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A Jestin

Publications and source records attributed to A Jestin.

51 records · Page 3Linked to original sources
PubMed1991

Detection of Newcastle disease virus RNA in infected allantoic fluids by in vitro enzymatic amplification (PCR).

The Polymerase Chain Reaction (PCR) procedure was applied in order to identify the Newcastle disease virus (NDV), an avian paramyxovirus (A-PMV 1). The sequence selected for amplification consists of 238 bp lying in the gene encoding the fusion protein F. A pair of 19-mer and 18-mer oligonucleotides, flanking this sequence, were used as primers. Following RNA extraction by the proteinase K method, a cDNA was prepared using the previous 19-mer oligonucleotide as the primer. The amplification reaction product was analyzed by electrophoresis and ethidium bromide staining, using the restriction enzymes HaeIII, Mbo II, and Nar I. The PCR was performed on cDNA prepared from 30 A-PMV 1 and 3 other strains (A-PMV2, A-PMV3, A-PMV4). It was thereby demonstrated that the selected sequence was highly specific and constant. However, two of the PMV1 strains isolated from feral ducks, are thought to present a deletion of about 25 bp inside this fragment as shown by the smaller length of the corresponding amplified product and the disappearance of the NarI restriction site. The advantages of this technique, as a first step in evaluating virulence by means of molecular biology, is discussed.

Allantois↗
PubMed1990

Restriction fragment pattern analysis of genomes from French isolates of suis herpes virus 1 (Aujeszky's disease virus).

Purified DNA from 45 isolates of suis herpes virus 1 (SHV1) collected between 1980 and 1987 from clinical outbreaks of Aujeszky's disease on French farms was compared by restriction fragment pattern (RFP) analysis. The BamHI generated RFPs were found to be distinguishable, confirming RFP analysis as a potential epidemiological tool. The RFP could be assigned to two established major electrophoretic types and different subtypes. The RFP analysis indicated that the majority of outbreaks were caused by ADV with a central European genome type. The heterogeneity of RFPs among PRV isolates recovered from the central nervous system, lung, and foetus was not restricted specifically to one clinical entity. Variations in the virulence of the 45 isolates studied in mice, chicks, or piglets were unrelated to the RFP subtypes. One unusual RFP has been described for one strain of low virulence.

Analysis of Variance↗
PubMed1990

Rapid detection of pseudorabies virus genomic sequences in biological samples from infected pigs using polymerase chain reaction DNA amplification.

The presence of the pseudorabies virus (PRV) genome in infected hosts has previously been studied by standard hybridization techniques, which showed the viral genome to be present at very low levels in infected tissues. The recently introduced polymerase chain reaction (PCR) procedure provides an alternative and rapid means of amplifying small quantities of specific DNA sequences. We applied this technique to a study of pigs infected by PRV. The sequence selected for amplification consisted of 222 base pairs lying in the gene coding for the glycoprotein gp50. We used a pair of 20-mer oligonucleotides flanking this sequence as primer and a cloned Stu-Nde fragment containing the sequence as target DNA. To avoid the tedious DNA extraction procedure we performed PCR directly on disrupted cells and detected specific amplification after 25 cycles of PCR with the thermostable Taq DNA polymerase. Amplified products were detected by gel electrophoresis directly. Nasal samples from experimentally and naturally infected pigs were tested by this PCR technique. When compared with tissue culture and serological tests, detection by gel electrophoresis of PCR amplified fragments provided excellent specificity and sensitivity. We concluded that PCR amplification will be a valuable tool for rapid diagnosis of PRV infection in pigs, taking less than 1 h to complete.

Animals↗
PubMed1990

Silver staining of DNA restriction fragments for the ultrarapid identification of pseudorabies virus strains.

Restriction endonuclease analysis of pseudorabies virus DNA has been used to study various virus strains. To make use of a rapid technique for the identification of viral strains, studies have been undertaken to facilitate purification of the DNA from viral particles present in cytoplasmic fractions. The ultrasensitive photochemical silver staining of nucleic acid, described by Beidler et al. (Analytic Biochemistry 1982: 126) has been adapted and applied to the detection of pseudorabies virus restriction fragments. In a period of 5 h more than 1 microgram of DNA can be extracted from a 25 cm2 plastic flask containing infected cells and purified without ultra centrifugation. Low molecular weight DNA was separated from high molecular weight DNA by polyacrylamide gel electrophoresis. The restriction fragments were selectively visualized by silver staining which can detect 0.025 micrograms of total pseudorabies virus DNA. The electrophoretic DNA pattern of vaccine and wild strains has been studied using these techniques and the results agree with previously published data.

DNA Restriction Enzymes↗
PubMed1990

Diagnosis of swine influenza with an immunofluorescence technique using monoclonal antibodies.

Direct diagnosis of swine influenza infection by an indirect immunofluorescence technique using anti-nucleoproteine monoclonal antibody was compared with virus isolation. Five 8-week-old pigs were inoculated with 2 x 10(7) EID50 of strain A H1N1Sw/4115/85. Clinical signs developed in only three pigs. Antigen was detected in nasal epithelial cells obtained from all animals the first day after inoculation; the antigen was detected in one pig 6 days after the infection. Fluorescence was present in the nucleus, nucleolus and cytoplasm of infected cells. The indirect immunofluorescence test was specific and as sensitive as virus isolation in embryonated eggs, allowing a rapid diagnosis that could be achieved within hours.

