Detection and genetic identification of Borrelia lusitaniae in questing Ixodes inopinatus tick from Tunisia.

Background: Until now, there has been limited information on the prevalence and the phylogeny of Borrelia burgdorferi sensu lato in Ixodes ticks in Tunisia, particularly in Ixodes inopinatus. Methods: The present study aimed to determine the prevalence and the phylogeny of B. burgdorferi s.l., in coexisted I. ricinus and I. inopinatus ticks collected from Northern Tunisia. One hundred questig ticks were collected during winter 2020 by tick-dragging method in Beja gouvernorate located in the north of Tunisia. Real-time PCR panel targeting B. burgdorferi s.l. 23S rRNA gene were performed. Positive DNA samples were subjected to conventional PCRs targeting 457 bp fragment of the Borrelia sp. flagellin (fla) gene using primers FlaF/FlaR. The identified Borrelia sp. isolate underwent partial sequence analysis to determine genospecies and evaluate their phylogenetic position. Results: The study revealed a prevalence rate of 28% (28/100) for B. burgdorferi sensu lato in the Ixodes ticks. The prevalence rates across tick species and genders did not show significant variations (p > 0.05). Interestingly, the study underlines the coexistence of I. inopinatus and I. ricinus sharing the same geographic areas in Northern Tunisia. Furthermore, DNA of B. lusitaniae was detected in I. inopinatus ticks for the first time in Tunisia. Revealed B. lusitaniae bacterium is similar to previously identified strains in Mediterranean region, but distinct from those isolated exclusively from countries of Eastern and Central Europe, such as Serbia, Romania, and Poland. This study highlights the prevalence of B. burgdorferi s.l. in I. ricinus/I. inopinatus ticks, and reveals B. lusitaniae in I. inopinatus ticks for the first time in Tunisia. Conclusion: These findings suggest the involvement of I. inopinatus as a potential vector of this pathogenic genospeciess in Tunisia. This may help understanding the ecology of Ixodes ticks, the natural infection and the transmission dynamics of Borrelia species in this country.

Serological and molecular survey of brucellosis and chlamydiosis in dromedary camels from Tunisia.

The present sero-epidemiological survey was designed and conducted to scrutinize the current status of camel-related brucellosis and chlamydiosis in Tunisia. Whole blood and serum samples were collected from 470 dromedaries (Camelus dromedarius) from eight different Tunisian governorates. Serum samples were subjected to indirect enzyme-linked immunosorbent assay (iELISA). The detection of Brucella and Chlamydia DNA was performed using conventional PCR targeting the bcsp-31 and 16 S rRNA gene, respectively. Overall, 10/470(2.12%) and 27/470 (5.75%) camels were revealed seropositive to Brucella and Chlamydia, respectively. Multivariate logistic regression analysis showed different risk factors associated with these infections. Meaningful high rates of seropositivity of brucellosis (9.5%; p = 0.000; OR=64.193) and chlamydiosis (22.6%; p = 0.000; OR=42.860) were noted among camels showing previous abortions in particular for aged females. Besides, Chlamydia seropositivity is significantly important during winter (12.5%; p = 0.009; OR= 27.533), and in camels raised in small farms (11.4%, p = 0.000, OR=86.052). Molecular analysis revealed no positivity from all analyzed blood samples. These findings indicate the involvement of camels in the epidemiology of these abortive infectious diseases. This raises awareness and serious public health concern for infectious camel diseases in order to develop further diagnostic improvements and effective control strategies.

Diversity and Phylogeny of Cattle Ixodid Ticks and Associated Spotted Fever Group Rickettsia spp. in Tunisia.

