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Relationships between trypanosome infection measured by antigen detection enzyme immunoassays, anaemia and growth in trypanotolerant N'Dama cattle
Veterinary Parasitology, 42 (1992) 213-223 Elsevier Science Publishers B.V., Amsterdam
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Relationships between trypanosome infection measured by antigen detection enzyme immunoassays, anaemia and growth in trypanotolerant N'Dama cattle J.C.M. Trail a, G.D.M. d'Ieteren a, P. Viviani b, G. Yangari b and V.M. Nantulya c alnternational Livestock Centre for Africa (ILCA), Nairobi, Kenya bOGAPROV, Moanda, Gabon Clnternational Laboratory for Research on Animal Diseases (ILRAD), NairobL Kenya (Accepted 23 October 1991 ) ABSTRACT Trail, J.C.M., d'Ieteren, G.D.M., Viviani, P., Yangari, G. and Nantulya, V.M., 1992. Relationships between trypanosome infection measured by antigen detection enzyme immunoassays, anaemia and growth in trypanotolerant N 'Dama cattle. Vet. Parasitol., 42." 213-223. Relationships were evaluated between trypanosome infection as measured by antigen detection enzyme immunoassays (antigen ELISA), anaemia as determined by average packed red cell volume (PCV), and animal performance as assessed by daily weight gain in 99 N'Dama cattle in Gabon exposed to natural tsetse challenge at I 1.5 months of age and recorded 14 times over a 13 week period. Approximately half the animals were found to be infected for an average of five of the 14 times that they were examined: 38% with Trypanosoma congolense, 13% with Trypanosoma vivax and 49% with a mixed infection. Trypanosoma congolense infections had significant deleterious effects on animal growth, while T. vivax infections did not. Animals found on several occasions to be infected with T. congolense had significantly lower PCV values than those demonstrated to be infected on fewer occasions. No relationship was found between mean optical density (OD) values in antigen ELISA and PCV values. Animals capable of maintaining PCV values, even when antigen ELISA positive on a high number of occasions, grew at the same rate as uninfected animals. Animals that could not maintain PCV values when infected had poorer growth. Antigen ELISA has the potential to increase the efficiency of selection oftrypanotolerant N'Dama cattle under tsetse challenge in the field, in three main ways. ( 1 ) Accurate identification of trypanosome species, especially in mixed species infections, clarifies relations between infection, anaemia and animal performance. (2) Detection of animals antigenaemic without patent parasitaemia could allow individuals with superior ability to control trypanosome infection to be identified. ( 3 ) More accurate measurement of the proportion of time an animal is infected allows more accurate evaluation of its anaemia control capability.
INTRODUCTION
The most important cattle breed expressing the trypanotolerance trait, the ability to survive and be productive in tsetse-infested areas without the aid of Correspondence to: G.D.M. d'Ieteren, International Livestock Centre for Africa (ILCA), P.O. Box 46847, Nairobi, Kenya. © 1992 Elsevier Science Publishers B.V. All rights reserved 0304-4017/92/$05.00
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trypanocidal drugs is the N'Dama. Major constraints to improving the breed's genetic resistance to trypanosomiasis have been related to difficulties in quantifying trypanotolerance traits. Previous studies reviewed by Murray et al. (1990) have concluded that the ability to control the development of anaemia and to control the level of parasitaemia are key indicators of the trypanotolerance trait and that these processes, although controlled genetically, are not necessarily directly linked to each other. In field studies, the degree of anaemia can be easily quantified by measuring packed red cell volume (PCV). In contrast, the level of parasitaemia is not so easily quantified, and has depended on demonstration of trypanosomes in peripheral blood by parasitological techniques. To date, the most sensitive practical field approach has been the detection of trypanosomes by the dark ground/phase contrast buffy coat method (Murray et al., 1977 ) and quantification of the level of infection on the basis of a parasitaemia score (Paris et al., 1982). However, a high proportion of infections go undetected as the level of parasitaemia fluctuates markedly and is often below the limit of detection of the technique (Masake and Nantulya, 1991 ). Recently, antigen detection enzyme immunoassays (antigen ELISA) for the diagnosis of Trypanosoma vivax, Trypanosoma congolense and Trypanosoma brucei infections in cattle have been developed and tested (Nantulya and Lindqvist, 1989; Nantulya, 1990). The assays are based on monoclonal antibodies which recognise trypanosome antigens specific for the three trypanosome species. Demonstration of trypanosomal antigens in the blood of the animal is therefore synonymous with parasitological diagnosis unless the animal had self-cured within the previous 3 weeks. Nantulya (1990) reported that significant numbers of cattle of a trypanosusceptible breed, that were not detected to be parasitaemic by routine parasite detection techniques, were found to be infected by the tests for antigen. Trail et al. ( 1992 ) reported similar results with trypanotolerant N'Dama cattle, and concluded that the ELISA could offer a practical possibility for selection oftrypanotolerant animals based on infection criteria. There are important areas where results of antigen detection enzyme immunoassays may clarify and quantify relationships between trypanosome infection, anaemia and animal growth in N'Dama cattle. (1) Animals which are antigenaemic without patent parasitaemia could represent individuals with the best ability to control the infection. These would be the target for selection and breeding under field conditions. (2) Animals found to be negative in the antigen detection test may provide more accurate comparators for evaluation of anaemia control under trypanosome infection, as past work on anaemia, using animals not detected as parasitaemic as controls, has suggested that a proportion of these animals was actually infected, but could not be identified by the phase contrast test used (Trail et al., 1992).
