molecular study on tick borne haemoprotozoa
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Accepted Manuscript
Title: A molecular study of tick-borne haemoprotozoanparasites (Theileria and Babesia) in small ruminants in
Northern Tunisia
Author: Youmna Mghirbi Amaia Ros-Garca Pilar Iribar
Adel Rhaim Ana Hurtado Ali Bouattour
PII: S0304-4017(13)00453-6
DOI: http://dx.doi.org/doi:10.1016/j.vetpar.2013.08.005
Reference: VETPAR 6933
To appear in: Veterinary Parasitology
Received date: 22-4-2013
Revised date: 11-7-2013
Accepted date: 6-8-2013
Please cite this article as: Mghirbi, Y., Ros-Garca, 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
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http://dx.doi.org/doi:10.1016/j.vetpar.2013.08.005http://dx.doi.org/10.1016/j.vetpar.2013.08.005http://dx.doi.org/10.1016/j.vetpar.2013.08.005http://dx.doi.org/doi:10.1016/j.vetpar.2013.08.005 -
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A molecular study of tick-borne haemoprotozoan parasites (Theileriaand1Babesia) in small ruminants in Northern Tunisia2
3Youmna Mghirbi
a c
, Amaia Ros-Garcab
, Pilar Iribarb
, Adel Rhaima
, Ana4Hurtadob, Ali Bouattoura c5
6aLaboratoire dpidmiologie et microbiologie vtrinaire, service dentomologie7mdicale, Institut Pasteur de Tunis, Tunis 1002, Tunisia8
9bDepartment of Animal Health, NEIKER - Instituto Vasco de Investigacin y10Desarrollo Agrario, Berreaga 1, 48160 Derio, Bizkaia, Spain11cUniversit Tunis El-Manar, Tunis, Tunisia12
13*Author for correspondence14Email: [email protected] Pasteur de Tunis, BP74, 1002 Tunis-Blvdre, TUNISIE16Tel : +216 71 893 34017Fax : +216 71 791 83318
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Abstract21In this study, the frequency of Theileriaand Babesiaspecies in sheep and goats22was assessed via reverse line blotting (RLB). A total of 263 apparently healthy23sheep and goats from 16 randomly selected flocks located in 9 localities situated24in 3 bioclimatic zones in Tunisia were investigated for the blood protozoans. RLB25hybridization with polymerase chain reaction detected only Theileria ovisin sheep26and goats, accounting for 22.4% (95% confidence interval [CI]: 17.627.1%)27
positive samples. The infection rate in sheep (28.1%; 95% CI: 23.832.3%) was28higher than in goats (4.7%; 95% CI: -10.920.4%). Neither Babesia nor mixed29infections were detected. Only two Ixodid tick species (Rhipicephalus turanicus30andRhipicephalusbursa) were collected from the examined sheep and goats in 531localities.R. turanicuswas the dominant species (95.5%) collected mainly in the32humid zone, while apparently rare in the sub-humid zone. R. bursawas the only33species collected in the semi-arid area. RLB analysis identified six different34
piroplasms in ticks, with an overall prevalence of 31.5% (95% CI: 28.134.9%).35Twenty percent (95% CI: 14.4-25.5%) of the collected ticks tested positive for36Theileria spp., 3% (95% CI: -5.611.6%) for Babesiaspp. and 0.9% (95% CI: -378.19.9%) of the ticks harbored both genera; several of these species are not38known to occur in small ruminants. This is the first report on the detection of39TheileriaandBabesiaspecies DNA in small ruminants and ticks in Tunisia.40Keywords:Theileriaspp.;Babesiaspp.; small ruminants; ticks; RLB; Tunisia41
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1. Introduction46Piroplasm species are tick-borne parasitic protozoa that are differentiated into the47genera Theileriaand Babesia. Some of these protozoa are highly pathogenic for48cattle, sheep, and goats, causing theileriosis and babesiosis, diseases that are49widely distributed in tropical, subtropical, and temperate countries, where they are50endemic. The economic impact of these diseases can be significant. Several51named and as yet unnamed Theileria species cause ovine theileriosis (Preston,522001); Theileria lestoquardi, Theileria luwenshuni and Theileria uilenbergi are53
