Serum Neutralization Assay Research Articles

Characterization of anti-AAV2 neutralizing antibody levels in sheep prior to and following intravitreal AAV2.7m8 injection

AbstractGene augmentation therapy is a promising treatment for incurable, blinding inherited retinal diseases, and intravitreal delivery is being studied as a safe alternative to subretinal injections. Adeno-Associated Viruses (AAV) are commonly-used vectors for ocular gene augmentation therapy. Naturally occurring pre-operative exposure and infection with AAV could result in presence of neutralizing antibodies (NAB’s) in patients’ serum, and may affect the safety and efficacy of treatment. Our aim was to characterize the humoral response against AAV pre- and post-intravitreal delivery of AAV2.7m8 vectors in a naturally-occurring sheep model of CNGA3 achromatopsia. Serial serum neutralization assays were performed to screen sheep for pre-exiting anti-AAV2 NAB’s, and to assess the effect of intravitreal AAV2.7m8 injection on post-operative NAB titers and intraocular inflammation in sheep. The effect of viral dose and transgene type were also assessed. Serological screening revealed pre-operative seropositivity in 21.4% of animals, with age being a risk factor for the presence of anti-AAV2 NAB’s. NAB titers increased following intravitreal AAV administration in the majority of sheep. There was no significant difference in the degree of post-operative serum neutralization between pre-operatively seronegative sheep and those with pre-existing antibodies. However, only sheep with pre-existing antibodies presented with signs of post-operative inflammation. We conclude that pre-existing anti-AAV2 NAB’s do not affect the level of post-operative NAB titers; however, they increase the risk of post-operative ocular inflammation. Our results could have implications for the management of AAV-mediated ocular gene therapies, a technology being increasingly studied and used in patients.

Dendritic cell-based biomimetic nanoparticles for foot-and-mouth disease induce robust cellular immunity

Foot-and-mouth disease (FMD) is a highly contagious and economically devastating viral disease of ruminants and swine, badly affecting the livestock industry worldwide. In clinical practice, vaccination is a frequently employed strategy to prevent foot-and-mouth disease (FMDV). However, commercial inactivated vaccines for FMD mainly rely on humoral immunity, exhibiting poor cellular immune responses and causing adverse reactions. Here, we use the double emulsion method to prepare poly (lactic-co-glycolic acid) nanoparticles (PLGA-NP) encapsulated with IL-2 cytokines, wrap the dendritic cell (DC) membrane carrying FMDV antigen information on the surface of the nanoparticles, obtaining a biomimetic nanoparticle vaccine Biom@DC with uniform size. This vaccine can effortlessly move through lymph nodes due to its nanoscale size advantage. It also possesses DC ability to present antigens, and antigen presentation can be made more effective with high biocompatibility. The sustained release of IL-2 encapsulated in the core of PLGA-NP in vivo can effectively promote the body's cellular immune response. Immune tests on mice have shown that Biom@DC may greatly increase T cell activation and proliferation both in vivo and in vitro, while also significantly reducing the fraction of inhibitory Treg cells. Furthermore, in the micro serum neutralization assay for FMDV, it has been demonstrated that the group vaccinated with Biom@DC exhibits a clear neutralizing effect. Given its strong immunogenicity, Biom@DC has the potential to develop into a novel, potent anti-FMDV vaccination.

Diversity in clinical isolates of Ictalurid herpesvirus 1 (IcHV1) from U.S. farm-raised catfish and virulence assessment in channel and channel × blue catfish hybrids.

