ABSTRACT
The exact role of animal reservoirs in the epidemiology of Trypanosoma brucei gambiense, a parasite ravaging affected rural parts of sub-Saharan Africa, still remains unclear. However, the existence of the parasite in animals is well documented in other parts of Africa. A randomised cross sectional study was conducted in an old sleeping sickness focus in Nigeria by screening 600 cattle and 600 pigs using the card agglutination test for trypanosomosis (CATT) and identifying trypanosomes using ITS 1 and TgsGP PCRs. Data generated were analysed using the Chi square test and odds ratio at 95% confidence interval. The overall infection rates for the CATT and TgsGP-PCR were 8.9 and 0.9%, respectively which varied significantly between cattle (7.2%) and pigs (10.7%) with the CATT. The CATT based infection rates in relation to study sites, breeds and management practices varied significantly (p < 0.05) between 2.0 and 17.0%, while the PCR based ranged between 0 and 1.7%. Trypanosomes of animal origin identified by ITS 1 PCR were T. brucei (4.2%), Trypanosoma congolense forest (3.2%), T. congolense savannah (2.0%), Trypanosoma vivax (2.2%) and mixed infections (1.5%) in cattle as well as T. brucei (4.8%), T. congolense forest (1.8%), T. congolense savannah (1.0%) and mixed infections (1.2%) in pigs. T. brucei gambiense and other animal trypanosomes were identified among animals in the focus, indicating the existence of animal reservoirs of human infective T. b. gambiense. This suggests that the inclusion of reservoir control component in T. b. gambiense control programmes will help in the control of this parasite in this focus.
Key words: Animal reservoirs, Trypanosoma brucei gambiense, sleeping sickness focus, card agglutination test for trypanosomosis (CATT), ITS-PCR, TgsGP-PCR, Gboko, Nigeria.
Trypanosoma brucei gambiense is the cause of Gambian sleeping sickness which is a chronic, debilitating, complex and fatal parasitic zoonosis ravaging affected rural parts of sub-Saharan Africa. The disease transmitted by tsetse flies is widespread, posing serious public health problems in the region and to visiting tourists (Conway-Klaaseen et al., 2002; Jelinek et al., 2002). Sleeping sickness is seriously neglected in the areas of drug development and sustainable control programmes.
Trypanosoma brucei gambiense and T. b. rhodesiense are both sub-species of T. brucei which are infective to man. In natural conditions, transmission of the parasite is cyclical through bites of infected tsetse flies including Glossina palpalis, Glossina tachinoides and Glossina fuscipes. These vectors are especially common at watering places like rivers or lakes where people frequently visit to collect water and do their washings, and animals visit to drink water (Service, 1980). The parasite is divided into two sub-types: type 1, causes a more chronic disease and represents over 90% of all cases of sleeping sickness (Cordon-Obras et al., 2010; Wombou et al., 2011), type 2, has not been isolated for ages, while T. b. rhodesiense is said to be associated with an acute like disease and represents the remaining 10% of the disease (Hide et al., 1990). Though, fewer than 10,000 cases are documented yearly from 2009 due to under reporting, the pathogen still ranks ninth of the 25 human infectious diseases in Africa based on its socio-economic impact (WHO, 2012).
Studies in animals revealed the involvement of numerous wildlife species such as primates, reptiles, antelopes and wild bovids (Mbaya et al., 2009; Njiokou et al., 2010), as well as domesticated animals such as pigs, goats, sheep and cattle (Nkinin et al., 2002; Simo et al., 2006) as suitable reservoirs for T. brucei gambiense and consequently their roles in the transmission of the disease.
In Nigeria, no attention has been given to the animal reservoirs of T. b. gambiense. However, human infection in the Gboko endemic area of Benue State was first reported in 1974, and since then no successful attempt has been made to control the disease in the affected region (Aiyedun and Amodu, 1974). This study conducted an active screening of T. b. gambiense to ascertain the possible role of animal reservoirs in the epidemiology of the parasite in the old Gboko sleeping sickness focus in Nigeria and also characterized isolates using TgsGP polymerase chain reaction.
