Pig Serum Research Articles

ELISA-assay for Pig Taenia solium Cysticercosis by using T18 recombinant antigen

Cysticercosis is caused by infection with the larval stage of tapeworm parasite Taenia solium. This zoonose is a major public health issue with high prevalence in Madagascar, around 21% and 7% to 20% respectively in swine and human. ELISA and Enzyme-linked Immuno-electro Transfer Blot (EITB) remained the main laboratory serological diagnostic tools available in the island. Four native glycoproteins are recognized by antibodies in a majority of sera from patients with cysticercosis and a similar pattern seems to be present in infected pigs. For a better management of this disease in Madagascar, the aim of this study is to set-up and validate the one majority glycoprotein antigen T18 (18KDa) for diagnosis test of pig cysticercosis, produced as recombinant protein. Using a cDNA library prepared from T. solium cysticerci obtained from different regions of Madagascar, we first analyzed the polymorphism of the T18 gene. Then, gene was cloned into the pMAL-c2X plasmid for expression as an N-terminal maltose-binding protein (MBP) fusion protein and C-terminal hexa-His tagged protein. Product protein was then validated by ELISA test of pig sera. Preliminary results show a strong conservation of T18 gene. And indeed larger amounts of soluble recombinant protein were obtained from these antigen. T18 recombinant-ELISA present 70% of sensitivity and 100% of specificity.

Synergistic fermentation of Cordyceps militaris and herbal substrates boosts grower pig antioxidant and immune function

BackgroundPathogenic infections can significantly impact the health of livestock. Traditionally, antibiotic growth promoters (AGPs) have been used in feed to enhance growth performance and disease control. However, concerns regarding antibiotic resistance have led to the exploration of traditional herbal medicine as a natural alternative, guided by the principle of medicine-food homology. The Taguchi method was employed to optimize the culture formula for cordycepin production, an active component of Cordyceps militaris (C. militaris). The influences of C. militaris supplementing solid-state fermentation (CMSSF) in feed on the growth performance and immune responses of grower pigs were evaluated in the present study.ResultsThe C. militaris ethanol extract (CME) displayed potent free radical scavenging activity against 2, 2-Diphenyl-1-picrylhydrazyl (DPPH) and 2,2’-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) after undergoing fermentation. Additionally, the antibacterial testing revealed that CME effectively inhibits the growth of common pig pathogens such as Glaesserella parasuis, Pasteurella multocida, Staphylococcus hyicus, and Streptococcus suis. In lipopolysaccharide (LPS)-treated intestinal porcine enterocyte cell line (IPEC-J2), CME significantly suppressed the production of pro-inflammatory cytokines, including tumor necrosis factor (TNF)-α and interleukin (IL)-6. In addition, higher antioxidative activity was detected as indicated by elevated concentration of superoxide dismutase (SOD) in pig serum. The levels of immunoglobulin M (IgM), IgA, and IgG antibodies, as well as classical swine fever virus (CSFV) antibodies (S/P ratio) in serum were all increased. Growth performance of pigs fed with dietary CMSSF supplementation was improved in comparison with the control.ConclusionsResults demonstrated that CMSSF has the potential to be used as a natural growth promoter to enhance immunity, antioxidation, as well as overall health and growth performance of grower pigs.

Dynamics of PCV2 and PCV3 in the Serum and Oral Fluids of Pigs After PCV2 Vaccination in a Commercial Farm.

This study investigated the dynamics of porcine circovirus type 2 (PCV2) and PCV3 on a commercial farm following PCV2 vaccination. Serum samples from 35 pigs, starting at 3 weeks of age, were collected weekly until 21 weeks of age. Oral fluids from six pens of pigs of the same age were also analyzed. Viral DNA was assessed in pooled sera and individual oral fluid samples, while antibodies (IgG and IgA) were measured in the serum and oral fluids. Productive parameters, including weekly mortality and cumulative mortality, were evaluated. The results revealed that PCV2 and PCV3 co-infection was detected in pigs at 8 weeks of age, with PCV3 being detected in oral fluids two weeks earlier. PCV3 DNA was detected in oral fluids at 4 weeks of age. PCV2 IgG antibodies in the serum increased gradually after vaccination, peaking at 7 weeks of age, then declined and stabilized until 21 weeks of age. PCV3 IgG antibodies fluctuated early but were uniformly positive after 13 weeks of age. In oral fluids, PCV2 IgG and IgA antibodies showed a strong response only at 3 and 23 weeks of age. In contrast, a strong and consistent IgG response was observed in oral fluids in the absence of PCV2 and PCV3 co-infection of pigs at 3 to 11 weeks of age. The farm's productive parameters remained stable throughout the study. These findings suggest that PCV2 and PCV3 co-infection, along with high PCV3 detection levels in serum and oral fluids, may have an impact on the efficacy of PCV2 vaccination.

