Diversity of Campylobacter coli genotypes in the lower porcine gastrointestinal tract at time of slaughter.

AIMS: To compare the genotypes of Campylobacter coli obtained from the rectal and ileal samples of pigs at the time of slaughter. METHODS AND RESULTS: Five animals were sampled following slaughter with ileal contents and anal swabs being taken post-evisceration. Swabs were directly plated onto charcoal cefoperazone desoxycholate agar (CCDA) while ileal contents were enriched in CCDA broth. Twenty isolates were picked from each site sampled and all 200 isolates were Camp. coli. Isolates were genotyped using random amplified polymorphic DNA (22 discrete types) and flaA (11 discrete types). Both methods found that 55% of the genotypes were unique to rectal samples. Only one animal yielded the same flaA type from ileal and rectal samples. CONCLUSIONS: Rectal sampling of pigs yielded a more diverse subset of Camp. coli genotypes than ileal contents, but failed to yield all of the genotypes carried by an individual animal. SIGNIFICANCE AND IMPACT OF THE STUDY: A small sample of pigs carried a very diverse population of Camp. coli genotypes; and sampling of a single site in the gut will recover only part of this population. Hence, any genotyping studies of Camp. coli in pigs must be interpreted with caution, and epidemiological studies could be confounded by the number of Camp. coli genotypes available.

Antibiotic resistance of retail food and human Campylobacter isolates on the island of Ireland from 2001-2002.

The antimicrobial resistance profiles of Campylobacter isolates recovered from a range of retail food samples (n=374) and humans (n=314) to eight antimicrobial compounds were investigated. High levels of resistance in food C. jejuni isolates were observed for ceftiofur (58%), ampicillin (25%) and nalidixic acid (17%) with lower levels observed for streptomycin (7.9%) and chloramphenicol (8.3%). A total of 80% of human C. jejuni isolates were resistant to ceftiofur, while 17% showed resistance to ampicillin and nalidixic acid, 8.6% to streptomycin and 4.1% to chloramphenicol. Resistance to clinically relevant antimicrobials such as erythromycin, ciprofloxacin and tetracycline was 6.7, 12, and 15% respectively for all food isolates and was similar to corresponding resistance prevalences observed for human isolates, where 6.4, 12 and 13% respectively were found to be resistant. Comparisons of C. jejuni isolates in each location showed a high degree of similarity although some regional variations did exist. Comparison of total C. jejuni and C. coli populations showed minor differences, with C. jejuni isolates more resistant to ampicillin and ceftiofur. Multidrug resistance patterns showed some profiles common to human and clinical isolates.

Occurrence of Campylobacter in retail foods in Ireland.

A surveillance study was carried out to determine the prevalence of Campylobacter in a range of retail foods purchased in three Irish cities over a 20-month period between March 2001 and October 2002. In total 2391 food samples were analysed during this period. Campylobacter was isolated from 444 raw chicken (49.9%), 33 turkey (37.5%) and 11 duck samples (45.8%). Lower isolation rates of 7/221 (3.2%), 10/197 (5.1%) and 31/262 (11.8%) were observed for raw beef, pork and lamb, respectively. One sample of pork paté from 120 samples analysed (0.8%) was Campylobacter-positive. A total of three shellfish samples (oysters) from 129 raw specimens examined (2.3%) were found to contain Campylobacter. Low prevalences of the organism (0.9%) were also isolated from fresh mushrooms. Of 62 raw bulk tank milk samples analysed, Campylobacter was recovered in a single sample (1.6%). Campylobacter was not detected in any of the comminuted pork puddings, prepared vegetables and salads, retail sandwiches or cheeses made from unpasteurised milk. In total, 543 Campylobacter were isolated from all of the food samples analysed, of which 453 (83.4%) were confirmed as Campylobacter jejuni and the remaining 90 (16.6%) as Campylobacter coli.

Inhibition of tumor necrosis factor-alpha (TNF-alpha) production and arthritis in the rat by GW3333, a dual inhibitor of TNF-alpha-converting enzyme and matrix metalloproteinases.

