Murine Models for Trypanosoma brucei gambiense Disease Progression—From Silent to Chronic Infections and Early Brain Tropism

Trypanosoma brucei gambiense is responsible for more than 90% of reported cases of human African trypanosomosis (HAT). Infection can last for months or even years without major signs or symptoms of infection, but if left untreated, sleeping sickness is always fatal. In the present study, different T. b. gambiense field isolates from the cerebrospinal fluid of patients with HAT were adapted to growth in vitro. These isolates belong to the homogeneous Group 1 of T. b. gambiense, which is known to induce a chronic infection in humans. In spite of this, these isolates induced infections ranging from chronic to silent in mice, with variations in parasitaemia, mouse lifespan, their ability to invade the CNS and to elicit specific immune responses. In addition, during infection, an unexpected early tropism for the brain as well as the spleen and lungs was observed using bioluminescence analysis. The murine models presented in this work provide new insights into our understanding of HAT and allow further studies of parasite tropism during infection, which will be very useful for the treatment and the diagnosis of the disease

PCR detection of Trypanosomes in the blood of BALB/c mice infected with 10³ parasites of the silent <i>Tbg</i>1135c isolate.

<p>Detection is based on the amplification of 3 <i>Trypanosoma brucei</i> specific gene targets. The + sign represents a positive result (2–3 out of 3 targets amplification). ± represents a doubtful result (1 out of 3 targets amplification).</p

Analysis of <i>T. b. gambiense</i> organs and central nervous system invasion in BALB/c-infected mice.

<p>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 <i>Tbg</i>945b isolate (1, 2, 3) and of paralyzed mice (n = 2) treated with cyclophosphamide before infection with either 5×10⁶ parasites of subchronic <i>Tbg</i>1122c or <i>Tbg</i>1166c isolates. Paralysis occurred 10 and 6 months PI with <i>Tbg</i>1122c and <i>Tbg</i>1166c isolates respectively. Only results from <i>Tbg</i>1122c 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 <i>Tbg</i>1122c) 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 <i>Tbg</i>1166c 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-<i>Tbg</i>1135b (n = 6) or LucR-<i>Tbg</i>1135c (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-<i>Tbg</i>1135b (11 weeks), LucR-<i>Tbg</i>1135c (10 weeks), LucR-<i>Tbg</i>1135b+cyclo (9 weeks), LucR-<i>Tbg</i>1135+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-<i>Tbg</i>1135b, (3) infected for 18 weeks with 10⁶ LucR-<i>Tbg</i>1135c, (4) pre-treated with cyclophosphamide and infected for 16 weeks with 10⁶ LucR-<i>Tbg</i>1135c. Quantification data of light emission signals for ROI delimitating each organ are given in photons/second/cm²/steradian (p/sec/cm²/sr).</p

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

<p>Four mice were infected with either a low (10³) or a high (10⁶) load of <i>Tbg</i>945b, <i>Tbg</i>1122b, <i>Tbg</i>1166b, <i>Tbg</i>1135b or <i>Tbg</i>1135c 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.</p

<i>In vitro</i> and <i>in vivo</i> growth characteristics of <i>T. b. gambiense</i> field isolates.

<p>A. <i>In vitro</i> culture of <i>Tbg</i>1122, <i>Tbg</i>1166 and <i>Tbg</i>1135 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 <i>Tbg</i>945 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<i>Tbg</i>1122c (n = 4), <i>Tbg</i>1166c (n = 6), <i>Tbg</i>1135c (n = 9) or <i>Tbg</i>1135b (n = 10) isolates. D. Represents the results of one representative BALB/c mouse infected with 1–5×10⁶<i>Tbg</i>1122b, <i>Tbg</i>1166b (n = 10) or <i>Tbg</i>1135b 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 <i>Tbg</i>1166 (▴ , ▵), <i>Tbg</i>1122 (▪ , □) and <i>Tbg</i>1135 (• , ○).</p

Biological properties of type 1 <i>T. b. gambiense</i> field isolates.

<p>Biological properties of type 1 <i>T. b. gambiense</i> field isolates.</p

Correction: Murine Models for <i>Trypanosoma brucei gambiense</i> Disease Progression—From Silent to Chronic Infections and Early Brain Tropism

Correction: Murine Models for <i>Trypanosoma brucei gambiense</i> Disease Progression—From Silent to Chronic Infections and Early Brain Tropis

Minisatellites and microsatellites analyses.

<p>The genotype of each isolate (<i>Tbg</i>945, <i>Tbg</i>1122, <i>Tbg</i>1166 and <i>Tbg</i>1135) was analyzed as described earlier <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0000509#pntd.0000509-Biteau1" target="_blank">[24]</a> by determining the number of repeats per allele and compared to group 1 and 2 genotypes.</p

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

<p>Different trypanosome protein extracts: (3A,D) <i>Tbg</i>1122b total lysate, (3B) <i>Tbb</i>427 total lysate (T) and <i>Tbb</i>427 fractions containing the soluble proteins (F1) or cytoskeleton/membrane proteins (F2), (3C) <i>Tbb</i>427 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 <i>Tbg</i>945b (1), <i>Tbg</i>1122b (2), <i>Tbg</i>1135b (3), <i>Tbg</i>1135c (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.</p