ELISA-based detection of fresh and frozen-thawed lamb meat: a promising analytical approach for meat authentication.

Fresh/chilled meat differs in quality from frozen-thawed meat, and consumers prefer fresh meat over frozen-thawed meat. Differentiating between the two types of meat is an important part of the meat quality control system. This study aimed to develop an ELISA based on cytoplasmic antioxidant enzyme superoxide dismutase (SOD) as a biomarker. IgGs were raised in experimental animals and purified using immunoaffinity chromatography. The assay was optimized with guinea pig anti-SOD pAb as the capture antibody and rabbit anti-SOD pAb as the detection antibody. The assay showed excellent performance for differentiation, as the ROC area under the curve values were >0.9. A sensitivity of 95.8% and specificity of 95.0% were observed at a positive percentage (PP%) criterion value of 52.752. Sandwich ELISA results showed a significant difference between the chilled and repeatedly frozen-thawed lamb samples. Lamb meat stored for five days in a chiller showed a PP% value below the threshold PP% value, indicating that the assay can differentiate meat until 5 days of chiller storage. In addition, a thawing time of more than 18 h at 4 ± 1 °C is required to differentiate between both types of lamb meat with a size of 500 g. The developed ELISA can be applied at various points in the meat value chain to differentiate fresh meat from frozen-thawed meat.

Design and assessment of a double antigen indirect ELISA for lumpy skin disease surveillance in India.

Lumpy skin disease (LSD), caused by the lumpy skin disease virus of the genus Capripoxvirus, is rapidly emerging across most countries in Asia. Recently, LSD has been linked to very high morbidity and mortality rates. Until 2019, India remained free of LSD, resulting in a lack of locally developed diagnostic kits, biologicals, and other tools necessary for managing the disease in a country with such a large livestock population. Therefore, this study aimed to design and validate an indigenous and cost-effective in-house ELISA for large-scale screening of cattle samples for antibodies to LSDV. The viral major open reading frames ORF 095 and ORF 103 encoding virion core proteins were expressed in a prokaryotic system and the recombinant antigen cocktail was used for optimization and validation of an indirect ELISA (iELISA). The calculated relative diagnostic sensitivity and diagnostic specificity of the iELISA were 96.6 % and 95.1 %, respectively at the cut-off percent positivity (PP≥50 %). The in-house designed double-antigen iELISA was found effective to investigate the seroprevalence of LSDV in various geographical regions of India.

Production and characterization of biologicals for disease diagnosis and pathological evaluation of elephant endotheliotropic herpesvirus (EEHV).

Elephant endotheliotropic herpesviruses (EEHV) belong to the family Herpesviridae and cause a highly fatal hemorrhagic infection in elephants. EEHV poses a global threat to the already endangered elephant population. Since EEHV is a non-cultivable virus, there is a scarcity of specific diagnostics, therapeutics, and vaccines. In this study, our objective was to develop biologicals for diagnosis and pathological studies against the most prevalent EEHV1A/1B. We expressed two truncated fragments of the DNA polymerase, glycoprotein B (gB), and glycoprotein (gL) of EEHV in the prokaryotic system. Hyperimmune serum against the purified antigens was raised in rabbits and guinea pigs. We validated the reactivity of this hyperimmune serum using western blotting, ELISA, and immune-histochemistry on known positive infected tissues. Samples collected from 270 animals across various states in India were evaluated with these biologicals. The raised antibodies successfully demonstrated virus in immune-cytochemistry. Additionally, all known positive samples consistently exhibited significant inhibition in the OD values when used in the competitive format of ELISA across all four antigens when compared to the serum collected from known negative animals. An apparent sero-prevalence of 10 % was observed in the randomly collected samples. In summary, our study successfully developed and validated biologicals that will be invaluable for EEHV diagnosis and control.

Development of superoxide dismutase based visual and spectrophotometric method for rapid differentiation of fresh and frozen-thawed buffalo meat.

Study aimed to develop biomarker-based assay for rapid detection of fresh and frozen-thawed buffalo meat in the supply chain. The method is based on development of a solvent system and identification of suitable substrate and developer for screening of biomarkers. For the confirmation column chromatography, gel electrophoresis and Western Blotting were carried out. Validation was done by intra- and inter-day validation, storability study, and determination of thermal history. Best results were shown with pH 8.0 Tris-HCl; extraction buffer, 205 µM nicotinamide adenine dinucleotide hydrogen; substrate, 184 µM Nitroblue tetrazolium, and 1.9 µM phenazine methosulfate; developer. The thermal history ranged from 0.14 to 0.17 during storage at -20 °C. The intra- and inter-day assay precision (CV %) ranged from 5.3 to 6.5 %; in chilled and 14.1 - 9.2 % in frozen-thawed samples. The study confirmed SOD as a viable biomarker. Developed method using SOD has significant potential for rapidly differentiating chilled or frozen-thawed meat.

Development and evaluation of recombinant gD protein based ELISA for sero-surveillance of BoHV-1 in India.

Bovine herpes virus-1 (BoHV-1) is responsible for production losses through decreased milk yields, abortions, infertility, and trade restrictions in the bovine population. The disease is endemic in many countries including India. As the virus harbors a unique feature of latency animals once infected with the virus remain sero-positive for lifetime and can re-excrete the virus when exposed to stressful conditions. Hence, identification and culling of infected animals is only the means to minimize infection-associated losses. In this study, an economical indigenous assay for the detection of BoHV-1 specific antibodies was developed to cater to the huge bovine population of the country. The viral structural gD protein, expressed in the prokaryotic system was used for optimization of an indirect ELISA for bovines followed by statistical validation of the assay. The diagnostic sensitivity and specificity of the indirect ELISA were 82.9% and 91.3% respectively. Systematically collected serum samples representing organized, unorganized and breeding farms of India were tested with the indigenously developed assay for further validation.