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Comparative serum biochemical changes in mongrel dogs following single and mixed infections of Trypanosoma congolense and Trypanosoma brucei brucei
Veterinary Parasitology 190 (2012) 56–61
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Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Comparative serum biochemical changes in mongrel dogs following single and mixed infections of Trypanosoma congolense and Trypanosoma brucei brucei R.C. Ezeokonkwo a,∗ , I.O. Ezeh a , J.I. Onunkwo b , I.W. Onyenwe a , C.N. Iheagwam a , W.E. Agu a a b
Department of Veterinary Parasitology and Entomology, University of Nigeria, Nsukka, Nigeria Department of Veterinary Public Health and Preventive Medicine, University of Nigeria, Nsukka, Nigeria
a r t i c l e
i n f o
Article history: Received 19 April 2011 Received in revised form 9 May 2012 Accepted 15 May 2012 Keywords: Comparative biochemistry Trypanosoma congolense T. brucei brucei Dogs
a b s t r a c t The serum activities of alkaline phosphatase (AP), alanine aminotransferase (ALT), aspartate aminotransferase (AST) and the serum levels of conjugated bilirubin (CB), blood urea nitrogen (BUN) and creatinine were studied following single and mixed infections of mongrel dogs with Trypanosoma congolense and Trypanosoma brucei brucei. Twenty mongrel dogs of both sexes aged between 3 and 6 months, and weighing between 2.5 and 5.9 kg were used for the study. The dogs were kept in clean metal cages in a flyproof house and were fed and given water ad libitum. The twenty dogs were divided into four groups of five dogs each. Group I dogs were uninfected control, group II were infected with T. congolense, group III were infected with T. brucei brucei and group IV were infected with both T. congolense and T. brucei brucei. Each dog in the infected groups II and III was inoculated intraperitonealy (i/p) with 1.0 ml of PBS diluted blood containing 1.0 × 106 trypanosomes whereas each infected dog in group IV (mixed infection) was inoculated with 0.5 ml of the PBS diluted blood containing 0.5 × 106 T. congolense and 0.5 ml of the PBS diluted blood containing 0.5 × 106 T. brucei brucei i/p. Parasites were detectable in the blood of the infected dogs in groups II, III, and IV 10–13 days post infection (PI) with the mean pre-patent period (PP) of 12, 10, and 11 days respectively. Trypanosome infection caused a significant (P < 0.05) increase in the serum activities of AP, ALT, AST and the serum levels of creatinine, CB, and BUN. The significant increases in the serum levels of CB, BUN, and creatinine and serum activities of AP and AST became noticeable from day seven PI in all the infected groups whereas that of ALT became noticeable from day 14 PI and increased continuously until the experiment was terminated. These increases however did not differ significantly (P > 0.05) between the infected groups in most cases. It was thus concluded that single or mixed infection of mongrel dogs with T. congolense and T. brucei brucei resulted in significant increases in the serum activities of AP, AST, ALT and serum levels of creatinine, CB and BUN which in most cases did not differ significantly (P > 0.05) among the infected groups. Crown Copyright © 2012 Published by Elsevier B.V. All rights reserved.
1. Introduction
∗ Corresponding author. Tel.: +234 8030919933/+234 8023469911. E-mail addresses: [email protected], [email protected] (R.C. Ezeokonkwo).
Trypanosomosis transmitted by tsetse flies is of great significance to human health and animal production in Africa. It is a serious hindrance to profitable livestock production and a major health hazard for dogs and other
0304-4017/$ – see front matter. Crown Copyright © 2012 Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.vetpar.2012.05.012
R.C. Ezeokonkwo et al. / Veterinary Parasitology 190 (2012) 56–61
companion animals in Nigeria (Anene and Omamegbe, 1987). The upsurge in the cases of canine trypanosomosis in eastern Nigeria is complicated by the fact that chemotherapy using very commonly available, diminazene aceturate is increasingly becoming ineffective in the treatment of the disease in dogs (Kaggwa et al., 1988; Chukwu et al., 1990; Kalu, 1995). Analyses of serum enzyme activities and substances like creatinine, CB and BUN are important in diagnosing certain pathologic conditions. Most of these enzymes act intracellularly, though few (lipase and amylase) act extracellularly. A pathological condition may result in increased cell destruction and death leading to the release of the intracellular enzymes and these substances into blood circulation which can be detected by various assays. Hitherto, changes in the serum enzyme activities and levels of CB, BUN, and creatinine of trypanosome-infected mongrel dogs have not been well studied. However it has been reported that trypanosomosis adversely affects vital organs like the liver, spleen and kidney leading to in most cases destruction of these organs and hence the increased release of these enzymes and substances contained within the cells into the blood circulation (Fiennes, 1970; Losos and Ikede, 1972; Taylor and Authie, 2004). The quantification of these enzymes and substances by end point assays can be used to determine the extent of the damage of the organs. This work was therefore undertaken to investigate and compare to what extent, single and mixed infections of Trypanosoma congolense and Trypanosoma brucei brucei alter the serum activities of AP, ALT, AST and serum levels of BUN, CB, and creatinine in mongrel dogs.
