Receptor-binding domain-based immunoassays for serosurveillance differentiate efficiently between SARS-CoV2-exposed and non-exposed farmed mink.

During the COVID-19 pandemic, infection of farmed mink has become not only an economic issue but also a widespread public health concern. International agencies have advised the use of strict molecular and serosurveillance methods for monitoring the SARS-CoV2 status on mink farms. We developed 2 ELISAs and a duplex protein microarray immunoassay (MI), all in a double-recognition format (DR), to detect SARS-CoV2 antibodies specific to the receptor-binding domain (RBD) of the spike protein and to the full-length nucleoprotein (N) in mink sera. We collected 264 mink serum samples and 126 oropharyngeal samples from 5 Spanish mink farms. In both of the ELISAs and the MI, RBD performed better than N protein for serologic differentiation of mink from SARS-CoV2-positive and -negative farms. Therefore, RBD was the optimal antigenic target for serosurveillance of mink farms.

Two serological approaches for detection of antibodies to SARS-CoV-2 in different scenarios: a screening tool and a point-of-care test.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has infected more than 8 million people worldwide, becoming a pandemic. Detecting antibodies against SARS-CoV-2 is of utmost importance and a good indicator of exposure and circulation of the virus within the general population. Two serological tools based on a double recognition assay [enzyme-linked immunosorbent assay (DR-ELISA) and lateral flow assay (DR-LFA)] to detect total antibodies to SARS-CoV-2 have been developed based on the recombinant nucleocapsid protein. A total of 1065 serum samples, including positive for COVID-19 and negative samples from healthy donors or infected with other respiratory pathogens, were analyzed. The results showed values of sensitivity between 91.2% and 100%, and specificity of 100% and 98.2% for DR-LFA and DR-ELISA, respectively. No cross-reactivity against seasonal coronavirus (HCoV-NL63, HCoV-229E, HCoV-HKU1, HCoV-OC43) was found. These results demonstrate the importance of serology as a complementary tool to polymerase chain reaction for follow-up of recovered patients and identification of asymptomatic individuals.

A lateral flow assay for the rapid diagnosis of Mycobacterium bovis infection in wild boar.

The native Eurasian wild boar (Sus scrofa) is a reservoir of Mycobacterium bovis, the causative agent of animal tuberculosis (TB), a chronic disease in livestock, companion animals and wild mammals. Cases of M. bovis infection in wild boar or feral pig have been reported worldwide, making early detection a priority in the eradication of the disease. Point-of-care diagnostic tests, such as low cost lateral flow assays, provide high specificity and sensitivity and can be performed on site, an essential requirement for a rapid screening of wildlife. A lateral flow assay, LFA, (INgezim TB CROM Ab) for the detection of M. bovis-specific antibodies in wild boar serum and blood has been developed based on MPB83, one of the major immunogenic antigens of the bacterium. A total of 140 samples of wild boar serum, well-characterized by Mycobacterium tuberculosis complex culture and TB compatible post-mortem lesions, have been analysed with LFA, and results were compared with one in-house and two commercial Enzyme-linked Immunosorbent Assays (ELISA), INgezim TB Porcine and INgezim Tuberculosis DR. In experimental samples, the achieved values of sensitivity of the different techniques ranged from 84.3% to 92.1% and the specificity was 100% in all of them. In field animals, specificity ranged from 96% to 100%, whereas sensitivity ranged from 48% to 64% in juvenile wild boar, increasing to 93.3%-100% in adult wild boar. In particular, the total sensitivity and specificity values obtained with the new LFA were 83% and 97%, respectively, indicating that INgezim TB CROM Ab could be used as a first approach for the surveillance of TB in wild boar, with a special applicability for animal-side testing.

Monitoring Anti-NS1 Antibodies in West Nile Virus-Infected and Vaccinated Horses.

