Characterization of Borrelia burgdorferi isolates by restriction endonuclease analysis and DNA hybridization.

Genomes of several Borrelia burgdorferi isolates from North America and Europe were characterized by restriction endonuclease analysis and DNA hybridization using labeled B. burgdorferi whole-cell DNA (strain ATCC 35210). Several different restriction and homology patterns were observed among these isolates, indicating genotypic heterogeneity within this genus and species. It was concluded from this study that restriction endonuclease analysis of B. burgdorferi whole-cell DNA may be a reliable and accurate method for identifying strains or genotypes of the Lyme disease agent.     

Characterization of orthopoxviruses isolated from man and animals in Germany

Summary.  Fourteen orthopoxvirus strains isolated from humans, cats, a dog, a cow, and an elephant in Germany were characterized. All were classified as cowpox virus based on haemorrhagic lesions induced on the chorioallantoic membrane of chicken eggs and reactivity of a 160 kDa protein with anti-A-type inclusion protein hyperimmun serum in a Western blot. More detailed comparison of the isolates by restriction endonuclease mapping using HindIII and XhoI demonstrated a close relationship between all isolates and confirmed them as cowpox viruses. However, some minor differences between the isolates were detected which proved to be of epidemiological value. One group consisting of five closely related isolates contained a unique 4.0 kb HindIII fragment. In a Southern blot this fragment failed to hybridize with other cowpox virus isolates including the reference strain. Received July 17, 1997 Accepted October 7, 1998     

Nucleoside analysis of DNA from Trypanosoma brucei and Trypanosoma equiperdum

We have digested trypanosome DNA with a combination of pancreatic DNase I, nuclease P1 and bovine alkaline phosphatase and fractionated the resulting nucleosides on a Supelcosil LC-18-S column by high pressure liquid chromatography. We find less than 0.1% unusual nucleosides, both in Trypanosoma brucei and in a Trypanosoma equiperdum stock, in contrast to a previous report of an unusual nucleoside replacing dC at 1.3% of total nucleosides in T. equiperdum. Our results agree with previous suggestions that the modification of inactive telomeric expression sites for variant-specific surface glycoprotein genes in T. brucei only affects a very small fraction of the total DNA.     

Differentiation of Mycobacterium bovis isolates from animals by DNA typing.

The insertion sequence IS6110 and the direct repeat (DR) specific to tuberculosis complex mycobacteria and the highly repeated DNA sequence, the polymorphic GC-rich repeat sequence (PGRS), were systematically used to identify restriction fragment length polymorphisms (RFLPs) within 210 isolates of Mycobacterium bovis. The isolates were primarily of bovine origin, but isolates from badgers, feral deer, sheep, humans, and a pig were included. The RFLP probes IS6110, DR, and PGRS individually identified 17, 18, and 18 different RFLP types, respectively, but in combination these probes identified a total of 39 different M. bovis RFLP types. The recommendations (J. D. A. van Embden, M. D. Cave, J. T. Crawford, J. W. Dale, K. D. Eisenach, B. Gicquel, P. W. M. Hermans, C. Martin, R. McAdam, T. M. Shinnick, and P. M. Small, J. Clin. Microbiol. 31:406-409, 1993) for a standardized RFLP analysis for M. tuberculosis were adapted to facilitate gel documentation, image analysis, and construction of a database of RFLP types. In the present study the same M. bovis RFLP types were evident in the various animal species included, indicating that the strains were not host restricted. Application of these techniques to defined field studies should help elucidate more accurately aspects of the epidemiology of bovine tuberculosis in different countries.     

Fibrinogen and fibrinogen/fibrin degradation products in the urine of rabbits infected with Trypanosoma (Trypanozoon) brucei

Summary Studies have been carried out on the urine of rabbits infected with Trypanosoma (Trypanozoon) brucei to determine whether fibrinogen or fibrinogen/fibrin degradation products (FDP) could be detected. No fibrinogen was found but during the last two weeks of this 7-week infection low levels of FDP were present in the urine which did not exceed 5 g/ml. Rabbit urine was shown to contain a potent proteolytic enzyme capable of breaking down rabbit fibrinogen and both early and late FDP were present in the cleavage products. No deposits of fibrin were detected in the kidney, but casts were present in the urine suggesting renal damage. The most likely explanation of the urinary FDP is that either an increase in the glomerular permeability occurs allowing filtration of plasma FDP or a local fibrinogenolysis in the kidney tubules.     

