Research Achievement

Equine herpes virus 1 (EHV1): A highly sensitive and specific neutralizing monoclonal antibodybased diagnostic kit namely Equiherpes B-ELISA was developed by the Centre for diagnosis of EHV-1 antibodies. Presently the kit is under the process of commercialization.

Equine herpes virus 4 (EHV4): A type-specific ELISA using EHV-1/4 recombinant glycoprotein G has been developed for differentiation of EHV-1 and EHV-4 infections. A multiplex PCR targeting glycoprotein C and G genes has also been developed for differentiation of EHV-1 and EHV-4 and is routinely used in the laboratory.

Equine rotavirus: A sandwich enzyme-linked immunosorbent assay (s-ELISA) was developed employing a monoclonal antibody (mAb) raised against VP6 of rotavirus, for detection of equine Rotavirus (ERV) from faecal samples. This assay has been validated by two external laboratories using bovine, sheep and equine rotavirus samples and detects rotavirus infection among different animals. An RT-PCR using VP6 gene primers was also developed, which compared well with the s-ELISA.

Equine influenza virus (EIV): EIV is routinely diagnosed by haemagglutination inhibition (HI) assay. Recently, RT-PCR and real-time RT-PCR based assay targeting M gene were developed for typing and diagnosis of EIV. Additionally, development of monoclonal antibody based sandwich ELISA for antigenic detection is under progress.

Theileria equi: For serodiagnosis of T. equi, a recombinant antigen based-ELISA has been developed using a truncated gene segment of a merozoite surface protein, EMA-2. The DSp and DSn of this assay in comparison to OIE-approved CIELISA kit were 0.97 and 0.96, respectively.

Trypanosomosis: An indirect ELISA has been standardized using whole cell lysate antigen of Trypanosoma evansi. RoTat 1.2 gene-specific PCR has also been standardized for sensitive detection of trypanosomal nucleic acid in the blood.

Japanese encephalitis virus (JEV): Serum neutralization test (SNT) and haemagglutination inhibition (HI) has been standardized for diagnosis of JE. Monoclonal antibodies against JEV have also been raised and are under trial for development of mAb-based capture ELISA.

Equine infectious anemia: Coggins test for EIA is routinely being used at the Centre. A recombinant protein from a synthetic gene of 26 kDa expressed in E. coli was evaluated for use in AGID/indirect ELISA in a pilot study for sero-diagnosis of EIA. The DSn and DSp for the assay were found to be 100% in comparison to Coggins test.

Equine viral arteritis: A standardized virus neutralization test is routinely used for serodiagnosis of EVA.
Small Animal models for understanding pathology and disease mechanisms

Equine influenza: ICAR-NRCE has developed a novel BALB/c mouse model for studying pathology and pathogenesis of equine influenza virus. The model will help in understanding disease mechanisms and host-pathogen interaction, while simultaneously working for screening of vaccine candidates for their protective efficacy and immune response.

Equine herpes Virus 1: ICAR-NRCE has standardized the BALB/c mouse model for EHV1 for respiratory infection as well as for abortion studies.The abortion model has been utilized in Pathology Laboratory of NRCE for immuno-protective efficacy of inactivated EHV-1 vaccine. Also the respiratory model has been used for immune-prophylactic studies of recombinant proteins of EHV-1.

Vaccines and Immuno-biologicals developed by NRCE

EHV1 vaccine: An equine herpes virus 1 (EHV1) killed vaccine namely “EquiherpAbort ” incorporating indigenous strain (Hisar-90-7) of EHV1 has been developed by the Centre. This killed vaccine has already undergone field trials in mares.The vaccine with a three dose schedule generates protective immune response in pregnant mares,which is comparable to that of commercially imported Pneumabort ‘K’ vaccine.

Update equine influenza vaccine: During 2008-09,India experienced another outbreak of equine influenza caused by antigenically and genetically divergent EIV strain and was significantly different from the previous (1987) isolate. Thus, the vaccine has been updated in 2010, incorporating the new strain {A/eq/Katra-Jammu.06/08 (H3N8)} responsible for EI outbreaks during 2008-09. The updated vaccine is safe and efficacious as evident by the protective immune response generated by the vaccine in field trials in equines.

Salmonella Abortus equi: Improved bacterin and outer membrane protein-based vaccines have been developed for Salmonella Abortus equi.

Monoclonal antibodies: Monoclonal antibodies have been developed for diagnosis and characterization of equine herpes virus 1, equine rotavirus, equine influenza, Japanese encephalitis & Trypanpsoma evansi.

Kits for disease diagnosis: HERP kit & Equiherpes B-ELISA kit (for EHV1 diagnosis), recombinant protein based ELISA for the diagnosis of Theileria equi & COFEB kit for diagnosis of Theileria equi have been developed by the Centre.

