Microbiological, chemical and physical quality of drinking water for commercial turkeys: a cross-sectional study

Microbiological, chemical and physical quality of drinking water for commercial turkeys: a... Abstract Drinking water for poultry is not subject to particular microbiological, chemical and physical requirements, thereby representing a potential transmission route for pathogenic microorganisms and contaminants and/or becoming unsuitable for water-administered medications. This study assessed the microbiological, chemical and physical drinking water quality of 28 turkey farms in North-Eastern Italy: 14 supplied with tap water (TW) and 14 with well water (WW). Water salinity, hardness, pH, ammonia, sulphate, phosphate, nitrate, chromium, copper and iron levels were also assessed. Moreover, total bacterial count at 22°C, presence and enumeration of Enterococcus spp. and E. coli, presence of Salmonella spp. and Campylobacter spp. were quantified. A water sample was collected in winter and in summer at 3 sampling sites: the water source (A), the beginning (B) and the end (C) of the nipple line (168 samples in total). Chemical and physical quality of both TW and WW sources was mostly within the limits of TW for humans. However, high levels of hardness and iron were evidenced in both sources. In WW vs. TW, sulphate and salinity levels were significantly higher, whilst pH and nitrate levels were significantly lower. At site A, microbiological quality of WW and TW was mostly within the limit of TW for humans. However, both sources had a significantly lower microbiological quality at sites B and C. Salmonella enterica subsp. enterica serotype Kentucky was isolated only twice from WW. Campylobacter spp. were rarely isolated (3.6% of farms); however, Campylobacter spp. farm-level prevalence by real-time PCR was up to 43% for both water sources. Winter posed at higher risk than summer for Campylobacter spp. presence in water, whereas no significant associations were found with water source, site, recirculation system, and turkey age. Low salinity and high hardness were significant risk factors for C. coli and C. jejuni presence, respectively. These results show the need of improving sanitization of drinking water pipelines for commercial turkeys. INTRODUCTION The quality of drinking water for livestock is a subject of utmost importance, as it can directly and indirectly affect animal health and productivity (Umar et al., 2014). Although general recommendations as well as specific guidelines for poultry water quality are available (Carter and Sneed, 1996; Amaral, 2004), farmers are often unaware of the importance of water quality (Umar et al., 2014). In the European Union (EU), this subject has received little attention in terms of EU legislation, as drinking water for poultry is not subject to particular microbiological, chemical and physical requirements. Regulation (EC) 852/2004 on the hygiene of foodstuffs establishes minimum requirements for livestock drinking water, but no qualitative parameters are listed (European Commission, 2004). Council Directive 98/58/EC on farmed animal welfare states that ‘all animals must have access to a suitable water supply or be able to satisfy their fluid intake needs by other means’, although no suitability thresholds are indicated (European Commission, 1998a). On the other hand, the Terrestrial Animal Health Code (Art. 6.4.5) of the OIE-World Organization for Animal Health states that ‘the drinking water supply to poultry houses should be potable according to the WHO or to the relevant national standard’ (OIE, 2016). This indication derives either from the possible transmission of pathogens, such as Salmonella and Campylobacter (Amaral, 2004), or from the possible infiltration of contaminants (Carter and Sneed, 1996). Recently, the European Food Safety Authority (EFSA, 2011) indicated farm water as one of the sources of direct contamination with Campylobacter for livestock and humans. Campylobacter is more susceptible than E. coli to water chlorination (Lund, 1996), and it was reported a 3.5-fold risk of infection for broiler flocks supplied with unchlorinated vs. chlorinated water (Kapperud et al., 1993). Moreover, C. jejuni has been isolated from the biofilm of nipple drinking systems for poultry when the birds were also colonized (Zimmer et al., 2003), although there is limited evidence for such drinking systems to be the source of Campylobacter colonization. This may be due to failure to detect Campylobacter in water as a result of insufficient water volumes processed or of microorganisms in a viable, but not culturable, state (Sparks, 2009). Data on the microbiological, chemical and physical quality of drinking water for livestock in Italy are scarce. Moreover, in the poultry sector, groundwater is frequently used, with no compulsory periodical water quality controls being requested for this source of water supply. Finally, turkeys are often treated with medicines, including antimicrobials, administered with drinking water, and some chemical and physical water properties like pH, hardness and iron levels may interfere with drug dissolution and stability in water (Scandurra, 2013). For these reasons, the present study aimed to assess the microbiological, chemical and physical quality of drinking water in commercial turkey farms supplied with either tap or well water. A number of factors putatively associated with water quality (e.g., environmental conditions, husbandry practices, season, water recirculation system, etc.) were also investigated, and water quality at different sampling sites along the farm water pipeline was assessed. MATERIALS AND METHODS Sample Collection Samples from 14 turkey farms supplied with well water (WW) and 14 supplied with tap water (TW) were analyzed. Both groups were randomly selected within the densely-populated poultry area (DPPA) of North-Eastern Italy. This area is characterized by the highest density of poultry in Italy and one of the greatest in Europe (Mulatti et al., 2011). Farms under study were operational for an average of 17 ± 12 years (SD) and consisted of 4 ± 2 sheds housing 10.000-70.000 turkeys in total; birds therein were 55 ± 35 days of age. Water wells were 65 ± 45 m deep underground. Seventeen farms had a system for water recirculation vs. 11 without such system. In order to guarantee pipeline hygiene, all farmers declared to apply a pipeline sanitization protocol as part of their normal operating procedures with stabilized hydrogen peroxide at each new production cycle (concentration of 2–3%) and continuously when the drinking system was operating (concentration of 25–50 ppm). Water samples were collected in 2012–2013 in late winter (February-March) and in mid-summer (July-August) at 3 sampling sites: the water source (A), the tank at the beginning of the nipple line where medicines are mixed for administration via water (B) and the end of the nipple line (C). Water temperature at the source and pH at A, B, C were also measured. Samples were collected in sterile containers, delivered to the laboratory at 4°C and processed within 24 h for microbiological analyses and 48 h for chemical and physical analyses. Chemical and Physical Analyses Ammonia concentration was determined by spectrometric indophenol assay (APAT, 2003), while the concentration of nitrate, sulphate and phosphate was determined by ionic chromatography (APAT, 2003). Chromium, copper and iron concentrations were quantified by atomic absorption spectrophotometry (APAT, 2003). The EDTA (ethylenediaminetetraacetic acid) titration method was used to determine the level of hardness (APAT, 2003). Microbiological Analyses Water samples were tested for total bacterial count at 22°C, enumeration of Enterococcus spp., enumeration of E. coli, presence of Salmonella spp. While specific legislation for animal drinking water is not available, analyses were performed according to European Council Directive 98/83/EC on the quality of water intended for human consumption (European Commission, 1998b). A national reference guideline (ISS, 2007), which set analytical methods for water intended for human consumption, was also considered. Analyses for enumeration of Enterococcus spp. and E. coli were performed according to UNI EN ISO 7899-2 (UNI, 2003) and UNI EN ISO 9308-1 (UNI, 2002), respectively. Enumeration of total bacterial count at 22°C and 37°C were performed according to UNI EN ISO 6222 for colony count (UNI, 2001), and according to ISO 6887-1 (ISO, 1999) and ISO 8199 (ISO, 2005a) for sample dilutions and for membrane filtration method. For the detection of Salmonella spp., 1 L samples were tested by membrane filtration method and then processed according to ISO 6579 (ISO, 2002). To detect the presence of Campylobacter spp., both cultivation and real-time PCR were performed. Isolation was performed according to the standard method for Campylobacter detection in water (ISO, 2005b), which was slightly modified (SCA, 2002; Williams et al., 2012). Briefly, water samples were filtrated through a sterile membrane with a pore size of 0.2 μm (Sartorius, Goettingen, Germania) in a vacuum pump system. Then, each membrane was inoculated into 50 mL of Exeter broth (Mast Diagnostics, Merseyside, UK) and incubated at 41.5°C for 48 h under microaerobic conditions. Two hundred μL of each broth culture were streaked onto Karmali agar (OXOID, Basingstoke, UK), after passive filtration (Giacomelli et al., 2012), and incubated at 41.5°C for 48 h under microaerobic conditions. Suspected Campylobacter colonies were examined by multiplex end-point PCR for genus and species identification (Yamazaki-Matsune et al., 2007). For real-time PCR, water samples were filtrated as described above and filters inoculated and vortexed in 5 mL of deionized water. Three mL of the suspension were centrifuged at 5,000 rpm for 10 min and the pellet resuspended in 200 μL of PBS. The DNA extraction was carried out by using the High Pure Template Preparation Kit (Roche Diagnostics, Mannheim, Germany) according to the manufacturer's instructions, and the multiplex real-time PCR assay by using Taqman® probes and specific primers for C. jejuni and C. coli detection, as previously described (Toplak et al., 2012). Statistical Analyses An explorative data analysis was performed to examine the distributions of chemical and physical parameters. Given the high skewness of all these parameters, median and interquartile range were used as descriptive statistics, and, for each parameter, linear quantile mixed model targeting the median was adopted to assess the association with the type of water source (TW or WW), the sampling site (A, B or C) and the season (winter or summer), that represented the fixed effects in the model. To take into account the hierarchical structure of the sampling design, the farm was included in the model as grouping factor, whereas sampling site and season were additionally declared in the random part of the model. A General Linear Model (GLM) was used to evaluate the association between mean temperature and type of water source, season and their interaction. The outcomes of microbiological analyses, expressed as presence/absence, were modelled using Generalized Linear Mixed Models (GLMMs), choosing the binomial distribution as the response distribution. Major water properties (pH, salinity, hardness and iron) and farm characteristics, such as water source, presence of a supply system with water recirculation and birds age, were included in the models as fixed effects as putative risk factors. According to sampling design, random effects of farms, sampling site and season