Pathobiochemical mechanisms involved in the control of the disease caused by Trypanosoma congolense in African grey duiker (Sylvicapra grimmia)

Veterinary Parasitology 96 (2001) 51–63

Pathobiochemical mechanisms involved in thecontrol of the disease caused byTrypanosoma

congolensein African grey duiker(Sylvicapra grimmia)

A.O. Ogunsanmia,∗, V.O. Taiwob

a Department of Wildlife and Fisheries Management, University of Ibadan, Ibadan, Nigeriab Department of Veterinary Pathology, University of Ibadan, Ibadan, Nigeria

Received 4 August 1999; received in revised form 18 September 2000; accepted 27 September 2000

Abstract

The course ofTrypanosoma congolenseinfections in African grey duiker (Sylvicapra grimmia)and sheep and goats were studied. Several parameters suggested that the grey duiker was much moreresistant to trypanosomosis than sheep and goats. They showed increases in weight during infection,had a much longer pre-patent period, and their peak parasitaemia levels were about 100-fold lowerthan those of sheep and goats. Parasites were no longer detected in grey duiker blood 35 daysafter infection. Anaemia, measured as drops in packed cell volume (PCV), haemoglobin (Hb)concentration and erythrocyte (RBC) counts were not observed in the grey duiker. In contrast,sheep and goats suffered severe weight losses and had continuously high parasitaemia levels. Sheepand goats developed progressively severe normocytic normochromic anaemia and leucopenia fromday 14 post-infection onwards.

Serum levels of total protein, globulin and albumin of grey duiker did not change significantlythroughout the course of infection, while the levels of total serum protein, globulin andg-globulinexhibited significant increases from day 21 post-infection onwards in sheep and goats, with peakvalues recorded on 28 and 35 days post-infection in sheep and goats, respectively. There wereinconsistent variations in albumin levels in sheep and goats throughout the course of infection.

There were no significant changes in erythrocyte activities of AST and ALT, while there weretransient but significant elevations of ALP level on day 35, and GGT levels between 14 and 35days post-infection in grey duiker. Conversely, the levels of all the enzymes were progressivelydepressed, especially from 14 to 49 days post-infection.

In vitro erythrocyte peroxidation remained relatively unchanged throughout the period of theexperiment in the grey duiker, except for slight but significant increase on day 42 post-infection.

∗Corresponding author.E-mail address:[email protected] (A.O. Ogunsanmi).

0304-4017/01/$ – see front matter © 2001 Elsevier Science B.V. All rights reserved.PII: S0304-4017(00)00410-6

52 A.O. Ogunsanmi, V.O. Taiwo / Veterinary Parasitology 96 (2001) 51–63

However, in vitro erythrocyte peroxidation increased significantly by between 100 and 300%of pre-infection levels from 14th to 42nd day p.i. both in sheep and goats, before returning topre-infection levels after 14 days of treatment.

Haematological values, serum and erythrocyte indices studied returned to near pre-infectionlevels 14 days after treatment with Berenil®.

It is concluded that the grey duiker is inherently trypanotolerant. This is shown by its ability tocontrol parasitaemia, suffer less severe anaemia, and to a relative degree resist pathobiochemicalderangements of some serum and erythrocyte metabolites and enzymes, as well as reduction ofinfection-induced erythrocyte lipid peroxidase damage than sheep and goats. © 2001 ElsevierScience B.V. All rights reserved.

Keywords: Trypanosoma congolense; Duiker; Control methods-protozoa; Pathology

1. Introduction

Knowledge of the physical characteristics of normal erythrocytes, their metabolic activi-ties, the changes that accompany ageing, and the mechanisms by which they are destroyedis requisite for a rational classification and for an understanding of the pathophysiologyand pathobiochemistry of haemolytic disorders (Liman, 1975; Fairbanks and Klee, 1989).In addition to proteins, lipids and carbohydrates, normal erythrocytes contain an impres-sive array of enzymes, anions and cations which are necessary for the optimum metabolicactivities and survival of the cells (Liman, 1975).

The major cause of anaemia in trypanosomosis has been attributable mainly to extravascu-lar haemolysis due to phagocytosis of erythrocytes by an expanded mononuclear phagocyticsystem (MPS) in the trypanosome-infected host (Anosa, 1988; Murray and Dexter, 1988).While intravascular haemolysis may be a minor feature (Esievo et al., 1984), haemolyticfactors (Huan et al., 1975), trypanosome protease and sialidase activities (Esievo et al., 1982;Knowles et al., 1989), auto-erythrocytic antibodies (Assoku and Gardiner, 1989) have beenreported to play various roles in erythrocyte lysis and/or destruction during trypanosomosis.

