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Biomedical subjects

J Mainil

Publications and source records attributed to J Mainil.

At least 37 records · Page 2Linked to original sources
PubMed1999

Reproduction of lesions and clinical signs with a CNF2-producing Escherichia coli in neonatal calves.

CNF2-producing necrotoxigenic E. coli (NTEC2) are associated with diarrhoea and septicaemia in calves. We orally inoculated neonatal calves with a NTEC2 strain in order to reproduce clinical signs and lesions. We observed diarrhoea in each inoculated calf, bacteraemia (80%), the presence of CNF2+ bacteria in the lungs (80%) and in the liver (20%). The observed lesions were inflammation of the entire gut, hypertrophy of the mesenteric lymph nodes and hepatisation of the lungs. We were unable to detect characteristic lesions that are classical signs of septicaemia.

Animals↗
PubMed1999

The locus for enterocyte effacement (LEE) of enteropathogenic Escherichia coli (EPEC) from dogs and cats.

Enteropathogenic Escherichia coli (EPEC) produce attaching and effacing lesions. The genes responsible for this lesion are clustered on the chromosome forming a 35.5 kilobase pathogenesis island called LEE. The LEE was identified, characterized and completely sequenced from the human EPEC strain E2348/69. The LEE carries genes coding for: a type III secretion system (genes esc and sep), the translocated intimin receptor (gene tir), the outer membrane protein intimin (gene eae) and the E. coli secreted proteins EspA, EspB, and EspD (genes esp). In addition to man and farm animals, EPEC are also isolated from dogs and cats. We studied structurally and functionally the LEE of dog and cat EPEC. First, we used four probes scattered along the LEE to identify the presence of a LEE in canine and feline EPEC isolates. Second, by PCR, we checked the presence of genes homologous to eae, sep, esp, and tir genes in these strains. Third, since the four types of eae and tir genes were described, we developed a multiplex PCR in order to determine the type of eae and tir genes present in each strain. Fourth, we determined by PCR the site of the LEE insertion on the chromosome. Fifth, we tested several of the canine EPEC in their capacity to induce attaching and effacing lesions in the rabbit intestinal loop assay. We can conclude from this study: first, that the a LEE-like structure is present in all tested strains and that it contains genes homologous to esp, sep, tir, and eae genes; second, that there is some preferential associations between the type of eae gene and the type of tir gene present in a strain; third, that the majority of the tested strains contained a LEE located elsewhere on the chromosome in comparison to the human EPEC strain E2348/69; and fourth that dog EPEC were able to induce attaching and effacing lesions in rabbit ileal loop assay.

Adhesins, Bacterial↗
PubMed1999

Heterogeneity of the eae genes in attaching/effacing Escherichia coli from cattle: comparison with human strains.

Enteropathogenic (EPEC) and enterohaemorrhagic (EHEC) Escherichia coli isolated from cattle were studied by DNA colony hybridization to subtype their intimin-encoding (eae) gene with probes derived from the variable parts of the eae alpha gene of the human EPEC strain E2348/69, the eae gamma gene of the human O157:H7 EHEC strain ATCC43888, and the eae beta gene of the bovine O26:H- EHEC strain 193, whose eae gene was first cloned and sequenced during this work. The EPEC and EHEC had been isolated from diarrhoeic calves (143 EPEC and 48 EHEC) and from healthy animals at the slaughterhouse (10 EPEC and 34 EHEC). The 191 bovine EPEC and EHEC isolated from diseased calves were positive with the Eae beta probe (55 and 27% respectively) and with the Eae gamma probe (9 and 73% respectively), whereas 52 EPEC (36%) were negative with the Eae alpha, Eae beta, and Eae gamma probes. The results were different for the 44 bovine EPEC and EHEC isolated from healthy cattle at slaughterhouses: most tested positive with the Eae gamma probe (80 and 82% respectively) and the remaining (20 and 18% respectively) with the Eae beta probe. Nine O26 human EHEC tested positive with the Eae beta probe and seven O111 with the Eae gamma probe. The bovine and human EPEC and EHEC belonging to these two serogroups gave identical results: the 18 bovine and human O26 isolates tested positive with the Eae beta probe, whereas the 13 O111 isolates were positive with the Eae gamma probe. In contrast, the isolates belonging to other serogroups (O5, O15, O18, O20, and O118) gave more variable results. The eae beta and eae gamma, but not the eae alpha, variants were thus distributed amongst bovine EPEC and EHEC. The eae beta variant seemed to be more frequently associated with the presence of clinical signs in calves, but one third of EPEC from diarrhoeic calves carried an eae gene variant other than the alpha, beta, or gamma variants. In addition, the use of these gene probes did not enable differentiation between bovine and human EHEC belonging to the same O serogroup.

