Molecular Identi cation and Prevalence of Animal African Trypanosomes Among Cattle Distributed within Jebba Axis of River Niger, Kwara State


 Background: Trypanosomiasis is a fatal disease that threatens the economy of at least 37 countries in Sub-Saharan Africa most especially livestock farming. In this study, we sought to investigate the prevalence of trypanosome infection in cattle, the potentials of these livestock as reservoirs of human-infective trypanosomes and the spatial distribution of trypanosome infected herds.Methods: The survey was conducted at the midland between the Northern and Southern part of Nigeria, an area perceived to have harboured migrating animals over the years due to insecurity in the north. A randomized cross-sectional study was conducted along the Jebba axis of river Niger, Kwara state by screening cattle from 36 herd clusters by nested PCR using ITS-1 generic primers. Data generated were analyzed using the Chi square test at 95% confidence interval. Results: Microscopic screening identified 3/398 samples representing 0.75% prevalence while twelve animals, representing 3.02% of the 398 sampled were detected as positive by PCR. Our result showed a decline in the PCV of infected animals (24.7%). The infection rate were categorized as single infection 11/12 (91.67%) and mixed infection 1/12 (8.33%). Animals were more susceptible to Trypanosoma congolense infection (50%) with T. congolense Savannah being the most prevalent Sub-specie (71.4%). Consequently, Trypanosome infections were more prevalent among female animals (4.30%), young animals (10.0%), White Fulani breeds (3.7%), animals with residency period of three years or less (3.18%), Transhumance animals (3.6%), animals with the diseases history (4.05%), animals with no history of drug administration (3.1%), animals close to river Niger (56.2%), larger herds (33.3%) and animals that have travelled to trypanosome endemic areas (50.0%). Aside age and distance of animals from river Niger, statistical difference in every other parameter tested were based on mere probabilistic chance. Spatial data showed that the disease is prevalent among herd located in less than 3Km distance from the river Niger which may represent a key risk factor. Conclusion: It is concluded that our study area may not be classified endemic but the epidemiological significance of this finding is that at least cattle populations may play a vital role in the maintenance and possible resurgence of the disease in the study area.


Introduction
African Trypanosomosis (AT) are parasitic diseases of public health concern, and limits agricultural productivity in almost all developing countries in Sub-Saharan Africa. The disease is caused by blood parasites, belonging to the genus Trypanosoma and widely transmitted in Africa by tsetse ies (1,2). In excess of 30 tsetse y species and subspecies pervade a territory of 33% of Africa's landmass and in uences animals and humans in at least 36 sub-Saharan Africa countries (2,3). It is generally classi ed as Neglected Tropical Disease (NTD), either as African Animal Trypanosomiasis (AAT) or Human African Trypanosomiasis (HAT) (4,5). In Africa, the disease affects around 100 million heads of cattle and in Nigeria, 6 million are estimated to be at risk out of a cattle population that is presently estimated to be Page 3/27 20 million (6). The World Health Organization (WHO) in the year 2000 gave an estimate of between 50 to 70 million individuals were at the risk of tsetse bites in Africa, which causes sleeping sickness. The disease advances rapidly from less virulent to a chronic animal disease. The actual number of cases on yearly estimate ranges from between 300,000 and 500,000 people and with a direct economic loss of between $4-5 billion in term of Gross Domestic Product (4,5,7).
In natural conditions, parasite transmission is majorly through nibbles of infected tsetse ies including Glossina species. In ruminants, three major species are responsible for these parasitic infections namely Trypanosoma congolense, Trypanosoma brucei and Trypanosoma vivax all of which are cyclically transmitted by tsetse ies and mechanically by other blood-feeding insects (8). In humans, Trypanosoma brucei gambiense causes chronic infection while Trypanosoma brucei rhodisiense causes acute cases (9), with recent reports showing cross infection of human-type trypanosomes in animals (10,11).
Identi cation of this parasite is very fundamental to evaluate the overall threat presented by tsetse specie (12). In recent years, epidemiology has been advanced tremendously with tools from molecular biology in the study of disease transmission and distribution. It has also permitted the zoonotic capability of unidenti ed agents to be determined (13). The utilization of conventional microscopic method is not adequate to recognize the trypanosomes in the host (14). A very sensitive diagnostic tool for speciation and identi cation of low cases of parasitaemia will help address the limitations associated with conventional strategies as regards parasite detection speci city and sensitivity (15). Though ELISA discovery was an incredible improvement in regards to sensitivity of pathogen determination, antigen detection using MAb-based ELISA is unreliable due to the presence of immune active agents in the blood even after animals are treated and cannot distinguish between active and cured infection (14).
Advent of PCR as a novel technique for distinguishing parasite DNA are more sensitive and reliable than those utilized previously. However, PCR with primer pairs does not differentiate band patterns in gels for speci c DNA segments and thus cannot differentiate isolates of the same species of trypanosomes and those that utilizes similar developmental sites such as the subspecies of T. brucei (16), and parasite species in the case of mixed and/or immature infections (17). Consequently, generic primers (12) has proven suitable for the identi cation of all known trypanosomes transmitted by tsetse in the ampli cation of ribosomal RNA gene loci within the internal transcribed spacer (ITS-1) region of the trypanosome genome. This is as a result of its inter-species length variation and high copy number. In this regards, species of trypanosome can be recognized by different band sizes produced due to DNA fragment ampli cation within the ITS-1 region (17,18). Various researches concentrating on trypanosomiasis transmission have been conducted in some parts of Nigeria. To date, there is no research interest that seeks to characterize and estimate the overall prevalence of AAT in Kwara State. Hence the need to study the occurrence and distribution of this parasitic infection in this agro-ecological zones of the middle belt.

