# The skin is a significant but overlooked anatomical reservoir for vector-borne African trypanosomes

Paul Capewell, Christelle Cren-Travaillé, Francesco Marchesi, Pamela Johnston, Taylor-Anne Gorman, Estefania Calvo-Alvarez, Aline Crouzols, Grégory Jouvion, Vincent Jammoneau, William Weir, et al.

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## To cite this version:

Paul Capewell, Christelle Cren-Travaillé, Francesco Marchesi, Pamela Johnston, Taylor-Anne Gorman, et al.. The skin is a significant but overlooked anatomical reservoir for vector-borne African trypanosomes. eLife, 2016, 10.7554/eLife.17716. pasteur-01371190

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Submitted on 24 Sep 2016

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1 The skin is a significant but overlooked anatomical reservoir for vector-borne African trypanosomes

3

4 Paul Capewell¹*, Christelle Cren-Travaillé²*, Francesco Marchesi³, Pamela Johnston³, Caroline

5 Clucas¹, Robert A Benson⁴, Taylor-Anne Gorman¹,⁴, Estefania Calvo-Alvarez², Aline Crouzols²,

6 Grégory Jouvion⁵, Vincent Jammoneau⁶, William Weir¹, M Lynn Stevenson³, Kerry O'Neill¹, Anneli

7 Cooper¹, Nono-raymond Kuispond Swar⁷, Bruno Bucheton⁶, Dieudonné Mumba Ngoyi⁸, Paul

8 Garside⁴, Brice Rotureau²** and Annette MacLeod¹**

9

10 ¹Wellcome Trust Centre for Molecular Parasitology, College of Medical, Veterinary and Life

11 Sciences, Henry Wellcome Building for Comparative Medical Sciences, Garscube Estate, Glasgow,

12 United Kingdom, G61 1QH

13 ²Trypanosome Transmission Group. Trypanosome Cell Biology Unit, INSERM U1201 & Department

14 of Parasites and Insect Vectors, Institut Pasteur, Paris, France

15 ³Veterinary Diagnostic Services, Veterinary School, University of Glasgow, Garscube Estate,

16 Glasgow, United Kingdom, G61 1QH

17 ⁴Institute of Infection, Immunology and Inflammation, College of Medical, Veterinary and Life

18 Sciences, Glasgow Biomedical Research Centre, University of Glasgow, Glasgow, United Kingdom,

19 G12 8TA

20 ⁵Human Histopathology and Animal Models Unit, Institut Pasteur, Paris, France

21 ⁶Institut de Recherche pour le Développement, Unité Mixte de Recherche IRD-CIRAD 177, Campus

22 International de Baillarguet, Montpellier, France

23 ⁷University of Kinshasa, Kinshasa, Democratic Republic of the Congo

24 ⁸Department of Parasitology, National Institute of Biomedical Research (INRB), Kinshasa,

25 Democratic Republic of the Congo

26

27 * Joint first authors

28 ** Joint last authors

29 The authors have no competing financial interests.

30 Keywords

31 Skin, blood, reservoir, transmission, infection, Human African Trypanosomiasis, trypanosomes,

32 *Trypanosoma brucei*, tsetse fly, vector-borne, evolution

33
The role of mammalian skin in harbouring and transmitting arthropod-borne protozoan parasites has
been overlooked for decades as these pathogens have been regarded primarily as blood-dwelling
organisms. Intriguingly, infections with low or undetected blood parasites are common, particularly
in the case of Human African Trypanosomiasis caused by *Trypanosoma brucei gambiense*. We
hypothesise, therefore, the skin represents an anatomic reservoir of infection. Here we definitively
show that substantial quantities of trypanosomes exist within the skin following experimental
infection, which can be transmitted to the tsetse vector, even in the absence of detectable
parasitaemia. Importantly, we demonstrate the presence of extravascular parasites in human skin
biopsies from undiagnosed individuals. The identification of this novel reservoir requires a re-
evaluation of current diagnostic methods and control policies. More broadly, our results indicate that
transmission is a key evolutionary force driving parasite extravasation that could further result in
tissue invasion-dependent pathology.
## Introduction

Understanding the process of parasite transmission is essential for the design of rational control measures to break the disease cycle and requires the identification of all reservoirs of infection. In a number of vector-borne diseases, it is becoming evident that asymptomatic individuals, be they humans or animals, can represent a significant proportion of the infected population and therefore an important reservoir of disease that requires targeting by control measures¹⁻⁴. The recent identification in West Africa of asymptomatic individuals with human trypanosomiasis (long-term seropositives) but undetected parasitaemia, raises the question of what role these individuals play in disease transmission³,⁵⁻⁷. Therapy is currently only directed towards microscopy-positive individuals and thus a proportion of the infected population remain untreated.

There is convincing evidence that seropositive individuals with low or undetected parasitaemia contain transmissible trypanosomes. Xenodiagnosis experiments, in which tsetse flies are fed on microscopy-negative infected humans⁸ or, more recently, experimentally-infected pigs⁹, have shown that these apparently aparasitaemic hosts contain the parasite since the tsetse flies became infected. It is uncertain where the trypanosomes reside in the host but, given the telmophagus (slash and suck) feeding habit of the tsetse fly, they could be skin-dwelling parasites ingested with the blood meal. Our findings suggest that parasites may be sufficiently abundant in the skin to allow transmission and therefore the skin may represent an anatomical reservoir of infection.

Detection of trypanosomes in the skin is not well documented, although there are descriptions of cutaneous symptoms associated with African trypanosomiasis and distinct 'trypanid' skin lesions¹⁰. Imaging data from mouse models of infection suggest that trypanosomes sequester to major organs such as the spleen, liver and brain¹¹,¹² and recent evidence has demonstrated trypanosomes in extravascular adipose tissue¹³. These adipose-associated trypanosomes appear to be a new life-cycle stage with a distinct transcriptional profile and, while tsetse bite-site associated transmission has been suggested¹⁴, and a historical study made a passing observation of localised deposition of trypanosomes in the skin matrix¹⁵, the broader role of skin-dwelling trypanosomes in transmission remains unclear. In this paper we report the investigation of a possible anatomical reservoir in the skin of the mammalian host. We provide conclusive evidence of *T.b. brucei*, (a causative agent of animal trypanosomiasis) and the human-infective trypanosome, *T.b. gambiense*, invading the extravascular tissue of the skin (including but not restricted to the adipose tissue) and undergoing onward transmission despite undetected vascular parasitaemia. We also provide evidence of localisation of trypanosomes within the skin of undiagnosed humans. The presence of a significant transmissible population of *T. brucei* in this anatomical compartment is likely to impact future control and elimination strategies for both animal and human trypanosomiasis.
## 83 Results

