Biomedical subjects
Publications and source records attributed to D G Newell.
In the past, research on zoonotic diseases has been fragmented. Teresa Belcher and Diane Newell discuss the formation of Med-Vet-Net, a "virtual institute", which aims to integrate activity between researchers in human and veterinary medicine across the European Union.
AIMS: Subspeciation of Campylobacter fetus subsp. fetus (CFF) and Campylobacter fetus subsp. venerealis (CFV) is important for international animal import regulations. Phenotyping can be unreliable, and genotyping by techniques like pulsed field gel electrophoresis is difficult in routine diagnostic laboratories. A PCR subspeciation technique has been reported [Aust Vet J (1997) 75, 827]; we aimed to develop this PCR and investigate its use on UK C. fetus isolates. METHODS AND RESULTS: We augmented the PCR with further primers, and tested 76 isolates of C. fetus and 16 isolates of other Campylobacter spp. PCR failed to correlate well with phenotyping, especially for CFV. We characterized the amplicon of the CFV-specific primers (reported as plasmid derived, but unavailable on the public databases); and predicted a parA gene sequence, anticipated to be plasmid-associated. However, although plasmid isolations from selected CFV isolates demonstrated the presence of several plasmids, there was no correlation between plasmid profile and PCR result. Further, the parA sequence was not detected by PCR in any of the plasmid bands. CONCLUSIONS: This PCR is not suitable for subspeciation of C. fetus in the UK. The results suggest that this is a reflection of the presence of an unusual clone of CFV currently present in cattle in this country. SIGNIFICANCE AND IMPACT OF THE STUDY: PCR cannot substitute for phenotyping of C. fetus isolates in the UK. The reasons for failure of PCR genotyping may reflect local strains and/or plasmid profiles. Further study is required to better elucidate molecular sub-speciation of C. fetus.
It has recently been shown that the enteropathogen Campylobacter jejuni has an N-linked general protein glycosylation pathway (Pgl) that modifies many of the organism's proteins. To determine the role of the N-linked general glycosylation in C jejuni, the authors studied the pglH gene, which shows high similarity to a family of sugar transferases. pglH mutants were constructed in strains 81116 and 11168H. Both mutants were shown to be deficient in their ability to glycosylate a number of C. jejuni proteins, but their lipooligosaccharide and capsule were unaffected. The pglH mutants had significantly reduced ability to adhere to and invade human epithelial Caco-2 cells. Additionally, the 81116 pglH mutant was severely affected in its ability to colonize chicks. These results suggest that glycosylation is important for the attachment of C. jejuni to human and chicken host cells and imply a role for glycoproteins in the pathogenesis of C. jejuni.
AIMS: To investigate the relationship between livestock carriage of Yersinia enterocolitica and human disease. The biotypes/serotypes of strains recovered from the faeces of pigs, cattle and sheep at slaughter during a national survey in Great Britain in 1999-2000, were compared with those of strains isolated from human cases of yersiniosis during the same period. METHODS AND RESULTS: The faecal carriage of Y. enterocolitica by cattle, sheep and pigs at slaughter was 6.3, 10.7 and 26.1%, respectively. Yersinia enterocolitica biotype (BT) 1a was the most frequently isolated biotype from livestock (58%) and was the predominant biotype (53%) isolated from human cases over the same period. The main recognized pathogenic Y. enterocolitica biotype isolated from livestock was BT3 (O:5,27) (35% of sheep, 22% of pigs and 4% of cattle) but this biotype was not detected in any of the human isolates investigated. The major pathogenic biotypes of strains isolated from humans were BT3 (O:9) (24%) and BT4 (O:3) (19%) whereas of the veterinary isolates investigated, only pigs (11%) carried BT3 (O:9) strains. CONCLUSIONS: Because of significant overlaps in phenotypes of the veterinary and human strains it is not possible to comment on the correlation between host and pathogenicity, especially of biotype 1a. SIGNIFICANCE AND IMPACT OF THE STUDY: The data suggest that further investigations using methods with greater discriminatory power are required. However the data also suggests that pigs may be the primary reservoir for human pathogenic Y. enterocolitica infection.
