Development of a Bead-Based Multiplex Fluorescent Immunoassay to Detect Antibodies against Maedi-Visna Virus in Sheep.

The Maedi-visna virus (MVV) causes a persistent infection in small ruminants, and its high genetic heterogeneity affects the performance of diagnostic tests when used in different populations. Therefore, the aim of this study was to develop a bead-based multiplex immunoassay tailored to detect antibodies against a Norwegian MVV strain. We used tissue samples from 14 PCR-positive sheep from a recent MVV outbreak in Norway to sequence the viral strain and produced recombinant antigens based on sequences from one animal. The assay included commercial TM-A and recombinant Norwegian p25, p16-25 and SU5 antigens. Cut-off values for each antigen were determined using receiver operating characteristic curves on 40 ELISA-negative and 67 ELISA-positive samples from the outbreak. The intraplate and interplate repeatability were investigated by testing a quadruplicate of five samples over three days, while the analytical sensitivity (aSe) and specificity (aSp) were measured in comparison to a commercial ELISA. The repeatability showed a coefficient of variation below 15% for most positive samples. The aSe was equal or higher for the multiplex assay than the ELISA, and the aSp of each antigen was 91.7, 93.3, 95.0 and 93.3% for p25, p16-25, SU5 and TM-A, respectively. The assay shows promising results; however, further evaluations of diagnostic characteristics are necessary before implementation in the Norwegian surveillance programme.

A semi-quantitative visual lateral flow immunoassay for SARS-CoV-2 antibody detection for the follow-up of immune response to vaccination or recovery.

The lateral flow immunoassay (LFIA) technique is largely employed for the point-of-care detection of antibodies especially for revealing the immune response in serum. Visual LFIAs usually provide the qualitative yes/no detection of antibodies, while quantification requires some equipment, making the assay more expensive and complicated. To achieve visual semi-quantification, the alignment of several lines (made of the same antigen) along a LFIA strip has been proposed. The numbering of the reacting lines has been used to correlate with the quantity of some biomarkers in serum. Here, we designed the first semiquantitative LFIA for detecting antibodies and applied it to classify the immune response to SARS-CoV-2 raised by vaccination or natural infection. We used a recombinant spike receptor-binding domain (RBD) as the specific capture reagent to draw two test lines. The detection reagent was selected among three possible ligands that are able to bind to anti-spike human antibodies: the same RBD, staphylococcal protein A, and anti-human immunoglobulin G antibodies. The most convenient detector, adsorbed on gold nanoparticles, was chosen based on the highest correlation with an antibody titre of 171 human sera, measured by a reference serological method, and was the RBD (Spearman's rho = 0.84). Incorporated into the semiquantitative LFIA, it confirmed the ability to discriminate high- and low-titre samples and to classify them into two classes (Dunn's test, P < 0.05). The proposed approach enabled the semiquantification of the immune response to SARS-CoV-2 by the unaided eye observation, thus overcoming the requirement of costly and complicated equipment, and represents a general strategy for the development of semiquantitative serological LFIAs.

Seroprevalence and Risk Factors Associated with Chlamydia abortus Infection in Sheep and Goats in North-Western Italy.

Chlamydia abortus, although poorly recognized as a human pathogen, is a zoonotic microorganism that can cause many different symptoms in humans, including subclinical infection and fatal illnesses in pregnant women. C. abortus is one of the most common causes of ovine and caprine infectious abortion worldwide, known as the causative agent of the enzootic abortion of ewes (EAE) or ovine enzootic abortion (OEA). To estimate C. abortus seroprevalence and the risk factors related to C. abortus in small ruminants, the sera from 3045 animals (both sheep and goat) belonging to 202 herds were tested and a questionnaire investigating flock management was administered. At the herd level, the true seroprevalence was 56.6% (CI95%: 46.9-66.3%), at sheep-farm and goat-farm level, the true seroprevalence was 71.4% (CI95%: 54.6-88.3%) and 44.8% (CI95%: 41.3-57.0%), respectively. The true seroprevalence was significantly higher among the sheep than the goats. The logistic regression model identified four factors associated with Chlamydia seropositivity: flock size (i.e., farms with >50 heads), contact with cattle, introduction of animals, and Coxiella seropositivity. The study evidenced a high seroprevalence of Chlamydia abortus in small ruminant farms in the Piedmont region. Considering its zoonotic potential and the health consequences in humans, communication to farmers on the importance of vaccination, as well as the sensibilization of farm vets, seem to be strategical.