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(3) More accurate determination of the prevalence of different trypanosome species, especially in mixed infections, is possible through the tests for antigen. Thus, Trail et al. (1992) found that while it was rare for field workers to report mixed infections when using the buffy coat technique, a high proportion of individual antigen tests showed this to be the case. This is of importance as trypanosome species can differ in different geographical areas in their pathogenicity in cattle (Stephen, 1986 ). (4) The level of parasitaemia, as measured by the score of Paris et al. ( 1982 ), has shown little relationship with animal performance (Trail et al., 1991 a,b ), possibly because the sensitivity of the test used is not high enough to exclude low grade infections. It has yet to be determined whether in the tests for antigen, the level of antigen titres can be used as an indicator of level of parasitosis. This present study was therefore undertaken to evaluate whether information from antigen detection enzyme immunoassays can be used to quantify relationships between infection, anaemia and animal growth in post-weaner N'Dama cattle exposed to a medium natural trypanosome challenge in Gabon. MATERIAL AND METHODS
Ninety-nine N'Dama cattle at the Government Ranch of the Office Gabonais d'Amelioration et de Production de la Viande (OGAPROV) in southeastern Gabon were exposed to a short-term, natural trypanosome challenge at an average age of 11.5 months. The tsetse challenge when recorded in 19841987 was medium, with a low fly density but high infection rate (Leak et al., 1991 ). Glossina tabaniformis was the main tsetse species while Glossina palpalis and Glossina nashi were also present. The study lasted 13 weeks, from October to December 1990. The animals had been born over a 3 month period in five separate multi-sire herds. The calves remained in these five herds until weaning at 8 months of age, when they were separated into male and female weaner herds; they were maintained in these two herds until the study had been completed. Blood samples were examined 14 times over the 13 weeks by the dark ground/phase contrast buffy coat method to detect the presence of trypanosomes (Murray et al., 1977 ). The degree of anaemia was estimated by measuring the PCV. Attempts were made to systematically control other possible causes of anaemia, ticks by weekly dipping and internal parasites through 3monthly dosing with anthelmintics. Animals were weighed weekly. Sera were prepared from the blood samples of all animals at the time of each weekly bleed and stored at - 15 ° C until transported to the International Laboratory for Research on Animal Diseases (ILRAD), Nairobi at the end of the test period. Antigen ELISA for T. congolense and T. vivax antigens was carried out as described by Nantulya and Lindqvist (1989), and optical density (OD)
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values determined. Three OD values, 0.05, 0.06 and 0.07 were separately regarded as positive thresholds for antigenaemia and compared throughout. Animals were first classified as to whether or not they had been detected parasitaemic, and whether or not they had been found to be antigenaemic in one or more of the 14 tests. Preliminary regression analyses showed T. vivax infections to have virtually no effect on animal performance, so data on T. congolense infections only were used. Animals positive by antigen ELISA were subdivided into those above and those below average for number of times positive, and above and below average for mean optical density of all positive samples. Animals were further subdivided, within classes of antigen-positive and antigen-negative animals, into those above and below average for average PCV over the 14 measurements. Analyses were carried out using least squares fixed model procedures (Harvey, 1977 ). The models fitted the effects of herd of origin, weaner herd (male or female herd ) and included the following where appropriate: whether detected as antigenaemic; number of times detected antigenaemic and average optical density nested within the antigen-positive class; average PCV nested within both antigen-positive and antigen-negative classes. Analyses were carried out on the anaemia control measure of individual animal's average PCV and the performance measure of daily weight gain over the 13 weeks. RESULTS
Trypanosoma congolense and T. vivax infections identified by antigen
ELISA When an OD of 0.05 which is recommended for use in bovine serum analysis (Nantulya et al., 1992), was taken as the threshold value regarded as positive, 283 (21% ) of the 1338 samples were shown to contain antigen and 55 (55%) of the 99 animals were found to be antigenaemic on one or more occasions. Animals identified as antigenaemic were positive on an average of 5.1 of the 14 occasions. The numbers of T. congolense, T. vivax and mixed infections are shown in Table 1 (a). Trypanosoma congolense and T. vivax were found as single infections in 64% and 22% of the samples respectively, while 14% were mixed. Overall, therefore, T. congolense was detected in 78% and T. vivax in 36% of the serum samples. When evaluated on an individual animal basis (Table 1 ( b ) ) , T. congolense and T. vivax were found as single species infections in 38% and 13% of the animals, while the other 49% had mixed infections. Overall, T. congolense was detected in 87% of infected animals and T. vivax in 62%. When the higher OD values of 0.06 and 0.07 were taken as the threshold, the percentages of samples regarded as antigen ELISA positive were reduced to 16% and 14% respectively, and the percentages of animals to 54% and 50%. Trypanosome species distributions did not change markedly (Table 1 ).
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TABLE1 Trypanosome species identified by antigen test using three different levels of optical density as the threshold for positivity Trypanosome species
Optical density required for antigen-positive test 0.05
(a) In individual samples T. congolense T. vivax Mixed I Total
0.07
No.
%
No.
%
No.
%
180 63 40 283
64 22 14 100
137 51 30 218
63 23 14 100
113 56 19 188
60 30 10 100
38
21
39
22
44
13
8
15
7
14
49 100
25 54
46 100
21 50
42 100
(b) In animals (14 samples per animal) T. congolense 21 T. vivax 7 Mixed ~ Total
0.06
27 55
tMixed T. congolense/T, vivax infections.
Trypanosoma congolense and T. vivax infections detected by both antigen
ELISA and buffy coat technique Eight animals only were found to be parasitaemic by the buffy coat technique on a total of 15 occasions out of 106 weekly tests. Seven of the animals were parasitaemic on a total of 14 occasions during this period but were antigenaemic on a total of 49 occasions. In contrast, the eighth animal, parasitaemic in 1 week, was never found to be antigenaemic. While 12 of the parasitaemias were reported as T. vivax and two as T. congolense, the 49 serum samples in which antigen was detected could be divided into two containing only T. vivax antigen, 15 mixed and 32 containing only T. congolense antigen.
Trypanosome species as determined by antigen ELISA and animal performance Preliminary analyses, using the 0.05 OD level, on the 27 animals with mixed
T. congolense and T. vivax infections ( T. congolense on an average of 5.6 occasions and T. vivax on an average of 3.2) revealed that T. vivax infections had virtually no effect on animal performance, while T. congolense infections had significant deleterious effects. The regression of daily weight gain on the number of T. vivax infections found in the animals infected with both species was - 1.0 + 3.3 g d a y - 1, while the regression of gain on the number of T. congolense infections found was - 6.2 +_2.9 g d a y - 1. When data on T. vivax and T. congolense infections were added together, the regression of gain on the
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total number of infections was reduced to - 3.6 _+2.1 g d a y - 1. The seven animals that were infected with T. vivax only (on an average of 2.5 occasions) had approximately the same daily weight gains over the 13 weeks as had the negative animals (86.8 + 18.3 g d a y - 1 compared with 88.8 + 7.7 g day- ~). Therefore, in all subsequent analyses involving performance, data referring to T. congolense infections only were considered. Thus the seven animals infected with T. vivax only were not considered as being infected, and the animals with mixed infections were evaluated on the basis of their T. congolense infection data only.
