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Abstract Background African animal trypanosomosis is a major constraint to the rearing of productive livestock in the sub-humid Sudan-Sahel zone of West Africa where cotton is grown. Trypanosomosis is mainly controlled using trypanocidal drugs, but the effective use of drugs is threatened by the development of widespread resistance. This study tested integrated best-bet strategies for containment and/ or reversal of trypanocide resistance in villages in south-east Mali where resistance has been reported. Methods Four sentinel villages each from an intervention area (along the road from Mali to Burkina Faso) and a control area (along the road from Mali to Côte d’Ivoire) were selected for the study. Tsetse control was based on deltamethrin-treated stationary attractive devices and targeted cattle spraying between March 2008 and November 2009. Trypanosome-positive cattle were selectively treated with 3.5 mg/kg diminazene aceturate. Strategic helminth control using 10 mg/kg albendazole was also undertaken. During the intervention, tsetse densities along drainage lines, trypanosome infections and faecal egg counts in risk cattle (3 to 12 months of age) were monitored. Results Catch reductions of 66.5 % in Glossina palpalis gambiensis and 90 % in G. tachinoides were observed in the intervention area. Trypanosome prevalence was significantly (p 0.05). Further studies are required before confirming the existence of albendazole resistant strongyles in the study area. Conclusion Integration of best-bet strategies in areas of multiple drug-resistance is expected to reduce trypanosome infection risk thus contributing to containment of trypanocidal drug resistance. Integrated best-bet strategies could therefore be considered a viable trypanosomosis control option especially in areas where multiple drug-resistance has been reported.

Abstract Background Tsetse fly-transmitted African animal trypanosomosis causes annual losses that run into billions of dollars. The disease is assumed to cause hunger and poverty in most sub-Saharan countries since it represents a serious impediment to sustainable livestock production. Both a cross-sectional and a longitudinal study were carried out from November to December 2007 to evaluate trypanosomosis risk and susceptibility of trypanosomes to trypanocidal drug treatment in village cattle populations in south-east Mali. Methods Eight purposively selected villages participated in the study. In each village, eight traps deployed along drainage lines over 24hour duration were used to catch tsetse. One hundred systematically selected cattle in the study villages were examined for trypanosomes. All trypanosome-positive cattle were randomly allocated into two treatment groups: a group treated with 0.5 mg/kg bw. isometamidium chloride (ISMM) and a group treated with 3.5 mg/kg bw. diminazene aceturate (DIM). The cattle were monitored for trypanosomes at day 14 and 28 post-treatment. Results Of the 796 cattle examined, 125 (15.7%) were trypanosome-positive. Village trypanosome prevalences ranged between 11% and 19%. There were no significant (p > 0.05) differences in the village trypanosome prevalences. Trypanosoma congolense was the dominant trypanosome species accounting for 73% (91/125) of the infections and T. vivax the remainder. Twenty (31.7%) of the 63 cattle on 0.5 mg/kg bw. ISMM treatment were still positive14 days post-treatment. Of the 43 aparasitaemic cattle monitored to day 28, 25.6% (11) became parasitaemic, resulting in a cumulative failure rate of 49.2% (31/63). Trypanosoma congolense accounted for 77.4% (24/31) of failed ISMM treatments. The 62 cattle treated with 3.5 mg/kg bw. DIM resulted in 30.6% (19/62) failed treatments. Although 42.2% (19/45) of T. congolense positive cattle did not respond to DIM treatment, all T. vivax positive cattle responded positively to DIM treatment. Conclusions The overreliance on trypanocides in the control of trypanosomosis will ultimately lead to multiple drug-resistant trypanosome populations as detected in villages in south-east Mali rendering the use of drugs doubtful. Effective alternative methods for trypanosomosis control ought to substitute chemotherapy to ensure sustainable cattle production in these villages. Since there is no single strategy for containing trypanocidal drug resistance, promotion of an integrated approach combining proven trypanosomosis control approaches in high trypanosomosis risk areas is most desirous. The best-bet strategy this study recommended for areas with multiple drug resistance included area-wide community tsetse control, control of co-infections to exploit self-cure against resistant trypanosome populations and the rational use of trypanocidal drugs which should be urgently promoted at all levels as a way of containing or reversing resistance.

