A novel S2-derived peptide-based ELISA for broad detection of antibody against infectious bronchitis virus.

Avian infectious bronchitis (IB) is a highly contagious disease caused by infectious bronchitis virus (IBV). Vaccination is an effective approach for controlling IBV. Therefore, reliable immune monitoring for IB is critical for poultry. In this study, a novel peptide derived from S2 protein was used to develop an enzyme-linked immunosorbent assay (ELISA) for the detection of broadly cross-reactive antibodies against IBV. The peptide-based ELISA (pELISA) showed good specificity and sensitivity in detecting IBV antibodies against different serotypes. A semilogarithmic regression method for determining IBV antibody titers was also established. Antibody titers detected by pELISA and calculated with this equation were statistically similar to those evaluated by indirect fluorescence assay (IFA). Moreover, the comparison analysis showed a 96.07% compatibility between the pELISA and IDEXX ELISA. All these data demonstrate that the pELISA generated here can be as a rapid and reliable serological surveillance tool for monitoring IBV infection or vaccination.

Detection of Influenza D-Specific Antibodies in Bulk Tank Milk from Swedish Dairy Farms.

Influenza D virus (IDV) has been detected in bovine respiratory disease (BRD) outbreaks, and experimental studies demonstrated this virus's capacity to cause lesions in the respiratory tract. In addition, IDV-specific antibodies were detected in human sera, which indicated that this virus plays a potential zoonotic role. The present study aimed to extend our knowledge about the epidemiologic situation of IDV in Swedish dairy farms, using bulk tank milk (BTM) samples for the detection of IDV antibodies. A total of 461 and 338 BTM samples collected during 2019 and 2020, respectively, were analyzed with an in-house indirect ELISA. In total, 147 (32%) and 135 (40%) samples were IDV-antibody-positive in 2019 and 2020, respectively. Overall, 2/125 (2%), 11/157 (7%) and 269/517 (52%) of the samples were IDV-antibody-positive in the northern, middle and southern regions of Sweden. The highest proportion of positive samples was repeatedly detected in the south, in the county of Halland, which is one of the counties with the highest cattle density in the country. In order to understand the epidemiology of IDV, further research in different cattle populations and in humans is required.

[Competitive ELISA test system for the detection of antibodies to the rabies virus in animals (Rhabdoviridae: Lyssavirus)].

INTRODUCTION: The main approach to the rabies prevention is the vaccination of domestic and wild carnivores. For the routine evaluation the anti-rabies vaccination effectiveness, World Organization for Animal Health (OIE) recommends various enzyme-linked immunosorbent assays (ELISA).The aim of the study was to design and validate a competitive ELISA (cELISA) test system for the detection of antibodies to the rabies virus (RABV). MATERIALS AND METHODS: The development of the cELISA was carried out following the OIE recommendations. RESULTS: The repeatability of the cELISA results within one laboratory was satisfactory (coefficient of variation 7.95-13.61%). The coefficient of determination (CD) between the results of the virus neutralization reaction (FAVN) and cELISA was 0.988, p < 0.001. The lower threshold for antibody detection was less than 0.02 IU/ml. The cELISA did not demonstrate cross-reactivity against antibodies to canine distemper virus, parainfluenza virus, parvovirus, coronavirus, and canine adenovirus (types I and II). During the study of 137 dog blood sera, diagnostic specificity (DSp) and diagnostic sensitivity (DSe) for the cELISA were 83.1% and 94.9%, respectively, and CD between the cELISA and FAVN results was 0.968, p < 0.001. DISCUSSION: Indirect ELISA test systems for determining the level of antibodies to RABV are not sensitive enough compared to reference tests, unlike cELISA. The developed test system is not inferior for its DSp and DSe to the commercial cELISA BioPro ELISA Rabies Ab (DSp 66.7%, DSe 94.4%). CONCLUSION: The developed cELISA test system can be used to detect antibodies to RABV in the blood serum of dogs for evaluating the effectiveness of mass vaccination programs.

