Article

Comparison of commercial enzyme-linked immunosorbent assays and fluorescent microbead immunoassays for detection of antibodies against porcine reproductive and respiratory syndrome virus in boars

Authors:
To read the full-text of this research, you can request a copy directly from the authors.

No full-text available

Request Full-text Paper PDF

To read the full-text of this research,
you can request a copy directly from the authors.

... Among the three antibody isotypes, IgG provides the highest diagnostic sensitivity and specificity and a period of detection exceeding 6 months (Henao--Diaz et al., 2020;Kittawornrat et al., 2010;Rotolo et al., 2018) Several commercial PRRSV oral fluid antibody ELISAs have been described (Henao-Diaz et al., 2019), but the PRRSV oral fluid antibody ELISA (IDEXX Laboratories, Inc., Westbrook, ME USA) evaluated in this study was the first marketed (2012) and is the most frequently described in the literature (Almeida et al., 2018;Woonwong et al., 2018). Using the manufacturer's recommended cut-off (sample-to-positive (S/P) ≥ 0.4), estimates of the test's diagnostic sensitivity based on experimental samples ranged from 86.7 % (13 of 15 positive samples) (Gerber et al., 2014) to 100 % (372 of 372 positive samples) (Henao-Diaz et al., 2019). Using PRRSV negative field samples, diagnostic specificity was reported as 97.1 % (194 of 200 negative samples) (Gerber et al., 2014), 97.4 % (281 of 289 negative samples) (Sattler et al., 2015), and 98.7 % (133 of 135 negative samples) (Croft et al., 2020). ...
... Using the manufacturer's recommended cut-off (sample-to-positive (S/P) ≥ 0.4), estimates of the test's diagnostic sensitivity based on experimental samples ranged from 86.7 % (13 of 15 positive samples) (Gerber et al., 2014) to 100 % (372 of 372 positive samples) (Henao-Diaz et al., 2019). Using PRRSV negative field samples, diagnostic specificity was reported as 97.1 % (194 of 200 negative samples) (Gerber et al., 2014), 97.4 % (281 of 289 negative samples) (Sattler et al., 2015), and 98.7 % (133 of 135 negative samples) (Croft et al., 2020). In the field, surveillance requires near-perfect diagnostic specificity because unexpected positive results cause a significant disruption and financial consequences, to presumed-negative production flows. ...
... However, users should be aware of "contradictory" results, i.e., positive for antibody and negative for RNA, which invariably occur during the course of the infection (Henao-Diaz et al., 2020). Other assays may be used for confirmation, but often have important shortcomings, i.e., may not provide timely turn-around, uniform diagnostic performance for all PRRSV isolates, and/or conditional independence (Gardner et al., 2000;Gerber et al., 2014). Given that routine surveillance is increasingly important in the control of economically important pathogens, producers should consider active, continuous analysis of surveillance results using statistical process control (SPC) or similar methods to provide context, determine the relevance of the diagnostic results, and thus characterize them as stable or unstable. ...
Article
Full-text available
Distinct from tests used in diagnostics, tests used in surveillance must provide for detection while avoiding false alarms, i.e., acceptable diagnostic sensitivity but high diagnostic specificity. In the case of the reproductive and respiratory syndrome virus (PRRSV), RNA detection meets these requirements during the period of viremia, but antibody detection better meets these requirements in the post-viremic stage of the infection. Using the manufacturer's recommended cut-off (S/P ≥ 0.4), the diagnostic specificity of a PRRSV oral fluid antibody ELISA (IDEXX Laboratories, Inc., Westbrook, ME USA) evaluated in this study was previously reported as ≥ 97%. The aim of this study was to improve its use in surveillance by identifying a cut-off that would increase diagnostic specificity yet minimally impact its diagnostic sensitivity. Three sample sets were used to achieve this goal: oral fluids (n = 596) from pigs vaccinated with a modified live PRRSV vaccine under experimental conditions, field oral fluids (n = 1574) from 94 production sites of known negative status, and field oral fluids (n = 1380) from 211 sites of unknown PRRSV status. Based on the analysis of samples of known status (experimental samples and field samples from negative sites), a cut-off of S/P ≥ 1.0 resulted in a diagnostic specificity of 99.2 (95% CI: 98.8, 99.7) and a diagnostic sensitivity of 96.5 (95% CI: 85.2, 99.2). Among 211 sites of unknown status, 81 sites were classified as antibody positive using the manufacturer's cut-off; 20 of which were reclassified as negative using a cut-off of S/P ≥ 1.0. Further analysis showed that these 20 sites had a small proportion of samples (18.0%) with S/P values just exceeding the manufacturer's cut-off (x̄ = 0.5). Whereas the remainder of positive sites (n = 61) had a high proportion of samples (76.3%) with high S/P values (x̄ = 6.6). Thus, the manufacturer’s cut-off (S/P ≥ 0.4) is appropriate for diagnostic applications, but a cut-off of S/P ≥ 1.0 provided the higher specificity required for surveillance. A previously unreported finding in this study was a statistically significant association between unexpected reactors and specific production sites and animal ages or stages. While beyond the scope of this study, these data suggested that certain animal husbandry or production practices may be associated with non-specific reactions.
... In several countries regional programs of PRRSV elimination are in place, while in Hungary a state-controlled eradication program is currently running (Olasz et al., 2016). There are several peer-reviewed publications showing generally good agreement between PRRSV ELISAs, but in many cases the evaluation was made using samples from experimentally infected, or vaccinated animals (Diaz et al., 2012;Gerber et al. 2014;Sattler et al., 2015Sattler et al., , 2014Seo et al., 2015;Sipos et al., 2009). Moreover, new products are being marketed, or improved versions of known ELISAs are being introduced. ...
... showing high specificity and sensitivity, are in agreement with the results of the earlier studies which also indicated excellent performance of this kit. The specificity comparison between Interestingly, the study of Gerber et al. (2014) showed similar sensitivity and specificity of IDEXX and Hipra ELISAs using experimental and diagnostic boar sera. ...
... ELISA is the most widely used method for PRRSV diagnosis and monitoring on a population level. There is a number of peer-reviewed publications comparing specificity and sensitivity of commercially available kits(Diaz et al., 2012;Gerber et al., 2014; Sattler et al., 2015, ...
Article
Porcine reproductive and respiratory syndrome (PRRS) is one of the most common infectious diseases of swine globally. Since the course of PRRS virus (PRRSV) infection is subclinical, laboratory diagnosis is necessary to detect the virus or specific antibodies. The aim of this study was to assess the sensitivity and specificity of IDEXX PRRS X3 Ab Test (IDEXX, USA), Civtest Suis E/S (Hipra, Spain), INgezim PRRS 2.0 (Ingenasa, Spain), VetExpert PRRS Ab ELISA 4.0 (BioNote, Korea), Pigtype PRRSV Ab (Qiagen, Germany) and PrioCHECK PRRSV Antibody ELISA (ThermoFisher, USA), using serum samples obtained from 5 conventional PRRSV-positive and 5 PRRSV-negative Polish pig farms. Specificity of ELISAs ranged from 94.2% (ThermoFisher) to 100% (IDEXX and Hipra). ThermoFisher ELISA had the highest detection rate and detected 67.2% samples from PRRSV-positive farms as positive but considering its low specificity some of the positive results may be incorrect. IDEXX ELISA considered as a reference detected 64.8% positive sera in PRRSV-positive farms. On the other hand Hipra Elisa identified only 51.8% of samples as positive. The diagnostic sensitivity of five ELISAs relative to IDEXX ranged from 80.3% (Hipra) to 96.3% (ThermoFisher). Our study showed significant differences in specificity and diagnostic sensitivity between the compared kits. The differences in the performance appeared to be practically negligible on farms where early infection with PRRSV occurred. However, on PRRSV-negative farms, or farms with PRRSV stable sow herds, some ELISAs can give results not reflecting the infection status in specific age groups.
