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The vervet monkey (Chlorocebus aethiops) as an experimental model for Trypanosoma brucei gambiense human African trypanosomiasis: a clinical, biological and pathological study
Transactions of the Royal Society of Tropical Medicine and Hygiene (2006) 100, 427—436
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The vervet monkey (Chlorocebus aethiops) as an experimental model for Trypanosoma brucei gambiense human African trypanosomiasis: a clinical, biological and pathological study O. Ouwe-Missi-Oukem-Boyer a,b,e,∗,1, J. Mezui-Me-Ndong b,1, C. Boda c, I. Lamine a, F. Labrousse d, S. Bisser b,e, B. Bouteille c a
Centre de Recherche M´ edicale et Sanitaire (CERMES), BP 10887, Niamey, Niger Centre International de Recherches M´ edicales de Franceville (CIRMF), BP 769, Franceville, Gabon c Institut d’Epid´ emiologie Neurologique et de Neurologie Tropicale, (EA 3174) Facult´ e de M´ edecine, 02, rue du Dr. Marcland, 87025 Limoges cedex, France d Centre Hospitalier Universitaire de Limoges, Hˆ opital Universitaire Dupuytren, Service d’Anatomie Pathologique, 02 avenue Martin-Luther-King, 87042 Limoges cedex, France e Minist` ere Franc¸ais des Affaires Etrang` eres, 244 bd Saint-Germain, 75007 Paris, France b
Received 10 May 2005 ; received in revised form 9 July 2005; accepted 11 July 2005 Available online 1 December 2005
KEYWORDS Human African trypanosomiasis; Trypanosoma brucei gambiense; Experimental model; Vervet monkeys; Chlorocebus aethiops
∗ 1
Summary It has long been known that the vervet monkey, Chlorocebus (C.) aethiops, can be infected with Trypanosoma rhodesiense, but this model has not been described for T. gambiense. In this study, we report the development of such a model for human African trypanosomiasis. Twelve vervet monkeys infected with T. gambiense developed chronic disease. The duration of the disease ranged between 23 and 612 days (median 89 days) in five untreated animals. Trypanosomes were detected in the blood within the first 10 days postinfection and in the cerebrospinal fluid, with a median delay of 120 days (n = 4, range 28—348 days). Clinical changes included loss of weight, adenopathy, and in some cases eyelid oedema and lethargy. Haematological alterations included decreases in haemoglobin level and transitory decreases in platelet count. Biological modifications included increased gamma globulins and total proteins and decreased albumin. Pathological features of the infection were presence of Mott’s cells, inflammatory infiltration of either mononuclear cells or lymphocytes and plasma cells in the brain parenchyma, and astrocytosis. These observations indicate that the development of the disease in vervet monkeys is similar to human T. gambiense infection. We
Corresponding author. Tel.: +241 67 70 92; fax: +241 67 72 95. E-mail address: [email protected] (O. Ouwe-Missi-Oukem-Boyer). These authors contributed equally to this work.
0035-9203/$ — see front matter © 2005 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.trstmh.2005.07.023
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O. Ouwe-Missi-Oukem-Boyer et al. conclude that C. aethiops is a promising experimental primate model for the study of T. gambiense trypanosomiasis. © 2005 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd. All rights reserved.
1. Introduction Human African trypanosomiasis (HAT), or sleeping sickness, is an endemic disease in 36 sub-Saharan countries. More than 60 million people living in some 200 microfoci are at risk of contracting the disease. HAT has recently re-emerged in southern Sudan and central Africa (Moore and Richer, 2001; Van Nieuwenhove et al., 2001), and the 45 000 new cases reported to WHO in 1999 are certainly underestimated (Anonymous, 1999). The disease is caused by infection with one of the two subspecies of Trypanosoma brucei: T. b. gambiense or T. b. rhodesiense. HAT is characterized by a haemolymphatic phase (also called ‘early phase’ or stage 1), in which trypanosomes invade the blood and lymphatic systems, and a meningoencephalitic phase (‘late phase’ or stage 2) characterized by progressive invasion of the central nervous system (CNS) by trypanosomes and inflammatory cells leading to neurological disorders and damages (Kennedy, 2004). If left untreated, the disease is always fatal. There are two major problems in the treatment of HAT: first, accurate diagnosis of the timing of CNS invasion is required because the stage of the disease determines the choice of treatment, and second, the molecules that are active against trypanosomes are toxic to the host. Few drugs are commercially available for the treatment of HAT: suramin and pentamidine for cases in early stages without CNS involvement; and melarsoprol, a highly toxic arsenical, and more recently eflornithine for the meningoencephalitic stage (Bouteille et al., 2003). Less toxic medications that are active against trypanosomes in both stages of the disease are required. These two problems have made it necessary to develop in-vivo experimental models. Laboratory rodents and sheep infected with T. b. gambiense and T. b. rhodesiense have been used as experimental models of HAT. However, murine models do not show obvious signs of meningoencephalitis, and it is impossible to obtain sufficient cerebrospinal fluid (CSF) for post-infection followup (Keita et al., 1997; Poltera et al., 1980), while sheep (Ovis aries) give a subacute illness of the CNS but are phylogenetically distant from humans (Bouteille et al., 1988a, 1988b). Vervet monkeys (Chlorocebus aethiops) infected with T. b. rhodesiense are therefore currently used in therapeutic research (Brun et al., 2001; Gichuki et al., 1994; Poltera et al., 1985; Schmidt and Sayer, 1982a). To date, no experimental primate model is available for T. b. gambiense. An ideal experimental primate model of HAT should be capable of developing chronic disease, including CNS invasion and obvious signs of meningoencephalitis. Such a model has not yet been developed (Bouteille et al., 1999), as vervets infected with T. b. rhodesiense show short survival without treatment, and significant CNS involvement can be achieved only by prolonging survival with an ineffective trypanocidal treatment (Schmidt and Sayer, 1982a, 1982b). Trypanosoma brucei gambiense, which produces a more chronic
infection, may prove to be a better model for studies of CNS involvement in HAT. We report here observations showing that infection of the vervet monkey with T. b. gambiense might constitute such an experimental model of HAT.
2. Materials and methods The study took place in two different centres, at two different times and using two different sets of study animals. However, the same protocol and the same parasite strain were used in both studies, and the data were therefore combined for this report.
2.1. Animals Thirteen adult vervet monkeys of both sexes were studied at: (1) Centre International de Recherches M´ edicales de Franceville (CIRMF), Gabon during 1996—1997 (eight monkeys originating from Ololua, Kenya); and (2) Centre de Recherche M´ edicale et Sanitaire (CERMES), Niamey, Niger during 2001—2003 (five monkeys originating from Hazyview, Republic of South Africa). Median body weight (range) at the beginning of the experiment was 3.3 (2.9—4.3) kg at CIRMF and 3.7 (2.5—5.1) kg at CERMES. All animals were quarantined for a 90-day period, during which monkeys were housed in individual cages and familiarized with the single-cage capture facilities. Before enrolment in the study, all animals tested negative for tuberculosis, simian immunodeficiency virus, simian T-cell lymphotropic virus type 1, protozoa (particularly trypanosomes) and helminth parasites via repeated stool, urine and blood examinations. The animals were maintained on a diet of fresh vegetables/fruits (40% of the daily energy contribution) and monkey biscuits (60% of the daily energy contribution). Food was given twice daily and water was provided ad libitum. Animals were also housed in individual cages during the experiment. This protocol was approved by the CIRMF Ethical Committee for Control of the Use of the Animals (CECUA) and animal handling was performed according to the Guidelines for the Care and Use of Laboratory Animals as adopted and promulgated by the Institute of Laboratory Animal Resources (1996). The experimental design of this study is presented in Figure 1. Because few monkeys were available, four animals (namely V1, V3, V5 and 1457) were initially used as controls before being infected.
