Epidemiological study on Ixodid tick infestation and tick borne haemopathogens on cattle in Awi Zone, northwest Ethiopia

Abstract Background Tick and tick borne haemopathogens are the main challenge of livestock production and productivity in Ethiopia particular in northwest Ethiopia due to favourable climate condition. Objectives The objectives of this study was to determining the prevalence of Ixodid tick infestation and tick borne haemopathogens on cattle, identifying the existing Ixodid tick species, assessing seasonal variation and major risk factors associated with tick infestation and tick borne haemopathogens. Methods A cross‐sectional study was conducted from December 2020 to july 2021 on Ixodid tick infestation and tick borne haemopathogens on cattle in the northwest Ethiopia. A toatal of 384 cattle were used for this study. Tick species were identified using morphological identification keys under a stereomicroscope. Thin blood smear examination were conducted to assess tick borne haemopathogens. Results The overall prevalence of Ixodid tick infestation and tick borne haemopathogens were 45% and 3%, respectively. Babesia bigemina was the only haemoparasite detected in the present survey. Potential risk factors were investigated for their association with tick infestation and B. bigemina using logistic regression and chi‐square test, respectively. Accordingly, age, body condition, agroecological systems and season were significantly (p < 0.05) associated with tick infestation whereas season and body condition were significantly (p < 0.05) associated with B. bigemina infection. A total seven tick species were identified. Amblyomma varigatum (55%) and Boophilus decoloratus (15.7%) were the predominant Ixodid tick species encountered. A total of 128 blood samples were collected from Jawi district and examined using thin blood smear. Of them, 3% were infected by the B. bigemina Conclusions Tick infestation in this study was high and seems to play vital role for the reduction of production, productivity and for the transmission B. bigemina. Therefore, sound and effective tick control and prevention strategies are needed to mitigate the risk factors for tick infestation and B. bigemina infection in Ethiopia.


INTRODUCTION
Ethiopia has an extremely diverse topography, a variety climatic features and agroecological zones that are expedient to host a very large animal population (Mekasha & Zewdie, 2014). Ethiopia is one of the countries with the largest number of livestock population in Africa (Alekaw, 2000). Despite the largest livestock population in Ethiopia, the economic benefits remain marginal due to prevailing diseases, poor management and low genetic performance (Dabassa et al., 2017;Jilo et al., 2017). In Ethiopia, ticks occupy the first place among the external parasites through mortality of animals, decreased production, downgrading and general rejection of skins and hides (Dabassa et al., 2017). The impacts of ticks on animals were either by inflecting direct damage or by transmission of tick-borne pathogens. They are responsible for severe economic losses both through the direct effects associated with their blood sucking behaviour (Kumsa et al., 2015a) and also indirectly act as reservoirs and vectors for a wide range of human and animal pathogens (Jongejan & Uilenberg, 2004). Ticks and tick-borne diseases affect 90% of the world's cattle population and are widely distributed throughout the world (Estrada-Peña et al., 2014). The country's environmental condition and vegetation are highly conducive for ticks and tick-borne disease perpetuation. The presence of diseases caused by haemoparasites is broadly related to the presence and distribution of their vectors. Ticks are more prevalent in the warmer climates, especially in tropical and sub-tropical areas (Ikpeze et al., 2015). Previous study conducted in different part of Ethiopia revealed that there are five genera and forty seven species of Ixodid ticks found on livestock (Hailu et al., 2008;Kumsa et al., 2016;Pegram et al., 2004;Tadesse & Sultan, 2014).
Tick borne haemopathogen have a serious economic impact on livestock sector due to decreased productivity, lowered working efficiency, increased cost for control measures and limiting introduction of genetically improved cattle in the area and death of livestock (Radostits et al., 2008;Uilenberg, 1995). In Ethiopia, anaplasmosis, babesoisis, cowdrosis and theileriosis have been reported as major tick borne diseases affecting domestic animals (Teshale et al., 2016). Among tick-borne pathogens of zoonotic importance spotted fever, Borrelia spp., Coxiella burnetii and Bartonella spp. have been documented from Ethiopia (Kumsa et al., 2015a).
There are different ways of classifying the climatic systems of Ethiopia, including the traditional, the Köppen's, the Throthwaite's, the rainfall regimes and the agroclimatic zone classification systems (Yohannes, 2003). The most commonly used classification systems are the traditional and the agroecological zones. According to the traditional classification system, which mainly relies on altitude and temperature, Ethiopia has five climatic zones such as 'Wurch' (upper highland), 'Dega' (highland), 'Weyna dega' (midland), 'kola' (lowland) and 'Berha' (Desert) (MoA, 2000). The seasonal variations within a bioclimatic zone may favour or hinder the development or activity of a tick species during certain periods (Latif & Walker, 2004). Dry environmental conditions are a serious danger to ticks, particularly to the questing larvae, which are very susceptible to drying out fatally (Walker et al., 2014). Also, the start and end of the rainy season may influence the different phases of the life cycle (Getachew et al., 2014). Other species are more restricted to a specific habitat where their specific hosts are present and where climatic conditions allow survival and reproduction (Mekonnen et al., 2007).

