Sushila Maan

Bluetongue virus, other orbiviruses and other reoviruses: Their relationships and taxonomy

Individual reoviruses can infect a wide variety of terrestrial and non-terrestrial vertebrates, terrestrial and non-terrestrial invertebrates, plants, and fungi. Bluetongue virus (BTV), which has been studied in the greatest depth and is the “type” species of the genus Orbivirus, includes 24 distinct serotypes, as well as many different topotypes (groups reflecting the geographical origins of each virus isolate) and nucleotypes (lineages), although these have no “formal” taxonomic status within the virus species itself. Clear overall similarities exist in the structure and replication strategies of the different reoviruses, which are reflected in the phylogenetic relationships of their genome segments. These relationships suggest that all of the reoviruses have evolved from a single common ancestor, in parallel with their different hosts (possibly originally from a marine species), a process known as co-speciation. Subsequent gene deletions or duplications, followed by gene rearrangements, have generated changes in both the number of genome segments and the proteins they encode. Reovirus particles are composed of up to three concentric shells of virus structural proteins (depending on the genus to which they belong) surrounding the segments of the virus genome. Although there are similarities in the innermost sub-core shell structure and protein (the T2 protein), the structure of the outer capsid and outer core layers varies significantly between members of different genera within the family.

Genome Sequence of a Reassortant Strain of Bluetongue Virus Serotype 23 from Western India

ABSTRACT The full genome sequence (19,177 bp) of an Indian strain (IND1988/02) of bluetongue virus (BTV) serotype 23 was determined. This virus was isolated from a sheep that had been killed during a severe bluetongue outbreak that occurred in Rahuri, Maharashtra State, western India, in 1988. Phylogenetic analyses of these data demonstrate that most of the genome segments from IND1988/02 belong to the major “eastern” BTV topotype. However, genome segment 5 belongs to the major “western” BTV topotype, demonstrating that IND1988/02 is a reassortant. This may help to explain the increased virulence that was seen during this outbreak in 1988. Genome segment 5 of IND1988/02 shows >99% sequence identity with some other BTV isolates from India (e.g., BTV-3 IND2003/08), providing further evidence of the existence and circulation of reassortant strains on the subcontinent.

Concurrent infection of bluetongue and peste-des-petits-ruminants virus in small ruminants in Haryana State of India

Bluetongue (BT) and peste-des-petits-ruminants (PPR) are major transboundary diseases of small ruminant, which are endemic in India. Testing of bluetongue virus (BTV) and peste-des-petits-ruminants virus (PPRV) from recent outbreaks (2015-2016) in different regions of Haryana State of India revealed that 27.5% of the samples showed the presence of dual infection of BTV and PPRV. Analysis of Seg-2 of BTV (the serotype-determining protein) showed the presence of BTV-12w in several isolates. However, analysis of N gene fragment amplicons showed that viruses belong to lineage IV were most closely related to a pathogenic strain of PPRV from Delhi. This is the first report of co-circulation of PPRV lineage IV and bluetongue virus serotype 12 in the state.

Molecular Cloning and Characterization of Mx2 for Early Pregnancy Diagnosis in Bubalus bubalis

India is the largest milk producing country in the world. This major achievement is only possible due to the presence of one of the species Bubalus bubalis (Buffalo). Early pregnancy diagnosis in buffalo is one of the major problems related to bovine reproduction system due to silent heat in this animal. Therefore, in the present study, one of the conceptus derived protein myxovirus resistance 2 (Mx2) released during 18–21xa0days of pregnancy was analyzed by conventional PCR, qPCR and characterized for its application as a biomarker for early pregnancy diagnosis in buffalo. Conventional PCR has shown remarkable amplification in the pregnant animal. Temporal expression of Mx2 revealed its significant increase (Pu2009<u20090.05) in transcription level during 18–21xa0days of pregnancy. 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 protein which are responsible for its interaction with other proteins. Physiochemical properties predicted the Mx2 protein nature during in vitro conditions which are required for any of the assays development. However, immunogenic studies revealed that Mx2 is strongly antigenic in nature and can be used for antibody production. In conclusion, Mx2 protein expression from buffalo may be a good indicator of conceptus implantation and can be used in early pregnancy diagnostic kits.

Bluetongue: Aetiology, Epidemiology, Pathogenesis, Diagnosis and Control

Bluetongue (BT) is an emerging and re-emerging vector-borne viral disease of domestic and wild ruminants, caused by viruses classified within the species bluetongue virus (BTV), genus Orbivirus, family Reoviridae. There are 27 recognized serotypes of BTV (with two more recently discovered ‘putative’ serotypes) as well as multiple geographic variants (topotypes) and many different strains and genotypes, most of which are transmitted between their vertebrate hosts by certain ‘vector-competent’ biting midges of the genus Culicoides. Bluetongue is an economically important transboundary disease that is listed by the World Organisation for Animal Health, reflecting the ability of BTV to (a) infect all ruminants, including important domesticated species; (b) cause severe disease, with high fatality rates in sheep and certain species of deer; and (c) cause large economic losses due to fatalities, reduced productivity and reproductive performance, animal movement and trade restrictions and surveillance and control strategies (including vaccination). The plurality of BTV serotypes and strains, the involvement of multiple host and vector species and the potential for (re)introduction of exotic BTV strains make the control and eradication of BT very complex and difficult to achieve. In this chapter, we review the current understanding of BTV biology, bluetongue epidemiology, pathogenesis and pathology, laboratory techniques to diagnose the disease and identify the virus, experimental animal models to study the disease and to evaluate vaccines and methods for the control and/or eradication of bluetongue.

