research article silent human trypanosoma brucei gambiense...

Research ArticleSilent Human Trypanosoma brucei gambiense Infectionsaround the Old Gboko Sleeping Sickness Focus in Nigeria

Karshima Solomon Ngutor1 Lawal A Idris2 and Okubanjo Oluseyi Oluyinka2

1Department of Animal Health Federal College of Animal Health and Production Technology PMB 001 Vom Nigeria2Department of Veterinary Parasitology and Entomology Ahmadu Bello University PMB 1045 Zaria Nigeria

Correspondence should be addressed to Karshima Solomon Ngutor torkarshimayahoocouk

Received 22 September 2015 Revised 30 December 2015 Accepted 3 January 2016

Academic Editor Emmanuel Serrano Ferron

Copyright copy 2016 Karshima Solomon Ngutor et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

Trypanosoma brucei gambiense causes Gambian trypanosomosis a disease ravaging affected rural parts of Sub-Saharan AfricaWe screened 1200 human blood samples for T b gambiense using the card agglutination test for trypanosomosis characterizedtrypanosome isolateswithTrypanosoma gambiense serumglycoprotein-PCR (TgsGP-PCR) and analyzed our data usingChi squareand odds ratio at 95 confidence interval for statistical association Of the 1200 samples the CATT revealed an overall infection rateof 18 which ranged between 00 and 35 across study sites Age and sex based infection rates ranged between 12 and 23We isolated 7 (333) trypanosomes from the 21 seropositive samples using immunosuppressed mice which were identified as T bgambiense group 1 by TgsGP-PCR Based on study sites PCR revealed an overall infection rate of 06 which ranged between 00and 15 Females and males revealed PCR based infection rates of 03 and 08 respectively Infection rates in adults (13) andchildren (01) varied significantly (119901 lt 005) We observed silent T b gambiense infections among residents of this focus Risksof disease development into the second fatal stage in these patients who may also serve as reservoirs of infection in the focus exist

1 Introduction

Trypanosoma brucei gambiense causes the Gambian sleepingsickness a very chronic debilitating complex and fatal par-asitic zoonosis ravaging affected rural parts of Sub-SaharanAfricaThe disease transmitted by tsetse flies is widespread inthe Sub-Saharan African region posing serious public healthproblems in the region and to tourists visiting tropical Africa[1 2] It is a prototype of a neglected zoonotic pathogenin terms of drug development and sustainable control pro-grammes

In natural conditions transmission of the parasite is cycli-cal through bites of infected tsetse flies includingGlossina pal-palis G tachinoides and G fuscipes These vectors are espe-cially common at watering places like rivers or lakes wherepeople frequently visit to collect water and do their washingand animals visit to drink water [3] The parasite is dividedinto two subtypes type 1 causes a more chronic disease andrepresents about 90 of all cases of Gambian trypanoso-mosis while type 2 is said to be associated with an acute

like disease and represents the remaining 10 of the disease[4 5]

The pathogen is ranked 9th of the 25 human infectiousdiseases in Africa based on its socioeconomic impact [6]and is incriminated in over 15000 new cases yearly with themajority of these cases ending fatally [7] Seventy millionpeople are continuously exposed to the risk of infection in38 Sub-Saharan African countries where active transmissionis reported and only 5ndash7 of the population at risk is coveredby surveillance [7] Some of these infectionsmay be latent andmay remain unnoticed until they get to the second fatal stage[8]

In Nigeria however human infection with T b gambiensein the Gboko sleeping sickness focus was first reported in1974 and since then no successful attempt has been made tocontrol the disease in the affected region [9] Considering thefact that this infection is still not yet routinely diagnosed inNigerian hospitals even in endemic areas and the risk associ-atedwith parasites invasion of the central nervous system andproducing fatal disease we designed this study to conduct

Hindawi Publishing CorporationJournal of Parasitology ResearchVolume 2016 Article ID 2656121 5 pageshttpdxdoiorg10115520162656121

2 Journal of Parasitology Research

LinksFontem SS focusAlkaleri

GashakaGboko

0 150 300 600

(km)North

Nigeria

Benue

BauchiYankari GR

Taraba

Fontem SSFocus of the RC

Cameroon

Figure 1 Links between the study sites and possible sources of T b gambiense infections GR game reserve RC Republic of Cameroon SSsleeping sickness

an active screening of T b gambiense in humans in the oldGboko sleeping sickness focus in Nigeria and characterizedisolates using TgsGP-polymerase chain reaction