... The feasibility of pen-based oral fluid has been assessed for growing pigs (Kittawornrat et al., 2012) and recently for a type of group-housed sows (Pierdon et al., 2016). Oral fluid sampling has also been evaluated at the individual level for boars (in boar studs) and growing pigs (Gerber et al., 2014;Decorte et al., 2015;Pepin et al., 2015). Even though the sow breeding herd may represent a non-negligible proportion of the population in a pig herd (particularly in farrow-to-finish herds), it needs to be tested to understand within-herd infection dynamics. ...
... When monitoring infection and immunity in sows or pigs, it is common practice to test for the presence of antibodies against PRRSV with a commercial ELISA. In the last few years, the ELISA has been adapted to the oral fluid matrix to detect antibodies against PRRSV (Kittawornrat et al., 2012;Gerber et al., 2014). Nevertheless, the diagnostic performance of this oral fluid ELISA for individual or pooled samples has not been investigated to date, whether for finishing pigs or sows. ...
... Since most of the breeding herds were PRRSV infected with or without PRRSV vaccination, the proportion of seropositive sows was considered to be higher than in growing-finishing pigs (more than 70%). Priors for the sensitivity and specificity of the serum ELISA carried out on growing-finishing pig samples were calculated from the results of experimental studies Gerber et al., 2014), from samples obtained in a PRRSV transmission trial (data available upon request) and the kit information (IDEXX Laboratory, Eragny sur Oise, France) (i.e. assumed sensitivity and specificity >0.9 with a mode at 0.99 and 95% certainty). ...
Article
The feasibility of using individual and pen-based oral fluid samples to detect PRRSV antibodies in growing-finishing pigs and group-housed sows was investigated. The diagnostic performances of a commercial oral fluid ELISA (OF-ELISA) and a serum ELISA (SER-ELISA) performed on individual or pooled samples from 5 or 10 pigs and sows was evaluated. The performance of the OF-ELISA was also assessed for pen-based oral fluids. Eight hundred and thirty-four pigs and 1,598 sows from 42 PRRSV-infected and 3 PRRSV-negative herds were oral fluid sampled and bled. PRRSV antibodies were detected by an OF-ELISA performed at individual, pool (5 or 10 samples) and pen levels. Serum samples were tested by a SER-ELISA at individual and pool levels. The sensitivity and specificity of ELISAs for individual samples were assessed by Bayesian analysis. The relative diagnostic performance for the pools was calculated by taking individual samples as the gold standard. SER-ELISA and individual OF-ELISA results were used as references for estimating OF-ELISA performance for pen-based samples. Individual oral fluid collection was feasible in all kinds of pigs, whereas pen-based samples were unsuccessful in 40% of the group-housed sow pens. High levels of sensitivity comparable to those of the SER-ELISA were found for the OF-ELISA when performed on individual, 5-sample pool or pen-based samples from pigs or sows. The OF-ELISA lacked specificity for individual samples from sows. Pooling 5 individual oral fluid samples or using pen-based samples increased test specificity.
... In one study using serum samples collected from challenged pigs, a sensitivity of 100% was found [5]. Measured with the IDEXX ELISA, an antibody response can be detected beginning between days 9 and 12 after PRRSV infection or vaccination with attenuated PRRSV live vaccines [2,[6][7][8][9]and lasting at least until day 120 after vaccination or challenge [10]. Although the application of certain inactivated PRRSV vaccines seems to prime the immune system and can, according to some studies, lead to a faster and more effective immune response after PRRSV infection [11, 12], no antibody response has been observed with the IDEXX ELISA after vaccination with inactivated PRRSV vaccines [10, 13]. ...
... This study tested the ability of six ELISAs for detection of PRRSV Ab after inactivated vaccination of piglets and subsequent infection with HP PRRSV. Data are published about the performance, sensitivity and specificity of the ELISAs tested in this study [1, 6, 15, 16]. They did, however, not concern serum of pigs vaccinated with an inactivated vaccine. ...
... This confirms the findings of another study, where no antibody response was detected with the IDEXX ELISA after inactivated PRRSV vaccination as well [10]. As expected from the results of other studies [2, 6, 16], the IDEXX ELISA tested most piglets PRRSV Ab positive from day 10 after infection onwards. According to a study by Zuckermann et al., the IDEXX ELISA was able to detect higher S/P values in pigs pre-vaccinated with an inactivated PRRSV vaccine than in non-vaccinated pigs on days 7 and 10 after the subsequent challenge [13]. ...
Article
Full-text available
Background In this study, six enzyme-linked immunosorbent assays (ELISA), intended for routine porcine reproductive and respiratory syndrome virus (PRRSV) herd monitoring, are tested for their ability to detect PRRSV specific antibodies in the serum of pigs after vaccination with an inactivated PRRSV type 1 vaccine and subsequent infection with a highly pathogenic (HP) PRRSV field strain. For this reason, ten piglets (group V) from a PRRSV negative herd were vaccinated twice at the age of 2 and 4 weeks with an inactivated PRRSV vaccine. Ten additional piglets (group N) from the same herd remained unvaccinated. Three weeks after second vaccination, each of the piglets received an intradermal application of an HP PRRSV field strain. Serum samples were taken before first vaccination as well as before and 3, 7, 10 and 14 days after HP PRRSV application. All serum samples were tested for PRRSV RNA by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) as well as for PRRSV antibodies with all six study ELISAs. ResultsAt the beginning of the study (before vaccination), all of the piglets were PRRSV antibody negative with all study ELISAs. They also tested negative for PRRSV RNA measured by RT-qPCR. From day 3 after HP PRRSV application until the end of the study, a viremia was detected by RT-qPCR in all of the piglets. On day 0 (day of HP PRRSV application), nine out of ten piglets of the pre-vaccinated group tested PRRSV antibody positive with one of the tested ELISAs, although with lower S/P values than after infection. On day 10 after HP PRRSV application, all study ELISAs except one had significantly higher S/P or OD values, respectively more positive samples, in group V than in group N. Conclusions Only one of the tested ELISAs was able to detect reliably PRRSV antibodies in pigs vaccinated with an inactivated PRRSV vaccine. With most of the tested ELISAs, higher S/P values respectively more positive samples after PRRSV infection were seen in the pre-vaccinated group than in the non-vaccinated.
... Detection of antibodies (Ab) against porcine reproductive and respiratory syndrome virus (PRRSV) is, in addition to a number of different established PCR methods [1, 2], one important tool for the monitoring and surveillance of PRRSV in pig farms [3, 4]. In addition to the cost-effective, simple and rapid analysis by ELISA, alternative methods, such as serum neutralization test, immunofluorescence assay or Western blot are used for special indications [3,567. In recent years, several ELISAs for detection of Ab against PRRSV in pig serum have been developed789, some of them with the intention of making them commercially available. ...
... Most of the ELISAs are able to detect Ab against PRRSV type 1 and type 2 [14] . However, some have been described as able to distinguish between PRRSV types [5, 7, 13] . The ELISAs presently used in routine analysis are usually based on the PRRSV nucleocapsid protein as antigen [15]. ...
