Br. vet. J . (1991) . 147, 1 5 5

DIMINAZENE ACETURATE RESIDUES IN THE TISSUES OF HEALTHY, TRYPANOSOMA CONGOLENSE AND TR YPANOSOMA BR UCEI BR UCEI INFECTED DOGS

P . A . ONYEYILI* and S . M . ANIKAt *Department of Veterinary Physiology and Pharmacology, Faculty of Veterinary Medicine, University of Maiduguri, P.M.B. 1069, Maiduguri, Nigeria; tDepartment of Veterinary Physiology and Pharmacology, Faculty of Veterinary Medicine, University of Nigeria, Nsukka, Nigeria

SUMMARY The tissue distribution and residue profile of diminazene aceturate was investigated in healthy dogs and in dogs infected with Trypanosoma congolense and Trypanosoma brucei brucei . The drug was administered at 3 .5 mg/kg i .m . and tissue samples were taken post mortem from the animals at 48, 72, 120, 168 and 240 h after injection . The drug was distributed to various organs and tissues of the body with the highest concentrations occurring in liver and kidney . Higher drug levels were obtained in the tissues of healthy dogs compared with trypanosome infected animals except in the brain . The levels of residues in the healthy animals were significantly different (P< 0 .05) from those of the infected dogs . The drug residues were still detectable in the tissues of the animals 10 days after drug administration .

INTRODUCTION Diminazene aceturate (Berenil R , Hoechst Farbwerk AG, Frankfurt am Main, West Germany) is a curative antitrypanosomal and antibabesial agent which is used in the control of animal trypanosomiasis and babesiosis (Milne et al., 1955 ; Bauer, 1963 ; Verma et al., 1973 ; Pandey & Mishra, 1978) . Few tissue distribution and residue studies have been carried out with this agent . Detectable amounts of the drug were observed in various tissues and organs of goats with mean concentrations of 60 .0 ± 4 .8, 45 .6 ± 2 .23, 1 .2 ± 0 .12 and 1 .0 ± 0 .7µg/g occurring in the liver, kidney, skeletal muscle and brain respectively 2 days after intramuscular administration at the rate of 3 .5 mg/kg (Onyeyili, 1982) . Gilbert (1983), working with carbon-labelled diminazene aceturate, observed that rabbits killed 7 days after intramuscular injection of 3 .5 mg/kg of the drug had 40 .53 ± 4 .0 and 3.27 ± 0 .31 ug/g in the liver and kidney respectively . Kellner et al. (1985) also working with carbon-labelled diminazene aceturate reported that the concentration of the drug was low in skeletal muscle and fat (below 1 ug/g), but higher in excretory organs .



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Although there have been tissue distribution and residue studies of this agent in some animal species we are unaware of any in dogs, especially those infected with African trypanosome species . The objective of the present study therefore was to determine the extent of tissue distribution and residue profile of the agent in healthy, Trypanosoma congolense and Trypanosoma brucei brucei infected mongrel dogs . This is important because dogs are used as a source of meat protein in some parts of Nigeria (Baba et al., 1983 ; Emehelu, 1988) .

MATERIALS AND METHODS Experimental animals and treatments

Forty-seven clinically healthy mongrel dogs of both sexes, weighing between 5 .0 and 7 .0 kg and between 10 and 12 months of age were used . On arrival the dogs were treated orally with fenbendazole (Panacure', Hoechst AG, Frankfurt Main, West Germany) at 100 mg/kg body weight and with disophenol (D .N .P .R, American Cyanamid Company, Princeton, NJ .) subcutaneously at the rate of 0 .2 ml/kg body weight . They were kept in a fly-proof house in separate cages and fed twice daily . Water was provided ad libitum . The dogs were screened for the presence of trypanosomes and other haemoprotozoa prior to the commencement of the experiments using haematocrit buffy-coat technique (Murray et al., 1977) . The animals were left to acclimatize for 1 month before the study commenced . They were separated into three groups of 15 animals each . The first group was not infected, while the second and third groups were infected intravenously with 1 ml of normal saline diluted rat blood containing Trypanosoma congolense strain TRIV 35 (5X105 trypanosomes/ml) and Trypanosoma brucei brucei strain 8/18 (5X10' trypanosomes/ml) respectively obtained from the Nigerian Institute of Trypanosomiasis Research, Vom . Trypanosomes were enumerated in a haemocytometer using the method of Brown & Losos (1977) . A 7% solution of diminazene aceturate in distilled water was administered intramuscularly to the dogs at 3 .5 mg/kg . The treatment was initiated 15 days post-infection when well established parasitaemia had been confirmed in the infected groups by assessment with the haematocrit buffy-coat technique (Murray et al., 1977) . Sample collection

