Animal and human enteric viruses in water and sediment samples from dairy farms

Agricultural Water Management 152 (2015) 135–141

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Animal and human enteric viruses in water and sediment samples from dairy farms Rodrigo Staggemeier a,∗ , Marina Bortoluzzi a , Tatiana Moraes da Silva Heck a , Roger Bordin da Luz a , Rafael Bandeira Fabres a , Mayra Cristina Soliman a , Caroline Rigotto a , Nelson Antonio Baldasso b , Fernando Rosado Spilki a , Sabrina Esteves de Matos Almeida a a b

Laboratory of Molecular Microbiology, Feevale University, RS-239, 2755, Novo Hamburgo, CEP 93352-000 RS, Brazil Empresa de Assistência Técnica e Extensão Rural do Rio Grande do Sul (ASCAR-EMATER/RS), Rolante, RS, Brazil

a r t i c l e

i n f o

Article history: Received 25 August 2014 Accepted 11 January 2015 Keywords: Infectious virus Dairy farms ICC-RT-qPCR

a b s t r a c t The detection of enteric viruses accompanied by a characterization of the viruses found in a given environmental matrix may inform about the sources of fecal contamination. In the present work, 55 water samples and 20 sediment samples were collected from 21 small farms in southern Brazil. Coliform counting was done as well as molecular detection of human enterovirus (EV), and human and animal adenoviruses. Viral detection was performed using real-time quantitative PCR (qPCR). Furthermore, the viral viability of human AdV (HAdV) by ICC-RT-qPCR in sediment and water samples was analyzed. Regarding to the coliforms, only 72.7% of the samples showed fecal contamination. HAdV was detected in 87.3% of water samples, followed by AvAdV (27.3%), CAV (20%), BAV (7.3%) and PoAdV or EV (1.8%). From the sediment samples, HAdV (80%) followed by CAV (20%), BAV (5%) and no positive results for PoAdV or EV. The viral loads ranged from 1.57 × 102 gc/L up to 6.68 × 109 gc/L (water), and from 1.97 × 103 gc/g to 2.18 × 108 gc/g (sediment). Most of these viral particles in water should be non-infectious, since after the ICC-PCR, HAdV was detected in only 4 samples (8.8%). On the other hand, it is noticeable that 5 sediment samples (25%) gave positive results for the presence of infectious viral particles. © 2015 Elsevier B.V. All rights reserved.

1. Introduction The overall quality of soils and sediments is highly impacted by fecal contamination. It is well known that these environmental matrices may harbor greater amounts of enteric viruses and other fecal microorganisms than water (Rao et al., 1986). The soil can act as an important reservoir of varied natural resources; however, it may also allow the permanence of various microorganisms that cause diseases (Nasser and Metcalf 1987; Santamaría and Toranzos, 2003). Sediment is the result of soil erosion, and it is suggested that the sediment from rivers, lakes and dams can act as a reservoir for pathogens (Alm et al., 2003). Viruses associated with particulate matter in suspension or in solid matrices tend to remain viable for a longer time than if they were dispersed in water (Lipson and Stotzky, 1984; Schenewski and Julich, 2001). Enteric viruses present in the soil as a result of the release of sewage, irrigation, and waste from agro-pastoral activities can migrate to

∗ Corresponding author. Tel.: +55 51 35868800x9043. E-mail address: [email protected] (R. Staggemeier). http://dx.doi.org/10.1016/j.agwat.2015.01.010 0378-3774/© 2015 Elsevier B.V. All rights reserved.

the deepest layers of the soil, reaching groundwater as a result of the successive adsorption–desorption phenomena (Schwartzbrod, 1995). The shedding of viral particles depends on the virus studied: it has been suggested that differences in the surface charge of the virions have an important role in the association of viral particles to solids (Gerba and Bitton, 1984). Retention of viral particles in the soil also depends on the soil type, temperature, pH, moisture level (Gerba et al., 1988), isoelectric point and hydrophobicity (Williamson et al., 2005). Detection of these pathogens in the sediments is an alternative environmental impact assessment (Greening et al., 2002). In developing countries, ever-expanding areas for farming and intensive land use have led to rapid soil degradation, especially in tropical and subtropical areas (Arshad and Martin, 2002). According to Shepherd and Wyn-Jones (1997), the risk of transmission for waterborne diseases by consuming water sources from rural properties is 22 times greater than the consumption of water from a public supply system. Water contamination in rural areas may triggers considerable losses in milk production by the involvement of these pathogens in their animals, besides causing diseases in human beings (Jacintho et al., 2005).

