epidemiological study
Onur Ceylan
*
, Ferda Sevinc
Selcuk University, Faculty of Veterinary Medicine, Department of Parasiyology, Konya, Turkey
A R T I C L E I N F O Keywords: Babesia ovis ELISA IFAT Sheep Endemic status A B S T R A C T
Ovine babesiosis is an endemic tick-borne disease of small ruminants in the Middle East, European, and some African and Asian countries, including Turkey. This study assessed whether the endemic status of this disease was stable or instable, which is important for disease control efforts. For this aim, 4115 sheep blood samples were collected from 81 cities in the seven geographical regions of Turkey. The diagnosis of Babesia ovis was made using microscopic and serological techniques. Thin blood smears were prepared from anticoagulated venous blood. Serum samples were screened for specific antibodies using an enzyme-linked immunosorbent assay (ELISA) and indirectfluorescent antibody test (IFAT). Recombinant Babesia ovis secreted antigen 1 (rBoSA1) was used in the ELISA. The antigen slides used in the IFAT were prepared from the B. ovis-infected blood at a high level of parasitemia (5 %). The animals were divided into three groups according to their age: group I (one to six months), group II (6–12 months), and group III (older than one year). The endemic status of B. ovis was de-termined according to the inoculation rate (h value) calculations. Babesia spp. merozoites were observed in 40 (0.97 %) of the slides. Seropositivity rates were 29.89 % (1230/4115) and 49.16 % (2023/4115) by the ELISA and IFAT, respectively. According to the IFAT results, 31.7 %, 33.6 %, and 52.8 % of the animals were ser-opositive in groups I, II, and III, respectively. The inoculation rates of the animals indicated that the endemic status of ovine babesiosis was mostly instable throughout the country. Endemic stability was found only in group I from four regions (Central Anatolia, Eastern Anatolia, Aegean, and Mediterranean). Based on these results, the risk of clinical infection due to tick infestation was high when the maternal immunity and non-specific age resistance weakens or disappears. Thus, vaccination is needed to protect sheep against B. ovis infections in Turkey.
1. Introduction
Ovine babesiosis, which is a hemolytic disease transmitted by ixodid ticks, occurs in small ruminants in many European, Asian, African, and the Far Eastern countries and causes significant economic losses in the livestock industry (Ahmed et al., 2006; Uilenberg, 2006; Inci et al., 2016; Sevinc and Xuan, 2015). The severity of infections varies ac-cording to host age, immune status, mixed infections with other pa-thogens, and genetic factors. The main clinical symptoms include fever, hemoglobinuria, anemia, anorexia, weakness, jaundice, and even death (Schnittger et al., 2012;Sevinc et al., 2013a;Sevinc and Xuan, 2015). Among the six species implicated in ovine babesiosis (B. crassa, B. fo-liata, B. motasi, B. ovis, B. taylori, and B. sp. Xinjiang), B. ovis is the most pathogenic species and is generally transmitted to hosts by ticks in the genus Rhipicephalus (Uilenberg, 2006;Shayan et al., 2007;Guan et al., 2012;Sevinc and Xuan, 2015).
Turkey acts as a wide land bridge between European and Asian countries where ovine babesiosis is of great importance due to favor-able geographical and climatic conditions for the maintenance of the vector ticks. Ovine babesiosis adversely affects small ruminant breeding in Turkey and causes serious economic losses due to control and treatment expenses (Sevinc and Xuan, 2015;Ozubek and Aktas, 2017). The most significant mechanisms of control include detecting and treating sick animals, identifying and eliminating the vector ticks, and protecting the healthy animals against the disease. In order to imple-ment these measures successfully, comprehensive investigations are needed in the places where the disease is observed (Sevinc and Xuan, 2015). Although the early diagnosis and subsequent treatments of the disease yield good results, protective measures must be taken to prevent the parasite from spreading. Vaccination is the most effective protective measure against vector-borne diseases (Healer and Cowman, 2016; Smit and Postma, 2016). To ascertain the necessity of a vaccine, the
https://doi.org/10.1016/j.vetpar.2020.109034
Received 17 December 2019; Received in revised form 17 January 2020; Accepted 18 January 2020
⁎Corresponding author.
