Visiting Scientists at the Gluck Equine Research Center

Dr. Rebecca Bellone

Assistant Professor

Department of Biology
The University of Tampa
Tampa, Florida

Albert and Lorraine Clay Fellowship

Support of a study-visit to the laboratories of Dr. E. Bailey and Dr. T.L. Lear by Dr. Rebecca Bellone of the University of Tampa; July 5, 2006 to August 6, 2006

Dr. Rebecca Bellone completed a PhD in the Veterinary Science Department of the University of Kentucky in August of 2001.   Currently she holds a faculty position at the University of Tampa with a primary responsibility is teaching courses in biological sciences including molecular genetics and basic biology courses.   Dr. Bellone’s research has focused on determining the genetic basis for the appaloosa patterns and an associated eye disease (congenital stationary night blindness) in horses. 

Project/Background

All appaloosa hair color patterns are controlled by a single dominant gene, designated as Leopard Complex, LP.  In addition to influencing hair color pattern this gene can cause eye problems; (night-blindness and uveitis) as well as weakness of keratin structures, such as hooves and hair. The goal of this study was to identify the gene responsible for this trait.

Previous work by Dr. Bellone led to mapping of the LP gene on horse chromosome 1 (Terry et al., 2004).  Based on comparison to the comparable human chromosome region and known gene function, two candidate genes were selected for further investigation (OCA2 and TRPM1).  Work conducted last summer under the auspices of the Clay Fellowship demonstrated that OCA2 was not responsible for the appaloosa trait or night blindness in appaloosa horses (Bellone et al., 2006). 

The purpose of this work was to investigate TRPM1 as a candidate gene for appaloosa color patterns and night-blindness in horses.  Other collaborators on the project included scientists from the University of Saskatoon, Texas A&M University, and a Canadian breeder of appaloosa horses.

Relevant Publications by the Visiting Scientist:

1. Bellone, R., Lawson, S., Hunter, N., Archer, S., & Bailey, E.  Analysis of a SNP found in exon 7 of Equine OCA2 and its exclusion as a cause for Appaloosa Spotting. Animal Genetics (in press).

2. Bellone, R., Lear, T., Adelson, D., and Bailey, E. (2006) Comparative mapping of oculocutaneous albinism type II (OCA2), transient receptor potential cation channel, subfamily M member 1 (TRPM1) and two equine microsatellites among four equid species by in situ hybridization. Cytogenetics and Genome Research. 114: ( DOI: 10.1159/000091935).

3. Terry, R.B., Archer, S., Brooks S., Bernoco D., & Bailey, E. (2004) The locus for appaloosa coat colour in horses (LP) maps to chromosome ECA1. Animal Genetics. 35:134-137. (JIF= 3.108).

4. Brooks, S.A., Terry, R.B., & Bailey, E. (2002) Association of PCR-RFLP for KIT with Tobiano coat color in horses. Animal Genetics. 33:301-303.

5. Terry, R.B., Bailey, E., Lear, T.L., and Cothran, E.G. (2001) Rejection of MITF and evidence against MGF as the genes responsible for appaloosa coat colour spotting patterns in horses. Animal Genetics. 33:82-83.

6. Terry, R.R., E. Bailey, D. Bernoco, and E.G. Cothran. (2000) Linked Markers Exclude KIT as the gene responsible for appaloosa coat color spotting patterns in horses. Animal Genetics. 32:98-102.

7. Terry, R.R., G. Cholewinski, and E.G. Cothran. (1999) Absence of the severe combined immunodeficiency disease gene among Arabian horses in Poland. Journal of Applied Genetics. 40(1):39-41.

Dr. Vijayachari Paluru

Assistant Director

Regional Medical Research Centre
(Indian Council of Medical Research)
WHO Collaborative Centre for Diagnosis, Research, Reference and Training in Leptospirosis
Post Bag No. 13
Port Blair ¨C 744 101
A & N Islands

vijayacharip@yahoo.com
Tel: 091-3192-251158 ,251164
Fax: 091-03192 -251163

Title of Project:

Development of antigen based detection system for early diagnosis of leptospiral infections.