Study area
This study was conducted along the Jebba axis of River Niger, Kwara State. Jebba is located on a geographical coordinate of 9°9′14″N 4°48′43″E with views of the River Niger. In light of its area, it is alluded as "Midland" and the "Door" between Southern and Northern parts of Nigeria (19). According to the latest census, the city's population in 2006 was 22,411 and it is approximately 500 Km away from Abuja, 306 Km and 600 Km from Lagos and Kaduna respectively.

Study population
Our study population consisted of mostly transhumance cattle, having the possibility of mixing with sentinel animals. For parasite identi cation, animals with recent administration of trypanocidal drugs were excluded from the study Study design A cross-sectional study was embarked upon that captures cattle distribution across the Jebba axis of river Niger, Kwara State and its tributaries to assess trypanosome distribution/infection across the geographical area, June 2019.

Sampling methods
Sample frame and sample size determination Systematic random sampling techniques was employed in this study. Herds of cattle in each coordinate were pooled and considered as a cluster from where animals were sampled by systematic randomisation.
Sampling frame was identi ed by listing herds (Myetti-Allah) locations across Jebba and samples were obtained in each cluster based on proportion to size of herd i.e. 6% per herd size is equalled to sample size per herd. Systematic random sampling technique was used to select animals in each of the randomized cluster whereby the sampling interval was generated by dividing the herd size by the sample size required for that herd. The rst study subject was randomly selected from among cattle, while others to be included in the sample were selected after every jth interval (20). Due to the complete absence of previous record on the prevalence of trypanosomiasis in our study area, 50% prevalence was assumed and the sample size was estimated in accordance with the method of (21). A minimum total sample size of 384 was drawn across all the identi ed clusters. The Sample size calculation was based on the assumption of a 95% con dence level, 50% assumed prevalence, 0.05 tolerable error. In each cluster, all animals regardless of the health status was considered in randomization so as to give an overall current infection rate status of the herd. Animals aged one year and younger were considered as young calves, while those over one year were regarded as adults. Dentition was used to determine the ages of animals while body conditions score (BCS) were assessed and adequately scored. Other parameters like breed, sex, source and location of the cattle were recorded. Herd data was collected to include the residency, travel history, herd size, history of trypanocidal treatment and history of disease.

