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Prophylactic activity of imidocarb against experimental infection with Babesia canis
veterinary parasitology ELSEVIER
Veterinary Parasitology63 (1996) 195-198
Prophylactic activity of imidocarb against experimental infection with Babesia canis F. Vercammen a,*, R. De Deken a, L. Maes b a VeterinaryDepartment, Institute of Tropical Medicine, Nationalestraat 155, 2000 Antwerp, Belgium b Department of Parasitology, Janssen Research Foundation, Turnhoutseweg30, 2340 Beerse, Belgium
Received 11 July 1995;accepted7 September 1995
Abstract A single subcutaneous injection of imidocarb at 6 mg kg-1 gave 2 weeks protection against experimental inoculation of Babesia canis merozoites in Beagle dogs. The prophylactic effect was evaluated by daily measurements of parasitaemia, rectal temperature, haematological values and specific antibodies. Keywords: Babesia canis; Dog; Control methods-Protozoa;Imidocarb;Prophylaxis
1. Introduction The prophylactic effect of imidocarb on canine babesiosis has been stated by some and refuted by others. Euze'by et al. (1980) found a prophylactic effect of 6 weeks when the drug was used at 3 mg kg -1 , whereas Uilenberg et al. (1981) found no prophylactic effect at all at the dose of 6 mg kg-1. The manufacturer of the drug claims a prophylactic period of 3 to 6 weeks (mean of 4 weeks). The present work was carried out to elaborate whether imidocarb offers any prophylactic effect against canine babesiosis.
2. Materials and methods 2.1. Experimental infections
Dogs were inoculated intraperitoneally with whole blood cryostabilates of Babesia canis, isolated in 1987 from a naturally infected dog in Gibraltar. Before inoculation, the
* Corresponding author. 0304-4017/96/$15.00 © 1996Elsevier Science B.V. All rights reserved SSDI 0304-401 7(95)0090 1-9
196
F. Vercammen et al. / Veterinary Parasitology 63 (1996) 195-198
stabilates were thawed rapidly in water at 37°C. Each inoculum contained about 1.5 × 105 merozoites.
2.2. Experimental design In an initial study, four male Beagle dogs (1.5 years old) were used. The dogs Nos. 1, 2 and 3 were administered imidocarb (Imizol ~, Mallinckrodt) at 6 mg kg-L subcutaneously, respectively 1, 2 and 3 weeks before inoculation. Dog No. 4 served as untreated control. Parasitaemia, rectal temperature and haematology were monitored daily for 18 days post-inoculation (Dpi). Serology was followed weekly for 6 months, using an indirect immunofluorescent antibody test (IFAT) (Vercammen et al., 1995). Parasitaemia was estimated by counting l 0 4 red blood cells (RBC) on May-Grfinwald-Giemsa stained blood-smears. The smears were made from the blood of the jugular vein and the ear capillaries. When less than 0.01% of the red blood cells were parasitized, the micro-haematocrit concentration technique was used to detect infection (Gothe et al., 1987). Dogs developing fever above 40.0°C were treated with 10 mg kg-~ oxytetracycline (Engemycine ® 10% DD, Mycofarm Belga) subcutaneously, once daily until temperature fell below 39.5°C. Values of packed cell volume (PCV, reference: 41.656.6%), haemoglobin (HB, reference: 13.8-19.1 g d1-1) and thrombocytes (reference: 195-440 10 3 /zl- l) were obtained by the blood analyser Technicon H-1E. In a follow-up confirmation study, three male Beagle dogs (1.5 years old) were used. The dogs Nos. 5 and 6 were administered imidocarb at 6 mg kg-I 2 weeks before inoculation. Dog No. 7 served as untreated control. After inoculation, the same protocol as in the initial study was used.