In order to investigate the potential for extravascular skin invasion by *T. brucei*, BALB/c mice were inoculated via IP injection with the low virulence STIB247 strain of *T. brucei* and skin sections were assessed over a 36-day time-course. The presence and relative quantities of extravascular parasites were evaluated by semi-quantitative scoring of the histological samples (Figure 1-source data 1) and compared to blood parasitaemia (Figure 1-source data 2). Extravascular parasites were first observed in the skin 12 days post-infection and remained throughout the experiment. Skin parasite numbers fluctuated to a lesser extent than blood parasitaemia and the apparent periodicity in the skin may be due to one particularly high data point on day 24 (Figure 1). Parasites were found in the dermis, subcutaneous adipose tissue (Figure 2) and in fascia beneath the panniculus carnosus muscle. We did not detect any particular clustering around dermal adipocytes. The presence of parasites in the skin was not associated with major inflammation (Supplementary File 1). To confirm that skin invasion by this parasite was not strain or sub-species specific, the more virulent TREU927 strain of *T. brucei* and the human-infective *T.b. gambiense* strain, PA, were used to infect mice. Extravascular skin invasion of the dermis, subcutaneous adipose tissue and fascial planes (Figure 2-figure supplement 1) was evident with associated mild to moderate inflammation (Supplementary File 2), in some instances the degree of skin invasion in the *T.b. gambiense* infected mice was far greater than *T.b. brucei*, perhaps suggesting a greater propensity for sequestration in this sub-species.

To confirm that the extravascular distribution of parasites was not an artefact of the route of inoculation, infections by natural vector transmission were carried out using a bioluminescent *T.b. brucei* strain, AnTat1.1E AMLuc/tdTomato. Mice were infected by a single infective bite of an individual *G.m. morsitans*. After 4 to 11 days and up to the end of the experiment, parasites were observed in the skin with a dynamic distribution (Figure 3A) and a variable density (Figure 3B). Parasites were first detected in the blood between 5 and 19 days after natural transmission and parasitaemia remained lower than $10^7$ parasites/ml. Observed bioluminescence directly reflects the total number of living parasites in the entire organism, including blood and viscera, but the intensity of the signal decreases with tissue depth. Therefore, at the end of each experiment, mice were sacrificed and their organs were checked for bioluminescence. The presence of extravascular parasites in cutaneous and subcutaneous tissues was first demonstrated by bioluminescence imaging in entire dissected skins (Figure 3C) and was not necessarily localised to the bite site. In addition to the skin, only the spleen, some lymph nodes and adipose tissue were observed to be positive for bioluminescence in several individuals (Figure 3-figure supplement 1). This suggests that the observed bioluminescence is likely to originate predominantly, but not solely, from parasites in the skin. In addition, only mild inflammation was observed after 29 days (Figure 3-figure supplement 2).
To confirm that trypanosomes in the skin are a viable population, fluorescent parasites were monitored by two intravital imaging methods following IP injection and natural transmission (Figure 4). First, IP-injected fluorescent *T. brucei* STIB247 were imaged *in vivo* using 2-photon microscopy (Figure 4A). Extravascular trypanosomes observed in the dermal layer of dorsal skin were highly motile, consistent with viability (Video 1). Second, naturally-transmitted fluorescent *T. brucei* AnTat1.1E AMLuc/tdTomato were imaged *in vivo* using spinning-disk confocal microscopy in the C57BL/6J-Flk1-EGFP mouse line that has green fluorescent endothelial cells in the lymphatic and blood vessels¹⁶ (Figure 4B). Extravascular trypanosomes were observed in the dermal layer of the ear and were highly motile (Videos 2, 3 and 4).

Differentiation of dividing trypanosomes to non-dividing stumpy forms is essential for transmission to the tsetse. Using the relative transcript abundance of the stumpy marker Protein Associated with Differentiation 1 (PAD1)¹⁷ to an endogenous control, Zinc Finger Protein 3 (ZFP3)¹⁸, we estimated that approximately 20% of skin-dwelling parasites were stumpy (Supplementary File 3). To directly determine the proportion of stumpy forms in skin sections, histological staining for PAD1 was also performed (Table 1-source data 4). PAD1-positive cells were observed in variable proportions (from 8 to 80%) in all bioluminescent skin samples examined after IP injection (Supplementary File 4). Following natural transmission, up to 38% of parasites detected using VSG surface markers (Figure 3-figure supplement 3A-B) also expressed PAD1 (Figure 3-figure supplement 3C-D) (n=441 cells from 8 skin sections). In all skin sections, stumpy parasites were homogenously distributed in the dermis and subcutaneous adipose tissues.

We next assessed the ability of skin-dwelling parasites to infect tsetse flies. Teneral flies (immature flies that have not yet taken a blood meal) were fed on different regions of skin from mice infected with AnTat1.1E AMLuc/TY1/tdTomato with differing levels of bioluminescence across the skin (Table 1-source data 1 and Table 1-source data 2). This was repeated in 20 mice with differing levels of parasitaemia. Flies were dissected and checked for the presence of fluorescent trypanosomes after two days (Table 1). In mice with undetectable or low parasitaemia (<5x10⁴ parasites/ml), no parasites were found in flies fed on non-bioluminescent regions. Conversely, a median of 36% (±23%, n=70) of flies that fed on bioluminescent regions of low parasitaemic mice became infected (Table 1 and Table 1-source data 3). This demonstrates that skin parasites, from mice without visible parasitaemia, can contribute to tsetse infection, possibly reflecting human asymptomatic infections. When parasites were detected in the blood and skin, tsetse infection rates increased up to 100% (median of 61% ±22%, n=120) (Table 1 and Table 1-source data 3). Parasites taken-up from the skin were able to further develop through the life-cycle to early procyclic forms in the fly as evidenced by GPEET-procyclin marker on the parasites surface (n=721 cells from 16 flies) (Table 1-source data 4 and Supplementary File 4).

To demonstrate that skin invasion by trypanosomes can occur in humans as well as the murine model, slides of historical skin biopsies, taken as part of a diagnostic screening programme for
*Onchocerca microfilaria* in the trypanosomiasis-endemic Democratic Republic of the Congo, were examined for trypanosomes. At the time of sampling, the incidence of trypanosomiasis was 1.5-2% and we hypothesised that some individuals may have harboured undiagnosed infections. Re-examination of 1,121 skin biopsies by microscopy revealed six individuals with trypanosomes in their skin (Figure 5). These individuals had not previously been diagnosed with human trypanosomiasis by clinical signs or the presence of blood parasites.

