Anaplasma capra in sheep and goats on Corsica Island, France: A European lineage within A. capra clade II?

Anaplasmosis is a tick-transmitted disease due to several species of the genus Anaplasma. In 2019, we demonstrated the presence of Anaplasma capra in two deer species at a zoological park in mainland France. As we suspected its presence in Corsica, we surveyed 11 geographically distant sheep or goat farms. Using molecular tools such as nested PCR targeting 16S ribosomal RNA (rRNA), citrate synthase (gltA) and heat-shock protein (groEL) genes, we detected the presence of A. capra on 5/11 farms, in 26/108 blood samples (24%), in sheep as well as in goats. Genotyping and phylogenetic analysis of A. capra revealed that isolates from Corsica island grouped closely with A. capra isolates reported in red deer and swamp deer from a zoological reserve in mainland France, as well as in roe deer from Spain, in a separate and well supported clade within A. capra clade II. This third report of the tick-borne bacterium A. capra in Europe suggests a potentially larger presence of this pathogen on the European continent, on domestic, native as well as wild ruminants, a broad host range already described in Asian countries for this species.

Human babesiosis in Alsace.

OBJECTIVES: Human babesiosis is a rare parasitic anthropozoonosis transmitted to humans by tick bites. Fifty-six cases of human babesiosis have been recorded in Europe. Two cases of babesiosis were reported in Alsace, France, in 2009. We performed a retrospective observational descriptive study to assess the epidemiology of the disease in Alsace. METHODS: Patients were included if they had a positive serology result for Babesia and/or a positive blood smear and/or a positive PCR result. The tests were performed in the microbiology laboratories of the university hospitals of Strasbourg, the civil hospitals of Colmar, and the hospital of Mulhouse between January 1, 2005 and December 31, 2015. Included patients were divided into three groups: definite case group (positive PCR or positive blood smear or seroconversion), possible case group (positive serology results without seroconversion with a compatible clinical picture and without other confirmed diagnoses), and incompatible case group (positive serology results without seroconversion, without compatible clinical picture and/or with other confirmed diagnoses). The compatible clinical picture was defined by the presence of flu-like symptoms and fever (≥38°C). RESULTS: Fifty-one patients had at least one positive result. Three cases were excluded (missing files). There were six definite cases, 12 possible cases, and 30 incompatible cases. All patients in the definite case group were immunocompetent. No deaths occurred. CONCLUSIONS: Human babesiosis is probably underdiagnosed due to its non-specific symptoms, lack of awareness about the disease, and the difficulty in making a diagnosis.

Low prevalence of zoonotic Babesia in small mammals and Ixodes ricinus in Brittany, France.

In order to evaluate the zoonotic risk due to Babesia spp., especially B. microti, we investigated their presence in 597 individuals of five small mammal species and in 2620 questing nymphs of Ixodes ricinus in rural landscapes of Western France (Brittany). Small mammals (rodents and shrews) are indeed suspected to be reservoir hosts for B. microti, and the tick I. ricinus is the vector of the three main zoonotic species in Europe, i.e. B. divergens, B. venatorum and B. microti. Only one bank vole carried B. microti (genotype "Munich") and only 13 and 2 nymphs of Ixodes ricinus ticks carried B. venatorum and B. capreoli respectively. According to these results, prevalences observed for zoonotic Babesia (0.17% for small mammals and 0.50% for ticks), indicate that exposure of humans to this infectious agent is probably low in western France.

Babesia divergens apical membrane antigen-1 (BdAMA-1): A poorly polymorphic protein that induces a weak and late immune response.

Babesiosis is an important veterinary and zoonotic tick borne disease caused by the hemoprotozoan Babesia spp. which infects red blood cell of its vertebrate host. In order to control the infection, vaccination that targets molecules involved in the invasion process of red blood cells could provide a good alternative to chemotherapy. Among these molecules, Apical Membrane Antigen-1 (AMA-1) has been described as an excellent vaccine candidate in Plasmodium spp. In this paper, we have investigated AMA-1 of Babesia divergens (BdAMA-1) as vaccine candidate by evaluating its polymorphism and by studying the humoral response against BdAMA-1 of sheep experimentally infected with B. divergens. Polymorphism of BdAMA-1 was investigated by sequencing the corresponding gene of 9 B. divergens isolates from different geographical areas in France. Two Bdama-1 haplotypes (A and B) could be defined based on 2 non-synonymous point mutations. In silico prediction of linear epitopes revealed that the antigenicity of the 2 haplotypes is very similar. Antibody production against the extracellular domain of BdAMA-1 is weak and late, between 1 and 5 months after the inoculation of parasites. Both IgG1 and IgG2 are components of the anti-BdAMA-1 response. These results indicate that while BdAMA-1 may not be an immuno-dominant antigen, it could induce a mixed type 1 and type 2 immune response. In light of these results, the potential of BdAMA-1 as vaccine candidate is discussed.

A retrospective serological survey on human babesiosis in Belgium.

