HAEMATOLOGY OF EXPERIMENTAL TRYPANOSOMA BRUCEI RHODESIENSE INFECTION IN VERVET MONKEYS 

HEMATOLOGIE DE L’INFECTION EXPERIMENTALE DE TRYPANOSOMA BRUCEI RHODESIENSE CHEZ LES SINGES VERVET

J.M. Kagira ,  J.K. Thuita, J.M. Ngotho,  D.M. Mwangangi & J.M. Ndung’u

Kenya Agricultural Research Institute-Trypanosomiasis Research Centre (KARI-TRC), PO BOX 362, Kikuyu, Kenya.

*Corresponding author: Tel. +254-733-986450, Email: jkagira@yahoo.com

            L’objet de cette étude était d’évaluer les changements hématologiques liés à l’infection de T. b. rhodesiense chez le modèle singe vervet de la maladie du sommeil à rhodesiense. Quatre singes ont été infectés par voie intrapéritonéale avec 104 T. b. rhodesiense et ont fait l’objet de surveillance pour déterminer les changements de leur profil sanguin à l’aide d’un analyseur d’hématologie. Une infection chronique a été observée sur 48 à 112 jours. Vers le 7ème jour après l’infection (pi), une anémie hypochromique microcytique, caractérisée par une baisse de l’hématocrite, du nombre de globules rouges, du volume corpusculaire moyen et de la concentration corpusculaire moyenne de l’hémoglobine s’est développée et a persisté tout au long de l’infection. La numération moyenne de thrombocytes a considérablement diminué de 3 x 105/µl (le jour de l’infection) à 6,8 x 104/µl (au 70ème jour pi), tandis que le volume moyen de thrombocytes s’est constamment accru tout au long de l’infection. Une baisse initiale du nombre total de leucocytes entre le 7ème et le 35ème jours pi, a été suivie d’une augmentation, notamment du nombre de lymphocytes. L’on a conclu que l’anémie hypochromique microcytique, la thrombocytopénie et un début de leucopénie constituent les principaux changements hématologiques liés à une infection chronique de T. b. rhodesiense chez les singes vervet.

Summary

            The objective of this study was to evaluate the haematological aberrations associated with an infection of T.b. rhodesiense in the vervet monkey model of the Rhodesian sleeping sickness. Four monkeys were infected intraperitoneally with 104 T.b. rhodesiense and monitored for changes in the blood profile using a haematological analyser. A chronic infection lasting between 48 and 112 days was observed. Microcytic hypochromic anaemia characterized by a decline in packed cell volume (PCV), red blood cell (RBC) numbers, mean corpuscular volume (MCV) and mean corpuscular haemoglobin concentration (MCHC) developed by day 7 post infection (pi) and persisted throughout the infection. The mean platelets counts declined drastically from 3 x 105/ml (day 0 pi) to 6.8 x 104/ml (day 70 pi) while the mean platelets volume rose consistently throughout the infection. An initial decline in total white blood cell counts between day 7 and 35 pi was followed by an increase, principally of lymphocyte numbers. It is concluded that the microcytic hypochromic anaemia, thrombocytopaenia and an initial leucocytopaenia are the main haematological changes associated with a chronic infection of T.b. rhodesiense infection in vervet monkeys.

Introduction

            African trypanosome infections are generally characterized by haematological aberrations, which drastically influences pathogenesis of the disease. Although a number of studies regarding this phenomenon have been conducted in the past, most of them focussed livestock trypanosomes with only a few on human infections. The rapid decline in red blood cell (RBC) counts, haemoglobin concentration and packed cell volume plus the clear sign of palour in mucous membranes in the infected hosts proves that anaemia is the most critical feature during the pathogenesis of trypanosomosis. For livestock trypanosomosis, the anaemia has been typed as either normocytic normochromic or macrocytic normochromic (Stephens, 1986). Thus, the ability of the erythropoetic system to continue to produce cells from the stem cell precursors is quite remarkable. Conversely,  studies on the type of anaemia caused by human infective T.b. rhodesiense and T.b. gambiense, have shown anaemia ranges between macrocytic normochromic to microcytic hypochromic. The difference in the type anaemia could be due to a number of factors, which includes: stage of the disease, pathogenicity of the trypanosomes and host species. The haematology studies of the human infective trypanosomes have been conducted in rats and mice but none in higher primates whose disease pathogenesis resembles that in man.

