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a Ecology and Health Research Centre, Department of Public Health, Wellington School of Medicine, PO Box 7343, Wellington South, New Zealand
b Freshwater Ecology Group, Department of Zoology, University of Otago, PO Box 56, Dunedin, New Zealand
c Spatial Information Research Centre, Department of Information Science, University of Otago, PO Box 56, Dunedin, New Zealand
* Corresponding author (reyles{at}wnmeds.ac.nz).
Received for publication November 6, 2002.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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Abbreviations: MPN, most probable number
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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Thermophilic Campylobacter are an important cause of gastrointestinal illness, accounting for approximately 10% of all diarrhea worldwide (Benenson, 1995). New Zealand has a high incidence rate of campylobacteriosis compared with other countries of similar socio–economic status (Hearnden et al., 2003). Notified cases of campylobacteriosis for the whole of New Zealand are generally highest in spring and summer, and decrease during winter. However, seasonal patterns of disease can vary among regions; for example, some areas exhibit high rates of disease in both summer and winter. Hearnden et al. (2003) concluded that spatiotemporal variation in disease rates in New Zealand indicates that campylobacteriosis has a complex ecology that requires further investigation.
Campylobacter are gram-negative, spiral-shaped bacteria that have been recognized as human pathogens since the 1970s (Skirrow, 1977). The optimum growth temperature for thermophilic Campylobacter is 42°C (Van Vliet and Ketley, 2001) and it requires an avian or mammalian host to replicate. Common reservoirs of Campylobacter include domestic stock (e.g., sheep, cattle, pigs, and poultry), wildlife (e.g., ducks, seagulls), and humans. Transmission of the bacteria to humans can occur through consumption of fecally contaminated food or water, contact with animals, and person-to-person contact (Blaser et al., 1983).
Campylobacter can survive in water for between 8 d (Buswell et al., 1998) and 120 d (Rollins and Colwell, 1986), but is not capable of multiplying in water. Detection of Campylobacter in surface waters is therefore an indication of recent fecal contamination (Jones, 2001). Since the first reported isolation of Campylobacter from freshwater by Knill et al. (1978), numerous studies have investigated the occurrence of Campylobacter in surface waters. Bolton et al. (1987) performed a survey of Campylobacter along a UK river system and detected serotypes of Campylobacter that were also common in humans, indicating that river water was a potential source of human infection in that area. Another study of Campylobacter in surface waters in the USA reported higher isolation rates in autumn and winter, and did not find a significant correlation between Campylobacter and microbiological or physical water quality parameters (Carter et al., 1987). Brennhovd et al. (1992) also reported increased recovery of Campylobacter in autumn, but in contrast to Carter et al. (1987), did detect a significant relationship between Campylobacter and three common microbiological indicators. In a more recent UK study, Obiri-Danso and Jones (1999b) measured Campylobacter and indicator levels at two freshwater bathing sites, and identified treated sewage effluent, agricultural runoff, streams, and waterfowl as likely sources of fecal contamination. Jones et al. (1990) found a negative correlation between the occurrence of Campylobacter in surface waters and the incidence of campylobacteriosis in Lancaster, UK.
Buswell et al. (1998) investigated the survival of Campylobacter in water using microcosm experiments, and found that in general, survival was increased at lower temperature and oxygenation levels. Obiri-Danso et al. (2001) reported that Campylobacter in sea and river water samples survived longer at lower temperatures (4°C) than at higher temperatures (37°C). Artificial sunlight (UVB) had a significant effect on survival of Campylobacter, resulting in nonculturability within 30 to 60 min, depending on the species tested. The ability of Campylobacter to assume a viable but nonculturable (VBNC) form under adverse environmental conditions has been identified (Rollins and Colwell, 1986). Koenraad et al. (1997) reviewed the epidemiology of Campylobacter in water-related environments, and argued that direct monitoring of Campylobacter in recreational waters is needed because of a lack of correlation with indicator organisms, and the low infectious dose of Campylobacter. Thomas et al. (1999a) reviewed the relative influences of various environmental factors on the survival of Campylobacter in aquatic systems. They concluded that survival was optimal at low temperatures, and was adversely affected at temperatures between 16 and 22°C. Survival did not appear to be affected by nutrient levels, but there was some evidence of reduced survival in the presence of autochthonous microflora, suggesting that Campylobacter may be subject to biotic interactions in the aquatic environment. In his review of Campylobacter in water, sewage, and the environment, Jones (2001) emphasized the seasonal variation in Campylobacter levels in surface waters, and also commented on a qualitative, but not quantitative association between Campylobacter and indicator organisms. All these authors emphasized the significant but poorly understood role that the aquatic environment plays in the epidemiology of campylobacteriosis.
