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Title: A molecular study of tick-borne haemoprotozoan parasites (Theileria and Babesia) in small ruminants in Northern Tunisia Author: Youmna Mghirbi Amaia Ros-Garc a Pilar Iribar Adel Rhaim Ana Hurtado Ali Bouattour PII: DOI: Reference: To appear in: Received date: Revised date: Accepted date: S0304-4017(13)00453-6 http://dx.doi.org/doi:10.1016/j.vetpar.2013.08.005 VETPAR 6933 Veterinary Parasitology 22-4-2013 11-7-2013 6-8-2013

Please cite this article as: Mghirbi, Y., Ros-Garc a, A., Iribar, P., Rhaim, A., Hurtado, A., Bouattour, A., A molecular study of tick-borne haemoprotozoan parasites (Theileria and Babesia) in small ruminants in Northern Tunisia, Veterinary Parasitology (2013), http://dx.doi.org/10.1016/j.vetpar.2013.08.005 This is a PDF le of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its nal form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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A molecular study of tick-borne haemoprotozoan parasites (Theileria and Babesia) in small ruminants in Northern Tunisia

Youmna Mghirbi a c, Amaia Ros-Garca b, Pilar Iribar b, Adel Rhaim a, Ana Hurtado b, Ali Bouattour a c

Laboratoire dpidmiologie et microbiologie vtrinaire, service dentomologie

Department of Animal Health, NEIKER - Instituto Vasco de Investigacin y

Desarrollo Agrario, Berreaga 1, 48160 Derio, Bizkaia, Spain Universit Tunis El-Manar, Tunis, Tunisia

*Author for correspondence

Email: ali.bouattour@pasteur.rns.tn

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Institut Pasteur de Tunis, BP74, 1002 Tunis-Blvdre, TUNISIE Tel : +216 71 893 340

Fax : +216 71 791 833

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mdicale, Institut Pasteur de Tunis, Tunis 1002, Tunisia

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Abstract In this study, the frequency of Theileria and Babesia species in sheep and goats was assessed via reverse line blotting (RLB). A total of 263 apparently healthy sheep and goats from 16 randomly selected flocks located in 9 localities situated in 3 bioclimatic zones in Tunisia were investigated for the blood protozoans. RLB hybridization with polymerase chain reaction detected only Theileria ovis in sheep and goats, accounting for 22.4% (95% confidence interval [CI]: 17.627.1%)

higher than in goats (4.7%; 95% CI: -10.920.4%). Neither Babesia nor mixed infections were detected. Only two Ixodid tick species (Rhipicephalus turanicus and Rhipicephalus bursa) were collected from the examined sheep and goats in 5 localities. R. turanicus was the dominant species (95.5%) collected mainly in the humid zone, while apparently rare in the sub-humid zone. R. bursa was the only species collected in the semi-arid area. RLB analysis identified six different piroplasms in ticks, with an overall prevalence of 31.5% (95% CI: 28.134.9%).

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Twenty percent (95% CI: 14.4-25.5%) of the collected ticks tested positive for Theileria spp., 3% (95% CI: -5.611.6%) for Babesia spp. and 0.9% (95% CI: 8.19.9%) of the ticks harbored both genera; several of these species are not known to occur in small ruminants. This is the first report on the detection of Theileria and Babesia species DNA in small ruminants and ticks in Tunisia. Keywords: Theileria spp.; Babesia spp.; small ruminants; ticks; RLB; Tunisia

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positive samples. The infection rate in sheep (28.1%; 95% CI: 23.832.3%) was

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1. Introduction Piroplasm species are tick-borne parasitic protozoa that are differentiated into the genera Theileria and Babesia. Some of these protozoa are highly pathogenic for cattle, sheep, and goats, causing theileriosis and babesiosis, diseases that are widely distributed in tropical, subtropical, and temperate countries, where they are endemic. The economic impact of these diseases can be significant. Several named and as yet unnamed Theileria species cause ovine theileriosis (Preston,

pathogenic for sheep and goats, and recently, Theileria sp. OT3, and Theileria sp. MK have been described in sheep and goats, but there is no reliable information about their pathogenicity (Preston, 2001; Nagore et al., 2004a; Ahmed et al., 2006; Altay et al., 2007b; Yin et al., 2007; Duh et al., 2008). Babesia ovis, Babesia motasi and Babesia crassa are recognized as the species causing ovine babesiosis: B. ovis is highly pathogenic to sheep and goats while the other two species are non-pathogenic or less pathogenic (Uilenberg, 2001).

