L.L. LOGAN, R.W. PALING, S.K. MOLOO and J.R. SCOTT
Introduction
Materials and methods
Results
Discussion
References
Genetic resistance to animal trypanosomiasis has been attributed to certain breeds of livestock, most notably to the indigenous West African N'Dama (Murray et al., 1979). This resistance is manifested by the N'Dama's ability to withstand the adverse effects of trypanosomiasis by regulating parasite growth; their ability to prevent or reduce the rate and degree of development of anaemia; their ability to sustain normal weight gains; and their ability to maintain normal ovarian function (Paling et al., in press). A better understanding of the mechanisms involved in trypanotolerance could aid in the search for genetic markers that could be used for the selective breeding of resistant cattle.
A study was initiated at ILRAD to compare changes in the parasitaemia levels and haematological values of previously uninfected N'Dama and Boran cattle sequentially challenged with four different serodemes of Trypanosoma congolense.
Animals
Ten N'Dama (B. taurus), transferred as frozen embryos from The Gambia and implanted into Boran heifers (Jordt et al., 1986) and 34 age and sex matched Boran (B. indicus) of Kenyan origin were raised in a trypanosomiasis-free area of Kenya.
Experimental Design
Eight yearling N'Dama (Group 1) and eight yearling Boran (Group 2), were challenged with Glossina morsitans centralis infected with T. congolense on five different occasions using eight infected fly bites per animal (Table 1). The first four T. congolense challenges were done using clones IL 1180, IL 2642, IL 1587 and IL 2079, respectively, belonging to four different serodemes of East African origin. The fifth challenge was with IL 1180, the same clone to which the cattle in groups 1 and 2 had been first exposed two years previously. Naive groups of eight Boran were included in the second to fourth challenges (Groups 3, 4, 5) and two naive Boran were included in the fifth challenge (Group 6).
Two naive N'Dama were maintained as controls throughout the five challenges.
Table 1. Experimental design for the repeated challenge of N'Dama and Boran cattle with Trypanosoma congolense-infected Glossina morsitans centralist
|
|
Infection |
Infection |
Infection |
Infection |
Infection |
|
1 |
2 |
3 |
4 |
5 |
|
|
Clone |
IL 1180 |
IL 2642 |
IL 1587 |
IL 2079 |
IL 1180 |
|
Groupsa |
1+2 |
1+2+3 |
1+2+4 |
1+2+5 |
1+2+6 |
|
Infection date |
6.5.85 |
11.11.85 |
19.5.86 |
17.11.86 |
27.4.87 |
|
Termination dated |
17.10.85 |
18.3.86 |
15.10.86 |
31.3.87 |
18.8.87 |
|
Duration (days) |
164 |
127 |
149 |
133 |
105 |
|
|
a Group 1 N'Dama |
: 5 females |
3 males |
|
|
Group 2 Boran |
: 5 females |
3 males |
|
|
Groups 3, 4 and 5 Boran |
: 4 females |
4 males |
|
|
Group 6 Boran |
: 1 female |
1 male |
|
|
b Treatment with Berenil 7 mg/kg | ||
Trypanosomes
T. congolense clones, IL 1180, IL 2642, IL 1587, IL 2079 from four different serodemes were used to infect the cattle. T. congolense IL 1180, a double-cloned derivative of STIB 212, was isolated in 1971 from a lion in the Serengeti region of Tanzania (Geigy et al., 1973; Nantulya et al., 1984). T. congolense IL 2642 was derived from IL 285 by two passages in irradiated mice. T. congolense IL 285 was a derivative of EATRO 209, isolated in 1962 from a cow in Busoga, Uganda (Emery and Moloo, 1981; Dwinger et al., 1987). T. congolense IL 1587 was a double cloned derivative of C24 (an isolate made in 1976 from a dog in Nairobi) (swinger et al., 1987). T. congolense IL 2076 was a clone derived from IL 20 E6 by two passages in irradiated mice. T. congolense IL 20 E-6 was a stock derived from STIB 249 by two passages in mice. T. congolense STIB 249 was originally isolated in 1971 from a lion in the Serengeti region of Tanzania (Geigy et al., 1973) and was known to be antigenically different from STIB 212 (Schlappi and Jenni, 1977; Nantulya et al., 1980).