were also included. To facilitate the evaluation of the results and the discussion, water parameters were classified into categories according to their observed distribution. The software R 3.3.0 was used to perform linear quantile mixed models, whereas software SAS v.9.4 was adopted for the other statistical analyses. RESULTS The results of microbiological, chemical and physical analyses by source of water supply, sampling site and season are given in Tables 1 and 2. Regarding water temperature, a significant interaction between water source and season was found (P < 0.01): WW was significantly colder than TW (17.4 ± 3.6 vs. 20.6 ± 2.8) in summer, but not in winter (14.6 ± 2.1 vs. 13.3 ± 2.7). Chemical, physical and microbiological quality profiles of WW and TW were mostly within the limits of TW for humans (Counc. Dir. 98/83/EC). Median values of pH, sulphate, nitrate, iron and salinity levels were significantly different between TW and WW (P < 0.05). Except for pH, the same was found between winter and summer. Concerning the sampling site, copper median concentration was significantly higher at site B (P < 0.001) and C (P = 0.041) compared to A. Both sources had a lower microbiological quality at site B and C (e.g., up to 103 CFU/100 mL for E. coli) than A (Tables 1 and 2). Table 1. Chemical and physical analyses of drinking water in 28 turkey farms supplied with either well or tap water during summer and winter of 2012–2013 in North-Eastern Italy. Water samples were collected twice in 2012–2013 (in winter and in summer) at 3 sampling sites: the water source (A), the beginning (B) and the end (C) of the nipple line. Data are expressed as median values with interquartile range (within parentheses). Significance (Sig.): *P < 0.05, **P < 0.01, ***P < 0.001.       Water source    Sampling site    Sampling season    Parameter  Limits of potability Dir.98/83/EC  Maximum acceptable level  Tap water  Well water  Sig.  A  B  C  Sig.  Summer  Winter  Sig.  pH  6.5–9.5  >6b; 5–8c  7.7 (0.4)  7.5 (0.4)  *  7.6 (0.5)  7.6 (0.4)  7.6 (0.4)    7.7 (0.4)  7.6 (0.4)    Sulphate (mg/L)  250  250b; 200c  31.1 (27.0)  36.8 (59.9)  *  30.8 (45.1)  32.0 (42.4)  31.8 (47.9)    27.5 (31.5)  33.4 (59.6)  **  Phosphate (mg/L)      0.0 (0.1)  0.1 (0.5)    0.1 (0.3)  0.0 (0.2)  0.0 (0.2)    0.0 (0.4)  0.0 (0.2)    Nitrate (mg/L)  50  25b,c  16.2 (11.8)  5.5 (17.8)  ***  13.3 (15.8)  12.3 (17.11)  12.3 (18.0)    11.9 (17.3)  15.0 (18.4)  **  Copper (μg/L)    600b,c  4.5 (12.6)  3.1 (22.2)    1.9 (3.1)  10.4 (22.3)  6.7 (26.5)  **  3.2 (11.4)  6.9 (18.5)    Chromium (μg/L)a  50    1.3 (1.8)  0.0 (0.1)    0.2 (1.7)  0.0 (1.4)  0.0 (1.3)    0.0 (1.2)  0.9 (1.8)    Ammonia (mg/L)a  0.5    0.0 (0.0)  0.0 (0.0)    0.0 (0.0)  0.0 (0.0)  0.0 (0.0)    0.0 (0.0)  0.0 (0.2)    Salinity (PSU)      0.3 (0.1)  0.4 (0.3)  ***  0.3 (0.2)  0.3 (0.2)  0.3 (0.2)    0.2 (0.1)  0.3 (0.2)  ***  Hardness (mg/L)    110c  240 (78)  245 (145)    240 (104)  252 (108)  240 (101)    260 (127)  240 (74)    Iron (μg/L)  200  300b,c  1.1 (4.8)  3.5 (19.4)  *  1.1 (4.8)  1.8 (7.1)  2.3 (7.7)    1.1 (4.8)  2.8 (9.4)  *        Water source    Sampling site    Sampling season    Parameter  Limits of potability Dir.98/83/EC  Maximum acceptable level  Tap water  Well water  Sig.  A  B  C  Sig.  Summer  Winter  Sig.  pH  6.5–9.5  >6b; 5–8c  7.7 (0.4)  7.5 (0.4)  *  7.6 (0.5)  7.6 (0.4)  7.6 (0.4)    7.7 (0.4)  7.6 (0.4)    Sulphate (mg/L)  250  250b; 200c  31.1 (27.0)  36.8 (59.9)  *  30.8 (45.1)  32.0 (42.4)  31.8 (47.9)    27.5 (31.5)  33.4 (59.6)  **  Phosphate (mg/L)      0.0 (0.1)  0.1 (0.5)    0.1 (0.3)  0.0 (0.2)  0.0 (0.2)    0.0 (0.4)  0.0 (0.2)    Nitrate (mg/L)  50  25b,c  16.2 (11.8)  5.5 (17.8)  ***  13.3 (15.8)  12.3 (17.11)  12.3 (18.0)    11.9 (17.3)  15.0 (18.4)  **  Copper (μg/L)    600b,c  4.5 (12.6)  3.1 (22.2)    1.9 (3.1)  10.4 (22.3)  6.7 (26.5)  **  3.2 (11.4)  6.9 (18.5)    Chromium (μg/L)a  50    1.3 (1.8)  0.0 (0.1)    0.2 (1.7)  0.0 (1.4)  0.0 (1.3)    0.0 (1.2)  0.9 (1.8)    Ammonia (mg/L)a  0.5    0.0 (0.0)  0.0 (0.0)    0.0 (0.0)  0.0 (0.0)  0.0 (0.0)    0.0 (0.0)  0.0 (0.2)    Salinity (PSU)      0.3 (0.1)  0.4 (0.3)  ***  0.3 (0.2)  0.3 (0.2)  0.3 (0.2)    0.2 (0.1)  0.3 (0.2)  ***  Hardness (mg/L)    110c  240 (78)  245 (145)    240 (104)  252 (108)  240 (101)    260 (127)  240 (74)    Iron (μg/L)  200  300b,c  1.1 (4.8)  3.5 (19.4)  *  1.1 (4.8)  1.8 (7.1)  2.3 (7.7)    1.1 (4.8)  2.8 (9.4)  *  a Statistical testing not performed because of insufficient non-zero observations. b Carter and Sneed, 1996 c Aviagen Turkey Inc., 2015 View Large Table 1. Chemical and physical analyses of drinking water in 28 turkey farms supplied with either well or tap water during summer and winter of 2012–2013 in North-Eastern Italy. Water samples were collected twice in 2012–2013 (in winter and in summer) at 3 sampling sites: the water source (A), the beginning (B) and the end (C) of the nipple line. Data are expressed as median values with interquartile range (within parentheses). Significance (Sig.): *P < 0.05, **P < 0.01, ***P < 0.001.       Water source    Sampling site    Sampling season    Parameter  Limits of potability Dir.98/83/EC  Maximum acceptable level  Tap water  Well water  Sig.  A  B  C  Sig.  Summer  Winter  Sig.  pH  6.5–9.5  >6b; 5–8c  7.7 (0.4)  7.5 (0.4)  *  7.6 (0.5)  7.6 (0.4)  7.6 (0.4)    7.7 (0.4)  7.6 (0.4)    Sulphate (mg/L)  250  250b; 200c  31.1 (27.0)  36.8 (59.9)  *  30.8 (45.1)  32.0 (42.4)  31.8 (47.9)    27.5 (31.5)  33.4 (59.6)  **  Phosphate (mg/L)      0.0 (0.1)  0.1 (0.5)    0.1 (0.3)  0.0 (0.2)  0.0 (0.2)    0.0 (0.4)  0.0 (0.2)    Nitrate (mg/L)  50  25b,c  16.2 (11.8)  5.5 (17.8)  ***  13.3 (15.8)  12.3 (17.11)  12.3 (18.0)    11.9 (17.3)  15.0 (18.4)  **  Copper (μg/L)    600b,c  4.5 (12.6)  3.1 (22.2)    1.9 (3.1)  10.4 (22.3)  6.7 (26.5)  **  3.2 (11.4)  6.9 (18.5)    Chromium (μg/L)a  50    1.3 (1.8)  0.0 (0.1)    0.2 (1.7)  0.0 (1.4)  0.0 (1.3)    0.0 (1.2)  0.9 (1.8)    Ammonia (mg/L)a  0.5    0.0 (0.0)  0.0 (0.0)    0.0 (0.0)  0.0 (0.0)  0.0 (0.0)    0.0 (0.0)  0.0 (0.2)    Salinity (PSU)      0.3 (0.1)  0.4 (0.3)  ***  0.3 (0.2)  0.3 (0.2)  0.3 (0.2)    0.2 (0.1)  0.3 (0.2)  ***  Hardness (mg/L)    110c  240 (78)  245 (145)    240 (104)  252 (108)  240 (101)    260 (127)  240 (74)    Iron (μg/L)  200  300b,c  1.1 (4.8)  3.5 (19.4)  *  1.1 (4.8)  1.8 (7.1)  2.3 (7.7)    1.1 (4.8)  2.8 (9.4)  *        Water source    Sampling site    Sampling season    Parameter  Limits of potability Dir.98/83/EC  Maximum acceptable level  Tap water  Well water  Sig.  A  B  C  Sig.  Summer  Winter  Sig.  pH  6.5–9.5  >6b; 5–8c  7.7 (0.4)  7.5 (0.4)  *  7.6 (0.5)  7.6 (0.4)  7.6 (0.4)    7.7 (0.4)  7.6 (0.4)    Sulphate (mg/L)  250  250b; 200c  31.1 (27.0)  36.8 (59.9)  *  30.8 (45.1)  32.0 (42.4)  31.8 (47.9)    27.5 (31.5)  33.4 (59.6)  **  Phosphate (mg/L)      0.0 (0.1)  0.1 (0.5)    0.1 (0.3)  0.0 (0.2)  0.0 (0.2)    0.0 (0.4)  0.0 (0.2)    Nitrate (mg/L)  50  25b,c  16.2 (11.8)  5.5 (17.8)  ***  13.3 (15.8)  12.3 (17.11)  12.3 (18.0)    11.9 (17.3)  15.0 (18.4)  **  Copper (μg/L)    600b,c  4.5 (12.6)  3.1 (22.2)    1.9 (3.1)  10.4 (22.3)  6.7 (26.5)  **  3.2 (11.4)  6.9 (18.5)    Chromium (μg/L)a  50    1.3 (1.8)  0.0 (0.1)    0.2 (1.7)  0.0 (1.4)  0.0 (1.3)    0.0 (1.2)  0.9 (1.8)    Ammonia (mg/L)a  0.5    0.0 (0.0)  0.0 (0.0)    0.0 (0.0)  0.0 (0.0)  0.0 (0.0)    0.0 (0.0)  0.0 (0.2)    Salinity (PSU)      0.3 (0.1)  0.4 (0.3)  ***  0.3 (0.2)  0.3 (0.2)  0.3 (0.2)    0.2 (0.1)  0.3 (0.2)  ***  Hardness (mg/L)    110c  240 (78)  245 (145)    240 (104)  252 (108)  240 (101)    260 (127)  240 (74)    Iron (μg/L)  200  300b,c  1.1 (4.8)  3.5 (19.4)  *  1.1 (4.8)  1.8 (7.1)  2.3 (7.7)    1.1 (4.8)  2.8 (9.4)  *  a Statistical testing not performed because of insufficient non-zero observations. b Carter and Sneed, 1996 c Aviagen Turkey Inc., 2015 View Large Table 2. Microbiological analyses of drinking water in 28 turkey farms supplied with either well or tap water during summer and winter of 2012–2013 in North-Eastern Italy. Water samples were collected twice in 2012–2013 (in winter and in summer) at 3 sampling sites: the water source (A), the beginning (B) and the end (C) of the nipple line. Significance for P < 0.05.     Water source  Sampling site  Sampling season  Parameter  Category  Tap  Well  P  A  B  C  P  Summer  Winter  P  Total microbial count 22°C  >103 UFC/mL (%)  60.7  46.4  0.29  5.4  71.4  83.9  <0.001  58.3  48.8  0.09  E. coli and Enterococcus  >102 UFC/100 mL (%)  15.5  35.7  0.44  7.1  35.7  33.9  <0.001  31.0  20.2  0.06  Campylobacter jejuni  PCR+ (%)  15.5  15.5  0.55  10.7  19.6  16.1  0.66  8.3  22.6  0.09  Campylobacter coli  PCR+ (%)  32.1  28.6  0.21  26.8  35.7  28.6  0.79  17.9  42.9  <0.001      Water source  Sampling site  Sampling season  Parameter  Category  Tap  Well  P  A  B  C  P  Summer  Winter  P  Total microbial count 22°C  >103 UFC/mL (%)  60.7  46.4  0.29  5.4  71.4  83.9  <0.001  58.3  48.8  0.09  E. coli and Enterococcus  >102 UFC/100 mL (%)  15.5  35.7  0.44  7.1  35.7  33.9  <0.001  31.0  20.2  0.06  Campylobacter jejuni  PCR+ (%)  15.5  15.5  0.55  10.7  19.6  16.1  0.66  8.3  22.6  0.09  Campylobacter coli  PCR+ (%)  32.1  28.6  0.21  26.8  35.7  28.6  0.79  17.9  42.9  <0.001  View Large Table 2. Microbiological analyses of drinking water in 28 turkey farms supplied with either well or tap water during summer and winter of 2012–2013 in North-Eastern Italy. Water samples were collected twice in 2012–2013 (in winter and in summer) at 3 sampling sites: the water source (A), the beginning (B) and the end (C) of the nipple line. Significance for P < 0.05.     Water source  Sampling site  Sampling season  Parameter  Category  Tap  Well  P  A  B  C  P  Summer  Winter  P  Total microbial count 22°C  >103 UFC/mL (%)  60.7  46.4  0.29  5.4  71.4  83.9  <0.001  58.3  48.8  0.09  E. coli and Enterococcus  >102 UFC/100 mL (%)  15.5  35.7  0.44  7.1  35.7  33.9  <0.001  31.0  20.2  0.06  Campylobacter jejuni  PCR+ (%)  15.5  15.5  0.55  10.7  19.6  16.1  0.66  8.3  22.6  0.09  Campylobacter coli  PCR+ (%)  32.1  28.6  0.21  26.8  35.7  28.6  0.79  17.9  42.9  <0.001      Water source  Sampling site  Sampling season  Parameter  Category  Tap  Well  P  A  B  C  P  Summer  Winter  P  Total microbial count 22°C  >103 UFC/mL (%)  60.7  46.4  0.29  5.4  71.4  83.9  <0.001  58.3  48.8  0.09  E. coli and Enterococcus  >102 UFC/100 mL (%)  15.5  35.7  0.44  7.1  35.7  33.9  <0.001  31.0  20.2  0.06  Campylobacter jejuni  PCR+ (%)  15.5  15.5  0.55  10.7  19.6  16.1  0.66  8.3  22.6  0.09  Campylobacter coli  PCR+ (%)  32.1  28.6  0.21  26.8  35.7  28.6  0.79  17.9  42.9  <0.001  View Large Salmonella enterica subsp. enterica serotype Kentucky was isolated only from WW at site A, both in summer and winter from the same farm. Campylobacter spp. was isolated only twice in a farm supplied with WW and from another one supplied with TW at site B and C in winter. Positivity at real-time PCR was found for C. coli in 30.4% of samples and for C. jejuni in 14.9% of samples. Table 2 shows the associations between Campylobacter spp. positivity at real-time PCR and source of water supply, season and sampling site; only season was found to be significant, with winter posing a higher risk for C. jejuni positivity in water. Table 3 shows the associations between putative risk factors for real-time PCR positivity to C. jejuni, C. coli and for total microbial count: low salinity and high hardness were identified as risk factors for presence of C. coli and C. jejuni, respectively. Table 3. Risk factors for RT-PCR positive samples to Campylobacter jejuni, Campylobacter coli and for total microbial count (TMC 22°C) >102 in 28 turkey farms sampled in North-Eastern Italy in summer and winter 2012–2013. Continuous variables were classified on the basis of the data distribution. Significance for P < 0.05.     