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J Nicolet

Publications and source records attributed to J Nicolet.

At least 91 records · Page 5Linked to original sources
PubMed1995

Identification and characterization of IS1296 in Mycoplasma mycoides subsp. mycoides SC and presence in related mycoplasmas.

IS1296, a new insertion sequence belonging to the IS3 family of insertion elements has been identified in Mycoplasma mycoides subsp. mycoides (Mmm) biotype small colony (SC), the agent of contagious bovine pleuropneumonia (CBPP). IS1296 is 1485-bp long and has 30-bp inverted repeats. It contains two open reading frames, ORFA and ORFB, which show significant similarities to the ORFs which encode the transposase function of IS elements of the IS3 family, in particular IS150 of Escherichia coli. IS1296 is present in 19 copies in Mmm SC-type strain PG1 and in 18 copies in a recently isolated field strain L2. It seems to transpose at low frequency in Mmm SC. IS1296 is also present in 5 copies in Mmm biotype large colony (LC)-type strain Y-goat, and in two copies in Mycoplasma sp. 'bovine group 7' reference strain PG50. It is, however, not present in other species of the 'mycoides cluster' or other closely related Mycoplasma sp. of ruminants.

Amino Acid Sequence↗
PubMed1995

Development of an efficient PCR method for toxin typing of Actinobacillus pleuropneumoniae strains.

A method has been developed which allows the determination of the activator, the structural and the secretion genes of the three toxins ApxI, ApxII and ApxIII in Actinobacillus pleuropneumoniae in only two PCR reactions. The oligonucleotide primers were designed to amplify a significant part of the activator and structural genes apxICA, apxIICA and apxIIICA together in a single PCR reaction giving amplification products which differ in length, in order to be clearly separated by agarose gel electrophoresis. Variations in the apxIA and apxIIIA genes which were found in different serotypes were taken into account in the design of the primers to give a uniform amplification product for both variants of the apxIA and the apxIIIA genes. The secretion genes apxIBD and apxIIIBD are also detected in a single PCR reaction containing two pairs of oligonucleotide primers which yield two differently sized fragments to differentiate between apxIBD and apxIIIBD genes. The reference strains of A. pleuropneumoniae serotypes 1-12 and 104 field strains representing all serotypes obtained from various laboratories worldwide were analysed for their content of apx genes. The two PCR reactions give toxin gene patterns which are characteristic for different groups of serotypes in A. pleuropneumoniae and allow the rapid differentiation of five toxin type groups, group 1 including serotypes 1, 5a, 5b, 9 and 11, group 2 including serotypes 2, 4, 6, 8, group 3 with serotype 3, group 4 with serotype 7 and 12 and group 5 with serotype 10. The method enhances and facilitates differentiation of A. pleuropneumoniae strains for diagnostics and epidemiology and allows the detection of serotypes with atypical toxin patterns.

Actinobacillus pleuropneumoniae↗
PubMed1995

Restriction fragment length polymorphisms among the flagellar genes of the Lior heat-labile serogroup reference strains and field strains of Campylobacter jejuni and C. coli.

Several typing systems have been described for Campylobacter jejuni and C. coli, to assess the complex epidemiology of these important enteric pathogens. In the present study two typing methods, slide agglutination according to the Lior scheme, and the demonstration of restriction-fragment length polymorphisms (RFLP) of flagellar genes, have been used in parallel on a set of 194 strains. This set comprised 118 sero-reference strains of C. jejuni and C. coli of the Lior scheme, as well as 76 clinical isolates. All isolates were serotyped and subjected to PCR for amplification of flagellar genes, and the PCR product was restricted with Alu I. Flagellar genes could be amplified in 152 strains. Among 85 seroreference strains, 74 different RFLP patterns were observed, and among 67 clinical isolates, there were 36 patterns. There was only limited correlation between flagellar RFLP and the Lior serogroup, and the variability of patterns in serogroups HL2 and HL4 were as marked as the variability between serogroups. Flagellar gene RFLP patterns are shown to be stable, highly discriminatory epidemiologic markers.

Animals↗
PubMed1995

Insertion element IS1296 in Mycoplasma mycoides subsp. mycoides small colony identifies a European clonal line distinct from African and Australian strains.

