Protection of rabbits against enteropathogenic Escherichia coli (EPEC) using an intimin null mutant.

BACKGROUND: Diarrhea and mortality resulting from infections with enteropathogenic Escherichia coli (EPEC) are of major economic importance in the rabbit meat industry. There is a growing need for an effective vaccine to cope with these problems and to reduce the use of antibiotics. EPEC are characterized by an attaching and effacing virulence mechanism. This is partly mediated by the intimate binding between an adhesin, called intimin, and a translocated receptor (Tir) of prokaryote origin. We constructed an intimin deletion mutant of the rabbit EPEC (REPEC) wild-type strain 97/241.6 (bio-/serogroup 3-/O15) and examined its protective capacity. RESULTS: After verifying its complete loss of virulence, we used the attenuated strain in vaccination-challenge experiments in which complete protection against a homologous, but virulent, strain was observed. The attenuated strain was able to persist in the intestinal lumen, where it elicited an immune response against EPEC-related virulence proteins, as was shown using an EspB-specific ELISA. Despite the priming of an immune response and the generation of specific antibodies, the intimin mutant was not able to fully protect rabbits against challenges with REPEC strains of other bio-/serogroups. CONCLUSION: These data indicate that protection against REPEC infections is at least partly bio-/serogroup dependent and a multivalent vaccine may be needed for protection against the full range of REPEC types. Such a combination vaccine may be developed using intimin null mutants, as the latter were clearly shown to be safe and effective against homologous infections.

Comparison of molecular techniques for the typing of Mycoplasma hyopneumoniae isolates.

In this study, we compared the potential of amplified fragment length polymorphism (AFLP), random amplified polymorphic DNA (RAPD) analysis, restriction fragment length polymorphism (RFLP) of the gene encoding lipoprotein P146, and the variable number of tandem repeats (VNTR) of the P97 encoding gene, as possible methods for typing an international collection of Mycoplasma hyopneumoniae isolates. All techniques showed a typeability of 100% and high intraspecific diversity. However, the discriminatory power of the different techniques varied considerably. AFLP (>0.99) and PCR-RFLP of the P146 encoding gene (>0.98) were more discriminatory than RAPD (0.95) and estimation of the VNTR of P97 (<0.92). Other, preferentially well spread, tandem repeat regions should be included in order for this latter technique to become valuable for typing purposes. RAPD was also found to be a less interesting typing technique because of its low reproducibility between different runs. Nevertheless, all molecular techniques showed overall more resemblance between strains isolated from different pigs from the same herd. On the other hand, none of the techniques was able to show a clear relationship between the country of origin and the fingerprints obtained. We conclude that AFLP and an earlier described PFGE technique are highly reliable and discriminatory typing techniques for outlining the genomic diversity of M. hyopneumoniae isolates. Our data also show that RFLP of a highly variable gene encoding P146 may be an equally useful alternative for demonstrating intraspecific variability, although the generation of sequence variability of the gene remains unclear and must be further examined.

Characterization of In Vivo acquired resistance of Mycoplasma hyopneumoniae to macrolides and lincosamides.

Macrolides and related antibiotics are used to control mycoplasma infections in the pig industry worldwide. Some porcine mycoplasmas, however, survive these treatments by acquiring resistance. The mechanism of acquired resistance to macrolides and lincosamides was studied in more detail for Mycoplasma hyopneumoniae by comparing both the phenotype and genotype of a resistant field isolate to five susceptible isolates. The MICs were significantly higher for the resistant strain for all antibiotics tested. The MICs for the 16-membered macrolide tylosin ranged from 8 to 16 microg for the resistant strain and from 0.03 to 0.125 microg/ml for the five susceptible strains. The MICs for the 15-membered macrolides and lincosamides were higher than 64 microg/ml for the resistant strain while only 0.06 to 0.5 microg/ml for the susceptible strains. Mycoplasma hyopneumoniae strains are intrinsically resistant to the 14-membered macrolides due to a G 2057 A transition (E. coli numbering) in their 23S rDNA. Therefore, high MICs were observed for all strains, although the MICs for the resistant strain were clearly increased. An additional, acquired A 2058 G point mutation was found in the 23S rRNA gene of the resistant strain. No differences linked to resistance were found in the ribosomal proteins L4 and L22. The present study showed that 23S rRNA mutations resulting in resistance to macrolides and lincosamides as described in other Mycoplasma spp. also occur under field conditions in M. hyopneumoniae.

