TECHNICAL PROBLEMS IN THE ESTIMATION OF TRYPANOTOLERANCE WITHIN CATTLE AND SMALL RUMINANT BREEDS

A. Verhulst and V.S. Pandey¹

SUMMARY

The availability of suitable methods for controlled challenge infections and evaluation of parameters of trypano-tolerance are the major problems in the genetic studies of trypanotolerance in ruminants.

Natural tsetse infection, experimental cyclical infection and syringe transmitted infection, are the available methods of challenge. Natural tsetse infection, although useful and widely used, suffers from drawbacks such as: - the inability to ensure standard and repeatable challenge (species, serodemes, virulence, dose,…), - the necessity to rear the animals in fly-proof stables or alternatively to move the experimental animals from a non-infected to an infected area, - the effect of environmental factors, - the impossibility of testing the animals outside the tsetse infected areas. The experimental cyclical infection may eliminate several of the difficulties of natural tsetse infection, but still there is a necessity to rear the animals in fly-proof stables or to keep them in an area with a very low trypanosome infection risk. Syringe transmitted infection avoids most of the problems encountered by other two methods of challenge infection. Using a well standardised infection procedure (species, single VAT such as AnTat 1.1 without cross reactivity with naturally occuring trypanosomes in the field, dose, route of infection) the test can be repeated anywhere and at any time allowing valid and reliable comparisons.

¹ Department of Animal Health and Production Institute of Tropical Medicine, Antwerp (Belgium).

At present, genetic markers for detecting trypanotolerance, are not available. The commonly used criteria for trypanotolerance are parasitaemia, PCV, production and reproduction para-meters. These parameters are influenced by several host, environ-mental and management factors. The syringe transmitted infection with a single VAT, AnTat 1.1, of Trypanosoma brucei brucei has been used by Institute of Tropical Medicine, Antwerp, Belgium. The parasitaemia, PCV, VAT specific and heterologous antibodies and complement have been measured The test appears to be a promising method of detecting trypanotolerant animals within a period of 30–40 days and trypano-sensitive animals in 15–20 days of infection. In the initial stages it will be interesting to apply this test on the animals that have already been tested for trypanotolerance by other methods, preferably on animals of different generations of known parentage. At a later stage it can be used on large scale to detect the level of trypanotolerance in ruminants.

It is also recommended to use the field test under natural conditions in the tsetse infected areas as developed by the team of International Livestock Centre for Africa (ILCA). This test envisages the use of one-year old cattle and the measurement of parameter of control of anaemia (PCV) for at least over 6 weeks of detected parasitaemia. It will not select the trypanotolerance trait sensu stricto, but several traits that altogether contribute to better productivity of cattle in tsetse infected areas. The test may be useful in big commercial ranches.

There is also a need to continue research on the role and use of complement, on trypanotoxin or detrimental factors which damage metacyclic trypanosomes in vitro and on genetic markers.

Genetic studies on the phenomenon of trypanotolerance in the domesticated ruminants require the availability of methods for controlled challenge infections and quantitative evaluation of trypanotolerance. A number of issues will be raised that must be considered in the development of such methodologies. These concern (i) the trypanosome challenge protocol and (ii) the parameters that will be taken as measures of trypanotolerance.

1. TRYPANOSOME CHALLENGE

Different challenge protocols are used: (i) natural infection, (ii) cyclical infection and (iii) syringe infection.

1.1 Natural Infection

A major issue centres around the question if the challenge should be through natural exposure in an area of high trypanosome presence, or if it, should be by means of controlled challenge under experimental conditions in a normally trypanosome-free area. It is recognised that the traits of animals, which are desirable for breeding purposes, are generally measured in the natural environment in which they are to be expressed. It is also recognised that in cases of field challenge, all factors concerned in trypanotolerance, including behavioral factors and attractiveness of Glossina spp., can be tested. But experience also shows that the host response to trypanosome infection depends, to a large extent, on physical health of the animal as well as factors of stress and other environmental factors, while the level of challenge and the type of serodeme in the field infection can hardly be repeated again and are difficult to evaluate.

In 1979, in the Gambia P.K. Murray et al. reported an accidental field challenge by G. palpalis of 31 Zebu and 37 N'Dama cattle. All the Zebu cattle became infected and 21 of them died. Only very few N'Dama cattle showed parasitaemia and none of them died.

In 1981 M.Murray et al. reported another experiment in Gambia in which 10 Zebu and 8 N'Dama were submitted to a natural challenge of G. morsitans submorsitans. All the Zebu cattle developed a severe parasitaemia and died. The N'Dama showed very low parasitaemia and only 3 of them died.

In 1983, in Burkina Faso, Roelants et al. have submitted successively 10 Zebu and 20 Baoule cattle to a severe natural challenge by G. m.submorsitans and G. palpalis. In this study also all the Zebu cattle died within a few weeks of exposure, while 9 Baoule cattle survived.