Previous investigations into the occurrence of Campylobacter in surface waters in New Zealand include a study of different sources of recreational and drinking waters by Savill et al. (2001). They found Campylobacter to be more prevalent in river water samples compared with shallow ground water, rainwater, and drinking water. A national study investigated the relationship between pathogens and indicators at 25 freshwater bathing sites throughout New Zealand and reported that Campylobacter levels were highest in catchments predominantly affected by either birds or sheep farming, and lowest in catchments affected by urban development (D. Till, personal communication, 2000), a finding similar to that of a study in Finland (Martikainen et al., 1990). Levels were highest in early autumn (April) and were correlated with turbidity and E. coli, but only in catchments with high Campylobacter levels.
Although there have been no New Zealand studies that have focused on a single river catchment, there have been several performed in other parts of the world (Bolton et al., 1987; Stelzer et al., 1989; Jones et al., 1990). By focusing on a single river catchment, this study aimed to investigate smaller-scale spatial variation in Campylobacter levels in freshwaters. As previously stated, spatiotemporal variation in campylobacteriosis disease rates is evident in New Zealand, suggesting that survival of Campylobacter in the environment may also vary by geographic region. Therefore, it may not be valid to compare Campylobacter levels in relation to land use by using sites from different regions. We argue that it is more appropriate to investigate the influence of environmental variables such as land use at the ecologically relevant scale of a single river catchment.
The Taieri River is an important recreational and drinking water resource for New Zealand's fifth largest city (Dunedin). It is also the subject of a considerable amount of research and monitoring attention, including the Taieri Catchment and Community Health Project, a collaborative project investigating the relationship between catchment management, freshwater ecosystems, and public health (M. Parkes, personal communication, 2002). The aim of the work presented in this paper was to examine the spatial and temporal fluctuations in Campylobacter contamination that underlie the public health risk from recreational use of the Taieri River. Specifically, we wished to identify
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MATERIALS AND METHODS |
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Campylobacter Analysis
Enumeration of thermophilic Campylobacter species was performed using a 1 x 500 mL, 3 x 100 mL, and 3 x 10 mL MPN format. This method has been used previously in New Zealand for recreational water samples (Savill et al., 2001). Each sample volume was filtered through a 0.45-µm membrane filter, which was then added to 25 mL of Preston Enrichment (PE) broth and incubated at 42°C for 48 h. Secondary selective enrichment was performed by streaking a loopful of growth onto modified CCDA-Preston agar plates and incubating under microaerophilic conditions at 42°C for a further 48 h. Presumptive Campylobacter colonies were confirmed microscopically and each plate was given a positive or negative score. A MPN table was used to estimate the concentration of Campylobacter in each sample, from a range of 0 to >11 MPN per 100 mL. This method only detects culturable Campylobacter cells and not viable but nonculturable cells.
Notification Data
In New Zealand, cases of campylobacteriosis are notifiable to the local Medical Officer of Health under the 1956 Health Act. Notification data were obtained from the local public health service (Public Health South) for the period June 2000–June 2001. The number of cases of campylobacteriosis occurring in the Taieri River catchment and nearby Dunedin City was calculated for each fortnight, beginning on the day of water sampling. Cases were included from this geographic area because they represent the population that is most likely to use the three bathing sites for contact recreation.
Statistical Analysis
Statistical analyses were performed using Systat Version 9 (SPSS, 1998). A Friedman test (the nonparametric equivalent of a repeated measures test) was used to test for significant differences among the 10 sampling sites. A paired sample sign test was used to test for significant differences in the medians of pairs of adjacent sites. Box plots were also produced in Systat (Campylobacter MPN results of >11 MPN per 100 mL were coded as 15 MPN per 100 mL for the purposes of visualization). Spearman's rank correlation coefficients (rs) between Campylobacter MPN and environmental variables (turbidity, salinity, temperature, and time of day) were computed. The Spearman rank correlation coefficient is a nonparametric equivalent of the Pearson's correlation coefficient, and can be used to estimate the association between two variables when normality of the data cannot be assumed.
