Environmental aspects of Campylobacter infections

Zentralbl. Mikrobiol. 146 (1991), 3-15 Gustav Fischer Verlag lena

[Forschungsinstitut fiir Hygiene und Mikrobiologie, Bad Elster, BRD]

Review

Environmental Aspects of Campylobacter Infections W.

STELZER, J. JACOB

and E.

SCHULZE

With 2 Figures

Key words: Campylobacter. detection, drinking water, surface water, sewage, survival

Summary Epidemiological data indicate high incidence of campylobacteriosis. Improperly prepared poultry-products, unpasteurized milk as well as non-chlorinated drinking water were shown to be the main vehicles of Campylobacter transmission to man. There is a lack of knowledge concerning the role of various environments in transmission of Campylobacter. The review summarizes the present knowledge about occurrence and survival of Campytobacters in various environments (sewage, sludge, surface water, drinking water). In conclusion risk assessment for public health is discussed.

Zusammenfassung Die epidemiologischen Daten zeigen eine Zunahme der Campylobacter-Infektionen. Als wichtigste Obertragungsmedien wurden Gefliigelfleischprodukte, nicht pasteurisierte Milch und ungechlortes Trinkwasser erkannt. Zur Rolle der Umwelt fiir die Verbreitung von Campylobacter gibt es zur Zeit nur liickenhafte Erkenntnisse. Die vorliegende Obersicht fal3t den gegenwiirtigen Wissensstand zum Vorkommen und Oberleben von Campylobacter in verschiedenen Umweltmedien (Abwasser, Kliirschlamm, Oberfliichengewiisser, Trinkwasser) zusammen. Mit den Daten wird das hygienische Risiko fur die menschliche Gesundheit diskutiert. I. 2. 3.

3.1. 4. 4.1. 4.2. 4.3. 4.4. 5. 6. 7. 8.

Introduction........ ... . . . . . . . . . . Epidemiological aspects . . . . . . . Isolation of Campytobacter from the environment. Basic principles Isolation of Campylobacter from water habitats Environmental sources of Campytobacter Drinking water . . Surface water Waste water and sewage sludge Slurry and soil . . . . . . . . Typing of environmental strains of Campylobacter Correlation between standard indicator bacteria and Campylobacter Survival of Campylobacter in the environment Conclusions and recommendations .

3 4

5 7 8 8 8 9

9 10 10 12 12

1. Introduction Campylobacter bacteria have been known since the beginning of this century. In 1913 and STOCKMAN reported on infectious abortion in cattle. SMITH and TAYLOR (1919) called the causative bacteria Vibrio fetus. JONES and LITTLE (1931) demonstrated that the McFADYEAN

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infectious abortion in cattle can be reproduced by pure cultures of Vibrio fetus. LEVY (1946) published one of the first reports on Vibrio-like organisms which are associated with human enteritis, however, without a further strain characterization. In the end of the 50's KING (1957) compared microaerophilic Vibrio-like organisms from patients with enterocolitis. One group of the bacterial isolates showed a striking similarity to Vibrio fetus but the other strains described as "related vibrios" were characterized by a higher optimal growth temperature. The "related vibrios" were preferably isolated in blood cultures from enteritis-patients (KING 1957). It was not until 1963 that the genus Campylobacter was introduced by SEBALD and VERON (1963). Nine years later, DEKEYSER et al. (1972) reported on Campylobacter isolates from stools of patients with enterocolitis. The same technique was utilized by BUTZLER et al. (1973) showing a high frequency of Campylobacter in children, as well as adults with acute enterocolitis (KAIJSER 1988). However, only when SKIRROW published his simple culture technique the research activity on Campylobacter infections exploded (SKIRROW 1977). In recent years considerable attention has been attached to campylobacters, since they represent an important cause of enteritis, gastritis and other human diseases (Table 1). At present, 12 species with medical importance for man are described (Table 1). As to diarrhoeal diseases, epidemiological data show a similar incidence of campylobacteriosis as reported for enteric infections caused by the classic pathogens (KAIJSER 1988). Most cases of Campylobacter enteritis are caused by C. jejuni, whereas the other species play only a minor role (Table 1). Improperly prepared poultry products like fresh chickens and unpasteurized milk as well as non-chlorinated drinking water were shown to be the main vehicles of Campylobacter transmission to man. Several large water-borne outbreaks, in which hundreds of persons were involved have been reported. Alltogether, there is still a considerable lack of knowledge concerning the assessment of the role of various environments: as sewage, sewage sludge, polluted surface waters, slurry and soil. This report gives a summary on the present knowledge in this field.