In 1987, at Centre de Recherche sur les Trypanosomiases Animales (CRTA), Bobo-Dioulasso (Burkina Faso), Clausen et al. introduced 20 Zebu and 76 Baoule cattle in a region under very high tsetse challenge. All Zebu had to be salvage treated with Berenil (Diminazene aceturate, Hoechst). Majority of the Baoule originating from another tsetse infected area could be classified as resistant. But 15 Baoule, out of a group of 17 animals, that had never been exposed to natural challenge before, had also to be salvage treated with a trypanicide. On the basis of a comparison of three clinical indicators of resistance, namely the duration of the disease, the total survival time and duration of stability of body weight, the Baoule cattle could be divided into three groups : (i) Highly susceptible Baoule, (ii) Baoulé of intermediate susceptibility and (iii) Resistant Baoulé. On the same group of animals Authié and Pobel (1990) demonstrated that the serum haemolytic complement activity in early infection was dependant on the intensity of parasite load and on the potential of each individual to control the disease. There was a significant correlation between the minimum complement activity, the minimum C3 and the minimum PCV in early infection. These three parameters correlated with individual resistance and might, therefore, be useful criteria for the identification of the most resistant individuals within a herd of trypanotolerant breed.

Recently, in field research under natural infection involving N'Dama cattle in Zaire and Gabon, Trail et al. (1991 a and b) investigated the effect of control of anaemia on animal productivity relative to that of other aspects of trypanotolerance, e.g. control of parasitaemia, with a view to evaluate feasibility of its assessment early in an animal's life.

In Zaire, breeding cows were maintained for 3 1/2 years under a mean monthly trypanosome prevalence (the percentage of animals detected as being parasitaemic at a monthly examination) of 10% per month (Trail et al., 1991 a). The comparative influences of time related parasitaemia, parasitaemia intensity (representing control of development of parasitaemia) and PCV value (representing control of development of anaemia) were measured on production parameters such as calving interval, calf weaning weight and cow productivity (weight of weaner calf per cow per annum). The findings were significant and showed that cows with transient parasitaemia had a 14% shorter calving interval and a 15% higher productivity than their contemporaries with prolonged parasitaemia. The effects of intensity of parasitaemia were not significant.

In contrast, animals maintaining a high PCV value had 11% shorter calving interval, a 9% heavier calf weaning weight and a 24% superior cow productivity over those maintaining a low PCV value. Control of development of anaemia, as measured by average PCV level, appeared to be the criterion of trypano-tolerance most closely linked to overall cow productivity. The repeatability of PCV values and the calving intervals was reasonably high (0.33) and almost equal to that of calf weaning weight.

On the other hand, the ability of individual N'Dama to maintain PCV values at sustained levels over several different challenge infections has also been demonstrated experimentally (Paling et al., 1987). The repeatabilities of the various traits measured between successive calving intervals set upper limits to their degrees of genetic determination and heritabilities (Falconer, 1981). Thus, the ability to control the development of anaemia, as indicated by PCV value, might be a reliable criterion to identify more trypanotolerant individual animals. The fact that calf PCV values were at least as important as dam PCV values in their effect on calf performance suggested that evaluation of this criterion of trypanotolerance in an animal might well be feasible before it reached maturity.

In Gabon, one-year old N'Dama cattle were examined following exposure to high natural tsetse challenge for varying lengths of time (Trail et al., 1991 b.). In three replicate examinations involving over 400 animals, the effects of PCV values on animal growth were confirmed. While a low grade parasitaemia score was associated with 10% superior growth rate, a high PCV value showed a 44% advantage. It was computed that the capacity to control anaemia could be determined within six weeks of detection of parasitaemia and it was proposed that this approach could be used as a field test to select young animals for trypanotolerance.

One can conclude that, although the natural tsetse challenge experiments have contributed considerably to our knowledge and understanding of the phenomenon of trypano-tolerance, in practice several technical problems still remain to be resolved such as:

• the necessity to move experimental cattle herds from a non infected area into an infected area, or alternatively to rear them first in fly-proof stables before natural exposure or to wait for the season of high exposure-risk (if this season can be clearly defined);

• the inability to ensure a standard and repeatable (in time and space) well known challenge : exact time of infection, infection dose and the features of the infecting trypanosomes such as species, serodemes, virulence, etc;

• the hardly repeatable environmental factors that may affect the physical condition of the animals and its response to trypanosoma infection; in this respect a natural challenge allows only the comparison of individuals belonging to the same experimental group and not between individuals belonging to different groups (in time and space);

• highly sensitive animals (e.g. Zebu) may die before efficient treatment can be resorted, thereby limiting the feasibility of mass application because of economic losses and ethical considerations;

• the impossibility of testing animals outside the tsetse infected areas of Africa.

