WATER RESEARCH A Journal of the International Water Association
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Occurrence of Mycobacterium avium subsp. paratuberculosis in raw water and water treatment operations for the production of potable water G. Aboagye a, M.T. Rowe a,b,* a b
Food Microbiology, The Queen’s University of Belfast, Belfast, Northern Ireland, United Kingdom Food Microbiology Branch, Agri-Food and Biosciences Institute, Newforge Lane, Belfast BT9 5PX, Northern Ireland, United Kingdom
article info
abstract
Article history:
Mycobacterium avium subsp. paratuberculosis (Map) causes Johne’s disease of cattle and is
Received 23 December 2010
implicated as a cause of Crohn’s disease in humans. The organism is excreted in animal
Received in revised form
faeces and can contaminate water catchment areas. This coupled with Map’s survival in
14 March 2011
the environment means that water destined for domestic use may be a source of exposure.
Accepted 15 March 2011
This work was designed to determine the occurrence of Map in Lough Neagh (the largest
Available online 21 March 2011
freshwater lake in the British Isles), used as a reservoir, and in two water treatment works (WTW1 and WTW2) which abstract from the lough and which have slow sand filtration
Keywords:
(SSF) and dissolved air flotation respectively as their principal treatment regimes. The
Mycobacterium avium subsp.
organism was not detected in lough water samples by culture (n ¼ 70) but 29% (20/70) were
paratuberculosis
positive by PCR. In the raw water to WTW1 and WTW2 no culture positives were detected
Water treatment
but 54% (13/24) and 58% (14/24) respectively were PCR positive. In WTW1 there were no culture positives at the SSF or final water but 31% (8/26) and 45% (9/20) respectively were PCR positive. In WTW2 similar results were obtained with 26% (6/23) and 48% (11/23) in the floccules and final water respectively. At WTW2 however one culture positive was detected in the final water. This latter finding is of concern. The inability to reach definitive conclusions indicates the need for further research, particularly in the detection methods for viable Map. ª 2011 Elsevier Ltd. All rights reserved.
1.
Introduction
Mycobacterium avium subspecies paratuberculosis (Map) is the known cause of Johne’s disease of ruminants, particularly affecting dairy cattle (Clarke, 1997). Although the disease is contracted in the early years of life of the animal overt clinical signs, such as emaciation and loss of milk yield are usually only manifested in later years (Fecteau and Whitlock, 2010). The animal disease is responsible for significant economic
losses prompting control programmes in many countries which have varying degrees of success (Bakker, 2010; Kennedy and Citer, 2010; Whitlock, 2010). In addition to the animal welfare and attendant agrieconomic issues Map has been implicated as a causal factor in a number of human conditions such as diabetes type 1 (Paccagnini et al., 2009; Rani et al., 2010) and in particular Crohn’s disease (Behr, 2010). This latter condition is incurable, causing abdominal pain and constipation which may
* Corresponding author. Food Microbiology Branch, Agri-Food and Biosciences Institute, Newforge Lane, Belfast BT9 5PX, Northern Ireland, United Kingdom. Tel.: þ44 (0)2890 255291; fax: þ44 (0)2890 255009. E-mail address:
[email protected] (M.T. Rowe). 0043-1354/$ e see front matter ª 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.watres.2011.03.029
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require surgical intervention (Dasari et al., 2010). Although the relationship between Map and Crohn’s disease is only putative it was deemed sufficiently strong for the UK Government to adopt the precautionary principle and advocate strategies to minimise the public’s exposure to the organism (Rubery, 2002). It has been shown that Map is excreted directly in the milk of infected animals and in high numbers in bovine faeces (Grant et al., 2002) that can contaminate the environment (Hermon-Taylor, 2009) and contribute to agricultural runoff which may be situated in water catchment areas (Pierce, 2009). Map has been detected in raw water sources by both culture and PCR in previous studies in the UK (Pickup et al., 2005; Whan et al., 2005a). However, the efficacy of water treatment processes in removing or killing the organism has not been determined to the best of the authors’ knowledge. The object of the work reported here was firstly to attempt both molecular and culture methods to detect Map in Lough Neagh over a seasonal cycle. Secondly, to test the incoming raw and outgoing final treated water from two water treatment works (designated WTW1 and WTW2) which abstract from the lough (Fig. 1). In addition, in the case of WTW1, the schmutzdecke or ‘dirty layer’ which is the biologically active site in a slow sand filter (SSF) and, in the case of WTW2, the surface floccules which contain the organic material as a result of dissolved air flotation (DAF) were also sampled.
2.
Materials and methods
2.1.
Lough Neagh and environs
It should be recognised that domestic grazing animals have access to the shoreline of Lough Neagh at many locations, there are comparatively few surrounding hills thus exposing it to the prevailing wind (south west with mean wind speed of 5 m s1) and it is comparatively shallow (maximum depth 31 m, average depth 10 m). These factors combined mean that although the two WTWs are not geographically proximal to each other (Fig. 1) they could be considered as having the same source water. This therefore allows, to some extent, a comparison between SSF, primary treatment process applied in WTW1 and DAF, primary treatment process employed in WTW2, for removal of or lethality for Map. Information on the Map infection status of herds contiguous to Lough Neagh was not available to the authors.
2.2.
Water treatment procedures at WTW1
The WTW1 (Fig. 2) abstracts raw water from the lough at a distance of 600 m from the shore at depths of 1.5 and 3 m. The raw water is pretreated with ozone (2.5 mg l1) or chlorine (concentration varied to have a minimum chlorine residual of
Fig. 1 e Geographical location of Lough Neagh showing the two water treatment sites.
w a t e r r e s e a r c h 4 5 ( 2 0 1 1 ) 3 2 7 1 e3 2 7 8
WTW1
WTW2
Raw untreated water
Raw untreated water
Initial flocculation Initial ozonation
Initial chlorination Initial chlorination
First stage filtration (RGF)
Final flocculation
Final ozonation
Dissolved-air flotation (DAF)
Second stage filtration (SSF)
Intermediate chlorination
pH bal.*/orthophosphorylation
pH bal.*/orthophosphorylation
Final chlorination
Final chlorination
Final treated water
Final treated water
* bal. = balance
Fig. 2 e Flow diagram of water treatment at water treatment works 1 and 2 (WTW1 and 2).