Ictalurid herpesvirus 1 (IcHV1) is the most significant viral agent in U.S. catfish aquaculture. Little is known regarding the genetic stability and antigenic variability of IcHV1. Herein, the genetic and antigenic diversity of IcHV1 field isolates was assessed by restriction fragment length polymorphism (RFLP) analysis and serum neutralization assays. RFLP analysis identified two distinct genotypes (IcHV1A and IcHV1B), both discrete from blue catfish alloherpesvirus (BCAHV). Neutralization assays with anti-IcHV1 monoclonal antibody Mab-95 indicate shared antigenic determinants for IcHV1A and IcHV1B that are absent from BCAHV, which Mab-95 did not neutralize. Virulence assessments with representative isolates demonstrate significant differences between isolates within RFLP groups and pooled RFLP group data suggest IcHV1B (pooled survival [mean ± SE]: 58.3% ± 2.6) may be more virulent than IcHV1A (survival: 68.6% ± 2.4). Rechallenges with representative IcHV1A and IcHV1B isolates indicate a cross-protective effect, with fish surviving initial exposure to IcHV1A or IcHV1B showing robust protection when subsequently re-exposed to IcHV1A or IcHV1B. This work demonstrated significant differences in virulence between case isolates, identifying two discrete IcHV1 lineages, distinct from BCAHV, with similar virulence in channel and channel × blue catfish hybrids and a cross-protective effect in catfish surviving exposure to either lineage.

A reporter virus particle seroneutralization assay for tick-borne encephalitis virus overcomes ELISA limitations.

Tick-borne encephalitis (TBE) virus is the most prevalent tick-transmitted orthoflavivirus in Europe. Due to the nonspecific nature of its symptoms, TBE is primarily diagnosed by ELISA-based detection of specific antibodies in the patient serum. However, cross-reactivity between orthoflaviviruses complicates the diagnosis. Specificity issues may be mitigated by serum neutralization assays (SNT), although the handling of clinically relevant orthoflaviviruses requires biosafety level (BSL) 3 conditions and they have highly divergent viral kinetics and cell tropisms. In the present study, we established a reporter virus particle (RVP)-based SNT in which the infectivity is measured by luminescence and that can be performed under BSL-2 conditions. The RVP-based SNT for TBEV exhibited a highly significant correlation with the traditional virus-based SNT (R2 = 0.8637, p < 0.0001). The RVP-based assay demonstrated a sensitivity of 92.3% (95% CI: 79.7%-97.4%) and specificity of 100% (95% CI: 81.6%-100%). We also tested the cross-reactivity of serum samples in RVP-based assays against other orthoflaviviruses (yellow fever virus, dengue virus type 2, Zika virus, West Nile virus and Japanese encephalitis virus). Interestingly, all serum samples which had tested TBEV-positive by ELISA but negative by RVP-based SNT were reactive for antibodies against other orthoflaviviruses. Thus, the RVP-based seroneutralization assay provides an added value in clinical diagnostics as well as in epidemiological studies.

Validation of a focus reduction neutralization test (FRNT) to rapidly titrate human SARS-CoV-2 neutralizing antibodies by using the CTL Immunospot S6 universal analyzer

In this paper we describe the validation of a focus reduction neutralization test (FRNT) to quantitate human SARS-CoV-2 neutralizing antibodies by using the CTL Immunospot S6 Universal Analyzer. We employed a previously published protocol and compared its performances to a well-established and traditional serum-neutralization assay (SN). To assess diagnostic sensitivity, a total number of 201 human sera positive by SN for SARS-CoV-2 NAbs were processed: 196/201 tested positive by FRNT50 (97.51 %). A diagnostic specificity of 100 % was obtained by evaluating 206 negative serum samples. Repeatability of the test was evaluated by determining the intra and inter-assay coefficient of variation (CV). A standard deviation of 0.83 and a CV of 13 % were evidenced demonstrating an acceptable reproducibility of the assay. Moreover, a Cohen's Kappa of 0.975 was obtained proving an extremely high level of agreement between the FRNT protocol and the SN. Despite an acceptable correlation between methods (p < 0.05), FRNT demonstrated a statistically significant increase in NAbs titres compared to SN as well as higher data variability and asymmetry. These discrepancies could be attributed to FRNT sensitivity or most probably to the subjective interpretation of SN, although this aspect needs to be further investigated with a more representative number of samples.Basing on our results, it is reasonable to replace the SN with the FRNT assay as, with this, fast processing time (less than 2 days) and operator bias-free results registrations are guaranteed.