West Nile virus (WNV) is a zoonotic arboviral pathogen affecting humans, birds, and horses. Vaccines are available for veterinary use, which efficiently prevent the infection in horses. Most common diagnostic tools rely on the identification of the agent (RT-PCR, virus isolation), or on the detection of antibodies (IgM and IgG) recognizing structural proteins of the virus or neutralizing virus infection in cell cultures (virus-neutralization tests). The recent emergence of WNV in different parts of the world has resulted in an increase in the vaccination of horses in many countries. Methods for differentiation between infected and vaccinated animals ("DIVA" assays) would be useful for surveillance and control purposes but are still not available. A usual approach in this regard is the use of antibodies to nonstructural proteins as markers of nonvaccinated, infected animals, and the nonstructural NS1 protein of WNV has been proposed as a candidate for such a marker. The aim of this study was to test the hypothesis that NS1 can be a useful antigen in DIVA assays for differentiating WNV vaccinated and infected horses in field conditions. For that, we examined serum samples from either vaccinated and infected horses both from experimental infections/vaccinations (under controlled conditions) and from the field, exposed to natural infection or vaccinated in response to a risk of infection. The overall conclusion of the study is that NS1 antigen can effectively differentiate WNV infected from vaccinated horses in experimental (controlled) conditions, but this differentiation might be difficult depending on the conditions prevailing in the field.

A monoclonal antibody to DIII E protein allowing the differentiation of West Nile virus from other flaviviruses by a lateral flow assay.

West Nile Virus (WNV) belongs to the Flaviviridae family, genus Flavivirus, which includes other emerging arthropod-borne viruses (arboviruses) pathogenic for animals and/or humans. West Nile Virus is a genetically diverse RNA virus with at least 7 different recognized lineages. Following its recent introduction and subsequent expansion to the Americas, WNV is currently one of the most widely spread arboviruses in the world having recently re-emerged in the Mediterranean basin, Central and Eastern Europe. Laboratory tests are essential to confirm WNV infection and monoclonal antibodies represent useful tools for the development of diagnostic assays. A monoclonal antibody, 1D11, recognizing an epitope in the domain III of the envelope glycoprotein of WNV, was selected for this study. Its suitability to detect a range of WNV variants representative of its whole genetic range, and to differentiate it from other flaviviruses and arboviruses, was assessed by means of an immunochromatographic assay in an LFA format. A panel of cell culture supernatants infected with 9 different WNV isolates representing a wide range of genetic lineages, and 16 non-WNV arboviruses, including flaviviruses closely related to WNV, were tested. The mAb correctly detected all WNV strains, and did not react with any of the non-WNV arboviruses.

Temporal analysis of the interference caused by paratuberculosis vaccination on the tuberculosis diagnostic tests in goats.

Vaccination against paratuberculosis (PTB) in goats is a cost-effective control strategy, and is also effective as regards preventing the onset of clinical cases. However, it causes interference in the diagnostic tests used in the control of tuberculosis (TB). A group of 99 goats from a herd with no history of TB or PTB infection was vaccinated against PTB at seven months of age. They then underwent consecutive intradermal tests [single (SIT) and comparative (CIT) intradermal tuberculin tests), interferon-gamma release assays (IGRA) and two serological tests (p22_CE and DR-ELISA) every three months, until the interference disappeared. When using the SIT test, a variable number of positive reactors were observed at 3 months (T3; 32.3%, 95% CI 23.9-42.1), 6 months (T6; 11.5%, 95% CI 6.5-19.4), 9 months (T9; 6.4%, 95% CI 3.0-13.2) and 12 months (T12; 0%, 95% CI 0-4.0) post-vaccination. In contrast, the CIT test had a specificity (Sp) of 100% (95%, CI 96.0-100, regardless of the time post-vaccination. The IGRA also obtained high Sp values throughout the study period. No significant interference in the serological tests was recorded at T3 [p22_CE, Sp = 96% (95% CI 90.1-98.4) and DR-ELISA, Sp = 98% (95% CI 92.9-99.4)], although an increase in antibody titers was observed in the following herd testing events. In conclusion, the use of the SIT test causes the onset of false-positive reactors if applied before 12 months post-vaccination in a TB-free/PTB-vaccinated herd. Nevertheless, the CIT test and IGRA obtained high Sp values under these epidemiological circumstances. The serological tests were also highly specific in the case of PTB-vaccinated goats, although their Sp decreased significantly after several intradermal tests.

