Abstract
Porcine rotavirus (RV) is a major viral agent associated with severe diarrhea in newborn piglets. RVA, RVB, RVC, and RVH are RV species that have already been identified in pigs. RVA is considered the most prevalent and relevant virus in pig production worldwide. This study aimed to evaluate the frequency of RV infection associated with diarrhea in suckling piglets from regular RVA-vaccinated Brazilian pig herds between 2015 and 2021. Therefore, 511 diarrheic fecal samples were collected from suckling piglets aged up to 3 weeks from 112 pig farms located in three main Brazilian pork production regions. All piglets were born to RVA-vaccinated sows. The nucleic acids of RVA, RVC, and RVH were investigated by RT-PCR assays and RVB by semi-nested RT-PCR assay. Of the diarrheic fecal samples analyzed, 221/511 (43.3%) were positive for at least one of the RV species. Regarding the distribution of RV species among the positive fecal samples that presented with only one RV species, 99 (44.8%), 63 (28.5%), and 45 (20.4%) were identified as RVB, RVC, and RVA, respectively. RVH was not identified in diarrheic piglets with a single infection. More than one RV species was identified in 14/221 (6.3%) of the diarrheic fecal samples evaluated. Co-detection of RVB + RVH (11/221; 5.0%), RVA + RVB (1/221; 0.4%), RVA + RVC (1/221; 0.4%), and RVB + RVC (1/221; 0.4%) was identified in fecal samples. The results demonstrated a significant increase in the RVC and, mainly, RVB detection rates in single infections. This study allowed us to characterize the importance of other RV species, in addition to RVA, in the etiology of neonatal diarrhea in piglets from pig herds with a regular vaccination program for RVA diarrhea control and prophylaxis.
Keywords: Pig, Neonatal diarrhea, Rotavirus A, Rotavirus B, Rotavirus C, Rotavirus H
Introduction
The main viral etiology associated with severe diarrhea in newborn piglets is linked to porcine rotavirus (RV). Rotaviruses belong to the Sedoreoviridae family, and the Rotavirus genus. The viral particle is characterized by an icosahedral capsid with three concentric protein layers with 60–80 nm diameter. The genome consists of 11 segments of double-stranded RNA (dsRNA) encoding six nonstructural proteins (NSP1–NSP6) and six structural proteins (VP1–VP4, VP6, and VP7) [1].
Based on the molecular and antigenic characteristics of the VP6 protein, present in the intermediate layer of the capsid, nine RV species were named RVA to RVD and RVF to RVJ [1]. The RV species identified in pigs are RVA, RVB, RVC, and RVH [2]. Among these RV species, the most frequently associated with outbreaks of diarrhea in suckling piglets is RVA, followed by RVB and RVC [3]. However, RVH has been sporadically detected in diarrheic piglets [4, 5].
Among the RV species, RVA was the first to be identified and is considered the most important because of its high pathogenicity and prevalence worldwide [3, 6]. First described in pigs in the 1980s, RVB has been reported as a secondary agent to porcine neonatal diarrhea [7]. However, a recent study identified RVB as a primary agent in villus atrophy, causing malabsorptive diarrhea and high mortality rates [8]. RVC is frequently diagnosed in fecal samples from piglets of different ages, including suckling piglets and post-weaning pigs [2]. RVH is associated with mixed infections, mainly those involving RVB [2].
Studies have shown the increased importance of RVC and, especially, RVB in relation to RVA infection in porcine neonatal diarrhea outbreaks [8, 9]. More recently, the number of Brazilian pig herds vaccinated with RVA has increased considerably. As a result, changes in the profile of RV infection have been observed with an increase in the frequency of diagnosis of non-RVA species. This retrospective study aimed to evaluate the frequency of RV species infection associated with diarrhea in suckling piglets from regular RVA-vaccinated Brazilian pig herds.
