A two-year study of the distribution of 'thermophilic' campylobacters in human, environmental and food samples from the Reading area with particular reference to toxin production and heat-stable serotype

The incidence of 'thermophilic' campylobacters in foods and environmental samples has been studied over a two-year period. Of 781 environmental samples, 529 (67%) were found to contain campylobacters, and campylobacters were isolated from 835 (39%) of 2116 food samples. Sewage was almost always contaminated with campylobacters (96.6% of samples) and of the food samples both poultry (55.5%) and offal (47.0%) were commonly contaminated. Determination of the heat-stable serotypes of all strains isolated from these sources and of 921 strains isolated from human faeces showed that there was a wide distribution of serotypes in most types of sample. Serotype Pen 2 was the commonest type found in human faeces (18.9%) and it was also commonest in offal (21.3%), beef (40.0%), sewage (17.7%) and was the third commonest type in poultry. A comparison of culture media and conditions for optimal production of both cytotoxic and cytotonic enterotoxins showed that Brucella Broth incubated under microaerobic conditions for 24 h at 42 degrees C was suitable for both toxins. Detection of cytotoxic activity was most sensitive using HeLa cells. The sensitivities of two ELISA systems and a Chinese Hamster Ovary tissue culture assay for detection of cytotonic enterotoxin were comparable. Not all strains isolated from cases of enteritis in human beings produced toxin; 23.1% produced cytotonic enterotoxin and 17.5% produced cytotoxin. There was no correlation between serotype and toxin production. The wide distribution of campylobacters, indistinguishable from those isolated from cases of enteritis in human beings, leads us to conclude that simplistic statements suggesting that one particular type of food is primarily responsible for cases of human disease should not be made.     

Campylobacter enteritis: a "new" disease.

By selective culture campylobacters (C jejuni and C coli) were isolated from the faeces of 57 (7-1%) out of 803 unselected patients with diarrhoea; none were isolated from 194 people who had not got diarrhoea. Specific agglutinins were found in the sera of 31 out of 38 patients with campylobacter enteritis and 10 of them had a rising titre. Half the patients were aged 15 to 44 years, but the incidence was highest in young children. All the patients with campylobacters had a distinctive clinical illness with severe abdominal pain. Campylobacters are a relatively unrecognised cause of acute enteritis, but these findings suggest that they may be a common cause. Spread of infection was observed within 12 out of 29 households, and in these cases children were usually implicated. Several patients were apparently infected from chickens, both live and dressed, and poultry may be the primary source of the organism. In two cases dogs with diarrhoea were found to be infected with strains indistinguishable from their human contacts. Ten patients acquired their infections while travelling abroad.     

Enterotoxin Production by Lecithinase-positive and Lecithinase-negative Clostridium perfringens Isolated from Food Poisoning Outbreaks and other sources

Strains of Clostridium perfringens from a variety of sources were examined for their ability to produce enterotoxin in vitro. Fifty-six of 65 (86%) strains isolated from separate outbreaks of food poisoning were found to be enterotoxigenic, only two of 174 strains from other sources produced enterotoxin. The ability to produce this toxin was not confined to particular serotypes: types frequently encountered as the cause of outbreaks were also isolated as enterotoxin-negative strains from faeces, minced beef and meat carcasses. Loss of toxigenicity was also observed in different serotypes. Five strains of lecithinase-negative Cl. perfringens produced high levels of enterotoxin. Four strains of Clostridium plagarum failed to produce enterotoxin although they were serologically typable with the Cl. perfringens antisera.     

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.     

Incidence of Enterotoxigenic Clostridium perfringens in Healthy Humans in relation to the Enhancement of Enterotoxin Production by Heat Treatment

Enterotoxin production was greatly enhanced in two of five food poisoning strains of Clostridium perfringens subjected to heat treatment prior to incubation in Duncan and Strong sporulation medium. Heating was carried out on three successive cultures of each strain, the optimum temperature for treatment being 85 °C for one strain and 95 °C for another: on each occasion cultures were heated for 20 min. The triple heat treatment procedure was used in testing strains of Cl. perfringens isolated from faeces of healthy human subjects for production of enterotoxin. Eleven of 35 (31%) individuals were found to be carriers of enterotoxigenic strains, the isolates producing more than 0·1 μ/ml of enterotoxin. Six of the 11 enterotoxigenic strains were killed by heating at 95 °C but one isolate produced more enterotoxin following treatment at this temperature than after heating at 75 °C.     