E-mail address:onurceylan@selcuk.edu.tr(O. Ceylan).
endemic status of the disease should be determined. Endemic stability and instability describe population immunity in epidemiology and are defined according to the inoculation rate, which is calculated using the number of seropositive individuals in a formula developed byMahoney and Ross (1972). Vaccination is required if the endemic status is in-stable. However, if it is stable, there is no need to vaccine to control the disease (Geleta, 2005;Ekici and Sevinc, 2011).
Although several epidemic and endemic B. ovis infections have been reported in all countries bordering the Mediterranean Sea, including Turkey and some Asian countries, limited information exists on the endemic status of the disease. This study aimed to determine the en-demic status of ovine babesiosis by analyzing its nationwide distribu-tion in Turkey.
2. Material and methods 2.1. Study area and sample collection
Turkey, which has a wide land area of 783.577 km2, is located between 36°-42° northern latitudes and 26°-45° eastern longitudes (Fig. 1) and has approximately 50 million small ruminants (Turkstat, 2019). The samples were collected from sheep in 81 cities across the seven geographical regions in Turkey between May and October in 2017. Blood samples were taken from 4115 apparently healthy sheep belonging to three age groups (I, II, and III comprised of 1–6, 6–12, and > 12 months, respectively) from various provincial locations,
districts, and villages where the sheep population was high. Overall, 836, 774, 763, 459, 458, 422, and 403 blood samples were collected from the Eastern Anatolia, Southeastern Anatolia, Central Anatolia, Marmara, Aegean, Black Sea, and Mediterranean regions, respectively. Blood samples (5−8 ml) were collected from different sheep herds (ranging from 1 to 8) in each city and taken to serum tubes using sterile needles. Blood was drawn from each animal, kept in a mini cooler box, and centrifuged (3000 rpm for 15 min) to obtain serum. All serum samples were stored at−20 °C until used in a serological test.Fig. 1 shows a map of sample collection sites.
2.2. Ethical statement
The owners of the sheep were informed about the study and their approval was obtained for sampling. All procedures used in this study were carried out according to the ethical guidelines for the use of an-imal samples permitted by Selcuk University, Veterinary Medicine (Permit for animal experiment: 2017/36, Date: 27.03.2017).
2.3. Blood smear preparation and microscopic examination
Thin blood smears from 4115 sheep were prepared from the antic-oagulated venous blood, dried in open-air,fixed with methanol, stained with Giemsa, and stored in storage boxes until microscopic examina-tion. In the staining process, after three minutes offixation with abso-lute methanol, the smears were stained with 10 % Giemsa solution for
Fig. 1. Map of Turkey showing sample collection sites (red dots) in all 81 cities. (For interpretation of the references to colour in thisfigure legend, the reader is referred to the web version of this article).
45 min, washed under tap water, and dried. After dropping immersion oil on the slides, the smears were examined for blood parasites (Babesia, Theileria, and Anaplasma) under high magnification (100X) of a light microscope. At least 20 microscopicfields were scanned to detect the pathogens.
2.4. Expression and purification of rBoSA1 and enzyme linked immunosorbent assay (ELISA)
A method previously described bySevinc et al. (2015a)was used to express and purify the rBoSA1, and the purified rBoSA1 content was determined using a spectrophotometer. rBoSA1 was put into ELISA microplate wells after reconstitution with 2μg/ml carbonate-bicarbo-nate buffer. Other test procedures were performed as previously de-scribed bySevinc et al. (2015a).
