Immunospecific immunoglobulins and IL-10 as markers for Trypanosoma brucei rhodesiense late stage disease in experimentally infected vervet monkeys


Corresponding Author Jann Hau, Department of Experimental Medicine, University of Copenhagen and University Hospital of Copenhagen, 3b Blegdamsvej 2200-K, Copenhagen, Denmark. Fax: +45 35 32 7399; E-mail:


Objective  To determine the usefulness of IL-10 and immunoglobulin M (IgM) as biomarkers for staging HAT in vervet monkeys, a useful pathogenesis model for humans.

Methods  Vervet monkeys were infected with Trypanosoma brucei rhodesiense and subsequently given sub-curative and curative treatment 28 and 140 days post-infection (dpi) respectively. Matched serum and CSF samples were obtained at regular intervals and immunospecific IgM, immunoglobulin G (IgG) and IL-10 were quantified by ELISA.

Results  There was no detectable immunospecific IgM and IgG in the CSF before 49 dpi. CSF IgM and IgG and serum IgM were significantly elevated with peak levels coinciding with meningoencephalitis 98 dpi. The serum IL-10 was upregulated in both early and late disease stage, coinciding with primary and relapse parasitaemia respectively. CSF white cell counts (CSF WCC) were elevated progressively till curative treatment was given. After curative treatment, there was rapid and significant drop in serum IgM and IL-10 concentration as well as CSF WCC. However, the CSF IgM and IgG remained detectable to the end of the study.

Conclusions  Serum and CSF concentrations of immunospecific IgM and CSF IgG changes followed a pattern that mimics the progression of the disease and may present reliable and useful biomarkers of the disease stage. Due to rapid decline, serum IgM and IL-10 are, additionally, potential biomarkers of the success of chemotherapy.

Immunoglobulines immunospécifiques et IL-10 comme marqueurs du stade tardif de la maladie àTrypanosoma brucei rhodesiense chez des singes Vervet infectés expérimentalement

Objectif:  Déterminer l’utilité de IL-10 et IgM comme biomarqueurs pour les stades de la maladie chez des singes Vervet, un modèle de pathogenèse utile pour l’homme.

Méthodes:  Les singes Vervet ont été infectés par T. b. rhodesiense et ensuite administrés soit des traitements sous-curatifs ou curatifs à 28 et 140 jours post-infection, respectivement. Des échantillons appariés de sérum et de LCR ont été prélevés à intervalles réguliers et les IgM, IgG et IL-10 immunospécifiques ont été quantifiés par ELISA.

Résultats:  Il n’y avait pas d’IgM et d’IgG immunospécifiques détectables dans le LCR avant 49 jours post-infection. Les IgM et IgG du LCR et les IgM du sérum étaient significativement élevés avec des pics de niveaux coïncidant avec la méningo-encéphalite 98 jours post-infection. L’IL-10 sérique était up réguléà la fois dans les stades précoces et avancés de la maladie, ce qui coïncide avec une parasitémie primaire et de rechute respectivement. Le taux de globules blancs du CSF (CSF WCC) était progressivement élevé jusqu’à l’administration du traitement curatif. Après traitement curatif, une baisse rapide et significative des taux sériques d’IgM et d’IL-10 ainsi que des CSF WCC était observée. Toutefois, les IgM et IgG du LCR demeuraient détectables jusqu’à la fin de l’étude.

Conclusions:  Les changements des concentrations sériques et du LCR en IgM immunospécifiques et les IgG du LCR suivaient un profil qui imite la progression de la maladie et peuvent représenter des biomarqueurs fiables et utiles du stade de la maladie. En raison de leur déclin rapide, les IgM et IL-10 sériques sont en plus des biomarqueurs potentiels du succès de la chimiothérapie.

Inmunoglobulinas inmuno específicas e IL-10 como marcadores para el estadio tardío de la enfermedad por Trypanosoma brucei rhodesiense en monos vervet infectados experimentalmente

Objetivo:  Determinar la utilidad del IL-10 y la IgM como biomarcadores para defnir el estadío de la Tripanosomiasis Humana Africana (THA) en monos vervet, un modelo útil de patogénesis para humanos.

Métodos:  Los monos Vervet fueron infectados con T. b. rhodesiense y subsecuentemente recibieron un tratamiento con dosis sub-curativas y curativas 28 y 140 días después de la infección(dpi), respectivamente. Se obtuvieron muestras de suero y líquido cefalorraquídeo (LCR) pareadas en intervalos regulares, y se cuantificaron los IgM, IgG e IL-10 inmunoespecíficos mediante ELISA.

