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Veterinary Microbiology 7 1982 295 306 295 Elsevier Scientific Publishing Company Amsterdam Printed in The Netherlands ENZYME LINKED IMMUNOSORBENT ASSAY FOR THE DETECTION OF THE CORONAVIRUS LIKE AGENT AND ITS ANTIBODIES IN PIGS WITH PORCINE EPIDEMIC DIARRHEA P CALLEBAUT P DEBOUCK and M PENSAERT Laboratory of Virology Faculty of Veterinary Medicine State University of Gent Casinoplein 24 B 9000 Gent Belgium Accepted 15 February 1982 ABSTRACT Callebaut P Debouck P and Pensaert M 1982 Enzyme linked immunosorbent assay for the detection of the coronavirus ike agent and its antibodies in pigs with porcine epidemic diarrhea Vet Microbiol 7 295 306 An enzyme linked immunosorbent assay ELISA was developed for the detection of the coronavirus like agent in feces of pigs naturally affected with porcine epidemic diarrhea PED or experimentally infected with the CV777 isolate The assay was specific and more sensitive than electron microscopy An ELISA blocking assay is described for the detec tion and titration of antibodies Specific antibody formation was demonstrated in pigs experimentally infected with CV777 and in swine naturally affected with PED INTRODUCTION A coronavirus like agent CVLA is associated with diarrhea affecting swine of all ages This type of diarrhea has been called porcine epidemic diarrhea PED Pensaert 1981 Based on its electron microscopic appearance the agent isolate CV777 has been proposed as a member of the Coronaviridae family Pensaert and Debouck 1978 To date infections by this agent have been diagnosed by the demonstration of the virus in intestinal epithelium using immunofluorescence IF on gut sections and in feces using electron microscopy EM Debouck et al 1981b Antibodies to CV777 have been demonstrated by indirect IF staining and by immunoelectron microscopy Pensaert et al 1981 All these methods are rather cumbersome and time consuming for large scale diagnostic and serological purposes The epizootiol ogy of CVLA has therefore not been examined in detail The ELISA has found wide application in the detection of antibodies and small amounts of antigen and seems to be particularly suitable for widespread epizootiological studies The assay has been used for the detection of bovine coronavirus in feces Ellens et al 1978a and for the serology of several coronaviruses including feline infectious peritonitis virus Osterhaus et al 1979 murine hepatitis virus Peters et al 1979 Kraaijeveld et al 1980a 0378 1135 82 0000 0000 02 75 1982 Elsevier Scientific Publishing Company 296 and human coronavirus strain 229E Kraaijeveld et al 1980a b In the present report an ELISA is described for the detection of CVLA antigens in crude fecal suspensions and the results are compared with those of EM It will also be shown that ELISA is useful for the detection of anti bodies against CV777 in swine sera MATERIALS AND METHODS Specimens of fecal material and of intestinal contents Specimens to be examined for CVLA antigens by ELISA consisted either of fecal material obtained directly from the rectum of live animals or of contents collected from the caecum at the time of killing The specimens originated not only from experimentally infected pigs but also from natural ly infected animals and from controls After collection all samples were stored at 70 C until further processing Specimens from experimental pigs Intestinal contents were obtained from two uninoculated cesarean derived colostrum deprived CDCD piglets and from 17 CDCD piglets orally inocu lated at the age of 2 days to 2 weeks with approximately 104 pig infective doses PID of CVLA isolate CV777 Debouck and Pensaert 1980 Debouck et al 1981a Specimens were collected from three piglets that were killed during the incubation period 1 and 2 days after inoculation from twelve piglets killed during the phase of severe diarrhea 2 to 5 days after inoculation and from two pigs killed during the stage of recovery 6 to 8 days after inocu lation Thirty six samples of fecal material were collected from seven CDCD pig lets Nos 1 to 7 during an 8 day observation period following oral administra tion of 104 PID of CV777 at the age of 2 weeks From each piglet a sample was collected prior to inoculation and during the incubation period 1 day after inoculation Further samples were collected during the stage of severe diarrhea 3 to 5 days after inoculation and towards the end of diarrhea 6 to 8 days as indicated in Table II Fecal specimens were also obtained from an uninoculated conventional fattening pig and from two conventional fattening pigs during the acute phase of diarrhea 2 days after oral inoculation with 104 PID of CV777 Specimens from affected swine in the field A fecal specimen was collected from each of 23 conventional pigs of vary ing ages originating from four different farms on which a natural outbreak of PED had been diagnosed by IF staining on gut sections of sacrificed animals At the time of collection 18 of these pigs had