key: cord-259193-ifdjyp5b
authors: Sato, K.; Inaba, Y.; Tokuhisa, S.; Miura, Y.; Kaneko, N.; Asagi, M.; Matumoto, M.
title: Detection of bovine coronavirus in feces by reversed passive hemagglutination
date: 1984
journal: Arch Virol
DOI: 10.1007/bf01315291
sha: 
doc_id: 259193
cord_uid: ifdjyp5b

A reversed passive hemagglutination (RPHA) method was developed for the detection of bovine coronavirus in fecal specimens. Sheep erythrocytes fixed with glutaraldehyde, and then treated with tannic acid were coated with anti-bovine coronavirus rabbit antibodies purified by affinity chromatography using bovine coronavirus linked to Sepharose 4B. The RPHA test was carried out by a microtiter method. Erythrocytes coated with purified specific antibodies were agglutinated by bovine coronavirus, but not by bovine rotavirus or enterovirus. The reaction was inhibited by antiserum to bovine coronavirus, confirming the specificity of the reaction. The RPHA test detected bovine coronavirus in 13 of 22 fecal specimens (59 per cent), from natural cases of diarrhea, while the positive rates were only 14 per cent (3/22) and 22 per cent (5/22) for immunofluorescent staining of primary cultures of calf kidney cells infected with the specimens, and immune electron microscopy respectively. The advantages of the RPHA method are its simplicity, high sensitivity and rapidity.

MEBUS and his associates (9, Ii, 17) have demonstrated an agent with the morphologic features of a coronavirus by electron microscopy of the feces of calves with neonatal diarrhea, and have proved it to be a causative agent of the disease. The virus multiplied in bovine embryonic kidney cell cultures, but failed to induce a readily recognizable cytopathic effect. Therefore although the virus may be assayed in these cultures, the method is rather cumbersome, as the presence of virus is detected either b y the microscopic examination of stained cultures, or b y immunofluorescence with a specific antiserum (10) . I~ABA et al. (7) have d e m o n s t r a t e d t h a t the virus replicates readily producing a m a r k e d cytopathic effect in cultures of the continuous cell lines. B E K -1 , derived from bovine embryonic kidney, thus providing a sensitive, practical assay m e t h o d for the virus and neutralizing antibody. A hemagglutination-inhibition test was also developed (14, 16) . More recently TAXAHASm et al. (18) have isolated a bovine coronavirus strain from feces in an o u t b r e a k of diarrhea a m o n g adult cows in p r i m a r y cultures of calf kidney cells. H o w e v e r a cytopathic effect was not detected until the 8th blind passage. The demonstration of the virus in diarrheal feces b y virus isolation and electron microscopy is therefore time consuming and the procedures are elaborate.

SA~EKATA et al. (12, 13) have reported t h a t the reversed passive hemagglutination ( R P t t A ) test is a useful and practical m e t h o d for the detection of rotavirus in the feces of infants with acute gastroenteritis. The advantages of the R P H A m e t h o d are its simplicity, high sensitivity compared with the electron microscopy, and its rapidity.

These observations p r o m p t e d us to investigate the application of R P H A to the detection of bovine coronavirus in the feces of cattle with diarrhea.

Primary bovine kidney (BK) cells were grown at 37°C in Eagle's minimum essential medium (MEM) containing 10 per cent tryptose phosphate broth (TPB) (Difco), 10 per cent calf serum, 100 units/ml penicillin, 100 ~g/ml streptomycin and 1 ~g/ml fungizone. The maintenance medium was MEM containing 10 per cent TPB. 0.05 per cent yeast extract, 0.5 per cent sodium glutamate, 0.1 per cent glucose and antibiotics.

The Kakcgawa strain of bovine coronavirus (18) was used. This strain was isolated in our laboratory from a cow with diarrhea and is identical to the bovine coronavirus of MEBUS et al. (10) by neutralization and immunofluorescence. The Shimane strain of bovine rotavirus (15) and the BFI strain of bovine enterovirus (6) were also used. Both were propagated in BK cells.