High-throughput screening for respiratory pathogens within pigs in Denmark; analysis of circulating porcine respiratory coronaviruses and their association with other pathogens

Porcine respiratory coronavirus (PRCV) typically causes subclinical or mild respiratory infections in pigs, but may lead to more severe disease with other factors. PRCV infection in Denmark was initially detected in 1984, but data are lacking about its current prevalence and diversity. Antibodies against PRCV were detected in about 75 % of recent pig sera from Denmark. In addition, pig nasal swab samples were screened for PRCV and 12 other respiratory pathogens using a high-throughput RT-qPCR system. All targeted pathogens were detected but at different prevalences. Significant associations were found between the presence of PRCV and certain other pathogens. From PRCV positive samples, partial spike gene sequences and complete nucleocapsid coding sequences were determined. In phylogenetic analyses, these PRCVs clustered with earlier European PRCVs and were distinct from transmissible gastroenteritis virus. We conclude that PRCV is widespread within the pig population in Denmark. Further studies on the significance of PRCV are warranted.

Identification of linear B cell epitopes on the E146L protein of African swine fever virus with monoclonal antibodies

The outbreak and spread of African swine fever virus (ASFV) have caused considerable economic losses to the pig industry worldwide. Currently, to promote the development of effective ASF vaccines, especially subunit vaccines, more antigenic protein targets are urgently needed. In this work, six transmembrane proteins (I329L, E146L, C257L, EP153R, I177L, and F165R) were expressed in mammalian cell lines and screened with pig anti-ASFV serum. It was found that the E146L protein was an immunodominant protein antigen among the six selected proteins. Moreover, the E146L protein induced antibody responses in all immunized pigs. To gain insight into the antigenic characteristics of the E146L protein, three monoclonal antibodies (mAbs; 12H12, 15G1, and 15H10) were generated by immunizing BALB/c mice with the purified E146L protein. The epitopes of the mAbs were further finely mapped through a peptide fusion protein expression strategy. Finally, the epitopes of the mAbs were identified as 48PDESSIAYMRFRN61 of the mAb 12H12, 138TLTGLQRII146 of the mAb 15G1, and 30GWSPFKYSKGNT41 of the mAb 15H10. Furthermore, the epitope of mAb 15H10 was validated as the immunodominant epitope with ASFV-infected pig sera. The chemically synthesized mAb 15H10 epitope peptide (EP1) exhibited the most extensive immunoreactivity with artificially or naturally ASFV-infected pig sera. The epitope 15H10 is located on the surface of the E146L protein and is highly conserved. These findings provide insight into the structure and function of the E146L protein of ASFV.

Application of Monoclonal Anti-Mycolate Antibodies in Serological Diagnosis of Tuberculosis.

Patient loss to follow-up caused by centralised and expensive diagnostics that are reliant on sputum is a major obstacle in the fight to end tuberculosis. An affordable, non-sputum biomarker-based, point-of-care deployable test is needed to address this. Serum antibodies binding the mycobacterial cell wall lipids, mycolic acids, have shown promise as biomarkers for active tuberculosis. However, anti-lipid antibodies are of low affinity, making them difficult to detect in a lateral flow immunoassay-a technology widely deployed at the point-of-care. Previously, recombinant monoclonal anti-mycolate antibodies were developed and applied to characterise the antigenicity of mycolic acid. We now demonstrate that these anti-mycolate antibodies specifically detect hexane extracts of mycobacteria. Secondary antibody-mediated detection was applied to detect the displacement of the monoclonal mycolate antibodies by the anti-mycolic acid antibodies present in tuberculosis-positive guinea pig and human serum samples. These data establish proof-of-concept for a novel lateral flow immunoassay for tuberculosis provisionally named MALIA-mycolate antibody lateral flow immunoassay.

