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. 2021 Jun 1;45(4):1071–1076. doi: 10.1007/s12639-021-01399-6

Changes in serum biochemical parameters of experimental Trypanosoma brucei brucei-infected Nigerian indigenous dogs administered multispecies probiotic and diminazene aceturate

Bamidele Jonathan Kolawole 1, Rosemary Ijeoma Ogechi Nwoha 1, Juwon Pius Erin 2, Ochuko Orakpoghenor 2,✉
PMCID: PMC8556426  PMID: 34789991

Abstract

Trypanosomosis is an infectious disease with great economic impact on livestock production. In this study, the effects of probiotic on serum biochemical changes of Nigerian indigenous dogs experimentally infected with Trypanosoma brucei brucei were investigated. Twenty (20) healthy dogs of both sexes (6–10 kg body weight) following screening, were randomly divided into 5 groups of 4 dogs each. Dogs in group I (uninfected and untreated); group II (uninfected + multi species (Pet dophillus®) probiotic); group III (infected + multi species probiotic); group IV (infected + diaminazene aceturate); and group V (infected + probiotic + diminazene aceturate). Dogs were fed probiotic for 21 days before infection, and up to the last day post-infection (PI). Dogs in infected groups were inoculated intraperitonially with 2.5 × 106 trypanosomes on day 21 and diminazene aceturate was administered on day 5 post-infection (PI). Prepatent period and parasitaemia were determined. Blood was collected in plain sample bottles and serum harvested for serum biochemical analyses. Prepatent periods of T. brucei brucei were 4.75 ± 0.25 days (group III), 4.00 ± 0.41 days (group IV) and 4.25 ± 0.49 days (groups V); and parasitaemia was significantly decreased (P < 0.05) in groups IV and V compared to group III post-treatment. Mean serum total protein was significantly increased in group III from day 28 up to the end of the study. No significant (P > 0.05) differences existed in the mean serum albumin and creatinine levels in all groups of dogs. The mean serum activities of alanine aminotransferase (ALT), alkaline phosphatase (ALP) and aspartate aminotransferase (AST) were increased significantly (P < 0.05) in group III post infection up to the end of the study. In conclusion, the multispecies probiotic enhanced the efficacy of diaminazene aceturate in mitigating the serum biochemical changes due to experimental T. brucei brucei infection in dogs.

Keywords: Probiotic, Trypanosoma brucei, Serum, Biochemical, Total protein

Introduction

African trypanosomosis is a very important disease of domestic animals in sub-Sahara Africa with a profound effect on economic development of the region (Ali and Bitew, 2011). Trypanosomes have been reported to cause trypanosomosis, known as sleeping sickness and nagana in man and animals respectively (WHO 2006; Sulaiman and Adeyemi, 2010). The species of trypanosomes documented to be affecting man and animals included two groups namely, haematinic group (Trypanosoma congolense, T. vivax) and tissue invading group (T. brucei, T. evansi, T. gambiense, T. rhodesiense and T. equiperdum). The former has been reported to remain in plasma and the latter found in extra and intra vascular spaces (Chretien and Smoak, 2005; Ngure et al. 2005; Sulaiman and Adeyemi, 2010). Canine trypanosomosis, first described in 1908, is primarily caused by T. brucei brucei and T. congolense (Bevan, 1913; Nwoha, 2013; Umeakuana et al. 2016) and relatively common in Nigeria due to high prevalence of Glossina sp. (Ahmed, 2007).

The control of trypanosomosis in livestock has been achieved widely by the use of antitrypanosomal drugs either therapeutically or prophylactically. The use of chemotherapeutic agents in the control of trypanosomosis is faced with challenges such as development of resistance to trypanocides and toxicity of drugs to patients (Ameen, 2013; Kobo et al. 2014) thus, prompting the search for newer drugs and new drug formulations that are safe, affordable, and effective against both early and late stages of the disease (Pink et al. 2005; Lawal et al. 2013).

Probiotics are viable, non-pathogenic microbial species generally considered as health promoting bacteria that support animals against invading pathogens via stimulation of the gastrointestinal immune responses (Fuller and Gibson, 1997; Mitsuoka, 2000; Lan et al. 2005). The beneficial effects of probiotics on health and nutrition of humans and animal are becoming increasingly recognized and are believed to play important roles in immunological, digestive and respiratory functions, and alleviation of infectious diseases (Eze et al. 2012). Hence in this study, the serum biochemical changes of Trypanosoma brucei brucei-infected Nigerian indigenous dogs treated with a multispecies probiotic and diminazene aceturate were investigated.

Materials and methods

Experimental animals

Twenty Nigerian indigenous dogs of both sexes, aged 1–2 years were used for this study following screening for gastrointestinal and hematological parasites, and examination for general health conditions. They were divided into 5 groups (I, II, III, IV and V), each comprising of 4 dogs and were acclimatized. They were fed once daily with household food, chicken and pork and water was provided ad libitum.

