Studies on the epidemiology and chemotherapy of trypanosoma evansi infection in camels

J.N. Waitumbi, 'Studies on the epidemiology and chemotherapy of trypanosoma evansi infection in camels', University of Nairobi, 2017

The field studies reported in this thesis on epidemiology and chemotherapy of Trypanosoma evansi in camels were conducted for a period of 18 months on two herds of camels located at Ngurunit and Olturot, in Marsabit District, Northern Kenya. In this area, camels provide subsistence for a nomadic population that own them. During the field studies, data on the disease incidence and patterns of the disease in different age groups was collected. Serum samples were also collected fortnightly and stored at -20oC, to be used later for serological analysis. The results of these studies show that trypanosomiasis was the most important disease complex in the area and epidemics occurred during and soon after the rains. Trypanosome infections were most severe in weaner and adult camels. The weaners developed severe clinical disease while in the adults, the effect of the disease was mainly recognised in the pregnant dams which aborted. Camel calves did not show infections until they were weaned and were over one year of age. Attempts to control the disease by individual animal treatment with quinapyramine sulphate (Trypacide sulphate, May and Baker Ltd, Dagenham, UK) failed while chemoprophylaxis using quinapyramine prosalt (Trypacide prosalt, May and Baker Ltd, Dagenham, UK) reduced infections to manageable level. Arising from the field studies, a number of questions needed to be answered. The first question was: which species of trypanosomes were responsible for the outbreak of trypanosomiasis in the study area? From morphometry, the trypanosomes isolated from the camels were of the brucei-type. Because attempts to show presence of tsetse in this area had failed, the trypanosomes would have been termed as ~. evansi, in line with Hoare's (1972) criteria of distinguishing T. evans1:- from other Trypanozoon. The tsetse map of Kenya shows that there are tsetse in the neighbourhood of the study area and because camels cover long distances in search of pasture and water, they could easily have traversed the tsetse infested areas and therefore acquired ~. brucei brucei infections. To investigate further the identity of the stocks collected the following characterization methods were used: 1) Tsetse transmissibility: Each of the trypanosome stocks was raised in irradiated rats and at peak parasitaemia, teneral Glossina morsitans mortisans were allowed to feed on them. The tsetse were then maintained by daily feeding on rabbits. On day 36, the flies were dissected and checked for trypanosome infections in the gut, proboscis and salivary glands. None of the 48 isolates was infective to ~.m.morsitans. In comparison, mature infections were found in the flies that had fed on rats infected with a defined ~.Q.bruce! reference strain (KETRI 2502) which had also been obtained from a camel. The rabbits used to maintain flies infected with the reference ~.h.brucei developed chancres and parasitaemia. Thus, by the criterion of tsetse trnsmissibility, the 48 isolates were most probably ~. eyansi. 2 Isoenzyme typing: Samples of soluble enzymes were prepared from each of the 48 stocks and analysed by thin layer starch gel electrophoresis for the ALAT, ASAT, PGM, lCD, ME and peptidases I and II. Except for PGM, none of the other enzymes revealed consistent differences between the 48 stocks and the reference ~.h.brucei strain. However, stocks of I. eyansi with a pattern similar to the one seen in ~.Q. brucei have been described before by Gibson, Marshall and Godfrey (1980). This approach was thus not useful for determining whether these isolates were I. evansi or I.Q. brucei. 3) Kinetoplast DNA (kDNA) minicircle analysis: Kinetoplast DNA minicircles were analysed using various restriction endonucleases. Digested samples were then analysed in agarose and polyacrylamide gels. The digested minicircles of the 48 stocks were homogeneous. In contrast, the T.Q. brucei reference strains used showed a complex of non-stoicheiometric bands irrespective of the endonuclease used. Analysis of kDNA minicircles was thus able to show unequivocally that the 48 stocks were T. evansi. 4) Chromosome-sized DNA analysis: Each of the trypanosome stocks was embedded in an agarose slab and chromosomes separated using contour-clamped homogeneous electric field gel electrophoresis. From the karyotype patterns, the intermediate chromosomes and the minichromosomes were bigger in the reference ~. evansi and the 48 stocks than those of the reference ~.R.brucei. The trypanosome stocks ana lysed could be grouped into 9 molecular karyotypes. Only one molecular karyotype was found in the herd that was kept under chemoprophylaxis. This herd had a long history of drug use and recurring parasitaemias were often found soon after treatment. When tested for drug sensitivity, the trypanosomes were shown to be four times less sensitive to quinapyramine sulphate than the sensitive stock. It is possible that, the trypanosomes in this herd could have been derived from one drug resistant type. with regard to the herd kept under individual treatment, nine molecular karyotypes were seen. The majority of the infections that occurred during the second epidemic could be traced to similar karyotypes seen at the beginning of the study. Thus, it appears that karyotyping is a sensitive method for revealing differences between T. evansi isolates and might be useful in revealing multiple re-isolation of the same trypanosome. The next question that needed to be answered was whether an antigen detection system would have been a better method of detecting infections than parasitological diagnosis. To answer this question, over 3000 serum samples, collected fortnightly for a period of 18 months, were analysed for the presence of antigens and results compared with the parasitological data. The results can be grouped into four categories: 1) Group one comprised cases in which the presence of trypanosomal antigens could be correlated with parasitological diagnosis. This was observed in 52 out of 61 (85%) instances in which trypanosomes were detected. On treatment, in most of the cases (80%), antigens disappeared from circulation within a period of 30 days further confirming the correlation noted above and also indicating the potential for use of this test to assess efficacy of treatment. In 20% of the instances, antigens remained detectable for a longer period of time, and in five cases even over 500 days. The reasons for 'persistence of the antigens in the few instances where they did persist could be due to failure of the trypanocides to effect a complete cure either because the trypanosomes were resistant to the drugs used or because the parasites were located in tissues inaccessible to the drugs. 