Characterization of axenic isolates of Giardia intestinalis established from humans and animals in Germany

A total of 15 isolates of Giardia intestinalis, the first axenic cultures of this organism to be described from Germany, were established in Bonn from faecal cysts obtained from human and animal stool specimens. Measurement of in vitro growth kinetics for 12 of the isolates revealed 3 phenotypes (`rapid', `medium-rate' and `slow' growers) characterized by generation times of 9–11 h (5 isolates), 12–15 h (5 isolates) and ≥18–20 h (2 isolates), respectively. Cloned sublines exhibited growth rates similar to those of the parent isolates. Genetic analyses involving use of the polymerase chain reaction to amplify segments of genes encoding variant-specific surface proteins or the enzyme glutamate dehydrogenase, coupled with the detection of restriction-fragment-length polymorphisms, identified genotypes belonging to three previously described genetic groups. Seven isolates (from humans, a calf and a chinchilla) were typed to genetic group I – a potentially zoonotic genotype belonging to assemblage A, one of two major genetic lineages defined by analysis of G. intestinalis from humans and animals. Six isolates (all from humans) showed identity with the group II genotype – recovered thus far only from humans and also belonging to assemblage A. Two isolates (one from a human, the other from a monkey housed at the Cologne zoo) were classified as assemblage B genotypes. The in vitro growth rates correlated strongly with genotype, group I or group II (assemblage A) genotypes accounting for all of the `rapid' and `medium-rate' cultures and both assemblage B isolates being `slow growers'. The data indicate that genetically based metabolic differences may determine how rapidly G. intestinalis isolates can grow in axenic culture. Received: 28 August 1997 / Accepted: 26 October 1997     

Autoimmunity in trypanosome infections: I. Tissue autoantibodies in Trypanosoma (Trypanozoon) brucei infections of the rabbit

Ten rabbits infected with Trypanosoma (Trypanozoon) brucei showed a substantial increase in a natural anti-tissue autoantibody and Wassermann antibody. Absorptions suggest that the liver and Wassermann antibodies are distinct. The liver antibody reacts equally well with homologous and autologous liver. Absorption with trypanosomes and liver show that cross-reacting trypanosomal antibodies are not responsible for the liver activity. These antibodies will contribute to the raised IgM levels of rabbit trypanosomiasis and may be important in this respect but the precise extent of the contribution is not known.

It is suggested that a depression of certain T-cell functions may release antibody secreting B-cell descendants from T-cell control resulting in elevated IgM.

     

Restriction endonuclease analysis of Aujeszky's disease (pseudorabies) virus DNA: Comparison of Northern Ireland isolates and isolates from other countries

Summary Aujeszky's disease (AD; pseudorabies) viruses isolated in Northern Ireland over a 20 year period were compared with isolates from other parts of the world using restriction endonuclease analysis of virus DNA. When the numbers of Bam H1, Kpn 1 and Sal 1 restriction sites were considered, pathogenic Northern Ireland isolates resembled viruses isolated in England, Hungary and the U.S.A. and could be differentiated from viruses isolated in Denmark, Belgium and the Netherlands. The avirulent Northern Ireland isolate NIA4 and the Bartha vaccine strain were very similar to each other and could be distinguished from pathogenic isolates.While almost all the pathogenic viruses isolated in Northern Ireland from 1963 to 1983 appeared to possess the same number of restriction sites none of the viruses, even those made at the same farm during one outbreak of infection, were identical. The differences were confined to variation in the sizes of certain fragments which map in variable regions of the genome.With 4 Figures     

Characterization of Paracoccidioides brasiliensis isolates by random amplified polymorphic DNA analysis.

We initially used 25 different random primers in order to test their ability to generate random amplified polymorphic DNA fragments from the dimorphic human pathogenic fungus Paracoccidioides brasiliensis. From the tested primers we chose five to distinguish between seven isolates of this microorganism. The DNA amplification patterns allowed clear differentiation of the seven isolates into two distinct groups with only 35% genomic identity. One of these groups contained two subgroups with 81% genetic similarity. The random amplified polymorphic DNA analysis method proved to be a good tool for analyzing and comparing different genomes of P. brasiliensis isolates.     

