Trop Anim Health Prod (2015) 47:237–241 DOI 10.1007/s11250-014-0685-3
SHORT COMMUNICATIONS
High frequency of porcine norovirus infection in finisher units of Brazilian pig-production systems Patrícia F. N. Silva & Alice F. Alfieri & Aline F. Barry & Raquel de Arruda Leme & Noemi R. Gardinali & Wim H. M. van der Poel & Amauri Alcindo Alfieri
Received: 14 July 2014 / Accepted: 16 September 2014 / Published online: 4 October 2014 # Springer Science+Business Media Dordrecht 2014
Abstract Norovirus (NoV) is a member of the Caliciviridae family and is considered an emerging human enteric pathogen. NoVs are detected in farm animals such as cattle, sheep and pigs. Porcine NoV (PoNoV) is widespread worldwide, but frequency of infection is often low. This study aimed to investigate the natural PoNoV infection from adult animals of an important Brazilian pig-production region. Faecal samples (n=112) of asymptomatic pigs aged 9 to 24 weeks old were collected from 16 grower-to-finish herds located in Paraná state, Brazilian Southern region, and evaluated for PoNoV presence. A reverse transcription-polymerase chain reaction (RT-PCR) assay was performed using specific primers that target a conserved region of the virus capsid gene (VP1). PoNoV was detected in 58 (51.8 %) of the 112 faecal samples and in 14 (87.5 %) of the 16 herds evaluated. Six of the obtained amplicons were submitted to phylogenetic genotyping analysis. The higher nucleotide (86.5–97.4 %) and amino acid (100 %) similarities of the sequences in this study were with the representative strains of the porcine NoV genogroup II genotype 11 (PoNoV GII-11). These results reveal that PoNoV infection is endemic in one of the most important pork production areas of Brazil and that the PoNoV GII-11 is prevalent in this region. Keywords Calicivirus . Enteric virus . Faeces . Intestinal health . Molecular epidemiology . Swine P. F. N. Silva : A. F. Alfieri : A. F. Barry : R. de Arruda Leme : N. R. Gardinali : A. A. Alfieri (*) Laboratory of Animal Virology, Department of Preventive Veterinary Medicine, Universidade Estadual de Londrina, Campus Universitário, PO Box 10011, Londrina, Paraná 86057-970, Brazil e-mail: [email protected] W. H. M. van der Poel Department of Virology, Central Veterinary Institute,, Wageningen University and Research Centre, PO Box 65, 8200 AB Lelystad, The Netherlands
Introduction Caliciviridae family is composed by the genera Lagovirus, Nebovirus, Sapovirus, Vesivirus and Norovirus. Norovirus genus has a single representative species, previously named Norovirus and recently renamed as Norwalk virus (NoV) (ICTV 2013). NoVs have been detected in human and in different animal species, such as bovine, swine, ovine, canine and feline (Martella et al. 2007, 2008; Patel et al. 2009; Wolf et al. 2009; Mijovski et al. 2010; Pinto et al. 2012). Human and animal NoVs are non-enveloped and have a positive-sense, single-stranded RNA genome of approximately 7.5 kb (Green 2007). The genome of NoV is organized into three open reading frames (ORFs). ORF1 encodes a polyprotein that origins six non-structural proteins after autocleavage. ORF2 encodes the major capsid protein-denominated VP1, and the ORF3 encodes a protein named VP2 with function in packing the genome into virions (Green 2007). Based on the complete VP1 amino acid (aa) sequence, NoVs are classified into five genogroups (GI–GV) and subdivided into genotypes. Human NoV strains are organized into GI, GII and GIV; the bovine and sheep isolates are in GIII; the canine and feline NoVs are classified as GIV, and the murine NoV is in the GV (Zheng et al. 2006; Pinto et al. 2012). Porcine NoV (PoNoV) strains are classified into GII and are closest to the most prevalent human NoV isolates. The PoNoV GII strains detected to date are distributed into the three genotypes GII-11, GII-18 and GII-19, while the human NoVs GII are classified into other distinct genotypes (Wang et al. 2007). NoV is considered the major cause of human non-bacterial gastroenteritis worldwide, including water- and food-borne outbreaks (Patel et al. 2009). For animals, the pathogenic role of NoV infection and its impact are not completely clear. Most
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of the epidemiological studies detected NoV in asymptomatic adult pigs worldwide (van der Poel et al. 2000; Keum et al. 2009; L’Homme et al. 2009). In Brazil, the epidemiological data regarding porcine enteric caliciviruses are limited. PoNoV genome was recovered from faecal samples of non-diarrhoeic finisher animals of pig farms located in Rio de Janeiro state, Brazilian Southeast region (Cunha et al. 2010a). The same research group reported the first detection of PoNoV GII-18 strain in Latin America (Cunha et al. 2010b). The circulation of PoNoV is poorly studied in Brazil. This country holds an important position in the international pork meat market, being the world’s fourth main producer and exporter of pig meat (ABPA 2014). To determine the frequency of natural infection by PoNoV in the major pig-producing regions of Brazil is important, since there is a chance that the virus might compromise the health and productivity of pig herds. The aim of this study was to evaluate the presence of PoNoV in asymptomatic grower-to-finish animals in Brazilian pig herds located in one of the largest Brazilian porkproducing areas with high animal population density.
