Biting Flies and Associated Pathogens in Camels in Amibara District of Afar Region, Ethiopia

Biting flies and associated pathogens are the major health constraints on camel production and productivity and are implicated in causing significant economic losses in the pastoralist community in Ethiopia. A cross-sectional study was conducted to estimate the prevalence of biting flies and their associated pathogens in relation to different risk factors in camels in the Amibara district, from October 2019 to April 2020. A total of 480 camels were examined for biting flies and associated pathogens. The study revealed that overall, 87% (418/480) and 18% (87/480) of camels were infested by one or more biting flies and infected with Trypanosoma evansi during the study period, respectively. The collected biting flies were identified into a total of 3 genera: Hippobosca, Stomoxys, and Tabanus under the stereomicroscope. In the present study, Hippobosca (40.4%) was the most prevalent biting fly, followed by Stomoxys (31%) and Tabanus (28.6%), which affected camels in the study area. Among camels infected with Trypanosoma evansi, 7.3% and 16% were positive for parasitological and serological tests, respectively. Age, body condition score, and season appeared to have a significant effect (p ≤ 0.005) on the prevalence of biting flies and T. evansi on dromedaries. According to the findings of this study, biting flies and Trypanosoma evansi were the most common limitations on camel health, production, and productivity in the study area. As a result of the possible threat of biting flies' infestation and Trypanosoma evansi on camels, all-around attention is required in terms of strategic acaricide application, proper antiprotozoal drug use, and raising knowledge about acaricide use to prevent and control biting flies' infestation.


Introduction
Ethiopia has the largest livestock population in Africa, but its contribution to the country's economy is still low, and diseases are a major constraint [1].Livestock population in Ethiopia is estimated at 70 million cattle, 57 million poultry, 52.5 million goats, 42.9 million sheep, 10.8 million donkeys, 8.1 million camels, 2.15 million horses, and 0.4 million mules [2].In addition to producing milk, meat, and skins and hides, animal resources play an important role in providing export commodities, such as live animals, hides and skins, and honey, contributing 8.3 percent of total GDP and about 20.2 percent of agricultural GDP [1].
According to the Central Statistical Agency's [3] report, Ethiopia is ranked sixth in Africa in terms of its camel population.Te distribution of camels is signifcantly infuenced by their ability to withstand extreme heat and desiccation.Camels are normally distributed throughout subtropical dry areas in Africa and Asian countries [4].In Ethiopia, camels are found in arid and semiarid parts of the southern, eastern, and northeastern parts of the country, mainly in the Borana, Ogaden, and Afar regions [5].
Dromedaries, the one-humped camel (Camelus dromedarius), is the only type of camel that exists in Ethiopia [6].Camellus dromedaries are an important animal's species in the pastoral economy in Ethiopia because of their extraordinary ability to perform in arid and semiarid environments where there is scanty vegetation, which is not sufcient for other livestock species.Te camel is a multipurpose animal especially adapted to arid and semiarid environments, enabling nomadic peoples of the world to live in difcult environments.Camel is primarily kept for milk production, meat production, draft power, transportation, and as a draft animal in agriculture [7].It also serves as a fnancial reserve and has a signifcant impact on social prestige and wealth.Despite its signifcant contribution to the pastoralist society's livelihood, there is very little scientifc information on the health and productivity of camels in Ethiopia [8].
A slow reproduction cycle, high calf mortality, and other health problems are among the major constraints contributing to the decreasing camel herd population and productivity [9].Ectoparasites are accountable for decreased camel production and reproduction, as well as leather quality deterioration, downgrading, and skin rejection [10].Biting fies and their associated pathogens are important constraints to the production, productivity, and performance of camels and other animals [11].
Tabanus and Stomoxy are hematophagous insects most commonly reported from camels and are responsible for noncyclical transmission of trypanosomes in camels in various parts of the world.Trypanosoma evansi is mechanically transmitted in camels by the bites of haematophagous fies such as Tabanus and Stomoxys [12].Te essential biting fies for the spread of T. evansi are Tabanus species [11].Biting fies belonging to the genera, Tabanus, Stomoxys, Chrysops, Hippobosca, and Lyperosia, have been reported to afect camels in Ethiopia [13,14].Rainfall, moisture-retaining clay soil, and surface water pools where Acacia senegal shrubs grow abundantly are all conducive to the vector's survival and reproduction [11].
One of the most common diseases in camels is trypanosomosis [13,15].Tis disease is caused by Trypanosoma evansi, which is mechanically transmitted by hematophagous biting fies, particularly tabanids [13].Te disease causes a signifcant productivity loss in camel herds and even mortality in the acute form [15]. Trypanosomosis is widespread throughout the world and a major constraint to camel productivity [13,16,17].
Early detection and taking major action are important to control blood-sucking arthropods instead of waiting till the problem gets severe [18].Biting fies can be efectively controlled by avoiding the fies' habitat, avoiding heavily wooded and grassy areas, using repellents, and performing frequent fy checks before they can transmit disease [19].Regular studies on the dynamics and species composition of biting fies and other parasitic arthropod populations, coupled with the present efcacy status of acaricides against the most prevalent and economically pertinent biting fies and other parasitic arthropod species of an area, are necessary to carry out effcient control [20].A comprehensive understanding of the genus identity, composition, seasonal dynamics and variation, and epidemiology is critical for preventing and controlling economically important parasitic arthropods [18].
Despite the existence of a large population of camels in Ethiopia and their great social and economic importance to the pastoralist community, there is no documented information on the genus of biting fies in camels, their associated pathogens, and associated risk factors for camels in the study area.As a result, the current study was designed with the objective of estimating the prevalence of biting fies and their associated pathogens in relation to diferent risk factors in camels in the Amibara district.

