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Strongylus vulgaris, a devastating parasitic nematode in equids, causes life-threatening verminous aneurysms that are challenging to diagnose early. This study pioneered integrating nanotechnology into an indirect enzyme-linked immunosorbent assay (i-ELISA) system to enhance the sensitivity and specificity for detecting S. vulgaris larval antigens in equine serum samples, with PCR confirmation of the species. A conventional i-ELISA and an innovative nano-based ELISA were developed using excretory-secretory antigens from adult S. vulgaris worms. The nano-ELISA incorporated gold nanoparticles (17.4–41.4 nm) conjugated with detection antibodies, enabling remarkable signal amplification. Of the 120 examined equines, 100 (83.33%) were positive for S. vulgaris infection. A conventional i-ELISA and an innovative nano-ELISA incorporating 17.4–41.4 nm gold nanoparticles were optimized using S. vulgaris excretory-secretory antigens. Both assays demonstrated high specificity, with no cross-reactivity against sera from animals infected with other helminth parasites. Remarkably, optical density (OD) readings from both i-ELISAs exhibited a positive quantitative correlation with infection intensity. The i-ELISA OD ranged from 0.45–0.74 (G3), 0.75–0.94 (G2), to 0.95–2.5 (G1). The nano-ELISA showed enhanced signal amplification, with OD ranging from 0.40–0.84 (G3), 0.85–0.99 (G2), to 1.0–3.5 (G1). This nanotechnology-amplified ELISA opens new, highly sensitive, and specific techniques for parasitic diagnosis in equine medicine. Its superior performance, facilitated by signal-amplifying gold nanoparticles, illuminates nanotechnology's potential in revolutionizing parasitological diagnostics for enhanced animal health and welfare management. [ABSTRACT FROM AUTHOR]

Sheeppox and Goatpox are highly contagious transboundary viral diseases of sheep and goats caused by Capripoxvirus, respectively. This study describes the development of indirect ELISA and its serodiagnostic potential as an alternative to the gold standard serum neutralization test (SNT). The homologue of vaccinia virus, ORF 117 (A27L) gene of the Romanian Fenner (RF) strain of Sheeppox virus (SPPV) was used for producing the full-length recombinant A27L (rA27L) protein (∼22 kDa) in a prokaryotic expression system. The immunogenic potential of purified rA27L was confirmed by detecting its specific reactivity with SPPV hyperimmune sera in Western Blot analysis. An indirect ELISA based on rA27L was developed and optimized. In comparison to SNT, the rA27L-ELISA assay exhibited a diagnostic specificity of 96% and 98%, while the sensitivity was 98% and 97%, with sheep and goat sera, respectively. No cross-reactivity was noted with other transboundary diseases such as Orf, Foot-and-Mouth disease, Peste des Petits Ruminants, Haemorrhagic septicemia and Brucella. A total of 508 serum samples were tested in total for the preliminary analysis. Among these, seropositivity rates for sheep (n = 270) and goats (n = 238) were 31.85% and 47.47%, respectively. Further, to determine accuracy of developed test a total of 1731 serum samples were analyzed from the states with high small ruminant populations in India which included Andhra Pradesh, Telangana, Karnataka, West Bengal, and Gujarat. Overall seropositivity recorded were 24% in sheep and 19.12% in goats. Among the population of sheep and goats examined, the females and animals in the age group of 1–2 years showed high Capripoxvirus antibodies. This study highlighted the potential utility of rA27L protein as a safe and alternative serodiagnostic reagent, unlike the SNT, for serosurveillance and seromonitoring of Capripoxvirus infection in sheep and goats. [ABSTRACT FROM AUTHOR]

Objectives: Keeping in view the economic and veterinary public health importance of brucellosis, this research was conducted to determine its seroprevalence and associated risk determinants in small ruminants in district Khanewal, Southern Punjab, Pakistan. Materials and Methods: Two-stage cluster sampling technique was used for sampling, and the sample size was calculated using C-survey 2.0. Accordingly, sera samples (n = 392) were collected from small ruminants in the study area from October 2022 to July 2023. All the samples were tested for the presence of anti-Brucella antibodies by Rose Bengal Plate Test (RBPT), followed by confirmation of all the samples using an enzyme linked immunosorbent assay (ELISA) kit (ID.vet®, France; sensitivity and specificity=100%, each). Results: The seropositivity rate of brucellosis was 7.14% [n = 28/392; 95% confidence interval (CI) = 4.87%–10.12%] by RBPT, whereas the results of ELISA showed an overall seroprevalence rate of 7.40% (n = 29/392; 95% CI = 5.11%–10.37%) in the study population. Univariate analysis of risk factors revealed that abortion history (AH), retained fetal membranes (RFMs), repeat breeding, flock size (FS), educational status of farmers (ESFs), awareness about brucellosis (AB), and farm hygiene had a significant association with the seroprevalence of brucellosis (p < 0.05). The multivariate analysis using a binary logistic regression model revealed that variables including tehsil, FS, AH, RFM, ESF, AB, and farming system were significant factors (p < 0.05) associated with brucellosis in the target population. Conclusion: Brucellosis is prevalent in small ruminants in Khanewal, Pakistan. The disease burden can be reduced by improving the reproductive health of animals, farm hygiene, and farmers' awareness about the diseases. Further studies are needed on a larger scale to devise stringent disease control strategies to avoid losses associated with brucellosis at regional, national, and global levels. [J Adv Vet Anim Res 2024; 11(1.000): 9-18]

