomonas foetus causes infertility and spontaneous abortion in cattle. In Saskatchewan, Canada, the culture prevalence of trichomonads was 65 of 1,048 (6%) among 1,048 bulls tested within a 1-year period ending in April 1994. Saskatchewan was previously thought to be free of the parasite. To confirm the culture results, possible T. foetus DNA presence was determined by the PCR. All of the 16 culture-positive isolates tested were PCR positive by a single-band test, but one PCR product was weak. DNA fingerprinting by both T17 PCR and randomly amplified polymorphic DNA PCR revealed genetic variation or polymorphism among the T. foetus isolates. T17 PCR also revealed conserved loci that distinguished these T. foetus isolates from Trichomonas vaginalis, from a variety of other protozoa, and from prokaryotes. TCO-1 PCR, a PCR test designed to sample DNA sequence homologous to the 5' flank of a highly conserved cell division control gene, detected genetic polymorphism at low stringency and a conserved, single locus at higher stringency. These findings suggested that T. foetus isolates exhibit both conserved genetic loci and polymorphic loci detectable by independent PCR methods. Both conserved and polymorphic genetic loci may prove useful for improved clinical diagnosis of T. foetus. The polymorphic loci detected by PCR suggested either a long history of infection or multiple lines of T. foetus infection in Saskatchewan. Polymorphic loci detected by PCR may provide data for epidemiologic studies of T. foetus.     

Identification of virus-like particles in Eimeria stiedae

When nucleic acid samples purified from sporozoites of Eimeria stiedae were analyzed by agarose gel electrophoresis, an ethidium-stainable band with an apparent electrophoretic mobility of 6.5 kb was consistently observed. The band was readily degradable upon RNAse treatment, and its susceptibility towards ribonuclease A on a decreasing ionic strength was suggestive of double-stranded RNA (dsRNA). Electron microscopy revealed spherical, probably icosahedral, virus-like particles (VLP) with a diameter of 35 nm in sporozoite lysates. The VLP were purified by CsCl buoyant density gradient centrifugation. Upon extraction, these particles yielded dsRNA molecules of a uniform length of 1.63 microns. The presence of the VLP was investigated in different Eimeria strains. All E. stiedae isolates contained the RNA virus, whereas the Eimeria intestinalis and Eimeria magna isolates tested did not. RNA/RNA hybridization experiments where the E. stiedae VLP dsRNA was probed to the genomes of the dsRNA viruses of Trichomonas vaginalis and Giardia intestinalis revealed a strong relatedness of the E. stiedae virus to the G. intestinalis virus, in contrast with the T. vaginalis virus, where no homology could be detected.     

Integration of herpes simplex virus type 1 DNA into the DNA of growth-arrested BHK-21 cells.

The ability of HSV-1 DNA to become associated with host cell DNA in an alkaline-stable form has been demonstrated following infection of a ts baby hamster kidney growth mutant (ts BTN-1), at the non-permissive temperature (39.5 depgrees C). After 8 h pre-incubation at 39.5 degrees C, ts BTN-1 cells infected at this temperature using m.o.i. ranging from 0.5 to 200 p.f.u./cell fail to replicate virus DNA even though transport of input virus genomes to the nucleus is the same at both permissive and non-permissive temperatures. Virions containing 3H-labelled DNA were used to infect ts BTN-1 cells at 39.5 degrees C, and the total cellular DNA isolated from these cells was resolved into host and virus material by repeated CsCl equilibrium gradient centrifugation. A significant amount of the input radioactivity was found as a distinct band in the host region in both neutral and alkaline CsCl gradients, strongly suggesting a covalent association between host and virus DNAs. Evidence for this association was strengthened by demonstrating that radioactive material (virus DNA) banding in the host region of CsCl gradients could be driven towards the density expected for virus DNA following degradation of the putative hybrid molecules by shearing.     

Natural macrophage cytotoxicity against Trichomonas vaginalis is mediated by soluble lytic factors

Mechanisms of lysis of the extracellular protozoan Trichomonas vaginalis by uninduced resident macrophages were analyzed. Supernatants obtained by culturing such macrophages with T. vaginalis were cytotoxic for the protozoa in a dose-dependent manner. Supernatants from macrophages cultured alone were cytotoxic at lower levels, whereas those obtained from T. vaginalis alone and from macrophages cultured with unrelated cells (B77) were not cytotoxic. Cytotoxic activity appeared after 4 h of contact between effectors and target cells and reached a plateau at 18 to 24 h. Microtubule disrupting agents (colchicine and vinblastine) enhanced protozoan lysis, whereas cytochalasin B, an inhibitor of microfilaments, completely blocked T. vaginalis lysis. Treatment of macrophages with protein synthesis inhibitors (cycloheximide and puromycin) impaired effector cytotoxicity. Lytic activity remained after dialysis of supernatants, treatment with 10% bovine fetal serum, and treatment at 56 degrees C for 1 h, but it was completely prevented by treatment at 90 degrees C for 10 min. In conclusion, our data show that natural cytotoxicity against T. vaginalis is performed by normal resident macrophages through the release of at least two heterogeneous soluble factors.     