Tumor necrosis factor-alpha (TNF)-converting enzyme (TACE) cleaves the precursor form of TNF, allowing the mature form to be secreted into the extracellular space. GW3333, a dual inhibitor of TACE and matrix metalloproteinases (MMPs), was compared with an anti-TNF antibody to evaluate the importance of soluble TNF and MMPs in rat models of arthritis. Oral administration of GW3333 completely blocked increases in plasma TNF after LPS for up to 12 h. In a model wherein intrapleural zymosan injection causes an increase in TNF in the pleural cavity, GW3333 completely inhibited the increase in TNF in the pleural cavity for 12 h. Under these dosing conditions, the plasma levels of unbound GW3333 were at least 50-fold above the IC(50) values for inhibition of individual MMPs in vitro. In a model wherein bacterial peptidoglycan polysaccharide polymers reactivate a local arthritis response in the ankle, a neutralizing anti-TNF antibody completely blocked the ankle swelling over the 3-day reactivation period. GW3333 administered b.i.d. over the same period also inhibited ankle swelling, with the highest dose of 80 mg/kg being slightly less active than the anti-TNF antibody. In a 21-day adjuvant arthritis model, the anti-TNF antibody did not inhibit the ankle swelling or the joint destruction, as assessed by histology or radiology. GW3333, however, showed inhibition of both ankle swelling and joint destruction. In conclusion, GW3333 is the first inhibitor with sufficient duration of action to chronically inhibit TACE and MMPs in the rat. The efficacy of GW3333 suggests that dual inhibitors of TACE and matrix metalloproteinases may prove therapeutic as antiarthritics.

Occurrence of Escherichia coli O157:H7, Listeria monocytogenes, Salmonella and Campylobacter spp. on beef carcasses in Northern Ireland.

A survey of beef carcasses was conducted in all 10 European community approved abattoirs in Northern Ireland to determine the incidence of Escherichia coli O157:H7. Analyses were based on excised samples of neck meat taken less than 48 h post-kill. Overall, 780 carcasses were sampled and all were negative for E. coli O157:H7. A sub-set of samples was analysed for the presence of Listeria monocytogenes (n=200), Salmonella (n=200) and Campylobacter spp.(n=100). L. monocytogenes was not detected but Listeria innocua was found on five carcasses and Listeria seeligeri on one. Three carcasses carried salmonellas; Salmonella Mbandaka was found on two and Salmonella Thompson on one. Campylobacter spp. were not detected on any carcasses. The results indicate that very few beef carcasses in Northern Ireland appear to carry any of the four pathogens sought, and this may help explain the low incidence of E. coli O157:H7 in the Northern Ireland human population, relative to the rest of the UK.

Optimising recovery of Campylobacter spp. from the lower porcine gastrointestinal tract.

To determine the incidence of campylobacters in Northern Ireland pigs, ileal contents and anal swabs were taken shortly after death. Direct streaking onto Preston agar, and modified charcoal cefoperazone desoxycholate agar (mCCDA), were compared, as was enrichment in selective broths prior to streaking onto the corresponding solid medium. For anal swabs direct plating on mCCDA was most efficient, with 100% of samples positive, whilst for ileal contents enrichment in mCCD broth was best with 86% of samples positive. Although only 34% of ileal samples enriched in Preston broths were positive they yielded three species not isolated from mCCD broth, and hence indicated that some pigs were infected by at least two species of Campylobacter. Overall, the number of samples found to contain campylobacters, and the range of species isolated, was seen to be markedly affected by both the choice of selective medium and the isolation procedures.

Frequency of occurrence of Campylobacter spp. in red meats and poultry in Northern Ireland and their subsequent subtyping using polymerase chain reaction-restriction fragment length polymorphism and the random amplified polymorphic DNA method.

Sampling of lamb (n = 100) and beef (n = 100) carcasses in abattoirs in Northern Ireland produced no evidence of Campylobacter spp. contamination and when retail packs of beef (n = 50) and pork (n = 50) were sampled these were also apparently free of Campylobacter spp. However, 38% of retail packs of chicken pieces (n = 120), yielded Campylobacter spp. These packs were purchased over a period of 1 year and came from a single local producer. After the species of the isolates had been determined (Campylobacter jejuni and Camp. coli were found in approximately equal numbers) they were subtyped using both polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and the random amplified polymorphic DNA (RAPD) method of typing. All of the poultry isolates were successfully typed by these methods, in contrast to the results obtained with serotyping where several isolates were found to be untypable. PCR-RFLP typing showed that specific subtypes were isolated repeatedly over a period of 1 year in the output of the producer studied. The more discriminating RAPD confirmed this observation, but with fewer isolates. This appears to indicate recurrent infection of broilers whose source can now be investigated using the methodologies developed.

