Rotavirus (RV) is a ubiquitous pathogen able to cause diarrhea in pigs of all ages, although suckling piglets are the most susceptible.^1-4 As animals age, most become protected from the disease by developing post-exposure immunity to RV coupled with maturation of the gut physiology and overall immunity.^1,5 Rotavirus groups A, B, and C are the most common in pigs, although E and H have also been demonstrated to cause disease in swine.¹ The RV groups are identified by the antigenicity of viral protein (VP) 6.¹ Sequencing of other structural viral proteins, such as VP7 and VP4, are employed to further type the virus into G (glycoprotein antigen) or P (protease-sensitive antigen) serotypes based on their antibody neutralization properties.^1,2,6

Group A was the first RV to be identified in pig production and has been considered the most critical and prevalent RV causing diarrhea in suckling piglets.⁶ Although RV groups B and C have been detected since the 1980s, the difficulty in growing these in cell culture did not allow for extensive investigation and analyses until recently.² Rotavirus C relevance as a diarrhea-causing pathogen in the pork industry was first thought sporadic. However, it has recently been recognized as endemic in most pig herds causing both subclinical disease and severe gastroenteritis in young piglets (78%, < 3 days of age).² Group B appears as a less prevalent RV and is sporadically found in pig herds and has been shown to have the ability to cause disease in piglets.^6,7 Due to the difficulty in culturing groups B and C, the only commercial vaccine available in Canada is based on the RV A G5 and A G9 subtypes. The prescription RNA particle vaccines are available for all three RV groups.

We aimed to understand the genetic diversity and geographical distribution of RV groups A, B, C, and G subtypes (VP7) infecting suckling piglets in Canadian farms. The determination of RV as cause of disease is not within the scope of this study, as not enough diagnostic data was collected, and the detection of RV does not imply infection and disease.

Animal care and use

This study used laboratory submission data from diagnostic veterinary submissions. Institutional animal use approval was not required.

Materials and methods

The animals were adequately housed and cared for in 290 commercial swine herds located in Alberta (AB), British Columbia (BC), Manitoba (MB), New Brunswick (NB), Ontario (ON), Quebec (QC), and Saskatchewan (SK). Fifty-eight swine veterinarians from 30 clinics submitted targeted (not random) enteric samples (fecal swab, intestinal content, or intestinal tissue) from suckling piglets presenting with rotaviral diarrhea between July 1, 2019 and December 31, 2023. Each sample collection was a result of the veterinarian investigating the cause of diarrhea in suckling piglets on their client’s farms. As they had previously eliminated other sources of pathogen-induced diarrhea, they submitted samples for RV sequencing to produce a prescription RV vaccine for each farm under the Sequivity RNA particle vaccine program (Merck & Co, Inc). Thus, samples, number of samples, and sample collection methods were not standardized among veterinarians and farms.

Samples received by the Animal Health Lab (AHL) at the University of Guelph were tested upon arrival for the RV group by polymerase chain reaction (qPCR), as previously described,⁸ followed by Sanger sequencing of the G type (VP7). If multiple samples within the same submission (case) were positive, only the sample with the lowest cycle threshold (Ct) on qPCR for each group (if more than one detected) was sequenced. Results were recorded matching the sequence to the clinic, farm, and veterinarian name (which remained confidential), province, date of collection, and age of pigs presenting clinical signs (only samples identified as suckling piglets were included). Sequencing results were analyzed using the Animal Health Sequivity Dashboard (Merck & Co, Inc), an RNA vaccine platform database and tool for sequence storage and analysis, as previously described by Sebo⁹ and followed by descriptive analysis.

Results

A total of 1117 samples from 614 cases of diarrhea were submitted to the AHL, where the samples with the lowest Cts (837 samples) were identified by sequencing the VP7 gene. Ontario had the highest representation in sequenced samples, with 22.6% (189 of 837) of the total samples, followed by AB with 21.8% (183 of 837), MB with 21.3% (178 of 837), SK with 17.7% (148 of 837), QC with 14.6% (122 of 837), BC with 1.2% (10 of 837), and NB with 0.8% (7 of 837). From all samples sequenced, RVA was present in 40.7% (341 of 837) of samples, RVB in 12.5% (105 of 837), and RVC in 46.8% (391 of 837) (Table 1). In most provinces (AB, BC, MB, NB, QC, and SK), RVC was the most detected group followed by RVA and RVB, while ON observed a higher presence of RVA followed by RVC and RVB. The number of farms and cases from each province are detailed in Table 1.