Development of an enzyme-linked immunosorbent assay based on viral antigen capture by anti-spike glycoprotein monoclonal antibody for detecting immunoglobulin A antibodies against porcine epidemic diarrhea virus in milk.

BACKGROUND: Porcine epidemic diarrhea (PED), caused by PED virus (PEDV), is a severe enteric disease burdening the global swine industry in recent years. Especially, the mortality of PED in neonatal piglets approaches 100%. Maternal antibodies in milk, particularly immunoglobulin A (IgA) antibodies, are of great importance for protection neonatal suckling piglets against PEDV infection as passive lactogenic immunity. Therefore, appropriate detection methods are required for detecting PEDV IgA antibodies in milk. In the current study, we prepared monoclonal antibodies (mAbs) against PEDV spike (S) glycoprotein. An enzyme-linked immunosorbent assay (ELISA) was subsequently developed based on PEDV antigen capture by a specific anti-S mAb. RESULTS: The developed ELISA showed high sensitivity (the maximum dilution of milk samples up to 1:1280) and repeatability (coefficient of variation values < 10%) in detecting PEDV IgA antibody positive and negative milk samples. More importantly, the developed ELISA showed a high coincidence rate with a commercial ELISA kit for PEDV IgA antibody detection in clinical milk samples. CONCLUSIONS: The developed ELISA in the current study is applicable for PEDV IgA antibody detection in milk samples, which is beneficial for evaluating vaccination efficacies and neonate immune status against the virus.

Comparison of the levels of selected specific antibodies in the immunoglobulin G of colostrum versus milk and serum in dairy cows (Bos taurus).

Commercial products containing immunoglobulin G (IgG) sourced from colostrum, milk, and/or serum may be used to supplement or replace maternal colostrum in newborn dairy calves. To determine if antibody specificities in bovine milk and serum IgG differ from colostrum IgG, we sampled serum, colostrum (1 to 2 hours post-partum), and milk (day 5 post-partum) from 24 dairy heifers or cows. Specific antibodies [IgG class (H&L)] to 8 common pathogens were measured using enzyme-linked immunosorbent assays (ELISAs). Immunoglobin G1 and IgG2 subclass-specific ELISAs were performed for 3 of these pathogens. Colostrum-derived IgG contained more specific antibodies to rotavirus [IgG (H&L) and IgG1] and to IgG (H&L) of bovine respiratory syncytial virus (BRSV), bovine parainfluenza-3 virus (BPI3V), Staphylococcus aureus, Escherichia coli F5 (K99), and bovine coronavirus than milk IgG. Colostral IgG contained more antibodies to BRSV (IgG1), rotavirus (IgG1), and IgG (H&L) specific for BRSV, bovine herpesvirus-1 (BHV-1), BPI3V, E. coli F5 (K99), and Streptococcus uberis than serum IgG. Compared to serum, milk contained more IgG (H&L) antibody to BRSV, BHV-1, and BPI3V, IgG1-specific BRSV, and rotavirus. These data indicate that IgG derived from colostrum delivers more specific antibodies to these endemic pathogens of calves compared to IgG sourced from milk or serum. In addition, the IgG1 subclass predominates in milk and colostrum, and both deliver a similar spectrum of antibodies.