Resultados:  No había niveles detectables de IgM e IgG immunospecíficos en el LCR antes de los 49 días post infección (dpi). Las IgM e IgG en LCR, y las IgM en suero eran significativamente elevadas, con los picos coincidiendo con meningoencefalitis 98 dpi. El nivel de IL-10 en suero era regulado por incremento, tanto en estadios tempranos como tardíos de la enfermedad, coincidiendo con la parasitemia primaria y la recaída respectivamente. Los conteos de leucocitos en LCR (CL_LCR) eran progresivamente más elevados hasta que se suministraba el tratamiento curativo. Después de recibir el tratamiento curativo, había una caída rápida y significativa en las concentraciones en suero de IgM e IL-10, al igual que en el CL_LCR. Sin embargo las IgM e IgG en líquido LCR se mantuvieron detectables hasta el final del estudio.

Conclusiones:  Las concentraciones en suero y LCR de IgM inmuno específica y los cambios de IgG en LCF seguían un patrón que imita la progresión de la enfermedad y podrían utilizarse como biomarcadores, útiles y fiables, del estadio de la enfermedad. Debido a la rápida caída en suero del IgM y el IL-10, estos serían adicionalmente potenciales biomarcadores para el éxito de la quimioterapia.


Human African trypanosomiasis (HAT, sleeping sickness) is a tropical parasitic disease caused by Trypanosoma brucei gambiense (chronic form) or T. b. rhodesiense (acute form). The disease is transmitted through the bite of tsetse flies and hence occurs only in sub-Sahara Africa, and has been classified as a neglected public health problem (WHO 2006). After active surveillance and vector control operations, the disease had been brought under control by the 1960s. However, a resurgence of sleeping sickness in many countries has been observed since the 1970s. It is estimated that 60 million people are at risk, but only 5 million are under active surveillance or have access to health centres. Prevalence is estimated at 400 000, incidence at 60 000 and the mortality at 40 000 per annum. In countries such as Angola, DR Congo, Sudan and Uganda the disease is considered epidemic due to a high prevalence and transmission level (Barrett 1998; Smith et al. 1998; Moore & Richer 2001; Fevre et al. 2005). There are indications that with the intensified interventions that have been undertaken recently, the number of cases is falling. It is now estimated that there are between 50 000 and 70 000 persons carrying the infection at any one time. Inadequate knowledge is considered a major challenge to successful control of sleeping sickness (WHO 2001, 2006).

Regardless of the causative parasites, the disease results in early haemolymphatic infection (stage 1) and a late stage meningoencephalitis (stage 2), when the trypanosomes invade the brain (WHO 1998; Kristensson et al. 2002). The disease is invariably fatal if untreated. Currently available treatments for HAT are few and limited due to toxicity, and lost efficacy in several regions (Van Nieuwenhove 1999; Barrett et al. 2007). Treatment is stage-specific, with the more toxic and difficult to administer treatments for stage 2 disease (Adams et al. 1986; Pepin & Milford 1994; Van Nieuwenhove 1999). Therefore, reliable early diagnosis of the disease stage is crucial.

The meningoencephalitis is characterised by parasite invasion of meninges and the choroid plexus, the infiltration of the leptomeninges and perivascular areas by plasma cells and in the late/terminal stages Mott cells (Mattern 1964; Poltera 1980). The disease is associated with elevated levels of immunospecific anti-parasite immunoglobulin M (IgM) antibodies in the CSF, characteristic of the meningoencephalitic stage of HAT (Mattern 1964; Greenwood & Whittle 1980), as are increasing concentrations of cytokines such as Interleukin 10 (IL-10) (Rhind et al. 1997; MacLean et al. 2001, 2006), IL-1β (Courtioux et al. 2006), anti-brain-specific proteins e.g. galactocerebrosides (Jauberteau et al. 1991; Chappuis et al. 2005) and Glial Fibrillary Acidic Protein (Hunter et al. 1992).

However, the utility for diagnosis of such biomarkers has not been investigated in detail. Most of the studies have been on the West African form which represents about 90% of reported cases of sleeping sickness (Lejon et al. 1998, 2002, 2003, 2007). Little information is available on the more severe and acute form caused by T. b. rhodesiense. Identification of clinical markers of disease stage would herald the safe application of available treatment and minimise the use of more toxic late stage drugs. Limited studies have been carried out in laboratory animal models to confirm and determine the usefulness and application of diagnostic markers and their potential contribution to choice of chemotherapy.