In Europe, most clinical babesiosis cases in humans have been attributed to Babesia divergens and Babesia sp. EU1. Babesia microti infection of humans occurs mainly in the United States; although a case of autochthonous B. microti infection and serological evidence of infection have been reported in Europe. The Indirect Fluorescent Antibody Test was used to screen sera from 199 anonymous Belgian patients with history of tick bite and clinical symptoms compatible with a tick-borne disease. The serological screen detected positive reactivity in 9% (n = 18), 33.2% (n = 66), and 39.7% (n = 79) of the samples against B. microti, B. divergens, and Babesia sp. EU1, respectively. Thus, evidence of contact among three potentially zoonotic species of Babesia and humans has been confirmed in Belgium. Preventive action and development of better diagnostic tools should help in prevention of clinical cases and to clarify the true burden of such infection for individuals and public health.

Identification of three CCp genes in Babesia divergens: novel markers for sexual stages parasites.

Babesia divergens, a tick-borne protozoan parasite of red blood cells, is the main agent of bovine and human babesiosis in Europe. Very few data are available concerning its life cycle and sexual reproduction inside the tick vector, Ixodes ricinus. The aim of this study was to define some markers of the B.divergens sexual stage. An in silico post-genomic approach was used to analyze genomic, transcriptomic and proteomic data and to select specific sexual stage proteins of the related apicomplexan genus Plasmodium. Three proteins, based on sequence identity between the available genomes of Plasmodium and Babesia spp., were chosen, as members of a highly conserved and apicomplexan sexual stages specific protein family (CCp) potentially involved in adhesive functions. Degenerate primers were used to amplify and clone three B.divergens orthologs (bdccp1, bdccp2, and bdccp3) corresponding to newly identified genes in this parasite. The opportunities offered by such markers to study parasite development in its vector are discussed.

Transstadial and transovarial persistence of Babesia divergens DNA in Ixodes ricinus ticks fed on infected blood in a new skin-feeding technique.

Although Babesia divergens is the the principal confirmed zoonotic Babesia sp. in Europe, there are gaps in our knowledge of its biology and transmission by the tick Ixodes ricinus. In order to reproduce the part of the parasite cycle that occurs in the vector, an in vitro animal skin feeding technique on blood containing in vitro cultivated B. divergens was developed. Parasite DNA was detected in all samples of salivary glands of nymphs and adults that had fed on parasitized blood as larvae and nymphs, respectively, indicating acquisition as well as a transtadial persistence of B. divergens. PCR performed on eggs and larvae produced by females that had fed on parasitized blood demonstrated the existence of a transovarial transmission of the parasite. Gorging B. divergens infected larvae on non-infected gerbils showed persistance of the parasite over moulting into the resulting nymphs. These results indicate that the parasitic stages infective for the vector (i.e. the sexual stages) can be produced in vitro. To our knowledge, this is the first report of artificial feeding of I. ricinus via membrane as well as in vitro transmission of B. divergens to its vector. The opportunities offered by the use of such a transmission model of a pathogen by I. ricinus are discussed.

Cell cycle arrest in archaea by the hypusination inhibitor N(1)-guanyl-1,7-diaminoheptane.

Hypusination is an essential posttranslational modification unique to archaeal and eukaryotic protein synthesis initiation factor 5A (aIF5A and eIF5A, respectively). We have investigated the effect of the efficient hypusination inhibitor N(1)-guanyl-1,7-diaminoheptane (GC(7)) on four archaeal and one bacterial species. We found that (i) archaea are sensitive to GC(7), whereas the bacterium Escherichia coli is not, (ii) GC(7) causes rapid and reversible arrest of growth of the archaeon Sulfolobus acidocaldarius, and (iii) the growth arrest is accompanied by a specific reversible arrest of the cell cycle prior to cell division. Our findings establish a link between hypusination and sustained growth of archaea and thereby provide the framework to study molecular details of archaeal cell cycle in connection with in vivo functions of hypusine and of aIF5A and eIF5A.

Nucleoid structure and partition in Methanococcus jannaschii: an archaeon with multiple copies of the chromosome.

We measured different cellular parameters in the methanogenic archaeon Methanococcus jannaschii. In exponential growth phase, the cells contained multiple chromosomes and displayed a broad variation in size and DNA content. In most cells, the nucleoids were organized into a thread-like network, although less complex structures also were observed. During entry into stationary phase, chromosome replication continued to termination while no new rounds were initiated: the cells ended up with one to five chromosomes per cell with no apparent preference for any given DNA content. Most cells in stationary phase contained more than one genome equivalent. Asymmetric divisions were detected in stationary phase, and the nucleoids were found to be significantly more compact than in exponential phase.

