Prevalence and distribution of pathogen infection and permethrin resistance in tropical and temperate populations of Rhipicephalus sanguineus s.l. collected worldwide.

The brown dog tick, Rhipicephalus sanguineus sensu lato (s.l.) Latreille (Acari: Ixodidae), is a peridomestic, cosmopolitan parasite of dogs known to vector numerous pathogens of veterinary and medical importance. Recent phylogenetic analyses separate this tick into temperate and tropical lineages. Populations of Rh. sanguineus s.l. have been reported to exhibit sodium channel target site insensitivity to permethrin and etofenprox, which is likely due to the prolonged use of pyrethroids against many pests in and around the home. In this study, populations collected in the Caribbean, Africa, Asia, Europe and North America, were tested to identify the distribution of a known resistance mechanism, pathogen-vector interactions and phylogeny in relation to latitude. Using molecular assays, populations from 29 distinct locations were simultaneously geographically typed and screened for bacterial infection by Rickettsia, Ehrlichia, Babesia and Hepatozoon species, and for the presence of a sodium channel single nucleotide polymorphism known to confer permethrin resistance. Implications of these results on Rh. sanguineus s.l. management in association with geographical distribution will be discussed.

Ehrlichia ruminantium in Amblyomma variegatum and domestic ruminants in the Caribbean.

The highly sensitive nested pCS20 polymerase chain reaction assay for Ehrlichia ruminantium was negative on 506 Amblyomma variegatum from Caribbean islands where clinical heartwater has not been reported, mainly the United States Virgin Islands (18), Dominica (170), Montserrat (5), Nevis (34), St. Kitts (262), and St. Lucia (17). Positive results were obtained with positive controls (Crystal Springs strain) and A. variegatum from countries in Africa where infections are endemic, mainly Tanzania (1/37) and Burkino Faso (2/29). Positive major antigenic protein-1 enzyme-linked immunosorbent assays for E. ruminantium were obtained on convenience samples of sera from apparently healthy cattle, sheep, and goats on Dominica (0/95, 0%; 3/135, 2%; 2/57, 4%), Grenada (0/4, 0%; 1/98, 1%; 1/86, 1%), Montserrat (0/12, 0%; 0/28, 2%; 5/139, 4%), Nevis (0/45, 0%; 0/157, 0%; 0/90, 0%), Puerto Rico (0/422, 0%; 0, 0%), St. Kitts (3/86, 4%; 1/25, 0%; 0/26, 0%), and St. Lucia (0/184, 0%; 0/15, 0%; 0, 0%), respectively. The pCS20 polymerase chain reaction results indicate E. ruminantium is not present on islands where clinical heartwater does not occur. The occasional positive major antigenic protein-1B enzyme-linked immunosorbent assay results appear, then, to be false-positive reactions, and serology appears to be of limited use in testing for E. ruminantium in the Caribbean, as is the case in Africa.

The hard-tick fauna of mainland Portugal (Acari: Ixodidae): an update on geographical distribution and known associations with hosts and pathogens.

This work is an updated revision of the available information on Portuguese ixodid tick species. It includes data on tick biology, ecology, taxonomy and host/pathogen-associations. The current list of Portuguese ixodid ticks comprises twenty species: Dermacentor marginatus (Sulzer, 1776), Dermacentor reticulatus (Fabricius, 1794), Haemaphysalis hispanica Gil Collado, 1938, Haemaphysalis inermis Birula, 1895, Haemaphysalis punctata Canestrini & Fanzago, 1878, Hyalomma lusitanicum Koch, 1844, Hyalomma marginatum Koch, 1844, Ixodes acuminatus Neumann, 1901, Ixodes bivari Dias, 1990, Ixodes canisuga Johnston, 1849, Ixodes frontalis (Panzer, 1798), Ixodes hexagonus Leach, 1815, Ixodes ricinus (Linnaeus, 1758), Ixodes simplex Neumann, 1906, Ixodes ventalloi Gil Collado, 1936, Ixodes vespertilionis Koch, 1844, Rhipicephalus (Boophilus) annulatus (Say, 1821), Rhipicephalus bursa Canestrini & Fanzago, 1878, Rhipicephalus pusillus Gil Collado, 1938, and Rhipicephalus sanguineus (Latreille, 1806).

