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volume 49 issue 5 (october 2015) : 607-611,   Doi: 10.18805/ijar.5571

Blood clotting response test for detecting resistance to second generation anticoagulant bromadiolone in house rat (Rattus rattus)

N
Nancy Garg
N
Neena Singla
1Department of Zoology, Punjab Agricultural University, Ludhiana-141 004, India.
Cite article:- Garg Nancy, Singla Neena (2025). Blood clotting response test for detecting resistance to second generation anticoagulant bromadiolone in house rat (Rattus rattus). Indian Journal of Animal Research. 49(5): 607-611. doi: 10.18805/ijar.5571.

ABSTRACT

Blood clotting response (BCR) test is a faster method with fewer ethical constraints than feed-testing and is widely used to monitor resistance. The second-generation anticoagulant, bromadiolone is being used to control rodents all over India for the last several years. To investigate whether its use has resulted in development of resistance in house rat, Rattus rattus, BCR test was standardized. Plasma samples of 15 rats of both the sexes were pooled separately and diluted with phosphate-buffered saline (pH 7.4). Ten different dilutions of plasma i.e. 4, 5, 6, 8, 10, 20, 40, 60, 80, 100% were tested for Prothrombin Time (PT) and International Normalized Ratio (INR) using rabbit brain thromboplastin reagent in coagulation analyzer. Time to clotting was converted into percent clotting activity (PCA) relative to plasma dilutions, to plot a standard curve. The PT of 15.7 and 17.6 sec for 100% plasma corresponded to INR of 0.8 and 0.9 in male and female rats, respectively. PCA of 22.3% corresponded with PT and INR of 70.3 sec and 3.8, respectively in male rats and PCA of 27.6% corresponded with PT and INR of 63.6 sec and 3.5, respectively in female rats. Data for PT and INR and standard curve for PCA vs PT prepared during present study can be further used to determine current status of bromadiolone resistance in R. rattus populations based on discriminating dose of bromadiolone.