pathogenic for sheep and goats, and recently, Theileriasp. OT3, and Theileriasp.54MK have been described in sheep and goats, but there is no reliable information55about their pathogenicity (Preston, 2001; Nagore et al., 2004a; Ahmed et al.,562006; Altay et al., 2007b; Yin et al., 2007; Duh et al., 2008). Babesia ovis,57
Babesia motasi and Babesia crassa are recognized as the species causing ovine58babesiosis: B. ovis is highly pathogenic to sheep and goats while the other two59species are non-pathogenic or less pathogenic (Uilenberg, 2001).60In Tunisia, theileriosis and babesiosis are the two main tick-borne haemoparasitic61diseases occurring in cattle and small ruminants. They have been extensively62studied in cattle (Bouattour et al., 1994; Mghirbi et al., 2008), but a paucity of63information exists concerning ovine theileriosis and babesiosis. The causative64agents of piroplasmosis, their actual geographic distribution and their vectors are65important components of the epidemiology of these diseases that need to be66studied to evaluate the impact and implementation of successful control67
programmes that include effective treatment of malignant theileriosis and/or68pathogenic babesiosis. The laboratory diagnosis of small ruminant piroplasmosis69was based on the microscopic detection of piroplasms in Giemsa-stained blood70
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smears. However, species identification by microscopy is difficult because71different parasites share a similar morphology, making identification particularly72difficult if mixed infections occur. In addition, identification can be difficult in73carrier animals where the presence of parasites is low and even in acute cases at74the onset of the disease. In recent decades, molecular techniques with high75sensitivity and specificity, such as species-specific polymerase chain reaction76(PCR) and PCR-based Reverse Line Blot (RLB) hybridization have been used for77detection and discrimination of ovine Theileria and Babesia species (Nagore et78al., 2004a; Schnittger et al., 2004; Aktas et al., 2005; Alhassan et al., 2005; Altay79et al., 2007b).80Here, we conducted a cross-sectional study to detect and differentiate Theileria81and Babesia species in small ruminants and ticks in three different bioclimatic82zones of Tunisia based on PCR amplification associated with RLB species-83specific hybridization.84
852. Materials and methods86
2.1Farm location and small r uminant populations87This cross-sectional study was carried out in 9 localities located in three different88
bioclimatic zones (humid, sub-humid and semi-arid) in northern Tunisia where89piroplasmosis is endemic (Fig. 1). All sites have a Mediterranean climate, with90cool, moist winters, and dry, hot summers. A total of 263 small ruminants were91randomly chosen following recommendations of the State Veterinary Office as92representative of the local management system, which was generally traditional,93that is, small flocks grazing on permanent pastures or bush.94
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The studied population of small ruminants (n=263) was composed of 19995Barbarine sheep (n=193 females and n=6 males) and 64 Arbi (local breed) goats96(females) from 16 randomly selected flocks (Table 1). Among these animals, 4297were younger than one year (n=26 lambs, n=16 kids) and there were 221 adults98(n=173 sheep, n=48 goats). The distribution of sheep and goats has no relation to99the bioclimatic zone; their breeding reflects regional habits and available pastures.100
1012.2Collection of blood and tick samples102Animals were bled once between April and June 2010, the period when they graze103in pastures and are exposed to ticks. Blood samples taken in ethylenediamine104tetraacetic acid (EDTA) containing tubes were used for subsequent DNA105extraction and hybridization analyses.106In addition, the entire body of each of the 263 animals was inspected for ticks,107
