A comparison of two culture media for the recovery of thermophilic campylobacters in broiler farm samples

A study to evaluate the performance of two different brands of media (Oxoid, Basingstoke, UK, and Mast Diagnostics, Merseyside, UK) for the isolation of thermophilic campylobacters from a range of broiler farm samples was undertaken. Oxoid media performed significantly better than the Mast formulations with overall Campylobacter recovery rates of 46% and 30.5%, respectively, observed from 213 samples tested (p< or =0.05). Consistently higher recoveries of campylobacters were observed from all samples when the results using both types of media were combined.     

Heat resistance and mechanism of heat inactivation in thermophilic campylobacters

The heat resistance of Campylobacter jejuni strains AR6 and L51 and the heat resistance of Campylobacter coli strains DR4 and L6 were measured over the temperature range from 50 to 60 degrees C by two methods. Isothermal measurements yielded D55 values in the range from 4.6 to 6.6 min and z values in the range from 5.5 to 6.3 degrees C. Dynamic measurements using differential scanning calorimetry (DSC) during heating at a rate of 10 degrees C/min yielded D55 values of 2.5 min and 3.4 min and z values of 6.3 degrees C and 6.5 degrees C for AR6 and DR4, respectively. Both dynamic and isothermal methods yielded mean D55 values that were substantially greater than those reported previously (0.75 to 0.95 min). DSC analysis of each strain during heating at a rate of 10 degrees C/min yielded a complex series of overlapping endothermic peaks, which were assigned to cell wall lipids, ribosomes, and DNA. Measurement of the decline in the numbers of CFU in calorimetric samples as they were heated showed that the maximum rate of cell death occurred at 56 to 57 degrees C, which is close to the value predicted mathematically from the isothermal measurements of D and z (61 degrees C). Both estimates were very close to the peak m1 values, 60 to 62 degrees C, which were tentatively identified with unfolding of the 30S ribosome subunit, showing that cell death in C. jejuni and C. coli coincided with unfolding of the most thermally labile regions of the ribosome. Other measurements indicated that several essential proteins, including the alpha and beta subunits of RNA polymerase, might also unfold at the same time and contribute to cell death.     

Evaluation of two automated enzyme-immunoassays for detection of thermophilic campylobacters in faecal samples from cattle and swine.

We evaluated the performance of two enzyme-immunoassays (EIA) for the detection of naturally occurring, thermophilic Campylobacter spp. found in faecal samples from cattle (n = 21 and n = 26) and swine (n = 43) relative to the standard culture method, and also assuming that none of the tests was the definitive standard. The primary isolation both for the culture and the EIA methods was carried out by overnight selective enrichment in Preston broth. The results showed good sensitivities for both EIA methods in cattle (95% and 84%) and swine (88% and 69%) samples. However, when testing cattle samples, EIA-2 method resulted in a rather low specificity (32%). This seemed to be partially due to the isolation of nonthermophilic species. In conclusion, EIA-1 method may provide a simple and fast tool with good accuracy in cattle and swine samples for automated screening of large number of samples.     

Determination of the optimal culture conditions for detecting thermophilic campylobacters in environmental water

This study evaluated alternative protocols for culturing thermophilic campylobacters in environmental water. All samples were filtered through a sterile 0.45 μm pore-size membrane, which was then incubated in Preston enrichment broth. Four variables were compared: water sample volume (2000 mL vs. 500 mL), enrichment broth volume (25 mL vs. 100 mL), enrichment incubation duration (24 h vs. 48 h), and number of enrichment passages (one vs. two). In addition, DNA extracts were prepared from all final broths and analyzed using three rRNA PCR assays. River water was collected at 3 sampling sites weekly for 9 weeks. Among these 27 collections, 25 (93%) yielded Campylobacter spp. under at least one of the 16 culture conditions. By univariate analysis, yields were significantly better for the 2000 mL sample volume (68.5% vs. 43.0%, p < 0.0001) and the 25 mL enrichment broth volume (64.5% vs. 47.0%, p < 0.0004). Neither of the enrichment period had a significant effect, although there was a trend in favor of 48 h incubation (59.5% vs. 52.0%, p = 0.13). The three PCR methods gave concordant results for 66 (33%) of the culture-negative samples and 103 (50%) of the culture-positive samples. Compared with culture results, Lubeck's 16S PCR assay had the best performance characteristics, with a sensitivity of 82% and a specificity of 94%. Of the 12 culture-negative samples positive by Lubeck's PCR assay, 11 (92%) samples were also positive by Denis' 16S PCR assay, suggesting that in these cases the culture might have been falsely negative. Based on our results, we conclude that the optimal conditions for detecting Campylobacter spp. in natural waters include 2000 mL sample volume and a single enrichment broth of 25 mL PB incubated for 48 h.     

