Abstract: A survey for the prevalence of trypanosomes species and the anemic status of trade cattle presented for slaughtering at Sokoto main abattoir, Northwest Nigeria was conducted between January and June, 2008. Blood samples were collected at ante-mortem via jugular vein and examined by Standard Trypanosome Detection Methods (STDM). Anaemic status was determined by Packed Cell Volume (PCV) and FAMACHA® Anaemic Guide technique while Polymerase Chain Reaction (PCR) technique was used to detect the presence of Trypanosoma brucei group. Out of 500 samples analyzed by STDM, 9 (1.8%) were positive out of which 6 (66.7%) had Trypanosoma vivax. The PCR technique detected 22 (4.4%) positive cases of Trypanosoma brucei group while 45 (9.0%) cattle were anaemic using the PCV and FAMACHA® techniques, respectively and White Fulani breed had the highest infection rate with 5 (55.6%) cases. These findings are significant as the study area has earlier been declared as tsetse free zone, transhumant activities as practice largely by the cattle herders may be responsible for these detections. Presence of other mechanical vectors may lead to rapid spread of the infection which may have adverse effects on productivity of the animals with resultant economic losses. The detection of the T. brucei group in the examined trade cattle may also portend danger to public health as some ruminants and pigs have been incriminated as reservoir hosts of the Human African Trypanosomosis (HAT) agents in some parts of Africa. Statewide surveillance is therefore, needed to establish the true prevalence of the infection in all domesticated animals in the study area.
INTRODUCTION
African trypanosomosis is a debilitating disease of man and domestic animals. It is caused by haemoflagelate of the genus: trypanosome, family: trypanosomatidae transmitted by tsetse flies (Glossina spo) (WHO, 1998) and its characterized by parasitaemia, fever, anemia, loss of condition reduced productivity and frequently high mortality which among the other factors limit the pace of rural development in tropical Africa (Abenga et al., 2002; Swallow, 2000; Fajinmi et al., 2007). Trypanosomosis complex is described by the World Health Organization (WHO) as serious diseases lacking effective control measure and all mammalian species are susceptible to the infection (Cattand et al., 2005). The lack of effective control measures may be attributed to the emergence of conventional trypanocidal drug resistant strains which may be overcome by the use of some medicinal plants and natural products (Bala et al., 2010; Faremi and Ekanem, 2011).
In Africa countries where trypanosomosis occurs, it is one of the major public health problems. The epidemiological trend indicates that it is wide spread in 36 sub-saharan countries for Human African Trypanosomosis (HAT) and 37 countries for Animal African trypanosomosis (AAT) covering over nine million square kilometers between Latitude 14°N and 29°S, approximately one third of Africa’s total land area (NITR Annual Report, 1989; Swallow, 2000; Steverding, 2008). About 60 million people and 50 million cattle are risk of being infected with up to three million livestock death annually. Less than 15% of cases in human are diagnosed and treated (Kristjanson et al., 1999; Kamuanga, 2003; Cecchi and Mattoli, 2009). The control of the disease has further been complicated by evidence of potential wild and domesticated animal hosts (Abenga and Lawal, 2005; Chreihen and Smoak, 2005). 21.3% of 402 small ruminants and pigs examined in Kenya were positive for trypanosomes of which 20 (5.0%) were positive for T.b. rhodiseiense through the identification of Serum Resistance Associated (SRA) gene by PCR technique (Ngayo et al., 2005).
The epidemic solely affects individual, families/communities and disrupts their economy posing a serious obstacle to agriculture and livestock development. Generally human population such as farmers, shepherds, herdsmen, hunters, tourists, merchants, researchers are at risk of exposure to trypanosomosis (PHAC, 2001; WHO, 1998). Human infection is caused by Trypanosoma brucei gambiense or Trypanosoma brucei rhodensiense resulting in sleeping sickness (Picozzi et al., 2002; 2005). Sleeping sickness is hallmarked by sleep and wakefulness disturbances with the sleep pattern fragmented (Lundkvist et al., 2004). T. vivax and T. congolense on the other hand affect the animals predominantly. The risk of transmission is primarily linked to the intensity of the encounters between vectors and host (Putt et al., 1988; Michel et al., 2002). Apart from tsetse flies which are major transmitter of the African animal trypanosomosis, other mechanical fly vectors have also been incriminated. Trypanosoma evansi in camels however are known to be transmitted mainly by haematophagus flies such as Tabanus, Lyperosae and Haematobia species (Kamal, 2008; Derakhshanfar et al., 2010).