Hepatitis E virus neutralization by porcine serum antibodies.

The consumption of raw or undercooked meat products poses a serious risk for human hepatitis E virus (HEV) infections. In many high-income countries, domestic pigs and wild boars represent the main animal reservoirs for HEV and are usually identified by reverse transcription-PCR and antibody enzyme-linked immunosorbent assay (ELISA). In order to characterize the humoral immune response in more detail, a cell culture-based serum neutralization assay using a culture-adapted HEV strain was established here. Measurement of neutralizing antibodies was only possible after removing the viral quasi-envelope by detergent treatment. Serum samples of 343 wild boars from Northern Germany were first analyzed for anti-HEV IgG using an in-house ELISA, resulting in 19% positive samples. Subsequently, a subset of 41 representative samples was tested with the neutralization assay, and the results correlated well with those obtained by ELISA. Not only the human HEV strain 47832c but also two porcine HEV strains were shown to be neutralized by porcine serum antibodies. Neutralizing activity was also found in samples containing both HEV-specific antibodies and HEV RNA. Testing of serum samples derived from two experimentally infected domestic pigs showed a steep increase in neutralizing activity at 24 or 51 days post infection, dependent on the used infectious dose. The developed assay can be useful for characterization of the humoral immune response after HEV infection and for assessing the efficiency of HEV vaccine candidates.

Cross-reactive antibody response to Monkeypox virus surface proteins in a small proportion of individuals with and without Chinese smallpox vaccination history

BackgroundAfter the eradication of smallpox in China in 1979, vaccination with the vaccinia virus (VACV) Tiantan strain for the general population was stopped in 1980. As the monkeypox virus (MPXV) is rapidly spreading in the world, we would like to investigate whether the individuals with historic VACV Tiantan strain vaccination, even after more than 40 years, could still provide ELISA reactivity and neutralizing protection; and whether the unvaccinated individuals have no antibody reactivity against MPXV at all.ResultsWe established serologic ELISA to measure the serum anti-MPXV titer by using immunodominant MPXV surface proteins, A35R, B6R, A29L, and M1R. A small proportion of individuals (born before 1980) with historic VACV Tiantan strain vaccination exhibited serum ELISA cross-reactivity against these MPXV surface proteins. Consistently, these donors also showed ELISA seropositivity and serum neutralization against VACV Tiantan strain. However, surprisingly, some unvaccinated young adults (born after 1980) also showed potent serum ELISA activity against MPXV proteins, possibly due to their past infection by some self-limiting Orthopoxvirus (OPXV).ConclusionsWe report the serum ELISA cross-reactivity against MPXV surface protein in a small proportion of individuals both with and without VACV Tiantan strain vaccination history. Combined with our serum neutralization assay against VACV and the recent literature about mice vaccinated with VACV Tiantan strain, our study confirmed the anti-MPXV cross-reactivity and cross-neutralization of smallpox vaccine using VACV Tiantan strain. Therefore, it is necessary to restart the smallpox vaccination program in high risk populations.

Genomic similarity and antibody-dependent enhancement of immune serum potentially affect the protective efficacy of commercial MLV vaccines against NADC30-like PRRSV