REFERENCES

  1. Beckmann, R. (1988). Mice on the farm. Rural Res., 138: 23-27.
  2. Endepols, S., Klemann, N., Jacob, J. and Buckle, A.P. (2012). Resistance tests and field trials with bromadiolone for the control of Norway rats (Rattus norvegicus) on farms in Westphalia, Germany. Pest Manage. Sci., 68: 348-354.
  3. Garedaghi, Y. and Khaki, A.A. (2014). Prevalence of Gastrointestinal and Blood Parasites of Rodents in Tabriz, Iran, with Emphasis on Parasitic Zoonoses. Crescent J. Med.Biol. Sci., 1: 9-12.
  4. Garg, N. and Singla, N. (2014). Toxicity of second generation anticoagulant bromadiolone against Rattus rattus: individual and sex specific variations. CIB Tech J. Zool., 3(2): 43-48.
  5. Gill, J.E., Kerins, G.M., Langton, S.D. and MacNicoll, A.D. (1993). The development of blood clotting response test for discriminating between difenacoum-resistant and susceptible Norway rats (Rattus norvegicusBerk.).Comp. Biochem. Physiol.,104: 29-36.
  6. Gill, J.E., Kerins, G.M.,Langton, S.D. and MacNicoll, A.D. (1994). Blood clotting response test for bromadiolone resistance in Norway rats. J. Wildl.Manage., 58: 454-461.
  7. Gratz, N.G. (1973). A critical review of currently used single-dose rodenticides. Bull.WHO, 48: 469-477.
  8. Greaves, J.H. and Ayres, P. (1967). Heritable resistance to warfarin in rats. Nature, 215: 877-878.
  9. Herbreteau, V., Bordes, F., Jittapalapong, S., Supputamongkol, Y. and Morand,S. (2012). Rodent-borne diseases in Thailand: targeting rodent carriers and risky habitats. Infect. Ecol. Epidemiol., 2, Article ID 18637.
  10. Hussain, I. (1998). Susceptibility to anticoagulants and the development of physiological resistance in Rattus norvegicus and Bandicota bengalensis. Ph.D. thesis, University of Reading, UK.: 250p.
  11. Kerins, G.M., Dennis, N., Atterby, H., Gill,J.E. and MacNicoll, A.D. (2001). Distribution of resistance to anticoagulant rodenticides in the Norway rat (Rattus norvegicus Berk.) in England 1995–98. In Pelz, H.J., Cowan, D.P., Feare, C.J. and Verlag, F. (Eds.) Advances in Vertebrate Pest Management I. Furth, Germany.: 149-159p.
  12. Lodal, J. (2001). Distribution and levels of anticoagulant resistance in rats (Rattus norvegicus) in Denmark. In Pelz, H.J., Cowan, D.P., Feare, C.J. and Verlag, F. (Eds.) Advances in Vertebrate Pest Management II. Furth, Germany.: 139-148p.
  13. MacNicoll, A.D. and Gill, J.E. (1993). Revised methodology for a blood clotting response test for identification of warfarin- resistant Norway rats (Rattus norvegicus). Bull. OEPP/EPPO, 23: 701-707.
  14. Martin, A.D., Steed, L.C., Redfern, R., Gill, J.E. and Huson, L.W. (1979). Warfarin-resistance genotype determination in the Norway rat (Rattus norvegicus). Lab. Animals, 13: 209-214.
  15. OEPP/EPPO. (1995). Guideline for the evaluation of resistance to plant protection products: Testing rodents for resistance to anticoagulant rodenticides. Bull. OEPP/EPPO, 25: 575-593.
  16. OEEP/EPPO. (1999). Guidelines for the efficacy evaluation of plant protection products: Testing rodents for resistance to anticoagulant rodenticides. Bull. OEPP/EPPO, 198: 131-137.
  17. Pelz, H.J. (2001). Extensive distribution and high frequency of resistance to anticoagulant rodenticides in rat populations from north-western Germany. In Pelz, H.J., Cowan, D.P., Feare C.J. and VerlagF. (Eds.) Advances in Vertebrate Pest Management II. Furth, Germany.: 161-170p.
  18. Pelz, H.J. and Klemann, N. (2004).Rat control strategies in organic pig and poultry production with special reference to rodenticide resistance and feeding behaviour. NJAS, 52: 173-184.
  19. Pelz, H.J., Rost, S., Hunerberg, M., Fregin, A., Heiberg, A.C., Baert, K., MacNicoll, A.D., Prescott, C.V., Walker, A.S., Oldenburg, J. and Muller, C.R. (2005). The genetic basis of resistance to anticoagulants in rodents.Genetics, 170: 1839-1847.
  20. Poller, L. and Hirsh, J. (1996). Laboratory monitoring of anticoagulants. In Poller, L. and Hirsh, J. (Eds.), Oral Anticoagulants, Hodder Headline Group, New York.: 49-64p.
  21. Prescott, C.V. and Buckle, A.P. (2000).Blood-clotting response tests for resistance to diphacinone and chlorophacinone in the Norway Rat (Rattus norvegicus Berk.). Crop Prot.,19: 291-296.
  22. Prescott, C.V., Buckle, A.P., Hussain, I. and Endepols, S. (2007). A standardized BCR resistance test for all anticoagulant rodenticides. Int. J. Pest Manage. 53: 265-272.
  23. Schelotto, F., Hernandez, E., Gonzalez, S., Del Monte, A., Ifran, S., Flores, K., Pardo, L., Parada, D., Filippini. M., Balseiro, V., Geymonat, J.P. and Varela, G. (2012). A ten-year follow-up of human leptospirosis in Uruguay: an unresolved health problem. Rev. Inst. Med. Trop. Sao Paulo, 54: 69-75.
  24. Singla, L.D., Singla, N., Parshad, V.R., Juyal, P.D. and Sood, N.K. (2008). Rodents as reservoir of parasites in India. Integr. Zool., 3: 21-26.
  25. Singla, N. and Babbar, B.K. (2010). Rodent damage and infestation in wheat and rice crop field: district wise analysis in Punjab State. Indian J. Ecol., 37: 184-188.
  26. Singla, N. and Babbar, B.K. (2012). Critical timings of rodenticide bait application for controlling rodents in sugarcane crop grown in situations like Punjab, India. Sugar Tech, 14: 76-82.
  27. Singla, N., Singla, L.D., Gupta, K. and Sood, N.K. (2013). Pathological alterations in natural cases of Capillaria hepatica infection alone and in concurrence with Cysticercus fasciolaris in Bandicota bengalensis. J. Parasit. Dis., 17: 16-20.
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    volume 49 issue 5 (october 2015) : 607-611,   Doi: 10.18805/ijar.5571

    Blood clotting response test for detecting resistance to second generation anticoagulant bromadiolone in house rat (Rattus rattus)

    N
    Nancy Garg
    N
    Neena Singla
    1Department of Zoology, Punjab Agricultural University, Ludhiana-141 004, India.
    Cite article:- Garg Nancy, Singla Neena (2025). Blood clotting response test for detecting resistance to second generation anticoagulant bromadiolone in house rat (Rattus rattus). Indian Journal of Animal Research. 49(5): 607-611. doi: 10.18805/ijar.5571.

