Parasitology (1979), 78, 271-286
271
With 2figuresin the text
Bovine trypanosomiasis: the red cell kinetics of Ndama and Zebu cattle infected with Trypanosoma congolense J. D. DARGIE,* P. K. MURRAY,t MAX MURRAY,* W. R. T. GRIMSHAW and W. I. M. MoINTYRE University of Glasgow Veterinary School, Bearsden Road, Glasgow {Received 9 June 1978) SUMMARY
The responses of susceptible Ndama and Zebu cattle to needle challenge with Trypanosoma congolense were followed using parasitological, haematological and radio-isotopic methods and compared with those of corresponding uninfected animals. In both breeds, infection became patent at the same time but peak parasitaemias were significantly lower, were attained later and were of short duration in the Ndama. All infected animals became anaemic, the severity of which correlated with the level and duration of parasitaemia. However, even when parasites could no longer be detected in the blood, packed cell volumes showed little tendency to recover. The anaemia was due to increased intravascular red cell destruction and was more pronounced in the Zebu. Haemodilution was not a feature. Increased red cell synthesis occurred in infected animals of both breeds but particularly in the Zebu; this accounted for the capacity to maintain packed cell volume levels following the initial drop associated with parasitaemia. However, in most cases red cell synthesis was less than expected from the degree of anaemia, suggesting impairment of bone marrow function. Measurement of red cell iron utilization indicated that this was due to defective iron re-utilization from degraded red cells arising from reticulo-endothelial blockade. I t is concluded that the anaemia in this disease and its underlying processes are broadly in line with the number of parasites in the blood and that the superior resistance of the Ndama cattle lies in their ability to control parasitaemia rather than their capacity to mount a more efficient erythropoietic response.
INTRODUCTION
The most significant factor in the disease process in naturally occurring and experimentally induced bovine trypanosomiasis is anaemia (reviewed by Murray, 1974), a fact well recognized by Hornby (1921). The factors underlying the anaemia have still to be properly delineated, although three mechanisms, acting singly or in concert, have been implicated. These are: haemodilution (Fiennes, 1954; Naylor, 1971; Holmes, 1976), increased red cell breakdown (Mamo & Holmes, 1975; * Present address: Animal Production and Health Section, International Atomic EnergyAgency, Vienna. f Merck, Sharp and Dohme Research Laboratories, Rahway, New Jersey. % International Laboratory for Research on Animal Diseases, P.O. Box 30709, Nairobi, Kenya. 0031-1820/79/0078-0611 $01.00 © 1979 Cambridge University PreS3
272
J. D. DARGIE AKD OTHERS
Holmes, 1976; Preston & Wellde, 1976) and reduced cell synthesis (Fiennes, 1954, 1970). In order to evaluate these properly it is necessary to measure directly and simultaneously the plasma and circulating red cell volumes of the animals concerned, and the rates at which red cells are added to and withdrawn from the circulation by synthesis and breakdown. To date only some of these parameters have been examined in infected cattle and even then only individually. The only information currently available on the erythrokinetics of bovine trypanosomiasis is that provided by Mamo & Holmes (1975), Holmes (1976) and Preston & Wellde (1976). These authors used animals infected with Trypanosoma congolense and, on the basis of data obtained with radio-isotopes, concluded that the anaemia was basically haemolytic in origin. However, whereas the former suggested that haemodilution was an important contributory factor and found no evidence of dyshaemopoiesis, the latter failed to demonstrate haemodilution but considered dyshaemopoiesis a complication in long-standing infections. In view of these differences it seemed appropriate to re-appraise the contribution made by each of these mechanisms to the anaemia oiT. congolense infections. This was done in groups of adult Ndama and Zebu cattle using 61Cr-labelled red cells, 12S I-labelled albumin and 59Fe-labelled transferrin. The results obtained are important from two standpoints. First, since all the measurements were made concurrently, they provide a comprehensive insight into the dynamics of the host-parasite interaction and thereby a better understanding of the disease process. Second, the investigation was carried out on cattle previously uninfected with trypanosomes. Since the Ndama are the more resistant to trypanosomes as judged by the outcome of bothflychallenge (Roberts & Gray, 1973) and needle challenge (Murray, Murray, Wallace, Morrison & Mclntyre, 1977) the present study provides new information on the question of trypanotolerance and its basis. MATERIALS AND METHODS
