Rapid and sensitive detection of peste des petits ruminants virus by a polymerase chain reaction assay.

A rapid and specific test was developed for the diagnosis of peste des petits ruminants disease. This assay is based on the rapid purification of RNA on glass beads followed by the reverse transcription-polymerase chain reaction (RT-PCR). To that effect, a set of primers (NP3/NP4) was used to amplify specifically a fragment of about 350 bp in the 3' end of the RNA messenger that encodes the nucleocapsid protein of the peste des petits ruminants virus. The PCR-product was detected by UV illumination after electrophoresis on agarose gel or by hybridisation with a digoxigenin-11-dUTP labelled oligonucleotide probe after a blot transfer. In comparison with the conventional titration technique on Vero cells, this RT-PCR assay was 1000-fold more sensitive. Compared with the popular Chomczynski and Sacchi's method [Anal. Biochem. 162 (1987) 156], the purification of the RNA on the glass beads offers the advantage of being more rapid and also avoiding the use of solvents.

Typing of sheep clinical isolates and identification of enterotoxigenic Clostridium perfringens strains by classical methods and by polymerase chain reaction (PCR).

A simple procedure was developed to identify toxitypes of Clostridium perfringens of different origins. Ninety strains of C. perfringens were identified by classical bacteriological methods, typing of the strains was done by a seroneutralisation test on mice. Production of enterotoxin was tested and all strains were analysed by PCR using gene of toxin alpha, gene of toxin E, gene of toxin beta and gene of enterotoxin. Simple amplification (amplifying one gene), and duplex and triplex amplification (amplifying two and three genes simultaneously) were performed. In the conditions of the experiment, the PCR method has proved efficacious. The specificity and sensitivity are excellent and superior to those of the classical methods. The prophylaxis of enterotoxaemia in animals is achieved by vaccination, the PCR technique can thus become a first-choice tool for the identification and typing of the C. perfringens strains which initiate these diseases. In turn, this would simplify the development of vaccines adapted to the epidemiological situation.

[Detection of viral hemorrhagic septicemia virus (VHSV) in rainbow trout (Oncorhynchus mykiss) by reverse transcription followed by polymerase chain reaction. Diagnostic validation]. / Détection du virus de la septicémie hémorragique virale (SHV) de la truite arc-en-ciel (Oncorhynchus mykiss) par une transcription inverse suivie d'une amplification en chaîne par polymérase. Validations diagnostiques.

Viral haemorrhagic septicaemia virus (VHSV) is a fish pathogen that attacks rainbow trout (Oncorhynchus mykiss) farming. The only diagnostic method recognised by the European community for VHS is based on the detection of the viral agent in cell culture followed by an immunological identification of the pathogen. Reverse transcription followed by a double amplification of the polymerase chain reaction (RT-PCR) for the gene encoding the viral glycoprotein G is proposed here as an alternative method to the virus assay in cell culture. The RT-PCR was found to have three advantages over the viral assay method. First, the RT-PCR was found to be more rapid than the virus assay method (24 h compared to 72-120 h). Second, this method was found to be more sensitive than the virus assay (2.10(-2) pfu of virus were detected per millilitre of viral suspension compared to 2.10(-1) pfu.mL-1 by inoculation of the cell lines EPC and RTG2). Third, the RT-PCR was shown to be specific towards the VHS virus assay (represented by its four serotypes VHS I, VHS II, VHS 23/75 and VHS IV). Moreover no amplification was obtained with the other rhabdoviruses used: infectious haematopoietic necrosis virus (American strain Amend 72, WRAC, RB, SRVC) and French reference strain 69/87, eel viruses, spring viraemia of carp virus and pike fry virus. The validation of this method was performed on organs removed from experimentally infected rainbow trout and ovarian fluid samples from farmed broodfish from D0 to D150. By using RT-PCR between D30 and D60, 13 samples from nine experimentally infected trout (ten kidney-spleen pools and three brains) tested positive, whereas only nine samples (four kidney-spleen pools and five brains) from six fish were positive at D30. The last positive response was obtained by RT-PCR at D60 for kidney-spleen pools from three fish. At D150, all the results were negative. From the 60 ovarian fluid samples tested, 28 were VHS positive by the RT-PCR versus 15 by the virus assay method. Eleven out of the 60 broodfish had neutralised anti-VHS, six were negative by RT-PCR and by the virus assay, four were positive by RT-PCR and negative by virus assay and one positive by both methods. The specificity, sensitivity and rapidity of the RT-PCR method makes it an attractive alternative to classical virological methods currently recommended by European Fish Health Surveillance Programmes.

