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EFFICACY OF PANTIZEN® IN THE TREATMENT OF SINGLE AND MIXED INFECTIONS OF Trypanosomabrucei AND Trypanosomacongolense IN ALBINO RATS

ABSTRACT

The efficacy of Pantizen®, a brand of diminazene aceturate in the treatment of single and mixed infections of Trypanosomabrucei and Trypanosomacongolense was studied in albino rats. Six groups (groups A, B, C, D, E, and F) of six albino rats each were used for the study. Groups A and B were single infections of T. brucei and T. congolense, respectively. Groups D and E were T. brucei first and T. congolense seven days after and T. congolense first and T. brucei seven days after, respectively, whereas Group C was mixed infection given the same day. Group F was uninfected control. Parameters monitored daily were clinical signs and parasitaemia whereas the rectal temperature, body weight changes, packed cell volume (PCV), haemoglobin concentration (Hb), red blood cell (RBC) counts, total and differential white blood cell counts (WBC), were monitored weekly. The infections were characterized by significant (P < 0.05) decrease in the red blood cell values (PCV, Hb and RBC counts) compared to Group F. The lowest values were seen in Group C. The mean pre-patent periods (PPP) of the infections were 4.33 ± 0.21, 9.50 ± 0.22, 2.00 ± 0.00, 4.33 ± 0.21 and 8.50 ± 0.22 days for groups A, B, C, D and E, respectively.  The respective mean levels of parasitaemia (x 106/ml) by day 14 post-infection (PI) when the animals started showing severe clinical signs were 303.43 ± 76.88, 36.86 ± 8.78, 313.65 ± 62.53, 188.55 ±28.02 and 73.54 ± 17.53 trypanosomes/ml of blood. By day 14 PI, the animals were treated with Pantizen® intraperitoneally at a dosage of 7mg/kg body weight. Relapse infections occurred in groups A, B, C and E while group D showed no relapse till the end of the experiment. The mean body temperature was significantly (p < 0.05) increased in all the infected groups when compared with the control group. Following treatment, no significant (p > 0.05) variation was observed in the body temperature among the groups on day 21 PI. The observed clinical signs (dullness, fever, pale mucous membranes and rough hair coat) occurred earliest in group C while in group B clinical signs occurred late and with a peculiar sign of bloody-diarrhoeic faeces. There was no significant (p > 0.05) variation in the mean body weight among the groups. The mean RBC counts, Hb concentration and PCV of the trypanosome- infected rats were significantly (p < 0.05) lower than the control by day 14 PI. The haematological values for group C differed significantly (p < 0.05) from that of other infected and treated groups. Total WBC counts decreased significantly (p < 0.05) in groups A, C and D and increased significantly (p < 0.05) in groups B and E on day 14 PI. Groups A, C and D had significantly (p < 0.05) lower lymphocyte counts on day 14 PI while groups B and E had significantly (p < 0.05) higher lymphocyte counts on days 14 and 21 PI. Significant (p < 0.05) decrease in neutrophil counts was recorded in all the infected groups on day 7 PI. Pathological lesions including vasculitis, myocardial, renal and hepatic necrosis, splenic hyperplasia and cellular infiltration of various tissues were observed in the infected groups.

It was thus concluded that:

The major clinical sign found in the study was anaemia.

Pantizen® significantly returned the values of PCV, RBC counts and Hb concentration to normal value ranges.

Pantizen® could not prevent relapse infection when given at 7mg/ kg body weight

TABLE OF CONTENTS

Title page –      –           –           –           –           –           –           –           –           –           –           i

Certification –  –           –           –           –           –           –           –           –           –           –           ii

Dedication –    –           –           –           –           –           –           –           –           –           –           iii

Acknowledgements –  –           –           –           –           –           –           –           –           –           iv

Table of contents –      –           –           –           –           –           –           –           –           –           vi

List of figures –           –           –           –           –           –           –           –           –           –           xiii

List of tables – –           –           –           –           –           –           –           –           –           –           xiv

List of plates – –           –           –           –           –           –           –           –           –           –           xvi

Abstract           –           –           –           –           –           –           –           –           –           –          xvii

CHAPTER ONE: INTRODUCTION –      –           –           –           –           –           –           1

CHAPTER TWO: LITERATURE REVIEW –     –           –           –           –           –           4

2.1                   Trypanosomosis –       –           –           –           –           –           –           –           4

2.1.1                The Trypanosomes      –           –           –           –           –           –           –           4

2.1.1.1             Salivaria          –           –           –           –           –           –           –           –           4

2.1.2.2             Stercoraria –   –           –           –           –           –           –           –           –           5

2.1.2                The impact of African animal Trypanososmosis on livestock Agriculture   5

2.1.3                The vector (Tsetse fly) –         –           –           –           –           –           –           6

2.1.4                Host susceptibility      –           –           –           –           –           –           –           8

2.1.5                Transmission of trypanosomosis –      –           –           –           –           –           9

2.1.6                Pathogenesis and clinical signs –        –           –           –           –           –           11

2.1.7                Antigenic variation –               –           –           –           –           –           –           13