## Discussion

We have shown that there exists a significant yet overlooked population of live, motile, extravascular *T. brucei* in the dermis and subcutis of animal models infected by artificial routes or by vector transmission. It is likely that once injected the parasites disseminate via the lymph and blood to the skin where they are ingested during tsetse fly pool-feeding and readily initiate cyclical development in the vector. Given the relative volume of the skin organ compared to the vasculature, it is possible, depending on the density of skin invasion, that more parasites exist in the skin than in the blood. The skin, therefore, represents an unappreciated reservoir of infection. The extravasation of trypanosomes was described previously in major organs such as liver, spleen<sup>11,12</sup> and visceral adipose tissue<sup>13</sup>, but the importance of these parasites in transmission was not investigated. Here we show that these skin-dwelling trypanosomes contribute to transmission and could explain the maintenance of disease foci, despite active screening and treatment. Skin invasion for enhanced transmission is likely a powerful evolutionary force driving extravasation, suggesting that the generalised tissue penetration underlying pathogenesis (i.e. splenomegaly, hepatomegaly, CNS invasion) is a secondary epiphenomenon. A skin reservoir also presents a novel target for diagnostics (e.g. skin biopsies), allowing the prevalence of infection to be accurately determined and the identification of any previously undetected animal reservoirs of human disease.

The skin as an anatomical reservoir of parasites is a recurring theme in arthropod-borne human diseases such as *Leishmania*<sup>19,20</sup> and *Onchocerca*<sup>21,22</sup>. Here we present evidence of trypanosomes in the skin of hitherto undiagnosed individuals. This anatomical reservoir may serve to explain how HAT foci re-emerge and persist despite low numbers of reported cases even in the absence of an animal reservoir<sup>23-25</sup>.

HAT was once widespread across much of sub-Saharan Africa but concerted control efforts brought it close to elimination during the 1960s<sup>26,27</sup>. However, disease foci persisted, with a resurgence in the number of reported cases to over 300,000 in the 1990s<sup>26-28</sup>. Currently, HAT is approaching elimination in many areas<sup>27,29</sup>. Understanding how HAT evaded elimination in the latter half of the 20th century and how it continues to persist is vital to efforts to eliminate the disease. For example, our results indicate that it may be necessary to develop novel therapeutics capable of accessing extravascular compartments. The current policy in most endemic countries is not to treat serologically positive individuals unless they demonstrate active infection, due to the long duration
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and high toxicity of treatment and the low predictive value of the serological tests. We suggest that this policy should be reconsidered in light of our compelling evidence that they represent a carrier population which may maintain HAT foci and explain previously thwarted efforts to eliminate this major pathogen.

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## Acknowledgments

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We acknowledge P. Solano, D. Engman, K. Matthews, M. Taylor, M. Carrington, R. Amino, E. Myburgh and K. Gull for providing various material, cell lines, antibodies and plasmids and to A. Tait for critically reviewing this manuscript.

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## Methods

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### Trypanosome strains and cultures

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All strains used in this study are pleomorphic. STIB247 is a low virulence *T.b. brucei* strain that induces a chronic infection and was isolated from a hartebeest in the Serengeti in 1971³⁰. TREU927 is the genome reference strain for *T. brucei* and is more virulent than STIB247. This strain was isolated from a tsetse fly in Kenya in 1969/1970³¹. PA is a human-infective group 1 *T.b. gambiense* strain isolated from a patient in the Democratic Republic of the Congo in 1975³². mCherry STIB247 was created by transfection of STIB247 with pHD1034-mCherry³³. This strain constitutively expresses fluorescent mCherry from the ribosomal RNA promoter and its expression is stable over repeated passage.

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The AnTat 1.1E clone of *T.b. brucei* was derived from a strain originally isolated from a bushbuck in Uganda in 1966³⁴. Bloodstream forms were cultivated in HMI11 medium supplemented with 10% foetal calf serum at 37°C in 5% CO₂. This strain was genetically engineered to produce two strains continuously expressing the red-shifted luciferase (PpyRE9H) and the tdTomato red fluorescent protein either individually or in combination.

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For the first AnTat 1.1E strain, the pTbAMLuc plasmid (M. Taylor, London School of Hygiene and Tropical Medicine, UK) was used for continuous cytosolic expression of the red-shifted luciferase PpyRE9H. The tdTomato coding sequence was cloned with *Hind*III and *Bam*HI into the pTSARib vector³⁵, generating the final pTSARib-tdTomato construct. The two plasmids were linearised with *Kpn*I and *Sph*I restriction enzymes, respectively, and used to transform procyclic parasites with an Amaxa Nucleofector (Lonza)³⁶. After 24h, transfected cells were selected by the addition of blasticidin or puromycin (10µg/ml). After one week, the population was examined (i) for red fluorescence by fluorescence microscopy and (ii) for both red fluorescence and bioluminescence by using a fluorimeter Infinite® 200 (Tecan). Cells were sub-cloned by limiting dilution, and clone selection was performed after 15 days by measuring both bioluminescence in a microplate reader

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Infinite® 200 (Tecan) and fluorescence with a Muse® cell Analyzer (Merck-Millipore). This strain, named AnTat1.1E AMLuc/tdTomato, was used for natural transmission experiments.

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The second AnTat 1.1E strain expressing the 3.1 Kb chimeric multiplex reporter protein PpyRE9H::TY1::tdTomato was named AnTat 1.1E AMLuc/TY1/tdTomato. This cytoplasmic reporter is composed of three distinct markers: the red-shifted luciferase (PpyRE9H) is fused to the tdTomato red fluorescent protein by a TY1 tag. Briefly, the 1.68 Kb optimised version of the North American firefly *Photinus pyralis* luciferase³⁷ was fused with a 10-bp sequence known as TY1-tag³⁸ and cloned into the pTSARib plasmid³⁵ by using *Xhol* and *Hind*III restriction enzymes to obtain the pTSARib-PpyRE9H-TY1 plasmid. Finally, the 1.4 Kb coding region of the tdTomato fluorescent protein was inserted downstream using *Hind*III and *Bam*HI. The resulting 8.9 Kb vector, containing a blasticidin S resistance cassette, was linearised with *Sphl* to integrate the rDNA promoter locus.