Campylobacter coli  Campylobacter jejuni  TMC 22°C >102  Parameter  Category  n.  %  P  n.  %  P  n.  %  P  Supply system with water recirculation  Yes  42  35.0  0.08  19  15.8  0.87  66  55.0  0.83    No  9  18.8    7  14.6    24  50.0    Birds age (days)  ≤30  13  31.0  0.35  4  9.5  0.45  20  47.6  0.58    30–50  18  37.5    10  20.8    27  56.3      51–75  12  28.6    6  14.3    26  61.9      >75  8  22.2    6  16.7    17  47.2    pH  ≤7.36  39  28.2  0.94  39  12.8  0.86  39  12.8  0.49    7.37-7.63  45  37.8    45  13.3    45  13.3      7.64-7.83  41  29.3    41  19.5    41  19.5      >7.83  43  25.6    43  16.3    43  16.3    Salinity (PSU)  ≤0.21  42  35.7  0.04  42  16.7  0.11  42  4.8  0.80    0.22-0,28  39  38.5    39  10.3    44  15.9      0.29-0,38  43  20.9    43  14.0    48  20.8      >0.38  44  27.3    44  20.5    34  20.6    Hardness (mg/L)  ≤191  42  21.4  0.40  42  4.8  0.05  42  16.7  0.37    192–240  44  34.1    44  15.9    39  10.3      241–300  48  29.2    48  20.8    43  14.0      >300  34  38.2    34  20.6    44  20.5    Iron (μg/L)  ≤ LOD  47  23.4  0.55  47  8,5  0.76  47  8.5  0.35    LOD – 2.28  37  40.5    37  21.6    37  21.6      2.29-12.22  42  26.2    42  11.9    42  11.9      >12.22  42  33.3    42  21.4    42  21.4        Campylobacter coli  Campylobacter jejuni  TMC 22°C >102  Parameter  Category  n.  %  P  n.  %  P  n.  %  P  Supply system with water recirculation  Yes  42  35.0  0.08  19  15.8  0.87  66  55.0  0.83    No  9  18.8    7  14.6    24  50.0    Birds age (days)  ≤30  13  31.0  0.35  4  9.5  0.45  20  47.6  0.58    30–50  18  37.5    10  20.8    27  56.3      51–75  12  28.6    6  14.3    26  61.9      >75  8  22.2    6  16.7    17  47.2    pH  ≤7.36  39  28.2  0.94  39  12.8  0.86  39  12.8  0.49    7.37-7.63  45  37.8    45  13.3    45  13.3      7.64-7.83  41  29.3    41  19.5    41  19.5      >7.83  43  25.6    43  16.3    43  16.3    Salinity (PSU)  ≤0.21  42  35.7  0.04  42  16.7  0.11  42  4.8  0.80    0.22-0,28  39  38.5    39  10.3    44  15.9      0.29-0,38  43  20.9    43  14.0    48  20.8      >0.38  44  27.3    44  20.5    34  20.6    Hardness (mg/L)  ≤191  42  21.4  0.40  42  4.8  0.05  42  16.7  0.37    192–240  44  34.1    44  15.9    39  10.3      241–300  48  29.2    48  20.8    43  14.0      >300  34  38.2    34  20.6    44  20.5    Iron (μg/L)  ≤ LOD  47  23.4  0.55  47  8,5  0.76  47  8.5  0.35    LOD – 2.28  37  40.5    37  21.6    37  21.6      2.29-12.22  42  26.2    42  11.9    42  11.9      >12.22  42  33.3    42  21.4    42  21.4    View Large Table 3. Risk factors for RT-PCR positive samples to Campylobacter jejuni, Campylobacter coli and for total microbial count (TMC 22°C) >102 in 28 turkey farms sampled in North-Eastern Italy in summer and winter 2012–2013. Continuous variables were classified on the basis of the data distribution. Significance for P < 0.05.     Campylobacter coli  Campylobacter jejuni  TMC 22°C >102  Parameter  Category  n.  %  P  n.  %  P  n.  %  P  Supply system with water recirculation  Yes  42  35.0  0.08  19  15.8  0.87  66  55.0  0.83    No  9  18.8    7  14.6    24  50.0    Birds age (days)  ≤30  13  31.0  0.35  4  9.5  0.45  20  47.6  0.58    30–50  18  37.5    10  20.8    27  56.3      51–75  12  28.6    6  14.3    26  61.9      >75  8  22.2    6  16.7    17  47.2    pH  ≤7.36  39  28.2  0.94  39  12.8  0.86  39  12.8  0.49    7.37-7.63  45  37.8    45  13.3    45  13.3      7.64-7.83  41  29.3    41  19.5    41  19.5      >7.83  43  25.6    43  16.3    43  16.3    Salinity (PSU)  ≤0.21  42  35.7  0.04  42  16.7  0.11  42  4.8  0.80    0.22-0,28  39  38.5    39  10.3    44  15.9      0.29-0,38  43  20.9    43  14.0    48  20.8      >0.38  44  27.3    44  20.5    34  20.6    Hardness (mg/L)  ≤191  42  21.4  0.40  42  4.8  0.05  42  16.7  0.37    192–240  44  34.1    44  15.9    39  10.3      241–300  48  29.2    48  20.8    43  14.0      >300  34  38.2    34  20.6    44  20.5    Iron (μg/L)  ≤ LOD  47  23.4  0.55  47  8,5  0.76  47  8.5  0.35    LOD – 2.28  37  40.5    37  21.6    37  21.6      2.29-12.22  42  26.2    42  11.9    42  11.9      >12.22  42  33.3    42  21.4    42  21.4        Campylobacter coli  Campylobacter jejuni  TMC 22°C >102  Parameter  Category  n.  %  P  n.  %  P  n.  %  P  Supply system with water recirculation  Yes  42  35.0  0.08  19  15.8  0.87  66  55.0  0.83    No  9  18.8    7  14.6    24  50.0    Birds age (days)  ≤30  13  31.0  0.35  4  9.5  0.45  20  47.6  0.58    30–50  18  37.5    10  20.8    27  56.3      51–75  12  28.6    6  14.3    26  61.9      >75  8  22.2    6  16.7    17  47.2    pH  ≤7.36  39  28.2  0.94  39  12.8  0.86  39  12.8  0.49    7.37-7.63  45  37.8    45  13.3    45  13.3      7.64-7.83  41  29.3    41  19.5    41  19.5      >7.83  43  25.6    43  16.3    43  16.3    Salinity (PSU)  ≤0.21  42  35.7  0.04  42  16.7  0.11  42  4.8  0.80    0.22-0,28  39  38.5    39  10.3    44  15.9      0.29-0,38  43  20.9    43  14.0    48  20.8      >0.38  44  27.3    44  20.5    34  20.6    Hardness (mg/L)  ≤191  42  21.4  0.40  42  4.8  0.05  42  16.7  0.37    192–240  44  34.1    44  15.9    39  10.3      241–300  48  29.2    48  20.8    43  14.0      >300  34  38.2    34  20.6    44  20.5    Iron (μg/L)  ≤ LOD  47  23.4  0.55  47  8,5  0.76  47  8.5  0.35    LOD – 2.28  37  40.5    37  21.6    37  21.6      2.29-12.22  42  26.2    42  11.9    42  11.9      >12.22  42  33.3    42  21.4    42  21.4    View Large DISCUSSION While surface water is a recognized vehicle of disease transmission for poultry (Amaral, 2004), the present study showed that WW given to commercial turkeys in North-Easter Italy is of satisfactory quality for the analysed parameters and therefore represents a suitable alternative to TW for livestock. Most turkey farms presented levels of nitrates lower than the limits for potability (50 mg/L). High levels of nitrates can be related to contamination with residential, industrial or agricultural waste. In humans, consumption of drinking water with high nitrate levels (i.e. higher than 50 mg/L) may cause hypertrophy of the thyroid (Van Maanen et al., 1994). The toxicity for poultry has been reported at levels higher than 50 mg/L for chickens and 75 mg/L for turkeys, while in broilers nitrate levels greater than 20 mg/L may have a negative effect on growth and feed conversion (as reviewed by Carter and Sneed, 1996). Also naturally occurring chemicals (i.e. copper and iron) were within the recommended limits for poultry (Carter and Sneed, 1996). Copper levels higher than 600 μg/L produce bitter flavor that may reduce water consumption (Carter and Sneed, 1996). Iron and copper are contained in valves, pipes and fittings and are present in coatings and alloys. This can explain the higher concentrations at site B and C compared to A, as the main source of contamination is often the corrosion of interior plumbing (WHO, 2011). Iron levels higher than 300 μg/L affect flavor, turbidity and color of water, as well as staining of plumbing fixtures (WHO, 2011). Although a high level of iron (600 ppm) in water was not found to affect broiler performances (Fairchild et al., 2006), it may form chelates with apramycin and tetracyclines, thus limiting the therapeutic effect of water medication (Scandurra, 2013). Well water had higher levels of hardness in comparison to TW. However, samples from both sources often presented levels higher than 200 mg/L, which is known to be critical for causing calcium deposits in the pipeline (Enne et al., 2006). Hard water has not been demonstrated to have either a positive or a negative direct impact on poultry health and performance (Carter and Sneed, 1996). However, hardness can decrease drugs solubility, preventing animals from receiving an effective dose (Scandurra, 2013). Although pH was within the range of acceptability for potable water (European Commission, 1998), it was significantly lower in WW. A level of pH lower than 5.5 can create problems to the urinary and digestive systems, bone demineralization and fragility, as well as corroding the pipeline and being incompatible with some medicines and vaccines (Enne et al., 2006). Water pH ranging between 6.0 and 6.3 is suspected of having negative effects on poultry performances (Carter and Sneed, 1996). On the other hand, Grizzle et al. (1996) found that a water pH of 6.25 did not negatively affect broiler growth in comparison with a water pH of 6.75. Rather, a water pH of 5.75 negatively affected it in comparison with a water pH of 6.25 and 6.75, respectively. Only a few samples had a high level of ammonia, whose contamination can derive from industrial and agricultural waste (WHO, 2011). To the best of our knowledge, we are not aware of any studies describing the effect of high levels of water ammonia on poultry health. However, ammonia can react with chlorine to reduce free chlorine and to form chloramines (WHO, 2011). Most samples presented a total microbial count (a general indicator of pipeline hygiene) under the limits for TW, and a very low fecal contamination (i.e. presence of E. coli and Enterococcus). Instead, a significant decrease in microbiological water quality was found at the nipple line. Water samples varied in hardness and iron levels. High levels of iron and hardness are known to be risk factors for biofilm deposition and bacterial proliferation in the pipeline (Wingender and Flemming, 2011). Specifically, iron promotes the growth of bacteria that derive their energy from the oxidation of ferrous iron to ferric iron (WHO, 2011). The formation of biofilms increases with the flow velocity of water (Lehtola et al., 2006); therefore, bird age is indirectly a further risk factor, as when birds are young the limited water consumption may be associated with a low water flow. Yet, poor microbiological water quality was not significantly associated with its physical and chemical properties, nor with the presence of a water recirculation systems and birds’ age. Taken together, these results call for improvements in microbiological water quality as directly related to sanitization procedures applied by farmers, which need to be reviewed. Farmers had also declared to routinely use different commercial products to guarantee pipeline hygiene. However, as previously suggested (Sparks, 2009), the efficacy of these products may largely differ depending on water properties. Isolation of potentially pathogenic microorganisms was generally uncommon. However, S. Kentucky was isolated from the same WW samples in different seasons at the water source, raising some concerns on the potential role of WW as vehicle of Salmonella transmission. Also Campylobacter was rarely isolated, although the positivity detected by real-time PCR ranged between 8% to 43%, irrespective of the sampling site. Pipeline hygiene was not influenced by season, while Campylobacter positivity was higher in winter. This is in accordance with previous findings which identified an optimal Campylobacter survival at low temperatures (around 4°C) (Thomas et al., 1998). The low frequency of Campylobacter isolation confirms the well know limitation of culture-based procedures in isolating the microorganism from water samples, which is likely to underestimate the true prevalence due to the high susceptibility of Campylobacter to suboptimal environmental conditions (Thomas et al., 1998; Chaisowwong et al., 2012). Moreover, the bacterium can be present in water also as viable but