1.2 Cyclical Infection

A number of above mentioned technical limitations of natural infection can be avoided by cyclical infection.

However, at CRTA (Bobo-Dioulasso), Pinder et al., (1987) could not find an adequate correlation between infection trials of cattle infected naturally and cyclically with an East-African clone of T. congolense.

Cattle reared in tsetse proof stables were submitted to cyclical infection with a West-African clone of T congolense (Duvallet et al., 1988). So far as PCV values and control of parasitaemia are concerned, classical significant differences were observed in the responses between Baoulé and Zebu cattle. But no clear distinction could be made between more sensitive and more resistant individuals within the Baoulé group.

At ILRAD, Paling et al., (1987) have submitted 8 N'Dama and 4 groups of 8 Boran Zebu to 4 successive cyclical infections using 4 different East-African clones of T. congolense transmitted by G. morsitans centralis. The Zebu had to be frequently salvage treated by a trypanicide drug, while the N'Dama did not need it. The Zebu developed a more intense initial parasitaemic wave and also a higher mean parasitaemia. They also showed a more pronounced drop of PCV values. The mean PCV values increased significantly in N'Dama cattle from the first to the fourth infection, indicating that these animals seem to develop a mechanism resulting in a better control of anaemia during successive infections. It is suggested that the lowest PCV value between days 32–52 post-infection could be used as a reliable indicator of anaemia control during a trypanosome infection.

While it may be concluded that these cyclical infections are helpful in eliminating a lot of technical problems encountered in case of natural challenge, the main technical difficulty still remains the necessity to rear the animals under experiment in a tsetse proof stable or to keep them in an area with very low trypanosome infection risk.

1.3 Syringe Transmitted Infection

In 1979, P.K. Murray et al. submitted 40 Zebu and 37 N'Dama cattle to a syringe infection with T. congolense and T. b. brucei that had been adapted to rodents. All animals became infected. While all the N'Dama survived and showed only a moderate decrease of PCV values, Zebu cattle showed severely decreased PCV values and 9 of them died.

In 1982, Guidot and Roelants infected Baoulé and Zebu cattle successively with syringe transmitted T. vivax and T. congolense. Those animals which had been reared in tsetse infested areas showed contradictory results. Both the Zebu and the Baoulé survived and most of Baoulé showed a more severe decrease of the PCV values than did the Zebu.

Syringe transmitted infection with a local clone of T. congolense has also been used in N'Dama cattle at the “Centre de Recherches et d'Elevage d'Avetenou / Togo (CREAT)” to evaluate different parameters of trypanotolerance e.g. PCV, parasitaemia, complement and lysozyme. Unfortunately this interesting programme was not completed or published.

Using well standardised syringe transmitted VAT-specific infection in goats of different breeds (or types) using the AnTat 1/1 clone of repertoire AnTAR 1 of T. b. brucei (MAVUBWE/66/EATRO/1125), it was possible to classify these breeds based on their mortality rate (M) and the mean survival time (S). The Djallonke goat of the extreme South of Benin showed to be the most resistant one (M=33% and S=31 days) (Demey, 1987; Verhulst and Hardouin, 1989), followed by the cross-bred toggenbourg & dwarf goat of the Kempen (Belgium) with M=83% and S=78 days (Verhulst, 1989), the Djallonke goat of the Savannah of the Central Province of Cameroon with M=100% and S=44 days (Demey, 1987; Verhulst and Hardouin, 1989) and finally the Saanen goat in Belgium with M=100% and S=44 days (Verhulst, 1990). The most trypanotolerant individuals in the cross-bred Toggenbourg x dwarf goat of the Kempen could be detected in an early stage by AnTat 1/1 VAT-specific trypanolytic antibody determination and by the Testryp-CATT (Verhulst, 1989).

More recently, using additional parameters of trypano-tolerance, e.g. complement activated by the classical pathway (CPW) and by the alternative pathway (APW), we developed a “Trypanotolerance Test” allowing a clear characterisation of the trypanotolerant individuals of goat (Baderha et al., 1990) and cattle (Doko et al., 1991) (Figures 1 and 2).

One can conclude that the syringe transmitted infection procedure avoids most of the problems encountered by the two other modes of challenge infections given above. Using a well standardised infection procedure (species of trypanosome, single VAT, dose and route of inoculation) the test can be repeated anywhere and at any time. The test can be used even in trypanosome infected areas of Africa without moving the experimental animals or rearing them in tsetse proof stables, provided that the trypanolytic antibodies against the VAT used for the test do not occur naturally in the farm animals (e.g. AnTat 1/1).

One can object that a syringe transmitted VAT-specific infection is a false reproduction of the natural challenge which exist under different environmental conditions.