Midichloria mitochondrii is widespread in hard ticks (Ixodidae) and resides in the mitochondria of phylogenetically diverse species.

The hard tick Ixodes ricinus (Ixodidae) is the sole animal thus far shown to harbour an intra-mitochondrial bacterium, which has recently been named Midichloria mitochondrii. The objectives of this work were (i) to screen ixodid ticks for Midichloria-related bacteria and (ii) to determine whether these bacteria exploit the intra-mitochondrial niche in other tick species. Our main goal was to discover further models of this peculiar form of symbiosis. We have thus performed a PCR screening for Midichloria-related bacteria in samples of ixodid ticks collected in Italy, North America and Iceland. A total of 7 newly examined species from 5 genera were found positive for bacteria closely related to M. mitochondrii. Samples of the tick species Rhipicephalus bursa, found positive in the PCR screening, were analysed with transmission electron microscopy, which revealed the presence of bacteria both in the cytoplasm and in the mitochondria of the oocytes. There is thus evidence that bacteria invade mitochondria in at least 2 tick species. Phylogenetic analysis on the bacterial 16S rRNA gene sequences generated from positive specimens revealed that the bacteria form a monophyletic group within the order Rickettsiales. The phylogeny of Midichloria symbionts and related bacteria does not appear completely congruent with the phylogeny of the hosts.

Population genetic structure of Ixodes ricinus in Switzerland from allozymic data: no evidence of divergence between nearby sites.

Ixodes ricinus is a vector and reservoir of numerous infectious agents, especially Borrelia burgdorferi, the agent of Lyme disease. In Switzerland, its ecology and physiology have been well studied. Moreover, the foci of some infectious agents transmitted by this tick are identified. They can display relatively to extremely small geographical size depending on the diseases considered. In order to understand how the gene flows occur and to characterise the genetic structure of Ixodes ricinus populations, we used an indirect method based on genetic markers: allozymes. The sampling was carried out in 5 localities. Eighteen loci were analysed and 2 appeared polymorphic. This shows the low allozymic variability displayed by Ixodes ricinus. Based on these 2 loci, the populations appeared panmictic in Switzerland. This may be explained by the wide range of vertebrate species this tick can infest, especially birds. However, the result is surprising if we consider the extreme localisation of the foci of some infectious agents. We conclude that more powerful genetic markers could be used in order to better understand the epidemiology of tick-borne diseases in Switzerland.

First isolation of Rickettsia slovaca from Dermacentor marginatus in France.

Three rickettsial strains isolated from Dermacentor marginatus ticks in southern France in 1991 were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blotting with polyclonal mouse antisera, microimmunofluorescence serologic typing, and the polymerase chain reaction followed by analysis of restriction fragment length polymorphisms. By these methods, the isolates appear to be identical to the spotted fever group rickettsia, Rickettsia slovaca, which has so far been found only in Czechoslovakia. The fact that this rickettsial strain of unknown pathogenicity may occur in the same regions where R. conorii is endemic is of particular epidemiologic importance.

Identification of spotted fever group rickettsiae isolated from Dermacentor marginatus and Ixodes ricinus ticks collected in Switzerland.

When 155 ticks collected in different regions of Switzerland were tested by the hemolymph test, 10.3% were found to contain spotted fever group rickettsiae. Six rickettsial isolates were made from Dermacentor marginatus ticks and three were made from Ixodes ricinus ticks. The polymerase chain reaction followed by restriction fragment length polymorphism analysis showed that the Dermacentor ticks were infected with Rickettsia length polymorphism analysis showed that the Dermacentor ticks were infected with Rickettsia slovaca and the Ixodes ticks were infected with a spotted fever group rickettsia. Microimmunofluorescence serologic type, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of proteins, and Western blot assay with polyclonal mouse antisera confirmed the results and determined that the Ixodes were infected with R. helvetica, the only previously described Swiss rickettsia. However, an additional new strain that could not be isolated was detected in one I. ricinus by hemolymph test and provisionally characterized by enzymatic restriction of its amplified DNA.

