Antibodies In Pig Sera Research Articles

Development of multiple elisas for the detection of antibodies against classical swine fever virus in pig sera

The major immunogenic proteins (E(rns), E2 and NS3) of classical swine fever virus (CSFV) (Shimen strain) were expressed in E. coli and purified by affinity chromatography. The recombinant antigens were applied to develop multiple enzyme-linked immunosorbent assays (ELISAs) for the detection of specific antibodies in pig sera. Optimum cut-off values were determined by receiver operating characteristic (ROC) analysis after testing 201 sera of vaccinated pigs and 64 negative sera of unvaccinated piglets. The multiple ELISAs were validated with 265 pig sera yielding high sensitivity and specificity in comparison with the virus neutralization results. The results demonstrated that multiple ELISAs can be a valuable tool for the detection of CSFV infection and serological surveys in CSFV-free countries or for the evaluation of the antibody responses in pigs induced by a live attenuated C-strain vaccination.

Expression of the putative ORF1 capsid protein of Torque teno sus virus 2 (TTSuV2) and development of Western blot and ELISA serodiagnostic assays: Correlation between TTSuV2 viral load and IgG antibody level in pigs

Porcine Torque teno virus (TTV) has a single-stranded circular DNA genome and is currently classified into a new genus Iotatorquevirus with two species in a newly established family Anelloviridae. Viral DNA of both porcine TTV species (TTSuV1 and TTSuV2) has a high prevalence in both healthy and diseased pigs worldwide and multiple infections of TTSuV with distinct genotypes or subtypes of the same species has been documented in the United States and in Europe. However, the prevalence of specific TTSuV antibodies in pigs remains unknown. In this study, the putative ORF1 capsid protein from TTSuV2 isolate PTTV2c-VA was expressed in Escherichia coli. The purified recombinant ORF1 protein was used as the antigen for the development of Western blot and indirect ELISA to detect TTSuV2-specific IgG antibodies in pig sera. The results revealed a relatively high rate of seropositivity to TTSuV2 in conventional pigs from different sources but not in gnotobiotic pigs. Overall, pigs with undetectable TTSuV2 viral load were more likely to have a lower anti-TTSuV2 antibody level. An analysis of 10 conventional pigs during a 2-month period showed that decreased viral loads or presumed virus clearance were associated with elevated anti-ORF1 IgG antibody levels. Interestingly, porcine circovirus associated disease (PCVAD)-affected pigs had a significantly lower level of TTSuV2 antibody than PCVAD-unaffected pigs ( p < 0.01). This is the first study to establish essential serodiagnostic tools for investigation of TTSuV seroprevalence and infection dynamics, which will help elucidate the potential pathogenicity of TTSuV infection in pigs.

Cloning, characterization and diagnostic performance of the salivary lipocalin protein TSGP1 from Ornithodoros moubata

The argasid tick Ornithodoros moubata is distributed throughout South and East Africa and Madagascar, where it colonizes wild and domestic habitats and feeds on warthogs, domestic swine, and humans. This argasid transmits the spirochete Borrelia duttonii, causing East African tick-borne relapsing fever in humans, and the African swine fever virus, which causes a highly lethal haemorrhagic disease in pigs. Tick surveillance and the elimination of O. moubata from synanthropic environments (human dwellings and pigsties) would facilitate the control and prevention of these two diseases. Since direct surveillance methods are impractical in this context, the development of an indirect method for the detection of specific antibodies against tick salivary proteins in samples taken from animal or human hosts living in the area under study would provide a more convenient surveillance and diagnostic tool. Previous work has indicated that the 20A1 salivary antigen of O. moubata could be an optimal candidate for the development of a specific serological test and identified it as an orthologue of the Ornithodoros savignyi TSGP1 lipocalin. The objectives of the present work were to clone, sequence, and molecularly characterize the O. moubata TSGP1, as well as its production as a recombinant protein in order to assess its usefulness as a diagnostic antigen in an ELISA test for tick surveillance. Our results show that O. moubata TSGP1 (OmTSGP1) conserves the tertiary structure of lipocalins and contains the biogenic amine-binding motif. We also show that OmTSGP1 shares 65% sequence identity with the O. savignyi TSGP1, demonstrating that they represent orthologous proteins and suggesting they share identical function as biogenic amine scavengers. A recombinant form of OmTSGP1 was produced, showing 100% sensitivity and 99.4% specificity in an ELISA test for the detection of anti-O. moubata antibodies in pig sera. This recombinant antigen represents a promising epidemiological tool for O. moubata surveillance that may help to implement control measures against O. moubata-borne diseases.