Les produits commerciaux contenant de l'immunoglobuline G (IgG) provenant du colostrum, du lait et/ou du sérum peuvent être utilisés pour compléter ou remplacer le colostrum maternel chez les veaux laitiers nouveau-nés. Pour déterminer si les spécificités des anticorps dans le lait de vache et les IgG sériques diffèrent des IgG du colostrum, nous avons prélevé du sérum, du colostrum (1 à 2 heures après le vêlage) et du lait (5 jours après le vêlage) de 24 génisses ou vaches laitières. Des anticorps spécifiques [classe IgG (H&L)] dirigés contre huit agents pathogènes courants ont été mesurés par dosages immuno-enzymatiques (ELISA). Des tests ELISA spécifiques aux sous-classes d'IG1 et d'IgG2 ont été effectués pour trois de ces agents pathogènes. Les IgG dérivées du colostrum contenaient plus d'anticorps spécifiques contre le rotavirus [IgG (H&L) et IgG1] et des IgG (H&L) contre le virus respiratoire syncytial bovin (BRSV), le virus parainfluenza bovin 3 (BPI3V), Staphylococcus aureus, Escherichia coli F5 (K99) et le coronavirus bovin que les IgG du lait. Les IgG du colostrum contenaient plus d'anticorps dirigés contre le BRSV (IgG1), le rotavirus (IgG1) et des IgG (H&L) spécifiques contre BRSV, l'herpèsvirus bovin-1 (BHV-1), le BPI3V, E. coli F5 (K99) et Streptococcus uberis que les IgG du sérum. Comparé au sérum, le lait contenait plus d'anticorps IgG (H&L) contre le BRSV, le BHV-1 et le BPI3V, des IgG1 spécifiques au BRSV et au rotavirus. Ces données indiquent que les IgG dérivées du colostrum fournissent des anticorps plus spécifiques contre ces agents pathogènes endémiques des veaux que les IgG provenant du lait ou du sérum. De plus, la sous-classe IgG1 prédomine dans le lait et le colostrum, et les deux fournissent un spectre similaire d'anticorps.(Traduit par Docteur Serge Messier).

Guest edited collection serological study of SARS-CoV-2 antibodies in japanese cats using protein-A/G-based ELISA.

BACKGROUND: Little is known about the epidemic status of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in cats in Japan due to insufficiently reliable seroepidemiological analysis methods that are easy to use in cats. RESULTS: We developed a protein-A/G-based enzyme-linked immunosorbent assay (ELISA) to detect antibodies against SARS-CoV-2 in cats. The assay was standardized using positive rabbit antibodies against SARS-CoV-2. The ELISA results were consistent with those of a conventional anti-feline-immunoglobulin-G (IgG)-based ELISA. To test the protein-A/G-based ELISA, we collected blood samples from 1,969 cats that had been taken to veterinary clinics in Japan from June to July 2020 and determined the presence of anti-SARS-CoV-2 antibodies. Nine cats were found to have SARS-CoV-2 S1-specific IgG, of which 4 had recombinant receptor-binding domain-specific IgG. Of those 9 samples, one showed neutralizing activity. Based on these findings, we estimated that the prevalence of SARS-CoV-2 neutralizing antibodies in cats in Japan was 0.05% (1/1,969 samples). This prevalence was consistent with the prevalence of neutralizing antibodies against SARS-CoV-2 in humans in Japan according to research conducted at that time. CONCLUSIONS: Protein-A/G-based ELISA has the potential to be a standardized method for measuring anti-SARS-CoV-2 antibodies in cats. The infection status of SARS-CoV-2 in cats in Japan might be linked to that in humans.

Development of an indirect ELISA to detect PEDV specific IgA antibody based on a PEDV epidemic strain.

BACKGROUND: Porcine epidemic diarrhea (PED), a swine epidemic disease caused by porcine epidemic diarrhea virus (PEDV), is characterized by severe watery diarrhea, vomiting, dehydration and high mortality in piglets, and has caused serious economic losses to the global porcine industry. The level of PEDV IgA antibody is a key marker to assess the extent of passive immunity of the resistance against PEDV infection. However, current commercial structure proteins-based kits for detection of PEDV antibody are not affordable, and those kits require complicated antigen preparation procedures, which cannot meet the scope of economic benefits of many large-scale pig companies in China. Therefore, there is an urgent need to develop an accurate, simple, and economical method for IgA detection in clinical samples. In this study, an indirect ELISA (i-ELISA) method was developed based on a purified PEDV epidemic strain (NH-TA2020). RESULTS: The results show that optimal working dilution ratios of PEDV antigen and HRP anti-swine IgA are at 1: 1000 and 1:15000 respectively. The sensitivity of this method is high with the maximum dilution of samples up to 1:160, and coefficients of variation (CV) of both the intra assays and inter assays were no more than 15%. In addition, the relative sensitivities of the i-ELISA were above 90% compared with values from commercial kits in both serum and oral fluid samples. CONCLUSIONS: Our results suggested that the i-ELISA developed in this study was an accurate, simple, and economical method for PEDV-IgA detection in clinical samples.

