PREVALENCE OF TICKS AND RISK FACTORS ASSOCIATED WITH THE INFESTATION OF SHEEP IN RIVER NILE STATE, SUDAN

A cross-sectional study was conducted in River Nile State, Sudan, be-tween June to August 2018 to determine the prevalence of tick infestation on sheep and the potential risk factors associated with the infestation. A total of 135 sheep from fi ve diff erent localities (Shendi, Al Matamah, Ad-Damer, Atbara, and Berber) were examined and, of these, 90 were tick-in-fested (66.7%). A total of 340 ticks (male 185, female 155) were collected and identifi ed using zoological taxonomic keys. Th e most dominant tick species collected in this survey were Rhipicephalus eversti Neumann, 1897 (38%), followed by Hyalomma anatolicum Koch, 1844 (23.8%), Rhipicephalus sanguineus Latreille, 1806 (20.6%), Rhipicephalus praetextatus Gerstack-er, 1873 (16.4%), and Hyalomma dromedarii Koch, 1844 (1.2%). Th e chi-square analysis showed that there was a signifi cant association ( p ≤ 0.05) between tick infestation and localities, housing type, sex, control of ticks, and removal of manure. Th e highest prevalence rate was recorded in At-bara, in the open housing type, in females, in farms that did not use acaricides and did not remove the manure frequently. On the other hand, there was no signifi cant association ( p > 0.05) between tick infestation and herd size, rearing system, breed, age and colour of coat ( p =0.846). Th is study expanded the knowledge on tick fauna and associated risk factors in the River Nile State, and it demonstrated that multiple tick species are infesting sheep with the potential to transmit several tick-borne diseases.


INTRODUCTION
Ticks, belonging to the family Ixodidae, are the most important vectors of a wide variety of pathogens including protozoa, bacteria, helminths, and viruses, which aff ect domestic animals and humans (Jongejan and Uilenberg, 2004). During feeding, ticks may cause direct or indirect eff ects on their hosts. Th e direct eff ects of ticks include the sucking of blood, which in turn leads to anaemia and damage to the skin. Consequently, these eff ects result in significant losses in productivity, fertility, body weight, milk and meat production, and mortality (Mapholi et al., 2014). Th e indirect losses of ticks are related to the infectious agents transmitted by them and the costs associated with the treatment and control (Hurtado and Giraldo-Ríos, 2018).
Ticks and tick-borne diseases are prevalent in Sudan, cause notable economic losses, and constitute major barriers to the development of animal production. Among these diseases, Th eileriosis, Babesiosis, and Anaplasmosis are considered the most important diseases (El Hussein et al., 2004).
In Sudan, more than 70 tick species were identifi ed including the most economically important ticks in Africa (Hassan, 2003).
According to Hoogstraal (1956) and Osman et al. (1982)  Although the distribution and prevalence rate of tick infestation have been documented previously in several states in Sudan, these data are changing dramatically due to climate changes and animal movement (Hassan and Salih, 2013). Th erefore, an annual investigation is recommended to update our data and to predict which kind of "new" or emergent infectious diseases could occur in the State. Additionally, only a few studies have investigated the risk factors associated with tick infestation on livestock farms in Sudan. Th erefore, this study was aimed to estimate the prevalence of tick infestation and to determine the potential risk factors associated with tick infestation in sheep in River Nile State.

Ethical approval
All animal procedures were carried out following the ethical standards established by the Institutional Ethics Committee of Sudan University of Science and Technology, Sudan.

Study area
Th e study was conducted in River Nile State, which is located in the northern part of Sudan between latitude 16 -22 °N and longitude 30 -32 °E. Th e state is bordered by Khartoum State to the south, the Arab Republic of Egypt to the north, Kassala State and the Red Sea State to the east and Kordofan State to the west. Generally, the climate is semi-desert, and the temperatures range between 47 °C in summer and 8 °C in winter. Th e mean rainfall is between 150 and 25 mm.

Study design
A cross-sectional study was performed over the time from June to August 2018 in River Nile State, Sudan ( Figure 1). Five diff erent localities, namely: Shendi, Al Matamah, Ad-Damer, Atbara, and Berber were conveniently selected and individual sheep were randomly sampled. Meanwhile, individual animal data including age, sex, breed and coat colour was recorded in a questionnaire. Moreover, the data regarding locality, herd size, housing type, rearing system, tick control, and removal of manure were also documented.
Th e animals were classifi ed into three age groups, young animals (age < 6 months), adults (age between 6 months and 3 years) and old (age > 3 years). Th e breed of sheep was classifi ed into two categories: crossbreed (Local breed × Saanen) and local breed. Th e coat colour of the examined sheep was classifi ed into four groups, white, black, brown, and mixed colour (more than one colour). Th e herd size was classifi ed into three groups: small (less than 70 animals), medium (70 -140 animals) and large (more than 140 animals). Th e housing type was categorized into two groups: the open and semi-closed. Th e rearing system was classifi ed into two groups: one-species rearing system (one species of animal) and a mixed rearing system (more than one species of animals).