Adhesins, Bacterial↗
PubMed1999

The long-term cytoskeletal rearrangement induced by rabbit enteropathogenic Escherichia coli is Esp dependent but intimin independent.

Attaching and effacing rabbit enteropathogenic Escherichia coli (REPEC) of the O103 serogroup adhere diffusely on HeLa cells and trigger a slow progressive cytopathic effect (CPE) characterized by the recruitment of vinculin and the assembly of actin stress fibres. In contrast to REPEC O103, the reference human EPEC strain E2348/69 is unable to trigger the CPE. In this study, we have shown first that the fimbrial adhesin AF/R2, which mediates the diffuse adhesion of REPEC O103, was not sufficient to induce the CPE capability upon E2348/69. Non-polar mutants of REPEC O103 for espA, espB, espD and eae were then constructed. The four mutants were unable to induce attaching and effacing lesions in the rabbit ileal loop model. The esp mutants were no longer able to induce the CPE, whereas the eae mutant still induced the CPE. Each espA, -B, -D mutant could be fully complemented in trans by the corresponding cloned esp genes from both the parental strain and the CPE-negative E2348/69 strain, indicating that no single esp encodes the information needed to confer the CPE phenotype. In conclusion, the CPE is the first example of an Esp-dependent but Eae (intimin)-independent alteration of the host cell cytoskeleton by certain EPEC strains.

Adhesins, Bacterial↗
PubMed1999

Use of a monoclonal antibody against an Escherichia coli O26 surface protein for detection of enteropathogenic and enterohemorrhagic strains.

A monoclonal antibody (MAb) was obtained from a mouse immunized with solubilized outer membrane proteins extracted from a bovine enterohemorrhagic strain of Escherichia coli (EHEC), O26. The MAb produced a strong immunoblot reaction at approximately 21 kDa for an O26 strain containing the intimin gene (eae) and verocytotoxin (VT), but not with an O26 eae- and VT-negative strain, or O157 eae- and VT-positive strains. The MAb was used in a sandwich enzyme-linked immunosorbent assay (ELISA) format to screen strains from animal and human sources, and all reactive strains were characterized for the presence of eae and the gene encoding VT factors by PCR. The antigen was detected in a group of strains containing a high proportion of O26, the majority of which were eae positive with or without VT; these were isolated mostly from animal enteritis cases but included a small number of human enteric isolates. Nonreactors included eae-positive (with or without VT) O157 strains and one O26 strain. In a survey of mixed cultures from both animal and human enteric disease, ELISA-positive reactions were obtained from 7.1 to 11.2% of samples from bovine, porcine, ovine, and human sources. The two human O8 and ten animal O26 ELISA-reactive pure strains obtained from these samples contained six eae- and/or VT-positive strains; the other six strains lost their ELISA positivity following storage at -70 degrees C, after which none were found to contain either eae or VT factors. The association of the antigen detected by the MAb with significant enteropathogenic E. coli and EHEC virulence factors in isolates from both animal and human enteric infections indicates a diagnostic potential for the assay developed.

Animals↗
PubMed1999

[Study of the role of Clostridium perfringens in bovine enterotoxemia].

Bovine enterotoxaemia is an acute to peracute syndrome occurring mainly in calves and characterized by the sudden or very rapid death of the calf, with colics, convulsions and nervous disorders as clinical signs, if any. The most pronounced lesion is a necrohaemorrhagic enteritis of the jejunum, the ileum, and sometimes the colon. Suckling beef calves are the most frequently affected ones. In 67% of the 78 field cases investigated, some kind of stress was observed 24 to 36 hours prior to the death: change in diet or pasture, vaccination... The most frequently isolated bacteria, and the one isolated in highest numbers, was non-sporulated non-enterotoxigenic toxinotype A Clostridium perfringens. Reproduction of the lesions was successful in a ligated intestinal loop assay in one calf with a few of these strains, more especially with one of them, which was shown later to produce another recently described toxin, the beta 2 toxin. A role for this beta 2 toxin in bovine enterotoxaemia is thus speculated for future research.

Animals↗
PubMed1998

Prevalence and molecular typing of attaching and effacing Escherichia coli among calf populations in Belgium.