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R. Staggemeier et al. / Agricultural Water Management 152 (2015) 135–141

Enteric viruses are a heterogeneous group of viral agents associated with subclinical infections and diseases in humans and animals, such as the viruses studied here: human adenovirus (HAdV), bovine adenovirus (BAV), canine adenovirus (CAV), avian adenovirus (AvAdV), porcine adenovirus (PoAdV) and enterovirus (EV). The agents mentioned above are characterized by their stability both in the gastrointestinal tract as in the environment as well as the characteristic of being excreted through humans and animals feces and can resist environmental contaminants (soil and water) for long periods of time (Katayama et al., 2002). Moreover, it is suggested that such viruses are important indicators of fecal contamination (Jiang et al., 2001; Carducci et al., 2008; Katayama et al., 2008; Ley et al., 2002; Hundesa et al., 2006; De Oliveira et al., 2012). Most of the enteric viruses are host-specific, thus being able to track the primary source of fecal contamination in a given environment (Ahmed et al., 2010; Jiang et al., 2007). Enteric viruses are often found in groundwater, and being waterborne, are responsible for a significant proportion of cases of gastroenteritis related to drinking water (Abbaszadegan et al., 1999; Borchardt et al., 2003). It is important to assess the viral viability for a proper evaluation of infection risks. ICC-RT-qPCR allows us to confirm the presence of infectious viruses by the analysis of the infection of cultured cells and subsequent transcription of viral mRNA, thus, viral detection of mRNA indicates the presence of infectious viral particles (Fongaro et al., 2013), the sensitivity is higher, since the cell culture prior to nucleic acid amplification increases the amount of infectious virus allowing viral detection before to produce observable cytopathic effect (Li et al., 2010). The aims of this article are the detection of coliforms and various fecal-oral transmission of viral agents (HAdV, BAV, CAV, PoAdV, AvAdV and EV) in water and sediment samples from springs, wells, dams and streams in rural properties from the cities of Rolante and Riozinho, southern Brazil, to quantify viral loads from both matrices and assess the viability of HAdV detected in the water and sediment samples on farms. The main goals of the present study are: (a) to gather information about the presence and diversity of viral markers of fecal contamination in water samples collected inside small farms in a populated watershed; and (b) tracing the main sources of contamination, from domestic animals to human beings, for water bodies located in these farms.

Table 1 Geographic coordinates (Sirgas Datum 69) of the farms chosen for the present study, municipalities of Rolante (1) and Riozinho (2), Brazil. Farms

Geographic coordinates

P11 P21 P31 P41 P51 P61 P71 P81 P91 P101 P111 P121 P131 P141 P12 P22 P32 P42 P52 P62 P72

S29◦ 38 26.0 S29◦ 38 19.5 S29◦ 38 30.4 S29◦ 38 29.1 S29◦ 39 42.9 S29◦ 39 55.8 S29◦ 38 50.9 S29◦ 38 52.1 S29◦ 39 14.1 S29◦ 38 18.0 S29◦ 36 23.7 S29◦ 35 11.0 S29◦ 37 17.5 S29◦ 39 02.6 S29◦ 37 58.7 S29◦ 38 03.2 S29◦ 37 05.4 S29◦ 36 48.6 S29◦ 35 54.0 S29◦ 37 31.5 S29◦ 37 28.7

W050◦ 35 18.9 W050◦ 34 31.3 W050◦ 34 37.9 W050◦ 34 43.1 W050◦ 35 13.7 W050◦ 34 46.0 W050◦ 32 09.5 W050◦ 32 07.2 W050◦ 32 53.9 W050◦ 32 08.1 W050◦ 31 43.3 W050◦ 34 03.6 W050◦ 34 17.2 W050◦ 34 21.5 W050◦ 28 27.9 W050◦ 28 48.1 W050◦ 26 55.0 W050◦ 26 06.5 W050◦ 27 27.1 W050◦ 25 49.1 W050◦ 24 55.8

of these are alongside a dunghill. Several present streams that originate in their own dams, springs and household sewage, are contaminated by the direct discharge of human and animal feces in water bodies, in all properties, cattle has direct access to ponds and rivers, and excreta from bovine and other species runs into water by superficial runoff. These are the most likely sources of contamination, and the same situation was found in all farms. 2.2. Coliform detection

2. Materials and methods

Fecal coliforms were detected by a Colilert® test kit following the manufacturer’s methodology within 24 h after collection. The specific nutrient indicators that make up the Colilert® are the substrate ONPG (ortho-nitrophenol-␤-galactopiranoside) and MUG (4-methyl-umbeliferil-␤-d-glucuronic). The test was considered positive for fecal coliforms when staining showed blue fluorescence when exposed to UV light. The test was considered negative in the absence of staining. The results were expressed in MPN (most probable number in 100 mL of water) according to the table provided by the manufacturer.