Study area
This study was carried out around the old Gboko sleeping sickness focus of Benue State which is located in the North-central zone of Nigeria between longitudes 7° 47’ and 10° 00’ East and latitudes 60 25’ and 8o 8’ North. It shares boundaries with five other states, namely: Nassarawa (north), Taraba (east), Cross River (south), Enugu (south-west) and Kogi (west) and with the Republic of Cameroon to the south-east (Figure 1). The major occupation in this region is agriculture particularly crop and livestock farming as well as fishing.
Study design
We conducted a randomised cross sectional study around the old Gboko sleeping sickness focus in Nigeria. Six Local Government Areas (LGAs) namely; Gashaka, Gboko, Ibi, Karim Lamido, Ukum and Vandikya were randomly selected around the focus using simple balloting. Three villages from each of the LGAs were randomly selected. Herds of animals were considered as clusters from which animals were sampled using stratified sampling technique. A total of fifteen cattle herds and fifteen piggeries were selected using simple balloting. From each selected herd, 30 or 50% of animals were sampled from herds with sizes of ≥50 or <50 animals, respectively. The selected animals were stratified based on breed, sex and age groups for the purpose of this study as follows: cattle ≤ 1year as young, > 1 year as adult; pigs ≤ 6 months as young, > 6 months as adult.
Blood sampling and the card agglutination test for T. b. gambiense
Two milliliters of blood was aseptically collected from cattle via the jugular vein using 5 ml syringe and 18 G needles and from pigs via the ear vein using 5 ml syringe and 21 G needles. These samples were immediately transferred into clean labelled sample bottles containing ethylene diamine tetra-acetic acid (EDTA) at 1 mg/ ml and mixed gently until the blood was properly mixed with the anticoagulant. Samples were then subjected to the card agglutination test for T. b. gambiense (CATT) within one hour of collection as described by Magnus et al. (1978). This test is based on the detection of T. b. gambiense specific Li Tat 1.3 antibodies using a purified T. b. gambiense variable surface antigen.
Isolation and purification of trypanosomes
The bloodstream form trypanosomes isolated from cattle and pigs were concentrated by centrifugation and separated from host blood using a DEAE 52 column (Whatman, Maidstone, Kent, UK) as described by Lanham and Godfrey (1970). These parasites were then stored at -20°C until needed for DNA extraction.
Extraction of DNA from purified trypanosomes
Trypanosome DNA extraction was done with GeneJET genomic DNA extraction kit (Thermo Scientific, Germany) using the method described by Oury et al. (1997). The extracted DNA was stored at -20°C until needed for PCR at the Biotechnology Laboratory of the Ahmadu Bello University, Zaria, Nigeria.
PCR detection of trypanosomes
Internal transcribed spacer 1 polymerase chain reaction (ITS 1-PCR) was used to identify Trypanosoma species using primer sets with forward sequence 5l–CCGGAAGTTCACCGATATTG-3l and reverse sequence 5l-TTGCTGCGTTCTTCAACGAA-3l (Inqaba Biotec, South Africa) targeting the variable length ITS 1 regions of trypanosome ribosomal DNA to identify trypanosomes species and sub-species with variable band sizes for different species and sub-species (Njiru et al., 2004). PCR was carried out in a final volume of 11 µl containing 5 µl phusion flash high fidelity PCR master mix (phusion flash II DNA polymerase, dNTPs, MgCl2), 1 µl each of forward and reverse primers, 1 µl of extracted DNA and 3 µl of PCR grade water. PCR conditions were as follows: an initial denaturation step for 10 s at 98°C to activate the phusion flash II DNA polymerase, four cycles of amplification with 1 s denaturation at 98°C, 5 s hybridization at 58°C and 15 s elongation steps at 72°C; eight cycles of amplification with 1 s denaturation at 98°C, 5 s hybridization at 56°C and 15 s elongation steps at 72°C; 23 cycles of amplification with 1 s denaturation at 98°C, 5 s hybridization at 54°C and 15 s elongation steps at 72°C; and a final extension step of 5 min at 72°C as described by the manufacturer.