Beta-1,3-glucan effect on sow antibody production and passive immunisation of progeny

β-glucans are glucose homopolymers known to modulate immunity. Here, the β-glucan effect on sow antibody production and passive immunisation of neonatal pigs was analysed. Treatments included: (1) corn-soy fed Control group; (2) β-glucan; (3) Actinobacillus pleuropneumoniae (App) vaccination; and (4) β-glucan + App vaccination. Birth and weaning weights were not affected (P>0.05) by treatment. Independent of treatment, IgA, IgG and IgM in colostrum were elevated and declined rapidly. Vaccination against App resulted in an increase (P<0.05) in IgG and IgA specific for App serotypes 1, 5 and 7 in colostrum and milk, and a corresponding increase (P<0.05) in serum in pigs at 4 d of age. However, β-glucan did not enhance specific App antibodies in pig serum. While the dosage used here did not enhance passive immunisation in pigs, β-glucan can be potentially efficacious as an oral adjuvant to enhance immunoglobulin production in response to vaccination.

A highly specific and sensitive sandwich blocking ELISA based on baculovirus expressed pseudorabies virus glycoprotein B

A direct sandwich blocking enzyme-linked immunosorbent assay (BacgB ELISA) based on the reaction between a monoclonal antibody (MAb) and a recombinant glycoprotein B (gB) of pseudorabies virus (PRV) was developed. This protein was obtained in large quantities from insect cells infected with a PRV gB recombinant baculovirus. Expression of the gB was confirmed by immunoperoxidase monolayer assay (IPMA) with gB specific MAbs. The specificity and sensitivity of the developed BacgB ELISA were evaluated and compared with two commercially available tests by using sets of sera of known PRV infection or vaccination history. For validation, 347 serum samples have been tested. The BacgB ELISA had a high sensitivity and specificity, which were comparable with those of the two commercial tests. In addition, the BacgB ELISA allows detecting anti-gB antibodies in pig serum as early as 7 days following infection. Also maternal antibodies in uninfected pig sera were detected. We conclude that the BacgB ELISA is a useful tool for the detection of as well vaccinated as infected pigs (including derivatives from gE negative vaccine strains), with the added advantage that it uses an antigen that can be produced safely and in large quantities.

A highly specific and sensitive competitive enzyme-linked immunosorbent assay (ELISA) based on baculovirus expressed pseudorabies virus glycoprotein gE and gI complex

A direct competition enzyme-linked immunosorbent assay (ELISA) based on baculovirus expressed complex of pseudorabies virus (PRV) glycoproteins E (gE) and I (gI) has been developed. For that purpose gE and gI genes of PRV were co-expressed in insect cells. Complex formation was confirmed by radioimmunoprecipitation assay. The specificity and sensitivity of the test were evaluated and compared with an ELISA using only gE as an antigen and a commercially available test. For validation, 245 negative sera and 165 positive sera have been tested. The gE/gI ELISA had a higher sensitivity and specificity when compared with the ELISA using only gE as the antigen. Both sensitivity and specificity were comparable with the commercially available test. Moreover, the test based on the baculovirus gE/gI complex allows the detection of anti-gE antibodies in pig serum as early as two weeks after infection. The gE/gI ELISA test is easy to perform; its additional advantage is that the gE/gI antigen can be produced in baculovirus system in large quantities without handling live pseudorabies virus.

Purification of larval Taenia solium antigens by isoelectric focusing

By preparative isoelectric focusing in a rotating ampholine column, crude cystic membrane (M) or fluid (F) antigens of larval Taenia solium were each separated into 20 fractions. M fractions were less specific and sensitive than F fractions in detecting cysticercosis antibodies in pig serum. Among the F fractions, F 15 showed the best potential to serve as a screening antigen. It contained 18 polypeptides, with pI 5.3–8.2 and a specific epitope of 25 kDa which was detected by immunoblotting. Although F 15 showed slight cross-reactions with heterologous antisera in double-antibody IgG enzyme-linked immunosorbent assays (ELISA), it yielded the highest absorbance values when tested against homologous antisera. The antigen was used to screen sera samples from 4870 pigs slaughtered in Hong Kong and five other Chinese cities for cysticercosis antibodies by double-antibody ELISA, Falcon Assay Screening Test (FAST)–ELISA and enhanced chemiluminescent immunoassay. The results varied significantly between assays. However, the samples collected from Shenzhen yielded the highest positive rates. Enhanced chemiluminescent immunoassay based on camera-luminometry was found suitable for use under field conditions.