2. Materials and methods 2.1. Animals and their management Twenty mongrel dogs of both sexes aged between 3 and 6 months and weighing between 2.5 and 5.9 kg were used for the study. They were kept in clean metal cages in a fly proof house. They were fed heavily once daily and were given clean drinking water ad libitum. The dogs were acclimatized for three weeks during which time they were dewormed and deticked with 0.2 mg/kg body weight of Kepromec® (Ivermectin, Kepro B. V., Deventer, the Netherlands) subcutaneously (s/c) and 300 mg Distocide® (Praziquantel, Shin Poong Pharm Co. Ltd., Seoul, Korea) per dog. They were also vaccinated with canine distemper, Adenovirus type 2, parainfluenza, Parvovirus and Leptospira bacterin vaccine (Fort Dodge Animal Health, Iowa, USA). Also the dogs were given OXY-TQ® long acting Oxytetracycline (H.Y. Pharmaceutical Ltd. Helve Province, China) at 1 ml/10 kg body weight intramuscularly (i/m). The dogs were screened and confirmed negative for trypanosomes by wet mount technique and buffy coat method (Murray et al., 1977). Pre-infection parameters were taken from the dogs at day 14 and day 21 post-arrival before the commencement of the experiment. Adequate cleanliness, thorough disinfection and hygiene were maintained in the experimental animal house throughout the experiment.
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2.2. Experimental design The 20 dogs were divided into four groups (I–IV) of five dogs each using stratified random sampling technique. Group I dogs were uninfected controls while groups II and III were each infected with 1.0 × 106 T. congolense and T. brucei brucei respectively. Dogs in group IV (mixed infection) were each infected with 0.5 × 106 T. congolense and 0.5 × 106 T. brucei brucei. The infected dogs were monitored daily for the development of parasitaemia by wet blood film examination and buffy coat technique (Murray et al., 1977). Parameters studied were; serum activities of AP, ALT, and AST, and serum levels of BUN, CB, and creatinine. The serum levels of these substances were determined on the day of the infection (day 0) and subsequently every 7 days up to the end of the experiment. 2.3. Trypanosome infections T. congolense and T. brucei brucei used in this work were obtained from the Nigerian Institute for Trypanosomiasis Research (NITR), Vom, Plateau State, Nigeria. T. brucei brucei was originally isolated from cattle during an outbreak that occurred at Federe in Jos East Local Government Area of Plateau State in 1997 while the T. congolense was originally isolated from cattle in Karu, Nasarawa State, Nigeria in 1995. Both parasites were cryo-preserved in liquid nitrogen. The stabilates had been severally passaged in albino rats prior to the infection of the experimental dogs. The donor rats were bled by puncture of the retrobulbar plexus at the median canthus of the eye and the infected blood serially diluted with PBS. The level of parasitaemia was determined by the rapid matching method of Herbert and Lumsden (1976). Each dog in groups II, and III was inoculated i/p with 1.0 ml of PBS diluted blood containing 1.0 × 106 T. congolense and T. brucei brucei respectively while each dog in group IV (mixed infection) was inoculated with 0.5 ml of the PBS diluted blood containing 0.5 × 106 T. congolense and 0.5 ml of the PBS diluted blood containing 0.5 × 106 T. brucei brucei. 2.4. Serum sampling technique Two microliters of blood was drawn from the cephalic vein of each dog after thorough cleaning of the area with 70% ethanol. The blood was put into a plastic test tube without an anticoagulant. The test tube containing the blood was kept in a slanting position in a bench for about 3 h to enable the blood clot to yield the serum. Thereafter, the coagulated blood on the wall of the test tubes was scooped to the bottom of the tubes and latter centrifuged for 5 min at 20,833 G to ensure clear separation of the serum from the clotted blood cells. The clear serum was later used for the assays. 2.5. Methods for determining the various parameters The standard colorimetric method of Reitman and Frankel (1957) using Randox test kits (Randox laboratories Ltd., UK) was used for the determination of the serum activities of ALT and AST. Phenolphthalein monophosphate
method (Klein et al., 1960) using Quimica Clinica test kits (QCA, Spain) was used for the determination of serum activity of AP. Urease Berthelot method (Fawcett and Scott, 1960) for quantitative in vitro determination of urea in serum and urine using Randox urea colorimetric kit was used to determine BUN. The modified Jaffe method (Fossati et al., 1983) for in vitro determination of creatinine in serum, plasma or urine using Randox creatinine colorimetric method with depolarization was used to determine urea creatinine. The bilirubin kits prepared by Biosystems reagents and instruments (Biosystems, S.A., Barcelona, Spain) were used to determine the serum level of conjugated bilirubin (Pearlman and Lee, 1974; Zoppi et al., 1976). The assays were done in triplicate for each substance and for each experimental dog per sampling period. Finally, the average value was taken. 2.6. Handling of experimental animals during the study The guidelines set out by the University of Nigeria, Nsukka Ethics Committee for Medical and Scientific Research (MSR) which include good, clean and hygienic housing, adequate feeding, provision of clean water and humane handling of animals during sample collection were strictly followed in handling the dogs during the experiment. Valid approval and ethical clearance were obtained from the Ethics Committee of the University before the commencement of the experiment.