Detection of Trypanosoma gambiense specific glycoprotein (TgsGP)
The TgsGP primer set designed by Radwanska et al. (2002) with forward sequence 5l-GCTGCTGTGTTCGGAGAGC-31 and reverse sequence 5l-GCCATCGTGCTTGCCGCTC-31 (Inqaba Biotec, South Africa) was used to characterize the T. brucei isolates identified using the ITS-PCR as T. b. gambiense type 1 by amplifying the TgsGP gene yielding band sizes at 308 bp. Cycling conditions for TgsGP PCR were: denaturation at 98°C for 10 s to activate the phusion flash II DNA polymerase, followed by 40 cycles at 98°C for 1 s, annealing at 63°C for 30 s, 30 s elongation at 72°C and a final extension at 72°C for 5 min. All the amplified products were analyzed by electrophoresis in a 2% agarose gel with 100 bp molecular makers and UV illumination after ethidium bromide staining. All amplifications were carried out using a Gene Amp PCR system 9700 (Applied Biosystems, UK).
Data analysis
All data obtained during the study were analyzed using Graph-Pad Prism 4.0. Infection rates of T. b. gambiense were calculated by dividing the number of infected animals by the total number of animals examined and expressed as percentages. This was done for different variables such as animal species, study sites, sex, age, breeds and management practices. The Chi square (X2) test and odds ratio were used where appropriate to compare the infection rates based on different variables and values of p < 0.05 were considered significant.
A total of 1200 animals including cattle (600) and pigs (600) were subjected to the CATT, ITS 1 and TgsGP-PCRs revealing overall infection rates of 8.9 and 0.9%, respectively. A significantly higher (p = 0.03) T. b. gambiense infection rate was observed in pigs (10.7%) than cattle (7.2%) using the CATT as shown in Table 1. However, very few sub-type 1 cases were detected using TgsGP-PCR and infection rate was statistically insignificant with this method (Table 1). CATT-based infection rate varied significantly (p < 0.001) between villages with the highest prevalence in Gboko (17.0%) and the lowest in Ibi (4.0%). The variations between the PCR-based infection rates across villages were statistically insignificant ranging between 0 and 2.0% (Table 2).
Age and sex based T. b. gambiense infection rates using the CATT showed no significant variations ranging between 7.0 and 10.2% (Table 3). PCR-based infection rates in relation to both age and sex ranged between 0.4 and 1.2% with no significant variation (Table 3). There were significant variations between the CATT-based infection rates recorded across cattle breeds (p =0.0023), pig breeds (p = 0.003) and management practices (p = 0.0117) with prevalence rates ranging between 2.0 and 14.3% with the White Fulani cattle, indigenous pigs and extensively managed pigs revealing highest infection rates (Table 4). PCR-base infection rates in relation to breeds and management practices ranged between 0 and 1.7% with no statistical association (Table 4).
The prevalence of T. brucei species by ITS 1 (including T. b. gambiense) was 4.2% in cattle and 4.8% in pigs. Majority of T. brucei infections were in pigs (4.8%) while majority of the T. congolense infections were in cattle (5.2%). Other species and sub-species identified by ITS 1-PCR were T. vivax, T. congolense forest and T. congolense savannah (Figures 2 and 3).