Prevalence and Genetic Variation Investigation of the Pseudorabies Virus in Southwest China.

In 2022, a significant PRV outbreak in a southwestern China pig farm led to a high incidence of sow abortion. A serological analysis using gE antigen-based ELISA revealed a high prevalence (69.30%) of PRV gE antibodies among the affected pigs, with a significant variation across different pig populations (1.11-76.12%). We collected additional 5552 pig serum samples and 580 pig cerebrospinal fluid (CSF) samples from various pig farms in Southwest China between 2022 and 2024. The seropositive rates for PRV gE antibodies ranged from 2.36% and 8.65% in the serum samples, while the positive detection rates for the PRV gE gene in the cerebrospinal fluid samples, as determined by PCR, were between 1.06% and 2.36%. The PCR analysis and sequencing of the PRV gB, gC, gE, and TK genes from eight randomly selected samples identified two distinct strains, CQ1 and CQ2. CQ1's gC gene showed similarity to the vaccine strain Bartha, while the other genes aligned with Chinese classical strains, suggesting its potential genetic recombination. CQ2 aligned with the Chinese classical strain SC. Although the overall PRV infection in Southwest China's pig farms is relatively low, occasional outbreaks with high positivity rates are observed. These findings highlight the necessity for increased surveillance and stringent control measures to safeguard the swine industry.

Molecular detection and genetic characteristics of porcine reproductive and respiratory syndrome virus in central China

Porcine reproductive and respiratory syndrome caused by porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most economically devastating viral diseases in the global pork industry. To further clarify the epidemic characteristics of the virus, 365 clinical samples were collected from diseased pigs suffering from abortion and respiratory disease from 2018 to 2023 on 63 pig farms in Henan and Shanxi provinces, and screened for the presence of PRRSV using reverse transcription-polymerase chain reaction (RT-PCR). A total of 62 clinical samples (62/365, 16.99 %) were positive for PRRSV, and subsequently, full-length ORF5 gene sequences of 29 PRRSV strains and the complete genome sequence of one PRRSV HeN-HC isolate were obtained and analyzed. Phylogenetic analysis based on the ORF5 gene showed that 22 of the 29 PRRSV2 strains belonged to sublineage 1.8 (NADC30-like), 5 belonged to sublineage 8.5 (HP-PRRSV), and 2 belonged to sublineage 5.1 (VR-2332-like), indicating that both HP-PRRSV and NADC30-like strains were mainly circulating in Henan and Shanxi provinces. Compared to VR-2332 strain, different types of amino acid mutations were found in the GP5 protein of these 29 strains, and the amino acid deletions were displayed in the Nsp2 protein of the HeN-HC isolate, leading to the variation of protein structures. It is noteworthy that recombination events were identified in the HeN-Ping and HeN-B strains. In addition, a total of 60, 094 pig serum samples from Henan province were collected, and the positive rate of specific antibodies against PRRSV was 86.37 % from 2019 to 2022, and 86.66 %, 84.85 %, 87.54 % and 86.30 % in 2019, 2020, 2021 and 2022, respectively. Overall, this study provides valuable insights into the molecular epidemiology and evolution of PRRSV circulating in central China.

Development of a blocking ELISA for detection of Japanese encephalitis virus antibodies in pig and horse sera

Japanese encephalitis virus (JEV) is a mosquito-borne virus that can infect pigs, horses, and other mammals, including humans. Sero-epidemiological investigations of JEV have been performed using hemagglutination inhibition (HI), virus neutralization (VN) tests and enzyme-linked immunosorbent assay (ELISA). A need exists for a new ELISA that can detect JEV antibodies in the sera of several animal species. We aimed to develop a blocking ELISA (B-ELISA) for detecting JEV antibodies in pig and horse serum samples. JEV antibodies in 218 pig and 315 horse serum samples were measured using HI and VN tests. The purified KV1899-306 strain was used as an antigen for B-ELISA. The purified antibody (7A13) was conjugated with horseradish peroxidase and used as a detector antibody. The sera of pigs and horses to measure antibody against JEV were subjected to B-ELISA and analyzed. The B-ELISA had a diagnostic sensitivity of 94.6% to 100%, a specificity of 91.2 to 100%, and an accuracy of 94.9 to 98.6% compared with those of the HI and VN tests in pig and horse sera. The B-ELISA had a higher correlation with pig sera (r = 0.89 and 0.90 for VN and HI) than with horse sera (r = 0.75 and to 0.79). The new B-ELISA could be useful in the sero-surveillance of JEV in pig and horse sera and replace indirect ELISA.

