Scand. J. Immunol. 36, Suppl. 11, 34-36, 1992
Experimental Infection with a Haemorrhage-Causing Trypanosoma vivax in N'Dama and Boran Cattle D. J. L. WILLIAMS, L. L. LOGAN-HENFREY, E. AUTHIE, C. SEELY & F. McODIMBA
Williams DJL, Logan-Henfrey LL, Authie E, Seely C, McOdimba F. Experimental Infection with a Haemorrhage-Causing Trypanosoma vivax in N'Dama and Boran Cattle. Scand J Immunol I992;36(Suppl.ll):34-6 N'Dama cattle control experimental infections with clones of Trypanosoma congolense of varying degrees of virulence, but nothing is known about their capacity to control infections caused by highly virulent. East African stocks of T. vivax. Thus four N'Damas and four trypanosusceptible Borans were infected with a tsetse-transmitted stock of T. vivax IL2337. In Ayrshire cattle this stock is known to cause severe haemorrhagic disease. No differences were observed in the parasitaemia between the two groups. Both groups became anaemic. The mean packed cell volume fell to 16.8 ±5.0% in the N'Dama cattle and to 24.2 + 2.2% in the Borans on day 26 post infection. These differences were not significant. Antibody responses to invariant trypanosome antigens were analysed. No differences were observed between the groups in the pattern of recognition or the isotype elicited. Antibody bound to the surface of erythrocytes was occasionally detected. No anti-platelet activity was observed. The results show that N'Dama cattle, which are known to be resistant to disease caused by T. congolense and by T. vivax stocks from West Africa, were highly susceptible to an infection of T. vivax which causes acute haemorrhagic disease. D. J. L. Williams. International Laboratory for Research on Animal Diseases. P.O. Box 30709. Nairobi. Kenya
Trypanosomiasis is considered to be one of the most important disease constraints on livestock production in sub-Saharan Africa. Some taurine breeds of cattle, notably the N'Dama, the Boaule and the Muturu, have survived in trypanosomiasis-endemic areas for nearly 5000 years [1]. These trypanotolerant breeds resist the pathogenic effects of the infection. They limit parasitaemia and anaemia and remain productive under trypanosomiasis challenge [2]. The N'Dama breed has been widely studied. Results from cattle exposed to natural challenge suggest that N'Damas control Trypanosoma brucei, T. congolense, T. vivax and mixed infections more effectively than Zebu breeds [3]. However, these data are difficult to interpret since in the field the weight of challenge may vary, and cattle are frequently subjected to multiple challenges with mixed infections and may have other, intercurrent, parasitic infections. Thus challenges under controlled experimental 34
conditions are required to fully characterize trypanotolerance. Whilst numerous experimental challenges in N'Dama and Zebu cattle have been carried out using T. congolense, there are few reports in which well-characterized stocks of T. vivax have been used. Therefore we selected a stock of T. vivax, which is known to cause an acute, haemorrhagic disease, and challenged a group of N'Dama and Boran cattle. Their response to infection was assessed.
M A T E R I A L S AND M E T H O D S Four male N'Damas and four male Borans, aged 3 years old, were infected by exposure to bites from 10 infected Glossina morsitans centralis which had been fed on a goat previously infected with 7". vivax IL2337. T. vivax 1L2337 was derived from stock EATRO 2430, isolated from a cow with acute haemorrhagic syndrome in the Kilifi district of Kenya in 1978 [4].