Experimented design
This study was part of a large-scale investigation involving 62 adult Ndama and 62 adult Zebu cattle; they averaged 4 years of age. The cattle of both breeds were believed to be free from previous exposure to trypanosomes. Thus, the Zebu cattle were purchased from Northern Senegal, well beyond the northern limits of the tsetse fly belt. The Ndama cattle were obtained from the Government Agricultural Experimental Station at Yundum, The Gambia, an area considered tsetse-free. Before inclusion in the experiment, cattle were screened by clinical, parasitological and haematological examination for the presence of trypanosomes and other intercurrent infections. Sera from all experimental animals were negative for trypanosomiasis on examination by indirect immunofluorescence. The work was conducted on 7 infected cattle of each breed inoculated subcutaneously with 3-6 x 107 T. congolense organisms. The stabilate was prepared from an isolate made 1 month previously from a naturally infected Ndama and subjected to 3 passages in rats prior to use. Three uninfected animals of each breed acted as controls. All the cattle were injected with radio-isotopically labelled materials
The erythrokinetics of bovine trypanosomiasis
273
7 weeks after challenge and during the following 2 months blood samples were collected and analysed for radioactivity. Apart from the day of injection, the animals were maintained under natural conditions in an area thought to be tsetsefree. During the day they grazed extensively and were tethered at night. They were also sprayed twice weekly with an insect repellant to minimize the possibility of mechanical transmission by biting flies and an anthelmintic was given at monthly intervals to control gastro-intestinal parasites. Determination of parasitaemia The assessment of parasitaemia was made in jugular blood and was based on the buffy coat-dark ground-phase technique of Murray, Murray & Mclntyre (1977). The level of infection was graded from 1+ t o 5 + ; a 5 + infection indicated more than 10 organisms/microscopic field at 250 x magnification and each step between 1 + and 5 + , a difference of about 1 log; this was calculated t o give an estimated log trypanosome/ml value (Murray, Murray, Murray, Morrison, Pyne & Mclntyre, 1977). Blood analyses Packed cell volumes (PCV), haemoglobin concentration and red cell counts were measured using respectively the capillary micro-method, the cyanmethaemoglobin procedure and an electronic particle counter (Coulter Electronics Ltd, Kenya). Measurements of serum iron concentration and total iron binding capacity (TIBC), which were performed using a commercial test kit (Roche Diagnostics, Roche Products Ltd), enabled calculation of latent iron binding capacity (LIBC) and the concentration and percentage saturation of transferrin. Preparation of labelled materials Bovine albumin was trace-labelled with 125I by the method of McFarlane (1958). Labelling of red cells with 51Cr was performed by treating 40 ml of heparinized blood with 3 mCi 51Cr as sodium chromate and incubating at 30 °C for 45 min. The labelled cells were washed with saline until free of unbound, isotope and suspended in saline for injection, each animal receiving its own labelled cells. [59Fe]transferrin was prepared by incubating 200 ml of normal plasma with 2 mCi [s9Fe]ferric citrate for 30 min. Injection and sampling procedures and measurements of radioactivity Amounts of labelled albumin, transferrin and autologous red cell suspensions containing respectively about 350 /iCi 125 I, 100 /id 59Fe, 2 mCi 51Cr were injected via a jugular catheter. Blood samples were collected 15, 30, 60, 90, 120 and 180 min later, twice daily over the following 6 days and thereafter daily for 50 days. One ml of plasma and blood was diluted to 10 ml with dilute NaOH for radioactivity determinations. These were made using an autogamma scintillation spectrometer (Packard Instruments, Illinois). Separation of isotopic mixtures was achieved by spectrometry and use of standard solutions of each isotope.
