Serological evidence of exposure to ebolaviruses in domestic pigs from Guinea

The genus Ebolavirus comprises several virus species with zoonotic potential and varying pathogenicity for humans. Ebolaviruses are considered to circulate in wildlife with occasional spillover events into the human population which then often leads to severe disease outbreaks. Several studies indicate a significant role of bats as reservoir hosts in the ebolavirus ecology. However, pigs from the Philippines have been found to be naturally infected with Reston virus (RESTV), an ebolavirus that is thought to only cause asymptomatic infections in humans. The recent report of ebolavirus‐specific antibodies in pigs from Sierra Leone further supports natural infection of pigs with ebolaviruses. However, susceptibility of pigs to highly pathogenic Ebola virus (EBOV) was only shown under experimental settings and evidence for natural infection of pigs with EBOV is currently lacking. Between October and December 2017, we collected 308 serum samples from pigs in Guinea, West Africa, and tested for the presence of ebolavirus‐specific antibodies with different serological assays. Besides reactivity to EBOV nucleoproteins in ELISA and Western blot for 19 (6.2%) and 13 (4.2%) samples, respectively, four sera recognized Sudan virus (SUDV) NP in Western blot. Furthermore, four samples specifically detected EBOV or SUDV glycoprotein (GP) in an indirect immunofluorescence assay under native conditions. Virus neutralization assay based on EBOV (Mayinga isolate) revealed five weakly neutralizing sera. The finding of (cross‐) reactive and weakly neutralizing antibodies suggests the exposure of pigs from Guinea to ebolaviruses or ebola‐like viruses with their pathogenicity as well as their zoonotic potential remaining unknown. Future studies should investigate whether pigs can act as an amplifying host for ebolaviruses and whether there is a risk for spillover events.     

Development of a Double‐Antigen Microsphere Immunoassay for Simultaneous Group and Serotype Detection of Bluetongue Virus Antibodies

Bluetongue viruses (BTV) are arboviruses responsible for infections in ruminants. The confirmation of BTV infections is based on rapid serological tests such as enzyme‐linked immunosorbent assays (ELISAs) using the BTV viral protein 7 (VP7) as antigen. The determination of the BTV serotype by serological analyses could be only performed by neutralization tests (VNT) which are time‐consuming and require BSL3 facilities. VP2 protein is considered the major serotype‐defining protein of BTV. To improve the serological characterization of BTV infections, the recombinant VP7 and BTV serotype 8 (BTV‐8) VP2 were synthesized using insect cells expression system. The purified antigens were covalently bound to fluorescent beads and then assayed with 822 characterized ruminant sera from BTV vaccinations or infections in a duplex microsphere immunoassay (MIA). The revelation step of this serological duplex assay was performed with biotinylated antigens instead of antispecies conjugates to use it on different ruminant species. The results demonstrated that MIA detected the anti‐VP7 antibodies with a high specificity as well as a competitive ELISA approved for BTV diagnosis, with a better efficiency for the early detection of the anti‐VP7 antibodies. The VP2 MIA results showed that this technology is also an alternative to VNT for BTV diagnosis. Comparisons between the VP2 MIA and VNT results showed that VNT detects the anti‐VP2 antibodies in an early stage and that the VP2 MIA is as specific as VNT. This novel immunoassay provides a platform for developing multiplex assays, in which the presence of antibodies against multiple BTV serotypes can be detected simultaneously.     

