Immunodiagnosis of bovine trypanosomiasis in Anambra and Imo states, Nigeria, using enzyme-linked immunosorbent assay: zoonotic implications to human health.
African animal trypanosomiasis caused by Trypanosoma brucei, T. congolense and T. vivax remains one of the major constraints to health and productivity of cattle and other domestic animals in the tsetse infested areas of tropical Africa owing to its varied clinical manifestations (1).
In Nigeria, it has continued to be a menace in the livestock industry despite the age-long attempts to control the disease (2). In the same vein, Human African Trypanosomosis (HAT) arising from T. brucei gambiense and T.b. rhodesiense, constitute a major problem arising from the controversial role of animal reservoir hosts. Although campaigns against HAT in the 1960s brought the disease below epidemic proportions, there is presently a dramatic resurgence in both forms of HAT which has been blamed on wide spread of civil disturbances and wars, declining economies, reduced health financing, dismantling of disease control programmes and animal reservoir hosts (3).
The HAT, sleeping sickness is a complex and debilitating disease of man. The disease still ravages in several parts of sub-Saharan Africa despite decades of efforts aimed at control (4). The HAT poses as an emerging public health crisis in several countries including Nigeria (5), and a major health risk to tourists coming to tropical Africa (1). Human animal trypanosomosis is caused by the parasitic protozoa of the Genus Trypanosoma (4). World Health Organization (WHO), had commented on the complex nature of the epidemiology of the human disease arising from two species of the infecting trypanosome in various parts of Africa (4-6).
The fact that many animals had T. brucei infection might be of zoonotic importance, since domestic animals are known reservoirs of T.b. gambiense in west Africa (7). A recent sero-prevalence study employing the standard direct sandwich technique of ELISA showed that of 320 sheep and goats, blood samples assayed in Ondo and Ekiti states, Nigeria, 47.8, 46.6 and 47.18% have had previous contact with T. brucei, T. congolense and T. vivax respectively (8). Trypanosomiasis seems to be remerging as an important livestock disease in Nigeria, assuming major clinical importance in small ruminants and extending to previously designated tsetse free zones (9). The reasons for this need are to be investigated in other to avoid animal human transmission.
Material & Methods
A total of 264 adult cattle of mixed breeds and sexes (male 220, female 44) from cattle markets in Awka and Orlu in Anambra and Imo states respectively were used for this study.
Serum sample collection:Appropriately labelled plain 10 ml vacutainer tubes with monoject needles were used to collect 5-8 ml of blood from the cattle via the jugular veins. The tubes with blood were kept in ice-packs in slanting position and transported to the laboratory, where the tubes were stored at 4[degrees]C overnight to allow for adequate retraction and clotting. The samples were centrifuged at 3000 rpm for 10 min and sera dispensed in duplicate into appropriately labelled sample tubes and stored at -20[degrees]C until used analysed.
Generation of monoclonal antibodies: This was carried out as per the procedure described by Milstein and Kohler (10), with little modifications. Succinctly, monoclonal antibodies were raised by immunising Balb/c mice with the T. congolense, T. vivax and T. brucei antigens lysate in Freud's adjuvant. The spleens were removed from the mice and the individual cells fused with constantly dividing (immortal) B-tumour cells in polyethylene glycol (PEG). The B-tumour cells were selected for a purine enzyme deficiency and often for their inability to secrete immunoglobulin (11). The resulting cells were distributed into micro-well plates in hypoxanthine aminopterin thymidine medium which killed off the fusion partners, in that each well was left with one hybridoma cell. The immunoglobulins were prepared from the ascetic fluid purified and tested by ELISA against different trypanosome species-specific antigens, Anaplasma marginale antigen, Babesia bijemina antigen Theeileria parva antigen, to establish their specificity.
Generation of conjugate: Briefly, 10 mg horseradish peroxidase (HRP) type VI was dissolved in 0.2 ml of 1.25% glutaraldehyde in phosphate buffered saline (PBS) (pH 7.2) and left at room temperature overnight. About 1.0 ml of 0.1 M carbonate buffer (pH 9.2) was added and dialysed changing twice in 4 h. To this 5 mg of purified monoclonal antibodies raised against different bovine trypanosome species were added in 0.25 carbonate buffer and incubated at room temperature overnight and 0.1 ml of 0.2M lysine was added followed by the addition of equal volume of glycerol and kept at -20[degrees]C. A chequer board titration was used to determine the dilution of the conjugates.
