Swine Antibodies Research Articles

Immunogenicity of novel variants of the chimeric chP3R99 anti-atherogenic antibody in insulin-resistant rats and swine

Immunogenicity of novel variants of the chimeric chP3R99 anti-atherogenic antibody in insulin-resistant rats and swine

Approaching the complexity of Crimean-Congo hemorrhagic fever virus serology: A study in swine

Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne zoonotic orthonairovirus of public health concern and widespread geographic distribution. Several animal species are known to seroconvert after infection with CCHFV without showing clinical symptoms. The commercial availability of a multi-species ELISA has led to an increase in recent serosurveillance studies as well as in the range of species reported to be exposed to CCHFV in the field, including wild boar (Sus scrofa). However, development and validation of confirmatory serological tests for swine based on different CCHFV antigens or test principles are hampered by the lack of defined control sera from infected and non-infected animals. For the detection of anti-CCHFV antibodies in swine, we established a swine-specific in-house ELISA using a panel of swine sera from CCHFV-free regions and regions with reported CCHFV circulation. We initially screened more than 700 serum samples from wild boar and domestic pigs and observed a correlation of ≃67% between the commercial and the in-house test. From these sera, we selected a panel of 60 samples that were further analyzed in a newly established indirect immunofluorescence assay (iIFA) and virus neutralization test. ELISA-non-reactive samples tested negative. Interestingly, only a subset of samples reactive in both ELISA and iIFA displayed CCHFV-neutralizing antibodies. The observed partial discrepancy between the tests may be explained by different test sensitivities, antibody cross-reactivities or suggests that the immune response to CCHFV in swine is not necessarily associated with eliciting neutralizing antibodies. Overall, this study highlights that meaningful CCHFV serology in swine, and possibly other species, should involve the performance of multiple tests and careful interpretation of the results.

Sequential vaccinations with divergent H1N1 influenza virus strains induce multi-H1 clade neutralizing antibodies in swine

Vaccines that protect against any H1N1 influenza A virus strain would be advantageous for use in pigs and humans. Here, we try to induce a pan-H1N1 antibody response in pigs by sequential vaccination with antigenically divergent H1N1 strains. Adjuvanted whole inactivated vaccines are given intramuscularly in various two- and three-dose regimens. Three doses of heterologous monovalent H1N1 vaccine result in seroprotective neutralizing antibodies against 71% of a diverse panel of human and swine H1 strains, detectable antibodies against 88% of strains, and sterile cross-clade immunity against two heterologous challenge strains. This strategy outperforms any two-dose regimen and is as good or better than giving three doses of matched trivalent vaccine. Neutralizing antibodies are H1-specific, and the second heterologous booster enhances reactivity with conserved epitopes in the HA head. We show that even the most traditional influenza vaccines can offer surprisingly broad protection if they are administered in an alternative way.

Comparative Analysis of Swine Antibody Responses following Vaccination with Live-Attenuated and Killed African Swine Fever Virus Vaccines.

African swine fever virus (ASFV) is circulating in many swine-producing countries, causing significant economic losses. It is observed that pigs experimentally vaccinated with a live-attenuated virus (LAV) but not a killed virus (KV) vaccine develop solid homologous protective immunity. The objective of this study was to comparatively analyze antibody profiles between pigs vaccinated with an LAV vaccine and those vaccinated with a KV vaccine to identify potential markers of vaccine-induced protection. Thirty ASFV seronegative pigs were divided into three groups: Group 1 received a single dose of an experimental LAV, Group 2 received two doses of an experimental KV vaccine, and Group 3 was kept as a non-vaccinated (NV) control. At 42 days post-vaccination, all pigs were challenged with the parental virulent ASFV strain and monitored for 21 days. All pigs vaccinated with the LAV vaccine survived the challenge. In contrast, eight pigs from the KV group and seven pigs from the NV group died within 14 days post-challenge. Serum samples collected on 41 days post-vaccination were analyzed for their reactivity against a panel of 29 viral structural proteins. The sera of pigs from the LAV group exhibited a strong antibody reactivity against various viral structural proteins, while the sera of pigs in the KV group only displayed weak antibody reactivity against the inner envelope (p32, p54, p12). There was a negative correlation between the intensity of antibody reactivity against five ASFV antigens, namely p12, p14, p15, p32, and pD205R, and the viral DNA titers in the blood of animals after the challenge infection. Thus, antibody reactivities against these five antigens warrant further evaluation as potential indicators of vaccine-induced protection.

