Accepted Manuscript

Title: Seroprevalence of Toscana virus infection in

Authors: Ons Fezaa Olfa Bahri Nissaf Ben Alaya Bouafif Henda Triki Ali Bouattour

PII: S1201-9712(13)00268-3 DOI: http://dx.doi.org/doi:10.1016/j.ijid.2013.08.008 Reference: IJID 1787

To appear in: International Journal of Infectious Diseases

Received date: 10-6-2013 Revised date: 2-8-2013 Accepted date: 13-8-2013

Please cite this article as: Fezaa O, Bahri O, Alaya Bouafif NB, Triki H, Bouattour A, Seroprevalence of Toscana virus infection in Tunisia, International Journal of Infectious Diseases (2013), http://dx.doi.org/10.1016/j.ijid.2013.08.008

This is a PDF file 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 final 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. Running heads:

Recto: Seroprevalence of Toscana virus infection, Tunisia

Verso: O. Fezaa et al.

Correspondence footnote:

*Corresponding author. Tel.: +216 71 893 340; fax: +216 71 791 833.

E-mail address: [email protected] (A. Bouattour).

[ARTICLE TYPE – FULL LENGTH ARTICLE]

Seroprevalence of Toscana virus infection in Tunisia

Ons Fezaaa, Olfa Bahrib, Nissaf Ben Alaya Bouafifc, Henda Trikib, Ali

Bouattoura,*

aLaboratoire d’Epidémiologie et Microbiologie Vétérinaire, Service d’Entomologie

Médicale, Institut Pasteur de Tunis, Université Tunis El Manar, BP74, 1002 Tunis-

Bélvédère, Tunisia bLaboratoire de Recherche “Diversité Génétique des Entérovirus et Virus des Hépatites”Accepted Institut Pasteur de Tunis, Faculté deManuscript Médecine de Tunis, Université Tunis El Manar, Tunis, Tunisia cObservatory of New and Emerging Diseases, Ministry of Health, Tunis, Tunisia

[Received date]

Corresponding Editor: Eskild Petersen, Aarhus, Denmark

Page 1 of 20

KEYWORDS

Toscana virus;

Phlebotomus;

ELISA;

IFA;

Tunisia

Summary

Objectives: The main objective of this study was to assess the prevalence of IgG antibodies against Toscana virus (TOSV) by an ELISA test and to determine the extent of its circulation in Tunisia.

Methods: An indirect ELISA test was performed to detect anti-TOSV IgG. The results were compared to those of an indirect fluorescent antibody (IFA) test.

Results: The survey tested 494 healthy people from various regions of Tunisia by

ELISA for anti-TOSV IgG; 47 people (9.5%) were found to be positive.

Seroprevalence varied by bioclimatic region and gender. Two hundred and twelve samples, randomly chosen from the same selected population and tested with ELISA, were retested using an IFA for IgG antibodies. An 85% concordance between the IFA and ELISAAccepted was obtained (kappa = 0.650). Manuscript

Conclusions: These serological data confirm the circulation of TOSV in different bioclimatic zones in Tunisia where the vector sand flies are found. The detection of

IgG against TOSV suggests that the diagnosis of TOSV infection is often neglected,

Page 2 of 20 as this virus often causes asymptomatic infections, with only a few patients developing severe illnesses involving neurological manifestations.

Introduction

Toscana virus (TOSV) is an arbovirus transmitted to humans by the bites of

Phlebotomine sand flies, mainly Phlebotomus perniciosus and Phlebotomus perfiliewi.1,2 TOSV belongs to the Phlebovirus genus of the Bunyaviridae family, and can cause brain injuries. Many reported cases have shown that TOSV has a tropism for the central nervous system (CNS) and represents a major cause of meningitis and meningo-encephalitis.1,3 In Italy, for example, the virus appears to be one of the three major viral pathogens involved in aseptic meningitis, whose clinical manifestations resemble those of other viral agents.4 The cases of meningitis or meningo-encephalitis caused by TOSV are regularly observed during the summer season, with a peak in

August when sand fly activity is high.5 The incidence of neurological damage varies from year to year and is directly related to differences in the annual density of the vector, which is greatly influenced by changes in climatic conditions.