non-culturable forms, which are able to survive under adverse conditions (Rollins and Colwell, 1986). For these reasons, real-time PCR is a valid support to detect the microorganism in water and to understand possible routes of transmission. While no significant association between Campylobacter spp. presence and source of water supply was found, low salinity and high hardness were identified as risk factors for presence of C. coli and C. jejuni, respectively. Moreover, there was a tendency towards significance (P = 0.08) in the association between presence of a water recirculation system and C. coli. As previously suggested (Sahin et al., 2015), Campylobacter transmission to birds is more likely to occur from the farm environment through water, rather than from the water source itself (Bull et al., 2006; Mughini-Gras et al., 2016). In conclusion, the results of the present study call for improvements in sanitization procedures for farm drinking water pipelines, highlighting also issues related to drinking water characterized by high levels of hardness and iron. While water recirculation systems, bird age, and most chemical and physical water properties did not seem to be associated with microbiological water quality, low salinity and high hardness were specific risk factors for C. coli and C. jejuni presence, respectively. Although Campylobacter spp. isolation from water samples was problematic, detection of Campylobacter spp. genetic material showed that this zoonotic pathogen is highly prevalent in the farm pipeline. SUPPLEMENTARY DATA Supplementary data are available at Poultry Science online. Acknowledgements This study was funded by the Italian Ministry of Health, Rome, Italy (Project RC IZSVe 08/11; B21J12000280001). REFERENCES Amaral L. 2004. Drinking water as a risk factor to poultry health. Rev. Bras. Cienc. Avic . 6: 191– 199. Google Scholar CrossRef Search ADS   Agenzia per la protezione dell’ambiente e per i servizi tecnici (APAT). 2003. Metodi analitici per le acque. Available from: http://www.isprambiente.gov.it/it/pubblicazioni/manuali-e-linee-guida/metodi-analitici-per-le-acque. Aviagen Turkeys Inc. 2015. Management Guidelines - Raising commercial turkeys. Available at: http://www.aviagenturkeys.us/uploads/2015/12/21/Aviagen%20Commercial%20Guide.pdfLast accessed on 5/3/2018. Bull S., Allen V., Domingue G., Jorgensen F., Frost J., Ure R., Whyte R., Tinker D., Corry J., Gillard-King J., Humphrey T.. 2006. Sources of Campylobacter spp. Colonizing Housed Broiler Flocks during Rearing. Appl. Environ. Microbiol.  72: 645– 652. Google Scholar CrossRef Search ADS PubMed  Carter T. A., Sneed R. E.. 1996. Drinking water quality for poultry. North Carolina Cooperative Extension Service . Available from: https://www.bae.ncsu.edu/bae/extension/ext-publications/water/drinking/pst42-wqg-poultry-sneed.pdf Chaisowwong W., Kusumoto A., Hashimoto M., Harada T., Maklon K., Kawamoto K.. 2012. Physiological Characterization of Campylobacter jejuni under Cold Stresses Conditions: Its Potential for Public Threat. J. Vet. Med. Sci.  74: 43– 50. Google Scholar CrossRef Search ADS PubMed  Enne G., Greppi G., Serratoni M.. 2006. The role of water in animal breeding. Ital J. Agronomy . 1: 519– 527. Google Scholar CrossRef Search ADS   European Commission. 1998a. Council Directive (EC) 98/58/EC of 20 July 1998 concerning the protection of animals kept for farming purposes. Off. J. Eur. Comm.  L221: 23– 27. European Commission. 1998b. Council Directive (EC) 98/83/EC of 3 November 1998 on the quality of water intended for human consumption. Off. J. Eur. Comm . L330: 32– 54 European Commission. 2004. Council Regulation (EC) No 852/2004 of the European Parliament and of the Council of 29 April 2004 on the hygiene of foodstuffs. Off. J. Eur. Union  L139: 1– 54. European Food Safety Authority. 2011. The European Union Summary Report on Trends and Sources of Zoonoses, Zoonotic Agents and Food-borne Outbreaks in 2009. EFSA Journal . 9: 2090. CrossRef Search ADS   Fairchild B., Batal A., Ritz C., Vendrell P.. 2006. Effect of Drinking Water Iron Concentration on Broiler Performance. The Journal of Applied Poultry Research . 15: 511– 517. Google Scholar CrossRef Search ADS   Grizzle J., Armbrust T., Bryan M., Saxton A.. 1996. Water quality I: The effect of water nitrate and pH on broiler growth performance. The Journal of Applied Poultry Research . 5: 330– 336. Google Scholar CrossRef Search ADS   Giacomelli M., Andrighetto C., Lombardi A., Martini M., Piccirillo A.. 2012. A Longitudinal Study on Thermophilic Campylobacter spp. in Commercial Turkey Flocks in Northern Italy: Occurrence and Genetic Diversity. Avian Dis . 56: 693– 700. Google Scholar CrossRef Search ADS PubMed  ISO. 1999. Microbiology of food and animal feeding stuffs. Preparation of Test Samples, Initial Suspension and Decimal Dilutions for Microbiological Examination. Part 1: General rules for the preparation of the initial suspension and decimal dilutions. in ISO 6887-1:1999 . International Organization for Standardization, Geneva, Switzerland. ISO. 2002. Microbiology of food and animal feeding stuffs - Horizontal method for the detection of Salmonella spp . in ISO 6579:2002 . International Organization for Standardization, Geneva, Switzerland. ISO. 2005a. Water quality - General guidance on the enumeration of micro-organisms by culture. in ISO 8199:2005 . International Organization for Standardization, Geneva, Switzerland. ISO. 2005b. Water quality - Detection and enumeration of thermotolerant Campylobacter species . in ISO 17995:2005 . International Organization for Standardization, Geneva, Switzerland. Istituto Superiore di Sanità. 2007. Reference analytical methods for water intended for human consumption according to the Italian Legislative Decree 31/2001. Microbiological Methods . In: L. Bonadonna, M. Ottaviani, (Eds.), Rapporti ISTISAN 07/5. Rome (Italy). Available from: http://www.iss.it/binary/aqua/cont/RappIstisan%2007%205.1204715346.pdf Kapperud G., Skjerve E., Vik L., Hauge K., Lysaker A., Aalmen I., Ostroff S., Potter M.. 1993. Epidemiological investigation of risk factors for Campylobacter colonization in Norwegian broiler flocks. Epidemiol. Infect.  111: 245– 256. Google Scholar CrossRef Search ADS PubMed  Lehtola M., Laxander M., Miettinen I., Hirvonen A., Vartiainen T., Martikainen P.. 2006. The effects of changing water flow velocity on the formation of biofilms and water quality in pilot distribution system consisting of copper or polyethylene pipes. Water Res.  40: 2151– 2160. Google Scholar CrossRef Search ADS PubMed  Lund V. 1996. Evaluation of E. coli as an indicator for the presence of Campylobacter jejuni and Yersinia enterocolitica in chlorinated and untreated oligotrophic lake water. Water Res.  30: 1528– 1534. Google Scholar CrossRef Search ADS   Mughini-Gras L., Penny C., Ragimbeau C., Schets F., Blaak H., Duim B., Wagenaar J., de Boer A., Cauchie H., Mossong J., van Pelt W.. 2016. Quantifying potential sources of surface water contamination with Campylobacter jejuni and Campylobacter coli. Water Res.  101: 36– 45. Google Scholar CrossRef Search ADS PubMed  Mulatti P., Ferrè N., Marangon S.. 2011. Spatial distribution of 2000–2007 low pathogenicity Avian Influenza Epidemics in Northern Italy. In: S.K. Majumdat, Brenner F. J., Huffman J. E., McLean R. G, Panah A. I, Pietrobon P. J, Keeler S. P, Shive S.. Pandemic Influenza Viruses: Science, Surveillance and Public Health , pp 232– 247. The Pennsylvania Academy of Science: Easton, PA. OIE Terrestrial Animal Health Code. 2016. Chapter 6.4. Biosecurity procedures in poultry production . 25th Edn. 2016:1-6. Available from: http://www.oie.int/index.php?id = 169&L = 0&htmfile = chapitre_biosecu_poul_production.htm Rollins D. M., Colwell R. R.. 1986. Viable but nonculturable stage of Campylobacter jejuni and its role in survival in the natural aquatic environment. Appl. Environ. Microb . 52: 531– 538. Sahin O., Kassem I. I., Shen Z., Lin J., Rajashekara G., Zhang Q.. 2015. Campylobacter in Poultry: Ecology and Potential Interventions. Avian Dis.  59: 185– 200. Google Scholar CrossRef Search ADS PubMed  Scandurra S. 2013. Veterinary drugs in drinking water used for pharmaceutical treatments in breeding farms. PhD Thesis, University of Bologna, Bologna (Italy). Sparks N. 2009. The role of the water supply system in the infection and control of Campylobacter in chicken. Worlds Poult. Sci. J.  65: 459– 474. Google Scholar CrossRef Search ADS   Standing Committee of Analysts. 2002 The Microbiology of Drinking Water - Part 3 - Practices and procedures for laboratories. Available from: https://www.gov.uk/government/publications/standing-committee-of-analysts-sca-blue-books Thomas C., Gibson H., Hill D., Mabey M.. 1998. Campylobacter epidemiology: an aquatic perspective. J. Appl. Microb . 85: 168S– 177S. Google Scholar CrossRef Search ADS   Toplak N., Kovač M., Piskernik S., Možina S., Jeršek B.. 2012. Detection and quantification of Campylobacter jejuni and Campylobacter coli using real-time multiplex PCR. J. Appl. Microbiol.  112: 752– 764. Google Scholar CrossRef Search ADS PubMed  Umar S., Munir M. T., Azeem T T., Ali S., Umar W., Rehman A., Shah M. A.. 2014. Effects of water quality on productivity and performance of livestock: A mini review. Veterinaria  2: 11– 15. UNI. 2001. Water quality - Enumeration of culturable micro-organisms - Colony Count by Inoculation in a Nutrient Agar Culture Medium. in UNI EN ISO 6222:2001 . Ente Nazionale Italiano di Unificazione Milan, Italy. UNI. 2002. Water quality - Detection and enumeration of Escherichia coli and coliform bacteria - Part 1: Membrane filtration method. in UNI EN ISO 9308-1:2002 . Ente Nazionale Italiano di Unificazione Milan, Italy. UNI. 2003. Water quality - Detection and enumeration of intestinal enterococci - Part 2: Membrane filtration method. in UNI EN ISO 7899-2:2003 . Ente Nazionale Italiano di Unificazione Milan, Italy. Van Maanen J., van Dijk A., Mulder K., de Baets M., Menheere P., van der Heide D., Mertens P., Kleinjans J.. 1994. Consumption of drinking water with high nitrate levels causes hypertrophy of the thyroid. Toxicol. Lett.  72: 365– 374. Google Scholar CrossRef Search ADS PubMed  Williams L. K., Sait L., Cogan T., Jørgensen F., Grogono-Thomas R., Humphrey T. J.. 2012. Enrichment culture can bias the isolation of Campylobacter subtypes. Epidemiol. Infect.  140: 1227– 1235. Google Scholar CrossRef Search ADS PubMed  Wingender J., Flemming H.. 2011. Biofilms in drinking water and their role as reservoir for pathogens. Int. J. Hyg. Environ. Health . 214: 417– 423. Google Scholar CrossRef Search ADS PubMed  World Health Organization. 2011. Guidelines for Drinking-water Quality  Fourth ed. WHO Press, Geneva, Switzerland. Yamazaki-Matsune W., Taguchi M., Seto K., Kawahara R., Kawatsu K., Kumeda Y., Kitazato M., Nukina M., Misawa N., Tsukamoto T.. 2007. Development of a multiplex PCR assay for identification of Campylobacter coli, Campylobacter fetus, Campylobacter hyointestinalis subsp. hyointestinalis, Campylobacter jejuni, Campylobacter lari and Campylobacter upsaliensis. J. Med. Microbiol.  56: 1467– 1473. Google Scholar CrossRef Search ADS PubMed  Zimmer M., Barnhart H., Idris U., Lee M.. 2003. Detection of Campylobacter jejuni Strains in the Water Lines of a Commercial Broiler House and Their Relationship to the Strains That Colonized the Chickens. Avian Dis.  47: 101– 107. Google Scholar CrossRef Search ADS PubMed  © The Author(s) 2018. Published by Oxford University Press on behalf of Poultry Science Association. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Poultry Science Oxford University Press