Porcine reproductive and respiratory syndrome (PRRS) is one of the most significant diseases affecting the pig industry worldwide. The PRRSV mutation rate is the highest among the RNA viruses. To date, NADC30-like PRRSV and highly pathogenic PRRSV (HP-PRRSV) are the dominant epidemic strains in China; however, commercial vaccines do not always provide sufficient cross-protection, and the reasons for insufficient protection are unclear. This study isolated a wild-type NADC30-like PRRSV, SX-YL1806, from Shaanxi Province. Vaccination challenge experiments in piglets showed that commercial modified live virus (MLV) vaccines provided good protection against HP-PRRSV. However, it could not provide sufficient protection against the novel strain SX-YL1806. To explore the reasons for this phenomenon, we compared the genomic homology between the MLV strain and HP-PRRSV or NADC30-like PRRSV and found that the MLV strain had a lower genome similarity with NADC30-like PRRSV. Serum neutralization assay showed that MLV-immune serum slightly promoted the homologous HP-PRRSV replication and significantly promoted the heterologous NADC30-like PRRSV strain replication in vitro, suggesting that antibody-dependent enhancement (ADE) might also play a role in decreasing MLV protective efficacy. These findings expand our understanding of the potential factors affecting the protective effect of PRRSV MLV vaccines against the NADC30-like strains.

Detection of Anti-Rift Valley Fever Virus Antibodies in Serum Samples of Patients with Suspected Arbovirus Infection.

The definitive diagnosis of the Rift Valley fever virus (RVFV) requires a form of testing that is available only in reference laboratories. It includes indirect immunofluorescence assay (IFA), the serum neutralization assay (NA), and real-time PCR. Therefore, often, no attempts are made to detect it, even among travelers from endemic areas. In this study, the presence of anti-RVFV IgG and IgM was retrospectively screened in stored serum samples from people who were admitted with arbovirus symptoms at the National Institute for Infectious Diseases (INMI) L. Spallanzani, Rome, Italy. Overall, 80 residual serum samples were anonymized, and sub-aliquots were prepared and tested for anti-RVFV IgG and IgM. A serum neutralization assay was used as a confirmatory test. There was a positive result in eight out of 80 samples (10%) for anti-RVFV IgG, with titers ranging from 1:40 up to 1:1280. Three of eight (2.6%) samples were confirmed as seropositive through an in-house serum neutralization assay, with antibody titers ranging from 1:10 to 1:160. All samples resulted negative for anti-RVFV IgM and RVFV RNA when tested by IFA and real-time RT-PCR, respectively. Our data highlight that several RVFV infections can possibly escape routine virological diagnosis, which suggests RVFV testing should be set up in order to monitor virus prevalence.

Development and Evaluation of an Enzyme-Linked Immunosorbent Assay Targeting Rabies-Specific IgM and IgG in Human Sera.

Immunity from rabies depends on rabies virus neutralizing antibodies (RVNA) induced after immunization; however, the influence of antibody isotype switching has not been extensively investigated. This has become particularly relevant with changes in World Health Organization (WHO) recommended rabies vaccine regimens that may influence RVNA isotype kinetics, potentially affecting the peak, and longevity, of RVNA immunoglobulin (IgG) levels. We developed rapid and reliable assays for quantifying the anti-rabies IgM/IgG class switch in human serum based on an indirect ELISA technique. The immune response was tracked in ten individuals naïve to the rabies vaccine by quantifying serum titers weekly, from day seven to day 42 post-immunization, using a serum neutralization assay and the ELISA IgM/IgG assays. The average RVNA IU/mL levels were at D0 ≤ 0.1, D7 0.24, D14 8.36, D21 12.84, D28 25.74 and D42 28.68. Levels of specific IgM antibodies to rabies glycoprotein (EU/mL) were higher, on average, at D7, 1.37, and from D14, 5.49, to D21, 6.59. In contrast, average IgG antibodies (EU/mL) predominated from D28, 10.03, to D42, 14.45. We conclude that levels of anti-rabies IgM/IgG at D28 characterize the isotype class switch. These assays, combined with serum neutralization assays, distinguished the RVNA levels in terms of the IgM/IgG responses and are expected to add to the diagnostic repertoire, provide additional information in establishing rabies vaccine regimens, both post- and pre-exposure prophylaxis, and contribute to research efforts.