Astrakhan fever rickettsiae: antigenic and genotypic analysis of isolates obtained from human and Rhipicephalus pumilio ticks.

Two spotted fever group rickettsia strains, A-108 and A-167, were isolated from the hemolymph of Rhipicephalus pumilio ticks collected in the Astrakhan region of Russia, which is area endemic for Astrakhan fever. These tick isolates were compared with a strain isolated from a patient suffering from Astrakhan fever and with reference spotted fever group rickettsiae strains. New tick isolates and the human strain were identical in their serologic, antigenic, and genetic characteristics by several methods: microimmunofluorescence, protein gel electrophoresis with immunoblotting, polymerase chain reaction followed by restriction endonuclease fragment length polymorphism analysis, and pulsed-field gel electrophoresis (PFGE). Astrakhan fever rickettsiae were found to be serologically and antigenically similar to Israeli spotted fever rickettsiae. Both of them probably belong to a single Rickettsia conorii pathotype complex. Only PFGE pattern analysis could clearly discriminate Astrakhan fever rickettsiae from other isolates.

An outbreak of acute bartonellosis (Oroya fever) in the Urubamba region of Peru, 1998.

During May 1998, we conducted a case-control study of 357 participants from 60 households during an outbreak of acute bartonellosis in the Urubamba Valley, Peru, a region not previously considered endemic for this disease. Blood and insect specimens were collected and environmental assessments were done. Case-patients (n = 22) were defined by fever, anemia, and intra-erythrocytic coccobacilli seen in thin smears. Most case-patients were children (median age = 6.5 years). Case-patients more frequently reported sand fly bites than individuals of neighboring households (odds ratio [OR] = 5.8, 95% confidence interval [CI] = 1.2-39.2), or members from randomly selected households > or = 5 km away (OR = 8.5, 95% CI = 1.7-57.9). Bartonella bacilliformis isolated from blood was confirmed by nucleotide sequencing (citrate synthase [g/tA], 338 basepairs). Using bacterial isolation (n = 141) as the standard, sensitivity, specificity, and positive predictive value of thin smears were 36%, 96%, and 44%, respectively. Patients with clinical syndromes compatible with bartonellosis should be treated with appropriate antibiotics regardless of thin-smear results.

Species-specific BALB/c mouse antibodies to rickettsiae studied by western blotting.

BALB/c mice were inoculated intraperitoneally either once only, or up to four times at weekly intervals, with viable Rickettsia rickettsii, Rickettsia conorii or the Israeli spotted fever group rickettsia. Sera collected one week after the last inoculation were tested for the presence of antibodies reactive with the above organisms by indirect fluorescent antibody testing and Western blot. With repeated inoculations there was a general progressive rise in homologous and heterologous immunofluorescence titers although the increase after the first inoculation was always the greatest. For each rickettsia, the homologous titers were higher than the heterologous titers. Western blots showed that the reactive antibodies were against rickettsial high molecular mass species specific protein antigens and homologous species-specific antibody reactions were detectable earlier than heterologous cross-reacting antibody reactions. Antibodies in mice sera did not react with the group specific lipopolysaccharide-like antigens of the rickettsiae although such reactivity was strong in Western blots with sera from patients suffering from acute Rickettsia conorii infections. Our findings suggest that the intraperitoneal route of inoculation of BALB/c mice can be used for the differentiation of spotted fever group rickettsiae.

A relapsing fever group spirochete transmitted by Ixodes scapularis ticks.