274
J. D. DABGIE AND OTHERS
Calculations and expression of results Blood volumes Plasma and red cell volumes were measured with [125I]albumin and [61Cr]red cells by dividing the respective total injected activities by the activity of the 15-min samples of plasma and of whole blood corrected for PCV. Blood volume was then calculated as the sum of the plasma and red cell volume. Red cell survival The 61Cr activity of each blood sample was converted t o activity/ml of red cells and expressed as a percentage of the 15-min value. A semi-log plot was made against time from which the apparent red cell half-life was obtained by regression analysis. Since 51Cr is rapidly eluted from bovine red cells for 5-7 days after injection, the radioactivity of samples collected during this time was excluded from the calculation. Red cell synthesis and life-span Red cell synthesis was assessed on the basis of 2 parameters, i.e. the rates of plasma iron turnover (PITR) and red cell iron utilization. The former was determined by expressing the 59Fe radioactivity of each plasma sample taken during the first 3 h as a percentage of the 15 min post-inj ection value and making a semi-log plot of activity against time. The rate of disappearance of isotope was expressed as a half-life {t\) value, which together with the serum iron concentration and plasma volume enabled calculation of PITR from the formula of Bothwell, Hurtado, Donohue & Finch (1957). T^T,™-. , n n i serum iron (me/ml) x plasma volume x 0-693 x 1440 7 , . . , , ^-rr . PITR (mg/kg/day) = ' ' t\ (mm) x body weight
The percentage incorporation of 59Fe into red cells was followed by making a linear plot of the activity/ml of red cells against time, calculated from the daily blood radioactivity and PCV. From the maximum activity (59Fe max), the percentage iron utilization was obtained from the formula: _ , .•*... 100 x red cell volume x 59Fe max r-^—: = — , Percentage utilization = total injected activity and the red cell iron incorporation rate calculated as the product of percentage utilization and PITR. Since PCV values were reasonably constant throughout the isotopic investigation it can be assumed that rates of red cell iron incorporation and red cell iron turnover (RCIT) were approximately equal. Hence the fraction of red cell iron renewed daily (RCIR) could be determined from: RCIT (mg/day) ' RCI (mg) where RCI equals the total red cell iron, obtained as the product of total blood volume, blood haemoglobin concentration and 3-4; 3-4 being mg Fe/g haemoglobin. The red cell life-span (days) was then calculated as the reciprocal of RCIR.
The erythrokinetics of bovine trypanosomiasis
275
Statistical methods The independent 't' test was used to evaluate data and significance was considered where P < 0-05. All results are expressed as mean + standard error (S.E.).
EESXTLTS
Anaemia and parasitaemia The PCV levels of both infected groups deteriorated rapidly between the 1st and 6th weeks (Fig. 1) but whereas the values of the Ndama dropped by only 25 % from a pre-infection mean of about 34 % to a mean of 26 %, those of the Zebu fell by 41 % from 34 to 20 %. This difference was highly significant (P < O-01). Subsequently, the average figures for both groups showed a slight tendency to increase but the Ndama maintained their superiority throughout (P < 0-02). T h e PCV levels of the controls fluctuated between 33 and 37 % and while the Ndama generally had higher values, the difference was never significant. Red cell counts and haemoglobin concentrations followed the pattern described for PCV and hence are not described further. The onset of anaemia correlated closely with the appearance of parasites in the blood, and its severity with the level and duration of the initial peaks of parasitaemia (Fig. 1). Thus, while infections in both breeds became patent by 4 days after inoculation, the peak parasitaemias were significantly lower and attained later in the Ndama; a peak level of 4-00 + 0-4 estimated log trypanosomes/ml was reached by day 13 in the Ndama whereas a peak level of 4-82 + 0-2 was recorded by day 10 in the Zebu (P < 0-05). Furthermore, whereas