Bovine trypanotolerance: A natural ability to prevent severe anaemia and haemophagocytic syndrome?

International Journal for Parasitology 36 (2006) 521–528 www.elsevier.com/locate/ijpara

Invited review

Bovine trypanotolerance: A natural ability to prevent severe anaemia and haemophagocytic syndrome? J. Naessens * International Livestock Research Institute, P.O. Box 30709, 00100 Nairobi, Kenya Received 10 November 2005; received in revised form 8 February 2006; accepted 15 February 2006

Abstract Trypanotolerance is the capacity of certain West-African, taurine breeds of cattle to remain productive and gain weight after trypanosome infection. Laboratory studies, comparing Trypanosoma congolense infections in trypanotolerant N’Dama cattle (Bos taurus) and in more susceptible Boran cattle (Bos indicus), confirmed the field observations. Experiments using haemopoietic chimeric twins, composed of a tolerant and a susceptible co-twin, and T cell depletion studies suggested that trypanotolerance is composed of two independent traits. The first is a better capacity to control parasitaemia and is not mediated by haemopoietic cells, T lymphocytes or antibodies. The second is a better capacity to limit anaemia development and is mediated by haemopoietic cells, but not by T lymphocytes or antibodies. Weight gain was linked to the latter mechanism, implying that anaemia control is more important for survival and productivity than parasite control. Anemia is a marker for a more complex pathology which resembles human haemophagocytic syndrome: hepatosplenomegaly, pancytopenia and a large number of hyperactivated phagocytosing macrophages in bone marrow, liver and other tissues. Thus, mortality and morbidity in trypanosome-infected cattle are primarily due to self-inflicted damage by disproportionate immune and/or innate responses. These features of bovine trypanotolerance differ greatly from those in murine models. In mice, resistance is a matter of trypanosome control dependent on acquired immunity. However, a model of anaemia development can be established using C57BL/6J mice. As in cattle, the induction of anaemia was independent of T cells but its development differed with different trypanosome strains. Identification of the molecular pathways that lead to anaemia and haemophagocytosis should allow us to design new strategies to control disease. q 2006 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved. Keywords: Trypanosomiasis; Trypanotolerance; Anemia; Haemophagocytic syndrome; Cattle; Murine models; Tumor necrosis factor alpha

1. African trypanosomes While other pathogens evade innate and adaptive responses in the plasma by hiding in a host cell, African trypanosomes are unique for being able to multiply and survive in the blood of their mammalian host. Trypanosomes elude antibody attack by sporadically varying their surface glycoprotein, forcing the host to mount a new cycle of antibody production each time a new variant appears. In this way, the parasite manages to survive and increase its chances of transmission by tsetse or biting flies. Unfortunately for the host, the disease often leads to a fatal outcome. Not all mammalian hosts are equally susceptible. For example, Trypanosoma brucei brucei is known to infect cattle * Tel.: C254 20 422 3000; fax: C254 20 422 3001. E-mail address: [email protected]

and mice, but not humans. A trypanolytic factor in human serum, apoL-I in high density lipoprotein (Vanhamme and Pays, 2004), is lethal for T. b. brucei, but not for the related parasite Trypanosoma brucei rhodesiense which has adapted to a life in the blood of its human host. Further, not all trypanosome strains living in the host’s blood are invariably lethal, and the disease severity is strain dependent. In Europe and North America, the parasite Trypanosoma theileri occurs in a high percentage of otherwise healthy cattle and appears in blood cell cultures, but as it does not cause pathology (Verloo et al., 2000) is not the object of control. There exists a variety of disease patterns associated with different host–trypanosome combinations. Patterns of susceptibility to infection and disease between trypanosome strains and different mammalian hosts may help us identify mechanisms that lead to higher resistance and potentially allow the design of new control measures.

