Pathogen Pathways − Contamination of water bodies via artificial drainage
5. Experiment 2: Pathogen contamination of artificial drainage and surface runoff - repeated simulated rainfall events (Objectives 3, 6 & 9)
5.1 Overview
Two plots (Plots C and D) were grazed overnight and the dairy cows were removed on the morning of 7 October 2003. In this experiment Plot C and D are the ‘Grazed plots’ and Plots A and B were left ungrazed (being last grazed 12 days prior to rainfall simulation). Rainfall was simulated once on Plots A and B at the start of the experiment to generate drainage and runoff. For each of the two Grazed plots (C and D), rainfall was simulated three times; immediately after grazing (Day 1), again 2 days after (Day 3) and finally 8 days after grazing (Day 9) (Figure 8). Natural rainfall, drainage and surface runoff also occurred on 13 October 2003.
Figure 8: Natural and simulated rainfall and drainage and runoff volumes for Campylobacter spiked plots (C and D), during the experimental period (25 September–16 October 2003) of this study
5.2 Sample collection
Five fresh dung spots on the grazed plots (C and D) were marked and samples of faeces taken for Campylobacter and E. Coli enumeration. The fresh dung spots were enriched with a cultured Campylobacter spike mixed into fresh faeces collected from the milking shed 10 minutes prior to enriching. Samples of spike-enriched faeces were also taken for Campylobacter and E. Coli enumeration. The spike-enriched faeces (faeces volume 2L) were applied on top of the pats deposited during grazing and, consequently, the population of Campylobacter at the surface of each pat should reflect the added spike. After rainfall simulation, drainage and runoff water samples were collected from the Grazed plots (Plots C and D) and plots grazed 12 days prior (Plots A and B) for enumeration of thermophilic Campylobacter and E. Coli, as well as screening and enumerating Cryptosporidium oocysts and Giardia Cysts. After each simulated rainfall event, dung pats were sampled by breaking their surface water-stable skin and the dung slurry from under the hard skin was sampled. The skin was replaced after sampling.
For the determination of Campylobacter, five drainage water samples (each 1 L) and three surface-runoff water samples (each 1 L) were collected from each plot at each rainfall simulation. The first sample was collected within 10 minutes of the start of either the drainage or surface-runoff event and subsequent samples were collected at intervals of 10-30 minutes, the time interval widening at each subsequent sampling time.
5.3 Results
Giardia and Cryptosporidium oocysts in faeces
A total of 22 faeces specimens were analysed but no Giardia cysts and no Cryptosporidium oocysts were detected in either fresh faeces or samples taken from the aged dung pats. This result indicates than none of the cows that deposited faeces on the runoff areas were shedding cysts at this time. It is not known whether other cows grazing the larger drainage were shedding cysts.
Prevalence of Campylobacter in fresh faeces and as effected by dung pat age
The prevalence of thermotolerant Campylobacter in the fresh dung spots was highly variable (Table 3) confirming earlier observations by Wu (2001) that cow shedding of Campylobacter is highly individualistic.
On Day 1, faeces spiked with laboratory-cultured organisms had concentrations of Campylobacter of 1.1 x 104 MPN/g and 3.3 x 103 MPN/g in the spikes added to plot C and D, respectively (Table 3). This difference probably resulted from the different populations of Campylobacter in the two buckets of faeces collected from the yard to make the spike-enriched faeces. As it turned out, some of the pats deposited during grazing had higher populations than the spike-enriched faeces. The spike-enriched faeces were applied on top of the pats deposited during grazing and, consequently, the population of Campylobacter at the surface of each pat should have reflected the spike-enriched faeces. At the subsequent samplings, on Day 3 and Day 9, Campylobacter concentrations of faeces sampled from just under the water-stable skin of the dung pats were highly variable. There was no consistent effect of dung pat age on Campylobacter concentrations. Campylobacter was able to survive for at least 8 days in the dung pat and in some pats concentrations increased (Table 3).
Figure 9: The change in dung pat surface physical characteristic from freshly deposited dung pat on Day 1 to a dung pat with a water-stable skin on Day 3
Day 1 Plot C
Freshly spiked dung patch No. 3 immediately after rainfall simulation.
Day 3 Plot C
Dung patch No. 3 with a water-stable skin still in place before rainfall simulation.
Day 3 Plot D
Dung patch No. 3 with a water-stable skin still in place after rainfall simulation
Table 3: Campylobacter concentrations (MPN/g wet weight) of spike-enriched faeces and of the freshly deposited cow faeces (in parenthesis), showing change in concentration with time after deposition
| Plot Number |
Dung pat number |
Day 1 Spike-enriched faeces mean Campy. conc. (MPN/g) |
Day 3 Dung Campy.conc. (MPN/g) |
Day 9 Dung Campy. conc. (MPN/g) |
|---|---|---|---|---|
| C | 1 | 1.1 x 104 (2.4 x 104)* | 4.6 x 103 | 9.3 x 103 |
| 2 | 1.1 x 104 (9.3 x 103)* | 2.4 x 104 | 7.5 x 103 | |
| 3 | 1.1 x 104 (2.4 x 104)* | 4.6 x 103 | 4.6 x 102 | |
| 4 | 1.1 x 104 (4.6 x 104)* | 1.5 x 104 | 1.1 x 105 | |
| 5 | 1.1 x 104 (4.6 x 103)* | 7.5 x 101 | 4.3 x 102 | |
| D | 1 | 3.3 x 103 (7)* | 2.4 x 103 | 4.3 x 102 |
| 2 | 3.3 x 103 (1.5 x 101)* | 2.3 x 101 | 9.3 x 103 | |
| 3 | 3.3 x 103 (4.3 x 101)* | 9.3 x 102 | 4.6 x 104 | |
| 4 | 3.3 x 103 (4.6 x 103)* | 2.4 x 104 | 4.6 x 103 | |
| 5 | 3.3 x 103 (2.3 x 101)* | 2.4 x 104 | 4.6 x 104 |
*Values in parenthesis are natural Campylobacter concentrations of the fresh dung pats just prior to spiking with the cultured Campylobacter.
