ugwu, christian emetueobi
TRANSCRIPT
1
CLINICAL AND HAEMATOLOGICAL STUDIES IN DOGS WITH
SINGLE AND MIXED EXPERIMENTAL Trypanosoma brucei AND
Ancyclostoma caninum INFECTION
BY
UGWU, CHRISTIAN EMETUEOBI
PG/MSC/02/33565
A DISSERTATION SUBMITTED TO THE DEPARTMENT OF
VETERINARY MEDICINE, UNIVERSITY OF NIGERIA, NSUKKA
IN PARTIAL FULFILMENT OF THE REQUIEMENTS FOR THE
DEGREE OF MASTER OF SCIENCE IN VETERINARY MEDICINE
FEBUARY, 2010
\
APPROVAL PAGE
2
This dissertation has been approved for the Department of Veterinary
Medicine, University of Nigeria, Nsukka.
BY
………………………. ……………………………..
PROF. B. M. ANENE DR. C. O. EMEHELU
Supervisor Head of Department
………………………. …………………………..
External Examiner Dean of Faculty
DEDICATION
3
MY LATE PARENTS, MRS AGNES ODOBO UGWU
AND
MR DAVID ASOGWA UGWU
ACKNOWLEDGEMENTS
4
I wish to express profound gratitude to my supervisor, Prof. B.M. Anene, who
tirelessly guided me throughout every stage of this work. Precisely at every
stage of this work, he was a motivator and teacher. The astuteness and
thoroughness in him during the course of this work are appreciated and worth
commending. Also, I wish to thank Dr. P. Nnadi, Dr. Ezeibe, Dr. I.J. Eze, Dr C.
Igbokwe, Dr. Iheagwam, Dr. Ezeokonkwo and all the staff of Department of
Veterinary Medicine and Department of parasitology who helped me. My
special regards and appreciation goes to Dr. Ikenna Eze for his brotherly gesture
during the course of the work. Finally, I thank Mr. Chimezie Aneke for his
kindness, Managing Director of Blue Bat Company, Limited (Dr. C. C. Ibe) for
his meekness and understanding and all the members of my family, especially
my wife, Mrs. Ada Phina Ugwu for their unflinching support.
LIST OF FIGURES PAGE
5
Fig. 1: Mean body weight (kg) of dogs infected with T. brucei and A. caninum
and mixed infection of T. brucei and A. caninum……………… 35
LIST OF TABLES PAGE
6
TABLE 1: Mean rectal temperature (
oC) ± standard error (SE) of dogs infected
with T. brucei, A. caninum and mixed infection of T. brucei and A. caninum …… 32
TABLE 2: Mean egg per gram (EPG) ± SE of dogs infected with T. brucei,
A. caninum and mixed infection of T. brucei and A. caninum ……………………. 38
TABLE 3: Mean Packed cell volume (%) ± SE of dogs infected with
T. brucei, A. caninum and mixed infection of T. brucei and A. caninum …………… 39
TABLE 4: Mean Total white blood cell counts (103/µl) ± SE of dogs infected
with T. brucei, A. caninum and mixed infection of T. brucei and A. caninum ..……. 40
TABLE 5: Mean Absolute neutrophil counts (10
3/µl) ± SE of dogs infected
with T. brucei, A. caninum and mixed infection of T. brucei and A. caninum ……… 42
TABLE 6: Mean Absolute lymphocyte counts (103/µl) ± SE of dogs infected
with T. brucei and A. caninum and mixed infection of T. brucei and A. caninum …… 43
TABLE 7: Mean Absolute eosinophil counts (10
3/µl) ± SE of dogs infected
with T. brucei and A. caninum and mixed infection of T. brucei and A. caninum …... 44
TABLE 8: Mean Absolute monocyte counts (10
3/µl) ± SE of dogs infected
with T. brucei and A. caninum and mixed infection of T. brucei and A. caninum ……. 45
7
LIST OF PLATES PAGE
PLATE 1: Dark tarry faeces ………………………………………………. 33
PLATE 2: The control dogs (uninfected) ………………………………….. 33
PLATE 3: Bilateral ocular discharge (B) and rough hair coat (C) ……… 34
PLATE 4: Inappetence (E) and bilateral corneal opacity (D) …………… 34
PLATE 5: Recumbency (F) and anorexia (G) ……………………………… 36
PLATE 6: Normal spleen (H) and splenomegaly (I) ………………………… 47
PLATE 7: Normal liver (O) and icteric liver (N) ……………………………… 47
PLATE 8: Heamorrhagic enteritis (M) ……………………………………… 48
PLATE 9: Intussusception (J) ………………………………………………… 48
PLATE 10: Normal lung (K) and fibrotic lung (L) ………………………… 49
TABLE OF CONTENTS
8
TITLE PAGE i
APPROVAL PAGE ii
DEDICATION iii
ACKNOWLEDGEMENT iv
LIST OF FIGURES v
LIST OF TABLES vi
LIST OF PLATES vii
TABLE OF CONTENTS viii
ABSTRACT xii
CHAPTER ONE 1
1.0 INTRODUCTION 1
CHAPTER TWO 4
2.0 LITERATURE REVIEW 4
2.1.0 African animal trypanosomosis 4
2.1.1 Aetiology 4
2.1.2 Transmission 4
2.1.3 Life cycle of the parasite 5
2.1.4 Pathogenesis and pathological manifestations 6
2.1.5 Clinical manifestations 10
2.1.6 Immunity in trypanosomosis 11
2.1.7 Immunosuppression in trypanosomosis 13
2.1.8 Haematology 15
9
2.2.0 ANCYLOSTOMA CANINUM 17
2.2.1 Aetiology 17
2.2.2 Life cycle of Ancylostoma caninum 18
2.2.3 Clinical signs and pathogenicity of Ancylostoma caninum 19
2.2.4 Immunity in helminth infections 20
2.2.5 Haematology 23
CHAPTER THREE 25
3.0 MATERIALS AND METHODS 25
3.1.0 Experimental Animals 25
3.1.1 Experimental design 25
3.1.2 Trypanosome infection 26
3.1.3 Ancylostoma caninum 26
3.1.4 Feacal culture 26
3.1.5 Ancylostoma caninum infection 27
3.1.6 Conjunct Trypanosoma brucei and Ancylostoma caninum infection 27
3.2.0 Detection of parasitaemia 28
3.2.1 Wet mount 28
3.2.2 Buffy coat 28
3.2.3 Giemsa stained thin films 28
3.3 Faecal egg count 29
3.4.0 Haematology 29
3.4.1 Blood collection 29
10
3.4.2 Packed cell volume 29
3.5 Post mortem examination 30
3.6 Statistical analysis 30
CHAPTER FOUR 31
4.0 RESULTS 31
4.1Course of infection 31
4.1.1 Ancylostoma caninum 31
4.1.2 Trypanosoma brucei 31
4.1.3 Mixed infection (T. brucei and A. caninum) 35
4.2 Faecal Egg Output (EPG) 37
4.3 Haematology 37
4.3.1 Packed cell volume 37
4.3.2 Total white blood cell counts 37
4.3.3 Absolute neutrophil counts 41
4.3.4 Absolute lymphocyte counts 41
4.3.5 Absolute eosinophil counts 41
4.3.6 Absolute monocyte counts 41
4.4 Post mortem findings 46
CHAPTER FIVE 50
5.0 DISCUSSIONS 50
6.0 CONCLUSIONS AND RECOMMENDATIONS 54
REFERENCES 56
11
APPENDIX 74
ABSTRACT
12
Trypanosomosis is one of the most devastating diseases of animals caused by
infection with a protozoan parasite trypanosome, which is transmitted by tse-tse fly.
Besides anaemia, which is a cardinal symptom of the disease, infection also impairs
the immune system of animals and renders them more susceptible to other. Under
natural field condition, in areas where trypanosome and helminth parasites are
endemic, mixed infection appears to be common. A study was conducted to
determine the clinico-haematological manifestations in dogs experimentally infected
with Trypanosoma brucei and Ancylostoma caninum singly and in combination.
Twenty young local dogs were used in the study. They were randomly grouped into 4
with 5 dogs in each group; group A (uninfected control), group B (infected with A.
caninum), group C (infected with T. brucei) and group D (mixed infection with A.
caninum/T. brucei). For the mixed infection, dogs were initially infected with A.
caninum and then T. brucei infection superimposed 23 days later by the time of
patency of Ancylostoma eggs in the stool. Results of this study showed that the
prepatent period (PP) of A. caninum infection in the dogs was 24.5±0.4 days. The PP
of T. brucei infection alone was 5.0±0.0 days, but was 4.6±0.22 days in the mixed
infection. Clinical signs of dullness, inappetence, anorexia, weakness, pale mucous
membrane due to anaemia, rough hair coat were encountered in both infections.