Moreover, reactive oxygen radicals and inorganic nitrogen oxides produced by acti-vated phagocytes during infection may cause damage, not only to trypanosomes but alsoto host erythrocytes leading to lysis or subsequent sequestration (Grosskinsky et al., 1983;Zinki, 1989).

Peroxides and oxygen radicals are aggressive cellular toxins that can destroy connectivetissue, damage biological membranes, oxidise sulphydryl groups, inactivate enzymes andcause peroxidative damage of nucleic acids (Braun et al., 1991). Ameh (1984) showed thatthere was a significant increase in the susceptibility of erythrocytes to oxidative haemolysisafter challenge with 30mM hydrogen peroxide 3–4 days afterTrypanosoma brucei gambi-enseinfection of rats. This was associated with a decrease in the levels of blood and liverglutathione. These observations suggested an oxidative stress and a free radical load in theinfected rats. Thus, free radical mediated erythrocytes membrane damage may be involvedin causing haemolysis during acute infection through peroxidation of the erythrocytes.

It has been shown that infections by theT. bruceigroup of parasites may alter the host’santioxidant defence against free radicals. Anosa and Kaneko (1983) showed that erythrocyte

A.O. Ogunsanmi, V.O. Taiwo / Veterinary Parasitology 96 (2001) 51–63 53

peroxidase and reductase activities increased and superoxide dismutase activity decreasedper unit weight of spleen inT. bruceiinfection of deer mice. The increase in the activitiesof the erythrocyte enzyme suggested an increased demand for their function to maintainglutathione in the reduced state due to free radicals. It has been reported that ascorbic acidis depleted in the tissue ofT. bruceiinfected guinea pigs (Roskin and Nastiukova, 1941) andT. evansiinfected rats (Nyden, 1948). Niki et al. (1988) showed that plasma ascorbic acidfunctioned as the primary defence against free radicals in the whole blood and that ascorbicacid was rapidly depleted in the process. Free radicals in infected animals may arise fromtrypanosome metabolism (Baltz et al., 1985) and macrophage activation in the expandedMPS (Klebanoff, 1982; Schwacha and Loegering, 1992).

Therefore, if there is reduction in the capacity of the erythrocytes ofT. bruceiinfected miceto prevent free radical-mediated membrane damage in vivo, as has been observed in vitro(Igbokwe et al., 1994), predisposition of erythrocytes to peroxidation may accelerate theirageing and increase their deformability and susceptibility to fragmentation (Hochstein andJain, 1981; Knight et al., 1992). Erythrocyte fragmentation has already been confirmed inT.bruceiinfected deer (Anosa and Kaneko, 1983). Gardiner et al. (1989) reported erythrocytemicroangiopathy inT. vivaxinfection of cattle. Thus, erythrocyte peroxidation may be oneof the factors which play a role in the pathogenesis of the anaemia in trypanosomosis.

Because the mechanisms involved in trypanosomosis may be complex, it will be of scien-tific importance to investigate possible pathobiochemical derangements including erythro-cyte peroxidation during trypanosome infection in wild and domesticated small ruminants,as this has not been reported in these animal species.

2. Materials and methods

2.1. Experimental animals

Six African grey duiker (three males and three females), born in captivity at the ZoologicalGarden, University of Ibadan, Ibadan, were used for this study. They were aged between20 and 30 months and weighed between 12 and 15 kg. Twenty Yankassa sheep (10 malesand 10 females), aged between 18 and 24 months, and 20 West African dwarf (WAD) goats(10 males and 10 females) aged between 20 and 27 months were also used. All the sheepand goats weighed between 17 and 20 kg. They were obtained from a private smallholderfarmer on the outskirts of Ibadan, Oyo State, Nigeria.

All the experimental animals groups were maintained separately according to their sexand ear-tagged for easy identification. The grey duiker were fed on leaves ofIpomea in-volucrata, Afzekia africanaandEucalyptus torolianaand supplemented with dried peels ofyam (Discorea rotundata), grains of millet and maize and fruits ofMilletia threoningii.Thesheep and goats were fed ration containing 14% crude protein, supplemented with maizeoffal, hay, Cynodon plectostachyumand Centrosema pubescens.Water and commercialmineral salt licks were provided ad libitum for the three groups of animals.