Tick-borne rickettsioses are mainly caused by obligate intracellular bacteria belonging to the spotted fever group (SFG) of the Rickettsia genus. So far, the causative agents of SFG rickettsioses have not been detected in cattle ticks from Tunisia. Therefore, the aim of this study was to investigate the diversity and phylogeny of ticks associated with cattle from northern Tunisia and their associated Rickettsia species. Adult ticks (n = 338) were collected from cattle in northern Tunisia. The obtained ticks were identified as Hyalomma excavatum (n = 129), Rhipicephalus sanguineus sensu lato (n = 111), Hyalomma marginatum (n = 84), Hyalomma scupense (n = 12) and Hyalomma rufipes (n = 2). After DNA extraction from the ticks, 83 PCR products based on the mitochondrial 16S rRNA gene were sequenced and a total of four genotypes for Rh. sanguineus s.l., two for Hy. marginatum and Hy. excavatum and only one for Hy. scupense and Hy. rufipes were recorded, with the occurrence of one, two and three novel genotypes, respectively, for Hy. marginatum, Hy. excavatum and Rh. sanguineus s.l. mitochondrial 16S rRNA partial sequences. The tick DNA was tested for the presence of Rickettsia spp. by using PCR measurements and sequencing targeting three different genes (ompB, ompA and gltA). Of the 338 analyzed ticks, 90 (26.6%), including 38 (34.2%) Rh. sanguineus s.l., 26 (20.1%) Hy. excavatum, 25 (29.8%) Hy. marginatum and one (50%) Hy. rufipes tick, were positive for Rickettsia spp. Based on 104 partial sequences of the three analyzed genes, the BLAST analysis and phylogenetic study showed the infection of Hy. excavatum, Hy. marginatum and Rh. sanguineus s.l. tick specimens with R. massiliae, R. aeschlimannii and R. sibirica subsp. mongolitimonae and one Hy. rufipes tick specimen with R. aeschlimannii. In addition, coinfection with R. massiliae and R. aeschlimannii was reported in one Hy. marginatum and one Rh. sanguineus s.l. tick specimen, while a coinfection with R. massiliae and R. sibirica subsp. mongolitimonae was recorded in one Rh. sanguineus s.l. tick specimen. In conclusion, our study reports, for the first time in Tunisia, the infection of cattle ticks belonging to Hyalomma and Rhipicephalus genera with zoonotic Rickettsia species belonging to the SFG group.

Anaplasma ovis Prevalence Assessment and Cross Validation Using Multiparametric Screening Approach in Sheep from Central Tunisia.

We conducted a 5-month-long screening of Anaplasma spp. and Anaplasma ovis infection in sheep from central Tunisia. During this longitudinal study, we investigated the infection dynamics using both direct and indirect assessments validated with a polymerase chain reaction (PCR) as the gold standard method. The experimental design included 84 male lambs aged from 6 to 8 months, and 32 ewes, both chosen randomly from June to November with a periodicity of 2 weeks approximately between June and September, and 1 month between September and November. A total of 9 field visits were carried out in this period during which animals were clinically examined and biological samples were extracted. Thus, a total of 716 blood smears, 698 sera from the nine sampling dates, as well as 220 blood samples from the first and the ninth sampling dates were collected from apparently healthy lambs and ewes, respectively, and analyzed by competitive enzyme-linked immunosorbent assay (cELISA) and polymerase chain reaction (PCR) assay, for the detection of Anaplasma antibodies and A. ovis DNA, respectively. Sera were analyzed by competitive enzyme-linked immunosorbent assay (cELISA) and PCR, for the detection of Anaplasma antibodies and A. ovis DNA, respectively. The Anaplasma spp. initial seroprevalence rate was 33.3% in lambs and 100% in ewes, and it then flowed in an upward trend to reach a maximum of 52.6% in lambs, whereas in ewes, the Anaplasma spp. seroprevalence rate remained unchanged and equal to 100%. Meanwhile, the A. ovis initial molecular prevalence was 22.6% at the first visit and 26.3% at the last visit in lambs, whereas in ewes, the molecular prevalence rates of A. ovis were higher in both the first and the last visit estimated at 100% and 85.7%, respectively. The Kappa coefficient between cELISA and PCR indicated a moderate level of agreement on the first sampling date (0.67) and a low agreement level on the last (0.43). Furthermore, an exploratory data analysis using a multimodal machine learning approach highlighted the underlying pattern of each analytical technique used in this study. In this prospect, we were able to establish the performance of each technique at detecting Anaplasma spp. in sheep. The combination of these approaches should improve the field assessment while promoting a data-based decision in precision epidemiology. The genetic follow-up test relevant to A. ovis msp4 sequences revealed three different genotypes, two of which were previously described in Italy.

Epidemiology and genetic characteristics of tick-borne bacteria in dromedary camels of the world.