0020-7519/$30.00 q 2006 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijpara.2006.02.012

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2. Bovine trypanosomosis African trypanosomosis in livestock is a serious hindrance to development and reduction of poverty. Estimates of the cost to consumers and producers on the African continent reach US$ 1 billion (Kristjanson et al., 1999). The main parasites that cause disease in livestock are tsetse-transmitted Trypanosoma congolense and Trypanosoma vivax and to a minor extent T. brucei. Their distribution is restricted to Africa, although T. vivax has crossed the Atlantic and spreads in South America via mechanical transmission by biting flies. Trypanosoma evansi is also transmitted by biting flies and infects a wide range of livestock, including camels and buffalo in parts of Asia. Cattle are also an epidemiologically important reservoir for the human-infective parasite T. b. rhodesiense (Hide et al., 1996; Welburn et al., 2001; Njiru et al., 2004). During the bite of an infected tsetse fly, metacyclic trypanosome forms are deposited in the skin of the mammalian host. An immune reaction to the metacyclic parasites causes a huge swelling in the skin, known as a chancre, and triggers the enlargement of the local draining lymph node. Metacyclics differentiate into bloodstream forms, migrate to the blood and cause a systemic infection. The most consistent clinical features in livestock are intermittent fever and anaemia. There is a general leukopenia, enlarged spleen and liver, and loss of weight. Chronically infected animals lose appetite, become lethargic and emaciated, and die usually of congestive heart failure. 3. Cerebral infections in cattle Both T. congolense and T. vivax are intravascular parasites, while the T. brucei ssp. and T. evansi can leave the blood vessels and invade solid tissues (Losos and Ikede, 1972). Trypanosoma congolense show a preference for microvascular sites, where they will occur in higher densities and may even bind to endothelial cells (Banks, 1978). They contribute to pathology by provoking dilation of the microvasculature, compromising capillary circulation and impairing nutrient and metabolite exchange. Because T. congolense and T. vivax do not leave the circulation, cerebral infection is not a major clinical feature in cattle infections, but it has been reported with T. brucei. About half of all cattle infected with T. b. rhodesiense developed fatal CNS disease, which is comparable with that found in man (Wellde et al., 1989). Further T. b. brucei has been reported to cause CNS abnormalities and can be found in CSF (Losos and Ikede, 1972; Morrison et al., 1983). Although figures are not available, the frequency of CNS involvement seems to be lower for T. b. brucei than for T. b. rhodesiense, and may depend on the particular strain. No cerebral infections have been observed with monospecific infections with T. congolense or T. vivax in cattle (Losos and Ikede, 1972; Masake et al., 1984) but a high frequency of CNS involvement was observed in concurrent infections (Masake et al., 1984). These authors suggested that T. congolense, because of its potential to bind microvascular walls (Banks, 1978), may partially damage the endothelial barrier either

mechanically or through inflammatory responses, allowing T. brucei to cross into the CNS. Most isolates from the CSF of multiply-infected cattle were T. brucei, but in one case T. congolense was recovered. The isolation of T. congolense from brain tissue of a multiply infected cow, was probably the result of a similar mixed infection (Haase et al., 1981). T. vivax is able to cross the blood barrier in goats and has also been found in the eye of infected goats and cattle, causing corneal cloudiness (Ilemobade and Schilhorn van Veen, 1974; Whitelaw et al., 1988). More recently, clear evidence for cerebral infections in cattle has been observed in an outbreak of surra, caused by T. evansi (Tuntasuvan et al., 1997). Even T. theileri, which is considered a non-pathogenic parasite, was reported in the CSF of a cow with encephalitis (Braun et al., 2002). The evidence available so far suggests that T. brucei strains, and in particular the subspecies T. b. rhodesiense, constituted the major cerebral infections in cattle. The presence of trypanosomes in immunoprotected tissues such as brain and eyes presents a problem for therapy since they are protected from drugs that do not cross the blood–brain barrier (Jennings et al., 1979; Whitelaw et al., 1988), and potentially develop reactive encephalitis (Jennings et al., 1993). The relative frequencies of cerebral infections in tolerant and susceptible cattle has not been investigated. 4. Trypanotolerance Cattle of taurine origin were first introduced in Africa around 6000 BC. From their origin of domestication in the Near East, taurine cattle spread through Egypt and the North African coast and expanded westward until they encountered the tsetse belt, which prevented further migration. Millennia of selection in tsetse-infested areas allowed some of these cattle breeds to develop a certain degree of ‘reduced susceptibility’ to trypanosomosis. It is possible that genes conferring this tolerance entered the population through cross breeding with an ancient population of African cattle, whose existence could be traced by DNA analysis in breeds from the continent (Hanotte et al., 2002). The term trypanotolerance was defined as the trait that confers the capacity to survive and remain productive after trypanosome infection (Murray et al., 1982). Despite the rapid and wide distribution of zebu cattle (Bos indicus) over the African continent since their first introduction around 700 AD, taurine breeds predominate in areas of the tsetse belt. Early studies described that certain taurine breeds in West Africa could cope better in tsetse-infested areas (reviewed in Murray et al., 1982; Murray and Dexter, 1988). Under natural conditions of tsetse challenge, trypanotolerant cattle had lower mortality, lower trypanosome levels, less severe anaemia, superior weight gain and better reproductive performance than more susceptible indicine breeds. The breeds were tolerant to both T. vivax and T. congolense, with a higher degree of resistance to T. vivax (Murray et al., 1981, 1982; Mattioli et al., 1999). Field studies suggested that control of anaemia, but not parasitaemia, had a major effect on overall productivity (Trail et al., 1991a) and had a significant degree of heritability (Trail et al., 1991b).