Additionally, specific signs of bloody diarrhea and sunken eyes were present in A.
caninum infected dogs while fever, swollen face, bilateral ocular discharge and
corneal opacity accompanied T. brucei infection. A combination of these signs in a
more severe form characterized the mixed infection of both parasites. There was a
significant decrease (P<0.05) in the packed cell volume (PCV) in all the infected
groups (B, C and D) as from day 19 post infection (p.i.). Infection with Ancylostoma
caninum caused significant increase (P<0.05) in the total leucocyte count of the dogs
in group B from day 29 p.i., whereas significant decrease were recorded in
trypanosome infected dogs group C and in mixed infection group (D) on days 38 and
34 p.i., respectively. The absolute neutrophil counts significantly increased (P<0.05)
in group B by day 29 p.i. whereas significant decreases were recorded in groups C and
D from day 34 p.i.. There was no variation in the absolute lymphocyte count except
on day 20 p.i. when an increase was detected only in the mixed infection group (D).
13
The absolute eosinophil counts significantly increased (P<0.05) in group B from day
14 p.i. in contrast to a significant decrease (P<0.05) in the mixed infection group (D)
by day 34 p.i. There was no significant variation (P<0.05) in the absolute monocyte
counts of the infected dogs. The results of the egg per gram (EPG) of faeces on days
28, 34 and 39 were 20300±12195, 81233±26410 and 67683±13971 for group B, and
34325±8044, 54425±24764 and 55800±12304 for group D, respectively, and showed
no significant difference (P>0.05). It was concluded that concurrent infection of T.
brucei and A. caninum resulted in enhanced pathogenicity manifested in remarkable
clinico-haematological alterations in the infected dogs.
14
CHAPTER ONE
1.0 INTRODUCTION
The trypanosome is a protozoan parasite transmitted by the bite of a tsetse
fly to people and to wild and domestic animals in which it causes
trypanosomosis (Maudlin, 2006; ILRAD, 1991). The disease is widespread
across more than a third of African continent infested with the tsetse fly vector
(Feldmann, et al., 2005; Torr et al., 2005). It is one of the most devastating
diseases of animals in sub-Sharan Africa with estimated losses due to its direct
and indirect consequences running into billions of dollars (Swallow, 1998;
Ng’ayo et al., 2005). Trypanosoma vivax, T. congolense and T. brucei are the
most important African animal trypanosome species. Their infections in
domestic animals cause anaemia, weight loss and reproductive disorders;
infected animals may die if not treated. Another striking feature of African
trypanosomosis is its profound suppression of immune system of the infected
mammalian host (Goodwin, 1970; Goodwin et al., 1972; Rurangirwa et al.,
1979; Griffin et al., 1980; ILRAD, 1992). This impairment of the immune
response due to trypanosomosis has given rise to increased susceptibility of
trypanosome-infected animals to other infections (Parkin and Hornby, 1930;
Mackenzie et al., 1975; Scott et al., 1977; Nantulya et al., 1982; Ikeme et al.,
1984) and ineffective vaccination of animals against a variety of diseases in
areas of endemic trypanosomosis (Holmes, et al., 1974; Ilemobade et al., 1982;
Sharpe et al., 1982; Rurangirwa et al., 1983). Indeed, it is believed that under
15
natural conditions, it is often opportunistic infections rather than
trypanosomosis itself that kills trypanosome-infected animals (Mackenzie et al.,
1975).
Responses of trypanosome-infected animals to other parasitic infections have
been studied by various workers. Urquhart et al. (1973) and Philips et al.
(1974) observed that the normal process of immune expulsion of adult worms
(immediate-type response) was suppressed in T. b. brucei infected rats and
mice. It was further observed that concurrent gastrointestinal nematodes
infection led to a more severe worm infection (Griffin et al., 1981 a,b;
Kaufmann, et al., 1992; Dwinger, et al., 1994; Fakae, et al., 1994; Goossens et
al., 1997; Wakelin and Onah, 1999, 2000). Gastrointestinal nematodes are
recognized as a major cause of impaired productivity in livestock and domestic
animals in the tropics (Chiejina, 1986; Fabiyi, 1987). Infections are mostly
subclincal probably due to acquired or innate resistance (Chiejina, 1987; Fakae,
1990). Goossens et al. (1997) confirmed that under natural conditions,
trypanosomosis and helminthosis often occur in mixed infections, and are
prevalent in sub-Saharan Africa, where they are endemic.
Trypanosomosis and ancylostomosis are parasitic diseases of considerable
veterinary importance in dogs in Nigeria (Omamegbe et al., 1984; Anene and
Omamegbe, 1987; Anene et al., 1996). With the reports that trypanosomes
cause non-specific depression of immune response to a variety of heterologous
antigen (Anene, et al., 1989 a; Murray, et al., 1980; Wakelin, 1984) concurrent
16
infections of trypanosomes and A. caninum will probably result in a
prolongation of worm survival and more severe disease.
The whole blood is an important and reliable medium for assessing the health
status of animals (Anosa and Isoun, 1978). Information obtained through blood
analysis is useful in the clinical assessment of animal patients. The changes in
blood parameters are indicative of ill-health, and they assist in the diagnosis,
severity and prognosis of disease conditions (Coles, 1986; Bush, 1991).
This work is therefore designed with the objective to determine the
haematological changes that occur in dogs experimentally infected with T.
brucei and A. caninum singly and in combinations. Further, the clinico-
pathological manifestations of the infection are also recorded.
17
CHAPTER TWO
2.0 LITERATURE REVIEW
2.1.0 AFRICAN ANIMAL TRYPANOSOMOSIS
2.1.1 Aetiology
African animal trypanosomosis is caused by protozoa in the family
trypanosomatidae, genus trypanosome (Soulsby, 1986). The most important
African trypanosome species include T. vivax, T. congolense and T. brucei.
These three species are considered the most important because they are the main
pathogens of domesticated animals in the areas in which the disease occurs
(Stephen, 1986; Katherin and Edith, 2004). They are all members of the
salivarian group of trypanosome and are transmitted via the mouth parts of
tsetse fly, hence the name salivarian trypanosomes.
2.1.2 Transmission
Trypanosomes have arthropod vectors in which transmission is either cyclical or
non cyclical (Urquhart et al., 2002). In cyclical transmission, the arthropod is a
necessary intermediate host, in which the trypanosomes multiply, undergoing a
series of morphological transformation before forms infective for the next
mammalian host are produced. When multiplication occurs in the digestive
tract and proboscis, so that the new infection is transmitted when feeding, the
process is known as anterior station development, as distinct from posterior
station development where multiplication and transformation occurs in the gut
and the infective forms migrate to the rectum and are passed with the faeces.
18
The various species of trypanosomes which use the process of anterior station
development are often considered as a group, the salivaria, while the posterior
station trypanosome species are grouped together as the stercoraria. The
salivarian trypanosomes are all transmitted by tsetse flies, the main species
being T. vivax, T. congolense and T. brucei. In non cyclical transmission,
trypanosomes are transferred from one mammalian host to another by the
interrupted feeding of biting insects, notably tabanids and stomoxys (Maxie et
al., 1979; Soulsby, 1986).
2.1.3 Life cycle of the parasite
According to Urquhart et al. (2002) tsetse flies ingest trypanosomes in the blood
or lymph while feeding on an infected host. Afterwards the trypanosomes loose
their glycoprotein surface coat and in the case of T. brucei and T. congolense,
become elongated and multiply in the midgut, migrating forward to the salivary
gland (T. brucei) and the proboscis (T. congolense). They subsequently,
undergo a transformation losing their typical trypanosome or trypamastigote
form and acquire an epimastigote form, characterized by the fact that the
kinetoplast lies just in front of the nucleus. After further multiplication of the
epimastigotes, they transform again into small typically trypamastigote forms
with a glycoprotein surface coat. These are the infective forms for the next host
and are called metacyclic trypanosomes. The entire process takes at least two to
three weeks and the metacyclic trypanosomes are inoculated into the new host
when the tsetse fly feeds (Hoare, 1972; ILRAD 1990).
19
2.1.4 Pathogenesis and pathological manifestations
Animals become infected with trypanosomes when they are bitten by tsetse fly.
In the process of taking a blood meal from an animal, infected fly deposits
saliva laden with trypanosomes in the connective tissue of the animal’s skin. At
this site of inoculation, the metacyclic forms multiply locally and differentiate
to the bloodstream form, which is specially adapted to live in mammalian blood,
producing within a few days raised cutaneous inflammatory swelling called
chancre (Urquhart et al., 2002). Thereafter, they enter the blood stream,
multiply by binary fission and a parasitaemia detectable in the peripheral blood
usually become apparent 1-3 weeks later. Once in the blood, the parasites have
access to most other major organs.
With the appearance of parasites in the blood, susceptible animals develop
intermittent fever and anaemia (ILRAD, 1990). Subsequently, the parasitaemia
may persist for many months, although its levels may wax and wane due to
immune response of the host. The fever is highest at the first peak of
parasitaemia and fluctuates thereafter with parasitaemic waves (Taylor and
Authie, 2004). Anaemia develops with the onset of parasitaemia and it is the
cardinal feature of the disease (Anosa, 1988; Murray and Dexter, 1988;
Urquhart et al., 2002; Naessens et al., 2005). The anaemia that occurs during
acute trypanosomosis is due primarily to accelerated destruction of red blood
cells (Jennings et al., 1974, 1977; Valli and Forsberg, 1979; ILRAD, 1990).
20
Red cells are phagocytosed by activated macrophages and the haemoglobin of
red cells is digested and stored in the macrophages as iron complexes.