TOSV has been reported in several European countries. Seroprevalence varies from country to country.5,6 In the southern Mediterranean region, however, the epidemiology of TOSV infection is still largely unknown. A study conducted in

Morocco Acceptedshowed the presence of TOSV in the sandManuscript fly vectors, but included no data on the epidemiology of the virus in humans.7 We have recently shown that TOSV is present in Tunisia and is responsible for approximately 10% of cases of neurological disease.8,9 In addition, sand flies taken from pools in northern Tunisia have been shown to carry strains of TOSV.10 The TOSV infection can be diagnosed using direct

Page 3 of 20 methods, such as virus isolation, vital antigen detection by immunofluorescence, and

RT PCR, or by indirect methods, detecting the presence of specific IgM and IgG antibodies using immunoenzymatic techniques.11

The main objective of this study was to assess the prevalence of IgG antibodies against TOSV in Tunisia by ELISA test and to determine the extent of its circulation in the country. As an alternative to the ELISA test, the indirect fluorescent antibody test (IFA) was used, and results were compared to those of the ELISA technique.

Materials and methods

Population tested

The seroprevalence study was conducted using sera collected from 494 anonymous, healthy individuals aged 5 to 85 years. Data related to age, sex, previous medical history, and geographical origin for each person were recorded, and subjects were classified into four age groups (Table 1). Sera were taken from volunteer blood donors from whom informed consent was obtained. Sera from persons younger than

20 years and older than 60 years came from the serum banks of regional hospitals of each governorate, where the individuals had attended appointments for various examinationsAccepted with non-infectious pathological indications.Manuscript

[Table 1 here]

Page 4 of 20 The population studied came from three governorates (Figure 1) – ,

Mahdia, and – in different bioclimatic zones. In , located in a humid bioclimatic zone12 that gets more than 800 mm of annual rainfall,

274 serum samples were taken from people living in five randomly selected localities:

Jbel el Jouza, Tbénia, Ain Draham, El Faija, and . In Governorate, located in central Tunisia in a lower semi-arid bioclimatic zone12 with annual rainfall between 200 and 300 mm, serum samples were taken from 112 individuals. In

Tataouine Governorate, located in the south in an arid bioclimatic area12 with annual rainfall between 88 and 157 mm, 108 samples were collected for antibody testing.

[Figure 1 here]

ELISA detection of IgG antibodies against TOSV

An indirect ELISA test was performed to detect anti-TOSV IgG antibodies, as described above.8 Wells of polystyrene plates were coated with a predetermined optimum quantity of positive and negative antigens (ISS Phl.3), in phosphate-buffered saline (PBS) (pH 7.2) and incubated overnight at 4 ºC. Human sera were diluted at

1:100 in PBS–TM (PBS, 0.05% Tween 20, 3% milk). Two negative and two positive control sera were included in each assay. Goat anti-human IgG (Fc-specific) peroxidaseAccepted conjugated antibody (Sigma) was dilutedManuscript at 1:1000 and added to each well. The color reaction was developed by adding a substrate solution containing

3,3′,5,5′-tetramethylbenzidine (TMB) (Sigma). At each step, the reaction mixture was incubated for 75 min at 37 ºC and washed extensively with PBS–Tween. H2SO4 was added at a final concentration of 1 N to stop the reaction. All serum samples were

Page 5 of 20 tested in duplicate with viral and negative control antigens. The IgG ELISA cut-off value was determined by the mean plus three standard deviations of the optical densities (OD) read, calculated as the average OD with antigen minus the average OD with negative control antigen. The borderline between positive and negative samples was calculated as the mean of the test results of 30 known negative sera plus three standard deviations. The OD of each sample was read at two wavelengths – 450 and

620 nm.

Comparison of ELISA and IFA techniques

Of the 494 samples tested by ELISA, only 212 were randomly chosen and re-tested by

IFA because of a limited number of slides and an insufficient quantity of serum left in some samples.

The IFA was performed in accordance with the procedure described by

Valassina et al. (1996).13 Briefly, Vero cells infected with a TOSV isolate (kindly provided by Dr M. Bouloy of the Institut Pasteur, Paris) were harvested and collected on spot slides (104 cells per spot); uninfected cells were collected in the same manner and used as negative control. The slides were air-dried and fixed in cold acetone for

10 min. When not immediately used, the slides were stored at −30 ºC. The sample sera were diluted at 1:100 and screened for specific IgG antibodies along with negative andAccepted positive control sera included in eachManuscript assay. Specimens were incubated for 30 min at 37 ºC, then washed three times with PBS for 10 min at room temperature. A goat anti-human IgG (whole molecule)−FITC antibody conjugate

(Sigma) was subsequently added at a final concentration of 1:1000, with Evans blue as a counter-stain. The slides were incubated for 30 min at 37 ºC and washed with

Page 6 of 20 PBS for 10 min. Two operators read each slide twice and positive results were characterized by small patches of intranuclear and intracytoplasmic fluorescence.

