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Concurrent Infection of Fascioliasis andTrypanosomosis and Associated Risk Factors in Local Zebu Breed Cattle of Western Ethiopia

Background A cross-sectional study was conducted from late October 2016 to June 2017, with the primary objective of estimating and analyzing the concurrent occurrence of both fascioliasis and trypanosomosis infections and associated risk factors along the tsetse-infested Didessa river basin. Methods The methodology applied was based on stratified sampling for the parasitological study, with entomological and malacological surveys, including fly dissection. Results The result of variance-ratio testing between trypanosomosis and fascioliasis infections (mean prevalence 0.117±0.322 and 0.283±0.451, respectively), was statistically significant (P[F>f]=0), with higher observed fascioliasis infection (n=147, 28.27%). Severe anemia was observed in trypanosomosis infection, with mean packed cell volume of 19.57 (OR=0.71, P>|z|=0.000), and vast fascioliasis infections identified among cattle with medium and poor body condition in terms of weight (n=91 [32.73%] and n=38 [21.47%]). On entomological study, 578 (62.62%) and 345 (37.38%) female and male Glossina tachinoides fly species were cached, respectively, with overall mean flies/trap/day of 5.19 (n=923). Despite the prevalence of trypanosomosis in infected cattle, of 130 G. tachinoides flies dissected, only three were found to be positive for an infection rate of 2.31%. Malacological study identified three snail species known to maintain fascioliasis: Lymnea truncatula (n=28, 45.16%), Lymnea natalensis (n=23, 37.10%), and Biomphalaria (n=11, 17.74%). Concurrent infection with fascioliasis and trypanosomosis was mainly associated with the co-occurrence of their intermediate host snails and Glossina flies, respectively, with 4.42% (n=23) prevalence. Conclusion This study clearly demonstrated that the former parasite was highly associated with emaciation, whereas the second was responsible for anemia. In future,researchers should focus solely on estimating meat and milk production of local cattle to assess the economic impact of the study parasites.


Introduction
Ethiopia has a huge and diverse livestock population that plays an important role in the economy and livelihoods of farmers and pastoralists. Despite the excess animal population, production and productivity is scanty and even below the average for most countries in sub-Saharan Africa.This may be due to poor nutrition, reproduction insufficiency, management constraints, and prevailing animal diseases. 1 Among the diseases, fascioliasis and trypanosomosis are the major impediments to livestock development and agricultural production, specifically for pastoralists living along the major river basins. In Ethiopia, about 180,000-200,000km 2 of agriculturally suitable land in the west, southwest, and northwestern lowlands and associated river basins of the country is infested by tsetse flies, trypanosomes,snails, and helminthic parasites. [2][3][4] Among the genera in the Fascioladae trematode family, Fasciola is a helminthic parasite affecting mainly ruminant livestock. 5 Thecommonly known species of F. hepatica and F. gigantica are the usual causes of fascioliasis in livestock, while rare in humans. However, sometimes they can affect humans through drinking contaminated water or consumption of aquatic plants containing encysted metacercaria. 6 The parasite lives part of its life in aquatic snails that act as intermediate hosts and are found in and around wet areas, such as irrigation, water holes, and waterlogged areas. 7 African animal trypanosomosis (AAT) is a parasitic infection caused by an extracellular flagellate. Trypanosomes are unicellular protozoan parasites of the phylum Sarcomastigophora, order Kinetoplastida, family Trypanosomatidae, and genus Trypanosoma. 8,9 Trypanosomosis in Africa is mainly restricted to areas in which the vector, the tsetse fly (Glossina spp.) can survive. However, it may exist outside the tsetse-belt areas and transmitted mechanically by biting flies of the genera Tabanus, Haematopota, Chrysops, and Stomoxys. This type of transmission has caused the acyclic (mechanical) spread of trypanosomosis outside tsetse-infested areas. 9 The most known pathogenic trypanosomes are T. congolense and T. vivax, which are responsible for the most important form of AAT and prevalent species in domestic animals. 10 Both fascioliasis and trypanosomosis probably lead to high mortality and poor productivity. They also cause considerable economic loss through organ condemnation at meat inspection, reduced productivity (weight gain, milk yield, and traction power), rendering large areas of Ethiopia totally unsuitable for cattle-based agriculture, and lowered immunity (resistance) of cattle to other concurrent disease infections. 11,12 Despite the concurrent occurrence of fascioliasis and trypanosomosis infection in the region, scientific investigation and information on the topic was very scanty. Therefore, the primary objective of this study was to investigate/estimate and analyze the concurrent occurrence of these infections and associated risk factors in the tsetse-infested Didessa river basin.