Aims and Objectives:

1. Identification of  antigens which are  specific, sensitive and expressed in host during natural infections.

2. Development of an effective antigen  capturing  system for the early diagnosis of leptospirosis.

Leptospirosis and Its Public Health Importance

Leptospirosis is a zoonotic disease of worldwide distribution caused by pathogenic spirochetes of the genus Leptospira.  It can affect multiple organ systems and can lead to variety of clinical syndromes. Hepato-renal involvement with jaundice, severe pulmonary hemorrhages, myocarditis or meningitis are some of the severe forms of the disease and frequently associated with high mortality and morbidity. Leptospirosis is an emerging public health problem in both developed and developing countries. Several large outbreaks were reported  in several parts of the world in recent years. Due to the complex nature of clinical manifestations, the disease cannot be diagnosed only on clinical grounds, requires laboratory support for confirmation.

Work Plan

Construction of genomic DNA library:   Genomic libraries will be constructed using LAMBDA ZAP cloning Kit (Stratagene).Genomic DNA will be prepared from leptospiral isolates and will be digested by restriction enzymes.  Digested fragments in the range of 2.5kb to 5kb will be cloned and expressed. 

Screening the library: The gene product of the cloned DNA fragment (construct of Lamda ZAP genomic library) will be screened against sera collected from leptospirosis patients to identify the proteins that are expressed during natural infections.

Development of antigen capturing ELISA:  Antibodies will be developed against the identified antigens which are specific and sensitive. These antibodies will be utilized for the development of antigen capturing ELISA.

Publications

1. P. Vijayachari, Niyaz Ahmed, A P Sugunan, S. Ghousunnissa, K. R. Rao, Seyed E. Hasnain and S.C. Sehgal, Use of Fluorescent amplified fragment length polymorphism for analysis of the molecular epidemiology of Leptospirosis in India. J Clin Microbiol 42, 1-6 (2004) 

2. Vijayachari P, Sharma S, Hartskeerl R.A, Terpstra W.J and Sehgal S.C. A unique strain of leptospira isolated from a patient with pulmonary haemorrhages in Andaman Islands: a proposal of serovar Portblairi of serogroup sehgali. Epidemiol Infect 132, 663--673 (2004)

3. P. Vijayachari, A.P. Sugunan, M.V. Murhekar, S. Sharma & S.C. Sehgal Leptospirosis among schoolchildren of Andaman & Nicobar Islands, India ¨C low levels of morbidity and mortality among pre-exposed children during an epidemic. Epidemiol Infect (2004) (in press)

4. A.P. Sugunan, K. Natarajaseenivasan, P. Vijayachari and S.C. Sehgal.  A cutaneous laceration leads to leptospiral infections ¨C a case report.  J Med Microbiol. (2004)  (In Press)

5. K. Natarajaseenivasan, P. Vijayachari, A.P. Sugunan, S. Sharma and S.C. Sehgal.  Leptospiral proteins expressed during Acute and Convalescent phases of human Leptospirosis .  Ind J Med Res. (2004) (In Press)

6. Roy S, Biswas D, Vijayachari P & Sehgal S.C. A 22-MER arbitrary primer enhances discriminatory powe of RAPD Fingerprinting technique in characterization of leptospires. J Trop Med Intnal Health.2004 (in press)

7. Vijayachari, P., Sehgal S.C, Marga G.A.Goris, Terpstra W.J, Hartskeerl R.A. Leptospira Interrogans serovar valbuzzi: a cause of sever pulmonary haemorrhages in the Andaman Islands. J of Med Microbiol 52, 913 ¨C 18 (2003)

8. Sharma S, Vijayachari P, Sugunan AP, Sehgal SC. Leptospiral carrier state and seroprevalence among the animal population - a cross sectional survey in Andaman and Nicobar islands. Epidemiol Infect 131 985-989 (2003).