3. Results
Parasites could never be detected in the dogs that were treated 1 week (Dog 1) and 2 weeks (Dogs 2, 5 and 6) before inoculation. In the challenge control dogs (Nos. 4 and 7) and the one treated 3 weeks (Dog 3) before inoculation, parasites became detectable from 4 Dpi onwards to reach a maximum parasitaemia of 0.12%, 0.009% and 0.02%, respectively. These dogs developed fever from 4 Dpi onwards. In Dog 3 treatment with oxytetracycline was carried out on Days 5 and 6 and again on Day 15 after infection. Dog 4 received treatment on Day 6 after infection and Dog 7 was not treated. Each treatment immediately reduced fever and parasitaemia. The evolution of PCV, HB and thrombocytes is shown in Fig. 1. Only Dogs 3, 4 and 7 showed decreased values. The maximum fall in PCV, HB and thrombocytes was on average 32.1 + 8.5%, 33.3 __+7.1% and 90.1 + 0.1%, respectively. Specific antibodies could be detected from 11 Dpi onwards in Dogs 4 and 7, and from 18 Dpi onwards in Dog 3. Serology of these three dogs remained positive during the whole study period with maximum titres of 1/512. The Dogs 1, 2, 5 and 6 remained serologically negative (titre below 1/32).
197
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Fig. l. Average packed cell volume (%), haemoglobin (g dl- l ) and thrombocytes( × 10 9 1- I) in healthy dogs (1, 2, 5, 6) and in sick dogs (3, 4, 7) after challenge with Babesia canis. Dog 1, Dogs 2, 5 and 6 and Dog 3 were challenged after receiving an imidocarb injection respectively 1, 2 and 3 weeks before. Dogs 4 and 7 were the challenge control animals. 4. Discussion The Dogs (Nos. 1, 2, 5 and 6) that were inoculated within 2 weeks of imidocarb administration did not become infected: they showed no symptoms, no parasitaemia and no antibody response and their haematological values remained within normal limits. Dog No. 3, inoculated 3 weeks after imidocarb injection, showed the same level of morbidity as the challenge control dogs (Nos. 4 and 7): all three dogs developed high
198
F. Vercammen et a l . / Veterinary Parasitology 63 (1996) 195-198
fever and parasitaemia after an incubation period of 3 days. These dogs presented a rather pronounced hypochromic anaemia with a remarkable fall in their thrombocyte values. A fall in the values of PCV, HB and thrombocytes is highly characteristic in the pathology of canine babesiosis (Jacobson and Clark, 1994). Seroconversion occurred after 11 to 18 days and the antibodies remained detectable for at least 6 months. Under the conditions of the present study, a single dose of imidocarb at 6 mg kg- 1 offers prophylaxis against B. canis infection for a period of 2 weeks. This period of chemoprevention is considerably shorter than the one mentioned by EuzEby et al. (1980), but contrasts with the results obtained by Uilenberg et al. (1981). The reason for these conflicting observations might be related to differences in the susceptibility of the dogs, the virulence of the parasite strain, the inoculation dose or route and the parasite development stage that was inoculated. This study confirms that dogs can be protected against babesiosis with imidocarb for a period in the range of 2 weeks. The latter can be of practical relevance when people take dogs into endemic infected areas during their holiday period.
Acknowledgements The authors wish to thank Prof. Dr S. Geerts, head of the Veterinary Department of the Institute of Tropical Medicine, for his comments and Mr R. Baelmans and Mrs R. Beudeker for their technical assistance.
References EuzEby, J., Moreau, Y., Chauve, C., Gevrey, J. and Gauthey, M., 1980. ExpErimentation des propri&Es antipiroplasmiques de I'imidocarb sur Babesia canis, agent de la piroplasmose canine en Europe. Bull. Acad. VEt. France, 53: 475-480. Gothe, R., Kraiss, A. and Kraft, W., 1987. Eine importierte Krankheit: Die Babesia canis- und Babesia gibsoni-lnfektion des Hundes. Kleintierpraxis, 32: 93-110. Jacobson, L. and Clark, I.A., 1994. The pathophysiology of canine babesiosis: new approaches to an old puzzle. J.S. Afr. Vet. Assoc., 65: 134-145. Uilenberg, G., Verdiesen, P.A.H.M. and Zwart, D., 1981. Imidocarb: A chemoprophylactic experiment with Babesia canis. Vet. Q., 3:118-123. Vercammen, F., De Deken, R. and Maes, L., 1995. Clinical and serological observations on experimental infections with Babesia canis and its diagnosis using the IFAT. Parasite, in press.