the levels of parasitaemia in the Ndama fell sharply between the 4th and 7th weeks to around an estimated 0-5 log trypanosomes/ml, remained depressed until week 11, and then fell t o zero, those of the Zebu were elevated until week 6 and then declined progressively over the following 5 weeks to a level of 0-5 log/ml. Subsequently the parasitaemia levels increased in the Zebu; this was attributed to re-infection as these animals came under a natural Glossina palpalis gambiensis challenge. By contrast, the Ndama were not obviously affected by the presence of the tsetse. Serum iron, binding capacities and transferrin saturation These measurements which were made on samples taken immediately prior to injection of radio-isotopes revealed few significant differences either between the infected cattle and their respective controls or between the two breeds (Table 1). Serum iron levels of the infected Zebus were generally higher than normal although 2 animals had abnormally low values (i.e. < 100 /ig %) and 1 of these died 7 days later. No such trend was observed in the Ndama but the values for this breed were lower than for the Zebu, and significantly so in the case of the infected groups (P < 0-05). TIBC levels were reduced in both infected groups relative to control values, particularly in the Zebu, and it is perhaps significant that the animal which succumbed to infection also had a very low value (i.e. 188/jg%). As a result of these differences, LIBC levels were 30 % lower than normal in the infected Zebu,
276
J. D. DAEGIE AND OTHEES
Zebu
8 Weeks after infection
16
12
Fig. 1. Packed cell volume and parasitaemia levels of Ndama and Zebu cattle after infection with Trypanosoma congolense. Table 1. Serum iron and iron-binding capacities (+ S.E.) of cattle 7 weeks after infection with Trypanosoma congolense
Cattle breed Ndama Infected Control P
Zebu Infected Control P
Serum iron (Ag%)
TIBC (/*g%)
120 ±7-1 122 + 5-1 N.S.*
322 + 12-3 368±21-7
149 ±13-2 137+7-8 N.S.
355 + 321 438+14-3 N.S.
N.S
LIBC
Transferrin saturation
im%)
(%)
Plasma transferrin (mg%)
202 ±15-5 246 + 19-4 N.S.
38 ± 3 0 33+1-7 N.S.
258 ±9-9 295 + 17-3 N.S.
206 ±22-4 287 ±2-0 N.S.
43 ±2-3 31 ±0-9
271
With 2figuresin the text
Bovine trypanosomiasis: the red cell kinetics of Ndama and Zebu cattle infected with Trypanosoma congolense J. D. DARGIE,* P. K. MURRAY,t MAX MURRAY,* W. R. T. GRIMSHAW and W. I. M. MoINTYRE University of Glasgow Veterinary School, Bearsden Road, Glasgow {Received 9 June 1978) SUMMARY
The responses of susceptible Ndama and Zebu cattle to needle challenge with Trypanosoma congolense were followed using parasitological, haematological and radio-isotopic methods and compared with those of corresponding uninfected animals. In both breeds, infection became patent at the same time but peak parasitaemias were significantly lower, were attained later and were of short duration in the Ndama. All infected animals became anaemic, the severity of which correlated with the level and duration of parasitaemia. However, even when parasites could no longer be detected in the blood, packed cell volumes showed little tendency to recover. The anaemia was due to increased intravascular red cell destruction and was more pronounced in the Zebu. Haemodilution was not a feature. Increased red cell synthesis occurred in infected animals of both breeds but particularly in the Zebu; this accounted for the capacity to maintain packed cell volume levels following the initial drop associated with parasitaemia. However, in most cases red cell synthesis was less than expected from the degree of anaemia, suggesting impairment of bone marrow function. Measurement of red cell iron utilization indicated that this was due to defective iron re-utilization from degraded red cells arising from reticulo-endothelial blockade. I t is concluded that the anaemia in this disease and its underlying processes are broadly in line with the number of parasites in the blood and that the superior resistance of the Ndama cattle lies in their ability to control parasitaemia rather than their capacity to mount a more efficient erythropoietic response.