Attaching and effacing Escherichia coli are involved in diarrhea in 2 to 8-week old calves. The virulence factors of these bacteria include: (i) the secretion of proteins (i.e. EspB) involved in microvilli effacement, (ii) the production of the intimin, a 94 kDa outer membrane protein encoded by the eaeA gene and involved in the intimate attachment of bacteria to epithelial cell and (iii) the production of verotoxins: VT1 and/or VT2. We investigated the presence and the pathotype of these strains in several calf populations by colony hybridization or by genetic amplification. Using the colony hybridization method we showed first that only 5% of calves who died from diarrhea presented EaeA+ E. coli strains and secondly that 19% of healthy calves showed an asymptomatic carriage. However, using colony hybridization and genetic amplification, we identified EaeA+ strains in 91% of calves living in farms with recurrent diarrhea problems. In 66% of the calves, there was a correlation between the presence of AEEC and diarrhea. At the pathotype level, most of the EaeA+ isolates were negative for VT probes. In VT+ bacteria, the majority were VT1+. The number of VT positive bacteria was significantly higher in calves who died from diarrhea than in healthy or sick calves. This underlined the aggravating role of verotoxins in the disease. Moreover, only 25% of the bovine AEEC were positive with the EaeB probe. Surprisingly, the proportion of EaeB+ strains was significantly higher in healthy calves than in other populations.

Adhesins, Bacterial↗
PubMed1997

Bovine attaching and effacing Escherichia coli possess a pathogenesis island related to the LEE of the human enteropathogenic Escherichia coli strain E2348/69.

Attaching and effacing Escherichia coli (AEEC) has been described as a cause of diarrhea in calves. The molecular pathogenesis of AEEC was mainly studied in human enteropathogenic E. coli strain E2348/69 in which the virulence correlated with the presence of a 35.4 kb pathogenesis island called LEE. We showed that several strains isolated from calves with diarrhea were able to produce attaching and effacing lesions in a rabbit ileal loop model and that they possess a pathogenesis island related to the LEE. Moreover, we showed that the LEE from bovine strains was inserted mainly at a different position in the chromosome compared to the human enteropathogenic E. coli strain E2348/69.

Animals↗
PubMed1996

Typing of bovine attaching and effacing Escherichia coli by multiplex in vitro amplification of virulence-associated genes.

Attaching and effacing Escherichia coli is a new causal agent of diarrhea in calves. Its major virulence factors are the intimin protein, encoded by the eaeA gene, and the Shiga-like toxins, encoded by slt genes. Because the sequences of these genes are available, we selected specific primers to amplify each virulence gene so as to develop a new identification test based on multiplex amplification of virulence-associated genes. Of 30 tested strains, 14 were eaeA+, 15 were eaeA+ slt-I+, 1 was eaeA+ slt-I+ slt-II+, and 1 was eaeA+ slt-II+. The method proved in our hands to be fast and specific and in perfect correlation with the hybridization method.

Adhesins, Bacterial↗
PubMed1996

Hybridization of 2,659 Clostridium perfringens isolates with gene probes for seven toxins (alpha, beta, epsilon, iota, theta, mu, and enterotoxin) and for sialidase.

OBJECTIVE: To genetically characterize Clostridium perfringens isolates for association of pathologic type with various diseases. DESIGN: Prospective study. SAMPLE POPULATION: 2,659 C perfringens isolates from various nonhuman animals species, human beings, and foods. PROCEDURE: Colony hybridization with DNA probes for 7 toxin (alpha, beta, epsilon, iota (subunits a and b), theta, mu, and enterotoxin) genes and 1 sialidase gene were performed to group the isolates by pathologic type. RESULTS: Enterotoxin-negative type-A isolates were the most common (2,575/2,659), were isolated from all sources, and were separated into 5 pathologic types. In cattle and horses with enterotoxemia, essentially only these pathologic types were identified. The enterotoxin-negative isolates of types C or D each had a single pathologic type. Type-C isolates were isolated only from swine with necrotic enteritis and type-D isolates from small ruminants with enterotoxemia, except that 1 type-D isolate was also found from a healthy fish. Type-B or -E isolates were not found. Among the 47 enterotoxin-positive isolates, 5 isolates from sheep or deer were type D and the other 42 were type A. These 42 isolates were grouped into 3 pathologic types: 1 type was isolated from samples of almost all origins, but the other 2 types were found in only 5 fish, 4 human beings, and 1 dog. CONCLUSIONS AND CLINICAL RELEVANCE: Genetic characterization of these isolates allowed identification of 11 different pathologic types. This approach may be useful in molecular diagnosis and prophylaxis of clostridial disease.

Animals↗
PubMed1994

Specific DNA probes to detect Escherichia coli strains producing cytotoxic necrotising factor type 1 or type 2.