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Bloodstream parasites were transformed with an Amaxa Nucleofector (Lonza)³⁶, sub-cloned by limiting dilution, and clone selection was performed by measuring both bioluminescence in a microplate reader Infinite® 200 (Tecan) and fluorescence with a Muse® cell Analyzer (Merck-Millipore). The selected AnTat 1.1E AMLuc/TY1/tdTomato sub-clone was comparable to the parental wild-type strain in terms of growth rate (Table 1-source data 1A), pleomorphism (Table 1-source data 4C), tsetse infectivity and virulence in mice (Table 1-source data 2). In order to verify the reliability of the bioluminescent marker as well as to define the bioluminescence detection threshold of the AnTat 1.1E AMLuc/TY1/tdTomato selected sub-clone, a parasite density / bioluminescence intensity analysis was performed in 96-micro-well plates with an IVIS® Spectrum imager (Perkin Elmer). Parasite density and bioluminescence intensity were correlated when bioluminescence levels were higher than 10⁴ p/s/cm²/sr, corresponding to about 10³ parasites, allowing estimation of the parasite density from *in vivo* imaging over this threshold (Table 1-source data 1B). This correlation was verified by quantification in a microplate reader Infinite® 200 (Tecan) at the very beginning of the first *in vivo* experiment as well as the end of the last one, demonstrating the stability of the triple reporter expression in the AnTat 1.1E AMLuc/TY1/tdTomato selected sub-clone over time, especially after at least one full *in vivo* parasite cycle in the tsetse fly and the mammalian host (Table 1-source data 1C). This strain was used for xenodiagnosis experiments and quantification of parasite densities.

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## Mouse strains

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BALB/c and C57BL/6J mice were used as models for chronic disease³⁹. In addition, to allow for further 3D intravital imaging of the lymphatic and blood systems, C57BL/6J-Flk1-EGFP mice expressing a GFP tagged *Kdr* (*Flk1*) gene encoding the vascular endothelial growth factor receptor 2 (VEGFR-2) were used¹⁶.
369 Ethical statements

370 This study was conducted under Home Office and SAPO regulations in the UK and in strict
371 accordance with the recommendations from the Guide for the Care and Use of Laboratory Animals
372 of the European Union (European Directive 2010/63/UE) and the French Government. The protocol
373 was approved by the "Comité d'éthique en expérimentation animale de l'Institut Pasteur" CETEA 89
374 (Permit number: 2012-0043 and 2016-0017) and undertaken in compliance with Institut Pasteur
375 Biosafety Committee (protocol CHSCT 12.131).

376

377 Skin invasion time-course

378 A total of 36 eight-week old BALB/c mice (Harlan, UK) were inoculated by intra-peritoneal (IP)
379 injection with 10⁴ parasites of strain STIB 247. Parasitaemia was assayed on each subsequent day
380 using phase microscopy⁴⁰. Twenty-four uninfected animals served as controls. Every three days for
381 36 days, three infected animals and two uninfected animals were culled and 2cm² skin samples
382 removed from the dorsum. Skin samples were fixed in 10% neutral buffered formalin prior to
383 histological analysis.

384

385 Natural infections using tsetse flies

386 Tsetse flies (Glossina morsitans morsitans) were maintained, infected and dissected at the Institut
387 Pasteur as described previously⁴¹. Flies were infected with AnTat1.1E AMLuc/tdTomato parasites.
388 Positive flies were selected first by screening the abdominal fluorescence (midgut infection) 15 days
389 after the infective meal, and then by a salivation test (mature salivary gland infection) after one
390 month. Single flies with salivary gland infections were used to infect the abdomen of mice
391 anaesthetised by IP injection of ketamine (Imalgene 1000 at 125mg/kg) and xylazine (Rompun 2%
392 at 12.5mg/kg) and feeding was confirmed by visual observation of the fly abdomen full of blood.
393 Control mice were either not bitten or bitten by uninfected flies. The presence and density of
394 parasites in the blood was determined daily by automated fluorescent cell counting with a Muse
395 cytometer (Merck-Millipore, detection limit 5.10² parasites/ml) or by direct examination under a
396 phase microscope with standardised one-use haemocytometers (Hycor Kova, detection limit 10⁴
397 parasites/ml), according to the manufacturer's recommendations.

398

399 Infection for xenodiagnosis

400 A total of 20 seven-week-old male C57BL/6J Rj mice (Janvier, France) were IP injected with 10⁵
401 AnTat 1.1E AMLuc/TY1/tdTomato bloodstream forms. Parasitaemia was assayed daily by
402 automated fluorescent cell counting with a Muse cytometer (Merck-Millipore, detection limit 5.10²
403 parasites/ml) according to the manufacturer's recommendations.
404 PAD1/ZFP3 relative expression

405 Three BALB/c were infected with *T.b. brucei* strain TREU927 and culled at day 11. The mice were
406 perfused and a 2cm² region of skin removed from the flank. Skin sections were lysed using a
407 Qiagen TissueLyzer LT and RNA extracted using a Qiagen RNAeasy kit following the manufacturer's
408 instructions. 100ng of RNA from each sample was reverse-transcribed using an Invitrogen
409 Superscript III RT kit. qPCR was performed on each sample using 5µl of cDNA using a protocol and
410 primers validated previously⁴² on an Agilent Technologies Stratagene Mx3005P qPCR machine.
411 The ratio of *PAD1* to *ZFP3*, and hence the proportion of cells transcribing the *PAD1* gene, was
412 estimated using the Agilent Technologies MXPro software.

413 Xenodiagnosis

414 Mice were first anaesthetised by IP injection of ketamine (Imalgene 1000 at 125 mg / kg) and
415 xylazine (Rompun 2% at 12.5 mg/kg). Batches of 10 teneral male tsetse flies (from 8 to 24 h post-
416 eclosion) were then placed in 50 ml Falcon tubes closed with a piece of net through which they
417 were allowed to feed directly on mouse skin regions of interest for 10 minutes. The selection of the
418 skin regions for fly feeding was based on mice bioluminescence profiles and parasitaemia. Unfed
419 flies were discarded and fed flies were maintained as previously described. Anaesthetised mice
420 were finally sacrificed by cervical dislocation and their skin was dissected for controlling
421 bioluminescence with an IVIS® Spectrum imager (Perkin Elmer). All the flies were dissected and
422 checked for the presence of trypanosomes either 2 or 14 days after their meal on mouse skin by
423 two entomologists blinded to group assignment and experimental procedures. Dissections were
424 performed as previously described⁴¹, entire midguts were scrutinised by fluorescence microscopy to
425 detect and count living red fluorescent parasites, and positive midguts were further treated for IFA.
426 A total of 420 flies were used in 3 independent xenodiagnosis experiments.