Dual-mode colorimetric-fluorometric immunoassay for foodborne parasitic infection

Immunoassays are the primary method used for detection of foodborne parasitic infection. The nanomaterials used in these assays often present challenges such as complex preparation requirements and low stability during long-term storage and transport. Herein, based on cost-effective K3[Fe(CN)6]-mediated in-situ generated CuFe Prussian blue analogues (CuFe-PBA) and quinoxaline derivatives with high fluorescence, a dual-mode immunoassay was proposed for foodborne parasitic infection. This platform is relied on the restoration of K3[Fe(CN)6] by ascorbic acid (AA). In addition to this process, K4[Fe(CN)6]-triggered the generation of CuFe-PBA, and the enhanced fluorogenic reaction of dehydrogenated AA and o-phenylenediamine (OPD) occur sequentially. Without complex probes preparation, the assay enabled the rapid on-site detection of ALP and foodborne parasitic infection through a colorimetric mode accessible via smartphone and offered enhanced sensitivity through a fluorescent mode. The method was used for the detection of ALP, achieving detection limits of 0.38 U/L and 0.03 U/L for colorimetric and fluorescence modes, respectively. Subsequently, the dual-mode immunoassay was applied to Trichinella spiralis infection. The sensitivity and specificity of both the colorimetric and fluorescence modes were 100 % in 950 pig serum samples. This immunosensor provides a universal analysis tool for the accurate, cost-effective and highly sensitive detection of foodborne parasitic infection

Immune responses induced by a recombinant C-strain of classical swine fever virus expressing the F317L protein of African swine fever virus

African swine fever (ASF), a highly infectious and devastating disease affecting both domestic pigs and wild boars, owes its etiology to African swine fever virus (ASFV). ASFV encodes more than 165 proteins. However, novel immunogenic proteins remain unknown. This study aimed to determine the antigenicity of the F317L protein (pF317L) of ASFV. The results revealed that pF317L was able to react with convalescent pig sera, indicating that pF317L could be a candidate antigen. The antigenic potential of pF317L expressed by rHCLV-F317L, a recombinant virus in the backbone of C-strain (a lapinized live attenuated classical swine fever virus) was further investigated in rabbits and pigs. The results revealed that antibodies and cell-mediated immune responses against pF317L were induced in either rabbits or pigs inoculated with rHCLV-F317L. Importantly, anti-pF317L antibodies from rabbits or pigs immunized with rHCLV-F317L significantly inhibited ASFV replication in vitro. In conclusion, pF317L demonstrates favorable immunogenic properties, positioning it as a promising candidate for the development of protective antigens in the ongoing endeavor to formulate efficacious ASF vaccine strategies.

Simple, rapid and enzyme-free assay for potentiometric determination of L-cysteine based on meso-2,3-dimercaptosuccinic acid self-assembled gold electrode

As a sulfur containing α-amino acid in nature, L-cysteine plays a crucial role in a variety of metabolic pathways with many important physiological functions. However, it is still a great challenge to construct a simple and rapid approach for L-cysteine up to now. In this paper, a simple, rapid and enzyme-free method was developed for potentiometric determination of L-cysteine by using meso-2,3-dimercaptosuccinic acid self-assembled monolayer on gold electrode. The surface morphology and property of self-assembled membrane with and without L-cysteine combined were characterized by means of scanning electron microscopy, X-ray photoelectron spectroscopy, electrochemical impedance spectroscopy, and cyclic voltammetry, respectively. The recognition mechanism may be attributed to formation of hydrogen bonds between meso-2,3-dimercaptosuccinic acid and the target molecule of L-cysteine, producing a double electric layer at the sensing interface, that the membrane potential decreases with increasing concentration of L-cysteine. The assay results showed that good linear potential response to L-cysteine in Tris-HCl buffer solution (pH=7.0) was obtained in a range of 1.0 × 10-8 to 1.0 × 10-3 mol/L, with a slope of −57.00 ± 0.63 mV/-pc (25 °C) and a detection limit of 7.9 × 10-9 mol/L. The electrode possessed fast response time (45 s), good reproducibility, and strong anti-interference performance. Moreover, the electrode can be well applied to the detection of L-cysteine in pig serums with a recovery rate of 94.3 ∼ 106.3%, indicating a promising application prospect.