Single RV detections (only one group or subtype involved) represented 72.3% (444 of 614) of the cases, while 170 of the 614 cases (27.7%) had more than one RV group and subtype detected. The 170 RV codetection cases were represented by 393 sequences, from which RVC was present in 40.5% (159 of 393), followed by RVA in 38.4% (151 of 393), and RVB in 21.1% (83 of 393). Thirty-two cases had all three groups (RV A, B, and C) detected, while 90 codetection cases had groups A and C present; other combinations of groups or subtypes were also identified (Table 2).

Eighteen RV subtypes were identified within all cases and included 5 RVA, 9 RVB, and 4 RVC (Figure 1). The most common RV was RVC G6 detected in 296 samples, with a mean homology of 90.8% (range: 69.06%-100%) among samples. Rotavirus A G9 was found in 205 samples with a mean homology of 94.5% (range: 86.76%-100%). Similar mean homology was found within provinces (Table 3). Some less common RV sequences were present only in a specific region or province, for example, RVB G8 was only detected in SK.

Discussion

Rotavirus-related diarrhea in suckling piglets is a concern for the pork industry due to its high prevalence and impact on preweaning mortality and piglet performance.¹⁰ Like other studies, we found that suckling piglet samples were mainly positive with only one RV, although multigroup/subtype RV codetections were present.^2,5,11 In this study, RVC was the most detected RV in Canadian provinces (except in ON), followed by RVA, which was similar to previous results from the United States where RVC has been detected in 76% of suckling piglets. As previously observed, RVB was the least detected yet most diverse group.⁶ Our results indicated that RVA was the most detected RV in ON, which is similar to past studies conducted in this province.^11,8 Buchan and colleagues¹¹ summarized three years of diagnostic reports involving diarrhea presentations in ON during the lactation and nursery phases. Rotavirus A was detected in 69% of diarrhea cases in suckling piglets, RVC in 37%, and RVB in 13%. Similarly, Tran et al⁸ found RVA in 56.4% of samples from suckling pigs, 10% of RVB, and 34.4% of RVC (93% of all samples were from Ontario and Quebec).

Marthaler et al¹² tested 7508 samples from pigs with diarrhea in Canada, the United States, and Mexico. They found that 83% of samples were qPCR positive for RVA, RVB, or RVC. Group A was detected at the highest percentage (62%). While RVB and RVC were seen at a lower frequency (33% and 53%, respectively), both were considered epidemiologically relevant. The study also reported that RV detection can be related to the age of the pig sampled. Rotavirus C was more frequently detected in pigs within the first 21 days of age, while RVA and RVB were suggested as the cause of diarrhea in pigs over 21 days of age,¹² which may explain the higher detection of RVC observed in our study which targeted samples from suckling piglets.

The reason why the prominent group detected differed in ON from other provinces is not apparent. However, RV group detection has been shown to vary geographically.¹ Furthermore, sow vaccination programs, age, diet, genetics, and farrowing room management can vary from province to province, potentially influencing RV distribution. A diversity of subtypes within groups were observed demonstrating the diversity of RV. The VP7 sequences can vary within the same group as was shown in the homology analyses within the two most detected RV subtypes (A G9 and C G6). It is unknown what percentage of homology of VP7 would offer cross-protective immunity within the same subtype, although different subtypes within the same group are known to have small to no cross-protection.^13,14 Higher mean homology was observed among the A G9 sequences than the C G6, which had lower mean homology both within the country as well as within provinces.

The results presented here were not paired and analyzed with qPCR Ct results, clinical signs, or specific diagnostic tests to confirm RV-related disease. However, samples were collected from farms presenting with diarrhea in suckling piglets, where the veterinarian had previously tested for other pathogens and eliminated them as the cause of disease. Our observations suggest two different primary RV groups in Canada, RVA in ON and RVC in the western provinces and QC, indicating the relevance of RVC and the classic RVA in the Canadian swine industry.

Implications

Under the conditions of this study:

• Rotavirus C was the most detected RV in Canadian suckling piglets.
• Most cases were single RV detections, although RV codetections were common.
• Knowledge of RV subtypes inform veterinarians on prevention programs.

Acknowledgments

The authors thank all the veterinarians and staff cooperating in this study.

Conflict of interest

Malgarin and de Grau are both employed by Merck Animal Health, which provided the funding for all diagnostic tests used in this study. All diagnostic tests were conducted by the AHL at the University of Guelph.