427

428 *In vitro* bioluminescence imaging

429 To perform the parasite density / bioluminescence intensity assay with AnTat 1.1E
430 AMLuc/TY1/tdTomato bloodstream forms, parasites were counted, centrifuged and resuspended in
431 fresh HMI11 medium at 10.10⁶ cells/ml. Then, 100µl (or 10⁶ parasites) of this suspension were
432 transferred into black clear-bottom 96-well plates and serial 2-fold dilutions were performed in
433 triplicate adjusting the final volume to 200µl of HMI11 with 300 µg/ml of beetle luciferin (Promega,
434 France). Luciferase activity was quantified after 10 minutes of incubation with a microplate reader
435 Infinite® 200 (Tecan), following the instructions of the Promega Luciferase Assay System. After
436 background removal, results were analysed as mean ±SD of three independent experiments.
437 **In vivo bioluminescence imaging**

438 Infection with bioluminescent parasites was monitored daily by detecting the bioluminescence in
439 whole animals with the IVIS® Spectrum imager (Perkin Elmer). The equipment consists of a cooled
440 charge-coupled camera mounted on a light-tight chamber with a nose cone delivery device to keep
441 the mice anaesthetised during image acquisition with 1.5% isofluorane. D-luciferin potassium salt
442 (Promega) stock solution was prepared in phosphate buffered saline (PBS) at 33.33 mg/ml, filter-
443 sterilised and stored in a -20°C freezer. To produce bioluminescence, mice were inoculated IP with
444 150 µl of D-luciferin stock solution (250mg/kg). After 10 minutes of incubation to allow substrate
445 dissemination, all mice were anaesthetised in an oxygen-rich induction chamber with 2%
446 isofluorane, and images were acquired by using automatic exposure (30 seconds to 5 minutes)
447 depending on signal intensity. Images were analysed with Living Image software version 4.3.1
448 (Perkin Elmer). Data were expressed in average radiance (p/s/cm²/sr) corresponding to the total flux
449 of bioluminescent signal according to the selected area (total body of the mouse here). The
450 background noise was removed by subtracting the bioluminescent signal of the control mouse from
451 the infected ones for each acquisition.

452 **2-photon microscopy**

453 Intravital multi-photon microscopy studies were carried out using a Zeiss LSM7 MP system
454 equipped with a tuneable titanium:sapphire solid-state two-photon excitation source (4W,
455 Chameleon Ultra II, Coherent Laser Group) coupled to an Optical Parametric Oscillator (Chameleon
456 Compact OPO; Coherent). Movies were acquired for 10 to 15 minutes with an X:Y pixel resolution
457 of 512 x 512 in 2µm Z increments producing up to 40µm stacks. 3D tracking was performed using
458 Volocity 6.1.1 (Perkin Elmer, Cambridge, UK). Values representing the mean velocity, displacement
459 and meandering index were calculated for each object. Mice were anaesthetised IP using
460 medetomidine (Domitor 0.5mg/kg) and ketamine (50mg/kg) and placed on a heated stage.
461 Following removal of hair with a depilatory cream, dorsal skin was imaged. An intravenous injection
462 of non-targeted quantum dots (Qdot705) (Life Technologies, UK) prior to imaging allowed
463 visualisation of blood vessels.

464 **Spinning-disk confocal microscopy**

465 AnTat1.1E AMLuc/tdTomato parasites were monitored in the ear of *Kdr* (*Flk1*) C57BL/6J Rj mice by
466 spinning-disk confocal microscopy as described previously⁴¹. Briefly, mice were first anaesthetised
467 by IP injection of ketamine (Imalgene 1000 at 125mg/kg) and xylazine (Rompun 2% at 12.5mg/kg).
468 Mice were wrapped in a heating blanket and placed on an aluminium platform with a central round
469 opening of 21mm in diameter. A coverslip was taped on the central hole and the mouse was
470 positioned so that the ear was lying on this oiled coverslip. Imaging was performed using an
471 UltraView ERS spinning-disk confocal system (Perkin Elmer) with a x40 oil objective (1.3 numerical
472 aperture). Movies were acquired by an EM-CCD camera (Hamamatsu) controlled by the Volocity
473
474

software (Perkin Elmer) with an exposure time of 500ms for a total of 30 to 120s. Images were
analysed using ImageJ 1.48v and its plugin Bio-formats importer (NIH).

475

Histological and immunohistochemical evaluation of the skin

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Paraformaldehyde-fixed skin samples were trimmed and processed into paraffin blocks. Sections were stained with Haematoxylin and Eosin (HE). Additional serial sections were processed for immunohistochemical staining using a polyclonal rabbit antibody raised against the invariant surface glycoprotein 65 (IGS65) (M. Carrington, Cambridge, UK) using a Dako Autostainer Link 48 (Dako, Denmark) and were subsequently counterstained with Gill's Haematoxylin.

481

Histopathological assessment of inflammation in the skin

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The extent of cutaneous inflammatory cell infiltration was assessed in haematoxylin and eosin stained sections with a semi-quantitative scoring system applied by two pathologists blinded to group assignment and experimental procedures. The extent of mixed inflammatory cell infiltration in the dermis and/or subcutis was assessed on a 0 to 3 grading scale (0 = no inflammation or only few scattered leukocytes; 1 = low numbers of inflammatory cells; 2 = moderate numbers of inflammatory cells; 3 = large numbers of inflammatory cells). Ten high-power fields were scored for each skin sample. An inflammation score calculated as the average of the scores in the 10 high-power fields was determined for each sample.

490

Semi-quantitative evaluation of the parasite burden in skin sections

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Parasite burden was assessed in skin sections stained with anti-IGS65 antibody by two pathologists blinded to group assignment and experimental procedures. Presence of parasites defined as intravascular (parasites within the lumen of dermal or subcutaneous small to medium-sized vessels) and extravascular (parasites located outside blood vessels, scattered in the connective tissue of the dermis or in the subcutis) was evaluated in 5 randomly selected high-power fields at x40 magnification with a 0 to 3 semi-quantitative grading scale (0 = no parasites detectable; 1 = low numbers of parasites (< 20); 2 = moderate numbers of parasites (20 < 50); 3 = large numbers of parasites (> 50). An average parasite burden score was calculated for each sample.

499

Immunofluorescence analysis

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Cells were treated for immunofluorescence after paraformaldehyde or methanol fixation as described previously¹⁷. Parasites were stained with one or two of the following antibodies: (i) the anti-CRD polyclonal rabbit antibody (1:300) to label the cross-reactive determinant of the glycosylphosphatidylinositol anchors of proteins, predominantly the variant surface glycoproteins⁴³, (ii) the anti-PAD1 polyclonal rabbit antibody (1:100) targeting the carboxylate-transporter Proteins Associated with Differentiation 1 (PAD1) (Keith Matthews, Edinburgh, UK)¹⁷, (iii) the anti-GPEET mouse IgG3 monoclonal antibody (1:500) targeting the *T. brucei* GPEET-rich procyclin (Acris Antibodies GmbH, San Diego, USA), (iv) the L8C4 mouse IgG1 monoclonal antibody labelling an
epitope of the PFR2 protein⁴⁴. Specific antibodies with minimal cross-reactions with mice and coupled to AlexaFluor 488, Cy3 or Cy5 (Jackson ImmunoResearch, USA) were used as secondary antibodies. DNA was stained with 4,6-diamidino-2-phenylindole (DAPI). IFA image acquisition was carried out on a Leica 4000B microscope with a x100 objective lens using a Hamamatsu ORCA-03G camera controlled by Micro-manager and images were normalised and analysed with ImageJ 1.49v (NIH).

