Field validation of a commercial blocking ELISA to differentiate antibody to transmissible gastroenteritis virus (TGEV) and porcine respiratory coronavirus and to identify TGEV-infected swine herds.

A commercially available blocking ELISA was analyzed for its ability to identify antibodies to porcine coronaviruses (transmissible gastroenteritis virus [TGEV] or porcine respiratory coronavirus [PRCV]), to differentiate antibodies to TGEV and PRCV, and to identify TGEV-infected herds. Nine sera from uninfected pigs, 34 sera from 16 pigs experimentally infected with TGEV, and sera from 10 pigs experimentally infected with PRCV were evaluated using both the TGEV/PRCV blocking ELISA and a virus neutralization (VN) assay. The ELISA was not consistently effective in identifying pigs experimentally infected with TGEV until 21 days postinfection. Sera from 100 commercial swine herds (1,783 sera; median 15 per herd) were similarly evaluated using both tests. Thirty of these commercial herds had a clinical history of TGEV infection and a positive TGEV fluorescent antibody test recorded at necropsy within the last 35 months, while 70 herds had no history of clinical TGEV infection. The blocking ELISA and the VN showed good agreement (kappa 0.84) for the detection of porcine coronavirus antibody (TGEV or PRCV). The sensitivity (0.933) of the ELISA to identify TGEV-infected herds was good when considered on a herd basis. The ELISA was also highly specific (0.943) for the detection of TGEV-infected herds when the test results were evaluated on a herd basis. When sera from specific age groups were compared, the ELISA identified a greater proportion (0.83) of pigs in herds with TGEV antibody when suckling piglets were used. In repeatability experiments, the ELISA gave consistent results when the same sera were evaluated on different days (kappa 0.889) and when sera were evaluated before and after heating (kappa 0.888). The blocking ELISA was determined to be useful for herd monitoring programs and could be used alone without parallel use of the VN assay for the assessment of large swine populations for the detection of TGEV-infected herds.     

Comparison of the antibody response to transmissible gastroenteritis virus and porcine respiratory coronavirus, using monoclonal antibodies to antigenic sites A and X of the S glycoprotein.

Pigs were inoculated with various strains of transmissible gastroenteritis virus (TGEV) or with porcine respiratory coronavirus (PRCV), and antigenic site-specific antibody responses were compared. A blocking-ELISA was used to study to what extent antibodies in convalescent sera interfered with the binding of monoclonal antibodies (MAB) 57.16 or 57.110 to the attenuated TGEV/Purdue virus. Monoclonal antibody 57.16 is directed against the A site on the peplomer, neutralizes virus, and recognizes TGEV and PRCV. Monoclonal antibody 57.110 is directed against the X site on the peplomer, but does not neutralize virus, and recognizes only TGEV. Antibodies directed against TGEV and PRCV could be detected in a blocking ELISA, using MAB 57.16 as a conjugate. Antibodies directed against both viruses were detectable as early as 1 week after inoculation. Antibody titers correlated well with those in a virus-neutralization test. Antibodies against TGEV could be detected in a blocking ELISA, using MAB 57.110 as a conjugate. Such antibodies were not induced by a PRCV infection. In the blocking ELISA, using MAB 57.110 as a conjugate, antibodies were detectable as early as 2 weeks after inoculation. There was a significant difference between antibody titers reached after infection with various TGEV strains, however. This difference is ascribed to a variation of the antigenic site defined by MAB 57.110 in TGEV strains. Conditions for a differential test for TGE serodiagnosis, and for serologic discrimination between TGEV- and PRCV-infected pigs, are discussed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evaluation of the baculovirus-expressed S glycoprotein of transmissible gastroenteritis virus (TGEV) as antigen in a competition ELISA to differentiate porcine respiratory coronavirus from TGEV antibodies in pigs.