... In a recently published study, the sensitivity of the IDEXX ELISA was stated at 80 % in the field samples tested, compared to the immunoperoxidase monolayer assay (IPMA) [15]. This is in contrast to other studies which stated a sensitivity of the IDEXX ELISA of 100 % in serum samples 21 days after PRRSV inoculation [5] and of 91.5 % in field samples [7]. The specificity of the QIAGEN ELISA was, at 98.1 %, lower than the IDEXX which had a 100 % specificity as has been published previously [5, 7, 12], respectively a 99.9 % specificity, according to the manufacturer. ...
Article
Full-text available
The aim of the study was to evaluate the performance of different newly developed and/or commercially available ELISAs for detection of PRRSV specific antibodies. Consequently, ten PRRSV negative piglets (group V) were vaccinated with a PRRSV type 2 vaccine. Blood samples were taken before as well as seven, 21 and 42 days after vaccination. At day 42 after vaccination (day 0 of the study) all of the piglets from group V and 10 non-prevaccinated PRRSV negative piglets (group N) were challenged with an HP PRRSV type 2 field strain. Blood samples were taken before and at days 3, 7, 10, 14, 21 and 28 after challenge. The success of vaccination and challenge was measured with RT qPCR. All serum samples were tested with six ELISAs for detection of PRRSV antibodies. Three of them are nucleocapsid-based, two use a glycoprotein extract and one uses inactivated whole virus as antigen. The specificity of the ELISAs was evaluated using 301 serum samples of piglets from PRRSV negative herds. The piglets from group V tested positive by RT qPCR at day 7 after vaccination and all piglets tested positive at day 3 after challenge. PRRSV specific antibodies were seen with all nucleocapsid-based ELISAs from day 21 after vaccination onwards in group V and from day 10 after challenge in group N. The glycoprotein-based ELISAs detected antibodies from day 42 after vaccination (group V) and day 21 after challenge (group N). The agreement according to kappa-coefficient was almost perfect. The glycoprotein-based ELISAs were able to distinguish PRRSV type 2, although with some cross reactions. Regarding specificity, the ELISAs performed differently (specificity between 97.4 % and 100 %), whereas most of the ELISAs with higher sensitivity had a slightly lower specificity. All tested ELISA were able to detect PRRSV antibodies in the serum of pigs vaccinated with a PRRSV type 2 vaccine and after challenge with an HP PRRSV type 2 field strain. The onset on antibody detection differed, depending on the type of antigen used in the ELISAs. Most of the ELISAs with a higher sensitivity had a lower specificity.
... One cost effective method is the serological detection of antibodies against PRRSV by ELISA. Several ELISAs have recently been developed, most of them detecting antibodies against both PRRSV type 1 and type 214151617. Some ELISAs, however, are intended to be able to differentiate between type 1 and 2 antibodies [16]. ...
... Several ELISAs have recently been developed, most of them detecting antibodies against both PRRSV type 1 and type 214151617. Some ELISAs, however, are intended to be able to differentiate between type 1 and 2 antibodies [16]. The IDEXX PRRS X3 Ab Test (IDEXX, Westbrook, USA) with a sensitivity of 98.8% and a specificity of 99.9%, according to the manufacturer, is the most often cited test [1,6,14] and is generally reckoned to be the de facto gold standard of the ELISAs for detection of antibodies against PRRSV [14,15,17]. ...
... No published data exists concerning the specificity and sensitivity or data of agreement for these ELISAs. One recent study is available that refers to ELISA IV in comparison to ELISA I in experimentally infected pigs and 205 samples of pigs from PRRSV negative herds [16]. However, no current data for this ELISA are available for seroconversion in vaccinated pigs, pigs from naturally infected herds or wild boars. ...
Article
Full-text available
Background In recent years, several new ELISAs for the detection of antibodies against the porcine reproductive and respiratory disease virus (PRRSV) in pig serum have been developed. To interpret the results, specificity and sensitivity data as well as agreement to a reference ELISA must be available. In this study, three commercial ELISAs (INgezim PRRS 2.0 - ELISA II, Priocheck® PRRSV Ab porcine ¿ ELISA III and CIVTEST suis PRRS E/S PLUS - ELISA IV, detecting PRRSV type 1 antibodies) were compared to a standard ELISA (IDEXX PRRS X3 Ab Test - ELISA I). The serum of three pigs vaccinated with an attenuated PRRSV live vaccine (genotype 2) was tested prior to and several times after the vaccination. Furthermore, serum samples of 245 pigs of PRRSV positive herds, 309 pigs of monitored PRRSV negative herds, 256 fatteners of assumed PRRSV negative herds with unknown herd history and 92 wild boars were tested with all four ELISAs.ResultsELISAs II and III were able to detect seroconversion of vaccinated pigs with a similar reliability. According to kappa coefficient, the results showed an almost perfect agreement between ELISA I as reference and ELISA II and III (kappa¿>¿0.8), and substantial agreement between ELISA I and ELISA IV (kappa¿=¿0.71). Sensitivity of ELISA II, III and IV was 96.0%, 100% and 91.5%, respectively. The specificity of the ELISAs determined in samples of monitored PRRSV negative herds was 99.0%, 95.1% and 96.4%, respectively. In assumed negative farms that were not continually monitored, more positive samples were found with ELISA II to IV. The reference ELISA I had a specificity of 100% in this study.Conclusions All tested ELISAs were able to detect a PRRSV positive herd. The specificity and sensitivity of the tested commercial ELISAs, however, differed. ELISA II had the highest specificity and ELISA III had the highest sensitivity in comparison to the reference ELISA. ELISA IV had a lower sensitivity and specificity than the other ELISAs.
... In recent years, the applicability of oral fluid samples for diagnostics of infectious disease like porcine reproductive and respiratory syndrome (PRRS), caused by the PRRS virus (PRRSV) has seen increased discussion in scientific literature. Several methods of detecting PRRSV RNA and PRRSV antibodies (Ab), using both different molecular diagnostic methods and serological techniques, were developed [1][2][3]. Sampling techniques were evaluated [4] and the effect of the stabilization of the oral fluid [5] and sample processing [6] was determined with the intention to improve the results. Different ropes for the oral fluid collection were tested [6,7]. ...
... Different ropes for the oral fluid collection were tested [6,7]. Some ELI-SAs, specifically developed for PRRSV Ab detection in oral fluid, show results comparable to serum ELISAs [2,8]. The usage of cotton ropes as chewing material for oral fluid collection was found to be the method of choice [6,7]. ...
Article
Full-text available
The monitoring of infectious diseases like the porcine reproductive and respiratory syndrome (PRRS) using pen-wise oral fluid samples becomes more and more established. The collection of individual oral fluid, which would be useful in the monitoring of PRRSV negative boar studs, is rather difficult. The aim of the study was to test two methods for individual oral fluid collection from pigs and to evaluate the specificity of a commercial ELISA for detection of PRRSV antibodies in these sample matrices. For this reason, 334 serum samples from PRRSV negative pigs (group 1) and 71 serum samples from PRRSV positive pigs (group 2) were tested for PRRSV antibodies with a commercial ELISA. Individual oral fluid was collected with a cotton gauze swab from 311 pigs from group 1 and 39 pigs from group 2. Furthermore, 312 oral fluid samples from group 1 and 67 oral fluid samples from group 2 were taken with a self-drying foam swab (GenoTube). The recollected oral fluid was then analysed twice with a commercial ELISA for detection of PRRSV antibodies in oral fluid. All serum samples from group 1 tested negative for PRRSV antibodies. The collection of oral fluid was sufficient in all samples. Sampling with GenoTubes was less time consuming than sampling with cotton gauze swabs. False positive results were obtained in 7 (measure 1) respectively 9 (measure 2) oral fluid samples recollected from cotton gauze swabs and in 9 and 8 samples from GenoTubes. The specificity of the oral fluid ELISA was 97.4% for cotton gauze swabs and 97.3% for GenoTubes. 70 out of 71 serum samples and all oral fluid samples from group 2 tested positive for PRRSV antibodies. The sensitivity of the oral fluid ELISA was 100%. According to the kappa coefficient, the results showed an almost perfect agreement between serum and oral fluid collected in both ways (kappa > 0.8). Both methods used for individual oral fluid collection proved to be practical and efficient and can be used for PRRSV antibody detection. It has to be considered, however, that false positive results may occur more often than in serum samples.
