Detection of Corona virus antigen by ELISA from diarrhoeic

Neonatal diarrhoea is one of the most important conditions of calves, associated with morbidity and mortalities. Diarrhoeal diseases have an adverse effect on calf health status, survival and productive performances. Corona virus is one of the etiological agents responsible for calf diarrhea worldwide. However there is paucity of literature stating the disease status in India. The present study was carried out to determine the prevalence of corona virus infection among cow calves in Mathura and adjacent regions. During the present study 63 diarrhoeic stool samples collected from cow calves were screened for corona virus. Of the 63 diarrhoeic samples 3 samples (4.76%) were found to be positive for corona virus by ELISA.


Introduction
the calf.Calves with bloody diarrhoea can die of hypovolemia within a few hours of the onset of Major etiological agents responsible for calf clinical signs [1].diarrhoea are bacteria (E.coli, Salmonella), Viral transmission can be through aerosols Viruses (Rotavirus, corona virus) and protozoa of respiratory secretions, via the faecal-oral (Cryptosporidia) [4].Corona viruses and rotaviruses route, or by mechanical transmission [1].are the most common viruses involved in neonatal calf diarrhoea.Coronaviruses belong to family Materials and Methods

Coronoviridae. Coronavirus particles are
Collection of specimens: A total of 63 diarrhoeic irregularly-shaped, 80-220 nm in diameter, with fecal samples were collected from calves from an outer envelope bearing distinctive, 'clubboth organized and non-organized dairy farms shaped' peplomers (20 nm long).This 'crownlocated in and around Mathura during winter like' appearance (Latin, corona) gives the family months of the study period, 2007-2009.The stool its name.
samples were collected in sterilized plastic The Viruses have non-segmented, singlecontainers, transported under ice and stored atstranded RNA with helical symmetry [5].Corona o 20 C till further processing.viral diarrhea in young calves is characterized by profuse watery or hemorrhagic diarrhoea lasting Screening by ELISA: ELISA was performed to for 2 to 6 days along with listlessness, anorexia, detect corona virus antigen in the fecal samples as pyrexia, and dehydration.Morbidity is high (30described by the kit manufacturer (Corona virus 100%) but mortality is influenced by the age of ELISA kit, Bio-X Diagnostics, Belgium).The 96 well plates provided in the kit contained two (EM) is very expensive and identifies only complete BCV particles accurately.Partial or different capture antibodies.Rows A, C, E and G were coated with corona virus specific capture complete loss of spikes on the viral envelope antibodies and rows B, D, F, H coated with nonwhich occurs during sample processing can specific antibodies, which acted as controls.mislead the diagnosis [11].In spite of this EM is These control rows allow the differentiation between still used as a basic test procedure [7].The results specific immunological reaction and non-specific of haemmaglutination test are affected by nonbindings so as to eliminate false positives.Faeces specific agglutinins present in the faeces [2].were diluted in the dilution buffer provided in the Virus isolation based tests are laborious and time kit.A volume of 100µl of diluted sample was consuming.RT PCR based tests are highly added to corresponding wells of specific and nonsensitive and widely accepted.For this, good specific antibody coated rows respectively.The RNA handling facilities, proper standardisation o plate was incubated for one hour at 25 C and and appropriate positive controls are required.washed 3 times with washing solution (diluted in The results of enzyme immunoassays largely the ratio 1:20 with distilled water) provided in depend on the quality of reagents.Polyclonal the kit.The conjugate, a corona virus specific antibody based tests produce high levels of nonmonoclonal antibody labeled peroxidase was specific back ground and cross reactions with used as such and poured in 100µl quantities per other antigens present.However, monoclonal well.The plate was incubated for one hour at antibody based tests overcome these difficulties o 25 C in a dark room and washed thrice with the and are widely used [7].In the present study we washing buffer.
have analyzed fecal samples obtained from single Then 100µl of chromogen (tetramehtyldiarrhoeic episodes.benzidine) were added and the plates allowed to Out of the 63 diarrhoeic stool samples stand at room temperature without excess light processed, 3 (4.76%)were found positive by for 10 minutes.Finally the reaction was stopped ELISA.Other studies also revealed that prevalence by adding stop solution (1M phosphoric acid) of corona virus in neonatal calf diarrhoea is provided in the kit.The optical density was slightly lower than that of rotavirus and varieed measured at 450nm after stopping the reaction between 3.64 to 54%.[8, 10 and 12].However, with 50µl of stop solution.The test was validated there is paucity of literature stating the corona using the positive control and data sheet provided virus prevalence status in India.As per Niture et by the kit.The net optical density of each sample al. [6] prevalence of rotavirus induced diarrhea in was calculated by subtracting the reading for each calves varied between 7.49 to 43% in India.sample well from corresponding negative Previous work by our group in the same Mathura control.
and adjacent regions during same study period Net optical density (O.D.) = O.D. of specific showed a rotavirus prevalence of 16.83% [3].Hence binding -O.D. of non-specific binding.Any prevalence of corona virus induced diarrhea is sample that yieldied an O.D difference of 0.15 or less compared to that of rotavirus in the studied regions during this study period (2007)(2008)(2009).In greater was considered positive.addition to neonatal diarrhea, bovine corona To cite this article : Dash SK, Krishna K, Goel A, Bhatia AK (2012) Detection of Corona virus antigen by ELISA from diarrhoeic cow calves in Mathura, India, Vet.World.5(3):166-168, doi: 10.5455/vetworld.2012.166-168.
dysentery in adult cattle and Till today a variety of methods are used to respiratory tract infections in calves and feed lot detect bovine corona virus (BCV) infection in cattle [1, 2 and 9 ].stool samples.Currently used methods include In conclusion, corona viruses are associated electron microscopy, haemagglutination test, with multiple bovine disease conditions and enzyme immune assay, virus isolation in cell should as a result not be neglected, but consistently monitored and controlled.Monoclonal antibody culture and RT PCR.Each method has its own advantage and disadvantage.Electron microscopy based ELISAs are simple and easy to perform, p. 1163-1185.In DM Knipe, PM Howley, and are ideal for utilization when generating DE Griffin, MA Martin, RA Lamb, B. Roziman important epidemiological data.