The use of serological tests in combination with the intradermal tuberculin test maximizes the detection of tuberculosis infected goats.

The diagnosis of tuberculosis (TB) in goats is based mainly on the single and comparative intradermal tuberculin (SIT and CIT) tests and, exceptionally, on the interferon-gamma (IFN-γ) assay, however they are not perfect in terms of sensitivity and specificity. Nevertheless, various serological assays that provide a potential cost-effective approach for the control of TB are also available or under development, and a variety of results have been reported regarding the ability of these tests to detect infected animals, particularly in the early stages of infection. In the present study, SIT/CIT and IFN-γ tests and three different serological assays were evaluated during two consecutive herd testing events in a recently infected caprine herd (n = 447) with a high prevalence of infection in order to evaluate their performance and provide field data with which to improve the TB control programs in this species. The proportion of infected animals that tested positive among all the infected goats (T+/I+ value) in the last herd testing event ranged from 26.2% (IC95%; 19.3-34.5) to 85.7% (IC95%; 78.5-90.7) using cell-based diagnostic tests. The SIT/SCIT tests detected more infected goats than the IFN-γ test, regardless of the interpretation criteria. The T+/I+ value of serology was 83.2 (IC95%; 75.2-89), although it increased significantly (P < 0.05) when using samples collected 15 days after the intradermal test (100%, IC95%; 97-100). In general, a parallel interpretation of intradermal tests with serology maximized the detection of infected goats. These results demonstrate that serological tests are valuable diagnostic tools to maximize the detection of TB infected goats, even in recent outbreaks, accelerating the eradication process.

Diagnostic aptitude of West Nile virus-like particles expressed in insect cells.

West Nile virus is a globally spread zoonotic arbovirus. The laboratory diagnosis of WNV infection relies on virus identification by RT-PCR or on specific antibody detection by serological tests, such as ELISA or virus-neutralization. These methods usually require a preparation of the whole virus as antigen, entailing biosafety issues and therefore requiring BSL-3 facilities. For this reason, recombinant antigenic structures enabling effective antibody recognition comparable to that of the native virions, would be advantageous as diagnostic reagents. WNV virions are enveloped spherical particles made up of 3 structural proteins (C, capsid; M, membrane and E, envelope) enclosing the viral RNA. This study describes the co-expression of these 3 proteins yielding non-infectious virus-like particles (VLPs) and the results of the initial assessment of these VLPs, used instead of the whole virus, that were shown to perform correctly in two different ELISAs for WNV diagnosis.

Absence of protection from West Nile virus disease and adverse effects in red legged partridges after non-structural NS1 protein administration.

The red-legged partridge (Alectoris rufa) is a competent host for West Nile virus (WNV) replication and highly susceptible to WNV disease. With the aim to assess in this species whether the inoculation of non-structural protein NS1 from WNV elicits a protective immune response against WNV infection, groups of partridges were inoculated with recombinant NS1 (NS1 group) or an unrelated recombinant protein (mock group), and challenged with infectious WNV. A third group received no inoculation prior to challenge (challenge group). The NS1 group failed to elicit detectable antibodies to NS1 while in the mock group a specific antibody response was observed. Moreover, no protection against WNV disease was observed in the NS1 group, but rather, it showed significantly higher viral RNA load and delayed neutralizing antibody response, and suffered a more severe clinical disease, which resulted in higher mortality. This adverse effect has not been observed before and warrants further investigations.

Evaluation of five serologic assays for bovine tuberculosis surveillance in domestic free-range pigs from southern Spain.

