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Title: Development and application of ELISAs to improve the chemoprophylactic control of African bovine trypanosomiasis
Author: Eisler, Mark Charles
Awarding Body: University of Glasgow
Current Institution: University of Glasgow
Date of Award: 1994
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The control of bovine trypanosomiasis in Africa continues to rely heavily on the chemoprophylactic drug isometamidium chloride (SamorinRTM). However, despite many years of use, no methods are available which are sufficiently sensitive to measure drug levels in treated cattle. Two new enzyme-linked immunosorbent assay (ELISA) procedures for the detection and quantification of isometamidium in bovine serum were developed and validated. The first of these, an indirect competition ELISA (ICE) was capable of detecting isometamidium to approximately 0.1 ngml-1. Following the treatment of cattle with isometamidium chloride by intramuscular injection at the recommended prophylactic dose rate of 1.0 mgkg-1, the drug could be detected in serum samples for up to four months. In a controlled laboratory experiment, Boran cattle injected intramuscularly with isometamidium chloride (dose 1.0 mgkg-1 body weight) were challenged at monthly intervals with Glossina morsitans centralis infected with one of three populations of T. congolense (IL 3893, IL 3889 or IL 1180) until all animals became infected. Untreated control cattle confirmed the infectivity of challenge. All cattle challenged with IL 3893 or IL 3889 developed infection following the first challenge, at which time the mean serum drug concentration was 6 ngml-1. Cattle challenged with IL 1180 became infected following 6 to 8 challenges. The mean serum drug concentration in these cattle at the time of their third challenge with IL 1180 was 0.75 ngml-1. Trypanosome populations IL 3893 and IL 3889 were considered to be highly resistant to isometamidium, while IL 1180, relatively sensitive. Hence, T. congolense persisting at serum isometamidium concentrations greater than 0.75 ngml-1 can be considered moderately resistant, and at concentrations greater than 6 ngml-1 markedly resistant. In a second, similar experiment isometamidium treated (1.0 mgkg-1) Boran cattle were with challenged monthly with T. congolense IL 1180 or a moderately isometamidium resistant T. congolense clone (IL 3343). The distribution of trypanosome challenge over a number of three month prophylactic periods was uneven, and almost ail challenge (based on the infection rate in untreated control cattle) occurred at the end of such periods. At the time of challenge, concentrations in the treated group were at their lowest and no inferences could be made about drug resistance in trypanosome infections, all of which were caused by T. vivax. A simpler, competitive enzyme immunoassay (CEIA) developed for isometamidium in bovine serum has several advantages over the ICE; fewer incubation steps; frozen storage of microtitre plates in batches; overnight competition incubation. The response variance of 57 untreated cattle was small (CV approximately 10%); partitioning showed 77% of this variance to be intrinsic to the samples, and 23% due to procedure (CV of duplicates wells approximately 5%). The CEIA could detect isometamidium in serum of treated cattle for up to ten weeks following treatment, with a high level of reproducibility. The limit of detection is approximately 0.5 ngml-1. The CEIA was used to investigate the pharmacokinetics of isometamidium in Friesian cattle (Bos taurus) treated intravenously and intramuscularly (dose rate 1.0 mgkg-1 body weight). The major pharmacokinetic parameters were calculated using standard pharmacokinetic equations. The large VSS (mean 24 lkg-1), and the prolonged MRT (83 h) and terminal phase half-life (136 h) following intravenous injection were consistent with extensive uptake of the drug into tissues such as liver, kidney and spleen. The relatively low bioavailability (60%), and prolonged MAT (7.8 days) following intramuscular injection was consistent with a primary depot at the site of injection. Isometamidium could be detected using the CEIA in the sera of all but one of 24 cattle treated intramuscularly (1.0 mgkg-1 body weight) in an area of natural tsetse challenge. Treated cattle were protected against trypanosome infections for at least 18 weeks; thereafter three trypanosome infections were detected, between 20 and 22 weeks following treatment. In contrast, in 18 untreated control cattle, nine trypanosome infections were detected over the first 18 weeks. While there was no evidence of drug-resistant trypanosomes, the CEIA was capable of quantifying drug-levels in 20 out of 23 cattle for at least 70 days. Finally, the CEIA was used to investigate the role of isometamidium in an experimentally induced wasting syndrome in Maasai Zebu cattle under a regimen of frequently repeated isometamidium treatments. Significant weight loss and mortality occurred in poorly nourished cattle after three or four isometamidium treatments and additional treatments with diminazene. Weight losses and glutamate dehydrogenase levels were correlated with isometamidium concentrations which showed small but significant elevations with successive treatments, but no marked increases. Such frequently repeated treatment regimens must be considered inadvisable. In conclusion, the two ELISAs are capable of determining isometamidium concentrations in sera from cattle in the field, and promise to provide a practical means of rationalising chemoprophylactic drug regimens, particularly where they can assist in identifying the development of trypanocidal drug resistance.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available