particularly on the ears and neck. Ticks were removed manually from the host108body, placed in bottles with 70% ethanol and labeled. They were identified using109published taxonomic keys (Bouattour, 2002).110
1112.3DNA extraction112DNA from whole-blood samples and semi-engorged adult ticks was extracted113using the Invitrogen (California, USA) Kit for DNA purification. DNA was eluted114in the elution buffer provided with the kit. DNA yields were determined with a115
NanoDrop ND-1000 Spectrophotometer (NanoDrop Technologies, DE, USA).116117118119
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2.4PCR amplif ication of 18S rRNA gene of Theileria/Babesia120To amplify the hyper-variable V4 region of the 18S ribosomal ribonucleic acid121(rRNA) gene of Theileria and Babesia species, RLB-F2 and RLB-R2,122respectively were used as forward and reverse primers were as adapted previously123
by Georges et al. (2001). Reactions were performed in 25 l volumes with 1124PCR buffer, 1.5 mM MgCl2, 200 M of each deoxyribonucleotide triphosphate,125200 nM of each primer and 1 U of Taq Platinium polymerase (Invitrogen,126California, USA). Cycling conditions were as described by Nagore et al. (2004a,127
b).128129
2.5 Reverse li ne blot hybr idization (RLB)130Oligonucleotide probes (Theileria/Babesia Catch-all TB, Theileria sp. OT1,131Theileria sp. OT3, T. ovis, T. lestoquardi, B. ovis, B. motasi, B. crassa),132containing a N-(trifluoroacetamidohexyl-cyanoethyl-N,N-diisopropyl133
phosphoramidite)-C6 amino linker, were as previously reported (Nagore et al.,1342004a). A subsequent RLB on adult ticks was performed with the above-135mentioned probes and 11 other specific probes for piroplasms (Babesia bigemina,136
Babesia bovis, Babesia divergens, Babesia major, Babesia occultans, Babesia137caballi, Theileria annulata, Theileria buffeli, Theileria equi, Babesia sp. EU1,138Theileria sp. 3185/02) (Gubbels et al., 1999, Nagore et al., 2004b, Garca-139Sanmartn et al., 2006, 2007, 2008). The preparation of the RLB membrane and140hybridization were carried out as previously reported by Nagore et al. (2004a).141Finally, after developing the film, the PCR products were stripped from the142membrane (Gubbels et al., 1999), and membranes were reused a maximum of143eight times. Plasmids including the amplicon of the V4 region of the 18S rRNA144
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gene of each of the species analyzed were used as positive controls. Cross-145contamination and false-positive results were prevented by using plugged tips,146
performing DNA extraction and setting PCR reactions in a room separate from147that used for post-PCR analysis, and using negative (water) controls during DNA148extraction and PCR amplification that were also subjected to RLB hybridization.149
1502.6Statistical analysis151The Chi-squared tests were used to compare proportions of positivity by host152(sheep and goats) in the three bioclimatic zones and age groups (adult vs153lamb/kid). Observed differences were considered to be significant when the154resultingPvalue was less than 0.05.155
1563. Results157
3.1 Detection and identification of Theileriaand Babesiaspecies in blood158samples by PCR-RLB159By RLB, the detection rate with the Theileria/Babesiacatch-all TB probe in small160ruminants was 22.4% (59/263; 95% confidence interval [CI]: 17.627.1%). The161overall piroplasm prevalence rate differed among the humid (14.7%; 95% CI: 6.516222.8%), sub-humid (29.0%; 95% CI: 18.239.7%) and semi-arid zones (35.3%;16395% CI: 29.641.0%). In addition, piroplasm prevalence was significantly higher164in sheep than in goats (P
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difference (P=0.04). Otherwise, Theileria oviswas identified in only 4.7% (95%170CI: -14.624.1%) of the goat samples distributed in the humid (2%; 95% CI: -17117.021.0%) and semi-arid (14.3%; 95% CI: -12.140.7%) zones, with significant172differences (P=0.009). In addition, a significant difference (P=0.0027) was173recorded between T. ovisinfected adults (25.8%; 95% CI: 21.330.3%), lambs and174kids (4.7%; 95% CI: -14.724.1%).175