Experimental design for the development of a multiplex antigen lateral flow immunoassay detecting the Southern African Territory (SAT) serotypes of foot-and-mouth disease virus.

Antigenic lateral flow immunoassays (LFIAs) rely on the non-competitive sandwich format, including a detection (labelled) antibody and a capture antibody immobilised onto the analytical membrane. When the same antibody is used for the capture and the detection (single epitope immunoassay), the saturation of analyte epitopes by the probe compromises the capture and lowers the sensitivity. Hence, several factors, including the amount of the probe, the antibody-to-label ratio, and the contact time between the probe and the analyte before reaching the capture antibody, must be adjusted. We explored different designs of experiments (full-factorial, optimal, sub-optimal models) to optimise a multiplex sandwich-type LFIA for the diagnosis and serotyping of two Southern African Territory (SAT) serotypes of the foot-and-mouth disease virus, and to evaluate the reduction of the number of experiments in the development. Both assays employed single epitope sandwich, so most influencing variables on the sensitivity were studied and individuated. We upgraded a previous device increasing the sensitivity by a factor of two and reached the visual limit of detection of 103.7 and 104.0 (TCID/mL) for SAT 1 and SAT 2, respectively. The positioning of the capture region along the LFIA strip was the most influent variable to increase the detectability. Furthermore, we confirmed that the 13-optimal DoE was the most convenient approach for designing the device.

Validation of a Commercial Indirect ELISA Kit for the Detection of Bovine alphaherpesvirus1 (BoHV-1)-Specific Glycoprotein E Antibodies in Bulk Milk Samples of Dairy Cows.

In this study, we validated a commercial indirect enzyme-linked immunosorbent assay (ELISA) to detect antibodies to glycoprotein E (gE) of Bovine alphaherpesvirus 1 (BoHV-1) in bulk milk (BM) samples using the OIE Manual of Diagnostic Tests and Vaccines for Terrestrial Animals. The assay performance characteristics were evaluated using a panel of positive (n = 36) and negative (n = 80) samples with known infectious bovine rhinotracheitis (IBR) status. The assay showed adequate repeatability (within-run and between-run), with a coefficient of variability (CV%) of replicates below 30%; only two 1:40 diluted samples had a CV% above 20%. Additionally, an agreement analysis of the qualitative results of replicates led to a Gwet's agreement coefficient of 0.99 (95% confidence interval (CI): 0.96−1.00, p < 0.001). The estimated diagnostic sensitivity (DSe) and diagnostic specificity (DSp) were 100% (95% CI: 90.3−100%) and 97.5% (95% CI: 91.3−99.7%), respectively. Overall, a good level of agreement was observed between the assay results and the true IBR status of samples (weighted Cohen's κ: 0.96, 95% CI: 0.78−1.00). The findings demonstrate that the indirect ELISA kit validated here is an easy-to-use and economical method to differentiate infected and gE-deleted marker vaccine-immunised animals using BM samples.

Detailed epitope mapping of SARS-CoV-2 nucleoprotein reveals specific immunoresponse in cats and dogs housed with COVID-19 patients.

Since the initial emergence in December 2019, the novel Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has been reported in over 200 countries, representing an unprecedented challenge related to disease control worldwide. In this context, cases of human to animal transmission have been reported, raising concern about the potential role of companion animals in the pandemic and stressing the need for reliable animal testing. In the study, a detailed epitope mapping of SARS-CoV-2 nucleoprotein, using both human and pet sera, allowed the identification of the most antigenic region in the C-terminus domain of the protein, which was used to develop an experimental double antigen-based ELISA. A panel of pre-pandemic sera and sera of animals immunized against (or naturally infected with) related coronaviruses was used to assess assay specificity at 99.5%. Positive sera belonging to animals housed with COVID-19 patients were confirmed with the experimental double-antigen ELISA using Plaque Reduction Neutralization test (PRNT) test as gold standard. The availability of a serological assay that targets a highly specific viral antigen represents a valuable tool for multispecies monitoring of Coronavirus Disease 2019 (COVID-19) infection in susceptible animals.

Design of multiplexing lateral flow immunoassay for detection and typing of foot-and-mouth disease virus using pan-reactive and serotype-specific monoclonal antibodies: Evidence of a new hook effect.

