Murine Models for Trypanosoma brucei gambiense disease progression--from silent to chronic infections and early brain tropism

Abstract

Background: Human African trypanosomiasis (HAT) caused by Trypanosoma brucei gambiense remains highly prevalent in west and central Africa and is lethal if left untreated. The major problem is that the disease often evolves toward chronic or asymptomatic forms with low and fluctuating parasitaemia producing apparently aparasitaemic serological suspects who remain untreated because of the toxicity of the chemotherapy. Whether the different types of infections are due to host or parasite factors has been difficult to address, since T. b. gambiense isolated from patients is often not infectious in rodents thus limiting the variety of isolates.

Methodology/principal findings: T. b. gambiense parasites were outgrown directly from the cerebrospinal fluid of infected patients by in vitro culture and analyzed for their molecular polymorphisms. Experimental murine infections showed that these isolates could be clustered into three groups with different characteristics regarding their in vivo infection properties, immune response and capacity for brain invasion. The first isolate induced a classical chronic infection with a fluctuating blood parasitaemia, an invasion of the central nervous system (CNS), a trypanosome specific-antibody response and death of the animals within 6-8 months. The second group induced a sub-chronic infection resulting in a single wave of parasitaemia after infection, followed by a low parasitaemia with no parasites detected by microscope observations of blood but detected by PCR, and the presence of a specific antibody response. The third isolate induced a silent infection characterised by the absence of microscopically detectable parasites throughout, but infection was detectable by PCR during the whole course of infection. Additionally, specific antibodies were barely detectable when mice were infected with a low number of this group of parasites. In both sub-chronic and chronic infections, most of the mice survived more than one year without major clinical symptoms despite an early dissemination and growth of the parasites in different organs including the CNS, as demonstrated by bioluminescent imaging.

Conclusions/significance: Whereas trypanosome characterisation assigned all these isolates to the homogeneous Group I of T. b. gambiense, they clearly induce very different infections in mice thus mimicking the broad clinical diversity observed in HAT due to T. b. gambiense. Therefore, these murine models will be very useful for the understanding of different aspects of the physiopathology of HAT and for the development of new diagnostic tools and drugs.

Figure 1. In vitro and in vivo growth characteristics of T. b. gambiense field isolates.

A. In vitro culture of Tbg1122, Tbg1166 and Tbg1135 isolates after adaptation to axenic culture conditions. Cultures were seeded with adapted trypanosomes (5.10⁴/ml) in supplemented MEM medium and enumerated every 24 h. The mean trypanosome densities±standard error of the mean of 4 independent cultures is presented. Similar doubling times were obtained with the three isolates (15.9 h, 15.8 h and 14.6 h respectively). B–D. Parasitaemia levels in immunocompetent (BALB/c), immunodeficient (NOD/SCID and cyclophosphamide-treated BALB/c) mice infected with the different field isolates. Parasitaemia was measured from tail-blood either by direct observation of the wet films under the microscope or by using a haemacytometer. The limit of detection was estimated at about 10⁴ parasites/ml. B. Represents the results of one representative BALB/c mouse (n = 6) infected i.p. with 10⁶ of the Tbg945 blood isolate. All infected mice showed successive waves of parasitaemia and died within 6–8 months PI. C. Represents the mean parasitaemia in NOD/SCID mice infected with 10³ Tbg1122c (n = 4), Tbg1166c (n = 6), Tbg1135c (n = 9) or Tbg1135b (n = 10) isolates. D. Represents the results of one representative BALB/c mouse infected with 1–5×10⁶ Tbg1122b, Tbg1166b (n = 10) or Tbg1135b blood (n = 6) isolates with (+cyclo) or without (−cyclo) prior administration (24 h before infection) of cyclophosphamide (200 mg/kg). Solid symbols indicate culture isolates, open symbols indicate blood isolates of Tbg1166 (▴ , ▵), Tbg1122 (▪ , □) and Tbg1135 (• , ○).

Figure 2. Time course of total serum IgM levels in BALB/c mice infected with different T. b. gambiense field isolates.

Four mice were infected with either a low (10³) or a high (10⁶) load of Tbg945b, Tbg1122b, Tbg1135b or Tbg1135c isolates then IgM levels were quantified by ELISA test 1 month (black bars), 3 months (white bars), 5 months (gray bars) and 12 months (hatched bars) after infection. IgM levels are expressed as a multiple (mean±standard error) of the level before infection.

Figure 3. Immunoblotting analysis of the trypanosome antigens recognised during BALB/c mice infections and identification of potential immunoreactive proteins.

