Brief Definitive Report

Antibody-mediated control of Trypanosoma vivax infection fails in

the absence of tumour necrosis factor

F. LA GRECA,1,2 C. HAYNES,1,2 B. STIJLEMANS,1,3 C. DE TREZ1,2 & S. MAGEZ1,2

1Research Unit of Cellular and Molecular Immunology, Vrije Universiteit Brussel, Brussels, Belgium, 2Department of Structural BiologyBrussels, VIB, Brussels, Belgium, 3Department of Myeloid Cell Immunology, VIB, Brussels, Belgium

SUMMARY

Trypanosoma vivax causes a wasting disease affecting live-stock breeding and agriculture in developing countries ofsub-Sahara Africa and South America. Being an extracellu-lar parasite, control of T. vivax has been proposed to bemediated by host antibodies. However, the use of a compara-tive infection model of wild-type (WT) and tumour necrosisfactor (TNF) knockout (TNF�/�) mice shows that the lat-ter is unable to control first-peak parasitaemia, despite thepresence of specific antitrypanosome antibodies. In contrast,WT mice parasitaemia peak control coincides with a com-bined early onset of TNF production and induction of spe-cific antibodies. TNF is mainly produced by liver-associatedmonocytes and neutrophils. In this study, no other correla-tion between cellular immunomodulations and peak parasita-emia control was observed, underscoring the importance ofthe role of TNF in the control of T. vivax infections.

Keywords antibody, innate immunity, parasitaemia, trypan-osomosis, tumour necrosis factor

INTRODUCTION

Salivarian trypanosomes are parasitic extracellular flagel-lated protozoa that survive in bloodstream and tissuefluids of the host. Among them, Trypanosoma congolenseand Trypanosoma vivax are the causative agents of the

animal-restricted disease known as nagana. Transmissionoccurs during the blood-feeding process of Glossina(tsetse) flies. However, T. vivax can also be spreadthrough mechanical transmission by alternative vectors,which has lead to its successful establishment outsidethe sub-Saharan tsetse belt, particularly in South Amer-ica (1).It is generally accepted that trypanosomes, being extra-

cellular parasites, have been evolutionary exposed to theselective pressure of their hosts humoral immune systemand hence have developed an evasion mechanism knownas antigenic variation that allows the parasite to escapefrom antibody (Ab)-mediated elimination (2). During thisprocess, the parasites switch their surface coat, primarilycomposed of a densely packed layer of a variant surfaceglycoprotein (VSG), presenting major variant antigenictypes (VATs) to the hosts immune system during succes-sive parasitaemia peaks (3). Despite antigenic variationbeing a common defence mechanism among the differentsalivarian trypanosome species, major differences havebeen reported between T. brucei/T. congolense and T. vivaxconcerning the VSG (4–6). To date, most insights into try-panosome immunobiology have come from experimentalstudies using mainly T. b. brucei and T. congolense, astheir rodent infectivity makes them suitable laboratoryworking models. In contrast, the poor infectivity ofT. vivax field isolates has resulted in little experimentaldata despite the great economic relevance of this species.So far, only two strains have been used for laboratoryinfection models, that is, T. vivax ILRAD700 (originallyderived from a naturally infected Zebu from Yakawada,Nigeria) (7) and the IL1392 stock that was derived fromthe Y486 strain (8). The latter has been used for the San-ger T. vivax genome project (9) and has been studied to alimited extent with respect to its course of infection in anumber of inbred mouse strains (10), its drug sensitivity(11), the hemato/parasitological parameters of infection

Correspondence: Stefan Magez, Laboratory of Cellular andMolecular Immunology, Vrije Universiteit Brussel, Pleinlaan 2,Building E8, 1050 Brussels, BelgiumandDepartment of Structural Biology Brussels, VIB, Pleinlaan 2,Building E8, 1050 Brussels, Belgium (e-mail: stemagez@vub.ac.be).Disclosures: None.Received: 17 September 2013Accepted for publication: 17 February 2014

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Parasite Immunology, 2014, 36, 271–276 DOI: 10.1111/pim.12106