Description of the Study Area.
Te Afar National Regional State is found in the eastern region of the country, situated between 39 °34′ to 42 °28′ E longitude and 8 °49′ to 14 °30′ N latitude.Te region covers approximately 270,000 square kilometers in terms of its overall geographic size, and over 90% of the people living there are engaged in pastoralism.Te other 10% consists of agropastoralists and commercial farmers who have recently emerged due to the development of small-scale irrigation along permanent and temporary rivers.Te area is situated alongside Eritrea in the northeast and Djibouti in the east.It is also delimited by the regional borders, with Tigray Regional State to the northwest, Amhara to the southwest, Oromia Regional State to the south, and on the southeast with the Somali Region of Ethiopia [21].
Te climate of Afar Regional State is arid and semiarid, with low and inconsistent rainfall.Te region's altitude ranges from 120 meters below sea level to 1500 meters above sea level.Te temperature ranges from 20 °C at higher elevations to 48 °C at lower elevations.Te region's rainfall is bimodal, with a mean annual rainfall of less than 500 mm in the semiarid western escarpments and less than 150 mm in the arid zones to the east.According to the Central Statistical Agency of Ethiopia (CSA), the region has a total population of 1.8 million people and is administratively divided into fve zones, 34 administrative districts, and 358 pastoral associations (PA) [21].
Amibara district is located about 250 km to the northeast of Addis Ababa and has 18 PAs (the least administrative units) with total population of ∼63,378, of which 35,374 are men and 28,004 women.Te districts have 2 small urban and 30 rural PAS.Te livestock populations of the Amibara district are composed of 103, 959 cattle, 122, 526 goats, 48,043 sheep, 3,888 donkeys, and 39,995 camels [21].
Te current study was conducted in the Amibara district (Figure 1) of the Afar region's Gabi Rasu zone (zone 3) in the Middle Awash Valley.Tis study area was selected purposefully based on the number of camels found in the area.Te district has arid and semiarid agroecologies, and livestock production is the community's main occupation.Te distribution of rainfall is inconsistent and occurs in two distinct periods.Te frst period, known as the long rainy season (kerma), happens from mid-June to mid-September.Te second period, called the short rainy season (Sugum), takes place between March and April.Te typical yearly precipitation ranges from 400 to 600 mm.

2
Veterinary Medicine International

Study Population.
Te study animal consists of camels of all ages and sex groups residing in the Amibara district of the Afar region.Individual camels selected with diferent criteria were included in the study.Te following information was collected: age, sex, body condition, season, herd composition, herd size, and previous treatment history.In the study area, certain camels are coherded (kept) with cattle, sheep, and goats.Body condition was determined using the references by the authors in [22,23], who classifed dromedary body condition scores as poor, medium, or good.Tis study included a total of 480 camels of various ages and both sexes.All camels included in this study were examined for biting fies and associated pathogens.Samples were collected from 160 animals during each of these three seasons: the cooler weather/wet/Gilal (November-December), shower/dry (Daba) January-February, and short rain/Sugum (March-April) seasons for seasonal variation of biting fies' distribution and their associated pathogens [24].