The three T. congolense clones, IL 1180, IL 2642 and IL 1587, were known to belong to different serodemes and to produce chancre reactions when transmitted by tsetse flies (Emery and Moloo, 1981; Akol and Murray, 1983; Dwinger et al., 1987).
Infection
Glossina morsitans centralis from the ILRAD colony were infected by feeding on flanks of goats infected with one of the above T. congolense clones. Infected tsetse flies were identified by the standard warm-probe method and eight infected tsetse were allowed to feed on the left flank of each animal (Emery and Moloo, 1981; Dwinger et al., 1986).
Jugular blood samples were collected into EDTA-coated 10 ml vacutainer tubes. Samples were collected daily for the first 31 days following each of the four challenges and thereafter twice weekly. During the five infection periods the following parameters were measured: development of parasitaemia by the dark ground method (DG) (Paris et al., 1982); anaemia as judged by the per cent blood packed cell volume (PCV); total erythrocyte counts and reticulocyte counts; total leukocyte counts; and weekly animal weights.
When the PCV of an animal dropped to 15% it was treated with Berenil (7 mg/kg) by deep intramuscular injection. At the end of each experimental time period, all the animals were treated with Berenil. A detailed study of leukocyte changes was made during the second challenge (Ellis et al., 1987). Observations on oestrus behaviour of N'Dama heifers started after treatment of the second T. congolense infection and continued through the third challenge. Progesterone profiles were determined (Lorenzini et al., in preparation).
Statistical analysis
Differences between breeds in treatment requirements, parasitaemia and PCV and between reactions to different parasite clones as well as differences between individual animals were tested for significance at probability levels of 0.05 or less.
None of the eight N'Dama required drug treatment during the course of five challenges with T. congolense. By contrast, the eight Boran (Group 2) that received five sequential challenges required Berenil treatment in 30 out of 40 (75%) T. congolense infections. The overall number of treatments required by all the Boran groups was 48 out of 66 infections or 73% (Table 2).
During the first four sequential infections in Group 2 Boran, there were no significant differences between numbers of animals requiring treatment during each trypanosomiasis infection or the average time (days) between the initial infections and treatment time, 42 (s.d. 10) days. Likewise of the four groups of naive Boran experiencing primary infections with T. congolense, treatment was required in 23 of 32 infections on an average of 49 (s.d. 13) days following infection. Again there was no significant difference in the number of animals requiring treatment between each of the four infection periods (Table 2).
Table 2. Requirements for trypanocidal drug treatmentsa of N'Dama and Boran infected by four antigenically different tsetse-transmitted clones of Trypanosoma congolense.
|
Breed |
T. congolense clone |
|||||
|
Group No |
IL 1180 |
IL 2642 |
IL 1587 |
IL 2079 |
IL 1180 Rechallenge |
|
|
N'Dama |
1 |
0/8b |
0/8 |
0/8 |
0/8 |
0/8 |
|
Boran |
2 |
6/8 (41)c |
8/8 (40) |
5/8 (39) |
7/8 (47) |
4/8 (63) |
|
Boran |
3 |
- |
5/8 (59) |
- |
- |
- |
|
Boran |
4 |
- |
- |
6/8 (44) |
- |
- |
|
Boran |
5 |
- |
- |
- |
6/8 (53) |
- |
|
Boran |
6 |
- |
- |
- |
- |
1/2 (84) |
a Berenil (7 mg/kg) was given when a PCV level of 15% was reached.
b Number of animals treated over number of animals infected.
c Average number of days post-infection (in brackets) after which treatment was required.
