Chemotherapeutic efficacy of secnidazole-diminazene aceturate combination therapy in experimental Trypanosoma brucei brucei infection in rats

1 Department of Veterinary Physiology and Pharmacology, University of Nigeria, Nsukka, Enugu State, Nigeria. 2 Department of Veterinary Parasitology and Entomology, University of Nigeria Nsukka, Enugu State, Nigeria. 3 Department of Veterinary Medicine, University of Nigeria, Nsukka, Enugu State, Nigeria. 4 Department of Veterinary Physiology and Pharmacology, Federal University of Agriculture, Markurdi, Benue State, Nigeria.


INTRODUCTION
The use of drugs for control and treatment of African animal trypanosomosis has been in practice for decades, but the rapidity with which trypanosomes develop resistance to these trypanocides has become a source of worry.Diminazene aceturate at the dose of 3.5 to 7 mg/kg has been recommended for the treatment of *Corresponding author.E-mail: ifeanyi.eke@unn.edu.ng.Tel: +2348037494699.
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License Trypanosoma brucei brucei infection.But nowadays it seems that this dose (3.5 mg/kg) is only able to get rid of clinical signs but fails to clear the parasites (Desquesnes and Gutiérrez, 2011).Relapse of infection occurred in dogs treated with 7 mg/kg DA (Chukwu et al., 1990).These treatment failures have been attributed to resistance by trypanosomes (Gutiérrez et al., 2013).Thus, due to increasing rates of treatment failure, relapse of previously treated cases where re-exposure has be precluded, drug combination has been suggested as an alternative treatment method to achieve greater efficacy (Onyeyili and Egwu, 1995).It has been suggested that combination of DA with other therapeutically active trypanocides or adjuvants may improve treatment outcome in animals (De dekens et al., 1989).However, effective combination therapies with good clinical application seem elusive.The treatment of dogs infected with T. b. brucei and Trypanosoma congolense with eflornithine and DA combination was not curative (Onyeyili and Anika, 1990).
Secnidazole (SEC) is effective against anaerobic micro-organisms and protozoa and appears particularly effective in the treatment of amoebiosis, giardiosis, trichomoniosis and bacterial vaginosis (Laurence et al., 2006).Secnidazole is widely distributed in the body and crosses the blood brain barrier in sufficient quantity (Rang et al., 1996).Diminazene aceturate is limited in its distribution in the brain due to its polarity, thus, it does not accumulate in therapeutic concentration (Onyeyili and Anika, 1991).Therefore, sequestered trypanosomes in the brain are not eliminated.This has been shown to be the source of relapse infection in animals (Soeiro et al., 2005).The in vitro and in vivo antitrypanosomal activities of SEC have been demonstrated, and SEC showed a concentration and dose dependent antitrypanosomal effects in vitro and in vivo (Eke et al., 2017).It is thought that combination of SEC and DA in the treatment of T. b. brucei infection in rats will produce additive synergistic effect, through the combination of their individual trypanocidal effects and pharmacokinetic properties.The aim of this study was therefore to evaluate the therapeutic efficacy of SEC-DA combination in the treatment of experimental T. b. brucei infection in rats.

Trypanosomes
The T. b. brucei used in this study was originally isolated from a dog presented at the Veterinary Teaching Hospital University of Nigeria Nsukka.Morphological identification and blood incubation infectivity test were by standard procedure (Rickman and Robson, 1970;Soulsby, 1982).These parasites were maintained in rats from which experimental animals were infected.

Animals
Twenty Sprague Dawley rats obtained from the laboratory animal facility of the Department of Veterinary Physiology and Pharmacology, University of Nigeria Nsukka were used for the study.They were housed in stainless steel rat cages.They were fed ad libitum with pelletized feed (Vital Feeds ® ).Clean drinking water was also provided ad libitum.They were acclimatized for one week before the study.The animal experimental protocol was approved by the Experimental Animal Ethics Committee of the Faculty of Veterinary medicine, University of Nigeria, Nsukka, August 2015 and in compliance with the Federation of European Laboratory Animal Science Association and the European Community Council Directive of November 24, 1986 (86/609/EEC).

Experimental
The rats were randomly assigned to 4 groups (n = 5).They were treated as follows: Group A: uninfected and untreated, served as the control; Group B: infected untreated; Group C: infected and treated with DA (3.5 mg/kg IP once); Group D: infected and treated with 3.5 mg/kg DA IP once and 200 mg/kg SEC orally.
Secnidazole (SEC) was administered for 6 days.Diminazene aceturate (Lobazene ® France) was giving immediately after administration of SEC (Secwid ® May and Baker Nigeria PLC) on the first day of treatment.The dose of secnidazole used in this study was based on the result of preliminary antitrypanosomal effects of SEC.