0.2 mg l1 above the sand) if the ozone is not in service. Preferential use is made of ozone because of the high levels of particulate matter in the raw water which may result in unacceptable levels of trihalomethane (formed by reaction between chlorine and organic matter) which poses a health risk. The rapid gravity filters (RGF) sieve the water through sand grains for the inter-ozonation (final ozonation) stage which seeks to remove algae and total organic carbon from the pretreated water (Fig. 2). The next stage of treatment is water clarification in the slow sand filtration (SSF) system where particulate matter in the water is removed, including microorganisms, by the top dirty layer (schmutzdecke) which is mainly a biological process resulting in metabolism of organic compounds and sieving of particulate matter by accumulated microorganisms that form a meshwork. This is supported by sand grains underlain with large gravels that serve as an underdrain, conveying the clarified water to the final treatment stage where free chlorine at a concentration of 1.2e1.4 mg l1 is added. The pH, before the final chlorination stage, is adjusted to 7.0 by adding soda ash and also orthophosphate (1.0e1.5 mg l1) to prevent lead leaching into the final treated water before the water is led to holding reservoirs and finally distributed to the general public.
2.3.
Water treatment procedures at WTW2
The WTW2 (Fig. 2) is situated about 800 m from the lough and initiates water treatment by a flocculation procedure
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employing aluminium sulphate before dosing the pretreated water with chlorine at 2 mg l1 to inhibit algal growth in storage tanks. An initial flocculation stage, where gross particulate matter is removed from the water occurs prior to the main flocculation stage which occurs in the dissolved air flotation chamber where smaller particles including microorganisms are removed. Here, clarified water is saturated with pressurised air (generated by a pressurising pump at 172e483 kPa) at a maximum retention time of 3 min and, with a drop in pressure of the system, the air is released from the water and the air bubbles generated attach themselves to the particulate matter including colloidal particles and suspended solids which float to the surface. This flotation occurs as a result of the entrapped air within the particles becoming lower in specific gravity than that of water. The floccules generated are removed by a skimming procedure. A final dosing with chlorine is performed at a concentration between 1.5 and 2 mg l1 (Fig. 2) after dosing with soda ash (to adjust the pH to 7.0) and orthophosphate (1.0e1.5 mg l1) to prevent leaching of lead into the treated water before the final water is pumped to holding reservoirs for distribution to the general public.
2.4.
Collection of samples
Raw water samples were collected from the three sampling sites (Lough Neagh, WTW1 and WTW2) over a period of 12 months e 3-month intervals for Lough Neagh to coincide with the four seasons and once every month for WTW1 and WTW2. The sampling sites were; Lough Neagh and proximal to the two abstraction sites at a depth of between 1.5 and 12 m using a manual corer (Hth-Teknik, Lulea, Sweden) which produced cores of approximately 10 mm thickness of sediment and contiguous water column, incoming raw and final treated water from both WTWs (1 l each), schmutzdecke (20 g) down to a depth of approximately 1 cm from drained SSFs in the case of WTW1 and flotation floccules (20 g) in the case of WTW2 as shown in Table 1. Sampling the schmutzdecke at WTW1 was determined by which SSF had been drained at the time of sampling and was therefore outside the control of experimental staff. There were five occasions on which a SSF was sampled twice at different chronological times. At WTW2 there were two DAF tanks and choice at sampling time was dependent on which was operational at the time.
2.5.
Pre-treatment of samples prior to analysis
The water samples (Lough Neagh, raw and final treated waters from WTW1 and 2) were membrane filtered (0.2 mm pore size, 47 mm diameter) and the membranes transferred to 20 ml 0.75% w/v cetylpyridinium chloride (CPC) solution (SigmaeAldrich Company Ltd, Dorset, UK) and incubated at room temperature for 4 h. Subsequently the membranes were transferred to 10 ml phosphate buffered saline plus 0.05% Tween 20 (PBS-T20, pH 7.3) and the CPC filtrate re-filtered as before to capture any Map cells dislodged from the filters during the decontamination phase. This second membrane was transferred to the PBS-T20 solution containing the first membrane and both abraded with forceps before being shaken vigorously by hand for 2 min
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Table 1 e IS900 and f57 PCR analyses of water and sediment samples obtained from Lough Neagh and two water treatment works. Sample location
Sample type
No. of samples taken
Number IS900 PCR positive/ total number of samples (%)
Number f57 Rt-PCR positive/ total number tested (%)
Lough Neagh
Water and sediment
70
20/70 (29%)
4/20 (20%)
WTW1
Raw water Schmutzdecke Final water
24 26 20 70
13/24 (54%) 8/26 (31%) 9/20 (45%) 30/70 (43%)
3/13 (23%) 3/8 (38%) 0/9 (0%) 6/30 (20%)
Raw water Floccules Final water
24 23 23 70
14/24 (58%) 6/23 (26%) 11/23 (48%) 31/70 (44%)
4/14 (29%) 3/6 (50%) 1/11 (9%) 8/31 (26%)
210
81/210 (39%)
18/81 (22%)
Total for WTW1 WTW2
Total for WTW2 Grand total
followed by vortex mixing for 2 min. The membranes were removed and the resulting suspension used for culture and PCR assay as described later in this paper. In respect of the samples from Lough Neagh these were treated as for the other water examples except that the filtering time was protracted, in some instances, because of the presence of excessive particulate matter. Twenty grams of the schmutzdecke and floccules were dispensed separately into 480 ml distilled water and stomached (Stomacher Model 400, Seward Ltd., Southdownview Way, Worthing, UK) at 260 rpm for 4 min and the resultant suspension subjected to double membrane filtration and CPC decontamination as described before.
2.6.