9. Sehgal, S.C., Sugunan, A.P, Vijayachari, P. Leptospirosis: Disease burden estimation and surveillance networking in India. Southeast Asian J  Tropical Medicine and Public Health,  34: 170-177 (2003)

10. Sehgal, S.C., Biswas D, Vijayachari, P. Sugunan, A.P and Roy S.  Molecular tools in leptospirosis diagnosis in characterization of isolates. Southeast Asian Journal of Tropical Medicine and Public Health, 34: 163-169 (2003)

11. Roy, S., Biswas, D., Vijayachari, P., Sugunan, A.P and Sehgal, S.C. Antigenic and genetic relatedness of Leptospira strains isolated from Andaman Islands in 1929 and 2001. Journal of Medical Microbiology 52; 909-911 (2003)

12. Sehgal, S.C., Vijayachari, P., Sugunan, A.P., Umapathi, T. Field application of Lepto lateral-flow for rapid diagnosis of leptospirosis. Journal of Medical Microbiology. 52: 897-901(2003)

13. Vijayachari P andSugunan, A.P. A book review on Human Leptospirosis (Word Health Organisation and international Leptospirosis Society) 2003,ISBN 92-4-154589-5, Indian J. Med. Res. 118: 251-252 (2003)  

14. Vijayachari, P, Sugunan, A.P and Sehgal, S.C. Evaluation of Lepto dri-dot as a rapid test for the diagnosis of leptospirosis. Epidemiol  Infect 129 : 617-621(2002)

15. Sehgal, S.C., Sugunan, A.P and Vijayachari, P. Outbreak of leptospirosis after the cyclone in Orissa. National Med J India. 15: 22-23 (2002)

16. Vijayachari. P, Sugunan, A.P, Sehgal, S.C. Role of Microscopic Agglutination Test (MAT) as a diagnostic tool during acute stage of leptospirosis in low and high endemic areas. Indian J. Med. Res. 114: 99-106 (2001)

17. Vijayachari, P., Sugunan, A.P, Umapati, T and Sehgal, S.C. Evaluation of Darkground microscopy as a rapid diagnostic procedure in leptospirosis. Indian J Med Res. 114: 54-58 (2001)

18. Sehgal, S.C., Vijayachari, P, Smythe, L.D et. al. Lai-like leptospira from the Andaman islands. Indian J. Med. Res. 112:135-39 (2000)  

19. Sehgal, S.C, Sugunan, A.P., Murhekar, M.V, Sharma, S., Vijayachari, P. Randomised controlled trial of doxycycline prophylaxis in an endemic area. International J. Antimicrob Agents. 13: 249-55 (2000)

20. Sehgal, S.C., Vijayachari, P., Murhekar, M.V., Sugunan, A.P., Sharma, S. and Singh, S.S. Leptospiral infection among primitive tribes of Andaman and Nicobar Islands. Epidemiol. Infect. 12 (1999)

21. Sehgal, S.C., Vijayachari, P., Sharma, S., Sugunan, A. P. LeptoDipstick - a rapid and simple method for serodiagnosis of leptospirosis in acute stage. Trans. Roy. Soc. Trop. Med. Hyg. 93:1-4 (1999)

22. Singh, S.S., Vijayachari, P., Sinha, A., Sugunan, A.P., Rasheed, M.A. and Sehgal, S.C. Clinico-epidemiological study of severe cases of leptospirosis in Andamans. Indian J. Med. Res. 109. 94-99 (1999)  

23. Murhekar, M.V., Sugunan, A.P., Vijayachari, P., Sharma, S. and Sehgal, S.C. Risk factors in the transmission of leptospiral infection. Indian J. Med. Res. 107: 218 (1998)  

24. Sehgal, S.C., Vijayachari, P. and Subramaniam, V. Evaluation of Leptospira Micro Capsular Agglutination Test (MCAT) for sero-diagnosis of Leptospirosis. Indian J. Med. Res. 106:504 (1997)  

25. Vijayachari, P., Lakshmi, N and Gururaj kumar A. Serodiagnosis of Pulmonary  Aspergillosis. Indian J. Med. Microbiol. 15(1), 23-24 (1997)

26. Lakshmi, N., Gururaj kumar A., Kailashanatha Reddy B., Sreenivasulu Reddy V. and Vijayachari, P. HIV Seroprevalence at Tirupati. Health Administrator 3(2), 29-32 (1992)