(12), the impact on B-cell immunobiology (13) and thecerebral invasion of T. vivax (14).As indicated above, control of trypanosomosis is mainly

being attributed to the interplay between antigenic varia-tion and the induction of host antibodies. Earlier studiesof the T. vivax infection model pointed to host antibodiesbeing crucially involved in the control of first-peak para-sitaemia (15). However, the same authors also reported acontribution of innate immune factors to T. vivax -trypan-osomosis control in a subsequent publication (16). Experi-mental T. brucei and T. congolense infections using themouse model have provided valuable insight into thegenetic determinants of trypanosomosis susceptibility (17)and allowed the identification of key immune mediators(18, 19). In particular, the tnf gene was found within aquantitative trait locus (QTL) for trypanoresistance inlivestock nagana (18), underscoring the validity of themouse model due to the presence of syntenic QTL (20).TNF is a key mediator in T. brucei and T. congolense-associated trypanosomosis outcome, affecting both hostsurvival and parasite control (18, 21). In this study, weshow the importance of TNF in the context of T. vivaxparasitaemia control using a comparative infection modelof WT and TNF�/� mice. In the absence of TNF, micefailed to control the first parasitaemia peak and suc-cumbed rapidly after infection. We have hypothesized thatthe hypersusceptibility of the latter does not rely on adefective Ab response, but rather, that a TNF-dependentinnate response contributes in a crucial manner to earlyparasitaemia control in WT mice. We have also examinedthe kinetics and source of TNF production in liver andspleen. Due to significant changes occurring during infec-tion at the level of cell numbers of all major splenic popu-lations, we focused on the spleen due to (i) its generalinvolvement in immune responses and (ii) its role in para-site retention during the first peak of parasitaemia (14).

MATERIALS AND METHODS

Mice and parasites

All experimental procedures were performed in accordancewith the recommendations in the Use of Laboratory Ani-mals in Research, Teaching and Testing of the Interna-tional Council for Laboratory Animal Science, VUB ethicscommittees # 10-220-4 and 12-220-4. C57BL/6 and AKRfemale mice (6–8 weeks old, Janvier, Le Genest-Saint-Isle,France) as well as age-matched C57BL/6 TNF�/� (22) andlMT mice (in-house bred) were used and housed underbarrier conditions in cages containing a maximum ofseven mice, with environment enrichment (nestable bed-ding and platforms) and unrestricted access to food and

water. Infections were initiated by intraperitoneal injectionof 2 9 103 viable parasites. Tail-snip 2�5 lL blood sampleswere used for parasitaemia counting by hemocytometer/light microscope. Parasites and VSG were purified as pre-viously described (23).

Ab-titre and cytokine ELISAs

Nunc� plates (Thermo Scientific, Aalst, Belgium) coatedwith 0�1 lg T. vivax VSG in PBS/well were blocked withprotein-free blocking buffer (Thermo Scientific). Threeindependent Ab-titre ELISA assays were performed usingserum samples from three different infections. Sera wereobtained from three infected mice on day 7 p.i. as well asfrom one na€ıve (N) control of each genetic background(WT and TNF�/�), and serial dilutions were prepared todetermine the isotype-specific Ab titres, which weredetected according to the manufacturers protocol (South-ern Biotech, SBA Clonotyping System-HRP, ImTec Diag-nostics N.V., Antwerp, Belgium).Tumour necrosis factor concentrations were measured

by sandwich ELISA (Mouse TNFa ELISA Ready-SET-Go! eBioscience, Viena, Austria; sensitivity: 8 pg/mL,standard curve range: 8–1000 pg/mL) on serially dilutedserum samples obtained at 0–3–5–7–12 days p.i. followingthe manufacturers protocol (infected WT, n = 10; infectedTNF�/�, n = 3; na€ıve WT/TNF�/�, n = 2). This experi-ment was repeated two additional times using serum sam-ples from different infections.