Neutralizing Antibodies against SARS-CoV-2 Beta and Omicron Variants Inhibition Comparison after BNT162b2 mRNA Booster Doses with a New PETIA sVNT Assay.

Monitoring antibody response following SARS-CoV-2 vaccination is strategic, and neutralizing antibodies represent the gold standard. The neutralizing response to Beta and Omicron VOCs was evaluated versus the gold standard by a new commercial automated assay. Serum samples from 100 healthcare workers from the Fondazione Policlinico Universitario Campus Biomedico and the Pescara Hospital were collected. IgG levels were determined by chemiluminescent immunoassay (Abbott Laboratories, Wiesbaden, Germany) and serum neutralization assay as the gold standard. Moreover, a new commercial immunoassay, the PETIA test Nab (SGM, Rome, Italy), was used for neutralization evaluation. Statistical analysis was performed with R software, version 3.6.0. Anti-SARS-CoV-2 IgG titers decayed during the first ninety days after the vaccine second dose. The following booster dose significantly (p < 0.001) increased IgG levels. A correlation between IgG expression and neutralizing activity modulation was found with a significant increase after the second and the third booster dose (p < 0.05. Compared to the Beta variant of the virus, the Omicron VOC was associated with a significantly larger quantity of IgG antibodies needed to achieve the same degree of neutralization. The best Nab test cutoff for high neutralization titer (≥1:80) was set for both Beta and Omicron variants. This study correlates vaccine-induced IgG expression and neutralizing activity using a new PETIA assay, suggesting its usefulness for SARS-CoV2 infection management.

Serological Protection Rates against TBEV Infection in Blood Donors from a Highly Endemic Region in Southern Germany

Tick-borne encephalitis (TBE) is the most significant tick-borne disease in Europe and Asia, with more than 10,000 cases per year worldwide. A surge of reported TBE cases can be observed despite the availability of highly efficient vaccines. There is little known about the serological immune protection rate of the population in Germany. The seroprotection rate is defined as the presence of neutralizing antibodies. In contrast, the vaccination rate, as defined by public health agencies, may differ from the true protection rate in a population. 2220 blood samples from inhabitants of the county Ortenaukreis in the Federal State of Baden-Württemberg in Germany were included in the study. These were tested for anti-TBEV IgG antibodies by an anti-TBEV-IgG-ELISA. Subsequently, all TBEV-IgG positive samples were confirmed for neutralizing antibodies in the micro serum neutralization assay. From the overall 2220 samples, 2104 were included in the comparison because of the selection of specific age groups (ages 20-69). In our sample size, we found an average serological protection rate (presence of neutralizing antibodies) of 57% (518/908) for the female blood donors and of 52% (632/1196) for the male blood donors. In this study, we present new findings on a highly endemic region in southern Germany. Additionally, we present current data regarding the serological TBEV protection rates in the Ortenaukreis in southern Germany and compare these with a dataset published by the RKI, which is based on vaccination reports of the primary care providers and health care insurers, and with a self-reporting study conducted by a vaccine manufacturer. Our results significantly exceed the official numbers of average active vaccination status by 23.2% for females and by 21% for males. This might indicate an even longer persistence of TBE-vaccination-induced antibody titers than previously assumed.

Comparison of antigenicity between recently isolated bovine rotaviruses A and vaccine strains by cross-neutralizing antibody tests and their significance for immunization.

Three bovine rotaviruses A (RVAs) isolated from a cattle farm in Japan were serotyped by serum neutralization assay, as compared with the RVA strains contained in a vaccine used on the same farm. Antisera were prepared against the three isolates and the vaccine strains of bovine RVA. The results of cross-neutralization tests revealed that the RVA isolates from this farm differed somewhat in serotype. Collected plasma from calves for 6 weeks after colostrum ingestion showed that maternal antibodies acquired against all isolates gradually decreased, but antibodies toward one isolate increased by 6 weeks after the mentioned decreasing. These results suggest that rotavirus vaccines administered to cows should include all serotypes commonly found in calves with diarrhea.