2.5. Preparation of the antigen slides and indirectfluorescent antibody test (IFAT)
The antigen slides were prepared from blood infected with B. ovis at a high level of parasitemia. After the blood was added to tubes in-cluding anticoagulant from naturally infected sheep, the blood cells were washed 3 times by centrifugation in phosphate buffer saline (PBS, pH 7.4) and buffy coat was removed each time. The volume was equilibrated to the initial volume by adding PBS. Then, 10μl blood was added to every well of multitest slides (ICN-6041505, 15-well) and dried at room temperature. The multitest slides were wrapped in paper and aluminum foil and stored at−80 °C until used in the IFAT, which was performed using the protocols described byEkici et al. (2012). The positive and negative control sera used in the present study were ob-tained from the previous study conducted bySevinc et al. (2007).
2.6. Calculation of the endemic status of B. ovis throughout Turkey The probability of babesiosis occurrence in an animal population can be calculated by determining the inoculation rate.Mahoney and Ross (1972)expressed the inoculation rate as‘h’ and formulated it as follows:
•
h = (-1)[ln(1-the rate of infected animal)]/mean age of animals (day)Mahoney and Ross (1972)stated that if the result obtained by the formula is between 0.05 and 0.005, the endemic status of the popula-tion is stable and if it is between 0.005 and 0.0005, there is an endemic instability in the population. They indicated that disease outbreaks occur at a very low probability in populations with an inoculation rate lower than 0.0005.
2.7. Statistical analysis
The categorical variables were presented in percentage frequency and the continuous data were indicated as mean ± standard deviation. The variables were pre-evaluated (using the Shapiro-Wilk and Levene’s tests) for normality and homogeneity of variances. Sensitivity and specificity of the diagnostic methods were calculated using the negative and positive predictive values. In cases where the expected cells fell below 20 %, the data were determined using the Monte Carlo Simulation Method for inclusion in the analysis. The significance level was indicated to beα = 0.05. The SPSS 25 (IBM Corp. Released 2017. IBM SPSS Statistics for Windows, Version 25.0. Armonk, NY: IBM Corp.) statistical package program was used to analyze the data. P values were calculated to compare the test performances and to determine the level of statistical significance between geographic regions and age groups according to the obtained data.
3. Results
3.1. Blood parasites diagnosed by microscopic examination
After the microscopic examination of the Giemsa stained blood smears, Babesia spp. merozoites were observed in 40 (0.97 %) animals (Fig. 2). One or more agents including Babesia spp., Anaplasma spp., and Theileria spp. were detected in 2296 (55.80 %) animals. In total, Ana-plasma spp. and Theileria spp. (Fig. 2) were detected in 1709 (41.53 %) and 1302 (31.64 %) of the animals, respectively. The number of single positive animals for Anaplasma spp. and Theileria spp. was 958 (22.55 %) and 564 (13.71 %), respectively.
3.2. Seroprevalence of B. ovis by ELISA
Of the 4115 serum samples, 1230 (29.89 %) were found to be po-sitive for anti-B. ovis antibodies using the ELISA test with rBoSA1 pro-tein as the antigen. The highest seroprevalence rate was in the Eastern Anatolia Region (53.83 %) and the lowest in the Marmara Region (9.80 %). Seroprevalence of B. ovis was the same (27.52 %) in the Central Anatolia and Southeastern Anatolia regions.Fig. 3provides information on other regions.
3.3. Seroprevalence of B. ovis by IFAT
By the IFAT, 2023 (49.16 %) samples were found to be positive for anti-B. ovis antibodies. The IFAT results showed the highest ser-opositivity rate in the Eastern Anatolia Region (65.19 %) and the lowest in the Marmara Region (24.18 %), similar to the ELISA results.Fig. 4 shows the seroprevalence rates in the other regions.
