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Relapses in dogs experimentally infected with Trypanosoma brucei and treated with diminazene aceturate or isometamidium chloride
Veterinary Parasitology, 27 (1988) 199-208 Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands
199
Relapses in D o g s E x p e r i m e n t a l l y Infected w i t h Trypanosoma brucei and Treated w i t h D i m i n a z e n e A c e t u r a t e or I s o m e t a m i d i u m Chloride E. KAGGWA1, W.K. MUNYUA2, and G.M. MUGERA2
1Department of Parasitology and Entomology, Ahmadu BeUo University, Zaria (Nigeria) 2Department of Pathology and Microbiology, University of Nairobi, P.O. Box 29053, Nairobi, (Kenya) (Accepted for publication 23 February 1987)
ABSTRACT Kaggwa, E., Munyua, W.K. and Mugera, G,M., 1988. Relapses in dogs experimentally infected with Trypanosoma brucei and treated with diminazene aceturate or isometamidium chloride. Vet. Parasitol., 27: 199-208. Twenty dogs of mixed local East African breeds were used. Five of the dogs were uninfected controls and 15 were infected with T. brucei (ILRAD 273). Five of the infected dogs were untreated controls, five were treated with a high curative dose of diminazene aceturate, (7 mg kg-1 body weight (wt.), and five were given a subcurative dose of isometimidium chloride (1 mg kg-1 body wt.). The drugs, given at 8 days post infection (d.p.i..), led to apparent recovery. The antibody titres, however, remained high in both groups and at 42-49 d.p.i, there was at least one relapse in each treatment group. Parasite populations from relapsed animals were more resistant to the drugs than the original infecting populations. The implications of these findings are discussed.
INTRODUCTION
The dog is susceptible to three Trypanosoma brucei subspecies, T. brucei brucei, T.b. rhodesiense and T.b. gambiense, which cannot be differentiated morphologically. The symptoms and pathology of T. b. brucei infections in dogs have been described previously ( Stephen 1970; Mwambu, 1977; Sayer et al., 1979; Morrison, et al., 1983; Kaggwa, et al. 1984, 1985). The pathogenesis, however, as in other animals, is under speculation. Drugs have been used for trypanosomiasis to study certain aspects of pathology which could contribute to the pathogenesis of the disease. Wilson and Cunningham (1971) studied antibody responses after chemotherapy with diminazene aceturate in cattle; Boreham (1966) used the same drug in cattle 0304-4017/88/$03.50
© 1988 Elsevier Science Publishers B.V.
200 infected with T. brucei to study blood kinin levels; Houba et al. (1969) used Suramin ( British Pharmacopias ) and Tryparsamide (British Pharmacopias ) to modify the disease course in monkeys infected with T. b. brucei and T.b. rhodesiense in studying heterophil antibodies and immunoglobulin. Neitz and McCully (1971) used subcurative doses of diminazene aceturate and Suramin B.P. (Antrypol) (ICI Pharmaceuticals, U.K. ) to prolong the disease course in horses so as to study the invasive potential of T. brucei for the cerebrospinal fluid. In dogs, the commonly used drug is diminazene aceturate (usually at 3.5 mg kg-~ body wt.) but sometimes isometamidium chloride is used at 0.5-1.0 mg kg- ~ where the parasites are sensitive ( Gitatha and Ogada 1969; Toure, 1970; Williamson, 1970). The aim of this study was to compare some parameters in T. brucei-infected dogs after treatment with either a high curative dose of diminazene aceturate, or a subcurative dose of isometamidium chloride, to show the difference, if any, in the recovery rate as indicated by these parameters. MATERIALSAND METHODS
The parasite Trypanosoma brucei (ILRAD 273) isolated in 1971 from a Kongoni Alcephalus buselaphus cokii (one of the East African antelopes) in Tanzania was used. Before carrying out the main experiment, the parasite was found to be sensitive in white albino mice to diminazene aceturate at 3.5 mg kg- 1 body wt. and to isometamidium chloride at >~2 mg kg- 1 body wt. Animals Fifteen adult dogs of mixed local East African breeds, kept singly in fly-proof kennels, were proved trypanosome-negative using serology and animal subinoculation. They were each intravenously infected with 104 trypanosomes, and randomly grouped as indicated in Table I. Five uninfected dogs were used as controls.
Procedure Clinical signs were observed in the control and infected dogs and blood samples were taken for haematology and serology. The indirect fluorescent antibody test (IFAT) was used to detect anti-trypanosome antibodies. Parasitaemia was detected using capillary buffer coat wet smears and trypanosome counts were made in a haemocytometer Neubauer chamber. Alanine and aspartate aminotransferases were determined colorimetrically using kits (Boeringer-
201 TABLE 1 Responses of control and T. brucei-infected dogs to the drugs Berenil1and Samorin2
Group No. of dogs Drug used Parasite sensitivity3( mg kg- ' body wt. ) Dosage given (mg k g - ' body wt. ) Dogs relapsed No days p.i.: Before relapses At death Relapse parasite sensitivity (mg kg- 1body wt. )
Control
Infected
1 5 None NA NA NA
2 5 Berenil 3.5 7 1
3 5 Samorin 2 1 2
4 5 None NA NA NA
NA
42 78 > 10
48,49 70.76 >2
NA 204 NA
NA
1Diminazene aceturate (Hoechst). 2isometamidium chloride (May & Baker). 3As tested in mice 4Longest survival date of this group. NA = not applicable; > = above stated amount.