2.2. Parasites The strain of T. b. gambiense MBA ITMAP 1811 used for both experiments is derived from a Congolese (RD Congo) patient and was kindly provided by Dominique Le Ray and Yves Claes of the Protozoology Laboratory, Tropical Medicine Institute (Antwerpen, Belgium). Stabilates were stored frozen in
Trypanosoma brucei gambiense in vervet monkeys
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Figure 1 Follow-up schedule of the 13 monkeys. Because few monkeys were available, four animals (V1, V3, V5, 1457) were initially used as controls before being infected. The cause for end of follow-up is indicated by a black (death), blue (treatment) or white (stop) arrow. Thin black arrows show the first day with trypanosomes in cerebrospinal fluid (CSF) in monkeys V1, V3, 553 and 1561. Survival after the first positive CSF test is reported in italics for the two untreated monkeys (V1 and 1561). * single oral administration of 100 mg/kg megazol alone (Enanga et al., 2000); # oral administration of 100 mg/kg megazol on three consecutive days (C. Boda et al., unpublished data); £ single oral administration of 100 mg/kg megazol immediately followed by intravenous injection of 20 mg/kg suramin (Enanga et al., 2000).
liquid nitrogen, then rapidly thawed and inoculated into Swiss mice by intra-peritoneal injection. Parasites were passaged several times in mice to ensure strain virulence. At day 0, blood was sampled from infected mice and trypanosome mobility verified via microscopical examination. Parasites were diluted in 250 l RPMI-1640 medium (Sigma, Saint Quentin Fallavier, France). Anaesthesia was carried out via intramuscular injection of 10 mg/kg ketamine hydrochloride (Imalgene, Rhone-M´ erieux, Lyon, France). Twelve sedated vervet monkeys were infected intravenously with 103 trypanosomes per animal.
2.3. Clinical follow-up The general behaviour and vigilance of vervet monkeys were recorded each week before anaesthesia. Animals were then sedated with ketamine hydrochloride and clinical parameters, including rectal temperature, body weight, adenopathy, oedema and splenomegaly, were recorded. Follow-up studies were interrupted for seven of the 12
infected monkeys, because these animals were included in a therapeutic protocol (Chauviere et al., 2003; Enanga et al., 2000) (C. Boda et al., unpublished data). The remaining five animals were monitored until death (see Figure 1).
2.4. Blood samples Parasitaemia was investigated daily via finger-prick blood tests, from infection until the test appeared positive. Blood was drawn from each monkey via inguinal venipuncture at regular intervals for parasitology and to determine the haematocrit, haemoglobin levels, and erythrocyte, leucocyte, platelet and differential leucocyte counts. All these analyses were performed immediately. Where available, remaining blood samples were stored at −20 ◦ C. Serum specimens were also obtained for biochemical and serological studies. Biochemical parameters measured included glucose, creatinine, serum glutamic oxalacetic transaminase (SGOT) and serum glutamic pyruvic transaminase (SGPT), total protein and albumin concentrations. Serum protein
−/− −/− −/− −/− −/− +/− −/− −/− +/− −/− +/+ +/− −/− 3.6 2.9 4.3 3.8 3.7 5.1 3.8 2.5 3.3 4.3 3.7 2.5
+ + + + + + + + + + + + +
+ + + + + + + + + + + + +
3 7 3 10 3 6 7 7 3 7 4 3
? ? 28 70 170 348 F F M F F M M F F F F F
Positive blood test (day)
Positive CSF test (day)
493 1457 CA5 CA6 1465 V5 V8 V10 V3 553 1561 V1 Controls
Initial weight (kg)
Clinical parameters
Haematological parameters
Parasitological parameters
Experimental subjects, controls, parameters evaluated and parasitology results
All infected animals showed weight loss after infection, while body weight was stable in uninfected controls (data not shown). Weight loss was continuous in six of 12 infected monkeys, and represented 21—37% of the initial body weight. Two animals showed minor (4—11%) and continuous weight loss, while two others showed minor (4—8%) and transient (2—6 weeks) weight loss. Follow-up for the latter two monkeys was very short, meaning that it was impossible to determine whether the early weight loss was transient or permanent. All animals showed enlargement of both the axillary and the inguinal lymph nodes, which increased in size by 50—400% (0.5—2 cm). Splenomegaly was observed in seven monkeys, during the entire follow-up in V1, and irregularly in six animals, persisting until the late phase of the disease in three monkeys. Splenomegaly was not observed in the five remaining monkeys. Transient eyelid oedema was observed in two CIRMF monkeys at days 42 and 49 post-infection, and repeatedly in two CERMES monkeys: at days 98 and 113 post-infection in one
Sex
3.1. Clinical parameters
Animal
3. Results
Table 1
At death, an autopsy was performed on three out of the five untreated monkeys, and on one treated monkey that died after the first day of treatment (Table 1). Organs were collected and fixed with formalin at CIRMF or at CERMES, then shipped to France. Post-mortem and histological examinations of the brain, spinal cord, heart, lungs, liver, kidneys, spleen, stomach and intestine were carried out. Fixation with formalin, embedding in paraffin and staining with haematoxylin and eosin were performed according to standard histological techniques (Keita et al., 1997). Immunohistochemical studies were made on one untreated monkey, as described previously (Keita et al., 1997).
CSF parasite load (tryp/ml)
2.6. Histology and immunohistochemistry
? ? 200 23 000 730 000 100—800
CSF cell count (n/l)
Lumbar punctures were performed at 2-week intervals under general anaesthesia. Cerebrospinal fluid (1 to 2 ml per animal) was collected and analysed immediately for parasitosis and cell count. Visibly haemorrhagic CSF samples were discarded. Where available, remaining CSF samples were stored at −20 ◦ C.
? ? 28—31 74 40 20—36
Biochemical analyses/serum protein electrophoresis +/+ +/+ +/+ +/+ +/+ +/− +/− +/− +/− +/+ +/− +/− +/−
2.5. CSF samples
− − − − − − − − − − + − −
electrophoresis and serological analysis were performed only on monkeys at CIRMF (Table 1). Biochemical analyses were performed immediately, while immunological experiments were performed on frozen (−20 ◦ C) samples, at the end of the follow-up. Indirect immunofluorescence tests were performed using fluorescein isothiocyanate (FITC)conjugated goat anti-human IgG and IgM (Bio-Sys, Compiegne, France). Spots were prepared with T. b. gambiense LiTat 1.3 (from lyophilized trypanosomes kindly provided by Pr N. Van Meirvenne and Dr P. B¨ uscher of the department of Tropical Medicine, Antwerpen, Belgium).
Histological/ immunohistochemical analyses
O. Ouwe-Missi-Oukem-Boyer et al.
Serological analysis
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Trypanosoma brucei gambiense in vervet monkeys
Figure 2 (B).
431
Face of an infected vervet monkey without eyelid oedema (A) and with obvious eyelid oedema (at day 181 post-infection)
monkey and days 181 and 433 in the other (Figure 2). Several other clinical symptoms were occasionally observed for individual subjects, including a stiff neck, lethargy, drowsiness, loss of appetite, dehydration and hair loss. In the five untreated animals, the median duration of the disease was 89 days (range 23—612 days; Figure 1). Control animals did not exhibit any detectable sign of alteration to their general behaviour during the experiment.
3.2. Parasitology 3.2.1. Parasitaemia Control animals were negative for trypanosomes during the entire follow-up. In infected monkeys, trypanosomes were detectable in the blood between the third and seventh day after infection, although one subject remained negative until the tenth day (Table 1). The time course of parasitaemia differed substantially between infected monkeys, and parasite loads also varied considerably between animals. The median curve for parasitaemia was thus uninformative (data not shown). However, although the parasitaemia curves were irregular, they always showed several peaks (at different times post-infection and of different intensity), separated by intervals (of varying length) without any trypanosomes (data not shown). Of the five infected monkeys that were monitored until death, two (vervets 493 and 1561) showed a high peak in parasitaemia just before death (21.6 × 106 and 16.1 × 106 trypanosomes per ml respectively), whereas detectable trypanosomes were no longer found in the blood of the other three monkeys, even when concentration techniques were used. 3.2.2. CSF parasitosis and CSF cell count During the experimental period, six of the 12 infected monkeys showed no detectable trypanosomes in their CSF, even when concentration techniques were used (Table 1). These animals were thus classed as being in the haemolymphatic phase of the disease. Parasites were found in the CSF of four infected subjects, with a median delay of 120 days, although large differences
occurred between animals in the timing of the first appearance of trypanosomes in the CSF (days 28, 70, 170 and 348). Parasite loads also differed considerably between animals: two showed low parasite loads (100—800 per ml), while two had considerably more trypanosomes (23 000—730 000 per ml). An increase in cell counts was also detected in these four animals, confirming that they were in the meningoencephalitic phase of the disease (Table 1). CSF parasitosis was not correlated with parasitaemia: three subjects had the same moderate blood parasite load (peaks reaching 106 trypanosomes per ml) but large differences in CSF parasite load, whereas the blood parasite load of the fourth subject was very high (peak reaching 35 × 106 trypanosomes per ml) and CSF parasite load low. Vervets V3 and 553 were treated, while vervets V1 and 1561 were monitored until death. Survival was considerably shorter in the vervet with a large number of CSF trypanosomes (vervet 1561; 19 days survival), while the animal with fewer parasites survived more than 8 months in stage 2 (V1; 264 days of survival following the first positive CSF examination) (Figure 1). For two monkeys, stage determination was uncertain because CSF parasite status was unclear. Erythrocytes were detected in the CSF, meaning that it was not possible to confirm whether the presence of trypanosomes and lymphocytes was due to contamination with blood, or to disease. A repeat examination was not possible because these animals were included in a therapeutic protocol that began before the disease stage could be clarified. These animals were therefore considered as being in an intermediate phase (between stage 1 and 2) for the purposes of this study.