Study Design and Sampling
Techniques.From October 2019 to April 2020, a cross-sectional study was conducted with the primary objective of identifying the genus of biting fies infesting camels and their associated pathogens, estimating their prevalence, and assessing risk factors associated with biting fies' infestation and associated pathogens in the study district.Te current study included only camels that had not been treated with any acaricides and antiprotozoals, for a month prior to sample collection.
According to Toma et al. [25], a combination of convenience and purposive sampling methods was used.First, the study district was chosen based on its camel population and accessibility to the area.A complete list of pastoral villages (sampling frame) was obtained from eight Amibara pastoral associations (PAs) chosen based on accessibility.Tree to six herds have been selected from among the volunteer herds.Te sampling method for camel herds was also purposeful (based on the owners' willingness) and involved simple random selection for the individual study animals.Depending on the number of camels available in Veterinary Medicine International each herd, an average of 8 (7-9) from herd sizes less than 20 (twenty), 15 (10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20) from herd sizes 20-40 (twenty to forty), and 25 (20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30) from herd sizes greater than 40 (forty) were randomly selected from each herd.Each of the animals selected as sampling units was examined for biting fy infestation and was categorized as positive or negative based on the presence or absence of biting fies on the animals' bodies.

Sample Size Determination of the Study Camels.
Because no previous estimate of the prevalence of biting fies' infestation and associated pathogens in the district existed, the sample size was calculated using a 50% expected prevalence, a 5% desired absolute precision, and a 95% confdence level or interval (CI) [26].Using the previous formula provided by [26] to calculate sample size (n) is as follows: where n � sample size, d � desired absolute precision (0.05), and Pex � expected prevalence (50%); thus, the desired sample size for Pex � 0.5 is n � 384.As a result, 384 were considered the minimum sample size required for biting fy infestation and their associated pathogens at the study site.However, to increase precision, a total of 480 camels from the district were examined for the presence of biting fies and for other associated pathogens during the whole study period.Accordingly, 160 camels were selected purposively from the district in each dry, wet, and short rainy season for biting fies as well as for the detection of associated pathogens.