Different trypanosome protein extracts: (3A,D) Tbg1122b total lysate, (3B) Tbb427 total lysate (T) and Tbb427 fractions containing the soluble proteins (F1) or cytoskeleton/membrane proteins (F2), (3C) Tbb427 cytoskeleton/membrane fraction (F2) were subjected to SDS-PAGE and tested by Western blotting against different sera from BALB/c (n = 4) mice infected with (10³) parasites, (3A–C): sera from Tbg945b (1), Tbg1122b (2), Tbg1135b (3), Tbg1135c (4); non-infected control mice (5) or (3D) antibodies specific for cytoskeleton or membrane proteins: rabbit polyclonal antibodies directed against ISG64 (6), ISG65 (7), ISG75 (8), mouse monoclonal antibodies recognizing PFR2 (9) or calflagin (10). A–C represents the results of one representative immunoblot out of 4 mice tested.

Figure 4. Reactivity patterns of immunoreactive invariant trypanosome proteins during BALB/c mice infections.

Four mice were infected with either a low (10³) or a high (10⁶) load of Tbg945b, Tbg1122b, Tbg1166b, Tbg1135b or Tbg1135c isolates and their sera collected at different time points were tested by Western blotting (1/100 dilution) against a strip loaded with recombinant protein: 0.5 µg PFR and ISG75, 1 µg ISG65, ISG64 and TgsGP and 2 µg calflagin. The data are representative of one immunoblot out of 4 mice tested. NI represents the control sera before infection.

Figure 5. Kinetics of antibody responses to native ISGs and calflagin in BALB/c mice infected with T. b. gambiense field isolates.

Sera from mice infected with 10³ parasites of Tbg945b (no symbol), Tbg1122c (▪), Tbg1122b (□), Tbg1135c (x), Tbg1135b (○) or with 10⁶ parasites of Tbg1135c (•) were collected at different time points PI and tested by ELISA for their reactivity against native ISG64, ISG65, ISG75 and calflagin. Results in Tbg1166b (data not shown) and Tbg1122b infections were similar. All isolates elicited an antibody response against the recombinant proteins, except the silent Tbg1135c (even with a high load of parasites). Each point represents the mean±standard error of 5 mice.

Figure 6. Analysis of T. b. gambiense organs and central nervous system invasion in BALB/c-infected mice.

A. Immunohistochemical detection of trypanosomes in the brains of mice (n = 2, only results from one mouse are shown) infected for 4 months with 10⁶ parasites of the Tbg945b isolate (1, 2, 3) and of paralyzed mice (n = 2) treated with cyclophosphamide before infection with either 5×10⁶ parasites of subchronic Tbg1122c or Tbg1166c isolates. Paralysis occurred 10 and 6 months PI with Tbg1122c and Tbg1166c isolates respectively. Only results from Tbg1122c are shown (4, 5, 6). A1 and A4 correspond to an olfactory bulb coronal section, A2 to a forebrain section, A3 and A5 to brain stem sections and A6 to a cerebellum section. Whole brain invasion was observed with the chronic isolate at an advanced stage of the disease (4 months PI, death within 6–8 months). Invasion was restricted to the olfactory bulb and the brain stem (including the cerebellum for Tbg1122c) in paralyzed mice infected with the sub-chronic isolates after treatment with cyclophosphamide. No invasion was observed in mice (n = 2) infected for 9 months with 5×10⁶ parasites of subchronic Tbg1166c isolate (data not shown). B. Spatial distribution of R-Luc activity in animals developing a sub-chronic or a silent infection and treated with or without cyclophosphamide (+/−cyclo). BALB/c mice were either directly infected with 10⁶ LucR-Tbg1135b (n = 6) or LucR-Tbg1135c (n = 2) or treated 24 h before infection with cyclophosphamide (n = 2). At different time PI, mice were anaesthetized and injected intravenously (i.v., retro-orbital) or intraperitoneally (i.p.) with coelenterazine and BLI signals were recorded in real time with a Biospace Imaging System. The panels show dorsal and ventral images of 2 representative mice infected for 8–11 weeks: LucR-Tbg1135b (11 weeks), LucR-Tbg1135c (10 weeks), LucR-Tbg1135b+cyclo (9 weeks), LucR-Tbg1135+cyclo (8 weeks). C. Spatial distribution of R-Luc activity in organs removed from LucR-Tbg1135 infected BALB/c mice. The different organs shown in this figure were isolated from mice: (1) non infected (control) (2) infected for 18 weeks with 10⁶ LucR-Tbg1135b, (3) infected for 18 weeks with 10⁶ LucR-Tbg1135c, (4) pre-treated with cyclophosphamide and infected for 16 weeks with 10⁶ LucR-Tbg1135c. Quantification data of light emission signals for ROI delimitating each organ are given in photons/second/cm²/steradian (p/sec/cm²/sr).