Culture protocols
One hundred microlitres of the PBS-T20 test cell suspensions were spread plated (British Standards Institution, 1984) onto Middlebrook 7H10 supplemented with VAN antibiotic cocktail (Sigma, Gillingham, Dorset, UK) and 2 mg l1 mycobactin J (Synbiotics Europe SAS, Lyon, France) and sealed with parafilm to avoid desiccation. One hundred microlitres of the suspension was also inoculated into triplicate tubes of M7H9 broth medium supplemented with PANTA antibiotic cocktail (Becton Dickinson and Company, Sparks, Maryland, USA) and mycobactin and triplicate Bactec 12B medium vials supplemented with PANTA and mycobactin. All samples were incubated at 37 C for at least 10 weeks and checked regularly for growth i.e. typical Map colonies on M7H10 plates, turbidity in M7H9 broths and positive growth index readings (30 units) for Bactec cultures. Middlebrook 7H10 was used in preference to Herrold’s Egg Yolk Medium, used previously for a similar study (Whan et al., 2005a), because it was found to give similar recovery rates, minimise the background microflora and allow better identification of Map colonies for further confirmation. In all cases if growth was detected an acid-fast stain and PCR assay were performed to confirm the presence of mycobacteria and Map respectively. In the case of the culture isolate obtained this was subjected to mycobactin dependency, f57 PCR assay and the IS900 PCR amplicon was sequenced and compared to Map using the NCBI BLAST GenBank database and also typed by mycobacterial interspersed repetitive unitvariable-number tandem-repeat (MIRU-VNTR). This method
is based on the polymorphism of tandemly repeated DNA sequences and has been used for genotyping several mycobacterial species (Supply et al., 2006). In this study, eight MIRU-VNTR loci were applied to differentiate Map strains (Thibault et al., 2007).
2.7.
Cell disruption for DNA release prior to PCR assay
To 1 ml of each test sample (PBS-T20 cell suspensions) in an Eppendorf tube, 10 ml of Map antibody beads (Matrix Pathatrix PM 50, Map test, Matrix Microscience, Newmarket, UK) were added and the solution agitated on a rotary mixer (LD-79, Labinco BV, The Netherlands) for 30 min at 10 m s1. The sample, after agitation, was placed in a magnetic particle concentrator (DYNAL MPC-S, Invitrogen Ltd, Paisley, UK) for 10 min and the resulting clear liquid aspirated and discarded. The pellet was re-suspended in 700 ml TEN lysis buffer (pH 8.0) containing 0.744 g l1 ethylenediaminetetraacetic acid (EDTA, sodium salt, Sigma), 23.376 g l1 sodium chloride (Fisher Scientific UK Ltd., Loughborough, UK), 1.576 g l1 TriseHCl (pH 8.0, Sigma) and 10 g l1 sodium dodecylsulphate (Amesham Biosciences, Uppsala, Sweden) supplemented with 25 mg ml1 proteinase K (Sigma) and filter sterilised (0.45 mm pore size). The suspension was incubated overnight at 37 C before being transferred to FastProtein Blue126 tubes (Qbiogene-ALEXIS Ltd, Carlsbad, California, USA) containing matrix B and subjected to ribolysation (Hybaid Ltd., Middlesex, UK) for 45 s at 6.5 m s1 before being placed on ice for 15 min to settle the foam generated. The DNA was extracted and purified as described below.
2.8.
Extraction and purification of DNA
Seven-hundred microlitres of phenol:chloroform:isoamylalcohol (25:24:1, pH 8.0, Sigma) was added to each FastProtein Blue tube and centrifuged at 7826 g for 10 min. The supernatant was aspirated into a micro-centrifuge tube containing 400 ml of isopropanol (99% v/v) and incubated at 20 C for 30 min to precipitate DNA. The centrifugation step was repeated followed by washing once with 500 ml of 70% v/v ethanol. Finally the ethanol was decanted off and the DNA pellet allowed to air dry for 30 min at room temperature before being re-suspended in 50 ml TriseEDTA (TE) buffer (pH 8.0) and stored at 20 C until required.
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2.9.
IS900 amplification of extracted DNA
An Adiavet Paratb PCR kit (Adiagene, 38 Rue de Paris, 22000 Saint Brieuc, France) which targets the IS900 amplicon of Map was employed for this PCR assay. This kit has been optimised for the detection of Map from bovine faecal, milk and tissue matrices. In the work reported here the assay protocol was further optimised for the detection of Map DNA in PBS-T20 and sediment matrices (data not shown) and the sensitivity equates to 10 cells per ml due to the presence of between 10 and 14 copies of the IS900 sequence per cell. The sensitivity was calculated using a range of dilutions of Map ATCC 43015 in PBS-T20 and plating out as described previously in conjunction with PCR assay. Two microlitres of extracted DNA was subjected to PCR amplification in a 50 ml reaction according to the manufacturer’s instructions (Adiagene) using a thermal cycler (MBS Satellite 0.2G Thermocycler; Thermo Electron Corp., Milford, MA, USA). The reaction conditions were as follows; 1 cycle of 37 C for 30 s, 94 C for 5 min followed by 45 cycles of 94 C for 15 s, 62 C for 30 s, 72 C for 40 s followed by final extension of 1 cycle of 72 C for 10 min and held at 4 C before analysis.
2.10.
f57 real time PCR (Rt-PCR) of extracted DNA
Only if the sample tested positive for the IS900 PCR was it subjected to f57 PCR for confirmatory purposes. By inference therefore there can be no IS900 PCR negative and f57 positive samples. The method of Donaghy et al. (2010) was used with the final PCR assay mix being as follows; 25 ml Taqman universal master mix, 5 ml 10 exo ipc mix (Applied Biosystems, Warrington, UK), 1 ml 50 ipc DNA, 9.5 ml water, 1.5 ml forward f57 primer, 1.5 ml reverse f57 primer, 1.5 ml probe and 5 ml of template DNA. The cycling conditions for the f57 Rt-PCR were as follows; 1 cycle of 50 C for 2 min, 1 cycle of 95 C for 10 min, followed by 40 cycles of 95 C for 15 s and final extension of 1 cycle of 60 C for 1 min. The primers and probe were as follows: JM111F, Forward, 50 -CCG CGA TCC CAA AAG TTG-30 ; JM249R, Reverse, 50 -CTC GTA GCT GCC GAT TCA TG-30 ; JM165P, Probe, 50 -FAM-TCA CGG ACT AGA CCG GT-MGB-30 . The 50 end was labeled with 6-carboxyfluorescein (FAM; Applied Biosystems) and quenched with a minor groove bonder (MGB; Applied Biosystems). A Ct value 35 was considered a negative. Using this Ct value as a threshold and performing tandem Rt-PCR and plate counts the sensitivity was estimated to be 103 cfu ml1.
3.
