Ali Bouattour

Detection and Identification of Ehrlichia spp. in Ticks Collected in Tunisia and Morocco

ABSTRACT A broad-range 16S rRNA gene PCR assay followed by partial sequencing of the 16S rRNA gene was used for the detection of members of the family Anaplasmataceae in ticks in North Africa. A total of 418 questing Ixodes ricinus ticks collected in Tunisia and Morocco, as well as 188 Rhipicephalus ticks from dogs and 52 Hyalomma ticks from bovines in Tunisia, were included in this study. Of 324 adult I. ricinus ticks, 16.3% were positive for Ehrlichia spp., whereas only 3.4 and 2.8% of nymphs and larvae, respectively, were positive. A large heterogeneity was observed in the nucleotide sequences. Partial sequences identical to that of the agent of human granulocytic ehrlichiosis (HGE) were detected in I. ricinus and Hyalomma detritum, whereas partial sequences identical to that of Anaplasma platys were detected in Rhipicephalus sanguineus. However, variants of Anaplasma, provisionally designated Anaplasma-like, were predominant in the I. ricinus tick population in Maghreb. Otherwise, two variants of the genus Ehrlichia were detected in I. ricinus and H. detritum. Surprisingly, a variant of Wolbachia pipientis was evidenced from I. ricinus in Morocco. These results emphasized the potential risk of tick bites for human and animal populations in North Africa.

Culex pipiens, an experimental efficient vector of West Nile and Rift Valley fever viruses in the Maghreb region.

West Nile fever (WNF) and Rift Valley fever (RVF) are emerging diseases causing epidemics outside their natural range of distribution. West Nile virus (WNV) circulates widely and harmlessly in the old world among birds as amplifying hosts, and horses and humans as accidental dead-end hosts. Rift Valley fever virus (RVFV) re-emerges periodically in Africa causing massive outbreaks. In the Maghreb, eco-climatic and entomologic conditions are favourable for WNV and RVFV emergence. Both viruses are transmitted by mosquitoes belonging to the Culex pipiens complex. We evaluated the ability of different populations of Cx. pipiens from North Africa to transmit WNV and the avirulent RVFV Clone 13 strain. Mosquitoes collected in Algeria, Morocco, and Tunisia during the summer 2010 were experimentally infected with WNV and RVFV Clone 13 strain at titers of 107.8 and 108.5 plaque forming units/mL, respectively. Disseminated infection and transmission rates were estimated 14–21 days following the exposure to the infectious blood-meal. We show that 14 days after exposure to WNV, all mosquito st developed a high disseminated infection and were able to excrete infectious saliva. However, only 69.2% of mosquito strains developed a disseminated infection with RVFV Clone 13 strain, and among them, 77.8% were able to deliver virus through saliva. Thus, Cx. pipiens from the Maghreb are efficient experimental vectors to transmit WNV and to a lesser extent, RVFV Clone 13 strain. The epidemiologic importance of our findings should be considered in the light of other parameters related to mosquito ecology and biology.

Reservoir Role of Lizard Psammodromus algirus in Transmission Cycle of Borrelia burgdorferi Sensu Lato (Spirochaetaceae) in Tunisia

Abstract To investigate the reservoir role of the lizard Psammodromus algirus for the Lyme disease spirochete, 199 lizards were trapped from April to October 2003 in El Jouza, northwestern Tunisia. In this site, the infection rate of free-living Ixodes ricinus (L.) by Borrelia was evaluated by immunofluorescence as 34.6% for adult ticks and 12.5% for nymphs. Eighty percent of P. algirus (117/146) captured during this study were infested by I. ricinus, the predominant tick species collected from lizards. The intensity of tick infestation of this host by larvae and nymphs ranged from 0.14 to 7.07 and from 1.5 to 6.58, respectively. These immature stages of I. ricinus were found on lizards in spring and the beginning of summer, with a peak of intensity during June (10.16 immature ticks by lizard). Tissue cultures from lizards and xenodiagnosis with larval I. ricinus were used to assess the infection and the ability, respectively, of infected lizards to transmit Borrelia to naive ticks. Seventeen percent of xenodiagnostic ticks (40/229) acquired B. lusitaniae while feeding on P. algirus. Therefore, we demonstrated the ability of the lizards to sustain Borrelia infection and to infect attached ticks, and we proved that P. algirus is a reservoir host competent to transmit B. lusitaniae.