Sample Collection and Laboratory Processing of Biting
Flies.In addition to the parasitological studies, biting fies were also collected from camels and the surrounding area to see if any of them could be used as vectors for the spread of Trypanosoma evansi and Trypanosoma Vivax.As it was believed to be representative of other comparable study localities, this collection was made in order to observe changes in the fy population over the course of various seasons.Flies that were actively feeding on the animals and hovering in the area of the animals were captured in the study area.Aerial sweeping nets were used to collect the fies from camels and their surroundings while they were fying (circling the area around the animals), as described by Barros and Barros [27].To put it briefy, the person holding the net walks beside the animals and sweeps it periodically to capture fies that are feeding on the animals and fying around the animals.Aerial sweeping nets were used to catch biting fies from camels while they were being fed, especially during the morning when the fies were actively feeding, resting, and circling on the camels.After each sampling, the nets used for collection were emptied before the next collection.Te total number of fies collected was counted and then transferred into a sample bottle.On every specimen that was collected in the feld, the following information was noted: location, date, season, and time of collection.Te captured fies were kept in sample bottles (screw-capped bottles) that were flled with 70% ethanol.Te sample was transported to the Veterinary Parasitology Laboratory at the College of Veterinary Medicine and Agriculture, Addis Ababa University, using these screw-capped bottles.In the laboratory, the process of identifying fies involved placing the sample on a Petri dish by using forceps.Trough the use of standard identifcation keys, the adult fies were then identifed to the level of genus under a stereomicroscope and counted manually under a stereomicroscope.Identifcation was done based on taxonomic features such as the arrangement of their wings, mouth parts, antennae, size of fies, the patterns on their abdomen, their color, and the separation between their eyes as described by [28][29][30].
2.6.Blood Sample Collection and Examination.Whole blood samples from 480 camels were collected via jugular vein puncture into 10 ml ethylene tetra-acetic acid (EDTA) and plain vacutainer tubes.Te Buf coat technique has been employed immediately after blood collection to determine the level of parasitemia and anemia at the collection site.Te blood was immediately transferred into capillary tubes, sealed at one end, and centrifuged at 12,000 rpm for 5 minutes using a microhaematocrit centrifuge [37].After that, each capillary tube was placed in a haematocrit reader, and the reading was expressed as a percentage of packed red cells to total volume (PCV) of whole blood.Te level of anemia was determined using a cutof PCV value of 27 and classifying animals with a PCV value less than 27 as anaemic and those with a PCV value greater than 27 as nonanaemic [37].
Buf coat method: with a diamond-tipped pencil, the capillary tube was cut 1 mm below the bufy coat.Te contents of the capillary tube were poured onto a clean slide and thoroughly mixed.It was covered by a 22 × 22 mm cover slip.A bright-feld microscope was used to examine the preparation, with the condenser top out and the diaphragm closed.For 30 minutes, the fxed blood smears were immersed in Giemsa stain solution in an upright position.After removing the stain, the slide was thoroughly washed in running tap water and allowed to drip-dry in an upright position before microscopic examination.At 100x magnifcation, the slides were examined under a microscope with oil immersion.According to Murray et al. [28,37], species identifcation was based on trypanosome morphological characteristics such as trypanosome size, posterior end shape, kinetoplast size and position, and fagellum presence or absence.
Tin blood smears were prepared using the method described by Basaznew et al. [38].Smears were air-dried and fxed in absolute methyl alcohol for 2-3 minutes.Te slides were stained with Giemsa for 20-25 minutes before being washed with tap water to remove excess stain.After airdrying, the slides were examined using an oil immersion objective lens (100x) to detect and identify Trypanosoma species [38].Furthermore, logistic regression was used to investigate the degree of association between a binary outcome (the presence or absence of biting fies) and various explanatory factors.Te chi-square tests were used to quantify the relationship between the variables and the presence of a biting fy infestation.To determine prevalence (percentage), a statistical analysis was performed.Kappa statistics (K) were used to determine the level of agreement between diagnostic tests.Te chi-square test, on the other hand, was used to examine the relationship between trypanosomosis positive camels in both tests and the assumed risk factors.Furthermore, the two-sample t-test was used to compare the mean PCV of parasitologically positive and negative camels, as well as serologically positive and negative camels, for trypanosomosis.For all statistical analysis, a statistical signifcance level of p ≤ 0.005 was considered.Ababa University, Bishoftu, Ethiopia) for the collection of blood samples from camels in an ethical manner.Camel sample collection did not jeopardize the animals' welfare.Te camel owners were informed about the study's purpose, risks, and benefts based on their level of understanding in order to provide complete information, including the duration of the study, biting fies, and blood sample collection from the camels.

Biting Flies' Infestation.
Of the total of 480 camels examined for fy infestation, 418 (87%) of them were infested with one or more biting fies.Overall, three genera of biting fies, Hippobosca, Stomoxys, and Tabanus, were encountered during the study period.Morphological identifcation of the collected biting fies revealed that Hippobosca (40.4%) was the most prevalent and predominant genus in the Amibara district, followed by Stomoxys (31%) and Tabanus (28.6%) as shown in Table 1.
Assessment of the abundances of the collected biting fies showed that a total of 1,706 adult fies (1,051 female fies and 655 male fies) were collected during the study period.Te number of biting fies that were collected from camels in the current study area during the wet season (536), short rain season (723), and dry season (447) is shown in Table 2.Moreover, the current study's fndings revealed that biting fies were present on camels throughout the study period.All biting fy genera were abundant during the short rainy season and scarce during the dry season (Table 2).
Te prevalence of biting fies on camels of diferent ages, sex, body condition score, herd composition, season, and herd size is shown in Table 3.Among the risk factors considered, age and season appeared to have a signifcant efect (p < 0.05) on the prevalence of biting fies on camels.However, the prevalence of fy infestation did not signifcantly difer among camels of diferent sex, body condition score, herd composition, and herd size (p > 0.05) (Table 3).