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Comparison of infections with T. congolense in laboratory conditions between trypanotolerant N’Dama calves (Bos taurus) with more susceptible Boran calves (B. indicus), confirmed observations in the field that N’Dama remained productive, continued to gain weight at the same rate as the uninfected controls, and females continued their oestrous cycle compared with the infected Boran cattle (Paling et al., 1991). Furthermore, N’Dama were better at controlling parasitaemia and the associated anaemia. No correlation between the degree of anaemia and parasitaemia was found in individual N’Dama, suggesting that the two processes were not linked to each other, despite earlier views to the contrary (Dargie et al., 1979). Previous exposure with heterologous trypanosome strains did not affect the course of infection, although it did reduce the severity of anaemia in N’Dama, but not Boran cattle (Williams et al., 1991). Several efforts have been made to identify genes that contribute to trypanotolerance and this understanding could help to discover the processes that confer resistance. In one type of approach, differential gene expression between tolerant and susceptible cattle allows the detection of genes whose up- or down-regulation is correlated with a tolerant phenotype. Using serial analysis of gene expression (SAGE), 187 genes that changed their expression were identified in N’Dama leukocytes after infection by T. congolense (Berthier et al., 2003; Maillard et al., 2004, 2005). Unfortunately, this technology has not been used to compare tolerant and susceptible animals. Gene expression in blood leukocytes was compared between N’Dama and Boran calves using gene array technology at different time points after infection (Hill et al., 2005). Thirty differentially expressed genes were described, including three members of the protein kinase C family. Confirmation of these observations is now needed, including data on purified cell populations and other tissues. A different, genomic approach aimed at identifying loci correlated with a trypanotolerant phenotype uses genotypic analysis of F2 calves derived from N’Dama and Boran grandparents (Hanotte et al., 2003). This approach had been successfully undertaken in a murine trypanotolerance model previously (Kemp et al., 1997; Kemp and Teale, 1998). In cattle 16 phenotypes, including anaemia, body weight and parasitaemia were used in the analysis of the bovine data and 18 quantitative trait loci (QTL) were identified (Hanotte et al., 2003). Trypanotolerance is thus highly polygenic, with each gene or locus explaining no more than 10–12% of the phenotypic variance of the trait. Most QTLs were linked to control of anaemia and only a few to parasitaemia, suggesting that anaemia control is complex and of major significance. An interesting observation was that five resistance alleles in the QTL originated from the susceptible Boran grandparent, suggesting that trypanotolerance as observed in N’Dama, could be improved upon by further crossbreeding (Hanotte et al., 2003). Genes located in a QTL and differentially expressed in tissues from trypanotolerant and susceptible animals should be particularly informative and are potential ‘resistance genes’.