INTRODUCTION
The most significant factor in the disease process in naturally occurring and experimentally induced bovine trypanosomiasis is anaemia (reviewed by Murray, 1974), a fact well recognized by Hornby (1921). The factors underlying the anaemia have still to be properly delineated, although three mechanisms, acting singly or in concert, have been implicated. These are: haemodilution (Fiennes, 1954; Naylor, 1971; Holmes, 1976), increased red cell breakdown (Mamo & Holmes, 1975; * Present address: Animal Production and Health Section, International Atomic EnergyAgency, Vienna. f Merck, Sharp and Dohme Research Laboratories, Rahway, New Jersey. % International Laboratory for Research on Animal Diseases, P.O. Box 30709, Nairobi, Kenya. 0031-1820/79/0078-0611 $01.00 © 1979 Cambridge University PreS3
272
J. D. DARGIE AKD OTHERS
Holmes, 1976; Preston & Wellde, 1976) and reduced cell synthesis (Fiennes, 1954, 1970). In order to evaluate these properly it is necessary to measure directly and simultaneously the plasma and circulating red cell volumes of the animals concerned, and the rates at which red cells are added to and withdrawn from the circulation by synthesis and breakdown. To date only some of these parameters have been examined in infected cattle and even then only individually. The only information currently available on the erythrokinetics of bovine trypanosomiasis is that provided by Mamo & Holmes (1975), Holmes (1976) and Preston & Wellde (1976). These authors used animals infected with Trypanosoma congolense and, on the basis of data obtained with radio-isotopes, concluded that the anaemia was basically haemolytic in origin. However, whereas the former suggested that haemodilution was an important contributory factor and found no evidence of dyshaemopoiesis, the latter failed to demonstrate haemodilution but considered dyshaemopoiesis a complication in long-standing infections. In view of these differences it seemed appropriate to re-appraise the contribution made by each of these mechanisms to the anaemia oiT. congolense infections. This was done in groups of adult Ndama and Zebu cattle using 61Cr-labelled red cells, 12S I-labelled albumin and 59Fe-labelled transferrin. The results obtained are important from two standpoints. First, since all the measurements were made concurrently, they provide a comprehensive insight into the dynamics of the host-parasite interaction and thereby a better understanding of the disease process. Second, the investigation was carried out on cattle previously uninfected with trypanosomes. Since the Ndama are the more resistant to trypanosomes as judged by the outcome of bothflychallenge (Roberts & Gray, 1973) and needle challenge (Murray, Murray, Wallace, Morrison & Mclntyre, 1977) the present study provides new information on the question of trypanotolerance and its basis. MATERIALS AND METHODS
Experimented design
This study was part of a large-scale investigation involving 62 adult Ndama and 62 adult Zebu cattle; they averaged 4 years of age. The cattle of both breeds were believed to be free from previous exposure to trypanosomes. Thus, the Zebu cattle were purchased from Northern Senegal, well beyond the northern limits of the tsetse fly belt. The Ndama cattle were obtained from the Government Agricultural Experimental Station at Yundum, The Gambia, an area considered tsetse-free. Before inclusion in the experiment, cattle were screened by clinical, parasitological and haematological examination for the presence of trypanosomes and other intercurrent infections. Sera from all experimental animals were negative for trypanosomiasis on examination by indirect immunofluorescence. The work was conducted on 7 infected cattle of each breed inoculated subcutaneously with 3-6 x 107 T. congolense organisms. The stabilate was prepared from an isolate made 1 month previously from a naturally infected Ndama and subjected to 3 passages in rats prior to use. Three uninfected animals of each breed acted as controls. All the cattle were injected with radio-isotopically labelled materials
The erythrokinetics of bovine trypanosomiasis
273