This was carried out in BK cell cultures prepared in 11 × 100 mm tubes. Serial 10-fold dilutions of the virus-containing material were made in maintenance medium, and each dilution was inoculated (0.1 ml/tube) into 3 tube cultures. The inoculated cultures were incubated in a roller drum apparatus at 37°C for 5 days and were examined for any cytopathic effect. The 50 per cent infectious dose (TCIDs0) of virus was calculated.

Bovine eoronavirus was purified from the culture fluid of B E cells infected with the Kakegawa strain which had been passaged eleven thnes in B E cells. Infectious culture fluid, clarified by centrifugation at 5,000 × g for 30 minutes was centrifuged at 100,000×g for 2 hours. The deposit was resuspended in 0.005 volumes of PBS (0.15M NaCl, 0.01 ~ phosphate buffer, pi 7.2) and centrifuged at 5,000×g for 30 minutes. The supernatant fluid from this centrifugation was mixed with a CsCl solution to a density of 1.224 g/ml and centrifuged in a Beckman SW50.1 rotor at i00,000 Xg for 18 hours. The gradient was collected in 0.3 ml volumes and fractions with a buoyant density of 1.235 to 1.250 were pooled, and dialyzed against PBS at 4 ° C for 18 hours. The resulting material was layered on to a 10--60 per cent sucrose density gradient and centrifuged in a Beckman SW25 rotor at I00,000 × g for 2 hours. Fractions (0.3 ml) were collected and those containing i0 ~g/ml or more protein as determined by optical density were pooled and dialyzed against PBS at 4°C for 62 hours. The resulting virus suspension was tested for infectivity, and for protein content by the method of LowRY et al. (8) . l~otavirus and enterovirus were concentrated by centrifugation of infectious culture fluid at I00,000 ×g for 2 hours. After resuspension in 0.01 volume of PBS the purified virus was clarified by centrifugation at 5000 × g for 30 minutes.

A n t i s e r a a g a i n s t t h e K a k e g a w a s t r a i n was p r e p a r e d in rabbits. Virus grown in B K cell cultures was purified u p to t h e step of CsC1 density gradient c e n t r i f u g a t i o n as described above. F r a c t i o n s w i t h a b u o y a n t d e n s i t y of 1.235 to 1.250 g/ml were pooled a n d dialyzed a t 4 ° C for 18 hours a g a i n s t PBS. Sufficient P B S was a d d e d to t h e resulting m a t e r i M to m a k e a suspension c o n c e n t r a t e d i00-fold from t h e original culture fluid. E a c h r a b b i t received one i n t r a v e n o u s dose of 1 ml of virus suspension, followed after 2 weeks b y a n i n t r a m u s c u l a r dose of 2 ml of equM volumes of virus suspension a n d F r e u n d ' s complete a d j u v a n t . A f u r t h e r i n t r a m u s c u l a r dose was given at 4 weeks, a n d s e r u m was o b t M n e d 2 weeks later. T h e a n t i s e r u m used in t h e present s t u d y h a d a n e u t r a l i z i n g a n t i b o d y titer of 1:4096, a n d a h e m a g g l u t i n a t i o n -i n h i b i t i n g a n t i b o d y titer of 1 : 2560.

Specific antibodies to t h e K a k e g a w a s t r a i n were purified b y affinity c h r o m a t og r a p h y from a r a b b i t a n t i s e r u m using C N B r -a c t i v a t e d Sepharose 4 B coupled to b o v i n e coronavirus, purified as described above. T h e m e t h o d was essentially t h a t of A x~N et aL (1) a n d CUATRECASAS et aL (3) . Three g r a m m e s of Sepharose 4 B ( P h a r m a c i a F i n e Chemicals, Sweden) a c t i v a t e d b y C N B r was a d d e d to 8 ml of d i l u e n t (0.5 NaC1, 0.i ~ NaHCO~) c o n t a i n i n g 50 ~g/ml of purified virus protein. The m i x t u r e was stirred a t room t e m p e r a t u r e for 2 hours, m i x e d w i t h 10 ml of 0.2 ~ glycine, p H 8.0 (Wako Chemicals, J a p a n ) a n d stirred for one additionM hour. This m a t e r i M was p a c k e d into a 9-ram d i a m e t e r column a n d w a s h e d r e p e a t e d l y w i t h a solution conraining 0.5 ~ NaCI and 0.I ~ NaHCO3 until the pH of the eluate was 8.3. The column was h~rther washed with 0.5 M NaCI in 0.i ~ acetic acid, pI-I 4.0 (until the pH of the filtrate became 4.0) and then equilibrated with PBS. The column was stored at 4 ° C after fi]]ing with 0.2 per cent NAN2.