Detection of Antibodies Against Capripoxviruses Using an Inactivated Sheeppox Virus ELISA

An indirect ELISA was developed to detect antibodies specific for capripoxviruses in goat, sheep and cattle sera. Heat‐inactivated Nigerian sheeppox virus was used as the ELISA antigen. Sera obtained from sheep and goats that were experimentally infected with different capripoxvirus isolates were used to develop and evaluate the sensitivity of the ELISA. Virus neutralization indexes were determined for the experimental sera in OA3.Ts cells. The specificity of the ELISA was determined using 231 sera from capripoxvirus naïve sheep and goats from Canada. In addition, the ELISA was tested for cross‐reactivity to anti‐orf virus antibodies using orf‐reactive sera and no cross‐reactivity was observed. Using experimentally generated sera obtained from animals infected with virulent sheeppox or goatpox virus isolates, the diagnostic sensitivity of the ELISA was 96% with a diagnostic specificity of 95%, where the diagnostic sensitivity of the virus neutralization assay was 96% with a diagnostic specificity of 100%. Further evaluation of this ELISA, using 276 cattle serum samples that were positive by virus neutralization assays, revealed a diagnostic sensitivity of 88% with a specificity of 97%. These results indicated that the inactivated capripoxvirus ELISA can detect capripoxvirus‐specific antibodies in sheep, goats and cattle that have been infected with virulent capripoxvirus isolates. Non‐virulent capripoxvirus isolates, in contrast, did not elicit positive (≥1.5 Log₁₀ neutralization index) antibody responses.     

The double‐antigen ELISA concept for early detection of Erns‐specific classical swine fever virus antibodies and application as an accompanying test for differentiation of infected from marker vaccinated animals

Emergency vaccination with live marker vaccines represents a promising control strategy for future classical swine fever (CSF) outbreaks, and the first live marker vaccine is available in Europe. Successful implementation is dependent on a reliable accompanying diagnostic assay that allows differentiation of infected from vaccinated animals (DIVA). As induction of a protective immune response relies on virus‐neutralizing antibodies against E2 protein of CSF virus (CSFV), the most promising DIVA strategy is based on detection of E^rns‐specific antibodies in infected swine. The aim of this study was to develop and to evaluate a novel E^rns‐specific prototype ELISA (pigtype CSFV E^rns Ab), which may be used for CSF diagnosis including application as an accompanying discriminatory test for CSFV marker vaccines. The concept of a double‐antigen ELISA was shown to be a solid strategy to detect E^rns‐specific antibodies against CSFV isolates of different genotypes (sensitivity: 93.5%; specificity: 99.7%). Furthermore, detection of early seroconversion is advantageous compared with a frequently used CSFV E2 antibody ELISA. Clear differences in reactivity between sera taken from infected animals and animals vaccinated with various marker vaccines were observed. In combination with the marker vaccine CP7_E2alf, the novel ELISA represents a sensitivity of 90.2% and a specificity of 93.8%. However, cross‐reactivity with antibodies against ruminant pestiviruses was observed. Interestingly, the majority of samples tested false‐positive in other E^rns‐based antibody ELISAs were identified correctly by the novel prototype E^rns ELISA and vice versa. In conclusion, the pigtype CSFV E^rns Ab ELISA can contribute to an improvement in routine CSFV antibody screening, particularly for analysis of sera taken at an early time point after infection and is applicable as a DIVA assay. An additional E^rns antibody assay is recommended for identification of false‐positive results in a pig herd immunized with the licensed CP7_E2alf marker vaccine.     

Influence of flavivirus co‐circulation in serological diagnostics and surveillance: A model of study using West Nile,Usutu and Bagaza viruses

This study aims at assessing the serological cross‐reactions existing between three mosquito‐borne flaviviruses with avian reservoirs co‐circulating in Europe: West Nile (WNV), Usutu (USUV) and Bagaza (BAGV). The study is useful for a better interpretation of serological results in diagnostics and surveillance. Serum samples obtained from a natural host, the red‐legged partridge (Alectoris rufa), experimentally infected with WNV, USUV or BAGV were analysed using two commercially available WNV competition ELISAs suitable for serological surveillance, and by the confirmatory virus neutralization test (VNT). The ELISAs examined showed different levels of specificity for WNV, as judged by cross‐reaction observed with the other flaviviruses. By VNT, virus‐specific antibodies were confirmed in 80%, 50% or 0% of sera from WNV‐, BAGV‐, or USUV‐inoculated birds, respectively. The results indicate how the co‐circulation of cross‐reacting flaviviruses may affect the outcomes of WNV serological surveillance when applying currently available serological tools. On the one hand, the choice of the ELISA test for antibody screening should consider the differences found in specificity, since one test is more specific for WNV while the other one is more suitable for detection of a broader range of flavivirus antibodies. On the other hand, besides corroborating that cross‐neutralization occurs between flaviviruses from different serocomplexes (WNV/USUV and BAGV), this study points out that cross‐neutralization between WNV and USUV is not symmetric, and reveals the difficulty to identify USUV infections serologically. This finding indicates that actual USUV infections might be underestimated in the current diagnostic schemes.     