Flow cytometry of liver-associated and splenic immunecells

One na€ıve and three infected animals of each backgroundwere euthanized (CO2 chamber) on days 0–3–5–7–13 p.i.(last time point only in WT), and na€ıve samples were pro-cessed first. Spleen and livers were harvested. The formerwere minced and cell suspensions were prepared, while thelatter were perfused with 10 mL of 100 U/mL collagenasetype III (Worthington Biochemical Corporation, GES-TIMED, Brussels, Belgium) in HBSS. After 300 incubationwith collagenase at room temperature, cell suspensionswere prepared in 20 mL of 2 mM EDTA/10% FCS/HBSSand passed through a 70-lm-nylon mesh filter, followedby 300 room temperature gradient centrifugation in 36%Percoll (GE Healthcare Life Sciences, Diegem, Belgium) at2000 rpm. The bottom layer as well as the splenic cell sus-pensions were harvested in 2% FCS/RPMI followingerythrocyte lysis (ACK lysis buffer: 0�15 M NH4Cl, 1�0 mM

KHCO3, 0�1 mM Na2-EDTA, pH 7�4).For extracellular staining, cells were blocked using an

Fc blocking antibody (CD16/CD32 Fcc III/II, BD Bio-

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F. La Greca et al. Parasite Immunology

sciences, San Jose, CA, USA). Cell viability was measuredusing 7-amino-actinomycin D (7AAD; BD Biosciences).For intracellular cytokine staining, cells were incubatedwith GolgiStop (1 lL/mL: BD Biosciences) for 5 h andsubsequently processed with the Fix/Perm staining kitaccording to the manufacturers guidelines (BD Bioscienc-es). Cells were Fc-blocked, and LPS (1 lg/mL; InvivoGen,Toulouse, France) was added when indicated. Antibodieswere added to 100 lL aliquots of 106 Fc-blocked leucocytes.Anti-CD1d-PE, anit-CD21-FITC, anti-B220-APCCy7, anti-IgM-PE, anti-B220-FITC, anti-CD19-APC-Cy7,anti-CD4-PE, anti-CD8-APC, anti-NK1�1-APCCy7, anti-Ly6G-PE,anti-IgG1-PE, anti-IgG2a-FITC, anti-CD138-APC, anti-IgG3-FITC, anti-CD11c-PerCP5�5, anti-CD5-APC, anti-TNF-PE, anti-Ly6C-APC, anti-Ly6G-APCCy7 and anti-7AAD-PerCP5�5 were purchased from BD Biosciences.Anti-AA4�1-APC, anti-CD23-PeCy7, anti-CD43-PE, anti-IgM-PeCy7, anti-CD5-FITC, anti-CD3-PeCy7, anti-CD11c-APCCy7, anti-CD11b-PerCp5�5, anti-F4/80 AlexaFluor 780, anti-CD3-FITC and anti-CD11b-PeCy7 werepurchased from eBiosciences. Anti-MHCII-Alexa Fluor 488and anti-MHCII-PerCP5�5 were purchased from Biolegend.Graphic representations and statistical data analysis

(unpaired t-test) were performed using GraphPad Prismsoftware. FACS analyses were performed using a FACSCanto II flow cytometer (BD Biosciences), and data wereprocessed using FLOWJO software (Tree Star Inc., Ash-land, OR, USA).

RESULTS

In C57BL/6 mice, the infection pattern of T. vivax IL-RAD700 shows a first parasitaemia peak between days 7–9 p.i., reaching a height of 4 9 108 parasites/mL blood(Table 1). A number of parasitaemia waves follow until

the animals eventually succumb to infection with a mediansurvival of 27 days. In accordance with earlier reports(15), host antibodies appear crucial for first-peak parasita-emia control, as C57BL/6 B-cell-deficient (lMT) mice rap-idly succumb to T. vivax infection due to uncontrolledparasitaemia on the first peak of parasitaemia (day 10).Interestingly, the absence of complement C5 factor doesnot prevent T. vivax control, as the median survival of C5-deficient AKR mice does not statistically differ from thatof immune competent WT mice (Table 1). Table 1 showsthat TNF is a crucial mediator in survival during T. vivaxinfections as TNF�/� mice succumb during the first peakof parasitaemia with a median survival of 10 days.To exclude the possibility of a defective Ab response in