2.1. Sampling

2.3. Sediment samples

The municipalities of Rolante and Riozinho, located in Vale do Paranhana, Rio Grande do Sul, have most of their populations living on small farms, and the economy is based on dairy production. In addition, some have cattle, poultry, swine or fish. The three main rivers in the region are Rolante, Areia and Riozinho Rivers. Fiftyfive water samples and 20 sediment samples from springs, wells, dams and streams from 21 farms located in the municipalities neighboring Rolante (14 farms) and Riozinho (7 farms) (Table 1). Samples were obtained from a single collection in the sites mentioned above (on 03/15/2011 and 03/22/2011, respectively). Each collection point had its location demarcated by Global Positioning System and its UTM coordinates annotated and plotted. Water samples (500 mL each) and sediment samples (100 g each) were collected aseptically from each point in sterilized glass bottles. The samples were transported to the laboratory under refrigeration, and were kept at 4 ◦ C until sample concentration. Water abstracted from wells and springs on farms from Rolante and Riozinho are used for both human and animal consumption, washing utensils (including those used for milk storage), personal hygiene, and crop irrigation. Five properties have dams for fish farming, and some

In order to detect viruses from soil samples, 1 g of the solid (sediment) was diluted 1 mL of Eagle’s minimum essential medium (E-MEM, Nutricell; pH 11.5). The solution was homogenized by vortexing it for 1 min and then it was centrifuged at 14,000 rpm for 10 min (Staggemeier et al., 2015). The supernatant was used for the DNA/RNA extraction. 2.4. Virus concentration Water samples were concentrated using an adsorption–elution method previously described by Katayama et al. (2002) with minor modifications. All procedures were conducted in biosafety cabinets to avoid sample contamination. Briefly, 0.6 g of MgCl2 ·6H2 O was mixed with 500 mL of each water sample and the pH was adjusted to 5.0 using a solution of 10% HCl. Subsequently, the resulting mixture was vacuum filtered through a negatively sterile membrane (type HA, 0.45 m pore size; 47 mm diameter). The membrane was rinsed with 87.5 mL of a 0.5 mM H2 SO4 (pH 3.0) solution followed by elution of viral particles adsorbed by the membrane with 2.5 mL of 1 mM NaOH (pH 10.5). The filtrate was then neutralized with

R. Staggemeier et al. / Agricultural Water Management 152 (2015) 135–141

12.5 ␮L of 50 mM H2 SO4 and 12.5 ␮L in a 100× Tris–EDTA (TE) buffer. The resulting mixture was aliquoted and stored at −80 ◦ C until further processing. This procedure has a concentration efficacy of 50% on average. 2.5. Viruses and cells Prototype viral strains from HAdV-5, HAdV-2, BAV-3, CAV-1 and -2, PoAdV-1, AvAdV (EDS-76 vaccine strain) and Poliovirus were used throughout the study. Viruses were cultivated in A549 (HAdV5 and -2), CRIB (BAV-3), MDCK (CAV-1 and -2), PK-15 (PoAdV-1) and Vero (Poliovirus) cells. These same control viruses were used throughout for both nucleic acid extraction and in parallel for concentration procedures. 2.6. Viral nucleic acid extraction The commercial RTP DNA/RNA Virus Mini Kit (InvitekTM , Germany) was used for viral nucleic acids (DNA, HAdV, BAV, CAV, PoAdV and AvAdV; RNA, EV) extraction, according to the manufacturer’s instructions, using an initial volume of 400 ␮L of each concentrated water and sediment sample for extraction through the silica filters. The viral DNA or RNA obtained was stored in a freezer at −80 ◦ C for later processing. 2.7. Quantitative PCR (qPCR) Prior to EV genome amplification, a previous step of cDNA synthesis was carried out. It was performed using the High Capacity cDNA Reverse TranscriptionTM commercial kit (Applied BiosciencesTM , USA), with the aid of random primers and RNAse Inhibitor (Applied BiosciencesTM , USA), following the manufacturer’s instructions. EV and the different AdV species analyses were performed by qPCR using a commercial kit SYBR® Green Platinun® qPCR Supermix-UDG (Life TechnologiesTM Corporation, Carlsbad, CA 92008, USA) in accordance with manufacturer’s instructions. The qPCR reactions have been optimized and carried out under the same conditions, using them as controls for absolute quantification of viral DNA/RNA from prototype samples of HAdV-2 (primer AdV), HAdV-5 (VTB2) and EV (5 UTR). qPCR reactions were conducted in a thermal cycler iQ5TM Bio-Rad (BioradTM , Hercules, CA 94547, USA). For each 25 ␮L reaction, 12.5 ␮L of the mix were used, 1 ␮L of each primer (20 pM), 5.5 ␮L of DNAse/RNAse free sterile water and 5.0 ␮L of the nucleic acid extracted from each sample. Each reaction was composed of a denaturation cycle at 95 ◦ C for 10 min, followed by: (a) HAdV: 40 cycles composed of one step at 95 ◦ C for 20 s, and a combined annealing/extension step at 55 ◦ C for 1 min; (b) AdV: 50 cycles at 95 ◦ C for 20 s and 58 ◦ C for 1 min; (c) EV: 35 cycles at 94 ◦ C for 20 s and 56 ◦ C for 1 min. The fluorescence data were collected during the annealing/extension step. After that, a denaturing curve was made to check the specificity of amplification products (melting step between 55 and 95 ◦ C). For generating standard curves, 10-fold serial dilutions of standard controls from 10−1 to 10−5 were prepared, starting at 6.01 × 107 genome copies per reaction (HAdV-5), 6.88 × 108 (HAdV-2) and 3.77 × 106 (EV), all standard controls and samples were run in duplicates. No template control (NTC) and negative control were used in each run to confirm that there was no contamination in the assay. All standard controls and samples were run in duplicates the limit of detection was found to be 40–60 gc/reaction, the efficiency was 96.5% (R2 = 0.99, slope = −3.41) using VTB2; 200–100 gc/reaction, efficiency was 98.6% (R2 = 0.99, slope = −3.35) using primer AdV; 200–100 gc/reaction, efficiency was 95.3% (R2 = 0.99, slope = −3.44) using primer EV. Melting curve analysis was done using High Resolution Melting electrophoresis (HRM) to verify PCR product