3.3. Biological analysis 3.3.1. Haematology All infected animals showed decreased haemoglobin values (Figure 3). Median values varied between 12.7 g/l and 8.0 g/l during the first month post-infection. The lowest median value was 7.4 g/l, which occurred 2—3 months after infection. Thereafter, haemoglobin levels
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Figure 3 Haemoglobin levels in vervet monkeys infected with Trypanosoma brucei gambiense. Boxes show the median and 25th and 75th percentiles, with 10th and 90th percentiles represented by error bars for 12 (days 0 and 14), 11 (day 28), 10 (day 41), 7 (day 56), 6 (day 70), 5 (day 84), 4 (days 97 and 112), 3 (days 125, 140 and 153) and 2 (days 170, 178 and 189) infected animals. Dashed horizontal lines indicate the range of normative values for haemoglobin in vervet serum (O. Ouwe-Missi-Oukem-Boyer et al., unpublished data).
remained stable during the course of the infection, but were always less than 10 g/l. By contrast, the median level of haemoglobin in control animals (range) was 12.5 (11.8—13.3) g/l during the study, and was always within the normal range (11.8—14.2 g/dl; O. Ouwe-Missi-Oukem-Boyer, unpublished data). The haematocrit and erythrocyte count followed a similar pattern, decreasing within the first month post-infection, then remaining at the low values of approximately 20% and 3 million, respectively. Leucocyte counts changed considerably during the course of infection, from initial median values of 5600 per mm3 to 2500 per mm3 , 3 weeks post-infection, and 9000—11 000 per mm3 after 3 months. However, individuals varied considerably, and peaks of 15 000—17 500 per mm3 were repeatedly recorded for one subject. Disturbances of the polynuclear/lymphocyte ratio were observed with an alternate prevalence of polynuclear and lymphocytes. The median absolute number of lymphocytes showed a moderate increase during the course of infection, reaching 4000—5000 cells per mm3 . The percentage of monocytes showed a similar trend, appearing stable and in the normal range (10—19%; O. Ouwe-Missi-Oukem-Boyer, unpublished data) during the first 3 months after infection, then increasing to 18—27%. However, again great variability occurred between animals. The two monkeys that were followed for the longest period showed an unexpected collapse in the percentage of monocytes a few days before death (2% for one subject, 5% for the other). Platelet counts decreased within the first weeks postinfection, attaining minimum values of 43 000—50 000 per mm3 in two animals. This decrease occurred in all infected subjects, but varied in time and intensity. In most animals, platelet counts remained low (normal range: 256 000—381 000 per mm3 ; O. Ouwe-Missi-Oukem-Boyer, unpublished data), fluctuating between 100 000 per mm3 and 240 000 per mm3 during the course of the disease.
3.3.2. Biology No significant modifications in glucose levels were observed in infected monkeys. Almost all animals showed a transient increase in creatinine, reaching a median of 11 mg/l early after infection, then a progressive return to normal median values of 5—6 mg/l until the end of the study. Substantial fluctuations of transaminases (SGOT and SGPT) occurred at the individual level, although these were not detectable when median curves were plotted (data not shown). Total serum proteins increased (Figure 4A), but albumin levels decreased as infection progressed (Figure 4B). The increase in proteins was due primarily to an increase in gamma globulins (Figure 5), while alpha 1, alpha 2 and beta globulin levels remained approximately stable. 3.3.3. Immunology The detection of specific antibodies was carried out using blood from monkey 1561, the subject that had the longest follow-up at CIRMF. This animal became positive for IgM within the first 2 weeks after inoculation. The maximum titre, 1:3200, was obtained 7 weeks after infection. Thereafter, titres oscillated between 1:200 and 1:800 until death, with the exception of two peaks of 1:1600, at days 70 and 170. IgG appeared 2 weeks after IgM detection. IgG titres increased from day 28 (1:1600) to day 86 (1:6400), then oscillated between 1:6400 and 1:3200 until day 170. Titres dropped dramatically from 1:6400 to 1:1600 during the meningoencephalitic phase (day 170 to death). 3.3.4. Histology and immunohistochemistry In vervet 1561, histological examination of brain tissues revealed perivasculitis with abundant infiltration of mononuclear cells in the brain parenchyma, indicating that HAT may have been responsible for the death of this monkey (Figure 6A). In vervet V1, autopsy revealed
Trypanosoma brucei gambiense in vervet monkeys
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Figure 4 Total serum proteins (A) and albumin (B) levels in vervet monkeys infected with Trypanosoma brucei gambiense. Boxes show the median and 25th and 75th percentiles, with 10th and 90th percentiles represented by error bars for 12 (days 0 and 14), 10 (day 41), 9 (days 7, 21 and 28), 7 (days 35 and 49), 5 (day 56), 4 (day 70), 3 (days 63 and 84) and 2 (days 77, 97, 112, 125 and 153) infected animals. Dashed horizontal lines indicate the range of normative values for each parameter in vervet serum (O. Ouwe-Missi-Oukem-Boyer et al., unpublished data).
extensive meningoencephalitis lesions. The brain showed serious inflammatory infiltration of lymphocytes and plasma cells. Mott’s cells were reported in perivascular sites and also in the white matter (data not shown). These two features are strongly suggestive of trypanosomiasis, which was considered the cause of death. Vervet V5 did not present any sign of encephalitis, but low intensity inflammatory infiltration of mononuclear cells was reported in the meninges. Low-level myocarditis was also observed in this animal, but was unlikely to be the cause of death. Vervet V3, which died a few hours after treatment, showed mild to severe inflammatory lesions in the myocardium, with a predominance of lymphocytes and plasma cells; lesions were less intense in the lungs and other organs and
were absent in the brain (data not shown). The general state of this monkey deteriorated considerably during the several days prior to death, and hyperthermia (42.8 ◦ C) and convulsive fits were recorded immediately before death. These symptoms may have been due to trypanosomiasis without obvious brain damage, despite the fact that the monkey was classed as stage 2, when brain lesions are expected. In vervet 1561, immunohistochemical examination of glial fibrillary acidic protein (GFAP) showed strong astrocytosis in the brain parenchyma and mild meningitis with the presence of mononuclear cells in the white and grey matter (Figure 6B). These inflammatory lesions correspond to degree 3 of meningoencephalitis (Keita et al., 1997).
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Figure 5 Distribution of the five fractions of serum proteins in vervet monkeys infected with Trypanosoma brucei gambiense. Each point of the curves represents the median for six animals.
4. Discussion
Figure 6 Brain histopathology in vervet monkey 1561 infected with Trypanosoma brucei gambiense for 189 days: perivasculitis (arrows) in the brain parenchyma, haematoxylin-eosin, ×120 (A); astrocytosis in the brain parenchyma (arrows) and mild meningitis with many mononuclear cells in the white and the grey matter (arrow heads), glial fibrillary acidic protein immunohistochemistry, ×120 (B).