Objectives: Keeping in view the economic and veterinary public health importance of brucellosis, this research was conducted to determine its seroprevalence and associated risk determinants in small ruminants in district Khanewal, Southern Punjab, Pakistan. Materials and Methods: Two-stage cluster sampling technique was used for sampling, and the sample size was calculated using C-survey 2.0. Accordingly, sera samples (n = 392) were collected from small ruminants in the study area from October 2022 to July 2023. All the samples were tested for the presence of anti-Brucella antibodies by Rose Bengal Plate Test (RBPT), followed by confirmation of all the samples using an enzyme linked immunosorbent assay (ELISA) kit (ID.vet®, France; sensitivity and specificity=100%, each). Results: The seropositivity rate of brucellosis was 7.14% [n = 28/392; 95% confidence interval (CI) = 4.87%-10.12%] by RBPT, whereas the results of ELISA showed an overall seroprevalence rate of 7.40% (n = 29/392; 95% CI = 5.11%-10.37%) in the study population. Univariate analysis of risk factors revealed that abortion history (AH), retained fetal membranes (RFMs), repeat breeding, flock size (FS), educational status of farmers (ESFs), awareness about brucellosis (AB), and farm hygiene had a significant association with the seroprevalence of brucellosis (p < 0.05). The multivariate analysis using a binary logistic regression model revealed that variables including tehsil, FS, AH, RFM, ESF, AB, and farming system were significant factors (p < 0.05) associated with brucellosis in the target population. Conclusion: Brucellosis is prevalent in small ruminants in Khanewal, Pakistan. The disease burden can be reduced by improving the reproductive health of animals, farm hygiene, and farmers' awareness about the diseases. Further studies are needed on a larger scale to devise stringent disease control strategies to avoid losses associated with brucellosis at regional, national, and global levels. [ABSTRACT FROM AUTHOR]

Abstract Tomato mosaic disease caused by Tomato mosaic virus (ToMV) reduces tomato crop production globally. Biocontrol measures using various rhizobacteria and algae have been developed to reduce the adverse effects of plant diseases. To this end, two rhizobacteria (probiotic bacteria) including Pseudomonas fluorescens, Bacillus subtilis, and aqueous extract of brown alga (Sargassum angustifolium) were applied. A certain concentration of bacterial suspension was added to the tomato rhizosphere along with the aqueous extract of brown alga and the plants were subsequently inoculated with ToMV. Semi-quantitative indirect-ELISA was performed to estimate the virus titer within inoculated plants. Also, the disease severity index was determined by visual scoring of the plants at 14 and 28 days post-inoculation. Growth indices of plants were evaluated and the data were statistically analyzed. The results showed that multiple treatments of the rhizobacteria and the aqueous extract of brown alga reduced the disease severity to 27.46%, and inhibit the ToMV accumulation up to 86.48% in tomato plants. Moreover, the growth indices of tomato plants pre-treated with a combination of the rhizobacteria and brown alga extract were significantly improved. Taken together, the results suggest that these biocontrol agents have a synergistic effect and their simultaneous application can, therefore, reduce the crop loss caused by ToMV.

In this study，the indirect-ELISA method was established based on the structural proteins VP0 and VP1 of porcine Kobuvirus （PKV）. The genes of structural proteins VP0 and VP1 of PKV were synthesized and ligated into the prokaryotic expression vector pET-32a， and then transferred into competent cells BL21. The expressions of two proteins were induced by IPTG and then were purified by Ni column. The two indirect ELISA assays were established while recombinant protein pET-32a-VP0 and pET-32a-VP as coating antigen through a checkerboard titration method for repeatability，sensitivity，specificity for experiments，and clinical testing. The results showed that the optimal coating conditions of recombinant protein pET-32a-VP0 and pET-32a-VP1 antigen were 2.0 mg/mL and 2.5 mg/mL at 37℃ for 1 h，respectively. The optimal conditions for the blocking solution were 5% skimmed milk，37℃，2 h ；the optimal incubation conditions for the serum were 1∶200， 37℃，1 h ；the optimal incubation conditions for the secondary antibody were 1∶20 000 and 1∶15 000，37℃，1 h ；optimal colour development time was 10 min. The critical values of the two indirect-ELISA assays were 0.306 and 0.277 respectively. The results of experiment indicated that the indirect-ELISA methods were successfully established，which are accurate and efficient for detecting serum specific antibody of PKV. The methods are characterized as good reproducibility，sensitivity，specificity and stability. It is very helpful for future clinical diagnosis and the development of antibody detection kits of PKV. [ABSTRACT FROM AUTHOR]

Tobacco mosaic virus (TMV) is one of the economically important plant viruses which causes disease in various crops throughout the world. It has been reported that plant growth-promoting rhizobacteria (PGPR) can be used as potential biocontrol agents against plant viruses. Herein, greenhouse experiments were conducted to undertake the trilateral interactions among PGPR, bell pepper, and TMV. To this end, four-leaf-stage bell pepper seedlings were pre-treated by the PGPR, including Pseudomonas fluorescens, P. putida, and Bacillus subtilis in single and multiple application methods. The plants were then mechanically inoculated with TMV and visually inspected for symptom development till 28 days post-inoculation (dpi). The TMV accumulation in inoculated plants was quantitatively measured by Indirect-ELISA 28 dpi. Analysis of the extinction values showed that application of the PGPR was associated with the least significant (p < 0.05) value (0.08) compared to the positive control (0.77). Inoculation of PGPR triggered the biosynthesis of the defense-related enzymes such as catalase, peroxidase, ascorbate peroxidase, and superoxide dismutase, mediating the biochemical protection against TMV in bell pepper plants. In addition to the disease control, a significant (p < 0.05) increase in growth parameters was observed in PGPR-treated plants compared to the control plants. In conclusion, these results indicated that multiple applications of PGPR strains enhanced the plant vigor and provided an increased level of TMV suppression in bell pepper plants.

Diagnostic assays that are able to detect foot-and-mouth disease (FMD) virus infection in the vaccinated population are essential tools in the progressive control pathway for the FMD. However, testing of serum samples using a single diagnostic assay may not completely substantiate freedom from the virus infection. Therefore, viral non-structural proteins (NSPs)-based various serological assays have been developed for the detection of FMD infection. Nevertheless, the NSPs-based ELISAs have been developed in the indirect-ELISA format, thereby necessitating the use of species-specific conjugated secondary-antibodies for the detection of anti-NSP antibodies in various FMD-susceptible species. Therefore, this study presents a novel recombinant 2B-NSP-based indirect ELISA, employing HRP-conjugated protein-A/G detection system which can detect anti-NSPs antibodies from multiple FMD-susceptible species in a single ELISA platform. Recombinant 2B (r2B) protein was expressed as His-SUMO tagged protein in the E. Coli cells and purified using NI-NTA affinity column chromatography. Using the r2B protein and HRP-conjugated protein A/G, an indirect ELISA was developed and validated for the detection of anti-2B antibodies in serum samples collected from multiple FMD-susceptible animal species with known FMD status. Further, a resampling based statistical technique has been reported for determination of optimal cut-off value for the diagnostic assay. Through this technique, the optimal cut-off of 44 percentage of positivity value was determined for the assay. At this optimal cut-off value, the developed diagnostic assay provided diagnostic sensitivity, specificity, and accuracy, positive and negative predictive values (PPV and NPV) of 92.35 %, 98.41 %, 95.21 %, 98.58 %, and 91.67 %, respectively. The assay was validated further by analyzing random serum samples collected across multi-locations in India. The assay can be used as a single platform for testing serum samples from different species of FMDV-susceptible animals and will be useful for NSP-based serosurveillance of FMDV. • A species-independent indirect ELISA using FMDV 2B-NSP and protein A/G-HRPO conjugate developed. • The assay can detect FMDV-2B NSP antibodies from various susceptible species on a single platform. • Resampling based statistical technique was used for determination of optimal cut-off of the assay. • At the cut-off value of 44 PP, a diagnostic sensitivity and specificity of 92.35 % and 98.41 % respectively, was determined. [ABSTRACT FROM AUTHOR]