Statistical analysis

To compare the seroprevalence obtained in each region, statistical differences among groups were determined by Pearson’s Chi-square test. Epi Info version 6.04 was used.

A probability (p) value of less than 0.05 was considered as statistically significant. In addition, the kappa coefficient measuring the agreement between two rates was used to evaluate the agreement between the ELISA and the IFA test. If the raters are in complete agreement then kappa = 1. If there is no agreement among the raters other than what would be expected by chance (as defined by Pr(e)), kappa = 0.

Results

Serological survey of TOSV

A total of 494 subjects (302 male, 192 female) with an average age of 45 years were tested. Of the 494 samples, 47 (9.5%, 95% confidence interval (CI) 7.07–12.45%) tested positive for anti-TOSV IgG by ELISA. The obtained seroprevalence varied between governoratesAccepted and sex, and no significant Manuscript difference was seen between age groups (p = 0.441).

Among the population tested, 14.6% of women (28/192, 95% CI 9.91–

20.38%) and 6.3% of men (19/302, 95% CI 3.38–9.65%) tested positive for TOSV infection; this difference is significant (p = 0.003).

Page 7 of 20 Regional differences were found to be statistically significant (p < 10−3, Table

1). Of the 274 serum samples tested from Jendouba, 15 were positive for IgG anti-

TOSV corresponding to a seroprevalence of 5.5% (95% CI 3.1–8.87%); this varied significantly by locality (Ain Draham, Jbel El Jouza, El Faija, Tbénia, Tabarka), with a significantly higher value in Ain Draham (n = 9/63; 95% CI 6.75–25.39%; p =

0.002). The seroprevalence rate in Jendouba was similar to that of Tataouine, where of 108 samples, six were positive for anti-TOSV IgG (5.6%, 95% CI 2.07–11.70%).

By contrast, in Mahdia, the prevalence was 23% (n = 26/112; 95% CI 15.76–32.14%).

Comparison between ELISA and IFA

Among the 212 serum samples tested with the two tests, 18 tested positive with both techniques. Of the remaining 194 samples, 12 were positive by IFA only and four by

ELISA only. A kappa equal to 0.650 (standard error of kappa = 0.080; 95% CI 0.493–

0.808) was obtained. The strength of the agreement is considered to be good (Table 2

[Au?1]).

[Table 2 here]

Discussion

Accepted Manuscript

Several studies have demonstrated TOSV infection to be endemic in Mediterranean countries.6 TOSV is considered to be the most frequent viral agent implicated in neuromeningeal human infections in endemic countries.14

Page 8 of 20 To investigate the prevalence of IgG antibodies against TOSV in Tunisia, serum samples were collected from three different bioclimatic zones. The ELISA test showed that 9.5% (47/494) of healthy people have antibodies against TOSV; this rate is roughly similar to that reported in blood donors from the south of France (12%),15 but lower than that observed in hyper-endemic areas such as Italy. Indeed, earlier serological surveys have shown that TOSV is endemic in the Mediterranean Basin with varying seroprevalences: 80% in Italy, 35–60% in Greece, 25% in Spain, and

20% in Cyprus.5,3,15 By contrast, the seroprevalence found in this investigation is higher than that observed in Portugal: 3.1% in the group showed no neurological signs and 4.2% of patients had neurological signs.16 In the absence of data concerning the

TOSV reservoir host, serological variations are strictly correlated with favorable habitats for the sand fly vectors,6 with a seroprevalence distribution based on socioeconomic status. Indeed, in Tuscany, TOSV seroprevalence was found to be higher in forestry workers (77.2%) than in the urban population (22.7%).5

In this study, we noted regional variations in seroprevalence: 5.5% and 5.6% in humid and arid zones, respectively, and 23% in the semi-arid zone. The significantly higher rate observed in Mahdia Governorate can be explained by the exposure of people in this region to diverse and abundant sand fly populations, predominantly P. perniciosus and P. perfiliewi species,17 considered to be the main vectors of TOSV; these species are more active in warm, humid environments. During the hot season,Accepted populations living in this area areManuscript active outdoors and therefore at greater risk of being bitten by sand flies.

