Recombinant interferon stimulated protein 15 (rISG15) as a molecular marker for detection of early pregnancy in Bubalus bubalis.

Early and accurate diagnosis of pregnancy in animals is important for improving the reproductive management of livestock. The buffalo (Bubalus bubalis) is the most important dairy animal in India, but there are reproductive problems resulting from extended calving interval and ovulation occurring in the absence of behavioral estrus. The lack of simple methods for early pregnancy diagnosis intensifies these problems. The present study, therefore, was conducted to ascertain the role of the interferon-stimulated gene, (ISG), 15 in pregnancy detection. The anti-ISG15 Mab based ELISA was developed that could be used for detecting pregnancy at 18 to 20 days after artificial insemination (AI). The ISG15 protein was isolated from a pregnant buffalo and was amplified, and cloned in Escherichia coli by using coding region primers. The ISG15 gene was expressed in the host Escherichia coli BL21 (DE3), and the protocol was standardized for optimum gene expression. Using immortal hybridoma (fused myeloma and B cells) cells, a highly specific and sensitive antibody, anti-ISG15 mAb, for detecting ISG15 (protein) in the serum of pregnant buffaloes was obtained. A blocking ELISA was developed using the anti-ISG15 mAb to detect pregnancy in buffalo within 18 to 21 days after AI. The ISG15 gene was upregulated (P < 0.05) in pregnant buffalo at 18 to 21 days of pregnancy. This assay has an overall diagnostic accuracy of 75.0%. It, therefore, is concluded that recombinant ISG15 retains the potential for detecting pregnancy in B. bubalis and may have applications in ELISA kits for pregnancy detection in closely related species.

Identification of serum protein markers for early diagnosis of pregnancy in buffalo.

Improper or delayed pregnancy diagnosis has significant impact over animal production, particularly in buffaloes which inherently suffer from several reproductive inefficiencies. Thus the present study has undertaken to identify serum protein markers pertaining to early pregnancy diagnosis in buffaloes. Serum samples were collected from 10 pregnant Murrah Buffalo heifers at weekly intervals from days 0-35 post-artificial insemination and from 12 inseminated non-pregnant cyclic buffalo heifers on days 0, 7, 14 and 21. Two-dimensional gel electrophoresis and densitometric analysis revealed the presence of five protein spots showing average density fold change of ≥4 during early pregnancy. Mass spectrometry analysis identified these up-regulated proteins as anti-testosterone antibody light chain, apolipoprotein A-II precursor, serum amyloid A, cytokeratin type II, component IV isoform 1, which are have established roles in embryogenesis, but over-expression of the fifth identified protein immunoglobulin lambda light chain in pregnancy has been elucidated as a novel finding in the current study. Further, with bioinformatics analysis, potential antigenic B-cell epitopes were predicted for all these five proteins. An antibody cocktail-based approach involving antibodies against all these five up-regulated entire proteins or their epitopes could be developed for early detection of pregnancy in buffaloes. © 2016 Japanese Society of Animal Science.

Development and evaluation of loop-mediated isothermal amplification assay for rapid detection of Capripoxvirus.

AIM: The present study was undertaken to develop a nucleic acid-based diagnostic assay loop-mediated isothermal amplification assay (LAMP) targeting highly conserved genomic regions of Capripoxvirus (CaPVs) and its comparative evaluation with real-time polymerase chain reaction (PCR). MATERIAL AND METHODS: Lyophilized vaccine strain of sheeppox virus (SPPV) was used for optimization of LAMP assay. The LAMP assay was designed using envelope immunogenic protein (P32) coding gene targeting highly conserved genomic regions of CaPV responsible for causing sheep pox, goat pox, and lumpy skin disease in sheep, goat and cattle respectively. Serial tenfold dilution of SPPV recombinant plasmid DNA was used for a calculating limit of detection. Analytical sensitivity and specificity were performed. RESULTS: The test described is quick (30 min), sensitive and specific for detection of CaPVs. The described assay did not show any cross-reactivity to other related viruses that cause apparently similar clinical signs. It was found to be ten times more sensitive than conventional PCR however, 100 times less sensitive than quantitative PCR (qPCR). LAMP assay results were monitored by color change method using picogreen dye and agarose gel electrophoresis. CONCLUSION: LAMP assay can be a very good alternative for CaPV detection to other molecular techniques requiring sophisticated equipments.

Isolation and molecular characterization of contagious pustular dermatitis virus from Rajasthan, India.

This article describes the isolation and identification of contagious pustular dermatitis virus/orf virus (ORFV) from an outbreak of contagious pustular dermatitis (orf) in flocks of goats, in the north western region of India (Rajasthan). The virus was isolated in Vero cell cultures from scab and swab suspensions and has been identified using GIF/IL-2 and B2L gene specific primers in PCR and sequencing. The virus showed high nucleotide identity with previously reported Chinese, far eastern, Brazilian and Indian isolates. This report described the use of molecular tools for fast, reliable and confirmatory diagnosis of ORFV infection.