Materials and methods
Sample collection
From January 2015 to December 2021 diarrheic fecal samples (n = 511) from suckling piglets aged up to three weeks: 0–7 days (n = 323), 8–14 days (n = 120), and 15–21 days (n = 68) were received by the Animal Virology Laboratory of the Universidade Estadual de Londrina specifically for the diagnosis of rotavirus infection in neonatal diarrhea outbreaks. The sampling was on pig herds from three (South n = 260; Southeast n = 109, and Midwest n = 142) main pork production regions in Brazil. Fecal samples were obtained from 112 pig farms with good management and nutritional practices. All pig herds used commercial vaccines for RVA (G4P[6] and G5P[7] genotypes), Clostridium perfringens C, and Escherichia coli (K88, K99, F41, and 987P) for neonatal diarrhea control. This retrospective study was performed with a collection of piglet fecal samples sent by large pig production integrators and pig companies of feed, genetics, and pharmaceuticals. The fecal samples collection was maintained at –20 °C until analysis.
Nucleic acid extraction and rotavirus detection
Fecal suspensions (10 to 20% w/v) were prepared in Tris/Ca++ buffer (50 mM Tris–HCl, 10 mM NaCl, 1.5 mM 2-mercaptoethanol, 3 mM CaCl2, pH 7.2) and centrifuged at 2,000 × g for 5 min at 4 °C. Aliquots of 500 µL of the supernatant were treated with sodium dodecyl sulfate 10%, homogenized, and incubated at 56 ºC for 20 min. Viral dsRNA was extracted using a combination of phenol/chloroform/isoamyl alcohol (25:24:1) and silica/guanidinium isothiocyanate methods [10]. Nucleic acid was eluted in 50 µL of water treated with diethyl pyrocarbonate-DEPC (Invitrogen™ Life Technologies, Carlsbad, CA, USA) and stored at –80 °C until analysis. Sterile ultrapure water aliquots were included as the negative control in all nucleic acid extraction and other amplification steps.
The presence of dsRNA of RVA, RVC, and RVH was investigated using RT-PCR assays, and that of RVB was investigated using semi-nested RT-PCR assay. Amplifications were performed using primers targeting the RVA VP7 and VP4 genes [11, 12], RVB NSP2 gene [13], RVC VP6 gene [14], and RVH VP6 gene [15]. Aliquots of sterile ultrapure DEPC-treated water were used as negative controls in all procedures. Fecal samples previously known to be positive for each of the RV species investigated were included as positive controls [2].
Statistical analysis
Statistical analysis was conducted using Fisher tests using RStudio v2022.02.0 software. A value of P < 0.05 was considered significant.
Results
Of the 511 diarrheic fecal samples analyzed over seven years (2015–2021), 221 (43.3%) samples were positive for at least one RV species. Regarding the RV species distribution in diarrheic fecal samples from suckling piglets with single (207/221, 93.7%) infections 99 (44.8%), 63 (28.5%), and 45 (20.4%) samples were positive for RVB, RVC, and RVA, respectively (Fig. 1). The frequency of single species of RV infection was much higher (n = 207; 93.7%) than mixed (n = 14; 6.3%) infections. RVH was not detected in cases of a single infection. Co-detection of different RV species was identified in 14/221 of the RV-positive samples, representing 6.3% of the positive fecal samples. RVB + RVH was simultaneously co-detected in 11 diarrheic fecal samples, while RVA + RVB, RVA + RVC, and RVB + RVC occurred in one fecal sample each. Concerning pig farms, at least one fecal sample was RV-positive in 75 (67%) herds.
Fig. 1.
Percentage of detection of porcine rotavirus species from diarrheic fecal samples collected in Brazilian pig herds between 2015 to 2021
Diarrheic fecal samples were categorized into three piglet age groups by week of age. In one-, two-, and three-week-old piglets, there were evaluated 323, 120, and 68 diarrheic fecal samples, respectively. RV infection was identified in 161 (49.8%), 42 (35.0%), and 18 (26.5%) fecal samples from piglets in the first, second, and third weeks, respectively. In one-week old piglets, RVB was detected with more frequency (n = 80; 24.8%); in the two-week old piglets, the proportion of diagnosis of RVA, RVB, and RVC was very similar, ranging from 10.0 to 11.7%. In the first week of age, there was a statistical difference (P < 0.01) in relation to the detected RV species (Table 1 – lowercase letters). RVB detection in the first week was significantly higher (P < 0.01) than in the second and third weeks. In addition, the detection of RVB and RVC was more frequent in the first than in the other weeks. No statistical difference was observed in relation to the detection of RVA in the evaluated weeks of age (Table 1 – uppercase letters).
