RAPID IDENTIFICATION OF BOVINE ROTAVIRUS BY ELECTROPHORESIS IN AGAROSE GEL

PUkalI., R. DvoUk, J. Franz, J. St!panek: Rapid Identification oj BOfJine RotafJirus by Electrophoresis in Agarose Gel. Acta vet. Smo, 60, 1991: 253-261., Horizontal electrophoresis of the rotavirus genome in aprose (EPA), indirect immunoenzymatic analysis (ELISA) and,counter-immunoelectroosJnOphoresis (CIEOP) were compared for the identification of .rotavirus in 94 feces .samples collected from scouring calves in 6 rearing premises. Positive results by all three methods were obtained in 48 animals (51 %), most effective being ELISA and EPA (both 43) and least effective CIEOP (29); The sensitivity of the rotavirus genome demonstration by EPA was equal to that of indirect ELISA (88 %), which demonstrates the group-specific. rotavirus antigen. The sensitivities of . EPA and BLISA were higher by 31 % than that of CIBOP. Identical results of BLlSAand EPA, CIBOP and EPA, and BLISA and CIBOP were. obtained in 89.3, 82.9 and 80.8 %, respectively. Compared with BLISA and CIBOP, EPA iSR straightforward procedure involving neither a complicated processing of samples, nor the preparation of a specific hyperimmune serum. The time required for BP.A equals to one half and one fifth of the time necessary for BLISA and CIBOP,respectively. BOfJine rotafJinu, diagnosis, electrophoresis -. EPA, ELISA, CIEOP Gastroenteral infections in calves, manifested by scours and associated with increased mortality, pose a serious problem in local cattle herds. They affect newborn calves mostly and are associated with considerable econOmic losses, resulting from retarded growth, impairment of the general condition and increased treatment ~ts and. mortality. • ' •.. .... . . In most cases, rotaviruses have been identified in the gut cOntents in many animal species and children affected with acute gastroenteritis (Bishop.et al. 1973; McNulty et al. 1976). Also attempts to demonstrate rotaviruses as causative a~ts of gastroenteritis in calves have been reported repeatedly. For a long time, however, such a~empts were limited to experimental infections and to virus propagation in cell cultures (Mebus et al. 1969, 1977; Bridger and Woode 1975). Owing to the poor adaptability of bovine rotaviruses to serial propagation in cell cultures, laborious and sophisticated procedures are necessary for this purpose and therefore rapid methods of direct and indirect detection and identification have been introduced into laboratory diagnostics. Originally, only electron and immunoelectron microscopy were used (Bridger and Woode 1975), later the techniques of counter-immunoelectroosmophoresis CIBOP (Middleton et al. 1976), complement fixation (Zissis et al. 1978), immunoenzymatic analysis BLISA (Bidwell et al. 1977; Bllens and de Leew, 1977), radioimmunoanalysis RIA (Sarkinen et al. 1980) and reverse passive haemagglutination and latex haemagglutination (Sanekata et al. 1981) were developed. Recently, methods based on the effects of the electric field on the rotavirus genome, extracted from clinical samples directly, have been· introduced. The arrangement of the. rotavirus genome, formed by segments of double-stranded RNA with molecular mass ranging between 0.2 x 10· and 2.0 x 10· (Clarke and McCrae 1981) .and consequently with . va,rious migration speeds in polyacrylamide and agarose .gels, results in· a: charactedstic distribution Called electrophorogramme (pedley et al. 1983). A comparisOn of RNA electrophoretogrammes of rotliviruses isolated during an epidemic outbreak of scours allows to use the genome analysis not Only for the aetiological diagnosis, but also for epidemiological investigations (Fijtman et al. 1981).

