Raj K Singh

National Research Centre on Equines, Hissār, Haryana, India

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Publications (22)41.19 Total impact

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    ABSTRACT: Virulence-associated plasmid (Vap) genes of clinical isolates of Rhodococcus equi were characterized. Isolates were identified by 16S rRNA, choE and traA gene PCR, followed by amplification, cloning and sequencing of 7 Vap genes. The isolates were found positive for vapA gene family. The comparative sequence analysis of vap genes revealed 99-100% similarity at both nt and aa levels with all sequences. The aa sequences of the predicted Vap proteins exhibited a high degree of similarity to each other, especially at the carboxy terminal ends. Invariable point mutations were observed in Vap proteins. The changes did not cause alteration in hydropathicity and secondary structures in VapA & H proteins. Few major changes in polarity and structures were observed in VapD, E and G proteins. Phylogenetic analysis revealed that all Vap genes followed similar branching pattern except isolate from Bahadurgarh (BBG163). We conclude that VapA is highly conserved and plays a major role in pathogenesis. This is the first report of sequence analysis and phylogenetic studies of Vap gene family of clinical virulent isolates of R. equi from India. Introduction Rhodococcus equi has been reported to infect a number of domestic animals and also as zoonoses in immunocompromised man
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    ABSTRACT: Objective. To express truncated TssB protein of Burkholderia mallei and to evaluate its diagnostic efficacy for serological detection of glanders among equines. Materials and Methods. In an attempt to develop recombinant protein based enzyme-linked immunosorbent assay (ELISA), N-terminal 200 amino acid sequences of B. mallei TssB protein-a type 6 secretory effector protein-were expressed in prokaryotic expression system. Diagnostic potential of recombinant TssB protein was evaluated in indirect ELISA using a panel of glanders positive (n = 49), negative (n = 30), and field serum samples (n = 1811). Cross-reactivity of the assay was assessed with equine disease control serum and human melioidosis positive serum. Results. In comparison to CFT, diagnostic sensitivity and specificity of ELISA were 99.7% and 100%, respectively. Conclusions. The indirect ELISA method using the truncated TssB offered safer and more rapid and efficient means of serodiagnosis of glanders in equines. These data highlight the use of TssB as potential diagnostic antigen for serological diagnosis of glanders.
    The Scientific World Journal 02/2014; 2014:469407. DOI:10.1155/2014/469407 · 1.73 Impact Factor
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    Emerging Infectious Diseases 02/2014; 20(2):324-6. DOI:10.3201/eid2002.130358 · 7.33 Impact Factor
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    ABSTRACT: Thermostabilizing effect of heavy water (D2O) or deuterium oxide has been demonstrated previously on several enzymes and vaccines like oral poliovirus vaccine and influenza virus vaccine. In view of the above observations, effect of heavy water on in situ thermostabilization of recombinant p26 protein on enzyme-linked immunosorbent assay (ELISA) for serodiagnosis of equine infectious anemia virus (EIAV) infection was investigated in the present study. The carbonate-bicarbonate coating buffer was prepared in 60% and 80% D2O for coating the p26 protein in 96-well ELISA plate and thermal stability was examined at 4°C, 37°C, 42°C, and 45°C over a storage time from 2 weeks to 10 months. A set of positive serum (n = 12) consisting of strong, medium, and weak titer strength (4 samples in each category) and negative serum (n = 30) were assessed in ELISA during the study period. At each time point, ELISA results were compared with fresh plate to assess thermal protective effect of D2O. Gradual increase in the stabilizing effect of 80% D2O at elevated temperature (37°C < 42°C < 45°C) was observed. The 80% D2O provides the thermal protection to rp26 protein in ELISA plate up to 2 months of incubation at 45°C. The findings of the present study have the future implication of adopting cost effective strategies for generating more heat tolerable ELISA reagents with extended shelf life.