Table 1.
Frequency of infection by different species of rotavirus distributed by age groups of piglets, Brazil, 2015–2021
| RV species | N° of positive samples (%) by week of age | Total (n = 511) | ||
|---|---|---|---|---|
| First (n = 323) | Second (n = 120) | Third (n = 68) | ||
| Single (#) | ||||
| RVA | 22 (6.8)c,A | 14 (11.7)a,A | 9 (13.4)a,A | 45 (8.8) |
| RVB | 80 (24.8)a,A | 12 (10.0)a,B | 7 (10.3)a,B | 99 (19.4) |
| RVC | 47 (14.5)b,A | 14 (11.7)a,AB | 2 (2.9)a,B | 63 (12.3) |
| Mixed (#) | ||||
| RVA + RVB | 1 (0.3) | - | - | 1 (0.2) |
| RVA + RVC | - | 1 (0.8) | - | 1 (0.2) |
| RVB + RVC | - | 1 (0.8) | - | 1 (0.2) |
| RVB + RVH | 11 (3.4) | - | - | 11 (2.1) |
| Total | 161 (49.8) | 42 (35.0) | 18 (26.5) | 221 (43.3) |
(#) Single and Mixed infections
a, b, c Different letters indicate statistical difference (P value < 0.01) of frequency of rotavirus species by week of age (columns)
A, B, C Different letters indicate statistical difference (P value < 0.01) of frequency species among the weeks of age (lines)
Regarding the RV species, the diagnosis of RVA (15.4%) was more significant (P < 0.01) in diarrheic feces from piglets from the southern region than other regions, while the detection of RVB (39.4%) was the Midwest region. On the other hand, for RVC, there was no statistical difference related to geographic region (Table 2 – uppercase letters). The rates of RVC infections occurred similarly in the three regions, ranging from 8.3 to 14.8%. The RVH was diagnosed in piglets from the three evaluated regions and was significantly detected (5.5%) in the southeast region.
Table 2.
Geographical distribution in Brazil of porcine rotavirus species identified in diarrheic fecal samples obtained between 2015 and 2021
| RV species (#) | Brazilian geographical region (%) | Total (n = 511) |
||
|---|---|---|---|---|
| South (n = 260) |
Southeast (n = 109) |
Midwest (n = 142) |
||
| RVA | 40 (15.4)a,A | 2 (1.8)b,B | 5 (3.5)c,B | 47 |
| RVB | 43 (16.5)a,B | 13 (11.9)a,B | 56 (39.4)a,A | 112 |
| RVC | 35 (13.5)a,A | 9 (8.3)ab,A | 21 (14.8)b,A | 65 |
| RVH | 2 (0.8)b,B | 6 (5.5)ab,A | 3 (2.1)c,AB | 11 |
| Total | 120 (46.2) | 30 (27.5) | 85 (59.9) | 235 (46.0) |
(#) Single and mixed infections
a, b, c Different letters indicate statistical difference (P value < 0.05) of the frequency of rotavirus species in the geographical region (columns)
A, B, C Different letters indicate statistical difference (P value < 0.05) of the frequency rotavirus species between the geographical regions (lines)
In the southern region, the frequencies of diagnosis of RVA (15.4%), RVB (16.5%), and RVC (13.5%) were similar. In the southeast and midwest regions, the RVB diagnosis rate was higher than that of the other RV species, especially in the midwest region, where this species was identified in 39.4% of the diarrheic fecal samples analyzed (Table 2 – lowercase letters).