Results and discussion
In the present study PCR positive results were found at all the sites sampled from Lough Neagh to final treated waters at the two WTWs (Table 1). The primary assay used was based on the IS900 insertion element, the specificity of which has been called into question (Kim et al., 2002; Taddei et al., 2008). However, the primer sequences used in the work reported here have been shown to give negative PCR assay results with Mycobacterium scrofulaceum (1 strain), Mycobacterium intracellulare (1 strain) and Mycobacterium SP2333 (Beatrice Blanchard, pers. comm.) which have been reported to give false
3275
positive responses with PCR assays based on the IS900 element. The IS900 element was chosen because it is present as multiple copies in the Map genome thereby making the assay sensitive enough to detect the low numbers of Map expected. The prepared DNA templates from samples which tested positive using the IS900 based primers were retested using primers directed at the f57 insertion element. This is reputed to be more specific (Mobius et al., 2008) but is present only as a single copy in the genome and therefore results in lower sensitivity (103 cells ml1) compared to IS900 (10 cells ml1). Bearing in mind that the f57 PCR assay was used as a confirmatory only on all IS900 positive PCR assays not all samples tested positive. Indeed only 18 out of 81 IS900 PCR positive samples (22%) tested positive using the f57 assay indicating that Map, if present, was likely to be only in low numbers or that the samples contained other mycobacteria, other than those mentioned previously, that possibly contained the IS900 insertion element. In Lough Neagh 20/70 (29%) of samples tested positive by PCR (Table 1) which is comparable to that observed by Pickup et al. (2005) who found an occurrence of 21/67 (31%) throughout lakes and water courses in the Lake District of England, UK that are used as a catchment area for the country. This number of IS900 PCR positives from Lough Neagh was lower than that from the raw water entering both WTWs (Table 1). This was unexpected since the Lough Neagh samples invariably contained sediment and Map is known to survive longer in sediment than in the water column (Whittington et al., 2005). There was no significant difference (P ¼ 1.0) in the raw untreated water entering the two WTWs i.e. 54 and 58% (Table 1) adding credence to the notion that the source water was essentially the same for both WTWs. Interestingly in a previous, albeit more limited study, of raw untreated waters entering WTWs from Lough Neagh Whan et al. (2005a) found 2/7 (28.8%) samples positive by IS900 PCR from a WTW distant from WTW1 and 2 and 0/26 (0%) from WTW2 itself. Although the IMSePCR methods used in the study of Whan et al. (2005a) and the work reported here were not directly comparable, the reported sensitivities were the same i.e. 10 Map cells ml1 (Whan et al., 2005b). To the authors’ knowledge there have been no significant changes in agricultural or other practices in the area surrounding WTW2 that would account for the difference in occurrence rates. In a study of the river Taff in Wales, UK Pickup et al. (2005) found that there was a significant association (1e5%) between rainfall and detection of Map. In the present study however no significant effect (binomial regression, P ¼ 0.544) of weather conditions (temperature, wind speed and rainfall) on occurrence of Map PCR positives was obtained from either the lough itself or the two WTWs. This disparity may be partly explained by the way the climate data was recorded in the two studies. In the case of Pickup et al. (2005) the mean rainfall values on days of PCR positive samples and on each of the preceding 7 days were compared with mean rainfall values on days of PCR negative samples. In the work reported here however the mean climatic parameters, compiled on a monthly basis, were used for comparison. These were obtained from a UK Met Office sited at Belfast International Airport situated approximately 4 and 22 km away from WTW1 and 2 respectively.
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A positive result with PCR is indicative of presence but not necessarily viability of Map cells. The finding of Map in the schmutzdecke layer of the slow sand filter (Table 1) was not unexpected since the biological action of the schmutzdecke resides in the bacteriocidal action of environmental protozoa and there is evidence that Map can survive such ingestion (Hermon-Taylor and Bull, 2002; Whan et al., 2006). This also applies to ingestion by nematode larvae and other invertebrates (Fischer et al., 2001; Lloyd et al., 2001; Whittington et al., 2001) associated with the schmutzdecke layer. In addition mycobacteria, in general, can reside and survive in biofilms (Falkinham et al., 2001; Pickup et al., 2006; Rowe and Grant, 2006) and the schmutzdecke could be expected to provide a suitable stratum. The schmutzdecke from a number of drained SSFs were sampled during the course of the study, one per month, so the findings are not likely to be due an aberration with one individual SSF. In respect of WTW1 the comparable percentage of PCR positive samples of final water compared to the schmutzdecke may be due to lysis of intact cells and leaching of naked Map DNA into the final water. Certainly no culture positive samples of final water were detected in WTW1. However the sensitivity of the PCR method used is approximately 10 cfu ml1 (data not shown) which is likely to be greater than the culture method. Published information on culture methods, albeit using different matrices, is 102 cfu per 102 g of faeces (Jorgensen, 1982; Reddacliff et al., 2003) and 102e103 cfu ml1 for milk (Grant et al., 2003). It should be recognised however that decontamination was employed (0.75% CPC for 4 h) and no resuscitation was attempted. This would undoubtedly have reduced recovery because of the likely presence of injured or dormant cells. If however the results obtained reflect significant lysis of Map cells and subsequent leaching of naked DNA into the final water this would indicate an efficient treatment. In WTW2 the percentage of PCR positive floccule samples, as a result of the DAF process, were fewer than either the incoming raw water or final treated water (Table 1). It should be noted that the formation of floccules, in contrast to the schmutzdecke, is a purely physiochemical process with no biological bioremediation involved. It is known that the cell wall of Map is hydrophobic (Brennan and Nikaido, 1995). This, it would be expected, would induce an affinity between Map and the air bubbles and result in concentration of Map in the surface floccule layer but this was not detected in the system investigated. It is of concern that a viable Map culture was obtained in the final treated water from WTW2 even with the difficulties of obtaining viable isolates from environmental sources. The fact that the isolate was mycobactin dependent, positive with f57 PCR and the IS900 amplicon and when sequenced showed 100% homology with Map using the NCBI BLAST GenBank database provides substantive proof of its identity. The culture was subjected to MIRU-VNTR typing analysis as described by Thibault et al. (2007) and was assigned the INMV number of 2 indicating that it is one of the two predominant bovine strains in the UK as well as other European countries such as Netherlands, Finland and Spain (Stevenson et al., 2009). In summary Map was detected by PCR in Lough Neagh which is in agreement with previous work on the lough (Whan et al., 2005a). It was also found by PCR assay throughout both
WTWs, even in the final water which indicates the presence of Map but not necessarily that of intact viable cells. There was no clear difference between the corresponding results from both WTWs which, because of the topography of the lough, essentially abstracted the same source water. This means that if PCR positive tests are used as the index then the efficacy of both treatments was the same. What was most concerning was the culture positive sample in final treated water from WTW2 which shows that viable Map, albeit probably in very low numbers, is entering the water distribution system. It is worthy of note that Pickup et al. (2006) detected Map using PCR in 1 of 54 domestic water cisterns. The need to present the interpretation of the results obtained in this study in the form of a range of explanations rather than a definitive conclusion highlights the need for further research in this area and particularly improvements in the culture of Map from environmental sources.