For the purification of specific antibodies, rabbit antiserum against the Kakegawa strain was inactivated at 56°C for 30 minutes and centrifuged at 27,000×g for 30 minutes. Eight ml of the supernatant fluid was applied to the column, which was then washed with PBS until buffer passing through had an optical density of zero. Specific antibody to bovine coronavirus was eluted with 3 ~ sodium thiocyanate solution in0.3 ml fractions, which were tested for optical density. Fractions containing i0 ~g/ml or more of protein were pooled and dialyzed against :PBS at 4 ° C for ]8 hours.

Sheep blood was collected in Alsever's solution. After 5 washes w i t h P B S the e r y t h r o e y t e s were fixed b y mixing equal volumes of a 10 per cent suspension and 1 per cent g l u t a r a l d e h y d e (Iwai Chemicals, J a p a n ) in P B S . The m i x t u r e was i n c u b a t e d at r o o m t e m p e r a t u r e for 20 hours w i t h occasional shaking. The fixed cells were w a s h e d 4 times w i t h PBS, s u s p e n d e d in P B S at a c o n c e n t r a t i o n of 10 per cent a n d m i x e d w i t h an equal volume of 50 ~g/ml tannic acid (Merck, W e s t Germany) in PBS. After incubation at 37 ° C for 15 m i n u t e s the cells were w a s h e d 5 times w i t h PBS, s u s p e n d e d in P B S at a c o n c e n t r a t i o n of 10 per cent and m i x e d w i t h an equal volume of P B S containing specific antibodies to bovine eoronavirus purified as described above. After incubation at room t e m p e r a t u r e for 1 hour, t h e cells were w a s h e d twice a n d a 1 per cent suspension was prepared. The diluent used for washing a n d p r e p a r a t i o n of the suspension was P B S containing 2 per cent calf serum a n d 0.2 per cent NAN2. The calf serum used was s h o w n to be free of antibodies to bovine eoronavirus and was absorbed w i t h glutarMdehyde-fixed e r y t h r o c y t e s at 37 ° C for 1 hour to r e m o v e antibodies to sheep red blood cells.

The test was carried out by a microtiter m e t h o d , using t h e diluent described above. 

This was carried out b y the indirect m e t h o d using r a b b i t a n t i s e r u m against the K a k e g a w a strain. I n f e c t e d B K cells in t u b e cultures were i n c u b a t e d at 37°C for 2 days in a roller d r u m a p p a r a t u s and t h e n scraped off the glass using a r u b b e r policeman. The cells, s u s p e n d e d in PBS, were smeared onto glass slides, air-dried a n d fixed in acetone at --20 ° C for 30 minutes. The fixed preparations were t r e a t e d at 37 ° C for 40 m i n u t e s w i t h specific r a b b i t antiserum, stained at 37 ° C for 40 m i n u t e s w i t h fluorescein i s o t h i o c y a n a t e -c o n j u g a t e d goat a n t i b o d y to r a b b i t IgG (Miles, U.S.A.) a n d e x a m i n e d for fluorescence.

A 20 per cent suspension of feces in P B S was centrifuged at 5,000 X g for 30 m i n u t e s a n d t h e s u p e r n a t a n t was filtered t h r o u g h ~o m e m b r a n e filter w i t h a pore size of 459 nm.

One m] of the filtrate was m i x e d w i t h 4 ml of a 1 : 10 dilution in P B S of t h e r a b b i t a n t i s e r u m against bovine coronavirus a n d i n c u b a t e d at 4°C for 18 hours. This m a t e r i a l was centrifuged at 100,000 × g for 2 hours, a n d the pe]lets r e s u s p e n d e d in 0.1 ml of P B S were e x a m i n e d b y the negative staining t e c h n i q u e w i t h p h o s p h o t u n gstate in a Jeol JE3/I-100Cx electron microscope (Jeol Ltd., J a p a n ) .