... ELISA has been used in PRRSV serological tests for its feasibility, sensitivity, rapidity and being suitable for large-scale use. At present, there are many studies on the specificity and sensitivity of commercial PRRSV ELISA kits [25][26][27][28]. The IDEXX PRRSV Ab ELISA kit and LSI PRRSV-Ab ELISA kit are two commonly used PRRSV antibody detection kits [29]. ...
Article
Full-text available
Background Porcine reproductive and respiratory syndrome (PRRS) is one of the most economically devastating diseases affecting the swine industry globally. Evaluation of antibody responses and neutralizing antibody titers is the most effective method for vaccine evaluation. In this study, the B cell line epitopes of PRRSV M protein were predicted, and two peptide ELISA assays were established (M-A110-129 ELISA, M-A148-174 ELISA) to detect antibodies against PRRSV M protein. Field serum samples collected from pig farms were used to validate the peptide ELISA and compare it with an indirect immunofluorescence assay. Results The sensitivity and specificity of M-A110-129 ELISA and M-A148-174 ELISA were (111/125) 88.80%, (69/70) 98.57% and (122/125) 97.60%, (70/70) 100%, relative to indirect immunofluorescence assay. This peptide ELISA could detect antibodies against different genotypes of PRRSV including type 1 PRRSV, classical PRRSV, HP-PRRSV, and NADC30 like PRRSV, but not antibodies against other common swine viruses. The results of ROC analysis showed that the area under the curve (AUC) of the M-A110-129 ELISA and M-A148-174 ELISA were 0.967 and 0.996, respectively. Compared the concordance of results using two peptide ELISA assays, the IDEXX PRRSV X3 Ab ELISA and a virus neutralization test, were assessed using a series of 147 sera from pigs vaccinated with the NADC30-like PRRSV inactivated vaccine. The M-A148-174 ELISA had the best consistency, with a Cohen’s kappa coefficient of 0.8772. The concordance rates of the Hipra PRRSV ELISA kit, M-A110-129 ELISA and M-A148-174 ELISA in the field seropositive detection results were 91.08, 86.32 and 95.35%, relative to indirect immunofluorescence assay. Conclusions In summary, compared with M-A110-129 ELISA, the PRRSV M-A148-174 ELISA is of value for detecting antibodies against PRRSV and the evaluation of the NADC30-like PRRSV inactivated vaccine, but the advantage is insufficient in serological early diagnosis.
... Our results showed that pig categories were significantly associated with PRRSV antibody levels in this study. Antibody detection methods mainly include indirect hemagglutination tests (IHAs), immunofluorescence assays and ELISAs (7). Each detection method has its own advantages and disadvantages and adapts to different scenarios. ...
Article
Porcine reproductive and respiratory syndrome (PRRS) is caused by the PRRS virus (PRRSV), and it is a widespread disease that severely affects swine production in all age groups. Detection of PRRSV antibody levels in pig farms is beneficial for immunity evaluations. In this study, a total of 1,206 serum samples of breeding boars, breeding sows, reserve pigs and commercial pigs from 16 large-scale swine farms in 4 different regions of Yunnan province in China were collected during 2019 and detected by indirect enzyme-linked immunosorbent assay (ELISA). The results showed that the average positive rate of PRRSV antibody was 88.32%, among which the antibody-positive rates were 89.03%, 89.18%, 92.11%, and 82.95% in East Yunnan (E. Yunnan), Central Yunnan (Cent. Yunnan), Northwest Yunnan (N.W. Yunnan) and Northeast Yunnan (N.E. Yunnan), respectively (P > 0.05). For the different pig categories, the reserve pigs (93.51%) showed much higher antibody-positive rates, followed by breeding sows (92.44%), commercial pigs (87.34%) and breeding boars (85.62%). Statistical analysis revealed that the rates were significantly different among different pig categories (P <0.05). These s results indicated that pig categories were significantly associated with PRRSV antibody levels in this study. All the positive rates in this study fulfilled the requirement of ≥ 70% set by the National Animal Disease Surveillance Plan of China (2011). The study could provide evidence of the antibody response of PRRSV at the farm level.
... (Biernacka et al., 2018).Gerber et al. (2014) stated that IDEXX kit has specifi city of 100%, which almost agrees with the manufacturer's declaration (99.9%). In this study, the highest number of false positives was acquired with Qiagen(9 samples), while the number of false negative samples obtained with Qiagen and INgezim was the same (2 samples). Our results for specifi city of I ...
Article
Full-text available
Porcine Reproductive and Respiratory Syndrome (PRRS) is one of the most economically important diseases in pigs, worldwide. Just in the US, the total costs to the swine industry have been estimated at $664 million per year. Therefore, the continuous and reliable monitoring of the PRRS status of a pig herd is required in order to prevent and reduce costs due to this infection. Mostly used methods for diagnosis of PRRS infection nowadays are serological (ELISA) and molecular (PCR) ones. This study aimed to assess the sensitivity and specificity of three different commercially available ELISA kits for detection of antibodies against PRRSV (IDEXX PRRS X3 Ab Test (IDEXX, USA), INgezim PRRS Universal (Ingenasa, Spain), Pigtype PRRSV Ab (Qiagen, Germany)) using 91 blood serum samples collected from pigs in Serbia. Our study showed no significant differences in specificity and sensitivity between three commercially available ELISA kits. However, IDEXX ELISA proved to be more reliable kit for detecting antibodies against PRRSV with sensitivity of 97,4% and specificity of 98,1%, considering INgezim and Qiagen kits specificity of 92,5% and 83%, respectively, and sensitivity of 94,7 % for both kits. In order to achieve maximal reliability of obtained results, ELISA diagnostic protocol for diagnosis of PRRS infection should be complemented with additional tests such as PCR and virus neutralization test.
... Specificity can vary, depending on the manufacturer, with reported ranges of between 90% and 100%. In one study, differences were also seen between ELISAs, in terms of how early seroconversion could be detected (Gerber et al., 2014). ...
Article
Full-text available
Porcine reproductive and respiratory syndrome (PRRS) has been assessed according to the criteria of the Animal Health Law (AHL), in particular criteria of Article 7 on disease profile and impacts, Article 5 on the eligibility of PRRS to be listed, Article 9 for the categorisation of PRRS according to disease prevention and control rules as in Annex IV and Article 8 on the list of animal species related to PRRS. The assessment has been performed following a methodology composed of information collection and compilation, expert judgement on each criterion at individual and, if no consensus was reached before, also at collective level. The output is composed of the categorical answer, and for the questions where no consensus was reached, the different supporting views are reported. Details on the methodology used for this assessment are explained in a separate opinion. According to the assessment performed, PRRS can be considered eligible to be listed for Union intervention as laid down in Article 5(3) of the AHL. The disease would comply with the criteria as in Sections 4 and 5 of Annex IV of the AHL, for the application of the disease prevention and control rules referred to in points (d) and (e) of Article 9(1). The animal species to be listed for PRRS according to Article 8(3) criteria are domestic pigs and wild boar.