Nevertheless, genetic studies require an accurate and repeatable protocol, that minimises, as much as possible, different environmental factors which may influence the phenotypical expression of a genetic trait.

2. MARKERS AND PARAMETERS OF TRYPANOTOLERANCE

2.1 Genetic Markers of Trypanotolerance

Till now, there is no genetic marker that can be used for selecting trypanotolerant cattle, sheep or goats. This means that selective breeding for trypanotolerance has to rely on laborious and time-consuming progeny-testing programmes and on subjective evaluation of the host phenotype. The phenotypic, assessment may also be a prerequisite for the identification of genetic markers.

However, the animal industry can greatly benefit from the knowledge gained as a result of research studies carried out in humans. Although the problems facing animal breeders are significantly different from those encountered by human geneticists, genetic markers can be used to rapidly identify genes of economic importance, thereby enabling cloning and characterisation. In addition, it is possible to define with these techniques a series of DNA markers closely linked to major genes which determine production traits or disease resistance. Such markers can be used to predict at birth or even in pre-implantation embryos (for example, immediately prior to embryo transfer) the phenotype of an animal, which until now could not be assessed until maturity.

Polymorphic markers will allow the “tagging” of specific segments of chromosomes. One can then follow the segregation in pedigrees of the tagged chromosomal regions and associate the marker with the presence of a desirable trait such as high productivity or disease resistance. Gelderman et al., (1985) demonstrated the feasibility of this approach for milk production in dairy cattle and Beever et al. (1989) for certain carcass characteristics in beef cattle.

The development of a series of DNA markers that will identify genes controlling traits of production or disease resistance requires a coordinated, international research effort. Given the rapid advancement of linkage analysis in man, the establishment of a gene linkage map in domestic animals is now realistic and possible. The use of “reverse genetics”, i.e. the identification of genes without prior knowledge of the causal mechanisms involved, has provided dramatic results in recent years.

But we must not be too optimistic for the prospects in the detection of the genes of trypanotolerance. The problem in practice is that measuring objectively and accurately a milk production or a carcass characteristics is much more easier than measuring trypanotolerance.

At the same time, there has been considerable advancement in our knowledge of the immunological processes involved in disease resistance. It is vitally important that studies aimed at elucidating the pathways determining disease resistance and trypanotolerance in particular be undertaken in conjunction with the molecular genetics.

2.2 Estimation of trypanotolerance by measuring phenotypical traits.

As stated above, the estimation of trypanotolerance by measuring phenotypical traits by using different parameters is probably a prerequisite to further progress in selective breeding or the development of polymorphic markers and DNA markers. Since there are different degrees of trypanotolerance, one must not only identify the phenomenon but also measure it both between animal breeds and within breeds. For that purpose, different parameters are used, but most of them present different problems of objectivity and/or accuracy, and/or sensitivity and/or feasibility.

In the practice, five different types of criteria could be used in an attempt to “measure” the trypanotolerance during natural or experimental trypanosomiasis: (i) direct pathological criteria e.g. parasitaemia, anaemia, survival time duration, prepatent period, (ii) indirect production criteria e.g. body weight and meat production, milk production, working capacity, reproductive performance, (iii) immunological parameters e.g. trypanolytic antibodies production, activation of complement by the classical pathway (CPW) and the alternative pathway (APW), cofactors of complement e.g. C₃ factor, B factor, different other effectors of immunity, e.g. interleukine², conglutinin, immunocompetent cells and the local cellular reactivity at the inoculation site of the trypanosomes, etc… (iv) non-immunological factors of resistance against trypanosomiasis e.g. trypanotoxin, (v) treatment index: frequency of trypanocide treatment necessary to maintain more or less trypanotolerant animals in optimal healthy condition under natural trypanosome challenge (e.g. Berenil index).

2.2.1 Pathological Criteria

Field and experimental studies carried out on several breeds of cattle have confirmed the superior trypanotolerance of the West African Shorthorn and N'Dama, as judged by their ability to resist the pathogenic effects of infection, i.e. not only to survive, but to gain normal body weight and to reproduce normally (Pierce 1906; Roelants 1986; Murray et al, 1982; Murray and Dexter, 1988). Anaemia is a normal consequence of trypanosome infection in domestic animals and cattle in particular (Hornby, 1921; Murray, 1979). Trypanotolerant cattle develop less severe anaemia (Murray and Dexter, 1988). It has also been noted that the intensity, prevalence and duration of the accompanying parasitaemia are less in trypanotolerant cattle breeds (Dargie et al., 1979; Murray et al., 1982). Thus, it has been concluded that the ability to resist anaemia and to control parasitaemia are key indicators of the trait of trypanotolerance.