West Nile and Usutu virus seroprevalence in Hungary: A nationwide serosurvey among blood donors in 2019.

In Hungary, West Nile virus (WNV) has been responsible for 459 laboratory confirmed human cases between 2004 and 2019, while the first human Usutu virus (USUV) infection was confirmed only in 2018. A comprehensive serosurvey was conducted among blood donors to assess the WNV and USUV seroprevalence in 2019, one year after the largest European WNV epidemic. Altogether, 3005 plasma samples were collected and screened for WNV and USUV specific Immunoglobulin G (IgG) antibodies by Enzyme-Linked Immunosorbent Assay (ELISA). All reactive samples were further tested for tick-borne encephalitis virus IgG antibodies by ELISA. Indirect immunofluorescence test and microneutralization assay were used as confirmatory methods. Overall, the WNV seroprevalence was 4.32%, and in five blood donors USUV seropositivity was confirmed. The highest seroprevalence was measured in Central, Eastern and Southern Hungary, while the Western part of the country proved to be less affected. There was a statistically strong association between the WNV seroprevalence of 2019 and the cumulative incidence in the period of 2004 and 2019 calculated for every NUTS 3 region. The last WNV serological screening was performed in 2016 and the prevalence of anti-WNV IgG proved to be 2.19%. One year after the 2018 WNV outbreak, a significant increase in seroprevalence was observed in the Hungarian population and evidence for USUV seropositivity was also obtained. The spatial pattern of seroprevalence can support the identification of high-risk areas raising awareness of the need for increased surveillance, such as screening vector, equine, and avian populations. The communication with general practitioners and other professionals in primary health care services can support the early identification of acute human cases. Education and awareness-raising on the importance of protection against mosquito vectors amongst residents are also important parts of preventive measures.

Prevalence of anti-severe acute respiratory syndrome coronavirus 2 antibodies in cats in Germany and other European countries in the early phase of the coronavirus disease-19 pandemic.

During the first months of the coronavirus disease (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), cases of human-to-cat transmission were reported. Seroconversion was shown in cats infected under experimental and natural conditions. This large-scale survey of 1,005 serum samples was conducted to investigate anti-SARS-CoV-2 antibody prevalence in domestic cats during the first 7 months of the pandemic in Germany and other European countries. In addition, we compared the sensitivity and specificity of two multispecies SARS-CoV-2 antibody enzyme-linked immunosorbent assays (ELISA). Results were confirmed by using an indirect immunofluorescence test (iIFT) and a surrogate virus neutralization test (sVNT). Sera that were highly positive for feline coronavirus (FCoV) antibodies (n = 103) were included to correct for cross-reactivity of the tests used. Our results showed an overall SARS-CoV-2 seropositivity of 1.9% (n = 19) in a receptor-binding domain (RBD)-based ELISA, additional 0.8% (n = 8) were giving inconclusive results. In contrast, a nucleocapsid-based ELISA revealed 0.5% (n = 5) positive and 0.2% (n = 2) inconclusive results. While the iIFT and sVNT confirmed 100% of positive and 50%-57.1% of the doubtful results as determined in the RBD ELISA, the nucleocapsid-based assay showed a high discrepancy and only one of the five positive results could be confirmed. The results indicate significant deficits of the nucleocapsid-based ELISA with respect to sensitivity and specificity. Due to a significantly higher rate (5.8%) of positive results in the group of highly FCoV antibody-positive samples, cross-reactivity of the FCoV-ELISA with SARS-CoV-2 antibodies cannot be excluded. Furthermore, we investigated the impact of direct contact of domestic cats (n = 23) to SARS-CoV-2 positive owners. Considering one inconclusive result, which got confirmed by iIFT, this exposure did not lead to a significantly higher prevalence (4.4%; p = .358) among tested subjects. Overall, we conclude that cats are a negligible entity with respect to virus transmission in Europe.