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5. The role of haemopoietic cells in trypanotolerance Once trypanotolerance was able to be measured under laboratory conditions, a question that arose was whether it was due to an improved acquired response, to a better acute innate response or to an innate disposition that confers a degree of resistance. Studies in mice infected with T. brucei parasites have repeatedly shown that antibodies, particularly those reacting with surface epitopes, were a major player in the control of parasitaemia and survival (Campbell et al., 1977; Reinitz and Mansfield, 1990). In contrast, mice with a defect in T cell maturation or depleted of T cells were not more susceptible to African trypanosomiasis and continued to produce antibodies, again correlating antibodies with trypanotolerance (Campbell et al., 1978; Rottenberg et al., 1993). Consequently, a lot of research effort has gone into finding differences in immune responses between tolerant and susceptible mouse strains. In contrast, studies on the resistance of African ruminant wildlife suggested a role for better innate responses. Control of parasitaemia in African buffalo was correlated with a decline in catalase activity and an increase in peroxide in the plasma capable of reducing trypanosome numbers (Wang et al., 2002). Human resistance to T. b. brucei is neither the result of a better acquired response nor of a better innate response, but is carried by apoL-I in human serum that is lytic to this trypanosome strain, but not to T. b. rhodesiense (Vanhamme and Pays, 2004). Bovine trypanotolerance might include any combination of such mechanisms. An elegant solution to check the involvement of cells of the haemopoietic system in bovine trypanotolerance, was to make use of the fact that cattle twins are haemopoietic chimeras. Twins composed of a male and female both carry male and female haemopoietic cells in the blood. This is because blood vessels in the placentas of bovine twin fetuses are known to fuse, allowing haemopoietic precursor cells to migrate into both siblings and populate their bone marrow. The proportion of haemopoietic cells from each co-twin is identical in both offspring, but is not necessarily 50%. Boran/N’Dama chimeric twins were created by putting a Boran and an N’Dama embryo in the same recipient mother (Naessens et al., 2003a). Analysis of the composition of T lymphocytes confirmed that all twins were chimeras, but that each pair of twins contained proportionately more cells of Boran origin (ranging from almost 100 to 70%) than N’Dama origin. We assumed that migration of haemopoietic precursor cells to the bone marrow occurred earlier in Boran foetuses, thus leading to a higher ratio of Boran haemopoietic cells in both twin calves. As expected, the ratio of Boran vs N’Dama cells was identical in each set of twin calves. Overall, the Boran chimeras were like normal Boran calves, except that they had some haemopoietic cells of N’Dama origin, while the N’Dama chimeras were like N’Dama calves, but had acquired the majority of their haemopoietic system from the susceptible Boran background. The responses of the twin chimeras were compared after infection with T. congolense with those of Boran and N’Dama singletons (Table 1). The Boran chimeras had similar levels of

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Table 1 Capacity (marked with a C sign) to gain weight and to better control parasitaemia and anaemia after infection with Trypanosoma congolense, of Boran and N’Dama singleton and haemopoietic chimeric calves Calves

Origin of haemopoietic tissue

Origin of all other tissues

Control of parasitaemia

Control of anaemia

Productivity (weight gain)

Boran N’Dama Chimera Boran Chimera N’Dama

Boran N’Dama Mainly Borana Mainly Borana

Boran N’Dama Boran N’Dama

K C K C

K C K K

K C K K

The capacity to control anaemia and to gain weight are associated with an N’Dama origin of haemopoietic tissues, while the capacity to control parasitaemia is associated with a N’Dama origin of non-haemopoietic tissues. a 70–98% of haemopoietic cells in chimeras were of Boran origin (Naessens et al., 2003a).

parasitaemia and anaemia as the Boran singletons, suggesting that the few haemopoietic cells that they had ‘inherited’ from their N’Dama co-twins were not capable of bestowing trypanotolerant traits on them. The N’Dama chimeras were able to control parasitaemia as well as the N’Dama singleton calves, but they developed severe anaemia, to the same extent as the susceptible Boran. Thus the two traits, better parasite control and better anaemia control, were not linked. The correlation between severe anaemia and absence of haemopoietic cells of trypanotolerant origin indicated a role for N’Dama haemopoietic cells in anaemia control. Importantly, the N’Dama chimeras also lost the capacity to gain weight compared with the singletons. This experiment thus confirmed that trypanotolerance is mediated by two independent mechanisms. The first is a better capacity to control parasitaemia and is not carried by cells from a haemopoietic lineage. This further suggested that this superior parasite control in N’Dama calves was not due to differences in their immune responses. The second mechanism is a better capacity to control the associated anaemia and is mediated by cells from the haemopoietic system. At this stage, we can only speculate about the exact mechanism: it could include an improved erythropoietic response in N’Dama or a reduced contribution of lymphoid and phagocytic cells in the development of anaemia. Since productivity, or the capacity to gain weight, was not associated with parasitaemia control but with anaemia control, further research focused primarily on the identification of the latter mechanism.