After a pilot study, we tested 443 cadavers using OraQuick Ebola rapid diagnostic tests during surveillance after the 10th Ebola outbreak in the Democratic Republic of the Congo. No false negative and 2% false-positive results were reported. Quickly returning results and engaging the community enabled timely public health actions.

In 2016, Mali reported a bacterial meningitis outbreak consisting of 39 suspected cases between epidemiologic weeks 9 and 17 with 15% case fatality ratio in the health district of Ouéléssebougou, 80 kilometers from the capital Bamako. Cerebrospinal fluid specimens from 29 cases were tested by culture and real-time polymerase chain reaction; 22 (76%) were positive for bacterial meningitis pathogens, 16 (73%) of which were Neisseria meningitidis (Nm). Of the Nm-positive specimens, 14 (88%) were N meningitidis serogroup C (NmC), 1 was NmW, and 1 was nongroupable. Eight NmC isolates recovered by culture from the outbreak were characterized using whole genome sequencing. Genomics analysis revealed that all 8 isolates belonged to a new sequence type (ST) 12446 of clonal complex 10217 that formed a distinct clade genetically similar to ST-10217, a NmC strain that recently caused large epidemics of meningitis in Niger and Nigeria. The emergence of a new ST of NmC associated with an outbreak in the African meningitis belt further highlights the need for continued molecular surveillance in the region., (Published by Oxford University Press for the Infectious Diseases Society of America 2019. This work is written by (a) US Government employee(s) and is in the public domain in the US.)

Laboratories play critical roles in bacterial meningitis disease surveillance in the African meningitis belt, where the highest global burden of meningitis exists. Reinforcement of laboratory capacity ensures rapid detection of meningitis cases and outbreaks and a public health response that is timely, specific, and appropriate. Since 2008, joint efforts to strengthen laboratory capacity by multiple partners, including MenAfriNet, beginning in 2014, have been made in countries within and beyond the meningitis belt. Over the course of 10 years, national reference laboratories were supported in 5 strategically targeted areas: specimen transport systems, laboratory procurement systems, laboratory diagnosis, quality management, and laboratory workforce with substantial gains made in each of these areas. To support the initiative to eliminate meningitis by 2030, continued efforts are needed to strengthen laboratory systems., (Published by Oxford University Press for the Infectious Diseases Society of America 2019. This work is written by (a) US Government employee(s) and is in the public domain in the US.)

Background: The MenAfriNet Consortium supports strategic implementation of case-based meningitis surveillance in key high-risk countries of the African meningitis belt: Burkina Faso, Chad, Mali, Niger, and Togo. We describe bacterial meningitis epidemiology in these 5 countries in 2015-2017., Methods: Case-based meningitis surveillance collects case-level demographic and clinical information and cerebrospinal fluid (CSF) laboratory results. Neisseria meningitidis, Streptococcus pneumoniae, or Haemophilus influenzae cases were confirmed and N. meningitidis/H. influenzae were serogrouped/serotyped by real-time polymerase chain reaction, culture, or latex agglutination. We calculated annual incidence in participating districts in each country in cases/100 000 population., Results: From 2015-2017, 18 262 suspected meningitis cases were reported; 92% had a CSF specimen available, of which 26% were confirmed as N. meningitidis (n = 2433; 56%), S. pneumoniae (n = 1758; 40%), or H. influenzae (n = 180; 4%). Average annual incidences for N. meningitidis, S. pneumoniae, and H. influenzae, respectively, were 7.5, 2.5, and 0.3. N. meningitidis incidence was 1.5 in Burkina Faso, 2.7 in Chad, 0.4 in Mali, 14.7 in Niger, and 12.5 in Togo. Several outbreaks occurred: NmC in Niger in 2015-2017, NmC in Mali in 2016, and NmW in Togo in 2016-2017. Of N. meningitidis cases, 53% were NmC, 30% NmW, and 13% NmX. Five NmA cases were reported (Burkina Faso, 2015). NmX increased from 0.6% of N. meningitidis cases in 2015 to 27% in 2017., Conclusions: Although bacterial meningitis epidemiology varied widely by country, NmC and NmW caused several outbreaks, NmX increased although was not associated with outbreaks, and overall NmA incidence remained low. An effective low-cost multivalent meningococcal conjugate vaccine could help further control meningococcal meningitis in the region., (Published by Oxford University Press for the Infectious Diseases Society of America 2019. This work is written by (a) US Government employee(s) and is in the public domain in the US.)