The spike (S) glycoprotein of the Miller strain of transmissible gastroenteritis virus (TGEV) was recently cloned and expressed in baculovirus. The recombinant S protein was used as the coating antigen in a competition (blocking) enzyme-linked immunosorbent assay (ELISA) in combination with monoclonal antibodies to the S protein epitope A (conserved on TGEV and porcine respiratory coronavirus [PRCV]) or epitope D (present on TGEV only) to differentiate PRCV- from TGEV-induced antibodies. One set (set A) of 125 serum samples were collected at different times after inoculation of caesarean-derived, colostrum-deprived (n = 52) and conventional young pigs (n = 73) with 1 of the 2 porcine coronaviruses or uninoculated negative controls (TGEV/PRCV/negative = 75/30/20). A second set (set B) of 63 serum samples originated from adult sows inoculated with PRCV and the recombinant TGEV S protein or with mock-protein control and then exposed to virulent TGEV after challenge of their litters. Sera from set A were used to assess the accuracy indicators (sensitivity, specificity, accuracy) of the fixed-cell blocking ELISA, which uses swine testicular cells infected with the M6 strain of TGEV as the antigen source (ELISA 1) and the newly developed ELISA based on the recombinant S protein as antigen (ELISA 2). The sera from set B (adults) were tested for comparison. The plaque reduction virus neutralization test was used as a confirmatory test for the presence of antibodies to TGEV/PRCV in the test sera. The accuracy indicators for both ELISAs suggest that differential diagnosis can be of practical use at least 3 weeks after inoculation by testing the dual (acute/convalescent) samples from each individual in conjunction with another confirmatory (virus neutralization) antibody assay to provide valid and complete differentiation information. Moreover, whereas ELISA 1 had 10-20% false positive results to epitope D for PRCV-infected pigs (set A samples), no false-positive results to epitope D occurred using ELISA 2, indicating its greater specificity. The progression of seroresponses to the TGEV S protein epitopes A or D, as measured by the 2 ELISAs, was similar for both sets (A and B) of samples. Differentiation between TGEV and PRCV antibodies (based on seroresponses to epitope D) was consistently measured after the third week of inoculation.     

A competitive inhibition ELISA for the differentiation of serum antibodies from pigs infected with transmissible gastroenteritis virus (TGEV) or with the TGEV-related porcine respiratory coronavirus

A competitive inhibition ELISA was developed to detect non-neutralizing antibodies to the peplomer protein of transmissible gastroenteritis virus (TGEV) in porcine sera using a monoclonal antibody as an indicator. It was demonstrated that field strains of the TGEV-related porcine respiratory coronavirus (PRCV) did not induce this antibody, whereas the Miller strain and field strains of TGEV did. The sensitivity of the competitive inhibition ELISA appeared to be similar to that of the virus neutralization (VN) test. The test enables differentiation of pigs which were previously infected with TGEV or PRCV and which cannot be distinguished by the classical anti-TGEV neutralization test. The present test is useful for selective serodiagnosis.     

Virological and serological studies of porcine respiratory coronavirus infection on a Japanese farm

We detected transmissible gastroenteritis virus (TGEV) antibodies in pig farms in Tochigi prefecture, although the farms had no past record of TGEV vaccination or TGE. Among the farms, Farm A showed a high antibody incidence. We could not confirm if either TGEV or porcine respiratory coronavirus (PRCV) induced the antibodies, since conventional tests failed to discriminate PRCV from TGEV. Therefore, we conducted virological and serological examinations of this farm for 4 years to establish the etiology - TGEV or PRCV. Although no TGEV was detected, PRCVs were isolated from the nasal samples of pigs. Using a commercial ELISA kit, it was found that the antibodies detected in pigs of all the raising stages and sows were raised against PRCV but not TGEV. The phylogenetic analysis of the nucleotide sequences of the isolates showed that they were closely related to each other, and formed a separate cluster apart from the U.S.A. and European strains. In Cesarean-derived, colostrums-deprived piglets inoculated with a PRCV isolate, no clinical signs were seen, and the viruses were mainly isolated from the nasal samples. Moreover, viral genes were detected from the nasal sample of the contact pig. The result suggested that PRCV infection was located in the nasal cavity of pigs, and horizontal transmission easily occurs. From these results, PRCVs with different origins from the exotic PRCVs might be prevalent in pig farms in Japan.     