Sandwich antigen capture ELISA: The monoclonal antibodies raised against T. brucei, T. congolense and T. vivax, separately were used at a dilution of 1 : 500 in coating buffer to coat each well of the flat bottom micro ELISA plates (Dynateck, Virginia, U.S.A.), and kept at 4[degrees]C overnight. The monoclonal antibody IgM raised specifically against T. brucei and IgG raised specifically against T. congolense and T. vivax were used (11). The plates were flipped empty and rinsed once with washing buffer and the excess buffer drained off the plates by gentle tapping of the inverted plate on a thick towel and 100 [micro]l of diluent buffer (washing buffer) was added in each well, then sera to be tested were added in duplicate in the wells using micro-pipettes with tips as follows 5 [micro]l in T. brucei plates and 10 [micro]l in the T. vivax and T. congolense plates. The wells in row A column 1 and 2 and wells in row B column 1 and 2 contain no sera and were left as reagent controls, while wells in row G column 11 and 12 contain positive controls and row H column 11 and 12 contain negative controls. The plates were incubated at 37[degrees]C for 15 min, then flipped empty and rinsed once with washing buffer. About 100 [micro]l of antis species monoclonal antibodies (IgM for T. brucei and IgG for T. congolense and T. vivax), separately raised against the species and conjugated to horseradish peroxidase and diluted at 1: 1000 in conjugate diluting buffer (phosphate buffer, 0.5% tween 80, 1% BSA) were added in each case. The plates were incubated at 37[degrees]C for 15 min, flipped empty and rinsed once. The plates were then filled up with washing buffer and soaked for 10 min and this was repeated twice. Then the substrate and chromogen solutions were added (100 [micro]l/well). The chromogen and substrate used consisted of 300 [micro] of hydrogen peroxide ([H.sub.2][O.sub.2]) in 30 ml of substrate buffer and 300 [micro]l of 22-azinobis (3-ethyl) benzthiaozoline 6-sulfonic acid (A.B.T.S) in same 30 ml of substrate buffer. The plates were incubated at 37[degrees]C in dark and the change in colour was observed and absorbance was read after 30 min at 405 nm wave length using a micro ELISA reader SFC 400 (Biotek Instruments, Inc, Vermount, U.S.A.). The wells with colour change were recorded. The quantitative value of absorbance of the colour intensity was extrapolated from the standard plot, indicating the quantity of antigen in the test serum.
All 264 different serum samples were collected from selected local governments in Imo and Anambra states. The animals were adults and of mixed breeds and sexes and were transported to the states from northern parts of Nigeria. From each state, 132 samples were collected. Out of the 264 a total of 110 (41.66%) different serum samples were positive for T. congolense while 108 (40.91%) were positive for T. vivax and 86 (32.58%) were positive for T. brucei in both the states.
Out of 110 (41.66%) sera positive samples for T. congolense, 48 (18.18%) were positive in Anambra state whereas 62 (23.48%) were positive samples in Imo state. Out of 108 (40.91%) positive for T. vivax in both the states, 50 (18.94%) were detected in Anambra and 58 (21.97%) in Imo state. Out of the 86 (32.58%) sera samples positive for T. brucei in both the states, 33 (12.5%) were detected in Anambra and 53 (20.08%) were detected in Imo state (Table 1).
Single infections were detected for these three species in both the states as 21 (7.95%) for T. congolense, 20 (7.58%) for T. vivax and 8 (3.03%) for T. brucei. In Anambra state alone, single infections were 11 (4.17%) for T. congolense, 10 (3.79%) for T. vivax and 5 (1.89%) for T. brucei. In Imo state, single infections were 10 each for T. congolense and T. vivax and 3 (1.14%) for T. brucei (Table 2).