This was carried out b y a microtiter m e t h o d using chicken e r y t h r o c y t e s (14) . Serum to be tested was inactivated at 56 ° C for 30 minutes and then treated with kaolin and packed chicken erythroeytes. The serum-antigen mixtures were incubated at room temperature for 1 hour, then mixed with chicken erythroeyte suspension and incubated at room temperature for a further hour. The HI titer was expressed as the reciprocal of the highest serum dilution showing complete inhibition of hemagglutination by 4 HA units.

Sheep erythrocytes were fixed with glutarMdehyde, treated with tannic acid and coated with various amounts of purified antibodies to bovine coronavirus. Using these erythrocytes the R P H A titer was determined for a concentrated suspension of purified bovine coronavirus with a titer of l0 s.2 TCIDs0/0.1 ml. The results are summarized in Table 1 . Erythrocytes treated with antibody concentrations from 1 to 60 izg/ml gave an R P H A titer of 1 : 64 whereas those treated with 70 to 100 tzg/ml antibody gave an R P H A titer of 1:128. Coated erythrocytes did not agglutinate in the absence of the virus, and virus did not agglutinate erythrocytes fixed with glutaraldehyde or fixed erythrocytes and treated with tannic acid. Based on these results erythrocytes coated with 80 ~g/ml of the purified antibodies were used in the following experiments. A concentrated suspension of bovine rotavirus with a titer of 10 s.2 TCIDs0/0.1 ml and a similar suspension of bovine enterovirus with a titer of 109.2 TCIDs0/0.1 ml gave negative results in the I~PHA test providing further confirmation of the specificity of the test. The fecal specimen from which the Kakegawa strain of bovine coronavirus was isolated (18) , gave an R P H A titer of 1:16. In the virus dilution method of R P H A inhibition, a 10-fold dilution of the rabbit antiserum against bovine coronavirus completely inhibited hemagglutination produced by the concentrated suspension of bovine coronavirus as well as that due to the fecal specimen which yielded the Kakegawa strain. The pre-immunization serum as well as antisera against bovine rotavirus and enterovirus did not inhibit the R P t I A reaction. In the serum dilution method of R P H A inhibition the antiserum had a titer of 1:512.

I~PHA tests were carried out on fecal specimens from cattle with diarrhea. Of the 22 specimens tested, 12 were collected from calves, 0.5 to Table 1 

Amount of antibody for coating RPI-IA titer~ 0 tzg/ml < 2 1 tzg/ml 64 5 Bg/ml 64 10~ 60 Bg/ml 64 70 ~-~100 ~zg/ml 128 R P t t A titer of a purified coronavirus suspension with 10 s-~-TCIDs0/0.1 ml and 2000 ~g/ml protein, using erythrocytes coated with indicated amount of purified antibody 1 month of age during an outbreak of acute gastroenteritis in the Fukushima Prefecture in March 1982. The remaining 10 specimens were obtained during an outbreak of diarrhea among milking cows in the Miyagi Prefecture in April 1982. All the specimens were also tested by immune electron microscopy and immunofluorescence. Acute and convalescent serum samples were available from 15 of the 22 animals and these were tested for • H I antibody against the Kakegawa strain. The results are summarized in Table 2 . In R P H A tests 12 of the 22 fecal specimens, or 59 per cent, were positive and the remaining 9 were negative ( < / : 2 ) . The positive specimens did not agglutinate erythrocytes fixed with glutara]dehyde or fixed erythrocytes treated with tannic acid. Of the 13 positives, 3 had titers equal to or higher than 1:8, 2 had a titer of 1:4 and the remaining 8 had a low titer of 1:2. The fecal specimens from calves had a higher positive rate (10/12, 83 per cent) than those from milking cows (3/10, 30 per cent). In the virus dilution method of R P H A inhibition a 10 fold dilution of the rabbit antiserum against bovine coronavirus inhibited the reaction of the positive specimens, whereas no inhibition was shown with the pre-immunization s e r u m .

I~PHA tests were carried out on 12 fecal specimens from healthy cattle. The specimens gave invariably negative results, supporting the specificity of the test.

