preliminary report on the clinical and hematological

7/31/2019 Preliminary Report on the Clinical and Hematological

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AUTHORS: OGUNWOLE, R. T.; MUHAMMAD, A. A.; KUGU, B.A.; SHUAIBU, Y.;

MOHAMMED, B. and AJAKAIYE, J. J.


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HAT and AAT are constraints to both human and animal health in sub-

Saharan Africa (Anderson 2011). Beside death, it causes a heavyeconomic loss mainly in Africa.

AAT is caused by tsetse fly transmitted Trypanosoma congolense,

Trypanosoma vivax orTrypanosoma brucei brucei or simultaneous

infection with one or more of these trypanosomes (Finelle, 1971).

HAT is caused by tsetse fly transmitted sub-species ofTrypanosoma

brucei

E.g: Trypanosoma brucei gambiense which is predominant in West Africawhile

Trypanosoma brucei rhodesiense is predominant in East Africa (Stich

et al., 2002).


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Both HAT and AAT are re-emerging public health problem of

epidemic proportions in many parts of Africa.

The problem is that extensive literature exists on both forms of the

disease but they are mostly confined to basic sciences, but neglect

clinical research and the impact of the disease on large parts of the

human and livestock population in rural Africa.

In addition, there is paucity of drugs for the treatment of this disease

especially that of HAT as no new drugs has been produced in the last

40 years (Steverding, 2010).

Furthermore, there is increased drug resistance in the re-emerging form

of this disease. This calls for new appraisal of the disease with modern

diagnostics in order to forge new horizon towards the manufacturing

of preventive biologicals and /or curative drugs.


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General Objective

To investigate and evaluate the clinical and hemato-biochemicalchanges in experimentally infected Wistar rats with trypanosomes, in

order to enhance proper clinical and chemotherapeutic management of

the disease in man and animals.

Specific objectives

To determine infectivity of both trypanosomes via appropriate evaluation of

parasitaemia and clinical manifestations.

To evaluate the haematological and biochemical changes observed in the

experimental animals.

To evaluate anatomo-pathological changes observed in the experimental animals.

To determine the gene infection-interaction locus(i) through the application of

appropriate molecular technique.


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2. MATERIALS AND METHODS

2. 1. Experimental site

The experiment was conducted at Nigerian Institute for Trypanosomiasis

Research (NITR), Kaduna, Kaduna State and is located between latitude

10 30 00 N and longitude 7 25 50 E of Nigeria.

2. 2. Experimental animals

Forty male Wistar albino rats, weighing between 180-210 0.2 g wereobtained from the rat colony of NITR, Kaduna. They were all housed in

standard cages, in the laboratory of trypanosomiasis department where a

13/11 h light/dark photoperiod cycle was maintained. They were fed with

commercial pellets obtained from Vital Feeds Plc, Jos, Nigeria. Both feedand water were given ad libitum throughout the experimental period.

Animals were allowed to acclimatise for two weeks before they were used

for the experiment.


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2. 3. Experimental design

Thirty-six rats were randomly divided into three groups of twelve each

and kept in standard cages with a dimension of 50 31 23 cm 3 as

follows:Group A was assigned as the control and was given 0.2 ml normal saline asplacebo.;

Group B was given 0.2 ml Trypanosoma brucei brucei (Federe strain) .

Group C was given 0.2 ml Trypanosoma congolense .

The remaining four rats were divided into two and used as replicate donor of both

parasites for the infected groups

T. b brucei and T. Congolense used were obtained from stabilates

maintained in liquid nitrogen at the Department of Vector and

Parasitology, of NITR, Kaduna, Nigeria. The concentration of both

inoculums in groups B and C was 1.85 107

/rat. The route of infection/rat. The route of infectionfor the ex erimental animal was intra eritoneal.for the experimental animal was intraperitoneal.


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2. 4. Measurement of indicators

2.4.1. Clinical Examination

Rectal temperature (RT): RT was measured daily by gently inserting astandard digital clinical thermometer (Philip Harris, UK) diagonally into

the rats colorectum at 2 cm depth; reading was recorded at 2 min. of

insertion and immediately after withdrawal.

Respiratory Frequency (RF): RF measurement was conducted using an

electronic chronometer (Puma, USA). Briefly, the chronometer was set at

15 sec. reading/bird and at stop time, values were multiplied by four to

arrive at respiration per minute (rpm).

Heart rate (HR) :Heart rate (HR) : Similarly,Similarly, HRHR was conducted with the same

instrument as the last one, while using cardiac palpation method, and

reading was recorded at the end of 15 sec. Through the multiplication of

value obtained by four to arrive at beats per minute (bpm).


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Weight measurement:Weight measurement: All animals in each group were weighed andAll animals in each group were weighed and

weight recorded once daily with a standard electronic weighing balanceweight recorded once daily with a standard electronic weighing balance

(Salter, Pocket Balance, England) with a maximum calibration of 1 kg(Salter, Pocket Balance, England) with a maximum calibration of 1 kg

and a precision of 0.1 g.and a precision of 0.1 g.

External clinical manifestation:External clinical manifestation: Clinical signs and symptoms wereClinical signs and symptoms were

physically observed and photo clips recorded for group B only.physically observed and photo clips recorded for group B only.

2.4.2. Determination of parasitaemia2.4.2. Determination of parasitaemia

Parasitaemia was monitored in blood obtained from the tail of the ratsParasitaemia was monitored in blood obtained from the tail of the rats

post-infection, pre sterilised with methylated spirit. 5 l of blood as wetpost-infection, pre sterilised with methylated spirit. 5 l of blood as wetmount was then examined under the microscope. The number of parasitemount was then examined under the microscope. The number of parasite

was determined microscopically at 40 magnification using the Rapidwas determined microscopically at 40 magnification using the Rapid

matching method of Herbert and Lumsden (1976).matching method of Herbert and Lumsden (1976).