In nature, animals often face conflicting demands. For example, breeding males must attract a mate but at the same time be ready to defend against rivals. The molecular mechanisms by which the brain resolves behavioural trade-offs are largely unknown. In this study, we compared the brain transcriptional responses of territorial male three-spined sticklebacks to a mating opportunity with a female and to a territorial challenge by a rival male. We focused on the diencephalon and the cerebellum, two regions of the brain implicated in courtship and aggression. There was a set of genes that were differentially expressed in response to both a courtship opportunity and a territorial challenge. Closer inspection of the direction of regulation revealed that genes that were downregulated in response to a courtship opportunity were upregulated in response to a territorial challenge and vice versa. Our study reveals some of the potential molecular mechanisms underlying behavioural trade-offs between sex and aggression, along with a possible solution to the conflict via social context-dependent gene regulation., (© 2016 John Wiley & Sons Ltd.)

Within many species, some individuals are consistently more aggressive than others. We examine whether there are differences in brain gene expression between aggressive versus nonaggressive behavioural types of individuals within a natural population of male three-spined sticklebacks ( Gasterosteus aculeatus ). We compared gene expression profiles of aggressive male sticklebacks to nonaggressive males in four regions of the brain (brainstem, cerebellum, diencephalon and telencephalon). Relatively few genes were differentially expressed between behavioural types in telencephalon, cerebellum and diencephalon, but hundreds of genes were differentially expressed in brainstem, a brain area involved in detecting threats. Six genes that were differentially expressed in response to a territorial intrusion in a previous study were also differentially expressed between behavioural types in this study, implying primarily non-shared but some shared molecular mechanisms. Our findings offer new insights into the molecular causes and correlates of behavioural plasticity and individual variation in behaviour.

Foxp3-expressing regulatory T cells (Tregs) are central regulators of immune homeostasis and tolerance. As it has been suggested that proper Treg function is compromised under inflammatory conditions, seeking for a pathway that enhances or stabilizes Treg function is a subject of considerable interest. We report that interleukin (IL)-27, an IL-12 family cytokine known to have both pro- and anti-inflammatory roles in T cells, plays a pivotal role in enhancing Treg function to control T cell-induced colitis, a model for inflammatory bowel disease (IBD) in humans. Unlike wild-type (WT) Tregs capable of inhibiting colitogenic T-cell expansion and inflammatory cytokine expression, IL-27R-deficient Tregs were unable to downregulate inflammatory T-cell responses. Tregs stimulated with IL-27 expressed substantially improved suppressive function in vitro and in vivo. IL-27 stimulation of Tregs induced expression of Lag3, a surface molecule implicated in negatively regulating immune responses. Lag3 expression in Tregs was critical to mediate Treg function in suppressing colitogenic responses. Human Tregs also displayed enhanced suppressive function and Lag3 expression following IL-27 stimulation. Collectively, these results highlight a novel function for the IL-27/Lag3 axis in modulating Treg regulation of inflammatory responses in the intestine.

Certain complex phenotypes appear repeatedly across diverse species due to processes of evolutionary conservation and convergence. In some contexts like developmental body patterning, there is increased appreciation that common molecular mechanisms underlie common phenotypes; these molecular mechanisms include highly conserved genes and networks that may be modified by lineage-specific mutations. However, the existence of deeply conserved mechanisms for social behaviors has not yet been demonstrated. We used a comparative genomics approach to determine whether shared neuromolecular mechanisms could underlie behavioral response to territory intrusion across species spanning a broad phylogenetic range: house mouse (Mus musculus), stickleback fish (Gasterosteus aculeatus), and honey bee (Apis mellifera). Territory intrusion modulated similar brain functional processes in each species, including those associated with hormone-mediated signal transduction and neurodevelopment. Changes in chromosome organization and energy metabolism appear to be core, conserved processes involved in the response to territory intrusion. We also found that several homologous transcription factors that are typically associated with neural development were modulated across all three species, suggesting that shared neuronal effects may involve transcriptional cascades of evolutionarily conserved genes. Furthermore, immunohistochemical analyses of a subset of these transcription factors in mouse again implicated modulation of energy metabolism in the behavioral response. These results provide support for conserved genetic "toolkits" that are used in independent evolutions of the response to social challenge in diverse taxa.