Serosurveillance for Severe Acute Respiratory Syndrome Coronavirus 2 Antibody in Feral Swine and White-Tailed Deer in Texas.

Background: Serological evidence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has been reported in white-tailed deer (WTD) in the United States and Canada. Even though WTD are susceptible to SARS-CoV-2 infection, there is no evidence of infection by this virus in other mammalian species that might interact with WTD in nature. Similar to WTD, feral swine are widely distributed and generally occupy the same range as WTD in Texas. The objective of this study was to determine the prevalence of SARS-CoV-2 neutralizing antibody in WTD during 2020 and 2021 and determine the prevalence of SARS-CoV-2 neutralizing antibody in feral swine during 2018 (prepandemic period) and from March 2020 to February 2021 (pandemic period) in Travis County, Texas. Materials and Methods: Sera samples were collected from hunter-killed WTD and feral swine during the prepandemic and pandemic period and tested for SARS-CoV-2 antibody by a plaque reduction neutralization assay in Vero cells. Results: SARS-CoV-2 antibody was not detected in any of the 166 feral swine sera samples, including 24 samples collected during the prepandemic and 142 samples collected during the pandemic period. Furthermore, SARS-CoV-2 antibody was not detected in the 115 WTD samples collected during late 2020, but antibody was detected in WTD in early 2021. Conclusions: The results indicated that SARS-CoV-2 infection of WTD occurred during early 2021 in Travis County, Texas, but serological evidence of SARS-CoV-2 infection was not detected in the feral swine samples collected from the same locality and during the same time period of the collection of WTD samples.

The Presence of Virus Neutralizing Antibodies Is Highly Associated with Protection against Virulent Challenge in Domestic Pigs Immunized with ASFV live Attenuated Vaccine Candidates.

African swine fever virus (ASFV) is currently producing a pandemic affecting a large area of Eurasia, and more recently, the Dominican Republic in the Western Hemisphere. ASFV is a large and structurally complex virus with a large dsDNA genome encoding for more than 150 genes. Live attenuated virus strains can induce protection in domestic swine against disease produced by homologous virulent parental viruses. The roles of the different immune mechanisms induced by the attenuated strains in protection still need to be understood. In particular, the role of ASFV neutralizing antibody in protection still is an important controversial issue to be elucidated. Here we present the development of a novel methodology to detect virus neutralizing antibodies based on the reduction of virus infectivity in a Vero cell adapted ASFV strain. The described method was used to assess levels of virus neutralizing antibodies in domestic swine inoculated with live attenuated ASFV. Results demonstrated a high association between the presence of virus neutralizing antibodies and protection in 84 animals immunized with the recombinant vaccine candidates ASFV-G-Δ9GL/ΔUK or ASFV-G-ΔI177L. To our knowledge, this is the first report demonstrating an association between virus neutralizing antibodies and protection against virulent challenge in such a large number of experimental individuals.

Hepatitis E virus in Cuba: A cross-sectional serological and virological study in pigs and people occupationally exposed to pigs.

Surveillance of hepatitis E virus (HEV) in risk groups is an important strategy to monitor its circulation pattern and to timely detect changes thereof. The aims of this cross-sectional study were to estimate the prevalence of HEV infections in pigs and humans from different regions of the country, to identify risk factors for increasing anti-HEV IgG prevalence and to characterize HEV strains. The presence of anti-HEV antibodies was assessed by commercial ELISA in serum samples from the general population, farm and slaughterhouse employees, as well as pigs sampled in the three regions of Cuba from February to September 2016. Overall, individuals with occupational exposure to swine or swine products (70/248, 28.2%) were 4 times more likely to be seropositive compared to the general population (25/285, 8.7%; OR: 4.18; p< .001). Within the risk group, risk factors included age, number of years working in a professional activity with direct exposure to swine, geographic region and distance between residence and closest professional swine setting, while wearing gloves had a protective effect. Prevalence of total anti-HEV antibodies in swine was 88.2% (165/187) and HEV RNA was detected by real-time RT-PCR in 9.2% (16/173) swine stools. All HEV strains sequenced clustered within genotype 3. Some strains clearly belonged to subtype 3a, while another group of strains was related with subtypes 3b and 3k but partial HEV sequences did not allow unequivocal subtype assignment. These findings suggest that the high HEV exposure in Cuban individuals with swine-related occupations could be due to enzootic HEV in certain regions, direct contact with infectious animals or their products as well as environmental contamination.