Evidence for a porcine respiratory coronavirus, antigenically similar to transmissible gastroenteritis virus, in the United States

A respiratory variant of transmissible gastroenteritis virus (TGEV), designated PRCV-Ind/89, was isolated from a swine breeding stock herd in Indiana. The virus was readily isolated from nasal swabs of pigs of different ages and induced cytopathology on primary porcine kidney cells and and on a swine testicular (ST) cell line. An 8-week-old pig infected oral/nasally with the respiratory variant and a contact pig showed no signs of respiratory or enteric disease. These pigs did not shed virus in feces but did shed the agent from the upper respiratory tract for approximately 2 weeks. Baby pigs from 2 separate litters (2 and 3 days old) also showed no clinical signs following oral/nasal inoculation with PRCV-Ind/89. In a third litter, 5 of 7 piglets (5 days old) infected either oral/nasally or by stomach tube developed a transient mild diarrhea with villous atrophy. However, virus was not isolated from rectal swabs or ileal homogenates of these piglets, and viral antigen was not detected in the ileum by fluorescent antibody staining even though the virus was easily recovered from nasal swabs and lung tissue homogenates. Swine antisera produced against PRCV-Ind/89 or enteric TGEV cross-neutralized either virus. In addition, an anti-peplomer monoclonal antibody, 4F6, that neutralizes TGEV also neutralized the PRCV-Ind/89 isolate. Radioimmunoassays with a panel of monoclonal antibodies indicated that the Indiana respiratory variant and the European PRCV are antigenically similar.     

Serological studies of transmissible gastroenteritis in Great Britain, using a competitive ELISA.

A competitive ELISA which differentiates between transmissible gastroenteritis virus (TGEV) and porcine respiratory coronavirus (PRCV) was used to detect non-neutralising antibodies to the peplomer protein of TGEV in porcine sera. The test was shown to be TGEV specific, having a relative specificity of 100 per cent, and to have a relative sensitivity of 94.9 per cent when compared with the virus neutralisation test. The prevalence of TGEV in Great Britain is low; only 0.6 per cent of sows sampled in 1990 were seropositive to TGEV. Seroconversion to the virus neutralisation test occurred in a closed herd in 1984, with no apparent spread, but later testing by the ELISA did not detect any blocking antibodies. The possibility of the existence of a less contagious strain of PRCV is discussed. All British isolates of TGEV tested by the indirect fluorescent antibody test were recognised by the monoclonal antibody 1D.B12, the indicator antibody in the ELISA.     

Detection of antibodies against porcine parvovirus nonstructural protein NS1 may distinguish between vaccinated and infected pigs

The humoral antibody response against the nonstructural protein NS1 and the structural protein VP2 of porcine parvovirus (PPV) was evaluated by immuno-peroxidase test (IPT) and enzyme linked immuno sorbent assay (ELISA) using recombinant PPV antigens. The coding sequence for NS1 and VP2 was inserted into the baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV) genome resulting in two recombinant baculoviruses AcNPV-NS1 and AcNPV-VP2, respectively. Sf9 cells (Spodoptora frugidiperda) inoculated with AcNPV-NS1 producing recombinant nonstructural protein (rNS1) and AcNPV-VP2 producing recombinant virion protein (rVP2) were used in IPT and ELISA to analyse serum antibodies. Pigs vaccinated with an inactivated whole virus vaccine and experimentally infected pigs were studied. Significant titers against rVP2 were obtained in both vaccinated and infected pigs. Specific antibodies against rNS1 could only be detected in infected pigs and NS1 may in this way allow the specific detection of infected animals. Analysis of serum samples collected up to 18 days post infection (p.i.) from four pigs experimentally infected with PPV showed that antibodies against rNS1 and rVP2 could in all cases be detected on day 9 p.i. Two individual pigs were inoculated twice with PPV and the antibody response was followed 89 days after second inoculation. Serum antibodies against borth rVP2 and rNS1 could be detected for this period of time.     