Growth temperature range, resistance to selective antibiotics, activities of 23 enzymes, protein fingerprints and fatty acids composition of the spirochaetal strain BR91, isolated from the Culex pipiens mosquito, were tested. The spirochaetes were grown in BSK-H Complete liquid medium. The optimal in vitro growth temperature of the strain was 33 °C. Strain BR91 was sensitive to trimethoprim, nalidixic acid, 5-fluorouracil, and tolerated phosphomycin. The strain produced acid and alkaline phosphatase, esterase (C4), esterase-lipase (C8), leucine arylamidase, naphthol-AS-BI-phosphohydrolase and α-fucosidase. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) assay revealed several major proteins in the size range of 13-16 kDa, 22-30 kDa and 37-131 kDa. Fatty acid methyl ester (FAME) analysis showed that C₁₄:₀, C₁₆:₀, C₁₈:₁ ω9c and summed feature 5 (C₁₈:₂ ω6,9c and/or C₁₈:₀ anteiso) are major fatty acids. This study highlights certain phenotypic differences between strain BR91 and the Lyme disease spirochaete Borrelia burgdorferi, and supports the hypothesis that strain BR91 represents a unique taxonomical entity in a system of spirochaetal species., (Copyright © 2013 Elsevier GmbH. All rights reserved.)

Aggressive behaviour associated with territorial defence is widespread and has fitness consequences. However, excess aggression can interfere with other important biological functions such as immunity and energy homeostasis. How the expression of complex behaviours such as aggression is regulated in the brain has long intrigued ethologists, but has only recently become amenable for molecular dissection in non-model organisms. We investigated the transcriptomic response to territorial intrusion in four brain regions in breeding male threespined sticklebacks using expression microarrays and quantitative polymerase chain reaction (qPCR). Each region of the brain had a distinct genomic response to a territorial challenge. We identified a set of genes that were upregulated in the diencephalon and downregulated in the cerebellum and the brain stem. Cis-regulatory network analysis suggested transcription factors that regulated or co-regulated genes that were consistently regulated in all brain regions and others that regulated gene expression in opposing directions across brain regions. Our results support the hypothesis that territorial animals respond to social challenges via transcriptional regulation of genes in different brain regions. Finally, we found a remarkably close association between gene expression and aggressive behaviour at the individual level. This study sheds light on the molecular mechanisms in the brain that underlie the response to social challenges.

Predation pressure represents a strong selective force that influences the development and evolution of living organisms. An increasing number of studies have shown that both environmental and social factors, including exposure to predators, substantially shape the structure and function of the brain. However, our knowledge about the molecular mechanisms underlying the response of the brain to environmental stimuli is limited. In this study, we used whole-genome comparative oligonucleotide microarrays to investigate the brain transcriptomic response to cues of a predator in the threespine stickleback, Gasterosteus aculeatus. We found that repeated exposure to olfactory, visual and tactile cues of a predator (rainbow trout, Oncorrhynchus mykiss) for 6 days resulted in subtle but significant transcriptomic changes in the brain of sticklebacks. Gene functional analysis and gene ontology enrichment revealed that the majority of the transcripts differentially expressed between the fish exposed to cues of a predator and the control group were related to antigen processing and presentation involving the major histocompatibility complex, transmission of synaptic signals, brain metabolic processes, gene regulation and visual perception. The top four identified pathways were synaptic long-term depression, RAN signaling, relaxin signaling and phototransduction. Our study demonstrates that exposure of sticklebacks to cues of a predator results in the activation of a wide range of biological and molecular processes and lays the foundation for future investigations on the molecular factors that modulate the function and evolution of the brain in response to stressors., (2011 S. Karger AG, Basel.)