Manhein GmbH Diagnostica, West Germany) which closely follow the method of Reitman and Frankel (1957). At 8 d.p.i, diminazene aceturate (7 mg k g - ' body wt.) was given intramuscularly to each of the five dogs in Group 2 (Table I), while isometamidium chloride (1 mg kg -1 body wt.) was given to each of the five in group 3. The remaining infected dogs were left as untreated controls. At specific d.p.i., one dog from each treatment group was killed for postmortem, histology and immunohistology. These results on pathology have been reported elsewhere (Kaggwa et al., 1985). After 27 d.p.i, there were only two dogs in each of the treated groups. The tests of drug sensitivity in mice of the parasite populations to the original drugs were then repeated. RESULTS
All infected dogs became parasitaemic in 2-3 days. The untreated control dogs showed high levels of parasitaemia (Fig. 1 ), high temperatures (Fig. 2) and anaemia (as indicated by decreased packed cell volume (PCV) percentages and haemoglobin (Hb) levels) (Figs. 3 and 4).There were slightly elevated serum alanine (ALAT) and aspertate aminotransferase (ASAT), levels (Figs. 5a,b ) and elevated antibody titres, (Fig. 6). Lymph node enlargement, eye lesions, oedema, lack of coordination, recumbency and finally death of all the control dogs were noted within 20 d.p.i.
202
lit
KEY : x -x
10
x x ....
S a m o r i n treatea dogs I n f e c t e d u n t r e a t e d dogs Berenil treated dogs Treatment
time
x/ ,.~x-X'x
/
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////
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/1111
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O 0
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20
30
I x 40 post
Days
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[
50 infected
60
70
80
Fig. 1. Mean parasitaemia, Log.lo trypanosomes per millilitre, in groups of five dogs, infected with T. b. brucei and treated with either Samorin or Berenil, or untreated. 43.0
KEY x,
42.C
x Infected
• .....
-O S a m o r i n
• ....
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untreated treated treated
o Control Treatment
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Fig. 2. Mean temperatures, °C, in groups of five dogs infected with T.b. brucei and treated with either Samorin or Bereni], or untreated.
Treatment with either of the drugs arrested the progress of clinical symptoms. The dogs regained their demeanour, body temperatures returned to normal (Fig. 2), the parasitaemia disappeared (Fig. 1 ) and serum enzyme levels decreased greatly ( Fig. 5 ). The PCV and Hb mean values remained within the normal range for both the treated groups up to 55 d.p.i, after which they dropped (Figs. 3 and 4). This drop coincided with the disease relapse where no treatments were attempted. The PCV and Hb mean values within the two groups
203 KEY x . . . . . x Berenil treated clogs o o Samorin treot.ed dogs
80
PC__Zv 70
~.
~. Infected untreated control
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A Uninfected control
Treatment 60
F
Upper and
L
Lower
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5C
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20 30 40 50 DAYS POST INFECTION
60
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BO
Fig. 3. Mean packed cell volume (PCV) % in groups of five dogs infected with T. b. brucei and treated with either Samorin or Berenil, or untreated. 17
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/--x--,/'\*/..//"
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Treatment time
Infected untreated Lower normal range Intact control Samorin treated e - - - - o Bereni[ treated
o L = x. . . . . x x x ~
6
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1012141618202224262830323436384042444648505254565860 Days and post infection
Fig. 4. Mean haemoglobin values (Hb) g/100 ml, in groups of five dogs infected with T. b. brucei and treated with either Samorin or Berenil, or untreated.
fluctuated uniformly (Figs. 3 and 4). An exception was at 53 d.p.i, when the isometamidium-treated group had a higher PCV (49.3%) than the diminazene-treated group (46.9%) but a lower Hb level (15.3 g/100 ml) than that of the diminazene group (15.6%). In order to test whether there was any significant difference, one-way analysis of variance was carried out. The two treated groups were compared for the four dogs that survived up to that period. There was no significant difference in the PCV between the two groups: the isometamidium group had a mean of 47.6% while the other group had a mean of 45.6%. Furthermore, the PCV values at 53 d.p.i, did not differ significantly from the overall means for each group subjected to the t-test at 0.05 level of
204
70}.--(A)
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x ~renil
601-
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=
Samorin
5 0 1_
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Untreated
J
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treated dogs treated clogs dogs
time
Treatment
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o
ro
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.x---x
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80
KEY
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,
........
Berenil treated dogs Samorin treated clogs
x Untreated dogs Treatment time
70 60
~o 4O
•
/x
\x
10' O !
' 10
' 20
' ' ' 30 40 50 DAYS POST INFECTION
~0
70'
8~
Fig. 5. Mean aminotransferase levels, international units per litre i.u. l - 1, in groups of five dogs infected with T. b. brucei and treated with either Samorin or Berenil, or untreated.
1280320
/ x~
20
//
r/~
.
~jl,'~Jl[/] ,
.....
• ---~,~,
-10 ~
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Treatment
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,
,
,
,
,
,
,
time
,
10 20 30 40 50 Days before and after infection
~
tr e~ed
,
80
,
-,
70
,
80
Fig. 6. Mean serum antibody levels shown in reciprocal titres in groups of five dogs infected with T. b. brucei and treated with either Samorin or Berenil, or untreated.
