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

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1 Indian J. Anim. Res., 49 (5) 215 : Print ISSN: / Online ISSN: AGRICULTURAL RESEARCH COMMUNICATION CENTRE Blood clotting response test for detecting resistance to second generation anticoagulant bromadiolone in house rat (Rattus rattus) Nancy Garg and Neena Singla* Department of Zoology, Punjab Agricultural University, Ludhiana-141 4, India. Received: Accepted: DOI: /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 phosphatebuffered saline (ph 7.4). Ten different dilutions of plasma i.e. 4, 5, 6, 8, 1, 2, 4, 6, 8, 1% 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 1% plasma corresponded to INR of.8 and.9 in male and female rats, respectively. PCA of 22.3% corresponded with PT and INR of 7.3 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. Key words: Blood clotting response, Bromadiolone, Prothrombin time, Rattus rattus, Resistance. INTRODUCTION Rodents have gained the reputation as one of the most persistent and ubiquitous vertebrate pests affecting human populations. They cause economic problems because of the damage they inflict to agricultural systems (Singla and Babbar, 21; Singla and Babbar, 212), social problems associated with their close proximity to human habitation (Beckmann, 1988), and health problems as carriers of zoonoses (Singla et al., 28; Herbreteau et al., 212; Singla et al., 213). The house rat, Rattus rattus Linnaeus (Rodentia: Muridae), is one of the most commonly encountered and economically important commensal rodents. It not only inflicts heavy damage to stored food but also have nuisance value being a disease carrier or vector (Schelotto et al., 212; Singla et al., 213; Garedaghi and Khaki, 214). Control of rodents currently relies on the use of rodenticides, including first and second generation anticoagulants. First generation anticoagulants, such as warfarin and pindone, were developed in the 195s. More potent second-generation anticoagulants, such as brodifacoum and flocoumafen, were developed from 197s onwards in *Corresponding author s neenasingla1@gmail.com. response to the development of resistance to some of the first generation anticoagulants (Gratz, 1973). Today, resistance to both first- and second-generation anticoagulants has been reported in the United Kingdom, Germany, Denmark, Belgium and the Netherlands (Pelz, 21; Lodal, 21; Kerins et al., 21; Pelz and Klemann, 24; Pelz et al., 25). In India, bromadiolone has been commercially available since 1988 for the control of agricultural and commensal rodents. Since then it is the only second generation anticoagulant being used commonly. Resistance to anticoagulants can develop in a population after 5-1 years of their sustained use. Thus, resistance tests are needed to identify resistant rodent populations. OEEP/EPPO (1995) guidelines propose lethal feeding period test and blood clotting response (BCR) test to detect resistance. Though laboratory feeding tests with anticoagulant bait are the final proof of resistance, but procedures are slow, unhumane and labour intensive (Prescott et al., 27). The BCR tests have several advantages over feeding tests (Gill et al., 1994). They are more quickly performed, are considered to be more