6. The role of T lymphocytes and antibodies in trypanotolerance To identify a potential role of lymphocytes in the induction of anaemia, calves were in vivo depleted for different T cell subpopulations using mouse monoclonal antibodies to bovine leukocyte antigens (Naessens et al., 1997), and the technique was refined to ensure that all cells of a particular phenotype would be removed from all peripheral lymphoid tissues and not just the blood (Naessens et al., 1998). Depletion of all CD8 T cells did not influence the progress of anaemia after T. congolense infection as compared with non-depleted control calves (Sileghem and Naessens, 1995). Similarly, using a combination of monoclonal antibodies to CD8 and WC1 to

completely deplete all CD8 and all g/d-T cells from the periphery did not influence anaemia development (De Buysscher E.V., Naessens J., unpublished data). Complete depletion of CD4 T lymphocytes was successfully obtained in Boran and N’Dama calves. In such depleted cattle no chancre formation was observed in the skin upon the bite of infective tsetse flies (Naessens et al., 2003b). The experiments suggested that the local skin reaction was an inflammatory response to metacyclic forms, specific for the metacyclic variant antigenic types (VAT), and mediated by CD4 T lymphocytes. However, the absence of a response in the skin had no effect on the subsequent migration of trypanosomes to the bloodstream and the progress of infection (Naessens et al., 2003b). After infection of CD4-depleted and non-depleted Boran and N’Dama calves with T. congolense, the antibody responses were found to be markedly reduced and delayed in the depleted animals (Naessens et al., 2002). This was the case for IgG and IgM antibodies to surface-exposed and internal trypanosome epitopes, as well for the natural IgM antibodies that react with non-trypanosome antigens (Buza and Naessens, 1999). In contrast to murine infections (Reinitz and Mansfield, 1990), the proportion of T-cell independent antibodies is very low in infected cattle. The extent of anaemia in the CD4depleted N’Dama and Boran calves did not differ from that in the non-depleted calves. The anaemia in the two Boran groups was more severe than that in the two N’Dama groups. Taken together, these data suggested that neither T cells, nor antibodies mediate the trypanosome-associated anaemia. The potential contribution of auto-antibodies (Kobayashi and Tizard, 1976; Facer et al., 1982; Assoku and Gardiner, 1992) and anti-trypanosome antibodies (Woo and Kobayashi, 1975; Rifkin and Landsberger, 1990) to removal of erythrocytes is therefore negligible in T. congolense infections in cattle.

7. Haemophagocytic syndrome in bovine trypanosomosis Thus far, three parameters were systematically used to monitor trypanotolerance: parasitaemia in blood, anaemia and weight gain, with the latter being the most indicative of overall productivity in a field situation. However, there are a number of additional pathological features associated with bovine trypanosomosis, including hepatosplenomegaly, pancytopenia and macrophage activation. A careful analysis reveals that this collection of features constitutes haemophagocytic syndrome

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Table 2 Summary of clinical and pathological signs of haemophagocytic syndrome (HPS)a with features observed in cattle with trypanosomosis Features associated with HPS Clinical manifestations High fever (Oweek) Hepatosplenomegaly CNS symptoms Biological manifestations Pancytopenia Macrophage activation Tissue infiltration of macrophages Liver dysfunction Hyper-triglyceridemia Hyper-ferritinemia Haemodilution Defective NK cytotoxicity a

Features associated with bovine trypanosomosis YES (many reports) YES (many reports) NO, except when trypanosomes can cross the BBB YES: anaemia, thrombocytopenia, neutropenia (Logan-Henfrey et al., 1999; many older reports) YES: in Trypanosoma congolense (Anosa et al., 1997, 1999) YES: in Trypanosoma vivax (Anosa et al., 1992) YES (many reports) No data? YES in cattle (Valli et al., 1980), marginally in sheep (Katunguka, 1995, 1999), up in mouse and rabbit YES in cattle (Mamo and Holmes, 1975), in sheep (Katunguka-Rwakishaya et al., 1992) YES, partial haemodilution (reviewed in Katunguka-Rwakishaya et al., 1992) No data

Me´nasche´ et al., 2005; Kumakura, 2005.

