Lilia Messadi

Geographic Distribution of MERS Coronavirus among Dromedary Camels, Africa

We found serologic evidence for the circulation of Middle East respiratory syndrome coronavirus among dromedary camels in Nigeria, Tunisia, and Ethiopia. Circulation of the virus among dromedaries across broad areas of Africa may indicate that this disease is currently underdiagnosed in humans outside the Arabian Peninsula.

First molecular survey and novel genetic variants' identification of Anaplasma marginale, A. centrale and A. bovis in cattle from Tunisia

Few data are available about the presence and distribution of Anaplasma species in cattle in North African countries. In this study prevalence, co-infections, risk factors and genetic diversity of Anaplasma species were evaluated in bovines from Northern Tunisia. A total of 232 cattle from 36 randomly selected farms in three Tunisian localities were investigated for the presence of Anaplasma species in blood by Real-time PCR and/or nested PCR. Overall infection rates of Anaplasma spp., Anaplasma marginale, Anaplasma centrale and Anaplasma bovis were 34.9%, 25.4%, 15.1%, and 3.9%, respectively. Anaplasma phagocytophilum was not detected in cattle. The most common co-infection pattern was an association of A. marginale and A. centrale (11.2%). Five cattle (2.1%) all reared in the sub-humid bioclimatic area, were co-infected by the three Anaplasma species. Molecular prevalence of Anaplasma infection varied significantly according to locality, bioclimatic area, tick infestation and type of breeding. Animals of the Holstein breed were less infected by A. marginale and A. centrale than other breeds. Genetic analysis of A. marginale msp4 gene indicated a high sequence diversity of Tunisian strains, suggesting a multiple introduction of infected cattle from different origins. Phylogenetic studies based on the 16S rRNA gene showed that the most prevalent A. centrale strains were closely related to the A. centrale vaccine strain. Moreover, all A. bovis variants clustered with other A. bovis sequences obtained from domestic and wild ruminant strains. This is the first molecular investigation on Anaplasma species in Tunisian cattle providing pivotal background for designing epidemiological studies and to develop control strategies in the country.

First molecular survey of Anaplasma bovis in small ruminants from Tunisia

To date, no information is available regarding the presence of Anaplasma bovis in the South Mediterranean area. In this study, prevalence, risk factors, and genetic diversity of A. bovis were assessed in small ruminants. A total of 563 healthy small ruminants (260 sheep and 303 goats), from 25 randomly selected flocks located in 5 localities from two bioclimatic areas in Tunisia, were investigated for the detection of A. bovis in blood by nested polymerase chain reaction (nPCR) assay. The overall infection rates of A. bovis were 42.7 and 23.8% in sheep and goats, respectively. Goats located in a sub-humid area were statistically more infected than those located in a humid area. A. bovis prevalence rate varied significantly according to sheep and goat flocks, and to the sheep breed. Infection with A. bovis was validated by sequencing. Sequence analysis based on the 16S rRNA gene showed that A. bovis from Tunisian goats and sheep clustered with other strain sequences detected from wild and domestic animals and published in GenBank. This study gives the first insight of presence of A. bovis DNA in small ruminants in Tunisia and suggests that these animal species may be playing an important role in the bovine anaplasmosis natural cycle caused by A. bovis in the South Mediterranean ecosystem.

Detection of novel strains genetically related to Anaplasma platys in Tunisian one-humped camels (Camelus dromedarius).

INTRODUCTION Little information is currently available regarding the presence of Anaplasma species in North African dromedaries. To fill this gap in knowledge, the prevalence, risk factors, and genetic diversity of Anaplasma species were investigated in Tunisian dromedary camels. METHODOLOGY A total of 226 camels from three different bioclimatic areas were sampled and tested for the presence of Anaplasma species by quantitative polymerase chain reaction (qPCR) and nested polymerase chain reaction (nPCR) assays. Detected Anaplasma strains were characterized by 16S rRNA sequence analysis. RESULTS Overall infection rate of Anaplasma spp. was 17.7%, and was significantly higher in females. Notably, A. marginale, A. centrale, A. bovis, and A. phagocytophilum were not detected. Animals were severely infested by three tick species belonging to the genus Hyalomma (H. dromedarii, H. impeltatum, and H. excavatum). Alignment, similarity comparison, and phylogenetic analysis of the 16S rRNA sequence variants obtained in this study suggest that Tunisian dromedaries are infected by more than one novel Anaplasma strain genetically related to A. platys. CONCLUSIONS This study reports the presence of novel Anaplasma sp. strains genetically related to A. platys in dromedaries from various bioclimatic areas of Tunisia. Findings raise new concerns about the specificity of the direct and indirect diagnostic tests routinely used to detect different Anaplasma species in ruminants and provide useful molecular information to elucidate the evolutionary history of bacterial species related to A. platys.