... Currently, serological testing still remains a reliable tool for the diagnosis of PRRSV infection [23]. Several commercial ELISA Kits for the detection of antibodies against PRRSV in serum are available in the market [24,25]. Each method has its advantages and drawbacks. ...
Article
Full-text available
Background Porcine reproductive and respiratory syndrome virus (PRRSV), the causative agent of PRRS, has two distinct and highly diverse genotypes (genotype 1 and genotype 2) in the field. Accurate diagnosis and differentiation of the two genotypes of PRRSV are critical to the effective prevention and control of PRRS. The non-structural protein 10 (Nsp10) plays a vital role in viral replication and is one of the most conserved proteins of PRRSV, thus constituting a good candidate for PRRSV diagnosis. Results In this study, we generated a monoclonal antibody (mAb) 4D9 against Nsp10 by immunizing BALB/c mice with purified recombinant Nsp10 expressed by an Escherichia coli system. Through fine epitope mapping of mAb 4D9 using a panel of eukaryotic expressed polypeptides with GFP-tags, we identified the motif ²⁸⁶AIQPDYRDKL²⁹⁵ as the minimal unit of the linear B-cell epitope recognized by mAb 4D9. Protein sequence alignment indicated that ²⁸⁶AIQPDYRDKL²⁹⁵ was highly conserved in genotype 2 PRRSV strains, whereas genotype 1 PRRSV strains had variable amino acids in this motif. Furthermore, a mutant of the motif carrying two constant amino acids of genotype 1 PRRSV, Cys290 and Glu293, failed to react with mAb 4D9. More importantly, the mAb 4D9 could differentiate genotype 2 PRRSV strains from genotype 1 PRRSV strains using Western blotting and immunofluorescence analysis. Conclusion Our findings suggest that Nsp10-specific mAb generated in this study could be a useful tool for basic research and may facilitate the establishment of diagnostic methods to discriminate between genotype 1 and genotype 2 PRRSV infection.
... Future studies should also assess oral fluids for suitability as a sample type to assess herd immunity. However, similar to fecal samples, inhibitors and low analyte concentrations may impair detection of antibodies in this sample type [19]. In addition, pen-based oral fluid samples represent composite samples from 25 up to 350 pigs and establishing the rates of individual protection appears not feasible. ...
... Future studies should also assess oral fluids for suitability as a sample type to assess herd immunity. However, similar to fecal samples, inhibitors and low analyte concentrations may impair detection of antibodies in this sample type [19]. In addition, pen-based oral fluid samples represent composite samples from 25 up to 350 pigs and establishing the rates of individual protection appears not feasible. ...
Article
Many assays for detection of antibodies against porcine epidemic diarrhea virus (PEDV) are based on detection of neutralizing antibodies or immunoglobulin (Ig) G in serum samples. However, due to the particular features of the mucosal immune system, presence of serum antibodies against enteric pathogens, such as PEDV, not always correlates with protection. In contrast, anti-PEDV IgA antibodies correlate with protection against subsequent challenges. An indirect PEDV IgA ELISA was previously developed to monitor IgA levels in colostrum and milk samples. In the present paper we describe an adaptation of the protocol for detection of IgA antibodies in serum and fecal samples. •The adapted protocol will aid in future assessment of protective levels of humoral response against PEDV infection by measuring IgA levels in serum and fecal samples. •Fecal samples are non-invasive and easy to collect at any time by animal caretakers and therefore offering advantages over the serum sample collection procedure. •A strong positive correlation between the anti-PEDV levels in fecal and serum samples was identified; however, detection of IgA antibodies was often more successful in serum than in paired fecal samples due to overall lower sample-to-positive (S/P) ratios for the latter sample type.
... Specifically, 64% (16/25) of the adult pigs were positive by FMIA but negative by the IDEXX ELISA. This is similar to a previous study where a high false positive rate in negative field samples was detected in adult boars when using the same antigen as in the present study (Gerber et al., 2014). High background reactivity is frequently observed using serum samples derived from sows (Denac et al., 1997). ...
Article
The objective of this study was to develop a fluorescent microbead-based immunoassay (FMIA) for simultaneous detection of IgG antibodies against Torque teno sus virus (TTSuV) species 1 (TTSuV1), TTSuV2, porcine reproductive and respiratory syndrome virus (PRRSV) type 1 (PRRSV-1) and PRRSV-2. Two-hundred serum samples were obtained over time from 20 PRRSV-free pigs with variable antibody titers to TTSuV. Twelve of 20 were exposed to TTSuV2 on day 0, 20/20 were vaccinated with a modified live-attenuated PRRSV-2 vaccine on day 35, and 20/20 were exposed to PRRSV-2 on day 63. Blood samples were collected weekly until day 77. Ninety field samples from 17 farms with unknown TTSuV and PRRSV status were also tested. Anti-TTSuV antibodies were detected in 30% of the experimental pigs on day 0 and 90% of the pigs were TTSuV2 seropositive by day 35. All PRRSV-2 vaccinated pigs had detectable anti-PRRSV-2 IgG 21 days after vaccination. The seroprevalence of both PRRSV and TTSuV increased with age and the seroprevalence of TTSuV2 and TTSuV1 were similar across age groups in the field. Comparison of the PRRSV-2 FMIA to the gold standard IDEXX ELISA revealed good correlation in young pigs but cross-reaction between PRRSV types in both experimental and field samples and high rate of false positives in older pigs. Overall results indicate that FMIA is a suitable platform for multiplex serological testing in pigs; however, the PRRSV antigens utilized in the current study had a false positive rate and considerable cross-reactions preventing reliable differentiation of PRRSV types.
Article
Full-text available
Latent class analysis is a widely used statistical method for evaluating diagnostic tests without any gold standard. It requires the results of at least two tests applied to the same individuals. Based on the resulting response patterns, the method estimates the test accuracy and the unknown disease status for all individuals in the sample. An important assumption is the conditional independence of the tests. If tests with the same biological principle are used, the assumption is not fulfilled, which may lead to biased results. In a recent publication, we developed a method that considers the dependencies in the latent class model and estimates all parameters using frequentist methods. Here, we evaluate the practicability of the method by applying it to the results of six ELISA tests for antibodies against the porcine reproductive and respiratory syndrome (PRRS) virus in pigs that generally follow the same biological principle. First, we present different methods of identifying suitable starting values for the algorithm and apply these to the dataset and a vaccinated subgroup. We present the calculated values of the test accuracies, the estimated proportion of antibody-positive animals and the dependency structure for both datasets. Different starting values led to matching results for the entire dataset. For the vaccinated subgroup, the results were more dependent on the selected starting values. All six ELISA tests are well suited to detect antibodies against PRRS virus, whereas none of the tests had the best values for sensitivity and specificity simultaneously. The results thus show that the method used is able to determine the parameter values of conditionally dependent tests with suitable starting values. The choice of test should be based on the general fit-for-purpose concept and the population under study.