Occurrence of campylobacters in small domestic and laboratory animals

Faeces samples from 156 healthy domestic pets and laboratory animals were examined for campylobacters with both a selective medium (Campylobacter Blood Free Medium; Oxoid, CM739) and selective filtration through a 0.65 μm pore size filter. Campylobacter spp. were isolated from 78 of the samples; filtration was the most effective method. Isolates were characterized by biochemical tests and DNA/DNA hybridization with whole chromosomal DNA from reference strains as probes. Campylobacter upsaliensis was the most common species isolated from cats (45 from 68 samples) whilst Camp. jejuni was more often isolated from dogs (19 from 56 samples). More attention should be paid to pets as a potential source of campylobacters capable of causing diarrhoea in human beings. Procedures other than those involving currently-used selective agents must be employed to efficiently detect all campylobacters.     

An investigation of the prevalence of the toxigenic types of Clostridium perfringens in horses with anterior enteritis: preliminary results

Equine anterior enteritis is an acute syndrome with unknown aetiology, although salmonellosis and infection with Clostridium perfringens have both been suggested as potential causes. The main aim of this preliminary study was to compare the prevalence of toxigenic types of C. perfringens in clinically healthy horses and in horses with anterior enteritis. From horses admitted with colic at Phillip Leverhulme Large Animal Hospital in 1995-1996, samples of gastric reflux, small intestinal contents and faeces were taken for isolation of C. perfringens. Five of those horses were admitted as anterior enteritis cases, of which C. perfringens was isolated in pure culture in all five horses. Two of the anterior enteritis cases from which viable bacterial counts had been performed revealed 10(6) CFU/g faeces C. perfringens. Samples of gastric reflux and small intestinal contents submitted from one of these horses revealed 10(4) CFU/mL and 10(5) CFU/mL respectively. The number of C. perfringens observed in the gastric reflux was considered significant as the total volume removed was 12 L. The counts observed in faeces taken from horses admitted with anterior enteritis, were significantly higher than the <10(2) CFU/g faeces observed in faeces from healthy horses and horse presenting with colic and with other diagnoses. The major toxigenic types of C. perfringens in both healthy and diseased horses are being investigated using the polymerase chain reaction (PCR) to amplify target DNA sequences of the toxin genes. Primers have been designed from the published DNA sequences of the enterotoxin, alpha, beta, epsilon and iota toxin genes. PCR products obtained from NCTC strains of C. perfringens have been cloned and the sequenced, to verify that the amplicon sequence is correct. Initial typing suggests that C. perfringens type A is the predominant toxin type isolated from healthy horses and horses with colic with other diagnoses.C. perfringens strains isolated from horses with anterior enteritis are of type D.     

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.     

Experimental Diarrhoea in Human Volunteers Following Oral Administration of Clostridium perfringens Enterotoxin

Peroral administration of purified enterotoxin to human volunteers provoked diarrhoea and abdominal pain, symptoms identical with those encountered in outbreaks of Clostridium perfringens food poisoning. Eight milligrams of enterotoxin caused diarrhoea in one of two volunteers. All of five subjects given 10 and 12 mg of purified enterotoxin or crude enterotoxin developed the classical symptoms of this food poisoning. Passive haemagglutination anti-enterotoxin titres in serum increased in only 5 of 9 volunteers after exposure to enterotoxin. As such levels of anti-enterotoxin can be detected in normal serum samples, titration of anti-enterotoxin may be of little use in diagnosing Cl. perfringens food poisoning. Enterotoxin was detected in all diarrhoeal faecal specimens, and the enterotoxin level varied from 0·2–16 μg/g. Detection of enterotoxin in diarrhoeal faeces may be the most reliable procedure in diagnosing outbreaks of Cl. perfringens food poisoning.     

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.     

Differences in production of several extracellular virulence factors in clinical and food Aeromonas spp. strains

C. PIN, M.L. MARÍN, D. SELGAS, M.L. GARCÍA, J. TORMO AND C. CASAS. 1995. Production of several extracellular virulence factors (lipase, protease and haemolysin) was compared in 15 Aeromonas spp. isolated from faeces of patients with Aeromonas -associated gastroenteritis and 81 strains isolated from food. Strains from food did not show differences in production of these factors when compared with strains isolated from faeces. However, if strains were considered in relation to autoagglutination (AA) character, the AA⁺ differed from AA^- strains in lipase and protease production. Supernatant fluids of AA⁺ food and human strains showed 2·5-fold more protease production than that observed in AA^- strains. These two characteristics of certain Aeromonas strains could be related with the more virulent capacity.     