Experimental Infection with Trypanosoma vivax The experiment was designed so that if the packed cell volume (PCV) of any animal fell below 15%, it was treated with 7 mg k g " ' diminazene aceturate (Berenil; Hoechst, Frankfurt, Germany). Blood samples were collected daily from the jugular vein into 2 mg ml"' ethylene diaminetetra-aeetate potassium salt (EDTA) for parasitological and haematological examination. Trypanosomes were enumerated by the buffy coat/phase contrast technique [5]. The PCV was determined using a microhaematocrit centrifuge (Damon/IEC Division, Needham Heights, USA) and a haematocrit reader (Gelman Hawksley Ltd, UK). Platelets were counted using a haemocytometer after blood was diluted 1:20 in 1% ammonium oxalate in distilled water. Erythrocyte and total leucocyte counts were determined using a Coulter Counter (Coulter Electronics Ltd, Harpenden, Herts., UK). Differential leucocyte counts were done on thin blood smears stained with Giemsa stain (Merck, Darmstadt, Germany). The absolute numbers of leucocytes, eosinophils, lymphocytes, neutrophils, basophils and monocytes per ml of blood were obtained by using the differential white cell count percentage and the total leucocyte count. Antibody responses to invariant 7". vivax antigens were measured using the Western blot technique described previously [6]. Anti-red blood cell antibodies were measured using a fluorescence activated cell sorter (FACScan; Becton-Dickinson, Belgium) or by ELISA. according to the methods of Assoku & Gardiner [7].
35
changes in the leucocyte count were caused by the alterations in the lymphocyte counts. No neutropaenia was detected; rather, a neutrophilia was detected on day 55 in the N'Damas and on days 7, 10 and 48 in the Borans. Antibodies bound to the surface of erythrocytes were detected in one Boran 15 dpi. These were of the IgM isotype. On day 24 pi all eight animals had antibodies bound to their erythrocyte surfaces. Both IgM and IgG were detected. By 32 dpi all the animals were negative. No antierythrocyte antibodies or anti-platelet antibodies were detected in sera from these cattle. Antibody responses to invariant trypanosome antigens were analysed. An immunodominant 69-kDa antigen was identified which elicited an IgM and an IgG response in both groups of animals. This antigen is probably homologous to a 69-kDa immunodominant antigen of T. congolense. A wide variety of other invariant antigens were also identified which elicited IgG antibodies. The majority of these antibodies were ofthe IgG 1 class but those of the IgG2 class were also detected. All antibodies to invariant antigens were detected 11-18 dpi.
RESULTS All eight animals became parasitaemic 8 to 9 days post infection (dpi). The peak of parasitaemia was reached 15 to 16 dpi and the level of parasitaemia decreased from 24 dpi until the experiment was terminated on 56 dpi. The PCV decreased from 7 dpi onwards and reached the lowest recorded level 25 dpi in the Borans and 26 dpi in the N'Damas. The lowest mean PCV was 16.8 ±5.0% in the N'Damas and 24.25 + 2.2% in the Borans. These differences were not significantly different. One N'Dama, whose PCV was 13% on day 26, was treated with diminazene aceturate. The remaining seven animals recovered without drug intervention. The reticulocyte counts reached a peak 28 to 30 dpi. The counts were higher in the N'Damas. The platelet count decreased in both groups and a thrombocytopaenia was observed 14-20 dpi. The counts remained below the preinfection level until the experiment was terminated 56 dpi. The leucocyte count decreased in both groups; the lowest count was detected 14 dpi when the N'Damas were leueopaenic. Thereafter the counts increased. A leucocytosis was observed 34 dpi in the Borans and 27 dpi in the N'Damas. The
DISCUSSION The results presented here show that four N'Dama and four Boran cattle were equally susceptible to challenge with T. uivax IL2337. Both the N'Damas and Borans experienced high levels of parasitaemia, an acute drop in PCV and a pronounced thrombocytopaenia. The ability of N'Damas to control infection of T. brucei, T. congolense and T. viva.x more efficiently than trypanosusceptible cattle has been widely reported [2, 8, 9]. The results presented in this paper are in direct contrast to those in previous publications and suggest that there was no difference between trypanotolerant and trypanosusceptible cattle in their ability to control this particular stock of T. vivax. This stock of T. vivax causes a severe disease with exaggerated pathological features which include rapid onset of anaemia, thrombocytopaenia and disseminated intravascular coagulation. However, the infection is short-lived and 7 out of the 8 cattle recovered from the infection without chemotherapeutic intervention. Thus the aetiology of the disease associated with this particular stock is
36
D. J. L. Williams et al.
very different to that described for other T. vivax and T. congolense isolates. Our results suggest that mechanisms other than those involved in trypanotolerance may be controlling this infection. ACKNOWLEDGMENT This is ILRAD Publication number 1050.