During the first exposure of the Boran to IL 1180 (Group 2) in May 1985, six of eight animals required Berenil treatment. Two years later this same group of cattle exposed to the same IL 1180 resulted in four of the eight requiring treatment. The average number of days to treatment in the first challenge was 41 days and 63 days in the second.
Figure 1. Parasitaemia score of N'Dama and Boran following challenge with four different Trypanosoma congolense clones.
During four challenges with different serodemes of T. congolense, trypanosomes were detected in the blood of all N'Dama and Boran 11.1 days on average (10.0-12.7) after infection. There was no significant difference in the prepatent periods of infections between the two breeds of cattle or between the four challenges (Figure 1). Similarly, there was no significant difference in the average number of days to the first peak of parasitaemia (average 14.3 days, range 13.9-16.0 days).
The average number of parasites per ml blood during the first parasitaemia peak was significantly lower (P<0.05) in N'Dama than in Boran (score average 4.00 versus 4.32, equivalent to approximately 2.5 and 7.8x10 trypanosomes/ml, respectively). The pattern of parasitaemia during each of the four infection periods in N'Dama was similar and could be divided into three phases (Figure 1). During the first phase (day 1-34), the onset of parasitaemia was followed by the first and highest peak thereafter the parasitaemia gradually dropped to 5.5x103 trypanosomes/ml (score of 2). A similar reduction in the level of parasitaemia took place in the Boran during this period. During a second phase (day 35-140) the six out of 32 Boran that did not receive treatment continued to have an average parasitaemia of approximately 5.5x10 trypanosomes/ml (score 2). During the second phase 26 of 32 Boran required treatment (day 42, std. 10). In contrast, the N'Dama entered a second phase (day 35-80) in which a further decline in parasitaemia occurred. By day 80, parasitaemia in N'Dama had dropped to very low levels (score below one) and intermittent parasitaemia continued until final treatment. Over the four infection periods, the overall mean parasitaemia score for the N'Dama was 2.10. This was significantly lower (P<0.05) than the score of the three untreated Boran in Group 2 (2.72). Within the group of N'Dama's, there were individual animals which consistently had significantly lower mean parasitaemia levels than the rest of the group. During the second IL 1180 challenge five of eight N'Dama became parasitaemia on day 18, peaked on day 22 post-infection and thereafter parasites were sporadically detected in the blood between days 22-67 (Figure 3). Between day 67 - 105 no parasites were detected in any of the N'Dama cattle. Six of eight Boran (Group 2) that were rechallenged with IL 1180 and both naive Boran (Group 6) became parasitaemia on day 12 and peaked on day 20. Four of the rechallenged Boran required treatment and two became aparasitaemic from day 87 onward. In the naive Boran group one animal was treated at day 80 and the second remained parasitaemia until the end of the 105-day period. The parasitaemia levels of the Boran in both challenges were not significantly different.
The fall in PCV in the N'Dama cattle during each of the four infection periods followed a similar pattern (Figure 2) which could be divided into five phases. Initially there was a short phase from day 10 to 15 during which the average daily PCV in N'Dama and Boran dropped at the same rate. In a second phase, from day 15 to 35, the average PCV in N'Dama decreased at a slower rate than that of the Boran. During the same period, the parasite levels of both groups were decreasing at a similar rate. During a third phase, from day 35 to 60, the average daily PCV in N'Dama remained static. By this time, most of the Boran had already required treatment. Phase 4 was the period in which the PCV level of N'Dama spontaneously rose to 30%. The average PCV of N'Dama had dropped to 22% during the first two infections and in the last two infections to 24 and 25%. The final time period (phase 5) was the period during which PCV values over 30% were recorded for all the N'Dama. This period started on day 120 during the first infection and on day 80 (s.d. 7 days) during the three infections that followed and it lasted until each infection was terminated with treatment.
Figure 2. Packed cell volume (PCV): changes in N'Dama and Boran following challenge with four different Trypanosoma congolense clones
Figure 3. Comparison of parasite members and packed cell volume (PCV) for N'Dama and Boran for the first and fifth challenge with T. congolense IL 1180 clone.