2.1.8                Immunosuppression in Trypanosomosis –      –           –           –           –           14

2.1.9                Mechanisms of immunosuppression induction in trypanosomosis –              14

2.1.10              Pathology of trypanosomosis –           –           –           –           –           –           15

2.1.11              Control of African animal trypanosomosis –              –           –           –           17

2.1.11.1           Host protection from vectors –            –           –           –           –           –           17

2.1.11.2           Control of the vectors –           –           –           –           –           –           –           17

2.1.11.2.1        The use of insecticides –         –           –           –           –           –           –           17

2.1.11.2.2        Live bait technique –   –           –           –           –           –           –           –           18

2.1.11.2.3        The use of traps –        –           –           –           –           –           –           –           18

2.1.11.2.4        Use of sterile male technique –           –           –           –           –           –           18

2.1.11.2.5        Bush clearing and destruction of wild animals         –           –           –           18

2.1.11.3           Parasite control –         –           –           –           –           –           –           –           19

2.1.11.3.1        Immunization –           –           –           –           –           –           –           –           19

2.1.11.3.2        Breeding of trypanotolerant animals –            –           –           –           –           19

2.1.11.3.3        Chemotherapy of animal trypanosomosis –    –           –           –           –           21

2.1.12              Trypanocidal drug resistance –           –           –           –           –           –           21

2.2                  Pantizen®–       –           –           –           –           –           –           –           –           22

2.2.1                Diminazene aceturate –           –           –           –           –           –           –           23

2.2.1.1             Physicochemical properties of Diminazene aceturate –          –           –           23

2.2.1.2             Mechanism of action of Diminazene aceturate –        –           –           –           23

2.2.1 .3            Elimination of Diminazene aceturate –           –           –           –           –           24

2.2. 1. 4           Side effects and toxicity of Diminazene aceturate –   –           –           –           24

2.2.2                Antipyrine –     –           –           –           –           –           –           –           –           27

2.2.2.1             Physical properties of Antipyrine –     –           –           –           –           –           27

2.2.2.2             Mechanism of action of Antipyrine – –           –           –           –           –           28

CHAPTER THREE: MATERIALS AND METHODS –             –           –           –           29

3.1                   Materials –       –           –           –           –           –           –           –           –           29

3.1.1                Experimental animals –           –           –           –           –           –           –           29

3.1.1.1             Housing and feeding of the experimental animals –   –           –           –           29

3.1.2                Trypanosomes –          –           –           –           –           –           –           –           29

3.1.3                The drug –       –           –           –           –           –           –           –           –           30

3.2                   Methods –        –           –           –           –           –           –           –           –           30

3.2.1                Experimental protocols –        –           –           –           –           –           –           30

3.2.2                Trypanosome infections of the experimental animals –         –           –           31

3.2.3                Drug administration    –           –           –           –           –           –           –           31

3.2.4                Blood collection from the experimental rats –            –           –           –           32

3.2.5                Parameters monitored –           –           –           –           –           –           –           32

3.2.5.1             Parasitaemia    –           –           –           –           –           –           –           –           32

3.2.5.1.1          Wet blood film examination   –           –           –           –           –           –           33

3.2.5.1.2          Microhaematocrit buffy coat (MBC) technique –       –           –           –           33

3.2.5.2            Temperature – –           –           –           –           –           –           –           –           34

3.2.5.3             Body weight – –           –           –           –           –           –           –           –           34

3.2.5.4             Haematology – –           –           –           –           –           –           –           –           35

3.2.5.4.1          Packed cell volume (PCV) –   –           –           –           –           –           –           35

3.2.5.4.2          Haemoglobin concentration (Hb) –     –           –           –           –           –           35

3.2.5.4.3          Red blood cell (RBC) counts –           –           –           –           –           –           36

3.2.5.4.4          Total white blood cell (TWBC) counts –        –           –           –           –           36

3.2.5.4.5          Differential white blood cell (DWBC) counts –         –           –           –           37

3.2.5.5   Histopathology –       –           –           –           –           –           –           –           –           38

3.2.6      Data analysis –          –           –           –           –           –           –           –           –           38

CHAPTER FOUR: RESULTS –     –           –           –           –           –           –           –           39

4.1       Onset of parasitaemia –           –           –           –           –           –           –           –           39

4.2       Mean levels of parasitaemia – –           –           –           –           –           –           –           39

4.3       Parasite clearance time and relapse –  –           –           –           –           –           –           40

4.4       Mean rectal temperature –       –           —          –           –           –           –           –           46

4.5       Clinical signs –            –           –           –           –           –           –           –           –           48

4.5.1    Infection with Trypanosomabrucei only –     –           –           –           –           –           48

4.5.2    Infection with Trypanosomacongolense only –         –           –           –           –           48

4.5.3    Infection with Trypanosoma brucei and Trypanosoma congolense same day –        48

4.5.4    Infection with Trypanosomabrucei first and T.congolense 7 days after-     –           49

4.5.5    Infection with T.congolense first and Trypanosomabrucei 7 days after-     –           49

4.6       Body weight changes –           –           –           –           –           –           –           –           54