(HPS) (Table 2). This set of clinical and pathological manifestations in humans occurs as a result of hyperactivation of the phagocytic system (Kumakura, 2005; Me´nasche´ et al., 2005). The condition is often fatal and is associated with an infection, malignancy, autoimmune disorder or with a genetic impairment in T and natural killer (NK) cells (Me´nasche´ et al., 2005). The fact that these pathological features occur together under apparently very different circumstances, suggests that they occur as a result of the deregulation of the same responses. Although the underlying events are not fully understood, it seems clear that more than one stimulus can give rise to hyperactivated, proliferating macrophages (Larroche and Mouthon, 2004). What causes the appearance of HPS in trypanosome infections is not known. But since infected calves quickly recover from anaemia and other pathologies after treatment with trypanocidal drugs (Murray and Dexter, 1988), the condition must be dependent on the continuous presence of trypanosomes or trypanosome factors. Since T lymphocytes did not seem to interfere with anaemia development, direct stimulation of macrophages probably causes this disease state. It is not known which parasite molecules trigger this pathway. Membrane variable surface glycoprotein (VSG), and to some extent soluble VSG, can trigger the secretion of tumour necrosis factor (TNF)-a in murine (Magez et al., 1998) and bovine macrophages (Sileghem et al., 2001) and could be the molecules that trigger anaemia. However, it is more likely that a number of trypanosome stimuli and host immune mediators synergise to produce anaemia and HPS.

trypanotolerant BL6 mice which continued to develop to a severe degree (Nakamura et al., 2003; Naessens, unpublished data). The same pattern held true in the two host strains for infections with the human-infective trypanosome T. b. rhodesiense (Naessens, unpublished data) and in BL6 and BALB/c mice infected with T. b. brucei (Magez et al., 2004). Thus parasitaemia control and anaemia control in mice are not correlated, as they were in bovine trypanosomosis. However, the data suggest that trypanotolerance as defined by longer survival in the mouse model, is very different from trypanotolerance in cattle, defined as higher productivity after infection. In the murine model, tolerance is correlated with parasitaemia control, but not with anaemia control, whereas in the bovine model, tolerance is primarily correlated with anaemia control, and to a lesser extent with parasitaemia control. Studies in tolerant mice have suggested that several mechanisms contribute to restriction of parasite growth. Comparison between infections in a collection of genedeficient C57BL/6 mice demonstrated that control of T. congolense parasites depended on at least two effector arms of the immune system: interferon (IFN)-g-dependent production of nitric oxide (NO) and TNF-a and presence of functional IgG antibodies (Magez et al., in press). The critical role of antibodies in murine trypanotolerance (Campbell et al., 1977; Magez et al., in press) but to a much lesser degree in bovine tolerance (see above) is evidence that the control of parasitaemia is also mediated by different mechanisms in the two species. We conclude from this that mice do not offer a good model to study bovine mechanisms of parasite control.

8. Murine trypanotolerance

9. A murine model for anaemia development

Trypanosoma congolense-infected C57BL/6 mice (BL6) die later than similarly infected A/J or BALB/c mice, providing us with a potential model to study trypanotolerance (Kemp et al., 1996). Susceptible A/J mice developed much higher parasitaemias than BL6, suggesting that mortality was correlated with high parasite numbers. However, the associated anaemia in A/J was mild and transient, in contrast to anaemia in the

Although mice may not provide good model systems to identify the responses that lead to resistance in a natural host, they may still offer good models to identify the molecular pathways that mediate particular resistance traits or pathological features, such as the trypanosome-associated anaemia. Since infected BL6 mice survived for a reasonably long time and developed severe anaemia after infection, we selected