Subgenotype analysis of Cryptosporidium parvum isolates from humans and animals in Japan using the 60-kDa glycoprotein gene sequences

Cryptosporidium parvum is a well-known intestinal parasite which is associated with severe acute diarrhea in humans and animals. This parasite is composed of morphologically identical but genetically different multiple genotypes. In humans, cryptosporidiosis is mainly caused by two C. parvum genotypes, human genotype (previously known as genotype 1 and recently proposed as new species C. hominis) and cattle genotype (previously known as genotype 2). However, recent molecular studies indicate the genetic heterogeneity among the isolates of C. parvum human or cattle genotype. Therefore, identification of the isolates at the subgenotype level is more useful for control of the Cryptosporidium infection or for understanding of the population structure of C. parvum genotypes. In the present study, we identified the subgenotypes of the C. parvum human or cattle genotype isolates from humans and animals in Japan using DNA sequencing analysis of the C. parvum 60-kDa glycoprotein gene (GP60) and showed the new subgenotype in a raccoon dog isolate. This study suggested that C. parvum cattle genotype might be composed of zoonotic and host-specific multiple subgenotypes.     

Infectivity and virulence of Trypanosoma (Trypanozoon) brucei for mice. IV. Dissociation of virulence and variable antigen type in relation to pleomorphism

Previous observations have indicated the possibility that the virulence of a trypanosome is closely associated with its variable antigen type. A set of clone populations has now been examined, all of which bear the same variable antigen. The clones isolated close to the point of isolation were found to be of moderate virulence to mice, whereas those isolated from the same stock following repeated passage were of extreme virulence. Virulence and variable antigen type need not, therefore, necessarily be associated and would appear to depend on the degree of pleomorphism. The degree of virulence exhibited by the clones was found not to be determined by their apparent rate of growth from inoculation to first patency.     

Characterization of Scedosporium prolificans clinical isolates by randomly amplified polymorphic DNA analysis.

Fingerprinting by randomly amplified polymorphic DNA (RAPD) analysis was used to differentiate Scedosporium prolificans isolates. A total of 59 arbitrary primers were screened with six unrelated S. prolificans isolates, and a panel of 12 primers was selected. The 12 primers were then used to detect DNA polymorphisms among 17 S. prolificans isolates from 11 patients with systemic S. prolificans infections diagnosed in three hospitals located in geographically different areas of Spain. Eight patients were diagnosed with S. prolificans infection in a single institution over a 6-year period, and two other patients were diagnosed with S. prolificans infection in a different hospital over a 1-year period. No single primer allowed for the discrimination of all the isolates from different patients, but this was possible by combining the RAPD patterns from three primers (UBC 701, AB1.08, and AB1.11 or UBC 701, AB1.08, and UBC 707). However, multiple isolates from the same patient were identical. In this study, we also compared a visual method and a computerized method for the analysis of the RAPD patterns. Both methods were satisfactory and gave few discordances, but given the advantages and disadvantages of each method, both systems should be used together. RAPD analysis provided a fast and economical means of typing S. prolificans isolates, with a high level of discrimination among unrelated isolates. Typing by RAPD analysis confirmed that the S. prolificans infections were epidemiologically unrelated.     

Quantitation of hepatitis B virus DNA (HBV DNA) in serum using the spot hybridization technique and scintillation counting

The simple spot hybridization technique to detect HBV DNA in serum was modified to concentrate Dane particles by pelleting. This minimised interference by serum components and allowed larger volumes of sera (up to 500 microliters) to be used in serial dilution on borderline positive samples and increased the efficiency of filtering through a "Hybri.Dot' system. Quantification of HBV DNA by 32P-scintillation Cerenkov counting based on serial standards of cloned HBV DNA processed through the "Hybri.dot' was easy to perform and the assay had good sensitivity (detection limit 2 pg, mode 6.25 pg), precision and accuracy. The pellet-simple spot quantitative assay proved more sensitive than those involving lengthier extraction procedures or direct application of serum to hybridization filters. Scintillation counting curves were linear over a wider range (0-800 pg HBV DNA) than optical densitometry measurements (0-50 pg) of autoradiographs. There was an excellent correlation with DNA polymerase activity (r = 0.948; P less than 0.001) but the assay proved more sensitive since HBV DNA (greater than 45 pg cloned equivalents) was detected in 4 out of 14 DNA polymerase negative sera and in 7 of 7 borderline (DNA polymerase cpm range 116-303) samples. This sensitive, quantitative HBV DNA assay should be of considerable value in studies on infectivity and effectiveness of antiviral therapies.     