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RESULTS |
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Temporal Variation in Campylobacter Concentrations in the River
Seasonal variation in Campylobacter concentrations was evident in the lower Taieri River (Fig. 2)
. The majority of sites (1–8) showed broadly similar seasonal patterns, with higher concentrations detected in summer (December–February). Seasonal variation was least evident at Sites 9 and 10, which generally showed low concentrations of Campylobacter. Seasonal variation was also evident when all sites were analyzed together (Table 2), with highest median Campylobacter levels in summer. The lowest median levels were observed in spring (September–November) and autumn (March–May). Autumn had the lowest proportion of positive samples (55%), compared with the other three seasons, which ranged from 84 to 91% positive. During summer, nine samples measuring greater than 11 MPN/100 mL were recorded. No samples were found to have concentrations greater than 11 MPN/100 mL during spring.
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Temporal Variation in Notified Cases of Campylobacteriosis
Notified cases of campylobacteriosis in the human population of the Taieri River catchment and nearby Dunedin City are shown in Fig. 4d. The pattern of disease for this region appears to be a gradual increase from winter to summer, with some random variation between fortnights. A comparison is made with Campylobacter levels at Site 2, because this site is the main recreational bathing site, and also showed the most temporal variation, compared with the other two bathing sites (9 and 10). During mid-summer (January) levels of Campylobacter at Site 2 were relatively high compared with other times of the year, and coincided with the two highest peaks of cases. There is a drop-off in cases during late summer and early autumn, which coincides with relatively low levels of Campylobacter at Site 2.
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DISCUSSION |
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After Campylobacter enter aquatic environments, they are subject to adverse biotic and abiotic conditions, including predation by protozoa, unfavorable temperatures, and lethal UV light levels (Thomas et al., 1999a). Campylobacter will therefore either settle out into the sediments or degenerate to the transitory viable but nonculturable form before cell death (Thomas et al., 2002). The spatial and temporal fluctuations in levels of culturable Campylobacter in the lower Taieri River will now be addressed in relation to the inputs and outputs of Campylobacter described above.
Temporal Variation in Campylobacter Concentration in the River
Overseas studies (Bolton et al., 1987; Carter et al., 1987; Jones et al., 1990; Brennhovd et al., 1992) have generally found Campylobacter concentration in surface waters to be higher in winter months, when lower water temperature and UV levels are thought to result in increased survival of Campylobacter in the environment (Jones et al., 1990). In addition, rainfall and river flow are generally higher in winter months, resulting in increased transport of pathogens from land stores to surface waters. In contrast, Campylobacter concentrations in the Taieri River were actually slightly higher in summer than in winter, an important observation in light of increases in human recreational exposure during this time. There are a number of possible reasons why Campylobacter levels were higher in summer in this study.
First, stock density is generally higher in summer, following lambing and calving, and this will result in increased fecal stores on land during this time. Minor flood events that occurred in summer (Fig. 4b) may have played an important role in the transfer of fecal material from land to surface waters. In addition, stock may be more likely to access streams and rivers to drink and cool themselves during summer. This may increase bacterial concentrations in the water column through both direct deposition and resuspension from sediments. Hunter et al. (2000) observed high fecal bacteria levels during summer low flow conditions in streams within agricultural land use in the UK and attributed this in part to higher stocking densities in summer months. A recent study in New Zealand found that E. coli concentrations increased by 400% after cattle had crossed a stream (Davies-Colley et al., 2002). The authors attributed this to direct deposition, wash-off of contaminants from legs, and disturbance of sediments by hooves.
Spatial Variation in Campylobacter Concentrations in the River
The variation in Campylobacter levels between different sites on the lower Taieri River reflects inputs from surrounding land and instream losses. Between Sites 1 and 2 (Fig. 3) the river passes through a small gorge, where native and planted forests provide a significant riparian buffer zone, such that inputs from grazing stock are likely to be low. The river is fast flowing through this section, and is therefore likely to be a zone of strong aeration, which would not favor survival of the microaerophilic Campylobacter species. The increase in Campylobacter levels between Sites 3 and 4 may be due to the inflow of a major tributary (Silver Stream) and two agricultural drains in this area. The drains serve surrounding dairy and sheep farms, and are likely to be contributing fecal material to the river and increasing Campylobacter concentrations. A study of the effect of agricultural land use on the river Condor in England found elevated Campylobacter levels downstream of the inflows of three streams subject to agricultural runoff (Jones and Hobbs, 1996).