Establishment of a peptide-based enzyme-linked immunosorbent assay for detecting antibodies against PRRSV M protein.

BACKGROUND: Porcine reproductive and respiratory syndrome (PRRS) is one of the most economically devastating diseases affecting the swine industry globally. Evaluation of antibody responses and neutralizing antibody titers is the most effective method for vaccine evaluation. In this study, the B cell line epitopes of PRRSV M protein were predicted, and two peptide ELISA assays were established (M-A110-129 ELISA, M-A148-174 ELISA) to detect antibodies against PRRSV M protein. Field serum samples collected from pig farms were used to validate the peptide ELISA and compare it with an indirect immunofluorescence assay. RESULTS: The sensitivity and specificity of M-A110-129 ELISA and M-A148-174 ELISA were (111/125) 88.80%, (69/70) 98.57% and (122/125) 97.60%, (70/70) 100%, relative to indirect immunofluorescence assay. This peptide ELISA could detect antibodies against different genotypes of PRRSV including type 1 PRRSV, classical PRRSV, HP-PRRSV, and NADC30 like PRRSV, but not antibodies against other common swine viruses. The results of ROC analysis showed that the area under the curve (AUC) of the M-A110-129 ELISA and M-A148-174 ELISA were 0.967 and 0.996, respectively. Compared the concordance of results using two peptide ELISA assays, the IDEXX PRRSV X3 Ab ELISA and a virus neutralization test, were assessed using a series of 147 sera from pigs vaccinated with the NADC30-like PRRSV inactivated vaccine. The M-A148-174 ELISA had the best consistency, with a Cohen's kappa coefficient of 0.8772. The concordance rates of the Hipra PRRSV ELISA kit, M-A110-129 ELISA and M-A148-174 ELISA in the field seropositive detection results were 91.08, 86.32 and 95.35%, relative to indirect immunofluorescence assay. CONCLUSIONS: In summary, compared with M-A110-129 ELISA, the PRRSV M-A148-174 ELISA is of value for detecting antibodies against PRRSV and the evaluation of the NADC30-like PRRSV inactivated vaccine, but the advantage is insufficient in serological early diagnosis.

Seroprevalence of antibodies against severe acute respiratory coronavirus 2 (SARS-CoV-2) in household dogs in Japan.

We investigated the seroprevalence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) among dogs in the Tokyo area via enzyme-linked immunosorbent assay (ELISA) using the spike protein as the target antigen. Plasma samples from 494 household dogs and blood-donor dogs were tested from July 2020 to January 2021. Of these samples, three showed optical densities that were higher than the mean plus two standard deviations of the mean of the negative-control optical densities (ODs). Of these three samples, only the sample with the highest OD by ELISA was confirmed positive by virus neutralization testing. The positive dog presented no SARS-CoV-2-related symptoms. The positivity rate of SARS-CoV-2 infections among dogs in the Tokyo area was approximately 0.2%.

Molecular detection of bat coronaviruses in three bat species in Indonesia.

Bats are an important reservoir of several zoonotic diseases. However, the circulation of bat coronaviruses (BatCoV) in live animal markets in Indonesia has not been reported. Genetic characterization of BatCoV was performed by sequencing partial RdRp genes. Real-time polymerase chain reaction based on nucleocapsid protein (N) gene and Enzyme-linked immunosorbent assay against the N protein were conducted to detect the presence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral RNA and antibody, respectively. We identified the presence of BatCoV on Cynopterus brachyotis, Macroglossus minimus, and Rousettus amplexicaudatus. The results showed that the BatCoV included in this study are from an unclassified coronavirus group. Notably, SARS-CoV-2 viral RNA and antibodies were not detected in the sampled bats.