Sample Size
Sample size was estimated as given below (21): Z = Con dence level/Z-score, p = Assumed Prevalence, q = Complementary probability, d = percentage error. Ninety ve percent con dence level was used which statistically equate to 1.96, and which also amount to a percentage error of 5%. Estimate for the sample size of the cattle is then given as: The assumed prevalence of trypanosome infection for the said region stood at P=50% Proportion to size of herd Sample collection and parasitological analyses Five milliliter (5 mL) of blood samples were collected from the jugular vein of each randomized animal using a sterile vacutainer needle into tubes containing anti-coagulant (Ethylenediaminetetraacetic acid) (7). Each sample were identi ed by a unique barcode system that correspond to the name of the village, herd cluster and sample number. The samples were transported in ice box to the laboratory and stored at 4 o C prior to laboratory analysis. Parasitological examination was done in the laboratory using the standard trypanosome detection Method i.e. Hematocrit Centrifugation Technique HCT (22), Buffy Coat Method, BCM (20), parasite load estimation (23) and Giemsa stained thick and thin lms (24). The Packed Cell Volume (PCV) of each animal was also determined while the parasites were identi ed (25,26) DNA template extraction and PCR cycling As prescribed by the manufacturer, Quick-gDNA™ Mini-Prep kit from Zymo Research Corporation, Irvine, CA, USA was used for gDNA extraction from the blood as prescribed by the manufacturer. DNA yield and purity assessment was done using Nanodrop ND-100 UV Spectrophotometer (Nanodrop Tech., Inc. DE, USA) while the DNA eluted was stored at -20 0 C until further use (20). PCR ampli cation was carried out with slight modi cation (12). In the rst round of the reaction, three microliter of DNA was added into the reaction mix. PCR was performed in a total reaction volume of 25 µL containing 2.5 µL standard Taq buffer, 1.0 µL dNTPs, 1.0 µL of each primer (25 µM), 0.25U µL Taq DNA polymerase, 3.0 µL template and nuclease-free water was added to a nal volume of 25 µL. Cycling conditions was set as follows: For nested PCR, two sequential runs were done using two primer sets. In the rst reaction run, TRYP 3 and 4 were used as outer primer followed by TRYP 1 and 2 which served as inner primers in the second reaction run. From the rst run, 2.0 µL of the PCR product from the rst run was added to 23 µL of the mix in the second round of the reaction and in a fresh PCR tube. Cycling conditions were the same as the standard PCR cycling (Except for 1 O C rise in annealing). Positive and a negative control was included in each set of reaction run (27). Reference DNA used in testing the sensitivity and speci city of primers was T. congolense specie identi ed by microscopy from the eld. The ampli ed DNA was resolved on 2% agarose gel, visualized under a UV trans-illuminator and photographed with a Gel documentation apparatus (Molecular Image Gel-doc with Image Lab Bio-Rad Lab. Inc. Framework V 3.0) for clear visualization and reference purposes. The nucleotide sequence of the primer used for the PCR is as shown below (12).

Inner primers
DNA Sequencing and phylogenetic analysis The PCR products were puri ed and 20µl of the PCR products was sequenced using Big Dye Terminator Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA). As described by Applied Biosystems, Product of PCR were sequenced using Applied Biosystems Cycle Sequencing Kits (BigDye Terminator v1.1 and v3.1 kits). The sequence data obtained were viewed on Finch Trace Viewer v.1.4.0 (Applied Biosystems, Foster City, CA, USA), while anking regions of high Noise-to-Signal ratio were trimmed off the sequence to improve the accuracy and precision of sequence data obtained. Ambiguous nucleotides were edited and replaced with conventional ones based on the highest peak recorded on the electropherogram. Each edited sequence were BLAST searched against the DNA sequence database (NCBI) and/or the published databases for various trypanosome species (Tri-Tryp) explicitly for trypanosomes. Sequences of the ITS-1 region were aligned using ClutalW against known sequences in order to con rm species identity. Molecular Evolution Genetic Analysis Version 7.0.2.6 (MEGA7) was used to construct phylogenetic tree to observe their evolutionary trend and variation over time.