Abstract Background Haemonchus contortus (H. contortus) is one of the most important parasites that cause huge economic losses to small ruminant industry worldwide. Effective prognosis and treatment depend upon the early diagnosis of H. contortus infection. To date, no widely-approved methods for the identification of prepatent H. contortus infection are available to identify prepatent H. contortus infection properly. The aim of this study was to evaluate the diagnostic potential of recombinant cold shock H. contortus protein (rHc-CS) during early and late infections of H. contortus in goat. Results Purified rHc-CS exhibited a clear band, with a molecular weight about 38 kDa. H. contortus eggs were not detected by fecal egg count technique from feces collected at 0 to 14 days post infection (D.P.I). However, eggs were detected at 21, 28 and 35 D.P.I. Hence, results of immunoblotting assay showed specific anti rHc-CS antibody detection in all goat sera collected at early stage (14 D.P.I) and late stage (21–103 D.P.I) of H. contortus infection. Furthermore, no cross reactivity was observed against Trichinella spiralis, Fasciola hepatica and Toxoplasma gondii or uninfected goats. Among several evaluated rHc-CS indirect-ELISA format variables, favorable antigen coating concentration was found 0.28 μg/well at 37 °C 1 h and overnight at 4 °C. Moreover, optimum dilution ratio of serum and rabbit anti-goat IgG was recorded as 1:100 and 1:4000, respectively. The best blocking buffer was 5% Bovine Serum Albumin (BSA) while the best time for blocking, serum incubation and TMB reaction were recorded as 60, 120 and 10 min, respectively. The cut-off value for positive and negative interpretation was determined as 0.352 (OD450). The diagnostic specificity and sensitivity of the rHc-CS, both were recorded as 100%. Conclusion These results validated that rHc-CS is a potential immunodiagnostic antigen to detect the specific antibodies during early and late H. contortus infections in goat.

Potato Leaf Roll Virus (PLRV) is one of the most serious viruses infecting potato plant (Solanum tuberosum L.) in Egypt. Indirect Enzyme Linked Immunosorbent Assay (Indirect- ELISA) results revealed that 70 % of the collected samples were infected with PLRV. A multiplex Polymerase Chain Reaction (PCR) was carried out using three different sets of primers, specific for both PLRV and Potato virus Y (PVY) isolates. For confirmation; the movement coat protein (MP) gene was isolated from the infected plant tissues, and a band with molecular size 336 bp was obtained using Reverse transcription-Polymerase chain reaction (RT-PCR). The DNA sequence of the Egyptian PLRV-Banha -MP gene was deposited in GenBank under an accession number of KR002119. Moreover, sequence analysis revealed that the Egyptian PLRV isolate was closely related to a New Zealand isolate of PLRV (GU002341), with identity of 100%. Transmission electron microscope (TEM) examination of PLRV showed isometric particles, with approximate size of 24-30 nm. The cytopathological examination of the potato plant infected with PLRV revealed many cellular effects such as; partially degraded and deformed chloroplast, starch with an increased size and color change, in addition to nucleus and cytoplasmic bridge. It could be concluded that PLRV is present in Egypt, infecting most of the potato cultivars. Moreover, four different strains of PLRV were detected based on the Single strand conformation polymorphism (SSCP) of the MP gene. The aims of the current study were to identify the PLRV infection of potato plant in Egypt using; molecular, serological and biological methods, in addition to studying the effect of this virus on potato cell organelles. This is the first record of the presence of four different strains of PLRV infecting potato in Egypt, using SSCP assay.

Onion yellow dwarf virus (OYDV) belonging to the genus Potyvirus, family Potyviridae, is one of the widely distributed viruses of Allium species worldwide. It causes dwarfing, yellow striping, crinkling and flaccidity of the leaves of onion and garlic. To see the occurrence and incidence of OYDV on Allium crop, an attempt was made to develop antibody based diagnostic assay which would be useful for routine indexing and screening of the germplasm. The total RNA was isolated from the symptomatic leaves of onion and the gene encoding coat protein (CP) was cloned. The nucleotide sequencing analysis of the cloned RT-PCR product revealed ~ 774 bp amplicon (OYDV CP) and it was further cloned in pET-28a (+) expression vector which yielded ~ 30 kDa fusion protein with Histidine tag (His6BP). The expression of fusion CP was primarily checked on SDS–PAGE and further confirmed by Western blot. The His6BP-OYDV-CP was obtained in soluble state after purification and was used to immunize New Zealand white rabbit for the production of polyclonal antibody (PAb). The produced PAb against the purified fusion protein successfully detected OYDV from onion and garlic samples at 1:2000 dilutions in indirect-enzyme linked immunosorbent assay (DAC-ELISA). Thus, this study presents first report that Histidine tag (His6BP) fusion OYDV-CP based antibody production and its successful application in identification of virus free onion and garlic genotypes. [ABSTRACT FROM AUTHOR]