However, at the International Laboratory for Research on Animal Diseases, Kenya (ILRAD), studies on N'Dama cattle, obtained as embryos from donors in the Gambia and implanted into surrogate Boran mothers (Jordt et al., 1986), showed no direct correlation between mean parasitaemia and PCV values computed for individual animals (Paling et al., 1987). Some N'Dama demonstrated higher PCV values during four consecutive trypanosome infections, while others showed better parasite control. Nevertheless, individual animals displayed the ability to resist anaemia or to control parasitaemia in a consistent and repeatable fashion in all the four experiments. It was therefore concluded that, while both processes are under genetic control, these are not directly linked.

In an experiment involving 18 Saanen goats and artificially infected with AnTat 1/1 at the Institute for Tropical Medicine, Antwerp-Belgium, (Verhulst, 1990), no significant correlation was observed between PCV values and parasitaemia and only a very low correlation between the relative decrease of PCV values between days 5–10 after challenge (PCV) and the daily cumulating parasitaemia (P), during the first parasitaemic wave (r=0,2).

There was also no correlation between PCV and the survival time (S), nor between PCV and P. However, S was correlated with P (r=0,49) (n=12). It was also interesting to note that the initial PCV values were correlated with the initial weight of the animals (r=0,5) (n=18).

Pinder et al., (1987), in studies on cattle subjected to experimental cyclic infection with T. congolense or to field challenge, were also unable to demonstrate any correlation among parasitaemia, anaemia and pathology.

The above findings on farm animals were also confirmed by observations on inbred mice. No significant correlation was observed between survival time, parasitaemia and anaemia neither by Seed and Sechelski (1988) after infection of irradiated inbred mice with pleomorphic Trypanosoma brucei rhodesiense, nor by Seed and Sechelski (1989) after infection of C₃H and B₁₀ inbred strains of mice with the LanTat-1 clone of T. brucei rhodesiense. Our studies also, using C₃H/He and CBA/Ca mice and metacyclic forms of Trypanosoma brucei brucei as infective material (Demey et al., 1985 a; Demey et al. 1985 a, b; Demey, 1987, Verhulst and Hardouin, 1989), confirmed these findings.

Considering these experimental results on farm as well as on laboratory animals, indicating a lack of correlation between PCV values and survival time or parasitaemia, the validity of PCV values as a criterion of individual selection for trypanotolerance sensu stricto could be questioned.

However, considering existing correlation between PCV values and animal productivity and reproduction performance, Trail et al. (1991 a and b) have investigated the possibility of use of PCV and parasitaemia parameters in field research involving N'Dama cattle in Zaire and Gabon, in order to examine the feasibility of assessing trypanotolerance early in animal's life (see below).

2.2.2. Productivity criteria (production and reproduction).

Production aspects of trypanotolerant cattle have been reviewed by Dwinger (1989) at the Trypanotolerance meeting of Banjul (Gambia) 25–28 September 1989. Reproduction aspects of trypanotolerant cattle have been reviewed at the same meeting by Chicoteau (1989).

A joint ILCA/FAO/UNEP study (1979) collected all the information obtainable at that time. A major finding of the study was that despite their small size, trypanotolerant animals were nearly as productive per unit of body weight as Bos indicus cattle when kept under similar conditions of trypanosomiasis risk and management. The Zebu productivity estimates were only at a 5% higher level per 100 kg of cow maintained per annum. It should be noted that under medium or high trypanosomiasis risk, no comparison on productivity levels would be feasible, since most of the Zebu would not survive (Murray et al., 1981).

The study also showed the influence of management and tsetse challenge on productivity levels. Cattle at research stations or ranches often receive intensive feeding and health care boosting their productivity above that of the village maintained animals. Similarly, it is not surprising that the productivity decreases when the level of trypanosomiasis risk increases.

Recently FAO (1987) published a summary of the data obtained during the 10 years that have passed since the appearance of the initial ILCA/FAO/UNEP study in 1979.

For Senegal and Sierra Leone, data were available both in 1979 and 1988 on the productivity of cattle kept at research stations. A comparison revealed that the productivity at Kolda following a careful analysis of the data and improvement of the management system (Fall et al., 1982), was much higher than previously estimated. In Sierra Leone, the productivity of N'Dama cattle at Teko research station was lower (Carew et al., 1986) than at the Mussaia station. This was due to a lower calf growth and lower reproductive performance (Table 4), the latter being characterized by age at first calving, length of calving interval and length of the productive life of the cow (Carew et al., 1986). In general the N'Dama has a high reproductive potential, which is frequently reduced by poor management and a harsh environment

(Starkey, 1982). Since many of the productivity indices calculated for the ILCA/FAO/UNEP study (1979) were based on estimated, ILCA and ILRAD jointly initiated the African Trypanotolerant Livestock Network.