Statistical Analysis
The results obtained from this study were subjected to descriptive statistics to determine the frequency and distribution of trypanosome infection across the study area. The prevalence rates among localities, breeds of cattle, age and sex of the animals was expressed as percentages of the total number of animals sampled. This was done by dividing the number of infected animals by the total number of animals examined and expressed as percentages. Categorical values were evaluated using the Chi Square to measure the strength of between variables at 95% con dence interval. All data obtained were analyzed using SPSS statistical software version 20.0. Values of P < 0.05 were considered signi cant

Results
Across the study area, seventy two (72) herd clusters were identi ed as sample frame from where 50% of the herds representing 36 clusters were balloted by simple random sampling. In all, 398 blood samples were obtained from across the study area, three of which were screened positive by microscopy, representing 0.75% prevalence, while twelve samples representing 3.02% were tested positive by nested PCR with distinct band sizes characteristic of the specie involved. Sequencing of the PCR products and bioinformatic analysis (http://blast.ncbi.nlm.nih.gov/Blast.cgi) further validated the species and subspecies of the Trypanosomes.

Discussion
Low prevalence reported in this study by microscopic screening (Table 1) is not surprising considering the low sensitivity imminent of parasitological diagnostic method (28, 29,30). This is especially so for eld animals characterized by low parasitaemia. The superiority of PCR over MHCT have been widely demonstrated in the epidemiological study of animal trypanosomosis (3,20,32,33). These differences are due to sensitivity thresholds of the techniques. As against the prevalence by microscopy, nested Polymerase Chain Reaction (PCR) method gave an overall prevalence of 3.02%. Each species tested produced amplicons of between 200-700 bp in length ( Figs. 1 and 2). The ITS-1 PCR product size of T. evansi was similar to that of T. brucei and sequencing analysis was key to differentiating between the two PCR products. Sample JT11 was further con rmed to be T. evansi (supplementary data) suggesting the possible role of cattle as reservoirs of T. evansi. Generally, bands obtained from the ampli cation result were in agreement with previous studies (12,31,34,35,36,38,39,40,41). Samples JH4 and JM8 resulted in band sizes of approximately 700bp and which were con rmed by Sequence analysis to be Trypanosoma congolense Savannah sub-specie. Molecular characterization of Trypanosoma species using ITS-1 generic primers and/or its slight modi cation gave an estimated range of ITS-1 band sizes with a maximum amplicon length of 640bp (12) but went further in noting that all species ampli cation using generic primers could lead to a bands size of between 150-750bp in length as evidenced in this study and those previously reported (17,31,39,42). An amplicon length of 210bp for T.vivax was not reported previously which may be an indication that T. vivax's 18 rRNA is fast evolving at 7 to 10 times the rate of non-salivarian trypanosomes and also signi cantly evolving faster than all other trypanosomes. Although, no living specimen of this trypanosome was isolated, our conclusion could only be based on DNA sequence analysis while its taxonomic relationships were deduced from phylogenetic analysis of the ampli ed ssuRNA (70). Further biological characterization will depend on isolation of a living specimen into culture. The ability to identify this trypanosome by the distinct size of the ITS-1 region has provided preliminary information on its distribution and prevalence that should help track it down in the eld. To make an evolutionary inference, all isolates aligned with the salivarian group except JO6 (Trypanosoma theileri) which fell in the stercoraria group. ssrRNA from JT11 (T. evansi), JAD7 (T. brucei), JAG2 (T. brucei) fell in the same branch but different clades which depicts a logical evolutionary event emanating from within these species (Fig. 3). Trypanosoma evansi is widely known to have evolved from Trypanosoma brucei and all of which were rooted on Trypanosoma vivax (16).
A drastic fall in PCV is traditionally considered a warning sign of the trypanosomiasis (43,44).
Classically, infection with trypanosome species that are pathogenic in local breeds of cattle result in retarded growth and anemia while nutritional status is a determining factor of infection (45,46). From our ndings, there is a PCV declined in trypanosome-positive cattle ( Table 2) possibly due to the effect of parasites on blood cells. Similarly, the average hematocrit values varied between cattle breeds. However, the very low PCV presented by the red bororo breed may not be a true re ection of the PCV trend as only one animal was screened positive. The type of trypanosome specie infection impacted differently on the PCV of animals with average falling below the standard obtainable for cattle (24%-46%) except for the animals infected with Trypanosoma brucei with an average Packed Cell Volume of 30.3 ± 0.92. The animal infected with Trypanosoma theileri, a non-pathogenic trypanosome of cattle had the lowest PCV value (19.2 ± 1.12) ( Table 3). From