Teschovirus and other swine and human enteric viruses in Brazilian watersheds impacted by swine husbandry

Abstract

Several emerging viral agents related to gastroenteritis are distributed in human and animal populations and may contaminate the environment due to anthropic activities. The objective of this study was to analyze the seasonal contamination by enteric virus and coliforms in water from streams in the Vale do Taquari, draining a large number of pig farms. Microbiological contamination was evidenced by the detection of total and thermotolerant coliforms, reaching their peak in December. Hepatitis E virus (HEV), Enterovirus-G (EV-G) genome, and Sapelovirus-A (SV-A) genome were not detected. On the other hand, Rotavirus (RV) was detected in 3% (1/32) of the samples, whereas Teschovirus-A (PTV) was detected in 6% (2/32). This is the first detection of PTV in environmental samples in Brazil, pointing that the virus is being shedded from swine herds to watersheds. Human mastadenovirus (HAdV) was the most frequent detected viral agent in 9.3% (3/32) with values of 2.54 × 10⁵, 7.13 × 10⁴, and 3.09 × 10⁵ genome copies/liter (gc/L). The circulation of coliforms and viral pathogens is noticeable due to anthropic activities and to the management of animal waste from the pig farming. In this way, enteric viruses can assist in monitoring the quality of watersheds and in tracking sources of contamination.

Introduction

The presence of viral pathogens excreted by the fecal route is often reported in aquatic ecosystems [1]. The dissemination of enteric viruses to the environment is facilitated by mismanagement of domestic sewage and animal manure due to inappropriate structure for agricultural activities, household septic systems, drainage of urban effluents, and bad practices when handling animal wastes [2–4]. The Vale do Taquari region (Southern Brazil) is characterized by intense pig farming activity. The intensive system for pig breeding with high animal density allows the transmission of pathogens between animals and their dissemination in the environment impacting hydric bodies [5]. Due to the high resistance of viral pathogens to inactivation under environmental conditions, some remain viable or potentially infectious for long periods in water [3, 6].

Some viral species can be used as markers of fecal contamination due to inadequate management of animal waste in rural areas [7]. Porcine teschovirus (PTV), Sapelovirus A (SV-A), and Enterovirus G (EV-G) are viruses belonging respectively to the genus Teschovirus, Sapelovirus, and Enterovirus of the Picornaviridae family. These swine pathogens were reported by others in water contaminated by swine manure in Spain and swine faces in Brazil [7–9], but there are no studies about the presence of these pathogens in environmental matrices from the southern region in Brazil. Due to previous studies realized describing the circulation of these pathogens in Brazil, its investigation is important.

PTV, SV-A, and EV-G are non-enveloped viruses with icosahedral symmetry and ssRNA positive polarity genome with approximately 6.2–8.5 Kb. They are transmitted by fecal route oral or vertical, replicating in the gastrointestinal epithelium and being eliminated in large quantities in the feces of infected animals [10]. These viruses may cause most often asymptomatic infections, but some serotypes can lead to clinical manifestations such as fever, diarrhea, pneumonia, reproductive failures, myocarditis, and polioencephalomyelitis in the extreme cases of PTV [10, 11].

Due to the lack of proper hygiene and sanitation in rural areas, other important human and zoonotic enteric viruses such as Human mastadenovirus C and F (HAdV-C and -F), Rotaviruses (RV), and Hepatitis E virus (HEV) are commonly found in the environment and are the main cause of waterborne diseases such as gastroenteritis, conjunctivitis, and hepatitis [3, 12] presenting risks to human and animal health. Data on the persistence of viruses in aquatic environments are relevant for estimating the health risk of the population, being an important tool to better understand its incidence and circulation, especially in developing countries, thus generating more information on the distribution, seasonality, and main circulating genotypes in certain geographic areas. With detection of this pathogens and other swine enteric viruses we can better determine the sources of contaminations [2, 13, 14]. The present work aims to analyze the contamination by enteric virus and coliforms in water from streams of Vale do Taquari, Rio Grande do Sul, covering pig farming areas, from September 2016 to June 2017 and to evaluate such viruses as specific indicators of contamination of water resources by pig farming effluents in the region.