The foot-and-mouth disease (FMD) is the most important transboundary viral disease of livestock in the international context, because of its extreme contagiousness, widespread diffusion, and severe impact on animal trade and animal productions. The rapid and on-field detection of the virus responsible for the FMD represents an urgent demand to efficiently control the diffusion of the infection, especially in low resource setting where the FMD is endemic. Colorimetric lateral flow immunoassay (LFIA) is largely used for the development of rapid tests, due to the extreme simplicity, cost-effectiveness, and on-field operation. In this work, two multiplex LFIA devices were designed for the diagnosis of FMD and the simultaneous identification of major circulating serotypes of the FMD virus. The LFIAs relied on the sandwich-type immunoassay and combined a set of well-characterised monoclonal antibodies (mAb) pairs. One LFIA aimed at detecting and identifying O, A and Asia-1 serotypes, the second device enabled the detection and differentiation of the SAT 1 and SAT 2 serotypes. Both devices also incorporated a broad-specific test line reporting on infection from FMDV, regardless the strain and the serotype involved. Accordingly, five and four reactive zones were arranged in the two devices to achieve a total of six simultaneous analyses. The development of the two multiplex systems highlighted for the first time the relevance of the mAb positioning along the LFIA strip in connection with the use of the same or different mAb as capture and detector ligands. In fact, the excess of detector mAb typically employed for increasing the sensitivity of sandwich immunoassay induced a new type of hook effect when combined with the same ligand used as the capture. This effect strongly impacted assay sensitivity, which could be improved by an intelligent alignment of the mAb pairs along the LFIA strip. The analytical and diagnostic performances of the two LFIAs were studied by testing reference FMDV strains grown in cell cultures and some representative field samples (epithelium homogenates). Almost equivalent sensitivity and specificity to those of a reference Ag-ELISA kit were shown, except for the serotype SAT 2. These simple devices are suitable in endemic regions for in-field diagnosis of FMD accompanied by virus serotyping and, moreover, could be deployed and used for rapid confirmation of secondary outbreaks after FMD incursions in free-areas, thus contributing to promptly implement control measures.

Cross-Sectional Serosurvey of Companion Animals Housed with SARS-CoV-2-Infected Owners, Italy.

We conducted a serologic survey among dogs and cats in Italy to detect antibodies against severe acute respiratory syndrome virus 2 (SARS-CoV-2). We found that SARS-CoV-2 seroprevalence was higher among cats (16.2%) than dogs (2.3%). In addition, seroprevalence was higher among animals living in close contact with SARS-CoV-2-positive owners.

A multi-target lateral flow immunoassay enabling the specific and sensitive detection of total antibodies to SARS COV-2.

A rapid test for detecting total immunoglobulins directed towards the nucleocapsid protein (N) of severe acute syndrome coronavirus 2 (SARS CoV-2) was developed, based on a multi-target lateral flow immunoassay comprising two test lines. Both test lines bound to several classes of immunoglobulins (G, M, and A). Specific anti-SARS immunoglobulins were revealed by a colorimetric probe formed by N and gold nanoparticles. Targeting the total antibodies response to infection enabled achieving 100% diagnostic specificity (95.75-100, C.I. 95%, n = 85 healthy and with other infections individuals) and 94.6% sensitivity (84.9-98.9, C.I. 95%, n = 62 SARS CoV-2 infected subjects) as early as 7 days post confirmation of positivity. Agreeing results with a reference serological ELISA were achieved, except for the earlier detection capability of the rapid test. Follow up of the three seroconverting patients endorsed the hypothesis of the random rise of the different immunoglobulins and strengthened the 'total antibodies' approach for the trustworthy detection of serological response to SARS CoV-2 infection.

Compartmentalized evolution of Bovine Viral Diarrhoea Virus type 2 in an immunotolerant persistently infected cow.

Bovine viral diarrhea virus (BVDV) is one of the most important pathogens of cattle worldwide. BVDV-1 is widely distributed in Italy, while BVDV-2 has been detected occasionally. BVDV can be classified in two biotypes, cytopathic (CP) or noncytopathic (NCP). The characteristic of the virus is linked with the infection of a pregnant dam with a NCP strain: due to viral establishment before maturation of the fetal immune system the calf remains persistently infected (PI) and immunotolerant to the infecting BVDV strain. Thanks to their immunotolerance, PI animals represent a unique model to study the viral distribution and compartmentalization in absence of immunoresponse in vivo. In the present study, NGS sequencing was used to characterize the BVDV2 viral strain infecting a PI calf and to describe the viral quasispecies in tissues. Even if the consensus sequences obtained by all the samples were highly similar, quasispecies was described evaluating the presence and the frequency of variants among all the sequencing reads in each tissue. The results suggest a high heterogeneity of the infecting viral strain suggesting viral compartmentalization. The quasispecies analysis highlights the complex dynamics of viral population structure and can increase the knowledge about viral evolution in BVDV-2 persistently infected animals.