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In Tunisia, theileriosis and babesiosis are the two main tick-borne haemoparasitic diseases occurring in cattle and small ruminants. They have been extensively studied in cattle (Bouattour et al., 1994; Mghirbi et al., 2008), but a paucity of information exists concerning ovine theileriosis and babesiosis. The causative agents of piroplasmosis, their actual geographic distribution and their vectors are important components of the epidemiology of these diseases that need to be studied to evaluate the impact and implementation of successful control programmes that include effective treatment of malignant theileriosis and/or pathogenic babesiosis. The laboratory diagnosis of small ruminant piroplasmosis was based on the microscopic detection of piroplasms in Giemsa-stained blood
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2001); Theileria lestoquardi, Theileria luwenshuni and Theileria uilenbergi are

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smears. However, species identification by microscopy is difficult because different parasites share a similar morphology, making identification particularly difficult if mixed infections occur. In addition, identification can be difficult in carrier animals where the presence of parasites is low and even in acute cases at the onset of the disease. In recent decades, molecular techniques with high sensitivity and specificity, such as species-specific polymerase chain reaction (PCR) and PCR-based Reverse Line Blot (RLB) hybridization have been used for

al., 2004a; Schnittger et al., 2004; Aktas et al., 2005; Alhassan et al., 2005; Altay et al., 2007b).

Here, we conducted a cross-sectional study to detect and differentiate Theileria and Babesia species in small ruminants and ticks in three different bioclimatic zones of Tunisia based on PCR amplification associated with RLB speciesspecific hybridization.

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2. Materials and methods

2.1 Farm location and small ruminant populations This cross-sectional study was carried out in 9 localities located in three different bioclimatic zones (humid, sub-humid and semi-arid) in northern Tunisia where piroplasmosis is endemic (Fig. 1). All sites have a Mediterranean climate, with cool, moist winters, and dry, hot summers. A total of 263 small ruminants were randomly chosen following recommendations of the State Veterinary Office as representative of the local management system, which was generally traditional, that is, small flocks grazing on permanent pastures or bush.

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detection and discrimination of ovine Theileria and Babesia species (Nagore et

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The studied population of small ruminants (n=263) was composed of 199 Barbarine sheep (n=193 females and n=6 males) and 64 Arbi (local breed) goats (females) from 16 randomly selected flocks (Table 1). Among these animals, 42 were younger than one year (n=26 lambs, n=16 kids) and there were 221 adults (n=173 sheep, n=48 goats). The distribution of sheep and goats has no relation to the bioclimatic zone; their breeding reflects regional habits and available pastures.

Animals were bled once between April and June 2010, the period when they graze in pastures and are exposed to ticks. Blood samples taken in ethylenediamine tetraacetic acid (EDTA) containing tubes were used for subsequent DNA extraction and hybridization analyses.

In addition, the entire body of each of the 263 animals was inspected for ticks, particularly on the ears and neck. Ticks were removed manually from the host body, placed in bottles with 70% ethanol and labeled. They were identified using

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published taxonomic keys (Bouattour, 2002).

2.3 DNA extraction

DNA from whole-blood samples and semi-engorged adult ticks was extracted using the Invitrogen (California, USA) Kit for DNA purification. DNA was eluted in the elution buffer provided with the kit. DNA yields were determined with a NanoDrop ND-1000 Spectrophotometer (NanoDrop Technologies, DE, USA).