Study Area
Bedele and Jimma Arjo districts are located 480 and 378.4 km west of Addis Ababa, respectively, at an elevation of 1,200-2,200 m above sea level. The climate alternates, with long summer rain (June-September), a short rainy season (March-April), and a dry winter season (October-February). The study area ( Figure 1) receives a mean annual rainfall of 1,200-1,-800 mm, with optimum temperature of 25°C. Agriculture is the mainstay of peoples' livelihoods, with a mixed farming system. Livestock plays an integral role in agriculture. 13

Study Design and Sample-Size Determination
This cross-sectional study was conducted from late October 2016 to June 2017 with the primary objective of estimating and analyzing the concurrent occurrence of fascioliasis and trypanosomosis infections and associated risk factors along the tsetse-infested Didessa river basin.
To obtain a representative sample area, stratified sampling was applied, as the target populations were all local zebubreed cattle of all age-groups and both sexes in each randomly selected study site. The sample size was determined using 15 expected disease prevalence in the area of 50%, since there had been no officially reported prevalence previously. A minimum 384 head of cattle was required, and 520 animals were included, increasing precision of the study: The methodology applied was based on stratified sampling for the parasitological study and entomological and malacological surveys, including fly dissection. Blood and fecal sample collection, laboratory presentation, diagnosis, recording of associated risk factors, and analysis of both study parasites were performed simultaneously to assess concurrent occurrence.

Blood-Sample Diagnostic Procedure
Paired blood samples were collected from the auricular vein (marginal ear vein) of each animal using two hematocrit capillary tubes after the animal had been restrained properly. Each tubewas filled to 75% of its height and sealed with crystal submit your manuscript | www.dovepress.com

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Veterinary Medicine: Research and Reports 2021:12 sealant. The first tube was used to measure packed cell volume (PCV) values for determination of anemia. 16 According to Murray et al, only cattle with PCV ≤24 are considered anemic. Then, the second tube was cut 1 mm below the buffy coat to include the top layer of red blood cells. The content of the tube was expressed onto a clean microscopic slide, mixed, and covered with a coverslip. Finally, prepared slides were examined for the presence of trypanosomes based on the type of movement in the microscopic field. Trypanosome species were confirmed after Giemsa staining and examination under oil-immersion microscopy (100× magnification) by morphological characteristics of the parasites. During sample collection, the body condition of each animal was categorized as poor, medium, and good, 17 and all associated risk factors, eg, age, sex, and altitude, were recorded.

Entomological Study and Fly Dissection
Monopyramidal traps (n =89) were deployed in the Didessa river basin of Bedele and Jimma Arjo districts. All traps were baited uniformly with octenol (1-octen-  19 The coordinates of each trap position were recorded using GPS, and a map of the study area was drawn. Dissection was carried out as described by Meharenet and Alemu, 20 starting by removing the wings and legs after wing-fray and ovary analysis had determined the age of male and female flies, respectively. Then, freshly killed tsetse flies were dissected under microscopy using 0.9% normal saline. Trypanosome infections in dissected body parts of tsetse flies (ie, midgut, salivary gland, and mouthpart or proboscis) were observed using a compound microscope (magnification 40×), and consequently parasite positive samples were again stained with Giemsa and examined under compound oil-immersion microscopy for trypanosome species identification based on morphology. 21,22 Coproscopic (Fecal Sample) Examination Fecal samples were collected directly from the rectum using sterile gloves after blood samples had been collected. The fecal samples were placed in a clean universal bottle preserved with 10% formalin and closed with a screw top in airtight condition. The bottles were labeled with unique identification numbers that matched the detailed data recorded using the standard format. The samples were transported to Bedele Regional Laboratory for gross and microscopic examination. For qualitative study, 23 a routinely used sedimentation-bead technique was used for identification of Fasciola eggs.

Malacological Study
Snails were collected by hand (gloved) or scooping from marshy grass lands and waterlogged areas around the Didessa river basin. In sum, 62 snails were collected, placed in plastic bags with fresh algae, aquatic vegetation, and aerated water, and transported to Bedele Regional Laboratory for identification. Snail identification was conducted at the laboratory based on morphology.