3.4. The results of statistical analysis
Considering the general distribution of the B. ovis infection throughout Turkey, a statistically significant difference was found be-tween the age groups according to the ELISA (P = 0.009,χ2= 9.659) and IFAT (P = 0.001, χ 2= 96.68) results (P < 0.05). By the ELISA, 22.5 %, 27.8 %, and 30.8 % seropositivities were determined in group I, II and III, respectively. There was no statistically significant difference between the age groups in the Southeastern Anatolia, Eastern Anatolia, Aegean, and Marmara regions (P > 0.05). Statistical differences be-tween the age groups in the other regions were significant (P < 0.05). By the IFAT, 31.7 %, 33.6 %, and 52.8 % seropositivities were de-termined in group I, II and III, respectively. The differences were not significant between the age groups in the Eastern Anatolia, Aegean, and Mediterranean regions (P > 0.05), while the difference was statistically significant in the other regions (P < 0.05).
According to the ELISA (Table 1) and IFAT (Table 2) results, there were statistically significant differences between the prevalence rates of B. ovis infections in the seven geographical regions.
The prevalence of B. ovis determined by microscopic examination was compared between the regions and a statistically significant re-lationship was found (Table 3).
The diagnostic performances of the three tests used in the study were compared. A statistically significant relationship was determined between the ELISA and IFAT (P = 0.001, χ 2= 383.009). A similar relationship was found between the ELISA and microscopy (P = 0.001, χ2= 27.264) and between the IFAT and microscopy (P = 0.001,χ2 = 23.755). The IFAT is the gold standard for the serodiagnosis of ba-besiosis. Therefore, the specificity of the ELISA was 84 % (0.82 to 0.85 at 95 % confidence interval) and the sensitivity was 44 % (0.42 to 0.46 at 95 % confidence interval). According to the comparison of the tests, the positive and negative predictive values were 73 % (0.70 to 0.75 at 95 % confidence interval) and 61 % (0.59 to 0.63 at 95 % confidence interval), respectively.
3.5. Determination of endemic status of B. ovis throughout Turkey To determine the endemic status of B. ovis infection, the inoculation rates of the groups were calculated based on the IFAT serological re-sults. At the regional level, the inoculation rates of the groups are shown inTable 4.
The inoculation rates of the animals from group II and group III were lower than 0.005 in all regions. According to thisfinding, the endemic status of B. ovis was instable in the age groups of 6–12 and > 12 months in the regions where the study materials were col-lected. However, the inoculation rates of group I were greater than 0.005 in four regions; Central Anatolia, Eastern Anatolia, Aegean, and Mediterranean. This shows a stable endemic status in the animals from the 1–6 months age group in these regions.
4. Discussion
Ovine babesiosis is a hemolytic disease of small ruminants trans-mitted to the hosts by ixodid ticks, which causes significant economic losses in the livestock industry in many European, Asian, African, and Far Eastern countries (Ahmed et al., 2006;Uilenberg, 2006;Inci et al., 2016;Sevinc and Xuan, 2015). Turkey is one of these countries and the disease impairs the economy every year during tick activity seasons (Ozubek and Aktas, 2017). Therefore, country-wide epidemiological research is necessary to create road maps for the control of the disease (Ekici et al., 2012;Sevinc and Xuan, 2015).
Most of the studies about the distribution of ovine babesiosis have been reported from the countries bordering the Mediterranean Sea (Southern Europe, North Africa, and the Middle East) and from some Asian and far Eastern countries, such as Turkey, Iran, Iraq, Israel, Tunis, Egypt, Pakistan, and Spain (Yeruham et al., 1995;Ferrer et al., 1998; Ekici et al., 2012;Haghi et al., 2013;Renneker et al., 2013;Shahzad et al., 2013;Rjeibi et al., 2016;Hussein et al., 2017;Zhou et al., 2017). These studies have generally focused on the prevalence of ovine
Fig. 3. The seroprevalence rates of B. ovis infection at the seven geographical regions of Turkey according to the ELISA results.
