Molecular characterization of OAS1 as a biomarker molecule for early pregnancy diagnosis in Bubalus bubalis

97-107OAS1 (2′-5′-oligoadenylate synthetase) is a type I interferon molecule which is responsible for antiviral activity and known for its action in innate immunity. It was also reported to be stimulated by the release of interferon tau (IFN-t) in the serum of pregnant animals during conceptus implantation. OAS1 is studied in most of the ruminants except for buffalo (Bubalus bubalis), which is well known for high milk production. Therefore, in the present study OAS1 mRNA was isolated from pregnant buffalo (B. bubalis) at 18-21 day of pregnancy. The ORF region of OAS1 was amplified and sequenced for characterization of this protein. In silico analysis of protein was done in order to find the suitability of protein as a candidate molecule for early pregnancy diagnostic kits. The functional characterization identifies various motifs present in the protein which are responsible for its interaction with other proteins. Physiochemical properties predict the protein nature during in vitro conditions. Further, immunogenic studies revealed OAS1 is highly antigenic nature and its suitability for use in vivo conditions. In conclusion, OAS1 protein expression in buffalo is a good indicator of conceptus implantation and has suitable properties for being used in early and precise pregnancy diagnostic kits

High throughput Luminex beads based multiplex assay for identification of six major bacterial pathogens of mastitis in dairy animals

IntroductionBovine mastitis is caused by over 150 different microorganisms. Specific identification and quantification of multiple bacteria in a single milk sample becomes essential for rapid intervention.MethodsIn the present study a Luminex beads based multiplex assay emphasizing on the precise identification of six major bacterial pathogens of mastitis was developed. Assay was developed in two triplex sets, triplex 1 comprised of Streptococcus agalactiae, Streptococcus dysgalactiae and Streptococcus uberis while triplex 2 consisted of Staphylococcus aureus, E. coli and Klebsiella pneumoniae.ResultsThe analytical sensitivity was 10 6 copies per reaction mixture for all the six bacteria. A 100% analytical specificity was observed for simultaneous detection of these bacteria. Clinical milk samples from 100 bovine quarters were tested for validation.DiscussionThe analytical sensitivity was similar to the findings reported earlier in real time PCR multiplex assay targeting the DNA of the 11 most common bacterial species or groups in mastitis. The analytical specificity of the optimized assay was 100% similar to reported earlier for simultaneous detection of Mycoplasma spp. and for seven entric viruses of humans.The developed assay indicates a concept proof of a rapid, cost effective high throughput diagnostic tool for identification of major bacteria causing mastitis

Full-genome sequencing as a basis for molecular epidemiology studies of bluetongue virus in India

Since 1998 there have been significant changes in the global distribution of bluetongue virus (BTV). Ten previously exotic BTV serotypes have been detected in Europe, causing severe disease outbreaks in naïve ruminant populations. Previously exotic BTV serotypes were also identified in the USA, Israel, Australia and India. BTV is transmitted by biting midges (Culicoides spp.) and changes in the distribution of vector species, climate change, increased international travel and trade are thought to have contributed to these events. Thirteen BTV serotypes have been isolated in India since first reports of the disease in the country during 1964. Efficient methods for preparation of viral dsRNA and cDNA synthesis, have facilitated full-genome sequencing of BTV strains from the region. These studies introduce a new approach for BTV characterization, based on full-genome sequencing and phylogenetic analyses, facilitating the identification of BTV serotype, topotype and reassortant strains. Phylogenetic analyses show that most of the equivalent genome-segments of Indian BTV strains are closely related, clustering within a major eastern BTV ‘topotype’. However, genome-segment 5 (Seg-5) encoding NS1, from multiple post 1982 Indian isolates, originated from a western BTV topotype. All ten genome-segments of BTV-2 isolates (IND2003/01, IND2003/02 and IND2003/03) are closely related (>99% identity) to a South African BTV-2 vaccine-strain (western topotype). Similarly BTV-10 isolates (IND2003/06; IND2005/04) show >99% identity in all genome segments, to the prototype BTV-10 (CA-8) strain from the USA. These data suggest repeated introductions of western BTV field and/or vaccine-strains into India, potentially linked to animal or vector-insect movements, or unauthorised use of ‘live’ South African or American BTV-vaccines in the country. The data presented will help improve nucleic acid based diagnostics for Indian serotypes/topotypes, as part of control strategies

Development and evaluation of real time RT-PCR assays for detection and typing of Bluetongue virus