Five ml of blood was collected from the right femoral vein of each of the dogs at 24, 36, 48, 60 and 72 h after diminazene aceturate administration using EDTA as an anti coagulant . The blood samples were centrifuged immediately at 1500 rpm for 15 min, to obtain the plasma . Three dogs from each of the groups were sacrificed at 48, 72, 120, 168 and 240 h post-administration and 10 g of tissue samples (liver, kidney, brain, heart and skeletal muscle) were collected . Two untreated and uninfected dogs were killed for the preparation of control tissues and tissue standards . The experimental work area and all utensils were cleaned thoroughly after each slaughter to prevent contamination . The plasma and tissue samples were stored frozen until analysed .



BERENIL IN HEALTHY AND TRYPANOSOME INFECTED DOGS

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Analysis of diminazene aceturate The diminazene aceturate content of the plasma was determined colorimetrically using the method of Klatt & Hajdu (1976a) . The method of Klatt & Hajdu (1976b) was used for the determination of the diminazene content of the tissues . In the procedure, 5 ml trichloroacetic acid (20%) was added to 10 g tissue and homogenized . Six ml of the clear supernatant was alkalized with 0 .5 ml of 5 M sodium hydroxide and extracted four times with 5 ml ethylacetate . The organic phase was evaporated to dryness after which 2 ml of 1 M hydrochloric acid was added and diazotized with 0 .2 ml sodium nitrite solution (0 .5%) . After 3 min, 0 .5 ml sulphaminic acid solution (1%) was added and shaken thoroughly . After a further 3 min period, I ml alpha-naphthylethylenediamine (0 .4%) was added and the developed colour measured at 540 nm with a spectrophotometer . Statistical analysis Linear regression analysis was performed on the mean tissue concentrations (three dogs at each time interval) and the slope of the elimination curves determined . The results were expressed as mean ± SEM . Tests for significance between mean parameters in respect of healthy, Trypanosoma congolense, and Trypanosoma brucei brucei infected dogs were performed, using an analysis of variance and the `null' hypothesis was rejected at the 5% level of probability .

RESULTS Intramuscular injection of diminazene aceturate at 3 .5 mg/kg body weight resulted in a measurable blood level for 24 h in healthy dogs and 36 h in trypanosome infected dogs . A mean plasma concentration (Table I) of 0 .2 ± 0 .08 ug/ml was obtained in healthy dogs while 0 .32 ± 0 .05 and 0 .28 ± 0 .07 ug/ml were obtained in T congolense and I b . brucei infected dogs respectively . Thirty-six hours post-drug administration mean values of 0.28 ± 0.06 and 0.22 ± 0 .08 pg/ml were recorded for T. congolense and T. b. brucei infected animals respectively . The plasma concentrations were observed to be below detectable levels at 48 h post-administration of the drug . Table I Mean plasma diminazene aceturate concentrations (µg/ml) in healthy and trypanosome infected dogs following intramuscular injection of the drug at the rate of 3 .5 mg/kg Concentrations (µg/ml) Time (h)

0 24 36 48

Healthy dogs

0 .00 0 .2±0 .08 0 .00 0 .00

*Mean±SEai based on three observations .

T . congolense

T . b . brucei

infected dogs

infected dogs

0 .00 0 .32 ± 0 .05 0 .28 ± 0 .06 0 .00

0 .00 0 .28 ± 0 .07 0 .22 ± 0 .08 0 .00



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The mean concentrations of the drug in the liver of healthy, T. congolense and T. b. brucei infected dogs 48 h after infection are shown in Table II . The highest concentrations of 81 ± 2.47, 39.6 ± 4.08 and 34 .5 ± 2 .77,ug/g were obtained in the healthy, T. congolense and T. b. brucei infected animals respectively . Table II Mean diminazene aceturate concentrations (ug/g) in the liver of healthy and trypanosome infected dogs injected intramuscularly with the drug at the rate of 3 .5 mg/kg Concentrations (,ug/g) Time (h)