Chapter
Systemic infectious diseases can be associated in some cases with high mortality rates. Thus, there is a need for early detection and diagnosis in order to initiate an appropriate treatment regime as soon as possible. Nowadays, the diagnosis of infectious diseases is still dependent on the evaluation of blood and/or tissue samples. Although they are effective, these procedures are invasive and expensive, moreover, depending on different clinical conditions, these types of tests may not be accessible for many patients and health care providers. For all these reasons saliva-based diagnostics have been the primary focus of investigation for a variety of infectious pathogens for several years. The aim of this chapter is to review the main methods that have been developed and evaluated in saliva for the diagnostic and monitoring of systemic infectious diseases of humans and domestic animals.
Chapter
Control of porcine reproductive and respiratory syndrome virus (PRRSV) remains problematic, and economic studies have uniformly shown that PRRSV inflicts major losses on swine health and productivity. A fuller picture of PRRSV genetic relationships and evolutionary origins may be facilitated by whole genome analyses and comparisons of multiple protein coding regions, including the polymerase gene, which is widely used in RNA viral evolutionary analyses. PRRSV viral infection can be divided into three distinct stages: acute infection, persistence, and extinction. Acute infection follows exposure and is characterized by rapid spread to primary sites of replication in lung and lymphoid tissues. PRRSV markedly alters innate immunity and inflammatory and immunoregulatory cytokines in a strain‐ specific manner. Infection with PRRSV induces immunity that eventually controls the initial infection, eliminates the virus, and establishes memory that is variably protective against future infection.
Article
Full-text available
Background: Effective vaccines against porcine reproductive and respiratory syndrome virus (PRRSV), especially against highly pathogenic (HP) PRRSV are still missing. The objective of this study was to evaluate the protective efficacy of an experimental live attenuated PRRSV 2 vaccine, composed of two strains, against heterologous challenge with a Vietnamese HP PRRSV 2 field strain. For this reason, 20 PRRSV negative piglets were divided into two groups. The pigs of group 1 were vaccinated with the experimental vaccine, group 2 remained unvaccinated. All study piglets received an intranasal challenge of the HP PRRSV 2 on day 0 of the study (42 days after vaccination). Blood samples were taken on days 7 and 21 after vaccination and on several days after challenge. On day 28 after challenge, all piglets were euthanized and pathologically examined. Results: On days 7 and 21 after vaccination, a PRRSV 2 viraemia was seen in all piglets of group 1 which remained detectable in seven piglets up to 42 days after vaccination. On day 3 after challenge, all piglets from both groups were positive in PRRSV 2 RT-qPCR. From day 7 onwards, viral load and number of PRRSV 2 positive pigs were lower in group 1 than in group 2. All pigs of group 1 seroconverted after PRRSV 2 vaccination. PRRSV antibodies were detected in serum of all study pigs from both groups from day 14 after challenge onwards. In group 2, moderate respiratory symptoms with occasional coughing were seen following the challenge with HP PRRSV 2. Pigs of group 1 remained clinically unaffected. Interstitial pneumonia was found in four piglets of group 1 and in all ten piglets of group 2. Histopathological findings were more severe in group 2. Conclusions: It was thus concluded that the used PRRSV 2 live experimental vaccine provided protection from clinical disease and marked reduction of histopathological findings and viral load in pigs challenged with a Vietnamese HP PRRSV 2 field strain.
Article
Full-text available
Two commercial PRRSV ELISA kits (IDEXX and Bionote) were evaluated for their sensitivity and specificity using 476 PRRS-positive serum samples collected from 7 animal challenge experiments and 1,000 PRRS-negative sera. Both ELISA kits exhibited 100% sensitivity with sera collected 14 to 42 days post-infection, and the results from the kits were highly correlated (R(2)=0.9207). The specificity of IDEXX or Bionote kit was 99.9% or 99.7%, respectively. In addition, the Bionote ELISA kit was used to examine 100 sera that were determined to be falsely positive either by IDEXX 2XR or 3XR ELISA, and only 7 of these samples were found to be positive. These results indicate that both ELISA kits exhibited similar levels of sensitivity and specificity and would complement one another for the verification of false-positive samples.
Article
Full-text available
The aims of this study were to compare three commercial porcine reproductive and respiratory syndrome virus (PRRSV) real-time RT-PCR assays for detection of genetically diverse PRRSV isolates in serum, semen, blood swabs, and oral fluids collected from experimentally-infected boars, and to evaluate the effects of sample pooling. Six groups of 3 boars negative for PRRSV were each inoculated with one of six PRRSV isolates (sharing 55-99% nucleotide sequence identity in ORF5). Samples were collected on days post-inoculation (dpi) -2, 1, 3, 5, 7, 14 and 21 and tested by one of three commercially available real-time RT-PCR assays (AB, TC, AD). At dpi 1, all assays detected at least one positive sample in each group. The highest detection rates were on dpi 3 and dpi 5. Between dpi 1 and 7, serum samples had the highest detection rate (90%) with 100% agreement between tests, followed by blood swabs (Kappa = 0.97) and semen (Kappa = 0.80). Oral fluids had the lowest detection rates (AB: 55%; TC: 41%; AD: 46%) and the highest disagreement between kits (Kappa = 0.63). Pools of five samples did not reduce the detection rates if there was one positive sample with a high amount of viral RNA in the pool. Serum and blood swab samples had shorter turn-around times for RNA extraction. The AB assay had a 1.6 times shorter PCR reaction time. In summary, serum and blood swabs had the best performance with highest detection rates and agreement between assays and shortest turn-around time.
Article
Full-text available
To compare the real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR) assays used for the diagnosis of Porcine reproductive and respiratory syndrome virus (PRRSV), a Europe-wide interlaboratory ring trial was conducted. A variety of PRRSV strains including North American (NA) and European (EU) genotype isolates were analyzed by the participants. Great differences regarding qualitative diagnostics as well as analytical sensitivity were observed between the individual RT-qPCR systems, especially when investigating strains from the EU genotype. None of the assays or commercial kits used in the ring trial could identify all different PRRSV strains with an optimal analytical and diagnostic sensitivity. The genetic variability of the PRRSV strains, which is supposed to hinder the diagnostic of the RT-PCR because of mutations at the primer binding sites, was also confirmed by sequencing and subsequent phylogenetic analysis. In summary, a major problem in PRRSV diagnostics by RT-qPCR is false-negative results. To achieve maximum safety in the molecular diagnosis of PRRSV, the combined usage of different assays or kits is highly recommended.
Article
Full-text available
The purpose of the present study was to evaluate the diagnostic performance of a commercial serum antibody enzyme-linked immunosorbent assay (ELISA) modified to detect anti-Porcine reproductive and respiratory syndrome virus (PRRSV) antibodies in pen-based oral fluid specimens. Experimental and field oral fluid samples of defined status in reference to exposure of swine with PRRSV were used to derive the kinetics of detectable concentrations of antibody against PRRSV. Immunoglobulin (Ig)M and IgA were readily detected in oral fluid specimens from populations in which PRRSV infection was synchronized among all individuals but not in samples collected in commecial herds. In contrast, IgG was readily detected at diagnostically useful levels in both experimental and field samples for up to 126 days. Estimates of the IgG oral fluid ELISA performance were based on results from testing positive oral fluid samples (n = 492) from experimentally inoculated pigs (n = 251) and field samples (n = 241) and negative oral fluid samples (n = 367) from experimentally inoculated pigs (n = 84) and field samples (n = 283). Receiver operating characteristic analysis estimated the diagnostic sensitivity and specificity of the assay as 94.7% (95% confidence interval [CI]: 92.4, 96.5) and 100% (95% CI: 99.0, 100.0), respectively, at a sample-to-positive ratio cutoff of ≥0.40. The results of the study suggest that the IgG oral fluid ELISA can provide efficient, cost-effective PRRSV monitoring in commercial herds and PRRSV surveillance in elimination programs.