Antigenaemia and animal performance The relationship between antigen ELISA positivity during the 13 weeks and average PCV and daily weight gain, using three OD thresholds for a positive antigen test, are shown in Table 2. A positive as compared with a negative antigen test appeared to be associated with a lower average PCV and lower weight gain at all three thresholds, but this was only significant for PCV at the 0.05 OD threshold. Table 3 shows the results of introducing the effects of low and high numbers of times positive and low and high OD values, into the analysis. In each case, low refers to below average and high to above average. Animals antigenaemic for a high number of times (approximately three times as many as those antigenaemic a low number of times) had significantly lower average PCV values at all three test thresholds. Differences in weight gains followed the same patterns but did not reach significance. Animals antigenaemic with a high average OD (approximately twice that of those with a low average) had the same PCV values at all three test levels. They tended to have lower weight gains, but differences did not reach significance. N u m b e r of times antigenTABLE2 Least s q u a r e s m e a n s ( + S E ) for effects o f a n t i g e n a e m i a , d e t e r m i n e d at three t h r e s h o l d levels, on average P C V a n d daily weight gain Optical density t h r e s h o l d level
Antigen test result
No. o f animals
PCV (%)
Gain (g d a y - ~)
0.05
+ + + -
48 51 45 54 43 56
31.1 + 0.36 32.1 + 0 . 3 7 31.3+0.38 32.1 __+0.37 31.4 + 0.39 32.0 + 0.36
74.3 + 6.89 87.7__+7.03 73.3+7.13 88.8+6.91 72.7 + 7.19 89.6__+ 6.67
0.06 0.07
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TABLE 3 Least squares means ( + SE) for effects of (a) number of times antigenaemic, and (b) mean optical density of antigenaemia, determined at three threshold levels, on average PCV and daily weight gain Optical density threshold level
Class
No. of animals
PCV (%)
Gain (g d a y - ~)
Low (2.46) High (7.55) Low (1.83) High (5.85) Low (1.30) High (4.15)
28 20 24 21 20 23
32.4+0.47 29.9+0.56 32.3+0.52 30.3+0.56 32.3+0.57 30.5+0.54
82.2+ 66.4+ 84.4+ 62.1 + 81.4+ 64.0+
9.01 10.62 9.62 10.41 10.51 9.84
Low (0.070) High (0.141) Low (0.082) High (0.157) Low (0.091) High (0.162)
24 24 23 22 21 22
31.2+0.53 31.1 +0.52 31.3+0.53 31.3+0.56 31.8+0.57 31.0+0.57
86.6+ 62.0+ 79.0+ 67.5+ 85.0+ 60.4+
10.01 9.94 9.87 10.45 10.16 10.40
(a) Number of times antigenaemic 0.05 0.06 0.07
(b) Mean optical density 0.05 0.06 0.07
TABLE 4 Least squares means ( + SE) for effects of average PCV within number of times antigenaemic, determined at three threshold levels, on daily weight gain AntiPCV 0.05 Antigen threshold genaemia class class No. of Mean Gain animals PCV (gday -~)
(%)
Negative
Low High Low Low frequency High High Low frequency High
25 26 14 14 10 10
29.8 33.9 30.7 33.8 27.5 32.1
84.1+ 9.46 90.6+ 9.47 73.6+12.28 96.3+ 12.18 35.7+14.56 90.6+14.51
0.06 Antigen threshold
0.07 Antigen threshold
No. of Mean Gain animals PCV (gday - l )
No. of Mean Gain animals PCV (gday -~)
27 27 12 12 11 10
28 28 10 10 11 12
(%)
29.8 33.8 30.8 33.6 27.7 33.0
86.7+ 9.28 90.2+ 9.36 73.0+13.29 96.0+ 13.29 40.7_+14.11 86.2+14.64
(%)
29.8 33.8 30.9 33.6 27.7 32.9
86.9+ 9.07 92.3+ 9.16 73.7+14.86 88.9+ 14.60 41.0_+14.15 85.4+13.53
aemic and average OD were independent of each other, the regression of OD on number o f infections being a non-significant 0.003 OD units per infection.
PCV values and animal performance The effects o f above or below average PCV values on daily weight gain relative to whether detected antigenaemic a high number of times, a low number o f times, or not at all, are given in Table 4. When animals were antigenaemic a high number of times, those with a high average PCV grew twice as fast as
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TABLE5 T i m e s taken for a n i m a l s to self cure j f r o m T. A n i m a l category
Self-cured, not infected at start o f period Self-cured, already infected at start o f period Did not self cure d u r i n g period C o u l d not be assessed
congolenseinfection
No. o f animals
No. o f positive tests per a n i m a l + SE
Weeks between first a n d last positive tests +SE
7
3.1 + 0.9
4.0 _+ 1.3
8
4.3 _+ 1.3
5.9 + 1.6
17 16
7.7 _+2.3 NA 2
11.6 _+ 1.9 NA
'Self cure defined as five negative tests after two or m o r e positive tests. 2Not applicable.
those with a low average PCV. This was the case at all three antigen test threshold values. When animals were antigenaemic a low number of times, the differences in daily weight gain between the high and low PCV groups averaged 27% and were not significant. When animals were never detected antigenaemic during the experimental period, the differences between the high and low PCV groups averaged 6%, again not significant.
