Lateral flow assay (LFA) detection of co-infection of predominant enteric pathogens from LFA positive Bovine rotavirus samples

Introduction

This disease is most commonly seen in young animals, while older animals tend to be less susceptible. This decreased sensitivity with age is likely due to changes in their physiology and/or the development of immunity from previous exposures (Estes and Kapikian, 2007) [5] . A variety of bacteria, protozoa, and viruses have been identified as potential causes of neonatal gastroenteritis, acting either individually or in combination (Snodgrass et al., 1990) [16] . Among bacteria and protozoa, Escherichia coli and Cryptosporidium spp. are the leading causes of diarrhoeal diseases. While Rotavirus are the primary viral agents responsible for severe gastroenteritis in both humans and animals, other viruses such as Coronavirus and Parvovirus also commonly contribute to diarrhoea (Lucchelli et al., 1992) [9] . Group A Bovine Rotavirus is a major cause of neonatal calf diarrhoea, a significant economic disease in calves that leads to high mortality rates. Globally, this condition results in annual losses of around $9.5 million USD (Niture et al., 2009; Silva et al., 2012) [12,13] . Bovine Rotavirus infection in neonatal calves leads to a mortality rate of approximately 5-20%. On average, the infection has an incidence and prevalence rate ranging from 30% to 40% (Dhama et al., 2009; Swiatek et al., 2010) [4,17] . Three countries in the subcontinent (India, Bangladesh and Pakistan) account for more than 30% of all Rotavirus related deaths worldwide (Miles et al., 2012) [10] . In India, prevalence of Rotavirus is between 10% to 52% of diarrhoeal cases in calves younger than one month old ( [, 14, 15, 7, 11] .

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) [18] . An aliquot of each faecal sample was diluted in phosphatebuffered 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 BoviD-5 Ag Test Kit used in the study is manufactured by BioNote, Inc. (South Korea). Disposable dropper 10

Principle

The Anigen Rapid BoviD-5 Ag Test Kit is a solid-phase immunochromatographic assay designed for the rapid and qualitative detection of Cryptosporidium antigen, Rotavirus antigen, Coronavirus antigen, Escherichia coli K99, and Giardia lamblia antigen in calf faecal samples.

Procedure of the Test

 Stool samples were collected using the sample collection swab.  The swab was placed into a tube containing the test solvent.  The swab was mixed thoroughly until the sample dissolved in the test diluent.  The tube was left undisturbed for about one minute to allow large particles to settle at the bottom.  Control devices were removed from their packaging and placed on a flat, dry surface.  Using the supplied disposable dropper, the extracted sample was aspirated.  Four drops of the sample were slowly added into the wells using the dropper.  The timer was started as the sample flowed across the result window. If the sample did not appear within one minute, an additional drop of the prepared sample was added to the sample well.  Test results were interpreted at 10 minutes, and readings taken after 20 minutes were disregarded.

Results and Discussion

In this study, four faecal samples that tested positive for Bovine Rotavirus using the Lateral Flow Assay (LFA) were further screened for co-infection with other predominant enteric pathogens, including Giardia, E. coli K99, Bovine Coronavirus, and Cryptosporidium. The results are summarized in the above table. All four samples confirmed positive for Rotavirus, as expected, since the selection was based on LFA-positive results. Notably, 2 out of 4 samples (50%) also tested positive for Bovine Coronavirus antigen, suggesting a significant level of viral co-infection among diarrhoeic calves. None of the samples tested positive for Giardia, E. coli K99, or Cryptosporidium (Fig. 3.1). The results of present study were similar with Zhu et al.

(2011) [19] tested 28 diarrhoeic faecal samples, the method [2] investigated multiple enteropathogens, including Bovine Rotavirus (BRV), Bovine Coronavirus (BCoV), Escherichia coli K99 (ETEC), and Cryptosporidium spp. (Crypto), which are common causes of diarrhoea in calves during their first 30 days of life. The analysis included 41 studies (with 94 substudies) from 21 countries assessing mixed infections in calves worldwide. The highest pooled global prevalence was found for BRV-Crypto co-infections at 6.69%, followed by BRV-BCoV at 2.84%, and BRV-ETEC at 1.64%. Irehan et al.

(2025) [6] analyzed 85 faecal samples from calves. The results showed 24 samples positive for BRV, 13 samples positive for BCoV, and 25 exhibiting mixed infections of both viruses.

Immunochromatographic tests identified antigens of E. coli K99 in 3 samples, Cryptosporidium spp. in 12 samples, and Giardia spp. in 28 samples. The presence of co-infection with Bovine Coronavirus and Rotavirus in 50% of cases is noteworthy, as these two viral are frequently implicated in multifactorial calf diarrhoea (Cho & Yoon, 2014) [3] . Co-infections can exacerbate disease severity, prolong viral shedding, and reduce the efficacy of treatment and vaccination (Al Mawly et al., 2015) [1] . The lack of detection of protozoal (Giardia, Cryptosporidium) and bacterial (E. coli K99) pathogens in this subset may be attributed to the small sample size or differences in the regional epidemiological distribution of pathogens. These findings underline the importance of routine screening for multiple enteric pathogens, especially in Rotaviruspositive calves, to guide effective management and control strategies. Broader surveillance using larger sample sizes is warranted to better understand the pattern and prevalence of co-infections in neonatal calf diarrhoea.

Conclusion

Rotavirus plays a crucial role as a major etiological agent of diarrhoea in bovine calves, leading to significant economic losses in both dairy and meat industries. For the detection of Bovine Rotavirus antigen, diarrhoeic samples should be screened using LFA. The findings indicated that 2 samples co-infected with both viruses (Bovine Coronavirus & Bovine Rotavirus).

Acknowledgement

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

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