Sample size
Th e overall number of animals to be included in the study was calculated using a formula of Th rusfi eld (2007): n = (1.96) 2 × P exp × (1-P exp ) / d 2 Accordingly, the sample size of 135 sheep was determined.

Collection and identifi cation of ticks
Ticks were collected from the predilection sites of sheep's bodies (preferred regions for ticks), which included ears, tails, udders and testicles. Th e collection was performed using a pair of blunt forceps, and the ticks were transferred into labelled tubes that contained 70% ethanol. Th e tick specimen was identifi ed using morphological keys (Hoogstraal, 1956;Walker et al., 2003).

Data analysis
Th e statistical soft ware program (SPSS version 16.0) was used to analyse the data. Th e association between the tick infestation and risk factors was analysed using the Chi-square test. In all analyses, a 95% the confi dence interval (CI) was held and the p-value less than 0.05 (p > 0.05) was set for statistical signifi cance.

Prevalence of tick infestation based on risk factors
Th ere was signifi cant variation (p ≤ 0.05) in the prevalence of tick infestation among the surveyed localities. Th e highest prevalence rate was reported in Atbara (100%), while the lowest prevalence was documented in Al Matamah (28%) ( Table 2). Both factors, i.e., tick control and removal of the manure, were also significantly associated with the tick infestation. Th e highest rate of ticks infestation was observed in farms that did not use acaricide (76.1%) compared with farms that used acaricide as a control measure (48.9%). Moreover, a higher infestation of ticks was detected in farms that did not remove the manure regularly (80%) as compared with other ones.
Risk factors such as herd size, rearing system, breed, age, and coat colour were not signifi cantly associated with the prevalence of tick infestation (p > 0.05) ( Table 2). However, the prevalence was higher in small heard size (70.8%) compared with medium (60%) and large size (66.7%). Th e highest prevalence rate of infestation was observed in one-species rearing system (77.1%) than in mixed rearing system (63%). Animals of the young age (age < 6 months) showed the lowest prevalence rate (60%) followed by adult animals (age between 6 months and 3 years) (67.6%), whereas animals of old age (age > 3 years) showed the highest prevalence rate (75%) ( Table 2).
During the sampling, all sheep examined were local breed. With respect to the coat colour, the prevalence rates of tick infestation ranged between 61.7% and 71.4% (Table 2).