2 68 INDIAN JOURNAL OF ANIMAL RESEARCH humane and are independent of the feeding behaviour of animals. They offer a useful primary tool for routine use for detecting physiological resistance against anticoagulants (Prescott et al., 27). BCR tests involve blood sampling and coagulation time assay of captive wild animals before and after an anticoagulant dose. After dosing, animals that show substantially reduced clotting activity are considered susceptible, while those showing smaller changes in clotting times after dosing are considered resistant (Kerins et al., 1993). The Prothrombin Time (PT) is a test that provides a measure of the extrinsic blood coagulation system, and is commonly used to indicate clinical effects of anticoagulants (Poller and Hirsh, 1996). Since the test is not lethal, rats can be used for breeding or confirmatory tests. The first BCR test, based on the 24 hr prothrombin response to a small dose of warfarin (Greaves and Ayres, 1967) was an entirely ad hoc procedure adopted in the course of a study of Welsh-type warfarin resistance. Later BCR tests were based on the methodology of OEEP/EPPO (1999), where a discriminating dose was calculated that would make susceptible animals respond; failure to respond was taken as evidence of resistance. BCR test methods are available for warfarin (Martin et al., 1979; MacNicoll and Gill, 1993) and second-generation anticoagulants such as difenacoum, bromadiolone, chlorophacinone and diphacinone (Gill et al., 1993 and 1994; Prescott and Buckle, 2) for few rodent species like Rattus norvegicus and Mus domesticus. For these species, it was proposed that an International Normalised Ratio (INR) equal to or greater than 5 be used as the response in the BCR resistance test (Prescott and Buckle, 2). These two rodent species typically have resting clotting times that are equivalent to an INR of 1.3, while Bandicota bengalensis have resting clotting times that are equivalent to INR between 2.4 and 3.3 (Hussain, 1998). The BCR test based on the use of the INR and baselines provided by Prescott et al. (27) are suitable for determining the incidence and, in particular, for assessing the level of resistance in populations of wild Norway rats (Endepols et al., 212). It will therefore be necessary to reconsider the INR as the response for the BCR test for each new species considered. BCR test has not yet been standardized for R. rattus. Further, different thromboplastin reagent test methods are used to assess clotting activity, but their sensitivities vary considerably. Standardization is therefore also desirable for a particular type of thromboplastin reagent. In present studies, rabbit brain thromboplastin was used for standardization of BCR test methodology for R. rattus. MATERIALS AND METHODS The present work was carried out in the Department of Zoology, Punjab Agricultural University, Ludhiana, India, located at 3º55 N latitude and 75º54 E longitude. Collection and maintenance of animals: For present studies, R. rattus of both sexes were trapped live from poultry farms in Ludhiana, India with the help of multi catch rat traps and housed individually in the laboratory cages ( cm). Food and water were provided ad libitum. Food consisted of a mixture of cracked wheat, powdered sugar and groundnut oil (WSO bait) in ratio 96:2:2. Rats were acclimatized for at least 15 days prior to experimentation. Metallic trays were kept under each cage for the collection and disposal of urine and faeces. Approval of Institutional Animal Ethics committee was obtained for the usage of animals. After acclimatization, mature and healthy rats of both sexes (n=15 each) were selected and weighed. Blood collection: Whole blood (up to 1 ml) from retro-orbital plexus of each rat was sampled after mildly anaesthetizing it using diethyl ether. Blood was collected in a clean tube containing 3.2% tri-sodium citrate solution as an anticoagulant and centrifuged at 3 rotations per minute for 15 minutes. The supernatant plasma was collected into a separate tube and used immediately. Calibration of clotting activity: Plasma samples of all the rats of two sexes were pooled separately and ten different dilutions of plasma i.e. 4, 5, 6, 8, 1, 2, 4, 6, 8, 1% were prepared with phosphate-buffered saline (ph 7.4). PT (sec) and INR of each dilution were determined in a coagulation analyzer. Rabbit brain thromboplastin reagent was taken in a clean and dry test cuvette and pre-warmed to 37º C for ten minutes in the incubation chamber of the coagulation analyser. 1µl of plasma was pipetted in a test cuvette and incubated at 37º C for three minutes. 2µl of pre-warmed thromboplastin reagent was added forcibly into the test cuvette by placed in the channel chamber of coagulation analyser. PT and INR for each dilution of plasma of male and female rats were recorded thrice and the mean of three observations was calculated. Data on PT for both the sexes were converted to percent coagulation activity (PCA) by considering 1% plasma having 1% PCA. A standard curve was derived by plotting time to clotting against PCA at each plasma concentration. RESULTS AND DISCUSSION Data for standard curve of clotting times for male and female rat plasma diluted with phosphate-buffered saline is summarized in Tables 1 and 2. For different dilutions of plasma, PT ranged from 15.7 to 7.3 sec in male rats and