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2.2 Collection of blood and tick samples

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2.4 PCR amplification of 18S rRNA gene of Theileria/Babesia To amplify the hyper-variable V4 region of the 18S ribosomal ribonucleic acid (rRNA) gene of Theileria and Babesia species, RLB-F2 and RLB-R2, respectively were used as forward and reverse primers were as adapted previously by Georges et al. (2001). Reactions were performed in 25 l volumes with 1 PCR buffer, 1.5 mM MgCl2, 200 M of each deoxyribonucleotide triphosphate, 200 nM of each primer and 1 U of Taq Platinium polymerase (Invitrogen,

b).

2.5 Reverse line blot hybridization (RLB)

Oligonucleotide probes (Theileria/Babesia Catch-all TB, Theileria sp. OT1, Theileria sp. OT3, T. ovis, T. lestoquardi, B. ovis, B. motasi, B. crassa), containing a

phosphoramidite)-C6 amino linker, were as previously reported (Nagore et al.,

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2004a). A subsequent RLB on adult ticks was performed with the abovementioned probes and 11 other specific probes for piroplasms (Babesia bigemina, Babesia bovis, Babesia divergens, Babesia major, Babesia occultans, Babesia caballi, Theileria annulata, Theileria buffeli, Theileria equi, Babesia sp. EU1, Theileria sp. 3185/02) (Gubbels et al., 1999, Nagore et al., 2004b, GarcaSanmartn et al., 2006, 2007, 2008). The preparation of the RLB membrane and hybridization were carried out as previously reported by Nagore et al. (2004a). Finally, after developing the film, the PCR products were stripped from the membrane (Gubbels et al., 1999), and membranes were reused a maximum of eight times. Plasmids including the amplicon of the V4 region of the 18S rRNA
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N-(trifluoroacetamidohexyl-cyanoethyl-N,N-diisopropyl

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California, USA). Cycling conditions were as described by Nagore et al. (2004a,

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gene of each of the species analyzed were used as positive controls. Crosscontamination and false-positive results were prevented by using plugged tips, performing DNA extraction and setting PCR reactions in a room separate from that used for post-PCR analysis, and using negative (water) controls during DNA extraction and PCR amplification that were also subjected to RLB hybridization.

2.6 Statistical analysis

(sheep and goats) in the three bioclimatic zones and age groups (adult vs lamb/kid). Observed differences were considered to be significant when the resulting P value was less than 0.05.

3. Results

3.1 Detection and identification of Theileria and Babesia species in blood samples by PCR-RLB

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By RLB, the detection rate with the Theileria/Babesia catch-all TB probe in small ruminants was 22.4% (59/263; 95% confidence interval [CI]: 17.627.1%). The overall piroplasm prevalence rate differed among the humid (14.7%; 95% CI: 6.5 22.8%), sub-humid (29.0%; 95% CI: 18.239.7%) and semi-arid zones (35.3%; 95% CI: 29.641.0%). In addition, piroplasm prevalence was significantly higher in sheep than in goats (P<0.05). Species-specific positive signals were only obtained with the T. ovis probe (Table 1). This Theileria species was detected in 7 localities (Table 1). The prevalence of this species was 28.1% (95% CI: 23.8 32.4%) in ovine samples; it ranged from 21.3% (95% CI: 13.129.5%) to 43.2% (95% CI: 40.146.3%) among the three bioclimatic zones, with a significant
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The Chi-squared tests were used to compare proportions of positivity by host

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difference (P=0.04). Otherwise, Theileria ovis was identified in only 4.7% (95% CI: -14.624.1%) of the goat samples distributed in the humid (2%; 95% CI: 17.021.0%) and semi-arid (14.3%; 95% CI: -12.140.7%) zones, with significant differences (P=0.009). In addition, a significant difference (P=0.0027) was recorded between T. ovis infected adults (25.8%; 95% CI: 21.330.3%), lambs and kids (4.7%; 95% CI: -14.724.1%).