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specificity; each viral specie has a specific temperature: (a) HAdV (86 ◦ C); (b) EV (86 ◦ C); (c) Using the primer AdV can distinguish the different AdV species, each of which has a specific melting temperature, HAdV (88 ◦ C), BAV (85.5 ◦ C), PoAdV (83.5 ◦ C), CAV (82 ◦ C) and AvAdV (80.5 ◦ C). The sequences of the primers and their location in the viruses’ genomes are given in Table 2.

2.8. Integrated cell culture-RT-qPCR assay (ICC-RT-qPCR) To quantify the number of infectious HAdV particles present in the water and sediment samples, an ICC-RT-qPCR assay (integrated cell culture – preceded by reverse transcriptase and qPCR) was conducted. This assay was performed as previously described by Fongaro et al. (2013) and aims to access viral mRNA following viral replication in cells (Chapron et al., 2000). Water and sediment samples, in a non-cytotoxic dilution, were inoculated in A549 cells for the ICC-RT-qPCR assay. After 1 h of incubation at 37 ◦ C with rotation every 15 min, the inoculum was removed and the cell layers were overlaid with high-glucose Dulbecco’s Modified Eagle’s Medium (DMEM) after being incubated at 37 ◦ C for 24 h. After incubation, the supernatant was recovered and 400 ␮L was used for genetic material extraction, as described above. Then, a reverse transcriptase reaction (RT) was used to generate cDNA from viral mRNA. The quantification of HAdV infectious particles was performed with qPCR, as described above.

3. Results 3.1. Coliform detection Fecal contamination markers were found in 72.7% (40/55) of the samples. There was a higher rate and load of contamination in surface waters (1471.7 MPN/100 mL) than in groundwater (49.9 MPN/100 mL). The springs had an average of 66.1 MPN/100 mL, higher than found in the well (11.7 MPN/100 mL) results. The highest average rate of coliform was found in streams (2009 MPN/100 mL). Only two dams (2/8) had coliforms, the largest amount of coliforms detected in a single dam (6300 MPN/100 mL), while the other contained 100 MPN/100 mL (Table 3).