The identification of the members of the Culex pipiens L. complex in arbovirus surveillance programs relies heavily on the use of morphology. In this work, we studied Cx. pipiens complex male mosquitoes collected from nine different locations, from northern, southern, and the hybrid zone sites in North America; Cairo, Egypt; and Nairobi, Kenya. Specimens were identified using DV/D ratio and also using amplification of the acetylcholinesterase (ACE.2) gene by both conventional and real-time polymerase chain reaction, and examination of the CQ11 locus. Consistent with previous findings, the morphological and molecular identifications did not always agree, particularly in regions of extensive introgression. There was an increased frequency of hybrid forms in late summer and early fall in Champaign Co., IL, that is north of the previously described Cx. pipiens complex hybrid zone. This represents an expansion of the North American hybrid zone. The biological and epidemiological relevance of the high degree of introgression and the late season increase in the proportion of intermediate forms is discussed.

A systemic disease occurred in a wild population of the common vole Microtus arvalis in South Moravia (Czech Republic) during the years 1999-2003. Acute infections were characterized by edema of extremities, occasionally with colliquating abscesses, arthritis, lymphadenitis, perforations of the skin resulting from colliquated abscesses, orchitis, and peritoneal granulomas. From the clinical samples, small Gram-negative coccobacilli were isolated and identified as Ochrobactrum intermedium by API 20NE and colistin sensitivity profiles. However, subsequent rrs (16S rRNA) and recA (recombinase A) gene sequencing analysis of two isolates (CCM 4915=CAPM 6434; CCM 4916=CAPM 6435) identified them as Brucella sp. with sequence identities of 100% to other Brucella spp. Analysis of the omp2a/b genes confirmed the two isolates as Brucella. In AMOS polymerase chain reaction (PCR), a 2000-bp fragment was generated that was not seen in other brucellae. Experimental infection of outbred ICR mice with these isolates resulted in a mortality rate of 50%. Based on the results of the molecular investigations and the mortality observed in experimentally infected mice we conclude that the epizootic was caused by Brucella sp. and not by Ochrobactrum intermedium. The study demonstrates the limitations of commercial biochemical test systems in accurately differentiating among Ochrobactrum and Brucella.

In North America, West Nile and St. Louis encephalitis viruses have been detected in a wide range of vector species, but the majority of isolations continue to be from pools of mixed mosquitoes in the Culex subgenus Culex. Unfortunately, the morphologic identification of these important disease vectors is often difficult, particularly in regions of sympatry. We developed a sensitive real-time TaqMan polymerase chain reaction assay that allows reliable identification of Culex mosquitoes including Culex pipiens pipiens, Cx. p. quinquefasciatus, Cx. restuans, Cx. salinarius, Cx. nigripalpus, and Cx. tarsalis. Primers and fluorogenic probes specific to each species were designed based on sequences of the acetylcholinesterase gene (Ace2). Both immature and adult mosquitoes were successfully identified as individuals and as mixed species pools. This identification technique provides the basis for a rapid, sensitive, and high-throughput method for expounding the species-specific contribution of vectors to various phases of arbovirus transmission.

Culex pipiens quinquefasciatus Say and Culex pipiens pipiens Linnaeus are sibling species incriminated as important vectors of emerging and re-emerging infectious diseases worldwide. The two forms differ little morphologically and are differentiated mainly based upon ecological, behavioural, physiological and genetic traits. Within the North American zone of sympatry, populations of Cx. p. quinquefasciatus and Cx. p. pipiens undergo extensive introgression and hybrid forms have been reported in nature. Both Cx. p. quinquefasciatus and Cx. p. pipiens are infected with the endosymbiotic bacteria Wolbachia pipientis. Here, we report the presence of a transposable element belonging to the IS256 family (IS256wPip) associated with Wolbachia in both Cx. p. quinquefasciatus and Cx. p. pipiens populations. Using reverse transcriptase PCR and sequence analysis, we show that IS256wPip has disrupted the wspB locus, a paralogue of the Wolbachia outer membrane protein (wspA) gene. The inactivation of the wspB appears to be specific to Cx. p. quinquefasciatus and to hybrids of the two forms, and was not observed in the surveyed Cx. p. pipiens mosquitoes. Our results support the hypothesis of a different origin of North American Cx. p. quinquefasciatus and Cx. p. pipiens populations. The flux of mobile genetic elements in the Wolbachia wPip genome could explain the high level of crossing types observed among different Culex populations. The insertion of IS256wPip into wspB may comprise a genetic candidate for discriminating Wolbachia symbionts in Culex.