Trypanosoma evansi in Camels.
Te card agglutination test indicated an overall seroprevalence of 16% (77/480) for the detection of agglutinating antibodies against Trypanosoma evansi (Table 4).Parasitemia ranged from a few parasites per slide to over fve trypanosomes per feld on the microscope feld.Te overall parasitological prevalence of Trypanosoma evansi in the Amibara district of the Afar region was 7.3% (35/480) (Table 4).
Te overall mean PCV of positive Trypanosoma evansi camels was 22.2%, which was lower than the overall mean PCV of negative camels, which was 26.7% (Table 5).
Out of 77 animals with positive serological test results, 25 were positive and 52 were negative using the Bufy coat method, respectively.Among the 35 animals with positive parasitological results, 25 were positive and 10 were negative using CATT/T.evansi, respectively, as shown in Table 7.A statistical test using kappa indicated a fair agreement between the two tests (k � 0.385).Te relative sensitivity of the CATT/T.evansi test used in this study was found to be 25/77 (32.5%) (Table 7).
Te prevalence of Trypanosoma evansi in camels of diferent ages, sex, body condition score, herd composition, season, and herd size is shown in Table 8.Among the risk Veterinary Medicine International factors considered, age, body condition score, and season appeared to have a signifcant efect (p ≤ 0.001) on the prevalence of Trypanosoma evansi in camels (Table 8).
Out of the camels infected with Trypanosoma evansi, 36.3% had Tabanus infestation, 33.3% had Hippobosca infestation, and 30.4% had Stomoxys infestation.Te fles were directly collected (caught) from the camels while the fies were feeding or resting on them by using aerial sweeping nets.T. evansi-infected camels were infested by at least one or more genera of biting fies.Tis indicates that the camels from which the fies were directly collected and blood samples tested highly positive for T. evansi.Tis fnding demonstrated that some camels were concurrently afected by Hippobosca, Stomoxys, and Tabanus.Tis means that some camels have only one fy infested them, some have two fies infested them, and some have three fies infested them at the same time.T. evansi infection may be more likely to infect camels that biting fies directly caught by using aerial sweeping nets while they are feeding on the animals.Te prevalence of the 3 genera was statistically signifcant (p < 0.05) (Table 9).
Te prevalence of trypanosomosis is determined by the seasonal and environmental factors, which are determined by the presence and number of biting fies.Te occurrence of this condition is notably enhanced by the wet and humid weather conditions, as well as the increased presence of biting fies during the short rain and wet season when the population of these fies is abundant [11,49].Tabanus studies conducted in diferent tropical regions have shown a clear association between periodic occurrences of T. evansi infections.Te study reveals variations in the distribution of camel biting fies among diferent fies' genera, consistent with previous research by [50].
During the rainy season, there is a rise in infections and an uptick in the fy population.Precipitation appropriate clay soil that retains moisture, as well as surface water pools, thus provides the necessary sustenance creating favorable environments for camels to graze, characterized by the abundant growth of Acacia senegal shrubs [11,51].
A large proportion of camels were found to be seropositive, in addition to the parasitological fndings.Te current serological result is consistent with previous fndings in diferent parts of Ethiopia (17.8%) in a similar study site by Weldegebrial et al. [42].However, the current fnding is lower than that of other researchers (24.9%) [44], 30% in Chad [52], 22% in India [53], 28% in Kenya [54], and 27.5% [55].Te present serological study result is higher than the previous study report conducted by the authors in [56] in Iran, which was 10%.
Trypanosome morphological identifcation in the current study showed that the amount of parasitemia varied from a few parasites per slide to over fve trypanosomes per feld of view of the microscope.Tese fndings coincide with the fnding of the authors in [50].Te overall parasitological prevalence of Trypanosoma evansi in the current study area was 7.3%.Tis result was highly in agreement with the fndings in [50], which reported 7.2% prevalence by using thin blood smears.Te higher seroprevalence compared to the parasitological result recorded in the current study could be due to the fact that demonstration of trypanosomes in blood is quite unreliable because a large proportion of infections in the feld (50 to 80%) do not develop detectable levels of parasitemia [57].Tis is due to the fact that trypanosome infection in camels is usually chronic, with very low parasitemia.Additionally, the inability of the CATT/ T. evansi test to distinguish current from cured infection [58], as detectable levels of antibodies can still be found in self-cured animals or after treatment with trypanocidal drugs [59], might contribute to the higher prevalence difference between the two tests.
CATT was sensitive in detecting 86 latent/aparasitaemic infections, but it failed to detect 10 of 35 (28.6%) patent/ parasiteaemic infections (Table 7).Te CATT/T.evansi test, a direct agglutination test, is the most widely used and has a proven record of reliability for various host species, including bufaloes and camels [60,61].Tis lower sensitivity of the CATT test observed in the current observation is consistent with previous studies in Kenya that reported a sensitivity of 65.5% [62] and 68.6% [15].Similarly, Hagos et al. [44] reported a 72% sensitivity of CATT/T.evansi from Ethiopia.Te occurrence of camel trypanosomes is more common in camels that are kept with other animals, such as cattle, sheep, and goats.Tis is similar to the observations 6 Veterinary Medicine International made by the authors in [50], that camels herded with small ruminant animals like sheep and goats were found positive for trypanosomes through DNA identifcation using molecular techniques like PCR.Te diverse range of animals that the biting fies feed on contributes to the observed epidemiological complexity of trypanosomosis [50].