3.2. Viral detection in water samples From the 55 water samples analyzed by qPCR (HAdV, BAV, CAV, PoAdV, AvAdV and EV), 87.3% showed positive for the presence of HAdV (48/55; mean 4.35 × 106 gc/L), 27.3% for AvAdV (15/55; mean 7.26 × 108 gc/L), 20% for CAV (11/55; mean 2.70 × 108 gc/L), 7.3% for BAV (4/55; mean 1.13 × 106 gc/L), 1.8% for PoAdV (1/55; 1.06 × 105 gc/L) and 1.8% for EV (1/55; 4.07 × 105 gc/L). In the groundwater (springs and wells) at least one viral agent was found in 94.6% (35/37) of the samples, HAdV being the most prevalent (89%), followed by AvAdV (35.1%), CAV (16.2%), EV (2.7%; 4.07 × 105 gc/L), BAV (2.7%) and PoAdV (2.7%). While for surface waters (dams and streams), viral detection was 83.3% (15/18), HAdV being the most prevalent (83.3%), followed by CAV (27.8%), BAV (16.7%) and AvAdV (11.1%). In the springs, general viral detection was of 92.3% (24/26), HAdV (92.3%), AvAdV (42.3%), CAV (19.2%), BAV (3.8%), PoAdV (3.8%) and EV (3.8%). All wells (11/11) had viral contamination, HAdV (100%), followed by AvAdV (18.2%) and CAV (9%). All dams (8/8) also showed viral contamination, HAdV (100%), followed by CAV (50%) and BAV (25%). Regarding the streams, viruses have been found in 70% (7/10), HAdV (70%), followed by BAV (10%), CAV (10%) and AvAdV (20%) (Table 3).

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Table 2 Primers and conditions used for PCR/qPCR amplification of genomic fragments from HAdV, BAV, CAV, PoAdV, AvAdV and EV used in the present study. Víruses

Target gene

HAdV

Hexon

EV

5 UTR

AdV

Hexon

a b c d

Primer Name

Sequence

Position

Polarity

VTB2-HAdVCf VTB2-HAdVCr ENT-F1 ENT-R2 ADV-F1 ADV-R1

5 -GAGACGTACTTCAGCCTGAAT-3 5 -GATGAACCGCAGCGTCAA-3 5 -CCTCCGGCCCCTGAATG-3 5 -ACACGGACACCCAAAGTAG-3 5 -CAGTGGTCGTACATGCACAT-3 5 -TCGGTGGTGACGTCGTGG-3

Sense Reverse Sense Reverse Sense Reverse

106–126a 190–207a 443–459b 541–559c 4–23d 67–86d

Annealing temperature

Amplicon length

55 ◦ C

101 bp

56 ◦ C

116 bp

58 ◦ C

130 bp

Primer sequences reported by Wolf et al. (2010). Primer sequences reported by Tsai et al. (1993). Primer sequences reported by Vecchia et al. (2012). this work, the position relative to AdV hexon gene and the length of amplicon is variable according to the virus species.

3.3. Viral detection in sediment samples Regarding the detection of nucleic acids in sediments, 80% of the samples were detected as positive for HAdV (16/20; mean 2.23 × 104 gc/g), followed by AvAdV (45%; 9/20; mean 6.1 × 107 gc/g), CAV (20%; 4/20; mean 3.12 × 107 gc/g), BAV (5%; 1/20; 1.80 × 107 gc/g) and none were positive for PoAdV and EV (Table 3). 3.4. Viral viability Viable HAdV were detected in 4 water samples (4/55 = 8.8% – 3 springs and 1 stream) and 5 sediment samples (5/20 = 25% – 2 springs, 2 dams and 1 stream), in 2 samples infectious virus in both matrices were detected, the soil matrix showed a higher infectious viral load (1.1 × 107 gc/g) while water showed (3.54 × 104 gc/L) (Table 4). 4. Discussion In rural environments, there is a great risk of waterborne disease outbreaks due the source of the water (wells and springs) (Stukel et al., 1990; Fayer et al., 2000). Agricultural activity can generate an environmental impact able to affect the water quality throughout the region. The manure of animals is poorly managed can pose a major environmental impact factor due to the contamination of the site by waterborne pathogens (Conboy and Goss, 2000; Fayer et al., 2000). Surface water and groundwater from rural properties may contain enteric viruses and the spread of these pathogens can be influenced by rain, since the excreta of animals are deposited in the soil to enrich the nutrient content of the same (De Oliveira et al., 2012). The management of excreta was inadequate in the farms studied. Most of the contamination found from animal origin could be associated to direct runoff of the excreta deposited in soils with no prior treatment; human sewage is not treated and no basic sanitary premises were found in these farms. HAdV was the most prevalent viral agent both matrices found in this study (87.3% and 80% respectively). HAdV is one of the principal etiological agents of gastroenteritis in children under 4 years old (Mehnert et al., 2001; Frost et al., 2002; Lee and Kim, 2002). This pathogen is included in the “Contaminant Candidate List 3” from the U.S. Environmental Protection Agency (USEPA) for its sanitary importance and its elevated incidence in water and sewage samples (USEPA, 2009). The viral load in water samples ranged from 102 gc/L to108 gc/L. The detection frequency found in the present study are higher in comparison to previous studies: Jiang and Chu (2004) reported 52%, Verheyen et al. (2009) 12.9%, Pina et al. (1998) 65%, and De Oliveira et al. (2012) found HAdV in up to 23.2% of water samples from dairy farms, depending upon the presence of accumulated days of rain. In the present study, viral loads of HAdV in groundwater varied between 103 gc/L and 104 gc/L