Development of an antigen-ELISA and a colloidal gold-based immunochromatographic strip based on monoclonal antibodies for detection of avian influenza A(H5) viruses.

Avian influenza A(H5) viruses (avian IAVs) pose a major threat to the economy and public health. We developed an antigen-ELISA (ag-ELISA) and a colloidal gold-based immunochromatographic strip for the rapid detection of avian A(H5) viruses. Both detection methods displayed no cross-reactivity with other viruses (e.g., other avian IAVs, infectious bursal disease virus, Newcastle disease virus, infectious bronchitis virus, avian paramyxovirus). The ag-ELISA was sensitive down to 0.5 hemagglutinin (HA) units/100 µL of avian A(H5) viruses and 7.5 ng/mL of purified H5 HA proteins. The immunochromatographic strip was sensitive down to 1 HA unit/100 µL of avian A(H5) viruses. Both detection methods exhibited good reproducibility with CVs < 10%. For 200 random poultry samples, the sensitivity and specificity of the ag-ELISA were 92.6% and 98.8%, respectively, and for test strips were 88.9% and 98.3%, respectively. Both detection methods displayed high specificity, sensitivity, and stability, making them suitable for rapid detection and field investigation of avian A(H5) viruses.

Development of an indirect ELISA for detecting swine acute diarrhoea syndrome coronavirus IgG antibodies based on a recombinant spike protein.

Swine acute diarrhoea syndrome coronavirus (SADS-CoV) is a newly identified swine enteropathogenic coronavirus that causes watery diarrhoea in neonatal piglets, leading to significant economic losses to the swine industry. Currently, there are no suitable serological methods to assess the infection of SADS-CoV and effectiveness of vaccines, making an urgent need to exploit effective enzyme-linked immunosorbent assay (ELISA) to compensate for this deficiency. Here, a recombinant plasmid that expresses the spike (S) protein of SADS-CoV fused to the Fc domain of human IgG was constructed to generate recombinant baculovirus and expressed in HEK 293F cells. The S-Fc protein was purified with protein G Resin, which retained reactivity with anti-human Fc and anti-SADS-CoV antibodies. The S-Fc protein was then used to develop an indirect ELISA (S-iELISA) and the reaction conditions of S-iELISA were optimized. As a result, the cut-off value was determined as 0.3711 by analyzing OD450nm values of 40 SADS-CoV-negative sera confirmed by immunofluorescence assay (IFA) and western blot. The coefficient of variation (CV) of 6 SADS-CoV-positive sera within and between runs of S-iELISA were both less than 10%. The cross-reactivity assays demonstrated that S-iELISA was non-cross-reactive with other swine viruses' sera. Furthermore, the overall coincidence rate between IFA and S-iELISA was 97.3% based on testing 111 clinical serum samples. Virus neutralization test with seven different OD450nm values of the sera showed that the OD450nm values tested by S-iELISA are positively correlated with the virus neutralization assay. Finally, a total of 300 pig field serum samples were tested by S-iELISA and commercial kits of other swine enteroviruses showed that the IgG-positive for SADS-CoV, TGEV, PDCoV and PEDV was 81.7, 54, 65.3 and 6%, respectively. The results suggest that this S-iELISA is specific, sensitive, repeatable and can be applied for the detection of the SADS-CoV infection in the swine industry.

Serological survey of antibodies against SARS-CoV-2 in dogs and cats, Thailand.

