Porcine group a rotaviruses with heterogeneous VP7 and VP4 genotype combinations can be found together with enteric bacteria on Belgian swine farms

https://doi.org/10.1016/j.vetmic.2014.04.002Get rights and content

Highlights

  • RT-qPCR is most sensitive for diagnosis of porcine RVA infections in Belgium.

  • RVA positive samples collected on Belgian swine farms were mostly co-infected with enteric bacteria.

  • A high genetic diversity was found for genes encoding VP7 and VP4 with 12 different G/P combinations.

  • G2, G3, G4, G5 and G11 genotypes were detected for VP7.

  • P[6], P[7], P[13], P[23] and P[27] genotypes were detected for VP4.

Abstract

Group A rotaviruses (RVA) are an important cause of diarrhea in young piglets, resulting in significant economic losses. However, the role of RVA in the etiology of piglet diarrhea on Belgian swine farms was previously unreported. In the present study, different techniques, including fast antigen detection tests, virus isolation, RT-PCR and RT-qPCR have been applied for detection of RVA in diarrheic (n = 28) and asymptomatic (n = 6) fecal samples collected on Belgian pig farms. RT-qPCR was shown to be most sensitive. Routine bacteriological analysis of the fecal samples showed that most diarrheic RVA positive samples were also co-infected with one or more bacterial species, such as Escherichia coli, Clostridium perfringens, Salmonella sp. and/or Brachyspira sp. Further genetic characterization of the VP7 and VP4 genes of 26 RVA strains resulted in the detection of six different G-genotypes (G2, G3, G4, G5, G9 and G11), and five different P-genotypes (P[6], P[7], P[13], P[23], P[27]), in a total of 12 different G/P combinations. A large intra-genotypic diversity was also apparent. In conclusion, results of the present study help us better understand the role of RVA in the pathogenesis of piglet diarrhea, and provide better insights into the vast genetic diversity present among circulating porcine group A rotaviruses.

Introduction

Diarrhea is one of the most important causes of mortality and growth retardation in young piglets and can be evoked by viruses, bacteria and parasites. Among these pathogens, rotavirus, transmissible gastroenteritis virus, porcine epidemic diarrhea virus, Escherichia coli, Salmonella, Clostridium perfringens and Isospora suis are considered most important. In general, porcine rotaviruses are an important cause of diarrhea during two critical time points in the pig's life. First, it is often involved in enteritis during the nursery period, mostly in piglets farrowed by gilts with a poor lactogenic immunity. Second, rotavirus also causes diarrhea shortly after weaning due to an abrupt disappearance of lactogenic immunity and the presence of a high infection pressure in the weaner shed (Bohl et al., 1978, Lecce and King, 1978). Co-infections between viruses, bacteria and parasites have been detected frequently in diarrheic piglets.

Genus rotavirus is divided in five species or groups (A to E) by the ICTV, and two tentative species (F and G). A potential new species (H) includes “new adult diarrhea rotavirus” isolated from a human in 1997, and a rotavirus isolated from a pig (Matthijnssens et al., 2012). Although rotavirus species A, B and C, and to a lesser extent also E and H can be found in feces from pigs, most research has been conducted on rotaviruses from species A (RVA) (Martella et al., 2010). The segmented dsRNA genome of RVA encodes six structural (VP1-4, VP6 and VP7) and six non-structural proteins (NSP1-NSP5/6). A full genome-based classification system for RVA has been established, giving all 11 gene segments a letter, followed by a number representing the genotype (Matthijnssens et al., 2008b). Genes encoding the outer-capsid proteins VP7 (G-genotypes) and VP4 (P-genotypes) are of utmost importance in this classification system, since these proteins induce neutralizing antibodies. To date, 27 G-genotypes and 37 P-genotypes have been identified in many species (Matthijnssens et al., 2011, Trojnar et al., 2013). So far, 12 G-genotypes (G1 to G6, G8 to G12 and G26) and 13 P-genotypes (P[1], P[5] to P[8], P[11], P[13], P[19], P[23], P[26], P[27], P[32], P[34]) have been disclosed from symptomatic and asymptomatic pigs (Martella et al., 2010, Papp et al., 2013). Geographical differences in strain predominance exist (Midgley et al., 2012), with G9P[13] for example being the most prevalent genotype combination detected in Ohio, United States (Amimo et al., 2013a). Moreover, genetically heterogeneous RVA strains can circulate in different units of the same pig farm, with a seasonally changing predominance of certain G/P genotype combinations (Miyazaki et al., 2012, Miyazaki et al., 2013).

