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Giant viruses of the Megavirales order have been recently isolated from aquatic environments and have long been neglected because they are removed from samples during viral purification for viral metagenomic studies. Due to bacterial overgrowth and susceptibility to high concentrations of antibiotics, isolation by amoeba co-culture has a low efficiency and is highly time-consuming. Thus, few environments have been exhaustively investigated to date, although the ubiquitous distribution of the Acanthamoeba sp. suggests that these viruses could also be ubiquitous. In this work, we have implemented a high-throughput method to detect amoebae lysis on agar plates that allows the testing of hundreds of samples in a few days. Using this procedure, a total of 11 new Marseilleviridae strains and four new Mimiviridae strains, including a virus infected with a virophage, were isolated from 1000 environmental samples from Tunisia. Of these, four corresponded to new genotypic variants. These isolates are the first African environmental isolates identified from these two families, and several samples were obtained from a hypersaline aquatic environment. These results demonstrate that this technique can be used for the evaluation and characterization of large collections of giant viruses to provide insight into understanding their ecology. [ABSTRACT FROM AUTHOR]

Background. Travelers are exposed to a variety of health risks in unfamiliar environments and fever is a common problem in patients returning from travel abroad. Rickettsial diseases are increasingly frequently being reported among international travelers. Here we present cases of Rickettsia typhi infection, the agent of murine typhus, that were identified in our laboratory the last year, in travelers from Tunisia. Methods. For each patient we tested an acute-phase serum sample and for one patient we tested a convalescent-phase serum sample. IgG and IgM antibody titers were estimated with use of the microimmunofluorescence (MIF) assay. Western blot (WB) assay was performed for all the patients. Results. We identified three cases of murine typhus after a travel in Tunisia. All cases were observed during late summer and early autumn and patients were suffering by persistent fever. None of them presented rash or inoculation eschar. MIF was positive for Rickettsia sp. in the acute-phase serum samples of two patients. In one patient, two acute-phase serum samples were Rickettsia sp. negative whereas a third convalescent-phase serum sample that was obtained 2 weeks after was Rickettsia sp. positive. By WB assay we identified infection by R typhi. A treatment was immediately started and patients became apyretic. Conclusions. In the countries of North Europe, although autochthones cases of murine typhus have not been described, sporadic cases of R typhi infection are identified in travelers who visited murine typhus endemic areas. Murine typhus should be considered in the diagnosis of febrile illness without rash in travelers returning from disease endemic areas, like the south Mediterranean area. [ABSTRACT FROM AUTHOR]

Rickettsia species are increasingly being recognized as a cause of infection among returning travelers. Murine typhus (MT) was mistakenly thought to have disappeared in the 1970s in Tunisia, yet recent serological data show that Rickettsia typhi, the causative agent of MT, still circulates in the Tunisian population. We report here a case of MT in a woman returning from Tunisia and hospitalized in France. Her presentation was nonspecific, with acute noneruptive fever. Diagnosis was confirmed by cross-adsorption and immunoblotting. Clinicians taking care of returning travelers with fever should be aware of MT, and know how to diagnose and treat it. [ABSTRACT FROM AUTHOR]

We report, for the first time, serologic evidence of Rickettsia felis and R. aeschlimannii infections acquired in Tunisia from 1998 to 2003. We found that most patients with antibodies against both R. conorii and R. typhi had serologic evidence of R. felis infection. [ABSTRACT FROM AUTHOR]

Objectives: Q fever has been reported throughout the African continent. The objective of this study was to detect the presence of Coxiella burnetii in febrile patients from Africa., Methods: Blood samples from febrile and non-febrile patients from six African countries and from France were investigated retrospectively for Q fever infection by molecular assays targeting the IS1111 and IS30A spacers., Results: We tested 1888 febrile patients from Senegal, Mali, Tunisia, Algeria, Gabon, and Morocco and found one male adult patient (0.3%) infected with C. burnetii in Algeria and six positive patients (0.5%) in Senegal. For one patient from Senegal we determined that the infection was caused by C. burnetii genotype 35. In Senegal, more patients were infected with C. burnetii in Keur Momar Sarr (p=0.002) than in the other locations. Blood samples taken from 500 (51% males) non-febrile people from Senegal and France were all negative., Conclusions: The installation of point-of-care laboratories in rural Africa can be a very effective tool for studying the epidemiology of many infectious diseases.

Marseillevirus is the founding member of the proposed family Marseilleviridae, which is the second discovered family of giant viruses that infect amoebae. These viruses have been recovered from environmental water samples and, more recently, from humans. Tunisvirus was isolated from fountain water in Tunis, Tunisia, by culturing on Acanthamoeba spp. and is a new marseillevirus. We describe here its 380,011 base-pair genome. A total of 484 proteins were identified, among which 320 and 358 have an ortholog in Marseillevirus and Lausannevirus (e-value<1e-2), respectively, and 259 and 299 have best reciprocal hits with a Marseillevirus and a Lausannevirus protein, respectively. In addition, a significant hit was found in organisms other than marseilleviruses for 144 Tunisvirus proteins, indicating extensive lateral gene transfers, as has been demonstrated previously for Marseillevirus. Finally, a total of 21 ORFans were identified. Phylogeny reconstructions and analysis of the gene repertoires of marseilleviruses, including the proportion of orthologs and the mean amino acid identity between genes in pairs, suggest that the proposed family Marseilleviridae encompasses three lineages. Lineage A is composed of Marseillevirus, Cannes 8 virus and Senegalvirus; lineage B is represented by Lausannevirus alone; and lineage C has Tunisvirus as its first member. Taken together, these findings suggest that the marseilleviruses display a substantial level of diversity.