Bluetongue virus – 23 stimulates inducible nitric oxide synthase expression and nitric oxide production in mononuclear cells of blood and/or regional lymphoid organs

Mononuclear leukocytes of peripheral blood mononuclear cells (PBMCs) and regional lymphoid organs (RLOs) play a critical role in primary BTV replication and subsequent viral dissemination to distant systemic organs. The lesions in animals develop primarily as a result of vascular insult, presumably induced by the activity of viral and/or proinflammatory vasoactive mediators. Hence, the current study was designed in sheep to investigate the responses of potent vasoactivators, inducible nitric oxide synthase (iNOS) and/or nitric oxide (NO) in mononuclear leukocytes of PBMCs and RLOs. The results show that BTV infection of sheep led to enhanced transcription of iNOS in PBMCs and in particular RLOs. The BTV RNAs and/or antigens were readily demonstrable in these mononuclear leukocytes, suggesting the possible role of BTV in iNOS induction. Moreover, upon in vitro infection of PBMCs with BTV-23, iNOS was up-regulated in time-dependent fashion and correlated with increased NO production. The results from these in vivo and in vitro studies thus suggest iNOS and NO produced by mononuclear leukocytes may potentially contribute to vascular-related pathology of BT.

Novel Molecular Diagnostics and Therapeutic Tools for Livestock Diseases

Recent novelties in diverse diagnostics and therapeutic tools in animal health sector have paved a brighter and clearer way ahead. These are proved to be better in detection, management, control and eradication of animal sufferings caused by various infectious and non-infectious diseases. These innovations have potential impact that extends beyond the animal health and welfare. The advancements have significantly contributed towards improvement in the economy of the country as well as food security. In the present competitive era of evolution, the organisms have inculcated a number of new strategies for survival and spread. Therefore, science needs to continuously evolve more sensitive, specific and high-throughput tools to overcome pathogen cleverness to escape from host immune surveillance. For visible or remarkable changes, it is necessary to use full potential of these advanced molecular techniques into current animal health standards and practices. Under ‘One Health’ concept, the health of animals and humans has to be taken care simultaneously. At present, these advanced molecular diagnostic methods play a significant role in the detection of new and emerging pathogens of livestock. The acquired information also helps to study the interrelationships of pathogens, their hosts and their surroundings. Additionally new vaccines bridging human and animal health development may be discovered. Latest developments in the field of diagnostics and vaccine design through genomics approach have also laid the foundation to enhance the diagnosis and surveillance and in turn helped in the control of infectious diseases. Latest high-throughput DNA sequencing platforms are currently being used for identification and detailed analysis of both disease pathogen and host genomes. The high-throughput data generated using these platforms need to be analysed adopting the bioinformatics and computational genomics that have taken a very high pace nowadays. In the context of animal health, the data analysis may provide some key opportunities for the development of better diagnostic and therapeutic tools for emerging or re-emerging diseases. Such novel and potent technologies put forward a significantly new scenario of disease knowledge, which enables more accurate predictions leading to faster and greater management responses to combat potentially devastating disease crises.

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 (Pu2009<u2009 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.

Comparative evaluation of polymerase chain reaction assay with microscopy for detection of asymptomatic carrier state of theileriosis in a herd of crossbred cattle

Aim: This study aims to develop and to standardize a polymerase chain reaction (PCR) assay that will diagnose clinical as well as carrier state of the disease and to compare the results with conventional microscopy technique. Materials and Methods: A herd of crossbred cattle with the previous history of theileriosis in village Lahli, district Rohtak, Haryana, was selected for this study. A total of 29 blood samples were collected randomly from cows including five clinically ill cattle. Blood smears from all animals and lymph node biopsy smears from animal with swollen lymph nodes were examined microscopically after conventional Giemsa staining. Phenol chloroform isoamyl alcohol method was used for extracting DNA from blood. Previously published primers targeting cytochrome b gene sequence of Theileria annulata were used in the PCR assay that was standardized to use in the laboratory. Results: Out of 29 samples tested,18 (62.06%) were found positive for theileriosis by PCR assay, whereas only 10 (34.48%) samples were detected positive by conventional microscopic technique using Giemsa staining method. Conclusions: On the basis results of comparative studies, it can be concluded that PCR assay is a more sensitive than microscopic examination for detection of theileriosis. This can be attributed to the ability of PCR assay to detect small amounts of genomic DNA of T. annulata or low parasitemia in cows. Therefore, PCR assay can serve as a more sensitive tool to detect Theileria for detection of theileriosis even in asymptomatic carrier cattle which is important for the implementation of successful control programs.

Isolation and Characterization of Mannheimia varigena from a Murrah Buffalo

The present study was aimed to isolate and characterize phenotypically as well as genotypically bacterial strain from tracheal froth of a Murrah buffalo on the Government livestock farm (GLF) located at Hisar, Haryana. The isolate showed the cultural, morphological and biochemical properties of Mannheimia haemolytica as per Bergey’s manual of determinative bacteriology and as confirmed by GENIII microplate (biolog). However, the isolate was further confirmed as Mannheimia varigena by 16S rRNA sequencing.