Te overall mean PCV of positive Trypanosoma evansi camels was 22.2%, which was lower than the overall mean PCV of negative camels, which was 26.7%.Te current study showed that parasitologically negative camels had signifcantly higher mean PCV than positive camels, which is consistent with previous reports [44,50,63].Tis suggests that the primary clinical fnding of trypanosomosis's was anemia.In serological tests, there was no signifcant diference in mean PCV between serologically negative and serologically positive camels.Tis could be due to the CATT test's lack of ability to distinguish antibodies from active infections from those from cleared or past infections, as previously suggested by [58].Terefore, the PCV values of cured camels from trypanosomosis (past infections) that are serologically detected as positive are not signifcantly diferent from seronegative ones [64], and highly reduced PCV values occur when trypanosome parasites are detectable in blood.
Te higher prevalence of T. evansi in adults than in young camels in both parasitological and serological tests in the present study is in agreement with previous reports in [16,60,63,65].Tis could be because of larger-scale movement, which increases the risk of infection in adult camels [52,66], heavy stress on adult male camels used for transportation of goods and possible poor management [67], and stress associated with pregnancy and lactation in adult camels [66].Te present study demonstrated that biting fies are widespread and the most signifcant ectoparasites of camels in the Amibara district, Gabi Rasu zone, Afar region, Ethiopia.Te observation of an overall prevalence of 87% biting fies in the present study is in line with the previous report of a 99.9% Stomoxys infestation in camels in and around the Fentale district [13].Tis study also showed that the prevalence of Trypanosoma evansi has a correlation with seasonal increments in the number of Tabanus during the rainy season, which is in agreement with the previous reports [51,68].Experiments have shown that over 20 diferent Tabanus species can transmit Trypanosoma evansi [49].Te presence of Tabanus species across all study seasons suggests that parasite transmission occurs wherever reservoir hosts and susceptible host's coexist.Te abundance of Tabanus species has been linked to sporadic outbreaks of the disease during the dry season and outbreaks during the rainy season [51].Some investigators state that Stomoxys and Hippobosca are possible vectors for transmission of Trypanosoma evansi [47,69].
Although Stomoxys has been implicated as a vector of Trypanosoma evansi, experimental trials involving transmission between horses, guinea pigs, and dogs have not proved that these fies are important vectors [36,70].However, it has been reported that the efciency of diferent   Veterinary Medicine International fies in transmitting Trypanosoma evansi varies depending on geographic conditions, as well as the interval between two successive feeds and the intensity of the fy challenge [49].Experimental trials conducted on Hippobosca camelina in Kenya to assess the mechanical transmission of Trypanosoma evansi showed that the fy failed to transmit the pathogen, but regurgitation is suspected to be the other possible means of transmission [29].However, Hippobosca camelina and Stomoxys calcitran are the primary carriers of trypanosomes in regions devoid of tsetse fies, according to research in the report of [50].Tis is because trypanosomes are consistently found in them, and they are abundant throughout the year.Additionally, in the current study, there is a notable preference for Hippobosca in camels, which corresponds to the fnding reported by [50].Te study mentioned above revealed that Hippobosca camelina had    In the present study, among the camels that were found to be infected with Trypanosoma evansi, 36.3% were infested with Tabanus, 33.3% were infested with Hippobosca, and 30.4% were infested with Stomoxys.During the study period, the fles were captured directly using aerial sweeping nets, and blood samples were simultaneously extracted from them.Camels infected with T. evansi were infested by one or more types of biting fies at the same time.Tis suggests that the camels, from which the fies were directly collected and blood samples were tested, had a high rate of positivity for T. evansi.Based on the current study fndings, T. evansi infection is more likely to occur in camels when they are bitten by fies that are directly caught using aerial sweeping nets.Te remaining fies were also collected by using aerial sweeping nets while the fies were fying or circling around the animals in the study area.In the result section of Table 9, we focus only on the biting fies collected by aerial sweeping nets directly from the camels.Tis is due to the fact that the research paper primarily focuses on biting fies and their associated pathogens in the study area.
Te fnding of a higher prevalence of biting fies as well as Trypanosoma evansi in adult camels than in the young camels in the present study might be most probably attributed to the fact that pastoralists kept young camels in the residential area and they do not go to distant areas where the fy burden is high.