用固定细胞阻断酶联免疫吸附试验鉴别诊断猪传染性胃肠炎病毒和猪呼吸道冠状病毒的感染

用固定细胞阻断酶联免疫吸附试验（ＥＬＩＳＡ）对来自丹麦猪的１８０份血清进行了猪传染性胃肠炎病毒（ＴＧＥＶ）和猪呼吸道冠状病毒（ＰＲＣＶ）感染的鉴别诊断，共检出了ＰＲＣＶ抗体阳性血清１０７份（５９．４％），ＴＧＥＶ抗体阳性血清０份。同时也检测了一些来自国内不同ＴＧＥＶ感染类型的猪场血清。该鉴别诊断方法在我国的建立和应用为从ＰＲＣＶ阳性国家进口猪的ＴＧＥ的检疫提供了一条有效途径。     

Prevalence of coronavirus antibodies in Iowa swine.

Three hundred and forty-seven serum samples from 22 Iowa swine herds were screened for TGEV/PRCV neutralizing antibody. Ninety-one percent of the sera and all 22 herds were positive. These sera were then tested by the blocking ELISA test to distinguish TGEV and PRCV antibody. The ELISA test confirmed the high percentage of TGEV/PRCV positive sera. By the blocking ELISA test, 12 herds were PRCV positive, 6 herds were TGEV positive and 4 herds were mixed with sera either positive for TGEV or PRCV antibody. The results suggest a recent increase in TGEV/PRCV seroprevalence in Iowa swine most likely due to subclinical PRCV infections.     

Comparison of two methods for detection of transmissible gastroenteritis virus in feces of pigs with experimentally induced infection

An indirect, double-antibody sandwich-type ELISA for detection of transmissible gastroenteritis virus (TGEV) was developed, using a solid phase of rabbit hyperimmune serum and a pool of 3 antipeplomer monoclonal antibodies to trap and to detect the virus, respectively. The technique was used to detect viral antigen in feces of pigs that had been infected with the virulent Miller strain, the attenuated Purdue strain, or the Erica strain (a Dutch field isolate) of TGEV. The results were compared with those of a solid-phase immunosorbent electron microscopy (SPIEM) technique for virus detection. Both techniques detected shedding of virulent virus in feces obtained from pigs on the first or second day after infection, and virus excretion continued for 6 to 8 consecutive days. Virus shedding started later in pigs infected with the attenuated Purdue strain of TGEV and lasted only 2 to 4 days. In comparison with the 2 virulent strains, infection with the attenuated strain appeared to be limited to a smaller portion of the small intestine. Of 242 fecal specimens that were tested by use of ELISA and SPIEM, 119 had positive results in both tests. Additionally, virus could be detected by ELISA in 21 and by SPIEM in 16 specimens. Fecal specimens obtained from pigs before infection always reacted negatively by ELISA for TGEV antigen; there was no cross-reactivity with fecal specimens containing porcine rotavirus or porcine epidemic diarrhea virus. The ELISA and SPIEM were found to be specific and sensitive for the detection of TGEV in feces.     

