18th Institute of Primate Research scientific conference, KIE Nairobi Biochemical changes in vervet monkeys experimentally infected with Trypanosoma brucei rhodesiense A. Gaithuma1,2, S.M. Karanja1, J.M. Kagira2, M. Ngotho2, and Maina1, N. JKUAT IPR Department of Biochemistry, Jomo Kenyatta University of Agriculture & Technology, Juja, Thika, Kenya Tropical and Infectious Diseases department, Institute of Primate Research, Karen, Nairobi, Kenya ABSTRACT This study evaluated various biochemical changes in blood and urine of T.b. rhodesiense infected vervet monkeys. A chronic infection lasting up to 62 dpi (days post-infection) was observed, marked by relapses starting from 52 dpi. The early stage infection (7-28 dpi) was characterized by significantly low blood glucose levels (P<0.05) and significantly high levels of triglycerides. Creatine kinase (CK) levels in blood were significantly high (P<0.05) after infection (7 dpi) but resumed baseline levels thereafter. High density lipoprotein (HDL) cholesterol significant decreased (P<0.05) after infection but resumed baseline values in late stage infection (post diminazene treatment) while Low density lipoprotein (LDL) cholesterol increased significantly (P<0.05) with disease progression. The late stage infection was particularly marked by significantly high (P<0.05) total cholesterol levels. In urine, ketone levels were significantly high (P<0.05) during infection while pH varied inversely with parasitaemia and directly with specific gravity. A panel of triglycerides, HDL, blood glucose could be used as a early stage disease indicators, and cholesterol levels could be exploited as an adjunct marker for late stage disease in addition to aiding humane end points in animal studies. Background Human African Trypanosomiasis (HAT) or sleeping sickness is a neglected disease of poverty caused by the protozoan parasites Trypanosoma brucei transmitted via tsetse fly vectors of the genus Glossina 60 million people in 36 sub-Sahara African countries are at a risk of HAT (WHO, 2006) Current field diagnostic methods are insensitive, unspecific, and require invasive/painful lumbar punctures The vervet monkey model of HAT has been shown to mimic the disease in man both clinically, immunologically and pathologically (Farah et al., 2005) Most unexplained biochemical changes observed during HAT might give an indication of the degree of damage to the host tissue as well as the severity of infection (Cano et al., 2004) Fig 1: Mean parasitaemia of infected vervet monkeys Fig 2: Mean changes in weekly glucose levels of control and infected vervet monkeys Fig 3: Mean changes in weekly creatine kinase levels Objectives To determine the levels of glucose, cholesterol and triglycerides and creatine kinase in vervet monkeys infected with T. b. rhodesiense IPR 001 To determine the levels of urine pH, specific gravity and ketones in vervet monkeys infected with T. b. rhodesiense IPR 001 Materials and methods Four monkeys were infected I.V. with 104 trypanosomes (Trypanosoma brucei rhodesiense isolate IPR 001 isolated from CSF of a late HAT patient in Palisa District, Uganda in 2008).The control group comprised of three monkeys The infected animals were given sub-curative treatment with Diminazene aceturate (DA) (Veriben®, Sanofi, France) at a dose of 5mg⁄kg bwt I.M. for 3 days, from 28 dpi Matched blood samples for plasma and serum separation and urine were taken at weekly intervals and parasitaemia was scored daily using the Herbert and Lumsden (1976) Ear prick blood for glucose assay was done using glucometer (Accu-Chek® Go, Roche Germany). Urinalysis was done on fresh urine (*semi-quantitative measurements) using dip-stick strips (ChoiceLine 10®, Roche Germany) Biochemical assays were performed on 32μL sample aliquots using Reflotron® plus (Roche, Germany) dry chemistry biochemical analyzer Data analysis Data analysis was done using Minitab version 15, descriptive statistics done using Ms Excel (XLSTAT Version 2010, Add-in). Means were deemed significant at (P < 0.05). Ethical review All protocols and procedures used in the current study were reviewed and approved by the Institutional Review Committee (IRC) of the Institute of Primate Research (IPR) Fig 4: Mean changes in weekly triglycerides levels Fig 5: Mean changes in weekly LDL and HDL cholesterol levels Fig 6: Mean changes in weekly ketone levels in urine* Fig 7: Mean changes in weekly specific gravity and pH levels in urine Fig 8: Mean changes in weekly total cholesterol Discussion The coma observed in infected monkeys and recovery after glucose infusion, pointed to a hypoglycaemic coma. This may explain comas observed in human patients The rise in creatine kinase levels observed in infected monkeys may have been as a result of muscle dystrophy (emaciation) which causes release of intracellular enzymes The elevation in triglycerides could be due acute phase reaction and disruption of fat metabolism (Beutler and Cerami, 1988) which may have manifested as ketosis (high urine ketone levels) Changes in urine pH levels may have been due to changes in energy metabolism that seem to be related to parasitaemia The rapid decline in HDL levels after infection may have been due to their suppression or utilization since the Trypanolytic factor (TLF) is an ApoL-1 (HDL-type) molecule The marked hypercholesterolemia in late stage disease was most probably due to release of LDL cholesterol from liver biosynthesis (strong correlation of LDL and total cholesterol) Further, disruption of the normal neural circuit (infection in this case) system in the brain has been found to directly affect the control of cholesterol metabolism by the liver (Perez-Tilve et al., 2010) Results The mean pre-patent period was 3 days with parasitaemia levels peaking first at 9 dpi and the second wave peaking at 13 dpi. Relapses occurred from 52 dpi (fig. 1) Hypoglycemia with blood glucose levels decreasing significantly (P<0.05) after infection (fig. 2). Two animals went into coma 9 dpi where one monkey promptly recovered after glucose i.v. infusion Creatine kinase (CK) levels Increased significantly (P<0.05) with onset of infection then decreased and remained low in advanced late stage infection (fig. 3) Triglyceride levels increased significantly (P<0.05) after infection (fig. 4),High density lipoprotein cholesterol (HDL) significantly decreased (P<0.05) from normal levels (mean= 1.55mmol/L) and increased during the advanced late stage infection while low density lipoprotein (LDL) cholesterol levels gradually increased from baseline levels (mean = 1.293 mmol/L) (fig. 5).There was a significant correlation (P<0.05; R2=79.3%) between LDL cholesterol and total cholesterol Urine ketone levels were significantly high (P<0.05) in early infection but gradually decreased after sub-curative treatment with DA. (fig.6) while urine pH and specific gravity showed variable changes where pH varied inversely with parasitaemia and directly with specific gravity (fig.7) The late stage disease was marked by a significant increase (P<0.05) in cholesterol levels after sub-curative treatment with DA to induce late stage (fig. 8) Conclusion A panel of triglycerides, HDL, blood glucose could be used as a early stage disease indicators, and total cholesterol levels could be used as an adjunct marker for late stage disease. References Ngure, R. M. et al., (2008). J. of Cell and Anim. Bio. 2(7):150-157 Perez-Tilve, D. et al., (2010). Nature Neuroscie. 13:877–882 Farah et al., (2005). Hndbk of lab. Ani. scie. Vol.III. New York: Press, pp. 169-224. Acknowledgements Research Production and Extension dept.-JKUAT and IPR for funding Staff of Animal Sciences Department (ASD-IPR) and staff of Biochemistry Department-JKUAT Correspondence email: faradysdr@yahoo.com