In this study, we present a chronic experimental model of HAT in C. aethiops infected by T. b. gambiense. Our results show that vervet monkeys are susceptible to T. b. gambiense MBA strain, confirming previous data on the absence of a trypanocidal factor in this primate species (Seed et al., 1990). All animals developed the haemolymphatic phase of the disease after a brief prepatent period. During the followup of the infected monkeys, trypanosomes were detected in the blood irregularly, and peaks of parasitaemia, when these occurred, varied in intensity. After a delay of variable length (28—348 days), the appearance of trypanosomes occurred simultaneously with an increased cell count in the CSF of four vervets, indicating that parasites may have reached the CNS in these animals. As in humans, both the haemolymphatic stage and the meningoencephalitic stage occurred in our experimental model, despite the fact that few infected monkeys were monitored. Two animals were classified as being in an intermediate stage because the presence of trypanosomes and the increased cell count may have been due to blood contamination of the CSF sample. Stage determination in human HAT patients may be also difficult when the presence of parasites in CSF is uncertain or when trypanosomes are detected but there is no evidence of an increased CSF cell count. Indeed, the criteria for stage 2 determination are highly controversial (Van Meirvenne, 1999), and the associated choice of treatment remains problematic, particularly in ‘borderline’ cases (Van Nieuwenhove, 1999). Since regular lumbar punctures are possible in vervets, T. b. gambiense-infected monkeys may be a helpful experimental model for the identification of alternative markers for stage determination. Experiments based on ‘lymphocyte subset typing’ in the blood and CSF of infected monkeys and IgM measurement in the CSF are currently under investigation in our laboratory. Adenopathy was found in both the axillary and inguinal regions as soon as the vervets were infected. Splenomegaly
Trypanosoma brucei gambiense in vervet monkeys was frequent, although it was not observed in all monkeys. Eyelid oedema was observed in several monkeys at different times during the follow-up. Cutaneous changes also occurred occasionally. These clinical symptoms are very similar to those described for HAT in humans (Dumas and Bisser, 1999). Weight loss, low haematocrit and anaemia occurred in all animals, and leucocyte counts changed considerably during the follow-up. Decreased weight, haemoglobin levels and haematocrit have also been reported in humans with HAT (Bisser et al., 1997; Van Meirvenne, 1999). Moreover, the reduced platelet counts observed are concordant with data from humans infected with T. b. gambiense (RobinsBrowne et al., 1975). The absence of hypoglycaemia in our subjects confirms a previous study in which subjects with Gambian trypanosomiasis were compared to controls for 29 biochemical analyses (Bisser et al., 1997). The modifications in protein levels that we observed, such as increased gamma globulin and total proteins and decreased albumin, are also characteristic of HAT in humans (Bisser et al., 1997; Van Meirvenne, 1999). Infection of C. aethiops with T. b. gambiense thus appears to resemble human T. b. gambiense infection closely. Among the four vervets that developed the late phase of HAT, two were monitored until death, giving a complete follow-up (V1 and 1561). During stage 2, several clinical symptoms were observed, including stiff neck, lethargy and drowsiness. In the days before death, these animals showed loss of appetite, lack of reflexes and a transient state of somnolence. In both animals, the circumstances of death were strongly suggestive of trypanosomiasis, as confirmed by microscopical and histological examinations. In these two monkeys, survival in the late phase (19 days for 1561 and 264 days for V1) appeared to be related to the speed with which the general state deteriorated (rapidly and slowly, respectively). Interestingly, the parasite load in CSF was much more elevated in vervet 1561 than in V1. In humans, the duration of HAT can also differ between individuals. In particular, stage 2 occurs at a highly variable time, depending on the virulence of the trypanosome genotypes and individual susceptibility (Dumas and Bisser, 1999; Jamonneau et al., 2000). To our knowledge, however, the relationship between parasite load and clinical outcome has never been demonstrated. The extended survival of the untreated vervet, V1, indicates that this model differs from the T. b. rhodesiense model, in which survival in the late stage is possible only when an ineffective trypanocidal treatment is administered (Schmidt and Sayer, 1982b). Consequently, our results suggest that T. b. gambiense-infected vervet monkeys may represent a more useful model for the study of CNS involvement in HAT than any previously studied animal model. The potential toxicity of trypanocidal drugs remains a major problem in the therapy of HAT. Experimental primate models are of particular interest for pharmacokinetics analysis, because regular sampling of all fluids (blood, CSF, urine, faeces) is possible, unlike in humans. A preliminary study of the kinetics, metabolism and excretion of megazol has been carried out using six T. b. gambiense-infected vervet monkeys (Enanga et al., 2000). More recently, we have used this experimental model to study the effect of megazol and a combination of megazol and suramin (Chauviere et al., 2003) (C. Boda et al., unpublished data), strengthening previous observations of the curative effect of these drugs in mice
435 and sheep (Boda et al., 2004; Bouteille et al., 1995; Enanga et al., 1998). The effects and/or effectiveness of new therapeutic protocols should be tested using T. b. gambienseinfected vervet monkeys. In conclusion, this new model of T. b. gambiense HAT in vervets is promising for studies of stage determination, CNS involvement and treatment perspectives for HAT. Conflicts of interest statement The authors have no conflicts of interest concerning the work reported in this paper.
Acknowledgements Drs Guy Dubreuil, Philippe Deloron, Audrey Morelli, Pascal Millet, Marie Claude Georges-Courbot and Alain Georges of the Centre International de Recherches M´ edicales de Franceville (CIRMF, Gabon); Drs Jean-Philippe Chippaux and Jean-Patrick Louboutin-Croc of the Centre de Recherche M´ edicale et Sanitaire (CERMES, Niger); and Pr. Michel Dumas of the Institut d’Epid´ emiologie Neurologique et de Neurologie Tropicale, Facult´ e de M´ edecine, Limoges, France are fully acknowledged. The authors also wish to acknowledge the teams responsible for daily primate maintenance at CIRMF and CERMES, the helpful technical assistance of CERMES technicians, and Dr Joanna Setchell for correcting the English text. This work was supported by Conseil R´ egional du Limousin (France) and the French Ministry of foreign affairs (Programme mobilisateur Trypanosomiase, grants TR 96-01 and TR 96-03, Projet FAC n◦ 9900092 and Projet FSP n◦ 97008500). CIRMF is supported by Total Gabon, the Gabonese government, and the French Ministry of foreign affairs. CERMES is funded by the government of Niger and the French Ministry of foreign affairs.
References Anonymous, 1999. Human African trypanosomiasis (sleeping sickness). Wkly. Epidemiol. Rec. 74, 245—246. Bisser, S., Bouteille, B., Sarda, J., Stanghellini, A., Ricard, D., Jauberteau, M.O., Marchan, F., Dumas, M., Breton, J.C., 1997. Contribution of biochemical tests in the diagnosis of the nervous phase of human African trypanosomiasis. Bull. Soc. Pathol. Exot. 90, 321—326 [in French]. Boda, C., Enanga, B., Dumet, H., Chauviere, G., Labrousse, F., Couquet, C., Saivin, S., Houin, G., Perie, J., Dumas, M., Bouteille, B., 2004. Plasma kinetics and efficacy of oral megazol treatment in Trypanosoma brucei brucei-infected sheep. Vet. Parasitol. 121, 213—223. Bouteille, B., Darde, M.L., Dumas, M., Catanzano, G., PestreAlexandre, M., Breton, J.C., Nicolas, A., N’Do, D.C., 1988a. The sheep (Ovis aries) as an experimental model for African trypanosomiasis. I. Clinical study. Ann. Trop. Med. Parasitol. 82, 141—148. Bouteille, B., Darde, M.L., Pestre-Alexandre, M., Dumas, M., Catanzano, G., Breton, J.C., Nicolas, A., Munoz, M., 1988b. The sheep (Ovis aries) as an experimental model for African trypanosomiasis. II. Biological study. Ann. Trop. Med. Parasitol. 82, 149—158. Bouteille, B., Marie-Daragon, A., Chauviere, G., de Albuquerque, C., Enanga, B., Darde, M.L., Vallat, J.M., Perie, J., Dumas, M., 1995. Effect of megazol on Trypanosoma brucei brucei acute and subacute infections in Swiss mice. Acta Trop. 60, 73—80. Bouteille, B., Keita, M., Enanga, B., Mezui Me Ndong, J., 1999. Experimental models for new chemotherapeutic approaches to
436 human African trypanosomiasis, in: Dumas, M., Bouteille, B., Buguet, A. (Eds), Progress in Human African Trypanosomiasis, Sleeping Sickness. Springer, Paris, pp. 289—300. Bouteille, B., Oukem, O., Bisser, S., Dumas, M., 2003. Treatment perspectives for human African trypanosomiasis. Fundam. Clin. Pharmacol. 17, 171—181. Brun, R., Burri, C., Gichuki, C.W., 2001. The story of CGP 40 215: studies on its efficacy and pharmacokinetics in African green monkey infected with Trypanosoma brucei rhodesiense. Trop. Med. Int. Health 6, 362—368. Chauviere, G., Bouteille, B., Enanga, B., de Albuquerque, C., Croft, S.L., Dumas, M., Perie, J., 2003. Synthesis and biological activity of nitro heterocycles analogous to megazol, a trypanocidal lead. J. Med. Chem. 46, 427—440. Dumas, M., Bisser, S., 1999. Clinical aspects of human African trypanosomiasis, in: Dumas, M., Bouteille, B., Buguet, A. (Eds), Progress in Human African Trypanosomiasis, Sleeping Sickness. Springer, Paris, pp. 215—234. Enanga, B., Keita, M., Chauviere, G., Dumas, M., Bouteille, B., 1998. Megazol combined with suramin: a chemotherapy regimen which reversed the CNS pathology in a model of human African trypanosomiasis in mice. Trop. Med. Int. Health 3, 736—741. Enanga, B., Ndong, J.M., Boudra, H., Debrauwer, L., Dubreuil, G., Bouteille, B., Chauviere, G., Labat, C., Dumas, M., Perie, J., Houin, G., 2000. Pharmacokinetics, metabolism and excretion of megazol in a Trypanosoma brucei gambiense primate model of human African trypanosomiasis. Preliminary study. Arzneimittelforschung 50, 158—162. Gichuki, C.W., Nantulya, V.M., Sayer, P.D., 1994. Trypanosoma brucei rhodesiense: use of an antigen detection enzyme immunoassay for evaluation of response to chemotherapy in infected vervet monkeys (Cercopithecus aethiops). Trop. Med. Parasitol. 45, 237—242. Institute of Laboratory Animal Resources/Commission on Life Sciences/National Research Council, 1996. Guide for the Care and Use of Laboratory Animals. National Academy Press, Washington, D.C., p. 125. Jamonneau, V., Garcia, A., Frezil, J.L., N’Guessan, P., N’Dri, L., Sanon, R., Laveissiere, C., Truc, P., 2000. Clinical and biological evolution of human trypanosomiasis in Cˆ ote d’Ivoire. Ann. Trop. Med. Parasitol. 94, 831—835.