4.
Conclusions
A limited survey (n ¼ 210) of Lough Neagh for M. avium subsp. paratuberculosis (Map) was performed along with two water treatment works (WTWs) that abstract from the lough using both PCR assay and culture. Map was detected by PCR in Lough Neagh and throughout the two WTWs, including the final treated water. One culture positive was confirmed and that was found in the final treated water. No difference was found in terms of Map removal between slow sand filtration (WTW1) and dissolved air floatation (WTW2). This work provides evidence that the public may be exposed to Map through water supplies.
Acknowledgements This work was supported by the following scholarships associated with Queen’s University of Belfast; Gibson, Harold Barbour and Mac Geough Bond. The authors would also wish to thank Northern Ireland Water for allowing access to their facilities, David Kilpatrick, Biometrics Branch, Agri-Food and Biosciences Institute (AFBI) for the statistical analyses and Dr. A. Gilmour, AFBI for helpful advice and guidance.
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paratuberculosis, SLC11A1 polymorphisms and type-1 diabetes mellitus. PLoS One 4 (9), e7109. Pickup, R.W., Rhodes, G., Arnott, S., Sidi-Boumedine, K., Bull, T.J., Weightman, A., et al., 2005. Mycobacterium avium subsp. paratuberculosis in the catchment area and water of the River Taff in South Wales, United Kingdom, and its potential relationship to clustering of Crohn’s disease cases in the city of Cardiff. Applied and Environmental Microbiology 71 (4), 2130e2139. Pickup, R.W., Rhodes, G., Bull, T.J., Arnott, S., Sidi-Boumedine, K., Hurley, M., Hermon-Taylor, J., 2006. Mycobacterium avium subsp. paratuberculosis in lake catchments, in river water abstracted for domestic use, and in effluent from domestic sewage treatment works: diverse opportunities for environmental cycling and human exposure. Applied and Environmental Microbiology 72 (6), 4067e4077. Pierce, E.S., 2009. Possible transmission of Mycobacterium avium subspecies paratuberculosis through potable water: lessons from an urban cluster of Crohn’s disease. Gut Pathogens 1 (1), 17. Rani, P.S., Sechi, L.A., Ahmed, N., 2010. Mycobacterium avium subsp. paratuberculosis as a trigger of type-1 diabetes: destination Sardinia, or beyond? Gut Pathogens 2 (1), 1. Reddacliff, L.A., Nicholls, P.J., Vadali, A., Whittington, R.J., 2003. Use of growth indices from radiometric culture for quantification of sheep strains of Mycobacterium avium subsp. paratuberculosis. Applied and Environmental Microbiology 69 (6), 3510e3516. Rowe, M.T., Grant, I.R., 2006. Mycobacterium avium ssp. paratuberculosis and its potential survival tactics. Letters in Applied Microbiology 42 (4), 305e311. Available from: Rubery, E., 2002. A Review of the Evidence for a Link Between Exposure to Mycobacterium paratuberculosis and Crohn’s Disease (CD) in Humans. A Report for the Food Standards Agency, January 2002, pp. 1e66 http://www.food.gov.uk/ multimedia/pdfs/mapcrohnreport.pdf (accessed 14.06.10). Stevenson, K., Alvarez, J., Bakker, D., Biet, F., de Juan, L., Denham, S., et al., 2009. Occurrence of Mycobacterium avium subspecies paratuberculosis across host species and European countries with evidence for transmission between wildlife and domestic ruminants. BMC Microbiology 9, 212. Supply, P., Allix, C., Lesjean, S., Cardoso-Oelemann, M., RuschGerdes, S., Willery, E., et al., 2006. Proposal for standardization of optimized mycobacterial interspersed repetitive unitvariable-number tandem repeat typing of Mycobacterium tuberculosis. Journal of Clinical Microbiology 44 (12), 4498e4510. Taddei, R., Barbieri, I., Pacciarini, M.L., Fallacara, F., Belletti, G.L., Arrigoni, N., 2008. Mycobacterium porcinum strains isolated from bovine bulk milk: implications for Mycobacterium avium subsp. paratuberculosis detection by PCR and culture. Veterinary Microbiology 130 (3e4), 338e347. Thibault, V.C., Grayon, M., Boschiroli, M.L., Hubbans, C., Overduin, P., Stevenson, K., Gutierrez, M.C., Supply, P., Biet, F., 2007. New variable-number tandem-repeat markers for typing Mycobacterium avium subsp. paratuberculosis and M. avium strains: comparison with IS900 and IS1245 restriction fragment length polymorphism typing. Journal of Clinical Microbiology 45 (8), 2404e2410. Whan, L., Ball, H.J., IGrant, I.R., Rowe, M.T., 2005a. Occurrence of Mycobacterium avium subsp. paratuberculosis in untreated water in Northern Ireland. Applied and Environmental Microbiology 71 (11), 7107e7112. Whan, L., Ball, H.J., Grant, I.R., Rowe, M.T., 2005b. Development of an IMSePCR assay for the detection of Mycobacterium avium ssp. paratuberculosis in water. Letters in Applied Microbiology 40 (4), 269e273. Whan, L., Grant, I.R., Rowe, M.T., 2006. Interaction between Mycobacterium avium subsp. paratuberculosis and environmental protozoa. BMC Microbiology 6, 63.
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Whitlock, R.H., 2010. Paratuberculosis control measures in the USA. In: Behr, M.A., Collins, D.M. (Eds.), Paratuberculosis. Organism, Disease, Control. CAB International, Oxfordshire, UK, pp. 319e329. Whittington, R.J., Lloyd, J.B., Reddacliff, L.A., 2001. Recovery of Mycobacterium avium subsp. paratuberculosis from nematode
larvae cultured from the faeces of sheep with Johne’s disease. Veterinary Microbiology 81 (3), 273e279. Whittington, R.J., Marsh, I.B., Reddacliff, L.A., 2005. Survival of Mycobacterium avium subsp. paratuberculosis in dam water and sediment. Applied and Environmental Microbiology 71 (9), 5304e5308.