Materials and methods The specimens evaluated in this study were selected from a collection of porcine faecal samples stored at −80 °C. The faecal samples were from adult animals of pig farms located in Toledo district, Western region of Paraná state (24° 42′ 49″ S; 53°44′ 35″ W). The animals were raised in multisite production systems with good nutritional and sanitary practices, including the “all-in-all-out” management. One hundred and twelve faecal samples of normal consistency from asymptomatic finisher (9 to 24 weeks old) pigs that were collected in December 2008 (summer, n=41) and June 2009 (winter, n=71) were included in this study. A total of 16 grower-to-finish units that were sampled in the summer (n=7) and winter (n=9) seasons were evaluated. The nucleic acids were extracted from 10 to 20 % (w/v) PBS faecal suspensions by using a combination of phenol/ chloroform/isoamyl alcohol (25:24:1) and silica/guanidinium isothiocyanate nucleic acid extraction methods (Boom et al. 1990; Alfieri et al. 2006). The RNA was eluted in 50 μl of ultrapure RNase-free diethylpyrocarbonate (DEPC)-treated sterile water. A sequenced wild-type Brazilian PoNoV GII11 strain from an adult pig with 90 % of nucleotide (nt) identity with the SwNV/swine43/JP strain and an aliquot of sterile ultrapure water were included as positive and negative controls, respectively, in all procedures. To perform the reverse transcription-polymerase chain reaction (RT-PCR) assay, forward (SwNV1, 5′-CGTACCAG AGGTCAACAAT-3′; nt 5124–5142) and reverse (SwNV2, 5′-AATCTAACAAAATCTCACCTG-3′; nt 5305−5285)
Trop Anim Health Prod (2015) 47:237–241
primers that were designed based on the SwNV/swine43/JP strain, GenBank accession number AB126320 (Reusken et al., Prevalence and genetic diversity of porcine caliciviruses in The Netherlands: identification of porcine sapoviruses closely related to human sapoviruses, Unpublished), were used. The primer pair targeted a 181 bp fragment size of the N-terminal capsid genomic region of PoNoV. RT reaction was performed using a denaturation solution of 12 μl containing 10 μl of the extracted RNA, 1 μl DMSO (5 % final concentration) and 1 μl (20 pmol) of SwNV2 primer, which was incubated at 97 °C/5 min. Subsequently, it was placed on ice for 5 min. A volume of 8 μl of RT mix solution containing 1× RT buffer (50 mM Tris–HCl pH 8.3, 75 mM KCl, 3 mM MgCl2), 0.1 mM each dNTP, 10 mM DTT and 100 U M-MLV Reverse Transcriptase (Invitrogen™ Life Technologies, Eugene, OR, USA) was mixed to the denaturation solution and incubated at 42 °C/30 min, followed by an inactivation reaction at 94 °C/5 min. For the PCR assay, the reaction was performed in a final solution of 50 μl containing 5 μl cDNA, 1.5× PCR buffer (30 mM Tris-HCl pH 8.4 and 75 mM KCl), 2 mM MgCl2, 0.2 mM of each primer (forward and reverse) and 2.5 U Platinum Taq DNA Polymerase (Invitrogen™ Life Technologies, São Paulo, SP, Brazil). The reactions were performed in a thermocycler (Swift™ MaxPro Thermal Cycler, Esco Healthcare Pte, Singapore) at 94 °C/3 min for denaturation followed by 40 cycles of 94 °C/1 min, 52 °C/1 min and 72 °C/ 1 min and a final extension at 72 °C/10 min. The amplified products were analysed by electrophoresis on a 2 % agarose gel in TBE buffer, pH 8.4 (89 mM Tris, 89 mM boric acid, 2 mM EDTA), stained with ethidium bromide (0.5 g/ml) and visualized under UV light. To confirm the specificity of the amplicons