Self cure from T. congolense infections Self cure was defined for this data set as five consecutive negative weekly tests after two or more positive tests (the lowest OD of 0.05 was used to indicate a positive antigen test). Table 5 shows that 32 of the 48 animals infected with T. congolense during the 13 weeks could be assessed as either having undergone self cure from infection during the period or as clearly not having self-cured. The remaining 16 animals could not be assessed, as infection occurred too late in the experimental period. Seven (21% ) of the animals not infected for 2 or more weeks at the start of the 13 weeks recovered after 3.1 positives over an average period of 4 weeks. Eight (25%) of the animals that had already been infected at the start of the 13 weeks recovered after 4.3 positive tests over an average period of 6 weeks (these last two figures would therefore be underestimates for the animals in this group). The 17 (53%) animals that clearly did not self cure during the 13 week period had 7.7 positive tests identified over an average period of 11.6 weeks. DISCUSSION
This carefully recorded situation, where animals positive for trypanosome antigens were identified as such in an average of five of the 14 weekly tests,
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and where 49% of antigenaemic animals had mixed T. congolense/T, vivax infections, allowed a major trypanosome species effect on animal performance to be detected. While T. congolense infections had significant deleterious effects on animal growth, the local T. vivax serodemes did not. The illustration of how, in mixed infections, the significant negative regression of weight gain on the number of T. congolense infections was obscured if the T. vivax data were not removed, shows the necessity for accurate trypanosome species identification, if infection effects and associations with other criteria of trypanotolerance are to be clarified and accurately quantified in the field. Past work has depended on species identification in the field using the buffy coat technique. When large numbers of animals have to be handled each day, determining the complete repertoire of trypanosome species infecting any one animal is very difficult using this technique. Antigen detection enzyme immunoassays could solve this problem. Because of the low number of animals detected parasitaemic by the buffy coat technique, follow-up work on trypanosome species identification by the two techniques must be carried out at a site and at a time when significant numbers of parasitaemias can be guaranteed. Animals found to be positive by antigen ELISA had lower average PCV values than those found to be negative, but this was only significant when the OD value of 0.05 was used as the threshold. When the number of times an animal was positive was included in the analysis, those with an above average number of positive tests had very significantly lower PCV values at all three of the OD thresholds. In contrast, when the mean OD value for all positive tests on an animal was included in the analysis, there was no association whatsoever with PCV values. In the only previously reported work on the association between trypanosome antigenaemia and PCV (Trail et al., 1992 ), it was only possible to conduct six antigen tests per animal over a 3 m o n t h period. In that study, animals positive for antigens were identified as such in an average of 2.3 tests. With these limited data, no significant effects on PCV or weight gain of antigen positivity, number of times an animal was positive, or mean OD value, were found. Follow-up work, at a site and time when animals are parasitaemic on a relatively large number of occasions will allow determination of whether the importance of the number of times an animal is antigenaemic is akin to the importance of number of times parasitaemic (Trail et al., 1991 a) and whether the lack of importance of mean optical density in the antigen tests is akin to the lack of importance of mean parasitaemia score (Trail et al., 1991 a). The very significant effects of above or below average PCV values on weight gain in the animals that were antigen-positive a high number of times, are akin to those of above or below PCV values in parasitaemic animals reported by Trail et al. ( 199 la). There may, therefore, be possibilities for selection on
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the basis of PCV when antigenaemic, similar to those for selection on the basis of PCV when parasitaemic. The absence o f a relationship between PCV and daily weight gain in the non-antigenaemic group suggests that the antigen test genuinely identified all infected animals, in contrast to previous findings with the buffy coat technique (Trail et al., 1992 ), where it appeared probable that a proportion o f animals, not identified by the technique, was infected. As reported a n u m b e r o f times (Trail et al., 1991a,b,c), infected N ' D a m a cattle grew at the same rate as uninfected N ' D a m a cattle if they could maintain normal PCV values. The finding that a proportion o f animals could be considered as having selfcured from infection after an average of 4 weeks (judged by being negative to the antigen test on five consecutive occasions) while a proportion had not self-cured after an average of 11.6 weeks, suggests that further work on the time taken by individual animals to self cure could be o f value. Two higher OD thresholds in the ELISA, 0.06 and 0.07, were compared with the 0.05 threshold normally regarded as positive (Nantulya et al., 1992 ). Both considerably reduced the n u m b e r o f samples detected as antigen-positive, but had very little effect on the n u m b e r of animals detected as positive during the experiment, or on any of the associations between infection, anaemia and growth parameters. The 0.05 O D threshold level would, therefore, appear to be a satisfactory indicator o f antigen positivity. ACKNOWLEDGEMENTS We are grateful to the m a n a g e m e n t of O G A P R O V Ranch for support in carrying out the field work in Gabon, S.H. Minja and J. Thuo for carrying out the assays at ILRAD and S. Nagda for sample preparation and assistance in data analysis. We thank Dr. E. Authie, Dr. A.S. Peregrine and Dr. G.J. Rowlands for helpful discussions.
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techniques. In: 15th Meeting of the International Scientific Council for Trypanosomiasis Research and Control. The Gambia, OAU/STRC. OAU/STRC, Nairobi, Kenya, Publication No. 110, pp. 247-254. Murray, M., Trail, J.C.M. and d'Ieteren, G.D.M., 1990. Trypanotolerance in cattle and prospects for the control of trypanosomiasis by selective breeding. Rev. Sci. Tech. Off. Int. Epizootiol., 9 (2): 369-386. Nantulya, V.M., 1990. Trypanosomiasis in domestic animals: the problems of diagnosis. Rev. Sci. Tech. Off. Int. Epizootiol., 9 (2): 357-367. Nantulya, V.M. and Lindqvist, K.J., 1989. Antigen-detection enzyme immunoassay for the diagnosis of Trypanosoma vivax, T. congolense and T. brucei infection in cattle. Trop. Med. Parasitol., 40: 267-272. Nantulya, V.M., Lindqvist, K.J., Stevenson, P. and Mwangi, E.K., 1992. Bovine trypanosomiasis at Ngurumen, Kenya: Diagnosis using monoclonal antibody-based antigen detection enzyme-linked immunosorbent assay (antigen ELISA). Ann. Trop. Med. Parasitol., submitted. Paris, J., Murray, M. and McOdimba, F., 1982. An evaluation of the sensitivity of current parasitological techniques for the diagnosis of bovine African trypanosomiasis. Acta Trop., 39: 307-316. Stephen, L.E., 1986. Trypanosomiasis: a Veterinary Perspective. Pergamon Press, Oxford, 551 pp. Trail, J.C.M., d'Ieteren, G.D.M., Colardelle, C., Maille, J.C., Ordner, G., Sauveroche, B. and Yangari, G., 1991a. Evaluation of a field test for trypanotolerance in young N'Dama cattle. Acta Trop., 48: 47-57. Trail, J.C.M., d'Ieteren, G.D.M., Feron, A., Kakiese, O., Mulungo, M. and Pelo, M., 1991b. Effect of trypanosome infection, control of parasitaemia and control of anaemia development on productivity of N'Dama cattle. Acta Trop., 48: 37-45. Trail, J.C.M., d'Ieteren, G.D.M., Maille, J.C. and Yangari, G., 1991c. Genetic aspects of control of anaemia development in trypanotolerant N'Dama cattle. Acta Trop., 48: 285-291. Trail, J.C.M., d'Ieteren, G.D.M., Maille, J.C., Yangari, G. and Nantulya, V.M., t992. Use of antigen-detection enzyme immunoassays in assessment of trypanotolerance in N'Dama cattle. Acta Trop., 50:11-18.