Predicting Antigenic Distance from Genetic Data for PRRSV-Type 1: Applications of Machine Learning.

The control of porcine reproductive and respiratory syndrome (PRRS) remains a significant challenge due to the genetic and antigenic variability of the causative virus (PRRSV). Predominantly, PRRSV management includes using vaccines and live virus inoculations to confer immunity against PRRSV on farms. While understanding cross-protection among strains is crucial for the continued success of these interventions, understanding how genetic diversity translates to antigenic diversity remains elusive. We developed machine learning algorithms to estimate antigenic distance in silico, based on genetic sequence data, and identify differences in specific amino acid sites associated with antigenic differences between viruses. First, we obtained antigenic distance estimates derived from serum neutralization assays cross-reacting PRRSV monospecific antisera with virus isolates from 27 PRRSV1 viruses circulating in Europe. Antigenic distances were weakly to moderately associated with ectodomain amino acid distance for open reading frames (ORFs) 2 to 4 (ρ < 0.2) and ORF5 (ρ = 0.3), respectively. Dividing the antigenic distance values at the median, we then categorized the sera-virus pairs into two levels: low and high antigenic distance (dissimilarity). In the machine learning models, we used amino acid distances in the ectodomains of ORFs 2 to 5 and site-wise amino acid differences between the viruses as potential predictors of antigenic dissimilarity. Using mixed-effect gradient boosting models, we estimated the antigenic distance (high versus low) between serum-virus pairs with an accuracy of 81% (95% confidence interval, 76 to 85%); sensitivity and specificity were 86% and 75%, respectively. We demonstrate that using sequence data we can estimate antigenic distance and potential cross-protection between PRRSV1 strains. IMPORTANCE Understanding cross-protection between cocirculating PRRSV1 strains is crucial to reducing losses associated with PRRS outbreaks on farms. While experimental studies to determine cross-protection are instrumental, these in vivo studies are not always practical or timely for the many cocirculating and emerging PRRSV strains. In this study, we demonstrate the ability to rapidly estimate potential immunologic cross-reaction between different PRRSV1 strains in silico using sequence data routinely collected by production systems. These models can provide fast turn-around information crucial for improving PRRS management decisions such as selecting vaccines/live virus inoculation to be used on farms and assessing the risk of outbreaks by emerging strains on farms previously exposed to certain PRRSV strains and vaccine development among others.

BNT162b2 Elicited an Efficient Humoral Response Against Different Strains of SARS-CoV-2 in People Living with HIV.

Vaccines have had a fundamental impact in containing the ongoing Coronavirus Disease 2019 (COVID-19) pandemic. However, there are few efficacy data relating to frail patients, including the HIV-positive patient. This study evaluated the Severe Acute Respiratory Syndrome Coronavirus 2 (SARSCoV- 2) serum neutralization in People Living with HIV (PLWH) compared to a cohort of healthy volunteers both vaccinated with BNT162b2. A serum sample was then withdrawn 14-21 days after the second dose of the vaccine and a serum neutralization assay was performed on Vero E6 cells. The experiments were performed using two strains of SARS-CoV-2 as 20A.EU1 and B.1.617.2. PLWH on Antiretroviral Therapy (ART) showed a vaccine response comparable to the healthy subjects. No correlation between CD4 count or CD4/CD8 and neutralizing antibodies (NTAbs) has been found. No differences in NT-Abs between patients with CD4 nadir above or under 200 cells/μl have been found. In both cohorts, vaccine-elicited serum better neutralized 20A.EU1 than B.1.617.2 strain. PLWH in ART and with good immuno-virological recovery showed a vaccine response comparable to that of healthy subjects and regardless of their immunological status at HIV infection diagnosis. However, larger studies are needed to confirm our results and to evaluate the vaccine response even in patients with low CD4 counts.