Vervet monkeys experimentally infected with human infective trypanosomes develop a disease that clinically mimics the disease in humans (Farah et al. 2004; Ouwe-Missi-Oukem-Boyer et al. 2006; Thuita et al. 2008). The vervet monkey therefore provides an excellent opportunity to conduct controlled laboratory studies on neuropathogenesis and identification of possible biomarkers of the disease stages. Three model disease stages have been described in vervets infected with T. b. rhodesiense (Schimdt & Sayer 1982; Gichuki & Brun 1999; Farah et al. 2004).

In the present study, we used the advanced late stage model in which vervets are infected and treated sub-curatively with diminazene aceturate (DA) 28 days after infection which results in clearance of the haemolymphatic infection in the animal. Since this drug does not cross the blood–brain barrier (BBB), the trypanosomes which have already crossed into the cerebrospinal fluid (CSF) proliferate and induce late-stage disease (Schimdt & Sayer 1982; Jennings & Gray 1983). When the parasites relapse in blood the animal is usually in the meningoencephalitic stages of the disease.

We used this model to study the changes in serum and CSF trypanosome-specific immunoglobulin concentrations after experimental infection. The vervet monkey is amenable to serial sampling allowing the analysis of paired serum and CSF samples of various molecules present in the two compartments. This allows precise and in-depth study of pathogenesis and the association between serum and CSF changes with clinical disease progression, especially events surrounding the entry and subsequent invasion of trypanosomes into the brain that culminates in meningoencephalitis. The identification of biomarkers would allow refinement and identification of humane end points to experimental studies in the model during drug trials.

The aim of the study was to investigate the concentration changes of immunospecific anti-parasite immunoglobulin G (IgG) and IgM antibodies, and IL-10 in serum and CSF during the course of infection and correlations with parasitaemia, CSF parasitosis and white cells changes.

Materials and methods


The monkeys were infected intravenously with approximately 104T. b. rhodesiense (isolate KETRI 2537) delivered in 1 ml of phosphate saline glucose (PSG). This trypanosome isolate was initially isolated from a sleeping sickness patient in Busoga region in Uganda in 1972, serially passaged in monkeys before cryopreservation (Fink & Schimdt 1980).


Eighteen vervet monkeys (Chlorocebus aethiops) of both sexes, weighing between 1.95 and 3.0 kg were used in the experiment. They were trapped from the wild in an area known to be non-endemic for human trypanosomiasis. The animals underwent to a 90-day quarantine, during which they were screened for zoonotic diseases and treated for ecto- and endoparasites before being subjected to the experiment. The monkeys were fed twice daily with commercial monkey pellets (Unga Feeds Kenya Ltd., Nakuru) and green maize, carrots, tomatoes and bananas. Water was provided ad libitum. They were housed in single stainless steel cages measuring 90 × 60 × 60 cm3 at room temperatures of 23–25 °C.

Ethical review

All protocols and procedures used in the current study were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of the Trypanosomiasis Research Centre (TRC).

Experimental design

Fourteen monkeys were infected while four comprised a non-infected control group. The vervets were sampled twice before the experimental infections to provide self-control data. A daily clinical evaluation of the appetite, clinical appearance and disease symptoms was conducted before and during the course of infection. The parasitaemia was scored using the method described by Herbert and Lumsden (1976).

The animals were anaesthetised at weekly intervals with ketamine hydrochloride (Rotexmedica®, Trittau, Germany) at a dosage of 10 mg/kg body weight (kg/bwt) and diazepam (Valium®, May and Baker, UK) at a dosage of 1 mg/kg bwt, weighed, and clinically examined. Simultaneous blood and CSF sample were obtained at regular intervals throughout the study. The blood sample was taken by syringe and needle from the femoral vein for haematology and serum separation. The CSF sample was obtained by lumbar puncture and presence of trypanosomes and white cells in the infected animals was first determined using the Neubauer cell counting chamber and an automated haematology analyser (Coulter Ac.T diff; Beckman coulter, Miami, FL, USA). A further 1 ml CSF sample was collected into a Pasteur pipette whose narrow end had been heat-sealed and examined for trypanosomes according to the method described by Gould and Sayer (1983). A third portion (0.2 ml) of the CSF sample was sub-inoculated into a mouse as the gold standard method to determine when the trypanosomes first relapsed in both blood and CSF following sub-curative treatment. The infected animals were given sub-curative treatment with diminazene aceturate (DA) (Veriben®, Sanofi, France) at a dose rate of 5 mg/kg body weight through intramuscular injection for 3 consecutive days, commencing on 28 dpi.