The Decoy Epitopes Elicit Swine Antibodies by Connecting to the Carboxyl-terminal Epitope of the Capsid Protein of Porcine Circovirus Type 2

The Decoy Epitopes Elicit Swine Antibodies by Connecting to the Carboxyl-terminal Epitope of the Capsid Protein of Porcine Circovirus Type 2

Development and Validation of Indirect Enzyme-Linked Immunosorbent Assays for Detecting Antibodies to SARS-CoV-2 in Cattle, Swine, and Chicken.

Multiple domestic and wild animal species are susceptible to SARS-CoV-2 infection. Cattle and swine are susceptible to experimental SARS-CoV-2 infection. The unchecked transmission of SARS-CoV-2 in animal hosts could lead to virus adaptation and the emergence of novel variants. In addition, the spillover and subsequent adaptation of SARS-CoV-2 in livestock could significantly impact food security as well as animal and public health. Therefore, it is essential to monitor livestock species for SARS-CoV-2 spillover. We developed and optimized species-specific indirect ELISAs (iELISAs) to detect anti-SARS-CoV-2 antibodies in cattle, swine, and chickens using the spike protein receptor-binding domain (RBD) antigen. Serum samples collected prior to the COVID-19 pandemic were used to determine the cut-off threshold. RBD hyperimmunized sera from cattle (n = 3), swine (n = 6), and chicken (n = 3) were used as the positive controls. The iELISAs were evaluated compared to a live virus neutralization test using cattle (n = 150), swine (n = 150), and chicken (n = 150) serum samples collected during the COVID-19 pandemic. The iELISAs for cattle, swine, and chicken were found to have 100% sensitivity and specificity. These tools facilitate the surveillance that is necessary to quickly identify spillovers into the three most important agricultural species worldwide.

Development of a chemiluminescence immunoassay to accurately detect African swine fever virus antibodies in serum

The outbreak of African swine fever (ASF) has caused significant economic losses to animal husbandry worldwide. Currently, there is no effective vaccine or treatment available to control the disease, and therefore, efficient disease control is dependent on early detection and diagnosis of ASF virus (ASFV). In this study, a chemiluminescent immunoassay (CLIA) was developed using the ASFV protein p54 as a serum diagnostic antigen and an anti-p54 monoclonal antibody. After optimizing the working parameters of the CLIA, the sensitivity of the established CLIA was 1:128, ASFV-specific serum antibody was identified, and there was no cross-reaction with other swine virus antibodies. After testing 49 clinical serum samples, the consistency rate between the CLIA and the World Organization for Animal Health (OIE) recommended commercial kit was 100 %. Thus, this CLIA had a high degree of specificity, sensitivity, and reliability, and could be used as a rapid detection method for epidemiological investigations of ASFV infection.

Rapid and visual detection of African swine fever virus antibody by using fluorescent immunochromatography test strip

African swine fever virus (ASFV) is a large and complex DNA virus that causes a highly contagious and often lethal swine viral disease, for which no vaccine and effective treatments are available yet. Hence, ASFV presents significant economic consequences for the swine industry. A rapid and simple diagnostic method is urgently needed to monitor ASFV-specific antibodies for controlling the spread of ASFV. In this study, we chose the truncated p54 protein as an antigen and combined it with Eu-doped fluorescent microspheres as tracers to detect anti-ASFV antibodies specifically. Results showed that the truncated p54 protein had high specificity to ASFV antibody and had no cross-reactions with other swine virus antibodies. The results between our fluorescent immunochromatography test strip (FICTS) and commercial ELISA kits showed high consistency. The proposed FICTS offers a rapid, sensitive, specific, and visual method for ASFV antibody detection and shows great potential for ASF epidemic surveillance and control.

