Immunological and molecular detection of Bovine rotavirus from diarrhoeic calves

Introduction

Rotavirus is a major cause of acute gastroenteritis in calves, humans, and several other species (Dhama et al. 2009; Martella et al. 2010) [6,17] . In calves, Bovine rotavirus is the leading infectious agent of neonatal calf diarrhoea (NCD), commonly termed "white scour," and it most frequently affects animals between 1-2 weeks of age (Dhama et al. 2009) [6] . Rotavirus, a member of the Reoviridae family, is a non-enveloped virus with a triple-layered capsid and an 11-segment double-stranded RNA genome (Estes and Kapikian, 2007) [7] . These segments encode structural proteins VP1-VP7 and non-structural proteins NSP1-NSP6. VP6, a highly conserved inner capsid protein, is used to classify rotaviruses into groups A-H, with evidence of a potential ninth group (Matthijnssens et al. 2012; Mihalov-Kovács et al. 2015) [18,19] . The outer capsid proteins VP4 (P type) and VP7 (G type) are key immunogenic markers, and their reassortment results in diverse circulating genotypes (Kadam et al. 2019) [12] . Group A Bovine rotavirus is the primary cause of NCD and contributes substantially to economic losses, estimated at around USD 9.5 million annually (Silva et al. 2012) [25] . Infection leads to 5-20% mortality in neonatal calves and shows a global prevalence of 30-40% (Dhama et al. 2009; Swiatek et al. 2010) [6] . In India, Rotavirus accounts for 10-52% of diarrhoeal cases in calves younger than one month (Nataraju et al. 2009) [20] .

Materials and Methods

Sample collection from calves

A total of 50 faecal samples or rectal swabs were collected from diarrhoeic calves. The samples were collected following the standard method described by Yilmaz (2016) [30] . An aliquot of each faecal sample was diluted in phosphate-buffered saline (PBS, pH 7.2). A 10% (w/v) faecal suspension was prepared using PBS. The suspension was centrifuged at 10,000 rpm for 30 minutes at 4 °C to remove coarse debris and bacteria. The resulting clarified supernatant was stored at -20 °C until further use for RNA extraction.

Lateral flow assay test (LFA)

The Anigen Rapid Rota Ag test kit used in the study was manufactured by BioNote, Inc. (South Korea).

Reverse transcriptase Polymerase Chain Reaction (RT-PCR) 2.3.1 Isolation of viral RNA

The extraction of viral RNA was a critical step in the detection of RNA viruses. Viral RNA was extracted from the collected faecal samples using the RNA Sure® Virus Kit (Genetix, Asia Biotech Pvt. Ltd., New Delhi), following the manufacturer's protocol. The extracted RNA was stored at -20 °C until further use.

Complementary DNA (cDNA) Synthesis

cDNA was synthesized using the First Strand cDNA Synthesis Kit following the manufacturer's instructions.

Amplification of VP6 & VP7 Gene of Rotavirus

Results and Discussion

The samples were screened for the presence of Bovine rotavirus using both Lateral Flow Assay (LFA) and Reverse Transcription Polymerase Chain Reaction (RT-PCR), targeting the VP6 and VP7 genes. Out of the 50 samples, 4 (8.00%) tested positive for Rotavirus by LFA, and the same number of samples (4/50, 8.00%) were confirmed positive by RT-PCR.

Detection of Bovine rotavirus by Lateral flow assay test (LFA)

Out of the 50 diarrhoeic faecal samples tested for Rotavirus using the Lateral Flow Assay (LFA), 4 samples (8.00%) tested positive. The positive results were indicated by the presence of clear lines in both the test and control areas of the device, while negative samples showed only the control line (Figure 1). The overall prevalence of Bovine rotavirus detected by LFA in the present study aligns with the findings of Uddin-Ahmed et al. (2022) [29] , who reported an overall prevalence rate of 8.54%. However, Malla et al. (2024) [16] observed a slightly lower prevalence of 7%. In contrast, several other studies have reported significantly higher prevalence rates: Klein et al. (2009) [14] -71.87%, Içen et al. (2013) [11] -25%, Hassan et al. (2014) [9] -36%, Abouelyazeed et al. (2021) [1] -16.2%, and Barua et al. (2021) [2] -10%.