Microbiological, chemical and physical quality of drinking water for commercial turkeys: a cross-sectional study

Loading next page...
 
/lp/ou_press/microbiological-chemical-and-physical-quality-of-drinking-water-for-DwsdotySs0
Publisher
Oxford University Press
Copyright
© The Author(s) 2018. Published by Oxford University Press on behalf of Poultry Science Association.
ISSN
0032-5791
eISSN
1525-3171
D.O.I.
10.3382/ps/pey130
Publisher site
See Article on Publisher Site

Abstract

Abstract Drinking water for poultry is not subject to particular microbiological, chemical and physical requirements, thereby representing a potential transmission route for pathogenic microorganisms and contaminants and/or becoming unsuitable for water-administered medications. This study assessed the microbiological, chemical and physical drinking water quality of 28 turkey farms in North-Eastern Italy: 14 supplied with tap water (TW) and 14 with well water (WW). Water salinity, hardness, pH, ammonia, sulphate, phosphate, nitrate, chromium, copper and iron levels were also assessed. Moreover, total bacterial count at 22°C, presence and enumeration of Enterococcus spp. and E. coli, presence of Salmonella spp. and Campylobacter spp. were quantified. A water sample was collected in winter and in summer at 3 sampling sites: the water source (A), the beginning (B) and the end (C) of the nipple line (168 samples in total). Chemical and physical quality of both TW and WW sources was mostly within the limits of TW for humans. However, high levels of hardness and iron were evidenced in both sources. In WW vs. TW, sulphate and salinity levels were significantly higher, whilst pH and nitrate levels were significantly lower. At site A, microbiological quality of WW and TW was mostly within the limit of TW for humans. However, both sources had a significantly lower microbiological quality at sites B and C. Salmonella enterica subsp. enterica serotype Kentucky was isolated only twice from WW. Campylobacter spp. were rarely isolated (3.6% of farms); however, Campylobacter spp. farm-level prevalence by real-time PCR was up to 43% for both water sources. Winter posed at higher risk than summer for Campylobacter spp. presence in water, whereas no significant associations were found with water source, site, recirculation system, and turkey age. Low salinity and high hardness were significant risk factors for C. coli and C. jejuni presence, respectively. These results show the need of improving sanitization of drinking water pipelines for commercial turkeys. INTRODUCTION The quality of drinking water for livestock is a subject of utmost importance, as it can directly and indirectly affect animal health and productivity (Umar et al., 2014). Although general recommendations as well as specific guidelines for poultry water quality are available (Carter and Sneed, 1996; Amaral, 2004), farmers are often unaware of the importance of water quality (Umar et al., 2014). In the European Union (EU), this subject has received little attention in terms of EU legislation, as drinking water for poultry is not subject to particular microbiological, chemical and physical requirements. Regulation (EC) 852/2004 on the hygiene of foodstuffs establishes minimum requirements for livestock drinking water, but no qualitative parameters are listed (European Commission, 2004). Council Directive 98/58/EC on farmed animal welfare states that ‘all animals must have access to a suitable water supply or be able to satisfy their fluid intake needs by other means’, although no suitability thresholds are indicated (European Commission, 1998a). On the other hand, the Terrestrial Animal Health Code (Art. 6.4.5) of the OIE-World Organization for Animal Health states that ‘the drinking water supply to poultry houses should be potable according to the WHO or to the relevant national standard’ (OIE, 2016). This indication derives either from the possible transmission of pathogens, such as Salmonella and Campylobacter (Amaral, 2004), or from the possible infiltration of contaminants (Carter and Sneed, 1996). Recently, the European Food Safety Authority (EFSA, 2011) indicated farm water as one of the sources of direct contamination with Campylobacter for livestock and humans. Campylobacter is more susceptible than E. coli to water chlorination (Lund, 1996), and it was reported a 3.5-fold risk of infection for broiler flocks supplied with unchlorinated vs. chlorinated water (Kapperud et al., 1993). Moreover, C. jejuni has been isolated from the biofilm of nipple drinking systems for poultry when the birds were also colonized (Zimmer et al., 2003), although there is limited evidence for such drinking systems to be the source of Campylobacter colonization. This may be due to failure to detect Campylobacter in water as a result of insufficient water volumes processed or of microorganisms in a viable, but not culturable, state (Sparks, 2009). Data on the microbiological, chemical and physical quality of drinking water for livestock in Italy are scarce. Moreover, in the poultry sector, groundwater is frequently used, with no compulsory periodical water quality controls being requested for this source of water supply. Finally, turkeys are often treated with medicines, including antimicrobials, administered with drinking water, and some chemical and physical water properties like pH, hardness and iron levels may interfere with drug dissolution and stability in water (Scandurra, 2013). For these reasons, the present study aimed to assess the microbiological, chemical and physical quality of drinking water in commercial turkey farms supplied with either tap or well water. A number of factors putatively associated with water quality (e.g., environmental conditions, husbandry practices, season, water recirculation system, etc.) were also investigated, and water quality at different sampling sites along the farm water pipeline was assessed. MATERIALS AND METHODS Sample Collection Samples from 14 turkey farms supplied with well water (WW) and 14 supplied with tap water (TW) were analyzed. Both groups were randomly selected within the densely-populated poultry area (DPPA) of North-Eastern Italy. This area is characterized by the highest density of poultry in Italy and one of the greatest in Europe (Mulatti et al., 2011). Farms under study were operational for an average of 17 ± 12 years (SD) and consisted of 4 ± 2 sheds housing 10.000-70.000 turkeys in total; birds therein were 55 ± 35 days of age. Water wells were 65 ± 45 m deep underground. Seventeen farms had a system for water recirculation vs. 11 without such system. In order to guarantee pipeline hygiene, all farmers declared to apply a pipeline sanitization protocol as part of their normal operating procedures with stabilized hydrogen peroxide at each new production cycle (concentration of 2–3%) and continuously when the drinking system was operating (concentration of 25–50 ppm). Water samples were collected in 2012–2013 in late winter (February-March) and in mid-summer (July-August) at 3 sampling sites: the water source (A), the tank at the beginning of the nipple line where medicines are mixed for administration via water (B) and the end of the nipple line (C). Water temperature at the source and pH at A, B, C were also measured. Samples were collected in sterile containers, delivered to the laboratory at 4°C and processed within 24 h for microbiological analyses and 48 h for chemical and physical analyses. Chemical and Physical Analyses Ammonia concentration was determined by spectrometric indophenol assay (APAT, 2003), while the concentration of nitrate, sulphate and phosphate was determined by ionic chromatography (APAT, 2003). Chromium, copper and iron concentrations were quantified by atomic absorption spectrophotometry (APAT, 2003). The EDTA (ethylenediaminetetraacetic acid) titration method was used to determine the level of hardness (APAT, 2003). Microbiological Analyses Water samples were tested for total bacterial count at 22°C, enumeration of Enterococcus spp., enumeration of E. coli, presence of Salmonella spp. While specific legislation for animal drinking water is not available, analyses were performed according to European Council Directive 98/83/EC on the quality of water intended for human consumption (European Commission, 1998b). A national reference guideline (ISS, 2007), which set analytical methods for water intended for human consumption, was also considered. Analyses for enumeration of Enterococcus spp. and E. coli were performed according to UNI EN ISO 7899-2 (UNI, 2003) and UNI EN ISO 9308-1 (UNI, 2002), respectively. Enumeration of total bacterial count at 22°C and 37°C were performed according to UNI EN ISO 6222 for colony count (UNI, 2001), and according to ISO 6887-1 (ISO, 1999) and ISO 8199 (ISO, 2005a) for sample dilutions and for membrane filtration method. For the detection of Salmonella spp., 1 L samples were tested by membrane filtration method and then processed according to ISO 6579 (ISO, 2002). To detect the presence of Campylobacter spp., both cultivation and real-time PCR were performed. Isolation was performed according to the standard method for Campylobacter detection in water (ISO, 2005b), which was slightly modified (SCA, 2002; Williams et al., 2012). Briefly, water samples were filtrated through a sterile membrane with a pore size of 0.2 μm (Sartorius, Goettingen, Germania) in a vacuum pump system. Then, each membrane was inoculated into 50 mL of Exeter broth (Mast Diagnostics, Merseyside, UK) and incubated at 41.5°C for 48 h under microaerobic conditions. Two hundred μL of each broth culture were streaked onto Karmali agar (OXOID, Basingstoke, UK), after passive filtration (Giacomelli et al., 2012), and incubated at 41.5°C for 48 h under microaerobic conditions. Suspected Campylobacter colonies were examined by multiplex end-point PCR for genus and species identification (Yamazaki-Matsune et al., 2007). For real-time PCR, water samples were filtrated as described above and filters inoculated and vortexed in 5 mL of deionized water. Three mL of the suspension were centrifuged at 5,000 rpm for 10 min and the pellet resuspended in 200 μL of PBS. The DNA extraction was carried out by using the High Pure Template Preparation Kit (Roche Diagnostics, Mannheim, Germany) according to the manufacturer's instructions, and the multiplex real-time PCR assay by using Taqman® probes and specific primers for C. jejuni and C. coli detection, as previously described (Toplak et al., 2012). Statistical Analyses An explorative data analysis was performed to examine the distributions of chemical and physical parameters. Given the high skewness of all these parameters, median and interquartile range were used as descriptive statistics, and, for each parameter, linear quantile mixed model targeting the median was adopted to assess the association with the type of water source (TW or WW), the sampling site (A, B or C) and the season (winter or summer), that represented the fixed effects in the model. To take into account the hierarchical structure of the sampling design, the farm was included in the model as grouping factor, whereas sampling site and season were additionally declared in the random part of the model. A General Linear Model (GLM) was used to evaluate the association between mean temperature and type of water source, season and their interaction. The outcomes of microbiological analyses, expressed as presence/absence, were modelled using Generalized Linear Mixed Models (GLMMs), choosing the binomial distribution as the response distribution. Major water properties (pH, salinity, hardness and iron) and farm characteristics, such as water source, presence of a supply system with water recirculation and birds age, were included in the models as fixed effects as putative risk factors. According to sampling design, random effects of farms, sampling site and season were also included. To facilitate the evaluation of the results and the discussion, water parameters were classified into categories according to their observed distribution. The software R 3.3.0 was used to perform linear quantile mixed models, whereas software SAS v.9.4 was adopted for the other statistical analyses. RESULTS The results of microbiological, chemical and physical analyses by source of water supply, sampling site and season are given in Tables 1 and 2. Regarding water temperature, a significant interaction between water source and season was found (P < 0.01): WW was significantly colder than TW (17.4 ± 3.6 vs. 20.6 ± 2.8) in summer, but not in winter (14.6 ± 2.1 vs. 13.3 ± 2.7). Chemical, physical and microbiological quality profiles of WW and TW were mostly within the limits of TW for humans (Counc. Dir. 98/83/EC). Median values of pH, sulphate, nitrate, iron and salinity levels were significantly different between TW and WW (P < 0.05). Except for pH, the same was found between winter and summer. Concerning the sampling site, copper median concentration was significantly higher at site B (P < 0.001) and C (P = 0.041) compared to A. Both sources had a lower microbiological quality at site B and C (e.g., up to 103 CFU/100 mL for E. coli) than A (Tables 1 and 2). Table 1. Chemical and physical analyses of drinking water in 28 turkey farms supplied with either well or tap water during summer and winter of 2012–2013 in North-Eastern Italy. Water samples were collected twice in 2012–2013 (in winter and in summer) at 3 sampling sites: the water source (A), the beginning (B) and the end (C) of the nipple line. Data are expressed as median values with interquartile range (within parentheses). Significance (Sig.): *P < 0.05, **P < 0.01, ***P < 0.001.       