Mixed infections were also detected. This was ob served during the plate reading. Since the three organisms were tested for in each serum sample, in separate plates, a serum appearing positive in the three plates is an indication of mixed infection of the three organisms. Positive in only two plates indicates mixed infection of the two organisms while positive in only one plate indicates single infection. The most predominant, involved three species--T. congolense, T. vivax and T. brucei and this occurred in 35 (13.26%) animals in Imo and 18 (6.81%) animals in Anambra, with a total of 53 (20.08%) in the two states. This is followed by the mixture of T. vivax and T. congolense which was detected in 14 (5.30%) animals in Anambra state and 6 (2.27%) in Imo state with a total of 20 (7.58%) in both the states. Mixed infections of T. vivax and T. brucei were detected in 8 (3.03%) cattles in Anambra and 5 (1.89%) cattles in Imo states making a total of 13 (4.92%) in two states. T. congolense and T. brucei mixed infections were also detected in 3 (1.14%) animals in Anambra and 10 (3.79%) animals in Imo states making a total of 13 (4.92%) in both the states (Table 2).
The absorbance values which determine the quantitative value of each trypanosome antigen used in each state were shown in Table 3. The readings that fall between 0.05 and 0.1 showed a very high quantity of antigen, hence depicting an active infection. T. vivax is found to have the highest number of high absorbance value with 14, followed by T. congolense and T. brucei having four each (Table 3).
Indications from the results show that mixed infections caused by T. congolense, T. vivax and T. brucei in Anambra and Imo states, were the prevalent infections in the two states. The findings depict a contrary view to that found by Dipeolu12, that the prevalence of mixed infection caused by T. congolense and T. vivax is the most significant. It also differs from the observations of Akinboade (13) who reported that a mixed infection due to T. congolense and T. brucei was most prevalent in cattle. However, the contrary observations could be attributed to differences in diagnostic methods used. Dipeolu (12) worked on "survey of blood parasites in domestic animals" using parasitological methods. The sensitivity of these methods especially in low parasitaemic conditions is unreliable compared to the reliable and highly sensitive ELISA technique used in this study. Supportably Losos and Ikede (14) reported a mixed infection of T. congolense, T. vivax and T. brucei in one cattle using DNA hybridization technique (14). This technique is also sensitive on its own although hybridisation signals failed to be obtained when samples that are to be used were kept at 4[degrees]C for up to four days where by the DNA gets degraded because of micro-condensation.
The failure to detect mixed infection of the three species by the parasitological method (which is based on the mortality and the morphology of the trypanosomes), is not unusual because of the fluctuating parasitaemic behaviour of blood stream trypanosomes (T. vivax and T. congolense). This means that the trypanosome species with highest proportion are likely to be diagnosed and infection attributed to these parasites only, where as the species which are low in numbers might not be identified microscopically.
The detection of mixed infection comprising T. congolense, T. vivax and T. brucei in the same group of animals and the high prevalence rate of trypanosomes in this study in spite of the increased use of trypanocides have highlighted the following--the stable occurrence of drug resistant strains of trypanosomes; the utmost need for more effort towards the control of the disease; and the distribution of animal reservoir and insect vectors. All these factors act either individually or in concert to maintain the infection in domestic animals and as reservoir for HAT.
In this study, the prevalent species among the three species of trypanosomes is T. congolense in both the states though the difference is not significant from T. vivax infection in between the states. In Anambra alone, the prevalent specie is T. congolense while equity is the case in Imo between T. congolense and T. vivax. One can significantly say that infections due to T. congolense and T. vivax are the main constraint on livestock development in the two states and in Nigeria as a whole considering the fact that preponderant majority of the livestocks raised for consumption in Nigeria are raised in the northern part of the country and transported to the southern part. However, in Africa, T. congolense infection usually manifests as a chronic form of the disease and milder than that caused by T. vivax (15). The importance of this work should not be over emphasised considering lack of current research on trypanosomiasis in Nigeria. Some of the factors that affect the prevalence of trypanosomiasis in Nigeria include animal breed, type of management, season of the year and the type of vegetation (16). It is also known that nomadism tends to expose animals to high tsetse challenge and hence trypanosome infection.
In Nigeria, cattle are considered as one of the principal livestock, and their survival and development are necessary to ameliorate the worsening situation regarding the supply of animal protein. Cattle production in the country has been restricted to the northern part because of the erroneous belief that the southern part (forest zone, including Anambra and Imo states) was highly infected by tsetse flies which transmit trypanosomiasis (17). Afterwards the cattles are transported to the south for marketing.