贵州省猪传染性胃肠炎及呼吸冠状病毒病血清学调查

用酶联免疫吸附试验对采自贵州省内88个县(市)的2906份血清进行了猪传染性胃肠炎病毒及呼吸冠状病毒抗体检测,结果检出猪传染性胃肠炎病毒抗体阳性血清12份,总体阳性率为0.41%;猪呼吸冠状病毒抗体阳性血清44份,总体阳性率为1.51%;2818份血清两种抗体均呈阴性(阴性率为96.97%),32份血清检测无效或无结论(无效率为1.10%)。     

Enzyme-linked immunosorbent assay for detection of transmissible gastroenteritis virus antibody in swine sera

An enzyme-linked immunosorbent assay (ELISA) was developed for detection and quantification of serum antibodies to transmissible gastroenteritis virus (TGEV) in swine. Sera from pigs inoculated with cell culture-origin TGEV or gut-origin TGEV were tested for anti-TGEV antibody by ELISA and by serum virus-neutralization test (NT). The ELISA detected antibody 3 days (av) sooner than did the NT when sera from pigs inoculated with cell culture-origin TGEV were tested and 1 day sooner than did the NT when sera from pigs inoculated with gut-origin TGEV were tested. The ELISA appeared to be more sensitive than the NT, since ELISA was more responsive to low-level antibody and ELISA titers exceeded NT titers.     

Porcine respiratory coronavirus in Quebec: Serological studies using a competitive inhibition enzyme-linked immunosorbent assay

Porcine respiratory coronavirus (PRCV) was identified for the first time in Quebec, using a blocking enzyme-linked immunosorbent assay (ELISA). Unlike the virus neutralization test (VNT), this ELISA was able to distinguish transmissible gastroenteritis virus (TGEV) from PRCV. Among the 15 seropositive fattening herds from group A, sera containing PRCV antibodies represented 74.8%, whereas those with TGEV antibodies represented only 7.2%. In group B, which consisted of 15 sow herds, nine herds expressed only PRCV-specific antibodies while the other herds had animals positive for TGEV-specific antibodies.     

Seroprevalence of economically important viral pathogens in swine populations of Trinidad and Tobago,West Indies

The objective of this study was to evaluate the seroprevalence and identify the strains of swine influenza virus (SwIV), as well as the seroprevalence of porcine parvovirus (PPV), transmissible gastroenteritis virus (TGEV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine respiratory coronavirus (PRCV), porcine circovirus type 2 (PCV-2), and classical swine fever virus (CSFV) in pigs in Trinidad and Tobago (T&T). Blood samples (309) were randomly collected from pigs at farms throughout T&T. Serum samples were tested for the presence of antibodies to the aforementioned viruses using commercial ELISA kits, and the circulating strains of SwIV were identified by the hemagglutination inhibition test (HIT). Antibodies against SwIV were detected in 114 out of the 309 samples (37%). Out of a total of 26 farms, 14 tested positive for SwIV antibodies. HI testing revealed high titers against the A/sw/Minnesota/593/99 H3N2 strain and the pH1N1 2009 pandemic strain. Antibodies against PPV were detected in 87 out of the 309 samples (28%), with 11 out of 26 farms testing positive for PPV antibodies. Antibodies against PCV-2 were detected in 205 out of the 309 samples tested (66%), with 25 out of the 26 farms testing positive for PCV-2 antibodies. No antibodies were detected in any of the tested pigs to PRRSV, TGEV, PRCV, or CSFV.     

Differentiation between transmissible gastroenteritis virus and porcine respiratory coronavirus using a cDNA probe.

A plasmid, pG3BS, containing a cDNA clone from the 5' coding region of the peplomer glycoprotein gene appears to be specific for enteric transmissible gastroenteritis virus (TGEV) strains and for live-attenuated TGEV vaccines. This cDNA probe is used to differentiate porcine respiratory coronavirus (PRCV) isolates from TGEV field and vaccine strains by a slot blot hybridization assay. Probe pG3BS also hybridizes to canine coronavirus (CCV) RNA but does not hybridize to antigenically related feline infectious peritonitis virus (FIPV) RNA. The RNAs of 13 enteric TGEV isolates from the United States, Japan, and England, 4 US-licensed live-attenuated TGEV vaccines, and antigenically closely related CCV were detected by pG3BS. The RNAs of FIPV and 3 US isolates of PRCV did not react with pG3BS but were detected by a TGEV-derived plasmid, pRP3. Pigs infected with either PRCV or TGEV test serologically positive for TGEV antibody by the serum neutralization test. Characterization of the virus circulating in a swine herd by the pG3BS probe will differentiate between an enteric TGEV and a respiratory PRCV infection.     