Specificity of six gene sequences for the detection of the genus Brucella by DNA amplification.

DNA extracted from all Brucella species, reference and vaccine strains were amplified by PCR using primers specific for the genes encoding a 31-kDa Brucella protein, the heat shock proteins (DnaJ, DnaK, HtrA and GroEL) and 16S RNA. No difference was found between Brucella species and biovars with all primer pairs used, even after restriction enzyme analysis of the amplified fragments. The specificity of the amplified products was confirmed by hybridization with a digoxigenin 3'-labelled specific probe and by PCR using 98 non-Brucella micro-organisms' DNA. Only Ochrobactrum anthropi and Phyllobacterium spp. yielded a PCR product by using 31-kDa DnaK, DnaJ, GroEL and 16S RNA primers. After hybridization and restriction analysis, 16S RNA fragments of 3301 and 3331 O. anthropi strains showed a total similarity to those from Brucella. A similar result was shown with DnaJ fragments obtained with 3301 strain of O. anthropi after EcoRI digestion.

PCR and gene probe identification of botulinum neurotoxin A-, B-, E-, F-, and G-producing Clostridium spp. and evaluation in food samples.

A degenerate primer pair was selected to amplify specifically a 260-bp DNA fragment from Clostridium botulinum types A, B, E, F, and G, and five individual probes allowed identification of each toxinotype by hybridization of the PCR products. The 72 strains of different Clostridium species tested and 11 other bacterial species commonly found in food samples gave an amplification product. This assay was able to detect 1 C. botulinum type A or B and 10 C. botulinum type E strains per reaction. With 184 artificially contaminated food samples, after an 18-h enrichment step, the sensitivity was 10 bacteria per g of sample and the correlation with the mouse bioassay reached 95.6%.

Polymerase chain reaction for the rapid identification of Clostridium botulinum type A strains and detection in food samples.

A polymerase chain reaction (PCR) was developed for the detection of Clostridium botulinum type A, a cause of human botulism. A two primer set and an oligonucleotide detection probe were used to specifically detect Cl. botulinum type A neurotoxin gene (BoNT/A). After 40 cycles of amplification, detection of a 798 bp amplified DNA fragment was carried out by agarose gel electrophoresis and Southern blot hybridization. This assay was able to detect 12.5 fg of purified target DNA or five bacteria per reaction. The sensitivity in artificially contaminated food samples after an 18 h enrichment step ranges from 10 to 10(3) bacteria per g according to the type of food samples. No cross-reactions were observed with the other Cl. botulinum toxinotypes and other bacteria found routinely in food. This PCR method may provide a suitable and rapid alternative to standard techniques for detection of Cl. botulinum type A in food samples.

[Use of the in vitro enzymatic amplification method for the detection of Mycobacterium paratuberculosis in feces]. / Utilisation de la méthode d'amplification enzymatique in vitro pour la détection de Mycobacterium paratuberculosis dans les fèces.