Clinical, serological, and molecular evidence of ehrlichiosis and anaplasmosis in dogs in Tunisia.

A seroepidemiological survey was conducted in five bioclimatic areas of Tunisia to determine the prevalence of antibodies to Ehrlichia canis and Anaplasma phagocytophilum antigens, surrogate markers of the agents of canine monocytic ehrlichiosis (CME) and canine granulocytic ehrlichiosis, respectively. Among 286 collected sera, 54.2% and 25.2% were seropositive for E. canis and A. phagocytophilum, respectively, by the indirect immunofluorescence antibody (IFA) test. Clinical and hematological tests were done only for 58 sick dogs from Tunis area. A reverse line blot (RLB) hybridization was then used to identify isolated Ehrlichia and Anaplasma species infecting dogs (n = 228). Among them, only two dogs were infected by A. phagocytophilum; ten sample dogs were demonstrated infected by E. canis and ten infected by Ehrlichia sp., from which one dog showed a mixed infection with A. phagocytophilum and E. canis and one with A. phagocytophilum and Ehrlichia sp. RLB findings were confirmed by sequencing; BLAST search against GenBank revealed high similarity of the sequence of Ehrlichia sp. PCR/RLB amplicons with Anaplasma platys 16S rRNA partial sequence.

A preliminary study on the attenuation of Tunisian schizont-infected cell lines of Theileria annulata

Abstract Four Theileria annulata cell lines were characterised at low passage levels using two polymorphic markers and then used to infect calves. Their virulence seemed to be related to the number of genotypes present within the cell line. In all, 3 of the 4 cell lines were cultured up to passage 100 or 200 and inoculated into calves. Their characterisation using the same markers indicated that the attenuation was related to a reduction in the parasite polymorphism down to a single genotype. The immunogenicity of the three attenuated cell lines was assessed in calves using two types of challenge. Optimal protection was observed against homologous challenges. The level of immunity to heterologous challenges appeared to decrease with attenuation and seemed to depend on the cell line used.

The epidemiology and geographic distribution of relapsing fever borreliosis in West and North Africa, with a review of the Ornithodoros erraticus complex (Acari: Ixodida).

Background Relapsing fever is the most frequent bacterial disease in Africa. Four main vector / pathogen complexes are classically recognized, with the louse Pediculus humanus acting as vector for B. recurrentis and the soft ticks Ornithodoros sonrai, O. erraticus and O. moubata acting as vectors for Borrelia crocidurae, B. hispanica and B. duttonii, respectively. Our aim was to investigate the epidemiology of the disease in West, North and Central Africa. Methods And Findings From 2002 to 2012, we conducted field surveys in 17 African countries and in Spain. We investigated the occurrence of Ornithodoros ticks in rodent burrows in 282 study sites. We collected 1,629 small mammals that may act as reservoir for Borrelia infections. Using molecular methods we studied genetic diversity among Ornithodoros ticks and Borrelia infections in ticks and small mammals. Of 9,870 burrows investigated, 1,196 (12.1%) were inhabited by Ornithodoros ticks. In West Africa, the southern and eastern limits of the vectors and Borrelia infections in ticks and small mammals were 13°N and 01°E, respectively. Molecular studies revealed the occurrence of nine different Ornithodoros species, including five species new for science, with six of them harboring Borrelia infections. Only B. crocidurae was found in West Africa and three Borrelia species were identified in North Africa: B. crocidurae, B. hispanica, and B. merionesi. Conclusions Borrelia Spirochetes responsible for relapsing fever in humans are highly prevalent both in Ornithodoros ticks and small mammals in North and West Africa but Ornithodoros ticks seem absent south of 13°N and small mammals are not infected in these regions. The number of Ornithodoros species acting as vector of relapsing fever is much higher than previously known.