Mean alkaline phosphatase (iu/l)
R.C. Ezeokonkwo et al. / Veterinary Parasitology 190 (2012) 56–61
80 60 40 20 0
0
3.1. Onset of parasitaemia Parasites were detectable in the blood of the infected dogs in groups II, III, and IV; 10–13 days PI with the mean PP being 12, 13, and 11 days PI for groups II, III, and IV, respectively. Parasitaemia persisted throughout the duration of the experiment. Figs. 1–3 show the weekly mean AP, ALT and AST of mongrel dogs infected with single and mixed infections of T. congolense and T. brucei brucei respectively. Trypanosome infections caused an increase in the serum activity of AP, ALT, and AST in the infected groups (II, III, and IV). The increase in AP and AST differed significantly (P < 0.05) between the uninfected control and the infected groups as from day seven PI up to the end of the experiment whereas the increases in ALT differed significantly (P < 0.05) from day 14 up to the end of the experiment. Comparatively, the increase in the AP levels did not differ significantly (P > 0.05) between the infected groups throughout the experiment. Also, the increases in the serum activities of ALT and ASP were not significantly different (P > 0.05) between the infected groups up to day 21 PI, but by day 28 PI the increase
21
28
Fig. 1. Weekly mean alkaline phosphatase of dogs infected with either T. brucei brucei or T. congolense or T. brucei brucei and T. congolense.
80 60 40 20 0
0
7
14
21
28
Days post infection Uninfected control Infected with T. congolense Infected with T. brucei brucei Infected with T. congolense + T. brucei brucei
Fig. 2. Weekly mean alanine aminotransferase of dogs infected with either T. brucei brucei or T. congolense or T. brucei brucei and T. congolense.
in group II (T. congolense infected) differed significantly (P < 0.05) from infected groups III and IV. Table 1 shows the weekly mean serum levels of conjugated bilirubin (CB), blood urea nitrogen (BUN), and creatinine of dogs infected with single and mixed infections of T. brucei brucei and T. congolense. Trypanosome infections increased the mean serum levels of these substances in the course of the experiment. The increase in Mean Alanine aminotransferase (iu/l)
3. Result
14
Uninfected control Infected with T. congolense Infected with T. brucei brucei Infected with T. congolense + T. brucei brucei
2.7. Statistical analysis Data were analyzed using one-way analysis of variance (ANOVA) and Duncan’s multiple range test (Duncan, 1966) using SPSS version 12.00 software package. The level of significance was considered at P < 0.05.
7
Days post infection
Mean Alanine aminotransferase (iu/l)
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80 60 40 20 0 0
7
14
21
28
Days post infection Uninfected control Infected with T. congolense Infected with T. brucei brucei Infected with T. congolense + T. brucei brucei
Fig. 3. Weekly mean aspartate aminotransferase of dogs infected with either T. brucei brucei or T. congolense or T. brucei brucei and T. congolense.
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Table 1 The weekly mean conjugated bilirubin, blood urea nitrogen, and creatinine (mg/dl) of dogs infected with single and mixed infections of T. brucei brucei and T. congolense. Days post infection
Treatment groups and mean conjugated bilirubin, blood urea nitrogen and creatinine (mg/dl) with standard error in bracket Group I (Uninfected control)
Conjugated bilirubin 0 7 14 21 28 Blood urea nitrogen 0 7 14 21 28 Creatinine 0 7 14 21 28
0.08a 0.08a 0.08a 0.08a 0.08a
(0.010) (0.004) (0.009) (0.009) (0.010)
Group II (Infected with T. congolense only) 0.09a (0.004) 0.09a (0.022) 0.20a (0.062) 0.11a (0.015) 0.56b (0.345)
Group III (Infected with T. brucei brucei only) 0.09a (0.002) 0.18a,b (0.050) 0.13a (0.028) 0.14a (0.021) 0.23a,b (0.027)
Group IV (Mixed infection of T.congolense and T. brucei brucei) 0.08a (0.007) 0.28b (0.079) 0.32a (0.153) 0.15a (0.029) 0.41a,b (0.010)
18.55a 18.50a 18.75a 18.75a 18.67a
(2.52) (1.71) (1.38) (1.25) (1.86)
18.68a (1.82) 43.67b (5.18) 64.83b (8.09) 64.50b (5.44) 69.50b (5.50)
18.62a (1.19) 39.83b (3.44) 62.50b (6.44) 63.33b (1.58) 63.00b (5.57)
18.62a (1.40) 45.80b (4.58) 69.60b (10.05) 65.00b (6.14) 78.00b (1.00)
0.88a 0.90a 0.88a 1.18a 0.87a
(0.05) (0.04) (0.04) (0.25) (0.67)
1.22a (0.32) 2.47b (0.47) 4.50b (0.92) 3.18a (0.13) 3.55b (0.55)
0.90a (0.15) 3.23b (0.41) 4.52b (0.88) 2.50a (0.49) 3.13b (0.62)
0.92a (0.06) 3.52b (0.75) 5.22b (0.93) 2.95a (1.20) 2.30a,b (0.50)
Different superscripts in a row (a,b,c ) indicate significant difference between the group means (P < 0.05). Data was analyzed using one-way analysis of variance (ANOVA).
