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Babesiosis in cattle and ixodid tick distribution in Dasenech and Salamago Districts, southern Ethiopia

Babesiosis, caused by protozoan parasites of the genus Babesia, and ixodid ticks are considered to be one of the most important causes that result in significant economic losses in cattle production worldwide, including in Ethiopia. A cross-sectional study was undertaken in the Dasenech and Salamago Districts of South Omo zone Ethiopia to determine the prevalence and associated risk factors of babesia infection and ixodid ticks of cattle using Giemsa-stained thin and thick film techniques and morphological identification keys for babesia species and tick identification, respectively. Out of 470 examined cattle, 102 (21.7%) were infected by Babesiosis (15.53% Babesia bigemina and 6.17% Babesia bovis). A statistically significant association (p < 0.05) was observed between babesia infection and season and tick infestation. However, cattle that were infected with the Babesia parasite revealed a lower mean PCV value (21.49%) than noninfected cattle (28.29%) and showed a statistically significant (p < 0.05) difference with the occurrence of Babesia infection. The overall prevalence of ixodid ticks was 53.8% (253/470) and revealed a statistically significant association (p < 0.05) between the season and origin of the animal. However, no statistically significant association (p > 0.05) was observed between sex, age, and body condition score of the animal with the occurrence of ixodid tick. A total of 8040 adult ticks belonging to four tick genera, Amblyomma, Rhipicephalus (Boophilus), Hyalomma, and Rhipicephalus, were collected from various body parts and identified. The high prevalence of Babesia infection and ixodid ticks in cattle at the study sites requires seroprevalence and molecular studies to identify the predominant Babesia species and to detect Babesia in tick hemolymph for the identification of tick genera responsible for the occurrence of Babesia infection. Additionally, tailoring suitable and coordinated tick management methods using chemotherapy as well as strategic treatment to overt clinical cases of bovine babesiosis is critical.

Determination of parasitemia and packed cell volume (PCV). The blood samples of 470 cattle were collected from the jugular vein and ear vein after disinfection with 70% alcohol for hematological analysis. Blood film examination was performed with Giemsa staining procedures, and microscopic examination of slides was conducted according to 35 . Blood samples were taken from the ear veins with piercing with a sterile lancet and then collected with heparinized capillary tubes after thin and thick blood smears were made and labeled. The slide was then air-dried and immediately fixed with absolute methyl alcohol for 3 min, and then, the smear was stained with 10% Giemsa stain for 45 min. Finally, a minimum of 50 fields of each stained blood smear was thoroughly examined under a compound microscope using oil immersion according to Foreyt 36 , to determine parasitemia and identify species of parasite based on a reference set by Soulsby 37 , and Moretti et al. 38 . Parasitemia was detected over Hemacolor ® -stained thin blood smears by counting at least 1000 red blood cells and noting the number that was infested with Babesia.
Blood collected by jugular venipuncture in 10% EDTA-containing Vacutainer tubes was subjected to estimation of packed cell volume (PCV) using microhematocrit centrifugation. The microhematocrit capillary tubes (up to ¾) were filled with blood with a sealed outermost end. The blood specimens were centrifuged at 12,000 rpm for 5 min using a hematocrit centrifuge 35 . After centrifugation, the capillaries were put into the hematocrit reader for hematocrit (PCV) measurements, and then cattle were classified as anemic based on a modification of the classification criteria of Reyers et al. (1998) cited by Jacobson 14 combined with that of Tvedten and Weiss 30 . Cattle with PCV of less than 24% were considered severely anemic, and those with PCV > 46% were considered nonanemic.
Tick collection and identification. The entire body surface of the cattle was inspected for the presence of ticks. After fully restraining the animals, all visible adult tick species were removed by gloved hands and using forceps holding the base capitulum so as not to lose the mouthparts of the ticks. Ticks from each animal were collected and kept in separate prelabeled universal bottles containing 70% ethyl alcohol until identification. Ticks were counted and subsequently identified to the genus level by using a direct stereomicroscope using key morphological characteristics, i.e., size of mouthparts, the color of the body, leg color, presence or absence of the eye, the shape of scutum, body, coxae one, festoon, and ventral plates were considered as described by Walker et al. 39 under a stereoscopic microscope.
Data management and statistical analysis. Data collected during the study period were stored in Microsoft Excel spreadsheets and analyzed using statistical software called STATA version 13 for Windows (Stata Corp. College Station, USA). Descriptive statistics (frequencies and percentages) were used to determine the prevalence of babesia infection and tick infestation in cattle. The overall prevalence of babesia infection and ticks was determined by dividing the number of positive animals by the total sample size and was expressed as a percentage. The chi-square test and multivariate logistic analysis were used to examine the relationship between potential risk factors, including origin, age, sex, body condition, season, and the occurrence of both Babesia infection and tick infestation. Effects were reported as statistically significant in all cases if the value is less than 0.05 at a 95% confidence interval (CI).
Ethics approval and consent to participate. Ethical approval for this research was obtained from the Wolaita Sodo University Research Ethics and Review Committee. Before collecting samples, informed consent was obtained from the cattle owners to take samples from their cattle and adopt strict hygienic measures.