## Histopathology of Historical Human Skin Samples

Historical human skin samples were collected from 1991 to 1995 as part of The National Onchocerciasis Task Force (NOTF)⁴⁵. Of this collection, 1,121 paraffin embedded skin samples were cut into 2.5 micron sections and stained with Giemsa (Sigma-Aldrich). Slides were screened for the presence of parasites by two pathologists independently and representative images taken at x100 magnification.
<figure><img src="image_3.png" /><figcaption>Figure 1. Parasite densities in the blood and in the extravascular tissue of the skin over a time-course</figcaption></figure>

The blood parasitaemia of *T.b. brucei* strain STIB247 (red) and the semi-quantitative score of extravascular parasites in the skin (blue) are shown over a 36-day time-course following infection in Balb/C mice. Blood parasitaemia was measured using phase microscopy using methodology outlined in ⁴⁰. Skin parasite burden is an average of five high-power fields scored by histological analysis (0 = no parasites detectable; 1 = low numbers of parasites; 2 = moderate numbers of parasites; 3 = large numbers of parasites). Standard error shown (n=3).
<figure><img src="image_4.png" /><figcaption>Day 12. Uninfected Control</figcaption></figure>

<figure><img src="image_5.png" /><figcaption>Day 24. Uninfected Control</figcaption></figure>

<figure><img src="image_6.png" /><figcaption>Day 12. Infected</figcaption></figure>

<figure><img src="image_7.png" /><figcaption>Day 24. Infected</figcaption></figure>

530

531 **Figure 2. Extravascular localisation of trypanosomes during an infection**

532 Histological sections of dorsal skin from uninfected and infected Balb/C mice stained with 533 trypanosome-specific anti-ISG65 antibody (brown), counterstained with Gill's Haematoxylin stain 534 (blue) at 12 days and 24 days post-inoculation with *T.b. brucei* strain STIB247. Parasites are visible 535 in extravascular locations of the skin including the deep dermis and subcutaneous adipose tissue 536 from day 12. The scale bar represents 20µm. 537

537
A

D6

<figure><img src="image_8.png" /></figure>

D12

<figure><img src="image_9.png" /></figure>

D20

<figure><img src="image_10.png" /></figure>

D26

<figure><img src="image_11.png" /></figure>

B

Bioluminescence intensity (p/s/cm²/sr)

<figure><img src="image_12.png" /></figure>

C

D29

<figure><img src="image_13.png" /></figure>

+

<figure><img src="image_14.png" /></figure>

<figure><img src="image_15.png" /></figure>

<figure><img src="image_16.png" /></figure>

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<caption>Figure 3. Dynamics of parasite distribution in the extravascular tissue of the skin and in the blood during a representative course of infection following natural transmission</caption>

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A total of seven mice were infected by the single infective bite of an individual *G.m. morsitans* on the belly with the *T.b. brucei* AnTat1.1E AMLuc/tdTomato strain. Panels A and C depict representative patterns. (A) Examples of bioluminescence profiles of 3 mice (+ bitten by an infected fly, - bitten by an uninfected fly and 0 not bitten) 6, 12, 20 and 26 days after the bite are shown. (B) Ventral (blue) and dorsal (green) bioluminescence (BL) intensities (in p/s/cm²/sr on the left Y-axis) and parasitaemia (in parasites/ml in red on the right Y-axis) were measured daily for 29 days and plotted as mean ±SD (n=7 mice). (C) The entire skins of mice (+) and (-) were dissected for bioluminescence imaging 29 days after the bite. For the mouse (+), Figure 3-figure supplement 1 shows the bioluminescence profile of dissected organs, Figure 3-figure supplement 2 presents the skin inflammation, and Figure 3-figure supplement 3 shows labelled parasites in skin sections.
<figure><img src="image_17.png" /><figcaption>Figure 4. Extravascular localisation of trypanosomes during an infection visualised using multi-photon microscopy (A) and spinning-disk confocal microscopy (B)</figcaption></figure>

(A) Still-image extracted from video (Video 1) of multi-photon live imaging of dorsal skin during a trypanosome infection. Intravenous non-targeted quantum dots (white) highlight blood vessels. *T.b. brucei* STIB 247 parasites transfected with mCherry to aid visualisation (red) are clearly visible and motile outside the vasculature and within the extravascular skin matrix (green). (B) Still-image extracted from (Video 3) of spinning-disk confocal live imaging of the ear of an *Kdr (Flk1) C57BL/6J Rj* mouse during a trypanosome infection. *T.b. brucei* AnTat1.1E AMLuc/tdTomato parasites expressing tdTomato (red) are moving in the extravascular region surrounding a vessel of the dermis (green).
563

<figure><img src="image_18.png" /></figure>

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<caption>Figure 5. Extravascular localisation of trypanosomes in previously unidentified human cases of trypanosomiasis</caption>

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Histological sections of skin collected from previously unidentified cases of human trypanosomiasis from the Democratic Republic of Congo, showing the presence of extravascular parasites in biopsies from three individuals (A, B and C). Skin biopsies were collected as part of a national onchocerciasis screening programme that took place in the same geographic region as an active trypanosomiasis focus. Slides were stained with Giemsa and examined under oil immersion at 100x magnification. In addition to visible slender forms (black arrows) in the extravascular tissue of the skin, a clearly identifiable stumpy transmission form with typical morphology and an unattached undulating membrane is also present in the skin of one individual (red arrow in A). The scale bar represents 5µm.
<table><caption>Table 1. Skin parasites are ingested during tsetse pool-feeding</caption><thead><tr><th>Fly batches</th><th>Parasites in blood (per ml)</th><th>Parasites in skin (per cm²)</th><th>Dissected flies</th><th>Fly infection rates (%)</th></tr></thead><tbody><tr><td>C1</td><td>0</td><td>0</td><td>32</td><td>0%</td></tr><tr><td>C</td><td>0</td><td>0</td><td>8</td><td>0%</td></tr><tr><td>A4B</td><td>&lt; 10<sup>4</sup></td><td>&lt; 10<sup>3</sup></td><td>16</td><td>0%</td></tr><tr><td>B1B</td><td>2.2x10<sup>4</sup></td><td>&lt; 10<sup>3</sup></td><td>13</td><td>0%</td></tr><tr><td>A4A</td><td>&lt; 10<sup>4</sup></td><td>6.6x10<sup>5</sup></td><td>17</td><td>35%</td></tr><tr><td>A2A</td><td>1.1x10<sup>4</sup></td><td>3.8x10<sup>6</sup></td><td>7</td><td>86%</td></tr><tr><td>B1A</td><td>2.2x10<sup>4</sup></td><td>4.6x10<sup>7</sup></td><td>16</td><td>31%</td></tr><tr><td>3B</td><td>4.4x10<sup>4</sup></td><td>2.6x10<sup>4</sup></td><td>14</td><td>36%</td></tr><tr><td>3A</td><td>4.4x10<sup>4</sup></td><td>2.6x10<sup>4</sup></td><td>16</td><td>38%</td></tr><tr><td>1B</td><td>1.8x10<sup>5</sup></td><td>8.0x10<sup>3</sup></td><td>12</td><td>67%</td></tr><tr><td>1A</td><td>1.8x10<sup>5</sup></td><td>8.0x10<sup>3</sup></td><td>14</td><td>79%</td></tr><tr><td>4B</td><td>2.2x10<sup>5</sup></td><td>1.2x10<sup>4</sup></td><td>17</td><td>53%</td></tr><tr><td>4A</td><td>2.2x10<sup>5</sup></td><td>1.2x10<sup>4</sup></td><td>18</td><td>56%</td></tr><tr><td>2B</td><td>1.6x10<sup>6</sup></td><td>8.0x10<sup>3</sup></td><td>14</td><td>36%</td></tr><tr><td>2A</td><td>1.6x10<sup>6</sup></td><td>3.2x10<sup>4</sup></td><td>18</td><td>39%</td></tr><tr><td>B4B</td><td>4.3x10<sup>6</sup></td><td>6.7x10<sup>7</sup></td><td>10</td><td>80%</td></tr><tr><td>B4A</td><td>4.3x10<sup>6</sup></td><td>6.7x10<sup>7</sup></td><td>17</td><td>100%</td></tr></tbody></table>