AIMS: In view of recent findings that a multidrug efflux pump CmeABC exists in Campylobacter jejuni, 391 C. jejuni and 52 Campylobacter coli of human and animal origin were examined for a multidrug resistance phenotype. MATERIALS AND METHODS: The MICs of ampicillin, chloramphenicol, ciprofloxacin, erythromycin, kanamycin, tetracycline, cetrimide, triclosan, acridine orange, paraquat and ethidium bromide were determined. Resistance to organic solvents and the effect of salicylate (known inducer of the marRAB operon in Escherichia coli and Salmonella) were also examined. RESULTS: Two C. coli and 13 C. jejuni isolates, mainly from pigs or poultry, were resistant to three or more antibiotics and 12 of these strains had reduced susceptibility to acridine orange and/or ethidium bromide. Strains (n = 20) that were less susceptible to acridine orange, ethidium bromide and triclosan were significantly more resistant (P < 0.05) to ampicillin, chloramphenicol, ciprofloxacin, erythromycin, nalidixic acid and tetracycline, with two- to four-fold increases in MIC values compared with strains (n = 20) most susceptible to acridine orange, ethidium bromide and triclosan. Growth of strains with 1 mM salicylate caused a small (up to two-fold) but statistically significant (P < or = 0.005) increase in the MICs of chloramphenicol, ciprofloxacin, erythromycin and tetracycline. CONCLUSIONS: These data indicate that multiple antibiotic resistant (MAR)-like Campylobacter strains occur and it may be postulated that these may overexpress cmeABC or another efflux system.
AIMS: To compare typeability, discriminatory ability, and inter-laboratory reproducibility of three flagellin PCR/RFLP (fla typing) methods previously described for Campylobacter. METHODS AND RESULTS: The sample set (n = 100) was diverse, including both C. jejuni (n = 85) and C. coli (n = 15). Two of the three flaA typing methods amplified flaA alone, whereas one, a multiplex assay, amplified flaB in addition to flaA. DdeI restriction enzyme was employed for all methods, but HinfI was also investigated. 98-100% typeability was obtained for flaA-based methods, but only 93% for the multiplex assay, due to inconsistent amplification of a non-specific product. In addition, there appeared to be selective amplification of flaA over flaB. More DdeI types were generated using a longer flaA PCR amplicon, whilst additional use of HinfI increased the number of types by ca 25%. Inter-laboratory reproducibility for both flaA-based methods was defined at 100%. CONCLUSIONS: Fla typing requires standardization with respect to PCR primers and restriction enzymes. This study identified an assay, employing the full flaA gene and DdeI digestion, as an appropriate method on which to standardize. 100% inter-laboratory reproducibility was demonstrated using that method. SIGNIFICANCE AND IMPACT OF THE STUDY: This work should facilitate progress towards inter-laboratory standardization of fla typing.
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Surface layer proteins (SLPs) are essential for induction of abortion by Campylobacter fetus subsp. fetus in experimentally challenged ewes. These proteins are encoded by multiple sap genes and vary in size and antigenicity. The role of SLP antigenic variation during experimental ovine infection was investigated. Following subcutaneous challenge, the SLPs were highly antigenic, and antibodies were detected in serum, milk, bile, and urine. Fecal anti-SLP antibodies were detected only in animals challenged orally. Ewes challenged with wild-type strain 23D with variable SLPs developed detectable circulating anti-SLP immunoglobulin G (IgG) antibodies by 2 weeks postchallenge. In contrast, ewes challenged with mutants of 23D that had fixed expression of a single SLP developed antibodies within 1 week postchallenge, suggesting that antigenic variation in SLPs may delay the host antibody response. Although not statistically significant, the data from challenge experiments in which vaccinated ewes were used suggested that SLP-expressing vaccines could protect animals from abortion and that this effect was independent of the SLP expressed, indicating involvement of conserved epitopes in the SLP. The conserved 184-amino-acid N-terminal region of the SLP, identified from previously published sequences, was epitope mapped with rabbit anti-SLP antisera by using overlapping synthetic 20-mer peptides. Two putative epitopes were identified at amino acids 81 to 110 and 141 to 160. Amino acids 81 to 100 also bound serum IgG antibodies from experimentally challenged sheep. Conserved antigenic regions of the SLP that induce protective immune responses may enable development of synthetic vaccine candidates for C. fetus subsp. fetus-associated ovine abortion.