the serum levels of the conjugated bilirubin was significantly different (P < 0.05) between the uninfected control (I) and infected groups (II, III, and IV) only on days 7 and 28 PI, but not on days 14 and 21. On day 7 PI, the increase in the infected groups III and IV was significantly different (P < 0.05) from group II. There was however no significant difference (P > 0.05) between the infected groups on day 28 PI. The increases in blood urea nitrogen was significantly different (P < 0.05) between the uninfected control (I) and infected groups (II, III, and IV) from day 7 PI up to the end of the experiment. There was also increases in the serum levels of creatinine between the uninfected control group (I) and infected groups (II, III, and IV) on days 7, 14 and 28 PI, but not on day 21 PI. It was further observed that the increases in the serum levels of the creatinine and blood urea nitrogen in the infected groups did not differ significantly (P > 0.05) from each other throughout the experiment. 4. Discussion The onset of parasitaemia in this experiment which is 10–13 days PI with the mean PP being 12, 13, and 11 days PI for groups II (T. congolense), III (T. brucei brucei), and IV (mixed infection of T. brucei and T. congolense), respectively, is consistent with the reports of Abenga et al. (2005) and Akpa et al. (2008). This result was however different from that of Onyeyili and Anika (1991) and Anene et al. (1999) who reported shorter PP of 7 days PI. The pattern of parasitaemia in this experiment is similar to the pattern seen in experimental trypanosomosis of albino rats and mice (Ezeokonkwo et al., 2007) but differs with that of cattle where there is fluctuating parasitaemia (Morrison et al., 1981). The nature of the infection
in this study can best be described as an acute infection since the observed clinical indices in all the infected dogs pointed to the fact the infected dogs if left untreated would have died within a short period of 4 weeks PI in contrast to the chronic disease syndrome characteristic of ruminant trypanosomosis characterized by low parasitaemia and many waves of parasitaemia in which the affected animals survive for many months without treatment (Anene and Ezekwe, 1995; Taylor and Authie, 2004). The result of the AP, ALT, AST, CB, BUN, and creatinine assays revealed that single infections of either T. brucei brucei or T. congolense and mixed infection of T. brucei brucei and T. congolense significantly (P < 0.05) increased the mean serum activities of AP, ALT, AST, and serum levels of BUN, CB and creatinine. The significant increase in the serum activities of AP, AST and serum levels of BUN and creatinine commenced seven days PI whereas that of ALT started 14 days PI and remained significantly high until the termination of the experiment. This increase in the serum levels of these substances did not differ significantly between the infected groups for most of the duration of the experiment except for ALT and AST in which the dogs infected with T. congolense (group II) alone differed significantly from the other groups by day 28 PI. The result above was substantially consistent with the findings of Obidike et al. (2005) and Akpa et al. (2008) who reported significant increases in the serum activities of AP, ALT, and AST in albino rats and mongrel dogs, respectively, following trypanosome infections. These significant elevations as reported in this work indicated that the integrity of the vital organs like the liver in the case of elevations in AP, ALT, AST, and kidney in the case of elevations in BUN and creatinine were compromised following infection of the
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dogs with the trypanosomes. This might have led to the increased destruction of hepatocytes and other cells of the body like the kidney cells, osteocytes, and skeletal muscle cells, giving rise to increased release of these substances in circulation hence the elevation in the serum. Specifically in dogs, it has been reported that the AP and ALT levels are relatively narrow in range under normal circumstances and hence a raise in the level in the serum is an indicator of hepatic malfunction (NeuschhwanderTerti, 1995). It had been reported that T. brucei brucei were sequestrated in several organs including the liver during infection (ILRAD, 1994). This sequestration in the liver might have led to the damage of the hepatocytes and the damaged hepatocytes in turn released the substances in large quantities leading to the elevation in the level of these substances in the serum (Omeke and Ugwu, 1991; ILRAD, 1994). Sequestration of the trypanosomes in the kidney might have led to the destruction of the kidney cells thereby interfering with the ability of the kidney to excrete creatinine and nitrogenous wastes from plasma and hence the significant elevation of these substances in the blood as seen in this work (January et al., 1991; Igbokwe and Mohammed, 1992; Ogunsanmi et al., 1994). Surprisingly, there was no significant difference between the elevations in these substances caused by single infections of either T. brucei brucei or T. congolense and mixed infection of T. brucei brucei and T. congolense for most of the duration of the experiment except for T. congolense in which the levels of ALT and AST differed significantly from the other infected groups almost at the end of the experiment (day 28 PI). One had expected T. brucei brucei to have caused more significant elevations of these substances throughout the duration of the experiment considering the fact the parasite as earlier reported causes an extensive degenerative changes in most organs of the infected host in addition to the anemia which was of secondary importance when compared to the T. congolense that primarily causes anemia in infected hosts (Losos and Ikede, 1972). 5. Conclusion The serum activities of AP, AST, ALT and serum levels of CB, BUN, and creatinine significantly increased following infection with either single or mixed infections of T. brucei brucei and T. congolense. These increases were noticed at the onset of the infection and were sustained until the end of the experiment. Acknowledgements We thank Mr. Augustine Ngene of the Department of Veterinary Medicine, University of Nigeria, Nsukka for technical assistance and Mrs Blessing Ugwuoke of the Department of Veterinary Parasitology and Entomology, University of Nigeria, Nsukka for taking adequate care of the experimental animals during the study.