Mice were IP infected with *T.b. brucei* AnTat1.1E AMLuc/TY1/tdTomato and the parasitaemia and bioluminescence were monitored daily until the day of xenodiagnosis. The number of parasites in the blood was determined using a haemocytometer or a flux cytometer. The number of parasites in the skin was estimated from the measured bioluminescence intensity by using a standard curve (Table 1-source data 1 and Table 1-source data 2). Batches of teneral flies were fed on different skin regions of mice infected with differing levels of bioluminescence across the skin and with differing levels of parasitaemia (Table 1-source data 2 and Table 1-source data 3). Fly batches A4A, A2A, B1A, 3B and 3A were used to assess tsetse transmission in hosts with low numbers of blood parasites but high numbers skin parasites, while fly batches 1B, 1A, 4B, 4A, 2B, 2A, B2B and B4A were used to investigate the impact of high numbers of parasites in both the skin and blood. Flies were dissected and their midguts checked for the presence of fluorescent trypanosomes after two days to determine the proportion of infected flies (Table 1-source data 4A-B). For some of these experiments, results of an in-depth quantification of parasite stages by IFA is provided in Supplementary file 4. Stumpy forms were observed only in the blood of mice with parasitaemia values highlighted in light grey. Bioluminescence was detected in the skin of mice with values highlighted in dark grey.
595 **Figure Supplements**

596

597 **Figure 2-figure supplement 1. Skin invasion by *T.b. brucei* strain TREU927 and *T.b.*

598 **gambiense** strain PA

599 Histological sections of dorsal skin from a mouse infected with *T.b. brucei* strain TREU927 at 20x magnification and two mice infected with *T.b. gambiense* strain PA at 40x magnification 10-days post-inoculation. Trypanosome-specific anti-ISG65 antibody reveals the presence of extravascular parasites (brown) and the slides were counterstained with Gill's Haematoxylin stain (blue) to reveal host skin structure.

604

605 **Figure 3-figure supplement 1. Bioluminescence mostly originates from parasites in the skin**

606 Mouse (+) was sacrificed and dissected for bioluminescence imaging 29 days after the infective bite. Fig 3C shows the bioluminescence profile of its entire skin and dissected organs are shown here.

609

610 **Figure 3-figure supplement 2. Mild inflammation of skin tissues one month after an infection**

611 by natural transmission

612 After 29 days, the most bioluminescent skin region of mouse (+) was dissected, fixed in paraformaldehyde, embedded in paraffin and stained with HE. Multifocal inflammatory infiltrates containing neutrophils were located in the dermis and subcutaneous tissue and associated with oedema. Inflammatory foci were generally centred on blood vessels (arrows).

616

617 **Figure 3-figure supplement 3. Extravascular parasites in the skin express both VSGs and**

618 PAD1 surface markers

619 After 29 days, the most bioluminescent skin region of mouse (+) was dissected, fixed in paraformaldehyde, embedded in paraffin and treated for IFA with the anti-CRD antibody that predominately labels parasites expressing VSGs (A-B), or the anti-PAD1 antibody specific to transmission form "stumpy" cells (C-D).
623 Source Data

624

625 Table 1-source data 1. Characterisation of the AnTat 1.1E AMLuc/TY1/tdTomato sub-clone

626 (A) The *in vitro* growth of the selected AnTat1.1E AMLuc/TY1/tdTomato sub-clone (red) was similar
627 to that of the parental wild-type strain (blue). Bloodstream forms were cultured in HMI11, counted
628 daily in a Muse cytometer (Merck-Millipore) and diluted after 4 days. (B) A parasite density /
629 bioluminescence intensity analysis was performed by measuring the bioluminescence in successive
630 2-fold dilutions in 96-micro-well plates with an IVIS® Spectrum imager (Perkin Elmer). When plotted
631 as mean ±SD (n=3), parasite densities and bioluminescence intensities were correlated when the
632 bioluminescence levels were higher than 10⁴ p/s/cm²/sr, corresponding to about 10³ parasites,
633 allowing estimation of the parasite density from *in vivo* imaging over this threshold. This standard
634 curve was used to estimate the number of parasites in the skin from measured values of
635 bioluminescence. (C) This correlation was verified by quantification in a microplate reader Infinite®
636 200 (Tecan) at the very beginning of the first *in vivo* experiment as well as the end of the last one
637 (mean ±SD, n=3).
638

638

639 Table 1-source data 2. Parasite densities in extravascular tissue of the skin and in the blood
640 of mice used for differential xenodiagnosis

641 Mice were injected IP with AnTat1.1E AMLuc/TY1/tdTomato and monitored daily for
642 bioluminescence and parasitaemia. (A) Bioluminescence profile of four mice (- uninfected control
643 and (1-3) three infected mice) four days after infection. (B) The entire skins of the uninfected control
644 mouse (-) and mouse 3 were dissected for bioluminescence imaging four days after infection. (C)
645 Parasite densities in the blood and in the skin (calculated from the mean dorsal bioluminescence
646 intensity measurement and from the standard curve in Figure 4-figure supplement 3B, in
647 parasites/cm² in blue) were calculated daily over one week and plotted as mean ± SD (n=13 mice).
648