Data Analysis
Data processing was carried out using both qualitative and quantitative data analysis. For qualitative testing, tables, frequency distribution, and means ± SD analysis were some of the methods employed. For quantitative testing, prevalence rates and differences in mean PCV based on disease prevalence were analyzed using independent t-tests, and Chi-square 2 tests were used to estimate significance with adjusted ORs. Test statistics were used to assess overall disease prevalence with precision of 5% and 95% CIs. We used StataCorp for analysis.
Among the associated risk factors considered, body condition and anemia were found to be statistically significant (AOR 1.70, 95% CI 1.10-2.68, P=0.01, and AOR 0.13, 95% CI 0.06-0.27, respectivelyP=0) for fascioliasis and trypanosomosis infections. As shown in Table 2, animals with good body condition had less chance of being infected by fascioliasis, while nonanemic animals had lower risk of having trypanosomosis infection than animals with poor body condition and anemia, respectively. Higher prevalence of Fasciola  Table 3).

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From the entomological study, 578 (62.62%) and 345 (37.38%) female and male G. tachinoides flies were collected for an overall FTD of 5.19 (n=923. Despite the prevalence of trypanosomosis in infected cattle (n=130), G. tachinoides flies were dissected, and three found positive (two were harboring T. congolense and the remaining T. vivax) for an infection rate of 2.31%. All were identified at different sampling points ( Table 4).

Discussion
Bovine fascioliasis is one of the most important parasitic diseases in cattle, causing mortality and production losses in various parts of Ethiopia. It is a priority disease in both highland and lowland areas. 24 This fluke's distribution is worldwide in areas where cattle, sheep, and goats are raised and there is a niche for the Lymnaeid snail as a maintenance or intermediate host. 25 Similarly, AAT and its primary vector, the tsetse, are among the biggest constraints to sustainable livestock production in Africa, specifically Ethiopia. 26 Although extensive trypanosomosis and tsetse (T&T)-control operations have been running since the beginning of the twentieth century, tsetse infestation in sub-Saharan Africa and Ethiopia has hardly receded. 27 In the context of AAT or T&T control, achieving eradication of the disease has been highly debated and many disease experts believe that sustained reduction in disease incidence to a locally acceptable level ("control") is a more realistic target. 28 However, geographic variation in T&T-species distribution and ecoepidemiology of the disease and concurrent occurrence with other gastrointestinal parasites, mainly fascioliasis, make it highly harmful to livestock production. 29 Mathewos et al 29 suggested that concurrent infection of Trypanosoma and Fasciola was highly pathogenic, resulting in anemia and even death in cattle if not treated early. Contrary to this, the current study showed that animals with concurrent infections had better PCV than those thathad trypanosomosis infection only. Inconsistently with the present findings, Dwinger et al and Kaufmann et al 30,31 confirmed an increase in pathogenicity when helminthosis is aggravated by trypanosome infections. Furthermore, Mathewos et al 29 confirmed an improvement in body-weight gain and PCV levels when infected cattle were treated with a combination of trichlabendazole and trypamidium/samorin, which was consistent with this finding, suggesting that each parasite is highly responsible for anemia and loss of body weight (condition).
The conducted entomological survey confirmed the presence of G. tachinoides with trypanosomosis infection. When Glossina flies are infected with trypanosomes,they gate infection throughout their lifetime, which makes them cyclic transmitters of the disease. 32 The trypanosomosis prevalence found was due to the high Glossina fly density at the study area and confirmed trypanosome infection in densely distributed flies.
One of the most important factors inthe occurrence and prevalence of Fasciola infection in the study area was the availability of suitable snail habitats. 33 This study demonstrated that L. truncatula, L. natalensis, and Biomphalaria species are highly dominant snail species, and they are known intermediate hosts for fascioliasis. Other studies were also in agreement with this finding, which confirms that the distribution and occurrence of Lymnaeid snails results in high fascioliasis prevalence. [33][34][35][36] Conclusion Concurrent infection of fascioliasis and trypanosomosis was mainly associated with the cooccurrence of their intermediate host snails and Glossina flies, respectively. This study clearly demonstrated that the former parasite was highly associated with emaciation, whereas the second was responsible for anemia. The major findings of the present study strongly suggest that concurrent infection with Trypanosoma and Fasciola is the most harmful form of parasitosis in animals within the study area. In future, it is recommended that researchers focus solely on estimating meat and milk

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production of the local cattle to access the economic impact of the study parasites.

Data-Sharing Statement
The data sets supporting the conclusions of this research article are available upon request to the corresponding author.

Ethics Approval and Consent to Participate
The National Institute for the Control and Eradication of Tsetse Fly and Trypanosomosis and Ministry of Agriculture, Ethiopia authorized the fieldwork. The purpose of the study was clearly explained to the cattle owners and veterinary officers, and consent was obtained verbally from the institute's technical team and cattle owners. Participants' involvement in the study was on a voluntary basis. Those who were unwilling to participateor wished to quit were informed that they may do so without any restriction. This study did not involve any human/animal trials or experiments.