Long-term protective immunity to goatpox in goats after a single immunization with a live attenuated goatpox vaccine

In this study, the duration of immunity following a single-dose vaccination using an attenuated live goatpox vaccine (GTPV/Uttarkashi/1978 strain) was evaluated in goatpox-seronegative goats for 52 months. Long-term immunity was evaluated by clinical protection upon virulent virus challenge and serum neutralization assay applied to serum samples. The rise in the level of GTPV-specific antibodies was found to reach a maximum at 21days post-vaccination, and these antibodies were maintained for 1 to 2years after immunization, with a steady decline. Upon virulent virus challenge at 12, 24, 42, and 52months post-vaccination, protection in all the vaccinated animals was evident (100%), whereas, the control animals developed severe clinical disease. This is the first time that the long-term immunity of a live goatpox vaccine has been investigated up to 52months after vaccination in goats by virulent virus challenge and demonstration of serum neutralization titres. This vaccine has immense potential for controlling and eradicating goatpox from an enzootic region.

West Nile and Usutu Virus Introduction via Migratory Birds: A Retrospective Analysis in Italy.

The actual contribution of migratory birds in spreading West Nile (WNV) and Usutu virus (USUV) across Europe and from Africa to old countries is still controversial. In this study, we reported the results of molecular and serological surveys on migrating birds sampled during peaks of spring and autumn migration at 11 Italian sites located along important flyways, from 2012 to 2014. A total of 1335 specimens made of individual or pooled sera, and organs from 275 dead birds were tested for WNV and USUV RNA by real time PCR (RT-PCR). Furthermore, sera were tested by serum neutralization assay for detecting WNV and USUV neutralizing antibodies. Molecular tests detected WNV lineage 2 RNA in a pool made of three Song Thrush (Turdus philomelos) sera sampled in autumn, and lineage 1 in kidneys of six trans-Saharan birds sampled in spring. Neutralizing antibodies against WNV and USUV were found in 5.80% (n = 72; 17 bird species) and 0.32% (n = 4; 4 bird species) of the tested sera, respectively. Our results do not exclude the role of migratory birds as potential spreaders of WNV and USUV from Africa and Central Europe to Mediterranean areas and highlight the importance of a more extensive active surveillance of zoonotic viruses.

A simple method for purification of epsilon toxin of Clostridium perfringens type D for serum neutralization assay

Enterotoxaemia, a disease that affects domestic ruminants, is caused by the epsilon toxin of Clostridium perfringens type D and B. Control and prophylaxis are based on systemic vaccination of small ruminant herds with epsilon toxoid. Purified epsilon toxin is an essential material for vaccine evaluation. It is also necessary for diagnosis of enterotoxaemia disease in the field by in vitro tests including ELISA. The aim of this study was to set up a method for preparation of functional purified epsilon toxin of C. perfringens type D to be used in serum neutralization test. In this study, epsilon toxin was prepared from C. perfringens type D culture precipitated with ammonium sulfate, dialyzed against phosphate buffered saline (PBS) buffer and then, purified using chromatography system. Then, the purified epsilon toxin was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Toxin function was confirmed by cell culture and minimum lethal dose (MLD) assays. Also rabbits were immunized by vaccine in two turns with a 28-day interval. Then, blood samples were collected, and serum neutralization (SN) test was carried out. Results showed that the purified toxin was suitable for SN assay. Our purification method was simple, fast and cost-effective for preparation of epsilon toxin.