648 Table 1-source data 3. Skin parasites are sufficient to initiate a tsetse infection

649 Schematics summarising the principal results from the xenodiagnosis experiment. In a mouse with
650 no detected transmissible parasites in the blood (absence of stumpy forms by IFA and absence of
651 infection of flies fed on a non-bioluminescent region of the skin), flies can ingest transmissible
652 parasites from the bioluminescent region of the skin (left panel). When a mouse presents
653 transmissible forms in the blood, fly infection rates increase with the concomitant ingestion of
654 parasites from the skin (right panel). Values correspond to those obtained for mouse A4 and B4.
655

656

656 Table 1-source data 4. Parasite stage determination by labelling of specific surface markers

657 Parasites recovered from infected tsetse midguts (A-B) or included in bloodsmears (C) were fixed in
658 methanol for 5 seconds and stained either with the anti-GPEET antibody detecting early procyclic
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forms (red in A-B) and the L8C4 antibody labelling the flagellum PFR (green in A-B), or with the
anti-PAD1 antibody detecting intermediate and stumpy forms (green in C), respectively.

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**Figure 1-source data 1. Semi-quantitative evaluation of the parasite burden in skin sections (STIB247)**

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Every three days for 36 days of a STIB247 *T.b. brucei* infection, five mice were culled (three infected, two control) and skin sections stained with parasite-specific anti-IGS65 antibody. Parasite burden was assessed by two pathologists blinded to group assignment and experimental procedures. Presence of parasites defined as intravascular (parasites within the lumen of dermal or subcutaneous small to medium-sized vessels) and extravascular (parasites located outside blood vessels, scattered in the connective tissue of the dermis or in the subcutis) was evaluated in 5 high-power fields at x40 magnification with a 0 to 3 semi-quantitative grading scale (0 = no parasites detectable; 1 = low numbers of parasites; 2 = moderate numbers of parasites; 3 = large numbers of parasites).

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**Figure 1-source data 2. Daily parasitaemia during STIB247 infection in Balb/C mice**

The daily parasitaemia during a 36-day STIB247 *T.b. brucei* infection was estimated using phase microscopy and methodology outlined in ⁴⁰.
678 Supplementary Files

679

680 Supplementary File 1. Histopathological assessment of inflammation in the skin during

681 STIB247 infection

682 The extent of cutaneous inflammatory cell infiltration during the 36-day STIB247 experiment was 683 assessed on haematoxylin and eosin stained sections with a semi-quantitative scoring system 684 applied by two pathologists blinded to group assignment and experimental procedures. The extent 685 of mixed inflammatory cell infiltration in the dermis and/or subcutis was assessed on a 0 to 3 686 grading scale (0 = no inflammation or only few scattered leukocytes; 1 = low numbers of 687 inflammatory cells; 2 = moderate numbers of inflammatory cells; 3 = large numbers of inflammatory 688 cells). Ten high-power fields (HPFs) were scored for each skin sample. 689

690

691 Supplementary File 2. Histopathological assessment of inflammation in the skin during

TREU927 infection

692 The extent of cutaneous inflammatory cell infiltration at day 10 of infection by strain TREU927 693 experiment was assessed on haematoxylin and eosin stained sections with a semi-quantitative 694 scoring system applied by two pathologists blinded to group assignment and experimental 695 procedures. The extent of mixed inflammatory cell infiltration in the dermis and/or subcutis was 696 assessed on a 0 to 3 grading scale (0 = no inflammation or only few scattered leukocytes; 1 = low 697 numbers of inflammatory cells; 2 = moderate numbers of inflammatory cells; 3 = large numbers of 698 inflammatory cells). Ten high-power fields (HPFs) were scored for each skin sample. 699

700

701 Supplementary File 3. Expression of PAD1 relative to ZFP3

702 The relative abundance stumpy cells in the skin of three BALB/c was estimated using qPCR at day 703 704 11 post-inoculation with *T.b. brucei* strain TREU927. Mice were culled and perfused to remove 705 blood parasites and a 2cm² region of skin removed from the flank. The tissue was homogenised and 706 RNA extracted. 100ng of RNA from each sample was reverse-transcribed and qPCR performed to 707 estimate the cycle thresholds (C~T~) of the stumpy marker PAD1 and the endogenous control ZFP3. 708 709 As C~T~ is inversely proportional to amount of target cDNA in the sample and PAD1 and ZFP3 have 710 similar qPCR efficiencies, a comparison of the delta (Δ) of C~T~ between PAD1 and ZFP3 transcripts 711 reveals the relative ratio of PAD1 to ZFP3 transcripts and hence the proportion of differentiated 712 parasites transcribing the PAD1 gene. 713

710

711 Supplementary File 4. Both the respective densities and the proportions of transmissible 712 forms of parasites in the skin and in the blood govern the tsetse infection rates during pool 713 feeding

714 For some of the xenodiagnosis experiments shown in Table 1, identification and quantification of
715

parasite stages was performed by IFA on blood smears and skin sections. Stumpy forms were

716

observed only in the blood of mice with parasitaemia values highlighted in light grey.

717

Bioluminescence was detected in the skin of mice with values highlighted in dark grey. The number

718

of parasites in the skin was calculated according to the values obtained in the standard *in vitro*

719

assay (Table 1-source data 1) and is therefore probably an underestimate. Tsetse flies were

720

dissected 2 days after xenodiagnosis. Populations of intermediate and stumpy form cells were

721

assessed in blood smears and in successive skin sections stained either with the anti-CRD antibody

722

or the anti-PAD1 antibody (see Method section). Populations of early procyclic cells were assessed

723

in dissected fly midguts stained with the anti-GPEET antibody (see Method section). ND: not

724

determined.

725

726

**Video 1. Extravascular trypanosomes visualised in the skin using 2-photon microscopy**

727

Intravital multi-photon imaging of the flank skin during trypanosome infection 10 days after IP

728

inoculation. An intravenous injection of non-targeted quantum dots prior to imaging allowed

729

visualisation of blood vessels. mCherry STIB247 *T.b. brucei* parasites (red) are observed moving in

730

the extravascular region surrounding blood vessels of the dermis (white). Collagen auto-

731

fluorescence is visible as green.

732

733

**Videos 2-4. Extravascular trypanosomes visualised in the skin using spinning-disk confocal microscopy**

734

735

Spinning-disk confocal live imaging of the ear of an *Kdr (Flk1) C57BL/6J Rj* mouse during a

736

trypanosome infection after natural transmission. *T.b. brucei AnTat1.1E AMLuc/tdTomato* parasites

737

expressing tdTomato (red) are observed moving in the extravascular region surrounding blood and /

738

or lymphatic vessels of the dermis (green).

739

740

741

742