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Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Comparative serum biochemical changes in mongrel dogs following single and mixed infections of Trypanosoma congolense and Trypanosoma brucei brucei R.C. Ezeokonkwo a,∗ , I.O. Ezeh a , J.I. Onunkwo b , I.W. Onyenwe a , C.N. Iheagwam a , W.E. Agu a a b
Department of Veterinary Parasitology and Entomology, University of Nigeria, Nsukka, Nigeria Department of Veterinary Public Health and Preventive Medicine, University of Nigeria, Nsukka, Nigeria
a r t i c l e
i n f o
Article history: Received 19 April 2011 Received in revised form 9 May 2012 Accepted 15 May 2012 Keywords: Comparative biochemistry Trypanosoma congolense T. brucei brucei Dogs
a b s t r a c t The serum activities of alkaline phosphatase (AP), alanine aminotransferase (ALT), aspartate aminotransferase (AST) and the serum levels of conjugated bilirubin (CB), blood urea nitrogen (BUN) and creatinine were studied following single and mixed infections of mongrel dogs with Trypanosoma congolense and Trypanosoma brucei brucei. Twenty mongrel dogs of both sexes aged between 3 and 6 months, and weighing between 2.5 and 5.9 kg were used for the study. The dogs were kept in clean metal cages in a flyproof house and were fed and given water ad libitum. The twenty dogs were divided into four groups of five dogs each. Group I dogs were uninfected control, group II were infected with T. congolense, group III were infected with T. brucei brucei and group IV were infected with both T. congolense and T. brucei brucei. Each dog in the infected groups II and III was inoculated intraperitonealy (i/p) with 1.0 ml of PBS diluted blood containing 1.0 × 106 trypanosomes whereas each infected dog in group IV (mixed infection) was inoculated with 0.5 ml of the PBS diluted blood containing 0.5 × 106 T. congolense and 0.5 ml of the PBS diluted blood containing 0.5 × 106 T. brucei brucei i/p. Parasites were detectable in the blood of the infected dogs in groups II, III, and IV 10–13 days post infection (PI) with the mean pre-patent period (PP) of 12, 10, and 11 days respectively. Trypanosome infection caused a significant (P < 0.05) increase in the serum activities of AP, ALT, AST and the serum levels of creatinine, CB, and BUN. The significant increases in the serum levels of CB, BUN, and creatinine and serum activities of AP and AST became noticeable from day seven PI in all the infected groups whereas that of ALT became noticeable from day 14 PI and increased continuously until the experiment was terminated. These increases however did not differ significantly (P > 0.05) between the infected groups in most cases. It was thus concluded that single or mixed infection of mongrel dogs with T. congolense and T. brucei brucei resulted in significant increases in the serum activities of AP, AST, ALT and serum levels of creatinine, CB and BUN which in most cases did not differ significantly (P > 0.05) among the infected groups. Crown Copyright © 2012 Published by Elsevier B.V. All rights reserved.
1. Introduction
∗ Corresponding author. Tel.: +234 8030919933/+234 8023469911. E-mail addresses: [email protected], [email protected] (R.C. Ezeokonkwo).
Trypanosomosis transmitted by tsetse flies is of great significance to human health and animal production in Africa. It is a serious hindrance to profitable livestock production and a major health hazard for dogs and other
0304-4017/$ – see front matter. Crown Copyright © 2012 Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.vetpar.2012.05.012
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companion animals in Nigeria (Anene and Omamegbe, 1987). The upsurge in the cases of canine trypanosomosis in eastern Nigeria is complicated by the fact that chemotherapy using very commonly available, diminazene aceturate is increasingly becoming ineffective in the treatment of the disease in dogs (Kaggwa et al., 1988; Chukwu et al., 1990; Kalu, 1995). Analyses of serum enzyme activities and substances like creatinine, CB and BUN are important in diagnosing certain pathologic conditions. Most of these enzymes act intracellularly, though few (lipase and amylase) act extracellularly. A pathological condition may result in increased cell destruction and death leading to the release of the intracellular enzymes and these substances into blood circulation which can be detected by various assays. Hitherto, changes in the serum enzyme activities and levels of CB, BUN, and creatinine of trypanosome-infected mongrel dogs have not been well studied. However it has been reported that trypanosomosis adversely affects vital organs like the liver, spleen and kidney leading to in most cases destruction of these organs and hence the increased release of these enzymes and substances contained within the cells into the blood circulation (Fiennes, 1970; Losos and Ikede, 1972; Taylor and Authie, 2004). The quantification of these enzymes and substances by end point assays can be used to determine the extent of the damage of the organs. This work was therefore undertaken to investigate and compare to what extent, single and mixed infections of Trypanosoma congolense and Trypanosoma brucei brucei alter the serum activities of AP, ALT, AST and serum levels of BUN, CB, and creatinine in mongrel dogs.