Seventeen species of sand fly circulate in the different bioclimatic zones of

Tunisia, where their distribution differs from one zone to another.18,19 In Jendouba, a humid zone, P. perniciosus and P. perfiliewi are the predominant species, but the

Page 9 of 20 seroprevalence of TOSV was only 5.5%. This might be explained by a low human population density and the lower sand fly densities previously reported in this zone.20

In Tataouine, Phlebotomus papatasi and Sergentomyia minuta [Au?2] are the dominant species, although P. perniciosus exists but at a lower rate.21 The low sand fly population density of this arid zone could explain the low seroprevalence of

TOSV. Indeed in the Mediterranean Basin, P. perniciosus is predominant in the sub- humid and semi-arid zones.22

TOSV is mainly transmitted by P. perniciosus,23,24 the dominant species in semi-arid and arid bioclimatic zones,19 which is also involved in transmitting

Leishmania infantum to dogs and humans in Tunisia. The incidence of this protozoan is also higher in the semi-arid zone.25,26

Our study showed no significant difference among age groups. There was, however, an age-dependent increase in infection in Jendouba and Mahdia, while in

Tataouine, infection was more frequent among children. A study conducted in

Granada, Spain showed an anti-TOSV IgG rate of 4% in persons <15 years to 60.4% in those aged >65 years, however this difference was not significant.24 In Tuscany the seroprevalence rate was found to be 19.8% in adults and 5.8% in children.27 A lower

TOSV seroprevalence in children could be related to lower exposure to the vector.27

The serological results showed that the infection seems to occur much less frequently in men than in women (p = 0.003). Women living in rural areas are usually most involvedAccepted in cleaning and feeding animal s andManuscript therefore near the habitats of sand flies; consequently they are much more exposed to Phlebotomus bites.

The prevalence of detection of IgG antibodies against TOSV suggests that the diagnosis of TOSV infection is often neglected, since this virus usually causes asymptomatic infections, with only a few patients developing severe illnesses that

Page 10 of 20 include neurological manifestations (meningitis and encephalitis).4 Indeed, our recent study showed that 10% of patients with neurological diseases (31 cases) tested positive for TOSV IgM.8

We used an ELISA, considered to be the most sensitive technique,28 to detect

IgG antibodies against TOSV, and then carried out an IFA test to compare the results.

These two techniques are sensitive for detecting anti-TOSV antibodies as compared to other serological tools. They are known to be easy to use and rapid in reporting results,29 bearing in mind that positivity for IgG antibodies does not distinguish between current and past viral infections.

We estimated a concordance greater than 85% between the results of the IFA and ELISA (kappa = 0.650). However, a discrepancy between the two techniques was observed and a positive result obtained for 12 of 212 sera by IFA. This could result from a false secondary reaction previously reported for IFA, especially inherent to reading microscope slides. This suggests the utility of routine double-reading by two technicians.

These serological data confirm the circulation of TOSV in different bioclimatic zones in Tunisia where the vector sand flies are found. Physicians should consider this neglected disease in patients with unexplained meningitis and aseptic cerebrospinal fluid. ELISA diagnostic assays are recommended to carry out the epidemiological study. In addition, the development of an RT-PCR is necessary for research onAccepted TOSV and other Naples virus complexes Manuscript (Naples and Sicily virus) to identify the viral strains circulating in patients and vectors. An investigation of sand fly vectors is key to understanding the epidemiology of the Phlebovirus-related diseases. Investigations of the bio-ecology and the behavior of each vector species and

Page 11 of 20 the identification of the reservoir hosts of such viruses, would help us understand and monitor these phleboviruses and develop appropriate control programs.

Acknowledgements

We are grateful to Dr Michele Bouloy for providing us with the IFI slides and the

Toscana antigen. We are also grateful to all the hospital medical staff of Jendouba,

Mahdia, and Tataouine for their help. We also thank Dr Sparagano O.A.E., Dr A.

Ghram [Au?3] and Dr Deborah Glassman for constructive comments and English corrections on early drafts of the manuscript. This work was conducted with financial support from Inter-Pasteurien Concerted Actions (ACIP-08).

Ethical approval: The Bioethics Committee of Institut Pasteur de Tunis approved this study.

Conflict of interest: The authors declare that they have no competing interests.