Coronavirus disease of 2019 (COVID-19) caused by severe acute respiratory syndrome virus type 2 (SARS-CoV-2) is an emerging severe acute respiratory disease affecting global human health. In this study, a large-scale serological survey of antibodies against SARS-CoV-2 in dogs and cats was conducted during the first and second waves of COVID-19 outbreaks in Thailand, from April to December 2020. A total of 3215 serum samples were collected from dogs (n = 2102) and cats (n = 1113) living in Bangkok and in the vicinities. Serum samples were tested for SARS-CoV-2 antibodies by using an indirect multispecies enzyme-linked immunosorbent assay (ELISA). Positive and suspected samples were additionally tested for neutralizing antibodies by the surrogate virus neutralization test (sVNT). The indirect ELISA results showed that 1.66% (35 out of 2103) of dogs and 0.36% (four out of 1112) of cats were positive for SARS-CoV-2 antibodies. The sVNT results showed that all ELISA-positive and suspected samples were negative for neutralizing antibodies. Positive serum samples (35 dogs and four cats) were obtained from clinically healthy animals and animals with mild respiratory signs aged <1-13 years living in Bangkok and Samutprakarn Provinces. In summary, a serological survey revealed evidence of anti-N-IgG antibodies suggesting SARS-CoV-2 exposure in both dogs and cats during the first and second COVID-19 outbreaks in Thailand.

Seroprevalence of SARS-CoV-2 (COVID-19) exposure in pet cats and dogs in Minnesota, USA.

The COVID-19 pandemic caused by the coronavirus SARS-CoV-2 is continuing to spread globally. SARS-CoV-2 infections of feline and canine species have also been reported. However, it is not entirely clear to what extent natural SARS-CoV-2 infection of pet dogs and cats is in households. We have developed enzyme-linked immunosorbent assays (ELISAs) using recombinant SARS-CoV-2 nucleocapsid (N) protein and the receptor-binding-domain (RBD) of the spike protein, and the SARS-CoV-2 spike-pseudotyped vesicular stomatitis virus (VSV)-based neutralization assay to screen serum samples of 239 pet cats and 510 pet dogs in Minnesota in the early phase of the COVID-19 pandemic from mid-April to early June 2020 for evidence of SARS-CoV-2 exposures. A cutoff value was used to identify the seropositive samples in each experiment. The average seroprevalence of N- and RBD-specific antibodies in pet cats were 8% and 3%, respectively. Among nineteen (19) N-seropositive cat sera, fifteen (15) exhibited neutralizing activity and seven (7) were also RBD-seropositive. The N-based ELISA is also specific and does not cross react with antigens of common feline coronaviruses. In contrast, SARS-CoV-2 antibodies were detected at a very low percentage in pet dogs (~ 1%) and were limited to IgG antibodies against SARS-CoV-2 N protein with no neutralizing activities. Our results demonstrate that SARS-CoV-2 seropositive rates are higher in pet cats than in pet dogs in MN early in the pandemic and that SARS-CoV-2 N-specific IgG antibodies can detect SARS-CoV-2 infections in companion animals with higher levels of specificity and sensitivity than RBD-specific IgG antibodies in ELISA-based assays.

A serological survey of severe acute respiratory syndrome coronavirus 2 in dogs in Wuhan.

The novel coronavirus disease (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in an unprecedented public health crisis and economic losses. Although several cases of cats and dogs infected with SARS-CoV-2 have been reported during this outbreak, the prevalence of SARS-CoV-2 in dog and its transmission among other companion animals are still unknown. Here, we report an extensive serological study of SARS-CoV-2 infection in dogs in Wuhan and analyse the infection rates at different stages of the pandemic outbreak. A total of 946 dogs serum samples were collected from Wuhan, of which 36 samples were obtained prior to the pandemic outbreak. Indirect enzyme-linked immunosorbent assay (ELISA) showed that 16 sera collected during the outbreak were detected as positive through the receptor-binding domain (RBD) of SARS-CoV-2. Of these 16 sera, 10 exhibited measurable SARS-CoV-2-specific neutralizing antibodies whose titres ranged from 1/20 to 1/180. No serological cross-reactivity was detected between SARS-CoV-2 and canine coronavirus (CCV). Furthermore, with the effective control of the outbreak, a decrease in the SARS-CoV-2 seropositive dog number was observed. Our results suggest that SARS-CoV-2 has infected companion dogs during the outbreak, and that COVID-19 patient families have a higher risk of dog infection. Our findings deepen our understanding of the infection of SARS-CoV-2 in dogs and provide an important reference for prevention of COVID-19.