Surveillance and monitoring of equine diseases in India ICAR-NRCE is involved in nation-wide monitoring and sero-surveillance of important equine infectious diseases with a view to manage, control and eradicate diseases. Important achievements of the Centre in disease surveillance are: Information generated by institute about the status of African horse sickness (AHS) in the country helped in declaring India free of AHS in 2006 by Office International des Epizooties (OIE). Outbreaks of glanders in equines have been detected since 2006-07 and control measures are being adopted for preventing its further spread. After a brief pause for two years, the disease again emerged in December 2010, in Chandpur area of Bijnor (UP). From 2012 onwards, various states such as Chattisgarh,Himachal Pradesh, Uttarakhand, Andhra Pradesh, Haryana, Maharashtra, Punjab, J&K and Gujarat have reported incidence of glanders. A few of these states have been able to control the disease with the help of NRCE. Presumably the disease spreads to other states from U.P. l NRCE diagnosed equine influenza (EI) in India in 2008 from Jammu region (July 2008) that subsequently affected equines in 13 different states. The biosecurity measures were implemented in collaboration with various state animal husbandry departments. No new cases of EI have been reported from India since May 2009, however, low antibody titres in serum from some parts of the country with no apparent signs are a matter of worry and thus surveillance and monitoring for the disease continues. NRCE has continuously been screening equines for equine infectious anaemia from 1998. One mule was found to be seropositive during 2009-10 followed by a horse detected positive in 2011-12 in Haryana state.

Molecular characterization of equine pathogens

Equine influenza virus (EIV): HA genes of EIV isolates from 2008 outbreak (A/eq/Jammu-K a t r a / 0 8 , A / e q / M y s o r e / 0 8 a n d A / e q /Ahmedabad/09) were cloned and sequenced,which were identical to the Clade 2 of American lineage of H3N8 subtype. Also, the genetic analysis and selection pressure of matrix (M) gene of the Indian isolates from 2008-09 outbreaks were studied and it was found that M1 and M2 proteins shared 98.41% and 99.54% homology with other Clade 2 viruses of Asian origin for M1 and M2 amino acid sequences, respectively. Phylogenetic analysis revealed clustering of Indian and Chinese isolates in a separate cluster designated as “Asian clade” for M gene.

Equine rotavirus (ERV): Sequencing of VP7 gene of ERV isolates indicated circulation of G10, G3 and G6 serotypes in India. Sequencing of outer surface proteins (VP4 and VP7) of equine rotaviruses for their genotyping and molecular epidemiology was done.

Japanese encephalitis virus (JEV): Whole genome sequencing has been done. Sequence analysis of Egene of JEV isolated from an equine indicates genotype 3 was responsible for causing the disease in equines and that the equine JEV isolate clustered with Vellore group of JE isolates responsible for JEV in humans in India.

In vitro culture of Trypanosoma evansi and Theileria equi: The Centre succeeded in in-vitro cultivation of bloodstream forms of T. evansi in artificial media by using specially formulated cell culture medium supplemented with 20% adult horse serum, however, Theileria equi has been successfully cultivated in microaerophilous stationary phase (MASP) culture from infected blood.

Biological resource Bank NRCE has a strong biological resource base having numerous pathogens, recombinant clones, reference sera, equine sera, monoclonal antibody secreting hybridomas, etc. Pathogenic isolates (viruses, bacteria and parasites) of equine origin available with NRCE include EHV1 (14 isolates), EHV4 (14), equine rotavirus (29), equine influenza (11), Japanese encephalitis virus (2), West Nile virus (1),Rhodococcus equi, Streptococcus equi, S.zooepidemicus, S. equisimilis, Burkholderia mallei, Salmonella Abortus equi, Enterobacter aerogenes, Escherichia coli, Staphylococcus aureus and Trypanosoma evansi. ICAR-NRCE has a number of hybridomas secreting monoclonal antibodies against equine herpes virus 1, equine influenza, equine rotavirus, Japanese encephalitis virus and West Nile virus.ICAR-NRCE has a repository of more than 15,000 equine serum samples collected from different geographical locations in its Equine Serum Bank. ICAR-NRCE has a collection of recombinant plasmid clones with recombinant genes of pathogens including equine influenza virus,equine rotavirus, EHV1, EHV4, EI, JEV, EIAV, R.equi, Burkholderia mallei, Trypanosoma evansiand Theileria equi.

Indigenous breed characterization

Phenotypic characterization of Indigenous horse and pony breedsPopulations of equines in the country has drastically decreased. Six breeds namely, Marwari, Kathiawari, Spiti, Zanskari, Bhutia and Manipuri were characterized phenotypically on the basis of their biometric indices and coat colour and significant differences were observed. On the basis of their heights at wither, Kathiawari and Marwari breeds were grouped under “horse” while Zanskari, Manipuri, Bhutia and Spiti fell under”pony” breeds.

