Area of study and survey samples
The study was carried out on the EEC (coordinates 29°44'27.1"S and 51°03'06.2"W) and in the city animal shelter (coordinates 29°44'20.06"S and 51°03'03.4"W) [Fig. 1]. EEC receives groups that do ecological tours and perform some workshops. The kennel receives abandoned dogs and cats in need of veterinary care, including vaccination and castration.
This study was performed in mid October, 2017 and late February, 2018. Both considered surface water, vegetables and dog stool samples.
Water samples were collected in one stream, three weirs and in one animal shelter septic tank, being the latter close to the kennel (see Fig. 1). The samples were named P1 – stream (29°44'29.2"S and 51°03'09.5"W), P2 – weir (29°44'23.5"S and 51°03.06.7"W), P3 – weir (29°44'23.1"S and 51°03.06.3"W), P4 – weir (29°44'23.4"S and 51°03'05.7"W) and P5 – septic tank (29°44'21.0"S and 51°03'04.4"W) [EEC in yellow highlighting; P1 at P5 in red in Fig. 1]. All samples were aseptically collected in 500mL sterile flasks, packed in isothermal boxes and sent to the Laboratory of Molecular Microbiology (LMM) of Feevale University to be processed.
The dog’s stools were collected in the kennel as soon as they were excreted (see Fig. 1 and the epidemiological aspects in Table 3). The use, handling and disposal of the stools used in this project are in agreement with the ethical principles for animal experimentation. The Ethics Committee of Animal Use of Feevale University (CEUA/Feevale) approved this research under registration number 01.17.055/2016.
Lettuce (Lactuca sativa) was collected in the EEC’s garden (closer to EEC in the Fig. 1). Lettuce samples were named H1 to H4, and three parts of each were analyzed (leaf, stem and root), renamed: H1L, H1S and H1R.
Sample were filed and named LMM with their respective number, following the sampling protocols.
Detection of TC and FC assay were performed by the Colilert® substrate enzyme method (Idexx®, USA) according to the manufacturer’s instructions. All samples (from P1 to P5) were assayed within 24 h after collection. The sample was considered positive for TC based on the yellow indicator. F or FC was considered positive if fluorescing blue following exposure to 300 nm UV light and negative in the absence of color and/or fluorescence. Results are expressed as most probable number in 100 mL of water (MPN/100mL), according to the table provided by the manufacturer.
Processing samples at viral analyzes
Samples were concentrated by applying the ultracentrifugation method , in which 36mL of each was used and centrifuged at a rate of 21,000 x g at 8° C for 3 hours. Products were resuspended in 1mL of Tris-EDTA buffer (pH 8.0) and homogenized under vigorous agitation for 1 minute, according to the standard protocol described in previous studies . The final volume was aliquoted and the DNA extracted.
Lettuce (from H1 to H4) from both collections (2017 and 2018) were organized in: leaves (HL), stem (HS) and roots (HR). Each one generated three samples, totaling 12 samples per collection and 24 in total. Each part was chopped and macerated separately. Subsequently, one gram of each was placed in 15 mL falcons and 9 mL of PBS buffer was added (Phosphate-buffered saline), then brought into the incubator at 22°C with constant stirring at 173 RPM for 1 hour. After, 1mL of supernatant was aliquoted into reaction microtubes and sent for DNA extraction using the protocols already described previously in the LMM at food processed pork analyzes that have been detected HEV .
For stool sample processing, 0.2 grams of diluted stools in 1 mL of Eagle’s Minimum Essential Medium (MEM) were used, then vortexed for 1 minute and centrifuged for 3 minutes at 120,000 RPM. From the supernatant, 1mL was used for viral DNA extraction, according to Heldt et al. .
Viral DNA extraction
Viral extraction of samples mentioned below was carried out using the Promega® extraction kit, following manufacturer’s instructions. Aliquots of 200μL of each sample were used, and the final elution was performed in microtubules free of DNAse and RNAse at analysis by Polymerase Chain Reaction (PCR).
Polymerase Chain Reaction
Nested-PCR, which DNAPol target gene, was used for the detection of different AdVs in all samples. In the first round, 1μL of each primer pol-F (5'-CAGCCKCKGTTRTGYAGGGT-3') and pol-R (5'-GCHACCATYAGCTCCAACTC-3') were used, both with 20 pmol concentration, 18μL of DNAse free water and RNAse, 25μL of mix (Promega®) and 5μL of extracted DNA, totaling 50μL of reaction volume. At the second round, the same reagents and volume of the first one were used. Only the 5μL of DNA extracted by the product of the first PCR reaction was replaced, as well as the set of oligonucleotides for the oligonucleotides sense pol-nF (5'GGGCTCRTTRGTCCAGCA-3') and reverse pol-nR
(5'-TAYGACATCTGYGGCATGTA-3') . The generated PCR product is approximately 300 bp. Each reaction had negative and positive control, water RNAse and DNAse free and HAdV-41 respectively.
The DNA extracted from the dog stool samples were concomitantly submitted to PCR, specific for CAV and CPV. A positive control was used for CAV analyses detected by the previous LMM. The standard PCR reaction for CAV was performed in a final volume of 50 μL containing 5 μL of DNA diluted in 25 μL of mix (Promega®), 18 μL of water and 20 pmol of each primer. The primer utilized for canine AdV was CAV-F1, 5'-CACGATGTGACCACTGAGAG-3' and CAV-R1, 5'-GGTAGGTATTGTTTGTGACAGC-3 (20 pmol dilution). The amplicon resulted 300 to 350 bp of the gene encoding a CAV-1 and CAV-2 hexon protein, respectively .
For CPV detection, the Vanguard® HTLP 5/CV-L vaccine was used as positive control, which contains the viruses of interest. Reactions of the conventional PCR for CPV were performed with the same final volume used for the CAV. The primer for CPV was: CPV-555-F, 5'-CAGGAAGATATCCAGAAGGA-3 'and CPV-555-R, 5'-GGTGCTAGTTGATATGTAAT3ACA-3' . The generated PCR product was 555 bp of the capsid protein gene (VP2 – viral protein).
Amplicon purification, sequencing and phylogenetic analyzes
All positive PCR samples were submitted at purified assays by the PureLink® kit (Invitrogen), according to manufacturer's instructions, and then subjected to sequencing. For that, ABIPrism 3100 system/company ACTGene equipment was used for to characterize species and/or type.
Nucleotide sequences resulting were analyzed by the CAP3 program implemented by BioEdit 7.0.5. The alignments were performed using Clustal Omega . The phylogenetic trees were obtained through the Neighbor Joining method  combined with Kimura 2 , MEGA7 software .