Detection of Actinobacillus pleuropneumoniae serotype 2 antibodies in pig sera by an inhibition enzyme immuno assay (EIA)

An inhibition Enzyme Immuno Assay (EIA) for detection of antibodies against A. pleuropneumoniae serotype 2 (App-2) in pig sera, based on the inhibition of the binding of an App-2 specific monoclonal antibody was established. The monoclonal antibody (mAb 102-G02) was found to be directed against an epitope on the O-chain of App-2 LPS. Some App-2 isolates did not react with the mAb 102-G02. These isolates are referred to as App-2X. In the inhibition EIA highly purified App-2 LPS was used to coat microtiter plates. Serial dilutions of pig sera were added to the plates prior to the mAb 102-G02. The degree of binding of App-2 antibodies from pig sera was determined as the percentage inhibition of the mAb 102-G02. Pig sera from specific pathogen free (SPF) herds, from experimentally infected animals, and from conventionel herds were tested. A serum dilution of 1 200 was found to be optimal, when using 50% inhibition as the discriminating inhibition percentage. Serum from App-2X infected herds showed a lower reactivity as compared to serum from App-2 infected herds. No crossreactivity was observed with serum from pigs infected with other App serotypes. The sensitivity and specificity were 100% and 98.9%, respectively.

Detection of pig farms with Ornithodoros erraticus by pig serology. Elimination of non-specific reactions by carbohydrate epitopes of salivary antigens

Ornithodoros erraticus is the European vector of African Swine Fever. It is therefore essential to know on which pig farms the tick is present in order to prevent contact with swine. Currently, studies are being made to ascertain this through the detection of anti- O. erraticus antibodies in the sera of swine, using three extracts from the salivary glands of the parasite (SGE): a complete extract (SGE-1), a soluble antigens extract (SGE-2), and a tissue antigens extract (SGE-3). The results of the present work show that SGE-2 gives the best differentiation between swine bitten by O. erraticus and unbitten swine in the enzyme-linked immunosorbent assay (ELISA). Using this extract, an optical density (OD) five-fold higher than the basal OD indicates that the pigs carry anti- O. erraticus antibodies. A serological study carried out in Salamanca with 8083 sera from 1756 pig farms revealed the presence of the parasite on 135 farms. However, during this study we noticed that some sera of unbitten animals gave false-positive reactions. Western blot analysis of SGE-2 of these false-positive sera demonstrated the same bands (except for two) as the real anti- O. erraticus sera. We observed, in ELISA and Western blot analysis, that such false-positive sera only recognised carbohydrate epitopes on SGE-2. This reactivity disappeared on deglycosylated SGE-2 (SGE-2-P). Therefore, SGE-2-P is the antigen that confers the greatest specificity to serology. In this study it was also observed that the low levels of anti- O. erraticus antibodies found in some cases may be because the swine were bitten some months previously on a different farm or that the current farm harboured only a few specimens of O. erraticus, so pig-tick contact is unlikely and hence the pigs either only develop a primary response or the time between contacts is very long and the levels of antibodies fall. Since pigs could be bitten on a different farm, the presence of low levels of anti- O. erraticus antibodies in pig sera do not necessarily indicate the presence of the tick on the farm where sampling was carried out.

Application of peroxidase labelled antibody assays for detection of porcine IgG antibodies to hog cholera and bovine viral diarrhea viruses