2. Materials and methods 2.1. Animals and their management Twenty mongrel dogs of both sexes aged between 3 and 6 months and weighing between 2.5 and 5.9 kg were used for the study. They were kept in clean metal cages in a fly proof house. They were fed heavily once daily and were given clean drinking water ad libitum. The dogs were acclimatized for three weeks during which time they were dewormed and deticked with 0.2 mg/kg body weight of Kepromec® (Ivermectin, Kepro B. V., Deventer, the Netherlands) subcutaneously (s/c) and 300 mg Distocide® (Praziquantel, Shin Poong Pharm Co. Ltd., Seoul, Korea) per dog. They were also vaccinated with canine distemper, Adenovirus type 2, parainfluenza, Parvovirus and Leptospira bacterin vaccine (Fort Dodge Animal Health, Iowa, USA). Also the dogs were given OXY-TQ® long acting Oxytetracycline (H.Y. Pharmaceutical Ltd. Helve Province, China) at 1 ml/10 kg body weight intramuscularly (i/m). The dogs were screened and confirmed negative for trypanosomes by wet mount technique and buffy coat method (Murray et al., 1977). Pre-infection parameters were taken from the dogs at day 14 and day 21 post-arrival before the commencement of the experiment. Adequate cleanliness, thorough disinfection and hygiene were maintained in the experimental animal house throughout the experiment.
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2.2. Experimental design The 20 dogs were divided into four groups (I–IV) of five dogs each using stratified random sampling technique. Group I dogs were uninfected controls while groups II and III were each infected with 1.0 × 106 T. congolense and T. brucei brucei respectively. Dogs in group IV (mixed infection) were each infected with 0.5 × 106 T. congolense and 0.5 × 106 T. brucei brucei. The infected dogs were monitored daily for the development of parasitaemia by wet blood film examination and buffy coat technique (Murray et al., 1977). Parameters studied were; serum activities of AP, ALT, and AST, and serum levels of BUN, CB, and creatinine. The serum levels of these substances were determined on the day of the infection (day 0) and subsequently every 7 days up to the end of the experiment. 2.3. Trypanosome infections T. congolense and T. brucei brucei used in this work were obtained from the Nigerian Institute for Trypanosomiasis Research (NITR), Vom, Plateau State, Nigeria. T. brucei brucei was originally isolated from cattle during an outbreak that occurred at Federe in Jos East Local Government Area of Plateau State in 1997 while the T. congolense was originally isolated from cattle in Karu, Nasarawa State, Nigeria in 1995. Both parasites were cryo-preserved in liquid nitrogen. The stabilates had been severally passaged in albino rats prior to the infection of the experimental dogs. The donor rats were bled by puncture of the retrobulbar plexus at the median canthus of the eye and the infected blood serially diluted with PBS. The level of parasitaemia was determined by the rapid matching method of Herbert and Lumsden (1976). Each dog in groups II, and III was inoculated i/p with 1.0 ml of PBS diluted blood containing 1.0 × 106 T. congolense and T. brucei brucei respectively while each dog in group IV (mixed infection) was inoculated with 0.5 ml of the PBS diluted blood containing 0.5 × 106 T. congolense and 0.5 ml of the PBS diluted blood containing 0.5 × 106 T. brucei brucei. 2.4. Serum sampling technique Two microliters of blood was drawn from the cephalic vein of each dog after thorough cleaning of the area with 70% ethanol. The blood was put into a plastic test tube without an anticoagulant. The test tube containing the blood was kept in a slanting position in a bench for about 3 h to enable the blood clot to yield the serum. Thereafter, the coagulated blood on the wall of the test tubes was scooped to the bottom of the tubes and latter centrifuged for 5 min at 20,833 G to ensure clear separation of the serum from the clotted blood cells. The clear serum was later used for the assays. 2.5. Methods for determining the various parameters The standard colorimetric method of Reitman and Frankel (1957) using Randox test kits (Randox laboratories Ltd., UK) was used for the determination of the serum activities of ALT and AST. Phenolphthalein monophosphate
method (Klein et al., 1960) using Quimica Clinica test kits (QCA, Spain) was used for the determination of serum activity of AP. Urease Berthelot method (Fawcett and Scott, 1960) for quantitative in vitro determination of urea in serum and urine using Randox urea colorimetric kit was used to determine BUN. The modified Jaffe method (Fossati et al., 1983) for in vitro determination of creatinine in serum, plasma or urine using Randox creatinine colorimetric method with depolarization was used to determine urea creatinine. The bilirubin kits prepared by Biosystems reagents and instruments (Biosystems, S.A., Barcelona, Spain) were used to determine the serum level of conjugated bilirubin (Pearlman and Lee, 1974; Zoppi et al., 1976). The assays were done in triplicate for each substance and for each experimental dog per sampling period. Finally, the average value was taken. 2.6. Handling of experimental animals during the study The guidelines set out by the University of Nigeria, Nsukka Ethics Committee for Medical and Scientific Research (MSR) which include good, clean and hygienic housing, adequate feeding, provision of clean water and humane handling of animals during sample collection were strictly followed in handling the dogs during the experiment. Valid approval and ethical clearance were obtained from the Ethics Committee of the University before the commencement of the experiment.