            There have also been inconsistent reports on the pattern of leucocyte changes in African trypanosomosis. In livestock, most workers report a distinct leucopenia, which correlates with neutropaenia and lymphopaenia. However, leucocytosis was reported in T. brucei and T. congolense infections in deer and cattle. Thrombocytopenia has also been reported consistently in trypanosomes infections possibly due to production of a platelet lytic product by the parasites.

The primate model of sleeping sickness provides a good opportunity to understand the pathogenesis of human infective T.b. rhodesiense on an animal whose physiology closely resembles that of the humans (Farah et al., 2005). The primate model of sleeping sickness at KETRI has reported on the various aspects of the disease, but only scanty information on haematological is available. The current study therefore evaluated the sequential haematological changes in vervet monkeys (Chlorocebus aethiops) infected with a clone of T.b. rhodesiense.

Material and methods

            All protocols and procedures used in the current study were reviewed and approved by the KETRI Institutional Animal Care and Use Committee.

Animals

            Four vervets monkeys of both sexes, weighing between 2.6-3.7 kgs were used in the current study. The monkeys were wild caught, after which they were subjected to a 90-days quarantine period, when they were screened for evidence of disease including zoonotics, Simian Immunodeficiency Virus (SIV), and helminths. During the period, they became accustomed to handling and staying in the squeeze back steel cages. Before the study, the vervets were transferred to experimental wards and allowed to settle for another two weeks. At quarantine and during the experiment, the animals were fed twice daily with commercial monkey pellets, green maize, carrots, tomatoes, and bananas. Water was provided ad libitum. They were also maintained at an ambient temperature of between 20 – 25 oC.

Trypanosomes

            T.b. rhodesiense stabilate KETRI 3741 used in this study was cloned from KETRI 2537. The latter is a stabilate which has beem used in the KETRI vervet monkey for years, and was a derivative of EATRO 1989 which was isolated in Uganda from a human patient, by direct inoculation of the patient’s blood and lymph node fluid into a monkey and later cryo-preserved (Fink and Schmidt, 1980).

Experimental design

            The four vervet monkeys (489, 534, 531 and 517) were infected by intravenous injection with approximately 104. Before and during the course of infection, a daily clinical evaluation was done. The parasitaemia levels were estimated using the rapid matching method described by Herbert and Lumsden (1976). The animals were anaethestized at weekly intervals with Valium (1.0 mg/kg) and ketamine hydrochloride (10-15 mg/kg), weighed, and detailed examination undertaken. Four millilitres of blood was collected by inguinal venupuncture and used for haematology and biochemistry. Only 1 ml of EDTA blood was used for haematology analysis reported in this study.

            The packed cell volume was determined using the standard microhaematocrit method. Detailed haematology analysis was conducted using an automated haematology analyser (Coulter Ac.T diff, Beckman coulter). For the erythrocyte indices, the parameters evaluated included: total RBC counts, haemoglobin (HB), haematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC) and red cell distribution width (RDW). Platelets values included total platelets counts and mean platelet volume (MPV). The total white blood cell counts and the differential counts were also provided by the analyser.

Parasitaemia

            The prepatent period was 3 days, which was followed by a rise in parasitaemia to a peak at 6 days post infection (dpi). The parasitaemia dropped to undetectable levels (below antilog 5.4) at 9-10 dpi, followed by rise. Thereafter the parasitaemia remained high and was characterized by fluctuations

Clinical signs

            All the animals developed classical symptoms of trypanosomosis, which included fever, raised hair coat, increased respiratory and pulse rates, pallor of mucous membranes, enlarged lymph nodes and spleen, increased aggressiveness and loss in weight. The monkeys were euthanised at extremis. Vervet 489, 534, 531 and 517 were euthanised on 48, 49, 70, and 113 dpi, respectively. Late stage disease signs were observed only in vervet 517.