Genotypic characterization of Indian equine breeds Genetic diversity analysis, population structure and relationship among six Indian horse(Kathiawari, Marwari) and pony breeds (Manipuri, Spiti, Zanskari and Bhutia), along with English Thoroughbred horses as an outgroup was carried out which indicated high genetic diversity in all India breeds except Spiti ponies. Individual assignment indicated admixture in all the breeds except Thoroughbred horses.The neighbor-joining dendrogram using the allele sharing distance clearly defined clusters for most of the breeds; Indian horse and pony breeds clustered separately while Thoroughbred formed a separate out-group. The Bayesian analysis revealed three distinctive clusters of Indian horse and pony breeds with Kathiawari as the most prominent cluster in horse breed, second of Zanskari, Spiti and Manipuri ponies and third one having Bhutia and a subpopulationof Marwari horses.

Establishment of nucleus herd Exotic Donkeys: Jennies and jacks of European breed (Poitou), imported from France through ODA, UK in 1990, are being maintained at EPC,Bikaner for the improvement of indigenous donkeys and production of superior mules. Marwari Horses: In an effort to conserve the true to breed equids, the Centre has also established a nucleus herd of Marwari horse at EPC, Bikaner. Indigenous donkey: The Centre has initiated the establishment of nucleus heard of small grey and large white donkeys found in India for conservation and improvement of donkeys. Equine sanctuary at EPC, Bikaner: ICAR-NRCE has initiated an in-vivo conservation programme in the form of developing an equine sanctuary
at EPC, Bikaner. Under this 12 Zanskari ponies(eight mares & four stallions) were brought from Zanskar valley, Kargil, Ladakh, Jammu & Kashmir in November, 2009. In 2014, a total of 11 Manipuri ponies (seven mares & four stallions) were brought from Imphal, Manipur.

Improvement in production potential of equines

Semen cryop reservation and artificial insemination (AI): In order to conserve the germplasm of indigenous equine breeds, the technique for cryopreservation of semen of Marwari stallions and donkeys have been standardized. The technique of artificial insemination using frozen semen for production of superior quality Marwari horses, superior mules and donkeys has been perfected. The pure germplasm of endangered indigenous breeds of horses is being conserved using this technology.

Pregnancy diagnosis: An eCG based sandwich ELISA has been developed for detection of pregnancy between days 30 to 150 of gestation in mares. The kit is cost effective, horse specific and animal friendly. Pregnancy diagnosis between days 14 and 18 post-insemination has been achieved using ultrasonography in donkey and horse mares.

Donkey fibre has been used to produce carpets by mixing with sheep fibres in the ratio of 40:60 in association of CSWRI, Avikanagar.

Utilization of Animal Energy with enhanced System Efficiency-l Single animal drawn matching plough, seed drill(two furrow) and harness were designed and developed for donkeys and mules for performing various agricultural operations. Animal energy potential was utilized successfully in agricultural operations namely ploughing and sowing for different work hours without any adverse effect on the animals. l Similarly, mules also used in different ploughing experiments indicated that these can also be used efficiently in agricultural operation as all resumed to normal physiological conditions by the next morning.

Sustainable utilization of Mule power for chaffing operation:The mules were successfully used for chaff cutting operation to reduce women drudgery. Average output capacity of chopped bajra straw in rotary mode chaff cutter was 660 kg/ hour. Deployment of mules for operating a chaff cutter in rotary mode of operation is a viable option for sustainable utilization of equine power during idle hours.

Utilization of equine dung for preparation of vermicompost: To overcome the problem of dung disposal,vermicompost is being prepared using equine dung in readymade vermibeds successfully and it is being applied in agricultural fields, lawns and plants.

Patents- A highly sensitive kit for detection of antibodies against Theileria equi in serum of equids. Application No. 2763/DEL/2012 dated 06.09.2012 Nano-drug delivery for quinapyramine sulphate. Application, No. 2560/DEL/2011, dated 06.09.2011. (NRCE, Hisar and GJUS &T,Hisar) Polynucleotide sequence, processes, composition and methods thereof- Application No. 1575/CHE/2010 and PCT/IB 2011/052475(IIsc Bangalore and NRCE, Hisar) l A recombinant haemagglutinin domain containing protein for the detection and diagnosis of glanders and method of preparation thereof. Application No.1328/ DEL/2010 dated 08.06.2010. (DRDE Gwaliorand NRCE, Hisar)

Services NRCE provides following services to the farmers and equine breeders: The Centre provides disease diagnostic services for various infectious and noninfectious equine diseases to equine owners,Breeders, state animal husbandry departments, police and army horses. Artificial insemination to augment the production of superior quality Marwari horses, mules and donkeys.Quality jacks and jennies are supplied to various states, breeding societies and farmers, for production of superior quality mules and donkeys.NRCE is providing health certification for movement of equines within and outside the country. This facility has helped in promotion of export of horses. Extension activities: The scientific and technical staff provides clinical and diagnostic (including pregnancy diagnosis) services and consultancy to the farmers on demand in the areas of equine health and production.Farmers are imparted trainings, supplied education materials for equine management, production and health and received feed back by organizing health camp, awareness and farmers meets on regular basis in different areas of the country.

Major Landmarks