Bacteriophages are commonly found in association with free-living bacteria, both as exogenic phages (virions) and as prophages integrated into the bacterial genome. In contrast, the observation of bacteriophages associated with obligate intracellular bacteria has been described infrequently. An exception is provided by Wolbachia endosymbionts, which harbor multiple phage elements that have been designated as WO phage. Wolbachia are maternally inherited bacteria that occur in the cytoplasm of many invertebrates, where they often manipulate host reproduction. Previously, the WO phage orf7 locus and ankyrin repeat-encoding genes have been observed to represent sources of genetic diversity between Wolbachia (wPip) strains infecting mosquitoes of the Culex pipiens complex and have been suggested as potential participants in the reproductive manipulations. We have characterized WO phage associated with multiple Wolbachia-infected Culex strains and an uninfected strain using electron microscopy and RT-PCR. For each strain, different developmental stages were examined for transcription of three WO phage orf7 genes. The results provide evidence for the presence of both actively transcribed virions and inactive prophages. Variable orf7 transcription patterns are observed in comparisons of differing Cx. pipiens strains. Variability includes both mosquito stage-specific and sexually dimorphic orf7 expression patterns. This report provides additional support for the hypothesis that bacteriophages play an important role in Wolbachia and host evolution.

Gene flow between populations of Culex pipiens L. is relevant to observed differences in disease transmission, insecticide resistance, behavior, and physiology. Intracellular Wolbachia bacteria have been hypothesized to affect gene flow in insects. Specifically, Wolbachia cause a form of embryonic mortality known as cytoplasmic incompatibility (CI) in crosses between individuals with different Wolbachia types. Incompatibility in Culex is exceptional in that it represents the most complex CI pattern known, and yet Culex populations are not infected with divergent Wolbachia strains. This has led to the hypothesis that extrachromosomal factors such as phages or mobile genetic elements may be involved in determining CI phenotype. Recent molecular characterization of Culex laboratory strains has identified variation in the orf7 locus of the Wolbachia-associated bacteriophage WO. Here, crosses between eight Culex strains differing in their orf7 type were conducted to examine for the hypothesized involvement of bacteriophage WO in determining CI in Culex. Although crossing results show examples of compatibility, partial compatibility, and incompatibility, the results fail to show a correlation between the CI phenotypes and orf7 type. Specific examples include high egg hatch resulting in crosses between Culex strains that differ significantly in their orf7 type and low egg hatch resulting in crosses between Culex strains with similar orf7 types. Thus, the phage orf7 locus alone cannot predict CI type in the Culex strains examined in this study. However, rejection of the hypothesized role of WO phage in Culex CI will require the characterization of additional phage loci.

The medically important members of the Culex pipiens species complex provide an enigma for systematists, evolutionary biologists, and vector biologists. The species complex is composed of forms that differ in their ecology, behaviour, physiology and vector competence. Cytoplasmic incompatibility (CI) caused by endosymbiotic Wolbachia bacteria is thought to play an important role in restricting gene flow and the evolution of the Culex complex. Here we describe the first molecular marker useful for discriminating between Wolbachia infections in Culex. A putative bacteriophage locus (orf7) varies between Culex forms in copy number and sequence. We provide evidence that the orf7 loci are strictly associated with Wolbachia and are maternally inherited.

A total of 27 ticks, comprising Rhipicephalus sanguineus (Latreille) (n = 21), Haemaphysalis leachi (Andouin) (n = 4) and Haemaphysalis paraleachi (Camicas, Hoogstraal & El Kammah) (n = 2) were recovered from two clinically healthy female dogs in the Democratic Republic of the Congo. DNA of Anaplasma platys was detected in a female R. sanguineus, using primers derived from the 16S rRNA gene, which amplify members of the family Anaplasmataceae . Anaplasma platys DNA was also detected in the blood of one of the dogs. Phylogenetic analysis based on partial sequences of the 16S rRNA, the gltA and the groEL genes ranged the detected agent within the Anaplasma clade. This is the first reported detection of A. platys in ticks in Africa. This finding raises the question of the possible involvement of R. sanguineus in A. platys infection of dogs.