REFERENCES 1 Payne WJA. The origin of domestic cattle in Africa. Empire J Exp Agric 1964;32:97-113. 2 Murray M, Morrison Wl, Whiteiaw DD. Host susceptibility to African trypanosomiasis: trypanotolerance. Adv Parasitol 1982;21:I-68. 3 Trail JCM, D'leteren GDM, Teale AJ. Trypanotolerance and the value of conserving livestock genetic resources. Genome 1989;31:805-12. 4 Gardiner PR, Assoku RKG, Whiteiaw DD, Murray M. Haemorrhagic lesions resulting from Trypanosoma vivax infection in Ayrshire cattle. Vet Parasitol 1989;39:187-97.
5 Paris J, Murray M, McOdimba F. A comparative evaluation ofthe parasitological techniques currently available for the diagnosis of African Trypanosomiasis in cattle. Acta Tropica I982;39:307-36. 6 Assoku RKG, Gardiner PR. Detection of antibodies to platelets and erythrocytes during infection with haemorrhage-causing Trypanosoma vivax in Ayrshire cattle. Vet Parasitol 1989;31:199-216. 7 Authie E, Muteti DK, Williams DJL. Antibody responses to invariant antigens of Trypanosoma congolense in cattle of differing susceptibility to trypanosomiasis. Parasite Immunol, (in press). 8 Paling RW, Moloo SK, Scott JR, Gettinby G, McOdimba FA, Murray M. Susceptibility of N'Dama and Boran cattle to sequential challenges with tsetse-transmitted clones of Trypanosoma congolense. Parasite Immunol l991;I3:427-45. 9 Saror DI, Ilemobade AA, Nuru S. The hematology of N'Dama and Zebu cattle experimentally infected with Trypanosoma vivax. In: Proceedings ofthe 16th Meeting of the International Scientific Council for Trypanosomiasis Research and Control, Yaounde, Cameroon 1979. Nairobi, Kenya: Organization of African Unity, Scientific Technical and Research Commission, 1979:287-94.
Experimental Infection with a Haemorrhage-Causing Trypanosoma vivax in N'Dama and Boran Cattle D. J. L. WILLIAMS, L. L. LOGAN-HENFREY, E. AUTHIE, C. SEELY & F. McODIMBA
Williams DJL, Logan-Henfrey LL, Authie E, Seely C, McOdimba F. Experimental Infection with a Haemorrhage-Causing Trypanosoma vivax in N'Dama and Boran Cattle. Scand J Immunol I992;36(Suppl.ll):34-6 N'Dama cattle control experimental infections with clones of Trypanosoma congolense of varying degrees of virulence, but nothing is known about their capacity to control infections caused by highly virulent. East African stocks of T. vivax. Thus four N'Damas and four trypanosusceptible Borans were infected with a tsetse-transmitted stock of T. vivax IL2337. In Ayrshire cattle this stock is known to cause severe haemorrhagic disease. No differences were observed in the parasitaemia between the two groups. Both groups became anaemic. The mean packed cell volume fell to 16.8 ±5.0% in the N'Dama cattle and to 24.2 + 2.2% in the Borans on day 26 post infection. These differences were not significant. Antibody responses to invariant trypanosome antigens were analysed. No differences were observed between the groups in the pattern of recognition or the isotype elicited. Antibody bound to the surface of erythrocytes was occasionally detected. No anti-platelet activity was observed. The results show that N'Dama cattle, which are known to be resistant to disease caused by T. congolense and by T. vivax stocks from West Africa, were highly susceptible to an infection of T. vivax which causes acute haemorrhagic disease. D. J. L. Williams. International Laboratory for Research on Animal Diseases. P.O. Box 30709. Nairobi. Kenya
Trypanosomiasis is considered to be one of the most important disease constraints on livestock production in sub-Saharan Africa. Some taurine breeds of cattle, notably the N'Dama, the Boaule and the Muturu, have survived in trypanosomiasis-endemic areas for nearly 5000 years [1]. These trypanotolerant breeds resist the pathogenic effects of the infection. They limit parasitaemia and anaemia and remain productive under trypanosomiasis challenge [2]. The N'Dama breed has been widely studied. Results from cattle exposed to natural challenge suggest that N'Damas control Trypanosoma brucei, T. congolense, T. vivax and mixed infections more effectively than Zebu breeds [3]. However, these data are difficult to interpret since in the field the weight of challenge may vary, and cattle are frequently subjected to multiple challenges with mixed infections and may have other, intercurrent, parasitic infections. Thus challenges under controlled experimental 34
conditions are required to fully characterize trypanotolerance. Whilst numerous experimental challenges in N'Dama and Zebu cattle have been carried out using T. congolense, there are few reports in which well-characterized stocks of T. vivax have been used. Therefore we selected a stock of T. vivax, which is known to cause an acute, haemorrhagic disease, and challenged a group of N'Dama and Boran cattle. Their response to infection was assessed.