Water source    Sampling site    Sampling season    Parameter  Limits of potability Dir.98/83/EC  Maximum acceptable level  Tap water  Well water  Sig.  A  B  C  Sig.  Summer  Winter  Sig.  pH  6.5–9.5  >6b; 5–8c  7.7 (0.4)  7.5 (0.4)  *  7.6 (0.5)  7.6 (0.4)  7.6 (0.4)    7.7 (0.4)  7.6 (0.4)    Sulphate (mg/L)  250  250b; 200c  31.1 (27.0)  36.8 (59.9)  *  30.8 (45.1)  32.0 (42.4)  31.8 (47.9)    27.5 (31.5)  33.4 (59.6)  **  Phosphate (mg/L)      0.0 (0.1)  0.1 (0.5)    0.1 (0.3)  0.0 (0.2)  0.0 (0.2)    0.0 (0.4)  0.0 (0.2)    Nitrate (mg/L)  50  25b,c  16.2 (11.8)  5.5 (17.8)  ***  13.3 (15.8)  12.3 (17.11)  12.3 (18.0)    11.9 (17.3)  15.0 (18.4)  **  Copper (μg/L)    600b,c  4.5 (12.6)  3.1 (22.2)    1.9 (3.1)  10.4 (22.3)  6.7 (26.5)  **  3.2 (11.4)  6.9 (18.5)    Chromium (μg/L)a  50    1.3 (1.8)  0.0 (0.1)    0.2 (1.7)  0.0 (1.4)  0.0 (1.3)    0.0 (1.2)  0.9 (1.8)    Ammonia (mg/L)a  0.5    0.0 (0.0)  0.0 (0.0)    0.0 (0.0)  0.0 (0.0)  0.0 (0.0)    0.0 (0.0)  0.0 (0.2)    Salinity (PSU)      0.3 (0.1)  0.4 (0.3)  ***  0.3 (0.2)  0.3 (0.2)  0.3 (0.2)    0.2 (0.1)  0.3 (0.2)  ***  Hardness (mg/L)    110c  240 (78)  245 (145)    240 (104)  252 (108)  240 (101)    260 (127)  240 (74)    Iron (μg/L)  200  300b,c  1.1 (4.8)  3.5 (19.4)  *  1.1 (4.8)  1.8 (7.1)  2.3 (7.7)    1.1 (4.8)  2.8 (9.4)  *        Water source    Sampling site    Sampling season    Parameter  Limits of potability Dir.98/83/EC  Maximum acceptable level  Tap water  Well water  Sig.  A  B  C  Sig.  Summer  Winter  Sig.  pH  6.5–9.5  >6b; 5–8c  7.7 (0.4)  7.5 (0.4)  *  7.6 (0.5)  7.6 (0.4)  7.6 (0.4)    7.7 (0.4)  7.6 (0.4)    Sulphate (mg/L)  250  250b; 200c  31.1 (27.0)  36.8 (59.9)  *  30.8 (45.1)  32.0 (42.4)  31.8 (47.9)    27.5 (31.5)  33.4 (59.6)  **  Phosphate (mg/L)      0.0 (0.1)  0.1 (0.5)    0.1 (0.3)  0.0 (0.2)  0.0 (0.2)    0.0 (0.4)  0.0 (0.2)    Nitrate (mg/L)  50  25b,c  16.2 (11.8)  5.5 (17.8)  ***  13.3 (15.8)  12.3 (17.11)  12.3 (18.0)    11.9 (17.3)  15.0 (18.4)  **  Copper (μg/L)    600b,c  4.5 (12.6)  3.1 (22.2)    1.9 (3.1)  10.4 (22.3)  6.7 (26.5)  **  3.2 (11.4)  6.9 (18.5)    Chromium (μg/L)a  50    1.3 (1.8)  0.0 (0.1)    0.2 (1.7)  0.0 (1.4)  0.0 (1.3)    0.0 (1.2)  0.9 (1.8)    Ammonia (mg/L)a  0.5    0.0 (0.0)  0.0 (0.0)    0.0 (0.0)  0.0 (0.0)  0.0 (0.0)    0.0 (0.0)  0.0 (0.2)    Salinity (PSU)      0.3 (0.1)  0.4 (0.3)  ***  0.3 (0.2)  0.3 (0.2)  0.3 (0.2)    0.2 (0.1)  0.3 (0.2)  ***  Hardness (mg/L)    110c  240 (78)  245 (145)    240 (104)  252 (108)  240 (101)    260 (127)  240 (74)    Iron (μg/L)  200  300b,c  1.1 (4.8)  3.5 (19.4)  *  1.1 (4.8)  1.8 (7.1)  2.3 (7.7)    1.1 (4.8)  2.8 (9.4)  *  a Statistical testing not performed because of insufficient non-zero observations. b Carter and Sneed, 1996 c Aviagen Turkey Inc., 2015 View Large Table 1. Chemical and physical analyses of drinking water in 28 turkey farms supplied with either well or tap water during summer and winter of 2012–2013 in North-Eastern Italy. Water samples were collected twice in 2012–2013 (in winter and in summer) at 3 sampling sites: the water source (A), the beginning (B) and the end (C) of the nipple line. Data are expressed as median values with interquartile range (within parentheses). Significance (Sig.): *P < 0.05, **P < 0.01, ***P < 0.001.       Water source    Sampling site    Sampling season    Parameter  Limits of potability Dir.98/83/EC  Maximum acceptable level  Tap water  Well water  Sig.  A  B  C  Sig.  Summer  Winter  Sig.  pH  6.5–9.5  >6b; 5–8c  7.7 (0.4)  7.5 (0.4)  *  7.6 (0.5)  7.6 (0.4)  7.6 (0.4)    7.7 (0.4)  7.6 (0.4)    Sulphate (mg/L)  250  250b; 200c  31.1 (27.0)  36.8 (59.9)  *  30.8 (45.1)  32.0 (42.4)  31.8 (47.9)    27.5 (31.5)  33.4 (59.6)  **  Phosphate (mg/L)      0.0 (0.1)  0.1 (0.5)    0.1 (0.3)  0.0 (0.2)  0.0 (0.2)    0.0 (0.4)  0.0 (0.2)    Nitrate (mg/L)  50  25b,c  16.2 (11.8)  5.5 (17.8)  ***  13.3 (15.8)  12.3 (17.11)  12.3 (18.0)    11.9 (17.3)  15.0 (18.4)  **  Copper (μg/L)    600b,c  4.5 (12.6)  3.1 (22.2)    1.9 (3.1)  10.4 (22.3)  6.7 (26.5)  **  3.2 (11.4)  6.9 (18.5)    Chromium (μg/L)a  50    1.3 (1.8)  0.0 (0.1)    0.2 (1.7)  0.0 (1.4)  0.0 (1.3)    0.0 (1.2)  0.9 (1.8)    Ammonia (mg/L)a  0.5    0.0 (0.0)  0.0 (0.0)    0.0 (0.0)  0.0 (0.0)  0.0 (0.0)    0.0 (0.0)  0.0 (0.2)    Salinity (PSU)      0.3 (0.1)  0.4 (0.3)  ***  0.3 (0.2)  0.3 (0.2)  0.3 (0.2)    0.2 (0.1)  0.3 (0.2)  ***  Hardness (mg/L)    110c  240 (78)  245 (145)    240 (104)  252 (108)  240 (101)    260 (127)  240 (74)    Iron (μg/L)  200  300b,c  1.1 (4.8)  3.5 (19.4)  *  1.1 (4.8)  1.8 (7.1)  2.3 (7.7)    1.1 (4.8)  2.8 (9.4)  *        Water source    Sampling site    Sampling season    Parameter  Limits of potability Dir.98/83/EC  Maximum acceptable level  Tap water  Well water  Sig.  A  B  C  Sig.  Summer  Winter  Sig.  pH  6.5–9.5  >6b; 5–8c  7.7 (0.4)  7.5 (0.4)  *  7.6 (0.5)  7.6 (0.4)  7.6 (0.4)    7.7 (0.4)  7.6 (0.4)    Sulphate (mg/L)  250  250b; 200c  31.1 (27.0)  36.8 (59.9)  *  30.8 (45.1)  32.0 (42.4)  31.8 (47.9)    27.5 (31.5)  33.4 (59.6)  **  Phosphate (mg/L)      0.0 (0.1)  0.1 (0.5)    0.1 (0.3)  0.0 (0.2)  0.0 (0.2)    0.0 (0.4)  0.0 (0.2)    Nitrate (mg/L)  50  25b,c  16.2 (11.8)  5.5 (17.8)  ***  13.3 (15.8)  12.3 (17.11)  12.3 (18.0)    11.9 (17.3)  15.0 (18.4)  **  Copper (μg/L)    600b,c  4.5 (12.6)  3.1 (22.2)    1.9 (3.1)  10.4 (22.3)  6.7 (26.5)  **  3.2 (11.4)  6.9 (18.5)    Chromium (μg/L)a  50    1.3 (1.8)  0.0 (0.1)    0.2 (1.7)  0.0 (1.4)  0.0 (1.3)    0.0 (1.2)  0.9 (1.8)    Ammonia (mg/L)a  0.5    0.0 (0.0)  0.0 (0.0)    0.0 (0.0)  0.0 (0.0)  0.0 (0.0)    0.0 (0.0)  0.0 (0.2)    Salinity (PSU)      0.3 (0.1)  0.4 (0.3)  ***  0.3 (0.2)  0.3 (0.2)  0.3 (0.2)    0.2 (0.1)  0.3 (0.2)  ***  Hardness (mg/L)    110c  240 (78)  245 (145)    240 (104)  252 (108)  240 (101)    260 (127)  240 (74)    Iron (μg/L)  200  300b,c  1.1 (4.8)  3.5 (19.4)  *  1.1 (4.8)  1.8 (7.1)  2.3 (7.7)    1.1 (4.8)  2.8 (9.4)  *  a Statistical testing not performed because of insufficient non-zero observations. b Carter and Sneed, 1996 c Aviagen Turkey Inc., 2015 View Large Table 2. Microbiological analyses of drinking water in 28 turkey farms supplied with either well or tap water during summer and winter of 2012–2013 in North-Eastern Italy. Water samples were collected twice in 2012–2013 (in winter and in summer) at 3 sampling sites: the water source (A), the beginning (B) and the end (C) of the nipple line. Significance for P < 0.05.     Water source  Sampling site  Sampling season  Parameter  Category  Tap  Well  P  A  B  C  P  Summer  Winter  P  Total microbial count 22°C  >103 UFC/mL (%)  60.7  46.4  0.29  5.4  71.4  83.9  <0.001  58.3  48.8  0.09  E. coli and Enterococcus  >102 UFC/100 mL (%)  15.5  35.7  0.44  7.1  35.7  33.9  <0.001  31.0  20.2  0.06  Campylobacter jejuni  PCR+ (%)  15.5  15.5  0.55  10.7  19.6  16.1  0.66  8.3  22.6  0.09  Campylobacter coli  PCR+ (%)  32.1  28.6  0.21  26.8  35.7  28.6  0.79  17.9  42.9  <0.001      Water source  Sampling site  Sampling season  Parameter  Category  Tap  Well  P  A  B  C  P  Summer  Winter  P  Total microbial count 22°C  >103 UFC/mL (%)  60.7  46.4  0.29  5.4  71.4  83.9  <0.001  58.3  48.8  0.09  E. coli and Enterococcus  >102 UFC/100 mL (%)  15.5  35.7  0.44  7.1  35.7  33.9  <0.001  31.0  20.2  0.06  Campylobacter jejuni  PCR+ (%)  15.5  15.5  0.55  10.7  19.6  16.1  0.66  8.3  22.6  0.09  Campylobacter coli  PCR+ (%)  32.1  28.6  0.21  26.8  35.7  28.6  0.79  17.9  42.9  <0.001  View Large Table 2. Microbiological analyses of drinking water in 28 turkey farms supplied with either well or tap water during summer and winter of 2012–2013 in North-Eastern Italy. Water samples were collected twice in 2012–2013 (in winter and in summer) at 3 sampling sites: the water source (A), the beginning (B) and the end (C) of the nipple line. Significance for P < 0.05.     Water source  Sampling site  Sampling season  Parameter  Category  Tap  Well  P  A  B  C  P  Summer  Winter  P  Total microbial count 22°C  >103 UFC/mL (%)  60.7  46.4  0.29  5.4  71.4  83.9  <0.001  58.3  48.8  0.09  E. coli and Enterococcus  >102 UFC/100 mL (%)  15.5  35.7  0.44  7.1  35.7  33.9  <0.001  31.0  20.2  0.06  Campylobacter jejuni  PCR+ (%)  15.5  15.5  0.55  10.7  19.6  16.1  0.66  8.3  22.6  0.09  Campylobacter coli  PCR+ (%)  32.1  28.6  0.21  26.8  35.7  28.6  0.79  17.9  42.9  <0.001      Water source  Sampling site  Sampling season  Parameter  Category  Tap  Well  P  A  B  C  P  Summer  Winter  P  Total microbial count 22°C  >103 UFC/mL (%)  60.7  46.4  0.29  5.4  71.4  83.9  <0.001  58.3  48.8  0.09  E. coli and Enterococcus  >102 UFC/100 mL (%)  15.5  35.7  0.44  7.1  35.7  33.9  <0.001  31.0  20.2  0.06  Campylobacter jejuni  PCR+ (%)  15.5  15.5  0.55  10.7  19.6  16.1  0.66  8.3  22.6  0.09  Campylobacter coli  PCR+ (%)  32.1  28.6  0.21  26.8  35.7  28.6  0.79  17.9  42.9  <0.001  View Large Salmonella enterica subsp. enterica serotype Kentucky was isolated only from WW at site A, both in summer and winter from the same farm. Campylobacter spp. was isolated only twice in a farm supplied with WW and from another one supplied with TW at site B and C in winter. Positivity at real-time PCR was found for C. coli in 30.4% of samples and for C. jejuni in 14.9% of samples. Table 2 shows the associations between Campylobacter spp. positivity at real-time PCR and source of water supply, season and sampling site; only season was found to be significant, with winter posing a higher risk for C. jejuni positivity in water. Table 3 shows the associations between putative risk factors for real-time PCR positivity to C. jejuni, C. coli and for total microbial count: low salinity and high hardness were identified as risk factors for presence of C. coli and C. jejuni, respectively. Table 3. Risk factors for RT-PCR positive samples to Campylobacter jejuni, Campylobacter coli and for total microbial count (TMC 22°C) >102 in 28 turkey farms sampled in North-Eastern Italy in summer and winter 2012–2013. Continuous variables were classified on the basis of the data distribution. Significance for P < 0.05.     Campylobacter coli  Campylobacter jejuni  TMC 22°C >102  Parameter  Category  n.  %  P  n.  %  P  n.  %  P  Supply system with water recirculation  Yes  42  35.0  0.08  19  15.8  0.87  66  55.0  0.83    No  9  18.8    7  14.6    24  50.0    Birds age (days)  ≤30  13  31.0  0.35  4  9.5  0.45  20  47.6  0.58    30–50  18  37.5    10  20.8    27  56.3      51–75  12  28.6    6  14.3    26  61.9      >75  8  22.2    6  16.7    17  47.2    pH  ≤7.36  39  28.2  0.94  39  12.8  0.86  39  12.8  0.49    7.37-7.63  45  37.8    45  13.3    45  13.3      7.64-7.83  41  29.3    41  19.5    41  19.5      >7.83  43  25.6    43  16.3    43  16.3    Salinity (PSU)  ≤0.21  42  35.7  0.04  42  16.7  0.11  42  4.8  0.80    0.22-0,28  39  38.5    39  10.3    44  15.9      0.29-0,38  43  20.9    43  14.0    48  20.8      >0.38  44  27.3    44  20.5    34  20.6    Hardness (mg/L)  ≤191  42  21.4  0.40  42  4.8  0.05  42  16.7  0.37    192–240  44  34.1    44  15.9    39  10.3      241–300  48  29.2    48  20.8    43  14.0      >300  34  38.2    34  20.6    44  20.5    Iron (μg/L)  ≤ LOD  47  23.4  0.55  47  8,5  0.76  47  8.5  0.35    LOD – 2.28  37  40.5    37  21.6    37  21.6      2.29-12.22  42  26.2    42  11.9    42  11.9      >12.22  42  33.3    42  21.4    42  21.4        Campylobacter coli  Campylobacter jejuni  TMC 22°C >102  Parameter  Category  n.  %  P  n.  %  P  n.  %  P  Supply system with water recirculation  Yes  42  35.0  0.08  19  15.8  0.87  66  55.0  0.83    No  9  18.8    7  14.6    24  50.0    Birds age (days)  ≤30  13  31.0  0.35  4  9.5  0.45  20  47.6  0.58    30–50  18  37.5    10  20.8    27  56.3      51–75  12  28.6    6  14.3    26  61.9      >75  8  22.2    6  16.7    17  47.2    pH  ≤7.36  39  28.2  0.94  39  12.8  0.86  39  12.8  0.49    7.37-7.63  45  37.8    45  13.3    45  13.3      7.64-7.83  41  29.3    41  19.5    41  19.5      >7.83  43  25.6    43  16.3    43  16.3    Salinity (PSU)  ≤0.21  42  35.7  0.04  42  16.7  0.11  42  4.8  0.80    0.22-0,28  39  38.5    39  10.3    44  15.9      0.29-0,38  43  20.9    43  14.0    48  20.8      >0.38  44  27.3    44  20.5    34  20.6    Hardness (mg/L)  ≤191  42  21.4  0.40  42  4.8  0.05  42  16.7  0.37    192–240  44  34.1    44  15.9    39  10.3      241–300  48  29.2    48  20.8    43  14.0      >300  34  38.2    34  20.6    44  20.5    Iron (μg/L)  ≤ LOD  47  23.4  0.55  47  8,5  0.76  47  8.5  0.35    LOD – 2.28  37  40.5    37  21.6    37  21.6      2.29-12.22  42  26.2    42  11.9    42  11.9      >12.22  42  33.3    42  21.4    42  21.4    View Large Table 3. Risk factors for RT-PCR positive samples to Campylobacter jejuni, Campylobacter coli and for total microbial count (TMC 22°C) >102 in 28 turkey farms sampled in North-Eastern Italy in summer and winter 2012–2013. Continuous variables were classified on the basis of the data distribution. Significance for P < 0.05.     Campylobacter coli  Campylobacter jejuni  TMC 22°C >102  Parameter  Category  n.  %  P  n.  %  P  n.  %  P  Supply system with water recirculation  Yes  42  35.0  0.08  19  15.8  0.87  66  55.0  0.83    No  9  18.8    7  14.6    24  50.0    Birds age (days)  ≤30  13  31.0  0.35  4  9.5  0.45  20  47.6  0.58    30–50  18  37.5    10  20.8    27  56.3      51–75  12  28.6    6  14.3    26  61.9      >75  8  22.2    6  16.7    17  47.2    pH  ≤7.36  39  28.2  0.94  39  12.8  0.86  39  12.8  0.49    7.37-7.63  45  37.8    45  13.3    45  13.3      7.64-7.83  41  29.3    41  19.5    41  19.5      >7.83  43  25.6    43  16.3    43  16.3    Salinity (PSU)  ≤0.21  42  35.7  0.04  42  16.7  0.11  42  4.8  0.80    0.22-0,28  39  38.5    39  10.3    44  15.9      0.29-0,38  43  20.9    43  14.0    48  20.8      >0.38  44  27.3    44  20.5    34  20.6    Hardness (mg/L)  ≤191  42  21.4  0.40  42  4.8  0.05  42  16.7  0.37    192–240  44  34.1    44  15.9    39  10.3      241–300  48  29.2    48  20.8    43  14.0      >300  34  38.2    34  20.6    44  20.5    Iron (μg/L)  ≤ LOD  47  23.4  0.55  47  8,5  0.76  47  8.5  0.35    LOD – 2.28  37  40.5    37  21.6    37  21.6      2.29-12.22  42  26.2    42  11.9    42  11.9      >12.22  42  33.3    42  21.4    42  21.4        Campylobacter coli  Campylobacter jejuni  TMC 22°C >102  Parameter  Category  n.  %  P  n.  %  P  n.  %  P  Supply system with water recirculation  Yes  42  35.0  0.08  19  15.8  0.87  66  55.0  0.83    No  9  18.8    7  14.6    24  50.0    Birds age (days)  ≤30  13  31.0  0.35  4  9.5  0.45  20  47.6  0.58    30–50  18  37.5    10  20.8    27  56.3      51–75  12  28.6    6  14.3    26  61.9      >75  8  22.2    6  16.7    17  47.2    pH  ≤7.36  39  28.2  0.94  39  12.8  0.86  39  12.8  0.49    7.37-7.63  45  37.8    45  13.3    45  13.3      7.64-7.83  41  29.3    41  19.5    41  19.5      >7.83  43  25.6    43  16.3    43  16.3    Salinity (PSU)  ≤0.21  42  35.7  0.04  42  16.7  0.11  42  4.8  0.80    0.22-0,28  39  38.5    39  10.3    44  15.9      0.29-0,38  43  20.9    43  14.0    48  20.8      >0.38  44  27.3    44  20.5    34  20.6    Hardness (mg/L)  ≤191  42  21.4  0.40  42  4.8  0.05  42  16.7  0.37    192–240  44  34.1    44  15.9    39  10.3      241–300  48  29.2    48  20.8    43  14.0      >300  34  38.2    34  20.6    44  20.5    Iron (μg/L)  ≤ LOD  47  23.4  0.55  47  8,5  0.76  47  8.5  0.35    LOD – 2.28  37  40.5    37  21.6    37  21.6      2.29-12.22  42  26.2    42  11.9    42  11.9      >12.22  42  33.3    42  21.4    42  21.4    View Large DISCUSSION While surface water is a recognized vehicle of disease transmission for poultry (Amaral, 2004), the present study showed that WW given to commercial turkeys in North-Easter Italy is of satisfactory quality for the analysed parameters and therefore represents a suitable alternative to TW for livestock. Most turkey farms presented levels of nitrates lower than the limits for potability (50 mg/L). High levels of nitrates can be related to contamination with residential, industrial or agricultural waste. In humans, consumption of drinking water with high nitrate levels (i.e. higher than 50 mg/L) may cause hypertrophy of the thyroid (Van Maanen et al., 1994). The toxicity for poultry has been reported at levels higher than 50 mg/L for chickens and 75 mg/L for turkeys, while in broilers nitrate levels greater than 20 mg/L may have a negative effect on growth and feed conversion (as reviewed by Carter and Sneed, 1996). Also naturally occurring chemicals (i.e. copper and iron) were within the recommended limits for poultry (Carter and Sneed, 1996). Copper levels higher than 600 μg/L produce bitter flavor that may reduce water consumption (Carter and Sneed, 1996). Iron and copper are contained in valves, pipes and fittings and are present in coatings and alloys. This can explain the higher concentrations at site B and C compared to A, as the main source of contamination is often the corrosion of interior plumbing (WHO, 2011). Iron levels higher than 300 μg/L affect flavor, turbidity and color of water, as well as staining of plumbing fixtures (WHO, 2011). Although a high level of iron (600 ppm) in water was not found to affect broiler performances (Fairchild et al., 2006), it may form chelates with apramycin and tetracyclines, thus limiting the therapeutic effect of water medication (Scandurra, 2013). Well water had higher levels of hardness in comparison to TW. However, samples from both sources often presented levels higher than 200 mg/L, which is known to be critical for causing calcium deposits in the pipeline (Enne et al., 2006). Hard water has not been demonstrated to have either a positive or a negative direct impact on poultry health and performance (Carter and Sneed, 1996). However, hardness can decrease drugs solubility, preventing animals from receiving an effective dose (Scandurra, 2013). Although pH was within the range of acceptability for potable water (European Commission, 1998), it was significantly lower in WW. A level of pH lower than 5.5 can create problems to the urinary and digestive systems, bone demineralization and fragility, as well as corroding the pipeline and being incompatible with some medicines and vaccines (Enne et al., 2006). Water pH ranging between 6.0 and 6.3 is suspected of having negative effects on poultry performances (Carter and Sneed, 1996). On the other hand, Grizzle et al. (1996) found that a water pH of 6.25 did not negatively affect broiler growth in comparison with a water pH of 6.75. Rather, a water pH of 5.75 negatively affected it in comparison with a water pH of 6.25 and 6.75, respectively. Only a few samples had a high level of ammonia, whose contamination can derive from industrial and agricultural waste (WHO, 2011). To the best of our knowledge, we are not aware of any studies describing the effect of high levels of water ammonia on poultry health. However, ammonia can react with chlorine to reduce free chlorine and to form chloramines (WHO, 2011). Most samples presented a total microbial count (a general indicator of pipeline hygiene) under the limits for TW, and a very low fecal contamination (i.e. presence of E. coli and Enterococcus). Instead, a significant decrease in microbiological water quality was found at the nipple line. Water samples varied in hardness and iron levels. High levels of iron and hardness are known to be risk factors for biofilm deposition and bacterial proliferation in the pipeline (Wingender and Flemming, 2011). Specifically, iron promotes the growth of bacteria that derive their energy from the oxidation of ferrous iron to ferric iron (WHO, 2011). The formation of biofilms increases with the flow velocity of water (Lehtola et al., 2006); therefore, bird age is indirectly a further risk factor, as when birds are young the limited water consumption may be associated with a low water flow. Yet, poor microbiological water quality was not significantly associated with its physical and chemical properties, nor with the presence of a water recirculation systems and birds’ age. Taken together, these results call for improvements in microbiological water quality as directly related to sanitization procedures applied by farmers, which need to be reviewed. Farmers had also declared to routinely use different commercial products to guarantee pipeline hygiene. However, as previously suggested (Sparks, 2009), the efficacy of these products may largely differ depending on water properties. Isolation of potentially pathogenic microorganisms was generally uncommon. However, S. Kentucky was isolated from the same WW samples in different seasons at the water source, raising some concerns on the potential role of WW as vehicle of Salmonella transmission. Also Campylobacter was rarely isolated, although the positivity detected by real-time PCR ranged between 8% to 43%, irrespective of the sampling site. Pipeline hygiene was not influenced by season, while Campylobacter positivity was higher in winter. This is in accordance with previous findings which identified an optimal Campylobacter survival at low temperatures (around 4°C) (Thomas et al., 1998). The low frequency of Campylobacter isolation confirms the well know limitation of culture-based procedures in isolating the microorganism from water samples, which is likely to underestimate the true prevalence due to the high susceptibility of Campylobacter to suboptimal environmental conditions (Thomas et al., 1998; Chaisowwong et al., 2012). Moreover, the bacterium can be present in water also as viable but non-culturable forms, which are able to survive under adverse conditions (Rollins and Colwell, 1986). For these reasons, real-time PCR is a valid support to detect the microorganism in water and to understand possible routes of transmission. While no significant association between Campylobacter spp. presence and source of water supply was found, low salinity and high hardness were identified as risk factors for presence of C. coli and C. jejuni, respectively. Moreover, there was a tendency towards significance (P = 0.08) in the association between presence of a water recirculation system and C. coli. As previously suggested (Sahin et al., 2015), Campylobacter transmission to birds is more likely to occur from the farm environment through water, rather than from the water source itself (Bull et al., 2006; Mughini-Gras et al., 2016). In conclusion, the results of the present study call for improvements in sanitization procedures for farm drinking water pipelines, highlighting also issues related to drinking water characterized by high levels of hardness and iron. While water recirculation systems, bird age, and most chemical and physical water properties did not seem to be associated with microbiological water quality, low salinity and high hardness were specific risk factors for C. coli and C. jejuni presence, respectively. Although Campylobacter spp. isolation from water samples was problematic, detection of Campylobacter spp. genetic material showed that this zoonotic pathogen is highly prevalent in the farm pipeline. SUPPLEMENTARY DATA Supplementary data are available at Poultry Science online. Acknowledgements This study was funded by the Italian Ministry of Health, Rome, Italy (Project RC IZSVe 08/11; B21J12000280001). REFERENCES Amaral L. 2004. Drinking water as a risk factor to poultry health. Rev. Bras. Cienc. Avic . 