3 from 17.6 to 63.6 sec in female rats with corresponding INR of in male rats and from.9 to 3.5 in female rats. The PT of 15.7 and 17.6 sec for 1% plasma corresponded to INR of.8 and.9 in male and female rats, respectively. With decreasing plasma concentrations, PT increased in case of both male and female rats (Figures 1 and 2). INR was also found increased with decreasing plasma concentrations in case of both male and female rats (Tables 1 and 2). With increasing PT and decreasing plasma concentration, the PCA decreased in both male and female rats (Tables 1 and 2). So, the PT and INR will be extensively prolonged in a susceptible rat while PCA will be decreased because with dilution of plasma, the concentration of active clotting factors decreases and reduces the ability of blood to clot, similar to the effect of anticoagulants on blood. During present studies, the INR of R. rattus at 1% plasma and PCA was found low as compared to INR reported for R. norvegicus, M. domesticus and Bandicota bengalensis (Hussain, 1998). To generate dose-response data it is necessary to specify the coagulation time that will be regarded as a response, indicating that the animals coagulation system had been compromised. The effects of both may be expressed in terms of PCA. For R. norvegicus, an animal is considered to be susceptible if, after a specified period of dosing, its plasma PCA is less than 17% (Martin et al., 1979; MacNicoll and Gill, 1993; Prescott and Buckle, 2) or 1% (Gill et al., 1994; Gill et al., 1993). In this rat species, PCA of 17% Volume 49 Issue 5 (October 215) corresponded approximately with an INR of 5 using Diagen rabbit brain thromboplastin reagent (Prescott et al., 27). In present studies, PCA of 22.3% corresponded with PT and INR of 7.3 sec and 3.8, respectively in male rats and PCA of 27.6% corresponded with PT and INR of 63.6 sec and TABLE 1: Blood clotting time (PT), international normalized ratio (INR) and percent coagulation activity (PCA) for different dilutions of plasma of male Rattus rattus. Plasma (%) Average Average PCA INR TABLE 2: Blood clotting time (PT), international normalized ratio (INR) and percent coagulation activity (PCA) for different dilutions of plasma of female Rattus rattus. Plasma (%) Average Average PCA INR Plasma (%) FIG 1: Prothrombin time corresponding to different plasma concentrations in Male Rattus rattus Plasma (%) FIG 2: Prothrombin time corresponding to different plasma concentrations in female Rattus rattus PT (Sec) PCA FIG 3: Standard curve for PCA vs PT in male Rattus rattus.

4 61 INDIAN JOURNAL OF ANIMAL RESEARCH PT (Sec) PCA FIG 4: Standard curve for PCA vs PT in female Rattus rattus. 3.5, respectively in female rats. From the standard curve for PCA vs PT obtained in present studies, the calibration of PT will be poor at still lower plasma concentrations and PCA values (Fig 3,4). Prescott and Buckle (2) and Prescott et al. (27) also found calibration curve replication to be poor when determined particularly at low dilutions. The LD 5 values of bromadiolone against R. rattus have been reported to be 2.1 and 3.67 g/1g bw of.5% bromadiolone bait (prepared using.25% bromadiolone powder) for male and female rats, respectively (Garg and Singla, 214). 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 twice the LD 5 value of bromadiolone. ACKNOWLEDGEMENTS The authors are thankful to University Grants Commission, New Delhi, India, for providing financial assistance and Professor and Head of the Department of Zoology, Punjab Agricultural University, Ludhiana, India, for the facilities provided. REFERENCES Beckmann, R. (1988). Mice on the farm. Rural Res., 138: Endepols, S., Klemann, N., Jacob, J. and Buckle, A.P. (212). Resistance tests and field trials with bromadiolone for the control of Norway rats (Rattus norvegicus) on farms in Westphalia, Germany. Pest Manage. Sci., 68: Garedaghi, Y. and Khaki, A.A. (214). Prevalence of Gastrointestinal and Blood Parasites of Rodents in Tabriz, Iran, with Emphasis on Parasitic Zoonoses. Crescent J. Med.Biol. Sci., 1: Garg, N. and Singla, N. (214). Toxicity of second generation anticoagulant bromadiolone against Rattus rattus: individual and sex specific variations. CIB Tech J. Zool., 3(2): 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.,14: 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: Gratz, N.G. (1973). A critical review of currently used single-dose rodenticides. Bull.WHO, 48: Greaves, J.H. and Ayres, P. (1967). Heritable resistance to warfarin in rats. Nature, 215: Herbreteau, V., Bordes, F., Jittapalapong, S., Supputamongkol, Y. and Morand,S. (212). Rodent-borne diseases in Thailand: targeting rodent carriers and risky habitats. Infect. Ecol. Epidemiol., 2, Article ID 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.: 25p. Kerins, G.M., Dennis, N., Atterby, H., Gill,J.E. and MacNicoll, A.D. (21). Distribution of resistance to anticoagulant rodenticides in the Norway rat (Rattus norvegicus Berk.) in England In Pelz, H.J., Cowan, D.P., Feare, C.J. and Verlag, F. (Eds.) Advances in Vertebrate Pest Management I. Furth, Germany.: p. Lodal, J. (21). 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.: p. MacNicoll, A.D. and Gill, J.E. (1993). Revised methodology for a blood clotting response test for identification of warfarinresistant Norway rats (Rattus norvegicus). Bull. OEPP/EPPO, 23: 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: OEPP/EPPO. (1995). Guideline for the evaluation of resistance to plant protection products: Testing rodents for resistance to anticoagulant rodenticides. Bull. OEPP/EPPO, 25:

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