Veterinary Parasitology63 (1996) 195-198
Prophylactic activity of imidocarb against experimental infection with Babesia canis F. Vercammen a,*, R. De Deken a, L. Maes b a VeterinaryDepartment, Institute of Tropical Medicine, Nationalestraat 155, 2000 Antwerp, Belgium b Department of Parasitology, Janssen Research Foundation, Turnhoutseweg30, 2340 Beerse, Belgium
Received 11 July 1995;accepted7 September 1995
Abstract A single subcutaneous injection of imidocarb at 6 mg kg-1 gave 2 weeks protection against experimental inoculation of Babesia canis merozoites in Beagle dogs. The prophylactic effect was evaluated by daily measurements of parasitaemia, rectal temperature, haematological values and specific antibodies. Keywords: Babesia canis; Dog; Control methods-Protozoa;Imidocarb;Prophylaxis
1. Introduction The prophylactic effect of imidocarb on canine babesiosis has been stated by some and refuted by others. Euze'by et al. (1980) found a prophylactic effect of 6 weeks when the drug was used at 3 mg kg -1 , whereas Uilenberg et al. (1981) found no prophylactic effect at all at the dose of 6 mg kg-1. The manufacturer of the drug claims a prophylactic period of 3 to 6 weeks (mean of 4 weeks). The present work was carried out to elaborate whether imidocarb offers any prophylactic effect against canine babesiosis.
2. Materials and methods 2.1. Experimental infections
Dogs were inoculated intraperitoneally with whole blood cryostabilates of Babesia canis, isolated in 1987 from a naturally infected dog in Gibraltar. Before inoculation, the
* Corresponding author. 0304-4017/96/$15.00 © 1996Elsevier Science B.V. All rights reserved SSDI 0304-401 7(95)0090 1-9
196
F. Vercammen et al. / Veterinary Parasitology 63 (1996) 195-198
stabilates were thawed rapidly in water at 37°C. Each inoculum contained about 1.5 × 105 merozoites.
2.2. Experimental design In an initial study, four male Beagle dogs (1.5 years old) were used. The dogs Nos. 1, 2 and 3 were administered imidocarb (Imizol ~, Mallinckrodt) at 6 mg kg-L subcutaneously, respectively 1, 2 and 3 weeks before inoculation. Dog No. 4 served as untreated control. Parasitaemia, rectal temperature and haematology were monitored daily for 18 days post-inoculation (Dpi). Serology was followed weekly for 6 months, using an indirect immunofluorescent antibody test (IFAT) (Vercammen et al., 1995). Parasitaemia was estimated by counting l 0 4 red blood cells (RBC) on May-Grfinwald-Giemsa stained blood-smears. The smears were made from the blood of the jugular vein and the ear capillaries. When less than 0.01% of the red blood cells were parasitized, the micro-haematocrit concentration technique was used to detect infection (Gothe et al., 1987). Dogs developing fever above 40.0°C were treated with 10 mg kg-~ oxytetracycline (Engemycine ® 10% DD, Mycofarm Belga) subcutaneously, once daily until temperature fell below 39.5°C. Values of packed cell volume (PCV, reference: 41.656.6%), haemoglobin (HB, reference: 13.8-19.1 g d1-1) and thrombocytes (reference: 195-440 10 3 /zl- l) were obtained by the blood analyser Technicon H-1E. In a follow-up confirmation study, three male Beagle dogs (1.5 years old) were used. The dogs Nos. 5 and 6 were administered imidocarb at 6 mg kg-I 2 weeks before inoculation. Dog No. 7 served as untreated control. After inoculation, the same protocol as in the initial study was used.