Strains of Mycoplasma mycoides subsp. mycoides small colony (SC) type, the agent of contagious bovine pleuropneumonia (CBPP), were analysed with respect to the polymorphism of distribution of a newly discovered insertion element, IS1296, on the chromosome. Analysis of 64 strains isolated from Europe, Africa and Australia, including four vaccine strains and the type strain PG1, revealed ten different IS patterns, forming two main clusters. The European strains originated from outbreaks of CBPP in different countries, and from various other sources such as semen and preputial washings from cattle, lungs from goats and buffalo, and milk from sheep. They showed identical IS1296 patterns, except one strain which had an additional IS1296 element, but the pattern belonged to the same cluster. This shows that the strains from Europe form a clonal lineage. The strains originating from different geographical parts of the African continent and from Australia showed four closely related IS1296 patterns which belong to a separate cluster. This indicates that strains from Africa and Australia form a clonal lineage different from that of the European strains, suggesting that the sporadic cases of CBPP that have re-emerged in Europe almost 15 years after the last declared endemic case in 1967 arose from an established reservoir within Europe rather than being the result of repeated importation from Africa and Australia. While most strains from Africa and Australia had the same IS1296 pattern, all vaccine strains could be distinguished by an individual pattern. The type strain PG1 also had a particular IS1296 pattern which belongs to the cluster of the strains from Africa and Australia. The molecular definition of clonality of M. mycoides subsp. mycoides SC strains with IS1296 represents a rapid and reproducible method for subtyping and differentiation of vaccine strains. It permits at the present time the definition of two main clonal lines, one including the strains from the European continent and a second with strains from Africa and Australia.

Africa↗
PubMed1995

Diagnosis of Clostridium perfringens type C enteritis in pigs using a DNA amplification technique (PCR).

Clostridium perfringens type C, which produces alpha- and beta-toxin, causes severe haemorrhagic and necrotic enteritis in animals and humans. A polymerase-chain-reaction (PCR) assay was developed for the specific detection of the genes encoding alpha-, beta-, epsilon- and entertoxin of C. perfringens for rapid typing of C. perfringens strains, and especially for the identification of type C strains. Both the alpha- and beta-toxin genes were detected directly in porcine C. perfringens type C cultures and also in type B and type C collection strains to a sensitivity of 10(3) cells without purification of the DNA. The alpha-toxin gene was detected in all types of C. perfringens. The epsilon-toxin gene was found in type B and type D, and the enterotoxin gene in some type A strains. Nine other species of Clostridium and a variety of intestinal pathogenic bacteria showed no signal for these toxin genes in this PCR assay. The alpha- and beta-toxin genes PCR assay were used to identify C. perfringens strains isolated from intestinal contents of 36 necropsied piglets that had suddenly died or died after premonitory signs of diarrhoea. At necropsy, 20 piglets showed necrotizing enteritis (15 acute and 5 chronic cases) and were suspected to have suffered from a C. perfringens type C infection. All of them had C. perfringens which gave a positive PCR signal for alpha- and beta-toxin genes, and, hence, were identified as type C strains. From the 16 other piglets with lesions other than necrotizing enteritis, C. perfringens strains with the alpha-toxin gene, but no beta-toxin gene, were isolated.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Prevalence and clinical significance of vero-cytotoxin-producing Escherichia coli (VTEC) isolated from cattle in herds with and without calf diarrhoea.

To determine the clinical significance and prevalence of Vero cytotoxin (VT)-producing E. coli (VTEC) in Swiss cattle, faecal swabs and milk samples of 93 cattle from two farms with calf diarrhoea, and of 54 cattle from two similar farms without clinical problems, were screened for VTEC by PCR and colony-blot hybridization. On average, 21% of all cows were positive for VTEC by PCR, without differences between farms with and without diarrhoea problems. By contrast, VTEC were detected by PCR in 60% of animals younger than 2 years from farms with diarrhoea problems, whereas only 32% of comparable control animals from farms without clinical problems had VTEC. No VTEC were detected in the milk samples. A variety of toxin types, antimicrobial-susceptibility patterns and serotypes were found by colony-blot hybridization, among the VTEC in individual herds. However, there was a significant relationship between the VT1-only toxin type and the presence of antimicrobial resistance and clinical disease in calves. No association could be found between levels of VTEC excretion and the presence of diarrhoea. This is the first study to report systematic quantitative data on numbers of VTEC in faecal samples of cattle positive for VT genes by PCR.

Animals↗
PubMed1995

Rapid and accurate identification of Escherichia coli K-12 strains.

A specific PCR for the identification of K-12 strains, based on the genetic structure of the O-antigen gene cluster (rfb) of Escherichia coli K-12, is described. The assay clearly differentiates E. coli K-12-derived strains from other E. coli strains used in the laboratory or isolated from human and animal clinical specimens, from food, or from environmental samples. Moreover, lineages of K-12 strains can be distinguished with a second PCR based on the same gene cluster. The method presents a useful tool in identifying K-12 for monitoring strains which are used as biologically safe vehicles in biotechnological research, development, and production processes.