Haematology

RBC count and Hb concentration (Fig. 1)

            The infection was characterized by a decrease in RBC counts. The mean RBC level dropped from 6000-cells/ml (day 0) to 2700 cells/ml (day 70). The Hb concentration showed the same trend as RBC, with the levels dropping from 15.6 g/dl (day 0) to 4.7 g/dl (day 70). The rate of drop was lower in the animals with a more chronic infection.

Haematocrit (Fig.1)

            During the study, it was observed that the PCV and haematocrit values from same samples differed, with PCV values being always slightly higher. The infection caused a significant decrease in haematocrit. There was consistent drop in mean PCV from 50 (day 0) to 20 (day 70). The rate of decrease was high between day 0 and 21 pi. Thereafter, there was only a slight decline but an accelerated decline was observed starting 56 dpi.

Red cell indices

            The MCV levels declined between 0 and 63 dpi from 81.3 fl to 71.3 fl (Fig. 2). However, there was a slight increase and fluctuations between day 70 and 112 pi. Day 98 and 112 pi values were close to the pre-infection values. The MCH values also decreased slightly between days 0 to 28 pi, but thereafter fluctuated between 22 and 23 pg (Fig. 3).

            Similarly, the MCHC also decreased slightly and remained low between 21 and 35 dpi (29 – 29.7 g/dl). Thereafter, MCHC values fluctuated between 30 and 32 dpi (Fi.g. 3) There were sharper fluctuations in MCHC than MCH levels. The RDW also increased significantly from day 0 (13 fl) to day 49 pi (21 fl.) (Fig. 2). A slight decline occurred starting from day 49 to 70 pi, and a consistent rise was observed in animal 517 starting 91-112 dpi.

            The platelets fall was very characteristic, marked by drastic fall in counts from 3 x 105/ml (day 0 pi) to 6.8 x 104/ml (day 7 pi). Although this was followed by fluctuations, the platelets level remained low, rarely going above 1 x 105 cells/ml. In contrast, the volume of the platelets (MPV) rose significantly after the infection, remaining elevated during the whole infection period. A slight decline in MPV was observed in animal 517 at 98-112 dpi.

Platelets (Fig. 4)

Leucocytes (Fig 5)

            There was a significant drop in total WBC count between day 7 and 35 pi. This was followed by a rise in WBC counts up to when the animals euthanised. There was a sharp increase in number of cells in animal 517 between 98 and 112 dpi. The changes in WBC were mainly due to lymphocyte counts, although between days 98 and 112 pi, the increase could also be attributed to both granulocytes and monocytes.

Discussion

            The present study illustrates the complex haematological changes that occur when monkeys are infected with T.b. rhodesiense. Previous studies using this model reported the occurrence of anaemia and leucopaenia without going into details (Schimdt and Sayer, 1982, Schimdt, 1984). That the occurrence of anaemia in this model is the main factor that curtails the longevity of the disease course in the monkeys was evident in this study. The total RBC counts, Hb, haematocrit, declined consistently, with the decline following a sigmoid curve pattern. The observed reduced rate of decline of these values between days 21-49 could reflect of a period when the bone marrow is able to withstand the effects of trypanosomosis only to be whelmed later. The pallor observed in the mucous membrane coincided with the decline in these values.

            The red cell indices i.e., MCV, MCH, MCHC, and RDW are used to determine the type of anaemia. Microcytic hypochromic type of anaemia was observed in the early stages of the disease well characterized by dramatic decline MCV and a slight decline in MCH and MCHC. However, as the disease progressed MCV values increased although they were characterised by fluctuations. A combination of MCV and RDW has revolutionized interpretations on the type of anaemia (Neiger et al. 2002; Monzon et al., 1987). The red cell distribution width measures the level of heterogeneity in RBC and is an equivalent of anisocytosis observed in blood smears. The consistent increase in RDW in the present study showed that there was an increase in variation of red cells released by the bone marrow as the disease progressed, although the sharp decline in RDW between days 49-70 pi, could indicate a temporary ability of the bone marrow to produce microcytic RBC with reduced variations. In chronic stages of the disease, the MCH and MCHC values improved slightly although not consistently, but were still below the pre-infections levels showing that the bone marrow was still compromised.