in 96.9% (32/33) of positive samples, while single spring samples had 2.08 × 108 gc/L. In surface water, 73.3% (11/15) had 103 gc/L and 13.3% was the rate for 102 or 104 gc/L. These results are similar to those found in surface waters: Albinana-Gimenez et al. (2009) (101 –104 gc/L), Choi and Jiang (2005) (102 –104 gc/L), and Haramoto et al. (2010) (103 –105 gc/L). However, other studies reported viral loads lower in 2 logs from those presented here: Hundesa et al. (2006) (101 –102 gc/L), Wyn-Jones et al. (2011) (mean 5 × 102 gc/L) and Van Heerden et al., 2005 (mean < 1 gc/L), pointing out that the HAdV viral loads present in water are highly dependent on the geographic level, environmental damage, and the techniques used for detection. The result of 108 gc/L found in a particular spring water sample is even higher than those reported sometimes for HAdV in sewage samples: Bofill-Mas et al. (2006) found 3.8 × 107 gc/L in urban sewage in Spain, and He and Jiang (2005) (USA) detected an average of 106 gc/L. Thus, it shows a massive contamination of this water source, possibly due to soil contamination and consequently groundwater. Regarding the sediment samples, the viral loads were from 103 to 104 gc/g, this result corroborates the ability of the virus to accumulate in soils. According to Williamson et al. (2005), agricultural soils may have a concentration of 8.7 × 108 to 1.1 × 109 viral particles. The farms in the present study have used animal excreta as fertilizers, there are also no proper sanitary conditions for the families, most have used septic tanks for several years on their properties and this may favor the human viral concentration and abundance in soils (Williamson et al., 2013). Furthermore, by complementary sequencing and molecular characterization, it was verified that many of the HAdV found in this study belong to the subgroup C that is responsible for respiratory infections (data not shown). Studies indicate that HAdV-C is one of the viral agents most commonly excreted in the feces by humans. Even if there are no clinical symptoms (Fongaro et al., 2013; Mena and Gerba, 2009), the presence of these viral agents indicates contamination, however, with a reduced risk to public health. In sediment samples higher positivity (infectious HAdV) was found in comparison with the water samples (Table 4), all samples containing viable viral particles were also detected earlier by qPCR. The load viral for HAdV was almost 3 times higher in sediment samples than in water. Sim and Chrysikopoulos (2000) have suggested sorption at the solid-water interface may enhance virus longevity, and Rao et al. (1986) suggested that absorbed viruses offer less surface for interaction with inactivating substances and this feature may protect the viral structure. The present results suggest that viral particles aggregated to sediment could be more protected and resistant to inactivation when compared to those distributed in the water column; this has certainly been seen in adenoviruses in an infectious state in various environments over long periods (WynJones et al., 2011). Charles et al. (2009) reported infectious HAdV in groundwater over one year. Fongaro et al. (2013) reported 52.7% of infectious HAdV in the Peri Lagoon, spring source water and

Table 3 Detection of AdV species and EV genomes and coliform quantification in water/sediment samples from farms in the municipality of Rolante. Quantitative viral results from water (gc/L) and sediment (gc/g). Farm RO1 RO2 RO3 RO4 RO5 RO6 RO7

RO9 RO10

RO11

RO12

RO13 RO141

RI1

RI2

RI3

RI4

RI5

RI6 RI7

Fecal coliforms

HAdVW

HAdVS

BAVW

BAVS

PoAdVW

PoAdVS

CAVW

CAVS

AvAdVW

AvAdVS

EVW

EVS

Spring 1 Spring 2 Dam Artesian well Tap water (Spring) Artesian well Spring Stream Spring Tap water (Spring) Artesian well Stream Artesian well Spring Dam Stream Artesian well Artesian well Spring Dam Tap water (Spring) Stream Spring Artesian well Spring Dam 1 Dam 2 Artesian well Spring Stream Artesian well Spring 1 Spring 2 Tap water (Spring) Stream 1 Stream 2 Tap water (Spring) Spring Artesian well Spring Tap water (Spring) Stream Spring Tap water (Spring) Dam Stream Spring Tap water (Spring) Stream Dam Spring Tap water (Spring) Dam Artesian well Spring

31 50.4 <1 <1 98 2 133.4 3100 3.1 <1 5.1 310 100 5.2 6300 2160 <1 <1 410 <1 1 1210 <1 <1 630 <1 <1 19.9 16.1 1600 <1 3.1 122.3 110.6 1440 1610 21.3 1 2 98 14.8 850 2 21.8 <1 7400 4.1 14.6 <1 410 <1 4.1 100 <1 12.1