Animals↗
PubMed1995

Identification and characterization of an immunogenic outer membrane protein of Campylobacter jejuni.

We cloned and expressed in Escherichia coli a gene encoding an 18-kDa outer membrane protein (Omp18) from Campylobacter jejuni ATCC 29428. The nucleotide sequence of the gene encoding Omp18 was determined, and an open reading frame of 165 amino acids was revealed. The amino acid sequence had the typical features of a leader sequence and a signal peptidase II cleavage site at the N-terminal part of Omp18. Moreover, the sequence had a high degree of similarity to the peptidoglycan-associated outer membrane lipoprotein P6 of Haemophilus influenzae and the peptidoglycan-associated lipoprotein PAL of E. coli. Southern blot analysis in which the cloned gene was used as a probe revealed genes similar to that encoding Omp18 in all species of the thermophilic group of campylobacters as well as Campylobacter sputorum. All campylobacters tested expressed a protein with a molecular mass identical to that of Omp18. The protein reacted immunologically with polyclonal antibodies directed against Omp18 from C. jejuni. PCR amplification of the gene encoding Omp18 with specific primers and subsequent restriction enzyme analysis of the amplified DNA fragments showed that the gene for Omp18 is highly conserved in C. jejuni strains isolated from humans, dogs, cats, calves, and chickens but is different in other Campylobacter species. In order to obtain pure recombinant Omp18 protein for serological assays, the cloned gene for Omp18 was genetically modified by replacing the signal sequence with a DNA segment encoding six adjacent histidine residues. Expression of this construct in E. coli allowed purification of the modified protein (Omp18-6xHis) by metal chelation chromatography. Sera from patients with past C. jejuni infection reacted positively with Omp18-6xHis, while sera from healthy blood donors showed no reaction with this antigen. Omp18, which is an outer membrane protein belonging to the family of PALs is well conserved in C. jejuni and is highly immunogenic. It is therefore a good candidate as an antigen for the serological diagnosis of past C. jejuni infections.

Amino Acid Sequence↗
PubMed1995

Identification of Campylobacter jejuni on the basis of a species-specific gene that encodes a membrane protein.

To facilitate discrimination between the closely related enteropathogens Campylobacter jejuni and C. coli, unique differences in antigenic surface structure were examined. A genomic library of C. jejuni 81116 was constructed in plasmid pBluescriptIISK- and expressed in Escherichia coli K-12. Rabbit hyperimmune serum raised against C. jejuni ATCC 29428 recognized a clone expressing a C. jejuni 24-kDa membrane-associated protein. Antiserum raised against sonicated recombinant E. coli expressing the 24-kDa protein reacted with C. jejuni, whereas C. coli did not react specifically. Determination of the nucleotide sequence of the DNA insert of this recombinant plasmid revealed an open reading frame encoding 214 amino acids; the gene was designated mapA; and its gene product was designated MAPA. The 18 N-terminal amino acid residues constitute a signal sequence characteristic of prokaryotic membrane lipoproteins. In a dot blot hybridization assay with a mapA probe, 120 clinical isolates of C. jejuni were unequivocally discriminated from 126 other campylobacters, including 34 C. coli isolates. A PCR test based on the mapA sequence was developed for identification of C. jejuni. A PCR product was obtained with all of the clinical isolates of C. jejuni tested from human, dog, cat, bovine calf, and chicken sources. Recombinant MAPA with an added C-terminal six-histidine tail was affinity purified and used to immunize rabbits. The rabbit anti-MAPA serum specifically recognized the protein in whole cells of C. jejuni on Western blots (immunoblots). The MAPA protein was present in all of the C. jejuni strains tested and was absent in C. coli and related campylobacters.

Amino Acid Sequence↗
PubMed1995

Surveillance of contagious bovine pleuropneumonia in Switzerland.

An active surveillance system for contagious bovine pleuropneumonia (CBPP) has been established in Switzerland. The system is based on the detection of typical gross pathological lesions in the lungs of slaughtered cattle, followed by microbiological analysis of the sampled organs and sero-epidemiological investigation of the herd of origin for each suspect case. The programme was tested over six months. The prevalence of lung lesions detected in carcasses during this period in the 108 participating abattoirs was 0.04%, but Mycoplasma mycoides subsp. mycoides SC was not isolated from any of these organs. On the basis of the results presented, there is no evidence of CBPP in Switzerland. The surveillance programme will continue, however, in order to document the situation and eventually obtain a disease-free status in accordance with international standards.