Accurate data are collected on productivity of trypano-tolerant animals kept under varying challenge and management conditions. Under village conditions in the Gambia and Senegal it was shown that different production and reproduction performances were greatly dependant on management, e.g. tethering of cattle overnight or herding during the rainy season (Dunsmore et al., 1976), continuous presence of males in village herds and fecundation of very young females, milking of cattle by contract herdsmen (Dwinger, 1989). The severity of tsetse challenge was another important factor influencing the different production and reproduction performances as shown in Table I.

from Dwinger (1989)

Reproductive performances of trypanotolerant cattle undergo severe seasonal variations because of quantitative and qualitative changes in available food (mainly influenced by rains, savannah fires, fresh growth or ripening of grasses, covering with bushes), seasonable variation in heat stress and changes in epidemiological risks, e.g. trypanosome risk (Landais, 1983; Steinbach et al., 1971).

The age at first calving within each trypanotolerant bree d may vary considerably depending on growth rate and nutritional level (Chicoteau, 1989). The postpartum anoestrus and the interval between calvings in trypanotolerant cattle are changing in function of the origin of the animals, the nutritional status, the month of mating and calving, the year, management, epidemiological situation, e.g. trypanosomiasis risk (Gyawu, 1988; Landais, 1983; Planchenault, 1987 a,b,; Olutogun and Dettmers, 1986; Thorpe et al., 1987).

Thus, it is clear that in practice, the production and the reproduction parameters are influenced by a lot of different factors, which have no relationship with trypanotolerance status in sensu stricto which may affect diversely the scores either between animals of the same herd. For this reason, these parameters should be considered as rather impractical and inaccurate criteria to discriminate between different levels of trypanotolerance in breeds, herds or individuals.

2.2.3 Immunological Parameters

2.2.3.1. Antibody Response

Chandler (1958) and Desowitz (1959) have observed a better immune response of N'Dama, using respectively a sero-neutralisation and a respiration inhibition test.

In cattle submitted to natural challenge at CRTA (Bobo-Dioulasso), Clausen et al. (1989) did not observe notable differences in the immune response between sensitive and resistant animals. Duvallet (1989) also arrived to the same conclusion at CRTA by measuring agglutinating antibody response to one single clone of T. b. brucei.

Akol et al. (1986) and Pinder et al. (1988) demonstrated a better neutralising antibody response against the first parasitaemic pic in Baoulé cattle challenged with metacyclic T. congolense.

The problems encountered in these contradictory results of experiments are possible previous contacts with trypanosomes and the hardly repeatable challenge protocols and environmental factors that may affect breed and individual responses in different ways. The different tests used to evaluate the immune response are measuring different types of antibodies, more or less closely related to resistance and of different sensitivity and specificity. In this regard the trypanolytic antibodies appear to be highly specific and more closely related to resistance.

The choice of the antigen to be used in the antibody assay is problematic when the challenging trypanosomes constitute a mixed population of different VATs, as it is the case in natural challenge, cyclical infection or syringe infection with non-cloned trypanosomes.

Most of those technical problems can be avoided by challenging through syringe infection with one single VAT having no cross-reactivity with trypanosomes naturally encountered in farm animals in Africa. Then, the trypanolytic antibody titration against the first parasitaemic wave can be made by using the same VAT as used for the challenge. Using AnTat 1/1 syringe challenge in inbred mice in Belgium and goats and cattle in Cameroon and Benin, we observed an earlier AnTat 1/1-specific trypanolytic antibody response in more resistant animals, appearing immediately after the elimination of the first parasitaemic wave (Demey, 1987; Doko et al., 1991). Comparing these antibody titers at the same day after challenge may lead to false conclusions since the pic of antibodies in more resistant animals may already have been bye-passed when the highest antibody titers are reached in less resistant animals. On the other hand, as shown in Figures 1. and 2., the antibody rise is very fast. Thus, a very narrow range of time may produce important variations in antibody titers. For these reasons, even the VAT-specific antibodies have to be considered as awkward criteria in practice.

2.2.3.2. Cellular Immune Response

Different investigations on cattle at CRTA (Fumoux, 1987) did not show differences in sensitive and resistant animals.

In susceptible and relatively resistant inbred mice, no useful selection criteria based on the cellular immune response could be identified by Makumyaviri and Demey (1985); Makumyaviri (1987) and Verhulst (1989).

2.2.3.3. Complement Activity

Complement is involved in the defense against a number of pathogens and recently there has been an increasing interest in the interaction between complement and different protozoa and trypanosomes in particular e.g. T. lewisi (Allbright and Allbright, 1985; Sturtevant and Balber, 1987) T. cruzi (Kipnis et al., 1985; Sher et al., 1986), T. brucei gam-biense (Devine et al., 1986) et T. congolense (Tabel, 1982; Malu and Tabel, 1986).