2. Epidemiological Aspects As mentioned above, epidemiological data indicate a similar or even higher incidence of campylobacteriosis as reported for enteric infections caused by the classic pathogens Salmonella and Shigella. In most of the epidemiological studies less than 10% of the patients seaking medical care for diarrhoea had Campylobacter infection (KAIJSER 1988). Wild life birds and poultry represent the most important reservoirs for campylobacters. Thermophilic campylobacters were detected in amounts of up to 10,000,000 per g faeces of birds (WHO 1986). The intestinal tract of domestic animals such as pigs, cattle, dogs and cats has also been recognized as a reservoir of campylobacters. Therefore, meat, in particular poultry products, represent an important source of Campylobacter infections. A general surface distribution of C. jejuni on fresh chicken carcasses was found (HOOD et al. 1988). The counts of Campylobaeter jejuni per fresh chicken varied widely ranging from 1,000 to 10,000,000. Poultry liver samples contained 10 to 100 campylobacters/g (BREUER 1986). In contrast to the high surface contamination of fresh chicken carcasses total absence or low carriage of Campylobaeter jejuni was found in the frozen chickens examined (HOOD et al. 1988). Approximately 50% of the serotypes of human origin were found among chicken isolates only (HOOD .et al. 1988). Increasing consumption of fresh chicken meat is considered to be a main reason of Campy/obaeter infections in Great Britain (HOOD et al. 1988). It has been shown that newly hatched chickens are free from Campylobaeter (LINDBLOM et al. 1986). But campylobacters will be spread repeatedly between the animals, if one animal of the group is infected (KAISER 1988). Unpasteurized milk represents a further common source of human infection. Raw milk was implicated in 61 % of Campylobacter enteritis outbreaks which were reported to the Center for

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5

Disease Control between 1980 and 1982 (FINCH and BLAKE 1985). Milk may be contaminated with faeces during milking or by secretion of organisms into milk by cows with mastitis (BIRKHEAD et al. 1988; FINCH and BLAKE 1985). BIRKHEAD et al. (1988) reported the fIrst milkborne outbreak in which the same two serotypes of Campylobaeter jejuni were isolated both, from patients and from implicated dairy cows. In Canada thermophilic campylobacters were isolated from 17.5 % of pork, 2.6 % of beef, 4.1 % of veal carcasses, 73.7% and 38.2 % of turkey and chicken carcasses. Similar results from other countries are known. Drinking-water is known as a further transmission route of Campylobaeter infection documented by several large water-borne outbreaks in the past decade. The low infection dosis of Campylobaeter demonstrated from contaminated milk (ROBINSON 1981) is one fact explaining waterborne campylobacteriosis. It was indicated that surface water obviously was the most probable source of infection (GONDROSEN et al. 1985). The cause of another important human disease, gastritis and peptic ulcer, is C. pylori (MARSHALL and WARREN 1984). There is a possibility that the faecal-oral-transmission of C. pylori 'really takes place (GRAHAM et al. 1989). However, at present no data are available on the occurrence, distribution and survival of C. pylori in the environment.