regions with different ecological and climatic conditions. Because most reports are from the Central and Eastern part of the country, current data is not sufficient to analyze the country-wide distribution of the disease. The research based on microscopic examination indicate that the prevalence of B. ovis ranges from 0 % to 67.38 % in different parts of Turkey (Cakmak et al., 1991; Sevinc and Dik, 1996; Dumanlı et al., 1997;Aktas et al., 2001;Cicek et al., 2004;Aktas et al., 2007;Altay et al., 2017). In the present study, microscopic and serological techni-ques were used to observe B. ovis merozoites and to analyze B. ovis-specific antibodies, respectively. Microscopic examinations revealed similar results (0.97 %) to the previous studies. Babesia parasites were detected in all regions except the Mediterranean Region through mi-croscopic examination. Approximately half of the mimi-croscopically po-sitive animals were detected in the Central Anatolia Region. This higher rate compared to other regions may have been observed because the time spent duringfield studies in this region, especially in Konya, co-incides with the active period of clinical cases in June.
Due to the limitations of microscopic detection, immunological detection plays a vital role as it is based on antibody detection. Antibody detection methods have commonly been used to provide epidemiological data in population screenings. In this study, the IFAT and ELISA were used to detect B. ovis-specific antibodies. Previous serological researches indicate that the prevalence of B. ovis varies between 24.3 % and 80.9 % according to regions and age groups in Turkey. In the present study, both the IFAT and the ELISA results were within the ranges recorded in previous studies (Cakmak et al., 1991; Duzgun et al., 1991;Sevinc and Dik, 1996;Dumanlı et al., 1997;Sayın et al., 1997;Aktas et al., 2001;Emre et al., 2001;Cicek et al., 2004; Ekici et al., 2012). Seroprevalence rates of B. ovis were found to be 29.89 % and 46.16 % by the ELISA and IFAT, respectively. The dif-ference between these two test results was attributed to rBoSA1, which is a secretory protein of B. ovis. Specific antibodies against rBoSA1 are detectable starting from the 7th and 8th days of infection and up to 2.5 months after infection (Sevinc et al., 2015a). No data could be found about the duration of rBoSA1 antibodies after natural infections. However, specific antibodies, which are detectable towards the end of an acute infection, can be detected by the IFAT for a very long time after infection (Habela et al., 1990a;Sevinc et al., 2013b).
Serological methods are commonly used to provide epidemiological data in population screenings. One disadvantage of serological tests is that cross-reactions may occur between different species (Bruning,
activity between ovine Babesia species and T. ovis using the IFAT. Ad-ditionally, no cross-reactivity was found between B. ovis and T. ovis or B. motasi or B. crassa using the IFAT (Habela et al., 1990b). However, there is no study indicating that rBoSA1 is a specific antigen only for B. ovis in the ELISA. Further studies are needed to determine whether there is a cross-reaction with the antibodies against ovine pathogens other than B. ovis (Sevinc et al., 2015a).
Thefirst ELISA-based serological study was carried out byDuzgun et al. (1991) in Turkey. They used a synthetically derived antigen (11C5) originating from B. bovis, a pathogenic bovine Babesia agent, in the ELISA. Hashemzadeh Farhang et al. (2006) developed an ELISA technique using antigens obtained from B. ovis-infected erythrocytes. To our knowledge, this is thefirst comprehensive surveillance to de-termine the seroprevalence of B. ovis by the ELISA test using a re-combinant protein. In the present study, rBoSA1 protein was used as an antigen in the ELISA to detect specific antibodies against B. ovis.Sevinc et al. (2015a)reported that this protein was abundant in the infected erythrocytes during infection. rBoSA1 and the immunoreactive proteins (BoSA2, BoSPD, and ovipain-2) reported in some other studies can become the main components of subunit vaccines against ovine babe-siosis (Sevinc et al., 2015a, Sevinc et al., 2015; Erster et al., 2015; Carletti et al., 2016).