A comparison of a new campylobacter selective medium (CAT) with membrane filtration for the isolation of thermophilic campylobacters including Campylobacter upsaliensis

The newly developed CAT campylobacter selective medium employing the blood-free charcoal-based agar containing cefoperazone (8 mg I⁻¹), amphotericin (10 mg I⁻¹) and teicoplanin (4 mg I⁻¹) was compared with the membrane filtration culture technique for isolation of Campylobacter spp. including Camp. upsaliensis. Nine hundred and fifty human, 275 dog and 65 cat faeces (in which modified CCDA medium was also compared) were tested. In addition, the recovery of Camp. upsaliensis from pure cultures and from spiked human faeces was examined after membrane filtration. A 50-fold reduction in recovery after filtration using the 0·65 μm filters and a 150-fold reduction using the 0·45 μm filters was found. Recovery of Camp. upsaliensis from spiked faeces was considerably improved using the CAT medium compared with filtration, especially with the lower concentration of organisms (approx. 10⁴ cfu ml⁻¹). Campylobacter upsaliensis was recovered from 91 specimens of animal faeces, with CCDA recovering 26 isolates (29%), CAT recovering 76 isolates (84%) and membrane filtration (0·65 μm) recovering 82 isolates (90%). CAT selective agar was found to be a suitable medium for the isolation of thermophilic campylobacters including Camp. upsaliensis from faecal samples.     

Identification of unusual Campylobacter-like isolates from poultry products as Helicobacter pullorum

Twenty-six unclassified Campylobacter -like strains previously isolated from 15 chicken carcasses and caecal contents, together with two more strains isolated from chicken faeces on a different occasion, were identified as Helicobacter pullorum using various phenotypic identification methods. API Campy identification kits and a 16-test identification scheme developed for campylobacters failed to identify these bacteria, or identified them as Campylobacter spp. Eighteen strains (including the two isolated on a different occasion) were chosen for examination using a more comprehensive probabilistic identification scheme. Using this method, 14 of the 18 strains were identified as H. pullorum with ID scores >95% ; two strains were also identified as H. pullorum with lower ID scores. Of the remaining two strains, one was not identified with this scheme and the other was misidentified to the H. acinonyx pylori complex. Whole cell protein profiling by SDS-PAGE confirmed the identity of these isolates as H. pullorum , affirming the value of a polyphasic approach for accurately identifying campylobacteria. The comparatively high prevalence of H. pullorum in poultry determined in this study (60%) suggests that routine isolation and identification methods should be amended to enable a thorough evaluation of its role in human gastroenteritis and avian hepatitis. Some phenotypic characters useful in identifying poultry campylobacteria are presented which could be utilized, along with other technique(s), for improved differentiation of the campylobacteria that are found in poultry.     

Quantitative isolation efficiency of O26, O103, O111, O145 and O157 STEC serotypes from artificially contaminated food and cattle faeces samples using a new isolation protocol

Aims:  A range of new differential and confirmation plating media for some non-O157 Shiga toxin producing Escherichia coli (STEC) serotypes (O26, O103, O111, O145) and both sorbitol-positive and -negative O157 were evaluated using artificially contaminated samples.
Methods and Results:  Dairy products (raw milk, cheese made from pasteurized milk and raw milk), meat (ground beef, fermented meat) and cattle faeces were artificially contaminated using clinical STEC strains. Isolation efficiency was 100%, 82·3%, 88·5%, 65·9%, 64·3% and 15·8%, respectively, for an inoculum size of ≤100 CFU 25 g⁻¹. The consecutive use of differential and confirmation media limited the incidence of false positive isolates from 0% for raw milk samples, cheese made from pasteurized milk and for fermented meat to 2·1% for cheese made from raw milk, and to 8·9% for ground beef.
Conclusions:  Data presented in this paper indicated that the efficiency of the applied isolation method was dependent on sample-to-sample variation but not on the inoculum size.
Significance and Impact of Study:  Data in this paper indicated that isolation of low levels of non-O157 and sorbitol-positive O157 STEC from food samples is possible.     

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.     