3. Isolation of Campylobacter from the environment. Basic principles In environmental studies microbiologists face the problem to detect a few campylobacters within a huge and diverse bacterial flora. Additional diffIculties emerge. from the necessity to recover environmentally stressed and non-culturable campylobacters. Therefore, extremly sensitive isolation methods are required. From this point of view, direct plating of environmental samples on solid selective media is less successful than enrichment procedures (ARIMI et al. 1988; BOLTON et al. 1987). Additionally, several other major factors influence the isolation of campylobacters. A pre-enrichment in non-selective media is recommended for injured campylobacters (ARIMI et al. 1988; HUMPHREY 1986, 1989). Damaged cells of Campylobaeter are signifIcantly more sensitive to selective agents like antibiotics. Additionally, the sublethal injury in Campylobaeter is an inability to grow at higher incubation temperatures, in this case at 43 DC (HUMPHREY 1989). Sub-lethally damaged cells are able to effect repair at 37 DC, and pre-enrichment at this temperature was found to increase the isolation of C. jejuni from water significantly (HUMPHREY 1989). However, the pre-enrichment is problematic in non-selective media without antibiotics due to over-growth by other organisms. From this point of view, a pre-enrichment is recommended in selective broth at 37 DC for 4 h (HUMPHREY 1989). We found that the preincubation of non-inoculated enrichment broth, when performed in a microaerophilic environment at 37 DC for at least 24 h, facilitates the isolation of campylobacters from water habitats (STELZER 1988). One of the fundamental problems is that the culturability of Campylobaeter declines rapidly in the environment. The data indicate clearly, that non-culturable campylobacters increase logarithmically in water microcosmes (ROLLINS and COLWELL 1986). Obviously, most of the nonculturable campylobacters are living organisms and survive in the environment, undetectable by routine laboratory methods for cultivation. Extrapolating these findings to the natural environment, it can be concluded that the present methods used to detect campylobacters do not provide adequate quantifIcation (ROLLINS and COLWELL 1986). At present proper conditions to transform campylobacters from the viable but non-culturable state to the culturable state are not yet available. Preliminary results show that animal passage affects this revival (ROLLINS and COLWELL 1986). The non-culturable state of campylobacters is likely corroborated in pointsource outbreaks of campylobacteriosis in which no organisms can be isolated from the suspected transmission vehicles (ROLLINS and COLWELL 1986).

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Another fundamental methodological problem concerns the fact, that the Campylobaeter species of human enteritis show considerable differences in the sensitivity to the antibiotics applied in selective media. The most commonly used antibiotics of growth media are vancomycin, trimthoprim, and polymyxin B. In environmental studies the isolation rate of campylobacters was improved by antibiotica supplements involving rifampicin and cephalothin (STELZER 1988). Often, the advantage of a high selective medium is accompanied by the isolation of a narrow spectrum of Campylobaeter species. For example, C. upsaliensis is sensitive to cephalothin and can not be isolated by media supplemented with this antibiotic (LASTOVICA et al. 1989). On the other hand, polymyxin B supplemented in the most commercial media influences the recovery rate of C. eoli (Ng et al. 1988). Indeed, the history of Campylobaeter research is in practice the history of the further development of the antibiotic composition of the growth media. Most of the Campylobaeter isolation techniques involve a variety of selective media working at an incubation temperature of 42-43 °C. Therefore, the isolated strains are referred to as thennophilic campylobacters (BOLTON et al. 1988). However, scientists in the U.S.A. also isolated strains of Campylobaeter being organisms which exclusively grow at 37°C. Furthennore, these unusual campylobacters may also be sensitive to some of the antimicrobial agents incorporated into most of the conventional Campylobaeter selective media (BOLTON et al. 1987). With regard to the incubation temperature a comparison between incubation at 37°C and 43 °C showed on average a lO-fold lower count for incubation at 37°C (HOELLER 1988). In another study incubation of campylobacter selective broth at 37°C for 48 h followed by selective plating and incubation at 43°C improved significantly the isolation rate of Campylobaeter jejuni from naturally contaminated samples of river water (HUMPHREY and MUSKAT 1989). DEKEYSER et al. (1972) were the first to isolate Campylobaeter jejuni from stool specimens of patients with enterocolitis by a selective technique using membrane filters. Campylobaeter are high motile S-shaped bacteria capable to pass through the pores of membrane filters. The evaluation of 0.45!-lm and 0.65 !-lm pore size membranes showed that more strains of campylobacters were to be isolated under application of larger pore size membranes (BOLTON et al. 1988). It is interesting, that not only the pore size but also the filter material influences the rate of penetration of campylobacters. Cellulose acetate filters provide better results as cellulose nitrate filters (KADAR 1988). Half an hour is sufficient for Campylobaeter to pass the membranes (KADAR 1988). A microaerophilic environment is another important factor for the successful isolation of Campylobaeter. Reliable results are obtained by using commercial gas incubators with a continuous gaserous atmosphere, containing 5 % of O 2 , 10 % of CO2 , and 85 % of N 2. The more simple method by PENNIE et al. (1984) with citric acid for the CO 2 production and ferrum oxidation as O 2 reduction will be useful, if the non-inoculated selective media are pre-incubated in a microaerophilic environment as mentioned above (STELZER 1988). The use of a simple candle jar system is not to be recommended for primary isolation of campylobacters. It has to be noted that a standard method for the isolation of campylobacters from environmental samples is not yet available, although some important methodological principles have been outlined. Generally, for the isolation of campylobacters from environmental samples a step-bystep enrichment in liquid media is most effective. The enrichment step should be perfonned at first at 37°C for 4 h and later on at 42°C. Isolation procedures can be improved by dropping aliquots of the enrichment broth on larger pore size membranes, which then have to be placed face-up onto selective agar (KADAR 1988). The membranes are removed after a short incubation period and the incubation of the plates is continued in a microaerophilic environment (DEKEYSER et al. 1972). Based on these principles we have elaborated a standard method for the isolation of thennophilic campylobacters from water habitats (KADAR 1988; STELZER 1988).