A Field Study Evaluating Humoral Immunity in Calves Vaccinated with Multivalent Bovine Respiratory Pathogen Vaccines

Abstract Bovine Respiratory Syncytial Virus (BRSV), Bovine Parainfluenza 3 (BPI3) and Mannheimia haemolytica (Mh) are major respiratory pathogens in the bovine respiratory disease complex. It is important to optimize passive and active immunity to these pathogens early in life to reduce clinical and subclinical productivity losses. The administration of inactivated, adjuvanted and multivalent vaccines, such as Bovilis® Bovipast RSP (Bovipast), and Bovalto® Respi 3 (Bovalto) to calves, may enhance cellular and humoral immunity against BRSV, BPI3 and Mh. A field trial evaluated the immune responses to these three agents in the first year of life in 12 Bovipast and 13 Bovalto vaccinated calves, and 5 negative control calves. Calves were vaccinated starting at 2 weeks of age and revaccinated 4 weeks later (primo vaccination). A booster vaccination was given at approximately 10 months of age. Serum samples were taken at time intervals up to 6 months after primo vaccination and up to 1 month after the booster vaccination. BRSV serum titres were evaluated using a serum neutralisation assay (SN), and BRSV, BPI3 and Mh titres were evaluated using a commercial enzyme linked immunosorbent assay (ELISA) test. Serum antibodies after primo and booster vaccinations in the individual calves were evaluated by calculating the areas under the curve (AUC) of the Log2 transformed BRSV SN titres and the optic density measures of the ELISA tests for BRSV, BPI3 and Mh. Multivariate general linear models were used to evaluate the influence of the vaccination on the AUC of the serum measures within 6 months after the primo vaccination. Similarly, models evaluated the AUC of the serum measures after the booster vaccination. The Bovipast vaccinated calves had significantly higher SN and ELISA titres AUC following the primo vaccination and booster vaccinations compared to the negative control calves and the Bovalto vaccinated calves. The Bovalto vaccinated calves did not have a significantly different BRSV SN and ELISA titres AUC response after the primo or booster vaccinations compared to the negative control calves. The serum antibody responses to BPI3 and Mh in the vaccinated calves were less pronounced than the Bovipast BRSV antibody response. Bovipast and Boval- to vaccinated calves mounted a significantly higher AUC ELISA OD for both BPI3 and Mh and the highest AUC was measured in the Bovipast vaccinated calves. This study indicated that early vaccinations of calves with multivalent adjuvanted inactivated BRD vaccines, such as Bovilis® Bovipast RSP can elicit a humoral response with a cellular-mediated memory effect as indicated by the booster vaccination.

Presence of Antibodies to SARS-CoV-2 in Domestic Cats in Istanbul, Turkey, Before and After COVID-19 Pandemic.

Recent studies demonstrated that domestic cats can be naturally and experimentally infected with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). This study was performed to investigate the presence of SARS-CoV-2-specific antibodies within the domestic cat population in Istanbul, Turkey, before the coronavirus disease 2019 (COVID-19) and during the COVID-19 pandemic. Overall, from 155 cat sera analyzed, 26.45% (41/155) tested positive in the spike protein-ELISA (S-ELISA), 28.38% (44/155) in the receptor-binding domain-ELISA (RBD-ELISA), and 21.9% (34/155) in both, the S- and RBD-ELISAs. Twenty-seven of those were also positive for the presence of antibodies to feline coronavirus (FCoV). Among the 34 SARS-CoV-2-positive sera, three of those were positive on serum neutralization assay. Six of the 30 cats before COVID-19 and 28 of the 125 cats during COVID-19 were found to be seropositive. About 20% of ELISA-positive cats exhibited mainly respiratory, gastrointestinal, and renal signs and skin lesions. Hematocrit, hemoglobin, white blood cells, lymphocyte, and platelet numbers were low in about 30% of ELISA-positive cats. The number of neutrophils and monocytes were above normal values in about 20% of ELISA-positive cats. The liver enzyme alanine aminotransferase levels were high in 23.5% ELISA-positive cats. In conclusion, this is the first report describing antibodies specific to SARS-CoV-2 antigens (S and RBD) in cats in Istanbul, Turkey, indicating the risk for domestic cats to contract SARS-CoV-2 from owners and/or household members with COVID-19. This study and others show that COVID-19-positive pet owners should limit their contact with companion animals and that pets with respiratory signs should be monitored for SARS-CoV-2 infections.