trypanosusceptible TNF�/� mice, the level of infection-elicited immunoglobulins was characterized by Ab-titreELISA and flow cytometry using serum and spleen sam-ples obtained in early time points of infection. In ELISA,no difference was observed in either IgM or IgG2b anti-VSG titres when serum samples of infected WT andTNF�/� mice were compared (Fig. 1a,b). Other isotypeIgG (IgG1, IgG2a, IgG3) titres remained negativethroughout infection in both mouse strains. These obser-vations coincided with the finding that a 28-fold increasein total B220+CD138+ plasma B cells by day 7 p.i.occurred both in WT and TNF�/� mice (Fig. 1c). Stainingof specific Ig isotypes (IgM, IgG1, IgG2a and IgG3)revealed no statistical differences in surface immunoglobu-lin expression between WT and TNF�/� on day 7 p.i.(data not shown). Considering the importance of TNF inthe control of first-peak parasitaemia, serum levels of thecytokine were measured and found to increase from8�8 � 11�9 pg/mL in noninfected mice to 187�8 �143�9 pg/mL on day 7 post-infection (10�5 � 14�7 pg/mLwere measured in infected TNF�/� mice). Next, the originof the cytokine in T. vivax-infected WT mice wasinvestigated by intracellular cytokine staining at peakparasitaemia stages. All the cells recovered after gradi-ent separation of liver-associated cells were assayedwith multiple staining combinations allowing for theidentification of B and T lymphocytes, monocytes andneutrophils. CD11b+Ly6C+Ly6Gint monocytes andCD11b+Ly6CintLy6G+ neutrophils were found to be themain TNF-producing cells (data not shown). The sameanalysis was repeated on purified spleen cells, where noTNF+ cells were detected except when ex vivo LPS-re-stim-ulation was applied. Also here the bulk of TNF+ cells hada neutrophil or monocyte phenotype (data not shown).During the analysis of cellular TNF production outlined

above, it was observed that T. vivax infection resulted in aTNF-independent destructive effect on the spleen, as grosschanges in cellular composition were observed during both

Table 1 T. vivax ILRAD700 infection parameters in differentmouse strains

Mousestrain

First parasitaemiapeak

N° parasitaemiapeaks

Mediansurvival,daysDay

Height, 9106

parasites/mL � SD

C57BI/6(WT)

9 402 � 264 3 27

lMT 10 450 � 158 1 14�5AKR 8 719 � 477 2–3 20TNF�/� 9 339 � 219 1 10

WT, Wild type (n = 17); lMT, B cell deficient (n = 10); AKR,complement protein 5 (C5) deficient (n = 10); and TNF�/�,tumour necrosis factor deficient (n = 8).

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WT and TNF�/� infections. As splenomegaly is one of theearliest reported clinical features of animal trypanosomosis(a six-fold increase in cellularity was observed by day 7p.i.), population kinetics of splenic immune cells were sub-sequently assessed (i.e. B and T lymphocytes, monocytesand neutrophils). Immature and mature (B220+CD1d+

marginal zone and B220+CD1d� follicular; MZB and FoB,respectively) B lymphocytes progressively increased in cellnumbers during the first week of infection in both WT andTNF�/� mice. By day 7 p.i., when TNF�/� mice sufferedfrom a loss of parasitaemia control, WT mice had signifi-cantly more cells at the level of the immature B cell andmarginal zone B cell populations (Fig. 1d). In contrast,FoB total cell numbers increased until day 7 p.i. both inWT and TNF�/� mice without a statistically significantimpact of the absence or presence of TNF. After this pointand coinciding with the clearance of the parasitaemia peakin WT mice, the FoB and MZB cell subsets undergo amarked depletion. Despite this severe reduction in FoB andthe virtual elimination of MZB cell numbers by day 13 p.i.

(Fig. 1e), WT mice survived up to 2½ weeks past this point,suggesting that the loss of these cells does not directlyimply the loss of parasitaemia control. All T-cell subsetssubjected to study were also found to undergo expansionupon T. vivax challenge. CD3+CD4+ T cells showed thegreatest increase in cell numbers without any statistical dif-ferences between WT and TNF�/� mice (Fig. 1d). A signifi-cant difference was observed on day 7 p.i. regardingCD3+CD8+ lymphocytes, with infected TNF�/� miceshowing much higher numbers on the day of peak parasita-emia. CD5+NK1�1+ natural killer T (NKT) cells increasedsimilarly in the two genetic backgrounds. While the neutro-phil (CD11b+Ly6CintLy6G+) population increased in num-bers as the infection progressed in both mouse strains used,a significant difference between WT and TNF�/� mice wasobserved on day 7 p.i. (Fig. 1d). Monocytes (CD11b+Ly6-C+Ly6Gint) showed an expansion very early after infectionin both mouse strains, although we found again a signifi-cantly higher amount of cells in spleens of TNF�/� mice ascompared to WT mice by day 7 p.i.