Starting 2 weeks post-treatment with DA, and fortnightly thereafter, two monkeys identified randomly at the beginning of the study, were sacrificed. The animals, under deep anaesthesia, were infused with physiological saline followed by 10% neutral buffered formalin in order to fix the brain in situ. Other organs were also preserved in formalin for histopathology. The three monkeys remaining at 98 days after infection were treated curatively with melarsoprol (MelB) (Arsobal®, Specia, France) at a dose rate of 3.6 mg/kg infused intravenously via the saphenous vein for four consecutive days starting 140 dpi when there was a relapse of parasitaemia. These animals were monitored for a further 300 days to ascertain cure.

Serum and CSF samples

Serum and CSF samples were collected weekly starting 1 week before infection to 28 days after treatment with MelB. Thereafter the samples were collected weekly till 28 days after treatment, then fortnightly till 100 days after MelB treatment. The sampling was then carried out monthly to the end of the experiment 300 days after treatment with MelB. All serum and CSF samples were aliquoted before storage at −20 °C. The samples were transported in dry ice from Muguga to Copenhagen, where the assays were done.

Preparation of antigen

Swiss White mice, infected with T. b. rhodesiense (isolate KETRI 2537) were euthanised at peak parasitaemia by CO2 inhalation. Cardiac blood was drawn and filtered through DEAE cellulose to remove the erythrocytes and white cells as described by Godfrey and Lanham (1971). The pellet was frozen at −20 °C. The frozen pellet was disintegrated by extrusion three times by means of an X-press (AB Biox, Göteborg, Sweden) using a pre-cooled (−30 °C) 5-ml cylinder operated with a maximum force of 80 Mpa (Edebo 1983). The product was centrifuged at 20 000 g at +5 °C for 1 h, thereby separating the water-soluble somatic antigens (protein fraction) and the cell wall residue as the supernatant and pellet respectively. The somatic fraction of the antigen was sterile filtered (Minisart, NML, Satorius, Göttingen, Germany). The protein concentration was estimated by reading the absorbance at 280 nm in a spectrophotometer. The concentration of antigen was adjusted to approximately 1.5 μg/ml, and stored in aliquots at −80 °C until use.

IL-10 and immunospecific IgM and IgG assays

The IL-10 ELISA assays were done as previously described (Ngotho et al. 2006). For quantification of immunospecific anti-parasite IgG and IgM, ELISA plates (Nunc, Denmark) were coated using a dilution of 1/1000 of the antigen preparation. Indirect sandwich enzyme linked immunosorbent assays (ELISA) were used to quantify the levels of immunospecific anti-parasite IgG and IgM antibodies in the serum and CSF samples. The target aliquots were thawed overnight at +4 °C before laying out the plates. A pooled serum sample from the infected monkeys taken between 14 and 21 days after infection was arbitrarily assigned to contain 100 AU/ml and diluted in twofold series to serve as standards in the ELISAs. To block non-specific binding, the plates were incubated with 10% Bovine Serum Albumin (BSA) in physiological buffered saline (PBS) pH7.4 for 1 h at room temperature. The serum and CSF samples were diluted in PBS 1:160 and 1:20, respectively and the plates incubated, rocking gently at room temperature, for 1 h. Detection antibody (HRP-conjugated polyclonal Rabbit anti-Human IgM or IgG, DakoCytomation, Glostrup Denmark), diluted 1/1000, was added and plates incubated at 37 °C for 1 h. A fresh solution of five 2 mg OPD tablets in 12 ml distilled water and 12.5 μl hydrogen peroxide was used as substrate. The plates were incubated in the dark and after sufficient colour development the reaction was stopped by adding 100 μl l m sulphuric acid to each well. The plates were read with an ELISA-reader (Multiscan RC, Labsystems, Sweden) at 492 nm. All assays were done in duplicate and the plates washed four to five times between incubated steps. The intra assay coefficient of variation was 13.6% (n = 284) and 13.7% (n = 284) for the IgM and the IgG assays respectively. The sensitivities of the IgG and the IgM assays were 10 AU/ml.

Brain pathology

Wax-mounted frontal serial sections, 14–15 μm thick, were collected from the level of the third ventricle containing the choroid plexus, suprachiasmatic nucleus and brain stem. They were cut on a microtome, stained with Haematoxylin and Eosin (H&E) and examined.

Data analysis

Data were managed using an Ms Excel spreadsheet and analysis was undertaken using Minitab version 13. For categorical data log transformation was undertaken to normalise the distribution. Paired clustered means were compared using the Student t-test. Multiple means were compared using analysis of variance (anova). Means were deemed significant when the indicated probability for test of equality was less than 5% (P < 0.05). Data are presented as mean ± SE.