2) Group two comprised those cases in which sera from parasitologically proven infections did not have antigens. This was observed in 9 camels, 7 of which were from a herd that was being examined for the presence of trypanosomes weekly. Two possible explanations were advanced. One was that the antigens might have been mopped up by antibody to form immune complexes and therefore the epitopes recognised by the trapping antibody masked. Secondly, the trypanosomes could have been detected too early before sufficient parasite destruction had occurred to give detectable levels of antigen in circulation. Attempts to detect immune complexes failed and the second possibility was thought more likely. 3) Group three comprised camels that were at no time parasitaemic despite the presence of antigens. In the herd where control of trypanosomiasis was by prophylaxis, such antigens were noted to disappear from circulation after trypanocide therapy, indicating that, the presence of antigen represented true cases of trypanosomal infections, which could not be detected by the parasitological methods used. That the antigens detected were indeed due to the presence of trypanosomal infection was confirmed by the presence of anti-trypanosomal antibodies in the sera of antigen positive camels. 4) Group four comprised camel calves, in which no trypanosome infections were detected during the early period of their lives. Most of the calves also did not have antigen during this period. The calves appeared to have some form of protection from trypanosome infections. Anti-trypanosome antibodies were not found during this early period. This was suprising for calves born in a trypanosomiasis endemic area. What then was the source of protection? Are there non-specific factors akin to those that contribute to calfhood immunity against babesiosis? These questions remain to be answered. In six out of 40 calves, occasional antigenaemia was detected but no corresponding antibodies were found indicating absence of a patent infection. This observation is intriguing in the light of the fact that cross-reaction has not been observed between the monoclonal antibody used in the antigen detection and other haemoparasites (Nantulya, Musoke, Rurangirwa, Saigar, and Minja 1987). Does this antigen represent disrupted trypanosomes that were unable to establish infection. Clearly, further work is needed to try and eludicate the nature of calfhood immunity.The field studies reported in this thesis on epidemiology and chemotherapy of Trypanosoma evansi in camels were conducted for a period of 18 months on two herds of camels located at Ngurunit and Olturot, in Marsabit District, Northern Kenya. In this area, camels provide subsistence for a nomadic population that own them. During the field studies, data on the disease incidence and patterns of the disease in different age groups was collected. Serum samples were also collected fortnightly and stored at -20oC, to be used later for serological analysis. The results of these studies show that trypanosomiasis was the most important disease complex in the area and epidemics occurred during and soon after the rains. Trypanosome infections were most severe in weaner and adult camels. The weaners developed severe clinical disease while in the adults, the effect of the disease was mainly recognised in the pregnant dams which aborted. Camel calves did not show infections until they were weaned and were over one year of age. Attempts to control the disease by individual animal treatment with quinapyramine sulphate (Trypacide sulphate, May and Baker Ltd, Dagenham, UK) failed while chemoprophylaxis using quinapyramine prosalt (Trypacide prosalt, May and Baker Ltd, Dagenham, UK) reduced infections to manageable level. Arising from the field studies, a number of questions needed to be answered. The first question was: which species of trypanosomes were responsible for the outbreak of trypanosomiasis in the study area? From morphometry, the trypanosomes isolated from the camels were of the brucei-type. Because attempts to show presence of tsetse in this area had failed, the trypanosomes would have been termed as ~. evansi, in line with Hoare's (1972) criteria of distinguishing T. evans1:- from other Trypanozoon. The tsetse map of Kenya shows that there are tsetse in the neighbourhood of the study area and because camels cover long distances in search of pasture and water, they could easily have traversed the tsetse infested areas and therefore acquired ~. brucei brucei infections. To investigate further the identity of the stocks collected the following characterization methods were used: 1) Tsetse transmissibility: Each of the trypanosome stocks was raised in irradiated rats and at peak parasitaemia, teneral Glossina morsitans mortisans were allowed to feed on them. The tsetse were then maintained by daily feeding on rabbits. On day 36, the flies were dissected and checked for trypanosome infections in the gut, proboscis and salivary glands. None of the 48 isolates was infective to ~.m.morsitans. In comparison, mature infections were found in the flies that had fed on rats infected with a defined ~.Q.bruce! reference strain (KETRI 2502) which had also been obtained from a camel. The rabbits used to maintain flies infected with the reference ~.h.brucei developed chancres and parasitaemia. Thus, by the criterion of tsetse trnsmissibility, the 48 isolates were most probably ~. eyansi. 