M A T E R I A L S AND M E T H O D S Four male N'Damas and four male Borans, aged 3 years old, were infected by exposure to bites from 10 infected Glossina morsitans centralis which had been fed on a goat previously infected with 7". vivax IL2337. T. vivax 1L2337 was derived from stock EATRO 2430, isolated from a cow with acute haemorrhagic syndrome in the Kilifi district of Kenya in 1978 [4].
Experimental Infection with Trypanosoma vivax The experiment was designed so that if the packed cell volume (PCV) of any animal fell below 15%, it was treated with 7 mg k g " ' diminazene aceturate (Berenil; Hoechst, Frankfurt, Germany). Blood samples were collected daily from the jugular vein into 2 mg ml"' ethylene diaminetetra-aeetate potassium salt (EDTA) for parasitological and haematological examination. Trypanosomes were enumerated by the buffy coat/phase contrast technique [5]. The PCV was determined using a microhaematocrit centrifuge (Damon/IEC Division, Needham Heights, USA) and a haematocrit reader (Gelman Hawksley Ltd, UK). Platelets were counted using a haemocytometer after blood was diluted 1:20 in 1% ammonium oxalate in distilled water. Erythrocyte and total leucocyte counts were determined using a Coulter Counter (Coulter Electronics Ltd, Harpenden, Herts., UK). Differential leucocyte counts were done on thin blood smears stained with Giemsa stain (Merck, Darmstadt, Germany). The absolute numbers of leucocytes, eosinophils, lymphocytes, neutrophils, basophils and monocytes per ml of blood were obtained by using the differential white cell count percentage and the total leucocyte count. Antibody responses to invariant 7". vivax antigens were measured using the Western blot technique described previously [6]. Anti-red blood cell antibodies were measured using a fluorescence activated cell sorter (FACScan; Becton-Dickinson, Belgium) or by ELISA. according to the methods of Assoku & Gardiner [7].
35
changes in the leucocyte count were caused by the alterations in the lymphocyte counts. No neutropaenia was detected; rather, a neutrophilia was detected on day 55 in the N'Damas and on days 7, 10 and 48 in the Borans. Antibodies bound to the surface of erythrocytes were detected in one Boran 15 dpi. These were of the IgM isotype. On day 24 pi all eight animals had antibodies bound to their erythrocyte surfaces. Both IgM and IgG were detected. By 32 dpi all the animals were negative. No antierythrocyte antibodies or anti-platelet antibodies were detected in sera from these cattle. Antibody responses to invariant trypanosome antigens were analysed. An immunodominant 69-kDa antigen was identified which elicited an IgM and an IgG response in both groups of animals. This antigen is probably homologous to a 69-kDa immunodominant antigen of T. congolense. A wide variety of other invariant antigens were also identified which elicited IgG antibodies. The majority of these antibodies were ofthe IgG 1 class but those of the IgG2 class were also detected. All antibodies to invariant antigens were detected 11-18 dpi.