Conclusion and Recommendations
Hippobosca and Stomoxys are the most prevalent biting fies in the study area.Trypanosoma evansi is one of the most common pathogens afecting camels in the study area.Terefore, it is necessary to make signifcant endeavors at various levels to minimize the adverse efects on the health, production, and output of camels in the district.Tis is crucial for enhancing the living standards of the pastoralist community residing in the research area, where camels are predominantly used for milk, meat, and as a source of income.Terefore, based on the above conclusion, the following recommendations are forwarded: (i) Practically applicable control interventions need to be implemented to reduce the negative impacts of biting fies and trypanosomosis in camels with the appropriate use of acaricides in the study area (ii) Advanced molecular studies on biting fies' identifcation to species level and their associated pathogens in animal diseases in the wider pastoralist community in various parts of Ethiopia are needed

Figure 1 :
Figure 1: Map of Ethiopia showing the present study area.Source: Diva GIS.
[41]40] agglutination for trypanosomosis test (CATT/T.evansi)wasusedtoinvestigatetheseroprevalence of camel trypanosomes.Te CATT/T.evansitest is a direct, rapid card agglutination test that employs formaldehyde-fxed, freeze-dried trypanosomes stained with Coomassie blue and expressing a predominant variable antigen type of T. evansi (RoTat 1.2).Positive samples were determined at cutof point dilutions of 1 : 4 and higher[39,40].As a result, 25 μl of camel serum was pipetted onto the reaction zone of a plastic-coated test card after being diluted 1 : 4 in CATT bufer.After adding one drop (about 45 μl) of CATT reagent, the reaction mixture was spread out with a stirring rod and allowed to react for 5 minutes at 70 rpm on a card test rotator.Blue granular deposits show a visible positive reaction to the naked eye[41].2.7.Data Analysis.Te collected data were entered and saved in the database using the Microsoft Excel spreadsheet (MS-2007) program.All the data were summarized, compiled, and coded before being stored in a Microsoft Excel 2007 spreadsheet.For statistical analysis, the data from MS Excel were imported into STATA version 13.0 statistical software (Stata Crop, 2013) and R software, Version 3.5.1.R Software, Version 3.5.1,was used to compute descriptive and analytic statistics.
Descriptive statistics were used to summarize the data, which was then displayed in tables.Poisson regression was employed to analyze the number (count) of biting fies on animals as a function of various explanatory variables (sex, age, body condition score, herd size, herd composition, and season).
Consideration.Ethical approval was obtained from the Animal Research Ethics and Review Committee (certifcate Ref. No: VM/ERC/23/01/12/2020 from the College of Veterinary Medicine and Agriculture, Addis

Table 2 :
Overall abundances of collected biting fies in camels throughout diferent seasons in the Amibara district.

Table 1 :
Relative abundance of camel biting fies at the genus level in the Amibara district.

Table 3 :
Prevalence of biting fies on camels in the Amibara district by diferent risk factors.

Table 4 :
Overall parasitological and serological prevalence of Trypanosoma evansi in camels in the Amibara district in Afar Region, Ethiopia.

Table 5 :
Overall mean PCV of parasitologically positive and negative camels in the Amibara district Afar Region, Ethiopia.

Table 6 :
Mean PCV of camels positive and negative for parasitological and serological trypanosomosis in the Amibara district, Afar Region, Ethiopia.

Table 7 :
Comparative positivity for Trypanosoma evansi in camels using parasitological and serological type of tests in the Amibara district, Gabi Rasu zone, Afar Region, Ethiopia.

Table 8 :
Overall prevalence of Trypanosoma evansi in camels with respect to diferent risk factors in the Amibara district.

Table 9 :
[50]ls were positive for biting fies and Trypanosome evansi simultaneously in the Amibara district.towardscamels,as most of them were found to feed on camels.According to Getahun et al.[50], it is confrmed that Hippobosca camelina, Stomoxys calcitrans, and Tabanus spp were identifed as the potential vectors of trypanosomes outside of the tsetse fy belt.