Neg 1.95E+04 8.88E+03 5.71E+03 Neg 9.49E+03 2.34E+03 4.08E+03 1.01E+03 Neg 6.13E+03 1.73E+04 7.19E+03 1.18E+04 1.08E+04 5.52E+03 1.87E+03 1.33E+03 Neg 5.84E+03 1.14E+03 Neg 1.78E+04 4.99E+03 2.35E+04 4.43E+03 4.85E+03 1.26E+04 2.08E+08 9.04E+03 1.86E+03 1.82E+03 7.17E+04 1.95E+04 1.98E+03 Neg 6.71E+04 5.23E+04 2.72E+04 9.57E+04 2.46E+04 9.68E+03 1.38E+03 8.23E+03 1.57E+02 1.94E+03 3.28E+04 7.52E+03 Neg 2.68E+02 2.16E+04 3.54E+03 2.08E+03 3.87E+03 8.53E+04

1.20E+04 Neg 1.97E+03 * * * 5.87E+04 4.27E+03 * * * 1.96E+04 * 1.26E+04 2.36E+04 8.20E+03 * * * * * Neg 1.41E+04 * 7.51E+03 * Neg * * * * * * * 5.80E+04 6.96E+04 * 1.66E+04 * * * 3.78E+04 * * Neg 5.34E+03 * * * * * * 7.67E+03 * *

Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg 2.34E+06 Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg 7.41E+05 Neg Neg 4.75E+05 Neg Neg Neg Neg Neg Neg 9.51E+05 Neg Neg

Neg Neg Neg * * * Neg Neg * * * 1.80E+07 * Neg Neg Neg * * * * * Neg Neg * Neg * Neg * * * * * * * Neg Neg * Neg * * * Neg * * Neg Neg * * * * * * Neg * *

Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg 1.06E+05 Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg

Neg Neg Neg * * * Neg Neg * * * Neg * Neg Neg Neg * * * * * Neg Neg * Neg * Neg * * * * * * * Neg Neg * Neg * * * Neg * * Neg Neg * * * * * * Neg * *

Neg Neg 2.45E+06 Neg Neg Neg 2.49E+09 Neg 1.87E+05 Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg 2.31E+07 Neg Neg Neg Neg Neg Neg 1.92E+06 Neg Neg Neg Neg Neg Neg 4.02E+07 1.16E+05 Neg Neg Neg 3.56E+08 Neg 4.91E+06 Neg Neg Neg Neg Neg 2.06E+07 Neg Neg 3.23E+07 Neg Neg Neg Neg Neg

Neg Neg Neg * * * 1.01E+08 6.77E+06 * * * Neg * Neg Neg Neg * * * * * Neg 1.52E+07 * Neg * Neg * * * * * * * Neg Neg * Neg * * * Neg * * 1.50E+06 Neg * * * * * * Neg * *

Neg Neg Neg Neg Neg Neg Neg Neg 1.87E+05 Neg Neg 1.67E+06 Neg 1.26E+09 Neg Neg Neg Neg Neg Neg Neg Neg Neg 5.78E+08 2.93E+08 Neg Neg Neg Neg Neg 1.01E+05 Neg 6.68E+09 Neg Neg Neg 1.36E+08 5.84E+07 Neg 1.56E+08 Neg Neg Neg 3.78E+08 Neg 4,75E+05 Neg 4.07E+08 Neg Neg 7.08E+07 Neg Neg Neg 8.84E+08

Neg 8.55E+06 8.01E+06 * * * Neg Neg * * * Neg * 4.25E+07 Neg Neg * * * * * Neg Neg * Neg * Neg * * * * * * * 2.57E+07 1.20E+08 * 2.18E+08 * * * 4.46E+07 * * Neg 6,35E+07 * * * * * * 1.95E+07 * *

4.70E+05 Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg

Neg Neg Neg * * * Neg Neg * * * Neg * Neg Neg Neg * * * * * Neg Neg * Neg * Neg * * * * * * * Neg Neg * Neg * * * Neg * * Neg Neg * * * * * * Neg * *

139

Rolante (RO); Riozinho (RI); water sample (W); sediment sample (S); negative (Neg); non collected (*); Human Adenovírus (HAdV); Bovine Adenovírus (BAV); Porcine Adenovirus (PoAdV); Canine Adenovirus (CAV); Avian Adenovirus (AvAdV); Human Enterovirus (EV).

R. Staggemeier et al. / Agricultural Water Management 152 (2015) 135–141

RO8

Sample

140

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Table 4 Results for viable AdV by ICC-RT-qPCR.