RotafJirus by Electrophoresis in Agarose Gel.Acta vet. Smo, 60, 1991: 253-261., Horizontal electrophoresis of the rotavirus genome in aprose (EPA), indirect immunoenzymatic analysis (ELISA) and,counter-immunoelectroosJnOphoresis (CIEOP) were compared for the identification of .rotavirus in 94 feces .samplescollected from scouring calves in 6 rearing premises.Positive results by all three methods were obtained in 48 animals (51 %), most effective being ELISA and EPA (both 43) and least effective CIEOP (29); The sensitivity of the rotavirus genome demonstration by EPA was equal to that of indirect ELISA (88 %), which demonstrates the group-specific.rotavirus antigen.The sensitivities of .EPA and BLISA were higher by 31 % than that of CIBOP.Identical results of BLlSAand EPA, CIBOP and EPA, and BLISA and CIBOP were.obtained in 89.3, 82.9 and 80.8 %, respectively.Compared with BLISA and CIBOP, EPA iSR straightforward procedure involving neither a complicated processing of samples, nor the preparation of a specific hyperimmune serum.The time required for BP.A equals to one half and one fifth of the time necessary for BLISA and CIBOP,respectively.BOfJine rotafJinu, diagnosis, electrophoresis -.EPA, ELISA, CIEOP Gastroenteral infections in calves, manifested by scours and associated with increased mortality, pose a serious problem in local cattle herds.They affect newborn calves mostly and are associated with considerable econOmic losses, resulting from retarded growth, impairment of the general condition and increased treatment ~ts and.mortality.• ' •.. .... . .In most cases, rotaviruses have been identified in the gut cOntents in many animal species and children affected with acute gastroenteritis (Bishop.et al. 1973;McNulty et al. 1976).Also attempts to demonstrate rotaviruses as causative a~ts of gastroenteritis in calves have been reported repeatedly.For a long time, however, such a~empts were limited to experimental infections and to virus propagation in cell cultures (Mebus et al. 1969(Mebus et al. , 1977;;Bridger and Woode 1975).Owing to the poor adaptability of bovine rotaviruses to serial propagation in cell cultures, laborious and sophisticated procedures are necessary for this purpose and therefore rapid methods of direct and indirect detection and identification have been introduced into laboratory diagnostics.Originally, only electron and immunoelectron microscopy were used (Bridger and Woode 1975), later the techniques of counter-immunoelectroosmophoresis -CIBOP (Middleton et al. 1976), complement fixation (Zissis et al. 1978), immunoenzymatic analysis -BLISA (Bidwell et al. 1977;Bllens and de Leew, 1977), radioimmunoanalysis -RIA (Sarkinen et al. 1980) and reverse passive haemagglutination and latex haemagglutination (Sanekata et al. 1981) were developed.Recently, methods based on the effects of the electric field on the rotavirus genome, extracted from clinical samples directly, have been• introduced.The arrangement of the.rotavirus genome, formed by segments of double-stranded RNA with molecular mass ranging between 0.2 x 10• and 2.0 x 10• (Clarke and McCrae 1981) .andconsequently with .va,rious migration speeds in polyacrylamide and agarose .gels,results in• a: charactedstic distribution Called electrophorogramme (pedley et al. 1983).A comparisOn of RNA electrophoretogrammes of rotliviruses isolated during an epidemic outbreak of scours allows to use the genome analysis not Only for the aetiological diagnosis, but also for epidemiological investigations (Fijtman et al. 1981).
The aim of our experiments was to develop and test the simplest procedures of horizontal electrophoresis in agar (EPA) for routine detection and characterization of the rotavirus RNA separated from feces collected from scouring calves.

Samples and their processing:
Feces samples, collected from scouring calves, and culture media, collected after repeated passages of rotavirus isolates in the cell lines MA 104 or MDBK, were examined for the presence of rotaviruses.The examinations were performed currently since January to May 1990, i. e. during a period when frequent scours were recorded in 3-to 14-day-old calves in 6 separated rearing premises in the South Moravian county.A total of 94 calves were examined.1) Feces: The samples were collected into plastic bottles, frozen on dry ice immediately and transported to the laboratory.A 20 % suspension was prepared in the.Eppendorf test tubes containing 400 pI of respective bUffered solution using and adapted bacteriological loop.An equal part of a phenol + chloroform mixture (1: 1) was added to the samples suspended in the extraction buffer 'and the test tubes were shaken gently for 60 seconds to obtain a homogeneous suspension before centrifugation.All suspensions were processed in the K -24centrifuge Ganetzki) at8 000 g for 10 minutes.The supernatants for ELISA and CIBOP were stored at +4 •C, while those for EPA (the aqueous phase containing the double-stranded RNA segments) were transferred into other test tubes and kept at room temperature until examined.
2) Culture media: Samples of Culture media were diluted •and centrifuged in the same way as the feces samples.The volume of the aqueous phase was measured and, after adding 3 parts of 96% ethanol to 1 part of the fluid, the mixture was left to precipitate at -20 •C overnight.Mter processing in the K-24 centrifuge Ganetzki) at 10000 g for 30 minutes, the precipitate was resuspended in SO pI of the extraction:• buffer and used for EPA.

Electrophoresis in agarose gel (EPA):
A modifica~~on of horizontal electrophoresis in a S-mm-thick layer of I.S% agarose, as described by Chudzio et a1. (1989), was used.The gel was prepared by boiling agarose (Sigma) in 0.09 M Tris-borate buffer pfl 8.2.Approx.3S ml of the Hquid gel were poured into an adapted microtitre plate lid 8.5 x 8 em, .into which a comb had been placed• to form starts.After solidification the comb was removed, the lid was put into an electrophoretic vessel (own design) and approx.300 ml of Tris-borate.buffer were poured into it.SO pI of the processed sample were applied on respective start by a micropipette.The electrophoretic .vesselwas then connected to a voltage generator so .thatcathode passed the vessel on the side of the agarose layer, where the samples were applied.The reaction was left to run at 100 V for I.S hours.After switching off the voltage generator, the gel was stained in ethidiumbromide solution (1 pg • ml-1 ) for 10 minutes.The stained segments were viewed under ultraviolet light at 312 nm (Transiluminator, UVP, Cambridge). .