Consequently, the prevalence of these parasites in cattle and other domestic animals gives room for concern, as indications have occurred that T.b. gambiense and T.b. rhodesiense, to which man is the natural host, have been detected in several animal reservoirs, using molecular techniques. HAT described as Rhodesian sleeping sickness is caused by T.b. rhodesiense, resulting to acute disease course leading to death of infected persons within few weeks or months (4). In west and central Africa, HAT is caused by T.b. gambiense and is transmitted principally by tsetse flies, Glossina palpalis and G. tachinoides resulting to a devastating, chronic form of the disease described as Gambian sleeping sickness (Gambian trypanosomosis).
For many years, pigs have been identified as animal reservoir hosts for T.b. gambiense and recently has been associated with the persistence and epidemics of sleeping sickness in Uganda (18), Equatorial Guinea and Cameroon (19). It has also been associated with maintenance of old sleeping sickness endemic foci in Nigeria (20). Use of standard procedures such as, resistance to human serum based on the blood incubation infectivity test (21) and molecular characterisation of T. brucei stocks from man and animals by isoenzyme electrophoresis (22) and polymerase chain reaction based methods have led to the identification of more mammalian hosts as reservoirs for T.b. gambiense. Such hosts include dogs, sheep, cattle and a range of game animals. An estimated 60 million people are believed to be at risk while about 300,000 new cases are reported each year. Higher number of cases are likely to occur in several countries in view of the current upsurge in both forms of sleeping sickness (23). Going by WHO statistics of 1998, Gambian trypanosomosis has a wider geographical spread than Rhodesian disease with about 77.8% of HAT endemic countries suffering from T.b. gambiense (4). In Nigeria, although the exact sleeping sickness situation is not wellknown, there is increase in number of volunteer cases presented for treatment each year (24). Apart from the old Gboko endemic focus remaining active, outbreak of sleeping sickness in the new Abraka focus presently constitute a major health risk (24), resulting to several deaths. During an outbreak, out of 3,583 volunteers from 24 communities scattered around this focus, 359 were seropositive and 104 parasitologically positive for T.b. gambiense (25).
Identification of serum-resistance-association (SRA) gene in eight Trypanozoon isolates resistant to human serum has been reported by (26) while such genes were absent in isolates sensitive to human serum. Although this technique identified human-infective trypanosomes in cattle as reservoirs in the sleeping sickness endemic foci in Uganda arising from T.b. rhodesiense, it may similarly be used for identification of animal reservoir hosts in T.b. gambiense endemic areas (27) using PCR methods reported 8% T.b. gambiense infection rate in wild animals in the Bipindi sleeping sickness focus of Cameroon which was believed to be responsible for resurgence and perpetuation of the disease in the area. High prevalence of T.b. gambiense was observed in rodents (Atherurus africanus and Cricetomys gambianus), monkeys (Cercopithecus and Cercocebus) and ungulates (Cephalophus spp). Two small carnivores (Genetta servalina and Nandinia binotala) also harboured trypanosomes infective to man.
Similarly dogs, pigs, wild animals and bovids were identified as reservoir hosts in Liberia, Cote d'Ivoire and Burkina Faso based on isoenzyme electrophoresis, resistance to human serum and DNA analysis. The capacity of T.b. gambiense to proliferate and persist in alternative hosts in the absence of symptoms with prolonged maintenance of infectivity to the vectors makes it maintain a wide range of reservoir hosts which may differ from one endemic area to another. Adaptation of PCR technique for identification of T.b. gambiense and blood meals of both animal and human origins in tsetse flies (5), has paved way for determination of the roles of animal reservoir hosts in epidemics of Gambian sleeping sickness. The reports have shown involvement of Glossina palpalis in cyclical transmission of sleeping sickness from pigs to man in Cote d'Ivoire (28). Pig-tsetse-human, cattle-tsetse-human and sheep-tsetse-human transmission cycles have similarly been reported in three endemic foci in south-eastern Uganda (5).