obtained in this study, nine amplicons from nine different pig herds collected in summer (n=6) and winter (n=3) were selected for sequencing analyses. The PCR amplicons were purified by GFX™ PCR DNA and Gel Band Purification Kit (GE Healthcare, Little Chalfont, UK), quantified with Qubit™ Fluorometer (Invitrogen™ Life Technologies, Eugene, OR, USA) and sequenced in both directions with forward and reverse primers in an ABI 3500 Genetic Analyser with the BigDye™ Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems®, Foster City, CA, USA). Sequence quality analyses and consensus sequences were assembled using Phred/Phrap/CAP3 software (http:// asparagin.cenargen.embrapa.br/phph/). Similarity searches were performed with sequences deposited in GenBank using the Basic Local Alignment Search Tool—BLAST software (http://blast.ncbi.nlm.nih.gov/Blast.cgi). A phylogenetic tree based on amino acid (aa) was obtained using MEGA version 6.06 (Tamura et al. 2013), with the neighbour-joining statistical method based on Poisson correction model, which provided statistical support via bootstrapping with 1000 replicates.
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Trop Anim Health Prod (2015) 47:237–241
Sequence identity matrix was performed using the BioEdit software version 7.1.11 (http://www.mbio.ncsu.edu/bioedit/ bioedit.html). Statistical analysis was performed with Epi InfoTM, using chi-square (χ2) or Fisher’s exact tests to compare the detection rates according to the seasons. The confidence limit for the statistical tests was set at 95 % (p
SHORT COMMUNICATIONS
High frequency of porcine norovirus infection in finisher units of Brazilian pig-production systems Patrícia F. N. Silva & Alice F. Alfieri & Aline F. Barry & Raquel de Arruda Leme & Noemi R. Gardinali & Wim H. M. van der Poel & Amauri Alcindo Alfieri
Received: 14 July 2014 / Accepted: 16 September 2014 / Published online: 4 October 2014 # Springer Science+Business Media Dordrecht 2014
Abstract Norovirus (NoV) is a member of the Caliciviridae family and is considered an emerging human enteric pathogen. NoVs are detected in farm animals such as cattle, sheep and pigs. Porcine NoV (PoNoV) is widespread worldwide, but frequency of infection is often low. This study aimed to investigate the natural PoNoV infection from adult animals of an important Brazilian pig-production region. Faecal samples (n=112) of asymptomatic pigs aged 9 to 24 weeks old were collected from 16 grower-to-finish herds located in Paraná state, Brazilian Southern region, and evaluated for PoNoV presence. A reverse transcription-polymerase chain reaction (RT-PCR) assay was performed using specific primers that target a conserved region of the virus capsid gene (VP1). PoNoV was detected in 58 (51.8 %) of the 112 faecal samples and in 14 (87.5 %) of the 16 herds evaluated. Six of the obtained amplicons were submitted to phylogenetic genotyping analysis. The higher nucleotide (86.5–97.4 %) and amino acid (100 %) similarities of the sequences in this study were with the representative strains of the porcine NoV genogroup II genotype 11 (PoNoV GII-11). These results reveal that PoNoV infection is endemic in one of the most important pork production areas of Brazil and that the PoNoV GII-11 is prevalent in this region. Keywords Calicivirus . Enteric virus . Faeces . Intestinal health . Molecular epidemiology . Swine P. F. N. Silva : A. F. Alfieri : A. F. Barry : R. de Arruda Leme : N. R. Gardinali : A. A. Alfieri (*) Laboratory of Animal Virology, Department of Preventive Veterinary Medicine, Universidade Estadual de Londrina, Campus Universitário, PO Box 10011, Londrina, Paraná 86057-970, Brazil e-mail: [email protected] W. H. M. van der Poel Department of Virology, Central Veterinary Institute,, Wageningen University and Research Centre, PO Box 65, 8200 AB Lelystad, The Netherlands