Cephalopina titillator (C. titillator) is a common worldwide nasal bot fly larval infestation of camels, which belongs to the family Oestridae. This study aimed to evaluate two new immunologic diagnostic techniques; indirect-ELISA and Dot-ELISA, for the screening of C. titillator infestation in camels. Thirty slaughtered camel heads were examined carefully for the presence of C. titillator larvae. One hundred, third-stage larvae (L3), were dissected for the collection of their salivary glands, for the preparation of the salivary gland antigen. Blood samples were obtained for hematological and serological examinations. Results revealed a true prevalence of C. titillator in the sampled camels being 80% (24/30). Infested camels showed a significant reduction in leukocytes (P < 0.0001) and neutrophils (P = 0.045), and a significant increase in eosinophils and monocytes (P < 0.0001). The serological examination estimated apparent prevalence as 80% (24/30) and 90% (27/30) by Dot-ELISA and indirect-ELISA, respectively. Dot-ELISA revealed 100% sensitivity, specificity, and accuracy. While, indirect-ELISA displayed 100% sensitivity, 50% specificity, and 90% accuracy. Dot-ELISA exhibited perfect agreement with the gold standard test, so it could be considered an ideal, simple, and accurate immunologic screening technique for the detection of C. titillator in camels. [ABSTRACT FROM AUTHOR]

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Background: Haemonchus contortus (H. contortus) is one of the most important parasites that cause huge economic losses to small ruminant industry worldwide. Effective prognosis and treatment depend upon the early diagnosis of H. contortus infection. To date, no widely-approved methods for the identification of prepatent H. contortus infection are available to identify prepatent H. contortus infection properly. The aim of this study was to evaluate the diagnostic potential of recombinant cold shock H. contortus protein (rHc-CS) during early and late infections of H. contortus in goat. Results: Purified rHc-CS exhibited a clear band, with a molecular weight about 38 kDa. H. contortus eggs were not detected by fecal egg count technique from feces collected at 0 to 14 days post infection (D.P.I). However, eggs were detected at 21, 28 and 35 D.P.I. Hence, results of immunoblotting assay showed specific anti rHc-CS antibody detection in all goat sera collected at early stage (14 D.P.I) and late stage (21–103 D.P.I) of H. contortus infection. Furthermore, no cross reactivity was observed against Trichinella spiralis, Fasciola hepatica and Toxoplasma gondii or uninfected goats. Among several evaluated rHc-CS indirect-ELISA format variables, favorable antigen coating concentration was found 0.28 μg/well at 37 °C 1 h and overnight at 4 °C. Moreover, optimum dilution ratio of serum and rabbit anti-goat IgG was recorded as 1:100 and 1:4000, respectively. The best blocking buffer was 5% Bovine Serum Albumin (BSA) while the best time for blocking, serum incubation and TMB reaction were recorded as 60, 120 and 10 min, respectively. The cut-off value for positive and negative interpretation was determined as 0.352 (OD450). The diagnostic specificity and sensitivity of the rHc-CS, both were recorded as 100%. Conclusion: These results validated that rHc-CS is a potential immunodiagnostic antigen to detect the specific antibodies during early and late H. contortus infections in goat. [ABSTRACT FROM AUTHOR]

• Natural antibodies (NAb) binding not-self antigens in kid serum are shown. • The synthetic hapten 2,4,6-trinitrophenyl (TNP) presents the higher immunoreactivity to Nab. • IgM-NAb and total IgM levels are highly correlated within each sampling time. • IgG- and IgM-NAb levels are correlated only on the 1st and the second sampling time. • Specific antibody response (SpAb) is not related to NAb and Ig levels. Natural antibodies (NAb) are antibodies that can bind to a particular antigen without any apparent antigenic stimulation. In this paper, a careful analysis has been carried out on NAb levels in goat kid serum; possible correlations with the total immunoglobulin (tot-Ig) levels and specific antibody (SpAb) response were considered. Twenty randomly chosen kids were submitted to a first blood sampling (day 0). After 60 and 100 days, new blood samplings were carried out in the same animals. On day 0, after blood collection, all animals were immunized with a commercial vaccine; the immunization was repeated 30 days apart. Some exogenous antigens were tested to verify their immunoreactivity to NAb. Among them, the synthetic hapten 2,4,6-trinitrophenyl (TNP) conjugated with bovine serum albumin, resulted as the antigen with the higher immunoreactivity to NAb. Tot-Ig levels increased over time (p < 0.001). On the contrary, NAb levels, both IgG- and IgM-isotypes, significantly decreased during the experimental period (p < 0.001 and <0.05, respectively). Linear regression analyses showed a high correlation between IgM-NAb and tot-IgM levels (p < 0.001) at all the evaluated sampling times. However, a significant correlation between IgG-NAb and IgM-NAb was found only at the 1st (p < 0.01) and at the 2nd sampling (p < 0.05). No significant correlations were found between SpAb response and the other assessed humoral immune parameters. The obtained results are discussed in the light of the possible use of NAb assessment for the evaluation of the immune system activity in goat. [ABSTRACT FROM AUTHOR]

Highlights • Production of recombinant non-structural protein 2 fragments of BTV; rNS2Nt and rNS2Ct. • Evaluation of diagnostic efficacies of rNS2Nt and rNS2Ct fusion proteins in ELISA format. • Detection of bluetongue viral NS2 specific antibodies in ruminant serum samples. • rNS2 N-terminus protein indicated better immuno-reactivity than rNS2 C-terminus protein. Abstract Bluetongue viral antibody detection assays are important for routine disease diagnosis and sero-surveillance in susceptible host's especially small ruminants. In the present study, a partial NS2 gene encoding for two non-structural protein-2 fragments; N-terminus (1 M-A 177 aa) and C-terminus (178 P-V 354 aa) of BTV-23, were cloned separately, expressed and purified as recombinant NS2Nt and NS2Ct fusion proteins (˜39 kDa each) from prokaryotic expression system (Escherichia coli). Subsequent to affinity chromatographic purification under non-denaturing condition, both rNS2Nt and rNS2Ct fusion proteins were obtained in sufficient quantity and quality. Further, both antigens were also found to possess good reactivity in detecting NS2 specific BTV antibodies irrespective of serotypes in ruminant serum samples by indirect-ELISA. Of two fragments, rNS2Nt was found to be more efficient as diagnostic candidate. However, in comparison to structural protein based VP7 c-ELISA and rVP7 i-ELISA, the sensitivity (5.1%, 8.2%) and specificity (2.5%, 3.2%) of rNS2Nt i-ELISA were found to be relatively lower, respectively. The study indicated that N-terminus of NS2 protein could be used either alone or in combination/fusion with other non-structural proteins (NS1/NS3) of BTV for potential utility of NS protein based indirect-ELISA as an alternate assay for routine sero-surveillance of BTV infection in ruminants. [ABSTRACT FROM AUTHOR]