Group I–uninfected + untreated; Group II–uninfected + multispecies probiotic (Pet dophillus®) only; Group III–infected + probiotic only; Group IV–infected + dimanzene aceturate; Group V–infected + probiotic + diminazene aceturate.

Probiotic was administered for 21 days before infection and till the last day of experiment. The infection of dogs in groups III, IV and V were carried out using 2.5 × 106 T. brucei brucei (Federi strain) suspended in 1ml of phosphate buffer saline intraperitoneally at day 21.

Determination of parasitaemia in infected dogs

After the dogs were infected on day 21, parasitaemia was determined by daily collection of blood unto a clean glass slide and covered with cover slip. This was followed by viewing of the wet mount under light microscope at × 40 magnification for the presence of T. brucei brucei as described by Herbert and Lumsden (1976).

Blood collection

Pre-infection of dogs with T. b. brucei (days 0, 7, 14 and 21), blood was collected from each dog into plain sample bottles, serum harvested and processed for serum biochemical analyses. Following infection and treatment (days 28, 35 and 42), blood was also collected and processed for total protein, albumin and creatinine levels, and activities of alanine aminotransferase, aspartate aminotransferase and alkaline phosphatase determination by following the manufacturer’s protocol.

Data analyses

Data were presented as mean ± standard error of mean (± SEM) using charts. The data obtained were subjected to Two-way analysis of variance using Graphpad Prism (Version 8.0). Values of P < 0.05 were considered significant.

Results

Prepatent period and parasitaemi

The mean prepatent periods were 4.75 ± 0.25, 4.00 ± 0.41 and 4.25 ± 0.49 days in groups III, IV and V respectively. Post-treatment, there was complete clearance of parasitaemia in groups IV and V.

Total protein concentration

There was no significant difference (P > 0.05) in the serum total protein of all dogs pre-infection and in the non-infected dogs till the end of the study (Fig. 1). Post-infection and following treatment, serum total protein was significantly increased (P < 0.05) in groups III (10.50 ± 1.12 g/dL), IV (8.43 ± 0.38 g/dL) and V (8.75 ± 0.19 g/dL) on day 28 of experiment and this continued to increase significantly (P < 0.05) till end of the study in group III (14.45 ± 0.85 g/dL). Meanwhile, in groups IV and V, the serum total protein returned to values with no significant difference (P > 0.05) compared to pre-infection value from day 35 till the end of the study (Fig. 1).

Fig. 1.

Fig. 1

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Serum total protein of dogs inoculated with T. brucei brucei and administered multi-species probiotic

Albumin concentration

The serum albumin levels showed no significant difference (P > 0.05) in all groups of dogs pre- and post-infection, and post-treatment till the end of the study (Fig. 2).

Fig. 2.

Fig. 2

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Serum albumin level of dogs inoculated with T. brucei brucei and administered multi-species probiotic

Creatinine concentration

There was no significant difference (P > 0.05) in the serum creatinine levels in all groups of dogs till the end of the study but with a non-significant increase (P > 0.05) in group III on day 35 (Fig. 3).

Fig. 3.

Fig. 3

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Serum creatinine level of dogs inoculated with T. brucei brucei and administered multi-species probiotic

Alanine aminotransferase activity

Pre-infection till day 21, the activities of alanine aminotransferase (ALT) showed no significant differences (P > 0.05) between infected, non-infected and treated groups of dogs (Fig. 4). Post-infection (on day 28), activities of ALT were significantly increased (P < 0.05) in groups III (45.25 ± 3.14 × 103/µL), IV (35.33 ± 5.45 × 103/µL) and V (31.25 ± 6.44 × 103/µL). This continued to increase significantly (P < 0.05) in group III till the end of the study (119.00 ± 7.00 × 103/µL), but decreased significantly to pre-infection values on days 35 and 42 in groups IV (13.67 ± 1.20 ± 5.45 × 103/µL; 14.00 ± 1.00 × 103/µL) and V (11.25 ± 2.26 × 103/µL; 11.00 ± 1.00 × 103/µL) with no significant differences (P > 0.05) between them (Fig. 4).

Fig. 4.

Fig. 4

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Activities of alanine aminotransferase of dogs inoculated with T. brucei brucei and administered multi-species probiotic

Aspartate aminotransferase activity

The activities of aspartate aminotransferase (AST) showed no significant differences (P > 0.05) in all groups of dogs till day 21 of experiment and to the end of the study in groups I and II (Fig. 5). There was significant increase (P < 0.05) in the activities of AST on day 28 in groups III (37.75 ± 3.27 × 103/µL), IV (28.25 ± 4.55 × 103/µL) and V (25.75 ± 5.32 × 103/µL), and this continued to increase significantly in group III to the end of the study (55.50 ± 5.00 × 103/µL). On days 35 and 42, the activities of AST in groups IV (22.67 ± 2.20 × 103/µL; 22.00 ± 1.87 × 103/µL) and V (20.75 ± 2.42 × 103/µL; 19.00 ± 1.43 × 103/µL) showed no significant difference compared to their pre-infection values and those of groups I (21.25 ± 1.93 × 103/µL; 21.50 ± 0.85 × 103/µL) and II (24.25 ± 4.14 × 103/µL; 23.00 ± 1.84 × 103/µL) (Fig. 5).