2 Materials and Methods

21 Study Area This study was carried out around theold Gboko sleeping sickness focus which is located in thenorthern part of Nigeria between longitudes 7∘471015840 and 10∘001015840east and latitudes 60∘251015840 and 8∘81015840 north It shares boundarieswith five other states namely Nasarawa (north) Taraba(east) Cross River (south) Enugu (southwest) and Kogi(west) and with the Republic of Cameroon to the southeast(Figure 1)Themajor occupation in this region is agricultureparticularly crop and livestock farming as well as fishing

22 Study Design We conducted a cross-sectional studyaround the old Gboko sleeping sickness focus in Nige-ria within six Local Government Areas (LGAs) namelyGashaka Gboko Ibi Karim Lamido Ukum and Vandeikyaconsidering their relationships with the Gboko and Fontemsleeping sickness foci and the Gashaka-Gumti and Yankarigame reserves Human subjects were sampled systematicallyfrom each household by selecting every 5th household fol-lowing the order in which houses were built and individualswere selected using balloting by names

23 Sample Collection

231 Blood Sampling of Human Subjects Two millilitres ofblood was aseptically collected from each human subject viathe median cubital vein using a 5mL syringe and 21G needle

and transferred immediately into clean labelled sample bot-tles containing ethylene diamine tetra-acetic acid (EDTA) at15mgmL of blood [10] and gently shaken until the bloodwas properly mixed with the anticoagulant and analyzedwithin 1-2 hours after collection [11] These samples weresubjected to the card agglutination test for T b gambiense asdescribed by Magnus et al [12] All CATT positive sampleswere then inoculated into mice immunosuppressed withcyclophosphamide to isolate trypanosomes

24 Laboratory Analysis of Samples

241TheCardAgglutinationTest for Trypanosomosis (CATT)The CATT test kits were obtained from the Institute forTropical Medicine Antwerp Belgium Blood samples wereanalyzed using the CATT as described by Magnus et al [12]This test is based on the detection of T b gambiense specificLiTat 13 antibodies using a purified T b gambiense variablesurface antigen

242 Mice Infection and Parasitaemia Estimation Mice wereobtained from the Small Animal Experimental Unit of theNational Veterinary Research Institute Vom Nigeria for thestudy The mice were appropriately labelled and screenedfor ectoparasites and endoparasites by standard techniques[13] and acclimatized for two weeks before inoculationThe mice were immunosuppressed using intraperitonealadministration of cyclophosphamide at 200mg per kg bodyweight About 05mL of each seropositive blood sample wasenriched with 05mL of phosphate saline glucose (PSG)buffer and 03mL of the PSG buffer enriched seropositiveblood from each seropositive samplewas then inoculated into

Journal of Parasitology Research 3

an intraperitoneally immunosuppressed mouse Tail-bloodsamples were collected from each mouse every 24 hours toestimate parasitaemia using the rapid matching technique asdescribed by Herbert and Lumsden [14]

243 Isolation and Purification of Trypanosomes The blood-stream form trypanosomes isolated from humans and prop-agated in mice were separated from the infected mice bloodusing a DEAE 52 column (Whatman Maidstone Kent UK)as described by Lanham and Godfrey [15] These parasiteswere then stored at 4∘C until needed for DNA extraction

244 Extraction of DNA from Purified Trypanosomes Try-panosomeDNAextractionwas done usingGeneJETgenomicDNA extraction kit (Thermo Scientific Germany) using themethod described byOury et al [16]The extracted DNAwasstored at minus20∘C until needed for PCR at the BiotechnologyLaboratory of the Ahmadu Bello University Zaria Nigeria

245 Detection of Trypanosoma gambiense Specific Glyco-protein (TgsGP) The TgsGP primer set with sequencesforward 51015840-GCTGCTGTGTTCGGAGAGC-31015840 and reverse51015840-GCCATCGTGCTTGCCGCTC-31015840 [17] was used to char-acterize the Trypanosoma isolates Cycling conditions forTgsGP-PCR were denaturation at 98∘C for 10 seconds toactivate the Phusion Flash II DNA Polymerase followed by40 cycles with denaturation at 98∘C for 1 second annealingat 63∘C for 30 seconds 30-second elongation at 72∘C anda final extension at 72∘C for 5 minutes as recommended bythe manufacturer All amplified products were analyzed byelectrophoresis in a 2 agarose gel and UV illumination afterethidium bromide staining