Early in a trypanosome infection, the number of macrophages increases
throughout the body. This expanded pool of macrophages actively remove red
cells within vessels and tissues in many sites, including the spleen, liver, lungs,
lymph nodes and bone marrow, thereby greatly reducing the half-lives of red
cells (Murray and Dexter, 1998). Physical alterations in the surface membrane
of red cells can lead to their early removal by macrophages. Febrile responses
lead to decreased erythrocyte half-life, due to increased osmotic fragility,
decreased plasticity and increased membrane permeability. In infections that
cause extremely high parasitaemia, disseminated intravascular coagulation
(DIC) may occur, resulting in an accelerated destruction of red cells. This
coagulation causes fibrin thrombi to be deposited in small vessels. Red cells are
damaged by these partially blocked capillaries, and such damaged red cells may
then be phagocytosed by macrophages (ILRAD, 1990). Aberrant antibodies
may bind to the hosts own blood cells, thus facilitating their removal by
macrophages (Kobayashi et al.1976; Rifkin and Landsberger, 1990; Assouku
and Gardiner, 1992).
Microcytosis and low plasma-iron turnover rates have been observed during
chronic trypanosomosis, suggesting impaired erythropoiesis (Tratour and Idris,
1973; Dargie et. al., 1979). In addition, the presence of massive haemosiderin
deposits within the mononuclear phagocyte system may be indicative of
21
defective iron utilization (Murray et al., 1974; Valli and Forsberg, 1979). .
Trypanosomes have been found in the bone marrow, where it is possible that
they damage precursor cells by signaling for their early removal by
macrophages (ILRAD, 1990). Increase in numbers of cells of the monocytic
linage in bone marrow with a resulting destruction of immature red blood cells
has been observed. These observations suggest that trypanosomal infection may
cause defective blood cell production (Anosa et al., 1997).
Histopathologically, there is cellular response and frequent demonstration of
trypanosomes in the tissues (Ikede and Losos, 1975) especially T. brucei which
localizes and multiplies outside blood vessel unlike T. congolense found only in
the blood vessles. Ikede and Losos (1977) noted that there is usually interstitial
and perivascular mononuclear cell infiltration associated with the extravascular
localization of these trypanosomes. Cellular degeneration and inflammatory
infiltration occur (Taylor and Authie, 2004) in many organs such as the skeletal
muscle and the central nervous system, perhaps mostly in the myocardium,
where there is separation and degeneration of the muscle fibers (Urquhart et al.,
2002). Mc Cully et al. (1971) also reported neurological lesions, and
perivascular oedema, degeneration of heart fibers resulting from prolonged
anaemia and resultant anoxia (Taylor and Authie, 2004). Lesions in the brain
similar to those described for fatal cases of human sleeping sickeness have been
observed (Morrison et al., 1983; Wellde et al., 1989), and included extensive
infiltration into the meninges and perivascularly throughout the brain and spinal
22
cord of cells composed predominantly of lymphocytes, plasma cells and
macrophages. Severe meningoencehalitis was also observed in pigs that were
infected with T. brucei, and trypanosomes were isolated from the brain of these
animals (Otesile et al., 1991).
Lymphoid enlargement and splenomegaly were associated with hyperplasia of
the plasms cells and hypergamaglobinaemia due to an increase of IgM
production. In cases of long duration of infection, the spleen becomes shrunken
due to cellular exhaustion (Urquhart et al., 2002).
The gross pathological lesions seen at post-mortem include oedema, serous
effusion and gelatinous appearance of cutaneous fat (Ikede, 1974).
Subcutaneous oedema is prominent, accompanied by ascites, hydropericadium
and hydrothorax with straw coloured fluid with fibrin flakes (OIE, 2008). The
pericardial fat was gelatinous and the lung was emphysematous with
haemorrhages in the trachea (Boreham and Kimber, 1970). The liver may be
enlarged and oedema of lymph nodes in acute phase, but reduced size in chronic
phase (Baker, 1962). The liver and spleen were swollen and congested, while
the kidneys were pale and on cut surface showed haemorrhages especially along
the corticomedullary junction (Boreham and Kimber, 1970). Further, some
workers recorded enlarged pale kidney, heart and spleen, and a lung having a
firm consistency. There were ocular lesions, necrosis of the kidney and heart
muscle and subcutaneous petechial haemorrhages as well as gastroenteritis (Mc
Cully and Neitz, 1971; Ikede, 1974). There was meningoencephalitis (Losos and
23
Ikede, 1972; Seiler et al., 1981; Morrison et al., 1983; Moulton, 1986; Wellde et
al., 1989).
2.1.5 Clinical manifestations
Animals suffering from trypanosomosis may manifest syndromes ranging from
subclinical, mild or chronic infections to acute fatal disease (Stephen, 1986,
Maudlin, 2006). The severity of the clinical response is dependent on the
species and the breed of affected animals and the dose and virulence of the
infecting trypanosomes. Stress, such as poor nutrition and concurrent diseases
plays a prominent role in the process (Holmes et al., 2000). Dog and cat are
susceptible to T. brucei and T. congolense (Nfon et al., 2000). Trypanosoma
congolense infection may result in peracute, acute or chronic disease in
domestic animal species (cattle, sheep, goat, horse and camel). Dogs commonly
suffer chronic T. congolense infection even though there are reports of acute
disease following experimental infections (Anene et al., 1989 b; Ezeokonkwo,
2009). Trypanosoma brucei manifests mild, chronic or subclinical infection,
except in horse, camel, dog and cat where it causes severe to fatal infection
which if untreated almost invariably end fatally (Jennings, et al., 1977b;
Moulton and Sollod, 1976; Anene, 1989 b; Ezeokonkwo, 2009). The prepatent
period (PP) is shorter for T. brucei (5-10 days) than T. congolense (7-24 days)
(Godffery, 1966; Anene et al., 1989; Ezeokonkwo, 2009). The PP is followed
by intermittent fever, depression, lethargy, weakness and anorexia. The
heartbeat and respiration may be increased; progressive anaemia (paleness of
24
mucous membrane) loss of body condition and generalized enlargement of
superficial lymph glands. Abortion is common in females (Anene and
Omamegbe, 1984; Anene et al., 1991). Notable features of the disease in dogs
are corneal opacity, belpharitis, conjunctivitis; keratitis caused by the
trypanosome invasion of the tissue and oedema of face, limbs and its ventral
sides especially male genitalia (Stephen, 1986). There may be heart failure and
neurological changes resulting in aggressive signs, ataxia or convulsion in the
advanced stages of the disease prior to death (Anene et al., 1989 b). In acute
cases death occurs in few weeks of infection (Fairlamb, 1989). Animals
become extremely weak at the terminal stage of the disease and death is
associated with congestive heart failure due to anaemia and myocarditis or
secondary bacterial or viral infections. The secondary infections are believed to
develop because immune defense mechanisms are compromised in trypanosome
infected animals (Griffin et al., 1981a, b; Ikeme et al., 1984; Anene et al.,
1989a).
2.1.6 Immunity in trypanosomosis
The immune response of the host probably plays an important role in the
pathogenesis of trypanosomosis (ILRAD, 1992). Normally, when mammals are
infected with most type of pathogens, their immune system quickly generates
large numbers of white blood cells, which are highly competent in clearing the
infectious agents from the body. Host antibodies can kill and clear all
trypanosomes from their circulation and stop the development of the disease,
25
but often are unable. This inability is partially due to a parasite mechanism
known as antigenic variation, which helps trypanosomes evade destruction by
the immune system of their animal host. The parasite repeatedly change the
kind of antigenic protein displayed on their surface membrane to which the
immune cells are directed and in this way prolong their survival and finally
exhaust the host’s immune responses. In animals infected with trypanosomes,
there is a proliferation of a major type of white blood cell called B lymphocytes
and a dramatic increase in the antibody molecules that B cells secrete (Urquhart
et al., 2002). Antibodies recognize and bind to molecules that make up the
surface coat of trypanosomes, known as variable surface glycoprotein of VSGs.
The binding of host antibody to parasites initiate a cascade of reactions
mediated by serum proteins of the host animal, which cause the rupture (lysis)
of parasites. The antibodies also help scavenger cells such as macrophages to
clear antibody-coated trypanosomes from the circulation. Due to this multiple
variation in their antigenicity, however, not all trypanosomes in an infected
animal have antibody bound to their surface. These parasites multiply and
create new waves of parasitaemia (ILRAD, 1992). This phenomenom of
antigenic variation has prevented the development of vaccine and permit
reinfection when animals are exposed to new antigenic type of trypanosome
even after treatment (Shapiro, 1986; Nantulya and Moloo, 1988; Borst, 2002).