Dilution

1/5 1/151/45

1/1351/405

1/1215

1/3645

1/10935

1/32805

1/98415Dilution

1/5 1/151/45

1/1351/405

1/1215

1/3645

1/10935

1/32805

1/98415

0·0

OD

val

ues

0·20·40·60·81·01·21·4

WT N

TNF–/– Ninfected WT

infected TNF–/–

0·0

0·5

1·0

1·5

2·0

OD

val

ues WT N

TNF–/– Ninfected WT

infected TNF–/–

IgM-isotype serum Abs Plasma B cells

WTTNF–/–

naive 3 5 7 13

Time (days p.i.)

0

5

10

15

Cel

l num

ber x

106

Cell types

NKTCD8 T

CD4 TMNMZB

FoBimB

02

610

Cel

l num

ber x

107

4

15

20 WTTNF–/–

Day 7 p.i.

Cell types

NKTCD8 T

CD4 TMNMZB

FoBimB

02

613

Cel

l num

ber x

106

4

2335

WT

Day 13 p.i.

818

5575

IgG2b-isotype serum Abs(a) (b)

(d) (e)

(c)

Figure 1 Trypanosoma vivax VSG-specific serum IgM (a) and IgG2b (b) titres in na€ıve and infected WT and TNF�/� mice on day 7 p.i..Representative experiment of three independent experiments. Values of infected samples are expressed as averages of groups of threeanimals � SD. The following data obtained from flow cytometric analyses of three independent infections are represented as mean of threeT. vivax-infected samples of WT and TNF�/� mice per group � SD, as well as the respective na€ıve (N) controls. (c) Representative plasmaB (B220+ CD138+) cell kinetics during T. vivax infections in WT and TNF�/� mice. (d) Representative values for splenic immune cellpopulations on day 7 p.i. in WT and TNF�/� mice: immature B (imB), follicular B (FoB) and marginal zone B (MZB) lymphocytes,neutrophils (N), monocytes (M), CD4 T, CD8 T and NKT lymphocytes. The dashed lines represent the respective average values ofnoninfected samples (no statistical differences were found between the na€ıve values for each cell type). (e) Representative values for splenicimmune cell populations on day 13 p.i. in WT mice. The dashed lines represent the respective average values of noninfected samples.

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DISCUSSION

In this study, we show for the first time that proper para-sitaemia control of early T. vivax infections is intrinsicallyrelated to an early type I immune response of the host,combined with an infection-induced specific antibodyresponse. In agreement with previous studies, our resultsshow that lMT mice lacking mature B cells are unable tocontrol the first wave of parasitaemia and hence succumbearly on during infection. Taken that C5-deficient AKRmice had no problem in controlling successive parasita-emia waves, it appears that complement-mediated lysis isnot crucial in parasitaemia control. TNF�/� mice werefound to be hypersusceptible, developing a very severe dis-ease with remarkable loss of locomotor activity and anae-mia (data not shown), as reported for T. congolenseinfections (18). We have further extended such findings byassessing B-cell function in both mouse strains. However,no differences in total plasma cell numbers were observedbetween WT and TNF�/� mice. In addition, no differencesin specific anti-VSG antibody concentrations weredetected, suggesting that the TNF�/� hypersusceptibilitywas not related to an abnormal or defective B-cell func-tion as compared to WT mice. Importantly, TNF�/� micehave been reported to exhibit a defect in germinal centre(GC) formation (22). However, due to the extensivesplenomegaly and the disruption of splenic architecture,T. brucei-infected WT mice have also been shown to suffera lack of GC formation, and hence, both WT and TNF�/�

mice resemble each other in this regard (25). Furthermore,the occurrence of similar specific IgG titres during infec-tion in both WT and TNF�/� mice suggests that the inca-pability to control peak parasitaemia in the latter is notsimply attributed to a lack of GC formation or defectiveantibody affinity maturation in the TNF�/� mice only.More likely, and similar to parasitaemia control in the