Bluetongue virus is the type species of the genus Orbivirus, family Reoviridae. Bluetongue viruses (BTV) are transmitted between their vertebrate hosts primarily by biting midges (Culicoides spp.) in which they also replicate. Consequently BTV distribution is dependent on the activity, geographic distribution, and seasonal abundance of Culicoides spp. The virus can also be transmitted vertically in vertebrate hosts, and some strains/serotypes can be transmitted horizontally in the absence of insect vectors. The BTV genome is composed of ten linear segments of double-stranded (ds) RNA, numbered in order of decreasing size (Seg-1 to Seg-10). Genome segment 2 (Seg-2) encodes outer-capsid protein VP2, the most variable BTV protein and the primary target for neutralising antibodies. Consequently VP2 (and Seg-2) determine the identity of the twenty seven serotypes and two additional putative BTV serotypes that have been recognised so far. Current BTV vaccines are serotype specific and typing of outbreak strains is required in order to deploy appropriate vaccines. We report development and evaluation of multiple ‘TaqMan’ fluorescence-probe based quantitative real-time type-specific RT-PCR assays targeting Seg-2 of the 27+1 BTV types. The assays were evaluated using orbivirus isolates from the ‘Orbivirus Reference Collection’ (ORC) held at The Pirbright Institute. The assays are BTV-type specific and can be used for rapid, sensitive and reliable detection / identification (typing) of BTV RNA from samples of infected blood, tissues, homogenised Culicoides, or tissue culture supernatants. None of the assays amplified cDNAs from closely related but heterologous orbiviruses, or from uninfected host animals or cell cultures

Detection of Leptospira in urine of apparently healthy dogs by quantitative polymerase chain reaction in Haryana, India

Background and Aim: Leptospirosis is a zoonotic disease caused by bacteria of the genus Leptospira. The organism can spread through the urine of infected animals, which can get into water or soil and can survive there for weeks to months. The study was undertaken to detect the pathogenic Leptospira in healthy dogs' urinary shedding by real-time polymerase chain reaction (qPCR). Materials and Methods: Leptospirosis is a zoonotic disease caused by bacteria of the genus Leptospira. To detect the pathogenic Leptospira organisms in dogs' urinary shedding, 239 urine samples were collected from healthy dogs from April 2018 to March 2019 from different areas of Haryana. All the urine samples were processed for DNA extraction and qPCR technique was used to detect the presence of Leptospira. Results: Out of 239 urine samples of dogs, none of the samples resulted in the detection of DNA of pathogenic Leptospira organisms. Conclusion: The present study indicated low risk of transmission of Leptospira organisms from dogs' urine to human beings in Haryana

Genomic diversity among eastern and western topotypes of bluetongue virus serotype 16 based on whole genome sequence analysis

Bluetongue (BT) is a noncontagious, vector-borne disease of domestic and wild ruminants. The causative agent bluetonguevirus (BTV) is a double stranded RNAvirus belonging to genus Orbivirus within family Reoviridae. Eradication of BTVfromendemic regions like India is not an easy task due to the widely distributed Culicoides spp. midge vectors, the ubiquitousdistribution of vertebrate hosts and existence of a large number of serotypes of the virus (at least 26 till date). The completegenomes (19,193 base-pairs) of several strains of bluetongue virus serotype 16 (BTV-16) originated from Australia, China,Indian subcontinent, Mediterranean basin, Middle East, Africa (Nigeria) and Europe, were compared. These analysis showedthat all ten genome segments of a Nigerian strain are derived from a western lineage, showing only 77% - 84% nt identity withthe eastern topotype reference strain 'RSArrrr/16' (and its derivative 'RSAvvvv/16', a vaccine strain) that was originallyisolated in Pakistan, 76.4% - 83% with eastern BTV-16 strain from Australia (DPP96) and 77% - 89% with a reassortant strainfrom India. These detailed comparisons involving global strains showed that there is a very high degree of variation (up to24%) between BTV-16 strains from eastern and western geographical regions. These data confirm the value of whole genomesequencing for characterization of novel BTV isolates and has helped to identify representative suitable 'reference-strain' ofeastern topotype (BTV-16e), western topotype (BTV-16w), as well as 'cross-topotype' reassortant strain (BTV-16r) that aregenerated in the field for further serological, phylogenetic and molecular epidemiology studies

Veterinary World, EISSN: 2231-0916 Available at www.veterinaryworld.org/Vol.6/Dec-2013/3.pdf REVIEW ARTICLE Open Access

Genomic diversity among eastern and western topotypes of bluetongue virus serotype 16 based on whole genome sequence analysi