25 Data Analysis All data obtained during the study wereanalyzed using GraphPad Prism 40 Infection rates of T bgambiensewere calculated by dividing the number of infectedindividuals by the total number of individuals examined andexpressed as percentagesThiswas done for different variablessuch as study sites sex and age The Chi square (1205942) testand odds ratio were used where appropriate to compare theprevalence rates based on different variables and values of119901 lt 005 were considered significant

3 Results

A total of 1200 human blood samples from 6 sites aroundthe old Gboko sleeping sickness focus were analyzed usingthe CATT and PCR for the presence of Trypanosoma bruceigambiense as shown in Table 1

TheCATT revealed an overall infection rate of 18 of the1200 samples studied Based on the 6 sites studied GashakaGboko Ibi Karim Lamido Ukum and Vandeikya recordedinfection rates of 35 (7200) 25 (5200) 05 (1200)25 (5200) 00 (0200) and 15 (3200) respectively(Table 1) Sex based infection rates were 12 (7600) and23 (14600) for females and males respectively (Table 2)

Table 1 PCR and CATT based prevalence of Trypanosoma bruceigambiense in relation to study sites

Study sites Numberexamined

CATTpositive

TgsGP-PCRpositive

Gashaka 200 7 (35) 3 (15)Gboko 200 5 (25) 2 (10)Ibi 200 1 (05) 0 (00)Karim Lamido 200 5 (25) 1 (05)Ukum 200 0 (00) 0 (00)Vandeikya 200 3 (15) 1 (05)Total 1200 21 (18) 7 (06)1205942 mdash 1032 5892119901 value mdash 0066 03169

Table 2 PCR and CATT based infection rates of Trypanosomabrucei gambiense in relation to sex

Sex Numberexamined

CATTpositive

TgsGP-PCRpositive

Female 600 7 (12) 2 (03)Male 600 14 (23) 5 (08)Total 1200 21 (18) 7 (06)1205942 mdash 2375 1293119901 value mdash 01233 02554Odds ratio mdash 04941 03980

Table 3 PCR and CATT based infection rates of Trypanosomabrucei gambiense in relation to age

Age Numberexamined

CATTpositive

TgsGP-PCRpositive

Adult (ge18 years) 450 10 (22) 6 (13)Children (le17) 750 11 (15) 1 (01)Total 1200 21 (18) 7 (06)1205942 mdash 09338 6946119901 value mdash 03339 00082Odds ratio mdash 1527 1008

Infection rates recorded by adults and children were 22(7200) and 15 (7200) respectively (Table 3)

Trypanosomes were isolated from 7 (333) of the 21 ser-opositive samples using immunosuppressed mice Theseisolates were characterized using theTrypanosoma gambienseserum glycoprotein- (TgsGP-) PCR as group 1 of T bgambiense PCR revealed an overall infection rate of 06of the 1200 samples analyzed PCR based infection rates inrelation to study sites were 15 (3200) and 10 (2200)for Gashaka and Gboko respectively (Table 1) Both KarimLamido and Vandeikya recorded 05 (1200) while 0(0200) infection rates were recorded by both Ibi and Ukum(Table 1) Females and males revealed infection rates of 03(2600) and 08 (5600)while therewas significant variation(119901 lt 005) between the 13 (6450) and 01 (1750) infec-tion rates recorded by adults and males respectively


4 Discussion

In our study we inoculated human seropositive samples intomice to propagate T b gambiense considering the low para-sitaemia associated with this parasite Mice were inoculatedwith cyclophosphamide at 200mg per kg body weight tosuppress mice immunity due to the low parasite isolationrate reported for this parasite [18] Isolated trypanosomeswere also purified to ensure that DNA from mice blood doesnot contaminate trypanosome DNA which was required forpolymerase chain reaction

We observed silent T b gambiense infections in thisold sleeping sickness focus Previous report showed thatGboko was endemic for the pathogen [9] The findings ofour study 4 decades after the first report in the region stillindicate that the area may still be endemic for HAT andtsetse probably due to lack of or inadequate stakeholdersrsquoefforts towards the control and eradication of the diseaseThisregion shares border with the Fontem sleeping sickness focusof the Republic of Cameroon thus making transboundarytransmission a possibility The riverine nature of this regionmakes it suitable for tsetse habitat [3] and therefore is aprobable reason for the occurrence of this infection Otherfactors which include availability of several wildlife species inthe Gashaka-Gumti and neighbouring Yankari game reservesand the occupational hazards through farming and fishing[19] might have contributed to the occurrence of the diseaseFurthermore evidence of lack of sustained vector controlmeasures due to lack of funds [20] the high cost andunavailability of trypanocides used against T b gambienseand the increasing trend of treatment failure [21] might havealso contributed to the occurrence of this infection in theregion