Cytotoxic necrotising factors type 1 (CNF1) and type 2 (CNF2) are produced by many Escherichia coli strains isolated from man and animals with intestinal or extra-intestinal colibacillosis. In most laboratories, CNF-producing strains are detected by a cell cytotoxicity assay and confirmed with a neutralisation assay or a mouse footpad assay. In this study, we sought to determine whether DNA probes could detect clinical isolates of E. coli producing CNF2 or CNF1, or both, without the need for cell cultures or animal assays. Two internal fragments of the gene encoding CNF2 were used as DNA probes: a 875-bp XhoI-PstI DNA fragment and an adjacent 335-bp PstI-ClaI fragment. A positive response with both DNA probes was associated with CNF2-producing strains, whereas a positive response with only the 335-bp probe was associated with CNF1-producing strains. Results of colony hybridisation experiments with 185 clinical isolates of E. coli demonstrated that these DNA probes detected CNF2-producing strains with a sensitivity and specificity of 100% and CNF1-producing strains with a sensitivity and specificity of 99%. These two DNA probes should greatly facilitate epidemiological studies to assess the importance of CNF-producing strains as agents of diarrhoea and septicaemia.

Animals↗
PubMed1994

Typing of Clostridium perfringens by in vitro amplification of toxin genes.

The strains of Clostridium perfringens are classified according to major toxins produced. Classically, this determination involves the seroneutralization of their lethal effect in mice. However, this method requires specific antisera and a large number of mice. In this work, a new typing method was developed based on the amplification of toxin genes by polymerase chain reaction (PCR). By combination of several pairs of primers, the toxinotype of a Cl. perfringens strain was determined by looking at the pattern of bands on an agarose gel electrophoresis. This mixture contained primers amplifying simultaneously a part of alpha-toxin, beta-toxin, epsilon-toxin and enterotoxin genes. In order to distinguish between toxinotype A and E, the l-toxin gene fragment must be amplified in a separate PCR reaction. Moreover, with the primers combination, in most cases, a PCR product corresponding to the alpha-toxin gene was obtained from direct enrichments of animal intestinal contents.

Animals↗
PubMed1993

Escherichia coli producing CNF1 and CNF2 cytotoxins in animals with different disorders.

Two DNA probes were used for the detection of CNF1- and CNF2-positive E coli strains in a collection of 553 E coli isolates from cattle, sheep, goats, pigs, horses, dogs, cats and poultry. CNF-positive E coli were frequently associated with septicaemia in cattle, dogs, and cats, with diarrhoea in calves, cats and dogs, and with abortion in bovine and porcine species. CNF2-positive strains were observed among adult healthy cattle. They were also found in cases of pneumonia, metritis, mastitis in cattle and in 1 case of metritis of a mare. The physiopathology induced by CNF-positive E coli strains remains to be elucidated. However, the impact of CNF strains on veterinary pathology is clear and the diagnosis of CNF-producing E coli should become routine in veterinary practice.

Animals↗
PubMed1992

[Virulence factors and phenotypes of sixty-one strains of Escherichia coli of bovine origin, producing cytotoxic necrotising toxin type 1 (CNF 1)].

Virulence factors and phenotypes of 61 strains CNF1+ were investigated. Eighty-nine percent of the strains produced an aerobactin and were resistant to the bactericidal activity of sheep serum, both of which are properties of septicemic strains of E coli. None of the strains reacted either with DNA probes corresponding to the enterotoxins STaP, STb, LT-I and LT-IIa, or to the verotoxins VT-I and VT-II. None produced the adhesins K99, Att25 (FY or F17) and Att111. The great majority (93.4%) of the CNF1+E coli possessed both properties. These properties allow CNF1+ to be distinguished from CNF-E coli.

Animals↗
PubMed1991

Virulence factors associated with cytotoxic necrotizing factor type two in bovine diarrheic and septicemic strains of Escherichia coli.

Forty-three bovine isolates of Escherichia coli producing a second type of cytotoxic necrotizing factor (CNF2) and three K-12 strains carrying different Vir plasmids coding for CNF2 were tested for the presence of several virulence factors. Most of the strains were serum resistant (79%), produced an aerobactin (70%), and adhered to calf villi (53%); some of them produced a colicin (32%) and a hemolysin (9%). These strains were also tested by a colony hybridization assay with gene probes for six toxins (classical heat-stable [STaP and STb] and heat-labile [LT-I and LT-IIa] enterotoxins and Shiga-like toxins [SLT-I and SLT-II]) and five adhesion factors (K99, K88, 987P, F17, and F41). Only two gene probes, LT-IIa (9%) and F17A (53%), hybridized with the CNF2 strains. However, antibodies raised against F17 fimbriae did not agglutinate the strains hybridizing with the F17A probe. In contrast, all except one of these strains adhered to calf villi. Interestingly, these two properties, F17A positivity and adherence to calf villi, were the only ones expressed by the K-12 strains carrying different Vir plasmids. In conclusion, this study confirmed that CNF2-producing strains are unrelated to previously described toxigenic E. coli strains and also demonstrated that in half of the strains the production of CNF2 was associated with an adhesion factor genetically related to, but different from, F17, which is more than likely encoded by Vir plasmids.

Animals↗