Healthy dogs

0 .00 81 .0 ± 2 .47 60.0 ± 3 .44 47 .1 ± 7 .88 36.6 ± 4 .14 24 .0±5 .21

0 48 72 120 168 240

T . congolense

T . b . brucei

infected dogs

infected dogs

0.00 39 .6 ± 4 .08 33 .7 ± 2 .33 29.9 ± 1 .50 19.5 ± 1 .09 10.4±2 .12

0.00 34.5 ± 2 .77 39.1 ± 2 .05 25 .5 ± 3 .66 17 .3±2 .04 10.3±1 .01

* Mean ±SEM based on three observations .

On a wet weight basis, this was almost 45% of the dose in the healthy, 30 .1% in T. T. b. brucei infected dogs . The concentrations in the liver showed a continuous decrease and at 240 h (10 days) post-injection the concentration had dropped to 24 .0 ± 5 .21, 10.4 ± 2 .12 and 10 .3 ± 1 .01 1ug/g respectively in healthy T congolense and T. b . brucei infected animals . The highest concentrations (at 48 h post-injection) of 44 .3 ± 3 .61, 30 .6 ± 1 .53 and 27 .3 ± 2 .14 ug/g (Table III) were obtained respectively in the kidney of healthy, TT congotense and I b. brucei infected animals representing 8 .1%, 6 .4% and 5 .3% (on wet basis) of congolense and 26 .3% in

Table III Mean diminazene aceturate concentration (µg/g) in the kidney of healthy and trypanosome infected dogs injected intramuscularly with the drug at the rate of 3 .5 mg/kg Concentrations (,ug/g) Time (h)

0 48 72 120 168 240

Healthy dogs

0 .00 44 .3±3 .61 37 .7 ± 2 .26 29 .3 ± 1 .54 20 .0 ± 2 .36 15 .3 ± 3 .41

* Mean ± scat based on three observations .

T. congolense

'l b, brucei

infected dogs

infected dogs

0 .00 30 .6±1 .53 21 .5 ± 0.89 17 .6 ± 1 .02 11 .3 ± 0 .68 8 .5 ± 0 .93

0 .00 27 .3±2 .14 20 .4 ± 2 .94 15 .0 ± 1 .13 10 .5 ± 1 .27 8 .9 ± 1 .52



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the administered dose in the healthy, T. congolense and T b. brucei infected dogs respectively . The concentrations in the kidney decreased to 15 .3 ± 3 .41 ug/g or 3 .3%, 8 .5 ± 0 .93 µg/g or 1 .7% and 8.9 ± 1 .52 Itg/g or 1 .6% at 240 h respectively in healthy, T. congolense and T. b . brucei infected animals . The mean concentrations of diminazene in the brain, skeletal muscle and heart of healthy and trypanosome infected dogs are presented in Tables IV, V and VI . The concentrations in these tissues were markedly lower than those in the liver and kidney in both healthy and infected animals . The brain at 48 h had concentrations of 0 .78 ± 0.06, 0 .91 ± 0 .19 and 0 .97 ± 0 .12 ug/g in healthy, T. congolense and T b . brucei infected dogs respectively, and these decreased to 0 .18 ± 0 .08, 0 .27 ± 0 .04 and 0 .29 ± 0 .03 µg/g at 240 h Table IV Mean diminazene aceturate concentration (ug/g) in the brain of healthy and trypanosome infected dogs treated intramuscularly with the drug at the rate of 3 .5 mg/kg

Concentrations (µg/g)* Time (h)

0 48 72 120 168 240

Healthy dogs

T . congolense

0 .00 0 .78±0 .06 0 .55±0 .05 0 .43+0 .07 0 .36 ± 0 .05 0 .18+0 .08

infected dogs

T . b . brucei infected dogs

0 .00 0 .91±0 .19 0 .70±0 .12 0 .53 ± 0 .12 0 .37 ± 0 .08 0 .27 ± 0 .04

0 .00 0 .97±0 .12 0 .73±0 .08 0 .61 ± 0 .02 0 .48 ± 0 .06 0 .29 ± 0 .03

* Mean ± Lyi based on three observations .