26
2.1.7 Immunosuppression in trypanosomosis
Antigen interacting with antibody produces an immune complex which is a
component of the normal immune response (Stephen, 1986). This complex is
protective (WHO, 1977) and it is only in uncommon injurious situations that
they can result in a disease state (Gell and Combs, 1974). The idea of
immunosuppression in trypanosomosis started with Carmichael (1937), Schwetz
(1930), Carpano (1932) and others who noted that domestic animals which have
been subjected to long water deprivation, climatic extremes and vaccination
reaction suffer most severely from trypanosomosis. Immunosupperession
appears to be a nearly universal feature of infection with African trypanosomes
and thus may represent an essential element of the host-parasite relationship,
possibly by reducing the host’s ability to mount a protective immune response
(Katherine and Bea Mertens, 1999). Immunosuppression is a well documented
feature of trypanosomosis in cattle, humans and mice (Mansfield, 1989, De
Baetselier, 1996, Taylor, 1998). There is evidence that infection-related
immunodepression compromises the ability of animals to control
trypanosomosis (Sternberg et al., 1994) secondary infections (Scott et al., 1977;
Rurngirwa et al., 1978) as well as mount effective immune response to
vaccines. Goodwin (1970) and Goodwin et al. (1972) have shown that mice
and rabbits carrying a chronic T. brucei infection failed to elicit a proper
27
immune response to an injection of sheep erythrocytes in that the haemagglutin
response was depressed or even inapparent. Anene et al. (1989 a) recorded
significant reduction in humoral antibody response of T. brucei infected dogs to
Brucella arbortus S19 vaccination. Rinderpest attenuated virus failed to protect
cattle immunologically in a trypanosome infected herd of cattle (Holmes et al.,
1974).
The fact that Trypanosoma infections induce immunosuppression in affected
animals may have profound significance in understanding the pathogenesis of
the disease (Urquarhart et al., 1973). It was demonstrated that in rats in which a
N. brasiliensis infection was superimposed on a previously existing T. brucei
infection of 3 weeks duration, the normal process of immune expulsion of adult
worms did not occur, the production of circulating protective antibody (IgG)
and of reaginic antibody (IgE) was grossly impaired and there was no increase
in the number of mast cells in the intestinal villi. Similar results were obtained
when Trichuris muris and T. brucei was studied also in rats and mice (Urquhart
et al., 1973, Phillip et al., 1974). Rurangirwa et al. (1978) demonstrated in
cattle with experimental concurrent infections of T. vivax and Mycoplasma
mycoides (CBPP), that there was involution of the thymus.
The exact cellular pathways or mechanism involved in trypanosome induced
immunosuppression is still unknown. However, Mansfield and Bagasra (1978)
in their work examined the nature and extent of immunosuppression in
28
laboratory animal infected with trypanosomes and came up with the following
theories on the occurrence of immunosuppression:
1. B - cell mitogen preempting response to antigens. This is supported by
Moulton and Coleman (1977).
2. Action of suppressor T- cells on macrophages.
3. Loss of suppressor cell function of free fatty acids.
4. Effects of immune-complex on phagocyte function.
5. Depletion of the lymphoid element in the mononuclear phagocyte system
(MPS).
2.1.8 Haematology
Anaemia is one of the most important disease manifestations in animal
trypanosomosis (Ikede et al., 1977; Anosa and Kaneko, 1983, Anosa and Isoun,
1980, Anosa and Obi, 1980, Logan-Henfrey et al., 2000, Naessens et al., 2005)
and is usually proportional to the degree of parasitaemia (Soulsby, 1986). The
onset of anaemia closely correlated with the onset of fever, and appearance,
intensity and duration of parasitaemia. By the second to the third week of
infection, a sharp drop in the red blood cell count and haemoglobin level
developed, accompanied by increased circulation of immature red blood cells.
Following the acute phase of trypanosomosis characterized by progressive
anaemia and a fluctuating parasitaemia of 4-12 weeks duration, the packed cell
volume (PCV) of infected animals dropped to 20% or lower. The PCV
continued to drop and resulted in the death of the animal. The PCV however
29
fluctuated at a low level during chronic disease; or gradually improved as the
animal recovered. Later in infections of several months duration, when the
parasitaemia often became low and intermittent, the anaemia resolved to a
variable degree. However in some chronic cases, it persisted despite
chemotherapy (Logan-Henfrey et al., 2000). The anaemia of T. congolense
infected cattle was macrocytic hypochromic, but was microcytic hypochromic
terminally. The PCV of infected dogs reduced by 50% and the anaemia was
normochromic (Morrison et al., 1981). Other important changes in the blood
during the acute phase of the disease involved white blood cells, platelets and
plasma factors and occurred simultaneously with the anaemia of
trypanosomosis. The number of white blood cells was reduced to about half the
normal number due to a reduction in numbers of neutrophils and lymphocytes.
Monocytes and eosinophils were less severely affected. Thrombocytopaenia
developed in human and animal trypanosomosis (Wellde et al., 1978; Logan-
Henfrey et al., 2000). The number of circulating blood platelets also decreased
early in the infection due to a shortened platelet life span (i.e. excessive removal
at coagulation sites or in the circulation by macrophages), but thrombocytosis
was reported in another study (Esien and Ikede, 1978). The total leucocyte
counts were usually depressed during the early acute or subacute phase of
trypanosome infections (Anosa, 1980; Anosa and Isoun 1980) but elevated
leucocyte values were present in T. brucei and T. congolense infected dogs
(Anene et al., 1989 b) and T. brucei infections of highly tolerant deer mice
30
(Anosa and Kaneko, 1983). During the chronic phase of infections, the blood
leucocyte values recovered gradually and sometimes attained preinfection
values (Anosa, 1980; Anosa and Isoun, 1980).
The lymphocytes usually decreased in the blood in the acute phase of
trypanosome infection (Maxie et al., 1979; Anosa, 1980; Anosa and Isoun,
1980). Although increased numbers were associated with T. brucei infection of
highly tolerant deer mice (Anosa and Kaneko, 1983) and with human
trypanosomosis (Anosa, 1988). Lymphopaenia occurred partly because of
depletion of lymphocytes from lymphoid nodules which occurred in acute T.
vivax infection (Anosa, 1977; Anosa and Isoun, 1980) and partly because of the
sequestration of many lymphocytes in the inflammatory reactions in T. vivax
infections of ruminants and T. brucei infection in mice (Anosa and Kaneko,
1984).
Monocytosis was a consistent finding in trypanosomosis (Isoun, 1975; Anosa,
1980; Anosa and Isoun, 1980). Monocytosis coexisted with marked
proliferation of macrophages in the tissues of infected animals.
2.2.0 ANCYLOSTOMA CANINUM
2.2.1 Aetiology
Ancylostoma caninum is a small intestinal worm of dogs, fox, wolf and other
wild carnivores. It is of the subfamily Ancylostomatidae and genus
Ancylostoma. It is a rigid worm, grey or reddish in colour depending on the
presence of blood in its alimentary canal (Soulsby, 1986). The bucal cavity is
31
deep and bears a pair of triangular dorsal teeth and a pair of centrolateral teeth
(Sherding et al., 1994). The male measures about 10-12 µm long while the
female about 14-16 µm long. The male has well developed bursae with about
0.8-0.95 µm long spicules while the female has vulva at the junction of the
second and last thirds of its body. It is cosmopolitan in distribution being very
common in the tropics and subtropics (Soulsby, 1986).
2.2.2 Life cycle of Ancylostoma caninum
The adult worms live in the small intestine (Boag et al., 2003) where they attach
themselves and feed on the blood and lay eggs that pass in faeces. The time
from the consumption of infective larvae (L3) to the appearance of eggs in the
faeces is about 15-26 days (Soulsby, 1986; Okewole and Oduye, 2000).
Hookworms have very high fecundity of about 10,000 eggs per-day and under
slight moist sandy soil, and temperature of about 23oC to 30
oC hatch to infective
larvae (L3) in about 3 weeks (Hendrix, 1998). These larvae are excellent
swimmers and can travel through rain drops or dews on leaves and vegetation
and wait for dogs (host) to come along. These infective larvae (L3) enter the
host through ingestion, intact skin penetration and through the uterus or milk
(Stone and Smith, 1973; Georgi and Georgi, 1992). Perorally, the L3 is
ingested through contaminated food or water and live in the small intestine of
host. However, a few of the larvae will migrate through the body tissues and
ultimately to the trachea where they are coughed up and swallowed, while some
will stop migration midway and encyst in the muscle. Percutaneously, the L3
32
enter through the intact skin and migrate through the blood stream to the lungs
and trachea where they are coughed up and swallowed to the small intestine.
The encysted larvae in muscle of the host can subsequently migrate to the uterus
of pregnant host and infect the foetus or migrate to the mammary gland of
lactating bitch and thus infect the nursing puppies (Soulsby, 1986).
2.2.3 Clinical signs and pathogenicity of Ancylostoma caninum
According to Pinney (2000) and Stoll (2002) factors affecting the pathogenicity
of A. caninum include, mode of infection, age and nutritional status of the dog,
the amount of worms present in the gut, resistance developed to previous
infections and intercurrent disease. Animals most severely affected are puppies
which acquire substantial worm burden by lactogenic and prenatal routes
(Foster, 1932). Moreover, the iron reserve in the milk for the young puppies are
low (Miller, 1974) and thus they can not cope with blood loss resulting from the
feeding habit of the adult worm. Ancylostoma caninum is a voracious blood-
sucking parasite and the principal consequence is anaemia (Miller, 1971;
Urquhart et al., 2002) which coincides with the development of the buccal
capsule of the fourth larval stage in the intestine of the host (Soulsby, 1986).