A polymerase chain reaction was developed, using as target sequence an insertion element of 1,451 base pairs (IS 900), specific for Mycobacterium paratuberculosis (15-20 copies per genome). The test was performed in three stages: (1) extraction of bacterial deoxyribonucleic acid (DNA), from faeces stored at +4 degrees C, -20 degrees C, in 70% ethanol or in a buffer solution; (2) amplification of the target DNA by means of thermostable DNA polymerase; (3) detection of the amplified DNA by electrophoresis, confirmed by dot blot assay after hybridisation with an internal labelled oligonucleotide of digoxigenin. Reproducible results were obtained with DNA extracted from faeces stored at -20 degrees C or in 70% ethanol. The sensitivity and specificity of the method used, particularly double amplification and hybridisation, are discussed by comparing the results obtained by bacterial culture from faeces.

Detection of Echinococcus multilocularis DNA in fox faeces using DNA amplification.

In order to identify Echinococcus multilocularis DNA in fox faeces for epidemiological purposes, we have developed a new method to prepare DNA suitable for PCR amplification. DNA isolation from fox excrement was performed according to a novel procedure involving lysis in KOH, phenol-chloroform extraction and a purification step on a matrix (Prep-A-Gene). The target sequence for amplification was the E. multilocularis U1 snRNA gene. PCR products were indistinguishable for 32 different E. multilocularis isolates and no signal was observed after ethidium bromide staining with DNAs from other tapeworm species, including E. granulosus. The sensitivity of amplification was monitored by the addition of E. multilocularis DNA or eggs to faeces free of E. multilocularis and was estimated to be 1 egg per 4 g of faeces. PCR products were blotted onto nylon membranes and hybridized with an internal oligonucleotide probe in order to confirm the results. Twenty nine faecal samples from foxes shot in Franche-Comté (East France) were tested. Out of 10 samples from foxes in which no E. multilocularis adult worms could be observed after necropsy, 7 were PCR positive, showing that the PCR test is more sensitive than microscopical examination. Out of 19 samples from foxes harbouring E. multilocularis adult worms, 18 were PCR positive. The remaining PCR-negative sample could be due either to the misidentification of the species of adult worm (E. granulosus and E. multilocularis), or to DNA variation between different isolates of E. multilocularis. Further work in the field should be initiated in order to confirm these results.

Differentiation of Trichinella isolates by polymerase chain reaction.

Oligonucleotide primers were synthesized for the polymerase-chain-reaction amplification of target DNA from two sequences of Trichinella spiralis. Six strains belonging to T. spiralis, T. nativa, T. britovi, T. pseudospiralis, and T. nelsoni were tested. Amplification products were obtained with T. spiralis, T. britovi, and T. nelsoni DNA from a 53-kDa antigen cDNA sequence and with T. spiralis and T. nelsoni DNA from a 1.6-kb repetitive DNA sequence. Differences in the length of the amplification products obtained from the repetitive sequence would enable a differentiation between T. spiralis and T. nelsoni, suggesting that the 1.6 kb repetitive DNA sequence would not be specific for T. spiralis. No amplification was detected for T. nativa or T. pseudospiralis DNA from the two sequences and for T. britovi DNA from the 1.6-kb repetitive DNA sequence.

Detection by in vitro amplification of the alpha-toxin (phospholipase C) gene from Clostridium perfringens.

A polymerase chain reaction (PCR) with thermostable DNA polymerase from Thermus aquaticus is described for the specific amplification of the phospholipase C (alpha-toxin) gene of Clostridium perfringens. A set of primers selected for their high specificity could detect Cl. perfringens in stools with a detection limit of approximately 5 x 10(2) bacteria, after bi-amplification. A modified PCR without thermal steps was performed to rapidly amplify, with a yield of 60%, the DNA template. With this PCR method Cl. perfringens alpha-toxin gene could be detected within 2 h. The PCR method detected alpha-toxin positive Cl. perfringens but did not react with phospholipase C-producing Bacillus cereus, Pseudomonas aeruginosa, Cl. sordellii and Cl. bifermentans. The amplified PCR products were screened through ethidium bromide agarose gel electrophoresis or, in only 1 h, with the PhastSystem (Pharmacia). This PCR satisfies the criteria of specificity, sensitivity and rapidity required for a useful tool in epidemiology and for the diagnosis of the pathogen Cl. perfringens as it may be used directly on stool samples.