Description of the study areas
The study was conducted in two selected districts of Awi Zone, Amhara National Regional State, northwest Ethiopia. Awi Zone is located within the latitude of 10.95 • N and longitude of 36.5 • E. It lies at an altitude range of 648-3100 m above sea level (m a.s.l.) with average altitude of 2300 (m a.s.l) (Yeshambel et al., 2011). It is predominantly inhabited by the Awi, ethnic group which belongs to the central cushitic subfamily and inhabits in northwest Ethiopia (Alamneh, 2004

Ankasha district
Ankasha Guagusa Woreda is located in Awi zone in the northwest-

Jawi district
Jawi district is found in Awi zone, Amhara regional state, northwest

Study population
The study population were local/zebu cattle breed kept under individual households with different age, sex and body condition scores found in the two selected districts. The animals are managed with extensive management system and depend on grazing throughout the year for their feed sources with little supplementation of crop residues.

Study design and sampling procedure
A cross sectional study was conducted from December 2020 to July 2021 to determine epidemiology of Ixodid tick infestation and tick borne haemoparasites on Awi Zone, northwest Ethiopia. For this study, two districts were selected purposely based on the agroecological systems, which favours tick infestation while the study animals were selected using simple random sampling. Jawi district has a lowland agroecological systems ranges from 648-1300 m a.s.l. Ankasha district has both midland and highland agroecologies ranges from 1200 to 2800 m a.s.l. Multistage sampling was used to select kebeles and household levels. In multistage sampling, the two districts are primary units and in districts kebeles were selected randomly which were considered as secondary units. Within kebeles households were selected randomly and considered as tertiary unit. 'Kebeles' corresponds to the smaller territorial administrative unit, which is a tight system of neighbourhood administration or a collection of peasant associations and households.
A group of kebeles form a district. Information about agroecological systems and proportionally equal sample were taken in dry and wet season. The study animals were classified into two groups based on age as young (≤2 years) and adult (>2 years) according to Okello-Onen et al. (1999) and body condition score was recorded after classifying the animals into poor, medium and good (Verhees et al., 2011).

Sample size determination
The sample size required for this study was determined according to Thrusfield (2005 Then, the skin of each selected cattle was inspected for the presence or absence of ticks from half body part. The favourable predilection anatomical sites where ticks were searched are scrotum/udder, Groin, dewlap, belly, tail, leg/hoof and neck. All visible adult ticks were manually collected by using forceps from half regions of the animals' body and care was taken to avoid decapitulation (Walker et al., 2014). The collected tick was placed in a labelled clean universal bottle using 70% alcohol and transported to Bahir Dar regional animal health disease investigation and diagnostic laboratory centre for tick species identification.

Blood sample collection
For blood sample collection, Jawi district was purposely selected due to financial limitation and a total of 128 blood samples were collected from ear vein using heparinised capillary tube and sealed from randomly selected cattle from Jawi district (lowland agroecological systems) following the standard protocol described by Urquhart et al. (1996). After labelling, it was kept in cold chain and examined at Jawi Woreda veterinary clinic laboratory by thin blood smear technique. All of 128 blood samples were examined by microscope at ×100 magnification power.

Haemoparasite examination
Thin blood smear: Giemsa staining procedures and microscopic examinations were conducted according to OIE (2010) and Zafar et al. (2006). Each stained slides were examined for identification of blood protozoa (Urquhart et al., 1996). The parasite was identified by the characters described by Soulsby (1982). The numbers of microscopic fields, which were examined per slide for the detecting haemoparasites in the sampled cattle, were six microscopic fields from one positive animal, two microscopic fields from the two positive samples and one microscopic field from one positive sample. However, after examination of the whole parts of the prepared microscopic slide and if we do not get any haemoparasites, an animal is negative.

Prevalence of Ixodid tick infestation and tick borne haemoparasite
A total of 384 cattle were examined for tick infestation. Of which, 173 cattle were found positive for tick infestation with overall prevalence of 45%. In the current study, 128 blood samples were collected from Jawi district for examining the presence of tick borne haemoparasite in both wet and dry season. Of them, four cattle were positive for B.
bigemina with overall prevalence of 3% (Table 1). The parasitaemia levels of B. bigemina were low and medium from the three and one positive sampled animals, respectively.