Mean alkaline phosphatase (iu/l)
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80 60 40 20 0
0
3.1. Onset of parasitaemia Parasites were detectable in the blood of the infected dogs in groups II, III, and IV; 10–13 days PI with the mean PP being 12, 13, and 11 days PI for groups II, III, and IV, respectively. Parasitaemia persisted throughout the duration of the experiment. Figs. 1–3 show the weekly mean AP, ALT and AST of mongrel dogs infected with single and mixed infections of T. congolense and T. brucei brucei respectively. Trypanosome infections caused an increase in the serum activity of AP, ALT, and AST in the infected groups (II, III, and IV). The increase in AP and AST differed significantly (P < 0.05) between the uninfected control and the infected groups as from day seven PI up to the end of the experiment whereas the increases in ALT differed significantly (P < 0.05) from day 14 up to the end of the experiment. Comparatively, the increase in the AP levels did not differ significantly (P > 0.05) between the infected groups throughout the experiment. Also, the increases in the serum activities of ALT and ASP were not significantly different (P > 0.05) between the infected groups up to day 21 PI, but by day 28 PI the increase
21
28
Fig. 1. Weekly mean alkaline phosphatase of dogs infected with either T. brucei brucei or T. congolense or T. brucei brucei and T. congolense.
80 60 40 20 0
0
7
14
21
28
Days post infection Uninfected control Infected with T. congolense Infected with T. brucei brucei Infected with T. congolense + T. brucei brucei
Fig. 2. Weekly mean alanine aminotransferase of dogs infected with either T. brucei brucei or T. congolense or T. brucei brucei and T. congolense.
in group II (T. congolense infected) differed significantly (P < 0.05) from infected groups III and IV. Table 1 shows the weekly mean serum levels of conjugated bilirubin (CB), blood urea nitrogen (BUN), and creatinine of dogs infected with single and mixed infections of T. brucei brucei and T. congolense. Trypanosome infections increased the mean serum levels of these substances in the course of the experiment. The increase in Mean Alanine aminotransferase (iu/l)
3. Result
14
Uninfected control Infected with T. congolense Infected with T. brucei brucei Infected with T. congolense + T. brucei brucei
2.7. Statistical analysis Data were analyzed using one-way analysis of variance (ANOVA) and Duncan’s multiple range test (Duncan, 1966) using SPSS version 12.00 software package. The level of significance was considered at P < 0.05.
7
Days post infection
Mean Alanine aminotransferase (iu/l)
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80 60 40 20 0 0
7
14
21
28
Days post infection Uninfected control Infected with T. congolense Infected with T. brucei brucei Infected with T. congolense + T. brucei brucei
Fig. 3. Weekly mean aspartate aminotransferase of dogs infected with either T. brucei brucei or T. congolense or T. brucei brucei and T. congolense.
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Table 1 The weekly mean conjugated bilirubin, blood urea nitrogen, and creatinine (mg/dl) of dogs infected with single and mixed infections of T. brucei brucei and T. congolense. Days post infection
Treatment groups and mean conjugated bilirubin, blood urea nitrogen and creatinine (mg/dl) with standard error in bracket Group I (Uninfected control)
Conjugated bilirubin 0 7 14 21 28 Blood urea nitrogen 0 7 14 21 28 Creatinine 0 7 14 21 28
0.08a 0.08a 0.08a 0.08a 0.08a
(0.010) (0.004) (0.009) (0.009) (0.010)
Group II (Infected with T. congolense only) 0.09a (0.004) 0.09a (0.022) 0.20a (0.062) 0.11a (0.015) 0.56b (0.345)
Group III (Infected with T. brucei brucei only) 0.09a (0.002) 0.18a,b (0.050) 0.13a (0.028) 0.14a (0.021) 0.23a,b (0.027)
Group IV (Mixed infection of T.congolense and T. brucei brucei) 0.08a (0.007) 0.28b (0.079) 0.32a (0.153) 0.15a (0.029) 0.41a,b (0.010)
18.55a 18.50a 18.75a 18.75a 18.67a
(2.52) (1.71) (1.38) (1.25) (1.86)
18.68a (1.82) 43.67b (5.18) 64.83b (8.09) 64.50b (5.44) 69.50b (5.50)
18.62a (1.19) 39.83b (3.44) 62.50b (6.44) 63.33b (1.58) 63.00b (5.57)
18.62a (1.40) 45.80b (4.58) 69.60b (10.05) 65.00b (6.14) 78.00b (1.00)
0.88a 0.90a 0.88a 1.18a 0.87a
(0.05) (0.04) (0.04) (0.25) (0.67)
1.22a (0.32) 2.47b (0.47) 4.50b (0.92) 3.18a (0.13) 3.55b (0.55)
0.90a (0.15) 3.23b (0.41) 4.52b (0.88) 2.50a (0.49) 3.13b (0.62)
0.92a (0.06) 3.52b (0.75) 5.22b (0.93) 2.95a (1.20) 2.30a,b (0.50)
Different superscripts in a row (a,b,c ) indicate significant difference between the group means (P < 0.05). Data was analyzed using one-way analysis of variance (ANOVA).