    ABSTRACT

    Blood clotting response (BCR) test is a faster method with fewer ethical constraints than feed-testing and is widely used to monitor resistance. The second-generation anticoagulant, bromadiolone is being used to control rodents all over India for the last several years. To investigate whether its use has resulted in development of resistance in house rat, Rattus rattus, BCR test was standardized. Plasma samples of 15 rats of both the sexes were pooled separately and diluted with phosphate-buffered saline (pH 7.4). Ten different dilutions of plasma i.e. 4, 5, 6, 8, 10, 20, 40, 60, 80, 100% were tested for Prothrombin Time (PT) and International Normalized Ratio (INR) using rabbit brain thromboplastin reagent in coagulation analyzer. Time to clotting was converted into percent clotting activity (PCA) relative to plasma dilutions, to plot a standard curve. The PT of 15.7 and 17.6 sec for 100% plasma corresponded to INR of 0.8 and 0.9 in male and female rats, respectively. PCA of 22.3% corresponded with PT and INR of 70.3 sec and 3.8, respectively in male rats and PCA of 27.6% corresponded with PT and INR of 63.6 sec and 3.5, respectively in female rats. Data for PT and INR and standard curve for PCA vs PT prepared during present study can be further used to determine current status of bromadiolone resistance in R. rattus populations based on discriminating dose of bromadiolone.