RESULTS All eight animals became parasitaemic 8 to 9 days post infection (dpi). The peak of parasitaemia was reached 15 to 16 dpi and the level of parasitaemia decreased from 24 dpi until the experiment was terminated on 56 dpi. The PCV decreased from 7 dpi onwards and reached the lowest recorded level 25 dpi in the Borans and 26 dpi in the N'Damas. The lowest mean PCV was 16.8 ±5.0% in the N'Damas and 24.25 + 2.2% in the Borans. These differences were not significantly different. One N'Dama, whose PCV was 13% on day 26, was treated with diminazene aceturate. The remaining seven animals recovered without drug intervention. The reticulocyte counts reached a peak 28 to 30 dpi. The counts were higher in the N'Damas. The platelet count decreased in both groups and a thrombocytopaenia was observed 14-20 dpi. The counts remained below the preinfection level until the experiment was terminated 56 dpi. The leucocyte count decreased in both groups; the lowest count was detected 14 dpi when the N'Damas were leueopaenic. Thereafter the counts increased. A leucocytosis was observed 34 dpi in the Borans and 27 dpi in the N'Damas. The
DISCUSSION The results presented here show that four N'Dama and four Boran cattle were equally susceptible to challenge with T. uivax IL2337. Both the N'Damas and Borans experienced high levels of parasitaemia, an acute drop in PCV and a pronounced thrombocytopaenia. The ability of N'Damas to control infection of T. brucei, T. congolense and T. viva.x more efficiently than trypanosusceptible cattle has been widely reported [2, 8, 9]. The results presented in this paper are in direct contrast to those in previous publications and suggest that there was no difference between trypanotolerant and trypanosusceptible cattle in their ability to control this particular stock of T. vivax. This stock of T. vivax causes a severe disease with exaggerated pathological features which include rapid onset of anaemia, thrombocytopaenia and disseminated intravascular coagulation. However, the infection is short-lived and 7 out of the 8 cattle recovered from the infection without chemotherapeutic intervention. Thus the aetiology of the disease associated with this particular stock is
36
D. J. L. Williams et al.
very different to that described for other T. vivax and T. congolense isolates. Our results suggest that mechanisms other than those involved in trypanotolerance may be controlling this infection. ACKNOWLEDGMENT This is ILRAD Publication number 1050.
REFERENCES 1 Payne WJA. The origin of domestic cattle in Africa. Empire J Exp Agric 1964;32:97-113. 2 Murray M, Morrison Wl, Whiteiaw DD. Host susceptibility to African trypanosomiasis: trypanotolerance. Adv Parasitol 1982;21:I-68. 3 Trail JCM, D'leteren GDM, Teale AJ. Trypanotolerance and the value of conserving livestock genetic resources. Genome 1989;31:805-12. 4 Gardiner PR, Assoku RKG, Whiteiaw DD, Murray M. Haemorrhagic lesions resulting from Trypanosoma vivax infection in Ayrshire cattle. Vet Parasitol 1989;39:187-97.
5 Paris J, Murray M, McOdimba F. A comparative evaluation ofthe parasitological techniques currently available for the diagnosis of African Trypanosomiasis in cattle. Acta Tropica I982;39:307-36. 6 Assoku RKG, Gardiner PR. Detection of antibodies to platelets and erythrocytes during infection with haemorrhage-causing Trypanosoma vivax in Ayrshire cattle. Vet Parasitol 1989;31:199-216. 7 Authie E, Muteti DK, Williams DJL. Antibody responses to invariant antigens of Trypanosoma congolense in cattle of differing susceptibility to trypanosomiasis. Parasite Immunol, (in press). 8 Paling RW, Moloo SK, Scott JR, Gettinby G, McOdimba FA, Murray M. Susceptibility of N'Dama and Boran cattle to sequential challenges with tsetse-transmitted clones of Trypanosoma congolense. Parasite Immunol l991;I3:427-45. 9 Saror DI, Ilemobade AA, Nuru S. The hematology of N'Dama and Zebu cattle experimentally infected with Trypanosoma vivax. In: Proceedings ofthe 16th Meeting of the International Scientific Council for Trypanosomiasis Research and Control, Yaounde, Cameroon 1979. Nairobi, Kenya: Organization of African Unity, Scientific Technical and Research Commission, 1979:287-94.