the serum levels of the conjugated bilirubin was significantly different (P < 0.05) between the uninfected control (I) and infected groups (II, III, and IV) only on days 7 and 28 PI, but not on days 14 and 21. On day 7 PI, the increase in the infected groups III and IV was significantly different (P < 0.05) from group II. There was however no significant difference (P > 0.05) between the infected groups on day 28 PI. The increases in blood urea nitrogen was significantly different (P < 0.05) between the uninfected control (I) and infected groups (II, III, and IV) from day 7 PI up to the end of the experiment. There was also increases in the serum levels of creatinine between the uninfected control group (I) and infected groups (II, III, and IV) on days 7, 14 and 28 PI, but not on day 21 PI. It was further observed that the increases in the serum levels of the creatinine and blood urea nitrogen in the infected groups did not differ significantly (P > 0.05) from each other throughout the experiment. 4. Discussion The onset of parasitaemia in this experiment which is 10–13 days PI with the mean PP being 12, 13, and 11 days PI for groups II (T. congolense), III (T. brucei brucei), and IV (mixed infection of T. brucei and T. congolense), respectively, is consistent with the reports of Abenga et al. (2005) and Akpa et al. (2008). This result was however different from that of Onyeyili and Anika (1991) and Anene et al. (1999) who reported shorter PP of 7 days PI. The pattern of parasitaemia in this experiment is similar to the pattern seen in experimental trypanosomosis of albino rats and mice (Ezeokonkwo et al., 2007) but differs with that of cattle where there is fluctuating parasitaemia (Morrison et al., 1981). The nature of the infection
in this study can best be described as an acute infection since the observed clinical indices in all the infected dogs pointed to the fact the infected dogs if left untreated would have died within a short period of 4 weeks PI in contrast to the chronic disease syndrome characteristic of ruminant trypanosomosis characterized by low parasitaemia and many waves of parasitaemia in which the affected animals survive for many months without treatment (Anene and Ezekwe, 1995; Taylor and Authie, 2004). The result of the AP, ALT, AST, CB, BUN, and creatinine assays revealed that single infections of either T. brucei brucei or T. congolense and mixed infection of T. brucei brucei and T. congolense significantly (P < 0.05) increased the mean serum activities of AP, ALT, AST, and serum levels of BUN, CB and creatinine. The significant increase in the serum activities of AP, AST and serum levels of BUN and creatinine commenced seven days PI whereas that of ALT started 14 days PI and remained significantly high until the termination of the experiment. This increase in the serum levels of these substances did not differ significantly between the infected groups for most of the duration of the experiment except for ALT and AST in which the dogs infected with T. congolense (group II) alone differed significantly from the other groups by day 28 PI. The result above was substantially consistent with the findings of Obidike et al. (2005) and Akpa et al. (2008) who reported significant increases in the serum activities of AP, ALT, and AST in albino rats and mongrel dogs, respectively, following trypanosome infections. These significant elevations as reported in this work indicated that the integrity of the vital organs like the liver in the case of elevations in AP, ALT, AST, and kidney in the case of elevations in BUN and creatinine were compromised following infection of the
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dogs with the trypanosomes. This might have led to the increased destruction of hepatocytes and other cells of the body like the kidney cells, osteocytes, and skeletal muscle cells, giving rise to increased release of these substances in circulation hence the elevation in the serum. Specifically in dogs, it has been reported that the AP and ALT levels are relatively narrow in range under normal circumstances and hence a raise in the level in the serum is an indicator of hepatic malfunction (NeuschhwanderTerti, 1995). It had been reported that T. brucei brucei were sequestrated in several organs including the liver during infection (ILRAD, 1994). This sequestration in the liver might have led to the damage of the hepatocytes and the damaged hepatocytes in turn released the substances in large quantities leading to the elevation in the level of these substances in the serum (Omeke and Ugwu, 1991; ILRAD, 1994). Sequestration of the trypanosomes in the kidney might have led to the destruction of the kidney cells thereby interfering with the ability of the kidney to excrete creatinine and nitrogenous wastes from plasma and hence the significant elevation of these substances in the blood as seen in this work (January et al., 1991; Igbokwe and Mohammed, 1992; Ogunsanmi et al., 1994). Surprisingly, there was no significant difference between the elevations in these substances caused by single infections of either T. brucei brucei or T. congolense and mixed infection of T. brucei brucei and T. congolense for most of the duration of the experiment except for T. congolense in which the levels of ALT and AST differed significantly from the other infected groups almost at the end of the experiment (day 28 PI). One had expected T. brucei brucei to have caused more significant elevations of these substances throughout the duration of the experiment considering the fact the parasite as earlier reported causes an extensive degenerative changes in most organs of the infected host in addition to the anemia which was of secondary importance when compared to the T. congolense that primarily causes anemia in infected hosts (Losos and Ikede, 1972). 5. Conclusion The serum activities of AP, AST, ALT and serum levels of CB, BUN, and creatinine significantly increased following infection with either single or mixed infections of T. brucei brucei and T. congolense. These increases were noticed at the onset of the infection and were sustained until the end of the experiment. Acknowledgements We thank Mr. Augustine Ngene of the Department of Veterinary Medicine, University of Nigeria, Nsukka for technical assistance and Mrs Blessing Ugwuoke of the Department of Veterinary Parasitology and Entomology, University of Nigeria, Nsukka for taking adequate care of the experimental animals during the study.
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