O. Ouwe-Missi-Oukem-Boyer et al. Keita, M., Bouteille, B., Enanga, B., Vallat, J.M., Dumas, M., 1997. Trypanosoma brucei brucei: a long-term model of human African trypanosomiasis in mice, meningo-encephalitis, astrocytosis, and neurological disorders. Exp. Parasitol. 85, 183—192. Kennedy, P.G., 2004. Human African trypanosomiasis of the CNS: current issues and challenges. J. Clin. Invest. 113, 496—504. Moore, A., Richer, M., 2001. Re-emergence of epidemic sleeping sickness in southern Sudan. Trop. Med. Int. Health 6, 342—347. Poltera, A.A., Hochmann, A., Rudin, W., Lambert, P.H., 1980. Trypanosoma brucei brucei: a model for cerebral trypanosomiasis in mice — an immunological, histological and electronmicroscopic study. Clin. Exp. Immunol. 40, 496—507. Poltera, A.A., Sayer, P.D., Brighouse, G., Bovell, D., Rudin, W., 1985. Immunopathological aspects of trypanosomal meningoencephalitis in vervet monkeys after relapse following Berenil treatment. Trans. R. Soc. Trop. Med. Hyg. 79, 527—531. Robins-Browne, R.M., Schneider, J., Metz, J., 1975. Thrombocytopenia in trypanosomiasis. Am. J. Trop. Med. Hyg. 24, 226—231. Schmidt, H., Sayer, P., 1982a. Trypanosoma brucei rhodesiense infection in vervet monkeys. I. Parasitologic, hematologic, immunologic and histologic results. Tropenmed. Parasitol. 33, 249—254. Schmidt, H., Sayer, P., 1982b. Trypanosoma brucei rhodesiense infection in vervet monkeys. II. Provocation of the encephalitic late phase by treatment of infected monkeys. Tropenmed. Parasitol. 33, 255—259. Seed, J.R., Sechelski, J.B., Loomis, M.R., 1990. A survey for a trypanocidal factor in primate sera. J. Protozool. 37, 393—400. Van Meirvenne, N., 1999. Biological diagnosis of human African trypanosomiasis, in: Dumas, M., Bouteille, B., Buguet, A. (Eds), Progress in Human African Trypanosomiasis, Sleeping Sickness. Springer, Paris, pp. 235—252. Van Nieuwenhove, S., 1999. Present strategies in the treatment of human African trypanosomiasis, in: Dumas, M., Bouteille, B., Buguet, A. (Eds), Progress in Human African Trypanosomiasis, Sleeping Sickness. Springer, Paris, pp. 253—280. Van Nieuwenhove, S., Betu-Ku-Mesu, V.K., Diabakana, P.M., Declercq, J., Bilenge, C.M., 2001. Sleeping sickness resurgence in the DRC: the past decade. Trop. Med. Int. Health 6, 335—341.
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The vervet monkey (Chlorocebus aethiops) as an experimental model for Trypanosoma brucei gambiense human African trypanosomiasis: a clinical, biological and pathological study O. Ouwe-Missi-Oukem-Boyer a,b,e,∗,1, J. Mezui-Me-Ndong b,1, C. Boda c, I. Lamine a, F. Labrousse d, S. Bisser b,e, B. Bouteille c a
Centre de Recherche M´ edicale et Sanitaire (CERMES), BP 10887, Niamey, Niger Centre International de Recherches M´ edicales de Franceville (CIRMF), BP 769, Franceville, Gabon c Institut d’Epid´ emiologie Neurologique et de Neurologie Tropicale, (EA 3174) Facult´ e de M´ edecine, 02, rue du Dr. Marcland, 87025 Limoges cedex, France d Centre Hospitalier Universitaire de Limoges, Hˆ opital Universitaire Dupuytren, Service d’Anatomie Pathologique, 02 avenue Martin-Luther-King, 87042 Limoges cedex, France e Minist` ere Franc¸ais des Affaires Etrang` eres, 244 bd Saint-Germain, 75007 Paris, France b
Received 10 May 2005 ; received in revised form 9 July 2005; accepted 11 July 2005 Available online 1 December 2005
KEYWORDS Human African trypanosomiasis; Trypanosoma brucei gambiense; Experimental model; Vervet monkeys; Chlorocebus aethiops
∗ 1
Summary It has long been known that the vervet monkey, Chlorocebus (C.) aethiops, can be infected with Trypanosoma rhodesiense, but this model has not been described for T. gambiense. In this study, we report the development of such a model for human African trypanosomiasis. Twelve vervet monkeys infected with T. gambiense developed chronic disease. The duration of the disease ranged between 23 and 612 days (median 89 days) in five untreated animals. Trypanosomes were detected in the blood within the first 10 days postinfection and in the cerebrospinal fluid, with a median delay of 120 days (n = 4, range 28—348 days). Clinical changes included loss of weight, adenopathy, and in some cases eyelid oedema and lethargy. Haematological alterations included decreases in haemoglobin level and transitory decreases in platelet count. Biological modifications included increased gamma globulins and total proteins and decreased albumin. Pathological features of the infection were presence of Mott’s cells, inflammatory infiltration of either mononuclear cells or lymphocytes and plasma cells in the brain parenchyma, and astrocytosis. These observations indicate that the development of the disease in vervet monkeys is similar to human T. gambiense infection. We
Corresponding author. Tel.: +241 67 70 92; fax: +241 67 72 95. E-mail address: [email protected] (O. Ouwe-Missi-Oukem-Boyer). These authors contributed equally to this work.
0035-9203/$ — see front matter © 2005 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.trstmh.2005.07.023
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O. Ouwe-Missi-Oukem-Boyer et al. conclude that C. aethiops is a promising experimental primate model for the study of T. gambiense trypanosomiasis. © 2005 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd. All rights reserved.
1. Introduction Human African trypanosomiasis (HAT), or sleeping sickness, is an endemic disease in 36 sub-Saharan countries. More than 60 million people living in some 200 microfoci are at risk of contracting the disease. HAT has recently re-emerged in southern Sudan and central Africa (Moore and Richer, 2001; Van Nieuwenhove et al., 2001), and the 45 000 new cases reported to WHO in 1999 are certainly underestimated (Anonymous, 1999). The disease is caused by infection with one of the two subspecies of Trypanosoma brucei: T. b. gambiense or T. b. rhodesiense. HAT is characterized by a haemolymphatic phase (also called ‘early phase’ or stage 1), in which trypanosomes invade the blood and lymphatic systems, and a meningoencephalitic phase (‘late phase’ or stage 2) characterized by progressive invasion of the central nervous system (CNS) by trypanosomes and inflammatory cells leading to neurological disorders and damages (Kennedy, 2004). If left untreated, the disease is always fatal. There are two major problems in the treatment of HAT: first, accurate diagnosis of the timing of CNS invasion is required because the stage of the disease determines the choice of treatment, and second, the molecules that are active against trypanosomes are toxic to the host. Few drugs are commercially available for the treatment of HAT: suramin and pentamidine for cases in early stages without CNS involvement; and melarsoprol, a highly toxic arsenical, and more recently eflornithine for the meningoencephalitic stage (Bouteille et al., 2003). Less toxic medications that are active against trypanosomes in both stages of the disease are required. These two problems have made it necessary to develop in-vivo experimental models. Laboratory rodents and sheep infected with T. b. gambiense and T. b. rhodesiense have been used as experimental models of HAT. However, murine models do not show obvious signs of meningoencephalitis, and it is impossible to obtain sufficient cerebrospinal fluid (CSF) for post-infection followup (Keita et al., 1997; Poltera et al., 1980), while sheep (Ovis aries) give a subacute illness of the CNS but are phylogenetically distant from humans (Bouteille et al., 1988a, 1988b). Vervet monkeys (Chlorocebus aethiops) infected with T. b. rhodesiense are therefore currently used in therapeutic research (Brun et al., 2001; Gichuki et al., 1994; Poltera et al., 1985; Schmidt and Sayer, 1982a). To date, no experimental primate model is available for T. b. gambiense. An ideal experimental primate model of HAT should be capable of developing chronic disease, including CNS invasion and obvious signs of meningoencephalitis. Such a model has not yet been developed (Bouteille et al., 1999), as vervets infected with T. b. rhodesiense show short survival without treatment, and significant CNS involvement can be achieved only by prolonging survival with an ineffective trypanocidal treatment (Schmidt and Sayer, 1982a, 1982b). Trypanosoma brucei gambiense, which produces a more chronic
infection, may prove to be a better model for studies of CNS involvement in HAT. We report here observations showing that infection of the vervet monkey with T. b. gambiense might constitute such an experimental model of HAT.
