Bovine ephemeral fever virus (BEFV) is a member of the genus Ephemerovirus in the family Rhabdoviridae. It is an arthropod-borne virus transmitted by many species of midges and mosquitoes. It can cause severe economic consequences due to losses in milk production and the general condition of cattle and water buffalo. BEF occurs in some tropical, subtropical and warm temperate regions of Africa, Australia, the Middle East and Asia with seasonal outbreaks, but its possible spread to other areas (e.g. Europe) cannot be excluded. Therefore, using and developing rapid diagnostic methods with optimal performance is essential for identifying emerging pathogens and their control. In the present study, we developed two competitive serological ELISAs based on monoclonal antibodies (mAbs), designed by using BEFV inactivated antigen and the BEF recombinant nucleoprotein (N), respectively. A panel of 77 BEF-positive and 338 BEF-negative sera was used to evaluate the two tests. With a diagnostic sensitivity of 97.4 % using the inactivated virus and 98.7 % using the recombinant N, and a diagnostic specificity of 100 % using both antigens, our results suggest that these tests are suitable for the serological diagnosis of BEF.

Brucella BP26 proves to be a highly immunogenic antigen with excellent specificity in brucellosis detection. In China, the authorized use of the Bp26-deleted vaccine M5ΔBP26 for preventing small ruminant brucellosis highlights the importance of developing accurate detection methods targeting BP26, particularly for the diagnosis of differentiation between infected and vaccinated animals (DIVA). Using the traditional mouse hybridoma technique, we successfully obtained 12 monoclonal antibodies (mAbs) targeting BP26. The efficacy of these mAbs in detecting various animal brucellosis cases using the competitive ELISA method was evaluated. Among them, only the E10 mAb exhibited significant efficiency, being inhibited by 100, 97.62, and 100% of brucellosis-positive sera from cattle, small ruminants, and canines, respectively. The E10-based competitive enzyme-linked immunosorbent assay (cELISA) outperformed the BP26-based indirect enzyme-linked immunosorbent assay (iELISA) in accuracy, particularly for cattle and small ruminant brucellosis, with cELISA sensitivity reaching 97.62% compared to 64.29% for iELISA for small ruminants. Although cELISA showed slightly lower specificity than iELISA, it still maintained high accuracy in canine brucellosis detection. The epitope of mAb E10 was identified in the amino acid sequence QPIYVYPDDKNNLKEPTITGY, suggesting its potential as a diagnostic antigen for brucellosis. In conclusion, the E10-based cELISA presents an effective means of detecting animal brucellosis, particularly significant for DIVA diagnosis in China, where the BP26-mutant vaccine is widely used.

Infection with bovine leukemia virus (BLV) leads to enzootic bovine leukosis, the most prevalent neoplastic disease in cattle. Due to the lack of commercially available vaccines, reliable eradication of the disease can be achieved through the testing and elimination of BLV antibody-positive animals. In this study, we developed a novel competitive ELISA (cELISA) to detect antibodies against BLV capsid protein p24. Recombinant p24 protein expressed by Escherichia coli, in combination with the monoclonal antibody 2G11 exhibiting exceptional performance, was used for the establishment of the cELISA. Receiver-operating characteristic curve analysis showed that the sensitivity and specificity of the assay were 98.85 % and 98.13 %, respectively. Furthermore, the established cELISA was specific for detecting BLV-specific antibodies, without cross-reactivity to antisera for six other bovine viruses. Significantly, experimental infection of cattle and sheep with BLV revealed that the cELISA accurately monitors seroconversion. In a performance evaluation, the established cELISA displayed a high agreement with Western blotting and the commercial BLV gp51 cELISA kit in the detection of 242 clinical samples, respectively. In conclusion, the novel p24 cELISA exhibited the potential to be a reliable and efficient diagnostic tool for BLV serological detection with a broad application prospect.

African swine fever virus (ASFV) poses a significant threat to the global swine industry. Currently, there are no effective vaccines or treatments available to combat ASFV infection in pigs. The primary means of controlling the spread of the disease is through rapid detection and subsequent elimination of infected pig. Recently, a lower virulent ASFV isolate with a deleted EP402R gene (CD2v-deleted) has been reported in China, which further complicates the control of ASFV infection in pig farms. Furthermore, an EP402R-deleted ASFV variant has been developed as a potential live attenuated vaccine candidate strain. Therefore, it is crucial to develop detection methods that can distinguish wild-type and EP402R-deleted ASFV infections. In this study, two recombinant ASFV-p72 and -CD2v proteins were expressed using a prokaryotic system and used to immunize Bactrian camels. Subsequently, eight nanobodies against ASFV-p72 and ten nanobodies against ASFV-CD2v were screened. Following the production of these nanobodies with horse radish peroxidase (HRP) fusion proteins, the ASFV-p72-Nb2-HRP and ASFV-CD2v-Nb22-HRP fusions were selected for the development of two competitive ELISAs (cELISAs) to detect anti-ASFV antibodies. The two cELISAs exhibited high sensitivity, good specificity, repeatability, and stability. The coincidence rate between the two cELISAs and commercial ELISA kits was 98.6% and 97.6%, respectively. Collectively, the two cELISA for detecting antibodies against ASFV demonstrated ease of operation, a low cost, and a simple production process. The two cELISAs could determine whether pigs were infected with wild-type or CD2v-deleted ASFV, and could play an important role in monitoring ASFV infections in pig farms.