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Relationships between trypanosome infection measured by antigen detection enzyme immunoassays, anaemia and growth in trypanotolerant N'Dama cattle J.C.M. Trail a, G.D.M. d'Ieteren a, P. Viviani b, G. Yangari b and V.M. Nantulya c alnternational Livestock Centre for Africa (ILCA), Nairobi, Kenya bOGAPROV, Moanda, Gabon Clnternational Laboratory for Research on Animal Diseases (ILRAD), NairobL Kenya (Accepted 23 October 1991 ) ABSTRACT Trail, J.C.M., d'Ieteren, G.D.M., Viviani, P., Yangari, G. and Nantulya, V.M., 1992. Relationships between trypanosome infection measured by antigen detection enzyme immunoassays, anaemia and growth in trypanotolerant N 'Dama cattle. Vet. Parasitol., 42." 213-223. Relationships were evaluated between trypanosome infection as measured by antigen detection enzyme immunoassays (antigen ELISA), anaemia as determined by average packed red cell volume (PCV), and animal performance as assessed by daily weight gain in 99 N'Dama cattle in Gabon exposed to natural tsetse challenge at I 1.5 months of age and recorded 14 times over a 13 week period. Approximately half the animals were found to be infected for an average of five of the 14 times that they were examined: 38% with Trypanosoma congolense, 13% with Trypanosoma vivax and 49% with a mixed infection. Trypanosoma congolense infections had significant deleterious effects on animal growth, while T. vivax infections did not. Animals found on several occasions to be infected with T. congolense had significantly lower PCV values than those demonstrated to be infected on fewer occasions. No relationship was found between mean optical density (OD) values in antigen ELISA and PCV values. Animals capable of maintaining PCV values, even when antigen ELISA positive on a high number of occasions, grew at the same rate as uninfected animals. Animals that could not maintain PCV values when infected had poorer growth. Antigen ELISA has the potential to increase the efficiency of selection oftrypanotolerant N'Dama cattle under tsetse challenge in the field, in three main ways. ( 1 ) Accurate identification of trypanosome species, especially in mixed species infections, clarifies relations between infection, anaemia and animal performance. (2) Detection of animals antigenaemic without patent parasitaemia could allow individuals with superior ability to control trypanosome infection to be identified. ( 3 ) More accurate measurement of the proportion of time an animal is infected allows more accurate evaluation of its anaemia control capability.
INTRODUCTION
The most important cattle breed expressing the trypanotolerance trait, the ability to survive and be productive in tsetse-infested areas without the aid of Correspondence to: G.D.M. d'Ieteren, International Livestock Centre for Africa (ILCA), P.O. Box 46847, Nairobi, Kenya. © 1992 Elsevier Science Publishers B.V. All rights reserved 0304-4017/92/$05.00
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trypanocidal drugs is the N'Dama. Major constraints to improving the breed's genetic resistance to trypanosomiasis have been related to difficulties in quantifying trypanotolerance traits. Previous studies reviewed by Murray et al. (1990) have concluded that the ability to control the development of anaemia and to control the level of parasitaemia are key indicators of the trypanotolerance trait and that these processes, although controlled genetically, are not necessarily directly linked to each other. In field studies, the degree of anaemia can be easily quantified by measuring packed red cell volume (PCV). In contrast, the level of parasitaemia is not so easily quantified, and has depended on demonstration of trypanosomes in peripheral blood by parasitological techniques. To date, the most sensitive practical field approach has been the detection of trypanosomes by the dark ground/phase contrast buffy coat method (Murray et al., 1977 ) and quantification of the level of infection on the basis of a parasitaemia score (Paris et al., 1982). However, a high proportion of infections go undetected as the level of parasitaemia fluctuates markedly and is often below the limit of detection of the technique (Masake and Nantulya, 1991 ). Recently, antigen detection enzyme immunoassays (antigen ELISA) for the diagnosis of Trypanosoma vivax, Trypanosoma congolense and Trypanosoma brucei infections in cattle have been developed and tested (Nantulya and Lindqvist, 1989; Nantulya, 1990). The assays are based on monoclonal antibodies which recognise trypanosome antigens specific for the three trypanosome species. Demonstration of trypanosomal antigens in the blood of the animal is therefore synonymous with parasitological diagnosis unless the animal had self-cured within the previous 3 weeks. Nantulya (1990) reported that significant numbers of cattle of a trypanosusceptible breed, that were not detected to be parasitaemic by routine parasite detection techniques, were found to be infected by the tests for antigen. Trail et al. ( 1992 ) reported similar results with trypanotolerant N'Dama cattle, and concluded that the ELISA could offer a practical possibility for selection oftrypanotolerant animals based on infection criteria. There are important areas where results of antigen detection enzyme immunoassays may clarify and quantify relationships between trypanosome infection, anaemia and animal growth in N'Dama cattle. (1) Animals which are antigenaemic without patent parasitaemia could represent individuals with the best ability to control the infection. These would be the target for selection and breeding under field conditions. (2) Animals found to be negative in the antigen detection test may provide more accurate comparators for evaluation of anaemia control under trypanosome infection, as past work on anaemia, using animals not detected as parasitaemic as controls, has suggested that a proportion of these animals was actually infected, but could not be identified by the phase contrast test used (Trail et al., 1992).