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journal homepage: www.elsevier.com/locate/watres
Bacterial pathogens in Hawaiian coastal streamsdAssociations with fecal indicators, land cover, and water quality Emily J. Viau a, Kelly D. Goodwin b, Kevan M. Yamahara a, Blythe A. Layton a,1, Lauren M. Sassoubre a, Siobha´n L. Burns c, Hsin-I Tong c, Simon H.C. Wong a, Yuanan Lu c, Alexandria B. Boehm a,* a
Stanford University, Department of Civil & Environmental Engineering, 473 Via Ortega, Stanford, CA 94305, United States National Oceanic and Atmospheric Administration (NOAA), AOML, Miami, FL (stationed at SWFSC, San Diego, CA 92037), United States c University of Hawai’i at Manoa, Departments of Public Health Sciences and Microbiology, Honolulu, HI 96822, United States b
article info
abstract
Article history:
This work aimed to understand the distribution of five bacterial pathogens in O’ahu coastal
Received 7 January 2011
streams and relate their presence to microbial indicator concentrations, land cover of the
Received in revised form
surrounding watersheds, and physicalechemical measures of stream water quality. Twenty-
23 February 2011
two streams were sampled four times (in December and March, before sunrise and at high
Accepted 16 March 2011
noon) to capture seasonal and time of day variation. Salmonella, Campylobacter, Staphylococcus
Available online 12 April 2011
aureus, Vibrio vulnificus, and V. parahaemolyticus were widespread d12 of 22 O’ahu streams had all five pathogens. All stream waters also had detectable concentrations of four fecal indica-
Keywords:
tors and total vibrio with log mean standard deviation densities of 2.2 0.8 enterococci,
Salmonella
2.7 0.7 Escherichia coli, 1.1 0.7 Clostridium perfringens, 1.2 0.8 Fþ coliphages, and 3.6 0.7
Campylobacter
total vibrio per 100 ml. Bivariate associations between pathogens and indicators showed
Staphylococcus aureus
enterococci positively associated with the greatest number of bacterial pathogens. Higher
Vibrio
concentrations of enterococci and higher incidence of Campylobacter were found in stream
Fecal indicator
waters collected before sunrise, suggesting these organisms are sensitive to sunlight. Multi-
Tropical streams
variate regression models of microbes as a function of land cover and physicalechemical water quality showed positive associations between Salmonella and agricultural and forested land covers, and between S. aureus and urban and agricultural land covers; these results suggested that sources specific to those land covers may contribute these pathogens to streams. Further, significant associations between some microbial targets and physicalechemical stream water quality (i.e., temperature, nutrients, turbidity) suggested that organism persistence may be affected by stream characteristics. Results implicate streams as a source of pathogens to coastal waters. Future work is recommended to determine infectious risks of recreational waterborne illness related to O’ahu stream exposures and to mitigate these risks through control of land-based runoff sources. ª 2011 Elsevier Ltd. All rights reserved.
* Corresponding author. Tel.: þ1 650 724 9128; fax: þ1 650 723 7058. E-mail address:
[email protected] (A.B. Boehm). 1 Present address: Southern California Coastal Water Research Project, 3535 Harbor Blvd., Suite 110, Costa Mesa, CA 92626, United States. 0043-1354/$ e see front matter ª 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.watres.2011.03.033
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1.
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Introduction
Each year exposures to marine waters contaminated by microbes cause an estimated 120 million gastrointestinal infections (GIs), 50 million acute respiratory infections (ARIs) (Shuval, 2003), and numerous skin infections (Yau et al., 2009). One source of microbial pollution to coastal waters is landbased runoff that discharges from rivers, streams and culverts to the nearshore. Runoff may contain pathogens from leaking sewage infrastructure, wild and domestic animal excreta, and other poorly understood environmental reservoirs such as soils and sands; in sum these are non-point sources of pollution (Boehm et al., 2009a). Several epidemiology studies have investigated health effects from recreational exposure to land-based runoff and the results are equivocal. Haile et al. (1999) found increased risks of GI and ARI for swimmers recreating near storm drains at a southern Californian marine beach and a correlation between risk and fecal indicator bacteria (FIB) concentrations. In contrast, Calderon et al. (1991) found no statistically significant association between swimmers’ illness risk and FIB in a freshwater pond contaminated by agricultural runoff. Dwight et al. (2004) found that Southern Californian surfers exposed to urban runoff had higher illness rates than Northern Californian surfers exposed to rural runoff. In these epidemiology studies, pathogen data were not readily available or limited and the exact source of fecal indicator organisms in the runoff was not known. Data on pathogens in runoff and insight into the factors that modulate their concentrations in runoff, would improve our ability to calculate and understand risks from exposure to terrestrial runoff. Most research on fecal pollution and risks from recreational swimming has been conducted in temperate climates. Both US and WHO standards for recreational water quality were promulgated using data collected in temperate zones (Boehm et al., 2009a). This is despite the fact that US tropical beaches receive more visitors than all temperate beaches combined (Leeworthy and Wiley, 2001). Furthermore, numerous studies find enterococci and Escherichia coli, the indicators used to assess water quality around the globe, in tropical soils and streams (Hardina and Fujioka, 1991; Hazen, 1988); there is concern that the presence of these organisms in tropical recreational waters may not indicate contamination or presence of pathogens. To improve waterborne pathogen surveillance in the tropics, researchers suggest monitoring alternative indicators, like Clostridium perfringens and Fþ coliphages (Fung et al., 2007; Luther and Fujioka, 2004). However, pathogen data are needed to corroborate whether or not an association exists between FIB, alternative indicators, and pathogens in the tropics. In the present study, we measured human bacterial skin and GI pathogens in tropical coastal streams discharging to marine waters, and tested their association with traditional and alternative indicator organisms, surrounding land cover, and physicalechemical water quality. Specifically, we documented the occurrence or concentrations of Salmonella, Campylobacter, Staphylococcus aureus, Vibrio parahaemolyticus and Vibrio vulnificus in 22 tropical streams of O’ahu, Hawai’i using a combination of culture-based and molecular methods.