Each worm removes about 0.1 ml of blood daily. Their head organs inject into
the host substances which prevent coagulation of the blood, so that in heavy
infestations of several hundred worms, there is free bleeding from the dog’s
bowel and puppies quickly become profoundly anaemic. Initially, the anaemia
33
is normocytic and nomochromic but as the animal becomes iron deficient,
microcytic hypochromic anaemia supervenes (Debuf, 1994).
Generally, hookworm infection is asymptomatic, but very heavy loads of the
parasite coupled with poor nutrition (inadequate intake of protein and iron) will
eventually lead to severe anaemia (Soulsby, 1986). According to Bailey et al.
(1968), Stone and Giraclecleau (1968) and Soulsby (1986), the prenatal
infection is characterized by a sudden onset of severe anaemia, coma and death
within 3 weeks of birth, while the oral and less commonly the per-cutaneous
infections show mainly anaemia (paleness of mucous membrane), anorexia,
bloody or tarry colored diarrhea due to enteritis, stunted growth, dehydration
and weakness. Other signs were vomiting, emaciation, ocular discharge
reflecting a secondary bacterial infection of the eyes, some abdominal tissue
oedema and ascites due to hypo-albuminaemia, starring hair coat (Okewole and
Oduye, 2000).
If the infection is not overwhelming in a well nourished adult dog which has
acquired some degree of resistance, the clinical sign may be absent or nil (Otto,
1941; Miller, 1964, Steve et al., 1973).
2.2.4 Immunity in helminth infections
Individuals vary markedly in their capacity to resist, control and/or reject
infection and in their susceptibility to disease in general (Doenhoff, 2000). The
mammalian organism has several immune mechanisms to protect itself from a
variety of pathogenic organism and these include the humoral and cellular
34
components which can act independently or in various combinations (Gasbarre,
1997; Hedeler et al., 2005). In particular the invasion of mammalian organisms
by helminth parasites, poses a particular problem to the host as, because of their
size, they are not able to be dealt within the isolated cellular component of the
phagocyte or infected cell (Else, 1999). As such extremely potent and toxic
mechanisms are required to affect these and resilient organisms (worms) (Bell et
al., 1992). Protection in gastrointestinal (GI) nematode infections is associated
with immune effectors responses in the gut mucosa. Generally, these responses
create an environment hostile to the parasites which results in their reduced
fecundity, as well as their damage and expulsion. On the other hand, helminth
parasites have themselves evolved several mechanisms to escape the hosts
immune response which include rapid shedding of surface molecule after
antibody binding, secretion of toxic and immunomodulatory molecules and the
active movement to different tissue sites and organs. Guinee et al. (2003) using
Strongyloides ratti and Nippostrongylus brasiliensis as models showed that host
immune status affects the maturation time and the fecundity of the nematode
species. Bhopale and Johri (1978) also noted that after repeated sublethal dose
of Ancylostoma caninum in mice, a subsequent lethal dose resulted in tolerance
state of reduced migratory activity to tissue and organs and allergic expulsion
and/or destruction of the larvae. Immunity to Ancylostoma caninum in dogs
particularly was studied by Herrick (1928), Sarles (1929), McCoy (1931),
Foster (1935), Otto and Kerr (1939) who demonstrated reduction in morbidity
35
and mortality, worm establishment/expulsion and output of eggs from the
resistant animals and these could be seen as indices for protective immunity in
GI nematode infections. However, different parasite species may not be
susceptible to the same immune responses (Charon, 2004). Fakae et al. (2004)
noted that the ability of animals to regulate their parasitic infections is
genetically determined and therefore varies between individuals and breeds
within a given host population. The traits of concentration of eggs in faeces,
packed cell volume, extent of eosinophillia in the peripheral blood,
concentration of antiparasite antibodies and growth rate of the animal can be
used to identify animals with increased resistance (immuned) to infection (Stear
and Wakelin, 1998). Else (1999) noted that immediate hypersensitivity Type 1
or IgE-mediated hypersensitivity is the fastest reacting immune effectors
response and is initiated in its early stage by the action of the mast cells and this
is responsible for the damage and expulsion of the worms from the host. In
general, GI nematode infections in mammals elicit very strong Th2 – like
responses characterized by high levels of interlukin 4 (IL4), high levels of IgG1
and IgE antibodies, and large numbers of mast cells (Gasbarre et al., 2001)
which ensures protective immunity to the host.
36
2.2.5 Haematology
Gastrointestinal parasitism is a recognized cause of chronic external blood loss
in animals especially hookworms (Loukas et al., 2005). The quantity of blood
loss caused by hookworm is proportional to the worm burden and may reach
100 ml/day. Blood evaluation has been reported to be a more reliable index of
the worm burden than the faecal egg counts especially in chronic helminthosis
(Saror et al., 1979). Anosa (1977) studied the blood picture in Haemonchus
contortus infection in lambs, and recorded normocytic – normochromic
anaemia, characterized by reticulocyte response in about 2 – 16%. Similarly,
Haroun et al. (1996) and Kyriazakis et al. (1996) noted that the blood picture in
infection with gastrointestinal Trichostrongylus in lambs and Haemonchus
contortus infection in sheep showed normocytic normochromic anaemia.
Albers et al. (1990) in their work on the effect of Haemonchus cotortus in
young sheep, recorded in addition to the above haematological findings,
reduction in serum iron concentration and erythrocyte potassium concentration.
Debuf (1994) reported that the initial normocytic and normochromic anaemia
changed to microcytic hypochromic anaemia as the animal became iron
deficient. Dunbar et al. (1994) in their work on Ancylostoma infection in panter
kitten, recorded low haematological values such as RBC, Hb and PCV, mean
corpuscular volume (MCV), mean corpuscular haemoglobin (MCH) and
37
increased eosinophils (eosinophilia). The leucocytes were usually normal but
slight neutropaenia occurred. Eosinophilia predominantly, is the leucocytic
response in helminthiasis (Coles, 1986; Meyer et al., 1992). Eosinophilia as a
response in helminthiasis occurs when there is an allergic state developed by the
parasite or its secretory products or migrating larvae (Moncol and Batte 1967).
The antigen (protein, secretory products or migrating larvae) and antibody
reaction results in the release of histamine from the mast cells which attacks the
eosinophils from the marrow in the blood leading to eosinophilia in both the
blood and tissues (Schalm et al., 1975). Fakae et al. (1999) in their work (the
response of Nigerian West African Dwarf goats to experimental infections with
Haemonchus contortus and rising circulating eosinophils as a parameter) noted
that all the helminth infected groups of animals produced eosinophilia
exceeding 334 cell/ml.
38
CHAPTER THREE
3.0 MATERIALS AND METHODS
3.1.0 Experimental animals
Twenty five (25) young local breeds of dogs of mixed sexes weighing between
1.4 - 3.0 Kg purchased from Orie Orba market Nsukka, Enugu State were used
for the study. They were kept in well ventilated fly-proof house, fed once daily
and water was provided ad libitum. The dogs were allowed to acclimatize for
three weeks before commencement of the experiment. During the period of
acclimatization, the animals were deticked with Carbaryl and dewormed with
Pyrantel pamoate at the dosage of 8.5% as constituent of dusting powder and
14.4 mg/kg per os respectively. Blood from each dog was also examined for the
presence of trypanosomes and confirmed negative by wet blood film, Giemsa-
stained thin blood smears, the haematocrit buffy coat method (Murray et al.,
1977).
3.1.1 Experimental design
The dogs were randomly divided into 4 groups of 5 dogs each. Each group
received the following treatment:
Group 1: Uninfected control
Group 2: Infected with Ancylostoma caninum
Group 3: Infected with Trypanosoma brucei
39
Group 4: Concurrent infection with T. brucei and A. caninum
Upon infection, patency and infection course and severity were determined by
parasitaemia estimation and faecal egg counts. They were also closely observed
throughout the study period for clinical signs and mortality. Packed cell volume
(PCV), rectal temperature, body weight, total WBC, differential leucocytes
counts and parasitaemia were determined on the day of infection (day 0) and
subsequently on days 0, 5, 13, 19, 28, 33 and 37.
3.1.2 Trypanosome infection
Trypanosoma brucei stock isolated from a clinically infected dog was used in
this study. The species identification was by morphological characteristics on
Giemsa-stained thin film preparation (Soulsby, 1986). Dogs were inoculated
intraperitoneally (i.p.) each with 5 x 105 trypanosomes suspended in 1 ml of
normal saline. The number of parasites was determined using the rapid
matching method of Herbert and Lumsden (1976).
3.1.3 Ancylostoma caninum
Faeces were collected from dogs screened from the local market around
Nsukka. Positive samples were thus cultured in the Department of Veterinary
Parasitology and Entomology, University of Nigeria, Nsukka.
3.1.4 Faecal culture
Homogenous faeces from the Ancylostoma positive samples were first washed
with water and passed through a sieve after mashing with a spatula. The
suspension was centrifuged at 3000 rpm for 5 minutes using a bench centrifuge.