1763.2 Detection and identification of Theileriaand Babesiaspecies in tick177samples by PCR-RLB178During the blood-sampling period, 279 ixodid adult ticks (Table 1) were collected179from 6 localities; 142 (54.0%; 95% CI: 53.354.7%) and the 263 examined sheep180were infested with at least one tick. Only 2 tick species were identified:181
Rhipicephalus bursa Canestrini and Fanzago, 1877 (n=11) and R. turanicus182Pomerantzev, 1940 (n=268). The latter tick was the dominant species (86.0%),183mainly collected in the humid zone (185 specimens), while apparently very rare in184the sub-humid zone (8 specimens).185Among collected ticks, only semi-engorged specimens (215 R. turanicusand 10186
R. bursa) were used for RLB macroarray analysis using a panel of probes for 18187piroplasm species. The Theileria/Babesia catch-all TB probe revealed positive188signals for 71 ticks (66R. turanicusand 5R. bursa), most of which corresponded189to Theileria(64.8%; 95% CI: 59.969.6%), whereas 9.8% (95% CI: -3.423.0%)190were positive for Babesia(Table 1). Two additional ticks tested positive for both191Theileria and Babesia. A further 16 ticks produced only a weak positive192hybridization signal with the Theileria/Babesia catch-all TB probe but did not193react with any of species specific probes.194
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In these tick samples, only two piroplasm species that affect small ruminants were195detected: T. ovis(4.0%; 95% CI: -4.512.5%) andB. motasi(0.9%; 95% CI: -8.21969.9%). Other piroplasm species were also detected in these ticks: B. bigemina, T.197buffeli,B. caballi and T. equi(Table 1).198
1994. Discussion200
A cross-sectional study was carried out using RLB to investigate piroplasm201infection in small ruminants and ticks in northern Tunisia. The prevalence rate202found was higher than that found in Turkey and Syria (Muslih et al., 1988;203Giangaspero et al., 1992). The only piroplasm species identified in sheep and204goats was T. ovis, a non-pathogenic Theileria (Uilenberg, 1981), which could205explain the absence of reported clinical theileriosis cases in the studied regions. T.206oviswas also reported in Greece, Spain, Egypt and Syria by microscopy, serology207or molecular methods (Papadopoulos et al., 1996; Alyasino and Greiner, 1999;208Mazyad and Khalif, 2002; Nagore et al., 2004a). The prevalence rates of T. ovisin209sheep and goat in eastern (Altay et al., 2007a) and central (Inci et al., 2010)210Turkey was higher than the one reported in our study. The higher prevalence of211Theileriaspecies in sheep than in goats observed here was also reported in Turkey212(Altay et al., 2007a) and in the Macedonia region of Greece (Papadopoulos et al.,2131996). It appears that goats are less easily infected by this parasite or that the214infestation of goats is lower.215A higher prevalence of T. ovis was observed in the adult small ruminants in216comparison to the lamb and kid groups. This event concurred with a previous217study by Little John and Walker (1979) who found that age, sex, and breed218influenced the prevalence of protozoa diseases. We observed a difference in219
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piroplasm prevalence rates in small ruminants corresponding to bioclimatic220regions. This difference, which has been reported in other studies, is probably221linked to the effect of bioclimatic conditions on the distribution of the hard tick222vectors (Yeruham et al., 1995; Zangana and Naqid, 2011).223The dominant species found, R. turanicus, was mainly collected from sheep, a224result consistent with previous data reported in Tunisia (Bouattour et al., 1999;225Darghouth, 2004). Moreover, the study period -- spring season -- corresponds to226the period when this tick species is at peak activity (Bouattour et al., 1999). In227contrast,R. bursa, which is considered the principal vector of B. ovis(Friedhoff,2281997), was less frequently collected, which