Table V Mean diminazene aceturate concentrations (µg/g) in the skeletal muscle of healthy and trypanosome infected dogs treated intramuscularly with the drug at the rate of 3 .5 mg/kg

Concentrations (ug/g) * Time (h)

0 48 72 120 168 240

Healthy dogs

0 .00 1 .47±0 .25 0 .92±0 .14 0,49+0 .07 0 .32+0 .06 0 .09 ± 0 .08

*Mean ±sLm based on three observations .

T . congolense

T . b . brucei

infected dogs

infected dogs

0 .00 1 .05±0 .13 0 .95±0 .14 0 .58 ± 0 .08 0 .37 ± 0 .04 0 .27 ± 0.02

0 .00 0 .96±0 .19 0 .74±0 .07 0 .41 ± 0 .06 0 .31 ± 0 .02 0 .18+0 .09

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Table VI Mean diminazene aceturate concentrations (µg/g) in the myocardium of healthy and trypanosome infected dogs treated with the drug intramuscularly at 3 .5 mg/kg Concentration ,ug/g) Time (h)

0 48 72 120 168 240

* Mean

Healthy dogs

0 .00 1 .36 ± 0 .25 0 .80 ± 0 .13 0 .56 ± 0 .14 0 .41 ± 0 .05 0 .18 ± 0 .09 ± SERI

T . congolense

T . b . brucei

infected dogs

infected dogs

0 .00 0.61±0.04 0.41 ± 0 .04 0 .34±0 .03 0 .29 ± 0 .03 0 .13±0 .17

0 .00 0 .71 ± 0 .18 0 .43 ± 0 .07 0 .40 ± 0 .03 0 .33 ± 0 .04 0 .17±0 .09

based on three observations .

post-administration . In the skeletal muscle, concentrations of 1 .47 ± 0 .25, 1 .05 ± 0 .13 and 0 .96 ± 0 .19 ug/g were recorded at 48 h after treatment in healthy, T congolense and T b . brucei infected dogs respectively . Peak concentrations of 1 .36 ± 0 .25 ug/g (healthy dogs), .61 ± 0 .04 ,ug/g (T. congolense infected dogs) and 0 .71 ± 0 .18 4ug/g (T. b. brucei infected dogs) were obtained from the heart at 48 h after injection . These amounts decreased to 0 .18 ± 0 .09, 0.13 ± 0 .17 and 0 .17 ± 0 .09,ug/g at 240 h post-administration . The half-lives of the drug in the liver of healthy, T congolense and T b. brucei infected animals were 2 .55, 4 .62 and 5 .59 h respectively while the elimination rate constants were 0 .272, 0 .150 and 0 .124 per hour . The half-lives of the drug in the brain were the same (231 h) in both healthy and trypanosome-infected groups . The percentages of the peak concentration of the drug (at 48 h) obtained at subsequent sampling times in various tissues were calculated . Forty-eight hours after diminazene administration, the amount of the drug present in the various tissues of the body of both healthy and trypanosome infected dogs were assumed to be 100% . Seventy-two hours after drug administration, the percentages of the peak concentrations obtained in the trypanosome infected animals were found to be higher than those of healthy dogs except for percentages of the peak level obtained in the kidney . Thereafter, a decreasing order of percentages occurred until 240 h which was the end of the experimentation period . 0

DISCUSSION The results presented here show that diminazene aceturate is readily distributed to various organs and tissues of the body . Forty-eight hours post-administration, highest concentrations occurred in the excretory organs (liver and kidney) in all the groups . This is in agreement with the observations of Kellner et al. (1985) and Onyeyili (1982) who observed that the drug accumulated mainly in the liver and kidney . Low diminazene aceturate concentrations were found in the brain and skeletal muscle of both the healthy and trypanosome infected animals, agreeing with the findings of