Materials and methods

Sampling

Water samples were collected in streams that drain agglomerates of pig farming from the Vale do Taquari region, Rio Grande do Sul, from September 2016 to June 2017, where there is possible contamination from effluent flow, drinking water, and washing of materials derived from the handling in these sites due to be a region of intense pig farming. Samplings were carried out in different seasons and in eight points covering the municipalities of Marques de Souza, Lajeado, Travesseiro, Capitão, Forquetinha, and Arroio do Meio (Fig. 1). Samples were collected in sterile 500-mL borosilicate bottles for viral analysis and 100 mL for coliform analysis.

Fig. 1.

Vale do Taquari water collection points (map generated in Google Earth®)

Viral concentration

Thirty-six milliliter of water samples were concentrated by ultracentrifugation (3 h, 41.000×g) following pre-standardized and validated methodology in the Laboratory of Molecular Microbiology at Feevale University (Girardi, 2018). The supernatant was discarded, and the concentrated sample precipitated and adhered to the bottom of the tube recovered with TE (Tris-EDTA) 100× and stored at − 80 °C for further extraction of the genomic material validated before [15].

Extraction of RNA and viral DNA and cDNA

For extraction of RNA from the samples, the TRIZOL protocol was performed, and then cDNA synthesis was performed with the commercial kit High Capacity cDNA synthesis (Applied Biosystems) following the manufacturer’s methodology using 10 μL of RNA. The DNA extraction was performed from the commercial kit Viral Mini Spin Plus extraction kit (Biopur®, Brazil) following the manufacturer’s instructions.

RT-nested PCR for detection of HEV

The detection of HEV by RT-nested PCR was performed according to the protocol previously proposed [16] using primers for the region ORF1 of HEV. The reaction had a final volume of 50 μL containing 25 μL of Promega GoTaq Green Master Mix, 18 μL of water DNase/RNase-free, 1 ul of each primer in the contraction of 20 pmol, and 5 μL of cDNA. For amplification, the program was used with initial temperature of 95 °C for 5 min, followed by 45 cycles of 95 °C for 30 s, 59 °C for 1 min, 72 °C for 1 min, and at the end of the cycles 72 °C for 7 min. The second running was performed under the same conditions. After, the reactions of the electrophoresis of the amplified products were done, and the results were visualized with UV light.

RT-PCR for detection of RV

For detection of RV, the RT-PCR reaction uses the primers previously proposed [17] for VP6 region. The reaction had a final volume of 50 μL containing 25 μL of Promega GoTaq Green Master Mix, 18 μL of water DNase/RNase-free, 1 uL of each primer in the contraction of 20 pmol, and 5 μL of cDNA. The program used consisted of initial temperature of 94 °C for 5 min, followed by 35 cycles of 94 °C for 1 min, 54 °C for 1 min, 72 °C for 1 min, and at the end of the cycles, 72.8 °C for 7 min. After the reactions, the electrophoresis of the amplified products was done and the results were visualized with UV light.

Real-time quantitative PCR for detection of HAdV

For detection of HAdV-C and HAdV-F, the reaction of q-PCR uses primers and the program of temperatures previously proposed [18] for region of hexon protein. The reaction had a final volume of 25 μL: 5 μL of extracted DNA, 10 pmol of each primer, 12.5 μL of SYBR® Green q-PCR Super Mixf, and 5.5 μL of water RNAse-free (Milli-Q RNAse-/DNAse-free water system; Millipore). It is performed on 96-well plates (MicroAmp Applied Biosystems) with controls positive, negative, and the standard curve from (GC/L), using the iQ5™ Bio-Rad equipment (Biorad™, Hercules, CA 94,547, USA).

RT-nested PCR for detection of PTV, SV-A, and EV-G

The detection of PTV, SV-A, and EV-G by RT-Nested PCR was performed using primers previously proposed [9]. The reaction had a final volume of 50 μL containing 25 μL of Promega GoTaq Green Master Mix, 18 μL of water DNase/RNase-free, 1 μL of each primer in the contraction of 20 pmol, and 5 μL of cDNA. For amplification, the program was used with initial temperature of 94 °C for 5 min, followed by 35 cycles of 94 °C for 1 min, 55 °C for 1 min, 72 °C for 1 min, and at the end of the cycles, 72 °C for 7 min. The second race was performed under the same conditions. After the reactions, the electrophoresis of the amplified products was done and the results were visualized with UV light.