Specimen Types, Collection, and Transport for Influenza A Viruses of Swine.

Detection of influenza A virus (IAV), viral antigen, nucleic acid, or antibodies in swine is dependent upon the collection of the appropriate specimen type, the quality of the specimen, and the proper storage and handling of the specimen. The diagnostic tests to be performed should be considered prior to specimen collection. Sera are acceptable specimens for ELISA or hemagglutination inhibition tests but not for real-time RT-PCR. Likewise, swabs, wipes, and/or tissues are acceptable for real-time RT-PCR and virus isolation. The specimen type will also depend on the age of the swine being tested; oral fluids can be successfully collected from weaned pigs usually greater than 3 weeks of age, whereas nasal or oral swabs should be collected from suckling pigs in the first weeks of life. The sensitivity of the RT-PCR test is such that IAV can be detected in not only the pig itself but also on surfaces that the pig contacts and in the air. This chapter will outline the collection of different specimen types and procedures for proper specimen handling.

Development of recombinant nonstructural 1 protein based indirect enzyme linked immunosorbent assay for sero-surveillance of Japanese encephalitis in swine

Japanese encephalitis virus (JEV) causes severe neurological disease in humans, especially among children. The disease is endemic in several South Asian countries including India. Swine play a major role as amplifier host for JEV and act as a source of infection to humans through mosquito bite. Early detection of either virus or antibodies in swine will aid to undertake control measures to prevent virus spread to humans. Swine seldom show symptoms of JEV infection and the viraemic phase lasts for a short period of 3 to 4 days indicating the potential of detection of antibodies, which remain for relatively longer period, as a suitable alternative. Cost effective and sensitive assays for the detection of JEV antibodies in swine are not available indigenously. Hence, we have developed a recombinant nonstructural protein 1 (rNS1) based enzyme linked immunosorbent assay for the detection of IgG antibodies against JEV in swine. The test is robust, highly sensitive (91%), specific (97%), reproducible and affordable. Field validation of the assay was done by screening 3628 swine Serum samples collected from different parts of India. The overall sero-positivity was found to be 32.22%. The developed ELISA can be readily incorporated into surveillance programs for detection of Japanese encephalitis virus activity in swine population thereby aiding in prediction of outbreaks in humans.

Development and application of a monoclonal-antibody-based blocking ELISA for detection of Japanese encephalitis virus NS1 antibodies in swine.

Japanese encephalitis virus (JEV) is a zoonotic pathogen transmitted by Culex mosquitoes and is the leading cause of viral encephalitis in humans. JEV infection of swine, which are the main amplifying hosts for JEV, can cause reproductive failure in sows; in boars it can cause testitis and infertility. The prevalence of JEV in swine is a continuous threat to human health. A practical diagnostic method for monitoring JEV infection in swine herds is essential for control of the disease in both swine and humans. Here, we have identified a high-affinity anti-JEV NS1 monoclonal antibody (mAb) by indirect ELISA and utilized it for the development of a blocking ELISA (bELISA). The optimal NS1 protein coating concentration (2 μg/mL) and mAb working concentration (1 μg/mL) were determined by checkerboard titration. One hundred ten JEV-antibody-negative serum samples were used to establish 34.03% inhibition as the cutoff value for a negative result. By the bELISA, seroconversion in 80% of newly JEV-vaccinated pigs was detected by 7 days post-immunization, while by the commercial envelope-protein-based iELISA, seroconversion was detected in 20% of the newly vaccinated pigs. We found 98.7% agreement between the bELISA and the commercial iELISA when we tested 157 field samples using both methods. From an epidemiological survey of swine serum collected between 2014 and 2016, we found that the average JEV seropositive rate in unvaccinated commodity pigs was 8.1%, and in vaccinated boars and sows, it was 67.6%.

