Goatpox and sheeppox are highly contagious and economically important viral diseases of small ruminants. Due to the risk they pose to animal health, livestock production, and international trade, capripoxviruses are a considerable threat to the livestock economy. In this study, we expressed two core proteins (A4L and A12L) and one extracellular enveloped virion protein (A33R) of goatpox virus in a baculovirus expression vector system and evaluated their use as diagnostic antigens in ELISA. Full-length A4L, A12L, and A33R genes of the GTPV Uttarkashi strain were amplified, cloned into the pFastBac HT A donor vector, and introduced into DH10Bac cells containing a baculovirus shuttle vector plasmid to generate recombinant bacmids. The recombinant baculoviruses were produced in Sf-21 cells by transfection, and proteins were expressed in TN5 insect cells. The recombinant proteins were analysed by SDS-PAGE and confirmed by western blot, with expected sizes of ~30 kDa, ~31 kDa, and ~32 kDa for A4L, A12L, and A33R, respectively. The recombinant proteins were purified, and the immunoreactivity of the purified proteins was confirmed by western blot using anti-GTPV serum. The antigenic specificity of the expressed proteins as diagnostic antigens was evaluated by testing their reactivity with infected, vaccinated, and negative GTPV/SPPV serum in indirect ELISA, and the A33R-based indirect ELISA was optimized. The diagnostic sensitivity and specificity of the A33R-based indirect ELISA were found to be of 89% and 94% for goats and 98% and 91%, for sheep, respectively. No cross-reactivity was observed with other related viruses. The recombinant-A33R-based indirect ELISA developed in the present study shows that it has potential for the detection of antibodies in GTPV and SPPV infected/vaccinated animals.

Foot-and-mouth disease (FMD) is one of the most important animal diseases of economic significance globally. It is a highly infectious and contagious disease of cloven-hoofed animals including sheep and goat. For sero-diagnosis of FMD, recombinant antigen-based assays are considered as alternatives to conventional approaches such as the liquid phase blocking ELISA (LPBE). The early interventions towards control measures cannot be implemented unless the disease gets promptly diagnosed. It is relatively difficult to clinically diagnose FMD in goat due to the usual milder form or unapparent nature of symptoms. Under such situations where clinical samples are not available, demonstration of infection-specific FMD virus (FMDV) antibodies in serum sample may help identifying the animals exposed to the virus in retrospect. Antibody to 3AB nonstructural protein (NSP) has been considered to be the most reliable indicator for FMD diagnosis. The current study extended the earlier designed recombinant 3AB3 protein-based indirect ELISA originally validated on bovine serum samples to testing serum samples of goat. The performance of the indirect ELISA was validated using internationally accepted PrioCHECK® FMDV NS kit. The overall diagnostic sensitivity (DSn) of the indirect ELISA was estimated to be 95.52% (619/648), while the diagnostic specificity (DSp) on naïve and vaccinated animals varied at 98.06% (557/568) and 94.15% (435/462), respectively. In India, where FMD is prevalent and the goat population is so high, this \'in-house\' optimized assay can be considered to be an adjunct in sero-epidemiological investigation of FMD in goat.

The Chinese government\'s reclassification of Classical Swine Fever (CSF) from a class Ⅰ to a class Ⅱ animal infectious disease, now also including CSF under the disease eradication program, reflects the significant progress made through extensive immunization with CSF vaccines. In light of this advancement, there is an imperative need for an expedient and accurate method to assess the levels of immunoprotection against classical swine fever virus (CSFV) in vaccinated pigs, a critical component in the campaign to eradicate the disease. This study develops an indirect enzyme-linked immunosorbent assay (iELISA) based on a highly glycosylated E2 protein stable expressed in CHO-K1 mammalian cells. Statistical analysis revealed strong positive correlations between the iELISA and VNT results (r = 0.9063, p < 0.0001) that were much greater than those between the IDEXX ELISA and VNT results (r = 0.8126, p < 0.0001). Taking the VNT data as the standard, the consistency of the iELISA (κ =0.880) was greater than that of the IDEXX ELISA (κ =0.699). In summary, the iELISA provides a more efficient and precise method for assessing CSFV immunity in pigs. Its reliable detection of immunoprotection levels against CSFV makes it an essential tool for optimizing CSF vaccination strategies. Consequently, its application can significantly support the ongoing efforts to eradicate CSF.