One goal was to test pathogen associations with indicators including E. coli, enterococci, C. perfringens, Fþ coliphage, and total vibrio to determine which indicators have predictive power of bacterial pathogens. A second goal was to build multivariate statistical models to understand how land cover and physicalechemical stream parameters controlled pathogens and indicators. The modeling is premised on a conceptual model where bacterial concentrations in streams (1) increase due to microbial fluxes from the surrounding land; fluxes are affected by land cover, and (2) change in response to physicalechemical characteristics of the stream that affect organism persistence. The work presented here is unique in that it investigates the distribution of both GI and non-GI pathogens in a tropical climate, two research needs specifically mandated in the US BEACH Act of 2000.
2.
Materials and methods
2.1.
Sampling sites
Twenty-two streams were identified on O’ahu, Hawai’i for sampling (Table 1). Streams were selected because they discharge to coastal waters adjacent to popular swimming beaches. We also selected streams that drained watersheds with diverse land covers. In all cases, there were no known sewage point sources to the streams; all watersheds had ) was hio separate storm and sewage conveyances. One site (Ku a storm drain.
2.1.1.
Land cover
Land cover was determined for stream watersheds using ArcGIS (ESRI, Redlands, CA) and Hawai’i Land Cover 2001 data (NOAA 2001). The data consist of Landsat Enhanced Thematic Mapper data at 30 m resolution for 18 land coverage classes. These classes were aggregated into four broad categories: urban, agriculture, forested, and other (unclassified, unconsolidated shore, water, and bare land). Watershed boundary data were obtained from the Hawai’i Statewide GIS program (Hawaii, 2010). The fraction of each watershed that was urban, agricultural, and forested was calculated by normalizing the areas, respectively, by the total watershed area.
2.1.2.
Field sampling
Two water sampling campaigns were conducted at the 22 sites over five days in December 2009 (14e18 Dec 2009) and March 2010 (28 Mare3 Apr 2010). Daily precipitation during and prior to the field campaigns was obtained from a centralized rain gauge on O’ahu (Moanalua USGS site 212359157502601). It should be noted that rainfall is spatially variable over the island, so data from this gauge represent approximate rainfall in the studied watersheds. The streams have very limited USGS stream gauge coverage, so flow data were not available. During each campaign, the 22 sites were visited twice (once before the sun rose and once at high noon); in sum each stream was sampled four times. Twenty-liter water samples were collected in triple rinsed, 10% HCl-washed plastic containers. Water was sampled at an accessible location near the intersection of the stream and coastal ocean (Table 1).
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Table 1 e O’ahu Stream Survey Site Description, Land Use. Hawai’i department of health water quality standards are provided where relevant. Stream values in exceedance of these standards are bolded. Location Stream
Watershed land cover
Ancillary Measurements
Latitude/ Associated FOR URB AG TEMP Salinity DO CHLa TURB NO2þNO3 DINd PO4 (mg/l) (mg/L)c (NTU)b,c (mg-N/L)c (mg-N/L)c (mg-P/L)c Longitude of beach ( C) sample collection
Ala Waia 21.288 N, 157.839 W Ala Moana Storm 21.271 N, 157.824 W hio hio Ku Ku Draina Wai’alae Kapakahi 21.270 N, 157.778 W Kahala Wai’alae Golf 21.273 N, 157.771 W Coursea 21.278 N, 157.750 W Wailupe Wailupea Moanalua 21.333 N, 157.894 W Ke’ehi lagoon Kalihi 21.332 N, 157.891 W Ke’ehi lagoon kua kua Ma Ma 21.530 N, 158.229 W Kaupuni 21.448 N, 158.193 W Poka’i Bay ’ili’ilia 21.429 N, 158.180 W Ma’ili Ma ’ilia 21.409 N, 158.177 W Ma’ili Ma na kuli na kuli Na Na 21.376 N, 158.140 W leakahana laekahana 21.673 N, 157.936 W Ma Ma Waimea Waimea 21.641 N, 158.063 W Hale’iwa Anahulu 21.594 N, 158.103 W Kaiaka Paukauila 21.580 N, 158.117 W Kaiaka Kiikii 21.579 N, 158.120 W nolo nalo 21.365 N, 157.709 W Waima Waima Kailua Ka’elepulu 21.398 N, 157.726 W Kailua Kawainui 21.426 N, 157.741 W Kahana Kahana 21.556 N, 157.869 W Punalu’u Punalu’u 21.579 N, 157.885 W All Stream Average Hawai’i DOH Water Quality Standardse
18% 79% 0% 18% 79% 0%
24.0 24.5
25.2 33.2
5.3 6.5
2.1 0.2
2.3 1.2
319 52
388 72
16 8.1
54% 45% 0% 67% 31% 0%
23.7 24.1
30.6 23.6
4.5 7.1
1.6 1.3
10 15
22 47
141 92
11 14
67% 79% 72% 97% 83% 78% 78% 85% 90% 98% 80% 38% 27% 81% 47% 72% 98% 95%
24.6 25.0 24.0 23.9 24.7 23.6 24.6 23.5 23.7 23.6 23.0 23.1 22.8 24.4 24.3 24.5 22.1 21.7 23.8 e
22.4 33.5 23.5 37.3 30.0 34.3 30.1 20.9 0.6 7.8 14.9 11.0 10.2 17.2 17.5 11.7 4.8 1.2 20.1 e
6.2 4.6 5.3 6.3 6.0 7.0 6.5 5.0 3.5 6.4 6.6 4.7 7.9 5.0 6.9 5.3 5.3 7.4 5.9 >5
1.1 4.0 2.6 7.9 1.1 0.7 0.6 6.1 0.5 1.4 0.5 3.6 7.4 1.8 3.3 2.2 0.3 0.3 1.5 e
7.9 7.7 5.1 4.4 4.7 1.8 3.3 6.7 5.7 3.5 1.8 5.4 6.7 3.9 1.8 2.8 1.9 1.8 3.9 0.05). Seasonal fluctuations were significant for ENT (P ¼ 0.048) and F þ PHAGE (P ¼ 0.001), with MAR densities being lower than DEC by 0.25e0.5 log CFU/PFU per 100 ml. ENT concentrations also showed time of day
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a
differences (P ¼ 0.001) with AM values averaging 0.5-log higher than PM values.