Animal trypanosomosis has been ravaging several parts of the agro-ecological zones of Nigeria and the distribution follows the pattern of tsetse fly spread covering about 80% of the land mass between latitude 4°N and 13°N. Jos, Mambilla and Obudu highlands which had been hitherto declared as tsetse and trypanosomosis free zones are now infested (Onyiah et al., 1983; Onyiah, 1997; Dede et al., 2005; Ahmed et al., 2005). A recent report from a North central state of Nigeria revealed an overall trypanosome infection rate of 1.5% among sheep and goat over three seasonal variations (Samdi et al., 2010).
In Sokoto State, the status of animal trypanosomosis is not well documented as the state had been unofficially declared a tsetse free zone. Trannshumant activities intra and inter-state and across the international borders abound and may encourage introduction of the infection to the hitherto declared free zone. There is thus the need to establish and update knowledge on the existence of the infection in the study area in other to prepare effectively for control and eradication.
MATERIALS AND METHODS
Study area: The study was conducted between January and June 2008
at the Sokoto main abattoir. The nearby feeder cattle market is a converging
area for animals from the twenty three local government areas of the state,
neighbouring states and some other West African countries. Sokoto is within
Sudan Savannah zone, lies between latitude 10° and 14° North and longitude
3° and 9° East with mean annual rainfall of 1022 mm, mean relative humidity
of 50% and daily temperature range of 27 and 48°C. Majority of the rural
dwellers are predominantly subsistent crop farmers and herdsmen who migrate
with their animals during hot dry season towards the middle belt areas of the
country in search of greener pasture. There are sedentary animal population
within major towns that are kept semi-intensively and allowed to scavenge for
food and water or herded together to a common grazing and watering sources on
daily basis. Sokoto state has a land area of 102,500 sq km-1 (39,5655
sq miles), human population of 4,392,400 and an estimated population of 1,772,830
cattle (RIM Report, 1992).
Sample size: The abattoir was visited daily for seven days to estimate slaughter capacity per day. The obtained figures were pooled and averaged to obtain the sample size frame. Animals to be sampled were selected by systematic sampling technique whereby every nth unit in the population is sampled (Putt et al., 1988). The sampling interval (K) was determined by the use of formula K = N/n (where N= total number of animals available and n= the number rpequired in the sample). A random number (r) falling between one and K is then chosen by the use of card shuffling physical randomization technique (Cameron, 1999; FAO Animal Health Manual, 1999).
Clinical examination: The selected animals were examined at ante-mortem for classical clinical manifestations of trypanosomiasis signs such as fever, emanciation, bi-ocular discharge, enlargement of superficial lymph nodes and palour of mucous membrane. Eye colour chart (FAMACHA Anaemia Guide) was also used to detect change in colour of eye mucous membrane to determine the anaemic status (Grace et al., 2007).
Sample collection: Five milliliters of blood was collected from the jugular vein of each sampled cattle into ethylene tetra-acetic (EDTA) bottles, kept in cooler box and transported immediately to the Veterinary Public Health laboratory, Usman Danfodio University, Sokoto for processing. Likewise, a drop of whole blood from each sampled animal was placed on DNA binding matrix (Whatman® FTA classic cards); air dried, placed in container with hydroscopic dessicant and then stored at room temperature before processing (Picozzi et al., 2002, 2005).
Parasitological analysis: The obtained blood samples were analysed using the following Standard Trypanosome Detection Methods (STDM): Wet blood film, thick blood film, thin blood films, microhaematocrit centrifugation technique and animal inoculation method (WOAH, 2004). Motility in wet film and morphological appearance in Giemsa stained films were used to identify the trypanosome species. Organism with a free flagellum, very well developed undulating membrane and a small subterminal kinetoplast was classified as T. brucei organism with a medium sized marginal kinetoplast but without a free flagellum and inconspicuous undulating membrane was identified as T. vivax while organism with no free flagellum, inconspicuous undulating membrane and marginal and sub-terminal kinetoplast was identified as T. congolense (Stephen, 1986; Onyiah, 1997; Abenga et al., 2002).