Article
Full-text available
Precise and rapid detection of porcine reproductive respiratory syndrome virus (PRRSV) infection in swine farms is critical. Improvement of control procedures, such as testing incoming gilt and surveillance of seronegative herds requires more rapid and sensitive methods. However, standard serological techniques detect mainly IgG antibodies. A double recognition enzyme-linked immunosorbent assay (DR-ELISA) was developed for detection of antibodies specific to European PRRSV. This new assay can recognize both IgM and IgG antibodies to PRSSV which might be useful for detecting in routine surveillance assays pigs that are in the very early stages of infection and missed by conventional assays detecting only IgG antibodies. DR-ELISA is based on the double recognition of antigen by antibody. In this study, the recombinant nucleocapsid protein (N) of PRRSV was used both as the coating and the enzyme-conjugated antigen. To evaluate the sensitivity of the assay at early stages of the infection, sera from 69 pigs infected with PRRSV were collected during successive days post infection (pi) and tested. While standard methods showed low sensitivity rates before day 14 pi, DR-ELISA detected 88.4% seropositive samples at day 7 showing greater sensitivity at early stages of the infection. Further studies were carried out to assess the efficiency of the new assay, and the results showed DR-ELISA to be a sensitive and accurate method for early diagnosis of EU-PRRSV infection.
Article
Full-text available
Early diagnosis of Porcine reproductive and respiratory syndrome virus (PRRSV) is critically important for control of the disease. Two new commercially available enzyme-linked immunosorbent assays (ELISAs) based on different methodologies have been developed. In the present report, the 2 ELISAs were compared using blood samples from experimentally and naturally infected pigs. One of the 2 ELISAs was shown to be more sensitive than the other. The higher sensitivity of one of the ELISAs could pose a problem in PRRS diagnosis in endemic farms, because it can detect maternally derived antibodies for a longer time, overlapping with the detection of antibodies developed after PRRSV infection. However, the ELISA with higher sensitivity could be suitable for early detection of PRRSV antibodies in individual pigs, especially in PRRS-free herds.
Article
Full-text available
For effective disease surveillance, rapid and sensitive assays are needed to detect antibodies developed in response to porcine reproductive and respiratory syndrome virus (PRRSV) infection. In this study, we developed a multiplexed fluorescent microsphere immunoassay (FMIA) for detection of PRRSV-specific antibodies in oral fluid and serum samples. Recombinant nucleocapsid protein (N) and nonstructural protein 7 (nsp7) from both PRRSV genotypes (type I and type II) were used as antigens and covalently coupled to Luminex fluorescent microspheres. Based on an evaluation of 488 oral fluid samples with known serostatus, the oral fluid-based FMIAs achieved >92% sensitivity and 91% specificity. For serum samples (n = 1,639), the FMIAs reached >98% sensitivity and 95% specificity. The assay was further employed to investigate the kinetics of the antibody response in infected pigs. In oral fluid, the N protein was more sensitive for the detection of early infection (7 and 14 days postinfection), but nsp7 detected a higher level and longer duration of antibody response (28 days postinfection). In serum, the antibodies specific to nsp7 and N proteins were detected as early as 7 days postinfection, and the responses lasted more than 202 days. This study provides a framework from which a more robust assay could be developed to profile the immune response to multiple PRRSV antigens in a single test. The development of oral fluid-based diagnostic tests will change the way we survey diseases in swine herds and improve our ability to cheaply and efficiently track PRRSV infections in both populations and individual animals.
Article
Full-text available
Porcine circovirus type 2 (PCV2) and porcine reproductive and respiratory syndrome virus (PRRSV) are major contributors to the porcine respiratory disease complex (PRDC). Routine serological diagnosis and surveillance play an important role in the prevention of PRDC, as it is a leading cause of economic losses to the swine industry. We herein describe an advanced microsphere-based immunoassay that permits the simultaneous detection of antibodies to PCV2 and PRRSV, thereby reducing the time and effort involved in testing. Recombinant PRRSV nucleoprotein antigen and the PCV2 capsid antigen were coupled to fluorophore-dyed beads with distinct spectral addresses. Weekly serum samples from 72 pigs that were experimentally exposed to either PCV2, PRRSV, or both PCV2 and PRRSV were used to validate the microbead assay (MBA) in comparison with the "gold standard" enzyme-linked immunosorbent assays. The kinetics of the PCV2- and PRRSV-specific antibody responses measured by the microbead assay were comparable to those of the standard assays; Spearman's rank correlations were 0.72 (P < 0.001) for PRRSV and 0.80 (P < 0.001) for PCV2. Diagnostic sensitivity and specificity were determined using field sera whose positive or negative status was determined by the standard tests. The diagnostic sensitivity and specificity were both 98% for PCV2 and were 91% and 93%, respectively, for PRRSV (kappa coefficients, 0.85 and 0.67 for PCV2 and PRRSV, respectively). Multiplexing did not interfere with assay performance or diagnostic sensitivity. Therefore, the described study demonstrates proof of concept for the development of more versatile and economical microbead array-based multiplex serological test panels for veterinary use.
Article
Full-text available
The putative membrane (M) protein (ORF 6) and nucleocapsid (N) protein (ORF 7) genes of five U.S. isolates of porcine reproductive and respiratory syndrome virus (PRRSV) with differing virulence were cloned and sequenced. To determine the genetic variation and the phylogenetic relationship of PRRSV, the deduced amino acid sequences of the putative M and N proteins from these isolates were aligned, to the extent known, with other PRRSV isolates, and also other members of the proposed arterivirus group including lactate dehydrogenase-elevating virus (LDV) and equine arteritis virus (EAV). There was 96-100% amino acid sequence identity in the putative M and N genes among U.S. and Canadian PRRSV isolates with differing virulence. However, their amino acid sequences varied extensively from those of European PRRSV isolates, and displayed only 57-59% and 78-81% identity, respectively. The phylogenetic trees constructed on the basis of the putative M and N genes of the proposed arterivirus group were similar and indicated that both U.S. and European PRRSV isolates were related to LDV and were distantly related to EAV. The U.S. and European PRRSV isolates fell into two distinct groups, suggesting that U.S. and European PRRSV isolates represent two distinct genotypes.
Article
Full-text available
The antibody responses of pigs to porcine reproductive and respiratory syndrome virus (isolate VR-2332) were evaluated by indirect immunofluorescence, virus neutralization, and immunoblotting. All pigs in each group were positive by indirect immunofluorescence 14-21 days postexposure (DPE), and antibodies to specific viral proteins (15, 19 or 26 kD) were initially demonstrated by immunoblotting at 7-21 days DPE. Neutralizing antibodies were detected in only 2 pigs that were inoculated intranasally and given additional parenteral injections with adjuvant. These antibodies appeared much later, 51-70 DPE, than did antibodies detected by indirect immunofluorescence. The titer of the neutralizing antibodies increased until 127 DPE, after which the titers decreased, and 1 animal became seronegative for neutralizing antibody by 262 DPE.