The zoonotic nature of T. b. gambiense had remained doubtful for over a century until recent reports on its detection from different animal hosts (Njiokou et al., 2010; Njitchouang et al., 2010). The present study revealed an overall animal CATT infection rate of 8.9% which is lower than earlier findings of 34.3% (Simo et al., 2006), 22.7% (Njiokou et al., 2010) and 64.0% (Njitchouang et al., 2010) from different human African trypanosomosis (HAT) foci in the Republic of Cameroon using the same technique. A PCR-based overall infection rate (0.9%) lower than that of earlier findings: 3.08% (Njiokou et al., 2010), 9.82% (Njitchounang et al., 2010) and 14.8% (Simo et al., 2006) was also observed using the same PCR. The possible explanations for these variations may include differences in the sensitivity and specificity of the diagnostic tests used by the various studies, vector densities and the vectoral capacities of the Glossina species infesting the different study areas.
The variations between the prevalence rates recorded by the two diagnostic techniques used may not be unconnected with the high specificity of polymerase chain reaction, absence of T. b. gambiense DNA due to inactive infections and possibilities of false positive results arising from CATT screening (Magnus et al., 1978). It is important to mention that majority of samples that were positive for the CATT were infected with other trypanosomes like T. vivax and T. congolense which are also shown to cause false positivity (Magnus et al., 1978).
Pigs were earlier said to be capable of acting as reservoirs of T. b. gambiense in Benue State (Stephen, 1986). The present finding may therefore be a confirmation to this speculation. The infection rate observed in pigs in both Ukum and Vandikya may not be unconnected with the free movement of livestock within the State. Local and International trade (particularly with the neighbouring Cameroon Republic) of pigs which is a delicacy in the state may be an additional factor. The role of trade pigs in the spread of human serum resistant Trypanosoma species was also reported earlier (Onah and Ebenebe, 2003).
It was not unexpected to have observed the highest infection rate in Gboko LGA probably due to the old HAT focus in the area and the negligence in the area of disease control (Aiyedun and Amodu, 1974). The high density of tsetse flies and wildlife species in the Gashaka-Gumti game reserve may be a possible explanation for the higher prevalence recorded in Gashaka LGA. Adult animals are known to release higher concentrations of insect attractants such as phenols in their urine (Kremar et al., 2006) as well as acetone and octenol in their breath (Kremar, 2007). These substances attract tsetse flies to the animals resulting in more frequent bites and subsequent infection. Tsetse flies are also known to be more attracted to larger animals than smaller ones (Torr et al., 2007). These may be possible reasons for the slight difference in the prevalence between adult and young animals.
Though, it was statistically insignificant, the infection rate in relation to sex was higher in females than males in accordance with earlier reports by Shah et al. (2004) in the Republic of Cameroon and Karshima et al. (2012) in Taraba State, Nigeria. This may be attributed to stress associated with hormonal imbalances during pregnancy and lactation. Also, because of the chronic nature of T. b. gambiense infection, the chances of detection in females might be higher because they are kept longer in herds for the purpose of breeding, thus allowing the chronic infection to manifest.
The Bokoloji and Muturu cattle breeds are known to be trypano-tolerant (Murray et al., 1983). This resistance to Trypanosoma species may explain the lower prevalence recorded in these two breeds. The higher prevalence observed among the White Fulani may be attributable to their trypanosusceptibility and perhaps due to their higher representation in the sampling. Of the three cattle breeds studied, the White Fulani are usually raised using the nomadic system of management. This may be another possible explanation for the higher prevalence recorded by this group.
Extensively managed animals usually visit swampy areas and streams in search of drinking water thereby being exposed to the risk of bites by riverine species of tsetse flies. This may be a probable explanation for the higher prevalence observed among extensively managed animals. However, teneral tsetse flies that breed and live around human settlements may be responsible for the low prevalence observed among intensively managed animals.
Though this study targeted animal reservoirs of T. b. gambiense, the authors also considered the distribution of Trypanosoma species of animal origin in the focus to estimate possible risk of atypical Trypanosoma infections in human as reported elsewhere (Truc et al., 2007; 2013; Deborggraeve et al., 2008). The species of trypanosomes identified in this study were earlier reported by other workers (Yanan et al., 2007; Fasanmi et al., 2014) indicating that they are endemic in Nigeria. Mixed infections with similar Trypanosoma species as those reported by this study have also been documented in Nigeria (Karshima and Bobbo, 2011; Majekodunmi et al., 2013). These mixed infections might be due to bites from tsetse flies carrying more than one Trypanosoma infections or successive bites from different tsetse flies infected with different Trypanosoma species.