may explain the absence ofB.ovisin229the analyzed blood samples.230Six piroplasm species were detected in the ticks collected from sheep and goats.231The overall piroplasm prevalence (31.6%) was much higher than that reported in232questing ticks in Spain (Garca-Sanmartin et al., 2008). Among the piroplasm233species detected in our sample ticks,B. bigemina, T. buffeli,B. caballi and T. equi234were previously reported in ticks, cattle, and horses in these same regions235(Mghirbi et al., 2008; 2010). Two ovine piroplasms were also detected; T. ovis,236also found in the animals blood, and B. motasi, which was not detected in the237
blood samples and had never been reported in Tunisia before. By contrast, B.238motasi was reported in other North African countries (Algeria, Morocco and239Libya), associated with severe clinical cases in sheep and goats (Mahin et al.,2401984; Euzby, 1990; Friedhoff, 1997).Haemaphysalis ticks are the natural vector241of thisBabesiaspecies (Uilenberg, 1997). Although ticks of this genus are known242to exist in Tunisia, none were found during this survey, probably because the243
period of their peak activity is the fall (November-October) (Bouattour et al.,244
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1999). Alternatively,R. turanicusmight be a vector for thisBabesia (Darghouth,2452004). The infection of R. bursaand R. turanicus ticks by T. equiwas expected246sinceRhipicephalusticks are the acknowledged vectors of T. equiand B. caballi247(Darghouth, 2004). T. buffeliwas the most frequently found piroplasm in tested248ticks. This result concurs with the high prevalence of this non-pathogenic249Theileria species in cattle and ticks (R. bursa, R. turanicus, R. (Boophilus)250annulatus, Haemaphysalis punctata and Ixodes ricinus) in Tunisia (Mghirbi et251al., 2008; 2010). The source of the various species of piroplasms detected in the252ticks may be the infected cattle or horses on whose blood the ticks feed.253Four ticks showed a mixed profile of different piroplasm species that included254double and triple combinations of species from the same genus or both Theileria255andBabesia (Table 1). This may have resulted from the infection of ticks that fed256on hosts co-infected with various species or on different single infected hosts at257different life stages. Surprisingly, mixed infections were not observed in the sheep258and goats tested in this study, but were reported in cattle (Mghirbi et al., 2008)259and horses (data not shown) in the same investigated zones.260In conclusion, using RLB with a panel of several piroplasm species-specific261
probes, we detected six piroplasm species in healthy small ruminants and in ticks262collected from different bioclimatic zones in Tunisia. This information is essential263for defining the zones of risk and establishing control measures. Further studies264are needed on the economic impact of small ruminant piroplasms and on265determining which ticks are their vectors.266
267Conflict of interest statement268The authors declare no conflicts of interest.269
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270271
Acknowledgements272We are grateful to Dr. Leila Saieh, Dr. Kamel Khlif, Dr. Zouheir Ktata and Dr.273Ridha Ben Omrane for their help in fieldwork. The authors would also like to274thank the farmers for their collaboration collecting samples. We also thank Dr. G.275Uilenberg and Glassman D. for constructive comments on early drafts of the276manuscript. This work was conducted with financial support from the Spanish277Agency for International Development Cooperation (AECID, Project No.278A/026818/09), the Spanish National Institute for Agricultural and Food Research279and Technology (INIA, Project No. RTA2009-000-18-00-00), the European280Regional Development Fund (ERDF) and the Ministry for Higher Education,281Scientific Research, and Technology in Tunisia. AR is the recipient of a INIA pre-282doctoral fellowship.283