Typing of Candida krusei clinical isolates by restriction endonuclease analysis and hybridization with CkF1,2 DNA probe.

The use of restriction endonuclease analysis and Southern hybridization with our new CkF1,2 DNA probe, cold labeled with peroxidase, for the typing of Candida krusei isolates has been investigated. Fifty-five clinical samples isolated from forty-five patients hospitalized in eight centers, one environmental strain, and two reference strains were evaluated. Patterns were analyzed by a computer-assisted method and compared by numerical analysis. Clearer and less ambiguous patterns were obtained by restriction with endonuclease HinfI. It generated 9 to 14 (average, 11) well-separated fragments in the range of 6.5 to 2.0 kb. Both their numbers and sizes varied greatly among the strains studied. The CkF1,2 probe hybridized with one to seven fragments of HinfI patterns. A total of 48 distinct types were distinguished among the 58 strains studied. HinfI and CkF1,2 patterns showed similarities of less than 83 and 75% for unrelated strains and more than 91 and 100% for related strains, respectively. The methods showed 100% typeability, 98% reproducibility, and a discriminatory power of 1. C. krusei isolates from each patient were distinct, whether from one hospital or from different hospitals. Multiple isolates from the same patient were identical, both over time and at different anatomic sites. An endogenous origin is suggested for the colonizing and infecting isolates among the 45 patients. The CkF1,2 probe enhanced discrimination of the strains and provided a definitive comparison for strain identity. Genetic linkages between isolates were assessed at the subspecies level, and 12 clusters were delineated. A typing scheme is proposed for epidemiological studies of C. krusei.     

Pathogenicity of Campylobacter jejuni isolates from animals and humans.

Fourteen isolates of Campylobacter jejuni of different serotypes and one Campylobacter coli isolate, from various human and animal sources, were tested for potential pathogenic mechanisms. Enterotoxin production was not detected in the infant mouse test or by calf and piglet ligated intestinal loop studies. Isolates were not invasive by the Sereny test. All isolates associated with and penetrated HeLa cells, although both actions occurred generally in a minor way under the conditions of our study. The C. coli isolate showed extensive HeLa cell association, but three other C. coli isolated tested did not. None of the 15 isolates produced diarrhea or death in 3-day-old chickens inoculated orally and observed for 3 days, nor did they consistently produce diarrhea and death in 9- to 10-day-old infant mice over a 3-day period after oral inoculation. Diarrheal disease and mortality were not observed when 3-day-old gnotobiotic chickens were infected with one of five isolates and observed over a 2-week period.     

Changes in the pathogenicity of relapsed diminazene aceturate (DA)-resistant Trypanosoma brucei brucei as the trypanosomes are transmitted from DA-treated hosts to another set of animals

This study investigated the changes in pathogenicity of relapsed diminazene aceturate (DA)-resistant Trypanosoma brucei brucei (TBB) as the trypanosomes are transmitted from DA-treated hosts to another set of animals. The Federe strain of T. brucei brucei which was known to be DA resistant was used to infect two groups of rats, one of which was treated at the peak of infection. There was temporary clearance of the trypanosomes from blood of the treated group and subsequent relapse of the infection. The relapsed trypanosomes were used to infect a group of rats (group A1), and the pathogenicity of the relapsed T. brucei brucei in this rat group was compared to that of a group infected with primary T. brucei brucei stock that was not treated with DA (group B1). Results showed that when compared with the B1 rats, the A1 group had a slower onset of parasitaemia, significantly lower (p < 0.01) level of parasitaemia all through the study, significantly longer (p < 0.01) time period to reach peak parasitaemia, significantly longer (p < 0.01) post-infection survival time and significantly higher (p < 0.05) packed cell volume from day 6 post-infection. Further sequential transmission of this same T. brucei brucei infection to another group of rats (A2 and B2) produced the same effects on the above parameters used to assess pathogenicity. It was concluded that beyond temporarily clearing trypanosomes from blood of treated animals, treatment with DA reduces the pathogenicity of DA-resistant T. brucei brucei, and this reduced pathogenicity is carried on as the T. brucei brucei is further sequentially transmitted to another set of animals.     

Diffuse noxious inhibitory controls (DNIC) in animals and in man.