Leishmaniasis in cat shelters: A serological,molecular and entomological study

An epidemiological Leishmania spp. and entomological Phlebotomine sandflies survey was performed in cat shelters at leishmaniasis endemic area of Brazil. Blood and conjunctival swab (CS) samples were collected from 94 cats in two animal protection shelters. These samples were subjected to serological tests using the indirect immunofluorescence antibody test (IFAT) and indirect enzyme‐linked immunosorbent assay (ELISA) and to molecular test by polymerase chain reaction (PCR). In addition, a Phlebotomine sandflies survey was performed in the same shelters. The analyses revealed a positivity of 31.91% (30/94) through ELISA and 29.79% (28/94) through IFAT. The two serological tests showed a positive association with perfect agreement (k = 0.925). None of the cats were positive by Leishmania spp. DNA. One Lutzomyia (Lutzomyia) longipalpis male was found in one of the cat shelters. The results and the implications of our findings are discussed below.     

Foot‐and‐Mouth Disease Seroprevalence in Cattle in Eritrea

Information about seroprevalence of foot‐and‐mouth disease (FMD) and virus serotypes in Eritrea is unavailable, but is very important as it may guide the choice of intervention measures including vaccination to be implemented. We carried out a cross‐sectional study from February to June 2011 in Eritrea with a two‐stage cluster design, sampling cattle in 155 villages with the objective of determining the seroprevalence of FMD in four administrative regions of the country. We analysed cattle sera (n = 2429) for FMD virus antibodies using the non‐structural ELISA (NS ELISA) and virus neutralization test (VNT). The overall seroprevalence was 26% and 30% for the NS ELISA and VNT, respectively. FMD virus serotypes O (14%) and A (11%) were the most prevalent. Gash Barka showed the highest (39%) seroprevalence both in NS ELISA and VNT compared to the other three administrative regions. Strategic FMD virus vaccination with type O and A (matching circulating strains) in combination of zoo‐sanitary measures would be the best control option for Eritrea which could be started in areas where the disease is less endemic.     

Evaluation of Screening Assays for the Detection of Influenza A Virus Serum Antibodies in Swine

Increased surveillance of influenza A virus (IAV) infections in human and swine populations is mandated by public health and animal health concerns. Antibody assays have proven useful in previous surveillance programmes because antibodies provide a record of prior exposure and the technology is inexpensive. The objective of this research was to compare the performance of influenza serum antibody assays using samples collected from pigs (vaccinated or unvaccinated) inoculated with either A/Swine/OH/511445/2007 γ H1N1 virus or A/Swine/Illinois/02907/2009 Cluster IV H3N2 virus and followed for 42 days. Weekly serum samples were tested for anti‐IAV antibodies using homologous and heterologous haemagglutination‐inhibition (HI) assays, commercial swine influenza H1N1 and H3N2 indirect ELISAs, and a commercial influenza nucleoprotein (NP)‐blocking ELISA. The homologous HIs showed 100% diagnostic sensitivity, but largely failed to detect infection with the heterologous virus. With diagnostic sensitivities of 1.4% and 4.9%, respectively, the H1N1 and H3N2 indirect ELISAs were ineffective at detecting IAV antibodies in swine infected with the contemporary influenza viruses used in the study. At a cut‐off of S/N ≤ 0.60, the sensitivity and specificity of the NP‐blocking ELISA were estimated at 95.5% and 99.6%, respectively. Statistically significant factors which affected S/N results include vaccination status, inoculum (virus subtype), day post‐inoculation and the interactions between those factors (P < 0.0001). Serum antibodies against NP provide an ideal universal diagnostic screening target and could provide a cost‐effective approach for the detection and surveillance of IAV infections in swine populations.     