205 significance (isometamidium-treated group: t=1.713, df=47, P=0.0896 (>0.05); diminazene-treated group: t=l.080, df--51, P--0.285 (>0.05). Therefore, the deviation at 53 d.p.i, was non-significant. The serum antibody titres, however, remained significantly high in both the treated groups (Fig. 6). Relapses were suffered by one of the dogs treated with diminazene aceturate and two of those treated with isometamidium. The symptoms of the three relapse cases were similar to those of the primary disease, but they showed more involvement of the CNS, with a combination of the following signs: poorer coordination, aggression, hyperaesthaesia, circling in the kennels, head tilted on one side, paddling of the feet, up and down jaw movements, frothing at the mouth and eventually death. The relapse parasite populations were resistant to the usual diminazene aceturate doses and those of 10 mg kg- 1 body wt., and to isometamidium chloride at 2 mg kg-1 body wt. DISCUSSION The progress of the primary disease was arrested by both of the drugs. After treatment the respective parameters started changing at approximately the same time in both groups. Furthermore, these parameters did not indicate any difference in rate of recovery between the two treated dog groups. The post-treatment changes in temperature, red blood cell parameters and parasitaemia up to 40 d.p.i, infection were indicative of recovery. Serum transaminase levels dropped in both treated and untreated dogs. It has been suggested that the decrease of these enzymes during the latter half of the disease course might be due to less destruction of the trypanosomes as the body defence mechanisms become overpowered or immunosuppressed by the infection ( Kaggwa et al., 1984). This was assuming Gray's suggestion ( Gray, 1963) that the elevated enzyme levels might be from dying trypanosomes, to be correct. However, there was no difference in transaminase levels between those dogs treated with either diminazene aceturate or isometamidium chloride. There were no significant differences in the other parameters used to assess recovery between the two groups of treated dogs. Thus, after curative or noncurative doses there can be a period of apparent recovery, followed by a possible relapse withT, brucei. The application of the Berenil index (Berenil (Hoechst) for diminazene aceturate ) in the determination of field trypanosome risks and challenge (Rogers, 1985) should, therefore,be carefully evaluated in the face of possible relapses after the use of curative doses of diminazene aceturate doses and in absence of tsetse challenge, at least for T. brucei. Relapses in dogs treated with diminazene aceturate cannot be due to drug resistance of the infecting parasite population or to under-dosage. It has been suggested (Jennings et al., 1977, 1979; Morrison et al., 1983) that relapses in mice and cattle might be due to parasites which invade the brain, thus escaping
206
the action of drugs which do not cross the blood-brain barrier. In infected dog brain, the first histological lesions were seen on Day 8 p.i. but no parasites were detected until Day 16 p.i. (Morrison et al., 1981 ). If parasites in the brain had been the cause of the relapses in our dogs, then this organ might have been affected earlier than 8 d.p.i. Alternatively, the diminazene aceturate dose may not be giving a sufficiently high tissue concentration to kill all the parasites. The persistence of significant antibody titres indicated the presence of parasites in the body. However, higher doses in dogs predispose them to CNS toxic symptoms, (Losos and Crockett, 1969; Oppong, 1969; Ogada, 1969; Schmidt et al., 1978). A treatment regime of divided doses for trypanosomiasis carried out in dogs ( Gitatha and Ogada, 1969) and in goats (Aliu et al., 1984 ) has the possible disadvantage that the first subcurative dose does not clear all the parasites, thus exposing and predisposing the brain to invasion. It has been found (Schmidt and Bafort, 1985) that non-curative doses of trypanocides in voles with T. b. rhodesiense provoke encephalitis and brain invasion. Relapsed parasites were refractory to the usual curative doses of either drug and they also induced more involvement of the CNS. The latter observation has been confirmed in goats (Whitelaw et al., 1985). An altered reaction to the blood incubation infectivity test (BIIT) has also been reported for other T. brucei strains (Jennings and Urquhart, 1985 ). Unfortunately, all the canine cases are not subjected to the BIIT to eliminate the possibility of having the human infective parasites T. b. gambiense and T. b. rhodesiense. In conclusion, either the high curative dose of diminazene aceturate or the subcurative dose of isometamidium chloride, given as early as 8 d.p.i., resulted in only temporary clinical recovery of some of the infected dogs, since relapses eventually occurred in both groups. In addition, there was no difference in either the recovery rates or the respective parameter values between the two treated groups during the period of clinical recovery. Failure of the maximum tolerable dose of diminazene aceturate, given quite early after infection, points to the need both for further studies on pathogenesis and for a drug formula that can prevent relapses when the parasite populations show altered characteristics. ACKNOWLEDGEMENTS The authors are grateful to the University of Nairobi for a research grant, and to the West German Academic Exchange Programme for sponsoring one of the authors. The technical co-operation from the Faculty of Veterinary Medicine, Nairobi, is greatly appreciated. REFERENCES Aliu, Y.O., Odegaard,S. and S~gnen,E., 1984. Diminazene/Berenil;bioavailabilityand disposition in dairy goats.Acta Vet. Scand.,25: 593-596.
