Isolation and identification of group A rotaviruses among neonatal diarrheic calves, Morocco

Abstract

Background: Group A rotaviruses (RVA) are the main cause of neonatal calve diarrhea (NCD) in Morocco. In this study, we isolated RVA strains from NCD clinical samples in order to support RVA disease control in Morocco. This isolation process constitutes a first step toward vaccine development.

Methods: Thirteen fecal samples were obtained from calves with a single episode of neonate calf diarrhea at three different dairies and two samples were collected from field during a severe NCD outbreak. Diagnosis of RVA infection was based on fecal immune-chromatographic rapid test and further evaluated for their hemagglutination (HA) activity. RVA isolation was carried out on MA104 cells after inoculates were treated with different concentrations of trypsin TPCK. All RVA isolates were confirmed by LSI VetMAX™ Triplex Ruminant Rotavirus & Coronavirus Real-Time PCR kit. G and P typing were determined by direct sequencing of the VP4 and VP7 amplicons.

Results: RVA isolation was achieved for nine clinical samples following one or two passages (60 %) and was properly depended on HA activity and trypsin treatment of inoculates. The first sign of CPE detected consisted of increased cell granularity, obscure cell boundaries, cell rounding, and eventual degeneration and detachment of cells. At lower TPCK concentration (3-10 μg/inoculum), no changes at the cellular level were observed, while cells activated with 25-30 μg of trypsin/inoculums, they degenerated and trypsin cytotoxicity was enhanced. Appreciable changes in cell's morphology were detected with optimal trypsin concentration of 15-20 μg trypsin/inoculums. Data from qRT-PCR confirmed that unsuccessful cultivations have No-Ct, and all nine isolates have Ct values ranged between 12.17 and 24.69. Analysis sequencing revealed that field isolates were of G6 P[5] serotype and isolates from the dairy NCD samples were of G10 P[14] serotype.

Conclusions: To our knowledge, this is the first study in Morocco which reports the circulation of G10P[14] in NCD on dairy farms and G6P[5] in the field. Our study constitutes a crucial and a necessary step allowing preventive and veterinary medicine to support RVA disease controls in the country.

Keywords: Bovine rotavirus; G10 P[14]; G6 P[5]; Isolation; Morocco.

Fig. 1

Changes observed at the cellular level after MA104 infection with different bovine RVA strains or controls activated with different concentrations of TPCK-Trypsin (0–30 µg/inoculum). a No changes at the cellular level were observed and the shape of the cells was similar to the control cell with 0–10 μg/inoculum. b, c Cell lysis and degeneration due to trypsin cytotoxicity with 25–30 μg/inoculum. d The first sign of cytopathic effect (CPE) with considerable changes in cell’s morphology at 15–20 μg/inoculum. e, f Cells destruction after 4 days post-infection with 15–20 μg/inoculum. g, h Cells were apically infected and CPE was optimally observed at 15 µg/inoculum