diarrhea while the remaining five pigs had been convalescent for more than 1 week 297 Con trol specimens Control fecal samples used to determine the limit between positive and negative absorbance values in ELISA consisted of 25 non diarrheal specimens obtained from conventional pigs of all ages Control samples to test the specificity of ELISA consisted of 23 fecal specimens from piglets with diarrhea caused by transmissible gastroenteritis virus TGEV or porcine rotavirus Serum specimens A total of 62 serum samples to be examined for antibody by ELISA blocking were collected from four CDCD piglets and seven conventional ex perimental fattening pigs prior to inoculation with CV777 and at various in tervals between 7 and 81 days thereafter Additionally 76 serum specimens were obtained from 42 conventional pigs of all ages on five different farms The latter samples were collected from 5 to 180 days after the onset of an outbreak of PED Control serum specimens consisted of a convalescent serum from two con ventional pigs recovered from TGE and rotavirus infection and of three hyperimmune pig sera prepared respectively against a virulent Belgian strain of TGEV hemagglutinating encephalomyelitis virus HEV strain VW572 Pensaert and Callebaut 1974 and porcine rotavirus strain RV277 Debouck and Pensaert 1979 These hyperimmune sera were prepared as described else where Pensaert et al 1981 All sera were stored at 20 C until used ELISA procedure for the detection of CVLA antigen For the detection of CVLA antigen by ELISA the double antibody sand wich form of the assay was used It was conducted essentially as described by Ellens et al 1978 b in flat bottomed microplates Cooke Microtitre M 129 B using 100 pl volumes of reagents per well Plates were coated with a 1 2000 dilution of anti CV777 globulin prepared according to the method of Purcell et al 1973 from a hyperimmune porcine antiserum obtained as described elsewhere Debouck and Pensaert 1980 Samples to be examined were first homogenized by shaking for 1 h at 4 C in four volumes of phos phate buffered saline pH 7 2 PBS containing 0 01 Tween 80 and each was then added to two coated wells Controls included PB8 and a series of twofold dilutions of a standard CV777 antigen preparation The latter preparation was intestinal perfusate Debouck and Pensaert 1980 Briefly a CDCD piglet was inoculated orally with a bac teria free filtrate of a 20 intestinal homogenate containing CV777 After 27 h when the diarrhea started the small intestinal lumen of the anesthetized piglet was perfused with 1500 ml Eagle s minimal essential medium during 12 h at 37 C The perfusate was subsequently centrifuged at 3000 X g at 4 C for 1 h The supernatant containing CV777 antigen was known to contain 320 ELISA units E U in previous titrations 298 Specificity testing was performed by a blocking assay The first sample well was treated with a porcine negative control serum diluted 1 10 to the second sample well a 1 10 dilution of porcine anti CV777 serum was added Both sera were obtained from pigs different from that which provided the serum used for preparation of the anti CV777 globulin fraction The conjugate used was the anti CV777 globulin preparation labelled with horseradish peroxidase Grade I Boehringer according to the method de scribed by Wilson and Nakane 1978 The optimal working dilution was 1 600 The amount of conjugate bound was determined by adding the enzyme substrate solution containing 1 mg ml of recrystallized 5 aminosalicylic acid 0 005 hydrogen peroxide 1 mM Na2 EDTA and 0 01 M sodium phosphate final pH 6 0 After overnight reaction at 4 C the absorbance of each well was measured at 450 nm against the PBS blank using a Multiskan colorimeter Flow Laboratories A sample was scored positive if the absorbance in the well treated with negative control serum was equal to or higher than that of the 1 320 dilution of the standard CV777 preparation and if the absorbance value was reduced by 50 in the well treated with anti CV777 serum ELISA procedure for the detection of antibody to CV777 Antibody to CV777 in serum was measured by an ELISA blocking assay performed in a manner similar to the blocking assay used to control the specif icity of the CV777 antigen ELISA The same anti CV777 globulin prepara tion diluted 1 500 was used to coat the plates Antigen for all assays was the standard CV777 preparation diluted 1 20 in PBS supplemented with 2 5 fetal bovine serum and 0 05 Tween 80 this amount of antigen repre sented 16 E U Test sera were serially diluted in twofold series starting from a 1 5 dilution in PBS containing 0 5 M NaC1 0 05 Tween 80 and 5 por cine negative control serum and were added to the plates to which antigen had been bound Diluent alone served as a control The plates were incubated for 1 h at 37 C and thereafter overnight at 4 C before the conjugate was added The anti CV777 antibody used for the preparation of this conjugate was obtained from a pig other than that which provided the