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3.1. Isolation of Campylobacter from water habitats For isolation of the Campylobacters from water, both, selective agar and selective enrichment broth are used. Selective agar consists of 90 ml nutrient agar II) (SIFIN) with 10 ml of sheep blood; 5 !J.g of trimethoprimlml; 2.5 !J.g of polymyxin B/ml; 1O!J.g of vancomycin/ml; l5!J.g of cefalexin/ml; and 5 !J.g of rifampicin/ml. Selective enrichment broth consists of 80 ml nutrient broth 12) (SIFIN), 10 ml thioglycolate broth3) (SIFIN), 10 ml of sheep blood, and the antibiotic supplement as mentioned above. Portions of 10 ml of the selective enrichment broth are pre-incubated in a microaerophilic environment at 37 DC for at least 24 h (STELZER et al. 1988). In general, triplicate water sample portions should be used (surface waters: 1-1,000 ml; waste water 0.0001-10 ml). Volumes of 10 ml and more are passed through membrane filters with a pore size of 0.45 !J.m. The filters are aseptically removed from the filtration unit, rolled, and transferred to a tube containing 10 ml of selective enrichment (broth). Sample volumes of 1 ml and less are transferred directly to the selective enrichment broth. The tubes are incubated in a microaerophilic environment at 42 DC for 24 h. Selective enrichment broth is plated directly or preferably, aliquots (4- 5 drops) are dropped on membrane filters (0.45 !J.m or larger pore sizes) which have to be placed face-up onto selective agar (KADAR 1988). These plates are incubated under microaerophilic conditions at 42 DC for one hour; the membranes are removed and incubation of the plates is continued in the micraerophilic environment at 42 DC for up to 48 h. The number of Campylobacter-positive plates in each group is determined, and the Campylobacter colony count is estimated by using an MPN table .

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Fig. I. Enrichment of C. jejuni and C. coli (______.) from waste water samples and the occurrence of other organisms (yeasts /:;" Pseudomonas spp. A).

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Figures 1 demonstrates the high selectivity of the selective enrichment broth used. In these experiments (STELZER 1988) waste water samples were incubated in the selective enrichment broth for 48 h. During this time aliquots of undiluted and diluted selective enrichment broth were plated on selective agar repeatedly. The results show high Campylobacter counts after an incubation period of 24 h. Other microorganisms, particularly yeasts and Pseudomonas spp. were obtained at concentrations which were at least 1 log lower. I) Nutrient agar I: pancreatic peptone 13.5 gIl, protein hydrolysate 3.5 gil, yeast extract 3.0 gil, NaCI 5 gil, agar 13 gil. 2) Nutrient broth 1: as above without agar. 3) Thioglycolate broth: pancreatic peptone 15 gIl, yeast extract 5.0 gIl, I-cystine 0.5 gil, NaC12.5 gil, thioglycol acid (80%) 0.4 gIl, resacurine 0.001 gIl, agar 0.5 gIl.