Enzymoimmunoanalysis:
The modification described by Bidwell et al. (1977) was used for rotavirus demonstration.The IgG fraction of rabbit antiserum to rotavirus (100 pg • ml-1 ) was pipetted into each well of a microtitre plate and left there for 18 hours.Mter a thorough washing of the wells, 0.1 ml of the examined suspension, 0.1 ml of the optimal dilution of the positive calf serum to rotavirus and 0.1 ml of a solution of swine antibody to bovine IgG, conjugated with horse-radish peroxidase (SwAB/Px, USOL, Prague, diluted 1 : 2 000), were pipetted into the wells sequentially.Each step was followed by a thorough washing and each component was left in the wells for 120 minutes.Finally, 0.1 ml of substrate (S-aminosalicylic acid + O.OOS% hydrogen peroxide) was pipetted into each well.
Results of combinations of two methods agreed in 89,3 % (89/94) in Group I (ELISA + EPA), in 82.9 % (78/94) in Group II (CIEOP + EPA) and in 80.8 % (76/94) in Group III (ELISA + CIEOP).Comparisons of the results obtained  The sensitivity of the rotavirus genome demonstration by EPA was equal to that of ELISA (88 %" 38/43)" although controversial results were obtained in 10 cases.However" higher numbers of controversial results were obtained from the comparison of EPA and CIEOP ( 16)" or ELISA and CIEOP (18).

Discussion
Horizontal electrophoresis of the rotavirus genome in agarose" immunoenzymatic analysis and counter-immuno-e1ectroosmophoresis were used for the identification of rotavirus in 94 feces samples collected from scouring calves in 6 rearing premises.Although all three methods detected the bovine rotavirus" they differed in sensitivity and specificity.The most reliable results were obtained by electrophoresis of the extracted RNA in agarose" allowing a direct identification of rotaviruses" based on the characteristic arrangement of the RNA segments in the gel and on evaluation of respective e1ectrophoretogrammes.Rotavirus was demonstrated in 43 of the examined 94 feces samples by this method.Moreover" comparative evaluations of the electrophoretogrammes allow to analyse positive results in the epidemiological context and in time sequence (Fijtman et al. 1987)" thus increasing the diagnostic value of EPA.Similarly as PAGE" EPA can be used for the differentiation of the rotavirus RNA not only within a single animal species" but also between various species (Rodger et al. 1979).On the other hand, in the form described here" EPA cannot be used for the differential diagnosis of further enteropathogenic viruses that may cause diarrhoea in calves either alone" or in combination of rotaviruses, such as bovine coronavirus" calicivirus" astrovirus" etc.The necessity to consider further enteropathogenic viruses in the aetiological diagnosis of calf scours is stressed by the fact that" in our experiments" rotavirus could not be detected by any of the methods used in 49 % of the diarrhoeic calves under study.
. While Kalica et al. (1978) described 11 segments resulting from PAGE of the rotavirus RNA" in our experiments only 8 and in one case 9 bands were dis-cernible in 1.5 % agarose.Basing on a closer analysis of our electrophoretogrammes and on data published by other authors, we presume that this reduction of the number of bands is due to the positions of the segments 2 and 3, and 7, 8 and 9, which are so close in agarose that the segments become indiscernible.Despite this reduction, we were able to demonstrate a certain number of variants of distribution of the rotavirus genome segments, extracted both from feces of scouring calves and from virus suspensions cultured in vitro (Fig. 1 and 2).The most marked migration speed deviations were seen in the genome of the strains BR-408, TM-207 and TM-I23, showing a slower migration of the 11th segment, thus forming the "shortened RNA electropherotype", which has been described for the bovine rotavirus by Paul et al. (1988).A closer analysis, using more effective methods of molecular biology and serological and biological comparisons with other rotavirus isolates and reference strains, will be necessary for the investigations of possible associations between changes of bovine rotavirus genome and serological properties.
The examinations of feces samples showed a good agreement of results and equal sensitivities of ELISA and EPA.A similar agreement in the identification of rotaviruses has been reported for PAGE, ELISA and electron microscopy by other authors (Ed wards et al. 1987).The fact than not only complete virions, but also their morphological protein subunits may be demonstrated in clinical samples by ELISA and CIEOP (Dea et al. 1980;Ellens et al. 1977) could explain the controversial results of the type "ELISA-positive, CIEOP-positive, EPA-negative".Although polyclonal antibodies to the inner bovine rotavirus capsid were used in ELISA and CIEOP (Franz et al. 1984), controversial results of the type "ELISA-positive, EPA-negative" require further analysis to demonstrate the possible existence of another serological group of bovine rotavirus, even if no deviation from the "long RNA electropherotype", typical for the Group A rotaviruses (Kalica et al. 1978) was observed in genome extracted from respective samples.
. The major advantages• of EPA are the straightforward procedure, involving no complicated sample processing and preparation of specific immune sera, and its speed, both making this method usable in routine investigations.

Table I
Comparisoa of resalts of ."..m1aadoas of 94 feces samples for, the preseace of boviDe rotavirus, obtaJDecI by EPA, ELISA aDd cmop