our ndings, it may be illogical to conclude that this comparative decrease in the PCV is due to T. theileri infection as only one animal was infected. However, it is possible that the parasite may have transited from a non-pathogenic to pathogenic form, hence the need to have a controlled experiment aimed at monitoring the PCV in the face of Trypanosoma theileri infection and other trypanosome species. This study showed 3.02% overall prevalence which compares well with 4.3% national prevalence as reported by European Economic Commission project of 1989 and 1996 (47), 3.9% in Ogbomosho (48), 4.69% in Oyo (49) and 9.4% in Kaduna (50) as against high prevalence of 53.4% in Kaura, Kaduna (51) and 46.8% in Jos (52). These contradictory ndings might re ect seasonal or local differences in tsetse populations, sample size and site, improved sensitization among nomads on grazing course, better implementation on the use of trypanocidal drugs and urbanization which may have perturbed the ecology of the transmitting vector, leading to ecological migration to a more favourable ecosystem, hence low prevalence recorded in our study.
Although there is no any signi cant difference in the infection rate between male and female animals (Table 4), our results showed that females were more infected. This observed differences may be attributed to livestock management adopted in the farming community where larger numbers of males are frequently sold off the herd at any early age while the rest are kept for breeding or animal traction. Also female animals persist longer in herds for the purpose of breeding, thus allowing the chronic infection to be maintained for very long period. As a result, the remaining males are more closely monitored while the females are readily exposed to hazard in the population vis a vis multiple copulation with limited male animals in the herd. Also the larger population of females (59%) obtained in this study by simple random sampling may account for this difference. Previously, 199 male cattle and 121 female were examined with no statistical difference in the infection prevalence (49). However, occurrence of any disease is dependent on many factors of which sex is just one of them. Factors other than sex relating to the host or its environment could therefore have played a role in in uencing the susceptibility of animals to infection which has been documented in several studies (8, 10, 53).
From our study, there is a decreases in disease prevalence as animals get older probably due to ageacquired immunity which could represent a key positive factor and bearing in mind that trypanocidal treatments are more frequently used on adults by local farmers. In addition, young animals are more vulnerable to tsetse bites due to their skin fragility. Moreover, they are not agile enough to ward-off insects away along the grazing route as the adults. The tsetse ies also frequently target weak animals as a source of food in order to avoid being crushed by moving animals (54). In this study, despite the very low number of young animals randomly sampled, prevalence of 10.0% (7/70) for younger animals and 1.5% (5/328) were recorded for adult which is statistically signi cant (P < 0.05) ( Table 4). This indicated that the incidence rate was not similar in young and adult animals (8). Although not signi cant, the infection rate differ among cattle breed. The prevalence of trypanosome infection was lowest in Sokoto gudali (2.3%), a breed not known for trypanotolerance (56, 57), and may have resulted from adaptability of this breed to its environment. The higher prevalence observed among the White Fulani breed may be attributed to their trypano-susceptibility and perhaps due to their higher representation in the sampling (52.8%). Of the four cattle breeds studied, the White Fulani are usually raised under the nomadic system of management. This may be another possible explanation for the higher prevalence recorded by this cattle group (10).
Although our test of statistic showed no signi cant difference in the infection rate among cattle in regards to period of residency, the infection was more prevalent among animals that were recently domiciled (3.18%). This observation may well be attributed to recent in ux of herders down south due to insecurity and ban on open grazing in some parts of Nigeria and which has forced nomadism away from the north. Similarly, our ndings revealed that infection was found to be more prevalent among transhumance animals (3.6%) probably due to exposure to tsetse bites while pervading territories of different endemic locals in a bid to having greener pastures. Sentinel animals appear to be more protected from tsetse bites due to livestock management style and guided path to grazing by cattle keepers. Despite the difference in infection rate, the statistical test showed that the observed difference was due to mere probabilistic chance (Table 4).