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4. Environmentl sources of Campylobacter 4.1. Drinking water Several waterborne outbreaks caused by campylobacters were reported in the past decade (GONDROSEN et al. 1985; MENTZING 1981; PALMER et al. 1985; ROGaL et al. 1983; VOGT et al. 1982; WHO 1986). The numbers for persons having been involved ranged from few to three thousands. Only in two of these outbreaks, campylobacters were isolated from patients and in the implicated water samples. Unchlorinated surface water and faecal contamination of water storage reservoirs caused by wild life birds were found to be the main sources of the dissemination of Campylobacter infections via drinking-water. Generally, improved hygienic precautions should be taken into consideration for drinking-water supply from oligotrophic surface waters or non-polluted rivers, if the water is distributed without chlorination (GONDROSEN et al. 1985; STELZER et al. 1989). As examples, three typically epidemic outbreaks caused by drinking water should be mentioned. The first water-borne outbreak of gastroenteritis associated with C. jejuni was reported by VOGT et al. (1982). Approximately 3,000 people in Bennington (Vermont, U.S.A.) had a diarrhoeal illness associated with drinking of unboiled water from the town water system. Investigation showed that the entire water system was probably contaminated and the source of contamination was a main unfiltrated water source (VOGT et al. 1982). C. jejuni was cultured from patients but environmental samples of water failed to be positive for Campylobacter (VOGT et al. 1982). In another outbreak which occurred in a kibbutz near Jerusalem about 150 out of 512 inhabitants were affected (ROGOL et al. 1983). Strong circumstantial evidence indicated an association between the outbreak of Campylobacter jejuni enteritis and the use of water from an unprotected reservoir, but no bacteriological confirmation was obtained from water samples (ROGOL et al. 1983). SACKS et al. (1986) reported about an outbreak and described the water-borne spread of C. jejuni from a chlorinated deep-well system with an open-top settling tank. The deep-well system had numerous deficiences including an unlicensed operator, a failure of chlorination and an open-top treatment tower. It was suggested, that water systems that are unprotected from contact with birds may become contaminated and there with a source of outbreaks of human campy10bacteriosis. Fecal coliforms were found in water samples, but Campylobacter was not recovered from water.

4.2. Surface waters The content of campylobacters in surface waters has proved to be strongly dependent on rainfalls, water temperature and the presence of water fowl (BOLTON et al. 1987; GONDROSEN et al. 1985; HOELLER 1989; REISINGER et al. 1984; STELZER et al. 1989). In the majority of river water samples tested, less than 10 campylobactersl1 00 ml were detectable (STELZER et al. 1989). CARTER et al. (1987) isolated campylobacters in lower concentrations of 4-2401101 from pond water samples. In river systems 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 highest frequency of isolation and highest counts (10-230 campylobacters/100 ml) were associated with sites adjacent to or downstream of sewage works (BOLTON et al. 1987). Surface water run-off from adjacent farmland increased the counts of campylobacters in the river system subsequent to heavy rainfall (BOLTON et al. 1987). Different concentrations of Campylobacter were found depending upon water temperature during a half-year study of surface waters in the Viennes area (REISINGER et al. 1984). The highest levels of contamination were found at a water temperature of between 5 °C and 10 °C (REISINGER et al. 1984). Similar results were confirmed by KADAR (1988) in the Danube river near Budapest. The consumption of untreated surface water concerns an additional human health risk of campylobacteriosis. AHO et al. (1989) reported about an outbreak associated with the consumption

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of untreated surface water in Finland. In this epidemic 75 of 88 soldiers had gastrointestinal symptoms after consuming untreated surface water. Campylobaeter jejuni, heat-stable serotype 3/43/59, was isolated from men and also twice from surface water source used during the infantry drill. This outbreak shows clearly the importance of treating all surface water used for human consumption even in clear and cold stream waters (AHO et al. 1989).