There was no correlation between concentrations of Campylobacter and E. Coli in fresh faeces (Figure 10), indicating that E. Coli counts cannot be reliably used as a surrogate index for Campylobacter prevalence.
Figure 10: Relationship between Campylobacter and E. Coli concentrations in freshly deposited cow faeces
Giardia cysts and Cryptosporidium oocysts in drainage and surface runoff
Giardia cysts were present in most of the drainage and surface runoff water samples collected during this experiment. Although Giardia cysts numbers were highly variable, the highest numbers tended to be in samples collected immediately after grazing (Day 1) compared to the subsequent drainage events on Day 3 and Day 9 (Table 4). The presence of Cryptosporidia oocysts was not confirmed for any of the drainage or surface runoff water samples generated from the simulated rainfall events.
Table 4: Concentration of Giardia cysts measured in drainage and surface runoff samples collected at separate rainfall events at different times following grazing (faeces deposition)
| Timing of rainfall simulation | Plot Name |
Giardia cysts concentration (MPN/100L) | |
|---|---|---|---|
| Drainage sample |
Surface runoff sample | ||
| Day 1 (immediately after grazing) | C | 3.3 x 102 | <1 |
| D | 5.7 x 101 | 3.3 x 102 | |
| Day 3 | C | 1.3 x 101 | 2.5 x 101 |
| D | <1 | 1.6 x 101 | |
| Day 9 | C | 2 | <1 |
| D | <1 | <1 | |
Prevalence of Campylobacter in drainage and surface runoff
As the time between grazing and rainfall event increased, the concentration of thermotolerant Campylobacter in drainage and runoff samples decreased over time with each subsequent simulated rainfall event (Tables 5 and 6). There was little difference between the concentrations of Campylobacter in drainage and runoff for all samples collected.
Table 5: The effect of grazing and timing of repeated rainfall simulations on Campylobacter and E. Coli mean concentrations in drainage
| Timing of rainfall simulation | Plot | Campylobacter mean conc. (MPN/100 mL) |
E. Coli mean conc. (MPN/100 mL) |
|---|---|---|---|
| Day 1 (immediately after grazing) | C | 6.3 x 103 | 1.0 x 105 |
| D | 4.6 x 103 | 5.1 x 104 | |
| Day 3 | C | 1.5 x 103 | Not sampled |
| D | 3.4 x 102 | Not sampled | |
| Day 9 | C | 1.6 x 102 | 1.9 x 104 |
| D | 1.4 x 102 | 2.4 x 104 |
The decreasing concentrations of Campylobacter in drainage can be seen more clearly in Figures 11 and 12, where the concentrations of Campylobacter and suspended solids in drainage and surface-runoff are plotted against the percentage of total drainage or surface-runoff that had occurred up to the time of sampling.
It is suggested that the development of water stable skins over dung spots, rather than a reduction of Campylobacter numbers in the dung, is the main reason for the prevalence of Campylobacter and E. Coli in drainage and surface-runoff decreasing markedly as the lag-time between grazing and the successive rainfall events increased (Figure 9).
It should be noted that the drop off in concentrations of Campylobacter in runoff samples with time after grazing is more rapid than in drainage samples (Table 5 cf Table 6). One hypothesis is that high UV and aerobic conditions at the pasture surface may be consistent with high organism kill rates. In contrast, organisms flushed into soil macropores with the first simulated rainfall may survive in cool, microaerophilic soil pores to be flush out with later drainage events.
Table 6: The effect of grazing and timing of repeated rainfall simulations on Campylobacter and E. Coli mean concentrations in runoff
| Timing of rainfall simulation | Plot | Campylobacter mean conc. (MPN/100 mL) |
E. Coli mean conc. (MPN/100 mL) |
|---|---|---|---|
| Day 1 (immediately after grazing) | C | 1.1 x 103 | 4.1 x 105 |
| D | 3.4 x 102 | 1.5 x 105 | |
| Day 3 | C | 2.4 x 101 | Not sampled |
| D | 3.1 x 101 | Not sampled | |
| Day 9 | C | 1.6 x 101 | 2.6 x 104 |
| D | 2.6 x 101 | 4.0 x 104 |
At first glance, there appears to be a relationship between concentrations of Campylobacter and suspended solids in drainage and runoff samples (Figures 11 and 12). However, when these relationships are analysed more closely (Figure 13), there is no consistent relationship between concentrations of Campylobacter and suspended solids. Also, the higher suspended solid loadings of drainage water, in this experiment, did not appear to be associated with higher E. Coli counts.
Figure 11a: The change in Campylobacter and suspended solids concentrations in drainage samples with the % of total accumulated drainage (for the period 7-15 October 2003)
Figure 11b: The variation of Campylobacter and suspended solids concentrations in drainage samples with time after grazing (faeces deposition) for the period 7-15 October 2003
Figure 12a: The variation of Campylobacter and suspended solids concentrations in surface runoff with time after grazing (faeces deposition) as the percent of total accumulated surface runoff increases during successive simulated rainfall events over the period 7-15 October 2003
Figure 12b: The variation of Campylobacter and suspended solids concentrations in surface runoff water with time after grazing (faeces deposition) with each subsequent simulated rainfall event over the period 7-15 October 2003
Figure 13: Investigation of associations between Campylobacter, E. Coli and suspended solid concentrations in drainage water and runoff samples for the 7–15 October 2003 simulated rainfall events
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