Swine production is one of the major agriculture activities in Belgium, with a herd size of approximately 6 million swine, with 1.6 million pigs belonging to the <20 kg weight category. In contrast to other countries, little information is available about the role of porcine RVA infections in the etiology of piglet diarrhea on Belgian pig farms. The use of fast antigen detection tests for diagnosis of RVA infections in fecal samples collected during the late phase of RVA infection, has been thought to hamper diagnosis of porcine RVA infections in Belgian veterinary practices, resulting in an underdiagnosis of porcine RVA in the etiology of piglet diarrhea. At present, nothing is known about the genetic constellation of Belgian porcine RVA strains. An exception is the completely characterized genome of the porcine-like human G9P[6] RVA strain, RVA/Human-wt/BEL/BE2001/2009/G9P[6], isolated from a diarrheic child in 2009 in Belgium (Zeller et al., 2012).

Increased surveillance of RVA infections in pigs will help us better understand their etiological role in the pathogenesis of piglet diarrhea. Therefore, different diagnostic approaches were used for detection of RVA in fecal samples, including fast antigen detection tests, virus isolation, RT-PCR and RT-qPCR. RVA strains found in positive samples were further genetically characterized for genes encoding VP7 and VP4. This characterization may help elucidating the evolutionary relationship between porcine and human RVA, and facilitating interpretation of rare interspecies transmission events detected during human RVA surveillance.

Section snippets

Collection and processing of fecal samples

A total of 34 fecal samples were collected on 21 different farms in Belgium. From these, 28 samples were obtained from diarrheic piglets, whereas six samples were from non-diarrheic piglets. Samples were collected between 2011 and 2012 by private laboratories for diagnosis of enteric pathogens involved in piglet diarrhea. In these laboratories, the presence of RVA antigens was first evaluated using a fast antigen detection test: Prospect® Rotavirus Microplate Assay (ThermoScientific,

Validation of new RT-qPCR assay for the detection of porcine RVA gene segment 11

RT-qPCR primers, named qPCR_fw and qPCR_rv (Table 1), were designed against conserved regions of the porcine RVA gene segment 11. In silico evaluation of the primers showed a minimal tendency for dimer formation, and the 116 bp target region was free from secondary structures at the primer annealing sites. BLAST search for the forward and reverse primers showed that these primers recognized a large variety of RVA strains. RT-qPCR efficiencies were derived from the slopes of the standard curves (

Discussion

Diarrhea in pigs is an important cause of increased mortality, growth impairment, and economic losses. The role of RVA in the etiology of piglet diarrhea in Belgium was unclear. It was hypothesized that RVA infections were underdiagnosed on Belgian swine farms due to an improper use of fast diagnostic tests. Therefore, samples from Belgian diarrheic and asymptomatic pigs were collected and analyzed for the presence of RVA using different diagnostic approaches, followed by further genetic

Conflict of interest statement

The authors declare not to have any conflicts of interest.

Acknowledgments

The authors are grateful to Ellen Van Driesche, Charlotte Brosse, Willem Van Praet (DGZ Vlaanderen), Philip Vyt (Dialab), Filip Barbé and Griet Vercauteren (Mediclab) for supplying fecal material of pigs. The authors thank Lieve Sys and Ytse Noppe for their excellent technical assistance. LMD was supported by a doctoral fellowship of The Research Foundation-Flanders (FWO Vlaanderen). MZ, AD and IR were supported by doctoral fellowship of the Institute for the Promotion of Innovation through

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