Experimental trypanosomiasis in susceptible cattle is characterised by persistent and marked hypocomplementaemia (Kobyaski and Tizard, 1976; Nielsen et al., 1978; Rurangirwa et al., 1980).

Authié and Pobel (1990) investigated the kinetics of serum complement and C₃ during natural infection with T. vivax and T. congolense in cattle of different susceptibility, in order to determine whether there was a relationship to trypanotolerance. They observed a significant correlation between minimum complement activity, C₃ and minimum PCV in early infection. These three parameters correlated with individual resistance and might therefore, be useful criteria for the identification of the most resistant individuals within a trypanotolerant breed. These findings are of highest interest for the future selective breeding of trypanotolerance. The problems remaining in the practice are: difficulties to set up the experimental design, by keeping cattle out of trypanosome risk for a certain period of time and moving them into a region with natural challenge, the duration of the experiment, death of highly susceptible animals, necessity of treatment of some animals during the experiment, the use of time-consuming techniques for the assessment of the total haemolytic activity of the alternative complement pathway as developed by Barta and Hubberts (1978), the extreme variability of the complement activity (APW and CPW) in different animals before the challenge influencing greatly the minimum complement activity values during the infection, and finally the classical disadvantages of a natural challenge, e.g. exact time of infection, characteristics of the infecting trypanosomes (species, serodemes, virulence, doses, possible cross-reactivity with trypanosomes of previous contacts).

We have also investigated the kinetics of APW and CPW in Saanen goats in Belgium (Baderha et al., 1990) and in Borgou and in West African Dwarf Shorthorn cattle in Benin (Doko et al., 1991), during artificial infection with T. b. brucei AnTat 1/1 associated or not with four other VATs of the same repertoire. A number of other parameters were evaluated at the same time, e.g. clinical symptoms, PCV, VAT-specific trypanolytic antibodies and heterologous agglutinating antibodies using the Testryp-CATT to evaluate the multiplication rate of infecting trypanosomes.

Highly susceptible animals (Figure 1) demonstrate a severe decrease of complement in APW and a complete exhaustion in the CPW some days prior to death. In tolerant animals (Figure 2), APW and CPW are decreasing during the parasitaemia phase but are restored to initial values a few days after the trypanosomes have been eliminated and may rise above the initial values in highly trypanotolerant animals. First results seem to indicate that all non-resistant animals may be identified as early as 15–20 days after inoculation, allowing early treatment with trypanocide. To discriminate between the trypanotolerant animals, the “Trypanotolerance Test” has to be continued till day 30–40. Recovery time (days) of APW and CPW are easy parameters to use. The mean PCV decrease is evident for the total group of animals being tested, but seems to be of limited value to discriminate between trypanotolerant individuals, nor to predict death in an early stage. Parasitaemia (duration of the first AnTat 1/1-specific wave, duration of parasitaemia, cumulated parasitaemia values) is a parameter that is directly linked with the ability of the animal to control or to eliminate the trypanosomes. The AnTat 1/1-specific trypanolytic antibody titers display a most characteristic curve, but awkward for interpretation as mentioned above.

Since antibodies against AnTat 1/1 do not occur naturally in farm animals in Africa (Demey, 1987), an adequately standardized test can be repeated at any time and at any place, either in tsetse infested areas, without moving cattle, or rearing in a tsetse proof stable or embryo transfers. A new method has also been developed for an easy assessment of APW and CPW in cattle, goat and sheep (Baelmans, Pandey, Doko, Demey and Verhulst, unpublished).

2.2.4. Non-Immunological Factors of Resistance.

Biochemical mechanisms limiting trypanosome multiplication in trypanotolerant cattle have been suggested since long time by Murray et al. (1982).

Using an inhibition test of blood and metacyclic forms of T. congolense, Traore-Leroux et al. (1987) did not observe significant differences between trypanotolerant and non-trypanotolerant taurine cattle.

Recent studies of Olubayo (1991) showed that the sera from buffalo and eland contains detrimental factors which damage metacyclic trypanosomes in vitro. He identified a trypanotoxin with a molecular weight of 150.000 daltons and thus outside the range of the molecular weights of immunoglobulin molecules. The buffalo toxin is different from the toxin in the serum of waterbuck which was shown to be an IgG VSG-specific toxin. This trypanotoxin may be responsible for killing of metacyclic trypanosomes in the skin and in the circulatory system and limiting the number of infective parasites for subsequent multiplication. Products of dead trypanosomes also induce activation of the immune system which triggers the production of neutralising antibodies (Grootenhuis et al., 1990; Olubayo et al., 1990; Sendoshonga and Black, 1982). This results in low parasitaemia and no anaemia in some high trypanotolerant wildlife of Africa. Consequently, it would appear that the non-immunological mechanisms play a primary role in trypanotolerance which is subsequently supported by a secondary defense mechanism mounted by the immune system. It would be of highest interest to extend these studies to trypanotolerant farm animals in order to identify and/or to isolate related molecules and/or to isolate cell lines producing the molecules.