The animals were screened for trypanosomes by the microhaematocrit dark phase-contrastmethod of Paris et al. (1982). Jugular venous blood samples of each animal were sub-inoculated into adult albino mice (Nantulya et al., 1984), and were found to be negative for


trypanosomes. Despite this, the animals were treated with trypanocidal drug-diminazeneaceturate (Berenil®, Hoechst AG, Frankfurt, W. Germany) at a dose of 7.0 mg/kg bodyweight. The animals were also dewormed with fenbendazole (Panacur®, Hoechst, Ger-many) against gastrointestinal parasites, bathed with coumaphos solution (Asuntol®, Bayer,Germany) against ectoparasites and placed on oxytetracycline hydrochloride (TerramycinQ®, Pfizer, USA) for 5 days before onset of the experiment.

All the animals were bled two times before infection and the data generated served aspre-infection data. Thereafter each animal was given 1.5 × 106 Trypanosoma congolense(IL1180) (Nantulya et al., 1984) in normal saline by intraperitoneal injection. During theexperiment the animals were kept in fly-proof isolation units. The animals were weighedbefore, weekly during and after the experiment to monitor weight changes. Clinical signswere routinely observed in the animals with emphasis on the state of the hair coat, dailyactivity, and the size of prescapular and prefemoral lymph nodes. The experiment lastedfor 9 weeks (63 days). The infected sheep and goats were treated with Berenil® (7.0 mg/kgbody weight) on the 49th day post-infection in order to prevent them from dying, when theywere in very bad condition.

2.2. Parasitology, haematology and serum biochemistry

Blood was collected weekly from the experimental animals by jugular venapuncture intoplain and disodium salt of ethylene diamine tetra-acetic acid (EDTA) tubes for serum bio-chemistry, and parasitological and haematological analyses, respectively. For parasitolog-ical examination, the detection and number of trypanosomes (parasitaemia) was estimatedby the semi-quantitative scoring method according to Paris et al. (1982). Packed cell vol-ume (PCV), haemoglobin (Hb) concentration, erythrocyte (RBC) counts were determinedas described by Jain (1986). The mean corpuscular volume (MCV) and mean corpuscularhaemoglobin concentration (MCHC) were calculated (Jain, 1986).

Serum total protein and albumin levels of the experimental animals were determined asdescribed by Ogunsanmi et al. (1994a). Serumg-globulin was determined by the method ofVarley et al. (1980). Serum enzyme activities of alkaline phosphatase (ALP), alanine amino-transferase (ALT), aspartate aminotransferase (AST) andg-glutamyl transferase (GGT)were determined as described by Oyewale et al. (1998).

2.3. In vitro erythrocyte peroxidation and erythrocyte enzyme activities

In vitro erythrocyte peroxidation, using various concentrations of hydrogen peroxide(H2O2), was measured on days 0, 14, 28, 42 and 63 post-infection according to the methodof Duthie et al. (1989). The concentration of thiobarbituric acid reactive substances re-leased from oxidatively-lysed 109 erythrocytes was measured spectrophotometrically at535 nm. For erythrocyte biochemistry, 109 erythrocytes were dispensed into 2 ml colddeionised water and mixed in a vortex machine to obtain a haemolysate. The ALP, AST,ALT and GGT activities in the erythrocyte lysates were assayed as described for serumenzymes.


2.4. Statistical analysis

The data obtained at pre-infection and from control and trypanosome-infected animalswere subjected to statistical analysis of variance (ANOVA) (SAS, 1987) and the means wereevaluated for significant differences using the Duncan’s multiple range test (Duncan, 1959).

3. Results

3.1. Clinical signs

The observed clinical signs included fever of up to 39.5◦C in sheep and goats from 14 daysp.i. onwards (data not presented), while no febrile reaction was observed in the grey duiker.Other clinical signs include pallor of the oral and conjunctival mucous membranes, roughhair coats, loss of body weight and anorexia which were observed in all the infected sheepand goats were absent in the infected grey duiker. In fact, infected grey duiker gained anaverage of 18.7% weight increase (data not shown) during the 63-day experimental period.