Abattoirs↗
PubMed1995

[Presence of Campylobacter spp., Clostridium difficile, C. perfringens and salmonellae in litters of puppies and in adult dogs in a shelter].

In order to ascertain the importance of Campylobacter spp., C.difficile, C.perfringens and Salmonella as agents of bacterial gastroenteritis in dogs, two groups of animals were studied prospectively. The first group consisted of 77 puppies in 14 litters, with fecal cultures performed weekly for 10 weeks, starting at birth. The second group consisted of a kennel population with every dog cultured at entry, and at two-month intervals thereafter. Incidence of Campylobacter spp. was 32 and 31 per 100 dog-month of observation for healthy pups and healthy adult dogs respectively, 46 and 0 for C.difficile, 51 and 36 for C.perfringens and 6.5 and 1.3 for Salmonella. The incidence of Campylobacter spp. in pups peaked at 8 weeks of age. This incidence (43 per 100 dog-months) was higher in pups reared together with older dogs than in pups reared without contact to other dogs (0 per 100 dog-months). Toxigenic strains of C.difficile were found in 61.5% of the healthy neonate dogs. None of the cases of non-watery and non-inflammatory diarrhea we observed was associated with any of the pathogens studied. Furthermore newly acquired colonization with Campylobacter spp. or Salmonella was never associated with episodes of diarrhea. No conclusions could be drawn about the role of bacterial pathogens for causation of watery or inflammatory diarrhea which were not observed in our study.

Animals↗
PubMed1995

[The epidemiology of contagious lung disease in Switzerland].

Although contagious bovine pleuropneumonia (CBPP) has not been diagnosed in Switzerland since 1895, anti-CBPP antibodies were detected in several cattle that had been imported from Switzerland to Italy. In order to clarify the current disease situation in Switzerland, detailed epidemiological investigations were performed, including the control of 200,000 slaughter cattle in 108 abattoirs and serological testing of more than 2200 animals from 134 farms. The data collected in the slaughterhouses showed a prevalence of suspect lung lesions of 0.04%. Yet, the causing agent of CBPP Mycoplasma mycoides subsp. mycoides SC could not be isolated from any of these 84 microbiologically analysed lungs. On the other hand, up to 3.5% positive results were obtained from the serological tests. These results are most probably caused by cross-reactions of other mycoplasma. Based on these data Switzerland is still to be considered free of CBPP. Nevertheless, a continued surveillance programme including routine checks of all cattle carcasses at the abattoir and subsequent epidemiological investigation of suspect cases are recommended.

Animals↗
PubMed1995

[The prevalence of paratuberculosis in the Plateau de Diesse region].

We analysed 595 cows in 20 herds for serological evidence of paratuberculosis with an ELISA procedure for which we had recently determined a sensitivity of 50% and a specificity of 98%, and we obtained an overall calculated real prevalence of 5.99%. In two herds with clinical cases of paratuberculosis, the calculated true prevalence of seroreactors was 21.47%. In 9 herds with clinical suspicion of Johne's disease, the rate was 4.23%, 9 herds without any clinical suspicion showed a mean prevalence of 0%. Whereas the trend was clear for groups of herds, the observed prevalence in individual herds varied from 0% to 17.9%, without obvious association with manifest clinical problems of paratuberculosis.

Animals↗
PubMed1994

Association of the CAMP phenomenon in Actinobacillus pleuropneumoniae with the RTX toxins ApxI, ApxII and ApxIII.

A non-hemolytic mutant of Actinobacillus pleuropneumoniae serotype 5 has a deletion spanning the entire apxI operon. Therefore it does not produce ApxI and is unable to secrete ApxII. This mutant also has lost the co-hemolytic CAMP effect which is characteristic of the species A. pleuropneumoniae. The CAMP effect is restored when the mutant is complemented in trans by the apxIBD genes cloned in a broad host range vector, thus permitting secretion of ApxII, or when the entire apxI operon is cloned in the mutant, thus restoring the original toxin phenotype ApxI+ ApxII+. When the toxins ApxI, ApxII or ApxIII individually are expressed and secreted from E. coli harboring recombinant plasmids containing the genes apxICA and apxIBD or apxIICA and apxIBD or apxIIICABD, respectively, the distinct CAMP phenomenon is produced by the recombinant strains. The CAMP phenomenon is strongest by the recombinant E. coli strain expressing the non-hemolytic ApxIII, somewhat less when ApxI is expressed, and weak when ApxII is expressed. In A. pleuropneumoniae the CAMP phenomenon is also strongest in those serotypes which express ApxIII. The CAMP phenomenon of A. pleuropneumoniae is assumed to be directly caused by any of the RTX-toxins ApxI, ApxII or ApxIII. A previously reported gene from A. pleuropneumoniae, named cfp or hlyX, which provides E. coli strains with a hemolytic character and a CAMP phenomenon, shows high similarity to the E. coli global regulation gene fnr, and which is able to complement a delta fnr mutant.(ABSTRACT TRUNCATED AT 250 WORDS)

