Interlaboratory testing of porcine sera for antibodies to porcine circovirus type 2.

A panel of 20 porcine sera was distributed to 5 laboratories across Europe and Canada. Each center was requested to test the sera for the presence of porcine circovirus type 2 antibodies using the routine assays, indirect immunofluorescence assay (IFA) and indirect immunoperoxidase monolayer assay (IPMA), and to determine the titer of each serum. Results from all centers were then compiled and correlated. They demonstrate a wide variation in the titers obtained between laboratories. These differences were dependent on the assay used and the choice of fixative. In general, IPMA gave higher titers than did IFA, and paraformaldehyde gave higher titers than did acetone or ethyl alcohol. This report highlights the need for standardized procedures and biologicals for this virus.     

An ELISA for porcine reproductive and respiratory syndrome: production of antigen of high quality.

A reliable method was developed to produce a viral antigen preparation from porcine reproductive and respiratory syndrome virus (PRRSV) infected MARC-145 cells by solubilizing the virus with Triton X-100. This method eliminated problems previously encountered with high background reactions associated with PRRSV antigen or cell control antigen. With this new antigen, an indirect enzyme-linked immunosorbent assay (ELISA) was adapted to detect swine serum anti-body against PRRSV. In the ELISA, non-specific reactions associated with test serum samples have been eliminated by utilizing an effective blocking serum diluent. The ELISA is more sensitive than an indirect immunofluorescent assay (IFA), particularly with late-infection sera, while maintaining a high diagnostic specificity. In a comparison of IFA and ELISA using sera collected from 250 pigs of various ages from 5 herds that had PRRS histories, IFA revealed 178 positive samples and 72 negative samples. All of the IFA-positive sera were proven to be ELISA reactors. However, nearly one-half (34/72) of the IFA-negative samples were also ELISA reactors. The diagnostic specificity and sensitivity of the ELISA were 100% and 96.6% with 257 serum samples collected from known healthy PRRS-negative swine herds and 57 sera collected from infected swine at 6 to 56 days after infection, respectively. The ELISA is technically superior to IFA, time-efficient and cost-effective, and suitable for testing of a large number of samples over a short period of time.     

A modified immunoperoxidase assay for detection of antibody porcine reproductive and respiratory syndrome (PRRS) virus in swine sera

MARC-145 cell monolayers infected with PRRS virus were fixed in 3% neutral buffered formalin and embedded in paraffin. The sections were stained by avidin-biotin complex immunoperoxidase (ABC) method. Test sera were applied to sections as primary antibodies. The positive reactions were detected by ABC method and indirect immunofluorescent assay (IFA). There was good correlation between ABC and IFA, and the titers in ABC were higher than those in IFA. The present results indicate that the immunohistochemical staining is a useful test for the detection and quantitation of PRRS virus antibody in swine sera as well as IFA.     

A comparative study of an indirect fluorescent antibody test and an immunoperoxidase monolayer assay for the diagnosis of porcine proliferative enteropathy.

The currently used indirect fluorescent antibody test (IFAT) for the detection of antibodies against porcine proliferative enteropathy (PPE) was compared to an immunoperoxidase monolayer assay (IPMA). Serum samples used in this comparison were collected from 5-week-old pigs on day 0 (pre-experimental challenge) and on days 7, 14, 21, and 28 after oral inoculation with intestinal homogenate from pigs affected by PPE (28 challenged pigs) and sucrose phosphate glutamate solution (2 control pigs). All animals were euthanized 4 weeks after inoculation. Immunohistochemistry staining was applied to formalin-fixed, paraffin-embedded sections of ileum for the detection of Lawsonia intracellularis antigen. The serology results with each method agreed in all samples, except on days 0 and 7 in 1 control animal, which was positive by IPMA, but negative by IFAT. The percentage of agreement between IFAT and IPMA was 98.6%.     