Epidemiological evidence suggests prior infection of humans by Campylobacter jejuni leads to protection against disease following further exposure. It is known that infections elicit strong antibody responses following the onset of disease and that antibody levels are elevated in putatively immune populations. To determine if systemic and mucosal antibodies induced by a confirmed infection remain at elevated levels for prolonged periods, repeat serum, saliva and urine samples were taken from campylobacter patients from 1 week and up to a year postinfection. Antibodies were monitored by ELISAs using three different antigen preparations: acid-glycine extracts (AE) of C. jejuni strain 81116 and an aflagellate mutant (R2), and a whole-cell R2 sonicate, and by Western blotting. Levels of serum IgG antibodies against 81116AE and R2 sonicate, but not R2AE, remained significantly raised over time when compared to a comparison population. Serum anti-sonicate IgA antibody levels were initially significantly raised but decreased over time to levels similar to the comparison group. There were no significant differences in levels of salivary IgA against the AEs. Anti-sonicate salivary IgA and IgG levels were initially significantly higher than in the comparison group. Both declined over time but the IgG levels remained significantly higher. Significant correlations were seen between serum IgG levels and age and duration of illness. Serum antibodies against flagellin, 40 kDa and 29 kDa antigens were still detectable in most patients up to a year postinfection, as were salivary antibodies to flagellin, the major outer-membrane protein and a 40 kDa antigen.
Sixteen European badgers (Meles meles) from three statutory removal operations were studied. Samples of tracheal aspirate, pooled lymph nodes and urine were cultured for mycobacteria. Seven of the badgers were infected with Mycobacterium bovis and had tuberculous pulmonary lesions which varied in severity from extensive granulomatous consolidation to microgranulomas which were not detectable grossly. Tuberculous lesions were also observed in the upper respiratory airways, intestines, kidneys, spleen, liver, thymus, pleura and lymph nodes. One badger had tuberculous bite wounds. The histopathological characteristics of the tuberculous reactions and the associated tissue damage in various organs, together with the gross pathology, indicate that both mildly and severely infected badgers have the potential to excrete M. bovis by several routes.
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AIMS: The aim of this investigation was to compare the usefulness of two previously published flagellin PCR-RFLP typing (Fla-typing) techniques for the subtyping of Campylobacter jejuni strains, in terms of ease of use and discriminatory power. METHODS AND RESULTS: Six groups of isolates, which were epidemiologically unrelated but with similar Fla-types, and five groups of epidemiologically related poultry isolates, with similar PFGE profiles, were used in the comparison. The Fla-typing methods used varied in the number and length of fla-genes amplified and the restriction enzymes used. In addition, the use of separately amplified PCR fragments of both the flaA and flaB genes to generate RFLP profiles was investigated. CONCLUSION: The results clearly demonstrated that both previously published methods exhibit some advantages over the other. However, optimal discrimination was obtained by the use of separately amplified PCR fragments of both fla-genes. SIGNIFICANCE AND IMPACT OF THE STUDY: The subtyping of Camp. jejuni isolates is considered essential for epidemiological purposes. Genotyping methods are now more frequently used but have yet to be standardized. Fla-typing is a rapid and easy to use method with acceptable discriminatory power. However, the discriminatory power of the currently published Fla-typing techniques may be further improved by incorporating RFLP profiles of both fla-genes.
The genetic stability of selected epidemiologically linked strains of Campylobacter jejuni during outbreak situations was investigated by using subtyping techniques. Strains isolated from geographically related chicken flock outbreaks in 1998 and from a human outbreak in 1981 were investigated. There was little similarity in the strains obtained from the different chicken flock outbreaks; however, the strains from each of three chicken outbreaks, including strains isolated from various environments, were identical as determined by fla typing, amplified fragment length polymorphism (AFLP) analysis, and pulsed-field gel electrophoresis, which confirmed the genetic stability of these strains during the short time courses of chicken flock outbreaks. The human outbreak samples were compared with strain 81116, which originated from the same outbreak but has since undergone innumerable laboratory passages. Two main AFLP profiles were recognized from this outbreak, which confirmed the serotyping results obtained at the time of the outbreak. The major type isolated from this outbreak (serotype P6:L6) was exemplified by strain 81116. Despite the long existence of strain 81116 as a laboratory strain, the AFLP profile of this strain was identical to the profiles of all the other historical P6:L6 strains from the outbreak, indicating that the genotype has remained stable for almost 20 years. Interestingly, the AFLP profiles of the P6:L6 group of strains from the human outbreak and the strains from one of the recent chicken outbreaks were also identical. This similarity suggests that some clones of C. jejuni remain genetically stable in completely different environments over long periods of time and considerable geographical distances.