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(3) More accurate determination of the prevalence of different trypanosome species, especially in mixed infections, is possible through the tests for antigen. Thus, Trail et al. (1992) found that while it was rare for field workers to report mixed infections when using the buffy coat technique, a high proportion of individual antigen tests showed this to be the case. This is of importance as trypanosome species can differ in different geographical areas in their pathogenicity in cattle (Stephen, 1986 ). (4) The level of parasitaemia, as measured by the score of Paris et al. ( 1982 ), has shown little relationship with animal performance (Trail et al., 1991 a,b ), possibly because the sensitivity of the test used is not high enough to exclude low grade infections. It has yet to be determined whether in the tests for antigen, the level of antigen titres can be used as an indicator of level of parasitosis. This present study was therefore undertaken to evaluate whether information from antigen detection enzyme immunoassays can be used to quantify relationships between infection, anaemia and animal growth in post-weaner N'Dama cattle exposed to a medium natural trypanosome challenge in Gabon. MATERIAL AND METHODS
Ninety-nine N'Dama cattle at the Government Ranch of the Office Gabonais d'Amelioration et de Production de la Viande (OGAPROV) in southeastern Gabon were exposed to a short-term, natural trypanosome challenge at an average age of 11.5 months. The tsetse challenge when recorded in 19841987 was medium, with a low fly density but high infection rate (Leak et al., 1991 ). Glossina tabaniformis was the main tsetse species while Glossina palpalis and Glossina nashi were also present. The study lasted 13 weeks, from October to December 1990. The animals had been born over a 3 month period in five separate multi-sire herds. The calves remained in these five herds until weaning at 8 months of age, when they were separated into male and female weaner herds; they were maintained in these two herds until the study had been completed. Blood samples were examined 14 times over the 13 weeks by the dark ground/phase contrast buffy coat method to detect the presence of trypanosomes (Murray et al., 1977 ). The degree of anaemia was estimated by measuring the PCV. Attempts were made to systematically control other possible causes of anaemia, ticks by weekly dipping and internal parasites through 3monthly dosing with anthelmintics. Animals were weighed weekly. Sera were prepared from the blood samples of all animals at the time of each weekly bleed and stored at - 15 ° C until transported to the International Laboratory for Research on Animal Diseases (ILRAD), Nairobi at the end of the test period. Antigen ELISA for T. congolense and T. vivax antigens was carried out as described by Nantulya and Lindqvist (1989), and optical density (OD)
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values determined. Three OD values, 0.05, 0.06 and 0.07 were separately regarded as positive thresholds for antigenaemia and compared throughout. Animals were first classified as to whether or not they had been detected parasitaemic, and whether or not they had been found to be antigenaemic in one or more of the 14 tests. Preliminary regression analyses showed T. vivax infections to have virtually no effect on animal performance, so data on T. congolense infections only were used. Animals positive by antigen ELISA were subdivided into those above and those below average for number of times positive, and above and below average for mean optical density of all positive samples. Animals were further subdivided, within classes of antigen-positive and antigen-negative animals, into those above and below average for average PCV over the 14 measurements. Analyses were carried out using least squares fixed model procedures (Harvey, 1977 ). The models fitted the effects of herd of origin, weaner herd (male or female herd ) and included the following where appropriate: whether detected as antigenaemic; number of times detected antigenaemic and average optical density nested within the antigen-positive class; average PCV nested within both antigen-positive and antigen-negative classes. Analyses were carried out on the anaemia control measure of individual animal's average PCV and the performance measure of daily weight gain over the 13 weeks. RESULTS
Trypanosoma congolense and T. vivax infections identified by antigen
ELISA When an OD of 0.05 which is recommended for use in bovine serum analysis (Nantulya et al., 1992), was taken as the threshold value regarded as positive, 283 (21% ) of the 1338 samples were shown to contain antigen and 55 (55%) of the 99 animals were found to be antigenaemic on one or more occasions. Animals identified as antigenaemic were positive on an average of 5.1 of the 14 occasions. The numbers of T. congolense, T. vivax and mixed infections are shown in Table 1 (a). Trypanosoma congolense and T. vivax were found as single infections in 64% and 22% of the samples respectively, while 14% were mixed. Overall, therefore, T. congolense was detected in 78% and T. vivax in 36% of the serum samples. When evaluated on an individual animal basis (Table 1 ( b ) ) , T. congolense and T. vivax were found as single species infections in 38% and 13% of the animals, while the other 49% had mixed infections. Overall, T. congolense was detected in 87% of infected animals and T. vivax in 62%. When the higher OD values of 0.06 and 0.07 were taken as the threshold, the percentages of samples regarded as antigen ELISA positive were reduced to 16% and 14% respectively, and the percentages of animals to 54% and 50%. Trypanosome species distributions did not change markedly (Table 1 ).
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TABLE1 Trypanosome species identified by antigen test using three different levels of optical density as the threshold for positivity Trypanosome species
Optical density required for antigen-positive test 0.05
(a) In individual samples T. congolense T. vivax Mixed I Total
0.07
No.
%
No.
%
No.
%
180 63 40 283
64 22 14 100
137 51 30 218
63 23 14 100
113 56 19 188
60 30 10 100
38
21
39
22
44
13
8
15
7
14
49 100
25 54
46 100
21 50
42 100
(b) In animals (14 samples per animal) T. congolense 21 T. vivax 7 Mixed ~ Total
0.06
27 55
tMixed T. congolense/T, vivax infections.
Trypanosoma congolense and T. vivax infections detected by both antigen
ELISA and buffy coat technique Eight animals only were found to be parasitaemic by the buffy coat technique on a total of 15 occasions out of 106 weekly tests. Seven of the animals were parasitaemic on a total of 14 occasions during this period but were antigenaemic on a total of 49 occasions. In contrast, the eighth animal, parasitaemic in 1 week, was never found to be antigenaemic. While 12 of the parasitaemias were reported as T. vivax and two as T. congolense, the 49 serum samples in which antigen was detected could be divided into two containing only T. vivax antigen, 15 mixed and 32 containing only T. congolense antigen.