40
The supernatant was poured off and the sediment mixed uniformly and lightly
spread onto moist filter paper (Whatman England) on petri dishes. The petri
dishes were kept at room temperature (25-30oC) and moistened daily to ensure
optimum conditions. The cultures were harvested after one week by spreading
jets of water from a wash bottle. The larval suspensions were extracted by
using a 10 ml syringe. The suspensions of infective larvae were stored in the
refrigerator in test tubes pending use.
3.1.5 Ancylostoma caninum infection
The method of Miller (1964) was used to infect the experimental animals. The
concentration of larval suspension was estimated using an automatic pipette;
small doses of 20 µl larval suspensions were placed as drops on a microscope
slide and counted under x4 objective of a light microscope. Estimated infective
doses were contained in a volume of approximately 1000 µl. Infection was per
os using a 2 ml syringe without needle. Animals were starved prior to infection
so as to establish infection. A dose of 120 infective L3 suspended in 1 ml of
distilled water were delivered per os per dog.
3.1.6 Conjunct Trypanosoma brucei and Ancylostoma caninum infection
Dogs were initially infected with A. caninum and then T. brucei infection
superimposed 23 days later, by the time of patency of Ancylostoma eggs in the
stool.
41
3.2.0 Detection of parasitaemia
3.2.1 Wet mount
A drop of blood was made on a slide, covered with a cover slip and then viewed
under the microscope for the presence of moving trypanosomes within the blood
(Woo, 1970).
3.2.2 Buffy coat
The buffy-coat used was obtained after the packed cell volume reading. The
capillary tube was cut a few millimeters below the buffy-coat level. The buffy-
coat and some plasma were gently expressed on the slide, carefully mixed and
covered with a cover slip and viewed under a microscope using x40 objective
for trypanosomes.
3.2.3 Giemsa stained thin films
A small drop of blood as in the wet mount method was thoroughly mixed and
placed at the end of a clean slide on a horizontal surface and with another
smaller slide in a beveled position near the drop, the beveled slide is then used
to spread the blood to an even film. The film was allowed to dry in the air and
then stained with Giemsa stain ensuring that the film was completely covered
with the stain. The stain was allowed to stay for about 20 minutes and then
thoroughly washed with running tap water. The slide was allowed to dry in the
air and examined under x100 objective with immersion oil.
42
3.3 FAECAL EGG COUNT
Faeces were collected per rectum from infected dogs into labeled containers.
One gram of homogenized faeces were weighed out and washed thoroughly,
sieved through a sieve aperture size of 1.0 mm with saturated salt solution (S.G.
1.18) and the volume made up to 15 ml. Well mixed aliquot were delivered into
standard McMaster chamber. The total number of eggs counted were multiplied
by 50 and expressed as egg per gram (EPG) of faeces (MAFF, 1986).
3.4.0 HAEMATOLOGY
3.4.1 Blood collection
Two milliliters (2 ml) of whole blood was collected from the dogs into an
ethylene diamine tetra acetic acid (EDTA) bottle for the packed cell volume,
total white blood cell count and differential leucocyte counts.
3.4.2 Packed cell volume
The microhaematocrit centrifuge method was used for the determination of
PCV. Microhaematocrit (Capillary) tube was inserted in the blood and blood
was allowed to rise in it by capillary action up to three quarter (75%) the length
of the tube. The body of the capillary tubes was cleaned to eliminate
contamination of the microhaematocrit centrifuge. The end with which blood
was collected was sealed with a plasticine and placed in the microhaematocrit
centrifuge (Hawkley, England). They were centrifuged at 3000 rpm for 5
43
minutes, after which the PCV was read using the PCV reader (Coles, 1986). The
result was expressed in percentage (%).
3.5 POST MORTEM EXAMINATION
Necropsy was performed on some of the dead dogs at the necropsy room of the
Department of Veterinary Pathology and Microbiology, University of Nigeria,
Nsukka. The gross pathological lesions present were recorded.
3.6 STATISTICAL ANALYSIS
Analysis of variance (ANOVA) and Duncans multiple range test was used to
analyze data obtained which was recorded as means ± standard error (SE) of
mean. Values less of P<0.05 were statistically considered significant.
44
CHAPTER FOUR
4.0 RESULTS
4.1 COURSE OF INFECTIONS
4.1.1 Ancyclostoma caninum
The pre patent period was 24.5±0.4 days (23 – 25 days). There was no
increase in temperature (fever) throughout the period of the experiment
(Table 1). By day 24 post infection (PI), there was dullness, diarrhoea
with blood tinged faeces, inappetence, and progressive weight loss.
Within days 36 - 39, there were weakness, pale mucous membrane, rough
hair coat, dark tarry faeces (Plate 1.A) and sunken eyes. The control dogs
were healthy (Plate. 2) throughout the period of the experiment.
4.1.2 Trypanosome brucei
The PP of trypanosome infection was 5±0.0 days. There was increased
temperature by day 5 PI (Table 1). By day 6 PI, there were dullness, in
appetence, bilateral ocular discharge (Plate 3.B) and loss of weight. By
day 11 PI, there were swollen face, rough hair coat (Plate 3.C) and pale
mucous membrane. By day 15 PTI, there was anorexia, emaciation and
bilateral corneal opacity (Plate 4. D)
45
Table 1: Mean rectal temperature of dogs infected with T. brucei, A.
caninum and mixed infection of T. brucei and A. caninum.
Mean(oC) ± standard error
* Different superscripts in a row indicate statistically significant difference between the
means; p< 0.05
ND=not done
Experimental
period (days)
Group A –
Normal (control)
Group B
(A. caninum) Group C
(T. brucei) Group D
(A. caninum &
T. brucei)
0
38.06 ± 0.08a 38.04 ± 0.25
a 38.05 ± 0.06
a 38.10 ± 0.09
a
5
37.93 ± 0.10 a ND 38.50 ± 0.15
b 38.26 ± 0.07
b
13
38.08 ± 0.11 a 37.78 ± 0.16
a 38.15 ± 0.06
a 39.44 ± 0.16
b
19
38.05 ± 0.03 a 37.78 ± 0.27
a 39.18 ± 0.40
b 39.24 ± 0.21
b
28
38.25 ± 0.06 a 37.87 ± 0.07
b 39.40 ± 0.11
c 39.14 ± 0.14
c
33
38.60 ± 0.19 a 37.83 ± 0.18
b 38.80 ± 0.35
a 38.66 ± 0.20
a
37
38.10 ± 0.13a 37.67 ± 0.12
a 38.80 ± 0.21
a 39.36 ± 0.14
a
46
A
PLATE 1: DARK TARRY FAECES (A)
PLATE 2: CONTROL DOGS
47
B C
+
PLATE 3: BILATERAL OCULAR DISCHARGE (B) AND ROUGH HAIR COAT (C)
D
E
PLATE 4: INAPPETENCE (E) AND BILATERAL CORNEAL OPACITY (D)
48
4.13 Mixed infection (T. brucei and A. caninum).
The prepatent period of infection for A. caninum was 24.5±0.04 days (23 -25
days) and by day 24 PI (i.e. day 1 post trypanosome infection) some of the dogs
had bloody diarrhoea, dullness and inappetence. The PP of T. brucei was
4.6±0.22 days (4 – 5 days). There was anorexia, bilateral ocular discharge (Plate
3.C) weakness, pale mucous membrane, increased temperature (Table 1) and
emaciation within 3 – 5 days post trypanosome infection (PTI) (Fig.1). By day
11 PTI, there were swollen face, sunken eyes, and rough hair coat and dark tarry
mucoid faeces. There were corneal opacity, anorexia (Plate 5 G) and
recumbence (Plate 5.F) by day 15 PTI.
0
1
2
3
4
0 10 20 30
bo
dy
we
igh
t o
f d
og
s (k
g)
days of post infection
Group A (Control) Group B (A. caninum)
Group C (T. brucei) Group D (A.caninum + T brucei)
Figure 1. mean body weight of dogs infected with T. brucei, A. caninum and
mixed infection of T. brucei and A. caninum
49
G F
PLATE 5: RECUMBENCY (F) AND ANOREXIA (G)
50
4.2 Faecal Egg Output (EPG)
The results of the EPG are represented in table 2. There was no significant
difference (P>0.05) in the faecal egg output between the A. caninum infected
dogs (group B) and the conjunct T. brucei and A. caninum infection (group D).
4.3 HAEMATOLOGY
4.3.1 Packed Cell Volume
The results of the PCV are shown in table 3. By day 5 PI, there was no
significant difference between the infected groups and the uninfected
control (group A) although group D differed significantly from group C.
By day 19 PI, there was a significant (P<0.05) decrease in the PCV of
groups B and D compared with the control. By day 37 PI, groups B, C
and D differed significantly from group A (P< 0.05).
4.3.2 Total white blood cell count
The results are presented in table 4. There was a significant (p< 0.05)
increase in the total leucocyte counts of A. caninum infected dogs (group
B) on day 29 PI, whereas significant decreases (P<0.05) were recorded in
trypanosome infected dogs (group C) and mixed infection (group D) on
days 38 and 34 PI, respectively.