Ningxiang Pig-Derived Microbiota Affects the Growth Performance, Gut Microbiota, and Serum Metabolome of Nursery Pigs.

The gut microbiota is crucial for maintaining the host's intestinal homeostasis and metabolism. This study investigated the effects of fecal microbiota transplantation (FMT) from Ningxiang pigs on the growth performance, fecal microbiota, and serum metabolites of the same-old DLY pigs. The results indicated that the average daily gain of FMT pigs was significantly greater than that of the control (CON) group. Compared to the CON group, the FMT group significantly improved the apparent digestibility of crude fiber, crude ash, gross energy, and calcium of the pigs. The analysis of serum antioxidant status revealed that the activities of total superoxide dismutase and catalase in the serum of pigs in the FMT group were significantly elevated, whereas the level of malondialdehyde was significantly reduced. Furthermore, 16S rRNA sequencing analysis revealed that the Ningxiang pig-derived microbiota altered the fecal microbiota structure and modulated the diversity of the gut microbiota in the DLY pigs. Untargeted LC-MS metabolomics demonstrated that pigs in the FMT group exhibited distinct metabolomic profiles compared to those in the CON group. Significant changes were observed in key metabolites involved in amino acid, lipid, and carbohydrate metabolism. Additionally, a correlation analysis between serum differential metabolites and the gut microbiota revealed that the relative abundance of Lachnospiraceae_NK4A136_group and Corynebacterium was highly correlated with lipid compounds. In conclusion, Ningxiang pig-derived microbiota can alleviate oxidative stress and enhance growth performance in DLY pigs by modulating their gut microbiota and metabolic features.

Seroprevalence of swine hepatitis E virus and the farmers' potential risk of infection in the Province of Bali, Indonesia.

Hepatitis E virus (HEV) infection formerly and predominantly occurred in rural areas. However, it has recently been spread to urban and peri-urban areas. This study aimed to estimate the seroprevalence of HEV in pigs collected from urban and rural areas in Bali. The potential of the pig farmers' risk level for being exposed to HEV and the virus transmitted to them in association with their pig-rearing practices was also assessed. A total of 183 pigs from 68 herds were sampled in this study, with 91 pigs collected from Denpasar as the representative samples of urban areas and 92 pigs from Karangasem Regency as the representative samples from rural areas. Sera from the sampled pigs were collected and immunoglobulin G antibodies against HEV were detected using a commercial enzyme-linked immunosorbent assay. A questionnaire was prepared for interviewing the farmers. Bivariable and multivariable logistic regression analyses were performed to identify the putative factors associated with seropositivity. Meanwhile, the potential risk-incurring practices of the farmers for HEV being transmitted to them from their pig-rearing practices were assessed by scoring their responses from the interview. Overall, 23.5% (43/183) (95% confidence interval [CI]: 17.6-30.3) pig sera tested were detected to have the antibodies against HEV. Among 68 pig herds, 36.8% (25) (95% CI: 25.4-49.3) of them had antibodies in at least one pig sampled from each herd. Pigs sampled from Karangasem were 5 times (Odds ratio [OR] 5.34, 95% CI: 2.27-13.54, p < 0.001) more likely to be seropositive than pigs collected from Denpasar. However, no difference was found in the seropositivity to HEV in pig herds between Denpasar and Karangasem (p = 0.05). In assessing the pig rearing management factors, pig farmers from Denpasar were 3 times (OR 3.0, 95% CI: 1.07-8.52, p = 0.05) more likely to rear pigs for economic investment compared to the farmers from Karangasem. Regarding anticipating pig diseases that can be transmitted to humans, farmers from Denpasar were 6 times (OR 5.72, 95% CI: 1.48-26.7, p = 0.0074) more likely to anticipate zoonotic diseases compared to the farmers from Karangasem. Similarly, pig farmers from Denpasar were 3 times (OR 3.29, 95% CI: 1.08-10.23, p = 0.035) more likely to anticipate pig diseases that could be transmitted to humans than the farmers from Karangasem. Pig farmers from Denpasar had 4 times the odds (OR 4.49, 95% CI: 1.11-18.19, p = 0.03) of washing their hands after going to the pigpens compared to the farmers from Karangasem. All the participants were categorized as being at high risk of HEV exposure and transmission. IgG antibodies against HEV were detected among pigs reared in rural areas of Karangasem and those reared in urban areas of Denpasar. This suggests that the risk of HEV exposure and transmission in these areas is not negligible. To minimize the risk, public education on zoonotic diseases, including HEV infection, transmission, and prevention, needs to be implemented and particularly targeted to local pig farmers.