Detection of Bovine rotavirus by Reverse Transcriptase Polymerase Chain Reaction (RT-PCR)

For determining the overall prevalence, RT-PCR results were considered more definitive. The successful amplification of VP6 and VP7 gene fragments in these 4 samples confirmed the presence of Rotavirus infection in the calves (Figure 2 and3). The overall Rotavirus prevalence of 8.00% observed in this study is comparatively lower than the findings of several earlier studies. For instance, higher prevalence rates have been reported by Basera et al. (2010) [3] -10.15%, Pourasgari et al. (2016) [23] -49.4%, Makwana et al. (2020) [15] -13.11%, and Chen et al. (2022) [4] -46.00%. In contrast, [28] -4.3%, Debelo et al. (2021) [5] -3.64%, Singh et al. (2021) [26] -5.02% and Nayak et al. (2018) [21] -1.93%. The findings differ from previous studies, which reported lower LFA sensitivity while maintaining high specificity.

Reported sensitivities and specificities include: 81.9% and 98.2% (Nemoto et al. 2010) [22] , 78.7% and 100% (Khamrin et al. 2011) [13] , 83% and 100% (Sakli et al. 2019) [24] , and 74.54% and 98.44% (Malla et al. 2024) [16] . These variations indicate that LFA performance may differ across populations and study conditions, emphasizing the need for broader evaluation. The species-wise prevalence of Bovine rotavirus, as detected by RT-PCR, was found to be higher in cattle calves compared to buffalo calves. The findings of the present study are consistent with those of Ade et al. and Makwana et al. (2020) [15] , who also reported a higher prevalence of Bovine rotavirus in cattle calves than in buffalo calves. However, the present results contrast with the observations of Malik et al. (2013) [28] , Udaykar et al. (2013) [28] and Sthevaan et al. who reported a slightly higher infection rate in buffalo calves compared to cattle calves, with prevalence rates of 22.01% in buffalo calves vs. 13.33% in cattle calves, 4.76% vs. 3.77%, and 24.24% vs. 10.34%, respectively. [15] , and Golaviya et al. who observed higher prevalence rates of Bovine rotavirus infection in the youngest age group (1-15 days), with reported rates of 38.46%, 11.58%, and 47.40% respectively. The prevalence among females was found to be 9.5% (2/21), while in males it was slightly lower at 6.9% (2/29). Although the prevalence was marginally higher in females, the difference was not statistically significant due to the limited sample size.

Species-wise prevalence of Bovine rotavirus

Age-wise prevalence of Bovine rotavirus

Sex-wise prevalence of Bovine rotavirus

Similar findings were reported by Makwana et al. (2020) [15] , who observed a higher susceptibility to Bovine rotavirus among female calves (10.61%) compared to male calves (6.81%). In contrast, Dash et al. (2011) reported a higher prevalence of Rotavirus in male diarrhoeic calves (20.40%) compared to female diarrhoeic calves (12.80%). [26] reported a higher prevalence of Rotavirus A (RVA) infection during the winter season compared to other seasons. Likewise, Ahmad Malla et al.

Season -wise prevalence of Bovine rotavirus

(2022) [16] observed the highest prevalence in winter (28.6%), followed by autumn (12.5%). In the present investigation involving 50 diarrhoeic calf samples, the prevalence of Bovine rotavirus varied based on the colour of the diarrhoeic faeces. The highest prevalence was observed in cases of white-coloured diarrhoea, where 3 out of 12 samples (25.0%) tested positive using both LFA and RT-PCR. Yellow-coloured diarrhoea showed a lower prevalence, with 1 out of 16 samples (6.25%) testing positive. No positive cases were detected among the samples with green (17 samples) or bloody (5 samples) diarrhoea. These findings contrast with those reported by Singh et al.

Feeding -wise prevalence of Bovine rotavirus

Symptoms (Colour of diarrhoea) -wise prevalence of Bovine rotavirus

(2021) [26] , who observed a higher prevalence of Rotavirus infection in calves exhibiting pasty yellow-coloured faeces compared to those with watery diarrhoea. Similarly, Debelo et al. (2021) [5] reported the highest prevalence of Rotavirus (30.8%) in calves with watery diarrhoea.

Conclusion

The present study highlights the importance of Rotavirus as one of the major etiological agents of diarrhoea in bovine calves, contributing to considerable economic losses in both the dairy and meat industries. For the detection of Bovine rotavirus antigen, diarrhoeic samples should be screened using LFA and RT-PCR. The findings indicated that there was no significant difference in the diagnostic performance of these two tests.

Acknowledgement

The authors are thankful to the Department of Veterinary Microbiology, College of Veterinary science and Animal Husbandry, Kamdhenu University, Junagadh for giving the opportunity to carry out a research study.

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