3.2. Parasitaemia and haematology

Parasitaemia became detectable in infected grey duiker parasitaemia 14 days post-infection (p.i.) as compared to sheep and goats which had demonstrable parasitaemia 7days p.i. (Table 1). The level of parasitaemia was lower in the duiker with a peak of(5.3 ± 2.8) × 103 trypanosomes/ml 21 days p.i. In contrast, the peaked parasitaemias insheep and goats ((5.1±1.2)×105 and(7.7±0.7)×105 trypanosomes/ml, respectively) on14 days p.i. were about 100–150-fold higher than that of the peak parasiataemia developedby grey duiker (Table 1). The sheep and goats were consistently parasitaemic and continuedto lose condition rapidly and had to be treated with diminazene aceturate on 49 days p.i.because of their low PCV values. Both sheep and goats developed progressively severenormocytic normochromic anaemia and leucopenia from day 14 post-infection onwards,with a near return to pre-infection levels on day 63 (14 days after treatment with Berenil®).

3.3. Serum proteins

The results of the serum proteins (total protein, albumin, globulin andg-globulin) ofgrey duiker, sheep and goats are presented in Table 2. The serum total protein, globulin andalbumin levels of grey duiker did not change significantly (P > 0.05) throughout the courseof infection. The levels of total serum protein, globulin andg-globulin exhibited significantincreases (P < 0.05) especially from day 21 post-infection in sheep and goats, with peakvalues recorded on 28 and 35 days post-infection in sheep and goats, respectively. Gener-ally, there were inconsistent variations in albumin levels in sheep and goats throughout thecourse of infection.

3.4. Erythrocyte biochemistry and in vitro peroxidation

The results of erythrocyte electrolyte changes and enzyme activities of ALP, ALT, ASTand GGT in grey duiker, sheep and goats are shown in Table 3. There were no significant

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Table 4Mean erythrocyte peroxidation in grey duiker, sheep and goats infected withT. congolensea

H2O2 concentration (%) 0 14 28 42 63

Grey duiker0 0.03± 0.01 ab 0.04± 0.02 a 0.03± 0.01 a 0.03± 0.01 a 0.03± 0.01 a0.9 0.07± 0.01 b 0.08± 0.07 b 0.10± 0.01 b 0.16± 0.01 a 0.09± 0.01 b1.5 0.11± 0.01 b 0.07± 0.04 b 0.12± 0.01 b 0.17± 0.01 a 0.12± 0.01 b

Sheep0 0.02± 0.01 a 0.03± 0.01 a 0.04± 0.02 a 0.04± 0.01 a 0.02± 0.01 a0.9 0.07± 0.02 c 0.11± 0.02 b 0.19± 0.03 a 0.19± 0.02 a 0.06± 0.03 c1.5 0.12± 0.02 c 0.19± 0.02 b 0.23± 0.02 a 0.22± 0.03 a 0.09± 0.05 c

Goats0 0.02± 0.01 a 0.05± 0.01 a 0.04± 0.02 a 0.02± 0.01 a 0.03± 0.01 a0.9 0.08± 0.02 c 0.14± 0.04 b 0.22± 0.04 ab 0.27± 0.02 a 0.09± 0.01 c1.5 0.13± 0.03 c 0.18± 0.02 b 0.21± 0.03 ab 0.29± 0.03 a 0.15± 0.01 c

a Values in the same row with different letters (a, b, c, etc.) differ significantly (P < 0.05).b Data expressed as mean± standard error of mean (O.D. of thiobarbituric acid reactants).

variations (P > 0.05) in erythrocyte activities of AST and ALT throughout the course ofinfection in grey duiker. However, there were transient elevations (P < 0.05) of ALP levelon day 35, and GGT levels between 14 and 35 days post-infection in grey duiker. Conversely,the levels of all the enzymes were progressively depressed (P < 0.05), especially from 14to 49 days post-infection, returning to near pre-infection levels on 63rd day in sheep andgoats.

The in vitro erythrocyte peroxidation (Table 4) remained relatively stable throughoutthe period of the experiment in the grey duiker, except for slight but significant increase(P < 0.05) on day 42 post-infection at both 0.9 and 1.5% H2O2 concentrations. However,the levels of thiobarbituric acid reactive substances increased significantly (P < 0.05) bybetween 100 and 300% of pre-infection levels from 14th to 42nd day p.i. both in sheepand goats, before returning to pre-infection levels on day 63 post-infection at all the H2O2concentrations used (Table 4).