207 Boreham P.F.L., 1966. Pharmacologically active peptides produced in the tissues of the host during chronic trypanosome infections. Nature (London), 212: 190-191. Gitatha, S.K. and Ogada, T., 1969. Treatment of dogs infected with T. brucei subgroup organisms. East Afr. Trypanosomiasis Res. Organ. (E.A,T.R.O.) Rep. pp. 123-124. Gray, A.R., 1963. Serum transaminase levels in cattle and sheep infected with Trypanosoma vivax Exp. Parasitol., 14: 374-381. Houba, V., Brown, K.N. and Allison, A.C., 1969. Heterophile antibodies, M-antiglobulins and immunoglobulins in experimental trypanosomiasis. Clin Exp. Immunol., 4: 113-123. Jennings, F.W., Whitelaw, D.D. and Urquhart, G.M., 1977. The relationship between duration of infections with Trypanosoma brucei in mice and efficacy of chemotherapy. Parasitology, 75: 143-153. Jennings, F.W., Whitelaw, D.D., Holmes, P.H., Chizyuka, H.G.B. and Urquhart, G.M., 1979. The brain as a source of relapsing Trypanosoma brucei infection in mice after chemotherapy. Int. J. Parasitol., 9: 381-384. Jennings, F.W., and Urquhart, G.M., 1985. Induction of human serum-sensitive Trypanosoma brucei stabilates into human serum resistant "T. rhodesiense". Trans. Roy. Soc. Trop. Med. Hyg., 79: 80-85. Kaggwa, E., Munyua, W.K., Mugera, G.M., 1984. Pathogenicity of Trypanosoma brucei brucei in dogs. Bull Anim. Health. Prod. Afr., 32,360-368. Kaggwa, E., Munyua, W.K., Mugera, G.M., 1985. The pathology of Trypanosoma brucei brucei in the dog. Bull. Anim. Health. Prod. Afr., 33: 69-75. Losos, G.J. and Crockett, E., 1969. Toxicity of Berenil in dogs. Vet,. Rec., 85: 196. Morrison, W.I., Murray, M., Sayer P.D. and Preston, J.M., 1981. The pathogenesis of experimentally induced T. brucei infection in the dog. 1. Tissue and organ damage. Am. J. Pathol., 102: 168-181. Morrison, W.I., Murray, M., Whitelaw, D.D. and Sayer, P.D., 1983. Pathology of infection with Trypanosoma brucei: disease syndromes in dogs and cattle resulting from severe tissue damage. In: P.L. Gigase and E.A.E. van Marck (Editors), From Parasitic Infection to Parasitic Disease. Contrib. Microbiol. Immunol. 7: 103-119. Mwambu, P.M., 1977. The symptomatology of experimental Trypanosoma brucei species infection in dogs as observed at E.A.T.R.O. Int. Sci. Coun. Trypanosomiasis Res. Control (OAU/STRC), 15th meeting, The Gambia, pp. 427-440. Neitz, W.O., and McCully, R.M., 1971. Clinicopathological study on experimental Trypanosoma brucei infections in horses, Part I. Development of clinically recognisable nervous symptoms in nagana infected horses treated with subcurative doses of Antrypol and Berenil. Onderst. J. Vet. Res., 38: 127-140. Ogada, T., 1969. Toxicity of intramuscular Berenil in dogs. East Afr. Trypanosomiasis Rep. pp. 121-123. Oppong, E.N.W., 1969. Toxicity of Berenil in the dog. Vet. Rec., 85: 370. Reitman, S. and Frankel, S., 1957. A colorimetric method for the determination of serum glutamic oxalacetic and glutamic pyruvic transaminases. Am. J. Clin. Pathol., 28: 56-63. Rogers, D.J., 1985. Trypanosomiasis 'risk' or 'challenge': a review. Acta Trop. 42: 5-23. Sayer, P.D., Morrison, W.I., Preston, J.M., Hird, S.F., Price, J.E. and Murray, M., 1977. African trypanosomiasis in the dog. Int. Sci. Coun. Trypanosomiasis Res. Control, (OAU/STRC) 15th Meeting, The Gambia, pp. 489-496. Schmidt, H. and Bafort, J.M., 1985. African trypanosomiasis: Treatment-induced invasion of brain and encephalitis. Am. J. Trop. Med. Hyg., 34: pp. 64-68. Schmidt, H., Kaduk, B. and Fink, E., 1978. Pathomorphology of side-effects of trypanocidic diamidines. Vet. Pathol., 15: 574. Stephen, L.E., 1970. Clinical manifestation of the trypanosomiasis in Livestock and other do-
208 mestic animals. In: H.W. Mulligan, (Editors), The African Trypanosomiases. Allen Unwin, pp. 775-792. Toure, S.M., 1970. Prothidium and isometamidium in the treatment of trypanosomiasis in a dog due to Trypanosoma brucei. Rev. Elev. Mgd. V~t. Pays Trop., 23: 321-326. Whitelaw, D.D., Moulton, J.E., Morrison, W.I. and Murray, M., 1985. Central nervous system involvement in goats undergoing primary infections with Trypanosoma brucei and relapse infections after chemotherapy. Parasitology, 90: 255-268. Williamson, J., 1970. Review of chemotherapeutic and chemoprophylactic agents. In: H.W. Mulligan (Editor), The African Trypanosomiases. Allen & Unwin, pp. 125-221. Wilson, A.J. and Cunningham, M.P., 1971. Immunological aspects of bovine trypanosomiasis. IV. Patterns in the production of common antibodies. Trop. Anim. Health. Prod., 3: 133-139.
