Seroprevalence and associated risk factors of leptospirosis in bovine dairy farms in Telangana state, India.

The current cross-sectional study aimed to determine the seroprevalence of Leptospira infection in bovine dairy farms in the Telangana state of India, as well as the associated risk factors, in order to implement effective preventive measures for disease control. A total of 469 blood samples were collected from 67 herds/farms in different areas, covering 20 administrative districts in the state. These samples consisted of 253 from cattle and 216 from buffaloes. Questionnaires were used to collect data on host and epidemiological factors. The collected sera were tested using the gold standard serological test, the Microscopic Agglutination Test (MAT), which employed a panel of 18 reference serovars for Leptospira exposure. The statistical analysis of epidemiological data was carried out to identify the risk factors associated with Leptospira exposure. The overall observed seroprevalence at the animal and farm levels was 41.4% and 77.6%, respectively. The most prevalent anti-leptospiral antibodies were observed against the serogroups Icterohaemorrhagiae (32.4%), Pomona (22.2%), Javanica (19.1%), Australis (17.0%), Bataviae (15.5%), Autumnalis (12.9%), Hebdomadis (12.9%), and others, in the total reacting samples. At the animal level, the significant risk factors associated with exposure to Leptospira species were breed (p = 0.03) and health status (p = 0.03). Furthermore, the multivariate statistical analysis of farm factors revealed that farm size (p = 0.05), presence of dogs (p = 0.04) and rodents (p = 0.01) on the farm, use of fodder from wet soils (p = 0.04), and proximity to water bodies (p = 0.04) were significantly associated with exposure to Leptospira in the studied region. This study provides the first report from India highlighting the important risk factors at the herd/farm and animal level associated with Leptospira infections in cattle and buffaloes. The findings contribute to strengthening the one-health strategy by facilitating the design and planning of appropriate control measures to alleviate the burden of leptospirosis in bovines.

Complete genome sequence of bluetongue virus serotype 9: implications for serotyping.

The second complete genome of bluetongue virus serotype 9 (BTV-9) is presented in this report. The sequence analysis points to continued circulation in India of a mixed topotype virus apparently belonging to the BTV-9 serotype, and it raises questions about approaches for serotyping bluetongue viruses.

Reverse transcription loop-mediated isothermal amplification assays for rapid identification of eastern and western strains of bluetongue virus in India.

Bluetongue virus (BTV) infects all ruminants, including cattle, goats and camelids, causing bluetongue disease (BT) that is often severe in naïve deer and sheep. Reverse-transcription-loop-mediated-isothermal-amplification (RT-LAMP) assays were developed to detect eastern or western topotype of BTV strains circulating in India. Each assay uses four primers recognizing six distinct sequences of BTV genome-segment 1 (Seg-1). The eastern (e)RT-LAMP and western (w)RT-LAMP assay detected BTV RNA in all positive isolates that were tested (n=52, including Indian BTV-1, -2, -3, -5, -9, -10, -16, -21 -23, and -24 strains) with high specificity and efficiency. The analytical sensitivity of the RT-LAMP assays is comparable to real-time RT-PCR, but higher than conventional RT-PCR. The accelerated eRT-LAMP and wRT-LAMP assays generated detectable levels of amplified DNA, down to 0.216 fg of BTV RNA template or 108 fg of BTV RNA template within 60-90min respectively. The assays gave negative results with RNA from foot-and-mouth-disease virus (FMDV), peste des petits ruminants virus (PPRV), or DNA from Capripox viruses and Orf virus (n=10), all of which can cause clinical signs similar to BT. Both RT-LAMP assays did not show any cross-reaction among themselves. The assays are rapid, easy to perform, could be adapted as a 'penside' test making them suitable for 'front-line' diagnosis, helping to identify and contain field outbreaks of BTV.

DNA barcoding and surveillance sampling strategies for Culicoides biting midges (Diptera: Ceratopogonidae) in southern India.

