MAJOR RESEARCH ACHIEVEMENTS

The Centre has been working on various aspects of equine health and production and has made landmark achievements in the fields of development of diagnostics and prophylactics for equine diseases. The Centre has also been recognized as national referral centre for diagnosis of important equine infectious diseases by Department of Animal Husbandry, Dairying & Fisheries, Ministry of Agriculture (Government of India). Besides, the Centre has been working towards characterization of breeds of equines, cryopreservation of semen and artificial insemination. The centre has developed many kits and technologies which have either been patented or under the process of patent. Some of the landmark achievements of the centre are enlisted below:

1. Equine Health

A. Diagnostics for equine diseases

· Equine herpes virus-1 (EHV-1): A highly sensitive and specific neutralizing monoclonal antibodies-based diagnostic kit named Equiherpes B-ELISA was developed by the Centre for diagnosis of EHV-1 antibodies. This kit tests the serum sample using single dilution (1:250) thus making it very economical. This kit was formally released by Hon'ble DG, ICAR on August 20, 2008.

· Equine herpes virus-4 (EHV-4): A type-specific ELISA and immunostck assay have been developed using EHV-1/4 recombinant glycoprotein G for differentiation of EHV-1 and EHV-4 infections. A multiplex PCR targeting glycoprotein G 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 stool samples. A RT-PCR using VP gene primers has also been developed and its results were comparable to s-ELISA.

· Equine influenza virus (EIV): EIV is routinely diagnosed by haemagglutination inhibition assay. RT-PCR for equine influenza diagnosis and typing has been developed. DIVA assay for differentiation of vaccinated and infected animals is under progress. Immunoglobulin isotyoing is being routinely conducted by the Centre on the field samples.

· Theileria equi: A recombinant antigen based-ELISA diagnostic kit has been developed for detecting Theileria equi specific antibodies in equine serum. The kit has been validated vis-à-vis OIE approved CI ELISA diagnostic kit and DSp and DSn values for CI ELISA and ELISA were 0.95 & 0.97 and 0.93 & 0.96, respectively. This kit has also been validated in internal and external laboratories of India. We are now routinely using this kit for screening antibodies against T. equi in serum samples.

· Trypanosomosis: An indirect ELISA has been standardized using whole cell lysate antigen of Trypanosoma evansi. PCR has also been standardized for sensitive detection of surra.

· Japanese encephalitis virus (JEV): Serum neutralization test (SNT) and haemagglutination inhibition (HAI) standardized for diagnosis of JE. RT-PCR employing primers targeted against E-gene and 3’NTR has been standardized. mAb-based ELISA for Japanese encephalitis is also being developed.

· Equine infectious anemia: Coggins test for EIA is routinely being used at the Centre. In addition, r-protein has been developed at the Centre which is being validated for replacing the kit available commercially.

· Equine viral arteritis: Virus neutralization for equine viral arteritis is routinely used for serodiagnosis of EVA.

· Monoclonal antibodies: Monoclonal antibodies have been developed for diagnosis and characterization of equine herpes virus-1, equine rotavirus and Japanese encephalitis.

· Kits for disease diagnosis: HERP kit & Equiherpes B-ELISA kit (For EHV-1 diagnosis), COFEB kit (For diagnosis of piroplasmosis) and kit for pregnancy diagnosis were developed by the centre.

B. Vaccines and Immuno-biologicals developed by NRCE

· EHV-1 vaccine: An equine herpes virus-1 (EHV-1) killed vaccine incorporating indigenous strain (Hisar-90-7) of EHV-1 has been developed by the Centre. Experimental as well as field trials of equine herpesvirus-1 inactivated vaccine have been completed. The vaccine generates protective immune response, which is comparable to that of commercial imported Pneumabort 'K' vaccine in pregnant mares. No EHV-1 associated abortions or adverse reactions are observed in vaccinated mares.

· Equine influenza vaccine: The Centre developed equine influenza vaccine using indigenous isolate (A/equi-2/Ludhiana/87). The vaccine has been updated in 2010 incorporating epidemiologically relevant isolate A/eq/Katra-Jammu/06/08(H3N8) responsible for equine influenza outbreaks during 2008-09. The updated vaccine is safe and efficacious as is evident by the protective immune response generated by the vaccine in equines in a limited experimental trial. A new cell culture-based inactivated equine influenza vaccine has been developed and is under validation.