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

Not Available

Not AvailableTo unravel equid trypanosomosis caused by Trypanosoma evansi in Punjab state of India, a cross sectional study was designed by utilizing parasitological and sero-molecular tools with objective to assess the prevalence of T. evansi in association with various risk factors in all agroclimatic zones of Punjab state of India. Parasitological Romanowksy stained thin blood smears (RSTBS) to detect patent infection, molecular techniques polymerase chain reaction I (PCR I; TBR 1/2 primers; targeting minichromosomal satellite DNA of T. evansi), polymerase chain reaction II (PCR II; TR 3/4 primers; targeting variable surface glycoprotein region DNA of T. evansi) & LAMP (Loop mediated isothermal amplification) assay to detect latent infection and serological assays card agglutination test (CATT/T. evansi) & ELISA (Enzyme linked immunosorbent assay) to detect exposure status of trypanosomosis were utilized in the present study. A total 429 equid blood and serum samples from all the five agroclimatic zones of Punjab state tested by these techniques showed a prevalence of 1.39% (CL: 0-15.28) by RSTBS, 6.52% (10.94-45.09) by both TBR 1/2 PCR and LAMP assay, 5.82% (11.57-38.42) by TR 3/4 PCR, 15.15% (36.57-135.42) with CATT/T. evansi and 22.84% (17.77-840.22) with ELISA. Interpretation of various risk factors revealed that the donkey/mules population (RR = 5.46, 95% [CI] = 0.15-15.56) was found to be at higher risk of T. evansi infection predominantly at 'unorganized' farms (RR = 4.06, 95% [CI] = 0.12-4.51). Animal used for commercial purposes (RR = 3.25, 95% [CI] = 0.06-7.42), rearing of equids with other domestic animals (RR = 2.36, 95% [CI] = 0.10-17.11) and farms without application of fly repellant/insecticides/net (RR = 3.68, 95% [CI] = 0.08-5.94) made them more prone to the disease. This comprehensive report utilizing the classical, serological and molecular diagnostic tools for epidemiology of T. evansi establishes the endemic stability of this infection in all agro climatic zones of Punjab with LAMP assay to be a promisingly sensitive and specific technique for the diagnosis of T. evansi under isothermal conditions in field situations.Not Availabl

Not Available

Not AvailableTo unravel equid trypanosomosis caused by Trypanosoma evansi in Punjab state of India, a cross sectional study was designed by utilizing parasitological and sero-molecular tools with objective to assess the prevalence of T. evansi in association with various risk factors in all agroclimatic zones of Punjab state of India. Parasitological Romanowksy stained thin blood smears (RSTBS) to detect patent infection, molecular techniques polymerase chain reaction I (PCR I; TBR 1/2 primers; targeting minichromosomal satellite DNA of T. evansi), polymerase chain reaction II (PCR II; TR 3/4 primers; targeting variable surface glycoprotein region DNA of T. evansi) & LAMP (Loop mediated isothermal amplification) assay to detect latent infection and serological assays card agglutination test (CATT/T. evansi) & ELISA (Enzyme linked immunosorbent assay) to detect exposure status of trypanosomosis were utilized in the present study. A total 429 equid blood and serum samples from all the five agroclimatic zones of Punjabstate tested by these techniques showed a prevalence of 1.39 % (CL: 0-15.28) by RSTBS, 6.52% (10.94-45.09) by both TBR 1/2 PCR and LAMP assay, 5.82% (11.57-38.42) by TR 3/4 PCR, 15.15 % (36.57-135.42) with CATT/T. evansi and 22.84 % (17.77-840.22) with ELISA. Interpretation of various risk factors revealed that the donkey/mules population (RR= 5.46, 95% [CI] = 0.15-15.56) was found to be at higher risk of T. evansi infection predominantly at ‘unorganized’ farms (RR =4.06, 95% [CI] = 0.12-4.51). Animal used for commercial purposes (RR = 3.25, 95% [CI] =0.06-7.42), rearing of equids with other domestic animals (RR =2.36, 95% [CI] =0.10-17.11) and farms without application of fly repellant/insecticides/net (RR =3.68, 95% [CI] =0.08-5.94) made them more prone to the disease. This comprehensive report utilizing the classical, serological and molecular diagnostic tools for epidemiology of T. evansi establishes the endemic stability of this infection in all agro climatic zones of Punjab with LAMP assay to be a promisingly sensitive and specific technique for the diagnosis of T. evansi under isothermal conditions in field situationsNot Availabl