Results
Frequency of babesiosis in cattle of the study area. Among

Determination of the babesia infection status and mean PCV in study cattle.
In the current study, the level of babesia infection status was determined by using thin and thick blood smears. Approximately 95% of cattle were parasitemic, and the remaining 5% were aparasitemic up on thin and thick blood smears, respectively. The mean PCV of anemic cattle due to babesia infection was 21.49% (CI = 21.09-21.89%). However, a statistically significant association (t = 29.67, p = 0.0001) was observed between the PCV of anemic and nonanemic cattle and the occurrence of babesia infection ( Table 2).   ). The infestation of the ixodid tick during the wet season (50.6%) was found to be higher than that during the dry season (49.4%). Moreover, a statistically significant association (p < 0.05) was observed with the occurrence of ixodid ticks among address and season of the year (Table 4). However, no statistically significant variation (p > 0.05) was observed with the occurrence of the ixodid tick among other putative risk factors, such as age, sex, and body condition score, of the study cattle ( Table 3).
The prevalence of ixodid tick genera identified in the study district. The Table 4. The ixodid tick genera identified in the study district.

Freq (%) X 2 (p value) Freq (%) X 2 (p value) Freq (%) X 2 (p value) Freq (%) X 2 (p value)
Origin www.nature.com/scientificreports/ of animals showed a statistically significant difference (p < 0.05) with the occurrence of Amblyomma spp. and Hyalomma spp. Although Amblyomma spp. and Rhipicephalus spp. showed a statistically significant difference (p < 0.05) with the season ( Table 4). The odds of cattle infested by a tick in the Dasenech area were 2.38 times higher (CI, 1.60-3.55) when those cattle infested by a tick from the Salamago area were kept constant. When dry seasons were held constant, the odds of cattle being affected by ixodid ticks increased by 0.37 times (CI, 0.248-0.561). However, a statistically significant association (p < 0.05) was observed between tick infestation and season and origin. However, there was no statistically significant association (p > 0.05) between sex, age, and body condition score of the animal and the occurrence of ixodid tick (Table 5).

Tick distribution across the animal's body.
A total of 8040 ticks were collected from various body parts of cattle in the current investigation from a total of 253 tick-infested cattle out of 470 investigated animals. Accordingly, the highest proportions of ticks were collected from the scrotum/udder (32.10%), followed by the dewlap and neck (22.06%), anus, and vulva (20.14%), and head and ear (13.84%). Additionally, the majority of Rhipicephalus species were collected from scrotum/udder (32.91%) and dewlap and neck (27.09%), although a high number of Amblyomma species were collected from scrotum/udder (43.9%) and brisket (29.03%). On the other hand, Rhipicephalus (Boophilus) species were found around the anus and vulva (35.01%) as well as the head and ear (22.75%), whereas Hyalomma species were the least collected type of tick genera during the current study and were mostly found around the anus and vulva region (29.50%) and brisket (23.40%) ( Table 6).