samples by PCR-RLB

During the blood-sampling period, 279 ixodid adult ticks (Table 1) were collected from 6 localities; 142 (54.0%; 95% CI: 53.354.7%) and the 263 examined sheep were infested with at least one tick. Only 2 tick species were identified: Rhipicephalus bursa Canestrini and Fanzago, 1877 (n=11) and R. turanicus Pomerantzev, 1940 (n=268). The latter tick was the dominant species (86.0%), mainly collected in the humid zone (185 specimens), while apparently very rare in

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the sub-humid zone (8 specimens). Among collected ticks, only semi-engorged specimens (215 R. turanicus and 10 R. bursa) were used for RLB macroarray analysis using a panel of probes for 18 piroplasm species. The Theileria/Babesia catch-all TB probe revealed positive signals for 71 ticks (66 R. turanicus and 5 R. bursa), most of which corresponded to Theileria (64.8%; 95% CI: 59.969.6%), whereas 9.8% (95% CI: -3.423.0%) were positive for Babesia (Table 1). Two additional ticks tested positive for both Theileria and Babesia. A further 16 ticks produced only a weak positive hybridization signal with the Theileria/Babesia catch-all TB probe but did not react with any of species specific probes.
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3.2 Detection and identification of Theileria and Babesia species in tick

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In these tick samples, only two piroplasm species that affect small ruminants were detected: T. ovis (4.0%; 95% CI: -4.512.5%) and B. motasi (0.9%; 95% CI: -8.2 9.9%). Other piroplasm species were also detected in these ticks: B. bigemina, T. buffeli, B. caballi and T. equi (Table 1).

4. Discussion

A cross-sectional study was carried out using RLB to investigate piroplasm

found was higher than that found in Turkey and Syria (Muslih et al., 1988; Giangaspero et al., 1992). The only piroplasm species identified in sheep and goats was T. ovis, a non-pathogenic Theileria (Uilenberg, 1981), which could explain the absence of reported clinical theileriosis cases in the studied regions. T. ovis was also reported in Greece, Spain, Egypt and Syria by microscopy, serology or molecular methods (Papadopoulos et al., 1996; Alyasino and Greiner, 1999; Mazyad and Khalif, 2002; Nagore et al., 2004a). The prevalence rates of T. ovis in

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sheep and goat in eastern (Altay et al., 2007a) and central (Inci et al., 2010) Turkey was higher than the one reported in our study. The higher prevalence of Theileria species in sheep than in goats observed here was also reported in Turkey (Altay et al., 2007a) and in the Macedonia region of Greece (Papadopoulos et al., 1996). It appears that goats are less easily infected by this parasite or that the infestation of goats is lower. A higher prevalence of T. ovis was observed in the adult small ruminants in comparison to the lamb and kid groups. This event concurred with a previous study by Little John and Walker (1979) who found that age, sex, and breed influenced the prevalence of protozoa diseases. We observed a difference in
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infection in small ruminants and ticks in northern Tunisia. The prevalence rate

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piroplasm prevalence rates in small ruminants corresponding to bioclimatic regions. This difference, which has been reported in other studies, is probably linked to the effect of bioclimatic conditions on the distribution of the hard tick vectors (Yeruham et al., 1995; Zangana and Naqid, 2011). The dominant species found, R. turanicus, was mainly collected from sheep, a result consistent with previous data reported in Tunisia (Bouattour et al., 1999; Darghouth, 2004). Moreover, the study period -- spring season -- corresponds to

contrast, R. bursa, which is considered the principal vector of B. ovis (Friedhoff, 1997), was less frequently collected, which may explain the absence of B. ovis in the analyzed blood samples.