4.3. Waste water and sewage sludge Recent studies have shown a high pollution of raw sewage with thermophilic campylobacters. The amounts detected ranged between 10 and 100,000/100 ml (ARIMI et al. 1988; HOELLER 1988; STELZER et al. 1988). Sewage treatment processes showed comparable elimination rates for campylobacters and indicator bacteria (colony count, total coliforms) (STELZER et al. 1988). However, the behaviour of campylobacters in sewage treatment processes differs from that of indicator bacteria. The latter are to be eliminated efficiently by the final sedimentation process only, whereas campylobacters are reduced in an efficient way in the course of the activated sludge process, possibly because of its sensitivity to aeration. A recent study (ARIMI et al. 1988) shows that primary sedimentation can remove more than 78 % of the incoming campylobacters. Nevertheless, campylobacters, as well as Salmonella, are able to pass sewage treatment processes. Therefore, sewage treatment plant effluents are considered to be one of the major sources of campylobacters occurring in rivers (ARIMI et al. 1988; BOLTON et al. 1987; STELZER et al. 1988). Another situation was found in a low-rate maturation pond system (STELZER et al. 1988). The raw sewage samples (sewage flow of about 1,100 m3/day) contained an average of 51 campylobactersl 100 ml. About 97,1 % of campylobacters were destroyed by primary treatment (Emscher tanks). The oxidation pond treatment plant effluent did not show campylobacters in any case. The clarification process in a two-stage wastewater treatment plant normally reduces campylobacters by more than 95 %. In comparison with Salmonella contamination, sludge samples show no heavy campylobacter contamination so that the observed reduction can be explained by bacterial die-off or transition to a non-culturable stage (HOELLER 1988). Seasonal variations were low and water temperature had no influence on Campylobaeter counts in sewage systems. [n conclusion, sewage can be heavily contaminated by Campylobaeter. but wastewater treatment process can reduce campylobacter counts considerably (ARIMI et al. 1988; HOELLER 1988; STELZER et al. 1988). The percentage of coccoid, non-typable and nonculture Campy/obaeter is increasing during clarification processes (HOELLER 1988). At present, there are only few data available on Campy/obaeter contamination in sewage sludge. The examination of crude sludge showed mean Campy/obaeter counts of 110 cfu/100 rnl (minimum < 30 cfu/l00 ml and maximum 930,000 cfu/l00 ml) (HOELLER 1988). Activated sludge samples contained a mean concentration of 930 cful 100 ml and in ripe sludge Campylobae.ter concentrations of at maximum 2,400 cfu/l00 ml were found. No Campylobacter could be found in digested conditioned sludge or filter effluent (HOELLER 1988). In a study of a high-rate municipal sewage treatment plant Campylobaeter was found only in raw sewage sludge samples. Campylobacters were isolated from 28.6% of the sewage sludge samples of grit tank and primary settling tank. Campylobacters were never found in digested sludge of a mean sludge age of more than 90 days (unplublished data). Therefore a low risk of the spread of Campy/obaeter in the environment due to agricultural application of digested sewage sludge is assumed. 4.4. Slurry and soil There are no data available on occurrence and survival of campylobacters in slurry and soil. Increasing land application of slurry and sewage sludge, however, might possibly pose hygienic risks with regard to the dissemination of Campylobacter infections. For example, more information is needed on the survival of campylobacters in sludge which is applied to land and the hazards

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that survivors might pose to man and animals (~MI et al. 1988). We can only speculate on the possibility that human beings might acquire Campylabaeter infection from raw crops obtained from agricultural land treated with sewage sludge (ARIMI et al. 1988). Preliminary results show a fast decline of inoculated counts of Campylabaeter in sewage sludge. In this field relevant studies should be promoted.

5. Typing of environmental strains of Campylobacter Until now, detailed knowledge on the epidemiological significance of Campylabaeter species, biotypes, and serotypes isolated from environmental samples is not yet available. C. jejuni was isolated most frequently from sewage and natural water habitats. But in few studies distinctly more C. eali than C. jejuni were isolated from sewage systems as well as sludge samples (HOELLER 1988). It is discussed that the bacteria typed as C. coli in water preferable are not C. eali but stressed C. jejuni that failed to hydrolyse hippurate, the only biochemical reaction to distinguish the two species (HOELLER 1988). Preliminary results of whole-cell protein profiles underline this hypothesis (JACOB 1990). Further reports indicate atypical biochemical reactions of campylobacters isolated from water samples. The discussion on the human health risk of such "environmental" strains is open. With the recent development of improved media and culture techniques several new Campylabaeter species, formerly not associated with human infection, have been reported (Table 1). But there is no information about the occurrence and distribution of the whole spectrum of Campylabaeter species in water habitats so far. The large number of serotypes of C. jejuni and C. eali isolated from water is remarkable. Serotyping confirmed the presence of Campylobacter serotypes known from human infections. Additionally, a considerable number of serotypes which are not common for human infections were isolated (BOLTON et al. 1987; STELZER et al. 1988; STELZER et al. 1989). Furthermore, about one third of the water isolates was not to be classified according to types. Virulence studies permit the conclusion that water strains are less virulent than clinical ones (NEWELL et al. 1985). Consequently, further research activities have to be directed towards this field, in detail epidemiological studies are required.