Circular plasmid DNAs from the red alga Gracilaria chilensis

Summary Total cellular DNA extracted from eight red algal species (from the genera Gracilaria, Gracilariopsis, Porphyra and Gymnogongrus) was centrifuged on Hoechst dye/CsCl gradients. In five species, plasmid-like DNAs banded with the A+T rich organellar DNAs in the CsCl gradients. Based on their electrophoretic migration in different agarose gels, the plasmid-like DNAs are circular. This is the first report of putative plasmid DNAs in the red algae outside the genus Gracilaria. Two similar Gracilaria chilensis plasmid-like DNAs of 3.8 and 3.4 kb (GC2 and GC3) were cloned in pUC19. The cloned GC2 DNA did not hybridize to either the organellar or nuclear genomes of G. chilensis, suggesting that GC2 is a true plasmid. GC2 did hybridize to the plasmid of one other red algal species, Gracilaria sordida.     

Optimizing the APC gene mutation analysis in archival colorectal tumor tissue.

Critical steps in the methodology of mutation analysis on routinely fixed, paraffin-embedded samples have been evaluated, including extraction and purification of DNA, amplification of gene fragments in various sizes, and screening for mutations. The DNA was extracted from tissue sections with proteinase K, using various procedures, and purified. The mutation cluster region of the APC gene was amplified with polymerase chain reaction to generate either two large or four small overlapping DNA fragments. The GC-clamped fragments were screened for mutations with temperature gradient gel electrophoresis, and mutations were identified with sequencing. The DNA was easily amplified as large fragments from fresh or unfixed-frozen samples. However, DNA amplification of large fragments from archival samples was successful in only 40 of the 114 tumor specimens analyzed (35%). Prolonged extraction, either at 55 degrees C or at 37 degrees C, gave no better results. Polymerase chain reaction of smaller fragments, with sizes between 200 and 270 base pairs (bp), was successful for 97% of the amplification reactions when using DNA that was purified with silica. Screening with temperature gradient gel electrophoresis was reproducible and sensitive with a detection limit of 5% mutated DNA in the presence of an excess of wild-type DNA.     

Purification and analysis of a phospholipase A2-like lytic factor of Trichomonas vaginalis

Trichomonas vaginalis produces soluble factors that have been reported to have the ability to damage target cells in vitro, and it has been hypothesized that these factors may play a role in the pathogenesis of human trichomoniasis. A lytic factor (LF) was purified from T. vaginalis, and the molecular characteristics of LF were determined. T. vaginalis extract was subjected to hydrophobic chromatography with a 10 to 60% N-propanol gradient in 0.1 M ammonium acetate, resulting in the elution of LF from the column at 30% N-propanol. Cytotoxicity assays revealed that LF was cytotoxic to WEHI 164 cells and bovine red blood cells, and inactivation of LF by treatment with trypsin suggested that the active component of LF was a protein. Size exclusion chromatography of LF produced two fractions at 144 and 168 kDa, and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of LF under reducing conditions revealed two subunits of 57 and 60 kDa. Results of a fluorescence assay of LF on carboxyfluorescein-labeled liposomes composed of phosphatidylcholine-cholesterol showed that liposomes were hydrolyzed, suggesting that LF had phospholipase activity. Thin-layer chromatography analysis of BODIPY (4,4-difluoro-3a,4adiaza-s-indacene)-labeled phosphatidylcholine treated with LF demonstrated products that migrated identically to the products produced by treatment with phospholipase A(2) (PLA(2)). These results suggest that LF is a PLA(2) and may be an important virulence factor of T. vaginalis mediating the destruction of host cells and contributing to tissue damage and inflammation in trichomoniasis.     

Repetitive sequences scattered throughout the genome of Trypanosoma cruzi

A clone bank from Trypanosoma cruzi DNA was constructed in the plasmid vector pBR325 and screened with total labelled DNA from the same parasite. The experimental conditions used enable recombinant clones containing repetitive sequences to be detected. 2% of the clones gave a strong positive signal. Half of them carried mini-circle sequences but the other half contained repetitive sequences from the nuclear genome. 50% of all colonies showed up more weakly suggesting that half of the trypanosome DNA fragments carried few repetitive elements. One family of repeats, present in two clones from different genomic regions, hybridized with a broad range of nuclear DNA fragment sizes. Moreover, one of these clones had at least two kinds of elements with no common sequences. A third clone, detected under the same conditions, hybridized with distinct nuclear DNA bands. The number of copies estimated for the latter was much lower than the number of homologous sequences detected in nuclear DNA with the former two. This third recombinant plasmid proved useful to differentiate among closely related trypanosome stocks. Neither poly(A)+ or poly(A)- RNA, nor the 50 kilobase pair band corresponding to the satellite DNA already described in trypanosomes, contribute to the repeats present within these recombinant DNAs. Sequences with some degree of homology were found in the nuclear genome of T. brucei and Crithidia fasciculata.