Endemic status, which has always been at the center of epidemio-logical studies, is an epidemioepidemio-logical term used to describe the im-munity of a population. Population imim-munity is described as stable or instable according to the inoculation rate, which is usually measured by serological tests, and calculated using a formula developed byMahoney and Ross (1972). Endemic stability describes a dynamic epidemiolo-gical situation in which clinical symptoms rarely occur despite the high infection rate in a population. This phenomenon occurs when the in-fection power is sufficient to achieve functional immunity in the ma-jority of the population at a relatively young age. The symptoms are usually mild when the disease is observed in older animals. Therefore, the prevalence of the clinical disease decreases in older groups due to the high protective immunity. In veterinary, the phenomenon of an endemic situation has been extensively described in relation to tick transmitted diseases. The control measures using vaccination are not suggested when an endemic stability situation occurs in a location. In endemically stable populations, although the use of acaricide is at a minimum level, the incidence of clinical diseases remains low (Hay, 2001;Geleta, 2005;Jonsson et al., 2012).
Table 2
Regional comparison of B. ovis seroprevalence determined by IFA test.
IFA Black Sea Mediterranean Aegean Marmara Central Anatolia Eastern Anatolia Southeastern Anatolia Total
Negative n 268a 262a 228b 348c 352b 291d 343b 2092 % 12.8 % 12.5 % 10.9 % 16.6 % 16.8 % 13.9 % 16.4 % 100.0 % Positive n 154a 141a 230b 111c 411b 545d 431b 2023 % 7.6 % 7.0 % 11.4 % 5.5 % 20.3 % 26.9 % 21.3 % 100.0 % Total n 422 403 458 459 763 836 774 4115 % 10.3 % 9.8 % 11.1 % 11.2 % 18.5 % 20.3 % 18.8 % 100.0 %
Age is the most important factor affecting susceptibility to babe-siosis. In endemic areas, lambs younger than three months are generally resistant to acute infections due to passive immunity passed through colostrum and non-specific natural resistance. Protective immunity provided by maternal antibodies and age resistance lasts only 3 months after birth. Therefore, in order to ensure endemic stability in sheep, it is recommended that lambs be infested by B. ovis-infected ticks until 3 months of age. If ticks infest lambs during this period, antibody titers rise and they gain resistance to infections because specific antibodies can prevent infection by binding and neutralizing sporozoites. On the other hand, if lambs are not exposed to ticks during this period, their resistance to the disease decreases over time after 3 months of age (Habela et al., 1990a;Yeruham et al., 1995,1998;Ekici et al., 2012).
An epidemiological study was conducted on the endemic status of B. ovis in the Central part of Turkey (Ekici et al., 2012), and the endemic status of B. ovis was determined to be instable in all age groups. Based on these results, the authors suggested that vaccine development stu-dies and comprehensive stustu-dies including other regions of Turkey were needed (Ekici et al., 2012). In the present study, the endemic status of B. ovis in sheep was also found to be instable in all age groups across Turkey according to the IFAT results. The infection rate was 31.7 %, 33.6 %, 52.8 % in the animals aged 1–6 months, 6–12 months, and older than 12 months, respectively. In order to determine the endemic status, inoculation rates were calculated for each group and the values were found to be lower than the minimum value of 0.005. This shows that the endemic status of B. ovis is instable for all three groups across Turkey. Therefore, a vaccine is needed to achieve protective immunity
against infections caused by B. ovis in sheep in Turkey and to prevent economic losses due to ovine babesiosis. Contrary to the study byEkici et al. (2012)regarding the age group of 1–6 months, the inoculation rates in the Central Anatolia (h: 0.0072), Eastern Anatolia (h: 0.0087), Aegean (h: 0.0098), and Mediterranean (h: 0.0055) regions were higher than 0.005 in the present study. This indicates that the endemic status is stable in these groups. This can be attributed to non-specific natural resistance and passive immunity obtained by colostrum, especially in animals up to 3 months old. Unlike the older groups, all of which are endemically instable, this group is considered to have a lower risk of clinical infection due to maternal antibodies and non-specific age re-sistance. Based on this result, it was concluded that the risk of clinical infection was high when the maternal immunity and non-specific age resistance weakens or disappears. The results of afield study reporting clinical B. ovis infections (Sevinc et al., 2013a) supports the hypothesis of increasing instability with age. In that study, most of the infected animals were between 15 and 18 months of age with an infection rate of 74.6 % in all animals, and 7.4 % in the 2–4 months old lambs.