2. Materials and methods The study took place in two different centres, at two different times and using two different sets of study animals. However, the same protocol and the same parasite strain were used in both studies, and the data were therefore combined for this report.
2.1. Animals Thirteen adult vervet monkeys of both sexes were studied at: (1) Centre International de Recherches M´ edicales de Franceville (CIRMF), Gabon during 1996—1997 (eight monkeys originating from Ololua, Kenya); and (2) Centre de Recherche M´ edicale et Sanitaire (CERMES), Niamey, Niger during 2001—2003 (five monkeys originating from Hazyview, Republic of South Africa). Median body weight (range) at the beginning of the experiment was 3.3 (2.9—4.3) kg at CIRMF and 3.7 (2.5—5.1) kg at CERMES. All animals were quarantined for a 90-day period, during which monkeys were housed in individual cages and familiarized with the single-cage capture facilities. Before enrolment in the study, all animals tested negative for tuberculosis, simian immunodeficiency virus, simian T-cell lymphotropic virus type 1, protozoa (particularly trypanosomes) and helminth parasites via repeated stool, urine and blood examinations. The animals were maintained on a diet of fresh vegetables/fruits (40% of the daily energy contribution) and monkey biscuits (60% of the daily energy contribution). Food was given twice daily and water was provided ad libitum. Animals were also housed in individual cages during the experiment. This protocol was approved by the CIRMF Ethical Committee for Control of the Use of the Animals (CECUA) and animal handling was performed according to the Guidelines for the Care and Use of Laboratory Animals as adopted and promulgated by the Institute of Laboratory Animal Resources (1996). The experimental design of this study is presented in Figure 1. Because few monkeys were available, four animals (namely V1, V3, V5 and 1457) were initially used as controls before being infected.
2.2. Parasites The strain of T. b. gambiense MBA ITMAP 1811 used for both experiments is derived from a Congolese (RD Congo) patient and was kindly provided by Dominique Le Ray and Yves Claes of the Protozoology Laboratory, Tropical Medicine Institute (Antwerpen, Belgium). Stabilates were stored frozen in
Trypanosoma brucei gambiense in vervet monkeys
429
Figure 1 Follow-up schedule of the 13 monkeys. Because few monkeys were available, four animals (V1, V3, V5, 1457) were initially used as controls before being infected. The cause for end of follow-up is indicated by a black (death), blue (treatment) or white (stop) arrow. Thin black arrows show the first day with trypanosomes in cerebrospinal fluid (CSF) in monkeys V1, V3, 553 and 1561. Survival after the first positive CSF test is reported in italics for the two untreated monkeys (V1 and 1561). * single oral administration of 100 mg/kg megazol alone (Enanga et al., 2000); # oral administration of 100 mg/kg megazol on three consecutive days (C. Boda et al., unpublished data); £ single oral administration of 100 mg/kg megazol immediately followed by intravenous injection of 20 mg/kg suramin (Enanga et al., 2000).
liquid nitrogen, then rapidly thawed and inoculated into Swiss mice by intra-peritoneal injection. Parasites were passaged several times in mice to ensure strain virulence. At day 0, blood was sampled from infected mice and trypanosome mobility verified via microscopical examination. Parasites were diluted in 250 l RPMI-1640 medium (Sigma, Saint Quentin Fallavier, France). Anaesthesia was carried out via intramuscular injection of 10 mg/kg ketamine hydrochloride (Imalgene, Rhone-M´ erieux, Lyon, France). Twelve sedated vervet monkeys were infected intravenously with 103 trypanosomes per animal.
2.3. Clinical follow-up The general behaviour and vigilance of vervet monkeys were recorded each week before anaesthesia. Animals were then sedated with ketamine hydrochloride and clinical parameters, including rectal temperature, body weight, adenopathy, oedema and splenomegaly, were recorded. Follow-up studies were interrupted for seven of the 12
infected monkeys, because these animals were included in a therapeutic protocol (Chauviere et al., 2003; Enanga et al., 2000) (C. Boda et al., unpublished data). The remaining five animals were monitored until death (see Figure 1).
2.4. Blood samples Parasitaemia was investigated daily via finger-prick blood tests, from infection until the test appeared positive. Blood was drawn from each monkey via inguinal venipuncture at regular intervals for parasitology and to determine the haematocrit, haemoglobin levels, and erythrocyte, leucocyte, platelet and differential leucocyte counts. All these analyses were performed immediately. Where available, remaining blood samples were stored at −20 ◦ C. Serum specimens were also obtained for biochemical and serological studies. Biochemical parameters measured included glucose, creatinine, serum glutamic oxalacetic transaminase (SGOT) and serum glutamic pyruvic transaminase (SGPT), total protein and albumin concentrations. Serum protein
−/− −/− −/− −/− −/− +/− −/− −/− +/− −/− +/+ +/− −/− 3.6 2.9 4.3 3.8 3.7 5.1 3.8 2.5 3.3 4.3 3.7 2.5
+ + + + + + + + + + + + +
+ + + + + + + + + + + + +
3 7 3 10 3 6 7 7 3 7 4 3
? ? 28 70 170 348 F F M F F M M F F F F F
Positive blood test (day)
Positive CSF test (day)
493 1457 CA5 CA6 1465 V5 V8 V10 V3 553 1561 V1 Controls
Initial weight (kg)
Clinical parameters
Haematological parameters
Parasitological parameters
Experimental subjects, controls, parameters evaluated and parasitology results
All infected animals showed weight loss after infection, while body weight was stable in uninfected controls (data not shown). Weight loss was continuous in six of 12 infected monkeys, and represented 21—37% of the initial body weight. Two animals showed minor (4—11%) and continuous weight loss, while two others showed minor (4—8%) and transient (2—6 weeks) weight loss. Follow-up for the latter two monkeys was very short, meaning that it was impossible to determine whether the early weight loss was transient or permanent. All animals showed enlargement of both the axillary and the inguinal lymph nodes, which increased in size by 50—400% (0.5—2 cm). Splenomegaly was observed in seven monkeys, during the entire follow-up in V1, and irregularly in six animals, persisting until the late phase of the disease in three monkeys. Splenomegaly was not observed in the five remaining monkeys. Transient eyelid oedema was observed in two CIRMF monkeys at days 42 and 49 post-infection, and repeatedly in two CERMES monkeys: at days 98 and 113 post-infection in one
Sex
3.1. Clinical parameters
Animal
3. Results
Table 1
At death, an autopsy was performed on three out of the five untreated monkeys, and on one treated monkey that died after the first day of treatment (Table 1). Organs were collected and fixed with formalin at CIRMF or at CERMES, then shipped to France. Post-mortem and histological examinations of the brain, spinal cord, heart, lungs, liver, kidneys, spleen, stomach and intestine were carried out. Fixation with formalin, embedding in paraffin and staining with haematoxylin and eosin were performed according to standard histological techniques (Keita et al., 1997). Immunohistochemical studies were made on one untreated monkey, as described previously (Keita et al., 1997).
CSF parasite load (tryp/ml)
2.6. Histology and immunohistochemistry
? ? 200 23 000 730 000 100—800
CSF cell count (n/l)
Lumbar punctures were performed at 2-week intervals under general anaesthesia. Cerebrospinal fluid (1 to 2 ml per animal) was collected and analysed immediately for parasitosis and cell count. Visibly haemorrhagic CSF samples were discarded. Where available, remaining CSF samples were stored at −20 ◦ C.