Clinical signs, parasitaemia and CSF parasitosis

The pre-patent period following experimental infection was 3–4 days in all 14 monkeys. The parasites multiplied rapidly, giving a first parasitaemia peak of approximately 1.2 × 108trypanosomes/ml 10 dpi. Thereafter, the parasitaemia remained high, characterised by minor fluctuations (Figure 1a). The infected animals developed symptoms of trypanosomiasis, which included fever, dullness, increased respiratory and pulse rates, pallor of mucous membranes, enlarged lymph nodes and spleen, and loss of body weight. The trypanosomes were detected in the CSF between 7 (first animal) and 14 (last animal) dpi. Treatment with DA 28 dpi resulted in clearance of the trypanosomes in the blood and CSF. The parasites reappeared in the CSF (63–98) and blood (127–135) dpi respectively. Animals with relapsed parasitaemia were treated immediately after relapses were observed and before clinical manifestation of late stage disease. Treatment with MelB led to clearance of both the CSF and blood trypanosomes by the last day of treatment. One vervet monkey died suddenly 42 days after MelB treatment. The remaining monkeys remained aparasitaemic and clinically normal during the 300-day post-treatment monitoring period when they were euthanised to terminate the study.

Figure 1.

 (a) Mean parasitaemia, (b) mean CSF WCC, and (c) mean IL-10 changes in vervet monkeys infected with Trypanosoma brucei rhodesiense.

Brain histopathology

Infected vervet monkeys euthanised 98 days after infection had spectacular and characteristic brain lesions typical of meningoencephalitis, despite the absence of indicative clinical signs. The lesions were characterised by diffuse perivascular cuffings with mononuclear cells within the brain parenchyma and meninges. The animals that were allowed to recover had chronic fibroplastic lesions with histiocytic infiltration. The pathological brain lesions are described in Table 1, and illustrated in Figure 2a–f.

Table 1.   Results of brain histopathology
Animal numberTime of euthanasia (dpi)Observations
1 & 242Meningitis noted in 2
356Meningitis but no lesions were noted in brain tissue and choroid plexus
456Choriomeningitis. No lesions on brain parenchyma
569 (sudden onset of clinical signs of late stage sleeping sickness)Severe and diffuse inflammation of choroid plexus, meninges and brain parenchyma i.e. choriomeningoencephalitis characterised by perivascular cuffings, encephalitis with mononuclear cellular infiltrations mainly macrophages and microglyosis
670Generalised choriomeningitis. No lesions on brain parenchyma
884Generalised choriomeningitis. No lesions on brain parenchyma
791Generalised choriomeningitis. No lesions on brain parenchyma
9 & 1098Severe and diffuse meningoencephalitis – perivascular cuffings with mononuclear cellular infiltration in meninges and brain parenchyma
12187 (sudden death 42 days post-MelB treatment)Chronic fibroblastic inflammation of meninges and brain parenchyma with mononuclear cells infiltration leading to meningoencephalitis and perivascular cuffings
13 & 14455 (>300 days post-MelB treatment)Chronic fibroplastic meningitis and slight inflammation of parenchyma with infiltration with histiocytes/macrophages
18455 daysNon-infected control. No lesions observed
Figure 2.

 Illustration of brain pathology in vervet monkeys experimentally infected with Trypanosoma brucei rhodesiense. (a) Perivascular cuffing in vervet no. 5 which had clinical manifestation of meningoencephalitis, (H&E) ×200; (b) perivascular cuffing in vervet no. 12 which died suddenly 42 days after treatment with MelB (H&E) ×200; (c) generalised choriomeningitis in vervet no. 3, euthanised 56 dpi (H&E) ×200; (d) meningoencephalitis in vervet no. 5, euthanised 69 dpi (H&E) ×200; (e) severe encephalitis in vervet no. 9, euthanised 98 dpi (H&E) ×100; (f) perivascular cuffing with histiocytes in vervet no. 14, euthanised 300 days post-treatment with MelB (H&E) ×200.

Immunospecific anti-parasite immunoglobulin M

The mean serum IgM level in the vervet monkeys prior to infection was 1200 (range: 0–3650; median 1350) AU/ml. An initial rise of 5240 (range: 320–12 900; median 5130) AU/ml was observed 21 dpi with the first peak levels of 26 000 (range: 2460–93 500; median: 9790) AU/ml 42 dpi. This was followed by a slight decline between 63 and 77 dpi where the levels of 12 780 (range: 860–38 900; median: 13 000) AU/ml and 15 400 (range: 1070–64 400; median: 5870) AU/ml were recorded respectively. A steady second elevation was noted starting 84 dpi to peak at 55 400 (range: 1420–282 000; median: 7040) AU/ml 98 dpi when most of the animals had been removed through serial sacrifice for pathology. In the three remaining vervets, a serum IgM rose consistently and peaked at 194 000 (range: 51 600–293 000; median: 238 000) AU/ml 133 dpi. After treatment with MelB starting 140 dpi the serum IgM levels fell rapidly. Pre-infection levels were attained within 30–40 days (Figure 3a).