Farm RO1 RO8 RO11 RI1 RI2 RI3 RI6

Sample Spring 1 Dam Spring Tap water (Spring) Spring Stream Dam

Water (gc/L)

Sediment (gc/g)

HAdV (ICC) Neg Neg 9.29E+03 5.70E+03 9.60E+03 1.17E+05 Neg

HAdV (ICC) 6.02E+02 9.92E+06 4.30E+07 * Neg 2.36E+05 2.11E+06

Rolante (RO); Riozinho (RI); Negative (Neg); not collected (*).

public supply system water. Wyn-Jones et al. (2011) found viable HAdV in marine water (47%) and freshwater (20%). The presence of infectious HAdV in the region can pose a risk to public health. The animal adenoviruses had high prevalence in this study and a higher viral load in positive samples when compared to HAdV, demonstrating extensive fecal contamination of animal origin. AvAdV was second most prevalent in both matrices and is a viral agent found in healthy and diseased poultry with high excretion in feces (Mcferran and Smyth, 2000; Hess, 2000). There was a greater positivity in the sediment than in water. Most viral loads (46.7%) in water were concentrated at 108 gc/L (with 2 peaks at 109 gc/L in springs) while the sediment (55.5%) at107 gc/g (1 spring and 1 stream with 108 gc/g). CAV was found in 20% of water and sediment samples, with a mean viral load of 2.7 × 108 gc/L and 3.12 × 107 gc/g respectively. This virus has already been found infecting domestic and wild dogs in the state of Rio Grande do Sul (Dezengrini et al., 2007). The larger CAV load in water (40%) was of 107 gc/L (with a peak of 109 gc/L in a spring) and in the sediment (50%) at 106 gc/g. BAV was detected in 7.3% of water samples, 75% of these positive samples with viral loads of 105 gc/L and only one positive sediment sample (1.80 × 107 gc/g). BAV virus has been detected and used to trace the sources of fecal contamination derived from sewage and cattle farms (Fong et al., 2005; Maluquer De Motes et al., 2004). Ahmed et al. (2010) detected 10% BAV in water samples in Australia. PoAdV was detected only in a single water sample (1.06 × 105 gc/L). PoAdV was found in wastewater, river water and sludge (Maluquer De Motes et al., 2004; Hundesa et al., 2006, 2009), and has been used as an indicator of fecal contamination of the environment by porcine (Hundesa et al., 2006). The virus associated with particulate material showed a smaller variation of viral quantification (106 –107 ) than water (105 –108 ). AvAdV had a higher viral load in both environmental matrices in comparison with other species of animal AdV, and it was noticeable that the avian viruses were present mostly in groundwater. PoAdV and BAV had the smallest animal viral load in the water. Most properties had cattle, swine and poultry simultaneously, and animal husbandry, specially dairy, is important for the economy of these farms. Regarding human EV, human EV (4.07 × 105 gc/L) was detected on a single property coming from a spring used for animal consumption and washing utensils intended for the use in milk storage. The virus is proposed as reliable indicator of water quality (Fong and Lipp, 2005). De Oliveira et al. (2012) investigated the presence of EV on farms in the same watershed municipalities as this study. As in the example from this study, the authors also found low prevalence of EV (9.3%) in water, suggesting that these pathogens should not be used as a marker for fecal contamination this region. 5. Conclusions All properties presented contamination by enteric viruses. These results demonstrate the great fecal contamination of animal and human origin in waters and sediments from farms, possibly due to inadequate management of human and animal waste, lack of

sanitation, the springs are not protected and the wells are left open without meeting technical requirements, improperly sealed and near potential sources of contamination such as drains and grazing areas occupied by animals. The detection of infectious viruses demonstrates risks to human health. The sediment analysis also collaborated with the environmental analysis, checking for the presence of enteric viruses in this kind of matrix which was greater than that detected in water. Beyond that, it was observed that adenovirus infectious viral particles are more often found in the sediment than in water samples on dairy farms. This study suggests that the viral ability to associate with the solid matrix enables it to maintain the virus in an infectious state for long periods in that environment, furthermore, the virus capacity of percolation through the soil enables the contamination of water bodies in the region offering a risk even to the Guarani Aquifer since the site is in an outcropping region, Rolante, Areia and Riozinho rivers also can be recharged by these sources contaminated compromising the quality of these rivers and therefore the rivers of which they are tributaries. The positivity of human adenovirus was greater than animal adenovirus, possibly due to farms having septic tanks over a long number of years. The problems due to this contamination can reach beyond farmers, occasioning considerable losses in dairy production by the involvement of their animals by these pathogens. This study suggests the animal viruses above as good indicators of animal fecal contamination.

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