References

1. Echevarria JM, de Ory F, Eulalia Guisasola M, Sanchez-Seco MP, Tenorio A,

Lozano A, et al. Acute meningitis due to Toscana virus infection among patients from both the SpanishAccepted Mediterranean region and the regionManuscript of Madrid. J Clin Virol 2003;26:79–84.

2. Peyrefitte CN, Devetakov I, Pastorino B, Villeneuve L, Bessaud M, Stolidi P, et al.

Toscana virus and acute meningitis, France. Emerg Infect Dis J 2005;11:778–80.

3. Charrel RN, Gallian P, Navarro-Marí JM, Nicoletti L, Papa A, Sánchez-Seco MP, et al. Emergence of Toscana virus in Europe. Emerg Infect Dis 2005;11:1657–63.

Page 12 of 20 4. Braito A, Corbisiero R, Corradini S, Marchi B, Sancasciani N, Fiorentini C,

Ciufolini MG. Evidence of Toscana virus infections without central nervous system involvement: a serological study. Eur J Epidemiol 1997;13:761–4.

5. Valassina M, Valentini M, Pugliese A, Valensin PE, Cusi MG. Serological survey of Toscana virus infections in a high-risk population in Italy. Clin Diagn Lab

Immunol 2003;10:483–4.

6. Cusi MG, Savellini GG, Zanelli G. Toscana virus epidemiology: from Italy to beyond. Open Virol J 2010;4:22–8.

7. Es-Sette N, Nourlil J, Hamdi S, Mellouki F, Lemrani M. First detection of Toscana virus RNA from sand flies in the genus Phlebotomus (Diptera: Phlebotomidae) naturally infected in Morocco. J Med Entomol 2012;49:1507–9.

8. Bahri O, Fazaa O, Ben Alaya-Bouafif N, Bouloy M, Triki H, Bouattour A. Rôle du virus Toscana dans les infections neuroméningées en Tunisie. Pathol Biol (Paris)

2010;59:125–7.

9. Sghaier W, Bahri O, Kedous E, Fazaa O, Rezig D, Touzi H, et al. Retrospective study of viral causes of central nervous system infections in Tunisia (2003–2009).

Med Sante Trop 2012;22:373–8.

10. Bichaud L, Dachraoui K, Piorkowski G, Chelbi I, Moureau G, Cherni S, et al.

Toscana virus isolated from sandflies, Tunisia. Emerg Infect Dis 2013;19:322–4.

11. Verani P, Nicoletti L. Phlebovirus infections. In: Portefield JS, editor. Kass handbookAccepted of infectious diseases: exotic viral infection. Manuscript London: Chapman and Hall;

1995, p. 295–318.

12. Institut National de Recherches Forestières. Carte bioclimatique de la Tunisie selon la classification d’Emberger. Etages et variantes. INRF; 1976. Available at:

Page 13 of 20 http://eusoils.jrc.ec.europa.eu/esdb_archive/eudasm/africa/images/maps/download/afr

_tnbicl.jpg (accessed April 2013).

13. Valassina M, Cusi MG, Valensin PE. Rapid identification of Toscana virus by nested PCR during an outbreak in the Siena area of Italy. J Clin Microbiol

1996;34:2500–2.

14. Zehender G, Bernini F, Delogu M, Cusi MG, Rezza G, Galli M, Ciccozzi M.

Bayesian skyline plot inference of the Toscana virus epidemic: a decline in the effective number of infections over the last 30 years. Infect Genet Evol 2009;9:562–6.

15. De Lamballerie X, Tolou H, Durand JP, Charrel RN. Prevalence of Toscana virus antibodies in volunteer blood donors and patients with central nervous system infections in southeastern France. Vector Borne Zoonotic Dis 2007;7:275–8.

16. Amaro F, Luz T, Parreira P, Marchi A, Ciufolini MG, Alves MJ. Serological evidence of Toscana virus infection in Portuguese patients. Epidemiol Infect

2012;140:1147–50.

17. Boudabous R, Amor S, Khayech F, Marzouk M, Bdira S, Mezhoud H, et al. The phlebotomine fauna (Diptera: Psychodidae) of the eastern coast of Tunisia. J Med

Entomol 2009;46:1–8.

18. Chelbi I, Zhioua E. Confirmation of the presence of Sergentomyia (Sintonius) clydei (Sinton, 1928) in Tunisia. B Soc Pathol Exot 2012;105:396–8.

19. Ghrab J, Rhim A, Bach-Hamba D, Chahed MK, Aoun K, Nouira S, Bouratbine A.

PhlebotominaeAccepted (Diptera: Psychodidae) of human Manuscript leishmaniosis sites in Tunisia.