Figure 3.

 (a) and (b) Mean weekly serum and CSF IgM and IgG changes in vervet monkeys infected with Trypanosoma brucei rhodesiense.

There was no detectable IgM in the CSF of the vervet monkeys before the initiation of the experiment. The earliest point at which IgM was detected in CSF was 49 dpi 150 (range: 0–750; median: 0) AU/ml (P < 0.05). This was followed by a strong and rapid elevation to attained peak levels of 5340 (range: 0–11 400; median: 4950) AU/ml 98 dpi. This level was stable with slight fluctuations up to 5780 (range: 150–14 800; median: 2380) AU/ml 127 dpi. A decline in the CSF IgM levels was noted before treatment with MelB was commenced 140 dpi. Treatment with MelB led to a rapid and significant decline within 30 days (P < 0.05). However, IgM remained detectable in CSF throughout the 300-day post-treatment monitoring period (Figure 3a).

Wide four variations in serum and CSF IgM levels were noted between monkeys. The animal that died suddenly 42 days post-treatment with MelB did not have detectable CSF IgM until 133 dpi when levels of 150 AU/ml were observed. This increased to a peak of 1590 AU/ml a week before the monkey died. Brain histology of this animal demonstrated encephalitis with characteristic perivascular cuffing comprised of mononuclear cells.

The magnitude of IgM concentration was always higher, up to ×49 at peak levels, in serum compared to CSF. In the control animals, the serum levels remained at the pre-infection levels while no CSF immunospecific anti-parasite IgM was observed throughout the study.

Immunospecific anti-parasite immunoglobulin G

The pre-infection mean serum IgG level was about 2650 (range: 0–11 600, median: 850) AU/ml. An initial concentration increase was recorded starting 35 dpi. However, there was no statistically significant change and the serum IgG levels fluctuated around the pre-infection levels throughout the rest of the study (Figure 2b).

CSF IgG was first detected 49 dpi in two of the 14 infected vervet monkeys. There was a statistically significant increase in CSF levels (P < 0.004) to an early peak of 2160 (range: 180–7080; median: 690) AU/ml 98 dpi. The IgG levels increased to the highest peak level of 3700 (range: 270–8770; median: 2100) AU/ml 140 dpi. After MelB treatment, there was a statistically significant drop (P < 0.05) within 30 days. However, the CSF IgG levels remained detectable throughout post-treatment monitoring (Figure 2b). Similar to observations in the IgM measurements, control animals had serum IgG levels remain at pre-infection levels while no CSF IgG was observed throughout the study.

Interleukin-10 (IL-10)

There was no detectable serum and CSF IL-10 in all the animals prior to experimental infection. The mean serum IL-10 levels increased rapidly and were significantly elevated starting 7 dpi when levels of 43.4 (range: 0–75.9; median 40.4) pg/ml were recorded, to peak levels of 164 (range: 23.4–1038; median: 77.1) pg/ml 21 dpi (P < 0.05). After treatment with DA 28 dpi the serum IL-10 levels fell rapidly at first and then more gradually to below detectable limits (2 pg/ml) by 84 dpi. The serum IL-10 levels were again elevated again starting 127 dpi when the highest levels of 27.03 (range: 0–108.1; median: 0) were recorded 133 dpi. A second increase of serum IL-10 coincided with relapse of parasitaemia. When the animals were treated with MelB, the serum IL-10 levels dropped below detection levels within 20 days. There was no detectable IL-10 in CSF of the infected monkeys. Similarly, no IL-10 was detected in serum and CSF samples obtained from non-infected control vervets (Figure 1c).

CSF white cell counts

The mean pre-infection white cell counts (WCC) in the cerebrospinal fluid was 26 (range: 10–50) cells/ml. The count remained at this level until a significant elevation was noted 35 dpi 80 (range: 10–270; median: 70) which peaked at 230 (range: 120–540; median: 180) cells/ml 49 dpi. This was followed by a significant decline to 125 (range: 20–390; median: 90) cells/ml 63 dpi before a gradual and sustained rise to peak counts of 340 (range: 160–610; median: 260) cells/ml was recorder 119 dpi. There was a gradual decrease in WCC to 326 (range: 60–840; median: 80) cells/ml 140 dpi, which was followed by a more rapid decline after treatment with MelB to mean pre-infection counts of 35 (range: 30–40) cells/ml by 201 dpi. The WCC counts in the non-infected control animals fluctuated around the same level throughout the study (Figure 1b).