? ? 28—31 74 40 20—36
Biochemical analyses/serum protein electrophoresis +/+ +/+ +/+ +/+ +/+ +/− +/− +/− +/− +/+ +/− +/− +/−
2.5. CSF samples
− − − − − − − − − − + − −
electrophoresis and serological analysis were performed only on monkeys at CIRMF (Table 1). Biochemical analyses were performed immediately, while immunological experiments were performed on frozen (−20 ◦ C) samples, at the end of the follow-up. Indirect immunofluorescence tests were performed using fluorescein isothiocyanate (FITC)conjugated goat anti-human IgG and IgM (Bio-Sys, Compiegne, France). Spots were prepared with T. b. gambiense LiTat 1.3 (from lyophilized trypanosomes kindly provided by Pr N. Van Meirvenne and Dr P. B¨ uscher of the department of Tropical Medicine, Antwerpen, Belgium).
Histological/ immunohistochemical analyses
O. Ouwe-Missi-Oukem-Boyer et al.
Serological analysis
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Trypanosoma brucei gambiense in vervet monkeys
Figure 2 (B).
431
Face of an infected vervet monkey without eyelid oedema (A) and with obvious eyelid oedema (at day 181 post-infection)
monkey and days 181 and 433 in the other (Figure 2). Several other clinical symptoms were occasionally observed for individual subjects, including a stiff neck, lethargy, drowsiness, loss of appetite, dehydration and hair loss. In the five untreated animals, the median duration of the disease was 89 days (range 23—612 days; Figure 1). Control animals did not exhibit any detectable sign of alteration to their general behaviour during the experiment.
3.2. Parasitology 3.2.1. Parasitaemia Control animals were negative for trypanosomes during the entire follow-up. In infected monkeys, trypanosomes were detectable in the blood between the third and seventh day after infection, although one subject remained negative until the tenth day (Table 1). The time course of parasitaemia differed substantially between infected monkeys, and parasite loads also varied considerably between animals. The median curve for parasitaemia was thus uninformative (data not shown). However, although the parasitaemia curves were irregular, they always showed several peaks (at different times post-infection and of different intensity), separated by intervals (of varying length) without any trypanosomes (data not shown). Of the five infected monkeys that were monitored until death, two (vervets 493 and 1561) showed a high peak in parasitaemia just before death (21.6 × 106 and 16.1 × 106 trypanosomes per ml respectively), whereas detectable trypanosomes were no longer found in the blood of the other three monkeys, even when concentration techniques were used. 3.2.2. CSF parasitosis and CSF cell count During the experimental period, six of the 12 infected monkeys showed no detectable trypanosomes in their CSF, even when concentration techniques were used (Table 1). These animals were thus classed as being in the haemolymphatic phase of the disease. Parasites were found in the CSF of four infected subjects, with a median delay of 120 days, although large differences
occurred between animals in the timing of the first appearance of trypanosomes in the CSF (days 28, 70, 170 and 348). Parasite loads also differed considerably between animals: two showed low parasite loads (100—800 per ml), while two had considerably more trypanosomes (23 000—730 000 per ml). An increase in cell counts was also detected in these four animals, confirming that they were in the meningoencephalitic phase of the disease (Table 1). CSF parasitosis was not correlated with parasitaemia: three subjects had the same moderate blood parasite load (peaks reaching 106 trypanosomes per ml) but large differences in CSF parasite load, whereas the blood parasite load of the fourth subject was very high (peak reaching 35 × 106 trypanosomes per ml) and CSF parasite load low. Vervets V3 and 553 were treated, while vervets V1 and 1561 were monitored until death. Survival was considerably shorter in the vervet with a large number of CSF trypanosomes (vervet 1561; 19 days survival), while the animal with fewer parasites survived more than 8 months in stage 2 (V1; 264 days of survival following the first positive CSF examination) (Figure 1). For two monkeys, stage determination was uncertain because CSF parasite status was unclear. Erythrocytes were detected in the CSF, meaning that it was not possible to confirm whether the presence of trypanosomes and lymphocytes was due to contamination with blood, or to disease. A repeat examination was not possible because these animals were included in a therapeutic protocol that began before the disease stage could be clarified. These animals were therefore considered as being in an intermediate phase (between stage 1 and 2) for the purposes of this study.
3.3. Biological analysis 3.3.1. Haematology All infected animals showed decreased haemoglobin values (Figure 3). Median values varied between 12.7 g/l and 8.0 g/l during the first month post-infection. The lowest median value was 7.4 g/l, which occurred 2—3 months after infection. Thereafter, haemoglobin levels
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Figure 3 Haemoglobin levels in vervet monkeys infected with Trypanosoma brucei gambiense. Boxes show the median and 25th and 75th percentiles, with 10th and 90th percentiles represented by error bars for 12 (days 0 and 14), 11 (day 28), 10 (day 41), 7 (day 56), 6 (day 70), 5 (day 84), 4 (days 97 and 112), 3 (days 125, 140 and 153) and 2 (days 170, 178 and 189) infected animals. Dashed horizontal lines indicate the range of normative values for haemoglobin in vervet serum (O. Ouwe-Missi-Oukem-Boyer et al., unpublished data).
remained stable during the course of the infection, but were always less than 10 g/l. By contrast, the median level of haemoglobin in control animals (range) was 12.5 (11.8—13.3) g/l during the study, and was always within the normal range (11.8—14.2 g/dl; O. Ouwe-Missi-Oukem-Boyer, unpublished data). The haematocrit and erythrocyte count followed a similar pattern, decreasing within the first month post-infection, then remaining at the low values of approximately 20% and 3 million, respectively. Leucocyte counts changed considerably during the course of infection, from initial median values of 5600 per mm3 to 2500 per mm3 , 3 weeks post-infection, and 9000—11 000 per mm3 after 3 months. However, individuals varied considerably, and peaks of 15 000—17 500 per mm3 were repeatedly recorded for one subject. Disturbances of the polynuclear/lymphocyte ratio were observed with an alternate prevalence of polynuclear and lymphocytes. The median absolute number of lymphocytes showed a moderate increase during the course of infection, reaching 4000—5000 cells per mm3 . The percentage of monocytes showed a similar trend, appearing stable and in the normal range (10—19%; O. Ouwe-Missi-Oukem-Boyer, unpublished data) during the first 3 months after infection, then increasing to 18—27%. However, again great variability occurred between animals. The two monkeys that were followed for the longest period showed an unexpected collapse in the percentage of monocytes a few days before death (2% for one subject, 5% for the other). Platelet counts decreased within the first weeks postinfection, attaining minimum values of 43 000—50 000 per mm3 in two animals. This decrease occurred in all infected subjects, but varied in time and intensity. In most animals, platelet counts remained low (normal range: 256 000—381 000 per mm3 ; O. Ouwe-Missi-Oukem-Boyer, unpublished data), fluctuating between 100 000 per mm3 and 240 000 per mm3 during the course of the disease.
3.3.2. Biology No significant modifications in glucose levels were observed in infected monkeys. Almost all animals showed a transient increase in creatinine, reaching a median of 11 mg/l early after infection, then a progressive return to normal median values of 5—6 mg/l until the end of the study. Substantial fluctuations of transaminases (SGOT and SGPT) occurred at the individual level, although these were not detectable when median curves were plotted (data not shown). Total serum proteins increased (Figure 4A), but albumin levels decreased as infection progressed (Figure 4B). The increase in proteins was due primarily to an increase in gamma globulins (Figure 5), while alpha 1, alpha 2 and beta globulin levels remained approximately stable. 3.3.3. Immunology The detection of specific antibodies was carried out using blood from monkey 1561, the subject that had the longest follow-up at CIRMF. This animal became positive for IgM within the first 2 weeks after inoculation. The maximum titre, 1:3200, was obtained 7 weeks after infection. Thereafter, titres oscillated between 1:200 and 1:800 until death, with the exception of two peaks of 1:1600, at days 70 and 170. IgG appeared 2 weeks after IgM detection. IgG titres increased from day 28 (1:1600) to day 86 (1:6400), then oscillated between 1:6400 and 1:3200 until day 170. Titres dropped dramatically from 1:6400 to 1:1600 during the meningoencephalitic phase (day 170 to death). 3.3.4. Histology and immunohistochemistry In vervet 1561, histological examination of brain tissues revealed perivasculitis with abundant infiltration of mononuclear cells in the brain parenchyma, indicating that HAT may have been responsible for the death of this monkey (Figure 6A). In vervet V1, autopsy revealed
Trypanosoma brucei gambiense in vervet monkeys
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Figure 4 Total serum proteins (A) and albumin (B) levels in vervet monkeys infected with Trypanosoma brucei gambiense. Boxes show the median and 25th and 75th percentiles, with 10th and 90th percentiles represented by error bars for 12 (days 0 and 14), 10 (day 41), 9 (days 7, 21 and 28), 7 (days 35 and 49), 5 (day 56), 4 (day 70), 3 (days 63 and 84) and 2 (days 77, 97, 112, 125 and 153) infected animals. Dashed horizontal lines indicate the range of normative values for each parameter in vervet serum (O. Ouwe-Missi-Oukem-Boyer et al., unpublished data).