One of the major objectives of this study was to characterize T. brucei isolated from serologically positive cattle and pigs. This was necessary because of the roles of domestic and wild animals in the epidemiology of T. b. gambiense as earlier reported (Abenga and Lawal, 2005). It has been established that the TgsGP gene is present in all group 1 T. b. gambiense isolates examined to date but not in T. b. brucei, T. b. rhodesiense or group 2 T. b. gambiense (Capewell et al., 2013). This study confirmed only group 1 T. b. gambiense isolates in cattle and pigs in the study area, which was not surprising since it has been shown to be associated with over 90% of all sleeping sickness cases with the remaining assigned to T. b. rhodesiense since type 2 has not been isolated for ages (Cordon-Obras et al., 2010; Wombou et al., 2011).
The clinical significance of detecting only one species of human infective trypanosome in the study area is that, the risk of treatment failure associated with mixed infections of T. b. rhodesiense, groups 1 and 2 T. b. gambiense which responds differently to sleeping sickness chemotherapy (Brun et al., 2001) will be overcome.
The study identified cattle and pigs as domesticated animal reservoirs of group 1 T. b. gambiense in this region. The epidemiological significance of this finding is that at least cattle and swine populations may play a role in the maintenance and possible resurgence of the disease in this region.
The study identified cattle and pigs as domesticated animal reservoirs of group 1 T. b. gambiense in this region. Other trypanosomes of animal origin identified were T. brucei, T. vivax, T. congolense forest and T. congolense savannah. The implication of this finding is that, these reservoir animals might be traded for breeding purposes thereby initiating and spreading new sleeping sickness foci in non-infected areas. It is therefore very pertinent to include reservoir control component in T. b. gambiense control programmes to curtail the maintenance and resurgence of this parasite by animal reservoirs.
The authors have not declared any conflict of interest.
The authors are grateful to the Director of the Biotechnology Centre, Ahmadu Bello University Zaria, Nigeria, Prof. Kabir Junaidu and all other staff of the centre for allowing them use of their facilities.
REFERENCES
|
Abenga JN, Lawal AI (2005). Implicating roles of animal reservoir hosts in the resurgence of Gambian trypanosomosis (Sleeping Sickness). Afr. J. Biotechnol. 4(2):134-137.
|
|
|
|
Aiyedun BA, Amodu AA (1974). Human sleeping sickness in the Gboko endemic areas in Nigeria. Acta Trop. 33(1):88-95.
|
|
|
|
|
Brun R, Schumacher R, Schmid C, Kunz C, Burri C (2001). The phenomenon of treatment failures in human African typanosomiasis. Trop. Med. Int. Health 6:906914.
Crossref
|
|
|
|
|
Capewell P, Cooper A, Duffy CW, Tait A, Turner CM (2013). Human and animal trypanosomes in Coˆte d'Ivoire from a single breeding population. PLoS ONE doi:10.1371/journal.pone.0067852.
Crossref
|
|
|
|
|
Conway-Klaaseen JM, Wyrick-Glatzel JM, Neyrinck N, Belair PA (2002). Sleeping Sicknees in young American tourist. Lab. Med. 33:783-788.
Crossref
|
|
|
|
|
Cordon-Obras C, Garcıa-Estebanez C, Ndong-Mabale N, Abaga S, Ndongo-Asumu P (2010). Screening of Trypanosoma brucei gambiense in domestic livestock and tsetse flies from an insular endemic focus (Luba, Equatorial Guinea). PLoS Neg. Trop. Dis. 4(6):e704.