Rapid, sensitive peroxidase labelled antibody (PLA) assays using microtiter systems, were developed for detection of hog cholera virus (HCV) and cross-reacting bovine viral diarrhea virus (BVDV) antibodies in pig sera. HCV-infected pig kidney cell line (PK 15) prepared in microtiter plates were fixed and used in PLA assays. After inoculation with test serum, bound antibodies (HCV/BVDV) were reacted with either horseradish peroxidase (HRP) conjugated anti-porcine immunoglobulin (H & L) or biotinylated protein A (BPA) and subsequent HRP labelled avidin (A). Positive reactions were easily visualized under an inverted light microscope as foci of brown colored cells after enzyme degradation of hydrogen peroxidase in the presence of amino-ethylcarbazole (AEC). The PLA assays were superior to the indirect fluorescent antibody (IFA) test in detecting anti-HCV antibodies in porcine sera collected early after inoculation of pigs with a lapinized HCV vaccine. The performances of the PLA, IFA and FA neutralization (FAN) tests in measuring the immune response in the vaccinated pigs were comparable. Cross-reacting anti-BVDV antibody, as measured by a microtiter serum neutralization (MTSN) test, was not demonstrable in vaccinated pigs until they were challenged with a virulent HCV, 13 weeks later. The PLA assays relative to the IFA test detected more reactive samples among porcine field sera collected from HC-free pigs in Canada. Of 795 samples, 24 (3.01%) were reactive in the PLA employing HRP anti-porcine IgG, and 21 (2.6%) in the PLA, using BPA-HRP-A. When 324 of these sera were screened by the IFA test (using HC antigen), only one sample (0.30%) was found reactive. All but one reactive serum contained demonstrable anti-BVDV antibody as measured by the standard MTSN. The PLA has potential application as a rapid, inexpensive and automated test for seroepidemiological studies of HCV and BVDV in pigs.

Physicochemical characterization of porcine pararotavirus and detection of virus and viral antibodies using cell culture immunofluorescence

A cell culture immunofluorescence (CCIF) assay was optimized for detection of porcine pararotavirus (group C rotavirus) in intestinal contents. The greatest viral infectivity was observed when MA104 cells (5 days after subculturing) were rinsed and refed in serum-free medium before inoculation, pancreatin was added to the inocula, and the inocula were centrifuged onto the cells. Gentamicin treatment of pararotavirus samples to reduce bacterial contamination also reduced the viral infectivity of these samples for MA104 cells. An indirect CCIF assay was used to determine the prevalence of pararotavirus and rotavirus antibodies in pig sera. In pigs from four herds, pararotavirus antibodies were detected in 100% (68 of 68) of adults and 59% (24 of 41) of weanling pigs, while 86% (24 of 28) of nursing pigs from 12 herds had pararotavirus antibodies. The physicochemical properties of pararotavirus were examined and compared with those of group A rotaviruses by using the CCIF assay to quantitate in vitro changes in viral infectivity. Pararotavirus was inactivated (greater than or equal to 99% reduction in titer) by heating to 56 degrees C for 30 min, was slightly labile at pH 3 (16 to 34% reduction in titer), and was stable at pH 5 (0 to 17% reduction in titer) and in either (3 to 19% reduction in titer). One group A rotavirus (Gottfried strain) was stable at 56 degrees C (0% reduction in titer), whereas the OSU strain of group A rotavirus was inactivated at this temperature (99% reduction in titer).

Prevalence of enteroviral and parvoviral antibodies in pig sera

Surveys of serum antibodies to enterovirus serotype 8 and parvovirus were conducted in pigs three to 21 weeks old using the virus neutralisation (VN), enzyme-linked immunosorbent assay (ELISA) and haemagglutination inhibition tests. Antibody levels to enterovirus were at their lowest at four to eight weeks old, increased at 14 to 16 weeks to high levels which were maintained until 21 weeks. Specific immunoglobulin G (IgG) values measured by ELISA paralleled VN values and were similar in two separate farm surveys. Measurement of immunoglobulin M (IgM) levels indicated that enterovirus infection occurred about four weeks old. Sera obtained from a large number of geographically separated farms, from abattoir-bled animals and from colostrum-deprived piglets raised for 33 weeks from birth in an isolated environment were tested by VN. Results revealed that porcine enterovirus serotype 8 is spread widely throughout northern Victorian piggeries and high antibody levels prevail. In contrast to the enterovirus antibody results, parvovirus antibody levels measured in piglets declined from high levels at three to four weeks to undetectable levels from 13 weeks old.

Aujeszky's disease: Blocking ELISA for the detection of serum antibodies

A blocking ELISA for the detection of serum antibodies to Aujeszky's disease virus has been developed. The test was designed in particular with a view to examining large numbers of blood samples. It has been found to be sensitive, specific and precise. In comparison with the serum neutralization test it was superior in detecting low levels of antibodies in pig sera. The blocking ELISA gave higher titre values in serum samples taken early after experimental infection of pigs with Aujeszky's disease virus, than did the serum neutralization test.