Discussion
Babesia spp. and ticks are major constraints of livestock production that have a worldwide distribution with greater economic importance through direct and indirect effects on their hosts in tropical and subtropical regions by Criado-Fornelio et al. 40 , Rahman et al. 41 ; Filia et al. 42 , and have been reported in Ethiopia by Sitotaw   52 and Sitotaw et al. 43 , who reported a prevalence of 16.9% in Teltele district, 12% in Bariso,11.4% in Arsi, 6.51% in Alle district, 9.9% in Kohat and Karak District, 6.6% in Lahore, 6% in Tiyo District, 3.64% in Meki and Batu Towns, 1.5% in Assosa district, 1.2% in Nigeria and 0.3% in Debre-Zeit, central Ethiopia. In contrast, the current study's findings were lower than those reported by Rahman et al. 53 and Mohammed and Ebied 54 , who found prevalence rates of 42% in Malaysia and 40% in Benha, Qalubia Governorate, northeastern Egypt, respectively. This variation in babesiosis prevalence in cattle could be attributed to differences in the study area, the use of acaricides during tick infestation, the sensitivity of diagnostic tests used, proper use of antiparasitic drugs, cattle management systems in the focus area, and sampling seasons of the year, as well as the land use of the area, the distribution of infected vectors, and the accessibility of animals to wildlife reserves and parks and forest areas containing tick vectors, which vary among the study areas 55,56 .
The overall prevalence of Babesia species was 15.53% (B. bigemina) and 6.17% (B. bovis). This result corresponded with the former description of Solomon and Tanga 46 , who reported a prevalence of 6.51% for Babesia bovis from Alle District, Southwestern Ethiopia. Contrary to this finding, a lower prevalence was reported by Waktole et al. 50 ; Wodajnew et al. 51 , and Abdela et al. 57  In the current study, a higher prevalence of bovine babesiosis was noted in adult age (66.7%), followed by young (18.6%) and old age cattle (14.7%). In contrast, Lemma et al. (2015) from Jimma town in southwestern Ethiopia, Waktole et al. 50 from the Maki and Batu areas, and Ayaz et al. 47 from Pakistan consistently reported a high prevalence of Babesia infection in old age cattle, with prevalences of 27%, 7.5%, and 13.4%, respectively. However, Amorim et al. 62 identified that calves were more susceptible to Babesia species than adult cows, which was also opposed to the present finding. Young animals, particularly calves under the age of six months, are more likely to be resistant to minor infestations than older animals because they develop acquired immunity by feeding Colostrum of the dam 63 . On the other hand, the low frequency in young animals is due to limited grazing by young animals, which may reduce the likelihood of exposure to this vector 64 .
The prevalence of babesia infection in males (54.9%) was slightly higher than that in female (45.1%) cattle. This finding was in agreement with the report of Solomon and Tanga 46 and Waktole et al. 50 , who found a high prevalence of babesiosis in male cattle (7.09%) and 4.4% compared to females. Nevertheless, this result disagrees with the report of Kocan et al. 65 , who found a higher prevalence of babesiosis in females (11.2%) than in male cattle (6.96%). The higher prevalence of babesia infection in male animals could be linked to their being employed for farming purposes (plowing), which could cause stress and make them immunocompromised, as well as being kept out of the door, where they could be infected by a tick vector.
The prevalence of the disease based on the body condition of the animals was 21.6%, 19.8%, and 68.6% for good, medium, and poor body condition scoring, respectively, which was comparable with reports described by Hamsho et al. 4  The odds of babesia infection in the wet season was 0.74 times when the dry season was kept constant and showed a statistically significant difference (p < 0.05) with the occurrence of babesia infection. Similarly, Kamani et al. 64 ; Solomon and Tanga 46 , and Waktole et al. 50 reported a high prevalence of Babesia infection in the wet season compared to the dry season. In the seasonal prevalence of the disease, the disease was more prevalent in wet conditions, probably the reason behind this trend may be correlated to the seasonal activities of the hard ticks, which are more abundant on the days after the rain peaks, thus resulting in a higher incidence of babesia infection, as reported by Soulsby 66 .
The availability of vectors is a potential risk factor for Babesia infections 67 . The multivariate logistic regression analysis showed that the risk of babesiosis was significantly (p < 0.005) higher in tick-infested (95.1%) cattle than in nontick-infested (4.9%) cattle. A similar observation was reported by Costa et al. 68 . The presence of Babesia infection in cattle in the study area is broadly related to the presence of suitable vectors (ticks).
In the current study, there was a statistically significant (p = 0.0001) variation in the mean PCV value between infected cattle (21.49%) and noninfected cattle (28.29%), which was in line with the previous report by Waktole et al. 50 and Sitotaw et al. 43 . This suggests that anemia is caused by the blood suck of ticks or babesiosis since the parasite invades red blood cells to cause hemolysis. Moreover, anemia develops as a result of blood hemolysis, and hemolysis occurs due to mechanical damage by trophozoites to RBCs when multiplied by binary fission, phagocytosis of infected RBCs by the host immune system, and toxic substances secreted by the parasites 69 65.5%, 59.6%, 61% and > 80% from different parts of the country, respectively. On the other hand, the overall prevalence of tick infestation in the current study was higher than the previous reports described by Solomon 50 and Zelalem et al. 26 , who reported a prevalence of 36.19% in Alle district, 42.2% in Hosana district, 25.64% in Holeta district, 29.4% in Maki and Batu area, and 38% in the Chiro district, respectively. This may be due to differences in study area coverage and environment. This can also be due to factors such as animal health practices, temperature, and humidity that promote the survival and growth of ticks during their development, acaricide application as a control method, a low level of awareness of farmers, and the proportion of ticks 83,84 .