Six piroplasm species were detected in the ticks collected from sheep and goats. The overall piroplasm prevalence (31.6%) was much higher than that reported in questing ticks in Spain (Garca-Sanmartin et al., 2008). Among the piroplasm species detected in our sample ticks, B. bigemina, T. buffeli, B. caballi and T. equi

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were previously reported in ticks, cattle, and horses in these same regions (Mghirbi et al., 2008; 2010). Two ovine piroplasms were also detected; T. ovis, also found in the animals blood, and B. motasi, which was not detected in the blood samples and had never been reported in Tunisia before. By contrast, B. motasi was reported in other North African countries (Algeria, Morocco and Libya), associated with severe clinical cases in sheep and goats (Mahin et al., 1984; Euzby, 1990; Friedhoff, 1997). Haemaphysalis ticks are the natural vector of this Babesia species (Uilenberg, 1997). Although ticks of this genus are known to exist in Tunisia, none were found during this survey, probably because the period of their peak activity is the fall (November-October) (Bouattour et al.,
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the period when this tick species is at peak activity (Bouattour et al., 1999). In

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1999). Alternatively, R. turanicus might be a vector for this Babesia (Darghouth, 2004). The infection of R. bursa and R. turanicus ticks by T. equi was expected since Rhipicephalus ticks are the acknowledged vectors of T. equi and B. caballi (Darghouth, 2004). T. buffeli was the most frequently found piroplasm in tested ticks. This result concurs with the high prevalence of this non-pathogenic Theileria species in cattle and ticks (R. bursa, R. turanicus, R. (Boophilus) annulatus, Haemaphysalis punctata and Ixodes ricinus) in Tunisia (Mghirbi et

ticks may be the infected cattle or horses on whose blood the ticks feed. Four ticks showed a mixed profile of different piroplasm species that included double and triple combinations of species from the same genus or both Theileria and Babesia (Table 1). This may have resulted from the infection of ticks that fed on hosts co-infected with various species or on different single infected hosts at different life stages. Surprisingly, mixed infections were not observed in the sheep and goats tested in this study, but were reported in cattle (Mghirbi et al., 2008)

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and horses (data not shown) in the same investigated zones. In conclusion, using RLB with a panel of several piroplasm species-specific

probes, we detected six piroplasm species in healthy small ruminants and in ticks collected from different bioclimatic zones in Tunisia. This information is essential for defining the zones of risk and establishing control measures. Further studies are needed on the economic impact of small ruminant piroplasms and on determining which ticks are their vectors.

Conflict of interest statement The authors declare no conflicts of interest.


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al., 2008; 2010). The source of the various species of piroplasms detected in the

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Acknowledgements We are grateful to Dr. Leila Saieh, Dr. Kamel Khlif, Dr. Zouheir Ktata and Dr. Ridha Ben Omrane for their help in fieldwork. The authors would also like to thank the farmers for their collaboration collecting samples. We also thank Dr. G. Uilenberg and Glassman D. for constructive comments on early drafts of the

Agency for International Development Cooperation (AECID, Project No. A/026818/09), the Spanish National Institute for Agricultural and Food Research and Technology (INIA, Project No. RTA2009-000-18-00-00), the European Regional Development Fund (ERDF) and the Ministry for Higher Education, Scientific Research, and Technology in Tunisia. AR is the recipient of a INIA predoctoral fellowship.

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Yeruham, I., Hadani, A., Galker, F., Rosen, S., 1995. A study of an enzootic focus of sheep babesiosis (Babesia ovis, Babes, 1892). Vet. Parasitol. 60, 349-54. Yin, H., Schnittger, L., Luo, J., Seitzer, U., Ahmed, J.S., 2007. Ovine theileriosis in China: a new look at an old story. Parasitol. Res. 101 (Suppl 2), S191-195. Zangana, I.K., Naqid, I.A., 2011. Prevalence of piroplasmosis (Theileriosis and Babesiosis) among goats in Duhok Governorate College of Veterinary Medicine\ University of Duhok Al-Anbar. J. Vet. Sci. 4, 50-57.

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Babesia parasites infecting small ruminants by reverse line blotting. Parasitol.