coating anti body in order to decrease the chance for aspecific reactions The optimal dilution was 1 600 in test serum diluent The amount of conjugate bound to each well was determined as above A given dilution of test serum was con sidered to be positive if it reduced the absorbance by at least 50 when com pared with the buffer control Titers are expressed as the reciprocal of the highest positive dilution Electron microscopy To compare the sensitivity of EM and ELISA for the detection of CVLA the specimens of fecal material and intestinal contents obtained from experi mentally infected CDCD piglets and experimental fattening pigs as described 299 in the section Specimens from experimental pigs were examined for coronavirus by EM The samples were processed as described elsewhere Pensaert and Debouck 1978 RESULTS Reading procedure for the detection of CV777 antigen by ELISA The absorbance profile obtained with serially diluted perfusate in the anti gen assay is shown in Fig 1A All 25 control non diarrheal fecal specimens used to determine the limit between positive and negative absorbance values showed values in the range of 0 00 to 0 05 and these values were not reduced in the blocking assay The mean absorbance of 0 02 plus 3 times the standard deviation resulted in an absorbance value of 0 06 Therefore an absorbance value of 0 06 was considered evidence of the presence of CV777 antigen in a sample Using this criterion the standard CV777 antigen preparation in Fig 1A was positive up to a dilution of 1 320 This preparation was there fore considered to contain 320 E U Specificity of the ELISA for antigen detection In the specificity tests all 23 fecal specimens from TGEV or rotavirus positive pigs were ELISA negative for CV777 antigen Additionally there was a good correlation between the ELISA results and clinical data in the experimentally inoculated swine As shown in Table I all preinfection fecal or intestinal samples obtained from experimental piglets and fattening swine were ELISA negative All the fecal samples obtained during the incubation period were ELISA negative However all three samples of intestinal contents collected during the same period were ELISA positive The twelve samples of intestinal contents and 13 of the 14 specimens of fecal material collected during the acute phase of diarrhea were ELISA positive TABLE I Detection of CVLA antigen by ELISA in fecal samples and in samples of intestinal contents collected from pigs before and at different times after inoculation with CV777 Time of Clinical signs at collection time of collection No positive No tested Fecal material Intestinal contents Preinoculation none 0 8 0 2 1 to 2 dpi a none 0 7 3 3 2 to 5 dpi severe diarrhea 13 14 12 12 6 to 8 dpi recovery period 3 10 0 2 adpi days postinoculation pigs were orally inoculated with 10 PID of CV777 0 8 Only three out of ten fecal specimens and none of two samples of intestinal contents obtained during the stage of recovery were ELISA positive for CV777 antigen Of the 23 fecal samples collected from naturally infected pigs of all ages 18 were ELISA positive all of which were obtained from diarrheic animals The remaining five samples in which no CVLA antigen could be demonstrated were collected from animals that had convalesced for more than 1 week The ELISA positive samples had absorbance values in the range of 0 07 to 0 58 median 0 25 After blocking by incubation of the specimens with CV777 antiserum the absorbance values were reduced in every instance by more than 50 ranging from 0 01 to 0 10 median 0 04 E 0 6 c 0 4 0 2 0 0 5 A 20 0 320 1280 5 20 Reciprocal of dilution 0 4 0 3 g e 0 2 c Q 0 1 1201 01 I I O OF 320 5 Reciprocal of dilution 300 Fig 1 ELISA absorbance values at 450 nm obtained with A serial dilutions of intestinal perfusate B serial dilutions of a porcine serum the buffer control measuring 0 35 Sensitivity of the ELISA for antigen detection compared to that of EM As shown in Table II the experimental piglets Nos 4 5 6 and 7 were found to excrete the coronavirus like agent in their feces both by EM and ELISA In the remaining piglets Nos 1 2 and 3 virus shedding was detected by ELISA only In total nine fecal specimens found negative for coronavirus like particles by EM were ELISA positive whereas only one EM positive speci men was negative by ELISA All the samples collected during the acute stage of diarrhea at 4 days after inoculation when coronavirus was regularly but not always detected by EM were ELISA positive Towards the end of the diarrhea 6 and 7 days after inoculation some EM negative samples were still ELISA positive TABLE II Detection of CV777 by EM and ELISA in fecal samples from seven experimentally inoculated piglets 301 Piglet Coronavirus detected by ELISA EM No Days after inoculation 0 1 3 4 5 6 7 8 1 I I I 2 3 4 I I 5 I I I 6 7 I I a coronavirus detected coronavirus not detected The fecal samples of the two experimentally inoculated fattening pigs were negative for CVLA by EM but positive