Trypanosome species as determined by antigen ELISA and animal performance Preliminary analyses, using the 0.05 OD level, on the 27 animals with mixed
T. congolense and T. vivax infections ( T. congolense on an average of 5.6 occasions and T. vivax on an average of 3.2) revealed that T. vivax infections had virtually no effect on animal performance, while T. congolense infections had significant deleterious effects. The regression of daily weight gain on the number of T. vivax infections found in the animals infected with both species was - 1.0 + 3.3 g d a y - 1, while the regression of gain on the number of T. congolense infections found was - 6.2 +_2.9 g d a y - 1. When data on T. vivax and T. congolense infections were added together, the regression of gain on the
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total number of infections was reduced to - 3.6 _+2.1 g d a y - 1. The seven animals that were infected with T. vivax only (on an average of 2.5 occasions) had approximately the same daily weight gains over the 13 weeks as had the negative animals (86.8 + 18.3 g d a y - 1 compared with 88.8 + 7.7 g day- ~). Therefore, in all subsequent analyses involving performance, data referring to T. congolense infections only were considered. Thus the seven animals infected with T. vivax only were not considered as being infected, and the animals with mixed infections were evaluated on the basis of their T. congolense infection data only.
Antigenaemia and animal performance The relationship between antigen ELISA positivity during the 13 weeks and average PCV and daily weight gain, using three OD thresholds for a positive antigen test, are shown in Table 2. A positive as compared with a negative antigen test appeared to be associated with a lower average PCV and lower weight gain at all three thresholds, but this was only significant for PCV at the 0.05 OD threshold. Table 3 shows the results of introducing the effects of low and high numbers of times positive and low and high OD values, into the analysis. In each case, low refers to below average and high to above average. Animals antigenaemic for a high number of times (approximately three times as many as those antigenaemic a low number of times) had significantly lower average PCV values at all three test thresholds. Differences in weight gains followed the same patterns but did not reach significance. Animals antigenaemic with a high average OD (approximately twice that of those with a low average) had the same PCV values at all three test levels. They tended to have lower weight gains, but differences did not reach significance. N u m b e r of times antigenTABLE2 Least s q u a r e s m e a n s ( + S E ) for effects o f a n t i g e n a e m i a , d e t e r m i n e d at three t h r e s h o l d levels, on average P C V a n d daily weight gain Optical density t h r e s h o l d level
Antigen test result
No. o f animals
PCV (%)
Gain (g d a y - ~)
0.05
+ + + -
48 51 45 54 43 56
31.1 + 0.36 32.1 + 0 . 3 7 31.3+0.38 32.1 __+0.37 31.4 + 0.39 32.0 + 0.36
74.3 + 6.89 87.7__+7.03 73.3+7.13 88.8+6.91 72.7 + 7.19 89.6__+ 6.67
0.06 0.07
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TABLE 3 Least squares means ( + SE) for effects of (a) number of times antigenaemic, and (b) mean optical density of antigenaemia, determined at three threshold levels, on average PCV and daily weight gain Optical density threshold level
Class
No. of animals
PCV (%)
Gain (g d a y - ~)
Low (2.46) High (7.55) Low (1.83) High (5.85) Low (1.30) High (4.15)
28 20 24 21 20 23
32.4+0.47 29.9+0.56 32.3+0.52 30.3+0.56 32.3+0.57 30.5+0.54
82.2+ 66.4+ 84.4+ 62.1 + 81.4+ 64.0+
9.01 10.62 9.62 10.41 10.51 9.84
Low (0.070) High (0.141) Low (0.082) High (0.157) Low (0.091) High (0.162)
24 24 23 22 21 22
31.2+0.53 31.1 +0.52 31.3+0.53 31.3+0.56 31.8+0.57 31.0+0.57
86.6+ 62.0+ 79.0+ 67.5+ 85.0+ 60.4+
10.01 9.94 9.87 10.45 10.16 10.40
(a) Number of times antigenaemic 0.05 0.06 0.07
(b) Mean optical density 0.05 0.06 0.07
TABLE 4 Least squares means ( + SE) for effects of average PCV within number of times antigenaemic, determined at three threshold levels, on daily weight gain AntiPCV 0.05 Antigen threshold genaemia class class No. of Mean Gain animals PCV (gday -~)
(%)
Negative
Low High Low Low frequency High High Low frequency High
25 26 14 14 10 10
29.8 33.9 30.7 33.8 27.5 32.1
84.1+ 9.46 90.6+ 9.47 73.6+12.28 96.3+ 12.18 35.7+14.56 90.6+14.51
0.06 Antigen threshold
0.07 Antigen threshold
No. of Mean Gain animals PCV (gday - l )
No. of Mean Gain animals PCV (gday -~)
27 27 12 12 11 10
28 28 10 10 11 12
(%)
29.8 33.8 30.8 33.6 27.7 33.0
86.7+ 9.28 90.2+ 9.36 73.0+13.29 96.0+ 13.29 40.7_+14.11 86.2+14.64
(%)
29.8 33.8 30.9 33.6 27.7 32.9
86.9+ 9.07 92.3+ 9.16 73.7+14.86 88.9+ 14.60 41.0_+14.15 85.4+13.53
aemic and average OD were independent of each other, the regression of OD on number o f infections being a non-significant 0.003 OD units per infection.
PCV values and animal performance The effects o f above or below average PCV values on daily weight gain relative to whether detected antigenaemic a high number of times, a low number o f times, or not at all, are given in Table 4. When animals were antigenaemic a high number of times, those with a high average PCV grew twice as fast as
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J.C.M. TRAIL ET AL.
TABLE5 T i m e s taken for a n i m a l s to self cure j f r o m T. A n i m a l category
Self-cured, not infected at start o f period Self-cured, already infected at start o f period Did not self cure d u r i n g period C o u l d not be assessed
congolenseinfection
No. o f animals
No. o f positive tests per a n i m a l + SE
Weeks between first a n d last positive tests +SE
7
3.1 + 0.9
4.0 _+ 1.3
8
4.3 _+ 1.3
5.9 + 1.6
17 16
7.7 _+2.3 NA 2
11.6 _+ 1.9 NA
'Self cure defined as five negative tests after two or m o r e positive tests. 2Not applicable.
those with a low average PCV. This was the case at all three antigen test threshold values. When animals were antigenaemic a low number of times, the differences in daily weight gain between the high and low PCV groups averaged 27% and were not significant. When animals were never detected antigenaemic during the experimental period, the differences between the high and low PCV groups averaged 6%, again not significant.