            Microcytic hypochromic anaemia has also been reported in rabbits infected with T.b. gambiense (Emeribe and Anosa, 1991). However, in the latter an initial macrocytosis was observed. The microcytic hypochromic type of anaemia has previously been closely associated with iron deficiency (Dacie and Lewis, 2001). It is possible that during T.b. rhodesiense infection, failure of iron incorporation into red cell precursors even in presence of adequate iron storage will precipitate the occurrence of this type of anaemia (Dargie et al., 1979). Trypanosomosis is also characterised by massive erythrophagocytosis by the mononuclear phagocytic system (Dargie et al., 1979). Inefficient recovery of iron from the phagocytosed RBC can also lead to an iron “deficiency” status in the body. In such a case, it will be imperative to consider evaluating level of iron stores in the body.

            The drastic fall in platelets counts immediately after trypanosome infection in this study has also been reported by other workers (Stephens, 1986, Robins-Browne, 1975). In contrast, the volume of the platelets (MPV) rose significantly, remaining elevated during the whole infection period. An increase in MPV is associated with an increased growth of megarkaryocytes in response to thrombopoetic stress especially where there is peripheral destruction of platelets (Thompson and Jakubowski, 1988, Brown, 1988). In this study, a low platelet count combined with increasing platelets volume observed could be indicative of hyper-estruction of platelets (Kriestensen et al., 1992). Indeed, Davies (1982) reported that T.b. rhodesiense has a heat labile toxic protein, which has direct effect on platelets. Low platelets counts could also be due to other factors which include: pooling of blood in the spleen, removal of platelets by mononuclear phagocytic system and increased ‘consumption’ of platelets by disseminated intravascular coagulation reactions which have been widely reported in trypanosomosis (Greenwood and Whittle, 1976, Robins-Browne, 1975);

            In studies involving white blood cell counts, most workers report a distinct leucopaenia (Naylor, 1971; Wellde et al., 1974; Maxie et al., 1979) following trypanosome infection. In the current study, low leucocyte counts were observed between day 7 and 35 pi, followed by a consistent rise thereafter. As in other studies, the pattern of white blood cells strongly coincided with that of lymphocytes showing that the latter plays active role in immunopathogenesis of trypanosomosis. The rise in lymphocytes counts after 35 dpi can be regarded as a good prognostic sign, although its effectiveness in limiting disease pathogenicity is doubtful. The neutrophil counts also declined by day 7 pi and remained low until day 98 pi when there was consistent rise. Severe neutropenia during trypanosomosis infection is thought to increase susceptibility of infected animals to concurrent infections (Stephens, 1986). The occurrence of leucopaenia has been attributed to factors such as leucophagocytosis as a result of trypanosomal antigen coating of leucocytes and depression of leucocyte production (Mackenzie et al., 1978; Kaaya et al., 1980). Other studies have reported occurrence of leucocytosis after trypanosome infection (Katunguka-Rwakishaya, 1992; Paling et al., 1991, Emeribe and Anosa, 1991). In these studies, leucocytosis due to lymphocytosis has been associated with trypanotolerance breeds of animals including N’Dama and Scottish blackface sheep. For example, a comparative study showed that the trypanotolerant N’Dama developed leucocytosis while susceptible Borans developed lymphopaenia after a T. congolense infection (Paling et al., 1991). Thus, the leucocyte response in trypanosomosis is mainly determined by the stage of disease, trypanosome species, and host involved.

            The current study has shown that a T.b. rhodesiense infection in the vervet monkey model of HAT leads to development severe anaemia, thrombocytopaenia and leucopaenia, although the latter improves as the disease progresses. The sharp fall in both platelets and leucocytes by 7 dpi coincided with the first peak in parasitaemia, showing that drop could be related to toxic products from the trypanosomes.

Acknowledgements

            The authors are grateful to the technical assistance provided by Messrs. Ben Kinyanjui, Tom Adino, Stephen Kagwima, Stephen Mwangi and John Oidho of the Primate Division, TRC.

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