Sub-typing of animal and human Campylobacter spp. using RAPD.

Based on a 10-mer primer (5'-CCTGTTAGCC-3'), a random amplified polymorphic DNA (RAPD) method for typing Campylobacter coli isolated from pigs was developed. The method proved effective with a high discrimination and good reproducibility. In contrast with serotyping no untypable strains were found out of a total of 269 isolates (veterinary, food and clinical) examined. The method was also successfully applied to typing Campylobacter jejuni from a similar range of sources.

Extracts of Ginkgo biloba leaves inhibit monoamine oxidase.

Extracts of Ginkgo biloba leaves produce reversible inhibition of rat brain monoamine (MAO). Both MAO-A and -B types were inhibited to a similar extent. The MAO inhibitory compound(s) were present in dried or fresh Ginkgo biloba leaves as well as in commercially available capsules of Ginkgo biloba and appear to be heat stable with relatively low molecular weight. MAO inhibition by Ginkgo biloba may be a mechanism underlying reported anti-stress and anxiolytic activities of this natural product.

Stimulation of carnitine acetyltransferase in PC12 cells by nerve growth factor: relationship to choline acetyltransferase stimulation.

The activity of carnitine acetyltransferase (acetyl-CoA:L-carnitine O-acetyltransferase) was found to be at least 50-fold higher than that of choline acetyltransferase in PC12 cells. Nerve growth factor stimulated both enzymes in a parallel manner with respect to concentration of NGF and culture time. The stimulation of both enzymes was completely inhibited by 10 microM 6-thioguanine, an inhibitor of protein kinase N. Results are discussed with reference to the hypothesis that the two enzymes may be functionally related in neuronal cells.

Effects of GTP?S and other nucleotides on phosphoinositide metabolism in crude rat brain synaptosomal preparations.

Crude nerve-ending preparations from rat brain were labeled with either [(32)P]phosphate or myo[2-(3)H]inositol in order to observe effects of guanosine 5?-[?-thio]triphosphate (GTP?S) and other nucleotides on phosphoinositides, phosphatidate and inositol phosphates. This system exhibited typical responses to muscarinic agonists, including acetylcholine-a decrease in net labeling of [(32)P]polyphosphoinositides, an increase in labeling of [(32)P]phosphatidate, and a stimulation of [(3)H]inositol phosphate formation. GTP?S and other nucleotides may not readily penetrate intact synaptosomal membranes to cause activation of phospholipase C via an interaction with G proteins, and, as might be expected, there was no indication that G-protein interaction occurred in these preparations. However, other effects were observed. GTP?S decreased net [(32)P] incorporation in phosphatidylinositol (PI) and polyphosphoinositides in a dose-dependent manner. GTP?S also caused an initial marked stimulation of [(32)P] labeling of phosphatidate, suggesting a possible inhibition in the conversion of phosphatidic acid to PI. Other nucleotides [GTP, ATP, Gpp(NH)p, GMP] produced qualitatively similar effects on phosphoinositides. Thus GTP?S and other nucleotides, at physiologically relevant concentrations, may influence phosphoinositide turnover via extracellular or other mechanisms, in addition to the proposed interaction of GTP with G-proteins within membranes.

Acetyl-L-carnitine as a precursor of acetylcholine.

Synthesis of [3H]acetylcholine from [3H]acetyl-L-carnitine was demonstrated in vitro by coupling the enzyme systems choline acetyltransferase and carnitine acetyltransferase. Likewise, both [3H] and [14C] labeled acetylcholine were produced when [3H]acetyl-L-carnitine and D-[U-14C] glucose were incubated with synaptosomal membrane preparations from rat brain. Transfer of the acetyl moiety from acetyl-L-carnitine to acetylcholine was dependent on concentration of acetyl-L-carnitine and required the presence of coenzyme A, which is normally produced as an inhibitory product of choline acetyltransferase. These results provide further evidence for a role of mitochondrial carnitine acetyltransferase in facilitating transfer of acetyl groups across mitochondrial membranes, thus regulating the availability in the cytoplasm of acetyl-CoA, a substrate of choline acetyltransferase. They are also consistent with a possible utility of acetyl-L-carnitine in the treatment of age-related cholinergic deficits.