Virus isolation

For isolation of HAdV, cell cultures of A549 were maintained with Eagle’s Minimal Essential Medium (E-MEM) containing 10% of fetal bovine serum and 1% of penicillin/streptomycin, maintained at 37 °C in an atmosphere of 5% CO₂. For the assay, 24-well plates are used. The samples were pre-diluted with MEM (1:2) and filtered on 0.22-μm membranes. Subsequently, 200 μL of inoculum was added in contact with the cell carpet and incubated in the greenhouse for 2 h with agitation every 15 min.

After the incubation period, the inoculum was withdrawn and E-MEM with 1% penicillin/streptomycin was added for maintenance at 37 °C in an atmosphere of 5% CO₂ for 5 days. Subsequently, the plate was frozen at − 80 °C and thawed 3 times, and the inoculum was transferred to a new plate by repeating the procedure 5 times. The samples were then extracted with the protocol previously described and the q-PCR was performed.

Coliform counting

For the detection of total and thermotolerant coliforms (Escherichia coli) in all samples, the Colilert enzymatic method (Iddex, USA) was used, following the manufacturer’s instructions and counting expressed as Most Likely Number per 100 mL (MPN/100 mL).

Sequencing and phylogenetic analysis

The sequencing of the nucleotides was performed on the positive samples for TV-A and RV. The fragments were purified using the kit PureLink Quick Gel Extraction & Purification Combo Kit (Ambion™, Life Technologies, Löhne, Germany), following the manufacturer’s instructions and sequenced at the ABI Prism 3700 Genetic Analyzer, and the fragments were analyzed using the software BioEdit 7.0.5, and further phylogenetic analysis was conducted using the MEGA 7 software (Kumar, 2016) using prototype sequences for comparison.

Results

Regarding the bacterial markers, the results showed the presence of total and thermotolerant coliforms (Table 1) in all of the 32 analyzed samples; however, in the second collection, higher rates for both total and thermotolerant coliforms was observed, being the points 8 (173.290 NMP/100 mL) and 2 (141.360 NMP/100 mL) with higher rates for total coliforms and points 6 (12.230 NMP/100 mL) and 4 (11.120 NMP/100 mL) for thermotolerant coliforms.

Table 1.

Total (TC) and thermotolerant coliform (FC) (MPN/100 mL) counts of Vale do Taquari water samples from September 2016 to June 2017

Collect point	September		December		March		June
	TC	FC	TC	FC	TC	FC	CT	CF
P1	7.760	520	98.040	4.140	8.840	300	15.290	1.080
P2	12.81	1.870	141.360	7.120	14.550	970	17.270	1.340
P3	6.890	410	77.010	730	8.860	410	17.260	410
P4	16.16	4.040	98.040	11.120	50.120	3.550	24.890	1.580
P5	4.110	200	9.340	750	13.960	1.090	5.290	0
P6	29.24	2.750	37.240	12.230	28.510	1.100	27.230	630
P7	11.53	520	15.290	1.350	16.790	410	12.960	100
P8	64.88	10.810	173.290	3.320	39.680	8.600	45.690	5.610

Thirty-two water samples were analyzed for the presence of HEV, RV, PTV, SV-A, EV-G, and HAdV genomes. In the four water samplings, the presence of HEV, EV-G, and SV-A genome was not detected in any of the analyzed samples; RV was detected only in point 6 (June 2017) corresponding to 3% (1/32), PTV was detected in points 3 and 7 (September 2016 and June 2017) corresponding to 6% (2/32), and HAdV was the most frequent with 9.3% (3/32) found in points 3, 4, and 7 only in the month of December. The HAdVs found in this study through q-PCR correspond to genogroup C, and their means of quantification obtained were 2.54 × 10⁵, 7.13 × 10⁴ and 3.09 × 10⁵ genome copies (GC/L). After viral isolation of HAdV, in the first passage three positive samples with values of 7.63 × 10¹ GC/5 μL, 1.22 × 10² GC/5 μL, and 9.48 × 101 GC/5 μL and in the fifth passage, only two samples with values of 1.51 × 10² GC/5 μL and 1.17 × 10² GC/5 μL were detected

From the results obtained through conventional PCR further submitted to sequencing and phylogenetic analysis, 2 positive samples were observed positive for PTV (Fig. 2), genotypes PTV-A 7 and PTV-A 3, and 1 positive sample for RV (Fig. 3) referring to genogroup A.