Challenges for Serology‐Based Characterization of Foot‐and‐Mouth Disease Outbreaks in Endemic Areas; Identification of Two Separate Lineages of Serotype O FMDV in Uganda in 2011

Control of foot‐and‐mouth disease (FMD) in Uganda by ring vaccination largely depends on costly trivalent vaccines, and use of monovalent vaccines could improve the cost effectiveness. This, however, requires application of highly specific diagnostic tests. This study investigated outbreaks of FMD in seven Ugandan districts, during 2011, using the PrioCHECK^® FMDV NS ELISA, solid‐phase blocking ELISAs (SPBEs) and virus neutralization tests (VNTs), together with virological analyses for characterization of the responsible viruses. Two hundred and eighteen (218) cattle and 23 goat sera as well as 82 oropharyngeal fluid/epithelial tissue samples were collected. Some 50% of the cattle and 17% of the goat sera were positive by the PrioCHECK^® FMDV NS ELISA, while SPBEs identified titres ≥80 for antibodies against serotype O FMD virus (FMDV) in 51% of the anti‐NSP positive cattle sera. However, 35% of the anti‐NSP positive cattle sera had SPBE titres ≥80 against multiple serotypes, primarily against serotypes O, SAT 1 and SAT 3. Comparison of SPBEs and VNTs for the detection of antibodies against serotypes O, SAT 1 and SAT 3 in 72 NSP positive cattle sera showed comparable results against serotype O (P = 0.181), while VNTs detected significantly fewer samples positive for antibodies against SAT 1 and SAT 3 than the SPBEs (P < 0.001). Detection of antibodies against serotype O was consistent with the isolation of serotype O FMDVs from 13 samples. Four of these viruses were sequenced and belonged to two distinct lineages within the East Africa‐2 (EA‐2) topotype, each differing from the currently used vaccine strain (EA‐1 topotype). The relationships of these lineages to other serotype O viruses in the Eastern Africa region are discussed. To enhance the control of FMD in Uganda, there is need to improve the specificity of the SAT‐SPBEs, perform vaccine matching and implement improved regional FMD control.     

Diagnostic specificity of the African swine fever virus antibody detection enzyme‐linked immunosorbent assay in feral and domestic pigs in the United States

African swine fever (ASF) is a highly contagious haemorrhagic disease of pigs that has the potential to cause mortality nearing 100% in naïve animals. While an outbreak of ASF in the United States’ pig population (domestic and feral) has never been reported, an introduction of the disease has the potential to cause devastation to the pork industry and food security. During the recovery phase of an outbreak, an antibody detection diagnostic assay would be required to prove freedom of disease within the previously infected zone and eventually nationwide. Animals surviving an ASF infection would be considered carriers and could be identified through the persistence of ASF viral antibodies. These antibodies would demonstrate exposure to the disease and not vaccination, as there is no ASF vaccine available. A well‐established commercial enzyme‐linked immunosorbent assay (ELISA) detects antibodies against ASF virus (ASFV), but the diagnostic specificity of the assay had not been determined using serum samples from the pig population of the United States. This study describes an evaluation of the World Organization for Animal Health (OIE)‐recommended Ingezim PPA COMPAC ELISA using a comprehensive cohort (n = 1791) of samples collected in the United States. The diagnostic specificity of the assay was determined to be 99.4% (95% confidence interval (CI): [98.9, 99.7]). The result of this study fills a gap in understanding the performance of the Ingezim PPA COMPAC ELISA in the ASF naïve pig population of the United States.     