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

C. Surveillance and monitoring of equine diseases in India

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 NRCE about the status of AHS in the country helped in declaring India free of African horse sickness in 2006 by World Organization for Animal Health (OIE).

· Outbreaks of equine glanders in country during 2006-07 were timely detected and its control measures were taken to prevent its further spread. After that, there were no reports of glanders for two years from India. However, from November 2009 to March 2010, ten fresh cases of glanders have been diagnosed from equines in Raipur (Chhatisgarh) and also from Himachal Pradesh.

· NRCE diagnosed the 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.

· NRCE has continuously been screening equines for equine infectious anemia from 1998. One mule has been found seropositive during 2009-10.

· Zoonotic camel pox virus outbreak: We reported a camelpox virus outbreak and its zoonoses in three human in camels in Barmer district (Rajasthan).

D. Molecular characterization of equine pathogens

· Equine influenza virus (EIV): HA genes of EIV isolates from 2008 outbreak (A/eq/Jammu-Katra/08, A/eq/Mysore/08 and A/eq/Ahmedabad/09) were cloned and sequenced. Phylogenetic analysis established that 2008 EI outbreak in India was due to equ/2 (H3N8) subtype and that Indian isolates were identical to the Clade 2 of American lineage of H3N8 subtype.

· 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): Sequence analysis of E-gene of JEV isolated from an equine indicates genotype 3 is responsible for causing disease in equine and that the equine JEV isolate clusters together with Vellore group of JE isolates responsible for JEV in humans in India.

· In vitro culture of Trypanosoma evansi: 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.

E. 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 EHV-1 (6 isolates), EHV-4 (3), equine rotavirus (29), equine influenza (7), Japanese encephalitis virus (2), West Nile virus (1), Rhodococcus equi, Stretptococcus equi, S. Zooepidemicus, Burkholderia mallei, Salmonella Abortusequi, S. equisimilis, Enterobacter aerogenes, E. coli, Staphylococcus aureus, Trypanosoma evansi (3).
• NRCE has a number of hybridomas secreting monoclonal antibodies against equine herpes virus-1, equine rotavirus, Japanese encephalitis virus, West Nile virus.
• NRCE has repository of more than 15000 equine serum samples collected from different geographical locations in its Equine Serum Bank.
• NRCE has a collection of more than 100 recombinant plasmid clones with recombinant genes of pathogens including equine influenza virus, equine rotavirus, EHV-1, EHV-4, JEV, EIAV, R. equi, Burkholderia mallei, Trypanosoma evansi, Babesia equi.
• Biological repository of NRCE has been further strengthened by establishment of National Veterinary Type Culture Collection Centre, which has repository of animal pathogens including viral isolates (camel poxvirus, buffalo poxvirus, bovine herpes virus-1, rotavirus), bacterial cultures (R. equi, E. coli, Bordetella, Streptococcus, Staphylococcus, Corynebacterium, Pseudomonas, Enterobacter). This repository is ever expanding day by day.

2. Equine Production

A. Indigenous breed characterization

Marwari: This breed of horses constitutes an elite group of indigenous horses, which are known for their sturdiness, swiftness, elegance and beauty. A total of 114 true-to-breed Marwari horses comprising of 98 mares and 16 stallions from seven different locations were evaluated. DNA polymorphism studies revealed high level of heterozygosity and low level of heterozygosity deficit in the Marwari horse population which reflect high genetic variability in Marwari equine population. The Centre in collaboration with Department of Animal Husbandry, Dairying & Fisheries (DAHDF), Government of India has finalized the breed descriptor for the Marwari breed of horses for entry of these animals in to the stud book. The regions of MHC class-II (DRB-2a and 2b) gene in Marwari horses exhibited polymorphism in 48.39% genotypes.

Kathiawari: Genotyping of Kathiawari breeds of horses employing 30 fluorescent-labeled microsat pairs in multiplex PCRs showed high heterozygosity which clearly indicated that there is adequate genetic diversity among these equines.