Infection of rats
Parasitemia was estimated using the rapid matching technique (Herbert and Lumsden, 1976).All rats were infected intraperitoneally using 1 x 10 6 Trypanosoma b. brucei suspended in 0.2 ml of phosphate buffered saline (PBS).Rats were monitored daily for onset of parasitemia.
Parasitemia was established 5 days post-infection (PI) and treatment starting from day 8 PI, when parasitemia was well established on examination of blood films in all the infected rats.The hematocrit buffy coat technique and stained thin smears were used to confirm total parasite clearance (OIE, 2008).

Parasitemia
This was monitored daily after treatment.Parasitemia level, time of clearance of parasitemia and time of relapse of infection were determined and recorded.

Hematological changes
Samples for hematology were collected into EDTA sample bottles.Samples were collected on days 0, 7, 14 and 21.Full blood count (FBC) was done using the method of Schalm et al. (1975).Packed cell volume (PCV) was done using the microhematocrit method (Coles, 1986).Hemoglobin (Hb) concentration was determined using the cyanomethaemoglobin method (Coles, 1986).Changes in these parameters were measured and recorded.

Alanine transaminase activity
Alanine transaminase (ALT) activity was determined on days 14 and 21 PI using Randox (R) kits (Randox Laboratories Ltd United Kingdom) according to the manufacturer's guidelines.This is to monitor the effect of treatment on the changes caused by T. b. brucei to the liver.

Statistical analysis
Data collected were presented as mean ± SEM in tables.Analyses of data were done using one way analysis of variance.Variant means were separated using least significant difference (LSD).
Significance was accepted at p < 0.05.

Parasitological findings
There was significant (p < 0.05) reduction in parasitemia in the two treatment groups following treatment when compared with the infected untreated control.Parasitemia cleared completely 3 days post-treatment (PT) in all the rats treated with SEC-DA combination, while it cleared 5 days PT in rats treated with DA alone.Parasitemia was progressive in the untreated group and all the rats in the group died by day 21 PI.Relapse of infection occurred in group C (DA alone) 13 days PT (day 21 post-infection).There was no relapse of infection in the combination treatment up to the end of the experiment (70 days PT) (Table 1).

White blood cells
There was significantly (p < 0.05) higher level of leukocytes in the uninfected untreated control when compared with the infected rats on day 7 PI.On day 14, PI, significantly (p < 0.05) lower level of WBC was observed in the infected untreated control when compared with the groups infected and treated with DA alone, the SEC-DA combination and uninfected untreated.However, by day 21, the WBC count in the group treated with the SEC-DA combination was significantly (p < 0.05) higher than that of the group infected and treated with only DA and the uninfected untreated (Table 2).
Lymphocytes were significantly (p < 0.05) higher in the uninfected untreated group on day 7 PI, while significant (p < 0.05) increase in the lymphocyte count was recorded in the SEC-DA group on days 14 and 21 when compared with both the uninfected untreated and infected and treated with DA alone.Significantly (p < 0.05) higher levels of neutrophil were observed in the uninfected untreated group on days 7 and 14 when compared with the other groups.Nevertheless, on day 21 PI, there was significant (p < 0.05) drop in neutrophil in the group infected and treated with only DA (Table 3).

Red blood cell counts, PCV and hemoglobin (Hb) concentration
There was significant (p < 0.05) reduction in the RBC number in all infected rats on day 7 PI.Treatment with either SEC-DA combinations or DA alone lead to significant (p < 0.05) increase in the number of RBC over the infected untreated group by day 14 PI and by day 21 PI, there was no significant (p > 0.05) difference between the uninfected untreated and the infected and treated groups.Significant (p < 0.05) reduction in the PCV was observed in all infected animals on day 7 PI when compared with the uninfected untreated control.This however, significantly (p < 0.05) increased in all the treated groups by day 14 PI more than the infected untreated.By day 21, there was no significant (p > 0.05) variation between the infected and treated groups and the uninfected untreated control.Hemoglobin (Hb) concentrations in all the infected animals were significantly (p < 0.05) lower than the uninfected untreated control on day 7 PI.Following treatment, there was significant (p < 0.05) increase in all the treated animals more than the infected untreated on day 14.On day 21 PI, the Hb concentration of the SEC-DA group was significantly (p < 0.05) higher than both the group treated with only DA and the uninfected untreated group (Table 4).

Serum alanine transaminase activity
The infected untreated rats showed significantly (p < 0.05) higher ALT activity on day 14 PI when compared with the uninfected untreated, infected and treated with SEC-DA and DA alone.Nevertheless, the SEC-DA group showed consistently significant (p < 0.05) lower levels of ALT activity on both days 14 and 21 PI when compared with the uninfected untreated, infected untreated and the group treated with only DA (Table 5).