High prevalence of Borrelia lusitaniae in Ixodes ricinus ticks in Tunisia

To investigate whether ticks of the genus Ixodes are infected by Borrelia burgdorferi complex, 490 unfed Ixodes ricinus ticks were collected by flagging in three different areas of Tunisia in 1998. DNAs extracted from 81 adults, 60 nymphs and 38 larvae were analysed after genic amplification of the noncoding spacer between the two copies of the rrl–rrf genes of B. burgdorferi sl. The prevalence of B. burgdorferi sl. in adults, nymphs and larvae was found to be 34, 33.3 and 2.6%, respectively. All DNAs (n = 61) but one were identified as belonging to different genotypes of B. lusitaniae by analysis of the restriction fragment length polymorphism of amplification products. In addition, 290 adults, 14 nymphs and 7 larvae were used to inoculate BSK-H medium to isolate spirochetes. Fifteen strains were isolated from adult ticks in the humid areas of Tunisia, whereas only one was obtained from larvae. Isolates were identified as B. lusitaniae (15/16) and B. garinii (1/16). These results provide new evidence for the existence of Lyme borreliosis in North Africa.

First serological investigation of peste-des-petits-ruminants and Rift Valley fever in Tunisia.

This study, carried out between September 2006 and January 2007, is the first cross-sectional serological investigation of peste-des-petits-ruminants (PPR) and Rift Valley fever (RVF) in Tunisia. The objective was to assess the potential need to develop a dual, recombinant PPR-RVF vaccine and how such a vaccine might be utilised in Tunisia. An overall PPR seroprevalence of 7.45% was determined, a finding supported by the high specificity (99.4%) and sensitivity (94.5%) of the ELISA used. On assessment of the diversity and density of mosquitoes in the sampling area, four species of RVF-vectors of the genus Aedes and Culex were identified. However, no serological evidence of RVF was found despite the use of a highly sensitive ELISA (99-100%). Larger scale investigations are underway to confirm these findings and the continuation of the emergency vaccination program against these two diseases remains valid.

Borrelia hispanica Relapsing Fever, Morocco

We found that 20.5% of patients with an unexplained fever in northwestern Morocco had tick-borne relapsing fever. Molecular detection specific for the 16S rRNA gene identified Borrelia hispanica. The noncoding intergenic spacer sequence domain showed high sensitivity and good resolution for this species.

Cattle infestation by Hyalomma ticks and prevalence of Theileria in H. detritum species in Tunisia.

Seventy-four cattle, from three farms endemic for tropical theileriosis in the north of Tunisia, were studied for tick populations from June 1991 to June 1992. Ticks were removed from cattle twice a month in the summer and every month the rest of the year. They were identified and assessed for Theileria infection. A total of 5083 Hyalomma adult ticks were collected and the major species found was H. detritum (84.3%). The activity of this species is limited between June and August with a peak in numbers observed at the end of June and the beginning of July. Amongst the 2356 Hyalomma ticks dissected, no evidence of salivary gland infection was found in either H. m. marginatum or H. a. excavatum. However, 12.4% (277/2230) of H. d. detritum dissected ticks were infected with Theileria species and amongst these, 62% had one to two sporoblasts in their salivary glands (range 1-91). The prevalence, but not the intensity, of infection was greater in females than in male ticks, and the cases of tropical theileriosis followed the peak of infected females. This suggests that female ticks have a more important role in theileriosis transmission than male ticks. A significantly lower number of adult H. detritum were collected from calves, at their first tick season, than from adult cattle. Finally, this study showed that the infestation level of cattle by H. d. detritum and the prevalence of Theileria-infection in these cattle varied between the three farms studied.