4. Discussion

Although the grey duiker in the experiment were susceptible toT. congolenseinfection,they were apparently unaffected by trypanosomosis. The grey duiker showed no clini-cal signs of the disease, they were not anaemic, but only exhibited mild to scanty para-sitaemia. Most serum and erythrocyte biochemical parameters except for serumg-globulinand erythrocyte GGT showed no significant change following infection. Their trypanotol-erance appeared to be associated with the development of relatively low parasitaemia, earlyg-globulin and erythrocyte GGT responses, the reduction of infection-induced erythrocytelipid peroxidase damage, self-cure and absence of anaemia.

The prepatent period in the duiker was much longer than in sheep and goats. The lev-els of parasitaemia were usually more than 100-fold below the parasitaemia levels in both


sheep and goats. These differences could be due to inhibition of trypanosome proliferationby non-immune parasite growth regulating factors or by limitation of trypanosome growthowing to an early immune response (Grootenhuis et al., 1990) as serum hypergammaglob-ulinaemia occurred before detection of parasitaemia in grey duiker. In sheep and goats,significant increase in serumg-globulin was not detectable until 7–14 days after the firstdetection of trypanosomes in the blood. In experimentalT. bruceiinfections in mice, dif-ferentiation from slender to stumpy senescent trypanosomes was required to activate theprotective immune response in the resistant mouse strain. The early accumulation of senes-cent trypanosomes correlated with an earlier priming of the immune system which appearedto be the basis for the relative resistance of the trypanotolerant mice (Sendashoga and Black,1982; Black et al., 1985). Similarly, the long prepatent period inT. congolense-infected greyduiker may be ascribed to this parasite-regulating phenomenon.

Infected grey duiker had significantly elevated erythrocyte GGT levels during the peak ofinfection. GGT is present in high concentration in young erythrocytes, in the serum and in allother body cells excluding muscle cells (Moss et al., 1987). Thus, the increased erythrocyteGGT observed in the infected grey duiker in this study may be related to a prompt releaseof young erythrocytes into peripheral circulation in response to trypanosome infection.This may indicate an earlier and more productive bone marrow erythroid response in greyduiker duringT. congolenseinfection than in sheep and goats. There was decrease in thelevels of erythrocyte GGT in infected sheep and goats and this may be related to ineffectiveerythropoiesis as earlier described by Anosa et al. (1992).

Hyperproteinemia reported in sheep and goats in this study agrees with the findingsof Ogunsanmi et al. (1994a) inT. brucei infected sheep and this has been attributed tohypergammaglobulinemia most especially IgM. The rise in ALP, ALT, AST and GGT levelsobserved in sheep and goats in this study may be related to organ damage, particularly theliver, heart, muscles and kidneys which have been reported to occur during trypanosomosis(Losos and Ikede, 1972). The use of changes in serum enzymes to monitor tissue damageand/or proliferation in response to disease has been documented (Zilva and Pannall, 1984;Moss et al., 1987). This also corrborates to similar observations in goats infected withT.congolense(Adah et al., 1992) and inT. bruceiinfection of dogs (Omotainse et al., 1994).Also, the increase in the levels of these enzymes may be related to the amount of lysedtrypanosomes in the infected host (Moon et al., 1968).

The loss of most erythrocyte constituents with erythrocyte ageing has been reported asphysiological, since little metabolism takes place within the matured erythrocytes (Jain,1986). The otherwise normal phenomenon has been reported to be hastened leading to re-duction in erythrocytic half-life, and their earlier removal from circulation and subsequentdestruction by cells of the monocytic phagocytic system (MPS) in the spleen, liver, lungsand bone marrow during trypanosomosis; and has been widely accepted as one of the majorcauses of anaemia during both human and animal trypanosomosis (Aminoff, 1988; Anosa,1988; Murray and Dexter, 1988). In the present study, the duiker were able to maintain thelevels of erythrocyte ALP, ALT and AST within normal levels throughout the course of in-fection. These results show that the erythrocytes of grey duiker were obviously superior andthey were better able to maintain the normal levels of these constituents than sheep and goats.