51
Table 2: Mean egg per gram ± Se (EPG) of dogs infected with T. brucei or
A. caninum alone and in conjunct T. brucie/A. caninum
Experimental
Periods
(DAYS)
Group
B
A. caninum
D
T. brucie & A.
caninum
28
20300 ± 12195a
34325 ± 8044a
34
81233 ± 26410a
54425 ± 24764a
39
67683 ± 13971a
55800 ± 12304a
52
Table 3: Packed Cell Volume. Mean (%)± standard error for dogs
infected with T. brucei, A. caninum and mixed infection of T. brucei and
A. caninum.
* Different superscripts in a row indicate statistically significant difference between the
means; p< 0.05
Mean (%) ± standard error Experimental
period (days)
Group A –
Normal
(control)
Group B
(A. caninum) Group C
(T. brucei) Group D
(A. caninum &
T. brucei)
0 33.25 ±
1.25a
31.00 ±
1.08a
33.25 ±
0.75a
31.60 ± 3.16a
5 33.50 ± 1.04
ab
ND 31.75 ±
0.63a
34.40 ± 0.40 b
13 33.25 ±
0.48a
30.25 ±
0.63a
32.25 ±
0.48a
31.4 ± 1.32a
19 33.25 ± 0.48
a
29.25 ±
0.85b
30.75 ± 0.48
ab
28.43 ± 1.86 b
28 29.50 ±
1.44a
28.00 ±
0.58a
29.00 ±1.47a 26.40 ± 1.21
a
33 28.25 ±
2.46a
23.00 ±
1.53a
27.00 ±
1.15a
20.80 ± 0.73a
37 29.25 ± 2.50
a
20.67 ±
0.88b
22.67 ± 1.86
b
17.60 ± 1.03 b
53
Table 4: Total white blood cell counts. Mean (103/ µl) ± standard error for
dogs infected with T. brucei, A. caninum and mixed infection of T. brucei
and A. caninum.
Mean(103/µl) ± standard error
* Different superscripts in a row indicate statistically significant difference between the means; p<
0.05
Experimental
period (days)
Group A –
Normal
(control)
Group B
(A. caninum) Group C
(T. brucei) Group D
(A. caninum &
T. brucei)
0
9.76 ± 2.93a 9.41 ± 0.73
a 9.69 ±1.70
a 8.95 ± 0.98
a
14
13.99 ± 3.99a 15.83 ± 1.26
a 15.23 ± 4.08
a 12.60 ± 2.56
a
20
8.25 ± 1.32a 13.03 ± 6.52
a 7.30 ± 1.86
a 10.08 ± 0.98
a
29
7.48 ± 3.08 a 20.88 ± 5.84
b 7.91 ± 1.78
a 8.89 ± 1.78
a
34
13.22 ± 3.76 a ND 7.55 ± 2.28
ab
6.05 ± 0.88 b
38
10.93 ± 2.23 a 19.63 ± 1.97
b 4.42 ± 1.86
c 10.97 ± 1.86
a
54
4.3.3 Absolute neutrophils counts
The results are presented in table 5. The absolute neutrophil counts
significantly increased in the T. brucei infected dogs (group C) by day 20
PI, compared with the control (group A). By day 29 PI, there was a
significant increase in group B, and a decrease by day 34 PI in groups C
and D. By day 38 PI, group C significantly decreased compared with
both the control group and D.
4.3.4 Absolute lymphocyte counts
The results are presented in table 6. There were no variations in the
absolute lymphocyte counts except day 20 PI when an increase was
detected in the mixed infection (group D).
4.3.5 Absolute eosinophil counts
The results are shown in table 7. The absolute eosinophil counts
significantly increased by day 14 PI, in contrast to a significant decrease
in the mixed infection (group D) by day 34 PI.
4.3.6 Absolute monocyte counts
There were no significant difference (P>0.05) in absolute monocyte count
across the various groups A, B, C and D throughout the duration of the
experiment (Table 8).
55
Table 5: Absolute neutrophil count. Mean (103/ µl) ± standard error for
dogs infected with T. brucei, A. caninum and mixed infection of T. brucei
and A. caninum.
Mean(103/µl) ± standard error
* Different superscripts in a row indicate statistically significant difference between the means; p<
0.05
Experimental
period (days)
Group A –
Normal
(control)
Group B
(A. caninum) Group C
(T. brucei) Group D
(A. caninum &
T. brucei)
0
5.67 ± 1.81a 5.07 ± 1.08
a 5.19 ±1.50
a 4.68 ± 0.65
a
14
8.35 ± 2.04a 8.96 ± 0.21
a 6.57 ± 4.31
a 6.28 ±1.17
a
20
5.13 ± 1.21 a 8.30 ± 2.65
a 0.74 ± 0.01
b 4.32 ± 0.40
a
29
4.92 ± 2.75 a 13.13 ± 1.61
b 3.81 ± 1.27
a 6.08 ± 1.28
a
34
8.90 ± 2.52 a ND 1.93 ± 0.97
b 2.57 ± 0.46
b
38
7.16 ± 1.63 a ND 2.24 ± 0.71
b 6.51 ± 1.84
a
56
Table 6: Absolute lymphocyte counts. Mean (103/ µl) ± standard error for
dogs infected with T. brucei, A. caninum and mixed infection of T. brucei
and A. caninum.
Mean(103/µl) ± standard error
* Different superscripts in a row indicate statistically significant difference between the
means; p< 0.05
Experimental
period (days)
Group A –
Normal (control)
Group B
(A. caninum) Group C
(T. brucei) Group D
(A. caninum &
T. brucei)
0
5.01 ± 1.87a 4.42 ± 1.10
a 3.98 ± 0.85
a 4.23 ± 0.74
a
14
4.58 ± 2.51a 4.23 ± 0.68
a 9.72 ± 3.08
a 4.28 ± 1.58
a
20
2.69 ± 0.42 a 2.71 ± 1.26
ab 3.90 ± 0.60
ab 4.94 ± 0.63
b
29
2.15 ± 0.36a 2.90 ± 0.04
a 3.75 ±1.49
a 2.73 ± 0.48
a
34
3.56 ±1.05a ND 3.52 ± 1.10
a 3.03 ± 0.50
a
38
2.78 ± 0.94a ND 1.91 ± 0.18
a 3.72 ± 0.16
a
57
Table 7: Absolute eosinophil counts. Mean (103/ µl) ± standard error of
dogs inffected with T. brucei, A. caninum and mixed infection of T. brucei
and A. caninum.
Mean (103/µl) ± standard error
* Different superscripts in a row indicate statistically significant difference between the
means; p< 0.05
Experimental
period (days)
Group A –
Normal
(control)
Group B
(A. caninum) Group C
(T. brucei) Group D
(A. caninum &
T. brucei)
0
0.21 ± 0.04a 0.24 ± 0.07
a 0.26 ± 0.08
a 0.22 ± 0.02
a
14
0.24 ± 0.04a 0.52 ± 0.11
b 0.28 ± 0.04
ab
0.34 ± 0.08 ab
20
0.42 ± 0.21a 0.37 ± 0.25
a 0.21 ± 0.19
a 0.35 ± 0.10
a
29
0.21± 0.09 a 1.33 ± 0.29
b 0.09 ± 0.03
a 0.26 ± 0.07
a
34
0.35 ± 0.12 a ND 0.04 ±0.01
a 0.10 ± 0.04
b
38
0.47 ± 0.14a ND - 0.36 ± 0.28
a
58
Table 8: Absolute monocyte counts. Mean (103/µl) ± standard error of dogs
inffected with T. brucei, A. caninum and mixed infection of T. brucei and A.
caninum.
Mean(103/µl) ± standard error
* Different superscripts in a row indicate statistically significant difference between the
means; p< 0.05
Experimental
period (days)
Group A –
Normal
(control)
Group B
(A. caninum) Group C
(T. brucei) Group D
(A. caninum &
T. brucei)
0
0.25 ± 0.06a 0.40 ± 0.11
a 0.35 ± 0.12
a 0.49 ± 0.12
a
14
0.47 ± 0.18a 0.48 ± 0.17
a 0.34 ± 0.14
a 0.30 ± 0.12
a
20
0.46 ±0.22a 0.65 ± 0.37
a 0.32 ± 0.06
a 0.46 ± 0.11
a
29
0.20 ± 0.02a 0.30 ±0.09
a 0.30 ± 0.05
a 0.30 ± 0.04
a
34
0.40 ± 0.16a ND 0.45 ± 0.19
a 0.36 ± 0.10
a
38
0.61 ± 0.18a ND 0.27 ± 0.11
a 0.60 ± 0.12
a
59
4.4 Post mortem findings
Trypanosome infection was associated with paleness of carcass, swollen
spleen (Plate 6.I) and icteric liver (plate 7.N).
Hookworm infection caused haemorrhagic enteritis (Plate 8.M),
intussusception (plate 9.J) and fibrosis of the lungs (Plate 10.L) in the
infected dogs.