6. Correlation between standard indicator bacteria and Campylobaeter At present, the only reliable data that are available come from water habitats. In river water and sewage studies a close correlation was found between the number of campylobacters and total coliforms (Fig. 2). ~

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Table 1. Campylobacter and their medical importance for humans. Species

Disease

References

C. fetus ssp. fetus

Systematic illness

BLASER, M. J., RELLER, L. B.: N. Eng. J. Med. 305 (1981),1444-1452

C. fetus ssp. venerealis

Systemic illness

COVER, T. L., BLASER, M. D.: The pathobiology of Campylobacter infections in humans. Ann. Rev. Med. 40 (1989), 269-285

C. jejuni

fever, enteritis

SKIRROW, M. B., BENJAMIN, F.: J. Hyg. 85 (1980), 427-442

appendicitic

MORLET, N., GLANEY, R.: Med. J. Austr. 45 (1986), 56-57

Fisher's syndrom

KOHLER, A., et al.: Eur. Neurol. 28 (1988), 150-151

pancreatitis

DE BOIS, H. M. W., et al.: Brit. Med. J. 298 (1989), 1004

C. coli

enteritis

SKJRROW, M. B., BENJAMIN, J.: J. Hyg. 85 (1980), 427-442

C. sputorum ssp Sputorum

Gingival crevice

Bergey's manual of systemic bacteriology. Vol. I Williams & Wilkins, Baltimore, London 1984, p. 116

C. concisus

gingivitis; periodontitis; periontosis

Williams & Wilkins, Baltimore, London 1984, p. 117

C. pylori

gastritis peptic ulcer

BUCK, G. E., et al.: J. Infect. Dis. 153 (1986), 664-669

C. laridis

enteritis

SIMOR, E. A., WILCOX, L.: J. Clin. Microbiol. 25 (1987), 10-12

C. hyointestinalis

enteritis

EDMONDS, P., et al.: J. Clin. Microbiol. 25 (1987), 185-691

C. cinaedi

bacteriemia

CIMOLAI, N., et al.: J. Clin. Microbiol. 25 (1987),942-943

gastroenteritis

GRAYSON, M. L., et al.: Med. J. Austr. 150 (1989), 214-218

C. fennelliae

bacteriemia

NG, V. L., et al.: J. Clin. Microbiol. 25 (1987), 2008-2009

C. cryoaerophila

enteritis

TEE, W., et al.: J. Clin. Microbiol. 26 (1988), 2469-2473

C. upsaliensis

bacteriemia

LASTOVICA, A. J., et al.: J. Clin. Microbiol. 25 (1987), 657-659

enteritis

STEELE, T. W., et al.: J. Clin. Microbiol. 22 (1989), 71-74

Obviously, in oligotrophic surface waters, as described by CARTER et al. (1987), a close correlation disappears. In contrast to the river water studies, campylobacters were isolated more frequently from ponds and lakes with contents of total coliforms of less than 10Imi (CARTER et al. 1987). We never isolated campylobacters from river water samples of 100 ml of volume, with total coliform contents of less than 10Iml (Fig. 2) (STELZER et al. 1989).

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et al.

7. Survival of CampylQbacter in the environment Poultry products, milk, and water are considered to be the main vehicles for transmission of Campylobacter infection to human beings. Gastric acid is the first potent barrier against ingested campylobacters. BLASER et al. (1980) found that HCl solutions with pH values of 3.0 had an extremely adverse effect on the survival of Campylobacter jejuni although solutions with pH values> 3.6 had virtually no effect on survival. These data give rise for speculations that Campylobacter infections may result from exposition to very large numbers of organisms (BLASER et al. 1980). Another situation is discussed for milk and water. Buffer capacity of milk protects campylobacters against gastric acid and water has a rapid wash-through (BLASER et al. 1980), both representing improved preconditions for a better survival of campylobacters during stomach passage. In stool specimens, survival of Campylobacter jejuni was better at 4°C than at 25°C. At the lower temperature survival of Campylobacter jejuni was detected for up to 3 weeks (BLASER et al. 1980). As mentioned previously campylobacters survive sewage treatment processes. In survival experiments (PICKERT und BOTZENHART 1985) viable Campylobacter jejuni counts declined from the initial value of 1O,OOO,OOO/ml to zero. The experiments were performed in untreated sewage at a temperature of 14-15 °c over a period of 36 h. The survival times of C. jejuni in sewage sludge samples range between some hours and 10 d (unpublished data). Generally, campylobacters - similar to other pathogens - survive better at low temperatures, what suggests that cold water may be a more effective vehicle, if there is a point source of contamination (BLASER et al. 1980). Comparable results were found in drinking-water and river water milieus when kept at different temperatures. At 20°C no viable campylobacters could be demonstrated after more than 2 d. But at a low temperature survival was detected for several weeks (GONDROSEN 1986). The published data indicate that standard chlorination procedures are probably sufficient to prevent the spread of campylobacters along aquaeducts and pipelines. Actually, the known water-borne outbreaks of campylobacteriosis originated from the consumption of unchlorinated or inadequately chlorinated water (GONDROSEN 1986). Turbidity of drinking-water is one of the most important factors of inadquate chlorination, because chlorine has non effect inside of flocks. As an outcome of all survival experiments it should be taken into consideration, that the behaviour of laboratory strains passaged and adapted to artificial substrates may not be identical to that of wild type strains under natural conditions (BLASER et al. 1980). Indeed, PICKERT and BOTZENHART (1985) showed better survival of a well-adapted laboratory strain in different water milieus. Another aspect concerns the limited number of strains tested. Examination of a greater variety of strains and species will be necessary to justify definite conclusions (GONDROSEN 1986). At present the viable, but non-culturab1e stage of Campylobacter is one of the main problems in evaluating the survival of Campylobacter in the natural aquatic environment. We do not exactly know the fractions of dead cells or viable, but non-culturable organisms under environmental conditions. Furthermore, no experiences exist about the human health risks posed by viable, but non-culturable types of Campylobacter.