In the erythrocyte peroxidation assays, the hydrogen peroxide promoted the formation offree radicals in the presence of oxides. The hydroxyl free radicals initiate a self-propagating


reaction of oxidative damage to the polyunsaturated fatty acid components of the erythrocytecell membrane. The degradation products of the peroxidation such as lipid hydroperoxideand malonaldehyde (MDA) (Slater, 1984), which are thiobarbituric acid reactive substances,are therefore estimated (Duthie et al., 1989). The increased production of thiobarbituricacid reactive substances by the erythrocytes in the infected sheep and goats indicated areduced ability of these small domestic animals than that of grey duikers to prevent freeradical-mediated lipid peroxidation in the erythrocyte membrane. It could also be suggestedthat grey duiker has a higher physiological capability to mop up free radicals in its systemthan sheep and goats. This observation is consistent with the report of Ameh (1984) whoshowed that there was a significant increase in the susceptibility of erythrocytes to oxidativehaemolysis in 30mM H2O2 3–4 days after challenge withT.b. gambiense. This phenomenonwas postulated to be due to the depletion of erythrocyte and liver glutathione (Ameh, 1984)and a reduction in the ability to prevent free radical-mediated erythrocyte membrane damagein acuteT. bruceiinfection of mice (Igbokwe et al., 1994). The secretion into plasma of freeradicals, such as peroxides, singlet oxygen, hydroxyl ions and products of nitrogen oxidesmetabolism have been shown to be increased during protozoan infections as a result ofincrease in oxidative burst of macrophages and neutrophils (Haidaris and Bonventre, 1982;Grosskinsky et al., 1983; Lima and Kierszenbaum, 1985; Wozencraft, 1986). The presenceand the magnitude of the production of these toxic products as well as trypanosome secretedlytic enzymes (Huan et al., 1975; Knowles et al., 1989) into the plasma during trypanosomeinfection in vivo (Grosskinsky et al., 1983) might have contributed to a simultaneous increasein in vitro erythrocyte damage and hence, anaemia as observed inT. congolense-infectedsheep and goats in this study and those of others (Ogunsanmi et al., 1994b).

5. Conclusions

This study has shown that grey duiker has demonstrated its ability to withstand the effectsof the disease caused byT. congolenseboth by limiting the level of parasitaemia as well ashaving erythrocyte properties that could withstand haemolysis better than sheep and goatsduring infection. These two factors may be part of genetically-endowed trypanotolerantcharacteristics of the grey duiker.

References

Adah, M.J., Otesile, E.B., Joshua, R.A., 1992. Changes in level of transminases in goats experimentally infectedwith Trypanosoma congolense. Revue Elev. Med. Vet. Pays Trop. 45, 284–286.

Ameh, D.A., 1984. Depletion of reduced glutathione and the susceptibility of erythrocytes to oxidative haemolysisin rats infected withTrypanosoma brucei gambiense. IRCS Med. Sci. 12, 130.

Aminoff, D., 1988. The role of sialoglycoconjugates in the ageing and sequestration of red cells in circulation.Blood Cells 14, 229–247.

Anosa, V.O., 1988. Haematological and biochemical changes in human and animal trypanosomiasis: Parts I & II.Rev. Elev. Med. Vet. Pays Trop. 41 (68–78), 151–164.

Anosa, V.O., Kaneko, J.J., 1983. Pathogenesis ofTrypanosoma bruceiinfection in deer mice (Peromyscusmaniculatus). Haematologic, erythrocyte biochemical and iron metabolic aspects. Am. J. Vet. Res. 44, 639–644.


Anosa, V.O., Logan-Henfrey, L.L., Shaw, M.K., 1992. A light and electron microscopic study of changes in bloodand bone marrow in acute haemorrhagicTrypanosoma vivaxinfections of calves. Vet. Pathol. 29, 33–45.

Assoku, R.K.G., Gardiner, P.R., 1989. Detection of antibodies to platelets and erythrocytes during infection withhaemorrhagic-causingTrypanosoma vivaxin Ayrshire cattle. Vet. Parasitol. 31, 199–216.

Baltz, T., Baltz, D., Giroud, C., Crockett, J., 1985. Cultivation in semi-defined medium of animal infective formsof Trypanosoma brucei, T. equiperdum, T. evansi, T. rhodesienseandT. gambiense. EMBO J. 4, 1273–1277.

Black, S.J., Sendashonga, C.N., O’Brien, C., Borowy, N.K., Naessens, J., Webster, P., Murray, M., 1985. Regulationof parasitaemia in mice infected withTrypanosoma brucei. Curr. Top. Microbiol. Immunol. 117, 93–118.

Braun, V., Furer, R., Lutz, H., 1991. Selenium and vitamin E in blood sera of cows from farms with increasedincidence of disease. Vet. Rec. 128, 543–547.