6: 191– 199. Google Scholar CrossRef Search ADS   Agenzia per la protezione dell’ambiente e per i servizi tecnici (APAT). 2003. Metodi analitici per le acque. Available from: http://www.isprambiente.gov.it/it/pubblicazioni/manuali-e-linee-guida/metodi-analitici-per-le-acque. Aviagen Turkeys Inc. 2015. Management Guidelines - Raising commercial turkeys. Available at: http://www.aviagenturkeys.us/uploads/2015/12/21/Aviagen%20Commercial%20Guide.pdfLast accessed on 5/3/2018. Bull S., Allen V., Domingue G., Jorgensen F., Frost J., Ure R., Whyte R., Tinker D., Corry J., Gillard-King J., Humphrey T.. 2006. Sources of Campylobacter spp. Colonizing Housed Broiler Flocks during Rearing. Appl. Environ. Microbiol.  72: 645– 652. Google Scholar CrossRef Search ADS PubMed  Carter T. A., Sneed R. E.. 1996. Drinking water quality for poultry. North Carolina Cooperative Extension Service . Available from: https://www.bae.ncsu.edu/bae/extension/ext-publications/water/drinking/pst42-wqg-poultry-sneed.pdf Chaisowwong W., Kusumoto A., Hashimoto M., Harada T., Maklon K., Kawamoto K.. 2012. Physiological Characterization of Campylobacter jejuni under Cold Stresses Conditions: Its Potential for Public Threat. J. Vet. Med. Sci.  74: 43– 50. Google Scholar CrossRef Search ADS PubMed  Enne G., Greppi G., Serratoni M.. 2006. The role of water in animal breeding. Ital J. Agronomy . 1: 519– 527. Google Scholar CrossRef Search ADS   European Commission. 1998a. Council Directive (EC) 98/58/EC of 20 July 1998 concerning the protection of animals kept for farming purposes. Off. J. Eur. Comm.  L221: 23– 27. European Commission. 1998b. Council Directive (EC) 98/83/EC of 3 November 1998 on the quality of water intended for human consumption. Off. J. Eur. Comm . L330: 32– 54 European Commission. 2004. Council Regulation (EC) No 852/2004 of the European Parliament and of the Council of 29 April 2004 on the hygiene of foodstuffs. Off. J. Eur. Union  L139: 1– 54. European Food Safety Authority. 2011. The European Union Summary Report on Trends and Sources of Zoonoses, Zoonotic Agents and Food-borne Outbreaks in 2009. EFSA Journal . 9: 2090. CrossRef Search ADS   Fairchild B., Batal A., Ritz C., Vendrell P.. 2006. Effect of Drinking Water Iron Concentration on Broiler Performance. The Journal of Applied Poultry Research . 15: 511– 517. Google Scholar CrossRef Search ADS   Grizzle J., Armbrust T., Bryan M., Saxton A.. 1996. Water quality I: The effect of water nitrate and pH on broiler growth performance. The Journal of Applied Poultry Research . 5: 330– 336. Google Scholar CrossRef Search ADS   Giacomelli M., Andrighetto C., Lombardi A., Martini M., Piccirillo A.. 2012. A Longitudinal Study on Thermophilic Campylobacter spp. in Commercial Turkey Flocks in Northern Italy: Occurrence and Genetic Diversity. Avian Dis . 56: 693– 700. Google Scholar CrossRef Search ADS PubMed  ISO. 1999. Microbiology of food and animal feeding stuffs. Preparation of Test Samples, Initial Suspension and Decimal Dilutions for Microbiological Examination. Part 1: General rules for the preparation of the initial suspension and decimal dilutions. in ISO 6887-1:1999 . International Organization for Standardization, Geneva, Switzerland. ISO. 2002. Microbiology of food and animal feeding stuffs - Horizontal method for the detection of Salmonella spp . in ISO 6579:2002 . International Organization for Standardization, Geneva, Switzerland. ISO. 2005a. Water quality - General guidance on the enumeration of micro-organisms by culture. in ISO 8199:2005 . International Organization for Standardization, Geneva, Switzerland. ISO. 2005b. Water quality - Detection and enumeration of thermotolerant Campylobacter species . in ISO 17995:2005 . International Organization for Standardization, Geneva, Switzerland. Istituto Superiore di Sanità. 2007. Reference analytical methods for water intended for human consumption according to the Italian Legislative Decree 31/2001. Microbiological Methods . In: L. Bonadonna, M. Ottaviani, (Eds.), Rapporti ISTISAN 07/5. Rome (Italy). Available from: http://www.iss.it/binary/aqua/cont/RappIstisan%2007%205.1204715346.pdf Kapperud G., Skjerve E., Vik L., Hauge K., Lysaker A., Aalmen I., Ostroff S., Potter M.. 1993. Epidemiological investigation of risk factors for Campylobacter colonization in Norwegian broiler flocks. Epidemiol. Infect.  111: 245– 256. Google Scholar CrossRef Search ADS PubMed  Lehtola M., Laxander M., Miettinen I., Hirvonen A., Vartiainen T., Martikainen P.. 2006. The effects of changing water flow velocity on the formation of biofilms and water quality in pilot distribution system consisting of copper or polyethylene pipes. Water Res.  40: 2151– 2160. Google Scholar CrossRef Search ADS PubMed  Lund V. 1996. Evaluation of E. coli as an indicator for the presence of Campylobacter jejuni and Yersinia enterocolitica in chlorinated and untreated oligotrophic lake water. Water Res.  30: 1528– 1534. Google Scholar CrossRef Search ADS   Mughini-Gras L., Penny C., Ragimbeau C., Schets F., Blaak H., Duim B., Wagenaar J., de Boer A., Cauchie H., Mossong J., van Pelt W.. 2016. Quantifying potential sources of surface water contamination with Campylobacter jejuni and Campylobacter coli. Water Res.  101: 36– 45. Google Scholar CrossRef Search ADS PubMed  Mulatti P., Ferrè N., Marangon S.. 2011. Spatial distribution of 2000–2007 low pathogenicity Avian Influenza Epidemics in Northern Italy. In: S.K. Majumdat, Brenner F. J., Huffman J. E., McLean R. G, Panah A. I, Pietrobon P. J, Keeler S. P, Shive S.. Pandemic Influenza Viruses: Science, Surveillance and Public Health , pp 232– 247. The Pennsylvania Academy of Science: Easton, PA. OIE Terrestrial Animal Health Code. 2016. Chapter 6.4. Biosecurity procedures in poultry production . 25th Edn. 2016:1-6. Available from: http://www.oie.int/index.php?id = 169&L = 0&htmfile = chapitre_biosecu_poul_production.htm Rollins D. M., Colwell R. R.. 1986. Viable but nonculturable stage of Campylobacter jejuni and its role in survival in the natural aquatic environment. Appl. Environ. Microb . 52: 531– 538. Sahin O., Kassem I. I., Shen Z., Lin J., Rajashekara G., Zhang Q.. 2015. Campylobacter in Poultry: Ecology and Potential Interventions. Avian Dis.  59: 185– 200. Google Scholar CrossRef Search ADS PubMed  Scandurra S. 2013. Veterinary drugs in drinking water used for pharmaceutical treatments in breeding farms. PhD Thesis, University of Bologna, Bologna (Italy). Sparks N. 2009. The role of the water supply system in the infection and control of Campylobacter in chicken. Worlds Poult. Sci. J.  65: 459– 474. Google Scholar CrossRef Search ADS   Standing Committee of Analysts. 2002 The Microbiology of Drinking Water - Part 3 - Practices and procedures for laboratories. Available from: https://www.gov.uk/government/publications/standing-committee-of-analysts-sca-blue-books Thomas C., Gibson H., Hill D., Mabey M.. 1998. Campylobacter epidemiology: an aquatic perspective. J. Appl. Microb . 85: 168S– 177S. Google Scholar CrossRef Search ADS   Toplak N., Kovač M., Piskernik S., Možina S., Jeršek B.. 2012. Detection and quantification of Campylobacter jejuni and Campylobacter coli using real-time multiplex PCR. J. Appl. Microbiol.  112: 752– 764. Google Scholar CrossRef Search ADS PubMed  Umar S., Munir M. T., Azeem T T., Ali S., Umar W., Rehman A., Shah M. A.. 2014. Effects of water quality on productivity and performance of livestock: A mini review. Veterinaria  2: 11– 15. UNI. 2001. Water quality - Enumeration of culturable micro-organisms - Colony Count by Inoculation in a Nutrient Agar Culture Medium. in UNI EN ISO 6222:2001 . Ente Nazionale Italiano di Unificazione Milan, Italy. UNI. 2002. Water quality - Detection and enumeration of Escherichia coli and coliform bacteria - Part 1: Membrane filtration method. in UNI EN ISO 9308-1:2002 . Ente Nazionale Italiano di Unificazione Milan, Italy. UNI. 2003. Water quality - Detection and enumeration of intestinal enterococci - Part 2: Membrane filtration method. in UNI EN ISO 7899-2:2003 . Ente Nazionale Italiano di Unificazione Milan, Italy. Van Maanen J., van Dijk A., Mulder K., de Baets M., Menheere P., van der Heide D., Mertens P., Kleinjans J.. 1994. Consumption of drinking water with high nitrate levels causes hypertrophy of the thyroid. Toxicol. Lett.  72: 365– 374. Google Scholar CrossRef Search ADS PubMed  Williams L. K., Sait L., Cogan T., Jørgensen F., Grogono-Thomas R., Humphrey T. J.. 2012. Enrichment culture can bias the isolation of Campylobacter subtypes. Epidemiol. Infect.  140: 1227– 1235. Google Scholar CrossRef Search ADS PubMed  Wingender J., Flemming H.. 2011. Biofilms in drinking water and their role as reservoir for pathogens. Int. J. Hyg. Environ. Health . 214: 417– 423. Google Scholar CrossRef Search ADS PubMed  World Health Organization. 2011. Guidelines for Drinking-water Quality  Fourth ed. WHO Press, Geneva, Switzerland. Yamazaki-Matsune W., Taguchi M., Seto K., Kawahara R., Kawatsu K., Kumeda Y., Kitazato M., Nukina M., Misawa N., Tsukamoto T.. 2007. Development of a multiplex PCR assay for identification of Campylobacter coli, Campylobacter fetus, Campylobacter hyointestinalis subsp. hyointestinalis, Campylobacter jejuni, Campylobacter lari and Campylobacter upsaliensis. J. Med. Microbiol.  56: 1467– 1473. Google Scholar CrossRef Search ADS PubMed  Zimmer M., Barnhart H., Idris U., Lee M.. 2003. Detection of Campylobacter jejuni Strains in the Water Lines of a Commercial Broiler House and Their Relationship to the Strains That Colonized the Chickens. Avian Dis.  47: 101– 107. Google Scholar CrossRef Search ADS PubMed  © The Author(s) 2018. Published by Oxford University Press on behalf of Poultry Science Association. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

Journal

Poultry ScienceOxford University Press

Published: Apr 17, 2018

There are no references for this article.

You’re reading a free preview. Subscribe to read the entire article.


DeepDyve is your
personal research library

It’s your single place to instantly
discover and read the research
that matters to you.

Enjoy affordable access to
over 18 million articles from more than
15,000 peer-reviewed journals.

All for just $49/month

Explore the DeepDyve Library

Search

Query the DeepDyve database, plus search all of PubMed and Google Scholar seamlessly

Organize

Save any article or search result from DeepDyve, PubMed, and Google Scholar... all in one place.

Access

Get unlimited, online access to over 18 million full-text articles from more than 15,000 scientific journals.

Your journals are on DeepDyve

Read from thousands of the leading scholarly journals from SpringerNature, Elsevier, Wiley-Blackwell, Oxford University Press and more.

All the latest content is available, no embargo periods.

See the journals in your area

DeepDyve

Freelancer

DeepDyve

Pro

Price

FREE

$49/month
$360/year

Save searches from
Google Scholar,
PubMed

Create lists to
organize your research

Export lists, citations

Read DeepDyve articles

Abstract access only

Unlimited access to over
18 million full-text articles

Print

20 pages / month

PDF Discount

20% off