In countries where bovine tuberculosis (bTB) is still prevalent the contact among different animal species in extensive systems contributes to the circulation of Mycobacterium bovis (M. bovis) and other members of the Mycobacterium tuberculosis complex (MTC). Thus, free-range pigs can develop subclinical infections and may contribute to disease spread to bovine and wildlife. Serodiagnosis has been proposed as a screening tool for detecting infected pig herds; however, the value of this method to obtain an accurate diagnosis in this species is still not clear. In this study, sensitivity (Se) and specificity (Sp) estimates of four ELISAs and a lateral flow immunochromatographic antibody assay based on different M. bovis antigens, including MPB70 and MPB83 proteins, were evaluated in naturally infected domestic free-range pigs. For this purpose, submandibular lymph nodes and blood samples from 217 pigs from both TB-infected and historically negative farms were sampled at slaughterhouse and analysed by gross examination, histopathology, bacteriological culture and qPCR. Se and Sp estimates of the 5 evaluated assays ranged from 66.1% to 78% (CI95 from 52.6 to 87.7%) and from 98.9% to 100% (CI95 from 93.8 to 100%), respectively. Results of our study suggest that all the evaluated assays could be used as a first screening tool to conduct bTB surveillance in domestic pigs at population level; however, animals from seropositive herds should later be surveyed by other methods in order to reduce false negative results.

Development of a duplex rapid assay for immunoglobulins M and G to evaluate the parvoviral immune status of clinically healthy dogs.

A duplex rapid assay for detection of serum antibodies to canine parvovirus (CPV) was developed. Canine immunoglobulin (Ig)M or IgG were captured in immunotubes with anti-canine IgM or IgG and detected with parvovirus VP2 recombinant protein followed by an anti-VP2 monoclonal antibody. The assay was tested using a collection of sera from dogs that were vaccinated against CPV on arrival at an animal shelter in Madrid, Spain. Results were compared with those of 2 commercial enzyme-linked immunosorbent assays (ELISAs) considered as reference techniques. A high correlation was found between the duplex rapid assay and the ELISAs, presenting an accuracy of 98% and 100% for IgG and IgM, respectively. According to the IgG and IgM levels at days 0-3 postvaccination, the samples were divided into 2 groups. One group of dogs showed high IgG and low IgM values at the first sampling post-vaccination and during the following 14 days, indicating that they had previously been in contact with the virus, either by vaccination or infection before arrival at the animal shelter. A second group of dogs appeared to be unvaccinated or uninfected before arrival at the animal shelter because they had negative IgM and IgG values soon after vaccination. These animals responded to vaccination, as demonstrated by seroconversion of both isotypes of immunoglobulins. The developed assay appears to be useful in determining the unknown immune status of dogs to CPV, especially in kennels and shelters where the rate of infection by CPV is relatively high.

Comparison of two commercial enzyme-linked immunosorbent assays for the diagnosis of Porcine reproductive and respiratory syndrome virus infection.

Early diagnosis of Porcine reproductive and respiratory syndrome virus (PRRSV) is critically important for control of the disease. Two new commercially available enzyme-linked immunosorbent assays (ELISAs) based on different methodologies have been developed. In the present report, the 2 ELISAs were compared using blood samples from experimentally and naturally infected pigs. One of the 2 ELISAs was shown to be more sensitive than the other. The higher sensitivity of one of the ELISAs could pose a problem in PRRS diagnosis in endemic farms, because it can detect maternally derived antibodies for a longer time, overlapping with the detection of antibodies developed after PRRSV infection. However, the ELISA with higher sensitivity could be suitable for early detection of PRRSV antibodies in individual pigs, especially in PRRS-free herds.

Development and evaluation of a new epitope-blocking ELISA for universal detection of antibodies to West Nile virus.

West Nile virus (WNV) is an emerging zoonotic pathogen with a wide range of hosts, including birds, horses and humans. The development and evaluation of the performance of a new enzyme-linked immunosorbent assay (ELISA) are described for rapid detection of WNV-specific antibodies in samples originating from an extensive range of vertebrates susceptible to WNV infection. The assay uses a monoclonal antibody (MAb) which binds whole virus particles and neutralizes infection in vitro by recognizing a neutralizing epitope within the envelope (E) glycoprotein of the virus. This MAb, labelled with horseradish peroxidase, was used to compete with WNV-specific serum antibodies for virus-binding in vitro. The epitope-blocking ELISA was optimized in a manner that enabled its validation with a number of experimental and field sera, from a wide range of wild bird species, and susceptible mammals. The new ELISA exhibited high specificity (79.5-96.5%) and sensitivity (100%), using the virus-neutralization test as reference standard. It also required a much lower volume of sample (10 µl per analysis) compared to other ELISAs available commercially. This new method may be helpful for diagnosis and disease surveillance, particularly when testing samples from small birds, which are available in limited amounts.