60
H I
PLATE 6: NORMAL SPLEEN (H) AND SPLENOMEGALY (I)
O
N
PLATE 7: NORMAL LIVER (O) AND ICTERIC LIVER (N)
61
M
PLATE 8: HAEMORRHAGIC ENTERITIS (M)
J
PLATE 9: INTUSSUSCEPTION (J)
62
K L
PLATE 10: NORMAL LUNG (K) AND FIBROTIC LUNG (L)
63
CHAPTER FIVE
5.0 DISCUSSIONS
Results of this study showed that the prepatent (PP) period of experimental
infection of young dogs with A. caninum was 23-25 days (mean ± se; 24.5 ± 0.4
days). Okewole and Oduye (2000) reported a shorter mean PP of 14 ± 1.0 days
in experimental orally infected eight week old puppies. The PP period is known
to be influenced by the route of infection, sex, age and the degree of acquired
resistance of the hosts (Herrick, 1928; Miller, 1965a). The shorter PP recorded
by Okewole and Oduye (2000) may be explained by the fact that puppies were
used in their study as against young dogs used in this present study that may
have acquired some resistance to infection through previous exposure to
infection.
The PP of T. brucei infection was 5 days (5 ± 0.0 days) for the single infection
and 4-5 days (4.6 ± 0.22 days) for the mixed T. brucei and A. caninum infection.
The PP is within the range reported by other workers for T. brucei (Kaggwa et
al., 1984; Anene et al., 1989b; Akpa et al., 2008; Ezeokonkwo, 2009). The
stress of the concurrent ancylostomosis may explain the shortened PP in the
combined infection.
The clinical signs of infection observed in this study were characteristic of
ancylostomosis (Urquhart et al., 2002; Okewole and Oduye, 2000; Lefkaditis et
al., 2006) and trypanosomosis (Anosa, 1977; Ikede et al., 1977; Kaggwa et al.,
1984; Anene et al., 1989b; Akpa et al., 2008; Ezeokonkwo, 2009). These signs
64
included dullness, inappetence, anorexia, weakness, pale mucous membrane due
to anaemia and rough hair coat. Additional specific signs of bloody diarrhoea
and sunken eyes were present in A. caninum infected dogs, while fever, swollen
face, bilateral ocular discharges and corneal opacity accompanied T. brucei
infection. A combination of these signs in a more severe form characterized the
mixed infection of both parasites. This therefore suggested that T. brucei and A.
caninum interacted in a manner that accentuated their pathogenic effects.
Similar observations were made by Griffin et al. (1981a, b) in goats with
conjunct infection of T. congolense and Haemonchus contortus, and the
immunosuppressive effects of the trypanosome was incriminated as the cause of
this enhanced susceptibility. However, the observed lack of pronounced body
weight decrease in the mixed T. brucei and A. caninum infection may be
attributed to tissue oedema and ascites due to hypo-albuminaemia commonly
seen in ancylostomosis (Soulsby, 1986; Okewole and Oduye, 2000).
It is remarkable that the faecal egg output (EPG) was not influenced by the
concurrent infection of ancylostomosis and trypanosomosis contrary to previous
reports (Griffin et al., 1981a; Kaufmann et al., 1992; Dwinger et al., 1994;
Goosens et al., 1997) who reported significant increases in animals with
combined trypanosome and helminth infections. A further contrary report by
Onah et al. (2004) showed a significant decrease in rats with concurrent
infection of T. brucei and Strongyloides ratti compared with the single
infection. They concluded that the two parasites interacted in a manner that
65
ameliorated their pathogenic effects resulting in a decrease in the level of EPG.
Furthermore, it has been reported that the faecal egg output is inversely
proportional to the number of worms established (Krupp, 1961). In this study, it
is thus conceivable that the enhanced ability of worms to establish and mature in
the intestine, facilitated by the immunosuppressive effects of intercurrent
trypanosomosis may have moderated the egg output of the worms.
The haemogram of each of the parasite infection revealed low PCV indicative
of anaemia which is a consistent finding in ancylostomosis (Soulsby, 1986;
Okewole and Oduye, 2000; Sushma and Suryanaratana, 2001; Britto et al.,
2002) and trypanosomosis (Anosa et al., 1974; Saror, 1979; Anosa, 1988;
Murray and Dexter, 1988; Anene et al., 1989a). A severe form of the anaemia
characterized the combined trypanosome and hookworm infection and is
attributed to the combined effect of the parasites on the blood vascular system
of the dogs. Ancylostoma caninum is an avid blood sucker in the intestine and a
cause of chronic external blood loss in infected dogs (Soulsby, 1986; Urquhart
et al., 2002; Levy, 2008) while trypanosomes caused accelerated destruction of
red blood cells in infected animals (Jennings et al., 1974; Valli et al., 1979;
Anosa and Kaneko, 1983; 1989; Anosa et al., 1992; 1997 a, b).
There was leucocytosis in the A. caninum infected dogs induced by eosinophilia
and neuthrophilia. A contrary report stated that leucocytes are usually normal
but slight neutropaenia may occur. However, the leucocytosis recorded in this
study agrees with the findings of Sushma and Suryanarayana (2001) in
66
ancylostomosis in dogs. Eosinophilia predominantly is the leucocytic response
in helminthiasis (Coles, 1986; Meyer et al., 1992) while neuthrophilia is
associated with acute infections (Schlams et al., 1975). Dunbar et al. (1994)
reported increase eosinophils (eosinophilia) in Ancylostoma caninum infected
panter kittens. Britto et al. (2002) in their study of the bone marrow of adult
mongrel dogs naturally infected with A. caninum observed eosinophilic
granulocytic hyperplasia. On the other hand, there was leucopaenia in the dogs
infected with trypanosomes alone, and in combination with hookworm. This
corroborated the report of Omamegbe and Uche (1985) in naturally infected
dogs but contrasts with those of Onyeyili and Anika (1989) and Anene et al.
(1989 b) who reported leucocytosis in experimentally T. brucei infected dogs.
Leucopaenia was also reported by Wellede et al. (1974) in cattle and by Anosa
(1988) in human and animal trypanosomosis. Anosa (1988) associated
leucopaenia with the early phase of the disease, observing that it regressed in
protacted infections. The results further showed that the leucopaenia in the dogs
infected with trypanosomes alone was induced essentially by neutropaenia,
while it was a combination of neutropaenia and eosinopaenia in the dogs with
mixed infection. Neutropaenia has also been reported in dogs (Onyeyili and
Anika 1989; Anene et al., 1989b) and in cattle (Moulton and Sollod, 1976;
Anosa, 1980, 1983; Anosa and Isoun, 1980) with experimental T. b. brucei
infections. According to Anosa (1983) neutropaenia in T. b. brucei infection
may be due to depression of bone marrow granulocyte precursors by
67
trypanosome toxins and massive elimination of neutrophils when they engulf
trypanosomes. Trypanosome infection per se in this study produced no changes
in the eosinophil and monocyte counts, except in combined infection where
there was eosinopaenia. Anosa (1983) seem to believe that eosinophil and
monocyte counts rarely change in trypanosomosis. However, some workers
have reported eosinophilia and monocytosis in pigs (Jibike and Anika, 2003)
and monocytosis and eosinopaenia in dogs (Anene et al., 1989 b)
experimentally infected with T. brucei. Monocytosis is thought to coexist with
marked proliferation of macrophages in tissues of trypanosome infected animals
(Isoun, 1975; Anosa, 1980; Anosa and Isoun, 1980). The lymphocyte count
was not affected by the T. brucei infection just as in the A. caninum infection.
This result differed from those of other workers who reported lymphopaenia
(Anosa, 1983; Kaggwa, 1984; Ezeokonkwo, 2009) or lymphocytosis
(Omamegbe and Uche, 1985; Anene et al., 1989 b; Onyeyili and Anika, 1989)
in T. brucei infected dogs.
6.0 CONCLUSIONS AND RECOMMENDATIONS
The manifestations of trypanosomosis and ancylostomosis in this experimental
infection were characteristic. The concurrent infection resulted in a more sever
disease manifested in remarkable clinico-hematological alterations which may
be useful in providing supportive evidence, as well as serve some prognostic
purposes.
68
It is thus recommended that medical management of either of the disease
conditions should eliminate concurrent infections (through appropriate tests) as
this may complicate the disease process and adversely affect the outcome of any
medical intervention.
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87
APPENDIX
Appendix 1: Body weight. Mean (Kg) ± standard error of dogs infected
with T. brucei, A. caninum and mixed infection of T. brucei and A. caninum.
Mean (Kg) ± standard error
* Different superscripts in a row indicate significant difference between the means; p< 0.05
Experimental
period (days)
Group A –
Normal
(control)
Group B
(A. caninum)
Group C
(T. brucei)
Group D (A. caninum &
T. brucei)
0
2.65 ± 0.10 a 2.80 ± 0.11
a 2.18 ± 0.10
b 2.26 ± 0.11
b
5
2.83 ± 0.48 a ND 2.13 ± 0.08
b 2.48 ± 0.07
c
13
2.93 ±0.03 a 2.88 ± 0.10
a 2.30 ± 0.11
b 2.52 ± 0.04
b
19
2.93 ± 0.03 a 2.93 ± 0.06
a 1.58 ± 0.34
b 1.86 ± 0.12
28
2.95 ± 0.03 a 2.10 ± 0.06
bd 1.45 ± 0.26
c 1.82 ± 0.08
cd
33
2.95 ±0.05 a 2.00 ± 0.12
bc 1.50 ± 0.29
cd 1.46 ± 0.12
d
37
2.95 ± 0.05 a 1.77 ± 0.15
b 1.50 ± 0.29
b 1.50 ± 0.08
b