A total of 360 ticks were removed from 353 asymptomatic subjects in Belluno Province, Italy and surrounding areas, from 1998 to 2001. Ticks were identified as Ixodes ricinus (357), Ixodes hexagonus (1), Rhipicephalus sanguineus (1), and Ixodes ventalloi (1). Tick DNA was investigated by PCR and subsequent sequencing of amplified products to identity associated bacterial agents. Primers targeting different genes of Rickettsia (gltA and OmpA), Borrelia (16S rDNA, rpoB), Francisella (16S rDNA), and all genera members of the Anaplasmataceae (16S rDNA), were used. DNA of bacterial agents was identified in 28 Ixodes ricinus specimens (7.8%). Rickettsia helvetica was detected in 7 ticks. Rickettsia sp. IRS4 and Borrelia afzelii was detected in 4 ticks each. B. garinii and B. valaisiana were identified in one tick each. Anaplasma phagocytophilum, the agent of human granulocytic ehrlichiosis, was identified in 1 specimen of I. ricinus. A new Ehrlichia sp. ("Candidatus Ehrlichia walkerii", sp. nov.) was identified in 10 I. ricinus specimens.

Ixodes ricinus harbors many infectious agents pathogenic for humans. A cause of fever is found in less than 50% of patients exposed to ticks. Investigations on 359 Ixodes ticks removed from asymptomatic patients in Northern Italy revealed the presence of a new ehrlichial agent in 10 ticks. Comparison of the 16S rRNA and the gltA gene sequences showed the organism is most closely related to Ehrlichia ruminantium. We propose this new Ehrlichia be named "candidatus Ehrlichia walkerii."

During the months of October and November 2000 a total of 70 Dermacentor marginatus ticks were removed from different game pigs Sus scrofa in southern France and investigated by PCR using primers derived from the citrate synthase (gltA) and the outer membrane protein (OmpB) genes of rickettsiae. Based on sequence analysis of 1,150 bp fragment of gltA, Rickettsia slovaca was identified in 11 ticks (15.7%). These results confirm that Rickettsia slovaca, an emerging pathogen is highly prevalent in Dermacentor marginatus ticks in France. Moreover, a new Rickettsia genotype was detected in one specimen (1.4%). The pathogenic role of this new rickettsia has yet to be demonstrated.

A total of 650 ticks, including 13 species from five genera, were collected from animals, from people, or by flagging of the vegetation at sites on the Thai-Myanmar border and in Vietnam. They were tested by PCR to detect DNA of bacteria of the order RICKETTSIALES: Three Anaplasma spp. were detected in ticks collected in Thailand, including (i) Anaplasma sp. strain AnDa465, which was considered a genotype of Anaplasma platys (formerly Ehrlichia platys) and which was obtained from Dermacentor auratus ticks collected from dogs; (ii) Anaplasma sp. strain AnAj360, which was obtained from Amblyomma javanense ticks collected on a pangolin; and (iii) Anaplasma sp. strain AnHl446, which was closely related to Anaplasma bovis and which was detected in Haemaphysalis lagrangei ticks collected from a bear. Three Ehrlichia spp. were identified, including (i) Ehrlichia sp. strain EBm52, which was obtained from Boophilus microplus ticks collected from cattle from Thailand; (ii) Ehrlichia sp. strain EHh324, which was closely related to Ehrlichia chaffeensis and which was detected in Haemaphysalis hystricis ticks collected from wild pigs in Vietnam; and (iii) Ehrlichia sp. strain EHh317, which was closely related to Ehrlichia sp. strain EBm52 and which was also detected in H. hystricis ticks collected from wild pigs in Vietnam. Two Rickettsia spp. were detected in Thailand, including (i) Rickettsia sp. strain RDla420, which was detected in Dermacentor auratus ticks collected from a bear, and (ii) Rickettsia sp. strain RDla440, which was identified from two pools of Dermacentor larvae collected from a wild pig nest. Finally, two bacteria named Eubacterium sp. strain Hw124 and Eubacterium sp. strain Hw191 were identified in Haemaphysalis wellingtoni ticks collected from chicken in Thailand; these strains could belong to a new group of bacteria.