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Babesia parasites infecting small ruminants by reverse line blotting. Parasitol.373Res. 92, 189-196.374Uilenberg, G., 1997. General review of tick-borne diseases of sheep and goats375world-wide. Parassitologia. 39, 161-165.376Uilenberg, G., 198l. In: Advances in the Control of Theileriosis (Ed.), A.D. Irvin,377M.P. Cunningham and A.S. Young, Martinus Nijhoff, The Hague pp. 437.378Uilenberg, G., 2001. Babesiosis. In: Service, M.W. (Ed.), Encyclopedia of379arthropod-transmitted infection of man and domestic animals. CAB International.380
pp. 5360.381Yeruham, I., Hadani, A., Galker, F., Rosen, S., 1995. A study of an enzootic focus382of sheep babesiosis (Babesia ovis, Babes, 1892). Vet. Parasitol. 60, 349-54.383Yin, H., Schnittger, L., Luo, J., Seitzer, U., Ahmed, J.S., 2007. Ovine theileriosis384in China: a new look at an old story. Parasitol. Res. 101 (Suppl 2), S191-195.385Zangana, I.K., Naqid, I.A., 2011. Prevalence of piroplasmosis (Theileriosis and386Babesiosis) among goats in Duhok Governorate College of Veterinary Medicine\387University of Duhok Al-Anbar. J. Vet. Sci. 4, 50-57.388
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Table 1. RLB detection and identification of piroplasm species in small ruminants and ticks in Northern Tunisia389R. turanicu R. bursa
Bioclimatic
zones
Localities
N flocks/N
Host
N infected /
N infected /
N collectedPiroplasms identified
N infected /
N collectedPiroplasms id
T. equi (1)Sheep (2/17) 5/41Catch-all TB (4)
0 -
T. buffeli (1)
Tabarka (1/21)
Goats (0/4) 4/9Catch-all TB (3)
0 -
B. caballi (3)T. equi (5)Catch-all TB (4)
Amdoun (1/24) Sheep (0/24) 13/46
T. buffeli (1)
0 -
Sheep (2/12) 0 - 0 -Sejnane (3/47)Goats (0/35) 18/19 T. buffeli (18) 0 -
B. bigemina (1)T. ovis (7)
B. motasi (1)
T. buffeli (1)Catch-all TB (2)
Sheep (16/28) 13/70
T. ovis +B. caballi + T. equi (1)
0 -Maaden (2/38)
Goats (1/10) 0 - 0 -
HUMID
Nefza (1/13) Sheep (0/13) 0 - 0 -
T. ovis (1)Sheep (17/19) 2/8Catch-all TB (1)
0 -Oued El Abid(2/20)
Goats (0/1) 0 - 0 -Mellegue (1/11) Sheep (2/11) 0 - 0 -
SUB-HUMID
Touiref (2/38) Sheep (1/38) 0 - 0 -
B. bigemina (2Sheep (16/37) 0 - 3/6Catch-all TB (
T. buffeli (9) Catch-all TB (
T. buffeli + T. equi (1) T. buffeli + T.
SEMI-ARID El Jouf (3/51)
Goats (2/14) 11/22
T. bu eli +B. motasi 1
2/4
Total9 localities
(16/263 animals)
22.4%(CI: 17.6
27.1%)(59/263)
30.7% (CI:27.433.9%)
(66/215)
50.0%(CI: )(5/10)
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Fig. 1 Map of Tunisia showing the location of sites where blood and tick samples391were collected.392
393394
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Table 1. RLB detection and identification of piroplasm species in small ruminants and ticks in394Northern Tunisia.395
396397398399
R. turanicu R. burs
Bioclimatic
zones
Localities
N flocks/N
Host
N infected /
N infected /
N collected Piroplasms identifiedN infected /
N collected Piropla
T. equi (1)Sheep (2/17) 5/41Catch-all TB (4)
0 -
T. buffeli (1)
Tabarka (1/21)
Goats (0/4) 4/9Catch-all TB (3)
0 -
B. caballi (3)T. equi (5)Catch-all TB (4)
Amdoun (1/24) Sheep (0/24) 13/46
T. buffeli (1)
0 -
Sheep (2/12) 0 - 0 -Sejnane (3/47)Goats (0/35) 18/19 T. buffeli (18) 0 -
B. bigemina (1)T. ovis (7)
B. motasi (1)T. buffeli (1)Catch-all TB (2)
Sheep (16/28) 13/70
T. ovis +B. caballi +T. equi(1)
0 -Maaden (2/38)
Goats (1/10) 0 - 0 -
HUMID
Nefza (1/13) Sheep (0/13) 0 - 0 -
T. ovis (1)Sheep (17/19) 2/8Catch-all TB (1)
0 -Oued El Abid(2/20)
Goats (0/1) 0 - 0 -Mellegue (1/11) Sheep (2/11) 0 - 0 -
SUB-HUMID
Touiref (2/38) Sheep (1/38) 0 - 0 -
B. bigeSheep (16/37) 0 - 3/6Catch-a
T. buffeli (9) Catch-aT. buffeli + T. equi(1) T. buffe
SEMI-ARID El Jouf (3/51)
Goats (2/14) 11/22T. bu eli +B. motasi 1
2/4
Total9 localities
(16/263 animals)
22.4%(CI: 17.6
27.1%)(59/263)
30.7% (CI:27.433.9%)
(66/215)
50.0%(CI: )(5/10)
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http://ees.elsevier.com/vetpar/download.aspx?id=194200&guid=be84ca6b-94c0-4c93-adf3-edb29f07efe8&scheme=1