Crossref
|
|
|
|
|
Deborggraeve S, Koffi M, Jamonneau V, Bonsu FA, Queyson R (2008). Molecular analysis of archived blood slides reveals an atypical human Trypanosoma infection. Diagn. Microbiol. Infect. Dis. 61:428-433.
Crossref
|
|
|
|
|
Fasanmi OG, Okoroafor UP, Nwufoh, OC, Bukola-Oladele OM, Ajibola ES (2014). Survey for Trypanosoma species in cattle from three farms in Iddo Local Government Area, Oyo State. Sok. J. Vet. Sci. 12(1):57-61.
Crossref
|
|
|
|
|
Hide G, Cattand P, Leray D, Barry JD, Tait A (1990). The identification of Tryponasoma brucei subspecies using repetitive DNA sequence. Mol. Biochem. Parasitol. 39:213-226.
Crossref
|
|
|
|
|
Jelinek T, Bisoffi BL, Van-Theil P, Bronne U, Defery A, Gunderson SG, McWhinney P, Ripamonti D (2002). Cluster of African trypanosomiasis in travellers to Tanzania national park. Emerg. Infect. Dis. 8:634-635.
Crossref
|
|
|
|
|
Karshima NS, Ajogi I, Lawal AI, Mohammed G, Okubanjo OO (2012). Detection of Trypanosoma brucei gambiense specific Li Tat 1.3 antibodies in humans and cattle in Taraba State, North-Eastern Nigeria. J. Vet. Adv. 2(12):580-585.
|
|
|
|
|
Karshima SN, Bobbo AA (2011). Prevalence of bovine trypanosomosis in Taraba State, North-Eastern Nigeria. Trop. Vet. 29(4):44-50.
|
|
|
|
|
Kremar S (2007). Responses of tabanidae (Diptera) to canopy traps baited with 4-methyl phenol, 3-isopropylphenol and naphthalene. J. Vect. Ecol. 32:188-192.
Crossref
|
|
|
|
|
Kremar S, Mikuska A, Merdic C (2006). Responses of tabanidae (Diptera) to different natural attractants. J. Vect. Eco. 31:262-265.
Crossref
|
|
|
|
|
Lanham SM, Godfrey DG (1970). Isolation of salivarian trypanosomes from man and other mammals using DEAE-cellulose. Exp. Parasitol. 28:521-534.
Crossref
|
|
|
|
|
Magnus E, Vervoot T, Van-Meirvenne N (1978). A card-agglutination test with stained trypanosomes (CATT) for the serological diagnosis of T. b. gambiense trypanosomiasis. Ann. Soc. Belg. Med. Trop. 58:169-176.
|
|
|
|
|
Majekodunmi AO, Fajinmi A, Dongkum C, Picozzi K, Thrusfield MV, Welburn SC (2013). A longitudinal survey of African animal trypanosomiasis in domestic cattle on the Jos Plateau, Nigeria: prevalence, distribution and risk factors. Parasitol. Vect. 6:239.
Crossref
|
|
|
|
|
Mbaya AW, Aliyu MM, Nwosu CO, Ibrahim U (2009). Effect of DL-α-Difluoromethylornithine on biochemical changes in Baboons (Papio anubis) experimentally infected with Trypanosoma brucei gambiense. Nig. Vet. J. 31(1):34-44.
|
|
|
|
|
Murray M, Trail JCM, Turner DA, Wissocq Y (1983). Livestock productivity and trypanotolerance. Network Training Manual. ILCA, Addis Abba. pp. 45-75.
|
|
|
|
|
Njiokou F, Nimpaye H, Simo G, Njitchouang GR, Asonganyi T, Cuny G, Herder S (2010). Domestic animals as potential reservoir hosts of Trypanosoma brucei gambiense in Sleeping Sickness foci in Cameroon. Parasite 17:61-66.