Detection of antibodies against Anaplasma marginale in milk using a recombinant MSP5 indirect ELISA

An indirect enzyme linked immunosorbent assay (iELISA) for diagnosis of anaplasmosis using undiluted individual milk samples from dairy cows was developed. The recombinant 19 kDa major surface protein 5 (rMSP5) of Anaplasma marginale was used as antigen. A monoclonal antibody against bovine IgG1 conjugated with peroxidase and the chromogen 3,5,3',5'-tetramethylbenzidine were used in the test. Strong and weak, positive and negative milk samples were set up as reference controls. Results were expressed as percentage of positivity (PP) contrasting with the strongest positive control. The test was evaluated in two groups (G1 and G2) of lactating dairy cows from herds located in A. marginale non-endemic areas of Argentina. The infection status of both groups, G1 (n=128) sampled after anaplasmosis outbreak, and G2 (n=216) free of anaplasmosis was established by polymerase chain reaction (PCR). Serum samples of cows from G1 and G2 were analyzed by card agglutination test (CAT) and competitive ELISA (cELISA), while the novel iELISA was evaluated in their corresponding milk samples. At a cutoff of 42 PP, the ELISA has 98% sensitivity and 95% specificity. A significant difference (P<0.0001) was found between the mean PP value of negative samples from G1 (17.4+/-14.9), and G2 (8.6+/-7.1). The agreement and kappa (kappa) value between iELISA and PCR was 96%, kappa=0.919; between iELISA and CAT was 97%, kappa=0.880; and between iELISA and cELISA was 97%, kappa=0.899. These results strongly support the usefulness of iELISA to detect A. marginale antibodies in milk. Additional studies are necessary to define the ability of the milk iELISA to detect not only acutely infected, but also carrier cattle.     

Validation of an immunoperoxidase monolayer assay as a serologic test for porcine proliferative enteropathy.

The sensitivity and specificity of an immunoperoxidase monolayer assay (IPMA) was evaluated in a blind serologic study of a group of disease-free pigs and a group of pigs experimentally infected with intestinal homogenate containing Lawsonia intracellularis organisms. Sixty pigs from the control group were kept in the source farm, and another 60 animals were transferred to an isolation unit aid challenged intragastrically. All animals were bled before and 21 days after challenge. Fecal samples were collected on the same dates. The IPMA results were tested for sensitivity and specificity in a 2 x 2 table using the challenged and nonchallenged status as gold standard. Sensitivity and specificity were evaluated for different cutoff points (serum dilutions). Specificities of 100% were obtained for all the serum dilutions tested (1:15, 1:30, 1:60, and 1:120). The sensitivity levels were 90.7%, 88.9%, 81.5%, and 75.9% for the serum dilutions 1:15, 1:30, 1:60, and 1:120, respectively. The sensitivity of the dilution 1:15 was slightly, but not significantly, higher than the dilution currently used as the cutoff point (1:30). Cross-reactivity of the IPMA test was evaluated using sera from pigs experimentally inoculated with Brachyspira pilosicoli and various Campylobacter species. All these samples were negative. Sera samples from 3 porcine proliferative enteropathy known negative populations, 40 growing pigs from 2 commercial farms and a group of 6 cesarean-derived and colostrum-deprived pigs, also tested negative by IPMA. The IPMA serologic test with the cutoff point of 1:30 showed specificity of 100% and sensitivity close to 90% and, therefore, is an appropriate diagnostic test for herd screening but not for diagnosing PPE on the individual level.     

Development and evaluation of an immunochromatographic strip for detection of Streptococcus suis type 2 antibody.