7 weeks after challenge and during the following 2 months blood samples were collected and analysed for radioactivity. Apart from the day of injection, the animals were maintained under natural conditions in an area thought to be tsetsefree. During the day they grazed extensively and were tethered at night. They were also sprayed twice weekly with an insect repellant to minimize the possibility of mechanical transmission by biting flies and an anthelmintic was given at monthly intervals to control gastro-intestinal parasites. Determination of parasitaemia The assessment of parasitaemia was made in jugular blood and was based on the buffy coat-dark ground-phase technique of Murray, Murray & Mclntyre (1977). The level of infection was graded from 1+ t o 5 + ; a 5 + infection indicated more than 10 organisms/microscopic field at 250 x magnification and each step between 1 + and 5 + , a difference of about 1 log; this was calculated t o give an estimated log trypanosome/ml value (Murray, Murray, Murray, Morrison, Pyne & Mclntyre, 1977). Blood analyses Packed cell volumes (PCV), haemoglobin concentration and red cell counts were measured using respectively the capillary micro-method, the cyanmethaemoglobin procedure and an electronic particle counter (Coulter Electronics Ltd, Kenya). Measurements of serum iron concentration and total iron binding capacity (TIBC), which were performed using a commercial test kit (Roche Diagnostics, Roche Products Ltd), enabled calculation of latent iron binding capacity (LIBC) and the concentration and percentage saturation of transferrin. Preparation of labelled materials Bovine albumin was trace-labelled with 125I by the method of McFarlane (1958). Labelling of red cells with 51Cr was performed by treating 40 ml of heparinized blood with 3 mCi 51Cr as sodium chromate and incubating at 30 °C for 45 min. The labelled cells were washed with saline until free of unbound, isotope and suspended in saline for injection, each animal receiving its own labelled cells. [59Fe]transferrin was prepared by incubating 200 ml of normal plasma with 2 mCi [s9Fe]ferric citrate for 30 min. Injection and sampling procedures and measurements of radioactivity Amounts of labelled albumin, transferrin and autologous red cell suspensions containing respectively about 350 /iCi 125 I, 100 /id 59Fe, 2 mCi 51Cr were injected via a jugular catheter. Blood samples were collected 15, 30, 60, 90, 120 and 180 min later, twice daily over the following 6 days and thereafter daily for 50 days. One ml of plasma and blood was diluted to 10 ml with dilute NaOH for radioactivity determinations. These were made using an autogamma scintillation spectrometer (Packard Instruments, Illinois). Separation of isotopic mixtures was achieved by spectrometry and use of standard solutions of each isotope.
274
J. D. DABGIE AND OTHERS
Calculations and expression of results Blood volumes Plasma and red cell volumes were measured with [125I]albumin and [61Cr]red cells by dividing the respective total injected activities by the activity of the 15-min samples of plasma and of whole blood corrected for PCV. Blood volume was then calculated as the sum of the plasma and red cell volume. Red cell survival The 61Cr activity of each blood sample was converted t o activity/ml of red cells and expressed as a percentage of the 15-min value. A semi-log plot was made against time from which the apparent red cell half-life was obtained by regression analysis. Since 51Cr is rapidly eluted from bovine red cells for 5-7 days after injection, the radioactivity of samples collected during this time was excluded from the calculation. Red cell synthesis and life-span Red cell synthesis was assessed on the basis of 2 parameters, i.e. the rates of plasma iron turnover (PITR) and red cell iron utilization. The former was determined by expressing the 59Fe radioactivity of each plasma sample taken during the first 3 h as a percentage of the 15 min post-inj ection value and making a semi-log plot of activity against time. The rate of disappearance of isotope was expressed as a half-life {t\) value, which together with the serum iron concentration and plasma volume enabled calculation of PITR from the formula of Bothwell, Hurtado, Donohue & Finch (1957). T^T,™-. , n n i serum iron (me/ml) x plasma volume x 0-693 x 1440 7 , . . , , ^-rr . PITR (mg/kg/day) = ' ' t\ (mm) x body weight
The percentage incorporation of 59Fe into red cells was followed by making a linear plot of the activity/ml of red cells against time, calculated from the daily blood radioactivity and PCV. From the maximum activity (59Fe max), the percentage iron utilization was obtained from the formula: _ , .•*... 100 x red cell volume x 59Fe max r-^—: = — , Percentage utilization = total injected activity and the red cell iron incorporation rate calculated as the product of percentage utilization and PITR. Since PCV values were reasonably constant throughout the isotopic investigation it can be assumed that rates of red cell iron incorporation and red cell iron turnover (RCIT) were approximately equal. Hence the fraction of red cell iron renewed daily (RCIR) could be determined from: RCIT (mg/day) ' RCI (mg) where RCI equals the total red cell iron, obtained as the product of total blood volume, blood haemoglobin concentration and 3-4; 3-4 being mg Fe/g haemoglobin. The red cell life-span (days) was then calculated as the reciprocal of RCIR.