Packed Cell Volume (PCV) was also determined for each of the blood samples examined as haematological index for anaemic conditions.
| Table 1: | Reaction mixture used for PCR amplification |
PCR analysis: DNA was extracted from Whatman® FTA cards that were spotted at the Sokoto abattoir as described by Picozzi et al. (2002, 2005). The samples were tested specifically against T.brucei using TBR-PCR due to its public health significance. T he TBR PCR uses the kinetoplast minicircle DNA of Trypanosoma brucei as a target and gives rise to a 177 bp PCR product. It can be used for the detection of Trypanosoma brucei infection in human, animals as well as tsetse flies (Katakura et al., 1997) and it is approximately equivalent to one parasite. The following oligo-nucleotide primers sequences were used in the PCR analysis:
| TBR1, 5, CGA ATG AAT AAA CAA TGC GCA CT 3. |
| TBR 2, 5- AGA ACC ATT TAT TAG CTT TGT TGC 3 (Artama et al., 1992). |
Amplification of DNA: PCR amplification was carried out in a 25 μL reaction mixture as shown in Table 1.
Thermal cycling: Thermal cycling was carried out in a Dyad Peltier Thermal Cycler using the following conditions.
| • | 94°C for 3 min |
| • | 94°C for 1 min |
| • | 55°C for 1 min |
| • | 72°C for 30 sec |
| • | Repeat steps 2-4, 35 times |
| • | 72°C for 5 min |
The PCR amplifications were conducted in 25 μL reaction mixtures containing as final concentrations: 10 mM Tris-HCI pH 8.3, 50 mM KCI, 1.5 mM MgC I2, 200 μM each of the four deoxynucleoside triphosphates (dNTPs) and 1 unit to Taq DNA polymerase (REDTaq SuperPak DNA Polymerase, Sigma). These concentrations were the same for both primer sets. 0.4 μM (TBR1/2). Two mililiter of DNA eluate extracted from a disk (2 mm) from the sample prepared on FTA® cards were added as template DNA to the final PCR reaction. A DNA-positive control, as well as a negative control, was used every time a set field samples was tested. Amplifications were carried out using a Peltier thermal Cycler (DNA engine DYAD). For TBR1/2, cycles consisted of an initial denaturing step at 94°C for 45 sec, an annealing step at 60°C for 1 min and an extension step at 72°C incubation for 5 min.
Gel electrophoresis: 1.5 g of agarose was added to 150 mL of 1x tris Borate EDTA (TBE) buffer (36 Tris, 30 mM NaH 2P0 4, 1 mM EDTA, pH 8.0) stained with Ethidium bromide (0.5 μg mL-1). This was poured into a 30 x 20 cm mould. 15 μL of the PCR product from the second round was run on this 1.5% Agalrose gel alongside a 100 bp graduation marker at 100 V for 1 h on a Bio Rad Power Pac 300 machine. The gel was examined on a Bio Rad Gel Doc 2000 using Bio Rad Quantity One software.
The total 25 μL of amplification mixture was loaded onto a 1.5% agarose gel and electrophoresis was processed for 40 min at 100 V. The gel was stained with Ethidium bromide and the samples were examined under UV light.
Data analysis: Spearman correlation coefficient (SAS, 1999 version) was used to examine the relationship between the eye scores, PCV and the trypanosomes. Arithmetic means and standard errors were also used to determine the relationship between trypanosome infected animals and their PCV.
RESULTS
Out of the total 500 cattle examined 199 (39.8%) were males with 5 (2.5%) positive cases and 301 (60.2%) females with 4 (1.3%) cases. The overall prevalence rate obtained was 9 (1.8%) out of which 6 (1.2%) were T. vivax, 2 (0.4%) T. brucei and 1 (0.2%) T. congolense. The haematocrit centrifugation technique detected more trypanosome species than others in the parasitological analysis (Table 2). The White fulani breed harboured most of the trypanosomes above other breeds encountered in the course of this study (Table 3).
The FAMACHA eye score chart rated as ordinal (ranking) scale (Thrusfield, 1997) indicating scores 1, 2 and 3 as non anaemic while 4 and 5 scores are anaemic. The obtained values for non-anaemic through FAMACHA corresponds to non-anaemic through the PCV values obtained and vice-versa for the anaemic animals (Table 4).