Introduction Caliciviridae family is composed by the genera Lagovirus, Nebovirus, Sapovirus, Vesivirus and Norovirus. Norovirus genus has a single representative species, previously named Norovirus and recently renamed as Norwalk virus (NoV) (ICTV 2013). NoVs have been detected in human and in different animal species, such as bovine, swine, ovine, canine and feline (Martella et al. 2007, 2008; Patel et al. 2009; Wolf et al. 2009; Mijovski et al. 2010; Pinto et al. 2012). Human and animal NoVs are non-enveloped and have a positive-sense, single-stranded RNA genome of approximately 7.5 kb (Green 2007). The genome of NoV is organized into three open reading frames (ORFs). ORF1 encodes a polyprotein that origins six non-structural proteins after autocleavage. ORF2 encodes the major capsid protein-denominated VP1, and the ORF3 encodes a protein named VP2 with function in packing the genome into virions (Green 2007). Based on the complete VP1 amino acid (aa) sequence, NoVs are classified into five genogroups (GI–GV) and subdivided into genotypes. Human NoV strains are organized into GI, GII and GIV; the bovine and sheep isolates are in GIII; the canine and feline NoVs are classified as GIV, and the murine NoV is in the GV (Zheng et al. 2006; Pinto et al. 2012). Porcine NoV (PoNoV) strains are classified into GII and are closest to the most prevalent human NoV isolates. The PoNoV GII strains detected to date are distributed into the three genotypes GII-11, GII-18 and GII-19, while the human NoVs GII are classified into other distinct genotypes (Wang et al. 2007). NoV is considered the major cause of human non-bacterial gastroenteritis worldwide, including water- and food-borne outbreaks (Patel et al. 2009). For animals, the pathogenic role of NoV infection and its impact are not completely clear. Most
238
of the epidemiological studies detected NoV in asymptomatic adult pigs worldwide (van der Poel et al. 2000; Keum et al. 2009; L’Homme et al. 2009). In Brazil, the epidemiological data regarding porcine enteric caliciviruses are limited. PoNoV genome was recovered from faecal samples of non-diarrhoeic finisher animals of pig farms located in Rio de Janeiro state, Brazilian Southeast region (Cunha et al. 2010a). The same research group reported the first detection of PoNoV GII-18 strain in Latin America (Cunha et al. 2010b). The circulation of PoNoV is poorly studied in Brazil. This country holds an important position in the international pork meat market, being the world’s fourth main producer and exporter of pig meat (ABPA 2014). To determine the frequency of natural infection by PoNoV in the major pig-producing regions of Brazil is important, since there is a chance that the virus might compromise the health and productivity of pig herds. The aim of this study was to evaluate the presence of PoNoV in asymptomatic grower-to-finish animals in Brazilian pig herds located in one of the largest Brazilian porkproducing areas with high animal population density.