Disclaimer

Scientific manuscripts published in the Journal of Swine Health and Production are peer-reviewed. However, information on medications, feed, and management techniques may be specific to the research or commercial situation presented in the manuscript. It is the responsibility of the reader to use information responsibly and by the rules and regulations governing research or the practice of veterinary medicine in their country or region.

References

1. Vlasova AN, Amimo JO, Saif LJ. Porcine rotaviruses: Epidemiology, immune responses and control strategies. Viruses. 2017;9(3):1-27. https://doi.org/10.3390/v9030048

2. Marthaler D, Rossow K, Culhane M, Collins J, Goyal S, Ciarlet M, Matthijnssens J. Identification, phylogenetic analysis, and classification of porcine group C rotavirus VP7 sequences from the United States and Canada. Virology. 2013;446(1-2):189-198. https://doi.org/10.1016/j.virol.2013.08.001

3. Naseer O, Jarvis MC, Ciarlet M, Marthaler DG. Genotypic and epitope characteristics of group A porcine rotavirus strains circulating in Canada. Virology. 2017;507:53-63. https://doi.org/10.1016/j.virol.2017.03.008

4. Chepngeno J, Diaz A, Paim FC, Saif LJ, Vlasova AN. Rotavirus C: Prevalence in suckling piglets and development of virus-like particles to assess the influence of maternal immunity on the disease development. Vet Res. 2019;50(1):84. https://doi.org/10.1186/s13567-019-0705-4

5. Almeida PR, Lorenzetti E, Cruz RS, Watanabe T, Zlotowski P, Alfieri A, Driemeier D. Diarrhea caused by rotavirus A, B, and C in suckling piglets from southern Brazil: Molecular detection and histologic and immunohistochemical characterization. J Vet Diagn Invest. 2018;30(3):370-376. https://doi.org/10.1177/1040638718756050

6. Marthaler D, Rossow K, Gramer M, Collins J, Goyal S, Tsunemitsu H, Kuga K, Suzuki T, Ciarlet M, Matthijnssens J. Detection of substantial porcine group B rotavirus genetic diversity in the United States, resulting in a modified classification proposal for G genotypes. Virology. 2012;433(1):85-96. https://doi.org/10.1016/j.virol.2012.07.006

7. Miyabe FM, Dall Agnol AM, Leme RA, Oliveira TES, Headley SA, Fernandes T, Gonçalves de Oliveira A, Alfieri AF, Alfieri AA. Porcine rotavirus B as primary causative agent of diarrhea outbreaks in newborn piglets. Sci Rep. 2020;10:22002. https://doi.org/10.1038/s41598-020-78797-y

8. Tran H, Friendship R, Ojkic D, Poljak Z. Assessment of seasonality of rotavirus PCR detection in swine from Ontario and Quebec between 2016-2020 using submissions to a diagnostic laboratory. Can J Vet Res. 2022;86(4):241-253.

*9. Sebo C, Kitikoon P, Donovan T, Morgan C, Knetter S, Dempsey H, Crawford K, Mogler M, Thacker B, Strait E. Updates on influenza A vaccination using the SEQUIVITY technology. In: Proceedings of the 51st AASV Annual Meeting. American Association of Swine Veterinarians; 2020:112-115.

*10. Groth D. Clinical management of rotavirus. In: Proceedings of the 45th AASV Annual Meeting. American Association of Swine Veterinarians; 2014:561-562.

*11. Buchan J, Jansen H, Surmak A, Vilaca K, Moser G, Marenger K, Moser L. Evaluation of porcine rotavirus prevalence and distribution within southwestern Ontario. In: Proceedings of the 52nd AASV Annual Meeting. American Association of Swine Veterinarians; 2021:248.

12. Marthaler D, Homwong N, Rossow K, Culhane M, Goyal S, Collins J, Matthijnssens J, Ciarlet M. Rapid detection and high occurrence of porcine rotavirus A, B, and C by RT-qPCR in diagnostic samples. J Virol Methods. 2014;209:30-34. https://doi.org/10.1016/j.jviromet.2014.08.018

13. Anderson AV, Shepherd F, Dominguez F, Pittman JS, Marthaler D, Karriker LA. Evaluating natural planned exposure protocols on rotavirus shedding patterns in gilts and the impact on their suckling pigs. J Swine Health Prod. 2023;30(1):10-19. https://doi.org/10.54846/jshap/1294

14. Chattha KS, Roth JA, Saif LJ. Strategies for design and application of enteric viral vaccines. Annu Rev Anim Biosci. 2015;3:375-395. https://doi.org/10.1146/annurev-animal-022114-111038

* Non-refereed references.