The haematological parameters obtained are as shown in Table 5 and there are significant variations in the normal values and the obtained values for infected and uninfected cattle.
Two of the rats that were inoculated with infected blood died four days later and the histopathology of the kidney revealed fatty degeneration, coagulative necrosis and liquefactive necrosis. Other haemoparasites encountered through parasitological analysis include Babesia bovis,
| Table 2: | Prevalence of bovine Trypanosomes species in slaughtered cattle (500) at Sokoto abattoir using STDM |
| Note: In parentheses are the percentages | |
| Table 3: | Breed distribution of Trypanosomes species in examined slaughtered cattle at Sokoto abattoir using STDM |
| Note: In parentheses are the percentages | |
| Table 4: | Presence of trypanosomes in relation to FAMACHA scores and PCV values in the examined slaughtered cattle at Sokoto abattoir |
| Table 5: | Average haematological values of trypanosome infected and uninfected slaughtered cattle at Sokoto |
| *p<0.05, **p<0.01, PCV = packed cell volume, TEC= Total erthrocytic count, TLC= Total Leucocyte count, DLC= Differential leucocyte count, Values are presented as Mean+SE | |
Babesia gibsoni, Babesia bigemina, Babesia divergene, Anaplasma bovis, Anaplasma centralis, Anaplasma maginale and Microfilaria species.
Out of the 500 blood samples examined for the presence of T. brucei group using the polymerase chain reaction technique, 22 (4.4%) were positive. The two positive samples from STDM were also positive at this level.
DISCUSSION
The study area is a converging place for trade animals coming from different local government areas of the State, neighbouring States and across the international borders of Republic of Niger, Republic of Benin, Republic of Chad and occasional from the Northern parts of other West African States. Also the animal rearing activities in the study area is basically on extensive method where there is little restriction to the movement of animals. All these make it difficult to ascertain the origin of the animals presented at the abattoir for slaughter, thus inability to trace back the infection (Thrusfield, 1997).
The Standard Trypanosome Detection Method (STDM) is a combination of different techniques used to avoid misdiagnosis of the infection. The microhaematocrit centrifugation technique detected most of the infection than others and this is in agreement with Omotainse et al. (2004) and Kalejaiye et al. (1995, 2004) that obtained a higher infection rates of between 8.5 and 15% in some ruminants by the use of buffy coat technique in some parts of Nigeria. Buffy coat and haematocrit centrifugation are still regarded as the best parasitological techniques for quick detection of the parasite as they concentrate the trypanosomes allowing for quick and clear visualization (Chappuis et al., 2005).
The obtained prevalence rate of 1.8% through the STDM is significant as the study area has been unofficially declared a tsetse and trypanosomosis free zone. This declaration might have been borne out of the fact that the study area is in the extreme northwest of the country and always experience high environmental temperature and low humidity all year round which is unfavourable for the development of tsetse flies (the major vector of the infection) (Ahmed et al., 2005). It has been found out that the Morsitans and Palpalis groups are most prevalent in the Savannah zones (covering mainly the northern parts of Nigeria) and are found along the drainage lines and riverine vegetation (Bourn et al., 2001). The study area is drain mainly by River Sokoto and River Rima and there is the tendency of tsetse flies that incure into this microclimate to survive and become focal source of infection. Biting flies such as Tabanids and Stomoxys have been incriminated also as mechanical vectors (Ahmed et al., 2005) and environmental conditions of the study area favour their development.
Most of the cattle reared within the study area are kept semi-intensive and extensively and there is seasonal migration of the Fulani herdsmen who are in possession of a large number of cattle herds during dry season towards the middle belt in search of greener pasture and water. The middle belt region is known to be endemic for trypanosomosis as there is abundance of tsetse flies. The exposed cattle at the beginning of rainy season in the northwest returned possibly with the infection and this can be spread by mechanical vectors on their arrival. Transhumant activities of the herdsmen encourages the long range flying tsetse to follow the animals along the cattle route thus spreading the infection among the sedentary herds closer to routes (Onyiah, 1997). Gibson and Brady (2008) reported that wild male and female tsetse are known to have the ability to travel at ground speed of 5-5-6.5 ms-1 and at angular velocity of 350-400° sec The white Fulani breed haboured most of the parasites in the examined cattle, this breed is the most predominant breed in the study area (RIM report, 1992) and are known to be highly susceptible to trypanosomosis (Putt et al., 1988).