In this study, a high frequency of tick infestation was observed in Dasenech (54.9%) compared to Salamago (45.1%), with a statistically significant association (p < 0.05) with the occurrence of Ixodidae ticks. This could be due to the differences in the agroclimatic conditions of the study areas and the season of sample collection. It was reported that tick activity can be influenced by rainfall, altitude, season, and atmospheric relative humidity 19 . In addition, a large livestock population and herd size contribute to tick infestation, as ticks can easily obtain access to hosts and complete their life cycle to continue rapidly, and poor veterinary service and less attention are given to cattle management practices employed by herders, which might also pave the way for tick infestation.
In terms of the sex of the host, the prevalence of ixodid ticks by sex revealed that males (52.6%) were more infected than female (47.4%) cattle, with a statistically insignificant difference (p > 0.05). This finding agrees with the finding of Wasihun and Doda 28 , who reported higher infestation in male animals than in females. However, contrary to our findings, Abdeta et al. 18 and Kassa and Yalew 22 reported a higher prevalence in female animals (68% and 18.8%) compared to males (82.06% and 14.23%), respectively. This could be attributed to female cattle being kept in the house with proper management for dairy purposes, while male cattle grazed on a field all day can be exposed to tick infestation 85 .
Cattle with poor (71.2%) and good (16.6%) body condition scores showed a high prevalence of tick infestation compared to moderate body-conditioned cattle (12.3%), which did not show a statistically significant (p > 0.05) difference with the occurrence of tick infestation. Similarly, Kassa and Yalew 86 and Shiferaw and Onu 34 reported a statistically insignificant correlation (p > 0.05) between the existence of Ixodidae ticks and the animal's body condition score. On the other hand, Tiki and Addis 82 reported a statistically significant difference (p < 0.05) between body condition scores and the existence of ticks. The high infestation rate of hard ticks in poor body condition scores compared to moderately conditioned animals could be due to the less resistant behavior of weak animals to tick infestation and lack of enough body energy to build resistance 36,87 .
The proportion of tick infestation was higher in the adult (70.7%) than in the old (11.5%) and young (17.8%) age groups, with a statistically insignificant (p > 0.05) difference in the occurrence of tick infestation. This finding was strengthened by the findings of Desalegn et al. 88 , who reported higher infestation in adult cattle, and agrees with the findings of different authors Kemal et al. 22 90 , and Okello-Onen et al. 91 , who reported a higher proportion of adult cattle from different parts of the country. A higher proportion may be due to the long-distance movement of adult cattle to search for food, which increases the chance of contact with ticks and low immunity in older animals.
In the current study, Amblyomma was found to be the most abundant tick genus, followed by Hyalomma, Rhipicephalu (Boophilus), and Rhipicephalus. This finding was strengthened by Kassa and Yalew 86 , Kemal et al. 22 , Ayalew et al. 19 , Wasihun and Doda 80 and Yehualashet et al. 92 , who reported that Amblyomma was the most common and widely distributed cattle tick in Ethiopia and African countries. In contrast to this result, Ayana et al. 70 and Waktole et al. 50 reported Rhipicephalus as the dominant tick genus. In addition, according to Shane et al. 49 , Boophilus was identified as the main tick species in Tiyo District. This may be due to the different seasons in which the survey was conducted 5,81 .
In the current study, a total of 8040 ticks were collected from various parts of the animal body. The highest tick frequency was gathered from the scrotum/udder (32.10%), followed by dewlap and neck (22.06%), anus and vulva (20.14%), and head and ear (13.84%). Additionally, the majority of Rhipicephalus species were collected from scrotum/udder (32.91%) and dewlap and neck (27.09%). However, Ayana et al. 70 from the Yabello district reported a large number of ticks from the head and ear (34.57%) region. Furthermore, Amblyomma species were collected in a high percentage from scrotum/udder and brisket, whereas Rhipicephalus species were collected from head and ear and anus and vulva, Hyalomma species were collected from scrotum/udder, anus and vulva and dewlap and neck, and Boophilus species were collected from anus and vulva part of the body.

Conclusion
The present study revealed a high prevalence of Babesia infection and ixodid ticks in cattle in the Dasenech and Salamago Districts. Babesia bigemina and Babesia bovis are the two most important Babesia species of cattle that were identified in the study area. A statistically significant difference (p < 0.05) was observed between season and tick infestation with the occurrence of Babesia infection. In this study, Ambylomma was found to be the most predominant tick genus, followed by Hyalomma, Rhipicephalus (Boophilus), and Rhipicephalus. Thus, seroprevalence and molecular studies to identify the predominant Babesia species and for the detection of Babesia in tick hemolymph for the identification of tick genera responsible for the occurrence of Babesia infection are crucial. Additionally, tailoring suitable and coordinated tick management methods using chemotherapy as well as strategic treatment to overt clinical cases of bovine babesiosis is critical. www.nature.com/scientificreports/