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this mouse strain for further experiments. Anemia developed unchanged in infected BL6 mice after administration of the immunosuppressive drug cyclosporin A, suggesting that anaemia in murine, as in bovine trypanosomosis, is not mediated by T lymphocytes (Naessens, unpublished data). Studies in malaria (Clark and Chaudhri, 1988) and other infectious diseases had suggested that TNF-a could be a major player in the development of anaemia associated with disease and inflammation in general (Jelkmann, 1998). Therefore, the effect of absence of TNF-a on progress of disease was studied by comparing T. congolense infections between TNF-a-knockout and wild-type BL6 mice. Survival after infection was significantly shorter for the knock-out mice than for the wildtype mice (Iraqi et al., 2001), while parasitaemia was significantly higher (Naessens et al., 2004). Since, the knockout mice still produced near normal amounts of specific antibodies (Naessens et al., 2004), it was unlikely that the reduced parasitaemia and shorter survival were due to a defect in antibody production. Anemia in TNF-a-knock-out and wildtype mice were similar when measured during a T. congolense infection (Naessens et al., 2005). In contrast, during a T. b. rhodesiense infection, TNF-a-deficient mice developed a very mild anaemia compared with the wild-type (Naessens et al., 2005), suggesting a crucial role for this cytokine in anaemia development in this host–parasite combination. A similar observation was described after an infection with T. b. brucei: TNF-a-knock-out mice did not develop anaemia (Magez et al., 1999). TNF-a may contribute to anaemia at different levels: it may act on haemopoiesis by lowering erythropoietin and inhibit proliferation of precursor cells (Jelkmann, 1998) or it may have a role in the hyperactivation of the macrophage system and HPS (Larroche and Mouthon, 2004). Using mice deficient for TNF-receptor genes, it was observed that TNF-a mediated its role via the TNF-R2 but not the TNF-R1 (Magez et al., 2004). No direct correlation between the level of TNF-a and disease-associated pathology was obvious. However, low TNF-a in combination with high soluble TNF-R2 was correlated with lack of immunopathology (Magez et al., 2004). These data suggest that TNF-a mediates anaemia in particular host–trypanosome combinations, but not in others. At least two mechanisms lead to anaemia: one TNF-adependent and another TNF-a-independent. We speculate that the higher numbers of parasites found in T. brucei infections compared with T. congolense infections will cause higher TNF-a secretion, which in turn will contribute to an enhanced rate of erythrocyte removal. We do not know why some strains of mice, such as A/J and Balb/C, do not develop severe anaemia when infected by T. congolense or T. b. rhodesiense, despite the presence of other pathological features such as hepatosplenomegaly (Naessens, unpublished data). Is it possible that similar TNF-a-dependent and independent mechanisms operate in bovine trypanosomiasis? There are some indications that infections with a haemorrhagic T. vivax strain may be more similar to murine infections with T. brucei. Some East African stocks of T. vivax develop a higher and persistent parasitaemia compared with West African T. vivax stocks (Gardiner, 1989). Infections with such East African strains

develop more severe pathology and are accompanied by high fever, profound anaemia and thrombocytopenia and haemorrhages of mucosal surfaces, particularly in the gastro-intestinal tract. Auto-antibodies could be observed on erythrocyte and platelet surfaces (Assoku and Gardiner, 1989). Monocytes from cattle infected with a haemorrhagic T. vivax manifested a strong ex vivo TNF-a secretion (Sileghem et al., 1994). In contrast, no TNF-a production was detected with monocytes from uninfected or T. congolense-infected cattle, suggesting a more important role for this cytokine in pathogenesis. Unfortunately, no TNF-a-neutralisation studies have yet been carried out in cattle. However, during an infection with haemorrhagic T. vivax, the N’Dama were as susceptible, if not more, than the Boran and had to be treated to survive (Williams et al., 1992). It is possible that N’Dama have developed a better capacity to control anaemia when it is caused by a TNF-a-independent mechanism, which would be the case during a T. congolense or mild T. vivax infection. In contrast, the trypanotolerance trait would give no protection when anaemia is caused by a TNF-adependent mechanism, which we speculate could be the case with a haemorrhagic T. vivax strain. 10. Conclusions The N’Dama cattle have acquired tolerance to trypanosomosis by natural selection, and possess a genetic capacity to better control parasitaemia. Yet, this trait seems less important to productivity than the genetic capacity to prevent anaemia, severe pathology and HPS. To identify this mechanism is now our priority. We should be aware that pathology is complex, that more than one mechanism contributes to the anaemia and that the relative contribution of each mechanism will differ for each host–parasite combination. Mice that are tolerant to trypanosomosis have not acquired this trait by natural selection and it is likely that in murine models we compare susceptible mice with even more susceptible ones. Indeed, the more ‘tolerant’ C57BL/6 mice develop titres of T. congolense in blood that are 100-fold higher than the most susceptible calf and eventually all of the mice die, suggesting that they should be rated ‘very susceptible’. It is not surprising then that antibodies are so important for their survival, because all else fails to protect these mice. The risk of studying tolerance in a mouse model is that we may find that the most susceptible mice have some genetic defect in a response mechanism that is of importance in this species but irrelevant in a more resistant species, such as N’Dama cattle and wildlife. It is important that potential murine models are tested carefully to determine whether they can deliver the desired objective, particularly for a complex trait like resistance. Nevertheless, murine models and the existence of a range of available mutations and knock-out mice strains should be extremely helpful in identifying the basis of more simple traits such as the pathways that lead to anaemia or HPS. Comparing host factors that are activated in severely anemic mice with mice that fail to develop anaemia may allow us to identify the series of events in the host that lead to this feature. Knowledge of how and why destructive pathologies develop may help us to

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