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389

Table 1. RLB detection and identification of piroplasm species in small ruminants and ticks in Northern Tunisia.
390
R. bursa N infected / Piroplasms identified N collected 0 0 0 Bioclimatic zones HUMID Localities (N flocks/N Tabarka (1/21) Host (N infected / Sheep (2/17) Goats (0/4) Amdoun (1/24) Sheep (0/24) N infected / Piroplasms identified N collected 5/41 4/9 T. equi (1) Catch-all TB (4) T. buffeli (1) Catch-all TB (3) B. caballi (3) T. equi (5) Catch-all TB (4) T. buffeli (1) T. buffeli (18) B. bigemina (1) T. ovis (7) B. motasi (1) T. buffeli (1) Catch-all TB (2) T. ovis + B. caballi + T. equi (1) -

Sejnane (3/47) Maaden (2/38)

Sheep (2/12) Goats (0/35) Sheep (16/28)

ed

M
13/46 0 18/19 13/70

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-

R. turanicus

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0 0 0 -

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pt

Nefza (1/13) SUB-HUMID

Goats (1/10) Sheep (0/13)

0 0 2/8 0 0 0 0 11/22

i
0 0 0 0 0 0 3/6 2/4 B. bigemina (2) Catch-all TB (1) Catch-all TB (1) T. buffeli + T. equi (1)

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El Jouf (3/51)

Oued El Abid (2/20)

Sheep (17/19)

Mellegue (1/11) Touiref (2/38)

Goats (0/1) Sheep (2/11) Sheep (1/38) Sheep (16/37) Goats (2/14) 22.4% (CI: 17.6 27.1%) (59/263)

T. ovis (1) Catch-all TB (1) -

SEMI-ARID

T. buffeli (9) T. buffeli + T. equi (1) T. buffeli + B. motasi (1)

Total

9 localities (16/263 animals)

30.7% (CI: 27.433.9%) (66/215)

50.0% (CI: ) (5/10)

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391 392 393 394

Fig. 1 Map of Tunisia showing the location of sites where blood and tick samples were collected.

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394 395

Table 1. RLB detection and identification of piroplasm species in small ruminants and ticks in Northern Tunisia.
R. turanicus N infected / Piroplasms identified N collected 5/41 4/9 13/46 T. equi (1) Catch-all TB (4) T. buffeli (1) Catch-all TB (3) B. caballi (3) T. equi (5) Catch-all TB (4) T. buffeli (1) T. buffeli (18) B. bigemina (1) T. ovis (7) B. motasi (1) T. buffeli (1) Catch-all TB (2) T. ovis + B. caballi + T. equi (1) R. bursa N infected / Piroplasm N collected 0 0 0 -

Bioclimatic zones HUMID

Localities (N flocks/N Tabarka (1/21)

Host (N infected / Sheep (2/17) Goats (0/4)

Sejnane (3/47) Maaden (2/38)

Sheep (2/12) Goats (0/35) Sheep (16/28)

0 18/19 13/70

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T. ovis (1) Catch-all TB (1) -

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Amdoun (1/24)

Sheep (0/24)

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0 0 0

SUB-HUMID

Oued El Abid (2/20) Mellegue (1/11) Touiref (2/38)

Sheep (17/19) Goats (0/1) Sheep (2/11) Sheep (1/38)

Nefza (1/13)

Goats (1/10) Sheep (0/13)

an
0 0

0 0 0 0 0 0 3/6 2/4

B. bigemin Catch-all T Catch-all T T. buffeli +

2/8 0 0 0

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SEMI-ARID

El Jouf (3/51)

Sheep (16/37)

0 11/22

Goats (2/14) 22.4% (CI: 17.6 27.1%) (59/263)

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T. buffeli (9) T. buffeli + T. equi (1) T. buffeli + B. motasi (1)

Total

9 localities (16/263 animals)

30.7% (CI: 27.433.9%) (66/215)

50.0% (CI: ) (5/10)

396 397 398 399

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Figure

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