The potential role of ticks as vectors of Bartonella species has recently been suggested. In this study, we investigated the presence of Bartonella species in 271 ticks removed from humans in Belluno Province, Italy. By using primers derived from the 60-kDa heat shock protein gene sequences, Bartonella DNA was amplified and sequenced from four Ixodes ricinus ticks (1.48%). To confirm this finding, we performed amplification and partial sequencing of the pap31 protein and the cell division protein ftsZ encoding genes. This process allowed us to definitively identify B. henselae (genotype Houston-1) DNA in the four ticks. Detection of B. henselae in these ticks might represent a highly sensitive form of xenodiagnosis. B. henselae is the first human-infecting Bartonella identified from Ixodes ricinus, a common European tick and the vector of various tickborne pathogens. The role of ticks in the transmission of bartonellosis should be further investigated.

Host-seeking ixodid ticks were sampled in a floodplain forest ecosystem along the lower reaches of the Thaya (Dyje) river in South Moravia (Czech Republic) and Lower Austria during the period 1989-2002. The ticks were examined by dark-field microscopy for borreliae (Borrelia burgdorferi sensu lato, the agent of Lyme borreliosis), and attempts were made to culture the spirochetes in BSK-H medium from preparations containing their high numbers. Isolated borreliae were identified by PCR-RFLP analysis using probes directed against ribosomal spacer genes. A total of 797 nymphal and 719 adult (391 female, 328 male) Ixodes ricinus were examined: 16.2% of nymphs, 28.6% of females and 29.0% of males were positive. Dermacentor reticulatus (70 females, 30 males) and Haemaphysalis concinna (12 nymphs, 8 females, 2 males) were negative for spirochetes. The overall prevalence rate of borreliae in I. ricinus from the floodplain forest is slightly higher than the mean European data (i.e., 14% for nymphs, 21% for adults). The difference in infection rate between nymphal and adult ticks was significant, including the proportion of heavily infected (with > 100 borreliae) nymphs (2.1%) vs. adults (7.6%). Prevalence of borreliae in I. ricinus showed a significant decrease during autumn in this ecosystem. Three strains of spirochetes, all of the Borrelia afzelii genomic group, were isolated from female I. ricinus. Moreover, Trypanosoma/Crithidia sp. protozoa and Dipetalonema rugosicauda nematodes were detected in 0.4% and 1.0%, respectively, of all I. ricinus.

Seven virus isolates were obtained from 11,334 mosquitoes after the 1997 Morava River flooding in South Moravia (Czech Republic): 6 strains of Tahyna bunyavirus, California antigenic group (5 from Aedes vexans, 1 from Ae. cinereus), and 1 strain of West Nile flavivirus (WNV) from Culex pipiens. In 1999, one isolate of Tahyna virus from Ae. vexans and one isolate of WNV from Cx. pipiens were recovered from a total of 14,354 mosquitoes examined in the same area, whereas no virus was detected there in 1,179 overwintering mosquitoes (mostly Cx. pipiens) in March 2000. The infection rate of mosquitoes with arboviruses was significantly higher in 1997, the year of the flood and an enormously high population density of mosquitoes. Antibodies neutralizing WNV were detected in 13 of 619 (2.1%) hospitalized patients or persons seeking outpatient clinics of the area in 1997. Five of the seroreactors revealed clinical symptoms compatible with West Nile fever: in 2 of them (children), recent infection with WNV was confirmed by a significant increase of antibody titer between acute and convalescent serum samples.

During the years 1995-1996, a total of 1,743 overwintering Culex pipiens biotype molestus female mosquitoes were tested for the presence of spirochetes in several localities in South Moravia, Czech Republic.The spirochetes were observed in 5% of the mosquitoes investigated. One of the five isolated strains of spirochetes (BR-84) was identified as Borrelia afzelii. The potential role of mosquitoes in the ecology and epidemiology of Lyme disease (LD) borreliae should be further investigated.