Parasite 2006;13:23–33.

20. Croset H, Rioux JA, Maistre ME, Bayar N. Les phlébotomes de Tunisie (Diptera,

Phlebotomidae), mise au point systématique, chorologique et éthologique. Ann

Parasitol Hum Comp 1978;53:711–49.

Page 14 of 20 21. Tabbabi A, Ghrab J, Aoun K, Ready PD, Bouratbine A. Habitats of the sandfly vectors of Leishmania tropica and L. major in a mixed focus of cutaneous leishmaniasis in southeast Tunisia. Acta Trop 2011;119:131–7.

22. Rioux JA, Rispail P, Lanotte, G, Lepart J. Relations phlébotomes—bioclimats en

écologie des leishmanioses. Corollaires épidémiologiques. L’exemple du Maroc. Bull

Soc Botanique Fr 1984;131:549–57.

23. Verani P, Nicoletti L, Ciufolini MG. Antigenic and biological characterization of

Toscana virus, a new Phlebotomus fever group virus isolated in Italy. Acta Virol

1984;28:39–47.

24. Sanbonmatsu-Gámez S, Pérez-Ruiz M, Collao X, Sánchez-Seco MP, Morillas-

Márquez F, de la Rosa-Fraile M, et al. Toscana virus in Spain. Emerg Infect Dis

2005;11:1701–7.

25. Dedet J, Ben Osman F, Chadli A, Croset H, Rioux JA. La leishmaniose canine en

Tunisie. Ann Parasitol Hum Comp 1973;48:653–60.

26. Diouani MF, Ben Alaya Bouafif N, Bettaib J, Louzir H, Jedidi S, Ftaiti A, et al.

Dogs L. infantum infection from an endemic region of the north of Tunisia: a prospective study. Arch Inst Pasteur Tunis 2008;85:55–61.

27. Terrosi C, Olivieri R, Bianco C, Cellesi C, Cusi MG. Age-dependent seroprevalence of Toscana virus in central Italy and correlation with the clinical profile. Clin Vaccine Immunol 2009;16:1251–2.

28. ErgünayAccepted K, Litzba N, Lo MM, Aydoğan S, ManuscriptSaygan MB, Us D, et al. Performance of various commercial assays for the detection of Toscana virus antibodies. Vector

Borne Zoonotic Dis 2011;11:781–7.

29. Ciufolini MG, Fiorentini C, Di Bonito P, Mochi S, Colomba G. Detection of

Toscana virus-specific immunoglobulins G and M by an enzyme-linked

Page 15 of 20 immunosorbent assay based on recombinant viral nucleoprotein. J Clin Microbiol

1999;37:2010–12.

Accepted Manuscript

Page 16 of 20 Table 1 Distribution of the positive cases to TOSV according to geographical origin, age group, and sex

Bioclimatic Governoratesa 95% CI Localitiesb Age, No. positive/No. tested Sex zones No. positive/No. tested No. positive/No. tested 5–19 20–39 40–59 ≥60 M F

(%) (%)

Humid Jendouba 15/274 (5.5%) 3.1–8.87% Jbel el Jouza 0/33 0 7 15 11 22 11

El Faija 5/89 (6%) 0 25 38 5/26 64 25

Tbénia 0/32 0 1 23 8 23 9

Ain Draham 9/63 (14%) 0 5 7/42 2/16 48 15

Tabarka 1/57 (2%) 1 16 1/25 15 40 17

Semi-arid Mahdia 26/112 (23%) 15.76–32.14% - 2/35 8/32 12/30 4/15 51 61

Arid Tataouine 6/108 (5.6%) 2.07–11.70% - 4/58 1/32 1/16 0/2 54 54

Total Accepted47/494 (9.5%) 7.07–12.45% Manuscript 6/94 10/118 21/189 10/93 302 192

CI, confidence interval; M, male; F, female. ap < 10−3. bp = 0.002.

Page 17 of 20 Table 2 Comparison of the results of the two serological techniques

ELISA

IFA Number positive Number negative

Number positive 18 12

Number negative 4 178

Total 22 190

Accepted Manuscript

Page 18 of 20

Figure caption

Figure 1 Map of Tunisia: location of the regions studied.

Accepted Manuscript

Page 19 of 20

Map 1 : Map of Tunsia : location of studied regions

Accepted Manuscript

Page 20 of 20