Association of potential risk factors with Ixodid tick infestation
A total of five potential risk factors were tested using multivariate logistic regression Accordingly, age, body condition, agroecological systems and season were statistically significant (p < 0.05) associated with tick infestation in the districts. Keeping the effect of other variables constant, the odd of tick infestation in younger aged cattle was 1.94 times more likely to be infested than their older counter parts.
Similarly, the odd of tick infestation in wet season was 15.65 times higher than dry season. The association between body condition and agroecological systems with tick infestation were indicated (Table 2).

Association of potential risk factors with tick borne haemoparasites
A total of four potential risk factors were tested using chi-square test (X 2 ). Accordingly, body condition and season were statistically TA B L E 2 Potential risk factors significantly associated to cattle tick infestation using multivariate logistic regression analysis haemoparasite in poor conditioned cattle was 11.9 times higher than well-conditioned cattle. The statistical significance of association between seasons and tick-borne haemoparasite is indicated in (Table 3).

Identification of Ixodid tick species
A total of 2047 (1439 males and 608 females) ticks were collected in the districts. In the current study, four tick's genera and seven tick species were identified. Amblyomma and Hyalomma were the predominant and the lowest tick genera with prevalence of 68.7% and 4.1%, TA B L E 4 Poisson regression analysis of significant value ticks' genera collected from the three agroecological systems

Agroecological distribution and variations of tick species
The result of the study prevailed that there was a highest significant (p < 0.05) difference in abundance and distribution of Ixodid tick species infesting cattle like A. variegatum, A. cohorense, B. decoleratus, R. evertsi evertsi, R. praetextatus and H. marginatum in lowland, highland and midland agroecological systems. Its distribution is highest in lowland and midland agroecological systems than highland agroecological systems. But in H. truncatum species, there was no significant (p > 0.05) difference in abundance and distribution in lowland, midland and highland agroecological systems (Figure 1 and Table 7).

Seasonal variations of tick species
The current study indicated that there is a high difference in the prevalence of Ixodid tick infestation in season. Higher infestation was occurred in wet season than dry season. There was no seasonal variation in the occurrence of H. truncatum species (Figure 2).