Field application of an indirect gE ELISA on pooled milk samples for the control of IBR in free and marker vaccinated dairy herds.

BACKGROUND: The aim of the present study was to assess the reliability of a new strategy for monitoring the serological response against Bovine Herpesvirus 1 (BoHV1), the causative agent of infectious bovine rhinotracheitis (IBR). Bulk milk samples have already been identified as cost effective diagnostic matrices for monitoring purposes. Nevertheless, most eradication programs are still based on individual standard assays. In a region of northwestern Italy (Piedmont), the voluntary eradication program for IBR has become economically unsustainable. Being the prevalence of infection still high, glycoprotein E-deleted marker vaccines are commonly used but gE blocking ELISAs are less sensitive on bulk milk samples compared to blood serum. RESULTS: A recently developed indirect gE ELISA showed high versatility when applied to a wide range of matrices. In this study, we applied a faster, cost effective system for the concentration of IgG from pooled milk samples. The IgG enriched fractions were tested using a gE indirect ELISA for monitoring purposes in IBR-positive and IBR-marker-vaccinated herds. Official diagnostic tests were used as gold standard. During a 3 years study, a total 250 herds were involved, including more than 34,500 lactating cows. The proposed method showed a very good agreement with official diagnostic protocols and very good diagnostic performances: only 37 positive animals were not detected across the entire study. CONCLUSIONS: The results highlighted the ability of the proposed method to support the surveillance of IBR in the Piedmont region, reducing the costs without affecting the diagnostic performances.

A versatile and sensitive lateral flow immunoassay for the rapid diagnosis of visceral leishmaniasis.

Visceral leishmaniasis (VL) is a zoonotic infectious disease with a severe impact on humans and animals. Infection is transmitted by phlebotomine sand flies, and several domestic and wild mammals act as reservoirs for the infection, so the prompt detection of infected hosts is crucial to preventing and controlling the spread of the disease and its transmission to humans. A rapid and portable tool for VL diagnosis based on the lateral flow immunoassay (LFIA) technology is described herein. The device exploits a highly specific chimeric recombinant antigen as the recognition element for capturing anti-leishmanial antibodies, and protein A labelled with gold nanoparticles as the signal reporter. The LFIA shows excellent diagnostic sensitivity (98.4%), specificity (98.9%), and agreement with serological reference methods for diagnosing canine VL. The long-term stability of the LFIA device was confirmed based on six months of storage at room temperature or 4 °C, and the qualitative response of the device was not affected by limited thermal stress. The use of the broadly specific protein A means that the LFIA can be readily adapted to diagnose VL in dogs (the main reservoir for human infection) and other mammals, thus further assisting efforts to control the spread of VL. Graphical abstract A rapid and portable diagnostic tool for visceral leishmaniasis (VL) based on lateral flow immunoassay (LFIA) technology. The presence of anti-leishmanial antibodies is revealed through the binding of these antibodies to a highly specific chimeric antigen. Employing a broadly specific signal reporter (protein A labelled with gold nanoparticles) enables the LFIA to be easily adapted to diagnose VL in different animals.

Validation of an indirect ELISA employing a chimeric recombinant gag and env peptide for the serological diagnosis of equine infectious anemia.

The National Reference Center for equine infectious anemia (EIA) validated a commercial ELISA (Eradikit® EIAV Indirect ELISA, In3diagnostic®, Turin, Italy) employing a chimeric recombinant gag and env peptide for the detection of EIA virus antibodies, following the guidelines of the World Organization for Animal Health. The validation parameters evaluated were: analytical sensitivity (Se) and specificity (Sp); diagnostic Se and Sp; precision, based on repeatability and reproducibility through the estimation of the standard deviation (SD) and the coefficient of variation (CV); accuracy, estimated from a multiple K and relative Sp and Se with respect to those of the agar gel immunodiffusion test (AGIDT). Positive and negative predictive values were also defined. The assay showed a high specificity and a limit of detection of 1.43 log10 major than AGIDT. Diagnostic Se was 100% and Sp was 99.3%, while SD values ranged from 1.58 to 5.01 with a CV between 2.8% and 28.8%. Multiple K was 0.98 and relative Se and Sp were respectively 99.1% and 100%. The assay proved to be robust and to possess a high sensitivity in detecting first antibodies produced at onset of infection as well as high analytic and diagnostic Se and Sp values, confirming it as a serological assay fit for purpose within EIA surveillance programs.