Antigen-capture blocking enzyme-linked immunosorbent assay based on a baculovirus recombinant antigen to differentiate Transmissible gastroenteritis virus from Porcine respiratory coronavirus antibodies.

A new commercially available antigen-capture, blocking enzyme-linked immunosorbent assay (antigen-capture b-ELISA), based on baculovirus truncated-S recombinant protein of Transmissible gastroenteritis virus (TGEV) and 3 specific monoclonal antibodies, was developed and evaluated by examining a panel of 453 positive Porcine respiratory coronavirus (PRCoV), 31 positive TGEV, and 126 negative field sera by using another commercially available differential coronavirus b-ELISA as the reference technique to differentiate TGEV- from PRCoV-induced antibodies. The recombinant S protein-based ELISA appeared to be 100% sensitive for TGEV and PRCoV detection and highly specific for TGEV and PRCoV detection (100% and 92.06%, respectively), when qualitative results (positive or negative) were compared with those of the reference technique. In variability experiments, the ELISA gave consistent results when the same serum was evaluated on different wells and different plates. These results indicated that truncated recombinant S protein is a suitable alternative to the complete virus as antigen in ELISA assays. The use of recombinant S protein as antigen offers great advantages because it is an easy-to-produce, easy-to-standardize, noninfectious antigen that does not require further purification or concentration. Those advantages represent an important improvement for antigen preparation, in comparison with other assays in which an inactivated virus from mammalian cell cultures is used.

Development of a sensitive and specific indirect enzyme-linked immunosorbent assay based on a baculovirus recombinant antigen for detection of specific antibodies against Ehrlichia canis.

An indirect enzyme-linked immunosorbent assay (ELISA) based on baculovirus recombinant P30 protein of Ehrlichia canis and the 1BH4 anticanine IgG monoclonal antibody was developed and evaluated by examining a panel of 98 positive and 157 negative sera using the indirect fluorescent antibody (IFA) test as the reference technique. The P30-based ELISA appeared to be sensitive and specific (77.55% and 95.54%, respectively) when qualitative results (positive/negative) were compared with those of the IFA test; the coefficient of correlation (R) between the 2 tests was 0.833. Furthermore, it was possible to establish a mathematical formula for use in comparing the results of both techniques. These results indicate that recombinant P30 antigen-based ELISA is a suitable alternative of the IFA test for simple, consistent, and rapid serodiagnosis of canine ehrlichiosis. Moreover, the use of this recombinant protein as antigen offers a great advantage for antigen preparation in comparison with other techniques in which the whole E. canis organism is used as antigen.

Optimization of the modification of carrier proteins with aminated haptens.

In this report we show that succinic groups are far more reactive to amino compounds than the carboxylic groups derived from Asp and Glu on the protein when using coupling via 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDCI) (even by an 8 fold factor). Accordingly, a new carrier-protein was designed where both natural amino and carboxylic moieties were transformed into succinic residues. To prepare this hypersuccinylated carrier, all exposed carboxylic acids were first transformed into amino groups by reaction with ethylendiamine after activation with EDCI. Secondly, all these residues together with the ones from Lys were succinylated to prepare a fully succinylated protein. This was even more relevant considering that the amount of Lysine was 2-4 fold lower than Asp and Glu in most of the proteins. These "hyper-succinylated" proteins (KLH or BSA) offer significant improvements in protein reactivity compared to the native proteins (by a factor of 8-10). The optimization of the reaction, in which the presence of dioxane was found to be influential, permitted further improvements in the modification of the protein. Finally, this new strategy was successfully used to develop antibodies against the commercial anti-tumor molecule, ET-637-NH2. Using native KLH no response was found, whereas 1/64,000 serum dilutions gave very high values in ELISA procedures when immunization was performed using the hyper-succinylated KLH.