For the development of a competitive ELISA (cELISA) to detect serum antibodies against the Mycoplasma mycoides subsp. Mycoides (Mmm) (strain PG1), the causative agent of contagious bovine pleuropneumonia (CBPP), all the proteins of this pathogen were analyzed. Then, a specific extracellular region of a transmembrane protein with the potential for diagnosis was identified. After that, a monoclonal antibody (Mab) named 3A8 was obtained using this extracellular region as an immunogen. Finally, a cELISA was established with the extracellular domain of this transmembrane protein as the coating antigen, Mab 3A8 as the competitive antibody, and HRP-labeled goat anti-mouse IgG as the enzyme-labeled antibody. This established method was used to detect the antibody dynamic regularity of goats which are artificially immunized Mmm and was also compared with a commercial ELISA kit. Further, the sera of 1011 different cattle from border provinces of China were monitored using a candidate Mab 3A8 cELISA. The detection results of known background sera used in this study indicate that a candidate diagnostic marker was successfully identified by analyzing all the coding proteins of Mmm in this research, and the cELISA established based on the Mab 3A8 against this protein can detect CBPP-positive serum with specificity and has no cross-reaction with other related epidemic disease-positive sera. In addition, we tested the sera collected from the border areas of China using the established ELISA, and no positive sample was detected. The research protocol of the CBPP cELISA established in this study is different from the traditional method, which can greatly reduce the investment of manpower and capital and save development time. We believe that this study\'s protocol could serve as a reference for the development of detection methods for mycoplasma and other complex pathogens. KEY POINTS: • A Mmm-specific diagnostic marker was obtained based on protein characteristics. • A cELISA was established for CBPP serum antibody detection. • The serological investigation was conducted for CBPP in the border areas of China.

BACKGROUND: Equine piroplasmosis is caused by two tick-borne protozoan parasites, Theileria equi and Babesia caballi,, which are clinically relevant in susceptible horses, donkeys, and mules. Moreover, equine piroplasmosis significantly constrains international trading and equestrian events. Rapidly diagnosing both parasites in carrier animals is essential for implementing effective control measures. Here, a rapid immunochromatographic test for the simultaneous detection of antibodies to T. equi and B. caballi was evaluated using samples from horses and donkeys collected in Greece, Israel, and Italy. The results were compared with an improved competitive enzyme-linked immunosorbent assay (cELISA) for detecting antibodies to both parasites using the same panel of samples.
METHODS: Blood samples were collected from 255 horses and donkeys. The panel consisted of 129 horses sampled at four locations in northern Greece, 105 donkeys sampled at four locations in Sicily, and 21 horses sampled at two locations in Israel. The rapid test and the cELISA were performed according to the manufacturer\'s instructions, and the results were subjected to a statistical analysis to determine the sensitivity and specificity of both tests and their association.
RESULTS: The immunochromatographic test provided a result within 15 min and can be performed in the field, detecting both pathogens simultaneously. The overall coincidence rate between the rapid test and the cELISA for detecting antibodies against T. equi was 93% and 92.9% for B. caballi. The rapid test\'s sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) for T. equi were above 91.5%. Sixteen samples were positive for both parasites in the rapid test and eight in the cELISA. Either test had no significant association between T. equi and B. caballi detection. The detection rates of both parasites were significantly higher in Italy than in Greece or Israel and in donkeys than in horses. The agreement for T. equi between the results of both tests was high in Greece (93.8%) and Italy (95.2%) and moderate in Israel (76.2%). For B. caballi, the specificity and NPV of the rapid test were high (94.2% and 98.3%, respectively), although the sensitivity and PPV were moderate (69.2% and 39.1%, respectively) due to the small sample size. However, for B. caballi, the sensitivity was higher with the rapid test.
CONCLUSIONS: The rapid test detected T. equi and B. caballi simultaneously in the field, potentially replacing laborious cELISA testing and is recommended for import/export purposes. The test can also be helpful for the differential diagnosis of clinical cases, since seropositivity may rule out equine piroplasmosis since it does not indicate current or active infection.

