The colonization of turkeys by thermophilic campylobacters

The rate at which five broods of turkey chicks became colonized by thermophilic campylobacters was investigated. Day-old chicks were normally free of campylobacters on arrival on the farm with colonization beginning within 7 d. The carriage rate was 100% by day 14 in three of the broods and by day 21 in the other two. Higher carriage rates were obtained with enrichment procedures than with direct plating. Two broods were investigated over an extended interval for the number of campylobacters shed in their faeces. In brood A, campylobacters increased from day 1 to day 14 concomitant with increases in the carriage rate and in the number of chicks with diarrhoea. By day 39, when the birds were sold to other farms, the excretion rate had reached 6 × 10⁷ campylobacters g⁻¹ fresh faeces. Brood B was monitored over 91 d and showed peaks in Campylobacter numbers on days 19 and 75, corresponding to peaks in the number of diarrhoeic samples. The introduction of new birds into the brood resulted in an increase in the Campylobacter population and in the number of birds with diarrhoea. Campylobacter jejuni was the only species isolated but comprised several different biotypes. Analysis of the number of campylobacters at different sites along the gastrointestinal tract of mature turkeys at slaughter showed that numbers increased with distance from the beak and were highest in the caeca.     

Seasonal variation of thermophilic campylobacters in lambs at slaughter

A survey of the isolation rate and population size of thermophilic campylobacters in lambs at slaughter was carried out to determine the seasonal variation of thermophilic campylobacters in ovine hosts. Isolation rates determined by enrichment methods were always higher than those using direct plating onto selective agar and showed that Campylobacter could be isolated from 91·7% ( n = 360) of samples from the small intestine of the lambs. Enumerations (MPN), done monthly over a 2-year period, averaged 4·00 log 10 ( n = 1080, S.D . 0·16) campylobacters g⁻¹ fresh weight (fw) intestinal contents with some samples giving values higher than 7 log MPN gfw⁻¹. These results show that the prevalence of thermophilic campylobacters in sheep intended for slaughter is much higher than previously reported. Statistical analyses showed that there was a significant seasonal periodicity in the Campylobacter populations in the small intestines of lambs at slaughter ( P = 0·004) but that there was no statistical relationship with environmental parameters such as minimum and maximum temperature, rainfall or hours of sunshine. In an analysis along the length of the intestinal tract of lambs, campylobacters were isolated from 80% of small intestine and 30% of rumen samples, but not from the true stomach, large intestine or caecal samples. A survey of sheep grazing on salt marsh, fell (upland) and farm pasture showed that the isolation rate of thermophilic campylobacters in fresh faeces was substantially lower (29·3% ( n = 420)) than that from the small intestine of lambs at slaughter. No significant difference (>0·05) was found between the isolation rate of campylobacters from faeces sampled in late spring/early summer and autumn, nor between the different types of grazing.     

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.     

Seasonal variation of thermophilic campylobacters in sewage sludge

The seasonal variation of thermophilic campylobacters in Lancaster's sewage sludge was studied over a 21 month period. The numbers in fresh sludge (from primary sedimentation) vary between approximately 200 and 5000/100 ml for most of the year but there was a large increase in May and June (in May 1988 there were 42,100 campylobacters/100 ml which is 17 times more than in the preceding April). In 1989 there was a similar May/June peak but with lower numbers. This seasonal variation, measured by environmental monitoring, reflects the incidence of infections in the community. The same pattern was found in 2-d old sludge but the numbers were substantially lower (40% lower over the experimental period). Thermophilic campylobacters were virtually absent from digested sludge and sludge prior to land distribution. Survival experiments confirm that campylobacters survive for only a few hours in both sterile and unsterile digested and undigested sludge. These results suggest that it is safe to dispose of Lancaster's digested sludge on land but there is still uncertainty about the ability of campylobacters to survive in sludge in the viable but non-culturable form.     