In this study, an immunochromatographic strip (ICS) was developed for the detection of antibody against Streptococcus suis serotype 2 (SS2). Colloidal gold particles labeled with staphylococcal protein A (SPA), which can bind to the F(C) fragment of mammalian immunoglobulin, were used as the detector reagent. The capsular polysaccharide (CPS) of SS2 and affinity-purified IgG from a healthy naive pig were immobilized on test and control regions of a nitrocellulose membrane, respectively. The ICS was used to 1) detect anti-CPS antibody in 14 sera taken from 4 SS2-infected pigs, 24 sera from pigs hyperimmunized with SS2, and 68 sera from pigs inoculated or infected with bacteria other than SS2; 2) determine anti-CPS antibody titers of 20 positive sera for comparison with enzyme-linked immunosorbent assay (ELISA); and 3) detect anti-CPS antibody in 226 clinical sera taken from diseased pigs also for comparison with ELISA. An ELISA used as a reference test determined the specificity and sensitivity of the ICS to be 97.1% and 86.3%, respectively. There was excellent agreement between the results obtained by ELISA and the ICS (kappa = 0.843). Additionally, there was strong agreement between the results of bacterial isolation from pig tonsils and ICS test (kappa = 0.658). Because it is rapid and easy to use, the test is suitable for the serological surveillance of SS2 at farms.     

Evaluation of a covalent mix-enzyme linked immunosorbent assay for screening of Salmonella antibodies in pig serum

In this study, a commercial Salmonella covalent mix-enzyme linked immunosorbent assay (ELISA) for serological detection of Salmonella infection in swine was evaluated by comparing it with the conventional fecal culture method and inter-laboratory proficiency testing, using a panel of sera tested in 5 laboratories from Europe and North America. Comparison with culture results showed that 88.5% of 26 culture-positive animals were ELISA positive, as were 55% of 60 animals from 2 culture-positive pig herds. Of 90 animals from 2 high health farms with no clinical symptoms of salmonellosis, 98.9% tested negative. The interlaboratory comparison study found a kappa value of 0.9 between our laboratory (using an automated system) and the manufacturer laboratory (using the manual method). Comparison of ELISA results from all 5 participating laboratories showed very good to excellent agreement, between 85% and 97.5%. We found this assay to be useful for the screening of antibodies against Salmonella present in swine serum. It agrees well with bacterial cultures, is reproducible, sensitive, specific, repeatable, and suitable for automation.     

Blocking ELISA's for the distinction between antibodies against European and American strains of porcine reproductive and respiratory syndrome virus

A double blocking ELISA was developed in order to satisfy the need for large scale serological screening for PRRS and simultaneous distinction between infection with European and American strains of PRRSV in pig herds. The Immunoperoxidase monolayer assay (IPMA) and the double blocking ELISA enabled distinction on serological basis between infection with European and American strains of PRRSV. The distinction was possible from about day 7 after infection of pigs with PRRSV. The double blocking ELISA enabled the distinction at later stages of infection compared to the IPMA, irrespective of the strain involved.     

猪圆环病毒2型免疫过氧化物酶单层细胞试验抗体检测试剂盒的研制及应用

本研究建立了一种免疫过氧化物酶单层细胞试验（Immunoperoxidase monolayer assay,IPMA）,用于猪圆环病毒2型血清抗体检测,通过对IPMA反应条件的优化,组装了诊断试剂盒。研究结果表明,用IPMA检测猪圆环病毒2型人工感染猪血清,于感染后3周抗体阳转,第3周～10周抗体阳性检出率为92.8%（52/56）,对照组猪血清抗体检测均为阴性（33/33）。试剂盒在-20℃稳定保存18个月与其他几种猪病毒参考血清无交叉反应,与用重组蛋白抗原建立的rcELISA符合率为89.2%。对来自黑龙江、吉林、河北、上海、内蒙古、云南、江西等地猪场健康成年猪血清480份和发病猪血清424份进行了检测,抗体检出率分别为91.7%和79.2%,表明我国猪群中猪圆环病毒2型污染相当严重。该试剂盒的研制为我国PCV2流行病学调查和疫苗免疫效果的评价提供了技术手段。     

Evaluation of an enzyme-linked immunosorbent assay kit to detect Babesia bovis antibodies in cattle