[Detection of the eggs of Echinococcus multilocularis Leuckart, 1863, in the feces of the fox (Vulpes vulpes Linnaeus, 1758) by the polymerase chain reaction]. / Détection des oeufs d'Echinococcus multilocularis Leuckart, 1863 dans les fèces de renard (Vulpes vulpes Linnaeus, 1758) par amplification en chaîne par polymérase.

The polymerase chain reaction (PCR) was applied to the identification of eggs of Echinococcus multilocularis in faeces from foxes. The test was positive in three of six faeces samples from foxes which were harbouring adult worms, and in one of four samples from foxes in which no adult E. multilocularis was found in the intestines. These initial results show that it is possible to use PCR to identify E. multilocularis eggs in faeces. PCR can be used to complement examination of intestinal contents, showing that the distribution of eggs in faeces is uneven. The sensitivity of the test was estimated to be 50 eggs in 5 g of faeces. Further work is needed to confirm these initial results before the test can be used more widely.

Biotinylated probes to detect Leptospira interrogans on dot blot hybridization or by in situ hybridization.

Total genomic biotinylated probes which can identify leptospires by hybridization on filters or by in situ hybridization are described in this study. According to the weak G + C content of the strains studied (35-39%) and owing to the decreasing melting temperature (Tm) due to overbiotinylation, hybridization and wash temperatures were optimized at 33 degrees C and at 42 degrees C respectively. Fourteen serovars of Leptospira interrogans belonging to 11 different serogroups and three serovars of Leptospira biflexa were used in this study. Cross-hybridization results show that it is possible, by means of such probes, specifically to recognize pathogenic strains. These probes did not hybridize with the three saprophytic strains: L. buenos-aires, L. patoc and L. andamana. We also ran a total genomic probe, specific to the serovar buenos-aires which hybridizes only with homologous DNA.

Taxonomic position of lecithinase-negative strains of Clostridium sordellii.

Eleven out of 43 strains of Clostridium sordellii from clinical sources did not produce lecithinase activity and were not toxic to mice. However, these strains did belong to the C. sordellii group and could readily be differentiated from C. bifermentans and C. difficile on the basis of DNA-DNA homologies, carbohydrate fermentation patterns, enzyme activities, GLC analysis of fatty acid fermentation products and the electrophoretic analysis of whole cell protein extracts.

[Study and differentiation of some hydrolases active on triglycerides and esters, in anaerobic bacteria, using gas liquid chromatography (author's transl)]. / Etude et différenciation de certaines hydrolases actives sur les triglycérides et les esters, chez les bactéries anaérobies, par la méthode de chromatographie en gaz-liquide.

Anaerobic bacteria are classified using presence or absence of phospholipase and lipase, among other criteria. Techniques are described for the qualitative and quantitative detection of bacterial esterases (carboxylic ester hydrolase) and lipases (triacylglycerol acyl hydrolase) endo- or -exocellular, using gas liquid chromatographic method. Results with representatives anaerobic species are briefly presented.

[Actinomyces odontolyticus, first study in France]. / Actinomyces odontolyticus, première étude réalisée en France.

The authors report the detailed study of a strain of Actinomyces odontolyticus isolated from a pleural liquid. Gas-liquid chromatography can specify the biochemistral metabolism of the germ and set off an endocellular lipidolytic activity.

[Lipase activity of anaerobic bacteria on glycerol-tributyrate determined by gas-liquid chromatography]. / Etude des lipases actives sur le glycérol-tributyrate chez les Bactéries anaérobies par la méthode de chromatographie gaz liquide

Anaerobic bacteria are classified among other criteria by the presence or absence of phospholipase and lipase. The liquid gas chromatographic method detects with great sensibility the lipasic activity of the anaerobic bacteria. A liidolytic action has been demonstrated in Cl. perfringens.