Crossref
|
|
|
|
|
Njiru ZK, Constantine CC, Guya S, Crowther J, Kiragu JM, Thompson RCA, Davila AMR (2005). The use of ITS1 rDNA PCR in detecting pathogenic African trypanosomes. Parasitol. Res. 95:186-192.
Crossref
|
|
|
|
|
Njitchouang GR, Njiokou F, Djeunga HCN, Fewou PM, Asonganyi GC, Simo G (2010). Analysis of the domestic animal reservoir at a micro-geographical scale, the Fontem Sleeping Sickness focus (South-West Cameroon). J. Cell Anim. Biol. 4(5):73-80.
|
|
|
|
|
Nkinin SW, Njiokou F, Penchenier L, Grebaut P, Simo G, Herder S (2002). Characterization of T. brucei sl by isoenzymes in domestic pigs from the Fontem Sleeping Sickness focus of Cameroon. Acta Trop. 81:225-232.
Crossref
|
|
|
|
|
Onah DN, Ebenebe OO (2003). Isolation of human serum resistant Trypanosoma brucei from naturally infected pigs in NsuKka area of Enugu. Nig. Vet. J. 24(1):37-43.
|
|
|
|
|
Oury B, Dutrait N, Bastrenta B, Tibayrene M (1997). Trypanosoma cruzi: use of a synapomorphic RAPD fragment as a species-specific DNA probe. J. Parasitol. 83:52-57.
Crossref
|
|
|
|
|
Radwanska M, Claes F, Magez S, Magnus E, Perez-morga D, Pays E, Büscher P (2002). Novel primer sequences for Polymerase Chain Reaction–based detection of Trypanosoma brucei gambiense. Am. J. Trop. Med. Hyg. 67:289-295.
|
|
|
|
|
Service MW (1980). A guide to medical entomology, third edition, published by Blackwell scientific publishers. pp. 95-101.
Crossref
|
|
|
|
|
Shah SR, Phulan MS, Memon MA, Rind R, Bhatti A (2004). Trypanosome infections in camels. Pak. Vet. J. 24(4):209-210.
|
|
|
|
|
Simo G, Asonganyi T, Nkinin SW, Njiokou F, Herder S (2006). High prevalence of Trypanosoma brucei gambiense group 1 in pigs from the Fontem Sleeping Sickness focus in Cameroon. Vet. Parasitol. 139(3):57-66.
Crossref
|
|
|
|
|
Stephen LE (1986). Trypanosomiasis:. Pergamon, Oxford. Vet. Perspect. pp. 480-487.
|
|
|
|
|
Torr SJ, Prior A, Wilson AJ, Schofield S (2007). Is there safety in numbers? The effect of cattle herding on biting risk from tsetse flies. Med. Vet. Entomol. 21:301-311.
Crossref
|
|
|
|
|
Truc P, Buscher P, Desquenes M (2013). Atypical human infections by animal trypanosomes. PLoS Negl. Trop. Dis. 7(9):e2258.
Crossref
|
|
|
|
|
Truc P, Gibson W, Herder S (2007). Genetic characterization of Trypanosoma evansi isolated from a patient in India. Infect. Genet. Evol. 7:305-307.
Crossref
|
|
|
|
|
Wombou TCM, Solano P, Bengaly Z, Jamonneau V, Bucheton B (2011). Experimental evaluation of xenodiagnosis to detect trypanosomes at low parasitaemia levels in infected hosts. Parasite 18:295-302.
Crossref
|
|
|
|
|
World Health Organization (WHO) (2012). Report of a WHO Meeting on Elimination of African Trypanosomiasis (Trypanosoma brucei gambiense) Geneva, 3-5 December 2012. Geneva, Switzerland: World Health Organization.
|
|
|
|
|
Yanan EG, Evuti MA, Shamaki BU, Sugun MY, Dongkun C, Ogunsan EA (2007). Epidemiology of small ruminant trypanosomosis in some communities in Jos East, Plateau State: A guinea Savana Zone, North Central Nigeria. J. Protozool. Res. 17:44-47.
|
|