The sensitivity and specificity of an enzyme-linked immunosorbent assay (ELISA) to detect antibodies; to Babesia bovis was evaluated in 1000 sera from Holstein heifers. Five hundred of them were from cattle naturally or experimentally infected with B. bovis and 500 from uninfected heifers born and raised in a region free of the vector of cattle babesiosis. Additionally, the ELISA was evaluated and compared with an indirect immunofluorescent antibody (IFA) test in 374 heifers inoculated with different kinds of B. bovis antigens in four trials. The cross-reaction was also evaluated in 50 heifers infected with Babesia bigemina and 50 heifers infected with Anaplasma marginale. The mean percentage positivity of negative sera in relation to the ELISA strong positive sera was 8%. The seropositive/seronegative cutoff point was set as twice the mean percentage positivity of negative cattle sera ( = 16%). The sensitivity of the ELISA was 98% with a 95% confidence interval (CI) of 96–99%. The specificity was 95% (CI 93–97%). The agreement was 97% and the kappa value was 0.93. The predictive values of positive and negative results were 95% and 98% respectively. ELISA showed a similar sensitivity to that of the IFA test to detect antibodies to different B. bovis antigens. Its sensitivity ranged from 97.1% to 100% (CI 89–100%), while the sensitivity of the IFA test ranged from 92.8% to 100% (CI 83–100%). ELISA cross-reacted in 8% and 6% of the sera carrying B. bigemina and A. marginale antibodies, respectively, while the IFA showed 4% cross-reaction in each situation. The ELISA evaluated has the advantages of a proper sensitivity, objectivity and capacity to be adapted to test large number of samples in a short period of time. The results indicate that the ELISA is a suitable replacement for the IFA test to detect B. bovis antibodies in cattle sera, especially in epidemiological studies.     

Accuracy of an indirect fluorescent-antibody test and of a complement-fixation test for the diagnosis of Babesia caballi in field samples from horses

We evaluated the indirect fluorescent-antibody (IFA) test and complement-fixation (CF) test for diagnosis of equine piroplasmosis in the absence of a gold standard. Using Evan's blue, we estimated the specificity of the IFA test on a parasite-free, field horse population to be 98% (95% confidence interval=97, 99). We observed an excellent test agreement (kappa=0.83) between two collaborating laboratories when the IFA test was performed on identical samples from an endemic area. Using Bayesian analysis with informative prior probability distributions, we estimated the sensitivity of the IFA test to be 92% (95% probability interval, PI=81, 98), and specificity to be 95% (95% PI=88, 99). The CF test sensitivity and specificity estimates were 28% (95% PI=15, 47) and 99% (95% PI=96, 100), respectively. We found the IFA to be superior to the CF test, and the inclusion of Evan's blue in test protocol improved the performance of the IFA test. We conclude that the IFA test for Babesia caballi is a sensitive and specific test for the diagnosis of equine piroplasmosis.     

Effect of genotypic and biotypic differences among PRRS viruses on the serologic assessment of pigs for virus infection

Genetic, antigenic, and pathogenic variability is known to exist among porcine reproductive and respiratory syndrome (PRRS) viruses and has garnered great attention for diagnostics and disease control/prevention. A comparative serologic study was conducted on five field and two cell-attenuated viruses to determine if serologic responses to PRRS virus infection could be influenced by biotypic and/or genotypic differences of the viruses. The isolates used for the study varied in their virulence to pigs and in genomic sequences. Ten pigs were inoculated with each isolate (1x10(3) TCID(50)) via the intranasal route. All inoculated animals became viremic during the study period. Some animals inoculated with the attenuated viruses remained seronegative until the end of the study (42 days post-inoculation (PI)), but all of the animals inoculated with field viruses developed enzyme-linked immunosorbent assay (ELISA)- and indirect fluorescent antibody (IFA)-detectable antibodies, regardless of the virus strain used in the IFA assay. In contrast, a great degree of variation in the onset and level of serum-virus neutralization (SVN) antibody was observed by individual pigs and by each virus. The reactivity of SVN antibody was highly specific for homologous viruses. Cross-neutralization titers were better correlated with sequence homology of open-reading frames (ORFs) 4 and 5 among the viruses than any other structural genes. We conclude that the biotypic difference among PRRS viruses may affect the kinetic of humoral immune response in infected pigs. The IFA test may be used as a confirmatory test when a false-positive ELISA result is suspected or vise-a-versa, but SVN antibody titers are highly affected by antigenic variability.     