199
Relapses in D o g s E x p e r i m e n t a l l y Infected w i t h Trypanosoma brucei and Treated w i t h D i m i n a z e n e A c e t u r a t e or I s o m e t a m i d i u m Chloride E. KAGGWA1, W.K. MUNYUA2, and G.M. MUGERA2
1Department of Parasitology and Entomology, Ahmadu BeUo University, Zaria (Nigeria) 2Department of Pathology and Microbiology, University of Nairobi, P.O. Box 29053, Nairobi, (Kenya) (Accepted for publication 23 February 1987)
ABSTRACT Kaggwa, E., Munyua, W.K. and Mugera, G,M., 1988. Relapses in dogs experimentally infected with Trypanosoma brucei and treated with diminazene aceturate or isometamidium chloride. Vet. Parasitol., 27: 199-208. Twenty dogs of mixed local East African breeds were used. Five of the dogs were uninfected controls and 15 were infected with T. brucei (ILRAD 273). Five of the infected dogs were untreated controls, five were treated with a high curative dose of diminazene aceturate, (7 mg kg-1 body weight (wt.), and five were given a subcurative dose of isometimidium chloride (1 mg kg-1 body wt.). The drugs, given at 8 days post infection (d.p.i..), led to apparent recovery. The antibody titres, however, remained high in both groups and at 42-49 d.p.i, there was at least one relapse in each treatment group. Parasite populations from relapsed animals were more resistant to the drugs than the original infecting populations. The implications of these findings are discussed.
INTRODUCTION
The dog is susceptible to three Trypanosoma brucei subspecies, T. brucei brucei, T.b. rhodesiense and T.b. gambiense, which cannot be differentiated morphologically. The symptoms and pathology of T. b. brucei infections in dogs have been described previously ( Stephen 1970; Mwambu, 1977; Sayer et al., 1979; Morrison, et al., 1983; Kaggwa, et al. 1984, 1985). The pathogenesis, however, as in other animals, is under speculation. Drugs have been used for trypanosomiasis to study certain aspects of pathology which could contribute to the pathogenesis of the disease. Wilson and Cunningham (1971) studied antibody responses after chemotherapy with diminazene aceturate in cattle; Boreham (1966) used the same drug in cattle 0304-4017/88/$03.50
© 1988 Elsevier Science Publishers B.V.
200 infected with T. brucei to study blood kinin levels; Houba et al. (1969) used Suramin ( British Pharmacopias ) and Tryparsamide (British Pharmacopias ) to modify the disease course in monkeys infected with T. b. brucei and T.b. rhodesiense in studying heterophil antibodies and immunoglobulin. Neitz and McCully (1971) used subcurative doses of diminazene aceturate and Suramin B.P. (Antrypol) (ICI Pharmaceuticals, U.K. ) to prolong the disease course in horses so as to study the invasive potential of T. brucei for the cerebrospinal fluid. In dogs, the commonly used drug is diminazene aceturate (usually at 3.5 mg kg-~ body wt.) but sometimes isometamidium chloride is used at 0.5-1.0 mg kg- ~ where the parasites are sensitive ( Gitatha and Ogada 1969; Toure, 1970; Williamson, 1970). The aim of this study was to compare some parameters in T. brucei-infected dogs after treatment with either a high curative dose of diminazene aceturate, or a subcurative dose of isometamidium chloride, to show the difference, if any, in the recovery rate as indicated by these parameters. MATERIALSAND METHODS
The parasite Trypanosoma brucei (ILRAD 273) isolated in 1971 from a Kongoni Alcephalus buselaphus cokii (one of the East African antelopes) in Tanzania was used. Before carrying out the main experiment, the parasite was found to be sensitive in white albino mice to diminazene aceturate at 3.5 mg kg- 1 body wt. and to isometamidium chloride at >~2 mg kg- 1 body wt. Animals Fifteen adult dogs of mixed local East African breeds, kept singly in fly-proof kennels, were proved trypanosome-negative using serology and animal subinoculation. They were each intravenously infected with 104 trypanosomes, and randomly grouped as indicated in Table I. Five uninfected dogs were used as controls.
Procedure Clinical signs were observed in the control and infected dogs and blood samples were taken for haematology and serology. The indirect fluorescent antibody test (IFAT) was used to detect anti-trypanosome antibodies. Parasitaemia was detected using capillary buffer coat wet smears and trypanosome counts were made in a haemocytometer Neubauer chamber. Alanine and aspartate aminotransferases were determined colorimetrically using kits (Boeringer-
201 TABLE 1 Responses of control and T. brucei-infected dogs to the drugs Berenil1and Samorin2
Group No. of dogs Drug used Parasite sensitivity3( mg kg- ' body wt. ) Dosage given (mg k g - ' body wt. ) Dogs relapsed No days p.i.: Before relapses At death Relapse parasite sensitivity (mg kg- 1body wt. )
Control
Infected
1 5 None NA NA NA
2 5 Berenil 3.5 7 1
3 5 Samorin 2 1 2
4 5 None NA NA NA
NA
42 78 > 10
48,49 70.76 >2
NA 204 NA
NA
1Diminazene aceturate (Hoechst). 2isometamidium chloride (May & Baker). 3As tested in mice 4Longest survival date of this group. NA = not applicable; > = above stated amount.