2.2.5. Treatment Index

Berenil, diminazene aceturate, has a short preventive effect, the shortest of all the drugs so far in use, as it is rapidly excreted. For practical purposes it may be used to establish the challenge in an area. An experimental herd of cattle is allowed to run in the area in which it is required to establish the challenge. Blood samples of each of them are examined at regular intervals. Infected cattle are treated by Berenil at the time of the examination. The number of treatments recorded reflects the amount of infection in the area and this, the challenge, is expressed as the average number of infections each animal is likely to contract per annum.

In the same manner in an experimental or a commercial herd, the registration of the number of treatments with Berenil necessary for each individual to control trypanosomiasis during an observation period of at least one year, may allow to detect more sensitive or more resistant individuals. Using this method at the Madina Diassa ranch in Mali, M.Traore (1989) could distinguish 3 different groups in an experimental N'Dama herd: sensitive animals (11%), tolerant animals (72%), resistant animals (17%).

This method can be used in commercial herds for identifying more sensitive animals and even more sensitive lines in order to eliminate them from selection programmes. (Verhulst, personal communication). But it seems not to be a practical tool for genetic research because of lack of sensitivity, objectivity, accuracy and feasibility because of slow results.

3. CONCLUSIONS

With regard to the evaluation of the genetics of trypanotolerance and the potential of its use in breeding programmes using relevant selection procedures, a number of priorities should be recommended.

3.1 Field Test under Natural Infection

A field test under natural infection as developed by Trail et al. (1991) in Gabon, may be of very high interest to improve the productivity of cattle maintained in areas of trypanosome risk. The test should be done on one-year old cattle and anaemia control measurements carried out over 6 weeks of detected parasitaemia.

The test may be useful in big commercial ranches, where sufficient young cattle of same age and same date of birth will be available. Animals can only be compared within the same test group since experimental and environmental factors (rainfall, bush fires, regeneration of grasses, calving and weaning periods, entomological and epidemiological situation) are weakly repeatable. One must notice that this test will not select the trypanotolerance trait sensu stricto, but several traits that all together contribute to a better productivity in tsetse infested areas and probably in others as well.

3.2 Experimental Test using a Single VAT Infection

A “Trypanotolerance test” using a single VAT infection, as developed with AnTat 1/1 in the STD programme of EEC at the Institute of Tropical Medicine Antwerp-Belgium, can be used very easily at any time and place, in tsetse infected areas of Africa. The duration of the test is approximatively 15–20 days for the non-resistant animals and 30–40 days for the more or less trypanotolerant individuals.

In the initial stages, it will be interesting to apply this test on farm animals that have already been tested for trypanotolerance by other methods. Only at a later stage the test should be applied on trypanotolerant cattle of known parentage. Although this test has to be examined on a larger number of animals, the first results indicate that the test will probably produce accurate data, that can be used in genetic studies and in the identification and the selection of genetically more resistant lines. The most useful criteria will probably be the recovery time of APW, the recovery time of CPW, the cumulative parasitaemia and the duration of parasitaemia. The AnTat 1/1-specific trypanolytic antibodies, the variation in PCV values and the heterologous antibodies (CATT) may provide additional information. Other parameters will possibly be added to the panel of criteria e.g. C₃ factor, B factor, interleukine 2 and conglutinin.

3.3 Treatment Index

The registration of the number of treatments with Berenil required for each animal of a herd under natural tsetse challenge to control trypanosomiasis during an observation period of at least one year may lead to the identification of more sensitive and more resistant individuals. In farms where parentages are known, this procedure could probably be helpful to select the more resistant lines and to eliminate the most sensitive ones from the breeding programmes.

3.4 Basic Research

In strict experimental conditions, special efforts should be made:

1. to detect trypanotoxin or detrimental factors which damage metacyclic trypanosomes in vitro; they have already been identified in the sera of buffalo, eland and waterbuck (Oluhayo, 1991) and possibly exist in serum of some domestic animals that have adapted to survive in tsetse infested Africa since several millennia;

2. to elucidate the role of the alternative pathway of complement that can operate without antibody and that comprises the interaction of 7 proteins: C₃, C₃b, B, D, H, I and P (Alper et al., 1981). The B factor in particular has been proven to have a heritability of 0.93 (p <0,1) in cattle (Tabel et al., 1984);

3. to screen the genetic markers (Bola, Ola, isoenzymes blood groups, etc…) on cattle and/or small ruminants that have proven to possess one or several genetic traits involved in trypanotolerance. Genetic markers could then be used to identify genes, enabling cloning and characterisation and possibly the definition of DNA markers.

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