Article
Full-text available
Two types of porcine reproductive and respiratory syndrome virus (PRRSV) have been reported, the European type (EU PRRSV) and the North American type (US PRRSV). We developed a dual enzyme-linked immunosorbent assay (ELISA) for the simultaneous detection and differentiation of serum antibodies directed against either of the two PRRSV types. This tandem PRRS ELISA is based on affinity-purified recombinant nucleocapsid protein expressed in Escherichia coli. Sensitivity and specificity were assessed by using the IDEXX HerdChek PRRS ELISA and the indirect immunofluorescence assay as reference tests. A total of 1571 sera originating from the United States, Europe, and two PRRS-free countries, i.e., Switzerland and New Zealand, were used for validation of the tandem PRRS ELISA. The new test performed at least as well as the reference tests in regard to sensitivity (0.94 for the US PRRS ELISA and 0.93 for the EU PRRS ELISA) and specificity (0.96 for the US PRRS ELISA and 0.99 for the EU PRRS ELISA). Positive sera were correctly differentiated in 582 of 591 cases, indicating a high differentiation capability of this dual ELISA. The robustness and repeatability of the test were assessed and found to be appropriate for diagnostic applications. Taken together, the data indicate that the tandem PRRS ELISA described here is the first differentiation ELISA for PRRSV serology based on recombinant antigen. It is convenient with respect to antigen production, and it is reliable, economical, and highly sensitive and specific. Thus, it is considered to be a powerful tool for routine diagnostics, epidemiological surveys, and outbreak investigations.
Article
Specimens were tested for porcine reproductive and respiratory syndrome virus by real-time reverse transcriptase polymerase chain reaction (RT-PCR) with two commercial agent-specific assays. Result discrepancy between the two tests was confirmed by conventional RT-PCR, sequencing, or both on six of 423 clinical cases. Neither assay detected every positive case.
Article
A total 91 serum samples and 51 pig tissue samples were collected between October 2009 and June 2010 from 30 herds, where a clinical picture of infection or/and porcine reproductive and respiratory syndrome (PRRS) antibody-positive pigs were detected. Of the 142 samples tested, 65 (45.8%) were identified as porcine reproductive and respiratory syndrome virus (PRRSV) positive by a one-step reverse transcription and polymerase chain reaction (RT-PCR). The sequencing results of 258 nucleotides in ORF7 from 30 herds with PRRSV-positive samples revealed the circulation of six genetically different strains of PRRSV, all belonging to the Subtype 1 (Type I). Twenty-three (76.6%) of the thirty positive herds were infected with a genetically identical cluster, with 98.9-100% nucleotide identity between the herds, representing the detection of a new strain of PRRSV in Europe, not published previously. From these 23 herds, positive PRRSV samples were detected with gel-based RT-PCR, but all gave false-negative results with two commercial real-time kits. When using a third commercial real-time kit, 28 (93.3%) of 30 positive samples in gel-based RT-PCR were detected as the Type I, confirming that the sensitivity of this real-time kit is much greater than the sensitivity of the previous two. The influence of new genetic variants of PRRSV circulating in Slovenia on molecular diagnosis and the control of the infection is discussed.
Article
Porcine reproductive and respiratory syndrome virus (PRRSv) can have a significant economic impact on swine herds due to reproductive failure, preweaning mortality and reduced performance in growing pigs. Control at the farm level is pursued through different management procedures (e.g. pig flow, gilt acclimation, vaccination). PRRSv is commonly eliminated from sow herds by a procedure called herd closure whereby the herd is closed to new introductions for a period of time during which resident virus dies out. However, despite thorough application of biosecurity procedures, many herds become re-infected from virus that is present in the area. Consequently, some producers and veterinarians are considering a voluntary regional program to involve all herds present within an area. Such a program was initiated in Stevens County in west central Minnesota in 2004. PRRSv has been eliminated from most sites within the region and the area involved has expanded to include adjacent counties. The program has been relatively successful and reflects local leadership, a cooperative spirit, and a will to eliminate virus from the region.
Article
Unexpected positive results from the widely used IDEXX ELISA for the detection of antibodies to porcine reproductive and respiratory syndrome virus (PRRSV) may confound investigations of the disease. Supplementing the ELISA with blocking agents and the use of IgG purified from serum samples had no effect on the unexpected positive results, suggesting that they were due to an antibody-antigen reaction. Simple competitive and blocking ELISAs were developed by modifying the IDEXX ELISA, and they and an indirect fluorescent antibody test (IFAT) were used to examine PRRSV antibodies in 33 antibody-negative, 88 antibody-positive and 73 unexpectedly positive sera. All the unexpectedly positive sera were negative by IFAT, and 89.0 per cent were negative by both the competitive and blocking ELISAs. The competitive ELISA (97.7 per cent) and the blocking ELISA (96.5 per cent) detected more positive sera than the IFAT (90.9 per cent). These results show that both ELISAs are capable of distinguishing positive and unexpectedly positive sera, and suggest that most of the unexpected positive signals are false-positives.
Article
This paper presents a general statistical methodology for the analysis of multivariate categorical data involving agreement among more than two observers. Since these situations give rise to very large contingency tables in which most of the observed cell frequencies are zero, procedures based on indicator variables of the raw data for individual subjects are used to generate first-order margins and main diagonal sums from the conceptual multidimensional contingency table. From these quantities, estimates are generated to reflect the strength of an internal majority decision on each subject. Moreover, a subset of observers who demonstrate a high level of interobserver agreement can be identified by using pairwise agreement statistics between each observer and the internal majority standard opinion on each subject. These procedures are all illustrated within the context of a clinical diagnosis example involving seven pathologists.
Article
The 15 kDa nucleocapsid (N) protein is the most abundant protein of the porcine reproductive and respiratory syndrome virus (PRRSV), and is highly antigenic, which therefore makes it a suitable candidate for the detection of virus-specific antibodies and diagnosis of the disease. In this study, complementary DNA corresponding to the entire N gene of the IAF-Klop strain of PRRSV was cloned into the pGEX-4T-1 vector, and the N protein was expressed in Escherichia coli fused to the glutathione S-transferase (GST) protein. The resulting GST-N recombinant fusion protein was purified by affinity chromatography and used as antigen for serological testing by indirect enzyme-linked immunosorbent assay (ELISA). Two anti-N specific monoclonal antibodies (MAbs) (IAF-K8 and IAF-2B4), obtained following fusion experiments with spleen cells of BAlb/c mice that were immunized with the purified virus, were used in a competitive assay to increase the specificity of the ELISA. Both MAbs were found to be directed against highly conserved conformational epitopes of North American isolates of PRRSV. Optimal concentration of GST-N protein was determined by checkerboard titration, using hyperimmune pig antiserum to the homologous PRRSV strain, and corresponded to a range of 0.1-0.5 microg protein per well. When tested on 95 sera from pigs that were experimentally infected with the IAF-Klop strain, the competitive ELISA (K8-ELISA) was capable of detecting anti-PRRSV antibodies in 86.7% (65/75) and 92.6% (63/68) of pig sera known to be seropositive by indirect immunofluorescence (antibody titers >16) and a currently used commercial ELISA (HerdCheck(R); Idexx), with specificity values of 100 and 96.2%, respectively. When tested on clinical samples (542 sera) from 28 positive and 28 negative pig herds, the K8-ELISA performed in a similar way to HerdCheck(R) and immunofluorescence (IF) tests as shown by kappa values of 0.762 and 0.803. The sensitivity and specificity of K8-ELISA were 100% on a herd basis, whereas sensitivity values of 80 and 82% with a specificity of 98.7% were determined on an individual basis in comparison with HerdCheck(R) and IF tests.