In conclusion, B. ovis infections adversely affect the economy due to animals’ death and treatment expenditures. The endemic status of B. ovis in Turkey was found to be instable via the IFAT in the present study. For this reason, there is a need for an effective vaccine to protect animals against ovine babesiosis. A commercial vaccine has not yet been developed for protection of sheep against B. ovis. At this point, various secretory and surface proteins of B. ovis such as rBoSA1, rBoSA2, BoSPD, and ovipain-2 produced by recombinant DNA tech-nologies seem to be promising for the development of such vaccines. These studies should be continued to discover more recombinant pro-teins, and ultimately, these proteins will be of great importance in the development of subunit protein vaccines for the prevention of babe-siosis and babebabe-siosis-induced economic losses.
Declaration of Competing Interest
The authors declare no conflict of interest. Acknowledgements
This study was derived from the PhD thesis prepared by thefirst author under the supervision of the second author. Financial support was supplied by the Academic Staff Training Program Coordination Unit, Selcuk University in Turkey (Number of project: 2015-OYP-109). The authors would like to thank Associate Professor Mustafa Agah Tekindal for statistical analysis, and Research Assistant Ceylan Ceylan and Research Assistant Ali Uslu for their help during sample collection. References
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Table 3
Regional comparison of B. ovis prevalence determined by microscopic examination.
Microscopic examination Black Sea Mediterranean Aegean Marmara Central Anatolia Eastern Anatolia Southeastern Anatolia Total
Negative n 417a, b, c, d, e 403f 455d, e, f 456c, e, f 744b 831a, c, d, e, f 769a, c, d, e, f 4075 % 10.2 % 9.9 % 11.2 % 11.2 % 18.3 % 20.4 % 18.9 % 100.0 % Positive n 5a, b, c, d, e 0f 3d, e, f 3c, e, f 19b 5a, c, d, e, f 5a, c, d, e, f 40 % 12.5 % 0.0 % 7.5 % 7.5 % 47.5 % 12.5 % 12.5 % 100.0 % Total n 422 403 458 459 763 836 774 4115 % 10.3 % 9.8 % 11.1 % 11.2 % 18.5 % 20.3 % 18.8 % 100.0 %
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Table 4
Inoculation rates of different age groups.
Region Group (month) Number of animal Arithmetic mean of ages(day) % positivity (I) Inoculation rate (h) Central Anatolia 1–6 50 90 48.0 0.0072 6–12 108 270 33.3 0.0015 > 12 605 720 58.0 0.0012 Southeastern Anatolia 1–6 19 90 15.8 0.0019 6–12 20 270 40.0 0.0018 > 12 735 720 57.1 0.0011 Eastern Anatolia 1–6 11 90 54.5 0.0087 6–12 29 270 48.3 0.0024 > 12 796 720 66.0 0.0014 Marmara 1–6 84 90 17.9 0.0021 6–12 25 270 0 0.0000 > 12 350 720 27.4 0.0004 Aegean Region 1–6 34 90 58.8 0.0098 6–12 35 270 42.9 0.0020 > 12 389 720 50.1 0.0009 Mediterranean 1–6 46 90 39.1 0.0055 6–12 99 270 39.4 0.0018 > 12 258 720 32.6 0.0005 Black Sea 1–6 40 90 10.0 0.0011 6–12 145 270 29.7 0.0013 > 12 237 720 45.1 0.0008 General 1–6 284 90 31.7 0.0042 6–12 461 270 33.6 0.0015 > 12 3370 720 52.8 0.0010
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