Trypanosoma vivax was found to occur mostly in this study and they are known to be dominant species in ruminants in most parts of northern Nigeria (Omotainse et al., 2004). This parasite is haematinin which are found mainly in the plasma. Its infestation leads to rapid destruction of erythrocytes which leads to anaemia and rapid weight loss thus reduction in market value of the animals (Losos and Ikede, 1972). In Nigeria an estimated 837.2 million Naira annual losses from cattle has been attributed to trypanosomosis (NITR Annual Report, 1996). Death of two mice at the fourth day of inoculation with infected blood is suggestive of the presence of T. brucei group. The vascular tissue destruction at histopathology also lends credence to their presence. T. brucei groups are usually found in extravascular and intravascular areas leading to destruction of the invaded tissues resulting in abortion, perforation of intestine, haemorrhagic anaemia, lower milk production and central nervous system involvement (Anosa et al., 1992).
The overall average mean value of haemoglobin, PCV, TEC and TLC for uninfected cattle was higher than the infected ones. This may be due to destruction of red blood cells by the hematinin trypanosomes in infected ones (Anosa, 1988) and may also be attributed to the inhibition of erythrocyte formation in the bone marrow or their lysis by endotoxin liberated by trypanosomes. High environmental temperature as obtainable in the study area may equally altered the biochemical and haematological values alongside the presence of the parasites (Azab and Abdel-Maksoud, 1999; Anosa, 1988). The total WBC counts in all the infected animals were higher than the uninfected ones, this is as a result of the response of the affected animals to the infection (Anosa, 1988). There were no appreciable changes in monocyte and basophil counts between the two groups and this is in agreement with Rajkhowa et al. (2003) who found out that during naturally occurring trypanosomosis the values of monocytes and basophils are only slightly affected.
| Fig. 1: | PCR-TBR gel images of some positive samples from cattle slaughtered at Sokoto abattoir (Note: Lanes 5,6,9 show clear bands at 177 bp as in the positive control) |
Out of 500 bovine blood samples examined for the presence of T. brucei groups using polymerase chain reaction (TBR-PCR) technique 22 (4.4%) were positive (Fig. 1). The obtained value is higher than the STDM values in multifold and this is in agreement with Clausen et al. (1998) and Picozzi et al. (2002). Though a study in some two eastern states of Nigeria used ELISA technique to detect 3.03% infection rate due to T. brucei group (Ezeani et al., 2008) the PCR technique is known to combine the advantages of a greater sensitivity than the parasitological or immunological techniques with the added advantage of differentiating trypanosomes which have a similar morphology but very different economic impacts (Duvallet et al., 1999; Picozzi et al., 2002; Thumbi et al., 2008). The T.brucei groups have severe medical and economic impact on human and animals in most parts of sub-saharan Africa. The T.b gambiense causes a chronic, slow wasting disease that can eventually lead to death in West and Central Africa while T.b rhodesiense is acute and rapidly fatal, found mostly in East Africa. T.b brucei is found throughout the sub-Saharan Africa and have profound effect on animal population but its not infective to man due to its rapid lyses by human serum (Krafsur, 2009).
The use of PCR technique in this study has revealed that there are far more trypanosomes infected animals circulating in the study area than previously thought of, thus the need for a comprehensive epidemiological survey to establish the true prevalence of the infection in other to prepare effectively for its control.
ACKNOWLEDGMENT
The authors wish to acknowledge and most grateful to the University of Edinburgh, U.K. for graciously allowing us to carry out the Polymerase Chain Reaction (PCR) analysis in their laboratory. We are particularly indebted to Prof. Sue Welburne, Dr. Kim Picozzi, Miss O.O. Majekodunmi of Centre for Tropical Veterinary Medicine, Royal (Dick) School of Veterinary Studies, University of Edinburgh, U.K. for assisting with Whatman FTA® Cards,FAMACHA® Cards and in sample analysis.
Our profound appreciation goes to Mr. Ibrahim Ajiboso, Mr. Kehinde David and Mr. Jerome Anere for their computer and statistical analysis.