Serological and molecular size characterization of flagellins of Pseudomonas syringae pathovars and related bacteria

Flagella from a total of 118 strains representing mostly pathovars of the phytopathogenic group Pseudomonas syringae, but also P. chlororaphis, P. cichorii, P. corrugata, P. fluorescens, P. fuscovaginae, P. stutzeri, P. viridiflava, as well as related phytopathogenic genera (Burkholderia cepacia and Ralstonia solanacearum) were characterized by immuno-fluorescent staining, SDS-PAGE, and immunoblotting. Eighty-six strains of the P. syringae group pathovars, P. cichorii and P. viridiflava were shown to possess flagella of serotypes H1 or H2, composed of a unique flagellin, whose molecular size varied between 31 and 31.5 kDa. Similarities between the P. syringae flagellin and a 31 kDa surface protein involved in pathogenicity are pointed out. The distribution of H1 and H2 antigens in the nine recently described genomospecies of P. syringae-P. viridiflava group suggested that flagellin would represent a phylogenetic marker within the arginin-dihydrolase-negative fluorescent pseudomonads. The characterization of flagellin was proposed as an identification tool at a level situated between genus and species.

DspA, an essential pathogenicity factor of Erwinia amylovora showing homology with AvrE of Pseudomonas syringae, is secreted via the Hrp secretion pathway in a DspB-dependent way.

In Erwinia amylovora, the dsp region, required for pathogenicity on the host plant but not for hypersensitive elicitation on tobacco, is separated from the hrp region by 4 kb. The genetic analysis reported in this paper showed that this 4kb region is not required for pathogenicity on pear seedlings. The environmental conditions allowing expression of a dsp::lacZ fusion were examined: expression was barely detected in rich medium at 30 degrees C, and the highest expression was observed in M9 galactose minimal medium at 25 degrees C. A dsp::uidA fusion appeared to be expressed only in a HrpL-proficient strain, indicating that the dsp region, like the hrp region, is positively controlled via the alternative a factor HrpL. Sequence analysis revealed that the dsp cluster encodes two genes, dspA (5517 bp) and dspB (420 bp), and that the insertions leading to the dsp::lacZ and the dsp::uidA fusions were within dspA. A HrpL-dependent promoter sequence (GGAACC-N15-CAACA) was identified upstream of dspA, and primer extension analysis detected four transcriptional starts 7, 8, 9 and 10 bp downstream of this sequence. A sigma70 promoter sequence (TTGCCC-N16-GATAAT) was observed upstream of dspB. The functionality of this second promoter was confirmed by complementation analysis. This promoter allowed constitutive expression of dspB, as measured by the expression of a dspB::uidA fusion in rich medium. In M9 galactose medium, however, HrpL was shown to activate dspB, as expression of the dspB::uidA fusion was twofold higher in a HrpL+ background than in a HrpL- background. Transposon insertions in either dspA or dspB led to a non-pathogenic phenotype. Thus, both DspA and DspB were required for E. amylovora pathogenicity, as dspB could be expressed independently of dspA. DspA and DspB were visualized as polypeptides with apparent sizes of 190 kDa and 15.5 kDa, respectively, when encoded in the T7 polymerase/promoter system. DspA, which showed homology with the protein predicted from the partial sequence of Pseudomonas syringae pv. tomato avrE transcriptional unit III, was shown to be secreted into the external medium via the Hrp secretion pathway. DspB was predicted to be acidic, like the Syc chaperone of Yersinia. A chaperone role for DspB was suggested further by the fact that DspA secretion required a functional DspB protein.

Distribution of Pseudomonas syringae pathovars into twenty-three O serogroups.

Serological reactions of Pseudomonas syringae and Pseudomonas viridiflava were studied by Ouchterlony double diffusion. A total of 55 polyclonal antisera, containing anti-lipopolysaccharide (anti-LPS) precipitating antibodies, were cross-tested against antigenic suspensions of 51 strains. Twenty-three O serogroups were defined, primarily on the reaction of the type strains. Two families of O serogroups showed antigenic crossreactivities (PHA, MOP1, MOP2, MOP3, HEL1, HEL2, and SYR1; PERSAVTOM1, PERSAVTOM2, DEL, POR, and SYR2). Ten O serogroups showed a clearcut specificity: APTPIS, TAB, VIR1, VIR2, VIR3, SYR3, SYR4, SYR5, HUS, and LAC. The last serogroup (RIB) contained strains with rough colony morphology and side chain-deficient LPSs, as evidenced by sodium dodecyl sulfate-polycrylamide gel electrophoresis. The LPS basis of the O serogroups was demonstrated by immunoblotting. Serological reference strains were designated for all of the O serogroups and correspondence was established between the O serogroups studied and seven previous serogroups (L. T. Pastushenko and I.D. Simonovich, Mikrobiol, Zh. 41:222-229 and 330-339, 1979). A total of 355 strains of P. syringae (sensu lato) belonging to 15 pathovars, not including pathovar syringae, were typed into the 23 described O serogroups. O serogroups were assigned after double-diffusion reactions, with each strain compared with serological references. The utility of O serogrouping to study P. syringae pathovar structure and diversity is discussed.