African swine fever (ASF) is an acute, febrile, and highly lethal infectious disease in pigs caused by the African swine fever virus (ASFV). Effective detection methods and strict biosecurity measures are crucial for preventing and controlling ASF, especially since there are currently no commercially available vaccines or antiviral drugs to combat ASFV infection effectively. However, the emergence of low-virulence strains of ASFV in recent years has led to false-positive results, highlighting the importance of early-produced antibody detection methods. Therefore, detecting antibodies against ASFV produced early in the infection can facilitate the prompt identification of infected pigs. This study focused on the p30 protein, an early expressed protein during ASFV infection, to develop an indirect ELISA. This method was established using the HEK293F suspension cell expression system, which has the ability to produce large quantities of correctly folded proteins with normal functionality. In this study, we developed an indirect ELISA test utilizing the p30 recombinant protein produced by the HEK293F suspension cell expression system as the antigen coating. The concentration of the p30 protein obtained from the HEK293F suspension cell expression system was measured at 4.668 mg/mL, serving as the foundation for establishing the indirect ELISA. Our findings indicate that the indirect ELISA method exhibits a sensitivity of 1:12800. Furthermore, it demonstrates high specificity and excellent reproducibility. Comparing our results to those obtained from the commercial kit, we found a coincidence rate of 98.148 % for the indirect ELISA. In summary, we have developed a sensitive method for detecting ASFV, providing a valuable tool for monitoring ASFV infection in pig herds.

Schistosoma species are the causative agent of schistosomiasis and shows worldwide distribution. There is a great need to develop a sensitive diagnostic approach for controlling the disease. Previously, we identified large numbers of Extracellular Vesicle (EV) proteins from Schistosoma japonicum (S. japonicum), but rarely these proteins have been evaluated for their diagnostic potential. In the present study, we performed bioinformatic analyses of S. japonicum identified EV-associated proteins from the previous study and then identified Schistosoma-specific proteins with potentially secreted capability. Among them, we selected SJCHGC02838 protein, SJCHGC05593 protein, SJCHGC05668 protein and a hypothetical protein (SJHYP) to evaluate their diagnostic potential for detecting S. japonicum infection. First, we determined the expression of these four proteins at the transcript levels using qRT-PCR and revealed that all these genes showed higher expression in adult stage. Then, we cloned the full-length cDNA for each protein into a prokaryotic expression vector and successfully generated the recombinant proteins. Upon the purification of recombinant proteins, we developed an indirect ELISA method to evaluate the diagnostic potential of these purified recombinant proteins. The results showed high sensitivity for detecting Schistosoma infection. Additionally, these proteins also displayed a good potential for detecting Schistosoma infection, especially SJCHGC05668 protein at an early stage. The diagnostic potentials of these recombinant proteins were further evaluated by Western blot and comparatively analyzed by our previously developed cfDNA methods.

Leptospirosis is a globally distributed infectious disease caused by pathogenic spirochetes of the Leptospira genus, often overlooked. It is estimated that the disease affects approximately one million people annually, resulting in more than 58,900 deaths. The gold standard for serodiagnosis of leptospirosis is the Microscopic Agglutination Test (MAT). However, the limitations of this technique necessitate the exploration of alternative diagnostic methods. In this study, we evaluated the ErpY-like recombinant protein (rErpY-like) in the development of a serologic diagnostic assay for human leptospirosis. Eighty-six human sera samples, characterized by MAT, underwent evaluation through indirect IgM-ELISA and IgG-ELISA. The sensitivity and specificity values obtained from IgM-ELISA were 60% and 76%, respectively, while those from IgG-ELISA were 96.4% and 100%, respectively. The use of the rErpY-like protein in both IgM-ELISA and IgG-ELISA proves to be a sensitive and specific method for antibody detection. This could potentially serve as a valuable alternative tool in the diagnosis of human leptospirosis.

African swine fever virus (ASFV) poses a significant threat to the global pig industry, necessitating accurate and efficient diagnostic methods for its infection. Previous studies have often focused on a limited number of epitopes from a few proteins for detecting antibodies against ASFV. Therefore, the current study aimed to use multiple B-cell epitopes in developing an indirect Enzyme-Linked Immunosorbent Assay (ELISA) for enhanced detection of ASFV antibodies. For the expression of recombinant protein, k3 derived from 27 multiple peptides of 11 ASFV proteins, such as p72, pA104R, pB602L, p12, p14.5, p49, pE248R, p30, p54, pp62, and pp220, was used. To confirm the expression of the recombinant protein, we used the Western blotting analysis. The purified recombinant K3 protein served as the antigen in our study, and we employed the indirect ELISA technique to detect anti-ASFV antibodies. The present finding showed that there was no cross-reactivity with antibodies targeting Foot-and-mouth disease virus (FMDV), Porcine circovirus type 2 (PCV2), Pseudorabies virus (PRV), Porcine reproductive and respiratory syndrome virus (PRRSV), and Classical swine fever virus (CSFV). Moreover, the current finding was sensitive enough to find anti-ASFV in serum samples that had been diluted up to 32 times. The test (k3-iELISA) showed diagnostic specificity and sensitivity of 98.41% and 97.40%, respectively. Moreover, during the present investigation, we compared the Ingenasa kit and the k3-iELISA to test clinical pig serum, and the results revealed that there was 99.00% agreement between the two tests, showing good detection capability of the k3-iELISA method. Hence, the current finding showed that the ELISA kit we developed can be used for the rapid detection of ASFV antibodies and used as an alternative during serological investigation of ASF in endemic areas.