extensive meningoencephalitis lesions. The brain showed serious inflammatory infiltration of lymphocytes and plasma cells. Mott’s cells were reported in perivascular sites and also in the white matter (data not shown). These two features are strongly suggestive of trypanosomiasis, which was considered the cause of death. Vervet V5 did not present any sign of encephalitis, but low intensity inflammatory infiltration of mononuclear cells was reported in the meninges. Low-level myocarditis was also observed in this animal, but was unlikely to be the cause of death. Vervet V3, which died a few hours after treatment, showed mild to severe inflammatory lesions in the myocardium, with a predominance of lymphocytes and plasma cells; lesions were less intense in the lungs and other organs and
were absent in the brain (data not shown). The general state of this monkey deteriorated considerably during the several days prior to death, and hyperthermia (42.8 ◦ C) and convulsive fits were recorded immediately before death. These symptoms may have been due to trypanosomiasis without obvious brain damage, despite the fact that the monkey was classed as stage 2, when brain lesions are expected. In vervet 1561, immunohistochemical examination of glial fibrillary acidic protein (GFAP) showed strong astrocytosis in the brain parenchyma and mild meningitis with the presence of mononuclear cells in the white and grey matter (Figure 6B). These inflammatory lesions correspond to degree 3 of meningoencephalitis (Keita et al., 1997).
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Figure 5 Distribution of the five fractions of serum proteins in vervet monkeys infected with Trypanosoma brucei gambiense. Each point of the curves represents the median for six animals.
4. Discussion
Figure 6 Brain histopathology in vervet monkey 1561 infected with Trypanosoma brucei gambiense for 189 days: perivasculitis (arrows) in the brain parenchyma, haematoxylin-eosin, ×120 (A); astrocytosis in the brain parenchyma (arrows) and mild meningitis with many mononuclear cells in the white and the grey matter (arrow heads), glial fibrillary acidic protein immunohistochemistry, ×120 (B).
In this study, we present a chronic experimental model of HAT in C. aethiops infected by T. b. gambiense. Our results show that vervet monkeys are susceptible to T. b. gambiense MBA strain, confirming previous data on the absence of a trypanocidal factor in this primate species (Seed et al., 1990). All animals developed the haemolymphatic phase of the disease after a brief prepatent period. During the followup of the infected monkeys, trypanosomes were detected in the blood irregularly, and peaks of parasitaemia, when these occurred, varied in intensity. After a delay of variable length (28—348 days), the appearance of trypanosomes occurred simultaneously with an increased cell count in the CSF of four vervets, indicating that parasites may have reached the CNS in these animals. As in humans, both the haemolymphatic stage and the meningoencephalitic stage occurred in our experimental model, despite the fact that few infected monkeys were monitored. Two animals were classified as being in an intermediate stage because the presence of trypanosomes and the increased cell count may have been due to blood contamination of the CSF sample. Stage determination in human HAT patients may be also difficult when the presence of parasites in CSF is uncertain or when trypanosomes are detected but there is no evidence of an increased CSF cell count. Indeed, the criteria for stage 2 determination are highly controversial (Van Meirvenne, 1999), and the associated choice of treatment remains problematic, particularly in ‘borderline’ cases (Van Nieuwenhove, 1999). Since regular lumbar punctures are possible in vervets, T. b. gambiense-infected monkeys may be a helpful experimental model for the identification of alternative markers for stage determination. Experiments based on ‘lymphocyte subset typing’ in the blood and CSF of infected monkeys and IgM measurement in the CSF are currently under investigation in our laboratory. Adenopathy was found in both the axillary and inguinal regions as soon as the vervets were infected. Splenomegaly
Trypanosoma brucei gambiense in vervet monkeys was frequent, although it was not observed in all monkeys. Eyelid oedema was observed in several monkeys at different times during the follow-up. Cutaneous changes also occurred occasionally. These clinical symptoms are very similar to those described for HAT in humans (Dumas and Bisser, 1999). Weight loss, low haematocrit and anaemia occurred in all animals, and leucocyte counts changed considerably during the follow-up. Decreased weight, haemoglobin levels and haematocrit have also been reported in humans with HAT (Bisser et al., 1997; Van Meirvenne, 1999). Moreover, the reduced platelet counts observed are concordant with data from humans infected with T. b. gambiense (RobinsBrowne et al., 1975). The absence of hypoglycaemia in our subjects confirms a previous study in which subjects with Gambian trypanosomiasis were compared to controls for 29 biochemical analyses (Bisser et al., 1997). The modifications in protein levels that we observed, such as increased gamma globulin and total proteins and decreased albumin, are also characteristic of HAT in humans (Bisser et al., 1997; Van Meirvenne, 1999). Infection of C. aethiops with T. b. gambiense thus appears to resemble human T. b. gambiense infection closely. Among the four vervets that developed the late phase of HAT, two were monitored until death, giving a complete follow-up (V1 and 1561). During stage 2, several clinical symptoms were observed, including stiff neck, lethargy and drowsiness. In the days before death, these animals showed loss of appetite, lack of reflexes and a transient state of somnolence. In both animals, the circumstances of death were strongly suggestive of trypanosomiasis, as confirmed by microscopical and histological examinations. In these two monkeys, survival in the late phase (19 days for 1561 and 264 days for V1) appeared to be related to the speed with which the general state deteriorated (rapidly and slowly, respectively). Interestingly, the parasite load in CSF was much more elevated in vervet 1561 than in V1. In humans, the duration of HAT can also differ between individuals. In particular, stage 2 occurs at a highly variable time, depending on the virulence of the trypanosome genotypes and individual susceptibility (Dumas and Bisser, 1999; Jamonneau et al., 2000). To our knowledge, however, the relationship between parasite load and clinical outcome has never been demonstrated. The extended survival of the untreated vervet, V1, indicates that this model differs from the T. b. rhodesiense model, in which survival in the late stage is possible only when an ineffective trypanocidal treatment is administered (Schmidt and Sayer, 1982b). Consequently, our results suggest that T. b. gambiense-infected vervet monkeys may represent a more useful model for the study of CNS involvement in HAT than any previously studied animal model. The potential toxicity of trypanocidal drugs remains a major problem in the therapy of HAT. Experimental primate models are of particular interest for pharmacokinetics analysis, because regular sampling of all fluids (blood, CSF, urine, faeces) is possible, unlike in humans. A preliminary study of the kinetics, metabolism and excretion of megazol has been carried out using six T. b. gambiense-infected vervet monkeys (Enanga et al., 2000). More recently, we have used this experimental model to study the effect of megazol and a combination of megazol and suramin (Chauviere et al., 2003) (C. Boda et al., unpublished data), strengthening previous observations of the curative effect of these drugs in mice
435 and sheep (Boda et al., 2004; Bouteille et al., 1995; Enanga et al., 1998). The effects and/or effectiveness of new therapeutic protocols should be tested using T. b. gambienseinfected vervet monkeys. In conclusion, this new model of T. b. gambiense HAT in vervets is promising for studies of stage determination, CNS involvement and treatment perspectives for HAT. Conflicts of interest statement The authors have no conflicts of interest concerning the work reported in this paper.
Acknowledgements Drs Guy Dubreuil, Philippe Deloron, Audrey Morelli, Pascal Millet, Marie Claude Georges-Courbot and Alain Georges of the Centre International de Recherches M´ edicales de Franceville (CIRMF, Gabon); Drs Jean-Philippe Chippaux and Jean-Patrick Louboutin-Croc of the Centre de Recherche M´ edicale et Sanitaire (CERMES, Niger); and Pr. Michel Dumas of the Institut d’Epid´ emiologie Neurologique et de Neurologie Tropicale, Facult´ e de M´ edecine, Limoges, France are fully acknowledged. The authors also wish to acknowledge the teams responsible for daily primate maintenance at CIRMF and CERMES, the helpful technical assistance of CERMES technicians, and Dr Joanna Setchell for correcting the English text. This work was supported by Conseil R´ egional du Limousin (France) and the French Ministry of foreign affairs (Programme mobilisateur Trypanosomiase, grants TR 96-01 and TR 96-03, Projet FAC n◦ 9900092 and Projet FSP n◦ 97008500). CIRMF is supported by Total Gabon, the Gabonese government, and the French Ministry of foreign affairs. CERMES is funded by the government of Niger and the French Ministry of foreign affairs.
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