BACKGROUND: Culicoides spp. biting midges transmit bluetongue virus (BTV), the aetiological agent of bluetongue (BT), an economically important disease of ruminants. In southern India, hyperendemic outbreaks of BT exert high cost to subsistence farmers in the region, impacting on sheep production. Effective Culicoides spp. monitoring methods coupled with accurate species identification can accelerate responses for minimising BT outbreaks. Here, we assessed the utility of sampling methods and DNA barcoding for detection and identification of Culicoides spp. in southern India, in order to provide an informed basis for future monitoring of their populations in the region. METHODS: Culicoides spp. collected from Tamil Nadu and Karnataka were used to construct a framework for future morphological identification in surveillance, based on sequence comparison of the DNA barcode region of the mitochondrial cytochrome c oxidase I (COI) gene and achieving quality standards defined by the Barcode of Life initiative. Pairwise catches of Culicoides spp. were compared in diversity and abundance between green (570 nm) and ultraviolet (UV) (390 nm) light emitting diode (LED) suction traps at a single site in Chennai, Tamil Nadu over 20 nights of sampling in November 2013. RESULTS: DNA barcode sequences of Culicoides spp. were mostly congruent both with existing DNA barcode data from other countries and with morphological identification of major vector species. However, sequence differences symptomatic of cryptic species diversity were present in some groups which require further investigation. While the diversity of species collected by the UV LED Center for Disease Control (CDC) trap did not significantly vary from that collected by the green LED CDC trap, the UV CDC significantly outperformed the green LED CDC trap with regard to the number of Culicoides individuals collected. CONCLUSIONS: Morphological identification of the majority of potential vector species of Culicoides spp. samples within southern India appears relatively robust; however, potential cryptic species diversity was present in some groups requiring further investigation. The UV LED CDC trap is recommended for surveillance of Culicoides in southern India.

Segment-2 sequencing and cross-neutralization studies confirm existence of a neutralization resistant VP2 phenotypic variant of bluetongue virus serotype 1 in India.

Segment-2 (seg-2) of a bluetongue virus seropype-1 (BTV-1) isolate WGV104/08/Ind of Indian origin was sequenced and its neutralization behavior was studied to understand the antigenic similarity and relationship with other BTV-1 isolates. Multiple alignments of the coding region of seg-2 of WGV104/08/Ind revealed 97.6-99.0% and 97.2-98.4% similarity with other Indian BTV-1 isolates at nucleotide and deduced amino acid sequence level respectively. Several conservative and non-conservative substitutions were observed on the deduced VP2 amino acid sequence of WGV104/08/Ind. Non-conservative substitution of Lys119Glu on the B-cell epitope and Arg330Gly on the neutralizing epitope of VP2 of this isolate was observed. Using isolate-specific heterologous hyperimmune serum (HIS) the phenotypic antigenic relationship (r) was determined between WGV104/08/Ind and other Indian BTV-1 isolates which ranged from 0.092 to 0.208. The relationship score ranged from 0.203 to 0.295 when neutralization behavior of other Indian BTV-1 isolates was studied with the HIS of WGV104/08/Ind. Antigenic similarity (R) between WGV104/08/Ind and other Indian BTV-1 isolates was estimated from a reciprocal cross-neutralization study and ranged from 14.70% to 24.80% indicating existence of major subtype antigenic divergence and neutralization resistant behavior of WGV104/08/Ind.

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.

Genome Sequence of Bluetongue Virus Type 2 from India: Evidence for Reassortment between Outer Capsid Protein Genes.

Southern Indian isolate IND1994/01 of bluetongue virus serotype 2 (BTV-2), from the Orbivirus Reference Collection at the Pirbright Institute (http://www.reoviridae.org/dsRNA_virus_proteins/ReoID/btv-2.htm#IND1994/01), was sequenced. Its genome segment 6 (Seg-6) [encoding VP5(OCP2)] is identical to that of the Indian BTV-1 isolate (IND2003/05), while Seg-5 and Seg-9 are closely related to isolates from South Africa and the United States, respectively.

Deep sequencing as a method of typing bluetongue virus isolates.

Bluetongue (BT) is an economically important endemic disease of livestock in tropics and subtropics. In addition, its recent spread to temperate regions like North America and Northern Europe is of serious concern. Rapid serotyping and characterization of BT virus (BTV) is an essential step in the identification of origin of the virus and for controlling the disease. Serotyping of BTV is typically performed by serum neutralization, and of late by nucleotide sequencing. This report describes the near complete genome sequencing and typing of two isolates of BTV using Illumina next generation sequencing platform. Two of the BTV RNAs were multiplexed with ten other unknown samples. Viral RNA was isolated and fragmented, reverse transcribed, the cDNA ends were repaired and ligated with a multiplex oligo. The genome library was amplified using primers complementary to the ligated oligo and subjected to single and paired end sequencing. The raw reads were assembled using a de novo method and reference-based assembly was performed based on the contig data. Near complete sequences of all segments of BTV were obtained with more than 20× coverage, and single read sequencing method was sufficient to identify the genotype and serotype of the virus. The two viruses used in this study were typed as BTV-1 and BTV-9E.