Validation of an indirect ELISA using recombinant proteins as antigen to identify animals exposed to Babesia bigemina

The objective of this study was to instrument a serological assay for the epidemiological diagnosis of bovine babesiosis in Mexico, using the Babesia bigemina recombinant protein RAP‐1 (rRAP‐1α) as antigen. rRAP‐1α, r12d3 and rGP45 were the three recombinant antigens initially tested. Based on the highest titres obtained in the indirect ELISA (iELISA) with the positive control serum, using similar antigen concentrations, rRAP‐1α was selected for further use. The diagnostic sensitivity and specificity rates estimated for the iELISA with rRAP‐1α as antigen were 89.9% and 86.5%, respectively, while for the Indirect Fluorescent Antibody Test (IFAT), the gold standard assay, the sensitivity was 86.66% and the specificity was 95%. The ĸ agreement value determined was 0.52, indicating a moderate agreement between the iELISA and IFAT assays. The instrumented iELISA with rRAP‐1α as antigen shows an excellent specificity rate and an acceptable sensitivity that allows for the detection of antibodies to B. bigemina in cattle naturally exposed to the vector tick Rhipicephalus microplus. By using the iELISA‐rRAP‐1α, along with an iELISA with recombinant Merozoite Surface Antigen (rMSA‐1) for antibody determination against Babesia bovis in the serum samples collected from cattle at ‘La Posta’ experimental station in Mexico, a seroprevalence of 20.3% was estimated for B. bigemina and 19.4% for B. bovis, while 36.89% of samples were positive for both Babesia species. The iELISA test promises to be a safe and low‐cost type of diagnosis available to cattle producers in Mexico and would facilitate the definition of herd immunity status to implement measures of control adapted for the prevention of bovine babesiosis outbreaks.     

Bovine Brucellosis in Argentina and Bordering Countries: Update

Bovine brucellosis is a zoonotic disease spread worldwide. The infection in cattle is predominantly caused by Brucella abortus and is usually detected in pregnant females through abortions. The disease is endemic in Argentina; however, infection in humans is underestimated and often not reported. The prevalence of bovine brucellosis in countries bordering Argentina is quite variable: 0.04% in Uruguay, 10.20% in the north and 0.06% in the south of Brazil, 0.2% in Chile, 3.15% in Paraguay and 2.27% in Bolivia. In 1999, the Argentine National Control and Eradication Program was implemented. Its strategies include identification of vaccinated animals, compulsory vaccination with B. abortus S19 of 100% of 3‐ to 8‐month‐old females, negative serological tests before animal movements and categorization of farms in terms of their brucellosis status. The epidemiological surveillance in milk is performed through the milk ring test and the indirect ELISA. The result of a national brucellosis survey performed in 2004 indicates that 12.4% (95% CI: 10.89–14.0) of Argentine beef farms are seropositive to Brucella and that the apparent prevalence in cattle is 2.10% (95% CI: 1.90–2.40). The official serological diagnostic tests are as follows: buffered plate antigen test, as screening, serum agglutination test, 2‐mercaptoethanol and fluorescence polarization assay, competitive ELISA, as confirmatory tests, and complement fixation test, as definitive test. Santa Fe and a district in Córdoba have ‘Outstanding Plans’. Tierra del Fuego is a ‘Zone free from bovine brucellosis’. One question arising when studying the Argentine situation is why the disease remains endemic if good regulations exist to control and eradicate it. In future, some different aspects might be evaluated to understand it, and further studies should be performed to prioritize, select and refine control strategies.     