This review presents updated knowledge on the main tick-borne bacteria infecting one-humped camels (Camelus dromedarius) around the world. Camels are increasingly the subject of several scientific investigations, showing that they are receptive and carriers of several zoonotic bacteria. An appraisal is also given of the relative public health importance of these bacterial infections according to One Health concept. Microscopic, serologic and molecular findings are appropriately generated in order to exploit epidemiological data, and phylogeographic specificities associated to each vector-borne bacterium. Indeed, camels and their ticks harbour similar species and genotypes of pathogenic bacteria commonly identified in other animals, e.g., Anaplasma spp.,Ehrlichia spp., Borrelia spp., Rickettsia spp., Coxiella burnetii, Bartonella spp. and hemotrophic mycoplasmas. This evidence suggests an epidemiological role of camels in the spread of these pathogens in their natural habitats. However, these infections are commonly asymptomatic in camels resulting in underestimation of the impact of these infections. Furthermore, camels have recently been proven to have their own specific unclassified strains, such as Candidatus Anaplasma camelii and Candidatus Bartonella camelii, implying that possible interactions may lead to the emergence of pathogenic and zoonotic bacteria. In camel-rearing areas of the world, spatial and temporal spread of these infections, due to climatic and ecological changes and human activities such as development projects and urbanization, is expected. Hence the data presented herein provides a basis for strategic frameworks for the research and the development of novel diagnosis and control strategies worldwide, which are needed to protect camels, other livestock, and people in contact with dromedaries from threats that arthropod-borne pathogens can pose.

gltA typing of Anaplasma strains related to A. platys: Taxonomical and one health implications.

Species belonging to the genus Anaplasma (Rickettsiales) include bacteria of veterinary and public health importance. Beside the zoonotic Anaplasma phagocytophilum, A. platys, the etiological agent of canine cyclic thrombocytopenia, has been sporadically reported in clinically ill human patients. The ongoing emergence of novel strains related to this species in vertebrate hosts emphasises the need for genetic comparisons among strains identified in different regions of the world. In this paper we developed a PCR test suitable for amplification of the still undescribed gltA gene of Anaplasma strains related to A. platys from Mediterranean ruminants and applied on a panel of 248 samples. gltA sequencing allowed phylogenetic comparison with strains related to A. platys recently identified in China, and strains representative of the Anaplasmataceae family. Results suggest the designation of Candidatus A. turritanum, including Mediterranean A. platys - like strains, and Candidatus A. cinensis, including strains isolated in China. Data generated in this study are a solid reference for future epidemiological studies of novel unclassified strains related to A. platys and for their diagnosis and raise concern on their potential veterinary and public health implications encouraging investigating the suspected unexplored diversity within the genus Anaplasma in animals and human.

Screening and Analysis of Anaplasma marginale Tunisian Isolates Reveal the Diversity of lipA Phylogeographic Marker and the Conservation of OmpA Protein Vaccine Candidate.

Bovine anaplasmosis caused by Anaplasma marginale is a disease responsible for serious animal health problems and great economic losses all over the world. Thereby, the identification of A. marginale isolates from various bioclimatic areas in each country, the phylogeographic analysis of these isolates based on the most informative markers, and the evaluation of the most promising candidate antigens are crucial steps in developing effective vaccines against a wide range of A. marginale strains. In order to contribute to this challenge, a total of 791 bovine samples from various bioclimatic areas of Tunisia were tested for the occurrence of A. marginale DNA through msp4 gene fragment amplification. Phylogeographic analysis was performed by using lipA and sucB gene analyses, and the genetic relationship with previously characterized A. marginale isolates and strains was analyzed by applying similarity comparison and phylogenetic analysis. To evaluate the conservation of OmpA protein vaccine candidate, almost complete ompA nucleotide sequences were also obtained from Tunisian isolates, and various bioinformatics software were used in order to analyze the physicochemical properties and the secondary and tertiary structures of their deduced proteins and to predict their immunodominant epitopes of B and T cells. A. marginale DNA was detected in 19 bovine samples (2.4%). Risk factor analysis shows that cattle derived from subhumid bioclimatic area were more infected than those that originated from other areas. The analysis of lipA phylogeographic marker indicated a higher diversity of Tunisian A. marginale isolates compared with other available worldwide isolates and strains. Molecular, phylogenetic, and immuno-informatics analyses of the vaccine candidate OmpA protein demonstrated that this antigen and its predicted immunodominant epitopes of B and T cells appear to be highly conserved between Tunisian isolates and compared with isolates from other countries, suggesting that the minimal intraspecific modifications will not affect the potential cross-protective capacity of humoral and cell-mediated immune responses against multiple A. marginale worldwide strains.

Zoonotic vector-borne bacteria in wild rodents and associated ectoparasites from Tunisia.