Pestivirus infection in cattle dairy farms: E2 glycoprotein ELISA reveals the presence of bovine viral diarrhea virus type 2 in northwestern Italy.

BACKGROUND: Bovine viral diarrhea virus (BVDV) types 1 and 2 are members of the Pestivirus genus of the Flaviviridae family. This genus also includes the HoBi-like virus, tentatively classified as BVDV type 3. BVDV-1 is widely distributed in Italy despite the extensive use of BVDV-1-based vaccines, while BVDV-2 and HoBi-like Pestivirus have been detected occasionally. Monitoring the occurrence of sporadic or atypical pestiviruses is a useful approach to evaluate the need for additional vaccine strains that can be used in BVDV control programs. RESULTS: In this study we developed a multiwell antibody ELISA based on the recombinant E2 protein of the three bovine pestiviruses. We evaluated the assay's applicability for surveillance purposes using pooled milk samples, each prepared from a maximum of 35 lactating cows and collected from 176 dairy herds. As expected, the majority of the pooled samples reacted to a greater extent against the BVDV-1 E2 antigen. All three milk pools from a single farm reacted to the BVDV-2 antigen, however. Further analysis using spot tests, antigen detection, and sequence analysis of the 5'-UTR region confirmed the presence of five persistently infected calves carrying a BVDV-2a strain. CONCLUSIONS: This study highlights for the first time that sporadic circulation of BVDV-2 can be predicted by immunoenzymatic methods in the absence of specific vaccination.

Surveillance of Infectious Bovine Rhinotracheitis in marker-vaccinated dairy herds: Application of a recombinant gE ELISA on bulk milk samples.

Infectious Bovine Rhinotracheitis (IBR) occurs worldwide, requiring significant resources for eradication programs or surveillance purposes. The status of infection is usually detected by serological methods using the virus neutralization test (VNT) or enzyme-linked immunosorbent assay (ELISA) on individual sera. The gE DIVA (Differentiating Infected from Vaccinated Animals) vaccines approach, adopted in order to reduce the virus circulation and prevent clinical signs, have tightened the range of available methods for the serological diagnosis. Different gE blocking ELISA could be performed to detect specific antibodies in sera of infected or whole virus-vaccinated animals but with less sensitivity if applied to bulk milk samples, especially in marker-vaccinated herds. A new rec-gE ELISA was recently developed in Italy and applied with good performances on blood serum samples. The present paper focuses on the application of a rapid protocol for purification/concentration of immunoglobulin G (IgG) from bulk milk and on the use of the new rec-gE indirect ELISA. The study involved three different partners and 225 herds (12,800 lactating cows) with different official IBR diagnostic statuses. The diagnostic specificity of the method was demonstrated closed to 100% while the diagnostic sensitivity was strictly related to the herd-seroprevalence. Considering 2.5% as the limit of detection of within-herd seropositivity prevalence, the diagnostic sensitivity showed by the proposed method was equal to 100%. A single reactivation of a whole strain vaccine in an old cow was detected inside a group of 67 lactating cows, showing the field applicability of the method.

Development and validation of an indirect ELISA as a confirmatory test for surveillance of infectious bovine rhinotracheitis in vaccinated herds.

BACKGROUND: Bovine herpesvirus 1 (BoHV1) is a member of the viral subfamily of Alphaherpesvirinae that infects various species, including cattle, sheep, and goats. The virus causes infectious bovine rhinotracheitis (IBR), which is included in a European list of diseases that may require control and eradication programs. The lack of confirmatory tests affects the validity of diagnostic tools, especially those used for vaccinated herds. In this study, we report the development and validation of an indirect enzyme-linked immunosorbent assay (ELISA) based on BoHV1 glycoprotein E, which was expressed as a secreted recombinant antigen in a mammalian cell system. The performance of the new rec-gE ELISA was compared with that of commercially available indirect and/or blocking ELISAs. RESULTS: The sample set included blood sera from animals from IBR-positive farms, IBR-free farms, and marker-vaccinated farms. The indirect ELISA proposed in this study is based on antibody reactivity against BoHV1 gE, and showed high sensitivity and specificity (98.41 and 99.76 %, respectively). CONCLUSIONS: The ELISA performed well, in terms of both its diagnostic sensitivity and specificity, and as a confirmatory methodology, and therefore should improve the diagnostic protocols used for IBR surveillance.