Première étude sérologique de la prévalence d’ Anaplasma phagocytophilum chez le dromadaire ( Camelus dromedarius ) en Tunisie

The aim of the study was to evaluate the seroprevalence of Anaplasma phagocytophilum in dromedary (Camelus dromedarius). Sera of 226 healthy dromedaries from three regions of Tunisia (Sidi Bouzid, Bouficha and Douz) were tested by indirect immunofluorescence (IFA). The overall infection rate was estimated at 29.2%. The study of risk factors showed that region, age, gender, presence of ticks and types of breeding had no influence on the seroprevalence of A. phagocytophilum. This study indicates for the first time in Tunisia that dromedary may be involved in the natural cycle of A. phagocytophilum.

Anaplasma platys-like strains in ruminants from Tunisia

Molecular diagnosis of Anaplasma platys and related strains (A. platys-like) in carnivores and ruminants is challenging due to co-infections with cross-reacting strains, and require post-amplification sequencing of the hemi-nested PCR products traditionally generated by targeting the groEL gene. In this study, a Restriction Enzyme Fragment Length Polymorphism (RFLP) assay coupled to hemi-nested groEL PCR was developed to discriminate among A. platys and genetically related strains. This novel approach was used for investigating A. platys-like infection in 963 domesticated ruminants (241 goats, 355 sheep, and 367 cattle) from 22 delegations located in North Tunisia. Overall prevalence rates of A. platys-like were 22.8, 11, and 3.5% in goats, sheep, and cattle, respectively. Alignment, identity comparison, and phylogenetic analysis of the groEL sequence variants obtained in this study confirmed RFLP data suggesting that Tunisian ruminants are infected by novel unclassified Anaplasma strains genetically related to A. platys. Compared to sequencing, RFLP assay allows fast detection of A. platys and A. platys-like pathogens in the same sample and has a potential value especially when screening ticks, cats and ruminants, which can be a common host for these two bacteria. This newly developed molecular technique would provide valuable molecular tool for epidemiological studies related to A. platys as well as remove concern over specificity of serological and molecular methods routinely used to identify diverse Anaplasma strains and species in wild and domestic ruminants.

Spatio-temporal variations and genetic diversity of Anaplasma spp. in cattle from the North of Tunisia

In cattle, anaplasmosis is a tick-borne rickettsial disease caused by Anaplasma marginale, A. centrale, A. phagocytophilum, and A. bovis. To date, no information concerning the seasonal dynamics of single and/or mixed infections by different Anaplasma species in bovines are available in Tunisia. In this work, a total of 1035 blood bovine samples were collected in spring (n=367), summer (n=248), autumn (n=244) and winter (n=176) from five different governorates belonging to three bioclimatic zones from the North of Tunisia. Molecular survey of A. marginale, A. centrale and A. bovis in cattle showed that average prevalence rates were 4.7% (minimum 4.1% in autumn and maximum 5.6% in summer), 7% (minimum 3.9% in winter and maximum 10.7% in autumn) and 4.9% (minimum 2.7% in spring and maximum 7.3% in summer), respectively. A. phagocytophilum was not detected in all investigated cattle. Seasonal variations of Anaplasma spp. infection and co-infection rates in overall and/or according to each bioclimatic area were recorded. Molecular characterization of A. marginale msp4 gene indicated a high sequence homology of revealed strains with A. marginale sequences from African countries. Alignment of 16S rRNA A. centrale sequences showed that Tunisian strains were identical to the vaccine strain from several sub-Saharan African and European countries. The comparison of the 16S rRNA sequences of A. bovis variants showed a perfect homology between Tunisian variants isolated from cattle, goats and sheep. These present data are essential to estimate the risk of bovine anaplasmosis in order to develop integrated control policies against multi-species pathogen communities, infecting humans and different animal species, in the country.