This study demonstrated the time-related progression of late stage trypanosomiasis in a non-human primate model using a consistent and repeatable procedure. When vervet monkeys were infected with 104 trypanosomes, treated subcuratively with DA 28 dpi, the animals developed meningoencephalitis by 98 dpi. This was characterised by peak CSF white cell counts, CSF and serum IgM and CSF IgG. The two serum IL-10 peaks coincided with peak parasitaemia in peripheral circulation, regardless of whether the animal was in late or early stage.

Earlier studies in our laboratory showed that infected monkeys exhibited a transient increase in serum IFN-γ concentration between 6 and 8 dpi followed by a sustained increase of TNF-α concentrations in serum (Maina et al. 2004). It would appear that following experimental trypanosomiasis infection, vervet monkeys have a transient elevation of IFN-γ which is quickly countered by the up regulation of IL-10 levels observed to occur starting 7 dpi (Ngotho et al. 2006). The negative feedback autoregulation of IL-10 (Malefyt Rde et al. 1991) is a likely reason for the declining IL-10 levels noted to occur during early-stage disease. The treatment with DA appeared to accelerate the declining IL-10 levels. During the late stage serum IL-10 levels fell rapidly below detection limits within 30 days of curative therapy with MelB. In rodent studies, IL-10 has been detected during early infection when only mild neuropathology was observed. Therefore, IL-10 has been ascribed a neuroprotective role through maintenance of a balance between pathogenic and protective immune responses during trypanosmiasis (Namangala et al. 2001; Magez et al. 2004; Sternberg et al. 2005).

In human studies, a chronic elevation of plasma IL-10 has been described during late stage. Due to its marked decline after curative treatment, IL-10 has been suggested as a potential diagnostic marker of late-stage disease and as a marker for monitoring the success of chemotherapy (Rhind et al. 1997; MacLean et al. 2001, 2006; Lejon et al. 2002). Our results confirm these observations, and indeed serum IL-10 levels were below detection limits for the entire 300-day post-treatment monitoring period.

There was a strong and sustained immunospecific IgM response in both the CSF and serum of infected animals with peak levels attained with relapse parasitaemia and meningoecephalitis. This was also the case with CSF IgG levels. By contrast, the serum immunospecific IgG response was weak with the serum IgG levels decreasing after the first peak. These results are in agreement with previous findings that African trypanosomiasis is accompanied by a dramatic increase in immunoglobulin levels, especially IgM, due to B-cell activation (Mattern 1964; Whittle et al. 1977; Greenwood & Whittle 1980). Antibodies specific to trypanosomes are induced by several parasite antigens including variant and invariant surface glycoprotein epitopes (Reinitz & Mansfield 1990; Vincendeau & Bouteille 2006). Antigenic variation by the trypanosome is considered the most important immune escape mechanism of this parasite (Vickerman 1978; Pays 1999). This is capable of activating the B-cells in a T-cell-independent manner which results in an anti-VSG-specific IgM response (Mansfield 1993; Mansfield & Paulnock 2005). In rodent studies, parasites were eliminated due to anti-VSG-specific IgM which appeared at high levels 3–4 days after infection. In contrast, anti-VSG-specific IgG does not seem to be involved in the destruction of trypanosomes (Dempsy & Mansfield 1983; Reinitz & Mansfield 1990).

It would appear that this is also the case in vervet monkeys, where serum IgG levels dropped after an initial rise, indicating an inability to switch from an IgM response to an IgG response perhaps because of the changes in VAT epitopes exposed on the surface of the parasites. However, immunospecific anti-IgG antibodies in the CSF, undetectable up to 49 dpi, increased consistently to peak levels which coincided with CSF IgM 140 dpi. Early reports indicated that IgM appears in the CSF following parasite invasion of the central nervous system (Mattern 1964). Our results indicate that despite trypanosomes being present in the CSF as early as 7 days after infection, there was no detectable IgM and IgG in the CSF until 49 dpi. This time point coincided with the elevation of white cells in the CSF compartment.

Intrathecal synthesis of IgM has been demonstrated (Greenwood & Whittle 1980; Lejon et al. 2003), and its usefulness as the most sensitive marker of neuroinflammation has been tested with promising results (Chappuis et al. 2005; Lejon & Buscher 2005; Lejon et al. 2008). Our results seem to indicate that CSF IgG, and not serum IgG, behaves in a similar pattern. While there was a significant decrease within 30 days of curative treatment, the CSF IgM and IgG remained above detection levels for the 300-day post-treatment monitoring period, similar to observations in sleeping sickness patients (Lejon & Buscher 2005). The concordance of our results with human studies shows that the vervet monkey model of late stage sleeping sickness, even though provoked by sub-curative treatment, mimics the human disease and provides a useful tool for pre-clinical studies.