Serum from 15 of the 22 animals were tested for H I antibody against the Kakegawa strain, and significant rises in titer were demonstrated in 10 of these (67 per cent). The remaining 5 animals had H I antibodies, but showed no rise in titer. Of the fecal specimens from the 10 animals showing a rise in H I titer, 7 were positive in RPI-IA tests and 3 were negative, whereas of the fecal specimens from the remaining 5 animals which did not have rises in titer, only one was positive in R P H A tests. However, the difference in the positive rate of R P H A reaction observed between the two groups was not statistically significant (Fisher's exact test). By the I F staining method only 3 specimens, or 14 per cent, were positive, while electron microscopy gave a positive rate of 6/22 or 27 per cent. The specimens positive by the IF staining method were also positive in the g P H A test and the electron microscopy. All the specimens positive by electron microscopy were also positive in the g P H A test. This observation, that the positive rate obtained by g P H A test was higher than those by the other methods, was not statistically significant.

In the present study the I~PHA method was shown to be useful and practicM for the detection of bovine coronavirus in the feces of cattle with acute gastroenteritis, although the number of fecal specimens examined in the present study was small. Bovine coronavirus has been demonstrated in fecal specimens by the immunofluorescent staining of cell cultures inoculated with the specimen (2, 10, 18) , and by electron microscopy (4, 5, 11, 17, 18) . However, these methods are time consuming and elaborate procedures. On the other hand, the R P H A method is simple and the results are obtained in a short time.

The R P H A method developed in this study was shown to be specific for bovine coronavirus. The R P H A reaction occurred only with bovine coronavirus, not with bovine rotavirus or enterovirus, and the reaction was inhibited by antiserum against bovine eoronavirus, whereas no inhibition could be demonstrated with pre-immunization sera or antisera against bovine rotavirus and enterovirus. Furthermore the fecal specimen which yielded the Kakegawa strain of bovine coronavirus (18) , had a R P H A titer of 1:16 and the reaction was specifically inhibited by antiserum against the virus. Bovine coronavirus was detected by R P H A more frequently in the feces of those cattle which had a rise in H I antibody to the virus than of those which did not. This observation provides further evidence of the specificity of R P H A test. However, the observed difference was not statistically significant, and further confirmation of this finding is required. The concentration of purified specific antibodies to bovine coronavirus used for the coating of erythrocytes was important in obtaining a high sensitivity for the test. The rate of detection of bovine coronavirus in the fecal specimen from natural cases was higher (59 per cent) by the R P H A method than by the I F staining technique (14 per cent) or electron microscopy (27 per cent). However, these results require further confirmation as the differences observed were not statistically significant. The R P H A test could find wide application in the study of bovine coronavirus infections in cattle. R P H A may also be applicable to other viral infections such rotavirus infection of cattle and transmissible gastroenteritis of swine, in which virus is excreted in abundance in the feces.

Chemical Coupling of peptides and proteins to polysaccharides by means of cyanogen halides

Isolation of coronaviruses from neonatal calf diarrhea in Great Britain and Denmark

Selective enzyme purification by affinity chromatography

Rotavirus and coronavirus associated diarrhea in domestic animals

A coronavirus-like agent present in feces of cows with diarrhea

BFI virus: a new cytopathogenic virus isolated from cattle. I. Isolation and properties

Replication of bovine eoronavirus in cell line BEK-1 culture

Protein measurement with the Folin phenol reagent

Neonatal calf diarrhea: results of a field trial using a reovirus-like virus vaccine

Neonatal calf diarrhea: propagation, attenuation and characteristics of a eoronavirus-like agent

Pathology of neonatal calf diarrhea induced by a eoronavirus-like agent

Detection of rotavirus from feces by reversed passive haemagglutination method

Detection of rotavirus from infantile gastroenteritis patient's stools by means of reversed passive haemagglutination

Hemagglutination by calf diarrhea coronavirus

Isolation of a reovirus-like agent (rotavirus) from neonatal calf diarrhea in

Characterization of a calf diarrheal eoronavirus

Neonatal calf diarrhea: purification and electron microscopy of a coronavirus-like agent

Epizootic diarrhea of adult cattle associated with a coronavirus-like agent

Authors' address: Dr. Y. I~ABA, National Institute of Animal Health, Tsukuba, Ibaraki, 305, Japan.I~eeeived June 27, 1983