An immunoperoxidase monolayer assay for the detection of antibodies against swine influenza virus.

An immunoperoxidase monolayer assay (IPMA) has been developed to detect antibodies against swine influenza A virus (SIV) in pig sera. The test was evaluated by using sequential sera from pigs experimentally infected with H1N1 subtype of SIV. Two hundred field serum samples that had been examined by the hemagglutination inhibition (HI) test were also tested. Antibodies specific to SIV were detected as early as 3 days postinoculation (dpi) in the IPMA test as compared with 7 dpi by the HI test. Unlike HI, no serum treatment was required in the IPMA test. Regardless of the virus used in the test, IPMA detected antibodies to both H1N1 and H3N2 subtypes of SIV whereas HI detects antibodies against either H1N1 or H3N2, depending upon the virus used in the test. Results of this study indicate that IPMA is a useful test for screening of pig sera for SIV antibodies.     

Application of competitive enzyme-linked immunosorbent assay for the serologic diagnosis of classical swine fever virus infection.

A competitive enzyme-linked immunosorbent assay (C-ELISA), based on a truncated E2 recombinant protein of the Alfort/187 strain of classical swine fever virus (CSFV) and a specific monoclonal antibody M1669, was evaluated using 2,000 sera from clinically healthy pigs in Canada (a CSFV-free country) and sera from experimentally infected pigs. The relative specificity and sensitivity of the C-ELISA were 100% and 86%, respectively, at a cutoff of 25% inhibition using negative and positive pig sera, as defined by the neutralizing peroxidase-linked assay (NPLA). A kappa value of 0.91 was obtained, indicating an excellent level of agreement between the NPLA and the C-ELISA. When sera from 120 infected pigs were used in the test at > or = 21 days postinfection, the sensitivity of the C-ELISA and the kappa value increased to 97% and 0.98, respectively. This C-ELISA will be useful when a large number of samples must be tested, as could occur during a disease outbreak or for surveillance or prevalence studies.     

Development of a sensitive and specific indirect enzyme-linked immunosorbent assay based on a baculovirus recombinant antigen for detection of specific antibodies against Ehrlichia canis.

An indirect enzyme-linked immunosorbent assay (ELISA) based on baculovirus recombinant P30 protein of Ehrlichia canis and the 1BH4 anticanine IgG monoclonal antibody was developed and evaluated by examining a panel of 98 positive and 157 negative sera using the indirect fluorescent antibody (IFA) test as the reference technique. The P30-based ELISA appeared to be sensitive and specific (77.55% and 95.54%, respectively) when qualitative results (positive/negative) were compared with those of the IFA test; the coefficient of correlation (R) between the 2 tests was 0.833. Furthermore, it was possible to establish a mathematical formula for use in comparing the results of both techniques. These results indicate that recombinant P30 antigen-based ELISA is a suitable alternative of the IFA test for simple, consistent, and rapid serodiagnosis of canine ehrlichiosis. Moreover, the use of this recombinant protein as antigen offers a great advantage for antigen preparation in comparison with other techniques in which the whole E. canis organism is used as antigen.     