Prevalence and strain diversity of thermophilic campylobacters in cattle, sheep and swine farms

AIMS: To determine prevalence and strain diversity of thermophilic campylobacters in healthy ruminants and swine. METHODS AND RESULTS: Faecal samples collected from 343 herds (120 sheep, 124 beef cattle, 82 dairy cattle and 17 swine) in the Basque Country were screened in pools for thermophilic campylobacters. Two hundred and three herds were positive (67.1% dairy cattle, 58.9% beef cattle, 55.0% sheep and 52.9% pig), and species-specific PCR identified Campylobacter jejuni in 20.7% of the herds and Campylobacter coli in 6.4%. Campylobacter coli was isolated from the four production systems and was the most prevalent species in swine, where C. jejuni was not found. Other thermophilic campylobacters were found in all production systems. Four hundred and ninety-three animals from 11 positive herds were individually analysed, detecting significantly higher within-herd prevalences in dairy cattle (66.7%) and swine (57.8%) than in sheep (8.8%) or beef cattle (5.4%). flaA PCR-RFLP and pulsed-field gel electrophoresis analysis of a selection of isolates showed high genetic diversity. CONCLUSIONS: Healthy swine, cattle and sheep are important reservoirs of thermophilic campylobacters of different species and high genetic diversity. SIGNIFICANCE AND IMPACT OF THE STUDY: Efficient farm-based intervention measures are needed to reduce risk of infection. Non-C. jejuni/C. coli species should be monitored to investigate their significance for infection.     

Biotypes and DNA ribopatterns of thermophilic campylobacters from faeces and seawater in Eastern Spain

Twelve strains of thermophilic campylobacters isolated in Valencia from faeces of infants and young children with diarrhoea, and from seawater, were biotyped and examined by analysis of chromosomal DNA Hae III ribopatterns. The strains were identified as Campylobacter jejuni and C. coli , and all had different ribopatterns except for three identical faecal isolates of C. jejuni biotype I. No correlation was found between Lior biotype and Hae III ribopattern but several bands within ribopatterns were identified as possible species markers for use in diagnostic and epidemiological studies.     

The seasonal variation of thermophilic campylobacters in beef cattle, dairy cattle and calves

The epidemiology of clinical cases of campylobacter in temperate climates shows a striking seasonality. In the search for a seasonal environmental reservoir changes in the carriage rate and population size of campylobacters in bovine hosts with time have been measured. Most probable number (MPN) methodology was used to enumerate thermophilic campylobacters in samples taken from the small intestines of beef cattle at slaughter and the fresh faeces of four dairy herds and new-born calves. Statistical analyses revealed significant evidence for seasonal periodicity in the data from dairy herds ( P = 0·044). Not only was there a departure from constancy within a 12-month interval but these data revealed a true seasonality, that is, the same periodicity in numbers from one year to the next. Each herd had two peaks per year, in approximately spring and autumn. Peaks coincided in herds on neighbouring farms but those on farms in the north preceded those on farms in the south by 2 and 1 months, respectively ( P = 0·0057). Intestinal carriage by beef cattle at slaughter was 89·4% ( n = 360) with an average MPN campylobacters per gram fresh weight (MPN gfw⁻¹) of 6·1 × 10². Average MPN gfw⁻¹ in faeces from the dairy herds and calves were 69·9 ( S.D. 3) and 3·3 × 10⁴ ( S.D. 1·7 × 10²). There was no evidence of seasonal periodicity in the size of the campylobacter population in beef cattle at slaughter. Calves were campylobacter free at birth but became colonized within a few days.     

Water temperature: A factor in the seasonality of two freshwater sponge species,Ephydatia fluviatilis and Spongilla alba

Two freshwater sponge species, Ephvdatia fluviatilis and Spongilla alba, were maintained in a continuous-flow laboratory culture system at several different water temperatures. Experimental results suggest that sponge growth rate is affected by water temperature and that it is affected differently in the two species. The results correlate well with field observations on species abundance at different water temperatures and thereby support the view that water temperature is a factor determining seasonality in these species.     

Influence of incubation temperature on the microbial reductive dechlorination of 2,3,4,6-tetrachlorobiphenyl in two freshwater sediments

Volumn 62, no. 11, p. 4174, Abstract, lines 12 and 13: "para dechlorination was restricted from 18 to 34(deg)C" should read "para dechlorination was restricted to temperatures from 18 to 34(deg)C." [This corrects the article on p. 4174 in vol. 62.].     