Discussion
High rates of RVA detection have been reported in neonatal diarrhea in pig herds worldwide. The frequency of RVA detection is higher than that of other RV species, such as RVB, RVC, and RVH, both in Brazil and other countries [2, 3, 16]. In the present survey, it was possible to observe a significant increase in the rate of RVB detection, higher than the diagnosis rates of RVC and RVA, in the fecal samples from diarrheic piglets.
RVB detection rates were also higher than those found in other Brazilian studies that identified RVB at rates lower than those of RVA and RVC [2, 16]. A probable explanation for these differences may be related to the number of diarrheal fecal samples analyzed. Most of the Brazilian studies cited previously included a small number (between 33 and 50) of fecal samples from a few pig farms (up to four) with neonatal diarrhea outbreaks [2, 16]. In this survey, the sampling (n = 511) was more representative and had greater geographic and temporal coverage. A retrospective study conducted in Brazil evaluated diarrheic fecal samples of piglets aged 1 and 4 weeks from non-vaccinated RVA pig farms, collected between 2005 and 2007, from herds located in the south and midwest regions of Brazil. The authors identified, including piglets with single and mixed infections, 74/588 (12.6%) fecal samples positive for RVA, 52/588 (8.8%) for RVB, and 9/588 (1.5%) for RVC [17]. The RVB detection rate found in this survey agrees with research conducted in other countries, which also identified high rates of RVB infection in piglets in Australia (107/243; 44%), Japan (190/735; 25.9%), and the US (2,388/7,508; 31.8%) [3, 7, 18].
Analyzing the period evaluated in this survey, an increase in the frequency of RVC infection diagnosis was observed. In 2021, the detection rate of RVC was higher (37.7%) than that of RVB (24.6%). Previous studies conducted in Brazil have evaluated diarrheic fecal samples obtained between the years 2004 to 2010 and between 2011 to 2014 in which the frequency of RVC detection was 15/588 (2.6%) and 42/1,370 (3.1%), respectively [19, 20]. Notably, the diagnostic rate of RVC in this study was higher than that reported in other Brazilian studies.
Both RVB and RVC, even during the peak of infection, are eliminated in feces at much lower viral titers than those of RVA [21]. Brazilian studies that conducted an epidemiological survey of a large number of fecal samples used the silver-stained polyacrylamide gel electrophoresis (ssPAGE) screening technique [17, 19, 20]. Despite its ease of use and low cost, ssPAGE has a lower sensitivity than nucleic acid amplification techniques [17]. Combining these two characteristics, RVB or RVC infection accompanied by the excretion of a lower viral titer and a screening test with low sensitivity can result in false-negative results in the ssPAGE technique. However, the fact that all diarrheic fecal samples obtained in this study were subjected to RT-PCR may have contributed considerably to the increase in the detection rates of RVC and, mainly, RVB.
Over the 7 years (2015–2021) of fecal sample collection, the RV diagnosis rates varied considerably. During this period, the lowest diagnosis rate (19.1%) occurred in 2019 and the highest (64.3%) in 2016. However, this was a cross-sectional epidemiological survey. The fecal samples were evaluated mainly by active demand from producers or veterinarians in cases of diarrhea outbreaks in piglets in their pig herds. Therefore, the sampling also varied over this period; that is, it was not uniform during the seven years of the evaluation. Another aspect, which can also interfere with the interpretation of results distributed by year of sample collection, is that the period analysis (2015–2021) unfortunately included two full years of the pandemic (COVID-19). In 2020 and 2021, the number of fecal samples sent by pig companies for rotavirus diagnosis and other infectious diseases reduced drastically. As a result, any analysis of the temporal distribution of porcine rotavirus infection in the period evaluated may present interpretation bias.
Regarding the different age groups, the greatest number of RV-positive fecal samples were concentrated in diarrheic samples from piglets who were one week old (49.8%). Among the RV species identified in this age group, RVB had the greatest frequency. Comparing our results with a study carried out in the US, an inversion in the frequency of detection of RV species was observed, with the detection of RVC being more common in the first three days, while RVB increased throughout the analyzed period, reaching the highest frequency (50%) in animals aged ≥ 55 days [3].