Seasonal variation of thermophilic campylobacters in sewage sludge

The seasonal variation of thermophilic campylobacters in Lancaster's sewage sludge was studied over a 21 month period. The numbers in fresh sludge (from primary sedimentation) vary between approximately 200 and 5000/100 ml for most of the year but there was a large increase in May and June (in May 1988 there were 42100 campylobacters/100 ml which is 17 times more than in the preceding April). In 1989 there was a similar May/June peak but with lower numbers. This seasonal variation, measured by environmental monitoring, reflects the incidence of infections in the community. The same pattern was found in 2-d old sludge but the numbers were substantially lower (40% lower over the experimental period).
Thermophilic campylobacters were virtually absent from digested sludge and sludge prior to land distribution. Survival experiments confirm that campylobacters survive for only a few hours in both sterile and unsterile digested and undigested sludge. These results suggest that it is safe to dispose of Lancaster's digested sludge on land but there is still uncertainty about the ability of campylobacters to survive in sludge in the viable but non-culturable form.     

A study of thermophilic campylobacters in a river system

Fifteen kilometres of a river system traversing rural and urban areas and subject to sewage works effluent discharge was studied during a 12 1/2 month period. A total of 312 samples was collected from 12 sites at 14 d intervals and tested by a glass microfibre filtration method and a most probable number (MPN) method. Campylobacters were found in 43% of samples by the filtration method and 21% by the MPN method. The lowest frequency of isolation and lowest counts (<10 campylobacters/100 ml) were associated with samples collected from rural sites and fast-flowing stretches of river. The greatest frequency of isolation and highest counts (< 10–230 campylobacters/100 ml) were associated with sites adjacent to or downstream of sewage works. There was an obvious seasonal trend; most isolations and highest counts were obtained in late autumn and winter, and fewest isolations and lowest counts in spring and summer. Surface water run-off from adjacent farmland following heavy rainfall also increased the counts of campylobacters in the river system. Biotyping of isolates demonstrated that the most prevalent Campylobacter sp. was Campylobacter jejuni but C. coli, C. laridis and a previously unrecognized group of campylobacters were also isolated. Serotyping differentiated 14 serotypes of C. jejuni , 11 of C. coli and two of C. laridis. Furthermore, serotypes of C. jejuni commonly isolated from enteritis in man were frequently found in river water tested during this study.     

A study of thermophilic campylobacters in a river system

Fifteen kilometres of a river system traversing rural and urban areas and subject to sewage works effluent discharge was studied during a 12 1/2 month period. A total of 312 samples was collected from 12 sites at 14 d intervals and tested by a glass microfibre filtration method and a most probable number (MPN) method. Campylobacters were found in 43% of samples by the filtration method and 21% by the MPN method. The lowest frequency of isolation and lowest counts (less than 10 campylobacters/100 ml) were associated with samples collected from rural sites and fast-flowing stretches of river. The greatest frequency of isolation and highest counts (greater than 10-230 campylobacters/100 ml) were associated with sites adjacent to or downstream of sewage works. There was an obvious seasonal trend; most isolations and highest counts were obtained in late autumn and winter, and fewest isolations and lowest counts in spring and summer. Surface water run-off from adjacent farmland following heavy rainfall also increased the counts of campylobacters in the river system. Biotyping of isolates demonstrated that the most prevalent Campylobacter sp. was Campylobacter jejuni but C. coli, C. laridis and a previously unrecognized group of campylobacters were also isolated. Serotyping differentiated 14 serotypes of C. jejuni, 11 of C. coli and two of C. laridis. Furthermore, serotypes of C. jejuni commonly isolated from enteritis in man were frequently found in river water tested during this study.     

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.     