Duncan, R.D., 1959. Multiple tests and multiple F tests. Biometrics 11, 1–41.Duthie, G.G., Arthur, J.R., Bremmer, P., Kikuchi, Y., Nicol, F., 1989. Increased peroxidation of erythrocytes of

stress-susceptible pigs. An improved diagnostic test for porcine stress syndrome. Am. J. Vet. Res. 50, 84–87.Esievo, K.A.N., Saror, D.I., Ilemobade, A.A., Hallaway, M.H., 1982. Variations in erythrocyte surface and free

serum sialic acid concentrations during experimentalTrypanosoma vivaxinfection in cattle. Res. Vet. Sci. 32,1–5.

Esievo, K.A.N., Saror, D.I., Adegoke, O.O., 1984. Depleted serum haptoglobin in acute bovine trypanosomiasis.Vet. Parasitol. 15, 181–185.

Fairbanks, V.F., Klee, G.G., 1989. Biochemical aspects of haematology. In: Tietz, N.W. (Ed.), Fundamentals ofClinical Chemistry, 3rd Edition. W.B. Saunders Company, Philadelphia, Chapter 24, pp. 789–824.

Gardiner, P.R., Assoku, R.K.G., Whitelaw, D.D., Murray, M., 1989. Haemorrhagic lesions resulting fromTrypanosoma vivaxinfection in Ayrshire Cattle. Vet. Parasitol. 31, 187–197.

Grootenhuis, J.G., Dwinger, R.H., Dolan, R.B., Moloo, S.K., Murray, M., 1990. Susceptibility of African buffaloand Boran Cattle toTrypanosoma congolensetransmitted byGlossina morsitans centralis. Vet. Parasitol. 35,219–231.

Grosskinsky, C.M., Ezekowitz, R.A.B., Berton, G., Gordon, S., Askonsas, B.A., 1983. Mactrophage activation inmurine African trypanosomiasis. Infect. Immunol. 39, 1080–1086.

Haidaris, C.G., Bonventre, P.F., 1982. A role of oxygen-dependent mechanisms in killingLeishmania donovanitissue forms by activated macrophages. J. Immunol. 129, 850–855.

Hochstein, P., Jain, S.K., 1981. Association of lipid peroxidation and polymerization of membrane proteins witherythrocyte aging. Fed. Proc. 40, 183–188.

Huan, C.N., Webb, L., Lambert, P.H., Miescher, P.A., 1975. Pathogenesis of anaemia in African trypanosomiasis:characterization and purification of a haemolytic factor. Schweiz. Med. Wschr. 105, 1582–1583.

Igbokwe, I.O., Esievo, K.A.N., Saror, D.I., Obagaiye, O.K., 1994. Increased susceptibility of erythrocytes to invitro peroxidation in acuteTrypanosoma bruceiinfection of mice. Vet. Parasitol. 55, 279–286.

Jain, N.C., 1986. In: Jain, N.C. (Ed.), Schalm’s Veterinary Haematology, 4th Edition. Lea and Febiger, Philadelphia,pp. 20–86.

Klebanoff, S.J., 1982. Oxygen-dependent cytotoxic mechanisms of phenotype. In: Gallin, J.I., Fauci, A.S. (Eds.),Advances in Host Defense Mechanisms, Vol. 1. Raven Press, New York, pp. 111–162.

Knight, J.A., Voorhees, R.P., Martin, L., Anstall, H., 1992. Lipid peroxidation in stored red cells. Transfusion 32,354–357.

Knowles, G., Abebe, G., Black, S.J., 1989. Detection of parasite peptidase in the plasma of heifers infected withTrypanosoma congolense. Mol. Biochem. Parasitol. 34, 25–34.

Lima, M.F., Kierszenbaum, F., 1985. Lactoferrin effects on phagocytic cell function. I. Increased uptake and killingof an intracellular parasite in murine macrophages and human monocytes. J. Immunol. 143, 4176–4183.

Liman, J.W., 1975. Hemolytic anemias. In: Lanman, J.W. (Ed.), Hematology: Physiologic, Pathophysiologic andClinical Principles. Macmillan Publishing Company, New York, Chapter 5, pp. 103–221.

Losos, G.J., Ikede, B.O., 1972. Review of the pathology of domestic and laboratory animals caused byTrypanosomacongolense, T. vivax, T. brucei, T. rhodesienseandT. gambiense. Vet. Pathol. (Suppl.) 9, 1–71.

