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.

The study was conducted on 30 true acyclic Sahiwal cows (15 cows, ≥90 days postpartum; 15 postpubertal heifers, ≥30 months of age) and a similar 20 untreated controls (10 cows, 10 heifers). An 'Eazi' breed Controlled Internal Drug Release (CIDR) device (containing 1.38 g progesterone) was inserted intravaginally for 7 days (days 0 to 7) followed by 500 IU eCG i.m. at CIDR removal in all the treated animals. Heifers also received 5 mg oestradiol valerate i.m at CIDR insertion. The reproductive performance of these animals was recorded in terms of oestrus induction response, conception and pregnancy rates. Plasma progesterone (P 4 ) and oestradiol-17β (E 2 ) profiles of 4 representative animals from each treatment group before, during and after CIDR treatment were also monitored. An oestrus induction response of 100% was observed in treated cows and heifers. The majority of cows (53.3%) and heifers (60%) were induced to oestrus within 24-36 and 36-48 h, respectively after CIDR withdrawal; with mean intervals of 44±3.18 and 48±2.35 h, respectively. The conception rate at induced oestrus was higher in cows (40%) than heifers (20%). The final pregnancy rates after 2 subsequent oestruses were 80 and 60% in cows and heifers, respectively (overall 70% for all treated animals). In comparison, only 10% of control animals (2 cows only, 2/20) showed oestrus and become pregnant (10%) during theentire study period. The pretreatment (day 0) mean plasma P 4 levels were statistically (p>0.05) similar in cows and heifers (0.40±0.04 and 0.49±0.11 ng/ml, respectively). The peak P 4 levels were observed on day 1 in cows (13.94±1.41 ng/ml) and day 2 in heifers (19.15±3.30 ng/ml) with a progressive decline up to the day of CIDR withdrawal (3.35±0.92 and 8.79±1.71 ng/ml, respectively). Mean P 4 levels on day 9 and 10 in cows and heifers did not differ significantly from their respective day 0 values and the lowest values were recorded on day 10 both in cows and heifers (0.13±0.03 and 0.14±0.02 ng/ml, respectively). Wide variations in individual pretreatment E 2 levels were observed both in the cows (range = 4-26, mean = 13.00±4.65 pg/ml) and heifers (range = 10-14, mean = 11.50±0.96 pg/ml). Thereafter also, E 2 levels in cows showed variation and reached a peak level (53.50±2.99 pg/ml) on day 8. In heifers, peak mean E 2 level (111.25±39.81 pg/ml) was recorded on day 1, followed by a non-significant decline on day 2, a significant fall on day 6 and a non-significant increase on day 9 and 10. However, mean E 2 levels on days 7 (p<0.05), 8 and 9 (p<0.01) were significantly higher in cows compared to heifers. The post-CIDR withdrawal mean highest P 4 and lowest E 2 levels coincided with the period when the majority of animals were induced to oestrus. CIDR and eCG treatment resulted in effective induction of oestrus with satisfactory pregnancy rates in true acyclic Sahiwal cows and heifers.

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.

Interferon-tau (IFN-τ)-induced molecular markers such as ubiquitin-like modifier (ISG15), 2',5'-oligoadenylate synthetase 1 (OAS1) and myxovirus resistance genes (MX1 and MX2) have generated immense attention towards developing diagnostic tools for early diagnosis of pregnancy in bovine. These molecules are expressed at transcriptional level in peripheral nucleated cells. However, their presence in the serum is still a question mark. This study reports sequential changes in expression of MX2 transcript in whole blood and serum MX2 protein level on days 0, 7, 14, 21, 28 and 35 in pregnant (n = 9) buffalo heifers, and on days 0, 7 and 14 in non-inseminated (n = 8) and inseminated non-pregnant (n = 10) control animals. In non-inseminated and inseminated non-pregnant heifers, the differential expression of MX2 transcript and MX2 protein level remained similar between day 7 and 14 post-oestrus. However, in pregnant heifers, on 14th and 28th day post-insemination MX2 transcript was 16.38 ± 1.57 and 28.16 ± 1.91 times upregulated as compared to day 0. Similarly, serum MX2 protein concentration followed analogous trend as MX2 transcript and increased gradually with the progression of pregnancy. Correlation analysis between expression of MX2 transcript and its serum protein level showed a significant positive correlation in pregnant animals, while it was random in other two groups. Therefore, MX2 surge at transcriptional and serum protein level after day 14-28 of pregnancy in buffalo holds potential for its use in early pregnancy detection.

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.

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Interferon stimulated protein 15 is a well-known ubiquitin cross-reactive protein which is released from the ruminant uterus in response to interferon-tau (IFN-t) during conceptus implantation. This protein increases significantly during the early stages of pregnancy. Therefore, in the present study, Interferon stimulated gene (ISG15) coding region was isolated from buffalo blood at day 18 of pregnancy after artificial insemination. ISG15 was cloned in pET100 vector, overexpressed in E.coli and purified to investigate its properties. The protein was immunoblotted with mouse anti-His antibody to evaluate its characteristics after purification. To conclude, elucidation of protein properties in the prokaryotic system will provide an opportunity for understanding the basic biology of pre and post-implantation and development of a tool for early pregnancy diagnosis.