First Detection of Antibodies Against African Swine Fever Virus in Faeces Samples

African swine fever (ASF) is a viral, highly lethal haemorrhagic disease of swine with no available vaccine or effective treatment. Introduction of ASF into a country triggers immediate restriction measures that cause significant economic losses and threatens spread to neighbouring countries. Wild boar populations have been recently assigned an essential role in the spread of African swine fever virus (ASFV) to European countries. Therefore, effective surveillance and monitoring of wild boar populations is required, but sampling wild boar is logistically challenging and expensive. This study assessed the feasibility of detecting antibodies against ASFV in faeces for later implementation in surveillance and control programmes. Two groups of pigs were experimentally infected with an attenuated ASFV isolate Ken05, and blood, oral fluid and faecal samples were tested for the presence of viral DNA using quantitative real‐time polymerase chain reaction (qPCR) to monitor infection progress. Faecal samples were analysed using two indirect enzyme‐linked immunosorbent assays (ELISAs) based on semipurified viral protein (vp) 72 or purified recombinant vp30 expressed in mammalian cells. Faecal samples from 9 of 10 pigs with non‐haemorrhagic diarrhoea tested positive for antibodies against ASFV using the two ELISA tests that showed a positive correlation. The serum sample results from the two indirect ELISAs were compared against results from the reference ELISA technique and the immunoperoxidase test. Our findings indicate the feasibility of faecal sampling for detecting anti‐ASFV antibodies, which may provide a practical non‐invasive alternative for sampling wild boar populations. In conclusion, the application of these ELISA tests to faecal field samples could be particularly useful to screen for the presence of ASF in field conditions.     

Circulation of a Simbu Serogroup Virus,Causing Schmallenberg Virus‐Like Clinical Signs in Northern Jordan

Schmallenberg virus (SBV)‐like clinical cases of abortions in northern Jordan in early 2013, together with the emergence of SBV in Europe in 2011, its rapid spread within the following years and the detection of this virus in Turkey, raised questions about the distribution of SBV or related orthobunyaviruses. To evaluate the occurrence of SBV or related members of the Simbu serogroup of orthobunyaviruses in Jordan, bulk milk (cattle) and serum samples (cattle, sheep and goat) collected in northern Jordan in 2013 were first tested by commercially available SBV antibody ELISAs. Indeed, 3 of 47 bulk milk samples and 57 of 115 serum samples provided positive results, but SBV specificity of the ELISA results could not be confirmed by virus neutralization assays. Instead, subsequent cross‐neutralization tests were able to further investigate the specificity of these antibodies. Here, a significant inhibition of Aino virus was observed. Thus, the causative agent was most likely a Simbu serogroup virus closely related to Aino virus. Consequently, these results confirm that members of this group of virus are not only present in Europe, Africa or Australia, but also in the Middle East.     

Coxiella burnetii (Q‐Fever) Seroprevalence in Prey and Predators in the United Kingdom: Evaluation of Infection in Wild Rodents,Foxes and Domestic Cats Using a Modified ELISA

Coxiella burnetii, the agent of Q‐fever, is recognized as a worldwide zoonosis with a wide host range and potentially complex reservoir systems. Infected ruminants are the main source of infection for humans, but cats and other mammals, including wild rodents, also represent potential sources of infection. There has been a recent upsurge of reported cases in humans, domestic ruminants and wildlife in many parts of the world, and studies have indicated that wild brown rats may act as true reservoirs for C. burnetii and be implicated in outbreaks in livestock and humans. However, investigation of reservoir systems is limited by lack of validated serological tests for wildlife or other non‐target species. In this study, serum samples from 796 wild rodents (180 bank voles, 309 field voles, 307 wood mice) 102 wild foxes and 26 domestic cats from three study areas in the UK were tested for the presence of antibodies to C. burnetii using a commercial indirect ELISA kit modified for use in multiple wildlife species. Test thresholds were determined for each species in the absence of species‐specific reference sera using a bi‐modal latent class mixture model to discriminate between positive from negative results. Based on the thresholds determined, seroprevalence in the wild rodents ranged from 15.6% to 19.1% depending on species (overall 17.3%) and was significantly higher in both foxes (41.2%) and cats (61.5%) than in rodents. This is the first report to quantify seroprevalence to C. burnetii in bank voles, field voles, wood mice, foxes and cats in the UK and provides evidence that predator species could act as indicators for the presence of C. burnetii in rodents. The study demonstrates that wildlife species could be significant reservoirs of infection for both livestock and humans, and the high seroprevalence in domestic cats highlights the potential zoonotic risk from this species.     