Self cure from T. congolense infections Self cure was defined for this data set as five consecutive negative weekly tests after two or more positive tests (the lowest OD of 0.05 was used to indicate a positive antigen test). Table 5 shows that 32 of the 48 animals infected with T. congolense during the 13 weeks could be assessed as either having undergone self cure from infection during the period or as clearly not having self-cured. The remaining 16 animals could not be assessed, as infection occurred too late in the experimental period. Seven (21% ) of the animals not infected for 2 or more weeks at the start of the 13 weeks recovered after 3.1 positives over an average period of 4 weeks. Eight (25%) of the animals that had already been infected at the start of the 13 weeks recovered after 4.3 positive tests over an average period of 6 weeks (these last two figures would therefore be underestimates for the animals in this group). The 17 (53%) animals that clearly did not self cure during the 13 week period had 7.7 positive tests identified over an average period of 11.6 weeks. DISCUSSION
This carefully recorded situation, where animals positive for trypanosome antigens were identified as such in an average of five of the 14 weekly tests,
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221
and where 49% of antigenaemic animals had mixed T. congolense/T, vivax infections, allowed a major trypanosome species effect on animal performance to be detected. While T. congolense infections had significant deleterious effects on animal growth, the local T. vivax serodemes did not. The illustration of how, in mixed infections, the significant negative regression of weight gain on the number of T. congolense infections was obscured if the T. vivax data were not removed, shows the necessity for accurate trypanosome species identification, if infection effects and associations with other criteria of trypanotolerance are to be clarified and accurately quantified in the field. Past work has depended on species identification in the field using the buffy coat technique. When large numbers of animals have to be handled each day, determining the complete repertoire of trypanosome species infecting any one animal is very difficult using this technique. Antigen detection enzyme immunoassays could solve this problem. Because of the low number of animals detected parasitaemic by the buffy coat technique, follow-up work on trypanosome species identification by the two techniques must be carried out at a site and at a time when significant numbers of parasitaemias can be guaranteed. Animals found to be positive by antigen ELISA had lower average PCV values than those found to be negative, but this was only significant when the OD value of 0.05 was used as the threshold. When the number of times an animal was positive was included in the analysis, those with an above average number of positive tests had very significantly lower PCV values at all three of the OD thresholds. In contrast, when the mean OD value for all positive tests on an animal was included in the analysis, there was no association whatsoever with PCV values. In the only previously reported work on the association between trypanosome antigenaemia and PCV (Trail et al., 1992 ), it was only possible to conduct six antigen tests per animal over a 3 m o n t h period. In that study, animals positive for antigens were identified as such in an average of 2.3 tests. With these limited data, no significant effects on PCV or weight gain of antigen positivity, number of times an animal was positive, or mean OD value, were found. Follow-up work, at a site and time when animals are parasitaemic on a relatively large number of occasions will allow determination of whether the importance of the number of times an animal is antigenaemic is akin to the importance of number of times parasitaemic (Trail et al., 1991 a) and whether the lack of importance of mean optical density in the antigen tests is akin to the lack of importance of mean parasitaemia score (Trail et al., 1991 a). The very significant effects of above or below average PCV values on weight gain in the animals that were antigen-positive a high number of times, are akin to those of above or below PCV values in parasitaemic animals reported by Trail et al. ( 199 la). There may, therefore, be possibilities for selection on
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the basis of PCV when antigenaemic, similar to those for selection on the basis of PCV when parasitaemic. The absence o f a relationship between PCV and daily weight gain in the non-antigenaemic group suggests that the antigen test genuinely identified all infected animals, in contrast to previous findings with the buffy coat technique (Trail et al., 1992 ), where it appeared probable that a proportion o f animals, not identified by the technique, was infected. As reported a n u m b e r o f times (Trail et al., 1991a,b,c), infected N ' D a m a cattle grew at the same rate as uninfected N ' D a m a cattle if they could maintain normal PCV values. The finding that a proportion o f animals could be considered as having selfcured from infection after an average of 4 weeks (judged by being negative to the antigen test on five consecutive occasions) while a proportion had not self-cured after an average of 11.6 weeks, suggests that further work on the time taken by individual animals to self cure could be o f value. Two higher OD thresholds in the ELISA, 0.06 and 0.07, were compared with the 0.05 threshold normally regarded as positive (Nantulya et al., 1992 ). Both considerably reduced the n u m b e r o f samples detected as antigen-positive, but had very little effect on the n u m b e r of animals detected as positive during the experiment, or on any of the associations between infection, anaemia and growth parameters. The 0.05 O D threshold level would, therefore, appear to be a satisfactory indicator o f antigen positivity. ACKNOWLEDGEMENTS We are grateful to the m a n a g e m e n t of O G A P R O V Ranch for support in carrying out the field work in Gabon, S.H. Minja and J. Thuo for carrying out the assays at ILRAD and S. Nagda for sample preparation and assistance in data analysis. We thank Dr. E. Authie, Dr. A.S. Peregrine and Dr. G.J. Rowlands for helpful discussions.
REFERENCES Harvey, W.R., 1977. User's Guide for Least-Squaresand Maximum Likelihood Computer Program. Ohio State University, Columbus, 59 pp. Leak, S.G.A., Colardelle, C., d'Ieteren, G., Dumont, P., Feron, A., Jeannin, P., Minengu, M., Mulungo, M., Ngamuna, S., Ordner, G., Sauveroche,B., Trail, J.C.M. and Yangari, G., 1991. Glossina fusca group tsetse as vectors of cattle trypanosomiasis in Gabon and Zaire. Med. Vet. Entomol., 5:111-120. Masake, R.A. and Nantulya, V.M., 1991. Sensitivity of an antigen detection enzyme immunoassay for diagnosis of Trypanosoma congolense infections in goats and cattle. J. Parasitol., 77:231-236. Murray, C., Murray, M., Murray, P.K., Morrison, W.I., Pyne, C. and McIntyre, W.I.M., 1977. Diagnosis of African trypanosomiasis in cattle. Improved parasitological and serological
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