Between Sites 4 and 7 (Fig. 3), median Campylobacter levels remained high, apart from a slight decrease at Site 7. Although land use surrounding Sites 4 through 7 was predominantly intensive agriculture (dairy and sheep farming), a downstream increase in Campylobacter levels along this reach was not observed. This contrasts with the study on the river Condor, which described an increase in Campylobacter load from upstream to downstream (Jones and Hobbs, 1996). The absence of such a pattern in the lower Taieri River may be due to a system of drainage channels on the lower Taieri Plain, which partly lies below sea level. The system of drains collect in a single drain, known as the "Main Drain," which is in turn pumped into Lake Waipori (Fig. 1). Hence, most runoff from farms on the lower Taieri Plain does not enter the river directly, but passes through Lake Waipori, before entering the Taieri River at its confluence with the Waipori River. The significant increase in median Campylobacter levels seen at Site 8 (directly below the confluence) may be due to the inflow of water from the Main Drain via Lake Waipori.
Median Campylobacter levels decreased significantly between Sites 8 and 9. Below Site 8 the river leaves the Taieri Plain and passes through a small gorge before entering the sea at Taieri Mouth (Site 9). The land cover along this gorge is predominantly forested, and there are likely to be fewer inputs of Campylobacter along this part of the river, because livestock are absent. Campylobacter has been found previously in coastal waters (Jones et al., 1990; Obiri-Danso and Jones, 1999a). A study of three bathing beaches in the UK concluded that nonpoint-source pollution was just as important as point-source (sewage effluent) pollution in contributing to Campylobacter contamination of coastal waters. Weather conditions, time of day, and season also influenced Campylobacter survival in coastal waters (Jones and Obiri-Danso, 1999). The low levels of Campylobacter detected at Site 9 (median = 0.36 MPN/100 mL) compared with other studies of coastal waters (Jones et al., 1990; Obiri-Danso and Jones, 1999a) suggest the coastal environment at Taieri Mouth may be less polluted compared with overseas coastal waters.
Site 10 was situated on Lake Waihola, and exhibited the lowest median Campylobacter levels. Lake Waihola is a shallow (maximum depth = 2 m), eutrophic, tidal lake with inflow of semisaline water from the Taieri River during high tide (Otago Regional Council, 1999). The low levels detected at this site may be due to Campylobacter cells settling out into the sediment at a faster rate than they might in a flowing system such as the river. Also, biotic and abiotic conditions may be less favorable for Campylobacter survival in the lake, due, for example, to predation by protozoa or zooplankton, or increased exposure to UV light. Mawer (1988) sampled ponds and lakes in the UK and detected Campylobacter on 46% of occasions, a result similar to that at Lake Waihola (52%). Brennhovd et al. (1992) detected Campylobacter on 12.5% of sampling occasions in an oligotrophic lake compared with 59% of occasions in a eutrophic (moderately polluted) lake in Norway. In a study of a river system in Germany, Stelzer et al. (1989) were unable to detect Campylobacter downstream of a man-made lake, despite high levels being observed upstream of the lake, suggesting the lake was acting as a sink. Hill and Grimes (1984) failed to detect Campylobacter in a large freshwater lake associated with the Mississippi River, despite the presence of a large migratory waterfowl population.
Environmental and Water Quality Variables and Campylobacter
During summer, river flows are generally lower, such that net inputs of Campylobacter will have a larger effect on the concentration of Campylobacter in stream water (since there is less water in the system for dilution). An alternative way of assessing the overall load of Campylobacter in freshwater systems is to consider the flux rate (Fig. 4c). Two main peaks in Campylobacter flux were observed, one in winter and one in summer. In terms of the flux of Campylobacter that could potentially reach the coastal environment, both winter and summer are associated with high loads. Consideration of the flux of Campylobacter reaching coastal areas is important in New Zealand, where shellfish gathering for consumption is popular. Shellfish have the ability to concentrate pollutants, including bacteria from seawater. Teunis et al. (1997) observed a peak in Campylobacter levels in shellfish in winter, and concluded that there was a significant risk of infection associated with consumption of raw shellfish from Dutch waters. Wilson and Moore (1996) also detected higher levels of Campylobacter in shellfish in autumn and winter compared with summer months, and detected Campylobacter when fecal coliforms and E. coli were absent.