Highlights • Orf virus truncated F1L fusion protein (∼50 kDa) was expressed in Escherichia coli. • Optimized rF1L-ELISA specifically detected orf viral antibodies in small ruminants. • rF1L-ELISA has high diagnostic sensitivity (>89%) and specificity (92%)over SNT. • rF1L-ELISA has potential for sero-surveillance of orf infections in sheep and goat. Abstract Orf or contagious ecthyma, is a highly contagious transboundary disease of sheep and goats. For sero-diagnosis of orf, recombinant antigen based assays are considered as alternatives to conventional approaches such as serum neutralization test (SNT) and counter-immuno-electrophoresis (CIE). A major envelope protein of orf virus (ORFV), F1L, is highly immunogenic and is a candidate for use in these assays. In this study, the F1L gene of the ORFV-59/05 strain encoding a recombinant mature F1L protein (1 M-D 302 aa) with a C- terminal truncation, was produced as a fusion protein (∼50 kDa) in Escherichia coli. The immunogenic potential of purified rF1L was confirmed by detecting specific anti-F1L antibody responses in sera collected from immunized rabbits and guinea pigs using ELISA and SNT. An indirect-ELISA based on rF1L was developed and optimized. In comparison to SNT by ROC analysis in the detection of ORFV specific antibodies, this new assay exhibited a diagnostic specificity of 94.04% and 92.53% with sheep and goat sera, respectively, while the sensitivity was 89.22% and 94.25%, for sheep and goat sera. No cross reactivity was noted with sera collected from small ruminants infected with other transboundary diseases (goatpox, sheeppox, peste des petits ruminants, foot–and-mouth disease and bluetongue). Furthermore, the rF1L-ELISA applied to screen the vaccinated/challenged goat sera resulted in better detection (30%) than by SNT (28%) in spite of lower levels of antibodies which could be due to predominant cell mediated immune response in vaccinated animals. This study highlighted the potential utility of rF1L protein as a safe and novel diagnostic reagent in comparison to live virus antigen, in the development of sero-diagnostic assay for surveillance of ORFV infection in sheep and goats. [ABSTRACT FROM AUTHOR]

Viral diseases are very important on tomato and pepper because of their impact on the quality and quantity of these crops. Potato virus Y (PVY) belongs to the genus Potyvirus and family Potyviridae is one of the most important viruses and has a worldwide distribution on Solanaceae. In order to detection of the virus and determine taxonomic status of strains on tomato and pepper, a total of 88 samples were collected during growing seasons of 2015 and 2016 from different fields of Ziveh, Nazloo, Par, Chongharalouye Pol, Balanej and AliAbad around Urmia and subjected to Indirect-ELISA. The virus was detected in 23 samples. Total RNA was extracted from positive samples in ELISA, and synthesized cDNA was used to amplify a part of the coat protein (CP) gene in RT-PCR using specific primers. Fragments of 609 bp and 549 bp were amplified and sequenced directly. Phylogenetic analysis results showed the existence of PVY strains including N, C and O on tomato samples and NTN on pepper. This is the first report of PVY O, C, N on tomato and PVYNTN on pepper occurrence in Urmia and West-Azarbaijan province of Iran. [ABSTRACT FROM AUTHOR]

Background Increasing dairy consumption in China has been accompanied by rising incidence of milk allergy. Here we analyzed profiles of specific immunoglobulin E (sIgE) against cow's milk proteins, and assessed their value for milk allergy diagnosis among infants and young children from northern China. Methods Sera collected from 48 patients with milk allergy and 27 negative control subjects was analyzed by enzyme-linked immunosorbent assay to measure sIgE to α-lactalbumin (Bos d 4), β-lactoglobulin (Bos d 5), α-casein (Bos d 9), β-casein (Bos d 11), and κ-casein (Bos d 12). Results Among milk-allergic individuals, most were sensitized to at least one milk protein; about half were sensitized to Bos d 5, Bos d 9, Bos d 11 and Bos d 12, respectively, while few had positive serum sIgE against Bos d 4. Bos d 12 sIgE had the largest area under curve (AUC) (0.878; 95% CI, 0.800–0.957) and thus showed the best diagnostic performance in discriminating between milk-allergic and non-milk allergic patients, with a sensitivity of 92.6% and specificity of 72.9% using a statistically optimal cut-off value (OD 450nm , 0.191). The combinations of Bos d 5 + Bos d 12 showed an AUC of 0.926, which was larger than for any individual components. Conclusions Our results revealed inter-individual variation in the sensitization to different milk allergen component. Bos d 12 sIgE showed best performance in diagnosing milk allergy. Milk allergy diagnostic accuracy was further improved using combinations of milk allergen components by application of ROC curves based on logistic regression. [ABSTRACT FROM AUTHOR]

An indirect-ELISA for the diagnosis of mixed gastrointestinal (GI) nematode infection comprising Oesophagostomum, Haemonchus and Trichuris species was standardized using crude somatic antigen of Oesophagostomum columbianum (CSAg-Oc) and sera of slaughtered goats with known parasitological status including Oesophagostomum, Haemonchus, and Trichuris (strong positive), Haemonchus and Trichuris (weak positive) and parasite free goats (negative). Two cut-off points, i.e. higher and lower cutoff were determined using the strong positive, weak positive and the negative control sera of goats. Thus the test sera having optical density (OD) values greater than the higher cut-off were considered positive for mixed infection with all the three nematode species, intermediate between the higher and the lower cut-off values were considered positive for mixed infection of Haemonchus and Trichuris, and less than the lower cut-off value were considered negative for any of these three nematode species. The sensitivity, specificity and accuracy of the ELISA for diagnosis of mixed GI nematodoses were 81.25, 93.18% and 90.00%, respectively, while it was 92.86% sensitive, 75.00% specific and 91.67% accurate for the diagnosis of mixed infection with Haemonchus and Trichuris. The ELISA, so standardized, detected 27.78% sero-prevalence of Oesophagostomum plus Haemonchus and Trichuris infection and 38.89% percent of Haemonchus and Trichuris infection in the field goats. The standardized assay might be exploited as a diagnostic tool and also for sero-epidemiological study of two important GI nematodes of goats. [ABSTRACT FROM AUTHOR]