    The Scientific World Journal 01/2014; 2014:620906. DOI:10.1155/2014/620906 · 1.73 Impact Factor
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    ABSTRACT: Equine infectious anemia (EIA) is a retroviral infection of horses. Horses infected by EIA virus (EIAV) become inapparent carriers that remain asymptomatic for the remainder of their life span and serve as infection source to other horses. In this study, agar gel immunodiffusion test and ELISA were used to investigate the presence of antibodies to EIAV in equines. A total of 67,042 equine serum samples from 19 states and two union territories were tested during April 1999 to September 2012. The results revealed that none of the animals were positive for antibodies to EIAV from 1999 to December 2009. However, two EIAV sero-positive cases one each from indigenous and thoroughbred equines were detected in 2010 and 2012, respectively. Occurrence of EIA after a long gap of 11 years is indicative of reemergence of EIA in India which warrants concerted efforts in nationwide surveillance and monitoring for detection and elimination of EIAV carrier animals to prevent EIA outbreak.
    Indian Journal of Virology 12/2013; 24(3). DOI:10.1007/s13337-013-0142-3 · 0.36 Impact Factor
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    ABSTRACT: Equine infectious anemia (EIA)-a retroviral disease caused by equine infectious anemia virus (EIAV)-is a chronic, debilitating disease of horses, mules, and donkeys. EIAV infection has been reported worldwide and is recognized as pathogen of significant economic importance to the horse industry. This disease falls under regulatory control program in many countries including India. Control of EIA is based on identification of inapparent carriers by detection of antibodies to EIAV in serologic tests and "Stamping Out" policy. The current internationally accepted test for diagnosis of EIA is the agar gel immune-diffusion test (AGID), which detects antibodies to the major gag gene (p26) product. The objective of this study was to develop recombinant p26 based in-house immunoassays [enzyme linked immunosorbent assays (ELISA), and AGID] for EIA diagnosis. The synthetic p26 gene of EIAV was expressed in Escherichia coli and diagnostic potential of recombinant p26 protein were evaluated in ELISA and AGID on 7,150 and 1,200 equine serum samples, respectively, and compared with commercial standard AGID kit. The relative sensitivity and specificity of the newly developed ELISA were 100 and 98.6 %, respectively. Whereas, relative sensitivity and specificity of the newly developed AGID were in complete agreement in respect to commercial AGID kit. Here, we have reported the validation of an ELISA and AGID on large number of equine serum samples using recombinant p26 protein produced from synthetic gene which does not require handling of pathogenic EIAV. Since the indigenously developed reagents would be economical than commercial diagnostic kit, the rp26 based-immunoassays could be adopted for the sero-diagnosis and control of EIA in India.
    Indian Journal of Virology 12/2013; 24(3):349-56. DOI:10.1007/s13337-013-0149-9 · 0.36 Impact Factor
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    ABSTRACT: Rapid climatic shifts across the last glacial to Holocene transition are pervasive feature of the North Atlantic as well as low latitude proxy archives. Our decadal to centennial scale record of summer monsoon proxy Globigerina bulloides from rapidly accumulating sediments from Hole 723A, Arabian Sea shows two distinct intervals of weak summer monsoon wind coinciding with cold periods within Ållerød inerstadial of the North Atlantic named here as IACP-A1 and IACP-A2 and dated (within dating uncertainties) at 13.5 and 13.3 calibrated kilo years before the present (cal kyr BP), respectively. Spectral analysis of the Globigerina bulloides time series for the segment 13.6-13.1 kyr (Ållerød period) reveals a strong solar 208-year cycle also known as de Vries or Suess cycle, suggesting that the centennial scale variability in Indian summer monsoon winds during the Ållerød inerstadial was driven by changes in the solar irradiance through stratospheric-tropospheric interactions.
    Scientific Reports 09/2013; 3:2753. DOI:10.1038/srep02753 · 5.08 Impact Factor
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    ABSTRACT: Equine piroplasmosis is a tick-transmitted protozoan disease caused by Theileria equi and/or Babesia caballi. In the present study, we expressed a 53kDa protein from the truncated EMA-2 gene of T. equi (Indian strain) and developed EMA-2ELISA using this expressed protein. This ELISA is able to detect T. equi-specific antibodies in experimentally infected animals as early as 9 days post-infection. The assay developed was validated with the OIE recommended competitive ELISA (cELISA) on 120 serum samples and significant agreement (kappa=0.93) was observed between results of both the ELISAs which indicates suitability of EMA-2ELISA for use in sero-diagnosis. Diagnostic sensitivity and specificity of EMA-2ELISA - as compared with cELISA - were 0.97 and 0.96, respectively. Analysis of 5651 equine serum samples - collected during 2007-2012 from 12 states of India representing eight agro-climatic zones - by EMA-2ELISA revealed 32.65% seroprevalence of T. equi in India. In conclusion, the EMA-2ELISA developed using the T. equi EMA-2 recombinant protein as antigen for detecting T. equi-specific antibodies has good diagnostic potential for sero-epidemiological surveys.