The overall seroprevalence rate of 18 revealed by thisstudy is lower than the earlier report by Karshima et al [22]in a region of Taraba StateThe higher prevalence observed inGashaka may be attributable to the presence of the Gashaka-Gumti National Park which harbours several wildlife speciesthat can serve as reservoirs of T b gambiense The extensionof the Yankari game reserve forest into the Jevjev and Binariareas of Karim Lamido may explain also the high prevalenceobserved in the areaThe riverine nature of this region mighthave contributed to the high prevalence observed as riverineareas are known to promote tsetse breeding

The variations in the prevalence of T b gambiense inrelation to sex are in agreement with the earlier report byKarshima et al [22] who also reported higher prevalence inmales than in females in a region of Taraba State This maybe due to the greater involvement of males in occupationssuch as farming fishing and hunting which put them atmore risk than the females This finding however contradictsthe report of Mohammed et al [23] who reported higherprevalence in females in Southern Sudan probably due to dif-ferences in vegetation tsetse density and vectorial capacitiesof tsetse flies in the Sudan Although there was no significantdifference in the seroprevalence in adult and children theprevalence in children may not be unconnected with theirpractice of swimming around rivers and streams which mayexpose them to tsetse bites

One of themajor objectives of this studywas to isolate andcharacterize trypanosome isolates from serologically positivehumans This objective was achieved by the detection of theTgsGP gene which is present in only one strain of the 3trypanosomes pathogenic to man This gene is specific togroup 1 T b gambiense and is shown to confer resistanceto human serum We were able to isolate and characterize7 stocks from 21 CATT seropositive humans The highspecificity of the PCR technique may be a reason for the lowinfection rate revealed by the technique On the other hand itwas also possible that the CATT detected inactive infectionswhere antibodies have not completely disappeared from theblood

The epidemiological and clinical significance of detectingonly one species of human infective trypanosomes in thestudy area is that the risk of treatment failure associated withmixed infections of T b rhodesiense and types 1 and 2 T bgambiense which respond differently to HAT chemotherapy[24] will be overcome It was also not surprising to havedetected only type 1 T b gambiense among all human isolatessince it has been reported to be associated with over 90 ofcases of human African trypanosomosis [4 5]

In conclusion we observed silent T b gambiense infec-tions among residents of this old sleeping sickness focusInfections were more prevalent in children and males Theepidemiological implication of this finding is the risk oftsetse flies spreading these silent infections to uninfectedpopulation

Ethical Approval

Ethical clearance with reference number JUTHDCSADM127XIX5944 was obtained from the Jos University TeachingHospital Jos Nigeria before the commencement of thisstudy The study also observed all the guidelines governingthe use of laboratory animals in research with permissionfrom the Animal Welfare Unit of the National VeterinaryResearch Institute Vom Nigeria

Consent

Prior to sampling of each human subject verbal consent wassought and individuals aged 18 years and above willing toparticipate were given written consent to sign Individualsbelow the age of 18 years whowerewilling to participate in thestudy also signed written consents which were countersignedby at least one of their parents

Conflict of Interests

There is no conflict of interests regarding the publication ofthis paper

Acknowledgment

The authors acknowledged the Centre for BiotechnologyAhmadu Bello University Zaria Nigeria for allowing themto use the facilities for molecular analysis


References

[1] J M Conway-Klaaseen J MWyrick-Glatzel N Neyrinck andP A Belair ldquoAfrican sleeping sickness in a young Americantouristrdquo Laboratory Medicine vol 33 pp 783ndash788 2002

[2] T Jelinek Z Bisoffi L Bonazzi et al ldquoCluster of Africantrypanosomiasis in travelers to Tanzanian national parksrdquoEmerging Infectious Diseases vol 8 no 6 pp 634ndash635 2002

[3] MW Service A Guide to Medical Entomology Blackwell Scien-tific Publishers 3rd edition 1980

[4] C Cordon-Obras C Garcıa-Estebanez N Ndong-Mabale etal ldquoScreening of Trypanosoma brucei gambiense in domesticlivestock and tsetse flies from an insular endemic focus (LubaEquatorial Guinea)rdquoPLoSNeglected Tropical Diseases vol 4 no6 article e704 2010