The lack of a significant correlation between turbidity and Campylobacter in the Taieri Catchment is unusual. Turbidity was found to be an important environmental variable associated with Campylobacter contamination of freshwaters in a study of 25 bathing sites around New Zealand (D. Till, personal communication, 2002), and has been related to bacterial measures of water quality such as E. coli (Nagels et al., 2002). There may be occasions when Campylobacter is not detected in freshwaters, even during periods of high turbidity, due to a lack of carriage by stock. Consequently, turbidity and Campylobacter were decoupled in our study, a finding that highlights the importance of direct testing of Campylobacter instead of using surrogate measures like turbidity. The absence of an association between salinity and Campylobacter levels at Sites 8 through 10 is not surprising, as previous studies have found Campylobacter in coastal waters at similar levels to river waters (Jones et al., 1990; Obiri-Danso and Jones, 1999a).
Thomas et al. (1999b) investigated the effect of temperature on the survival of Campylobacter in water microcosms, and found survival was highest at temperatures of around 5°C. Survival was significantly reduced at temperatures of 15°C and above. In natural aquatic systems, the observation of higher levels of Campylobacter in winter months (Bolton et al., 1987; Carter et al., 1987; Jones et al., 1990; Brennhovd et al., 1992) has been attributed to increased survival at low temperatures. In the Taieri Catchment, Campylobacter concentrations were highest during late summer when water temperatures were warmest (10–18°C), possibly due to an increase in direct fecal contamination of waterways and minimum dilution due to low river flow. This observation may distort any correlation between temperature and Campylobacter in this study.
A weak negative correlation between Campylobacter levels and time of sampling was observed, suggesting that Campylobacter levels were lower in the afternoon compared with the morning. Diurnal variation in Campylobacter levels has been observed previously (Obiri-Danso and Jones, 1999a), and is thought to be due to the negative effect of UV light on the survival of Campylobacter.
Implications for Public Health
The enumeration method chosen for this study gave a range of 0 to >11 MPN/100 mL. Black et al. (1988) reported a 50% probability of infection in healthy adult volunteers challenged with 800 Campylobacter organisms. On the majority of sampling occasions (93%), Campylobacter concentrations were at relatively low levels (<11 MPN/100 mL) and would be unlikely to pose a health risk to healthy adult bathers. However, individuals may show marked variation in their responses to ingestion of Campylobacter, and people with smaller body sizes (e.g., children) could be at increased risk of infection at lower Campylobacter concentrations. Only 19 out of 270 (7%) samples were recorded as having Campylobacter concentrations of greater than 11 MPN/100 mL. The health risk to bathers on these occasions could be significantly greater, particularly if the actual concentration was several orders of magnitude higher.
Campylobacteriosis is a food and waterborne disease, and for the majority of notified cases it is not possible to determine a source of infection. The cases shown in Fig. 4d represent all cases of campylobacteriosis, and include those arising from both food and waterborne transmission. The epidemiology of campylobacteriosis in New Zealand is characterized by a summer peak in disease incidence, commonly attributed to an increase in foodborne cases associated with undercooked meat from barbeque cooking. In addition, people are more likely to participate in contact recreation in freshwaters during summer months. The Taieri River may be a source of waterborne cases of campylobacteriosis in the Taieri Catchment and Dunedin City, through contact recreation. Although some of the drinking water supply for Dunedin City comes from upper parts of the Taieri Catchment, this water is chlorinated before distribution, and therefore is unlikely to be a source of Campylobacter.
Although there was some correspondence between Campylobacter levels at Site 2 and cases of campylobacteriosis in the community, given the inherent variability in both variables between fortnights, it is difficult to draw any firm conclusions from the patterns observed. The sharp drop-off in cases in late summer is unusual, especially because this is the time of year when foodborne cases are expected to be high. The authors are not aware of any special food safety campaigns run during that time that may have led to a decrease in disease incidence.
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CONCLUSIONS |
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ACKNOWLEDGMENTS |
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REFERENCES |
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