The origins of Atlantic salmon (Salmo salar L.) recolonizing the River Mersey in northwest England

By the 1950s, pollution had extirpated Atlantic salmon in the river Mersey in northwest England. During the 1970s, an extensive restoration program began and in 2001, an adult salmon was caught ascending the river. Subsequently, a fish trap was installed and additional adults are now routinely sampled. In this study, we have genotyped 138 adults and one juvenile salmon at 14 microsatellite loci from across this time period (2001–2011). We have used assignment analysis with a recently compiled pan‐European microsatellite baseline to identify their most probable region of origin. Fish entering the Mersey appear to originate from multiple sources, with the greatest proportion (45–60%, dependent on methodology) assigning to rivers in the geographical region just north of the Mersey, which includes Northwest England and the Solway Firth. Substantial numbers also appear to originate from rivers in western Scotland, and from rivers in Wales and Southwest England; nonetheless, the number of fish originating from proximal rivers to the west of the Mersey was lower than expected. Our results suggest that the majority of salmon sampled in the Mersey are straying in a southerly direction, in accordance with the predominantly clockwise gyre present in the eastern Irish Sea. Our findings highlight the complementary roles of improving water quality and in‐river navigability in restoring salmon to a river and underlines further the potential benefits of restoration over stocking as a long‐term solution to declining fish stocks.     

Occurrence and strain diversity of thermophilic campylobacters in cattle of different age groups in dairy herds

AIMS: To investigate the occurrence and numbers of thermophilic campylobacters excreted by cattle in dairy herds, and to assess the strain diversity within herds. METHODS AND RESULTS: Faecal samples from 15 animals at each of 24 cattle farms were cultured quantitatively for thermophilic campylobacters and 23% of animals and 83% of farms were positive for Campylobacter jejuni. Young animals had a higher prevalence and higher faecal concentration than older animals. Serotyping and pulsed field gel electrophoresis (PFGE) of isolates showed that the most common serotypes were 2, 4-complex and 11. Serotype 2 was especially prevalent among calves (68% of the positive calves). In eight of the 20 positive herds, all isolates had the same sero- and PFGE type while, in the other herds, two to five different types were isolated. CONCLUSIONS: Significant differences were found between age groups in relation to the prevalence and numbers of excreted campylobacters, serotype distribution and strain diversity. The relatively few different strains in each herd indicate that transmission between animals is common. SIGNIFICANCE AND IMPACT OF THE STUDY: The high prevalence on cattle farms of the human pathogen C. jejuni and the wide distribution of serotype 2, the most common serotype among Danish patients, indicate that cattle might be an important reservoir for human infections. The ability of this serotype to colonize calves in high numbers further indicates that serotype 2 strains may have an advantage over other serotypes.     

Correlation between environmental monitoring of thermophilic campylobacters in sewage effluent and the incidence of Campylobacter infection in the community

Environmental monitoring of thermophilic campylobacters in liquid sewage effluent (primary settlement only) during 1988 and 1989 showed a prominent seasonality with distinct peaks in May and June (the average number of bacteria per 100 ml of effluent in months other than May and June was 2244 and the average for the peak months was 50,778). Apart from September 1989, this seasonality coincided precisely with the seasonal variation of campylobacter enteritis in the community with similar distinct peaks in May and June (the incidence of infection in May and June was twice or three times that in the other months). Sampling of sewers showed that the campylobacters in the sewage effluent came mainly from abbatoir and animal processing plants with only a minor input from the community. Therefore, the seasonal peaks in the sewage effluent and in the community may not be dependent on human infections but on zoonotic infections which may also peak in May and June.     

Correlation between environmental monitoring of thermophilic campylobacters in sewage effluent and the incidence of Campylobacter infection in the community

Environmental monitoring of thermophilic campylobacters in liquid sewage effluent (primary settlement only) during 1988 and 1989 showed a prominent seasonablity with distinct peaks in May and June (the average number of bacteria per 100 ml of effluent in months other than May and June was 2244 and the average for the peak months was 50 778). Apart from September 1989, this seasonality coincided precisely with the seasonal variation of Campylobacter enteritis in the community with similar distinct peaks in May and June (the incidence of infection in May and June was twice or three times that in the other months).
Sampling of sewers showed that the campylobacters in the sewage effluent came mainly from abbatoir and animal processing plants with only a minor input from the community. Therefore, the seasonal peaks in the sewage effluent and in the community may not be dependent on human infections but on zoonotic infections which may also peak in May and June.     