Differences in intestinal and renal Ca and P uptake in three different breeds of growing-finishing pigs

This study investigated the differences in bone growth and turnover and calcium (Ca) and phosphorus (P) uptake among three different breeds of growing-finishing pigs. Ninety healthy Duroc, Xiangcun black (XCB), and Taoyuan black (TYB) pigs (30 pigs per breed) at 35 day-old (D) with the average body weight (BW) of their respective breed were assigned and raised to 185 D. The results showed that Duroc pigs had higher bone weight and length than the XCB and TYB pigs at 80, 125, and 185 D and the bone index at 185 D (p < 0.05). Duroc pigs had higher bone mineral densities (femur and tibia) compared with the other two breeds at 80 D and 125 D, whereas TYB pigs had higher mineral content and bone breaking load (rib) compared with the other two breeds at 185 D (p < 0.05). The bone morphogenetic protein-2 and osteocalcin concentrations were higher, and TRACP5b concentration was lower in serum of TYB pigs at 125 D (p < 0.05). Meanwhile, 1,25-dihydroxyvitamin D3, parathyroid hormone, thyroxine, and fibroblast growth factor 23 concentrations were higher in serum of TYB pigs at 185 D (p < 0.05). The TYB pigs had higher apparent total tract digestibility of P at 80 D and 185 D and bone Ca and P contents at 185 D in comparison to the Duroc pigs (p < 0.05). Furthermore, gene expressions related to renal uptake of Ca and P differed among the three breeds of pigs. Collectively, Duroc pigs have higher bone growth, whereas TYB pigs have a higher potential for mineral deposition caused by more active Ca uptake.

Fibronectin type III domain-containing protein 5 (FNDC5)-like immunoreactivity and mRNA abundance in domestic animal tissues

Irisin is a 112-amino acid peptide hormone that is cleaved from fibronectin type III domain-containing protein 5 (FNDC5), a type I transmembrane protein abundantly found in muscle tissue. Irisin is a putative mediator of the benefits of exercise, neuroprotection, bone growth, and cardiac health. However, few studies have focused on irisin in domestic animals. Further, whether processed irisin is detectable in domestic animal tissues remains uncertain. To address this, we determined FNDC5 mRNA and protein concentration in anatine (duck) and porcine (pig) skeletal muscle, and in equine (horse), swine, and anatine serum samples. RT-PCR analysis identified FNDC5 mRNA in all pig and duck skeletal muscle samples. An approximately 25 kDa band representing FNDC5 was detected in both pig and duck skeletal muscle. Fluorescence immunohistochemistry using a rabbit monoclonal FNDC5/irisin primary antibody and a goat polyclonal anti-rabbit secondary antibody localized FNDC5/irisin-like immunoreactivity in both the glandular and muscular regions of pig stomach. FNDC5/irisin-like immunoreactivity was also identified in horse, pig, and duck serum using a multispecies irisin ELISA. The average values of irisin-like immunoreactivity were 13.7 (duck), 15.4 (horse), and 7.0 (pig) ng/mL in samples tested. Our results support the presence of irisin precursor in several domestic animals. Processed irisin, however, was not detectable. Further studies are required to validate reliable tools to detect and quantify processed irisin in domestic animals.