[5] C M Wombou Toukam P Solano Z Bengaly V Jamonneauand B Bucheton ldquoExperimental evaluation of xenodiagnosisto detect trypanosomes at low parasitaemia levels in infectedhostsrdquo Parasite vol 18 no 4 pp 295ndash302 2011

[6] A Geiger G Simo P Grebaut J-B Peltier G Cuny andP Holzmuller ldquoTranscriptomics and proteomics in humanAfrican trypanosomiasis current status and perspectivesrdquo Jour-nal of Proteomics vol 74 no 9 pp 1625ndash1643 2011

[7] OIE ldquoStandardized techniques for the diagnosis of tsetsetransmitted trypanosomiasisrdquo in OIE Terrestrial Manual OIERome Italy 2008

[8] S L Wastling K Picozzi C Wamboga et al ldquoLatent Try-panosoma brucei gambiense foci in Uganda a silent epidemicin children and adultsrdquo Parasitology vol 138 no 12 pp 1480ndash1487 2011

[9] B A Aiyedun and A A Amodu ldquoHuman sleeping sickness inthe Gboko endemic areas in Nigeriardquo Acta Tropica vol 33 no1 pp 88ndash95 1974

[10] S M Lewis and C T Stoddart ldquoEffects of anticoagulants andcontainers (glass and plastic) on the blood countrdquo LaboratoryPractice vol 20 no 10 pp 787ndash792 1971

[11] J B Kennedy K TMaehara and AM Baker ldquoCell and plateletstability in disodium and tripotassium EDTArdquo The AmericanJournal of Medical Technology vol 47 no 2 pp 89ndash93 1981

[12] E Magnus T Vervoot and N Van-Meirvenne ldquoA card-agglutination test with stained trypanosomes (CATT) forthe serological diagnosis of T B gambiense trypanosomiasisrdquoAnnales de la Societe Belge de Medecine Tropicale vol 58 no 3pp 169ndash176 1978

[13] O W Schalm N C Jain and E J Carrol Veterianry Haema-tology Lea and Febihger Publishers Philadelphia Pa USA 3rdedition 1975

[14] W J Herbert and W H R Lumsden ldquoTrypanosoma brucei arapid lsquomatchingrsquo method for estimating the hostrsquos parasitemiardquoExperimental Parasitology vol 40 no 3 pp 427ndash431 1976

[15] S M Lanham and D G Godfrey ldquoIsolation of salivarian try-panosomes from man and other mammals using DEAE-cellu-loserdquo Experimental Parasitology vol 28 no 3 pp 521ndash534 1970

[16] B Oury N Dutrait B Bastrenta and M Tibayrenc ldquoTry-panosoma cruzi evaluation of a RAPD synapomorphic frag-ment as a species-specific DNA proberdquoThe Journal of Parasitol-ogy vol 83 no 1 pp 52ndash57 1997

[17] M Radwanska F Claes S Magez et al ldquoNovel primersequences for polymerase chain reaction-based detection ofTrypanosoma brucei gambienserdquo American Journal of TropicalMedicine and Hygiene vol 67 no 3 pp 289ndash295 2002

[18] P P Pyana I N Lukusa D M Ngoyi et al ldquoIsolation ofTrypanosoma brucei gambiense from cured and relapsed sleep-ing sickness patients and adaptation to laboratory micerdquo PLoSNeglected Tropical Diseases vol 5 no 4 Article ID e1025 2011

[19] H M Chapman S M Olson and D Trumm ldquoAn assessmentof changes in the montane forests of Taraba State Nigeria overthe past 30 yearsrdquo Oryx vol 38 no 3 pp 282ndash290 2004

[20] S O Omotainse J O Kalejaiye P Dede and A J Dadah ldquoThecurrent status of tsetse and animal trypanosomosis in NigeriardquoVom Journal of Veterinary Sciences vol 1 no 1 pp 1ndash7 2004

[21] D M Ngoyi V Lejon P Pyana et al ldquoHow to shorten patientfollow-up after treatment for Trypanosoma brucei gambiensesleeping sicknessrdquo The Journal of Infectious Diseases vol 201no 3 pp 453ndash463 2010

[22] N S Karshima I Ajogi A I Lawal G Mohammed andO O Okubanjo ldquoDetection of Trypanosoma brucei gambiensespecific LiTat 13 antibodies in humans and cattle in TarabaState North-Eastern Nigeriardquo Journal of Veterinary Advancesvol 2 no 12 pp 580ndash585 2012