Manhein GmbH Diagnostica, West Germany) which closely follow the method of Reitman and Frankel (1957). At 8 d.p.i, diminazene aceturate (7 mg k g - ' body wt.) was given intramuscularly to each of the five dogs in Group 2 (Table I), while isometamidium chloride (1 mg kg -1 body wt.) was given to each of the five in group 3. The remaining infected dogs were left as untreated controls. At specific d.p.i., one dog from each treatment group was killed for postmortem, histology and immunohistology. These results on pathology have been reported elsewhere (Kaggwa et al., 1985). After 27 d.p.i, there were only two dogs in each of the treated groups. The tests of drug sensitivity in mice of the parasite populations to the original drugs were then repeated. RESULTS
All infected dogs became parasitaemic in 2-3 days. The untreated control dogs showed high levels of parasitaemia (Fig. 1 ), high temperatures (Fig. 2) and anaemia (as indicated by decreased packed cell volume (PCV) percentages and haemoglobin (Hb) levels) (Figs. 3 and 4).There were slightly elevated serum alanine (ALAT) and aspertate aminotransferase (ASAT), levels (Figs. 5a,b ) and elevated antibody titres, (Fig. 6). Lymph node enlargement, eye lesions, oedema, lack of coordination, recumbency and finally death of all the control dogs were noted within 20 d.p.i.
202
lit
KEY : x -x
10
x x ....
S a m o r i n treatea dogs I n f e c t e d u n t r e a t e d dogs Berenil treated dogs Treatment
time
x/ ,.~x-X'x
/
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tx /
////
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/1111
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O 0
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20
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Days
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50 infected
60
70
80
Fig. 1. Mean parasitaemia, Log.lo trypanosomes per millilitre, in groups of five dogs, infected with T. b. brucei and treated with either Samorin or Berenil, or untreated. 43.0
KEY x,
42.C
x Infected
• .....
-O S a m o r i n
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-• Berenil
o
untreated treated treated
o Control Treatment
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and a f t e r i n f e c t i o n
Fig. 2. Mean temperatures, °C, in groups of five dogs infected with T.b. brucei and treated with either Samorin or Bereni], or untreated.
Treatment with either of the drugs arrested the progress of clinical symptoms. The dogs regained their demeanour, body temperatures returned to normal (Fig. 2), the parasitaemia disappeared (Fig. 1 ) and serum enzyme levels decreased greatly ( Fig. 5 ). The PCV and Hb mean values remained within the normal range for both the treated groups up to 55 d.p.i, after which they dropped (Figs. 3 and 4). This drop coincided with the disease relapse where no treatments were attempted. The PCV and Hb mean values within the two groups
203 KEY x . . . . . x Berenil treated clogs o o Samorin treot.ed dogs
80
PC__Zv 70
~.
~. Infected untreated control
A
A Uninfected control
Treatment 60
F
Upper and
L
Lower
time
normal range
5C
40:
3: 2C
t 10
20 30 40 50 DAYS POST INFECTION
60
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BO
Fig. 3. Mean packed cell volume (PCV) % in groups of five dogs infected with T. b. brucei and treated with either Samorin or Berenil, or untreated. 17
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o L = x. . . . . x x x ~
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8
1012141618202224262830323436384042444648505254565860 Days and post infection
Fig. 4. Mean haemoglobin values (Hb) g/100 ml, in groups of five dogs infected with T. b. brucei and treated with either Samorin or Berenil, or untreated.
fluctuated uniformly (Figs. 3 and 4). An exception was at 53 d.p.i, when the isometamidium-treated group had a higher PCV (49.3%) than the diminazene-treated group (46.9%) but a lower Hb level (15.3 g/100 ml) than that of the diminazene group (15.6%). In order to test whether there was any significant difference, one-way analysis of variance was carried out. The two treated groups were compared for the four dogs that survived up to that period. There was no significant difference in the PCV between the two groups: the isometamidium group had a mean of 47.6% while the other group had a mean of 45.6%. Furthermore, the PCV values at 53 d.p.i, did not differ significantly from the overall means for each group subjected to the t-test at 0.05 level of
204
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=
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treated dogs treated clogs dogs
time
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o
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80
KEY
x------x a a
80
,
........
Berenil treated dogs Samorin treated clogs
x Untreated dogs Treatment time
70 60
~o 4O
•
/x
\x
10' O !
' 10
' 20
' ' ' 30 40 50 DAYS POST INFECTION
~0
70'
8~
Fig. 5. Mean aminotransferase levels, international units per litre i.u. l - 1, in groups of five dogs infected with T. b. brucei and treated with either Samorin or Berenil, or untreated.
1280320
/ x~
20
//
r/~
.
~jl,'~Jl[/] ,
.....
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-10 ~
0
t
x
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Treatment
c, Sam orinBereni[treated
~-- ----~
,
,
,
,
,
,
,
time
,
10 20 30 40 50 Days before and after infection
~
tr e~ed
,
80
,
-,
70
,
80
Fig. 6. Mean serum antibody levels shown in reciprocal titres in groups of five dogs infected with T. b. brucei and treated with either Samorin or Berenil, or untreated.