8. Conclusions and recommendations 1. With regard to Campylobacter infections the role which is played by the environment can not yet be defined exactly. There is a lack of knowledge on numbers and survival of campylobacters in slurry and soil. The available environmental data concern water habitats only. 2. In cold groundwater or non-chlorinated tap water campylobacters can survive for one week or more. However, standard procedures of chlorination will prevent the spread of campy10bacters via drinking-water, if the distributed water has a low turbidity. The consumption of non-

Environmental Aspects

13

chlorinated and inadequately chlorinated surface waters causes a considerable risk for outbreaks of campylobacteriosis. The contamination of drinking-water storage reservoirs by wild life birds has to be regarded as an additional risk element. Therefore, any contamination of drinking-water reservoirs must be prevented. Improved hygienic precautions should be taken into consideration for the supply of drinking-water, even if obtained from high-quality surface water, in case the water is distributed without chlorination. 3. The contamination of surface waters with campylobacters has proved to be higWy dependent on sewage effluents, rainfalls, water temperature and the presence of waterfowl. In the majority of river water samples tested, less than 10 campylobacters/IOO ml were to be detected. There should be hardly any risk for infection posed by bathing and recreational activities performed in surface waters, if the water quality corresponds to the set-up regulations for bathing water (WEBER et al. 1987)., 4. High correlation coefficients indicate a close relationship between the number of campylobacters and that of faecal indicators. 5. In municipal raw sewage 10-100,000 campylobacters/lOO ml were to be found on average. Primary treatment (sedimentation) and the activated sludge process are effective in the reduction of campylobacters. In conventional biological sewage treatment plants a Campylobacter reduction of approximately one log can be found. Stable elimination rates of more than 99% can be reproduced in low-rate oxydation pond systems. In sewage sludge low Campylobacter concentrations were found. No detection of Campylobacter could be demonstrated in digested conditioned sludge or filter effluent. The enrichment of Campylobacter, as known for Salmonella, can be neglected in sewage sludge. In wastewater the occurrence of campylobacters is comparable with the data available for the occurrence of non-typhoid Salmonella (Enteritis-Salmonella). Obviously, a higher health risk results for waterborne campylobacteriosis what can be derived from the lower infection dose of campylobacters which is comparable to that of Salmonella typhi. 6. Sewage effluents represent a major source for the spread of pathogens in the environment. Therefore, the complete treatment of sewage, including primary, secondary, and tertiary treatment steps, must be enforced. Low-rate oxydation pond system are recommended for sewage treatment in rural areas. They also represent a possibility for a further treatment step for the effluents of biological sewage treatment plants. The complete sewage treatment technology improves the protection of the population against biological health hazards, but does not completely prevent the dissemination of pathogens, campylobacters included, in the environment. In particular, the agricultural application of water polluted with sewage represents a main source of the contamination of forage, vegetables and fruits. The possibility of Campylobacter transmission from the sewage and polluted surface waters via agricultural applications to plants, animals and back to the human population via the food chain still has to be elucidated in the near future. A low risk of the spread of Cympylobacter in the environment due to agriultural application of digested sewage sludge is assumed.

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