Fig. 5.

Fig. 5

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Activities of aspartate aminotransferase of dogs inoculated with T. brucei brucei and administered multi-species probiotic

Alkaline phosphatase activity

Alkaline phosphatase (ALP) activities showed no significant difference (P > 0.05) in all groups of dogs before day 21 (pre-infection) of experiment (Fig. 6). At day 28 following infection and treatment, ALP activities increased significantly (P < 0.05) in groups III (37.75 ± 3.27 × 103/µL), IV (28.25 ± 4.55 × 103/µL) and V (25.75 ± 5.32 × 103/µL), and continue to increase significantly (P < 0.05) to the end of the study in group III (39.25 ± 4.90 × 103/µL; 55.50 ± 5.00 × 103/µL). In groups IV and V, there was return of ALP activities to values with no significant difference compared to groups I and II from day 35 till the end of the study (Fig. 6).

Fig. 6.

Fig. 6

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Activities of alkaline phosphatase of dogs inoculated with T. brucei brucei and administered multi-species probiotic

Discussion

The serum biochemical changes due to experimental T. brucei brucei (Federi strain) infection in indigenous dogs administered probiotic were investigated in this study. The prepatent period recorded in infected dogs was similar in dogs administered probiotic (group V) and those not administered probiotic (groups III and IV) as reported in other studies (Anene et al. 1989; Ezeokonkwo, 2009; Nwoha and Anene, 2016). This suggests that probiotic administration had no effect on the establishment of clinical trypanosomosis in dogs.

In infected dogs (groups III, IV and V), increased serum total protein and no significant changes in albumin and creatinine levels were observed post-infection and following treatment (day 28) in this study. Sulaiman and Adeyemi (2010) reported an increase in total serum protein and albumin concentrations in rats experimentally infected with T. brucei. Rajora et al. (1986) reported similar increase in serum total protein following experimental T. evansi infection in buffalo calves. Sadique et al. (2001) reported a decrease in serum total protein due to T. congolense in Zebu cattle in trypanosome-endemic zone. These variations in reaction to trypanosomes infections could be due to differences in either the strain or species of trypanosomes and animals used. The increase in serum total protein in this study might have been due to increase in synthesis of serum immunoglobulins resulting from enhanced antibody production for the control of the infection (Orhue et al. 2005). The excessive significant increase observed in group III could be due to antigenic variation resulting from the variable surface antigen of the parasite. Also, the increase in total serum protein in infected dogs could be due to cell derived proteins resulting from haemolysis secondary to infection. The increase in parasite mass proteins and/or released intracellular enzymes due to increasing parasitaemia or lysis of parasites by host immune system as well as protein loss from damaged tissues could also be responsible for the increased serum total protein (Sulaiman and Adeyemi, 2010). This increase in total serum protein due to infection was decreased following administration of DA and probiotic. Hence, it is suggested that the probiotic had immune stimulatory effect which enhanced the therapeutic effect of DA on the parasite as observed in group V in this study.

Serum AST, ALT and ALP were observed to increase significantly in this study following infection in the treated group with probiotic only (group III) and these are in agreement with earlier reports of Taiwo et al. (2003), Oyewole and Malomo (2009) and Ezeokonkwo et al. (2012). Otesile et al. (1991) reported similar increase in AST and ALT in boars experimentally infected with T. brucei. The increases in these enzymes may be due to tissue breakdown and inflammation in the host, particularly of the liver, heart, muscle and kidney (Taiwo et al. 2003; Yakubu et al. 2005; Omeje and Anene, 2012). The result showed that probiotic had no effects on tissue destruction by trypanosome and hence the rise in the levels of liver enzyme in the infected group III administered probiotic. The non-significant change in groups IV and V are clearly due to the impact of drugs in removing the parasites from blood and thus arresting their destruction effect on the cells of the liver. The similarity in the results of group IV and V is a further confirmation that probiotic had no effects on the liver enzyme concentrations in this study.

Hence in this study, it was observed that probiotic had no direct effect on the parasite or the disease pathogenesis but rather stimulated the host immune system to control the infection and enhanced the therapeutic efficacy of DA on the parasite.

Acknowledgements

Adeniji Kayode John in California (USA) and Dr John Ofoedu (FMC Umuahia) for their roles in purchase and shipment of the Pet Dophilus® probiotic used.

Authors’ contributions

RION conceptualized and designed the experiment; BJK and JPE performed the experiment; BJK, JPE and OO drafted the manuscript.

Funding

No fund was received by any of the authors for this study.

Declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

The use of dogs in this study was provided by Michael Okpara University of Agriculture Ethical Committee, and experiment has been performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki and its later amendments.

Consent for publication

All authors have read and agreed to the publication of the manuscript.

Footnotes

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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