Bluetongue, an arthropod borne non-contagious disease of ruminants especially sheep, is caused by bluetongue virus (BTV). Detection of BTV antibodies in susceptible hosts is considered to be of significance in disease diagnosis and differentiation. In the present study, a partial NS3 gene encoding for non-structural protein-3 N-terminus (1MT117 aa) of BTV-23, produced as purified recombinant NS3Nt fusion protein (~32 kDa) using prokaryotic expression system (Escherichia coli), was evaluated as a candidate antigen in an indirect-ELISA (rNS3Nt-ELISA) to measure the serologic response to NS3 protein in small ruminants. The rNS3Nt fusion protein obtained in sufficient quantity and quality has good reactivity in detecting NS3 specific antibodies in field serum samples by indirect-ELISA. As NS3 protein is highly conserved, rNS3Nt-ELISA has potential for NS3 specific detection of antibodies in BTV affected animals irrespective of different viral serotypes. In comparison to structural protein (VP7) based c-ELISA kit and i-ELISA kit, the diagnostic sensitivity (85.1%, 86.2%) and specificity (92.5%, 93.2%) of rNS3Nt-ELISA were found to be relatively lower, respectively. Nevertheless, the study indicated the potential utility of rNS3Nt- ELISA as an alternate assay in routine sero-diagnosis of BTV infection and possible sero-surveillance of ruminants under DIVA strategy. [ABSTRACT FROM AUTHOR]

The aim of present study was to detect the specific Hypoderma IgG-antibodies in blood samples of cattle by using of indirect-ELISA. For this purpose, an overall of 476 cattle from different regions in Wasit province / Iraq, were submitted for blood samples collection and then for serological assay that revealed on 249 (52.31%) as a total result for serologically positive cattle. Also, the study was discussed the seropositive prevalence of hypodermosis with a number of epidemiological risk factors (age, sex, breed, and herd management), which showed that the highest seropositive rates have been reported in >3 years group (59.39%) more than ≤3 years group (43.72%) for age factor, in females group (56.72%) more than males group (36.54%) for sex factor, and in bad group (59.48%) more than good group (32.81%) for herd management factor. Statistically, at a level of (P≤0.05) the significant differences were reported within the age, sex, and herd management factors; but it was not detected between both groups of breed factor (local and cross-breed). [ABSTRACT FROM AUTHOR]

The present work was carried out to study the sero-prevalence of Haemonchus contortus infection in sheep by Indirect-Enzyme Linked Immuno Sorbent Assay (Indirect-ELISA) using somatic antigen. Out of 100 abomasums screened, 57 found positive for H. contortus adult worms. A total of 250 serum samples which includes, 100 serum samples from local abattoir in and around shimoga region from the animals from which the abomasums were collected and 150 serum samples from migratory sheep were used to detect the circulating antibody against H. contortus by Indirect-ELISA using somatic antigen. Of the 57 sheep harboring adult worms in their abomasums, the serum samples showed positive reaction by Indirect-ELISA with somatic antigen. However, among 43 sheep which are not showing any adult worms of H. contortus in their abomasums, but their 21 serum samples showed positive reaction by Indirect-ELISA. The sensitivity and specificity of Indirect-ELISA was found to be 100 and 67.18 %, respectively. Also, the sero-prevalence of H. contortus infection was found to be 58.66 % out of 150 migratory sheep serum samples screened for detecting circulating antibodies of H. contortus by Indirect-ELISA using somatic antigen in and around shimoga region. [ABSTRACT FROM AUTHOR]

Infection of viruses on Cucurbitaceae may cause high yield and economic losses. Squash mosaic virus is a seed borne virus and among the most important virus infecting Cucurbitaceae. The aims of these research was to detect infection of several viruses on Cucurbitaceae and to examine seed transmission efficiency of SqMV. Detection of Cucumber mosaic virus (CMV), Squash mosaic virus (SqMV), Watermelon mosaic virus-2 (WMV-2), Zucchini yellow mosaic virus (ZYMV), and Tobacco ringspot virus (TRSV) from field samples and seeds was conducted using Indirect-ELISA method. Infection of CMV, SqMV and ZYMV was detected from field samples. Seed transmission of SqMV on commercial seeds of bottle gourd, watermelon, zucchini, cabocha, cucumber, and melon was 13, 13, 33, 73, 100, and 100%, respectively. Seed transmission of ZYMV was only occurred on bottle gourd and zucchini, i.e. 13.3% and 26.67%, respectively. Infection of SqMV through F2 seed was determined from cucumber, bottle gourd, and melon, i.e. 93, 100, and 100%, respectively. Therefore, the status of SqMV as quarantine pest should be evaluated since SqMV was already found in West Java.

An indirect enzyme-linked immunosorbent assay (ELISA) capable of detecting walnut proteins in commercial products was developed. Polyclonal antibodies against soluble walnut protein were used. Specific epitopes of walnut protein were analyzed for cross activity with peanut, soybean, sesame, rice, wheat, jujube, and defatted milk protein. Epitopes of walnut proteins had no cross activity. The indirect ELISA was highly specific for soluble walnut proteins. Recovery values from walnut protein solutions ranged from 88.47 to 115%, and low coefficients of variation (<10%) indicated good repeatability. Intra and inter-assay precisions were <6 and <7 %, respectively. The limit of detection was 10 ng/mL of soluble walnut protein. The method was applied to detect some commercial food products in China and the results showed that indirect ELISA could be promisingly used to quantify adulteration of walnut processed foods. [ABSTRACT FROM AUTHOR]

Buffalopox virus (BPXV) , an Indian variant of vaccinia virus (VACV), is a zoonotic agent and affects buffaloes, cattle and humans. A27L is one of the conserved major immuno-dominant envelope proteins of orthopox viruses (OPVs) involved in viral entry/maturation and elicits neutralizing antibodies. In this study, the A27L gene of BPXV-Vij/96 strain encoding recombinant mature A27L ( 21 S to E 110 ) and C-terminal truncated A27L-LZD ( 21 S to N 84 aa) proteins were cloned and over-expressed in Escherichia coli as fusion proteins. Structurally, A27L of BPXV was similar to that of VACV and found to contain four regions including a potential coiled-coil motif (CCM) in the centre (43 to 84aa). Oligomerization of recombinant A27L fusion protein (∼30 kDa) leads to the formation of dimer/trimers/tetramers under non-reducing conditions. Further, the purified rA27L protein was used for active immunization of rabbit (250 μg/rabbit) and adult mice (10 μg and 50 μg/mice) with or without adjuvants (FCA, alum and CpG). Immune response measured by using indirect-ELISA and SNT revealed a gradual increase in antigen specific serum IgG as well as neutralization antibody titers. Upon challenge with virulent BPXV strain, a protection of 60% was observed in suckling mice passively administered with anti-rA27L sera. No cross-reactivity of rA27L protein with hyperimmune sera against ORFV, GTPV, SPPV, PPRV, FMDV and BTV was noticed in indirect-ELISA. The study indicated that the rA27L protein is a safe and potential prophylactic as well as diagnostic antigen for buffalopox. [ABSTRACT FROM AUTHOR]