Fig. 2.

Phylogenetic reconstruction by the Neighbor-joining method based on partial sequences of the 5′UTR gene of PTV. Sequences obtained in the present study are marked with lozenges referring to genotypes TV-A7 and TV-A3

Fig. 3.

Phylogenetic reconstruction by the Neighbor-joining method based on partial sequences of the VP6 gene of RV. Sequences obtained in the present study are marked with lozenges referring to genotype RV-A

Discussion

Inadequate management of swine manure and possible draining and percolation to nearby hydric bodies may impact the quality of surface water in the study’s regions like reported before in other countries [19]. In order to evaluate the quality of water, the analysis of thermotolerant coliforms (E. coli) is widely used, but its detection together with other pathogens such as viruses can help to better evaluate the quality of water bodies and even help to determine the possible sources of fecal contamination [4]. In all of the 32 water samples analyzed, both total and thermotolerant coliforms were found, with higher rates in the second collection in December, when fertilization and soil preparation were observed in the region, which may have contributed to the increase of coliform rates in this period.

The PTV circulation was evidenced in the hosts in the southern region of Brazil in two studies carried out on herds of swine and wild boars [9, 10]. However, different genotypes of this pathogen have been found endemically in many countries in faces of symptomatic and asymptomatic animals [20–22]. In Brazil, no study, up to the present moment, has evidenced the presence of this virus in environmental matrices. However, in the present work, this virus was found in 6% of the analyzed water samples, as well as in a study in Spain, also carried out in pig farming areas, where its detection in water bodies was reported [7]. These discoveries potentiate the use of these viruses as a tool to trace contamination in the hydric bodies by effluents from rural properties.

Several studies have demonstrated that in the occurrence of outbreaks, PTV frequency in faces and serum samples from infected animals occurs in high prevalences of 36%, 37%, and 47% in the USA, Dominican Republic, and Spain [5, 23, 24].

Regarding the other viruses analyzed in this study, HEV, SV-A, and EV-G genomes were detected in none of the water sample. In fact, HEV occurs variably in water in zones of pig farming, despite the ubiquity of the virus in herds of many regions. Although the presence of HEV in the analyzed streams was not observed, the circulation of genotype 3 in pig herds of rural properties of the studied region is described previously [25]. Studies report very low detection rates, as reported in water in Slovenia where 2/60 samples were positive for HEV genomes [26], and in Brazil where they also did not detect the virus in superficial water in the Vale dos Sinos region [16].

The presence of EV-G in aquatic environments is cited in Taiwan related to the discharge of effluents from pig farming near the analyzed local [27] and in the Philippines noting its circulation both in waters from rural and urban environments, having frequency in all seasons [28]. Although in Brazil, the EV-G and SV-A circulations in herds are described [9, 10], few are reported with the presence of these pathogens in hydric bodies. However, it should be considered that the samples from the present study were taken far from the farms in the drainage basin, which may indicate that they do not remain at detectable levels along the way.

HAdV and RV were found in 9.3 and 3%, respectively. Other studies also report the presence not only of effluents, groundwater, and surface waters but also of water treated more frequently with HAdV compared with other RNA viruses [4]. Due to their prevalence of these pathogens in the environment, population and resistance, some Adenovirus species are good markers of fecal contamination of human and animal origin [29], frequently detected in water sources that are within the bacteriological standards currently used as a parameter of evaluation of water potability [1, 12, 30]. After isolation, HAdV was detected, proving to be infectious, thereby this analysis are important to determine the real risk to human health.

Thus, with the results obtained, it is possible to observe the circulation of these viral agents derived from pig farming and the impact generated by this activity on the water quality of the water bodies of the Vale do Taquari region. Thereby, the monitoring of viral agents circulating in environmental matrices becomes important for the establishment of profiles of the circulating genotypes enabling strategic planning of control and prevention.