205 significance (isometamidium-treated group: t=1.713, df=47, P=0.0896 (>0.05); diminazene-treated group: t=l.080, df--51, P--0.285 (>0.05). Therefore, the deviation at 53 d.p.i, was non-significant. The serum antibody titres, however, remained significantly high in both the treated groups (Fig. 6). Relapses were suffered by one of the dogs treated with diminazene aceturate and two of those treated with isometamidium. The symptoms of the three relapse cases were similar to those of the primary disease, but they showed more involvement of the CNS, with a combination of the following signs: poorer coordination, aggression, hyperaesthaesia, circling in the kennels, head tilted on one side, paddling of the feet, up and down jaw movements, frothing at the mouth and eventually death. The relapse parasite populations were resistant to the usual diminazene aceturate doses and those of 10 mg kg- 1 body wt., and to isometamidium chloride at 2 mg kg-1 body wt. DISCUSSION The progress of the primary disease was arrested by both of the drugs. After treatment the respective parameters started changing at approximately the same time in both groups. Furthermore, these parameters did not indicate any difference in rate of recovery between the two treated dog groups. The post-treatment changes in temperature, red blood cell parameters and parasitaemia up to 40 d.p.i, infection were indicative of recovery. Serum transaminase levels dropped in both treated and untreated dogs. It has been suggested that the decrease of these enzymes during the latter half of the disease course might be due to less destruction of the trypanosomes as the body defence mechanisms become overpowered or immunosuppressed by the infection ( Kaggwa et al., 1984). This was assuming Gray's suggestion ( Gray, 1963) that the elevated enzyme levels might be from dying trypanosomes, to be correct. However, there was no difference in transaminase levels between those dogs treated with either diminazene aceturate or isometamidium chloride. There were no significant differences in the other parameters used to assess recovery between the two groups of treated dogs. Thus, after curative or noncurative doses there can be a period of apparent recovery, followed by a possible relapse withT, brucei. The application of the Berenil index (Berenil (Hoechst) for diminazene aceturate ) in the determination of field trypanosome risks and challenge (Rogers, 1985) should, therefore,be carefully evaluated in the face of possible relapses after the use of curative doses of diminazene aceturate doses and in absence of tsetse challenge, at least for T. brucei. Relapses in dogs treated with diminazene aceturate cannot be due to drug resistance of the infecting parasite population or to under-dosage. It has been suggested (Jennings et al., 1977, 1979; Morrison et al., 1983) that relapses in mice and cattle might be due to parasites which invade the brain, thus escaping
206
the action of drugs which do not cross the blood-brain barrier. In infected dog brain, the first histological lesions were seen on Day 8 p.i. but no parasites were detected until Day 16 p.i. (Morrison et al., 1981 ). If parasites in the brain had been the cause of the relapses in our dogs, then this organ might have been affected earlier than 8 d.p.i. Alternatively, the diminazene aceturate dose may not be giving a sufficiently high tissue concentration to kill all the parasites. The persistence of significant antibody titres indicated the presence of parasites in the body. However, higher doses in dogs predispose them to CNS toxic symptoms, (Losos and Crockett, 1969; Oppong, 1969; Ogada, 1969; Schmidt et al., 1978). A treatment regime of divided doses for trypanosomiasis carried out in dogs ( Gitatha and Ogada, 1969) and in goats (Aliu et al., 1984 ) has the possible disadvantage that the first subcurative dose does not clear all the parasites, thus exposing and predisposing the brain to invasion. It has been found (Schmidt and Bafort, 1985) that non-curative doses of trypanocides in voles with T. b. rhodesiense provoke encephalitis and brain invasion. Relapsed parasites were refractory to the usual curative doses of either drug and they also induced more involvement of the CNS. The latter observation has been confirmed in goats (Whitelaw et al., 1985). An altered reaction to the blood incubation infectivity test (BIIT) has also been reported for other T. brucei strains (Jennings and Urquhart, 1985 ). Unfortunately, all the canine cases are not subjected to the BIIT to eliminate the possibility of having the human infective parasites T. b. gambiense and T. b. rhodesiense. In conclusion, either the high curative dose of diminazene aceturate or the subcurative dose of isometamidium chloride, given as early as 8 d.p.i., resulted in only temporary clinical recovery of some of the infected dogs, since relapses eventually occurred in both groups. In addition, there was no difference in either the recovery rates or the respective parameter values between the two treated groups during the period of clinical recovery. Failure of the maximum tolerable dose of diminazene aceturate, given quite early after infection, points to the need both for further studies on pathogenesis and for a drug formula that can prevent relapses when the parasite populations show altered characteristics. ACKNOWLEDGEMENTS The authors are grateful to the University of Nairobi for a research grant, and to the West German Academic Exchange Programme for sponsoring one of the authors. The technical co-operation from the Faculty of Veterinary Medicine, Nairobi, is greatly appreciated. REFERENCES Aliu, Y.O., Odegaard,S. and S~gnen,E., 1984. Diminazene/Berenil;bioavailabilityand disposition in dairy goats.Acta Vet. Scand.,25: 593-596.