IL 1180 - First challenge
IL 1180 - Second challenge
A comparison of the PCV values of the first and fifth challenge (Figure 3) show that in the second IL 1180 challenge the N'Dama began with a PCV of 37% and remained stable over the 105-day period with the lowest level reached being 29%. Six of eight Boran in Group 2 required treatment when their PCV dropped to 15%.
The observations made on parasitaemia level and PCV during the first four phases were used to calculate the individual and group means during each of the infections in the N'Dama; observations recorded after the groups daily average PCV reached 30% were not included. For the Boran the same time periods as for the N'Dama were used to calculate the means.
The overall mean PCV during the four successive infections with T. congolense was compared between the eight N'Dama (29.2%) and the three of eight Boran of Group 2 (24.7%) which on six occasions did not require treatment. The PCV of the N'Dama was significantly higher (P < 0.05) than that of the Boran.
The lack of a vaccine and the expense as well as practical difficulties of implementing control measures for the control of African trypanosomiasis in livestock has in recent years generated considerable interest in the exploitation of trypanotolerant breeds of cattle such as N'Dama and West African Shorthorn. N'Dama cattle have been shown to possess a marked degree of resistance to African trypanosomiasis (Roberts and Gray, 1973). This resistance is manifest in the N'Dama cattle's ability to control parasitaemia and their ability to recover from anaemia without total parasite clearance.
Our studies, conducted under controlled conditions, demonstrate that naive yearling N'Dama are able to control and recover from tsetse-transmitted T. congolense infections without treatment and are able to gain weight and maintain normal ovarian functions during trypanosome infection. None of the N'Dama required treatment during five successive challenges with T. congolense. Three quarters of the Group 2 Boran required treatment to avoid possible death. Following each sequential infection the N'Dama controlled their parasitaemias and recovered from anaemia in shorter intervals. This capacity to rapidly recover from anaemia was not related to an improvement in the control of the parasitaemia nor to a difference in virulence of the T. congolense clones used.
The rate of development and the degree of anaemia in N'Dama compared to zebu following trypanosome infection is considered to be related to a better control of the mean number of parasites in circulation by the N'Dama breed of cattle (Murray et al., 1981). The same general conclusion can be drawn from our study. However when comparing mean parasitaemia and PCV level of the N'Dama group during the subsequent infections, no direct correlation between the parameters was evident. Similarly, there was no direct correlation between the mean parasitaemia and PCV levels of individual N'Dama measured during a total of 32 infections. Some N'Dama demonstrated a consistently better ability to control the development of anaemia than others during the four infections, while others demonstrated a better parasite control. This might indicate that both processes are genetically controlled but are not directly linked to each other. Packed cell volume levels of trypanotolerant livestock in tsetse infested areas are considered to be positively correlated with production traits like reproductive performance and growth (ILCA, 1986). Based on these preliminary studies, N'Dama with the highest mean PCV during primary trypanosome infection could probably be regarded as the most tolerant and therefore also the most productive when exposed to trypanosomiasis risk.
The superior response of N'Dama to trypanosomiasis may be the result of a superior erythropoietic response, more resistant erythrocytes, or differences in their monocytic phagocytic system in terms of parasite and cell clearance mechanisms. Comparative studies of N'Dama and Boran conducted in each of these areas may reveal some of the genetic characteristics that constitute trypanotolerance and may lead to the identification of genetic markers that could be used for selective breeding of trypanotolerant livestock.
As suggested earlier (Paling et al., in press), under controlled experimental conditions the mean PCV of an animal within a group of age matched N'Dama, measured during primary infection with T. congolense, might serve as a selection criterion for trypanotolerance.
Acknowledgements
We are grateful for the technical assistance and excellent care of experimental animals provided by Messrs. F. McOdimba, D. Muteti, G. Lamb, J. Kamau and to the staff of the tsetse vector laboratory and the large animal unit. This is ILRAD publication No. 594.
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