DISCUSSION
Combination therapy of SEC-DA was more effective than mono-therapy of DA in rats experimentally infected with T. b. brucei.It caused faster clearance of parasitemia than single treatment with DA.The SEC-DA combination therapy also prevented relapse of infection, unlike single treatment with DA.Parasites were absent in the SEC-DA group after 3 days of treatment, while it cleared 5 days post-treatment (PT) with DA alone.Faster clearance of parasites by SEC-DA combination therapy could be attributed to the additive trypanocidal effects of both SEC and DA.The absence of relapse of infection in the SEC-DA combination could be as a result of the extensive distribution of SEC into organs and tissues were trypanosomes sequestrate and were DA accumulation is limited (Onyeyili and Anika, 1989;Onyeyili and Anika, 1991).These findings give credence to possible chemotherapeutic synergy between SEC and DA.
Trypanosomosis is characterized by severe reduction of the WBCs and subsequent immunosuppression (Allam et al., 2011).In the present study, there was massive depletion of total leucocyte counts of all infected rats which persisted in the untreated rats.However, after 7 days PT, there was gradual recovery of the leukocytes.The leukocytic response was stronger in the SEC-DA combination therapy.The lymphocytes are the most important cells in the immunological response to trypanosomosis in animals (Emeribe and Anosa, 1991).In this study, the SEC-DA combination therapy produced higher lymphocytic response than DA alone.This strong immunological response could be responsible for the earlier clearance of parasitemia and prevention of relapse infection by SEC-DA combination therapy leading to faster recovery of the animals.It could also suggest possible immunostimulatory effect of SEC.Anemia is a regular and an important feature of trypanosomosis in animals (Abenga et al., 2016).
Trypanosomes cause massive destruction of RBCs and subsequent depletion of the RBC number, severe reduction of the PCV and hemoglobin concentrations.These features were prominent in infected rats in this study.There was recovery of the RBC number, improvement of PCV and increased Hb concentration after treatment with SEC-DA combination and with DA alone.There was no significant difference in the rate of recovery of the hematocrit between the SEC-DA combination therapy and DA alone throughout the period of the study except in the Hb concentration, where there was significant increase in the SEC-DA group over the other groups on day 21 PI.Trypanosomosis caused by T. b. brucei is characterized by tissue invasiveness.This adversely affects the serum biochemical parameters (Allam et al., 2011).These changes are associated with some organ damages and monitoring of these changes after treatment could be helpful in prediction of treatment outcome and effectiveness of treatment.Increase in the ALT activity in T. b. brucei infection has been reported by various authors (Ezeokonkwo et al., 2012).In this study, expectedly, ALT activity was elevated in infected untreated rats by 14 days post infection.This finding is consistent with the report of Taiwo et al. (2003), who reported elevated ALT in sheep experimentally infected with T. b. brucei.However, treatment with DA and SEC-DA combination lowered the ALT activity in infected and treated rats.Nevertheless, lower ALT activity was recorded in the SEC-DA treated rats compared with DA treated rats.On the other hand, on day 21 PI, elevation of ALT activity was observed in rats treated with DA alone and this coincided with relapse infection in the rats treated with DA alone on the same day.The observed effect of the SEC-DA combination therapy on the serum ALT activity could be due to enhanced parasite clearance from the liver, due to extensive distribution of SEC in the liver thereby preventing further liver damage.
It is therefore concluded that SEC-DA combination therapy in rats was more effective therapy than DA alone in experimental T. b. brucei infection.This is because the combination therapy effectively reversed some of the hematological and biochemical changes associated with trypanosomosis.The combination therapy also caused faster clearance of parasitemia and prevented relapse of infection.

Table 1 .
Mean log parasitemia in rats infected with T. b. brucei and treated with either SEC-DA combination or DA alone.

Table 2 .
Mean total WBC (x 10 3 /µl) count of T. b. brucei infected rats treated with SEC-DA combination or DA alone.

Table 3 .
Mean lymphocyte and neutrophil counts of T. b. brucei infected rats treated with SEC-DA combination or DA alone.

Table 4 .
Mean RBC, PCV and hemoglobin (Hb) concentration of T. b. brucei infected rats treated with SEC-DA combination or DA alone.Different superscripts on the same row vary significantly (p < 0.05).AD: all rats in this group died before day 21.Rats were infected on day 0 and treatment started on day 8. A: uninfected untreated, B: infected untreated, C: infected and treated with diminazene aceturate (3.5 mg/kg), D: infected and treated with secnidazole (200 mg/kg) + diminazene aceturate (3.5 mg/kg). b