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Kellner et al. (1985) and Onyeyili (1982) who recorded low concentration in the brain tissues and skeletal muscles of healthy calves and goats respectively . The findings further showed that higher concentrations of diminazene aceturate were found in the liver, kidney, myocardium and skeletal muscle of healthy dogs compared with the trypanosome infected animals . The lower concentrations in the tissues of infected dogs may have resulted from reduced blood flow to organs and tissues of the body resulting from impaired circulatory function and changes in membrane permeability (Losos & Ikede, 1972) associated with trypanosomal infections . Changes in membrane permeability and concomitant circulatory disturbances could be induced by physiologically active peptides (kinins) which are potent capillary vasodilators . These kinins upon release by the trypanosome parasites act as non-specific inflammatory agents and cause inflammatory `stasis' and obstruction of local circulation (Boreham, 1970 ; Honigberg, 1986) . During infection, trypanosomes may be present in large numbers to produce significant biochemical changes in the host, including the production of large amounts of lactic and pyruvic acids (Goodwin, 1974) . These metabolites are capable of causing damage to small blood vessels, thereby interfering with cardiac function (Goodwin, 1974) . The lower concentrations of the drug observed in the liver, kidney and myocardium of the diseased animals may also have resulted from trypanosomal hepatitis, nephrosis and myocarditis which reduced the functional capabilities of these organs to concentrate the drug . In the case of brain tissue, higher drug concentrations were observed in the infected compared with healthy animals . This may be a result of inflammation caused by trypanosomal infections leading to enhanced penetration of the brain . There were apparently little differences in the levels of residues in T. congolense and T. b. brucei infected dogs except for the skeletal muscle which showed significant differences (P< 0 .05) . This may not be unconnected with the fact that T. congolense are strictly blood parasites (Maxie & Losos, 1977) with no extensive degenerative lesions while I brucei are found localized extravascularly in the connective tissues and produce extensive degenerative lesions (Ikede & Losos, 1972) . The present findings finally indicate that diminazene aceturate persists in the tissues of the dog for more than 240 h (10 days) after intramuscular administration . This should be given due consideration in the estimation of the withdrawal period for the agent .

REFERENCES BABA, S . S ., OGUNKOYA, A . B . & EZEOKOLI, C . D . (1983) . Tropical Veterinarian 1, 98 . BAUER, F . (1963) . Veterinary Preparations `Hoechst'. The Blue Book for the Veterinary Profession 7, BOREHAM, P . F . L . (1970) . Transactions of the Royal Society of Tropical Medicine and Hygiene 64, BROWN, L . A . & Losos G. J . (1977). Research in Veterinary Science23, 196 . EMEHELU, C . O . (1988) . MSc Thesis, University of Nigeria, Nsukka . GILBERT, R . J . (1983) . British Journal of Pharmacology 80, 133 . GOODWIN, L . G . (1974) . In Trypanosomiasis and Leishmaniasis, eds . K . Elliot, M . O'Connor

26 .

394 .

& G . E. W . Wolstenholme, p . 107 . Ciba Foundation Symposium No . 20 . Amsterdam : Elsevier . HoNIGBERG, B . M . (1986). Insect Science and its Application 7, 363 . 1KEDE, B . O . & Losos, G . J . (1972) . Veterinary Pathology 9, 278 . KLLLNER, H . M ., ECKERT, H . G . & VOLZ, M . H . (1985) . Tropical Medicine and Parasitology 36, 199 . KLATT, P . & HAJDU, P. (1976a) . Veterinary Record 99, 372 . KLATT, P . & HAJDU, P . (1976b) . Personal communication .



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Losos, G . J . & IKEDE, B . O. (1972) . Veterinary Pathology 9, (Suppl .), 1 . MAXIE, M . G. & Losos, G . J. (1977) . Veterinary Parasitology 3, 277 . MILNE, A . H ., ROBSON, J . & LWEBANDIZA, T . (1955) . Veterinary Record 67, 280 . PANDEY, N . N . & MISHRA, S . S . (1978) . Indian Veterinary journal 55, 144 . MURRAY, M ., MURRAY, P . K . & MCINTYRE, W . I . M . (1977) . Transactions of the Royal Society for Tropical Medicine and Hygiene 77, 325 . ONYEYILI, P. A. (1982) . MSc Thesis, Tuskegee Institute, Alabama . VERMA, B . B ., GAUTAM, O . P . & MALIK, P . D . (1973) . Veterinary Record 93, 465 . (Accepted for publication 9 August 19510

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Diminazene aceturate residues in the tissues of healthy, Trypanosoma congolense and Trypanosoma brucei brucei infected dogs.

The tissue distribution and residue profile of diminazene aceturate was investigated in healthy dogs and in dogs infected with Trypanosoma congolense ...
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