A species of Borrelia spirochetes previously unknown from North America has been found to be transmitted by Ixodes scapularis ticks. Infected ticks are positive for Borrelia spp. by DFA test but negative for Borrelia burgdorferi by polymerase chain reaction (PCR) using species-specific primers for 16S rDNA, outer surface protein A, outer surface protein C, and flagellin genes. A 1,347-bp portion of 16S rDNA was amplified from a pool of infected nymphs, sequenced, and compared with the homologous fragment from 26 other species of Borrelia. The analysis showed 4.6% pairwise difference from B. burgdorferi, with the closest relative being Borrelia miyamotoi (99.3% similarity) reported from Ixodes persulcatus in Japan. Phylogenetic analysis showed the unknown Borrelia to cluster with relapsing fever group spirochetes rather than with Lyme disease spirochetes. A 764-bp fragment of the flagellin gene was also compared with the homologous fragment from 24 other Borrelia species. The flagellin sequence of B. burgdorferi was 19.5% different from the unknown Borrelia and showed 98.6% similarity with B. miyamotoi. A pair of PCR primers specifically designed to amplify a 219-bp fragment of the flagellin gene from this spirochete was used to survey field-collected I. scapularis nymphs from five northeastern states (Connecticut, Rhode Island, New York, New Jersey, and Maryland). Positive results were obtained in 1.9-2.5% of 712 nymphs sampled from four states but in none of 162 ticks collected from Maryland. Transovarial transmission was demonstrated by PCR of larval progeny from infected females with filial infection rates ranging from 6% to 73%. Transstadial passage occurred from larvae through adults. Vertebrate infection was demonstrated by feeding infected nymphs on Peromyscus leucopus mice and recovering the organism from uninfected xenodiagnostic larvae fed 7-21 days later. Considering the frequency of contact between I. scapularis and humans, further work is needed to determine the potential public health significance of yet another zoonotic agent transmitted by this tick species.

Ehrlichial DNA amplified from Ixodes ricinus (Acari: Ixodidae) in France.

Granulocytic ehrlichia 16S rDNA was amplified for the 1st time from an Ixodes ricinus (Linne) tick collected in Europe. Sequence analysis of polymerase chain reaction products from the 16S rRNA gene demonstrated the organism from which it originated to be closely related to the agent of human granulocytic ehrlichiosis, an emerging disease that was recently described in the United States; Ehrlichia phagocytophila, the agent of tick-borne fever of ruminants in Europe; and Ehrlichia equi. the agent of the worldwide equine granulocytic ehrlichiosis. These granulocytic ehrlichiae have been associated with Ixodes spp. ticks that may act as vectors. It remains to be determined if each of these granulocytic ehrlichiae, that may constitute variants of the same species, is responsible for a specific disease in animals and in humans.

Prevalence of rickettsia-like organisms and spotted fever group rickettsiae in ticks (Acari: Ixodidae) from Zimbabwe.

The prevalence of rickettsia-like organisms in ticks from Zimbabwe was determined using the hemolymph test. Amblyomma hebraeum had the highest prevalence of rickettsia-like organisms. Other species with rickettsia-like organisms included Amblyomma sparsum, Amblyomma variegatum, Hyalomma marginatum rufipes, Ripicephalus simus, Haemaphysalis leachi, Amblyomma rhinocerotis, and Hyalomma truncatum. Ticks with no demonstrable rickettsia-like organisms infection were Boophilus decoloratus, Haemaphysalis spinulosa, Rhipicephalus appendiculatus, Rhipicephalus evertsi evertsi, and Rhipicephalus sanguineus. Polymerase chain reaction followed by restriction fragment-length polymorphism analysis on samples of hemolymph-positive ticks showed the agent of African tick-bite fever to be present in A. hebraeum, Rickettsia conorii to be present in Rhipicephalus simus and Haemaphysalis leachi, and a spotted fever group rickettsia similar to that in Hyalomma marginatum marginatum ticks from Morocco and Portugal to be present in Hyalomma marginatum rufipes.

Serosurvey for Cowdria ruminantium, Coxiella burnetii, and Spotted fever group rickettsiae in ostriches (Struthio camelus) from Zimbabwe.