[23] Y O Mohammed K H Elmali M M Mohammed-Ahmedand I Elrayah ldquoFactors influencing the sero-prevalence of Try-panosoma brucei gambiense sleeping sickness in Juba DistrictCentral Equatorial State Southern Sudanrdquo Journal of PublicHealth and Epidemiology vol 2 no 5 pp 100ndash108 2010

[24] R Brun R Schumacher C Schmid C Kunz and C BurrildquoThe phenomenon of treatment failures in Human AfricanTrypanosomiasisrdquo Tropical Medicine and International Healthvol 6 no 11 pp 906ndash914 2001

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

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Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


LinksFontem SS focusAlkaleri

GashakaGboko

0 150 300 600

(km)North

Nigeria

Benue

BauchiYankari GR

Taraba

Fontem SSFocus of the RC

Cameroon

Figure 1 Links between the study sites and possible sources of T b gambiense infections GR game reserve RC Republic of Cameroon SSsleeping sickness

an active screening of T b gambiense in humans in the oldGboko sleeping sickness focus in Nigeria and characterizedisolates using TgsGP-polymerase chain reaction

2 Materials and Methods

21 Study Area This study was carried out around theold Gboko sleeping sickness focus which is located in thenorthern part of Nigeria between longitudes 7∘471015840 and 10∘001015840east and latitudes 60∘251015840 and 8∘81015840 north It shares boundarieswith five other states namely Nasarawa (north) Taraba(east) Cross River (south) Enugu (southwest) and Kogi(west) and with the Republic of Cameroon to the southeast(Figure 1)Themajor occupation in this region is agricultureparticularly crop and livestock farming as well as fishing

22 Study Design We conducted a cross-sectional studyaround the old Gboko sleeping sickness focus in Nige-ria within six Local Government Areas (LGAs) namelyGashaka Gboko Ibi Karim Lamido Ukum and Vandeikyaconsidering their relationships with the Gboko and Fontemsleeping sickness foci and the Gashaka-Gumti and Yankarigame reserves Human subjects were sampled systematicallyfrom each household by selecting every 5th household fol-lowing the order in which houses were built and individualswere selected using balloting by names

23 Sample Collection

231 Blood Sampling of Human Subjects Two millilitres ofblood was aseptically collected from each human subject viathe median cubital vein using a 5mL syringe and 21G needle

and transferred immediately into clean labelled sample bot-tles containing ethylene diamine tetra-acetic acid (EDTA) at15mgmL of blood [10] and gently shaken until the bloodwas properly mixed with the anticoagulant and analyzedwithin 1-2 hours after collection [11] These samples weresubjected to the card agglutination test for T b gambiense asdescribed by Magnus et al [12] All CATT positive sampleswere then inoculated into mice immunosuppressed withcyclophosphamide to isolate trypanosomes

24 Laboratory Analysis of Samples

241TheCardAgglutinationTest for Trypanosomosis (CATT)The CATT test kits were obtained from the Institute forTropical Medicine Antwerp Belgium Blood samples wereanalyzed using the CATT as described by Magnus et al [12]This test is based on the detection of T b gambiense specificLiTat 13 antibodies using a purified T b gambiense variablesurface antigen

242 Mice Infection and Parasitaemia Estimation Mice wereobtained from the Small Animal Experimental Unit of theNational Veterinary Research Institute Vom Nigeria for thestudy The mice were appropriately labelled and screenedfor ectoparasites and endoparasites by standard techniques[13] and acclimatized for two weeks before inoculationThe mice were immunosuppressed using intraperitonealadministration of cyclophosphamide at 200mg per kg bodyweight About 05mL of each seropositive blood sample wasenriched with 05mL of phosphate saline glucose (PSG)buffer and 03mL of the PSG buffer enriched seropositiveblood from each seropositive samplewas then inoculated into







3 Results





CATTpositive

TgsGP-PCRpositive



Sex Numberexamined

CATTpositive

TgsGP-PCRpositive



Age Numberexamined

CATTpositive

TgsGP-PCRpositive





4 Discussion








Ethical Approval


Consent




Acknowledgment



References
































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology







3 Results





CATTpositive

TgsGP-PCRpositive



Sex Numberexamined

CATTpositive

TgsGP-PCRpositive



Age Numberexamined

CATTpositive

TgsGP-PCRpositive





4 Discussion








Ethical Approval


Consent




Acknowledgment



References
































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


4 Discussion








Ethical Approval


Consent




Acknowledgment



References
































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


References
































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

research article silent human trypanosoma brucei gambiense...

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