207 Boreham P.F.L., 1966. Pharmacologically active peptides produced in the tissues of the host during chronic trypanosome infections. Nature (London), 212: 190-191. Gitatha, S.K. and Ogada, T., 1969. Treatment of dogs infected with T. brucei subgroup organisms. East Afr. Trypanosomiasis Res. Organ. (E.A,T.R.O.) Rep. pp. 123-124. Gray, A.R., 1963. Serum transaminase levels in cattle and sheep infected with Trypanosoma vivax Exp. Parasitol., 14: 374-381. Houba, V., Brown, K.N. and Allison, A.C., 1969. Heterophile antibodies, M-antiglobulins and immunoglobulins in experimental trypanosomiasis. Clin Exp. Immunol., 4: 113-123. Jennings, F.W., Whitelaw, D.D. and Urquhart, G.M., 1977. The relationship between duration of infections with Trypanosoma brucei in mice and efficacy of chemotherapy. Parasitology, 75: 143-153. Jennings, F.W., Whitelaw, D.D., Holmes, P.H., Chizyuka, H.G.B. and Urquhart, G.M., 1979. The brain as a source of relapsing Trypanosoma brucei infection in mice after chemotherapy. Int. J. Parasitol., 9: 381-384. Jennings, F.W., and Urquhart, G.M., 1985. Induction of human serum-sensitive Trypanosoma brucei stabilates into human serum resistant "T. rhodesiense". Trans. Roy. Soc. Trop. Med. Hyg., 79: 80-85. Kaggwa, E., Munyua, W.K., Mugera, G.M., 1984. Pathogenicity of Trypanosoma brucei brucei in dogs. Bull Anim. Health. Prod. Afr., 32,360-368. Kaggwa, E., Munyua, W.K., Mugera, G.M., 1985. The pathology of Trypanosoma brucei brucei in the dog. Bull. Anim. Health. Prod. Afr., 33: 69-75. Losos, G.J. and Crockett, E., 1969. Toxicity of Berenil in dogs. Vet,. Rec., 85: 196. Morrison, W.I., Murray, M., Sayer P.D. and Preston, J.M., 1981. The pathogenesis of experimentally induced T. brucei infection in the dog. 1. Tissue and organ damage. Am. J. Pathol., 102: 168-181. Morrison, W.I., Murray, M., Whitelaw, D.D. and Sayer, P.D., 1983. Pathology of infection with Trypanosoma brucei: disease syndromes in dogs and cattle resulting from severe tissue damage. In: P.L. Gigase and E.A.E. van Marck (Editors), From Parasitic Infection to Parasitic Disease. Contrib. Microbiol. Immunol. 7: 103-119. Mwambu, P.M., 1977. The symptomatology of experimental Trypanosoma brucei species infection in dogs as observed at E.A.T.R.O. Int. Sci. Coun. Trypanosomiasis Res. Control (OAU/STRC), 15th meeting, The Gambia, pp. 427-440. Neitz, W.O., and McCully, R.M., 1971. Clinicopathological study on experimental Trypanosoma brucei infections in horses, Part I. Development of clinically recognisable nervous symptoms in nagana infected horses treated with subcurative doses of Antrypol and Berenil. Onderst. J. Vet. Res., 38: 127-140. Ogada, T., 1969. Toxicity of intramuscular Berenil in dogs. East Afr. Trypanosomiasis Rep. pp. 121-123. Oppong, E.N.W., 1969. Toxicity of Berenil in the dog. Vet. Rec., 85: 370. Reitman, S. and Frankel, S., 1957. A colorimetric method for the determination of serum glutamic oxalacetic and glutamic pyruvic transaminases. Am. J. Clin. Pathol., 28: 56-63. Rogers, D.J., 1985. Trypanosomiasis 'risk' or 'challenge': a review. Acta Trop. 42: 5-23. Sayer, P.D., Morrison, W.I., Preston, J.M., Hird, S.F., Price, J.E. and Murray, M., 1977. African trypanosomiasis in the dog. Int. Sci. Coun. Trypanosomiasis Res. Control, (OAU/STRC) 15th Meeting, The Gambia, pp. 489-496. Schmidt, H. and Bafort, J.M., 1985. African trypanosomiasis: Treatment-induced invasion of brain and encephalitis. Am. J. Trop. Med. Hyg., 34: pp. 64-68. Schmidt, H., Kaduk, B. and Fink, E., 1978. Pathomorphology of side-effects of trypanocidic diamidines. Vet. Pathol., 15: 574. Stephen, L.E., 1970. Clinical manifestation of the trypanosomiasis in Livestock and other do-
208 mestic animals. In: H.W. Mulligan, (Editors), The African Trypanosomiases. Allen Unwin, pp. 775-792. Toure, S.M., 1970. Prothidium and isometamidium in the treatment of trypanosomiasis in a dog due to Trypanosoma brucei. Rev. Elev. Mgd. V~t. Pays Trop., 23: 321-326. Whitelaw, D.D., Moulton, J.E., Morrison, W.I. and Murray, M., 1985. Central nervous system involvement in goats undergoing primary infections with Trypanosoma brucei and relapse infections after chemotherapy. Parasitology, 90: 255-268. Williamson, J., 1970. Review of chemotherapeutic and chemoprophylactic agents. In: H.W. Mulligan (Editor), The African Trypanosomiases. Allen & Unwin, pp. 125-221. Wilson, A.J. and Cunningham, M.P., 1971. Immunological aspects of bovine trypanosomiasis. IV. Patterns in the production of common antibodies. Trop. Anim. Health. Prod., 3: 133-139.