Analysis of swine antigen-specific antibody responses to Mycoplasma hyopneumoniae infection determined by protein microarray

Pigs harbor several different species of mycoplasmas, of which Mycoplasma hyopneumoniae presents the most significant economic impact on the swine industry. While ELISAs are the predominant diagnostic assay to measure antibody responses during infection with M. hyopneumoniae, the assay itself is only a rough estimate of the total antibody response. It lends little information on pathogen-wide antigen-specific responses. In addition, antibody responses to M. hyopneumoniae as measured by ELISA are slow to develop in infected swine. Our goal was to determine if a protein microarray could be more sensitive and informative of the serological responses of pigs to M. hyopneumoniae infection. The gene sequences of approximately 50 M. hyopneumoniae surface proteins or protein fragments were cloned, mutated to remove UGA codons, expressed in Escherichia coli and purified. The arrays were used to interrogate pig sera from various sources. Sera from naturally-infected swine gave some variability in antigen-specific responses, but, unexpectedly, the responses against the C-terminal portion of the major adhesin P97 was weak in all animals, including those that were experimentally infected. In two of four 118-day experimentally-infected caesarian-derived colostrum-deprived pigs, the strongest antibody responses occurred on days 30 and 54 against members of the P97/P102 paralog families. Our Day 0 results in the other two animals indicate that although thought to be mycoplasma free by all known criteria (serology and PCR), they may have harbored an inapparent Mycoplasma infection. In summary, the protein microarray has the potential to identify new targets for assay development to enhance sensitivity of antibody-based assays.

Expression and characterization of a recombinant porcinized antibody against the E2 protein of classical swine fever virus.

Classical swine fever virus (CSFV) is the causative agent of classical swine fever (CSF), a highly contagious and economically important disease of pigs. The envelope glycoprotein E2 of CSFV is the major antigen that induces neutralizing antibodies and confers protection against CSFV infections. Previously, we developed a murine monoclonal antibody (MAb), HQ06, against the E2 protein of CSFV. To produce the antibody conveniently and stably, the genes coding for the variable regions of the heavy and light chains of HQ06 and constant region genes from the swine antibody were fused and cloned into lentiviral expression vectors to express a recombinant porcinized MAb (rHQ06Sw) in mammalian cells. rHQ06Sw was able to react with the E2 protein or the CSFV virions specifically in different assays. Notably, rHQ06Sw could neutralize CSFV infection in a dose-dependent manner. Taken together, the functional porcinized MAb rHQ06Sw was generated, which can be used to develop novel diagnostic assays or to investigate the structure and functions of the E2 protein.

Seroprevalence of anti-hepatitis E virus antibodies in domestic pigs in Mexico

BackgroundHepatitis E virus (HEV) infection is one of the most common causes of acute liver diseases in humans worldwide. In developing countries, HEV is commonly associated with waterborne outbreaks. Conversely, in industrialized countries, HEV infection is often associated with travel to endemic regions or ingestion of contaminated animal products. Limited information on both, human and animal HEV infection in Mexico is available. As a consequence, the distribution of the virus in the country is largely unknown. Here, we assessed the seroprevalence of HEV among swine in different geographical regions in Mexico.MethodsSeroprevalence of anti-HEV antibodies in swine herds in Mexico was evaluated in a representative sample including 945 pig serum specimens from different regions of the country using a commercial enzyme-linked immunosorbent assay (ELISA).ResultsThe overall prevalence of anti-HEV antibodies in swine was 59.4%. The northern region of Mexico exhibited the highest seroprevalence in the country (86.6%), while the central and southern regions in Mexico showed lower seroprevalence, 42.7% and 51.5%, respectively.ConclusionsIn Mexico, HEV seroprevalence in swine is high. Importantly, northern Mexico showed the highest seroprevalence in the country. Thus, further studies are required to identify the risk factors contributing to HEV transmission among pigs in the country. Assessment of HEV human infection in the context of viral transmission in swine is required to better understand the epidemiology of hepatitis E in Mexico.