    Veterinary Parasitology 09/2013; DOI:10.1016/j.vetpar.2013.08.030 · 2.55 Impact Factor
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    ABSTRACT: Toll-like receptor 9 (TLR9) has been characterized as a receptor that recognizes unmethylated CpG motif and triggers a pro-inflammatory cytokine response that influences both innate and adaptive immunity. Buffalo is an economically important livestock species in many Asian and Mediterranean countries, but there is little information available on its TLR9 structure and response to stimulation with its agonist CpG-ODNs. Hence in this study, we report the analysis of newly sequenced buffalo TLR9 gene fragment. In this study, buffalo TLR9 amino acid sequence revealed close association of TLR9 proteins within other bovines and small ruminants; but high divergence from other species.Multiple alignment of deduced amino acid sequence of Bubalus bubalis TLR9 with other species showed that 156/201 (74.28%) amino acids were conserved in all species. Leucine rich repeat (LRR) motifs in the ectodomain of TLR9 are responsible for molecular recognition of its agonist. The LRR pattern of Bubalus bubalis TLR9 protein was predicted towards N-terminal sequence and was found to be conserved among all species except Rattus norvegicus and Equus caballus. Blast analysis of buffalo TLR9 sequence with single nucleotide polymorphisms (SNPs) database revealed 13 SNPs out of which 7 were cds-synonymous and 6 were of the functional significance. Furthermore, kinetics of TLR9 and proinflammatory IL-1βand TNF-α cytokine expression by buffalo PBMCs influenced by CpG-ODN is also discussed.
  • Swati Verma, Anil K. Gupta, Raj K. Singh
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    ABSTRACT: Seventy three core samples of 10 cc volume were analysed from Ocean Drilling Program (ODP) Site 756, Hole B with an average interval of 236 kyr per sample. Factor and cluster analyses of 37 high ranking species of benthic foraminifera enabled us to identify four clusters representing four biofacies. Biofacies Sl-Om is characterised by Stilostomella lepidula, Orthomorphina modesta, Robulus gibbus, Bulimina miolaevis and Cibicides cf. lucidus. This biofacies dominates the latest Oligocene to early Miocene interval (24–21 Ma and 18.75–16.5 Ma), indicating low to intermediate dissolved oxygen, organic carbon rich environment, warm and sluggish deep waters. During 21–18.75 Ma, the biofacies Gp-Nu (Globocassidulina pacifica, Nuttallides umbonifera, Trifarina angulosa, Quinqueloculina weaveri, Robulus gibbus and Pullenia bulloides) dominates the benthic assemblage at Hole 756B. This biofacies also has a short lived presence at ~ 16.5 Ma. This was an interval of intense circulation of cold and corrosive deep water similar to Antarctic Intermediate Water (AAIW), and intermediate to high flux of organic matter to Hole 756B. The Antarctic Circumpolar Current (ACC) initiated during this time. From 16.5 to 10.6 Ma, the benthic assemblage at Hole 756B is dominated by biofacies Ub-Gs (characteristic species Uvigerina buzasi, Globocassidulina subglobosa, Laticarina pauperata), indicating intermediate to high food supply and cold deep water. This interval coincides with the middle Miocene positive oxygen isotope shift and permanent build up of Antarctic ice sheets. The late Miocene interval (10.6–6.5) is characterised by biofacies Ec-Bs (dominant species Ehrenbergina carinata, Bolivina spathulata, Osangularia culter and Pullenia osloensis) indicating low oxygen condition with high food supply in the study area. This corresponds to an interval of global biogenic bloom.