The majority of animals in the present study had brain lesions 98 dpi: diffuse perivascular cuffings, meningitis and encephalitis. These findings were not accompanied by overt clinical manifestations, except in one animal which had a sudden onset of late-stage clinical signs such as dozing and paresis of limbs. These findings of advancing brains lesions and lack of clear neurological signs, even in animals with late -stage lesions, agree with previous studies in this model (Rudin et al. 1983; Schimdt 1983). Our studies further strengthen the observation that biological data offer more clues to when the disease has progressed to late stage than clinical manifestations which are variable, non-specific and inconsistent (Dumas & Bisser 1999; Kennedy 2004; Lejon & Buscher 2005; Bisser et al. 2006).

Using rodent models, it has been shown that the tight junctions between the endothelial cells are preserved. This indicates that the spread of trypanosomes from the blood into the brain parenchyma is an active process rather than a passive diffusion after BBB dysfunction (Mulenga et al. 2001; Kristensson et al. 2002; Masocha et al. 2007). Earlier studies in the vervet monkey model reported an early and consistent accumulation of parasites in the choroid plexus which have a limited BBB. It was postulated that from these sites the trypanosomes infested the perivascular spaces before the establishment of meningoencephalitis (Schimdt 1983; Schimdt & Bafort 1985).

In contrast, recent studies in mouse model have reported that no parasite gradient was observed around the ventricles which might indicate that the invasion of the brain parenchyma occurs by penetration of trypanosomes through the BBB, the site of which is determined by the composition of basement membranes of the vessels, rather than spread of the parasites from the circumventricular organs or through the CSF (Masocha et al. 2004).

We did not observe accumulation of parasites in the choroid plexus, even in animals with severe choriomeningoencephalitis. This was possibly due to the partial treatment with DA decribed by Rudin et al. (1983), which led to drastic reduction or even elimination of the parasites within the choroid plexus in rodent and non human primate studies. There were, however, increases in white cells numbers and trypanosomes counted in the CSF of animals with meningoencephalitis in our study. Considering that the studies by Schimdt (1983) and Rudin et al. (1983) were conducted using a similar protocol, there is a need for further investigations to clarify this contradiction.

The present observations of immunospecific IgM and IgG in serum, but not in CSF in the early stages of infection are surprising. The animals used in this study were trapped from a region of Kenya where human trypanosomiasis is not endemic (Schimdt & Sayer 1982; Kagira et al. 2007). However a survey at the time of capture revealed that about 1% of wild trapped monkeys do harbour trypanosomal antibodies (Jenneby et al. 2002). It is not unexpected since the area is endemic for T. brucei and T. congolense which are non-pathogenic to humans. Cross-reactivity between antibodies reacting against these two parasites and the parasite used to experimentally infect the animals of the present study cannot be ruled out. However, given the low prevalence found by Jenneby et al. (2002), and that all animals in our study followed a similar clinical course, an immunological memory to these two other parasites does not seem likely to have been present in the monkeys of this study.

In conclusion, the neuropathogenesis caused by T. b. rhodesiense is an immunopathological reaction involving cytokine, antibody and brain interactions. The disease pattern is associated with disease stage-related changes in concentrations of IL-10 and immunospecific IgM and IgG in serum and CSF that provide opportunities for the use of these molecules as biomarkers of disease progression and management. Using the late-stage vervet monkey model of T. b. rhodesiense, we have established that peak serum IL-10 is related to primary parasitaemia as well as relapse parasitaemia following subcurative treatment. In addition, this molecule is eliminated quickly from peripheral circulation providing a useful marker of success of chemotherapy. Immunospecific serum and CSF anti-parasite IgM as well as CSF immunospecific anti-parasite IgG are elevated in concentration to peak levels that coincide with meningoencephalitis and relapse parasitaemia.

Immunospecific serum and CSF IgM provide a most sensitive biomarker of late stage trypanosomiasis, while the serum immunospecific IgM may be a useful marker of success of chemotherapy. However, due to their persistent elevation post-treatment, CSF IgM and IgG are unlikely to be usable indicators of success of chemotherapy. The timing when neuroinvasion of parasites occurs in the vervet monkey model is now clearer. This allows more focussed studies of events involved in neuropathogenesis and development of intervention measures in the management of sleeping sickness.


This work received financial support from the government of Kenya and University of Copenhagen. The technical assistance from IPR, University of Copenhagen and TRC is gratefully acknowledged. This work is dedicated to the loving memory of Ben Kinyanjui, fondly BiKi; for he cared for experimental animals.