Sera from 216 ostriches on nine farms around Zimbabwe were examined by indirect fluorescent antibody (IFA) testing for the presence of antibodies reactive with Cowdria ruminantium, Coxiella burnetii, and Rickettsia africae, a spotted fever group rickettsia. Although no reactive antibodies could be detected to C. ruminantium or C. burnetii, 51/216 (35%) sera reacted with R. africae. The seroprevalence in ostriches from the south of Zimbabwe was significantly higher than in birds from the north (P < 0.01). Immunoblots of four sera positive by IFA (> 1/160) showed antibodies reactive with antigens of R. africae that also were recognized by pooled sera from mice inoculated with the organism. No reactive antibodies could be detected in six sera negative by IFA.

Analysis of the systematic relationships among ticks of the genera Rhipicephalus and Boophilus (Acari: Ixodidae) based on mitochondrial 12S ribosomal DNA gene sequences and morphological characters.

A portion of mitochondrial 12S rDNA sequences (337-355 base pairs) and 63 morphological characters of 36 hard-tick species belonging to 7 genera were analyzed to determine the phylogenetic relationships among groups and species of Rhipicephalus and between the genera Rhipicephalus and Boophilus. Molecular and morphological data sets were first examined separately. The molecular data were analyzed by maximum parsimony (MP), maximum likelihood, and neighbor-joining distance methods; the morphological data were analyzed by MP After their level of congruence was evaluated by a partition homogeneity test, all characters were combined and analyzed by MP. The branches of the tree obtained by combining the data sets were better resolved than those of the trees inferred from the separate analyses. Boophilus is monophyletic and arose within Rhipicephalus. Boophilus species clustered with species of the Rhipicephalus evertsi group. Most of the clustering within Rhipicephalus was, however, consistent with previous classifications based on morphological data. Morphological characters were traced on the molecular reconstruction in order to identify characters diagnostic for monophyletic clades. Within the Rhipicephalus sanguineus complex, the sequences of specimens morphologically identified as Rhipicephalus turanicus were characterized by a high level of variability, indicating that R. turanicus-like morphology may cover a spectrum of distinct species.

The ticks (Acari: Ixodida: Argasidae, Ixodidae) of Paraguay.

The ticks reported in Paraguay, which are here reviewed, can be categorized as 'endemic or established' (Argas persicus or a sibling species, Ornithodoros hasei, O. rostratus, O. rudis, O. talaje/O. puertoricensis, Amblyomma aureolatum, Am. auricularium, Am. brasiliense, Am. cajennense, Am. calcaratum, Am. coelebs, Am. dissimile, Am. dubitatum, Am. incisum, Am. longirostre, Am. nodosum, Am. ovale, Am. pacae, Am. parvum, Am. pseudoconcolor, Am. rotundatum, Am. scutatum, Am. tigrinum, Am. triste, Dermacentor nitens, Haemaphysalis juxtakochi, H. leporispalustris, Ixodes loricatus, Rhipicephalus microplus, and Rh. sanguineus), 'probably endemic or established' (Ar. miniatus, Ar. monachus, Am. argentinae, Am. humerale, Am. naponense, Am. oblongoguttatum, Am. pseudoparvum, I. aragaoi/I. pararicinus, I. auritulus, I. luciae), or 'erroneously reported from Paraguay' (O. coriaceus, Am. americanum and Am. maculatum). Most Paraguayan tick collections have been made in the Chaco phyto-geographical domain, in the central part of the country. Argas persicus or a related species, Am. cajennense, D. nitens, Rh. microplus and Rh. sanguineus are important parasites of domestic animals. Ornithodoros rudis, Am. aureolatum, Am. brasiliense, Am. cajennense, Am. coelebs, Am. incisum, Am. ovale and Am. tigrinum have all been collected from humans. In terms of public health, the collections of Am. cajennense and Am. triste from humans may be particularly significant, as these species are potential vectors of Rickettsia rickettsii and Ri. parkeri, respectively.

Ticks (Ixodidae) on humans in South America.