Expression and antiviral activity of a chimeric porcinized monoclonal antibody (cHQ06) against E2 protein of classical swine fever virus

Classical swine fever (CSF), one of OIE-listed diseases, is a highly contagious and economically important disease of pigs. Classical swine fever virus (CSFV) is the causative agent of CSF. The capsid (C) protein and the glycoproteins Erns, E1 and E2, are structural components of the virus. E2 is the most immunogenic protein of the CSFV glycoproteins, inducing neutralizing antibodies that provide protection against lethal CSFV challenge. In a previous study, we developed a murine MAb HQ06 against the E2 protein of CSFV. In this study, the variable region genes from HQ06 and constant regions gene of swine antibody are fused and cloned into the eukaryotic expression vectors to establish a cell line which can stably express a chimeric porcinized MAb (cHQ06) against E2 in CHO cell. The purified cHQ06 antibody protein was determined to be successfully generated, which exhibited high reactivity between cHQ06 and the E2 protein of CSFV by enzyme-linked immunosorbent assay (ELISA) and Western blotting. More importantly, we investigated the neutralizing activity of cHQ06 against CSFV. In conclusion, this study generated cHQ06 for efficient and stable production which can be used against to develop novel diagnostic assays, investigate the structure and function of the E2 protein and generate novel preparations of diagnosis and treatment.

Detection of Serum Antibodies to Hepatitis E Virus Based on HEV Genotype 3 ORF2 Capsid Protein Expressed in Nicotiana benthamiana.

BackgroundHepatitis E virus (HEV) causes epidemics in developing countries and is primarily transmitted through the fecal-oral route. There have been recent reports on the zoonotic spread of the virus, and several animal species, primarily pigs, have been recognized as reservoirs of HEV. Because of its possible spread, there is an urgent need of a method for the cost-effective production of HEV proteins that can be used as diagnostic antigens for the serological detection of anti-HEV antibodies.MethodsThe HEV open reading frame (ORF)2 protein was purified from plant tissue by using immobilized metal-anion chromatography (IMAC). The recombinant protein was used to develop an in-house ELISA for testing anti-HEV antibodies in both human and swine sera. Thirty-six serum samples collected from patients with serologically proven HEV infection with commercial kits were tested for anti-HEV IgG antibodies by using the plant-expressed protein. Forty-five serum samples collected from apparently healthy pigs in Bulgarian farms were also tested.ResultsWe confirmed the transient expression and purification of a truncated version of the HEV genotype 3 capsid protein in Nicotiana benthamiana and its usefulness as a diagnostic antigen. ELISA showed the presence of anti-HEV IgG antibodies in 29 of the 36 human samples. The in-house ELISA showed anti-HEV IgG antibodies in 34 of the 45 pigs.ConclusionsWe describe a method for the production of HEV ORF2 protein in N. benthamiana and the usefulness of this protein for the serological detection of anti-HEV antibodies in both humans and swine.

Improvement of indirect enzyme-linked immunosorbent assay for detection of Japanese encephalitis virus antibodies in swine sera

Japanese encephalitis (JE) is an important zoonosis caused by the mosquito-transmitted JE virus (JEV), which is a causative agent of reproductive failure in pregnant sows. Detection of JEV antibodies in swine is performed by hemagglutination inhibition (HI), virus neutralization (VN), and the plaque reduction neutralization test (PRNT). The most stringent PRNT is the 90% endpoint PRNT (PRNT90). These conventional assays are difficult to carry out in diagnostic laboratories with insufficient instruments or cell culture systems. An alternative assay that is easily conducted and time efficient is required. In this study, we improved the indirect enzyme-linked immunosorbent assay (I-ELISA) with clarified antigen for the detection of JEV antibodies. The I-ELISA results obtained from 175 swine serum samples were compared with HI, VN, and PRNT90 results. The sensitivity of I-ELISA was 91.8%, 95.0%, and 94.7% compared with HI, VN, and PRNT90 results, respectively. The specificity of I-ELISA was 92.2%, 94.7%, and 94.7% compared with HI, VN, and PRNT90 results, respectively. Moreover, the I-ELISA results were significantly correlated with the HI (r = 0.93), VN (r = 0.95), and PRNT90 (r = 0.92) results. These results suggest that the improved I-ELISA is useful for serosurveillance of JEV in swine.