The apparent gluten concentration profiles of 47 hydrolyzed foods (barley malt, sprouted grains, and hydrolyzed wheat proteins (HWP)) were evaluated using a multiplex-competitive ELISA that utilizes the G12, R5, 2D4, MIoBS, and Skerritt antibodies from commercial sources. Cluster analysis was conducted to evaluate similarities or differences in the gluten protein/peptide response profiles among the hydrolyzed foods and their similarities or differences with fermented foods analyzed previously by the ELISA. The gluten protein/peptide response profiles of the hydrolyzed foods mainly depended on the grain source (wheat, rye, or barley) of gluten. Some hydrolyzed foods presented profiles similar to those of certain fermented foods (e.g., barley malt and gluten reduced barley beers), whereas others presented unique profiles (e.g., HWP and sprouted wheat). Additional analysis using wheat gluten-incurred yogurts indicated that while not suitable for the barley- or rye-containing foods tested, a newly developed gluten-incurred yogurt calibrant shows promise for the possible use in the quantitation of several wheat-containing fermented and hydrolyzed foods.

Background: West Nile virus (WNV) infection, caused by a flavivirus, emerged in Europe and America in the past two decades. The etiological agent causes asymptomatic to life-threatening infection in humans and in some animal species. The objective of this study was to evaluate the seroprevalence of WNV among donkeys and mules in Bulgaria. Methods: A total of 200 archived serum samples were tested by competitive enzyme-linked immunosorbent assay (ELISA). Positive samples were additionally analyzed by virus neutralization assay. Results: Seroprevalence of 7% (14/200) was established among tested animals by ELISA. Two samples were subsequently verified for the presence of virus neutralizing antibodies; thus, the seroprevalence against WNV was determined to be 1% (2/200 [confidence interval = 0.12-3.61]). Positive results among mules included in the study were not found. Conclusion: The findings in the present research demonstrate that donkeys are exposed to WNV infection and seroconvert, which adds to the understanding of virus circulation among donkeys in settlements in north and south Bulgaria.

Rift Valley fever (RVF) is a mosquito-borne viral zoonosis caused by the Rift Valley fever virus (RVFV). The present work aims to investigate the epidemiological status and identify the risk factors associated with RVFV infection in dromedary camels (Camelus dromedarius) from southern Algeria. A total of 269 sera of apparently healthy camels was collected and tested using a competitive Enzyme-Linked Immunosorbent Assay (ELISA). Overall, 72 camels (26.7 %, 95 % CI: 21.4-32) were seropositive to RVFV. IgG antibodies were found to be most prevalent in camels from south-western areas, particularly in Tindouf wilaya (52.38 %, p < 0.0001), and in camels introduced from bordering Sahelian countries (35.8 %) (OR = 8.75, 95 %CI: 2.14-35.81). No anti-RVFV antibodies were detected in sera collected from local camels (0 %). Adult (5-10 years) and aged (>10 years) camels have a significantly higher risk of being infected by RVFV (OR = 2.15; 95 %CI = 1.21-3.81, OR = 2.05; 95 %CI = 1.03-4.11, respectively). This report indicated that dromedaries imported to the south-western areas are exposed to RVFV and may contribute to its spread in Algerian territories.

Analogs of immunodominant myelin peptides involved in multiple sclerosis (MS: the most common autoimmune disease) have been extensively used to modify the immune response over the progression of the disease. The immunodominant 35-55 epitope of myelin oligodendrocyte glycoprotein (MOG35-55 ) is an autoantigen appearing in MS and stimulates the encephalitogenic T cells, whereas mannan polysaccharide (Saccharomyces cerevisiae) is a carrier toward the mannose receptor of dendritic cells and macrophages. The conjugate of mannan-MOG35-55 has been extensively studied for the inhibition of chronic experimental autoimmune encephalomyelitis (EAE: an animal model of MS) by inducing antigen-specific immune tolerance against the clinical symptoms of EAE in mice. Moreover, it presents a promising approach for the immunotherapy of MS under clinical investigation. In this study, a competitive enzyme-linked immunosorbent assay (ELISA) was developed to detect the MOG35-55 peptide that is conjugated to mannan. Intra- and inter-day assay experiments proved that the proposed ELISA methodology is accurate and reliable and could be used in the following applications: (i) to identify the peptide (antigen) while it is conjugated to mannan and (ii) to adequately address the alterations that the MOG35-55 peptide may undergo when it is bound to mannan during production and stability studies.