4.7       Haematology – –           –           –           –           –           –           –           –           –           56

4.7.1    Packed cell volume (PCV) –   –           –           –           –           –           –           –           56

4.7.2    Red blood cell (RBC) counts –           –           –           –           –           –           –           58

4.7.3    Haemoglobin concentration (Hb) –     –           –           –           –           –           –           60

4.7.4    Total white blood cell (TWBC) counts –        –           –           –           –           –           62

4.7.5    Differential white blood cell (DWBC) counts –         –           –           –           –           64 4.7.5.1            Lymphocyte counts –  –           –           –           –           –           –           –           –           64

4.7.5.2 Neutrophil counts –     –           –           –           –           –           –           –           –           66

4.7.5.3 Monocyte counts –      –           –           –           –           –           –           –           –           68

4.7.5.4 Eosinophil counts –     –           –           –           –           –           –           –           –           70

4.8       Histopathology –         –           –           –           –           –           –           –           –           72

4.8.1    Brain – –           –           –           –           –           –           –           –           –           –           72

4.8.2    Heart   –           –           –           –           –           –           –           –           –           –           75

4.8.3    Liver   –           –           –           –           –           –           –           –           –           –           80

4.8.4    Spleen –           –           –           –           –           –           –           –           –           –           84

4.8.5    Kidney            –           –           –           –           –           –           –           –           –           87

CHAPTER FIVE: DISCUSSION AND CONCLUSION –           –           –           –           90

5.1       Discussion       –           –           –           –           –           –           –           –           –           90

5.2       Conclusion      –           –           –           –           –           –           –           –           –           96

REFERENCES          –           –           –           –           –           –           –           –           –           98

APPENDICES           –           –           –           –           –           –           –           –           –           119

CHAPTER ONE

INTRODUCTION

African Animal Trypanosomosis (AAT) is a disease complex caused by tsetse transmitted Trypanosoma congolense, Trypanosoma vivax or Trypanosoma brucei or simultaneous infection with one or more of these trypanosomes (Maré, 2004). It is transmitted cyclically by tsetse flies. Other haematophagous vectors such as Tabanus (most encountered), Stomoxys, Hippobosca, Liperosia, Haematopota and Chrysops have also been incriminated in the non-cyclical transmission of trypanosomes (Mbaya et al., 2010). For several decades, trypanosomosis has continued to contribute adversely to the economic and social well being of sub-Saharan Africans (WHO, 2006). It causes a disease known as ‘sleeping sickness’ in man and ‘nagana’ in animals where its major clinical signs are fluctuating body temperature and anaemia manifesting as pale mucous membrane. There is usually anorexia, dullness and enlargement of the superficial lymphnodes in dogs (Anosa and Kaneko, 1983; Ezeokonkwo et al., 2010).

The trypanosome species affecting man and domestic animals have been classified into two groups: the haematic group (Trypanosoma congolense and Trypanosoma vivax) which are intravascular organisms and the humoral (tissue invading) group (T. brucei, T. evansi, T. gambiense and T. rhodesiense) which dwell both in extra and intravascular spaces (Losos and Ikede, 1972; Abubakar et al., 2005; Ngure et al., 2008).

The use of trypanocidal drugs is the most widely adopted means of controlling trypanosomosis (Feldmann and Hendrichs, 2001). Chemotherapy of animal trypanosomosis depends on isometamidium chloride, homidium chloride and diminazene aceturate (Geerts and Holmes, 1998) and quinapyramine which was re-introduced to combat camel trypanosomosis (ILRAD, 1990). While Isometamidium and Homidium are mainly used as prophylaxis, diminazene aceturate exerts its action mainly as a curative measure (Mamman et al., 1995). The therapeutic index of diminazene aceturate in most animals is relatively high (Fairclough, 1963) but its use in equine trypanosomosis has been limited because of severe reaction mostly observed following intramuscular administration (ILRAD, 1990). Toxicity and even death had been reported in dogs, camels and donkeys injected with recommended doses of this drug (Anene et al., 1989b; ILRAD, 1990).

Multiple generic variants of trypanocides are available and the variations in the quality of these products is striking. Pantizen® is one of the recent formulations of diminazene aceturate present in Nigerian Market. It is composed of 445 mg of diminazene acerturate and 555 mg of antipyrine (Pantex B.V., Holland). It is necessary that evaluation of effectiveness of trypanocides used in the treatment of trypanosomosis be carried out especially for the new products before their acceptance in a given area.

The objectives of this study are therefore:

Specific objective:

To determine the efficacy of Pantizen® in the treatment of albino rats infected with single and mixed infections of brucei and T. congolense.

General objectives:

To determine the specific clinical signs of single and mixed infections of Trypanosoma brucei and Trypanosoma congolense in albino rats.

To determine to what extent the single and mixed infections of Trypanosoma brucei and T. congolense would alter the haematological indices of infected albino rats.

To study the pathological lesions in albino rats infected with single and mixed infections of brucei and T. congolense.

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