The erythrokinetics of bovine trypanosomiasis
275
Statistical methods The independent 't' test was used to evaluate data and significance was considered where P < 0-05. All results are expressed as mean + standard error (S.E.).
EESXTLTS
Anaemia and parasitaemia The PCV levels of both infected groups deteriorated rapidly between the 1st and 6th weeks (Fig. 1) but whereas the values of the Ndama dropped by only 25 % from a pre-infection mean of about 34 % to a mean of 26 %, those of the Zebu fell by 41 % from 34 to 20 %. This difference was highly significant (P < O-01). Subsequently, the average figures for both groups showed a slight tendency to increase but the Ndama maintained their superiority throughout (P < 0-02). T h e PCV levels of the controls fluctuated between 33 and 37 % and while the Ndama generally had higher values, the difference was never significant. Red cell counts and haemoglobin concentrations followed the pattern described for PCV and hence are not described further. The onset of anaemia correlated closely with the appearance of parasites in the blood, and its severity with the level and duration of the initial peaks of parasitaemia (Fig. 1). Thus, while infections in both breeds became patent by 4 days after inoculation, the peak parasitaemias were significantly lower and attained later in the Ndama; a peak level of 4-00 + 0-4 estimated log trypanosomes/ml was reached by day 13 in the Ndama whereas a peak level of 4-82 + 0-2 was recorded by day 10 in the Zebu (P < 0-05). Furthermore, whereas the levels of parasitaemia in the Ndama fell sharply between the 4th and 7th weeks to around an estimated 0-5 log trypanosomes/ml, remained depressed until week 11, and then fell t o zero, those of the Zebu were elevated until week 6 and then declined progressively over the following 5 weeks to a level of 0-5 log/ml. Subsequently the parasitaemia levels increased in the Zebu; this was attributed to re-infection as these animals came under a natural Glossina palpalis gambiensis challenge. By contrast, the Ndama were not obviously affected by the presence of the tsetse. Serum iron, binding capacities and transferrin saturation These measurements which were made on samples taken immediately prior to injection of radio-isotopes revealed few significant differences either between the infected cattle and their respective controls or between the two breeds (Table 1). Serum iron levels of the infected Zebus were generally higher than normal although 2 animals had abnormally low values (i.e. < 100 /ig %) and 1 of these died 7 days later. No such trend was observed in the Ndama but the values for this breed were lower than for the Zebu, and significantly so in the case of the infected groups (P < 0-05). TIBC levels were reduced in both infected groups relative to control values, particularly in the Zebu, and it is perhaps significant that the animal which succumbed to infection also had a very low value (i.e. 188/jg%). As a result of these differences, LIBC levels were 30 % lower than normal in the infected Zebu,
276
J. D. DAEGIE AND OTHEES
Zebu
8 Weeks after infection
16
12
Fig. 1. Packed cell volume and parasitaemia levels of Ndama and Zebu cattle after infection with Trypanosoma congolense. Table 1. Serum iron and iron-binding capacities (+ S.E.) of cattle 7 weeks after infection with Trypanosoma congolense
Cattle breed Ndama Infected Control P
Zebu Infected Control P
Serum iron (Ag%)
TIBC (/*g%)
120 ±7-1 122 + 5-1 N.S.*
322 + 12-3 368±21-7
149 ±13-2 137+7-8 N.S.
355 + 321 438+14-3 N.S.
N.S
LIBC
Transferrin saturation
im%)
(%)
Plasma transferrin (mg%)
202 ±15-5 246 + 19-4 N.S.
38 ± 3 0 33+1-7 N.S.
258 ±9-9 295 + 17-3 N.S.
206 ±22-4 287 ±2-0 N.S.
43 ±2-3 31 ±0-9