Antigenic variation among transmissible gastroenteritis virus (TGEV) and porcine respiratory coronavirus strains detected with monoclonal antibodies to the S protein of TGEV.

Five nonneutralizing monoclonal antibodies (MAb) generated to the virulent Miller strain of transmissible gastroenteritis virus (TGEV) and specific for the S protein were characterized. Competition assays between purified and biotinylated MAb indicated that MAb 75B10 and 8G11 mapped near a new subsite, designated V and 2 MAb, 44C11 and 45A8, mapped to a previously designated subsite D. A fifth MAb mapped between subsites V and E. These MAb were tested with 3 previously characterized MAb to subsites A, E, and F in fixed-cell ELISA and cell culture immunofluorescent assays against 5 reference and 9 field strains of TGEV and 2 US strains (ISU-1 and ISU-3) of porcine respiratory coronavirus (PRCV). Subsites A, E, and F were conserved on all TGEV and PRCV strains examined. The 2 MAb to subsite V, 8G11 and 75B10, reacted only with the Miller TGEV strains (M5C, M6, and M60), except that 75B10 also recognized field strain U328. The MAb 11H8 did not react with 4 field strains or the Purdue strains of TGEV. The 2 MAb to subsite D reacted with all TGEV strains examined, but not with 2 US PRCV strains, 2 European PRCV strains, 1 feline infectious peritonitis virus strain, and 1 canine coronavirus strain. Because of this specificity for TGEV, but not PRCV, these latter 2 subsite D MAb may be useful for the development of competition ELISA to differentiate serologically between TGEV and PRCV infections in swine, similar to the currently used European subsite D MAb.     

Seroprevalence of porcine respiratory coronavirus in selected Korean pigs

A total of 446 serum samples from 88 herds in Korea were examined for antibody to porcine respiratory coronavirus (PRCV) using blocking enzyme-linked immunosorbent assay (ELISA). All serum samples were collected from 24- to 26-week-old finishing pigs between December 1998 and June 1999. By ELISA, 237 out of 446 sera tested (53.1%) and 54 out of 88 sampled herds (61.3%) were positive against PRCV. Of 446 sera from 88 herd tested, 185 (41.5%) serum samples from 22 (25%) herds were seronegative against PRCV and transmissible gastroenteritis virus infection. Our data suggested that seropositive herds for PRCV are distributed diffusely throughout South Korea.     

A blocking ELISA using a monoclonal antibody for the serological detection of Mycoplasma hyopneumoniae

A blocking ELISA was developed by using a monoclonal antibody (4082-05-344-18) which specifically detected an epitope on the Mycoplasma hyppneumoniae 40 kDa membrane protein without cross-reacting with M flocculare or M hyorhinis. The results obtained with sera from specific pathogen-free pigs inoculated with M flocculare or M hyorhinis confirmed the specificity of the assay. An immunoblotting procedure was used to characterise the antibody response of pigs experimentally infected with M hyopneumoniae. Antibodies to the 40 kDa antigen were detected two weeks after infection and remained as major markers for at least 20 weeks. Cross-reacting antibodies to this antigen were not detected in convalescent sera from piglets infected with M flocculare or M hyorhinis. Sera from experimentally infected pigs were compared by means of the blocking ELISA and an indirect ELISA. The kinetics of ELISA antibodies after experimental inoculation were also studied. The detection of antibody was rather more stable for a longer time with the blocking ELISA than with the indirect ELISA. In an evaluation of more than 1000 sera from the field there was excellent agreement between the two methods.