Aflatoxin B1 (AFB1) is most frequently found in plant substrates, which has shown the highest toxigenic potential. Based on previous studies, the IARC has classified AFB1 as a class 1A human carcinogen. Several impacts on consumers, such as loss of human and animal lives; health care and veterinary care costs; contaminated foods and feeds disposal costs; and investment in research and management of the myco-toxin problem. Fourteen maize seed samples comprising of recommended and local varieties were collected from three maize growing zones (Zone I- Almora, Kullu, Bilaspur, Dhaulakuan, Kangara, Saharanpur, Zone II- Karnal, Ludhiana, Pantnagar, New Delhi and Zone III- Begusarai, Varanasi, Sabour-1 and Sabour 2). In our studies AFB1 toxin range were noticed Zone-I (0.0294- 153.5081 ppb), Zone-II (0.1761- 161.0537 ppb ppb) and Zone-III (3.8366- 53.1256 ppb) collected seed samples.This indicate that ELISA technique could be applied to the monitoring of Aflatoxin contamination in a lot of samples in a cost, accuracy, simplicity and time effective manner. [ABSTRACT FROM AUTHOR]

Phalaenopsis is one of the most popular orchid plants in the global market. Several viruses have been reported to negatively impact its growth, yield and quality. A mixed infection of Odontoglossum ringspot virus and Cymbidium mosaic virus was detected in Phalaenopsis hybrid “V3” (Phal. Yukimai × Phal. Taisuco Kochdian) plants in Taiwan. In the present communication, we report a relatively simple protocol for elimination of these two viruses using shoot-tip culture, and early isolation and selection of virus free protocorm-like body (PLB) through subcultures. The indexing of viruses in the field grown plants, PLBs and tissue culture plants was carried out by the Indirect enzyme-linked immunosorbent assay (ELISA), and one-step multiplex reverse transcription polymerase chain reaction (RT-PCR). The induction of PLBs in shoot-tips was achieved on 1/2× Murashige and Skoog's basal medium supplemented with 1% sucrose and 0.9% Bacto-agar. PLBs could be maintained, proliferated and converted to plantlets on 1 g/L Hyponex medium supplemented with 1% sucrose, 6% potato pulp, 0.5 g/L tryptone, 0.25% activated charcoal and 0.6% Bacto-agar. The regenerated plantlets from virus-free PLBs acclimatized easily in a greenhouse and showed 100% survival rate. All the tissue culture-raised Phalaenopsis plants in the greenhouse tested negative for the two viruses. Our study demonstrates that some PLB lines selected at the first subculture as virus-free were found to be infected with virus at second subculture, however, re-occurrence of virus was never found in PLB lines at third subculture onwards. Hence, at least 3 subcultures are necessary to authenticate that the cultures are free of viruses. Simple culture media without plant growth regulators used in the present study minimizes the chances of somaclonal variations and ensures genetic uniformity of cultured plantlets, a highly desirable trait in the orchid industry. The method developed in the present study has potential of virus elimination, mass propagation of genetically uniform and virus-free Phalaenopsis plants. [ABSTRACT FROM AUTHOR]

Classical pathogenic strains of Yersinia enterocolitica produce a 17 kDa outer membrane protein, Ail (attachment-invasion locus), which mediates bacterial attachment to some cultures epithelial cell lines and invasion of others. In the present study, hybridomas were developed for the production of monoclonal antibodies (MAbs) against Ail protein of Y. enterocolitica. A set of five stabilized hybridoma cell lines were generated, of which, two MAbs, YEA 302 and YEA 303, exhibited specific reaction to the native Ail protein (17 kDa) present in whole cell lysate of Y. enterocolitica strains beside having reaction with rAil. The other three MAbs, YEA 5, 17 and 32, had some cross reactions with proteins other than Ail. Two out of five MAbs were IgG1, two were IgG2b and one in IgM in nature. MAbs (YEA 302 and YEA 303) did not show any cross-reaction with whole cell lysate of Brucella abortus, Vibrio cholerae, Salmonella typhimurium and Escherichia coli and other species of Enterobacteriaceae including Y. pestis in ELISA and Western blot analysis. The presence of Ail protein among the strains recovered from pork and milk samples was evaluated by these sets of MAbs and the results were compared with the duplex PCR. Collectively, the data suggest that these MAbs may have the potential for their use in the detection of pathogenic Y. enterocolitica reliably, rapidly and at a relatively low cost. [ABSTRACT FROM AUTHOR]

Abstract: The greater amberjack, Seriola dumerilii (Risso 1810) is a semi-pelagic fish and a worldwide species; it is considered a promising candidate for the diversification of Mediterranean aquaculture. In this paper an experimental injection with Listonella (Vibrio) anguillarum was performed to study the immune response of S. dumerilii. Antibody titres to L. anguillarum O1 were determined with indirect-ELISA at different times over a period of 42 days. Results showed that the antibody levels against L. anguillarum were significantly higher in the challenged fish compared to the control. They started developing since the 5th day reaching the highest peak on day 20 after injection, indicating a fast response of the immune system. The observed antibody titre was very high versus L. anguillarum if compared to other fish species. [Copyright &y& Elsevier]