    Palaeogeography Palaeoclimatology Palaeoecology 04/2013; 376:172–183. DOI:10.1016/j.palaeo.2013.02.034 · 2.75 Impact Factor
  • Dipankar Saha, S. N. Dwivedi, Raj K. Singh
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    ABSTRACT: A significant component of domestic demand for water of urban areas located in the Gangetic plains is met by heavy pumping of groundwater. The present study is focused on the Patna municipal area, inhabited by 17 million people and spanning over 134 km2, where entire urban water demand is catered from pumping by wells of various capacities and designs. The present study examines the nature of the aquifer system within the urban area, the temporal changes in the water/piezometric level and the recharge mechanism of the deeper aquifers. The aquifer system is made up of medium-to-coarse unconsolidated sand, lying under a ~40-m-thick predominantly argillaceous unit holding 8- to 13-m-thick localised sand layers and continues up to 220 m below ground. Groundwater occurs under semi-confined condition, with transmissivity of aquifers in 5,500–9,200 m2 day−1 range. Hydraulic head of the deeper aquifer remains in 9–19 m range below ground, in contrast to 1–9 m range of that of the upper aquitard zone. The estimated annual groundwater extraction from the deeper aquifer is ~212.0 million m3, which has created a decline of 3.9 m in the piezometric level of the deeper aquifer during the past 30 years. Unregulated construction of deep tube wells with mushrooming of apartment culture may further exacerbate the problem. The sand layers within the aquitard zone are experiencing an annual extraction of 14.5 million m3 and have exhibited stable water level trend for past one and half decades. This unit is recharged from monsoon rainfall, besides contribution from water supply pipe line leakage and seepage from unlined storm water drains.
    Environmental earth sciences 02/2013; 71(4). DOI:10.1007/s12665-013-2577-7 · 1.57 Impact Factor
  • Raj K. Singh, Anil K. Gupta, Moumita Das
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    ABSTRACT: Diversity parameters of Neogene deep-sea benthic foraminifera were measured at Ocean Drilling Program (ODP) Hole 752A, southeastern Indian Ocean (water depth of 1086.3 m) using Information Function (H), Equitability (E), number of species (S) and Sander's rarefaction values. These parameters combined with population abundance of dominant benthic foraminifera (Bulimina macilenta, Nuttallides umbonifera, Cibicides wuellerstorfi, Cibicides lobatulus, Bolivina pusilla, Ehrenbergina carinata, Gavelinopsis lobatulus, Cassidulina laevigata, Globocassiulina subglobosa) reveal significant paleoceanographic changes in the southeastern Indian Ocean during the Neogene. The values of all the diversity parameters show a decrease from 25 to 23 Ma and thereafter an increase with peak values at ~ 13.5 Ma. The Late Oligocene to Earliest Miocene was an interval of more unstable conditions at Hole 752A dominated by species characteristic of low organic carbon, well-ventilated, carbonate corrosive high energy conditions. The highest values of diversity parameters coincide with the early Middle Miocene climatic optimum. All these parameters show a declining trend and gradual decrease from 13.5 to 4.5 Ma coinciding with the major build up of ice sheets in the Antarctic region. Major increase in B. pusilla and E. carinata population during this time suggests high nutrient levels and low oxygen conditions at Hole 752A. This interval corresponds with the so-called “biogenic bloom” and an intense Oxygen Minimum Zone as observed throughout the Indo-Pacific region. Deep waters were warmer from 4.5 to 3 Ma marked by an increase in species diversity values, coinciding with the early middle Pliocene warmth. The species diversity values abruptly decreased in the younger interval, contemporaneous with the major Northern Hemisphere glaciation. During this time species characteristics of high-energy bottom currents and relatively cold deep water were dominant.