Prevalence and serotypes of Salmonella associated with goats at two Australian abattoirs

Aims:  This study was carried out to determine the prevalence and serotype of Salmonella in goats presented for slaughter.
Methods and Results:  A total of 121 goats were examined for the presence of Salmonella in matching rumen, faecal and carcass samples. Samples were analysed for the presence of Salmonella following the Australian Standard AS 1766.2.5-1991. Salmonella was isolated from 56 (46·3%) faecal samples, 55 (45·5%) rumen samples and 35 (28·9%) carcass samples. The dominant serotypes isolated were Salmonella serotype Saintpaul (31%), Salmonella serotype Typhimurium (13%) and Salmonella serotype Chester (11%).
Conclusions:  Salmonella was isolated from at least one of the three sample sites in 68% of animals. Carcase contamination with faeces, compared with rumen liquor, is a greater hazard for Salmonella contamination of goat carcases. Goat meat is a potential source of Salmonella serovars associated with human disease.
Significance and Impact of the Study:  Goat carcases contaminated with Salmonella during slaughter could be a source of food-borne disease if consumed raw or inadequately cooked, or may be a source of cross-contamination to other foods.     

Improved method for the isolation of Campylobacter jejuni and Campylobacter coli bacteriophages

A centrifugation and filtration method of isolating Campylobacter phages has been developed. Forty-nine Campylobacter phages were isolated from 272 effluent samples of which 42 produced lysis with Campylobacter jejuni strains and seven with C. coli strains. Phages were recovered from pig manure, abattoir effluents, human faeces, sewage and poultry manure. Phages were not isolated from water samples, cattle and sheep faeces or farm pasture soil.     

Shigellosis in Eastern Nigeria: Etiological frequency of serotypes and resistance to antibiotics

Between 1984 and 1986 a total of 1000 faecal samples and rectal swabs were cultured from patients presenting with diarrhoea. Shigellae were isolated from 5·6% of these samples. Shigella flexneri was the commonest isolated (68%), followed by Sh. sonnei (23%). A total of 22·8% of children (0–12 years of age) were positive for shigellae. Twenty-seven (48·2%) strains were sensitive to all the five antibiotics tested (streptomycin, chloramphenicol, tetracycline, ampicillin and sulphonamide). A total of 22 (39·2%) were resistant to three or more antibiotics. Nine (16%) were resistant to all five. The public health significance of the study is discussed.     

Serotyping of Campylobacter jejuni, Campylobacter coli, and Campylobacter laridis from domestic and wild animals.

By using 50 unabsorbed antisera, we were able to serotype 272 (65.7%) of 414 thermotolerant campylobacters from wild and domestic animals, on the basis of heat-stable antigens identified by means of passive hemagglutination. Forty-two serotypes were recognized. The pattern of serotypes detected in the various animal species was compared to human clinical isolates by using the Czekanowski index (proportional similarity index). The highest degree of similarity to the clinical isolates was observed for the poultry isolates, followed by strains from wild birds, flies, and pigs (in order of decreasing similarity). The serotypes recovered most frequently from poultry (LAU 1 and LAU 2) were also most prevalent in Norwegian patients. In contrast, serotype LAU 35/44, the predominant porcine serotype, was never recovered from human clinical specimens. Flies captured in chicken farms and in piggeries harbored serotypes which were also commonly seen in chickens and pigs, respectively. Nine of the strains included in this study could not be ascribed to any defined species. All of these were resistant to nalidixic acid and did not produce H2S.     

The prevalence of campylobacters and arcobacters in broiler chickens

Chicken carcasses from a supermarket and from a poultry abattoir were examined using methods designed to isolate as many strains of campylobacters and related organisms as possible. Strains of arcobacter, but no campylobacters, were isolated from every carcass after enrichment. Campylobacter jejuni subsp. jejuni was isolated from all carcasses examined by direct plating and other Campylobacter -like strains were isolated from nine out of 15 abattoir carcasses by direct plating but not after enrichment. Only the Camp. jejuni subsp. jejuni strains could be identified to species level using a readily available identification scheme and/or a commercial identification kit. Examination of caecal contents from the 15 abattoir poultry yielded Camp. jejuni subsp. jejuni and Campylobacter -like strains from 15 and eight by direct plating, and from six and nine after enrichment, respectively. Four sites in the intestine of the abattoir birds (60 samples) were examined for arcobacters and only one strain was isolated. This indicates that arcobacters are probably not normal inhabitants of the poultry intestine. Poultry is a rich source of other campylobacteria besides the thermophilic Campylobacter spp.