2 Isoenzyme typing: Samples of soluble enzymes were prepared from each of the 48 stocks and analysed by thin layer starch gel electrophoresis for the ALAT, ASAT, PGM, lCD, ME and peptidases I and II. Except for PGM, none of the other enzymes revealed consistent differences between the 48 stocks and the reference ~.h.brucei strain. However, stocks of I. eyansi with a pattern similar to the one seen in ~.Q. brucei have been described before by Gibson, Marshall and Godfrey (1980). This approach was thus not useful for determining whether these isolates were I. evansi or I.Q. brucei. 3) Kinetoplast DNA (kDNA) minicircle analysis: Kinetoplast DNA minicircles were analysed using various restriction endonucleases. Digested samples were then analysed in agarose and polyacrylamide gels. The digested minicircles of the 48 stocks were homogeneous. In contrast, the T.Q. brucei reference strains used showed a complex of non-stoicheiometric bands irrespective of the endonuclease used. Analysis of kDNA minicircles was thus able to show unequivocally that the 48 stocks were T. evansi. 4) Chromosome-sized DNA analysis: Each of the trypanosome stocks was embedded in an agarose slab and chromosomes separated using contour-clamped homogeneous electric field gel electrophoresis. From the karyotype patterns, the intermediate chromosomes and the minichromosomes were bigger in the reference ~. evansi and the 48 stocks than those of the reference ~.R.brucei. The trypanosome stocks ana lysed could be grouped into 9 molecular karyotypes. Only one molecular karyotype was found in the herd that was kept under chemoprophylaxis. This herd had a long history of drug use and recurring parasitaemias were often found soon after treatment. When tested for drug sensitivity, the trypanosomes were shown to be four times less sensitive to quinapyramine sulphate than the sensitive stock. It is possible that, the trypanosomes in this herd could have been derived from one drug resistant type. with regard to the herd kept under individual treatment, nine molecular karyotypes were seen. The majority of the infections that occurred during the second epidemic could be traced to similar karyotypes seen at the beginning of the study. Thus, it appears that karyotyping is a sensitive method for revealing differences between T. evansi isolates and might be useful in revealing multiple re-isolation of the same trypanosome. The next question that needed to be answered was whether an antigen detection system would have been a better method of detecting infections than parasitological diagnosis. To answer this question, over 3000 serum samples, collected fortnightly for a period of 18 months, were analysed for the presence of antigens and results compared with the parasitological data. The results can be grouped into four categories: 1) Group one comprised cases in which the presence of trypanosomal antigens could be correlated with parasitological diagnosis. This was observed in 52 out of 61 (85%) instances in which trypanosomes were detected. On treatment, in most of the cases (80%), antigens disappeared from circulation within a period of 30 days further confirming the correlation noted above and also indicating the potential for use of this test to assess efficacy of treatment. In 20% of the instances, antigens remained detectable for a longer period of time, and in five cases even over 500 days. The reasons for 'persistence of the antigens in the few instances where they did persist could be due to failure of the trypanocides to effect a complete cure either because the trypanosomes were resistant to the drugs used or because the parasites were located in tissues inaccessible to the drugs. 2) Group two comprised those cases in which sera from parasitologically proven infections did not have antigens. This was observed in 9 camels, 7 of which were from a herd that was being examined for the presence of trypanosomes weekly. Two possible explanations were advanced. One was that the antigens might have been mopped up by antibody to form immune complexes and therefore the epitopes recognised by the trapping antibody masked. Secondly, the trypanosomes could have been detected too early before sufficient parasite destruction had occurred to give detectable levels of antigen in circulation. Attempts to detect immune complexes failed and the second possibility was thought more likely. 3) Group three comprised camels that were at no time parasitaemic despite the presence of antigens. In the herd where control of trypanosomiasis was by prophylaxis, such antigens were noted to disappear from circulation after trypanocide therapy, indicating that, the presence of antigen represented true cases of trypanosomal infections, which could not be detected by the parasitological methods used. That the antigens detected were indeed due to the presence of trypanosomal infection was confirmed by the presence of anti-trypanosomal antibodies in the sera of antigen positive camels. 4) Group four comprised camel calves, in which no trypanosome infections were detected during the early period of their lives. Most of the calves also did not have antigen during this period. The calves appeared to have some form of protection from trypanosome infections. Anti-trypanosome antibodies were not found during this early period. This was suprising for calves born in a trypanosomiasis endemic area. What then was the source of protection? Are there non-specific factors akin to those that contribute to calfhood immunity against babesiosis? These questions remain to be answered. In six out of 40 calves, occasional antigenaemia was detected but no corresponding antibodies were found indicating absence of a patent infection. This observation is intriguing in the light of the fact that cross-reaction has not been observed between the monoclonal antibody used in the antigen detection and other haemoparasites (Nantulya, Musoke, Rurangirwa, Saigar, and Minja 1987). Does this antigen represent disrupted trypanosomes that were unable to establish infection. Clearly, further work is needed to try and eludicate the nature of calfhood immunity