Bhutia: Characterization of true to breed animals of Bhutia breed ponies (n=35) in Sikkim revealed their mean heights at withers as 126.5 cm. In these ponies, bay was the most common colour (69%) followed by chestnut (23%), grey and other colours. Average body length and heart girth were recorded as 129 and 148.5 cm, respectively without any significant difference due to sex.

Spiti: In India, distribution of this breed is confined to Lahoul & Spiti, Kinnaur, Kullu, Mandi, limited areas of Kangra and Shimla district in Himachal Pradesh; Ladhakh division of J & K and Uttranchal but the true breeding tract is confined to 15 villages of two Panchayats (Kungri and Sagnam) in Pin Valley of Spiti sub-division of Distt. Lahoul & Spiti. The total population of Spiti ponies in H.P. is approximately 4000. True-to-breed phenotypic characters of this breed have been recorded by NRCE.

Manipuri : The phenotypic characterization of Manipuri ponies indicated that height at wither in both the sexes of Manipuri ponies ranged from 119-134 cm. In addition, heart girth (143.6 cm), hind leg length (82.3 cm), fore leg length (78.4 cm) and height at knee (39.4 cm), etc were also recorded. DNA of representative animals of this breed has been analyzed for molecular characterization of the breed.

Zanskari : Biometric indices of true-to-Zanskari breed of horses were recorded in their home tract in and around Leh, Laddakh (Jammu & Kashmir). Average height at wither of Zanskari breed was 126 cm. Average animal height was slightly higher in stallions (127.21 ± 7.57) then mares (125.45 ± 4.74 cm) but the differences were non significant. Beside this, average body length (123.07 vs 129.5 cm), heart girth (144.4 vs 148.9 cm), hind leg length (80.11 vs 79.95 cm), canon length (16.18 vs 15.80 cm), height at knee (37.57 vs 36.95 cm), face length (53.79 vs 53.75cm), face width (15.68 vs 15.25 cm), etc. were almost same in both stallions and mares, respectively without any significant difference. A nuclear herd of Zanskari ponies at our Bikaner Campus, after translocating these animals from their native herd at Ladakh in J and K.

B. Improvement in production potentials of equines

· Semen cryopreservation and artificial insemination (AI): In order to conserve the germplasm of indigenous equine breeds, the technique for cryopreservation of semen of Marwari, Kathiawari 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.

· Early pregnancy diagnosis: 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.

· Kit for pregnancy diagnosis: An eCG s-ELISA kit.

3. Patents

· Patent has been granted by the Patent Office, Government of India entitled "A method for preparation of a diagnostic kit useful for forecasting Equine Herpes Virus-1 disease".

· A patent has been filed for “COFEB-Kit for diagnosis of Babesia equi infection in equines”.

· A patent has been filed for “A method for preparing complement fixation test based (COFEB) kit for the diagnosis of Babesia equi infection in equines”.

· The Centre has filed a patent for “A kit for detection of pregnancy in equines and assay thereof”.

4. Services

NRCE provides following services to the farmers and equine breeders:

• The centre provides disease diagnostic services for various infectious and non-infectious 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.
• Assessment and transfer of technology using the latest know-how of information technology is also given due importance to extend the technologies to the end-users. 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 and supplied education materials for equine management, production and health.
• Extension activities: To receive feedback from the equine owners, various activities like health camp, awareness and farmers meets are organized on regular basis in different areas of the country.

5. Veterinary Type Cultures Facility at NRCE

· Indian Council of Agricultural Research entrusted NRCE the responsibility of establishing Veterinary Type Culture Centre (VTCC) during the X plan period. The Veterinary Type Cultures became functional in June 2005 for establishing national repository of microorganisms of animal origin including recombinant cultures and plasmids; and identification, characterization, conservation, maintenance and utilization of microorganisms. Approximately 250 bacterial isolates and 25 viral isolates have been collected in VTCC. In addition, a phage display library of single-domain antibodies of Indian desert camel and the twenty seven antigen-binder clones selected from the library have been deposited in the VTCC repository.