Wild rodents are considered as potential carriers of several zoonotic vector-borne bacteria but their epidemiology is poorly understood in Tunisia. A total of 305 biological samples (100 spleens, 100 livers, 100 kidneys, and 5 pooled ectoparasites (Xenopsylla cheopis, Laelaps echidninus, Ornithonyssus sp., Hoplopleura sp. and eggs of the rat fleas)) were collected from 100 wild rodents from three Tunisian governorates. Molecular screening was performed to reveal infections with main vector-borne bacteria. Captured rodents belonged to three rodent genera and species including Rattus rattus (n = 51, 51%), Meriones shawi (n = 24, 24%) and Mus musculus (n = 25, 25%). Examined rodents were found to be heavily infested by the rat flea X. cheopis (n = 32, 47%) and the rat mite L. echidninus (n = 22, 32.3%). However, the rat mite Ornithonyssus sp. (n = 13, 19.1%) and the rat lice Hoplopleura sp. (n = 1, 1.5%) were rarely identified. Based on 16S rRNA and msp4 genes, infection with Anaplasmataceae bacteria was detected in six specimens of R. rattus and one M. shawi. Pathogenic A. phagocytophilum (n = 1), A. phagocytophilum-like 1 (Anaplasma sp. Japan) (n = 1), and A. ovis (n = 5) were identified. On the basis of ompB, ompA and gltA genes, infection with Rickettsia spp. was identified in three specimens of R. rattus and one of M. shawi. Five Rickettsia species of the spotted fever group, corresponding to R. monacensis, R. helvetica, R. massiliae, R. africae, and R. aeschlimannii, were detected in mixed infections. Bartonella henselae DNA was also found in two R. rattus, based on rpoB partial sequences. All revealed Anaplasma, Rickettsia and Bartonella bacteria were detected in spleen samples. Ehrlichia, Coxiella and Borrelia spp. were not identified in any of the tested samples. In Tunisia, this is the first report indicating infections with Anaplasma, Rickettsia and Bartonella spp. in wild rodents, particularly present alongside domestic livestock and human. This represents a serious risk of potential bacterial transmission. Thus, controlling rodent population in animal herds, residential areas and sensitizing local people to this risk seem absolutely necessary.

Zoonotic Rickettsia Species in Small Ruminant Ticks From Tunisia.

Tick-borne rickettsioses present a significant public health threat among emerging tick-borne diseases. In Tunisia, little is known about tick-borne Rickettsia pathogens. Therefore, the aim of this study was to investigate the presence of Rickettsia species in small ruminant ticks from Tunisia. Adult ticks (n = 694) were collected from goats and sheep in northern Tunisia. Obtained ticks were identified as Rhipicephalus turanicus (n = 434) and Rhipicephalus sanguineus sensu lato (n = 260). Selected ticks (n = 666) were screened for the presence of Rickettsia spp. by PCR targeting a partial sequence of the ompB gene followed by sequence analysis. Rickettsial DNA was detected in 122 (18.3%) tested tick samples. The infection rates in Rh. turanicus and Rh. sanguineus s.l. ticks were 23.4 and 9.5%, respectively. The overall prevalence of rickettsial DNA was markedly higher in ticks collected from goats (23.2%) compared to those infesting sheep (7.9%). The detection of rickettsial DNA was significantly higher in ticks from the governorate of Beja (39.0%) than those from the governorate of Bizerte (13.9%). Two additional genes, the outer membrane protein A gene (ompA) and the citrate synthase gene (gltA), were also targeted for further characterization of the detected Rickettsia species. Genotyping and phylogenetic analysis based on partial sequences (n = 106) of the three different genes revealed that positive ticks are infected with different isolates of two Spotted Fever Group (SFG) Rickettsia, namely, Rickettsia massiliae and Rickettsia monacensis, closely related to those infecting camels and associated ticks from Tunisia, and humans and small ruminant ticks from neighboring countries like Italy, France, and Spain.

Molecular phylogeny and genetic diversity based on msp1a, groEL and gltA genes of Anaplasma ovis Tunisian isolates compared to available worldwide isolates and strains.