3. Results
Parasites could never be detected in the dogs that were treated 1 week (Dog 1) and 2 weeks (Dogs 2, 5 and 6) before inoculation. In the challenge control dogs (Nos. 4 and 7) and the one treated 3 weeks (Dog 3) before inoculation, parasites became detectable from 4 Dpi onwards to reach a maximum parasitaemia of 0.12%, 0.009% and 0.02%, respectively. These dogs developed fever from 4 Dpi onwards. In Dog 3 treatment with oxytetracycline was carried out on Days 5 and 6 and again on Day 15 after infection. Dog 4 received treatment on Day 6 after infection and Dog 7 was not treated. Each treatment immediately reduced fever and parasitaemia. The evolution of PCV, HB and thrombocytes is shown in Fig. 1. Only Dogs 3, 4 and 7 showed decreased values. The maximum fall in PCV, HB and thrombocytes was on average 32.1 + 8.5%, 33.3 __+7.1% and 90.1 + 0.1%, respectively. Specific antibodies could be detected from 11 Dpi onwards in Dogs 4 and 7, and from 18 Dpi onwards in Dog 3. Serology of these three dogs remained positive during the whole study period with maximum titres of 1/512. The Dogs 1, 2, 5 and 6 remained serologically negative (titre below 1/32).
197
F. Vercammen et al. / Veterinary Parasitology 63 (1996) 195-198 Paoked
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Fig. l. Average packed cell volume (%), haemoglobin (g dl- l ) and thrombocytes( × 10 9 1- I) in healthy dogs (1, 2, 5, 6) and in sick dogs (3, 4, 7) after challenge with Babesia canis. Dog 1, Dogs 2, 5 and 6 and Dog 3 were challenged after receiving an imidocarb injection respectively 1, 2 and 3 weeks before. Dogs 4 and 7 were the challenge control animals. 4. Discussion The Dogs (Nos. 1, 2, 5 and 6) that were inoculated within 2 weeks of imidocarb administration did not become infected: they showed no symptoms, no parasitaemia and no antibody response and their haematological values remained within normal limits. Dog No. 3, inoculated 3 weeks after imidocarb injection, showed the same level of morbidity as the challenge control dogs (Nos. 4 and 7): all three dogs developed high
198
F. Vercammen et a l . / Veterinary Parasitology 63 (1996) 195-198
fever and parasitaemia after an incubation period of 3 days. These dogs presented a rather pronounced hypochromic anaemia with a remarkable fall in their thrombocyte values. A fall in the values of PCV, HB and thrombocytes is highly characteristic in the pathology of canine babesiosis (Jacobson and Clark, 1994). Seroconversion occurred after 11 to 18 days and the antibodies remained detectable for at least 6 months. Under the conditions of the present study, a single dose of imidocarb at 6 mg kg- 1 offers prophylaxis against B. canis infection for a period of 2 weeks. This period of chemoprevention is considerably shorter than the one mentioned by EuzEby et al. (1980), but contrasts with the results obtained by Uilenberg et al. (1981). The reason for these conflicting observations might be related to differences in the susceptibility of the dogs, the virulence of the parasite strain, the inoculation dose or route and the parasite development stage that was inoculated. This study confirms that dogs can be protected against babesiosis with imidocarb for a period in the range of 2 weeks. The latter can be of practical relevance when people take dogs into endemic infected areas during their holiday period.
Acknowledgements The authors wish to thank Prof. Dr S. Geerts, head of the Veterinary Department of the Institute of Tropical Medicine, for his comments and Mr R. Baelmans and Mrs R. Beudeker for their technical assistance.
References EuzEby, J., Moreau, Y., Chauve, C., Gevrey, J. and Gauthey, M., 1980. ExpErimentation des propri&Es antipiroplasmiques de I'imidocarb sur Babesia canis, agent de la piroplasmose canine en Europe. Bull. Acad. VEt. France, 53: 475-480. Gothe, R., Kraiss, A. and Kraft, W., 1987. Eine importierte Krankheit: Die Babesia canis- und Babesia gibsoni-lnfektion des Hundes. Kleintierpraxis, 32: 93-110. Jacobson, L. and Clark, I.A., 1994. The pathophysiology of canine babesiosis: new approaches to an old puzzle. J.S. Afr. Vet. Assoc., 65: 134-145. Uilenberg, G., Verdiesen, P.A.H.M. and Zwart, D., 1981. Imidocarb: A chemoprophylactic experiment with Babesia canis. Vet. Q., 3:118-123. Vercammen, F., De Deken, R. and Maes, L., 1995. Clinical and serological observations on experimental infections with Babesia canis and its diagnosis using the IFAT. Parasite, in press.