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2.4. 3. Haemato-biochemical examination2.4. 3. Haemato-biochemical examination

Collection of blood samples:Collection of blood samples: Two drops of blood samples were collectedTwo drops of blood samples were collected

from the tail of each rat for immediate haematological analysis, while 4 mlfrom the tail of each rat for immediate haematological analysis, while 4 ml

were collected for biochemical analyses through humane perfusion fromwere collected for biochemical analyses through humane perfusion from

two rats per group every 3 days into previously sterilised glass tubestwo rats per group every 3 days into previously sterilised glass tubes

without anti-coagulant. Each tube was gently mixed by repeated inversionwithout anti-coagulant. Each tube was gently mixed by repeated inversionfor 2 min. Samples were immediately submitted for analysis. In addition,for 2 min. Samples were immediately submitted for analysis. In addition,

1 ml of blood was collected from one rat in each of from each group into1 ml of blood was collected from one rat in each of from each group into

previously sterilised glass tubes with EDTA as anti-coagulant andpreviously sterilised glass tubes with EDTA as anti-coagulant and

immediately sent to Centre for Biotechnology and Research Training,immediately sent to Centre for Biotechnology and Research Training,ABU, Zaria, for PCR analysis.ABU, Zaria, for PCR analysis.


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Haematological Analysis:Haematological Analysis: Packed cell volume (PCV) was determinedPacked cell volume (PCV) was determined

by microhaematocrit method (Schalmby microhaematocrit method (Schalm et al.,et al., 1975). Total red blood cell1975). Total red blood cellcounts (TRBC) was estimated using haemocytometer (Schalmcounts (TRBC) was estimated using haemocytometer (Schalm et al.,et al.,

1975).1975).

Blood smears were made on duplicate glass slides. These smears wereBlood smears were made on duplicate glass slides. These smears were

stained with Leishman stain and leucocytes counts were performedstained with Leishman stain and leucocytes counts were performed

using a manual haemocytometer method. Blood was diluted into anusing a manual haemocytometer method. Blood was diluted into an

eosinophil Unopette system (Becton Dickson and Co., Franklin Lakes,eosinophil Unopette system (Becton Dickson and Co., Franklin Lakes,

New Jersey, USA) using phloxine dye for counting granulocytesNew Jersey, USA) using phloxine dye for counting granulocytes(Campbell, 1995). One hundred leucocytes, including neutrophils,(Campbell, 1995). One hundred leucocytes, including neutrophils,

eosinophils, basophils, monocytes and lymphocytes were counted oneosinophils, basophils, monocytes and lymphocytes were counted on

each slide.each slide.


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Biochemical Analysis:Biochemical Analysis: Macro and microelements were determinedMacro and microelements were determined

through atomic absorption spectroscopy (Milesthrough atomic absorption spectroscopy (Miles et al.,et al., 2001), with the SP-92001), with the SP-9equipment from the PYE UNICAM firm. All minerals were analysed withequipment from the PYE UNICAM firm. All minerals were analysed with

reagents from Helfa diagnosticos, according to the manufacturersreagents from Helfa diagnosticos, according to the manufacturers

specifications.specifications.

Metabolites concentrations of : Creatine phosphokinase (CPK); SerumMetabolites concentrations of : Creatine phosphokinase (CPK); Serum

glutamic-oxaloacetic Transferase (SGOTglutamic-oxaloacetic Transferase (SGOTastast); Serum glutamic pyruvic); Serum glutamic pyruvic

transferase (SGPTtransferase (SGPTaltalt); Cholesterol; Total protein (TP); Conjugated total); Cholesterol; Total protein (TP); Conjugated total

protein (CTP) were measured spectrophotometrically (Hollands andprotein (CTP) were measured spectrophotometrically (Hollands and

Logan , 1966), with a biochemical analyser SP-9 equipment (PYELogan , 1966), with a biochemical analyser SP-9 equipment (PYE

UNICAM, Germany)UNICAM, Germany)


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Statistical AnalysesStatistical Analyses

The PC STATISTICA 8.0 package shall be used, and all data shall beThe PC STATISTICA 8.0 package shall be used, and all data shall be

subjected to a one-way ANOVA multifactorial variable analysis throughsubjected to a one-way ANOVA multifactorial variable analysis through

the general-linear-models procedure of the statistical Analysis Systemthe general-linear-models procedure of the statistical Analysis System

(SAS Users Guide, 1985). Duncans(SAS Users Guide, 1985). Duncans post-hocpost-hoc test will be employed totest will be employed to

identify means that differs at P < 0.05 (Duncan, 1955).identify means that differs at P < 0.05 (Duncan, 1955).


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3. Results3. Results

All results fromAll results from clinicalclinical measurements;measurements; haematology and serumhaematology and serumbiochemistry/electrolytesbiochemistry/electrolytes have all been collated and are currentlyhave all been collated and are currently

undergoing statistical analyses.undergoing statistical analyses.

We are still awaiting results of molecular analysis from CBRT andWe are still awaiting results of molecular analysis from CBRT and

Histopathology fixation both from ABU, Zaria.Histopathology fixation both from ABU, Zaria.

Below are photo clips of external physico-clinical manifestations andBelow are photo clips of external physico-clinical manifestations and

anatomo-pathological features of the experimental animalanatomo-pathological features of the experimental animals.s.


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