Materials and methods The specimens evaluated in this study were selected from a collection of porcine faecal samples stored at −80 °C. The faecal samples were from adult animals of pig farms located in Toledo district, Western region of Paraná state (24° 42′ 49″ S; 53°44′ 35″ W). The animals were raised in multisite production systems with good nutritional and sanitary practices, including the “all-in-all-out” management. One hundred and twelve faecal samples of normal consistency from asymptomatic finisher (9 to 24 weeks old) pigs that were collected in December 2008 (summer, n=41) and June 2009 (winter, n=71) were included in this study. A total of 16 grower-to-finish units that were sampled in the summer (n=7) and winter (n=9) seasons were evaluated. The nucleic acids were extracted from 10 to 20 % (w/v) PBS faecal suspensions by using a combination of phenol/ chloroform/isoamyl alcohol (25:24:1) and silica/guanidinium isothiocyanate nucleic acid extraction methods (Boom et al. 1990; Alfieri et al. 2006). The RNA was eluted in 50 μl of ultrapure RNase-free diethylpyrocarbonate (DEPC)-treated sterile water. A sequenced wild-type Brazilian PoNoV GII11 strain from an adult pig with 90 % of nucleotide (nt) identity with the SwNV/swine43/JP strain and an aliquot of sterile ultrapure water were included as positive and negative controls, respectively, in all procedures. To perform the reverse transcription-polymerase chain reaction (RT-PCR) assay, forward (SwNV1, 5′-CGTACCAG AGGTCAACAAT-3′; nt 5124–5142) and reverse (SwNV2, 5′-AATCTAACAAAATCTCACCTG-3′; nt 5305−5285)
Trop Anim Health Prod (2015) 47:237–241
primers that were designed based on the SwNV/swine43/JP strain, GenBank accession number AB126320 (Reusken et al., Prevalence and genetic diversity of porcine caliciviruses in The Netherlands: identification of porcine sapoviruses closely related to human sapoviruses, Unpublished), were used. The primer pair targeted a 181 bp fragment size of the N-terminal capsid genomic region of PoNoV. RT reaction was performed using a denaturation solution of 12 μl containing 10 μl of the extracted RNA, 1 μl DMSO (5 % final concentration) and 1 μl (20 pmol) of SwNV2 primer, which was incubated at 97 °C/5 min. Subsequently, it was placed on ice for 5 min. A volume of 8 μl of RT mix solution containing 1× RT buffer (50 mM Tris–HCl pH 8.3, 75 mM KCl, 3 mM MgCl2), 0.1 mM each dNTP, 10 mM DTT and 100 U M-MLV Reverse Transcriptase (Invitrogen™ Life Technologies, Eugene, OR, USA) was mixed to the denaturation solution and incubated at 42 °C/30 min, followed by an inactivation reaction at 94 °C/5 min. For the PCR assay, the reaction was performed in a final solution of 50 μl containing 5 μl cDNA, 1.5× PCR buffer (30 mM Tris-HCl pH 8.4 and 75 mM KCl), 2 mM MgCl2, 0.2 mM of each primer (forward and reverse) and 2.5 U Platinum Taq DNA Polymerase (Invitrogen™ Life Technologies, São Paulo, SP, Brazil). The reactions were performed in a thermocycler (Swift™ MaxPro Thermal Cycler, Esco Healthcare Pte, Singapore) at 94 °C/3 min for denaturation followed by 40 cycles of 94 °C/1 min, 52 °C/1 min and 72 °C/ 1 min and a final extension at 72 °C/10 min. The amplified products were analysed by electrophoresis on a 2 % agarose gel in TBE buffer, pH 8.4 (89 mM Tris, 89 mM boric acid, 2 mM EDTA), stained with ethidium bromide (0.5 g/ml) and visualized under UV light. To confirm the specificity of the amplicons obtained in this study, nine amplicons from nine different pig herds collected in summer (n=6) and winter (n=3) were selected for sequencing analyses. The PCR amplicons were purified by GFX™ PCR DNA and Gel Band Purification Kit (GE Healthcare, Little Chalfont, UK), quantified with Qubit™ Fluorometer (Invitrogen™ Life Technologies, Eugene, OR, USA) and sequenced in both directions with forward and reverse primers in an ABI 3500 Genetic Analyser with the BigDye™ Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems®, Foster City, CA, USA). Sequence quality analyses and consensus sequences were assembled using Phred/Phrap/CAP3 software (http:// asparagin.cenargen.embrapa.br/phph/). Similarity searches were performed with sequences deposited in GenBank using the Basic Local Alignment Search Tool—BLAST software (http://blast.ncbi.nlm.nih.gov/Blast.cgi). A phylogenetic tree based on amino acid (aa) was obtained using MEGA version 6.06 (Tamura et al. 2013), with the neighbour-joining statistical method based on Poisson correction model, which provided statistical support via bootstrapping with 1000 replicates.
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Trop Anim Health Prod (2015) 47:237–241
Sequence identity matrix was performed using the BioEdit software version 7.1.11 (http://www.mbio.ncsu.edu/bioedit/ bioedit.html). Statistical analysis was performed with Epi InfoTM, using chi-square (χ2) or Fisher’s exact tests to compare the detection rates according to the seasons. The confidence limit for the statistical tests was set at 95 % (p