The diversity of Mycoplasma hyopneumoniae within and between herds using pulsed-field gel electrophoresis.

Over the years, pulsed-field gel electrophoresis (PFGE) has been proven a robust technique to type isolates with a high resolution and a good reproducibility. In this study, a PFGE protocol is described for the typing of Mycoplasma hyopneumoniae isolates. The potential of this technique was demonstrated by comparing M. hyopneumoniae isolates obtained from the same as well as from different herds. The use of two different restriction enzymes, SalI and ApaI, was evaluated. For each enzyme, the resulting restriction profiles were clustered using the unweighted pair group method with arithmetic means (UPGMA). For both obtained dendrograms, the included isolates of the related M. flocculare species clustered separately from all M. hyopneumoniae isolates, forming the root of the dendrograms. The PFGE patterns of the M. hyopneumoniae isolates of different herds were highly diverse and clustered differently in both dendrograms, illustrated by a Pearson's correlation coefficient of only 0.33. A much higher similarity was observed with isolates originating from different pigs of a same herd. The PFGE patterns of these isolates always clustered according to their herd and this for both dendrograms. In conclusion, the results indicate a closer relationship of M. hyopneumoniae isolates within a herd compared to isolates from different herds and this for both restriction enzymes used. Since the described PFGE technique was shown to be highly discriminative and reproducible, it will be a helpful tool to further elucidate the epidemiology of M. hyopneumoniae.

Evaluation of amplified rDNA restriction analysis (ARDRA) for the identification of Mycoplasma species.

BACKGROUND: Mycoplasmas are present worldwide in a large number of animal hosts. Due to their small genome and parasitic lifestyle, Mycoplasma spp. require complex isolation media. Nevertheless, already over 100 different species have been identified and characterized and their number increases as more hosts are sampled. We studied the applicability of amplified rDNA restriction analysis (ARDRA) for the identification of all 116 acknowledged Mycoplasma species and subspecies. METHODS: Based upon available 16S rDNA sequences, we calculated and compared theoretical ARDRA profiles. To check the validity of these theoretically calculated profiles, we performed ARDRA on 60 strains of 27 different species and subspecies of the genus Mycoplasma. RESULTS: In silico digestion with the restriction endonuclease AluI (AG--CT) was found to be most discriminative and generated from 3 to 13 fragments depending on the Mycoplasma species. Although 73 Mycoplasma species could be differentiated using AluI, other species gave undistinguishable patterns. For these, an additional restriction digestion, typically with BfaI (C--TAG) or HpyF10VI (GCNNNNN--NNGC), was needed for a final identification. All in vitro obtained restriction profiles were in accordance with the calculated fragments based on only one 16S rDNA sequence, except for two isolates of M. columbinum and two isolates of the M. mycoides cluster, for which correct ARDRA profiles were only obtained if the sequences of both rrn operons were taken into account. CONCLUSION: Theoretically, restriction digestion of the amplified rDNA was found to enable differentiation of all described Mycoplasma species and this could be confirmed by application of ARDRA on a total of 27 species and subspecies.

Specific bovine antibody response against a new recombinant Cryptosporidium parvum antigen containing 4 zinc-finger motifs.

A Cryptosporidium parvum sporozoite and oocyst lambda gt11 cDNA library was screened with a hyperimmune rabbit serum that was developed against insoluble fragments of ultrasonicated oocysts. A clone named Cp22.4.1 encoding a protein of 231 amino acids with 4 zinc-finger domains characterized by a Cys-X2-Cys-X4-His-X4-Cys motif was isolated and characterized. There was a complete match between the sequencing data of the coding region of Cp22.4.1 and the corresponding gene at chromosomal level. Cloning in a pBAD-TOPO-TA expression vector permitted to evaluate the antigenicity of the recombinant His-tagged antigen. This antigen was recognized by 2 out of 5 sera from Cryptosporidium immune calves and not by sera from parasite naive animals.