Animals↗
PubMed1990

Detection of Aujeszky's disease virus in nasal cells of fattening pigs by immunoassay.

Both conventional and specific pathogen free pigs were inoculated intranasally with a strain of Aujeszky's disease virus (ADV). Nasal cells were collected daily by swab, aspiration or wash. The nasal cells were examined for ADV by isolation on cell culture, direct or indirect immunofluorescence and immunoperoxidase staining by monoclonal antibodies. The infected pigs were studied for nasal shedding of infected cells until 30 days after infection. The study was also extended to naturally infected farm pigs. Swabbing, washing and aspiration proved effective methods of collecting between 10(5) and 10(8) pavement or columnar epithelial cells and non-epithelial cells. Macrophages and polymorphonuclear leucocytes were also identified. Infected nasal cells were detected by immunofluorescence and immunoperoxidase from one to 21 days after infection. The viral antigen was detected in both epithelial and non-epithelial cells, the fluorescence was nuclear and, or, 'cytoplasmic', in the latter case only the cell membrane was stained. ADV antigens were detected in nasal cavity cells in pigs infected with a virulent and a hypovirulent strain. Nasal swabs proved effective in confirming infection both by virus isolation and immunological assay, and the latter was shown to be a useful experimental tool for the rapid diagnosis of Aujeszky's disease virus infection in fattening pigs suffering from acute respiratory distress.

Animals↗
PubMed1989

Rapid diagnosis of TGEV-like coronavirus in fattened pigs by indirect immunofluorescence labelling in nasal cells.

Diagnosis of TGEV-like coronavirus was made by an immunofluorescence technique using monoclonal antibodies directed against TGEV. Six, 15 weeks-old pigs were inoculated intratracheally with the strain P 6008 ST 4. No clinical sign was observed during the course of the infection. Viral antigens were detected in the cytoplasm of nasal cells obtained from swab in five animals as early as one day p.i. and during several days. The sensitivity and specificity of this method appeared to be comparable to virus isolation of this cell-adapted TGEV-like strain.

Animals↗
PubMed1987

Effects of experimental pseudorabies virus infection on vaccinated pregnant sows.

In regard to field problems, a study was done to determine the effects of an infection with virulent Pseudorabies virus (PRV) on pregnant sows vaccinated with an inactivated virus vaccine. Three routes of viral inoculation have been tested: intranasal, intra-uterine and intra-fetal. The nasal infection did not allow to induce pregnancy disorders observed on the field on pregnant sows. After insemination with semen mixed with PRV, abortion was observed on one of the two immune sows. In this one, viral antigen has been identified on vagina, sacral and trigeminal ganglia. The second sow, slaughtered 2 months after infection presented a normal pregnancy. The results of intra-fetal inoculations showed that the vaccination does not prevent the spread of the virus from fetus to fetus and the pregnancy failure. These trials allowed to study the spread and repartition of the virus in the genital tract. PRV antibodies have been detected in the vaginal and uterine washing fluids as well as the follicular fluids of infected animals, vaccinated or not.

Animals↗
PubMed1985

Enteropathogenicity of purified Clostridium perfringens enterotoxin in the pig.

In an assay procedure, purified Clostridium perfringens enterotoxin induced the accumulation of fluid in the ileal loop of the axenic pig. The smallest amount of enterotoxin causing a positive response was 25 micrograms (1:32 titer by counterimmunoelectrophoresis [CIEP] ), and 100 micrograms (1:128 CIEP titer) caused a marked response. Possibly, diarrhea in pigs with 1:32 CIEP titer or more of enterotoxin in the feces may be associated with enterotoxigenic C perfringens.

Animals↗
PubMed1985

Epizootiologic investigations of a diarrheic syndrome in fattening pigs.

A diarrheic syndrome linked to Clostridium perfringens was observed in fattening pigs. A good correlation was observed between the onset and the severity of diarrhea and the fecal passage of C perfringens enterotoxin. Intestinal fluids from affected pigs had activity comparable with that detected in a purified C perfringens enterotoxin. Seven pigs excreting enterotoxin were shown to develop serum antibodies to C perfringens enterotoxin.

Animals↗
PubMedDate not supplied

Use of flow cytometry to monitor infection and recombinant human alpha-1,3/4 fucosyltransferase production in baculovirus infected Sf9 cell cultures.

This paper describes the setup and the use of a flow cytometric method for monitoring Sf9 insect cell infection by a recombinant baculovirus expressing the human alpha1,3/4 fucosyltransferase Fuc-TIII. Using side scattered light coupled to green fluorescence detection after immunolabeling of the recombinant protein, this method made it possible to monitor baculovirus infection of Sf9 cells grown in batch cultures and infected at different cell densities and multiplicities of infection. The method was able to precisely assess the extent of infection of the insect cells from 60 h postinfection. In asynchronously infected Sf9 cell cultures, the two-step infection process (primary and secondary infection) was well-characterized using this technique. Finally, a reduced sensitivity to baculovirus infection was observed for cells infected at the end of the growth phase compared to the cells infected during exponential growth phase.

Animals↗