Abstract: Mauritius is one of the largest world producers of Anthurium cut flowers but outbreaks of bacterial blight have never been reported on the island. This work was about the characterisation and identification of bacterial strains isolated from Anthurium andreanum, Dieffenbachia maculata and Aglaonema simplex in Mauritius. Fifteen strains, that showed the morphological properties of Xanthomonas on conventional media, were tested on two semi-selective media (Esculin-trehalose and cellobiose-starch). ELISA tests using a panel of monoclonal antibodies were carried out and three out of 15 strains reacted with a Xanthomonas-specific monoclonal antibody (MAb XII). Analysis using four sets of ribosomal primers revealed that the same three Mauritius strains shared conserved PCR products with reference xanthomonads including virulent strains of Xanthomonas axonopodis pv. dieffenbachiae (Xad). BIOLOG tests and the Sherlock Microbial Identification system (MIDI) identified these three new strains at the species level as X. axonopodis. The complementary tests that were carried out clearly confirmed that the three strains are xanthomonads and, moreover, a DNA probe which showed specificity to Xad strains suggested that the three Mauritius strains are non-virulent forms of the pathogen causing Anthurium blight. [Copyright &y& Elsevier]

An indirect enzyme-linked immunosorbent assay (ELISA) with Dicrocoelium dendriticum excretory/secretory antigens was used to evaluate the presence of serum antibodies against the trematode in 738 sheep randomly chosen in Sardinia (Italy). Coprological sedimentation was used to discover egg-output. Seropositivity was detected in 86.2% tested sheep, whereas faecal prevalence was 6.7%; all that were faecal-positive also were ELISA-positive. [Copyright &y& Elsevier]

A method for detecting tobamoviruses from field soils was developed using non-precoated indirect enzyme-linked immunosorbent assay (Id-ELISA). Absorbance values in Id-ELISA were relatively low after directly applying Pepper mild mottle virus (PMMoV)-infested soil extract. However, heat treating the soil extract before application greatly enhanced the absorbance values. The heat treatment was essential for the Id-ELISA detection of tobamoviruses from infested soil, although the efficiency of virus recovery varied depending on the properties of soil. The number of local lesions in the infectivity assay was consistent with the absorbance values in Id-ELISA. Moreover, the absorbance values in Id-ELISA were correlated with the incidence of soil transmission of PMMoV. Thus, Id-ELISA combined with heat treatment is a practical technique for the diagnosis of infestation with Tobamovirus in field soils, Gray Lowland soil and Sand-dune Regosol. [ABSTRACT FROM AUTHOR]

Peste-des-petits-ruminants and bluetongue are two of the most important viral diseases of small ruminants. In the present study, a combined indirect enzyme-linked immunosorbent assay (CI-ELISA) was developed, which can be run parallelly on the same plate for simultaneous detection of antibodies against peste-des-petits-ruminants virus and bluetongue virus. A total of 503 goat sera samples were used for developing the CI-ELISA. The relative sensitivity and specificity of CI-ELISA for detection of antibodies against PPRV were 90% and 88.8%, respectively at cut off level of 34.52% positivity. In case of BTV, 95.9% relative sensitivity and 98.6% specificity values were obtained at cut-off level of 40.42% positivity. These results indicate that the single plate CI-ELISA reported here could be used as an alternate method to existing dual plate competitive ELISAs for diagnosis/sero-surveillance of both the diseases. [ABSTRACT FROM AUTHOR]

Avian mycoplasmosis, mainly caused by Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS) is an economically important disease of the poultry industry. The present study was aimed to develop whole cell based indirect-ELISA (i-ELISA) and DOT blot assay (DOT-ELISA) as rapid, sensitive, specific and economical sero-detection tests for MG and MS. A total of 306 blood samples were collected from birds slaughtered at local meat shops of different districts of Haryana, India to detect MG and MS antibodies. Sonicated antigens prepared from freshly grown culture of MG and MS were used to develop i-ELISA and DOT blot assay. In i-ELISA, 50.32% and 61.76% serum samples were found to be positive for MG and MS antibodies, respectively. However in DOT blot assay, 41.83% and 53.92% serum samples were found positive for MG and MS antibodies, respectively. The relative diagnostic sensitivity and specificity of DOT-ELISA were measured considering i-ELISA as a reference test. The relative diagnostic sensitivity of the DOT blot assay was found to be 69.48% and 82.01%; whereas relative diagnostic specificity was 86.18% and 91.45% for the detection of MG and MS antibodies, respectively. The developed serological assays may be used as rapid and economical diagnostic tools for large scale screening of poultry sera for MG and MS antibodies. • Indirect-ELISA and DOT blot assay was developed for serodetection of MG and MS antibodies in poultry. • The relative diagnostic sensitivity and specificity of both the assay was measured. • Both assays may be used as rapid and economical tool for serodetection of MG and MS in poultry. [ABSTRACT FROM AUTHOR]

Psittacosis is a zoonotic disease caused by Chlamydia psittaci (C. psittaci), leading to high risk for animal industry and human health. Lack of reliable commercial kits and effective vaccines is hampering control of C. psittaci infection. Polymorphic outer membrane protein Gs (PmpGs) are enriched in diverse C. psittaci , and its role are unclear during C. psittaci infection. In the present study, pmp20G gene was cloned into pET-28a vector and then the constructed plasmid was transferred into Escherichia coli Rossetta (DE3). After denaturation and renaturation, the recombinant Pmp20G-N was identified by SDS-PAGE and Western blot. Afterwards Pmp20G-N was used as the coating antigen to develop an indirect ELISA (I-ELISA) assay. Both the specificity and sensitivity of Pmp20G-N ELISA were 100%, while the MOMP-ELISA had 93.65% sensitivity and 98.94% specificity, respectively. The concordance between MOMP-ELISA and Pmp20G-N ELISA assay was 98.1%. Hence, Pmp20G-N ELISA has the potential to be a diagnostic antigen for detection C. psittaci antibody. However, further studies are needed to be done for differentiating C. psittaci from Chlamydia spp. and other C.psittaci- specific serovars using Pmp20G-N ELISA. • Polymorphic outer membrane protein 20G of C. psittaci is firstly identified as biomarker of diagnosis. • Pmp20G coated ELISA yields highly sensitivity and specificity compared to MOMP-ELISA. • It has been proved that Pmp20G coated ELISA has the potential for clinical detection of Chlamydia psittaci. [ABSTRACT FROM AUTHOR]

The article examines the effect of zinc on piglets' humoral immune response following classical swine fever (CSF) virus vaccination. Study shows the increase of antibody level that is specific to CSF virus in the serum samples of the piglets. It points out that no antibody increase was found in the controlled group of piglets with zinc in their diet, but without vaccine intake.