Multi-species ELISA for the detection of antibodies against SARS-CoV-2 in animals.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a pandemic with millions of infected humans and hundreds of thousands of fatalities. As the novel disease - referred to as COVID-19 - unfolded, occasional anthropozoonotic infections of animals by owners or caretakers were reported in dogs, felid species and farmed mink. Further species were shown to be susceptible under experimental conditions. The extent of natural infections of animals, however, is still largely unknown. Serological methods will be useful tools for tracing SARS-CoV-2 infections in animals once test systems are evaluated for use in different species. Here, we developed an indirect multi-species ELISA based on the receptor-binding domain (RBD) of SARS-CoV-2. The newly established ELISA was evaluated using 59 sera of infected or vaccinated animals, including ferrets, raccoon dogs, hamsters, rabbits, chickens, cattle and a cat, and a total of 220 antibody-negative sera of the same animal species. Overall, a diagnostic specificity of 100.0% and sensitivity of 98.31% were achieved, and the functionality with every species included in this study could be demonstrated. Hence, a versatile and reliable ELISA protocol was established that enables high-throughput antibody detection in a broad range of animal species, which may be used for outbreak investigations, to assess the seroprevalence in susceptible species or to screen for reservoir or intermediate hosts.

Seroprevalence of influenza D virus in bulls in Argentina.

Influenza D virus (IDV) is considered a new agent involved in bovine respiratory disease (BRD). Based on seroprevalence studies or isolation from clinical samples, this virus has been detected on several continents and in several animal species, including cattle, pigs, camel, horses, and goats. We used an indirect in-house ELISA to detect anti-IDV antibodies in 165 serum samples from bulls on 116 farms in the province of La Pampa, Argentina. Eighty-five of 116 (73%) farms had at least 1 positive animal, and 112 of 165 (68%) of the analyzed samples were positive. There were no significant differences in the proportion of seropositive samples depending on the geographic region in which the samples were taken. Our results suggest that IDV infection is endemic in La Pampa; the clinical importance of IDV in Argentina remains to be investigated.

Agrodiag PorCoV: A multiplex immunoassay for the differential diagnosis of porcine enteric coronaviruses.

Three different porcine enteric coronaviruses (PECs), i.e., porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV) and porcine Deltacoronavirus (PDCoV) are currently circulating in U.S. commercial swine herds. Differential diagnosis of PECs relies on laboratory methods. This study describes the development of an ELISA-like multiplex planar immunoassay based on virus-specific recombinant S1 proteins printed in an array of spots at the bottom of a 96-well microplate for simultaneous detection differential serodiagnosis of PEDV, TGEV, PDCoV in a single sample. The technology overall format and working principle is similar to the solid-phase standard ELISA. After the three typical incubation steps, the reaction was visualized as blue spots which intensity correlated with antibody levels to specific viral antigen target in the array. The diagnostic performance of the assay was evaluated on known status serum samples (n = 480) collected over time (day post-inoculation -7, 0, 7, 14, 21, 28, 35, and 42) from pigs inoculated with PEDV, TGEV Purdue, TGEV Miller, PDCoV (USA/IL/2014), or mock inoculated with culture media under experimental conditions. Antigen-specific cut-offs were selected to ensure 100% diagnostic and analytical specificity for each given antigen target. The overall diagnostic sensitivity was 92% (44/48 positives, 95% confidence interval (CI) 98,100) for PEDV S1, 100% (95/95 positives, 95% CI 98, 100) for TGEV S1, and 98% (47/48 positives, 95% CI 97, 100) for PDCoV S1. The results of this study demonstrate that the AgroDiag PEC multiplex immunoassay is an efficient and reliable test for differential detection and serodiagnosis of PEDV, TGEV and PDCoV.