Serological Surveillance and Direct Field Searching Reaffirm the Absence of Ornithodoros Erraticus Ticks Role in African Swine Fever Cycle in Sardinia

African swine fever (ASF), one of the most important diseases of swine, has been endemic in the Italian island of Sardinia for more than 35 years. During these decades, several strategies and eradication efforts have been implemented in the island with limited success. Strong climatic and ecological similarities exist between Sardinia and one area of the Iberian Peninsula where Ornithodoros erraticus ticks were involved in the persistence of ASF from 1960 to 1995. This fact leads to the hypothesis that, potentially, Ornithodoros ticks could be also involved in the ASF cycle in Sardinia, thus accounting for some of the reoccurring ASF outbreaks in this island. Initial efforts aimed at detection of Ornithodoros ticks in Sardinia were performed during the 1970s/1980s with no positive results. Accordingly, the absence of Ornithodoros ticks in Sardinia has been generally accepted. However, since a new and reinforced ASF eradication programme has been recently launched in Sardinia, it is essential to clarify the presence and role of these soft ticks in the epizootiology of ASF in this island. For that purpose, 1767 porcine serum samples collected from all around the island (1261 from domestic and 506 from wild boar) were analysed by ELISA for antibodies to salivary antigens of Ornithodoros erraticus. In addition, Ornithodoros ticks were directly searched in a number of pig premises that have suitable habitats for these ticks and were located in areas repeatedly affected by ASF. Only one serum sample resulted positive in the serological assay, and no Ornithodoros ticks were collected in none of the premises. These results indicate that these soft tick species are not involved in the epizootic cycle of ASF in Sardinia and highlight the importance of controlling other risk factors still present in the island for effectively eradicate the disease.     

Detection of Influenza A Virus Nucleoprotein Antibodies in Oral Fluid Specimens From Pigs Infected Under Experimental Conditions Using a Blocking ELISA

In commercial swine populations, influenza is an important component of the porcine respiratory disease complex (PRDC) and a pathogen with major economic impact. Previously, a commercial blocking ELISA (FlockChek^™ Avian Influenza Virus MultiS‐Screen^® Antibody Test Kit, IDEXX Laboratories, Inc., Westbrook, ME, USA) designed to detect influenza A nucleoprotein (NP) antibodies in avian serum was shown to accurately detect NP antibodies in swine serum. The purpose of this study was to determine whether this assay could detect NP antibodies in swine oral fluid samples. Initially, the procedure for performing the NP‐blocking ELISA on oral fluid was modified from the serum testing protocol by changing sample dilution, sample volume, incubation time and incubation temperature. The detection of NP antibody was then evaluated using pen‐based oral fluid samples (n = 182) from pigs inoculated with either influenza A virus subtype H1N1 or H3N2 under experimental conditions and followed for 42 days post inoculation (DPI). NP antibodies in oral fluid were detected from DPI 7 to 42 in all inoculated groups, that is, the mean sample‐to‐negative (S/N) ratio of influenza‐inoculated pigs was significantly different (P < 0.0001) from uninoculated controls (unvaccinated or vaccinated‐uninoculated groups) through this period. Oral fluid versus serum S/N ratios from the same pen showed a correlation of 0.796 (Pearson's correlation coefficient, P < 0.0001). The results showed that oral fluid samples from influenza virus‐infected pigs contained detectable levels of NP antibodies for ≥42 DPI. Future research will be required to determine whether this approach could be used to monitor the circulation of influenza virus in commercial pig populations.