Giant viruses and amoebae are common in freshwater, where they can coexist with other living multicellular organisms. We screened leeches from the species Hirudo medicinalis for giant viruses. We analyzed five H. medicinalis obtained from Tunisia (3) and France (2). The leeches were decontaminated and then dissected to remove internal parts for co-culture with Acanthamoeba polyphaga. The genomes of isolated viruses were sequenced on a 454 Roche instrument, and a comparative genomics analysis was performed. One Mimivirus was isolated and the strain was named Hirudovirus. The genome assembly generated two scaffolds, which were 1,155,382 and 25,660 base pairs in length. Functional annotations were identified for 47% of the genes, which corresponds to 466 proteins. The presence of Mimividae in the same ecological niche as wild Hirudo may explain the presence of the mimivirus in the digestive tract of the leech, and several studies have already shown that viruses can persist in the digestive tracts of leeches fed contaminated blood. As leeches can be used medically and Mimiviruses have the potential to be an infectious agent in humans, patients treated with leeches should be surveyed to investigate a possible connection.

Objective: Giant viruses and amoebae are common in freshwater, where they can coexist with various insects. We screened insect larvae to detect giant viruses using a high-throughput method., Methods: We analyzed 86 Eristalis tenax larvae obtained from stagnant water reservoirs in Tunisia. The larvae were decontaminated and then dissected to remove internal parts for coculture with Acanthamoeba polyphaga. Genome sequencing of isolated viruses was performed on a 454 Roche instrument, and comparative genomics were performed., Results: One Marseillevirus, named Insectomime virus, was isolated. The genome assembly generated two scaffolds, which were 382,776 and 3,855 bp in length. Among the 477 identified predicted proteins, the best hit for 435 of the identified proteins was a Marseillevirus or Lausannevirus protein. Tunisvirus was the most closely related to Insectomime, with 446 orthologs. One Insectomime protein shared with Lausannevirus and Tunisvirus showed the highest similarity with a protein from an aphid., Conclusion: The isolation of a Marseillevirus from an insect expands the diversity of environments in which giant viruses have been isolated. The coexistence of larvae and giant viruses in stagnant water may explain the presence of the giant virus in the larva internal structures. This study illustrates the putative role of amoeba in lateral gene transfer not only between the organisms it phagocytoses, but also between organisms living in the same environment. © 2013 S. Karger AG, Basel.

Two isolates of intra-amoeba-growing bacteria, LegA(T) ( = DSM 24804(T) = CSUR P146(T)) and LegM(T) ( = DSM 24805(T) = CSUR P145(T)), were characterized on the basis of microscopic appearance, staining characteristics, axenic growth at different temperatures and the sequences of the mip, rpoB, 16S rRNA and rnpb genes, as well as the 23S-5S region. Phylogenetic analysis showed that these two isolates lay within the radius of the family Legionellaceae. Furthermore, the analysis of these genes yielded congruent data that indicated that, although strain LegM(T) clusters specifically with Legionella feeleii ATCC 35072(T) and LegA(T) clusters with Legionella nautarum ATCC 49596(T), the divergence observed between these species was greater than that observed between other members of the family. Taken together, these results support the proposal that these two isolates represent novel members of the genus Legionella, and we propose to name them Legionella tunisiensis sp. nov. for LegM(T) ( = DSM 24805(T) = CSUR P145(T)) and Legionella massiliensis sp. nov. for LegA(T) ( = DSM 24804(T) = CSUR P146(T)).

Murine typhus, caused by Rickettsia typhi and transmitted mainly by the rat fleas, Xenopsylla cheopis, has emerged in the field of travel medicine. We analyzed retrospectively the epidemiological, clinical, and biological characteristics of the 32 murine typhus cases that were diagnosed during the past 3 years at the World Health Organization Collaborative Center for Rickettsial diseases, Marseille, France. All of the cases occurred in travelers and most of them had returned from Africa (N = 13 of 32) and South-east Asia (N = 12 of 32). Exposure to rats was reported only in a few (N = 2 of 32) patients. Almost half of the cases were diagnosed in August and September. Only four patients presented the classic triad: fever, rash, and headache. Moreover, we report the first known cases of a hemophagocytic syndrome associated with this disease. Murine typhus must be considered as an etiologic agent of febrile illness in returning travelers, particularly in those with unspecific symptoms.

Bartonella quintana is a fastidious microorganism associated with blood culture negative endocarditis. In this study, 40 sera with cross-reactivity between Chlamydia species from patients from Sfax, Tunisia, were serologically tested for Bartonella. Thirteen sera were positive for Bartonella with IgG titers >/=1:800. Western blot and cross-absorption confirmed the diagnosis of Bartonella quintana endocarditis in 12 cases and Bartonella henselae endocarditis in 1 case. These sera were also positive by LightCycler nested PCR amplification for the rnpb (7 of 13) and fur (11 of 13) genes. Eleven patients had a definite diagnosis of endocarditis, which represents 9.8% of all endocarditis. Because Bartonella endocarditis seems to be very common in Tunisia, we suggest that its serology be performed systematically whenever endocarditis is suspected.