    REFERENCES

    1. Beckmann, R. (1988). Mice on the farm. Rural Res., 138: 23-27.
    2. Endepols, S., Klemann, N., Jacob, J. and Buckle, A.P. (2012). Resistance tests and field trials with bromadiolone for the control of Norway rats (Rattus norvegicus) on farms in Westphalia, Germany. Pest Manage. Sci., 68: 348-354.
    3. Garedaghi, Y. and Khaki, A.A. (2014). Prevalence of Gastrointestinal and Blood Parasites of Rodents in Tabriz, Iran, with Emphasis on Parasitic Zoonoses. Crescent J. Med.Biol. Sci., 1: 9-12.
    4. Garg, N. and Singla, N. (2014). Toxicity of second generation anticoagulant bromadiolone against Rattus rattus: individual and sex specific variations. CIB Tech J. Zool., 3(2): 43-48.
    5. Gill, J.E., Kerins, G.M., Langton, S.D. and MacNicoll, A.D. (1993). The development of blood clotting response test for discriminating between difenacoum-resistant and susceptible Norway rats (Rattus norvegicusBerk.).Comp. Biochem. Physiol.,104: 29-36.
    6. Gill, J.E., Kerins, G.M.,Langton, S.D. and MacNicoll, A.D. (1994). Blood clotting response test for bromadiolone resistance in Norway rats. J. Wildl.Manage., 58: 454-461.
    7. Gratz, N.G. (1973). A critical review of currently used single-dose rodenticides. Bull.WHO, 48: 469-477.
    8. Greaves, J.H. and Ayres, P. (1967). Heritable resistance to warfarin in rats. Nature, 215: 877-878.
    9. Herbreteau, V., Bordes, F., Jittapalapong, S., Supputamongkol, Y. and Morand,S. (2012). Rodent-borne diseases in Thailand: targeting rodent carriers and risky habitats. Infect. Ecol. Epidemiol., 2, Article ID 18637.
    10. Hussain, I. (1998). Susceptibility to anticoagulants and the development of physiological resistance in Rattus norvegicus and Bandicota bengalensis. Ph.D. thesis, University of Reading, UK.: 250p.
    11. Kerins, G.M., Dennis, N., Atterby, H., Gill,J.E. and MacNicoll, A.D. (2001). Distribution of resistance to anticoagulant rodenticides in the Norway rat (Rattus norvegicus Berk.) in England 1995–98. In Pelz, H.J., Cowan, D.P., Feare, C.J. and Verlag, F. (Eds.) Advances in Vertebrate Pest Management I. Furth, Germany.: 149-159p.
    12. Lodal, J. (2001). Distribution and levels of anticoagulant resistance in rats (Rattus norvegicus) in Denmark. In Pelz, H.J., Cowan, D.P., Feare, C.J. and Verlag, F. (Eds.) Advances in Vertebrate Pest Management II. Furth, Germany.: 139-148p.
    13. MacNicoll, A.D. and Gill, J.E. (1993). Revised methodology for a blood clotting response test for identification of warfarin- resistant Norway rats (Rattus norvegicus). Bull. OEPP/EPPO, 23: 701-707.
    14. Martin, A.D., Steed, L.C., Redfern, R., Gill, J.E. and Huson, L.W. (1979). Warfarin-resistance genotype determination in the Norway rat (Rattus norvegicus). Lab. Animals, 13: 209-214.
    15. OEPP/EPPO. (1995). Guideline for the evaluation of resistance to plant protection products: Testing rodents for resistance to anticoagulant rodenticides. Bull. OEPP/EPPO, 25: 575-593.
    16. OEEP/EPPO. (1999). Guidelines for the efficacy evaluation of plant protection products: Testing rodents for resistance to anticoagulant rodenticides. Bull. OEPP/EPPO, 198: 131-137.
    17. Pelz, H.J. (2001). Extensive distribution and high frequency of resistance to anticoagulant rodenticides in rat populations from north-western Germany. In Pelz, H.J., Cowan, D.P., Feare C.J. and VerlagF. (Eds.) Advances in Vertebrate Pest Management II. Furth, Germany.: 161-170p.
    18. Pelz, H.J. and Klemann, N. (2004).Rat control strategies in organic pig and poultry production with special reference to rodenticide resistance and feeding behaviour. NJAS, 52: 173-184.
    19. Pelz, H.J., Rost, S., Hunerberg, M., Fregin, A., Heiberg, A.C., Baert, K., MacNicoll, A.D., Prescott, C.V., Walker, A.S., Oldenburg, J. and Muller, C.R. (2005). The genetic basis of resistance to anticoagulants in rodents.Genetics, 170: 1839-1847.
    20. Poller, L. and Hirsh, J. (1996). Laboratory monitoring of anticoagulants. In Poller, L. and Hirsh, J. (Eds.), Oral Anticoagulants, Hodder Headline Group, New York.: 49-64p.
    21. Prescott, C.V. and Buckle, A.P. (2000).Blood-clotting response tests for resistance to diphacinone and chlorophacinone in the Norway Rat (Rattus norvegicus Berk.). Crop Prot.,19: 291-296.
    22. Prescott, C.V., Buckle, A.P., Hussain, I. and Endepols, S. (2007). A standardized BCR resistance test for all anticoagulant rodenticides. Int. J. Pest Manage. 53: 265-272.
    23. Schelotto, F., Hernandez, E., Gonzalez, S., Del Monte, A., Ifran, S., Flores, K., Pardo, L., Parada, D., Filippini. M., Balseiro, V., Geymonat, J.P. and Varela, G. (2012). A ten-year follow-up of human leptospirosis in Uruguay: an unresolved health problem. Rev. Inst. Med. Trop. Sao Paulo, 54: 69-75.
    24. Singla, L.D., Singla, N., Parshad, V.R., Juyal, P.D. and Sood, N.K. (2008). Rodents as reservoir of parasites in India. Integr. Zool., 3: 21-26.
    25. Singla, N. and Babbar, B.K. (2010). Rodent damage and infestation in wheat and rice crop field: district wise analysis in Punjab State. Indian J. Ecol., 37: 184-188.
    26. Singla, N. and Babbar, B.K. (2012). Critical timings of rodenticide bait application for controlling rodents in sugarcane crop grown in situations like Punjab, India. Sugar Tech, 14: 76-82.
    27. Singla, N., Singla, L.D., Gupta, K. and Sood, N.K. (2013). Pathological alterations in natural cases of Capillaria hepatica infection alone and in concurrence with Cysticercus fasciolaris in Bandicota bengalensis. J. Parasit. Dis., 17: 16-20.
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