RVH is the most recently described species in piglets with diarrhea. RVH was first described in Brazil in 2012 in diarrheic piglets with mixed infections with other RV species [4]. In this retrospective study, RVH was identified at a lower frequency than other RV species, being identified only in co-detection with the other species, mainly with RVB, and exclusively in first-age piglets. Since RVH has always been identified in co-detection with other porcine RV species, its role as a primary agent of diarrhea in piglets remains unknown [4, 22].
In this study, it was possible to characterize a marked variation in the frequency of RV detection, including the RV species identified according to the regional distribution, that is, the Brazilian geographic region evaluated. The midwest and south regions had the highest frequencies of RV diagnoses with 57 and 44.6%, respectively, compared with the southeast region (22.0%). Although the creation system was not evaluated in this study, this variable may have influenced the results. In the south and, mainly, in the midwest region, pig herds predominate with a large number of sows, created in an integrated way with pig companies. In greater herds, the risk factors for the occurrence of diarrhea, including the most susceptible population and greater sanitary challenge, can predispose piglets to early enteric infections and subsequently, diarrhea. In the southeast region, predominate pig herds of independent creators, with a smaller number of sows and, consequently, lower sanitary risks for the occurrence and dissemination of infections caused by RV.
Finally, another aspect to be considered in the recent variations in the frequency of diagnosis of porcine RV may be the immune pressure induced by RVA vaccination. Currently, pregnant sows from many herds for commercial pig production are vaccinated to enhance the passive immunity of piglets and consequently reduce the number and intensity of neonatal diarrhea episodes. Commercial vaccines for the control and prophylaxis of neonatal diarrhea in piglets available in the Brazilian market, in addition to bacteria, consist of one or two G (VP7) and P (VP4) genotypes of RVA strains. Thus, the relative frequency of neonatal diarrhea caused by RVA infection tended to decrease. On the other hand, worldwide, there are no vaccines containing RVB and RVC strains in their formulation, mainly due to the difficulty of growing these two RV species in cell culture. In summary, the possibility of controlling neonatal infections caused by RVA through vaccination and the vaccine’s inability to control RVB and RVC infections have caused considerable increases in the frequency of RVB and RVC diagnoses in more recent RV surveys.
Conclusion
This study on the frequency of RV species diagnosed in diarrheic piglets showed a significant increase in the rate of RVB and RVC infections in suckling piglets. Currently, RVB and RVC infections are widely disseminated in pig herds of the main geographical regions of Brazilian pork production. Immune pressure promoted by RVA vaccination may favor an increase in diarrhea outbreaks caused by other RV species. Finally, this survey allowed us to characterize the importance of RV species other than RVA in the etiology of neonatal diarrhea in piglets. Additionally, these data highlight the importance of adopting effective internal and external biosecurity measures to minimize production losses resulting from RV infection in pig herds.
Author contributions
All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by AMD, NSG, RAL and ARDC. The first draft of the manuscript was written by AMD, NSG, and RAL. AFA and AAA reviewed the manuscript. All authors read and approved the final manuscript.
Funding
The authors thank the following Brazilian Institutes for their financial support: the National Council of Technological and Scientific Development (CNPq), the Brazilian Federal Agency for Support and Evaluation of Graduate Education (CAPES), the Financing of Studies and Projects (FINEP), and the Araucaria Foundation (FAP/PR). AAA and AFA are recipients of CNPq fellowships.
Data availability
The data presented in this study are available within the article.
Declarations
Ethics approval
This study was submitted to the Ethics Committee on Animal Experiments of the Universidade Estadual de Londrina (CEUA / UEL) and approved under identification number 11363.2015.16. All applicable international, national, and institutional guidelines for the care and use of animals were followed.
Consent to participate
All authors give their consent to participate in this article.
Consent of publication
All authors consent to publication of this manuscript.
Competing of interest
Authors declare they have no competing interest.
Footnotes
Publisher's Note
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Data Availability Statement
The data presented in this study are available within the article.