Actinobacillus pleuropneumoniae↗
PubMed1994

Sequence analysis and transcription of the apxI operon (hemolysin I) from Actinobacillus pleuropneumoniae.

The DNA sequence of the entire apxI operon from Actinobacillus pleuropneumoniae serotype 1 reference strain 4074 has been determined. This 8292-bp fragment of the chromosomal DNA contains four open reading frames (ORFs) of the strongly hemolytic ApxI toxin. These ORFs correspond to the genes apxIC, apxIA, apxIB and apxID, encoding the activator, the structural toxin protein and the two secretion proteins, respectively. Each of the four ORFs is preceded by a consensus sequence for a putative ribosome-binding site (RBS). The region upstream from apxIC contains several sites that could act as promoters. The transcription start point (tsp) of the apxI operon in A. pleuropneumoniae has been determined by primer extension analysis and was found to be located 133-bp upstream from the translation start codon. The tsp is preceded by sequences matching the -10 and -35 consensus sequence of promoters from Escherichia coli. This is the first promoter identified in A. pleuropneumoniae. The same tsp was used when the expression of apxI was induced by a high concentration of free Ca2+ in the growth medium, as well as when the expression of apxI was not induced by growing the cells in medium depleted of free Ca2+ ions. However, the signal strength of the primer extension was approximately tenfold stronger in Ca(2+)-grown cells. The leader sequence of the transcript is unusually long and very A+U rich (75% A+U).

Actinobacillus pleuropneumoniae↗
PubMed1994

Inducible L-arginine-dependent nitric oxide synthase activity in bovine bone marrow-derived macrophages.

In rodent macrophages, cytokines or bacterial constituents induce a Ca(2+)-independent nitric oxide (NO) synthase which plays a key role in antimicrobial and tumoricidal activity. Firm evidence for expression of a similar enzyme in other mammals has been lacking. Here we show that bovine bone marrow-derived macrophages produce nitrite in an L-arginine-dependent manner upon stimulation with heat-killed gram-positive or gram-negative bacteria. NO2- production was markedly diminished by arginase, and by the arginine analogue, NG-monomethyl-L-arginine. Bacteria-induced NO2- production was enhanced by concomittant exposure to interferon-gamma, tumor necrosis factor-alpha or both combined, although these cytokines alone (in the absence of bacteria) induced little NO2-. This is one of the first demonstrations of NO2- production by non-rodent macrophages.

Amino Acid Oxidoreductases↗
PubMed1994

Immune response against the L-lactate dehydrogenase of Mycoplasma hyopneumoniae in enzootic pneumonia of swine.

The L-lactate dehydrogenase (LDH) of Mycoplasma hyopneumoniae, formerly named protein P36, belongs to the predominant immunogenic proteins in pigs which were naturally or experimentally infected with M. hyopneumoniae. The antigenic reaction against M. hyopneumoniae LDH has been shown to be species specific. Recombinant M. hyopneumoniae LDH, which was genetically engineered to contain six histidine residues at its C-terminal end, was expressed in E. coli and purified to a high degree using Ni-chelate affinity chromatography. The genetically engineered LDH still showed the same biochemical activity and immunological specificity as the wild-type LDH and was used as an antigen for a M. hyopneumoniae LDH ELISA. Using this assay, we showed that pigs experimentally infected with M. hyopneumoniae raised antibodies against LDH in two steps. An early, relatively weak anti-LDH response was detected between 5 to 10 weeks post-infection when clinical signs and lung lesions occur. This first minor raise of anti-LDH antibodies occurred simultaneously with the strong appearance of antibodies against an antigen consisting of membrane proteins of M. hyopneumoniae prepared with Tween 20 extraction. A second, strong raise in anti-LDH antibodies was observed from the twelfth week after infection, at a time when the disease signs and the infectious agent disappeared. The high anti-LDH titer persisted until 21 weeks post-infection, in contrast to the antibody titer against the membrane proteins which started to decrease after its peak at 12 weeks post-infection. A LDH-ELISA may also be useful for detecting past infections.

Amino Acid Sequence↗