Distribution, abundance, and roles of freshwater clams (Bivalvia, Unionidae) in the freshwater tidal Hudson River

1. An extensive series of PONAR grabs was used to determine the distribution and abundance of unionid clams in the freshwater tidal Hudson River. 2. The five species of unionids collected were distributed very unevenly within the river. Mean river-wide density and biomass of unionids were 8.0m^?2 and 6.2 g DM m^?2 (shell-free)., respectively. 3. The environmental variables that we measured (water depth, distance from shore, sediment granulometry and organic content, presence or absence of macrophytes, and the chlorophyll a and particulate organic matter content of the water) explained little of the variation in abundance of unionids. 4. The distributions of the various species of clams did not differ significantly with respect to the environmental variables measured. 5. We estimate that unionids filter a significant amount of water (0.14m³ m^?2 day^?1, on average) in the Hudson River estuary, roughly equivalent in magnitude to downstream flushing. 6. We project that unionids will serve as a major substratum for the settlement of the zebra mussel (Dreissena polymorpha), which is now invading the estuary. We emphasize that unionids may play important non-trophic roles in large river ecosystems.     

Climatic governance of the latitudinal cline in seasonality of freshwater phytoplankton production

Data on the intra-annual variation in phytoplankton production for 114 southern and northern hemisphere lakes (42°S to 75° N) were combined to examine broad-scale latitudinal trends. Much of the seasonal variation in production can be statistically explained through the climatic factors of mean annual range of atmospheric temperature and annual variance in incident solar radiation, of which the amplitude of both increases with distance from the equator. Quantification of this latitudinal-climatic trend is an important step in understanding the hierarchial regulation of variability in lake trophic dynamics.     

Distribution and seasonality of desmids in the Maarsseveen Lakes area

The desmid flora of mesotrophic Lake Maarsseveen I, eutrophic Lake Maarsseveen II, as well as the interconnecting broads was studied during the period 1987–1991. Seasonal perodicity of euplanktonicStaurastrum andCosmarium species in the two Maarsseveen lakes fitted the well-known general pattern of desmid abundance peaks in (late) summer time. The deviating seasonality of two euplanktonicClosterium species (C. aciculare andC. acutum var.variabile) could be related to special nutritional demands. The tychoplanktonic desmid flora encountered in the interconnecting system of peat pits and canals proved to be more diversified as the influence of nutrient-poor seepage on the habitat (appearing from conductivity, transparency and the occurrence of particular macrophyte species) was greater. The rare speciesEuastrum germanicum andHeimansia pusilla had been only incidentally recorded in The Netherlands. Hanny Kooyman-van Blokland untimely died at the age of forty, in March 1994.     

Numerical analysis of electrophoretic protein patterns of Campylobacter laridis and allied thermophilic campylobacters from the natural environment

Twenty-one strains comprising Campylobacter laridis (nine), nalidixic acid sensitive campylobacters (NASC) (four), and urease-positive thermophilic campylobacters (UPTC) (eight) were characterized by one-dimensional SDS-PAGE of cellular proteins. The UPTC and NASC strains included six from river water, two from mussels and four from sea water. The type strains of three other Campylobacter species were included for reference. The protein patterns, which contained 45–50 discrete bands, were highly reproducible and were used as the basis for two numerical analyses. In the first, which included all the protein bands, the 21 strains formed nine clusters at the 80% similarity (S) level. The typical C. laridis strains were restricted to two phenons (2 and 5); the atypical strains being distributed among the remaining phenons. In the second analysis, which excluded the principal protein bands (40–48.5 kD range), the 21 strains formed five clusters at the 80% S level. The typical C. laridis strains were relatively homogeneous and fell into a single phenon (2) within which two subgroups were discernable. The atypical strains were more heterogeneous with respect to background protein pattern, with representatives appearing in all five phenons. An electropherotyping scheme comprising six electropherotypes, and based on both analyses is proposed. The high within-group S level and separation from reference strains of Campylobacter in the second analysis, suggested that UPTC and NASC strains belonged within C. laridis possibly as biovars.     

Distribution of the human faecal bacterium Bacteroides fragilis, its bacteriophages and their relationship to current sewage pollution indicators in bathing water

Although several bacteria are currently used as possible indicators of human pathogens in sewage-polluted sea water, they are often viewed as inadequate and especially inadequate as indicators of viral pathogens. This study investigates the distribution of Bacteroides fragilis and closely related Bacteroides spp. and their associated bacteriophages in sea water frequently used for recreational purposes. These organisms may provide a potentially more appropriate indicator. Bacteroides fragilis is one of about 10 species which are loosely placed together in the 'B. fragilis' group. Samples down-current from a sewage outfall were examined for the presence of B. fragilis group organisms and associated bacteriophages. Numbers were correlated with current bacterial and possible viral indicators at these sites. These B. fragilis group isolates were used as hosts to successfully isolate bacteriophages. The host range of these bacteriophages was investigated. It is hoped to expand this study by using these B. fragilis group hosts and their bacteriophages to identify a more suitable, European-wide, indicator of bacterial pathogens which can also be used to detect bacteriophages which are suitable as viral indicators.