by ELISA In intestinal contents of experimentally infected piglets CVLA was de tected at various times after inoculation by both methods In all three speci mens collected during the incubation period CVLA was detected by both EM and ELISA Nine out of twelve specimens obtained during the stage of diarrhea were also positive by both methods However the three remaining specimens obtained on day 2 3 and 4 after inoculation respectively were negative for coronavirus by EM but positive by ELISA Finally the two samples collected at the time of recovery were negative both by EM and ELISA ELISA blocking assay for the detection of antibody to CV777 The absorbance profile obtained with a serially diluted porcine serum sample in the ELISA blocking test is shown in Fig 1 B By comparison with the buffer control having an absorbance value of 0 35 the titer of this sample was estimated to be 160 The sensitivity and specificity of the ELISA blocking assay in detecting a sero conversion to CV777 infection was first established by testing pre and post inoculation sera obtained from experimentally infected piglets and fat tening swine As shown in Table III upper part all preinfection and early postinfection sera from the CDCD piglets were negative titer 5 Starting from 43 days after inoculation the ELISA blocking assay detected CV777 antibodies in all the piglet sera and titers ranged from 40 to 1280 Of the seven conventional experimental fattening pigs Table III lower part three animals had no detectable antibody titers in their preinfection 302 TABLE III Prevalence and titers of CV777 antibodies in sera collected from four piglets up and seven fattening pigs down at different times after inoculation with CV777 as deter mined by ELISA blocking assay Time of ELISA seropositive animals collection No No tested Mean titer Range of titer a Preinoculation 0 4 15 dpi b 0 4 43 dpi 4 4 60 40 80 73 dpi 4 4 240 80 640 81 dpi 4 4 460 80 1280 Preinoculation 4 7 16 5 40 7dpi 7 7 1486 160 2560 14 dpi 7 7 4023 1280 10240 21 dpi 7 7 777 320 1280 29 dpi 7 7 366 320 640 41 dpi 7 7 366 160 640 atiters expressed as the reciprocal of the highest dilution with absorbance fourfold rise in antibody titer were detected in the sera from the latter animals 7 days after inoculation when the ELISA blocking titers ranged from 160 to 2560 The highest titers were found on day 14 after inoculation The results of the serological examination of the pigs of all ages naturally infected in the field are given in Table IV Fourteen sera were collected be tween 5 and 14 days after the onset of the diarrheal outbreak and were ELISA negative All the sera except 3 collected 45 days or later after the start of the disease contained antibodies detectable by ELISA blocking The titers of these positive sera varied from 10 to 160 Finally the specificity of the ELISA blocking assay was indicated by the negative results obtained with convalescent and hyperimmune antisera directed towards other viruses including TGEV rotavirus and HEV DISCUSSION The results obtained with intestinal contents and fecal material of experi mentally and naturally infected swine demonstrate the efficacy of ELISA for the detection of CVLA antigen The specificity of the test was proven by the results on control material containing other viruses Further evidence TABLE IV ELISA detection of CV777 antibodies in sera of swine of all ages naturally affected in the field with PED 303 Days after ELISA seropositive animals start of diarrheal outbreak No No tested Mean titer Range of titer a 5 0 8 14 0 6 30 8 25 68 5 160 45 10 12 44 10 160 60 8 8 26 10 80 90 6 6 23 10 40 120 5 6 48 10 80 180 5 5 60 20 160 atiters expressed as the reciprocal of the highest dilution with absorbance 50 of the control of the specificity is obtained by the correlation of the ELISA results with the clinical data virus shedding is detected with high consistency during the acute phase of disease but much less consistently during the incubation period and during the recovery phase The ELISA results on samples collected during the latter two periods appear to depend upon the type of material tested All samples of intestinal contents collected the first and second day after inoculation were positive whereas all fecal samples collected 1 day after inoculation were negative During the phase of recovery on the con trary some fecal samples were positive while all intestinal samples were negative It is likely that this is a reflection of the time delay between the production and release of virus progeny in the intestine and its excretion in the feces However as only a small number of specimens have been tested more extensive examination is needed to corroborate the present findings The sensitivity of ELISA compares favourably to that of EM More fecal and intestinal specimens from experimentally infected pigs were positive by ELISA than by EM ELISA is more reliable than EM as shown by the finding that during the acute phase of diarrhea virus excretion coul