Even though the role of animal reservoir hosts in the transmission of T.b. gambiense have been controversial current biotechnologies that differentiate between T.b. brucei, T.b. gambiense and T.b. rhode-siense in the tsetse vector and animals confirm the true zoonosis of Gambian trypanosomosis. Going by such findings maintenance of animal-tsetse-animal T.b. gambiense transmission cycle is not unlikely. The zoonotic nature of the disease seems to be further enhanced by the feeding pattern of G. palpalis group which apart from feeding on man, feeds on domestic pigs, wild ruminants and lizards (29). Animal reservoir hosts for T.b. gambiense has been identified as one of the principal factors associated with the persistence of Gambian trypanosomosis in endemic areas in spite of chemotherapeutic campaigns (27). For example, the presence of pigs in Mbini focus of Equatorial Guinea is believed to be responsible for the persistence of infection despite several years of treatment (30). This is supported by the ability of G. palpalis group to cyclically transmit T.b. gambiense from unlimited number of animal reservoir hosts, to man (28,5) and the difficulty associated with the control of trypanosomosis in game animals. Based on observations using human serum resistance, isoenzyme electrophoresis and DNA-test for molecular characterisation of trypanozoans leading to more definitive identification of T.b. gambiense in pigs and other domestic and wild animals, the role of animals as reservoir hosts for Gambian sleeping sickness and resurgence of the disease can not be questioned any longer. Going by the feeding habit of G. palpalis group on unlimited range of animal hosts, and the ability of T.b. gambiense to perpetuate in such hosts without clinical symptoms supports the fact that animal reservoirs indeed constitute important complications militating against eradication of Gambian sleeping sickness in sub-Saharan Africa. An integrated approach to the control of human and animal trypanosomosis is essential in the control of the current upsurge in human trypanosomosis and in limiting the present economic impact on Africa and tourism potentials of sleeping sickness endemic countries.
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M.C. Ezeani (a), H. Okoro (b), V.O. Anosa (c), C.C. Onyenekwe (d), S.C. Meludu (e), C.E. Dioka (f) & C.C. Azikiwe (g)
(a) Department of Immunology, College of Health Sciences, (b,c) Department of Veterinary Pathology, University of Ibadan, Oyo State; (d,e,f) Department of Chemical Pathology, (g) Department of Pharmacology, Faculty of Medicine, Madona University, Elele Campus; (a,d,e,f) Faculty of Medicine, Nnamdi Azikiwe University, Nnewi Campus, Anambra State, Nigeria
Corresponding author: Dr Michael C. Ezeani, Department of Immunology, College of Health Sciences, Faculty of Medicine, Nnamdi Azikiwe University, Nnewi Campus, Anambra State, Nigeria.
Received: 14 March 2008
Accepted in revised form: 14 September 2008
Table 1. Prevalence of trypanosome species in sera of cattle in Anambra and Imo states Species Anambra Imo Total No. of samples 132 132 264 T. congolense 48 (18.18) 62 (23.48) 110 (41.66) T. vivax 50 (18.94) 58 (21.97) 108 (40.91) T. brucei 33 (12.5) 53 (20.08) 86 (32.58) Figures in parentheses indicate percentage. Table 2. Prevalence of single and mixed infections of trypanosomes in sera of cattle in Anambra and Imo states, Nigeria Species Anambra Imo Total No. of samples 132 132 264 T. congolense 11 (4.17) 10 (3.79) 21 (7.95) T. vivax 10 (3.79) 10 (3.79) 20 (7.58) T. brucei 5 (1.89) 3 (1.14) 8 (3.03) T. vivax/T. brucei/T. congolense 18 (6.82) 35 (13.26) 53 (20.08) T. vivax/T. congolense 14 (5.30) 6 (2.27) 20 (7.58) T. vivax/T. brucei 8 (3.03) 5 (1.89) 13 (4.92) T. congolense/T. brucei 3 (1.14) 10 (3.79) 13 (4.92) Figures in parentheses indicate percentage. Table 3. Optical density range, showing prevalence of trypanosome antigen quantity among the species used in this study O.D. range T. congolense T. vivax T. brucei Anambra Imo Anambra Imo Anambra Imo 001-002 20 26 31 24 29 28 003-004 3 10 8 5 4 4 005-006 0 1 0 5 0 3 007-008 0 0 2 2 0 1 009-0.1 0 3 2 3 0 0 0.05-0.1 0 4 4 10 0 4