DISCUSSION
The current study revealed that hard ticks are important external parasites and highly distributed in Ankasha and Jawi districts of Ethiopia.
In the present study, a total of 2047 adult ticks were collected from 173 cattle with over all prevalence of 45% of tick infestation. This finding is in line with the previous report of 40.26% around Haramaya town (Yalew et al., 2017). Similarly, Kindalem (2015) reported 48.44% in and around Kombolcha. However, the current finding disagrees with Misgana (2017 who reported overall prevalence of 91.5% in Adaa and Boset districts Oromia regional state. The variation of these findings may be due to different management system, seasonal variation, agroecology, study design, different target animals. The present study revealed that overall prevalence of 3% In this study, the prevalence of tick infestation was significantly (p < 0.05) highest (56.2%) in poor conditioned cattle. This finding is in line with the previous reports performed by Amsalu et al. (2015) in Dangla, northwest Ethiopia with the prevalence of 62.9%. Because poor body conditioned animals had lower resistance to tick infestation and they exposed tick infestation during grazing on the field than medium and good body conditioned animals.
The variations in prevalence were higher in females than males but there was no statistical (p > 0.05) difference during the study period. It agrees with the report of Fentahun and Mohammed (2012) in Assosa, Ethiopia but it disagrees with the report of Zerihun and simeon (2013) in Benchimagi zone, Ethiopia. This might be due to the fact that most of the time males enter to feed lot and thus they have less accessibility to be infested with tick. Feedlot animals are most likely with reduced tick infestation since the environment is not suitable for the free-living stages of tick (Jonsson, 2004). The sensitivity of thin blood smear is low and adequate for acute cases of Babesia bovis infection as compared to other techniques like IFAT, ELISA, complement fixation test and PCR (Salih et al., 2015). Blood smears are not reliable for detection of carrier animals. In these cases, molecular methods or serological diagnostic procedures are required demonstrate specific antibodies (Pohl, 2013). Polymerase chain reaction (PCR) assays can detect and differentiate Babesia species and are particularly useful in carriers than blood smear techniques (CFSPH, 2008). In the current study, R. annulatus and R.
microplus were not recorded.
The rate of tick infestation was significantly (p = 0.04) higher in adult animals than young animals. This report coincides with the report Tadele et al. (2016) in Jabitennan woreda, West Gojjam, Ethiopia. The reason might be decreased contact of young animals with other groups of animals, which can be source of transmission. Most of the time many adult cattle graze in the pasture and forest, and the chance of getting tick infestation is increasing (Ramsi et al., 2007).
In the present investigation, there was a significant (p < 0.05) association between body condition of cattle with the risk of tick borne haemoparasite infection. This could be due to the fact that animals with poor body condition have lower immunity which encourages infection of animal by different organisms like babesia. Among the infected animals, three of them displayed a clinical sign of high weight loss and congested mucous membranes. The weight loss of infected animals might be related to anorexia and the rapidly dividing parasites in the red blood cells produce rapid destruction of the erythrocytes because the packed cell volume falls below 20%, which will lead to anaemia (Demessie & Derso, 2015). In addition, during this study, it was very common to see high burden of ticks in animal with poor body condition unlike those animals with good body conditions and this can increase rate of infection from babesia. This is in agreement with the previous reports of Bihonegn et al. (2015) in Benshangul Gumez Region, Western Ethiopia.
According to seasonal infection rate of bovine babesiosis, the seasonal prevalence of babesiosis infection using thin blood smears examination revealed the overall infection rate of cattle was recorded during the wet season. These results were similar with results of Kamani et al. (2010) Kumsa et al. (2012).
In the present investigation, tick abundance count was significantly (p = 0.000) higher in wet season than dry season. This report was in consistent with the report of Kumsa et al. (2012) that high humidity and temperature are crucial factors that influence the seasonal variation of ticks. However, this study is in disagreement with the study done by Kemal et al. (2016) who reported that there was no considerable difference in the prevalence of ticks within the wet and dry season. Additionally, this finding is in agreement with the study done by Mekonnen et al. (2007) that ticks were found on cattle throughout the study period, although higher tick counts were observed during the rainy than dry season. This might be attributed that the dry season results in lower relative humidity and higher environmental temperature, which influences the mortality of ticks due to desiccation .  Shiferaw (2005) in Wolayita, SNNP, Ethiopia who stated that Boophilus decoloratus was the most abundant tick species. The observation of high tick counts in lowland agroecological systems on cattle in the present study is most probably attributed to the vast and seasonal availability of grazing land and unrestricted cattle movement from place to place in the lowland than in both the midland and highland agroecological zones .
Amblyomma variegatum was significantly (p = 0.000) higher in count during the wet season than dry season. It agrees with the previous study reports of Misgana (2017)  This difference might be attributed to the differences in climates and altitude among the study areas.
Amblyomma cohorense was the third most abundant tick species in the study areas with a prevalence of 17%. This finding is consistent with the previous research done by Mekonnen (2017) (2012). It was also consistent with the report of Tamirat et al. (2015) in Bedele, Ethiopia. But it was in disagreement with the previous report of Huruma et al. (2015) with the prevalence of 53.4%, which was done in Sebeta, Ethiopia.
In the present investigation, H. truncatum was the least abundant tick recorded in the study areas with the prevalence of 0.2%, which is in agreement with the previous report of Walker and Koney (1999) in Ghana. It is also in line with the report of Biru et al. (2017) who reported that H. truncatum is the least abundant tick species in Harerge, southeast Ethiopia. Based on the finding of the current study, the lowest number of T. truncatum was recorded in all agroecologies and during the wet and dry season with nonsignificance (p > 0.05) variations.

CONCLUSION
The current study revealed that there was high prevalence of Ixodid tick infestation and tick borne haemoparasite. A total of five potential risk factors were evaluated to determine the association with tick infestation and tick borne haemoparasite. Age, body condition, agroecological systems and season were significantly associated with tick infestation whereas body condition and season were significantly associated with the risk of tick borne haemoparasite. In this study, a total of four genera and seven tick species were identified. Of them A. variegatum and B. decoloratus were the predominant species encountered.
From this study result, it is possible to conclude that agroecological systems and season play great role in the variation in distribution and abundance of tick species and dynamics. Tick infestation of cattle was significantly higher in lowland than midland and highland. The tick burden was also higher in wet period than dry period. The total tick burden was also higher in wet period compared to dry period. This high abundance of tick infestation of cattle and their tick borne haemoparasite in the study areas require high attention at all concerned levels to minimise the impacts on the health and productivity of cattle to improve the living standards of farmers of the study areas. Therefore, strategically and effective tick control programs depending on the distribution pattern of ticks and factors responsible for their distribution should be implemented. Likewise, further research using molecular diagnostic techniques should be conducted for investigation of tick-borne haemopathogens on livestock in Ethiopia.