Twenty eight species of Ixodidae have been found on man in South America (21 Amblyomma, 1 Boophilus, 2 Dermacentor, 2 Haemaphysalis, 1 Ixodes and 1 Rhipicephalus species). Most of them are rarely found on man. However, three species frequently parasitize humans in restricted areas of Argentina (A. neumanni reported from 46 localities), Uruguay (A. triste from 21 sites) and Argentina-Brazil (A. parvum from 27 localities). The most widespread ticks are A. cajennense (134 localities in Argentina, Bolivia, Brazil, Colombia, Ecuador, French Guiana, Guyana, Paraguay, Suriname and Venezuela), A. ovale (37 localities in Argentina, Brazil, Ecuador, French Guiana, Guyana, Paraguay, Suriname and Venezuela) and A. oblongoguttatum (28 sites in Brazil, Colombia, French Guiana, Guyana, Suriname and Venezuela). Amblyomma aureolatum (18 localities in Argentina, Brazil, French Guiana and Paraguay), A. cajennense, and A. triste are vectors of rickettsioses to man in South America. A better understanding of the respective roles of these and other tick species in transmitting pathogens to humans will require further local investigations. Amblyomma ticks should be the main subjects of these studies followed by species of Boophilus, Dermacentor, Haemaphysalis and Rhipicephalus species. In contrast with North America, Europe and Asia, ticks of the genus Ixodes do not appear to be major players in transmitting diseases to human. Indeed, there is only one record of an Ixodes collected while feeding on man for all South America.

Rickettsia massiliae sp. nov., a new spotted fever group Rickettsia.

We propose the name Rickettsia massiliae sp. nov. (type strain, Mtu1 in the Collection of the World Health Organization Collaborative Center for Rickettsial Reference, Marseille, France) for a spotted fever group rickettsia determined to be distinct from previously recognized species by the serotyping method (L. Beati, J.-P. Finidori, B. Gilot, and D. Raoult, J. Clin. Microbiol. 30:1922-1930, 1992). This rickettsia has biological characteristics similar to those of the other spotted fever group rickettsiae. In addition, a sodium dodecyl sulfate-polyacrylamide gel electrophoresis protein analysis, a polymerase chain reaction followed by a restriction fragment length polymorphism analysis of DNA fragments, and pulsed-field electrophoresis of the genome of R. massiliae revealed unique migration patterns distinct from those of all previously recognized spotted fever group rickettsiae. These additional characteristics (Beati et al., J. Clin. Microbiol. 30:1922-1930, 1992), together with the data usually considered sufficient for description of rickettsiae, are crucial to the proposal of this new species and should be helpful in species identification.

Characterization of and application of monoclonal antibodies against Rickettsia africae, a newly recognized species of spotted fever group rickettsia.

Rickettsia africae is a newly described species which causes African tick bite fever. Mediterranean spotted fever caused by R. conorii is endemic in the same regions of Africa as tick bite fever, and differentiation of the two syndromes by characterization of their etiological agents is important for epidemiological studies. R. africae and R. conorii are, however, difficult to distinguish, and therefore, our aim was to produce monoclonal antibodies to address this problem. Monoclonal antibodies were produced against R. africae by fusing splenocytes from BALB/C mice immunized with purified rickettsial organisms and SP2/0-Ag14 myeloma cells. A total of 355 hybridomas producing monoclonal antibodies to R. africae were identified by initial screening with six different antigens by microimmunofluorescence assay. A panel of 23 representative monoclonal antibodies were selected and subcloned. This panel was screened with a further 17 different spotted fever group (SFG) rickettsial reference antigens. Of these 23 monoclonal antibodies, 1 cross-reacted with only R. parkeri, whereas the others cross-reacted with more than two different antigens. Immunoblotting indicated that all the monoclonal antibodies were directed against the epitopes on two major high-molecular-mass heat-labile proteins, of which the molecular masses were 128 and 135 kDa, respectively. This monoclonal antibody panel was used successfully to identify R. africae in the blood culture of an infected patient, in infected cells within shell vials, and in infected ticks collected from Africa. Furthermore, the cross-reactivity of each SFG rickettsia with each of these 23 monoclonal antibodies was scored and was used to build a dendrogram of taxonomic relatedness between R. africae and the other SFG rickettsiae on the basis of Jaccard coefficients and unweighted pair group method with arithmetic mean analysis. The relatedness was generally consistent with that obtained by other methods of comparison.