T. congolense model, antibody-mediated phagocytosis byactivated liver macrophages is a more efficient way ofparasite elimination (24). Important to mention is that inorder to efficiently kill T. congolense parasites, a

combination of antibodies, TNF and nitric oxide isrequired (19). Hence, this study subsequently addressedthe role of TNF in the control of experimental T. vivaxinfections. Liver-associated monocytes and neutrophilswere identified as the main TNF-producing cells, both inthe liver and (to a lesser extent) spleen of infected animals.With respect to the early death of TNF�/� mice, other fac-tors besides uncontrolled parasitaemia growth might beinvolved. Indeed, TNF�/� mice showed significantlyincreased neutrophils, monocytes and CD8+ T cells intheir spleen prior to the endpoint of infection. Hence, itcould be that these cells mediate pathological parametersthat were not discovered in this study. However, wehypothesize that it is most likely the variant-specific IgGs,combined with inflammatory activators such as TNF, thatare responsible for the bulk of the parasitaemia controlduring peak infection, allowing prolonged survival of fullyimmunocompetent hosts. In this case, TNF possiblyaffects the parasite fitness in a similar way as observed inthe TNF/T. congolense relation (26). This would subse-quently facilitate antibody/parasite opsonization and mac-rophage-driven Fc-mediated phagocytosis (24).

ACKNOWLEDGEMENTS

FLG and CH performed the research. FLG, CDT andSM designed the research study. BS contributed essentialreagents or tools. FLG analysed the data. FLG and SMwrote the paper.

FUNDING

This work was supported by the FWO grant G.0028.10Nand the Geconcerteerde Onderzoeks Actie GOA65/VUB.

CONFLICTS OF INTEREST

The authors declare no conflict of interests.

REFERENCES

1 Jones TW & Davila AM. Trypanosoma vivax– out of Africa. Trends Parasitol 2001; 17:99–101.

2 Vickerman K. Antigenic variation in trypan-osomes. Nature 1978; 273: 613–617.

3 Barry JD. Antigenic variation during Try-panosoma vivax infections of differenthost species. Parasitology 1986; 92(Pt 1):51–65.

4 Vickerman K & Preston TM. Comparativecell biology of the kinetoplastid flagellates.

In Lumsden WHR & Evans DA (eds):Biology of the Kinetoplastida. London,Academic Press, 1976: Vol. 1:35–130.

5 Gardiner PR, Nene V, Barry MM, ThatthiR, Burleigh B & Clarke MW. Characteriza-tion of a small variable surface glycoproteinfrom Trypanosoma vivax. Mol BiochemParasitol 1996; 82: 1–11.

6 Jackson AP, Berry A, Aslett M, et al. Anti-genic diversity is generated by distinct evolu-tionary mechanisms in African trypanosome

species. Proc Natl Acad Sci U S A 2012;109: 3416–3421.

7 Geysen D, Delespaux V & Geerts S. PCR-RFLP using Ssu-rDNA amplification as aneasy method for species-specific diagnosis ofTrypanosoma species in cattle. Vet Parasitol2003; 110: 171–180.

8 Leeflang P, Buys J & Blotkamp C. Studieson Trypanosoma vivax: infectivity and serialmaintenance of natural bovine isolates inmice. Int J Parasitol 1976; 6: 413–417.

© 2014 John Wiley & Sons Ltd, Parasite Immunology, 36, 271–276 275

Volume 36, Number 6, June 2014 Antibody-mediated control of Trypanosoma vivax

9 http://www.sanger.ac.uk/resources/down-loads/protozoa/trypanosoma-vivax.html.

10 De Gee AL, Shah SD & Doyle JJ. Trypano-soma vivax: courses of infection with threestabilates in inbred mouse strains. ExpParasitol 1982; 54: 33–39.