3.4.
b
Each pathogen tested (except STAPH and CCOLI) showed a positive, significant association (P 0.05) to at least one indicator based on GEE models (Table 3). SAL, CAMPY, and CJEJ were positively associated with ENT concentrationsdENT was 0.6e1 log higher when one of these pathogens was present compared to when it was absent (ANOVA, P < 0.05). ENT was marginally positively associated with STAPH (P < 0.1). CAMPY was marginally positively associated with CPERF e CPERF was 0.3 log unit higher when CAMPY was detected (ANOVA, P ¼ 0.09). SAL was also marginally associated with CPERF (P < 0.1). VVUL was positively associated with all indicators tested, while VPARA was positively associated with TOTVIB, CPERF, and EC.
3.5.
c
Pathogen associations with indicators
Multivariate models of microorganisms
Multivariate models of microorganisms were created using GEE models (Table 4, Fig. S1). The land cover variables URB, AG and physico-chemical characteristics TEMP, TURB, and PO3 4 were included as independent variables. SAL occurrence was marginally associated with AG, URB, TEMP, and PO3 4 (P < 0.10)eassociations were positive for all variables except URB. CAMPY occurrence was negatively associated with TEMP (P < 0.001) and marginally negatively associated with AG (P ¼ 0.1). STAPH was positively associated with both AG and URB (P < 0.05). No significant water quality or land use associations were found for VVUL while VPARA was marginally positively associated with URB and TURB (P < 0.1). For indicators, ENT, TOTVIB, and F þ PHAGE were significantly associated with more than one independent variable. ENT had a positive association with TURB and a negative association with TEMP (P < 0.05). While only of marginal significance (P < 0.1), ENT was also positively associated to both AG and PO3 4 . TOTVIB showed a significant negative relationship to AG and positive relationship to TEMP (P < 0.05). F þ PHAGE was negatively associated with AG (P < 0.05) and TEMP (P¼0.1), and positively associated with PO3 4 (P < 0.1). One stream parameter was significant for each of the other indicatorsdEC was positively related to PO3 4 (P < 0.05) and CPERF was positively associated with TURB (P < 0.05).
3.6. Comparison between ENT and CPERF as pollution indices Fig. 1 e Presence/absence of a) Salmonella, b) Campylobacter, and c) Staphylococcus aureus in 22 O’ahu coastal streams (shaded [ present, white [ absent) by time of day (AM/ PM) and season (DEC/MAR). Circles denote DEC with top half and MAR samples with bottom half. AM presence is on the left and PM presence is on the right. For CAMPY (1b), positive C. jejuni are lines, positive C. coli are dots, positive C. jejuni and other CAMPY are black, while other CAMPY are shaded. For STAPH (1c), positive MRSA in March is indicated by stripes while STAPH analyses not completed are black in that part of the circle.
Some researchers suggest that ENT found in pristine tropical soils interfere with their ability to predict pollution from fecal sources (Boehm et al., 2009a). Fujioka and colleagues suggest that CPERF may be a better indicator of fecal pollution than ENT for tropical waters and that CPERF concentrations can discern pollution sources based on a “Fung/Fujioka scale” of pollution (Fung et al., 2007). According to this scale, when CPERF is greater than 100 CFU/100 ml, sewage is the pollution source. When CPERF is between 10 and 100 CFU/100 ml, nonpoint pollution is the source. Finally, waters are considered
3285
w a t e r r e s e a r c h 4 5 ( 2 0 1 1 ) 3 2 7 9 e3 2 9 0
Fig. 2 e Log fecal indicator and total vibrio concentrations in 22 O’ahu coastal streams by time of day (AM/PM) and season (DEC/MAR)dindicators include E. coli (CFU), enterococci (CFU), C. perfringens (CFU), FD coliphages (PFU), and total vibrio (CFU). Box- and-whisker plots represent the median (inner box line), 25th and 75th percentiles (lower and upper outer box lines), whiskers cover 10th and 90th percentiles and data outliers are represented by open circles. Geometric means are indicated with a black circle (n [ 22).
uncontaminated when CPERF is present at less than 10 CFU/ 100 ml. We plotted CPERF versus ENT (Fig. 3) to gain insight on how the CPERF pollution scale compares to the USEPA ENT standard of 104 CFU/100 ml. Using the Fung/Fujioka scale, 53 of 88 samples indicated non-point source pollution while 6 samples indicated sewage pollution. Forty-four of these 59 “contaminated” samples (e.g. CPERF10 CFU/100 ml) also exceeded the ENT standard. Agreement between the Fung/ Fujioka scale for contamination and single-sample exceedance for ENT, as well as the pairwise correlation between ENT and CPERF (rp ¼ 0.50, P < 0.05) suggests that similar information can be obtained from ENT as CPERF in these tropical streams.
4.
Discussion
4.1.
Bacterial pathogens
Salmonella, Campylobacter, S. aureus, V. vulnificus, and V. parahaemolyticus, are implicated in recreational water and shellfish
outbreaks (USEPA 2009b). In this study, multiple isolations of these organisms in a given stream were frequent (Fig. 1, Table S2). STAPH and Vibrio spp. were most commonly detected (STAPH was present in 19/22 streams, while Vibrio spp. were detected in all streams) followed by CAMPY (18/22) and SAL (15/22); all pathogens were isolated from 12/22 streams. Salmonella, a leading cause of gastroenteritis in the US (USEPA 2009b), are frequently isolated from surface waters (Haley et al., 2009; Walters et al., 2011; Wilkes et al., 2009). We found SAL in the majority of O’ahu streams at concentrations greater than 1 MPN/1 L. SAL occurrence was positively associated with higher temperature (thus higher DO and salinity as well), nutrients, and AG; while negatively associated with URB (Table 4). Relationships between water quality and SAL may imply increased Salmonella persistence in warm, eutrophic, relatively saline water. Previously, Salmonella in microcosms showed decreased persistence in warm relative to cool waters and no real trends in persistence in variable saline waters (Evison, 1988; Wait and Sobsey, 2001), while Evison (1988) reported increased Salmonella persistence in waters
Table 3 e Bivariate pathogeneindicator relationships. GEE logistic regression (logit function) was used for dichotomous variables while GEE linear regression was applied for continuous variables. b is the parameter coefficient; P-value is provided. Microorganism
Enterococci b
Salmonella Campylobacter C. jejuni C. coli S. aureus V. vulnificus V. parahaemolyticus a P0.05. b 0.05 < P < 0.1.
1.09 1.12 2.23 0.10 0.49 0.43 0.16
E. coli
P-value a
0.012