Moon, A.P., Williams, J.S., Witherspoon, C., 1968. Serum biochemical changes in mice infected withTrypanosomarhodesienseandT. dutoni. Expl. Parasitol. 22, 112–121.

Moss, W.D., Henderson, A.R., Kachmar, J.F., 1987. Enzymes, analytes, methods, pathophysiology, andinterpretation. In: Teitz, N.W. (Ed.), Fundamentals of Clinical Chemistry, 3rd Edition. W.B. Saunders Company,Philadelphia, pp. 346–417.


Murray, M., Dexter, T.M., 1988. Anaemia in bovine trypanosomias: a review. Acta Trop. 45, 389–432.Nantulya, V.M., Musoke, A.J., Rurangirwa, F.R., Moloo, S.K., 1984. Resistance of cattle to tsetse-transmitted

challenge with Trypanosoma bruceior Trypanosoma congolenseafter spontaneous recovery fromsyringe-passaged infections. Infect. Immunol. 43, 735–738.

Niki, E., Yamamoto, Y., Takahashi, M., Yamamoto, Y., Kumuro, E., Miki, M., Yasuda, H., Mino, M., 1988. Freeradical mediated damage of blood and its inhibition by antioxidants. Vitamins 62, 200.

Nyden, S.J., 1948. Changes in ascorbic acid metabolism of the rat during infection withTrypanosoma hippicum(T. evansi). Proc. Soc. Exp. Biol. Med. 69, 206–210.

Ogunsanmi, A.O., Akpavie, S.O., Anosa, V.O., 1994a. Serum biochemical changes in West African dwarf sheepexperimentally infected withTrypanosoma brucei. Rev. Elev. Med. Vet. Pays. Trop. 47, 195–200.

Ogunsanmi, A.O., Akpavie, S.O., Anosa, V.O., 1994b. Haematological changes in ewes experimentally infectedwith Trypanosoma brucei. Rev. Elev. Med. Vet. Pays. Trop. 47, 53–57.

Omotainse, S.O., Anosa, V.O., Falaye, C., 1994. Clinical and biochemical changes in experimentalTrypanosomabrucei infection of dogs. Israel J. Vet. Med. 49, 36–39.

Oyewale, J.O., Ogunsanmi, A.O., Ozegbe, P., 1998. Plasma electrolyte, enzyme, protein and metabolite levels inadult African white-bellied pangolin (Manis tricuspis). Vet. Arhiv. 67, 261–266.

Paris, J., Murray, M., McOdimba, F., 1982. A comparative evaluation of the parasitology techniques currentlyavailable for the diagnosis of African trypanosomiasis in cattle. Acta Trop. 39, 307–316.

Roskin, G.I., Nastiukova, O., 1941. Vitamin C in the protozoic cell. C.R. (Dokl.) Acad. Sci. USSR 32, 566–568.SAS, 1987. Statistical Analysis System, Users’ Guide, Version 6.03. SAS Institute, Cary, NC, USA.Schwacha, M.G., Loegering, D.J., 1992. Respiratory burst capacity of activated macrophages is resistant to

depression by erythrocyte phagocytosis. Inflammation 16, 285–294.Sendashoga, C.N., Black, S.J., 1982. Human responses againstTrypanosoma bruceivariable surface glycoproteins

are induced by degenerating parasites. Parasite Immunol. 4, 245–257.Slater, T.F., 1984. Free radical mechanisms in tissue injury. Biochem. J. 222, 1–15.Varley, H., Gowenlock, A.H., Bell, M., 1980. General topics and common tests. In: Practical Clinical Biochemistry,

Vol. 1. William Heinemann Medical Books Limited, London, pp. 551–557.Wozencraft, A.O., 1986. Damage of malaria-infected erythrocytes following oxidant-generating systems.

Parasitology 92, 559–568.Zilva, J.F., Pannall, P.R., 1984. Clinical Chemistry in Diagnosis and Treatment, 4th Edition. Lloyd-Luke Medical

Books Ltd., London, pp. 367–382.Zinki, J.G., 1989. Leukocyte function. In: Kaneko, J.J. (Ed.), Clinical Biochemistry of Domestic Animals, 4th

Edition. Academic Press, San Diego, CA, pp. 316–337.

Documents

Pathobiochemical mechanisms involved in the control of the disease caused by Trypanosoma congolense in African grey duiker (Sylvicapra grimmia)