Seasonal dynamics, spatial distribution and genetic analysis of Anaplasma species infecting small ruminants from Northern Tunisia

To date, there have been no reports on seasonal variations of Anaplasma spp. in South Mediterranean small ruminants. In this longitudinal field study, single and mixed Anaplasma spp. infections in small ruminants from five different governorates belonging to three bioclimatic zones from the North of Tunisia were evaluated according to seasons. A total of 1685 blood small ruminant samples were collected in spring (355 sheep and 241 goats), summer (249 sheep and 202 goats), autumn (236 sheep and 186 goats) and winter (132 sheep and 84 goats). Molecular survey of A. ovis and A. bovis showed that average prevalence rates were 35.6% (minimum 30.7% in spring and maximum 43.6% in autumn) and 7.4% (minimum 0.9% in spring and maximum 18.1% in summer), respectively, in sheep, and 46% (minimum 21.7% in summer and maximum 65.5% in winter) and 10.1% (minimum 2.2% in autumn and maximum 23.8% in summer), respectively, in goats. A. phagocytophilum was not detected in all investigated animals. The infection profiles of A. ovis and A. bovis show that anaplasmosis caused by A. ovis is endemic in small ruminants from all investigated bioclimatic areas during the four seasons but conversely, A. bovis infection is highly intensified only in the summer. A. ovis and A. bovis infections were validated by sequencing. The comparison of the 16S rRNA sequences of A. bovis variants showed 100% identity between Tunisian variants isolated from goats, sheep and cattle. The analysis of A. ovis msp4 sequences revealed two different genetic variants previously described in Italy. This is the first survey outlining seasonal dynamics of Anaplasma spp. infections in Tunisian small ruminants. This situation should to be taken into account if anaplasmosis control programs in these domesticated animals are envisaged.

First molecular evidence of Borrelia burgdorferi sensu lato in goats, sheep, cattle and camels in Tunisia

Borrelia burgdorferi sensu lato (s.l.) are tick-transmitted spirochaetes of veterinary and human importance. Molecular epidemiology data on ruminants are still lacking in most countries of the world. Therefore, the aim of this study was to estimate the rate of B. burgdorferi s.l. infection in ruminants from Tunisia. A total of 1,021 ruminants (303 goats, 260 sheep, 232 cattle and 226 camels) from different bioclimatic areas in Tunisia were investigated for the presence of B. burgdorferi s.l. DNA in blood by real time PCR. Prevalence rates were 30.4% (92/303) in goats, 6.2% (16/260) in sheep, 1.3% (3/232) in cattle, and 1.8% (4/226) in camels. Only tick species belonging to Rhipicephalus and Hyalomma genera were found on the investigated animals. In small ruminants, the prevalence of B. burgdorferi s.l. varied significantly according to localities and farms. Goats located in humid areas were statistically more infected than those located in sub-humid areas. Prevalence rates varied significantly according to age and breed in sheep, and age and tick infestation in goats. This study provides the first insight into the presence of B. burgdorferi s.l. DNA in ruminants in Tunisia, and demonstrates that host species such as goats and sheep may play an important role in natural Lyme disease cycles in this country.

Coxiella burnetii in Tunisian dromedary camels (Camelus dromedarius): Seroprevalence, associated risk factors and seasonal dynamics

Q fever, caused by Coxiella burnetii, is a zoonotic disease responsible of abortion in ruminants. Few studies have investigated the prevalence of this infection in camels (Camelus dromedarius). The present report aimed to highlight the epidemiological status and identify the risk factors associated with C. burnetii infection in one-humped dromedary that is the most productive livestock species in arid areas. A total of 534 sera of healthy camels were collected in eight governorates from southern and central Tunisia. Samples were tested by an indirect Enzyme linked Immunosorbent Assay (ELISA). Results were analyzed using the Chi-square test and logistic regression. Overall, 237 camels (44%, 95%CI: 0.40-0.49) were seropositive to C. burnetii. Statistical analysis pointed out four potential risk factors associated with infection. A meaningful high seropositivity was observed in female camels with a previous history of abortion (70%) (OR = 4.186, 95%CI: 2.05-8.51). Seroprevalence was higher in aged camels (>10 years-old) (48%) (OR = 2.91, 95%CI: 1.37-6.17). Besides, camels, intended for meat production from small herds showed a high level of infection (52%) (OR = 2.43, 95%CI: 1.3-4.5). Coxiellosis evolved in dromedary herds throughout the year, however infection was significantly important in autumn (60%) (OR = 4.13, 95%CI: 1.86-9.17) and winter (56%) (OR = 5.52, 95%CI: 2.50-12.16). Bioclimatic stage, gender, tick infestation and contact with other ruminants were not risk factors in camels infection by C. burnetii. Our reports confirm that Tunisian one-humped dromedaries had been exposed to this bacterium and could contribute to its dissemination among farmers and other livestock animals. Furthers studies are required to evaluate the prevalence of Q fever among people professionally exposed like farmers, veterinarians and slaughterhouse workers.