Having excluded animals with recent administration of trypanocidal drugs, a high prevalence of the disease was noted among animals having disease history (4.05%). An explanation to this could be that animals having the disease history may have not been well treated to clear the parasite in their blood in the rst place or it may be that the treatment administered during the last infection may not be effective/e cacious or the parasite itself may have developed resistance to the administered drugs. During our survey, 92% of sampled animal had history of trypanocidal drugs treatment (Table 4) which may explain the low infection prevalence generally recorded with possible indications of drug resistance as seen in animals known to have had history of the disease and treatment but still reported in the study as infected animal.
The prevalence of trypanosome infection was signi cantly higher in locations closer to river Niger as compared to those further away (Table 5 and Fig. 6). This may be attributed to differences in herd management practices, grazing route which predisposes the herd to tsetse bites, herd composition and frequent exposure to trypanocidal drugs which may differ in each herd. The river could be a positive factor for the vector transmitting the disease as well as a source of water for grazing animals which could expose them to risks of bite by riverine species of the ies (58).
Although not signi cant, the disease rate was high among larger herds (33.3%) as compared to smaller ones (23.8%) ( Table 5). It may be that smaller herds are more closely monitored and easily managed and treated before the transmission sets in as compared to larger herds where animals are seen as a single entity. Furthermore, animals that had travel history to trypanosomiasis endemic zone of the country (Benue, Jos, Kaduna, Delta, Oyo and Ogun as published in literatures) were more infected (50.0%) probably due to contact of travelling animals with infected sentinels in endemic zones and exposure to tsetse bite during trans-boundary movement.
Majority of the trypanosomes in cattle were T. congolense and T. brucei which accounted for 50.0% (6/12) and 16.67% (2/12) (Fig. 5) respectively with nearly half of the overall infection due to Trypanosoma congolense Savannah sub-specie (Fig. 6), possibly as a result of large host range or probably due to the fact that riverine species of tsetse are generally considered susceptible to T. congolense infections (69). High prevalence of T. congolense infection is an indication of the dominance of G. mosrsitans species of the y (7,8,18,20,52,59,60,52) and could be that its transmission is highly favoured by the obligate cyclical vector or the T. vivax and T. brucei respond better to the trypanocidal drugs, diminazene aceturate and homidium chloride administered by farmers. A high prevalence of the Savannah subgroup in cattle may also indicate that the parasites were introduced recently into the tested herds coupled with its reported virulence as compared to other sub-specie (55). The low prevalence of T. brucei infection may relate to reported resistance of indigenous West African cattle to the parasite (61). However, the detection of T. brucei and T. evansi in Nigerian cattle might portend serious danger not only to cattle and other livestock but also to livestock owners and the host communities at large as T. evansi infection has been reported in cattle and humans in India (62, 63). Low prevalence of T. simiae infection is an indication of low transmission of the parasite as animals infected with this species will probably not survive the acute and severe nature of this parasite (64, 65). Double infections in animals are a normal occurrence in the eld (66). This study identi ed T. congolense Kili and T.vivax mixed infection in only one of the herd clusters. In Nigeria, previous surveys identi ed mainly T. congolense and T. vivax as animal pathogenic trypanosomes (67) and co-circulation has been reported in studies conducted in northern Nigeria (20,41,68). Co-infections with multiple Trypanosoma species have also been documented previously due to bites from tsetse ies carrying more than one Trypanosoma infections or successive bites from ies with different Trypanosoma species (8, 16, 37).

Conclusion
Within the Jebba axis of River Niger, an overall prevalence of cattle trypanosomosis by PCR was 3.02% as against 0.75% recorded by microscopy. Despite the low prevalence reported in this study, the present ndings should be of interest to veterinarians and health workers. Sex, breed, animal stay period, animal origin, disease and treatment history did not signi cantly in uence the rate of trypanosome infections (Tables 4 and 5). However, the test of statistics showed that age and relative distance of the herds to River Niger may be a contributory risk factor in the disease prevalence. This study has evidenced the circulation of six trypanosome species with all isolates having appreciable homology (> 80%) with what was already established in the NCBI database. Comparatively with set threshold of EEC (4.3%), the study area may not be classi ed endemic but the epidemiological signi cance of this study is that at least cattle population may play important role in the possible resurgence of the disease in this region. Factors such as geographical distribution of all trypanosome species can be used as a guide to improve control measures. The knowledge and awareness of trypanosome infection will also enhance concrete humanbased control measures in this local. This situation has determined the potential zone to be placed under surveillance in the case of disease outbreak in the country.

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