11 Kaminsky R, Chuma F, Zweygarth E, KitosiDS & Moloo SK. The effect of isometamidi-um chloride on insect forms of drug-sensitiveand drug-resistant stocks of Trypanosomavivax: studies in vitro and in tsetse flies.Parasitol Res 1991; 77: 13–17.

12 Chamond N, Cosson A, Blom-Potar MC,et al. Trypanosoma vivax infections: pushingahead with mouse models for the study ofNagana. I. Parasitological, hematologicaland pathological parameters. PLoS NeglTrop Dis 2010; 4: e792.

13 Blom-Potar MC, Chamond N, Cosson A,et al. Trypanosoma vivax infections: push-ing ahead with mouse models for the studyof Nagana. II. Immunobiological dysfunc-tions. PLoS Negl Trop Dis 2010; 4: e793.

14 DArchivio S, Cosson A, Medina M, Lang T,Minoprio P & Goyard S. Non-invasive invivo study of the Trypanosoma vivax infec-tious process consolidates the brain commit-ment in late infections. PLoS Negl Trop Dis2013; 7: e1976.

15 Mahan SM, Hendershot L & Black SJ. Con-trol of trypanodestructive antibody responsesand parasitemia in mice infected with Try-

panosoma (Duttonella) vivax. Infect Immun1986; 54: 213–221.

16 Mahan SM & Black SJ. Differentiation, mul-tiplication and control of bloodstream formTrypanosoma (Duttonella) vivax in mice. JProtozool 1989; 36: 424–428.

17 Antoine-Moussiaux N, Magez S & Des-mecht D. Contributions of experimentalmouse models to the understanding of Afri-can trypanosomiasis. Trends Parasitol 2008;24: 411–418.

18 Iraqi F, Sekikawa K, Rowlands J & Teale A.Susceptibility of tumour necrosis factor-alpha genetically deficient mice to Trypano-soma congolense infection. Parasite Immunol2001; 23: 445–451.

19 Magez S, Radwanska M, Drennan M, et al.Interferon-gamma and nitric oxide in combi-nation with antibodies are key protectivehost immune factors during Trypanosomacongolense Tc13 infections. J Infect Dis 2006;193: 1575–1583.

20 Hanotte O, Ronin Y, Agaba M, et al. Map-ping of quantitative trait loci controlling try-panotolerance in a cross of tolerant WestAfrican NDama and susceptible East Afri-can Boran cattle. Proc Natl Acad Sci U S A2003; 100: 7443–7448.

21 Magez S, Lucas R, Darji A, Songa EB, Ha-mers R & De Baetselier P. Murine tumournecrosis factor plays a protective role duringthe initial phase of the experimental infec-

tion with Trypanosoma brucei brucei. Para-site Immunol 1993; 15: 635–641.

22 Marino MW, Dunn A, Grail D, et al. Char-acterization of tumor necrosis factor-defi-cient mice. Proc Natl Acad Sci U S A 1997;94: 8093–8098.

23 Cross GA. Identification, purification andproperties of clone-specific glycoprotein anti-gens constituting the surface coat of Trypan-osoma brucei. Parasitology 1975; 71:393–417.

24 Pan W, Ogunremi O, Wei G, Shi M & TabelH. CR3 (CD11b/CD18) is the major macro-phage receptor for IgM antibody-mediatedphagocytosis of African trypanosomes:diverse effect on subsequent synthesis oftumor necrosis factor alpha and nitric oxide.Microbes Infect 2006; 8: 1209–1218.

25 Radwanska M, Guirnalda P, De Trez C,Ryffel B, Black S & Magez S. Trypanosomi-asis-induced B cell apoptosis results in lossof protective anti-parasite antibodyresponses and abolishment of vaccine-induced memory responses. PLoS Pathog2008; 4: e1000078.

26 Kaushik RS, Uzonna JE, Zhang Y, GordonJR & Tabel H. Innate resistance to experi-mental African trypanosomiasis: differencesin cytokine (TNF-alpha, IL-6, IL-10 and IL-12) production by bone marrow-derivedmacrophages from resistant and susceptiblemice. Cytokine 2000; 12: 1024–1034.

276 © 2014 John Wiley & Sons Ltd, Parasite Immunology, 36, 271–276

F. La Greca et al. Parasite Immunology