Some neurones in the dorsal horn of the spinal cord are strongly inhibited when a nociceptive stimulus is applied to any part of the body, distinct from their excitatory receptive fields. This phenomenon was termed "Diffuse Noxious Inhibitory Controls" (DNIC). DNIC influence only convergent neurones: the other cell types which are found in the dorsal horn, including specific nociceptive neurones, are not affected by this type of control. In normal conditions, these inhibitions can be triggered only by conditioning stimuli which are nociceptive. The inhibitions are then extremely potent, affect all the activities of the convergent neurones and persist, sometimes for several minutes, after the removal of the conditioning stimulus. In fact, only activity of A delta- or A delta- and C-peripheral fibres can trigger DNIC. DNIC are sustained by a complex loop which involves supraspinal structures since, unlike segmental inhibitions, they can not be observed in animals in which the cord has previously been transsected at the cervical level. The ascending and descending limbs of this loop travel respectively through the ventro-lateral and dorso-lateral funiculi respectively. We proposed that DNIC result from the physiological activation of some brain structures putatively involved in descending inhibition. However, lesions of the following structures did not modify DNIC: Periaqueductal grey (PAG), Cuneiform nucleus, Parabrachial area, locus coeruleus/subcoeruleus, rostral ventromedial medulla (RVM) including Raphe Magnus, Gigantocellularis and Paragigantocellularis nuclei. By contrast, lesions of Subnucleus Reticularis Dorsalis (SPD) in the caudal medulla strongly reduced DNIC. Both electrophysiological and anatomical data support the involvement of SRD neurones in spin-bulbo-spinal loop(s). Indeed, they are unresponsive to visual, auditory or proprioceptive stimulation but are preferentially or exclusively activated by nociceptive stimuli with a "whole-body receptive field"; they encode precisely the intensity of cutaneous and visceral stimulation within the noxious range and are exclusively activated by cutaneous A delta- or A delta- and C-fibre peripheral volleys; they send descending projections through the dorsolateral funiculus that terminate in the dorsal horn at all levels of the spinal cord. In man, exactly analogous results have been obtained by means of combined psychophysical measurements and recordings of nociceptive reflexes. Electrical stimulation of the sural nerve at the ankle simultaneously induces a nociceptive reflex in a flexor muscle of the knee (the RIII reflex) and a painful sensation from the territory of the nerve. Painful heterotopic conditioning stimuli, no matter whether thermal, mechanical or chemical in nature, depress both the reflex and the associated painful sensation, with stronger effects being observed with more intense conditioning stimuli.(ABSTRACT TRUNCATED AT 400 WORDS)     

Sensitivity of in situ hybridization techniques using biotin- and 35S-labeled human papillomavirus (HPV) DNA probes

In order to evaluate the sensitivity of our modified in situ DNA hybridization technique using biotinylated probes, formalin fixed, paraffin embedded biopsies from 20 cervical lesions known to contain human papillomavirus (HPV) DNA were re-examined by the technique using both 35S-labeled- and biotinylated HPV DNA probes. The probe concentrations as well as the detection limits of biotin probing were screened by spotting known amounts of HPV 16 DNA on nylon filter, and allowed to hybridize with biotinylated HPV 16 DNA probe. By this method, 4 pg of HPV 16 DNA could be detected using a probe concentration of 0.2 micrograms/ml. HPV DNA could be demonstrated in all 20 biopsies with both hybridization techniques. However, signals in subrabasal cells were detected more frequently with biotin- than with 35S-labeled probes. Additional experiments were performed using three cervical cancer cell lines (with known copy numbers of HPV DNA), to assess the detection limits of HPV infections by the in situ hybridization techniques. The CaSki cells (500-600 HPV 16 copies/cell) were unequivocally positive with both labelling systems. HeLa cells (10-50 HPV 18 copies/cell) were positive with the biotin probing in 10/10 smears, as compared to 7/10 smears when 35S-labeled probes were used. Radioactive probing was inferior to biotinylated probing in detecting the signals in SiHa cells (1-2 HPV 16 copies/cell). This is because even weak background signals could mask true positive signals when 35S-labeled probes are used. In contrast, no background is generated with the biotinylated probes, detected with streptavidin-biotinylated alkaline phosphatase complex. In situ hybridization with biotinylated DNA probes is as sensitive as techniques using 35S-labeled probes for detecting HPV infections in routine cervical biopsies or smears.