DISCUSSION
In Sudan, out of 70 tick species documented, 34 species are known to infest sheep and goats. Th ese species belong to the genera Amblyomma, Hyalomma, Rhipicephalus (Boophilus) (Osman, 1997). During the current study, fi ve species of ticks were found to infest sheep in River Nile State, Sudan. Th ese include (in order of abundance), R. eversti, H. anatolicum, R. sanguineus, R. praetextatus, and H. dromedarii. Th ese species were previously reported in sheep from River Nile State (Ahmed et al., 2005) and from Khartoum (Gad Elrab, 1986). On the other hand, Yagoub et al., (2015) identifi ed ten tick species that infesting sheep and goats in Nyala town, South Darfur. A similar fi nding was also reported in North Kordofan and Kassala States, in which ten species of ticks that infest sheep were recognized (Springer et al., 2020). It is evident that the climate and ecological conditions of River Nile State do not allow for the existence and reproduction of several other ticks species that infest sheep such as Rhipicephalus (Boophilus) species and Amblyomma species, which exist in other states of Sudan (Osman et al., 1982).
Th e overall prevalence of tick infestation in sheep in the Nile River State was 66.7%. Th is result was comparable to those reported by Mathewos et al., (2021) in Ethiopia (68.33%) and higher than the 51.97% reported by Khan et al., (2022) in Pakistan. Th e variation in the infestation rate may be attributed to varying environmental conditions, production and management factors that in turn aff ect the ticks' population (Norval et al., 1992).
In general, male ticks represented the majority of all species collected except for H. dromedarii and R. sanguineus. Th is confi rms the fi nding of Ahmed et al. (2005) who reported that the number of male ticks usually exceeded the number of females among all species infesting sheep.
In the current study, risk factors such as locality, housing type, sex, control of ticks and removal of manure were signifi cantly associated with tick infestation. A signifi cant variation in the tick infestation was observed among the localities where the highest prevalence was observed in Atbara (100%), while the lowest one was reported in Al Matamah (28%). Th is fi nding confi rms the results of Fesseha et al. (2022) who observed a high frequency of tick infestation in Dasenech in Ethiopia (54.9%) compared to results of Salamago (45.1%), with a statistically signifi cant association. Th is could be due to the distinctions in the agroclimatic conditions of the study areas, and also to the time of sample collection. It is well recognized that tick activity can be infl uenced by altitude, season, rainfall, and atmospheric relative humidity (Ayalew et al., 2014).
Higher tick infestation was reported in the open type (77.8%) compared with the semi-closed type (44.4%) housing. Contrary to that, a lower prevalence of tick infestation has been observed on farms with open houses type in Pakistan (Rehman et al., 2017). One possible explanation to this diff erence is the construction of open housing types in Sudan. Namely, the construction of these houses is very poor, using traditional materials such as mud and wood full of cracks, which provides an optimal environment for the ticks to survive. Moreover, the animals that were raised in close or semi-closed housing types in Sudan received a proper veterinary service and more attention since the majority of them was owned by the companies. Th is hypothesis was confi rmed by our results revealing that the prevalence of tick infestation was higher in farms that did not apply acaricide (76.1%) compared with the farm that used acaricide (48.9%). In addition, the prevalence was higher in the farms that did not regularly remove manure (80%) compared with other ones. As we know, ticks have a free-living stage, in which they are dropping down to the ground in order to complete their life cycle (Walker et al., 2003). During this time, ticks become more susceptible to many factors such as hygiene measures, which in turn reduce the tick population.
Th e analysis of infestation according to sex revealed that there were more tick infestations on female sheep than on male animals. Similar fi nding was also reported in previous study, which established that the tick infestation was higher in females (78.1%) than in males (58.42%) (Mathewos et al., 2021). Th e logical explanation for this fi nding is that the stress factors such as pregnancy and lactation may have made the female animals more susceptible to infestation with ticks as compared with males. Adding to that, the females are generally kept for a long time for birth-giving pursuits, which raises the possibility of being infested with ticks (Mathewos et al., 2021).
Th is study found no signifi cant association (p > 0.05) between tick prevalence and factors such as herd size, rearing system, breed, age, and coat colour. However, the proportion of tick infestation was higher in old animals (age 3 years) (75%) than in the adult (67.6%) and young (60%) age groups. Th is fi nding was strengthened by the fi ndings of Asmaa et al. (2014) who reported higher infestation in animals aged more than 3 years. A higher prevalence of tick infestation may be due to low immunity in older animals and the longmileage movement of older animals searching for food, which increases the probability of infesting with ticks (Fesseha et al., 2022).
Although there is no association between the herd size and tick infestation, the prevalence of infestation was higher in the small-size herds (less than 70 animals) (70.8%) as compared with the medium (60%) and large size (66.7%) herds. Th is was comparable with the study conducted by Sajid et al. (2020) in which they found that herds having 40 -60 goats showed the highest ticks infestation. Th is might be due to the fact that a large number of ticks were fed on a few animals, which increased the rate of infestation.
No signifi cant diff erences in the tick infestation rate were found between the sheep reared with other ruminants (mixed rearing system) and those reared separately (one species rearing system). Th is fi nding was in agreement with the report of Sajid et al. (2020) who found no association between the tick infestation and rearing type (with other ruminants or separately).
In the present study, sheep coat colour did not signifi cantly infl uence the prevalence of tick infestation since the prevalence ranged between 61.7% and 71.4%. Contrary to that, Hayati et al., (2020) and Hassan (1997) found that the coat colour of cattle had a signifi cant infl uence on the tick burdens, where animals with light coat colour carried more ticks compared to the animals with dark coat colour. Th e author suggested that ticks picked by cattle with dark coat colours such as black and brown die or leave before the attachment, due to the rather increased temperature in the animal skin microenvironment produced by the dark coat colour (Hayati et al., 2020). Probably such kind of eff ect does not exist in sheep due to the diff erence between sheep coats and cattle coats.

CONCLUSION
Th e current study revealed a high prevalence of tick infestation in sheep in River Nile State, Sudan, and this causes a major health restraint, which results in huge economic losses. Th is study showed that Rhipicephalus eversti Neumann, 1897 was the predominant tick species followed by Hyalomma anatolicum Koch, 1844, Rhipicephalus sanguineus Latreille, 1806, Rhipicephalus praetextatus Gerstacker, 1877, and Hyalomma dromedarii Koch, 1844. Th e risk factor analyses showed that localities, housing type, sex, control of ticks, and removal of manure signifi cantly aff ected the prevalence of tick infestations. While factors such as herd size, rearing system, breed, age and colour of coat had no infl uence on the prevalence of tick infestation. Due to the high prevalence of sheep ticks in the study area, the State requires prompt attention at all levels to decrease the impact of tick and tick-borne disease on the health and production of animals and thereby enhance their productivity.

АCKNOWLEDGEMENT
Th e authors would like to acknowledge animal owners and animal health professionals for their cooperation during tick collection in the fi eld.

Author's Contribution:
Th is work was carried out in collaboration among all authors. H.K.K. carried out the experiments. S.B.M. planned the experiments, analysed the data, wrote the manuscript and supervised the project. K.M.T contributed to the fi nal version of the manuscript. All authors read and approved the fi nal manuscript.