An ORF2 protein-based ELISA for porcine circovirus type 2 antibodies in post-weaning multisystemic wasting syndrome

Porcine circovirus type 2 (PCV2) plays a crucial role in the pathogenesis of post-weaning multisystemic wasting syndrome (PMWS) in swine. As PCV2 displays significant homology with PCV1 (a non-pathogenic virus) at the nucleotide and amino-acid level, a discriminative antigen is needed for specific serological diagnosis. The ORF2-encoded capsid protein from PCV2 was used to develop an indirect enzyme-linked immunosorbent assay (ELISA). GST-fused capsid protein from PCV2 and GST alone (both expressed in recombinant baculovirus-infected cells) were used as antigens for serodiagnosis. The specificity of the ELISA for detection of PCV2 antibodies was demonstrated in sera from pigs experimentally infected with PCV1, PCV2 and other swine viruses. The semi-quantitative nature of the test was evaluated versus an immunoperoxidase monolayer assay (IPMA). The ELISA was performed on 322 sera from pigs in eight Brittany herds and compared with IPMA. The sensitivity (98.2%) and specificity (94.5%) of this test were considered suitable for individual serological detection. High PCV2 seroprevalence was found in sows and pigs at the end of the growth phase (18-19 weeks) in all eight herds. The seroprevalence in piglets (11-17 weeks) was statistically correlated with clinical symptoms of PMWS (93% in affected versus 54%, in non-affected farms). A cohort study performed in PMWS-free farms showed that 57% of piglets exhibited active seroconversion after 13 weeks, indicating that PCV2 infection occurred earlier in PMWS-affected piglets.     

Porcine reproductive and respiratory syndrome virus: antigenic and molecular diversity of British isolates and implications for diagnosis

Porcine reproductive and respiratory syndrome (PRRS) is an endemic disease of pigs, caused by PRRS virus, a member of the Arteriviridae family. First seen in Britain in 1991, the disease continues to be a significant economic and welfare problem for pig producers. To date, only PRRSV genotype 1 has been found in Britain. At the genetic level, a considerable increase has been reported in the diversity of PRRS viruses isolated in Britain between 2003 and 2007, versus the early 1990 s. In this study, the diversity has been shown to extend to the antigenic level too, with potential consequences for diagnostic methods. Antigenic diversity was assessed using a panel of twelve monoclonal antibodies, only one of which reacted with all isolates tested. Nine diverse viruses were compared as potential antigens in immunoperoxidase monolayer assays, where each one produced quite different results for a common panel of sera. As a single virus is used in each diagnostic assay, results must therefore be interpreted cautiously. For a real-time RT-PCR assay, published oligonucleotide primer and probe sequences were evaluated against available genetic sequences of British and European viruses, and were re-designed where considerable mismatches were found. The multiplex assay incorporating these modified primers to detect genotype 1 and 2 PRRS viruses was then validated for use with diagnostic sera and tissues. As the increasing degree of diversity exhibited by British strains is mirrored in other countries, PRRSV will continue to provide an ongoing challenge to diagnosis at a global, as well as national level.     

Development of an ELISA based on the baculovirus-expressed capsid protein of porcine circovirus type 2 as antigen

The genome of porcine circovirus type 2 (PCV2) contains two major open reading frames, which have been shown to encode the virus capsid and replication-associated proteins. The capsid protein is a major structural protein of the virus; it can be a suitable target antigen for detecting PCV2-specific antibodies to monitor PCV2 infection. To produce the antigen, the capsid protein coding sequence was cloned into a baculovirus transfer vector, and a recombinant capsid (rC) protein of PCV2 was expressed as a combined fusion protein in frame with a C-terminal peptide of six histidines. The affinity-purified rC protein was used as coating antigen to develop an ELISA for detecting the virus-specific antibodies in swine sera. The rC protein-based ELISA (rcELISA) was evaluated by examining a panel of 49 PCV2-positive and 49 PCV2-negative swine sera. In comparative experiments of immunoperoxidase monolayer assay (IPMA) using 102 field sera, there was 89.2% coincidence between data obtained by the rcELISA and IPMA. The rcELISA achieved 88.5% specificity and 89.4% sensitivity for detection of PCV2 antibody in the field sera. The assay showed no cross-reactivity with antibodies to PCV type 1, porcine reproductive and respiratory syndrome virus and porcine parvovirus. The results suggest that the rcELISA is suitable for routine serodiagnosis and epidemiological surveys of PCV2-associated diseases.