The recent development of simple, rapid genotyping techniques for Campylobacter species has enabled investigation of the determinative epidemiology of these organisms in a variety of situations. In this study we have used the technique of fla typing (PCR-restriction fragment length polymorphism analysis of the flaA and flaB genes) to identify the sources of strains contaminating the carcasses of five campylobacter-positive and two campylobacter-negative broiler flocks during abattoir processing. The results confirmed that, in the United Kingdom, individual broiler flocks are colonized by a limited number of subtypes of Campylobacter jejuni or C. coli. In some but not all cases, the same subtypes, isolated from the ceca, contaminated the end product as observed in carcass washes. However, the culture methodology, i.e, use of direct plating or enrichment, affected this subtype distribution. Moreover, the number of isolates analyzed per sample was limited. fla typing also indicated that some campylobacter subtypes survive poultry processing better than others. The extent of resistance to the environmental stresses during processing varied between strains. The more robust subtypes appeared to contaminate the abattoir environment, surviving through carcass chilling, and even carrying over onto subsequent flocks. From these studies it is confirmed that some campylobacter-negative flocks reach the abattoir but the carcasses from such flocks are rapidly contaminated by various campylobacter subtypes during processing. However, only some of these contaminating subtypes appeared to survive processing. The sources of this contamination are not clear, but in both negative flocks, campylobacters of the same subtypes as those recovered from the carcasses were isolated from the crates used to transport the birds. In one case, this crate contamination was shown to be present before the birds were loaded.
A collection of Campylobacter fetus strains, including both C. fetus subsp. fetus and C. fetus subsp. venerealis, were phenotypically identified to the subspecies level and genotypically typed by PCR and amplified fragment length polymorphism (AFLP) analysis. Phenotypic subspecies determination methods were unreliable. Genotyping of the strains by PCR and AFLP showed a clear discrimination between the two subspecies.
Occupational exposure of susceptible humans to Campylobacter jejuni appears to result in resistance to disease. This is believed to be due to acquired protective immunity. To support this hypothesis the levels of C. jejuni-specific IgG and IgM antibodies were determined in sera from poultry abattoir workers. Such individuals are persistently exposed to C. jejuni, but apparently rarely acquire campylobacteriosis. Sera from 43 short-term workers (employed < or = 1 month), 78 long-term workers and 40 blood donors were investigated by ELISA. In 51 individuals a second serum sample, taken at least 1 month after the first, was also investigated. Eight workers had C. jejuni-positive faecal cultures and only one, a short-term worker, had symptoms of campylobacteriosis. There were significantly higher levels of specific IgG antibodies in long-term workers than in either of the other groups. There was no significant difference detectable in specific IgM antibody levels between any of the groups. The results provide supporting evidence that long-term exposure to C. jejuni induces circulating antibodies which reflect apparent reduced susceptibility to disease. Western blotting showed flagellin and polypeptides of 45, 40, 32 and 30 kD bound antibodies significantly more frequently by sera from long-term workers than short-term workers and blood donors. The most commonly detected antigens were the 40-kD (80%) and flagellin (55%). The results indicate that specific serum IgG responses induced by endemic exposure to C. jejuni might be directed towards a small number of protein antigens with apparently conserved epitopes.
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Phase variation of Campylobacter fetus surface layer proteins (SLPs) occurs by inversion of a 6.2-kb DNA segment containing the unique sap promoter, permitting expression of a single SLP-encoding gene. Previous work has shown that the C. fetus sap inversion system is RecA dependent. When we challenged a pregnant ewe with a recA mutant of wild-type C. fetus (strain 97-211) that expressed the 97-kDa SLP, 15 of the 16 ovine-passaged isolates expressed the 97-kDa protein. However, one strain (97-209) expressed a 127-kDa SLP, suggesting that chromosomal rearrangement may have occurred to enable SLP switching. Lack of RecA function in strains 97-211 and 97-209 was confirmed by their sensitivity to the DNA-damaging agent methyl methanesulfonate. Southern hybridization and PCR of these strains indicated that the aphA insertion into recA was stably present. However, Southern hybridizations demonstrated that in strain 97-209 inversion had occurred in the sap locus. PCR data confirmed inversion of the 6.2-kb DNA element and indicated that in these recA mutants the sap inversion frequency is reduced by 2 to 3 log(10) units compared to that in the wild type. Thus, although the major sap inversion pathway in C. fetus is RecA dependent, alternative lower-frequency, RecA-independent inversion mechanisms exist.