    Palaeogeography Palaeoclimatology Palaeoecology 11/2012; s 361–362:94–103. DOI:10.1016/j.palaeo.2012.08.008 · 2.75 Impact Factor
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    ABSTRACT: The complete genome of the Japanese encephalitis virus (JEV) strain JEV/eq/India/H225/2009(H225), isolated from an infected horse in India, was sequenced and compared to previously published JEV genomes. H225 genome was 10,977-nucleotides long, comprising a single ORF of 10,299-nucleotides, a 5′-UTR of 95 nucleotides and a 3′-UTR of 582 nucleotides. The H225 genome showed high levels of sequence identity with 47 fully sequenced JEV genomes, ranging from 99.3 % to 75.5 % for nucleotides and 99.2 % to 91.5 % for amino acid sequences. Phylogenetic analysis of the full-length sequence indicated that the H225 strain belongs to genotype III and is closely related to the Indian JEV strain Vellore P20778. A comparison of amino acids associated with neurovirulence in the E proteins and non-structural proteins of known virulent and attenuated JEV strains suggested H225 to be a highly virulent strain. This is the first report of whole-genome sequencing of a genotype III JEV genome isolated from equines.
    Archives of Virology 09/2012; 158(1). DOI:10.1007/s00705-012-1474-9 · 2.28 Impact Factor
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    ABSTRACT: Buffalopox virus (BPXV), a close variant of vaccinia virus (VACV) has emerged as a zoonotic pathogen. The host tropism of poxviruses is governed by host-range genes. Among the host-range genes: E3L, K3L, and C7L are essential for virus replication by preventing interferon resistance, whereas B5R is essential for spread of the virus and evasion from the host's immune response as in VACV. We report sequence analysis of host-range genes: E3L, K3L, C7L, and membrane protein gene (B5R) of BPXVs from buffalo, cattle, and human from recent outbreaks in India-their phylogenetic relationship with reference strain (BP4) and other Orthopoxviruses. BPXVs revealed a sequence homology with VACVs including zoonotic Brazilian VACV-like viruses. The aa sequences of E3L and K3L genes were 100 % similar in buffalo, cattle, and human isolates. However, four significant point mutations (I11K; N12K and S36F in C7L gene and D249G in B5R gene) were observed specific to buffalo isolate only. This signifies that different strains of BPXV were circulated during the outbreak. The mutations in C7L and B5R could play an important role in adaptation of BPXV in human and cattle which needs further functional studies. The strain of BPXV isolated from buffalo may not be adopted in human and cow. Various point mutations were observed in the host-range genes of reference strain (BPXV-BP4) which may be due to several passages of virus in cell culture. The phylogeny constructed based on concatenated gene sequences revealed that BPXVs are not as closely related to vaccine strain (Lister and Lister-derived strain-LC16m8), as hypothesized earlier, rather they are more closely related to reference strain (BPXV-BP4) and other vaccinia and vaccinia-like viruses such as Passatempo and Aracatuba viruses. The availability of information regarding host tropism determinants would allow us to understand molecular mechanism of species tropism of poxviruses which would be useful in unveiling new strategies to control zoonotic poxviral infections.
    Virus Genes 08/2012; 45(3). DOI:10.1007/s11262-012-0788-8 · 1.84 Impact Factor
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    ABSTRACT: Japanese encephalitis (JE) is an important vector-borne viral disease of humans and horses in Asia. JE outbreaks occur regularly amongst humans in certain parts of India and sporadic cases occur among horses. In this study, JE seroprevalence and evidence of JE virus (JEV) infection among horses in Haryana (India) is described. Antibodies against JEV were detected in 67 out of 637 (10.5%) horses screened between 2006 and 2010. Two foals exhibiting neurological signs were positive for JEV RNA by RT-PCR; JEV was isolated from the serum of one of the foals collected on the second day of illness. This is the first report of JEV isolation from a horse in India. Furthermore, a pool of mosquitoes collected from the premises housing these foals was positive for JEV RNA by RT-PCR. Three structural genes, capsid (C), premembrane (prM), and envelope (E) of the isolated virus (JE/eq/India/H225/2009) spanning 2,500 nucleotides (from 134 to 2,633) were cloned and sequenced. BLAST results showed that these genes had a greater than 97% nucleotide sequence identity with different human JEV isolates from India. Phylogenetic analysis based on E-and C/prM genes indicated that the equine JEV isolate belonged to genotype III and was closely related to the Vellore group of JEV isolates from India.