Anaplasma ovis, the causative agent of ovine anaplasmosis in tropical and subtropical countries, is a tick-borne obligatory intraerythrocytic bacterium of sheep, goats and wild ruminants. In Tunisia, data about the molecular phylogeny and the genetic diversity of A. ovis isolates are limited to the analysis of msp4 and groEL genes. The aim of this study was to genetic characterize 40 A. ovis isolates infecting 28 goats, 10 sheep, one camel and one Rhipicephalus turanicus tick located in different geographic regions of Tunisia on the basis of 3 partial genes (gltA, groEL and msp1a). Sequence analysis revealed 6 and 17 different genotypes in the partial gltA and groEL genes, respectively. Phylogenetic analysis revealed, as expected for the groEL gene, that sequences from small ruminants and their infesting ticks clustered separately from those isolated from camels. The analysis of amino-acid Msp1a sequences identified 18 novel genotypes of Msp1a repeats from 20 A. ovis isolates. These Msp1a repeats were highly variable with 33-47 amino-acids, and the number of repeats is one for 19 isolates infecting 18 goats and one R. turanicus tick, and 4 for a single isolate found in one sheep. Phylogenetic trees based on Msp1a partial sequences revealed that the N-terminal region of Msp1a protein appear to be relatively more informative phylogeographically compared to other markers especially according to countries. The presented data give a more detailed knowledge regarding the molecular phylogeny and the genetic diversity of A. ovis isolates occurring in different animal species and their associated ticks in Tunisia.

Molecular epidemiology of Anaplasma spp. related to A. phagocytophilum in Mediterranean small ruminants.

The genus Anaplasma currently comprises 6 bacterial species mostly pathogenic to animals and/or human, including the zoonotic species Anaplasma phagocytophilum, the causative agent of tick-borne fever (TBF) of ruminants, and of granulocytic anaplasmosis of horses, dogs and human. Recently, novel potentially non-pathogenic strains related to A. phagocytophilum have been identified in Japan, China, and Tunisia. This paper reports the identification, molecular typing, and evolutionary history of novel Anaplasma strains (A. phagocytophilum-like 1 and 2), related to but distinct from A. phagocytophilum in Mediterranean area of Europe and Africa. PCR-RFLP and phylogenetic analyses based on 16S rRNA provided evidence for the circulation of A. phagocytophilum-like 1 strains in Europe. Phylogeny based on groEL gene showed the inclusion of Sardinian and Tunisian A. phagocytophilum-like 1 strains in a unique clade distinct from, but related to that of Japanese strains. Results suggest that genetic diversity within the genus Anaplasma is much greater than expected and provide information useful for the development of specific and effective diagnostic and prophylactic tools.

Computational selection of minimum length groESL operon required for Anaplasma species attribution and strain diversity analysis.

Anaplasmosis is a tick-borne rickettsial disease caused by Anaplasma marginale, A. centrale, A. phagocytophilum, A. bovis, A. ovis and A. platys. Understanding the phylogenetic relations among these species is fundamental to perform an accurate identification and an informative intra-specific analysis. Heat shock groESL operon is frequently employed in phylogenetic analysis of Anaplasma species and, for the most cases, the use of partial sequences of this operon is randomly done without knowing the most appropriate regions to be used either in species attribution or in intra-specific diversity analysis. In this study, on the basis of all fully and nearly complete groESL sequences available in the GenBank, we firstly selected a minimum partial length sequence which allows species delineation and gives a similar topology to that found by analyzing the complete sequence. By using other in silico analyses, we obtained two minimal partial sequences that are the most interesting to describe intra-specific diversity within A. ovis and A. centrale. Our results raise concern on the use of randomly selected partial sequences of groESL operon employed for the detection and the characterization of Anaplasma species and provide additional background about minimum length groESL operon required for Anaplasma species attribution and strains diversity analysis.

Molecular detection, genotyping and phylogeny of Anaplasma spp. in Rhipicephalus ticks from Tunisia.

In Tunisia, most of Anaplasma species and unclassified strains have been detected in several animals, but data on the occurrence of Anaplasma spp. in ticks are still lacking. In this study, we report the molecular evidence, genetic characterization and phylogeny of Anaplasma spp. in ticks collected from small ruminants. A total of 395 ticks (178 males and 179 females) were collected from sheep (n = 215) and goats (n = 180). Tick species were identified as 232 Rhipicephalus turanicus, 99 Rhipicephalus sanguineus sensu lato, 34 Rhipicephalus bursa and 30 Rhipicephalus annulatus. Overall infection rate of Anaplasma spp. was 5.6% (20/357 analyzed ticks). All positive ticks were collected from goats and found to be infected by A. ovis. R. turanicus is the most infected tick species by A. ovis (7.9%) followed by R. sanguineus s.l. (2.5%) with an absence of infection in R. bursa and R. annulatus. A. ovis prevalence rate varied significantly according to bioclimatic areas and geographic regions. GroEL typing and phylogenetic analysis revealed that these analyzed ticks are infected with various and novel strains of A. ovis. The use of PCR-RFLP method complemented with sequencing and phylogenetic analysis based on 16S rRNA gene confirm that one R. turanicus tick, positive to A. ovis, is co-infected with A. phagocytophilum-like 2 (0.3%). Specific A. phagocytophilum, A. phagocytophilum-like 1, A. marginale, A. centrale, A. bovis, and A. platys and related strains were not detected in any of the tested ticks. Present data expand knowledge about tick-borne bacteria present in ticks and further clarify the transmission cycles of these bacteria and their different elements in Tunisia.