Timely and accurate diagnosis of Schistosoma infection is important to adopt effective strategies for schistosomiasis control. Previously, we demonstrated that Schistosoma japonicum can secret extracellular vesicles and their cargos may serve as a novel type of biomarkers for diagnosing schistosomiasis. Here, we developed a Gaussia luciferase immunoprecipitation assay combined with S. japonicum extracellular vesicle (SjEV) protein to evaluate its potential for diagnosing schistosomiasis. A saposin-like protein (SjSLP) identified from SjEVs was fused to the Gaussia luciferase as the diagnostic antigen. The developed method showed good capability for detecting S. japonicum infection in mice and human patients. We also observed that the method could detect Schistosoma infection in mice as early as 7 days of post-infection, which showed better sensitivity than that of indirect ELISA method. Overall, the developed method showed a good potential for detecting Schistosoma infection particularly for early stage, which may provide an alternative strategy for identify Schistosoma infection for disease control.

Cystic echinococcosis (CE) is an emergent neglected disease affecting human and animals in Egypt with a wide distribution and incidence. This study aimed to evaluate the use of a polyclonal antibody-based sandwich ELISA in the detection of Echinococcus granulosus antigen in human and camel sera. Hydatid cyst protoscoleces antigen (PsAg) was isolated from hydatid cysts collected from naturally infected camel livers and lungs. PsAg was used for immunization of rabbits to raise IgG polyclonal antibodies (IgG PsAb). IgG PsAb were then precipitated, purified using Protein-A Sepharose gel and labeled with horseradish peroxidase enzyme. We assayed the purity of the IgG PsAb, and the two prepared E. granulosus antigens CPsAg from camel cysts and HPsAg from human cysts by Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The resulted protein bands of the prepared CPsAg appeared at different molecular weights: 180, 90, 68, 54, 42 and 22 kDa while, HPsAg shared with it in 4 common bands at 68, 54, 42, and 22 kDa. The purified IgG PsAb had been resolved at two bands at 52 kDa and at 32 kDa. Sandwich ELISA were performed for the detection of circulating E. granulosus antigens in sera of human (n = 183) and camels (n = 190). The purified IgG PsAb showed strong reactivity against E. granulosus infected human and camel samples and no cross reactivity neither with free-healthy negative sera nor with others parasitic diseases (Schistosomiasis, Fascioliasis, Toxoplasmosis, Ancylostomiasis for human samples and Fascioliasis, ticks\' infestation, Eimeriosis, Cryptosporidiosis, Nasal myiasis, Toxoplasmosis for camel samples). The sensitivity of the assay was 98.25% (56/57) and 96.9% (31/32) against human and camel samples, respectively. Specificity was 100% in both human and camel samples. Sandwich ELISA detected CE in 33.3% (24/72) and 55.6% (50/90) random human and camel samples, respectively. Indirect ELISA, using CPsAg, was used for detection of antibodies in positive human and camels\' sera and detected 96.5% (55/57) and 93.8% (30/32) of human and camel samples, respectively. In our study, Genomic DNA was extracted from protoscoleces fluid of human liver hydatid cysts to identify the Echinococcus sp. isolate based on NADH dehydrogenase subunit 1 (NAD1) gene by Polymerase Chain Reaction (PCR) and the isolate (GenBank: OP785689.1) were identified as E. granulosus sensu lato genotype. In conclusion, Sandwich ELISA technique was found to be a potent and sensitive assay for detection of hydatid antigen in both human and camel samples.

Human-to-animal SARS-CoV-2 transmission was observed, including a veterinarian contracting COVID-19 through close contact with an infected cat, suggesting an atypical zoonotic transmission. This study investigated the prevalence of SARS-CoV-2 antibodies in cats during human outbreaks and elucidated the correlation between cat infections and human epidemics. A total of 1107 cat serum samples were collected and screened for SARS-CoV-2 antibodies using a modified indirect ELISA human SARS-CoV-2 antibody detection kit. The samples were confirmed using a cPass™ neutralization test. The SARS-CoV-2 seropositivity rate was 22.67% (199/878), mirroring the trend observed in concomitant human case numbers. The waves of the epidemic and the provinces did not significantly impact ELISA-positive cats. Notably, Chon Buri exhibited a strong positive correlation (r = 0.99, p = 0.009) between positive cat sera and reported human case numbers. Additionally, the cPass™ neutralization test revealed a 3.99% (35/878) seropositivity rate. There were significant differences in numbers and proportions of positive cat sera between epidemic waves. In Samut Sakhon, a positive correlation (r = 1, p = 0.042) was noted between the proportion of positive cat sera and human prevalence. The findings emphasize the need for ongoing surveillance to comprehend SARS-CoV-2 dynamics in both human and feline populations.