Development of an enzyme-linked immunosorbent assay using a monoclonal antibody to a dominant epitope in non-structural protein 4 of porcine reproductive and respiratory syndrome virus

Porcine reproductive and respiratory syndrome (PRRS) caused by the PRRS virus (PRRSV) is one of the most severe swine diseases causing great economic losses for the international swine industry. Non-structural protein 4 (NSP4) is critical to the life cycle of PRRSV and contains dominant B cell epitopes. This study prepared a monoclonal antibody against Nsp4, and 2D11, which contained the sequence 138KQGGGIVTRPSGQFCN153, was confirmed as the epitope. A 2D11-based double antibody sandwich enzyme-linked immunosorbent assay (dasELISA) was next developed with a cut value of 0.1987. A total of 1354 pig serum samples were detected by dasELISA and compared to a commercial ELISA kit (N-coated iELISA), resulting in a positive coincidence rate of 98.8% and negative coincidence rate of 96.9%. A total of 119 sera were positive by dasELISA while negative by iELISA. Higher positive rates by dasELISA were found in pig farms where PRRSV antibody levels varied widely. These results indicated that the dasELISA was a useful tool to detect PRRSV antibody in clinical samples.

Establishment of an ELISA Based on a Recombinant Antigenic Protein Containing Multiple Prominent Epitopes for Detection of African Swine Fever Virus Antibodies.

African swine fever virus (ASFV) poses a significant threat to the global pig industry, necessitating accurate and efficient diagnostic methods for its infection. Previous studies have often focused on a limited number of epitopes from a few proteins for detecting antibodies against ASFV. Therefore, the current study aimed to use multiple B-cell epitopes in developing an indirect Enzyme-Linked Immunosorbent Assay (ELISA) for enhanced detection of ASFV antibodies. For the expression of recombinant protein, k3 derived from 27 multiple peptides of 11 ASFV proteins, such as p72, pA104R, pB602L, p12, p14.5, p49, pE248R, p30, p54, pp62, and pp220, was used. To confirm the expression of the recombinant protein, we used the Western blotting analysis. The purified recombinant K3 protein served as the antigen in our study, and we employed the indirect ELISA technique to detect anti-ASFV antibodies. The present finding showed that there was no cross-reactivity with antibodies targeting Foot-and-mouth disease virus (FMDV), Porcine circovirus type 2 (PCV2), Pseudorabies virus (PRV), Porcine reproductive and respiratory syndrome virus (PRRSV), and Classical swine fever virus (CSFV). Moreover, the current finding was sensitive enough to find anti-ASFV in serum samples that had been diluted up to 32 times. The test (k3-iELISA) showed diagnostic specificity and sensitivity of 98.41% and 97.40%, respectively. Moreover, during the present investigation, we compared the Ingenasa kit and the k3-iELISA to test clinical pig serum, and the results revealed that there was 99.00% agreement between the two tests, showing good detection capability of the k3-iELISA method. Hence, the current finding showed that the ELISA kit we developed can be used for the rapid detection of ASFV antibodies and used as an alternative during serological investigation of ASF in endemic areas.