Genetic characterization of Anaplasma marginale strains from Tunisia using single and multiple gene typing reveals novel variants with an extensive genetic diversity.

Anaplasma marginale, which is responsible for bovine anaplasmosis in tropical and subtropical regions, is a tick-borne obligatory intraerythrocytic bacterium of cattle and wild ruminants. In Tunisia, information about the genetic diversity and the phylogeny of A. marginale strains are limited to the msp4 gene analysis. The purpose of this study is to investigate A. marginale isolates infecting 16 cattle located in different bioclimatic areas of northern Tunisia with single gene analysis and multilocus sequence typing methods on the basis of seven partial genes (dnaA, ftsZ, groEL, lipA, secY, recA and sucB). The single gene analysis confirmed the presence of different and novel heterogenic A. marginale strains infecting cattle from the north of Tunisia. The concatenated sequence analysis showed a phylogeographical resolution at the global level and that most of the Tunisian sequence types (STs) formed a separate cluster from a South African isolate and from all New World isolates and strains. By combining the characteristics of each single locus with those of the multi-loci scheme, these results provide a more detailed understanding on the diversity and the evolution of Tunisian A. marginale strains.

Anaplasma spp. in North Africa: A review on molecular epidemiology, associated risk factors and genetic characteristics.

The genus Anaplasma belonging to the Anaplasmataceae family (order Rickettsiales) comprises obligate intracellular Gram-negative bacteria of veterinary and public health importance. Six species and five types of strains genetically related are currently assigned to the genus Anaplasma including Anaplasma marginale, A. centrale, A. bovis, A. phagocytophilum, A. ovis and A. platys as classified species, and "A. capra", A. odocolei sp. nov., A. phagocytophilum-like 1 (Anaplasma sp.-Japan), A. phagocytophilum-like 2 (Anaplasma sp.-China) and A. platys-like (also named Candidatus Anaplasma camelii) as unclassified strains. Most of these Anaplasma species and strains have been molecularly identified in several animal and/or tick species in the north of Africa. The aim of this review is to summarize the current knowledge about molecular epidemiology, associated risk factors and genetic diversity of Anaplasma species and related strains infecting animals and/or their incriminated tick vectors in North Africa. All these data should be considered when establishing of common management and control programs for anaplasmosis infecting humans and different animal species in North African countries.

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.

Molecular typing and diagnosis of Anaplasma spp. closely related to Anaplasma phagocytophilum in ruminants from Tunisia.

Accurate diagnosis of animal and zoonotic diseases, such as granulocytic anaplasmosis, is crucial to estimate risk during control programs. In this study, 16S rRNA nested PCR and RFLP assay were combined to investigate the presence of Anaplasma phagocytophilum and genetically related strains (namely A. phagocytophilum-like 1 and 2) in 936 Tunisian ruminants. By using this method, A. phagocytophilum was not detected in any of the tested animals, while A. phagocytophilum-like 1 and A. phagocytophilum-like 2 were detected at variable prevalence rates in sheep, goats and cattle at coinfection rates respectively of 3.9, 2.5 and 0.5%. Sequence analysis validated RFLP data, and confirmed the co-occurrence of two potentially novel species closely related to A. phagocytophilum in Tunisian ruminants. Phylogeny indicated the presence of genetic variants shared by different ruminant species for each type of A. phagocytophilum-like strains. Results raise concern on the use and interpretation of indirect and direct tests traditionally employed for detecting pathogenic A. phagocytophilum strains in ruminants and in other vertebrates' species, and provide additional background to improve classification of bacterial species closely related to A. phagocytophilum, and to reconstruct their evolutionary history.

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.

First molecular evidence of [i]Borrelia burgdorferi[/i] 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.