Ribotypes and AP-PCR fingerprints of thermophilic campylobacters from marine recreational waters

Thirty-two strains of thermophilic campylobacters isolated from marine recreational waters and seven reference strains were biotyped and analysed by chromosomal DNA Hae III ribopatterns and AP-PCR profiles based on a random 10-mer primer (5'-CAA TCG CCG T-3'). The majority of seawater isolates (90%) were Campylobacter coli , and three strains were Camp. jejuni. Southern blot hybridization analysis showed differences between the strains, and in a numerical analysis three main clusters were formed at the 45% similarity level, that corresponded to Camp. jejuni subsp. jejuni, Camp. coli , and a combination of Camp. coli and Camp. jejuni subsp. doylei. AP-PCR profiles also differentiated between the species but were less discriminatory than ribotyping because six strains (17%) could not be typed by this method. Numerical analysis gave four main clusters at the 45% similarity level, corresponding to Camp. jejuni subsp. jejuni, Camp. coli (two clusters) and Camp. lari. The study shows that strains within each species are diverse genomically. Both molecular methods were highly discriminatory, although some strains with identical ribotypes could be distinguished by AP-PCR, and they are valuable new alternatives to traditional typing in epidemiological studies of environmental campylobacters.     

Intermittent tracheostomy in sheep

A modified tracheostomy was designed for respiratory function studies in sheep. Our primary objective was to prevent development of severe tracheal stenosis, which was observed with procedures designed for canine tracheostomy. Surgery consisted of an intercartilaginous tracheal slit, with apposition of tracheal mucosa to skin. This procedure produced no significant stenosis at postmortem evaluation in 19 sheep. In contrast, 5 of 20 sheep with excision of the ventral 1/3 of the third and fourth tracheal rings, had severe tracheal stenosis.     

Longitudinal molecular epidemiological study of thermophilic campylobacters on one conventional broiler chicken farm

Improved understanding of the ecology and epidemiology of Campylobacter in the poultry farm environment is key to developing appropriate farm-based strategies for preventing flock colonization. The sources of Campylobacter causing broiler flock colonization were investigated on one poultry farm and its environment, from which samples were obtained on three occasions during each of 15 crop cycles. The farm was adjacent to a dairy farm, with which there was a shared concreted area and secondary entrance. There was considerable variation in the Campylobacter status of flocks at the various sampling times, at median ages of 20, 26, and 35 days, with 3 of the 15 flocks remaining negative at slaughter. Campylobacters were recoverable from various locations around the farm, even while the flock was Campylobacter negative, but the degree of environmental contamination increased substantially once the flock was positive. Molecular typing showed that strains from house surroundings and the dairy farm were similar to those subsequently detected in the flock and that several strains intermittently persisted through multiple crop cycles. The longitudinal nature of the study suggested that bovine fecal Campylobacter strains, initially recovered from the dairy yard, may subsequently colonize poultry. One such strain, despite being repeatedly recovered from the dairy areas, failed to colonize the concomitant flock during later crop cycles. The possibility of host adaptation of this strain was investigated with 16-day-old chickens experimentally exposed to this strain naturally present in, or spiked into, bovine feces. Although the birds became colonized by this infection model, the strain may preferentially infect cattle. The presence of Campylobacter genotypes in the external environment of the poultry farm, prior to their detection in broiler chickens, confirms the horizontal transmission of these bacteria into the flock and highlights the risk from multispecies farms.     

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.     

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.     

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 occurrence of thermophilic cellulolytic fungi in a pasture land soil

Previous studies on the isolation of thermophilic fungi from soil have indicated their relative rarity. In the present investigation, however, many of the confirmed thermophilic fungi have been isolated from one pasture land, using soil enrichment methods and selective isolation media. These results point towards a wide distribution of thermophiles in soil and the cellulolytic activity of many of them.     

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.     

Concentration and prevalence of Escherichia coli O157 in sheep faeces at pasture in Scotland

AIMS: To study the presence, numbers and virulence profiles of Escherichia coli O157 in sheep faeces and validate the microbiological methods used to attain these data. METHODS AND RESULTS: Flock level prevalence was found to be 40% (six from 15) and 6.5% of faecal samples tested were found to be positive. Two farms gave samples defined as high shedding (>10(4) CFU g(-1)), one of which comprised 91% positive samples with 13/33 at the high shedding level. CONCLUSIONS: These data confirmed that sheep are an important reservoir of E. coli O157. SIGNIFICANCE AND IMPACT OF THE STUDY: Sheep play a significant role in the maintenance and dispersal of E. coli O157 in the farming environment and are an important source of human infection.     

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.