272 Effects of increasing folic acid on nursery pig growth performance and serum homocysteine

Abstract Barrows (n = 350; DNA 200×400; [n = 2,172; initial body weight (BW) = 6.0 ± 0.06 kg] were used in a 38-d growth trial to determine the effects of increasing folic acid on nursery pig growth performance and serum homocysteine. Pigs were weaned at 21 d of age and randomly allotted to 1 of 5 dietary treatments in a completely randomized design. A total of 70 pens were used with 5 pigs per pen and 14 replications per treatment. Dietary treatments were corn-soybean meal-based and consisted of increasing added folic acid: 0, 5, 10, 20, or 40 mg/kg. Treatment diets were fed in three phases from d 0 to 10 (phase 1), d 10 to 23 (phase 2), and d 23 to 38 (phase 3) after weaning. For phase 1, there were no differences (P &amp;gt; 0.10) in BW, average daily gain (ADG) and average daily feed intake (ADFI) across treatments (Table). However, increasing folic acid resulted in decreased gain to feed ratio (G:F; linear, P = 0.032). For phase 2, there was a marginally significant response in BW, ADG, and ADFI where performance was decreased as folic acid increased with the poorest performance observed when pigs were fed 20 mg/kg (quadratic, P ≤ 0.079). No treatment differences (P &amp;gt; 0.10) were observed for G:F. For phase 3 and overall (d 0 to 38), final BW, ADG, ADFI, and G:F were reduced with increasing folic acid with the poorest performance observed when pigs were fed 20 mg/kg (quadratic, P ≤ 0.049). No differences (P &amp;gt; 0.10) were observed for mortality or removals across treatments with a very low amount of overall mortality. On d 10 and 23, blood samples were collected from 70 pigs to determine serum homocysteine concentrations. A marginally significant treatment × d interaction was observed (linear folic acid × d, P = 0.069). An increase (linear, P = 0.037) in homocysteine concentrations was observed as folic acid increased from 0 to 40 mg/kg in the diet on d 10; however, no differences were observed across increasing folic acid treatments on d 23 (P = 0.450). Pigs had increased (P &amp;lt; 0.001) homocysteine concentrations on d 10 compared with d 23. In summary, the addition of folic acid resulted in reduced growth performance with the greatest impact observed when pigs were fed 20 mg/kg.

PSI-16 Effect of HiPhorius phytase on finishing pig growth performance and bone mineralization with the use of two diet formulation strategies

Abstract Two experiments were conducted to determine the effect of HiPhorius (dsm-firmenich, Parsippany, NJ) phytase on finishing pig growth performance, serum chemistry, bone mineralization, and carcass characteristics. In Exp. 1, 1,161 pigs (PIC 337 × 1050, initially 36.7 ± 0.48 kg) were used in a 105-d trial. There were 27 pigs per pen and 10 or 11 replications per treatment. Treatments consisted of: 1) Control diet with no added phytase and formulated to NRC (2012) requirement estimates for standardized total tract digestible (STTD) P; 2) 600 FYT/kg added phytase formulated to the same STTD P as the control diet considering a release of 0.13% STTD P and 0.095% STTD Ca; 3) 1,000 FYT/kg added phytase formulated to the same STTD P as the control diet considering release of 0.16% STTD P and 0.107% STTD Ca, and 4) high STTD P (no phytase; approximately 22% above NRC requirement estimates). All diets were formulated to a 1.30:1 STTD Ca:STTD P ratio, but analysis suggested all phases were greater than the formulated target. Overall, pigs fed NRC (2012) or increased STTD P had increased average daily gain (ADG) and hot carcass weight (HCW; P &amp;lt; 0.05) compared with pigs fed the treatments with added phytase. Pigs fed NRC (2012) STTD P had greater (P &amp;lt; 0.05) gain to feed ratio (G:F) compared with those with 1,000 FYT/kg with other treatments intermediate. In Exp. 2, 1,160 pigs (PIC 337 × 1050, initially 75.9 ± 1.32 kg) were used in a 58-d trial. There were 27 pigs per pen and 11 replications per treatment. Treatments were the same as in Exp.1 except diets were formulated to the same total Ca:P ratio [1.15:1 from 75 to 100 kg and 1.12:1 from 100 to 136 kg body weight (BW)] without a STTD Ca release consideration from phytase; however, diets analyzed with a wider Ca:P compared with the expected value (Table 1). Overall, there were no differences in ADG, average daily feed intake (ADFI) or G:F among treatments (P &amp;gt; 0.10). For pigs fed NRC or increased STTD P, there was an increase (P &amp;lt; 0.05) in metacarpal bone density, and a tendency for increased (P &amp;lt; 0.10) bone ash weight and percentage bone ash compared with pigs fed treatments containing phytase. In conclusion, regardless of diet formulation strategy, pigs fed diets with phytase had decreased growth performance (Exp.1) and bone mineralization (Exp.2), which was unexpected and could be a result of the nutrient release values attributed to the phytase being overestimated, not enough phytate-bound P that is phytase-sensitive present in the diets, or too wide of a Ca:P ratio leading to reduced phytase activity in the analyzed diet samples compared with the intended ratio based on diet formulation.