    Journal of veterinary science (Suwŏn-si, Korea) 06/2012; 13(2):111-8. DOI:10.4142/jvs.2012.13.2.111 · 1.14 Impact Factor
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    ABSTRACT: Glanders, a bacterial disease of equines caused by Burkholderia mallei, is a fatal infectious disease of equines and has zoonotic significance. The disease has been eradicated from many countries by statutory testing, elimination of infected animals and import restrictions. However, it is still endemic in parts of Africa, Asia, the Middle East and Central and South America. In India, major glanders outbreaks were reported from different parts of the country between 1976 and 1982. Later, sporadic cases of the disease were reported in 1988, 1990 and 1998. The country remained free of glanders for about eight years until the recent outbreaks occurred in eight States from 2006 to 2007. Recurrent episodes have occurred in Himachal Pradesh and Uttar Pradesh, whereas fresh outbreaks occurred in Chhattisgarh from 2009 to 2010. A total of 164 equines were declared positive; a majority of the positive cases (n=77) were from Uttar Pradesh, followed by Maharashtra (n=23), Uttarakhand (n=21) and Andhra Pradesh (n=16). Under the provision of Prevention and Control of Infectious and Contagious Disease in Animals Act, 2009, all the infected animals were euthanised and bio-security measures were implemented to curb the further spread of the disease.
    Veterinaria italiana 04/2012; 48(2):167-78. · 0.68 Impact Factor
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    ABSTRACT: The seroprevalence of Japanese encephalitis virus (JEV) among equines was evaluated from January 2006 to December 2009 in 13 different states of India by hemagglutination inhibition (HI) test and virus neutralization test (VNT). Antibodies against JEV were detected in 327 out of 3,286 (10%) equines with a maximum prevalence reported in the state of Manipur (91.7%) followed by Gujarat (18.5%), Madhya Pradesh (14.4%), and Uttar Pradesh (11.6%). Evidence of JEV infection was observed in equines in Indore (Madhya Pradesh) where a 4-fold or higher rise in antibody titer was observed in 21 out of 34 horses in November 2007 to October 2006. In March 2008, seven of these horses had a subsequent 4-fold rise in JEV antibody titers while this titer decreased in nine animals. JEV-positive horse sera had a JEV/WNV (West Nile virus) ratio over 2.0 according to the HI and/or VNT. These results indicated that JEV is endemic among equines in India.
    Journal of veterinary science (Suwŏn-si, Korea) 12/2011; 12(4):341-5. DOI:10.4142/jvs.2011.12.4.341 · 1.14 Impact Factor
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    ABSTRACT: Equine influenza is a contagious viral disease that affects all members of the family Equidae, i.e., horses, donkeys and mules. The authors describe the pattern of equine influenza outbreaks in a number of states of India from July 2008 to June 2009. The disease was first reported in June 2008 in Katra (Jammu and Kashmir) and spread to ten other states within a year. All outbreaks of equine influenza in the various states were confirmed by laboratory investigations (virus isolation and/or serological confirmation based on haemagglutination inhibition [HI] assays of paired samples) before declaring them as equine influenza virus-affected state(s). The virus (H3N8) was reported from various locations in the country including Katra, Mysore (Karnataka), Ahmedabad (Gujarat), Gopeshwar and Uttarkashi (Uttarakhand) and was isolated in 9- to 11-day-old embryonated chicken eggs. The virus was confirmed as H3N8 by HI assays with standard serum and amplification of full-length haemagglutinin and neuraminidase genes by reverse transcriptase-polymerase chain reaction. Serum samples (n = 4 740) of equines from 13 states in India screened by HI revealed 1074 (22.65%) samples as being positive for antibodies to equine influenza virus (H3N8).
    Veterinaria italiana 01/2010; 46(4):449-58. · 0.68 Impact Factor

Publication Stats

104 Citations
41.19 Total Impact Points

Institutions

  • 2010–2014
    • National Research Centre on Equines
      Hissār, Haryana, India
  • 2013
    • Wadia Institute of Himalayan Geology
      Dehra, Uttarakhand, India
  • 2004–2012
    • IIT Kharagpur
      • Department of Geology & Geophysics
      Kharagpur, Bengal, India