Molecular identification of Rickettsia parkeri infecting Amblyomma triste ticks in an area of Argentina where cases of rickettsiosis were diagnosed

Specimens of the hard tick Amblyomma triste were found infected with Rickettsia parkeri in an area of Argentina (General Lavalle, Buenos Aires Province) where cases of human illness attributed to this microorganism have been reported. Molecular detection of R. parkeri was based on polymerase chain reactions that amplify a ca. 400-bp fragment of the 23S-5S intergenic spacer and a ca. 500-bp fragment of the gene encoding a 190-kDa outer membrane protein. Three (6.97%) of 43 A. triste ticks were determined to be positive for R. parkeri. These results provide strong evidence that A. triste is the vector of R. parkeri in the study area. The findings of this work have epidemiological relevance because human parasitism by A. triste ticks has been frequently recorded in some riparian areas of Argentina and Uruguay and new cases of R. parkeri rickettsiosis might arise in the South American localities where humans are exposed to the bites of this tick species.     

Rickettsia amblyommii infecting Amblyomma sculptum in endemic spotted fever area from southeastern Brazil

The Rickettsia bacteria include the aetiological agents for the human spotted fever (SF) disease. In the present study, a SF groupRickettsia amblyommii related bacterium was detected in a field collected Amblyomma sculptum (Amblyomma cajennense species complex) tick from a Brazilian SF endemic site in southeastern Brazil, in the municipality of Juiz de Fora, state of Minas Gerais. Genetic analysis based on genes ompA,ompB and htrA showed that the detected strain, named R. amblyommii str. JF, is related to the speciesR. amblyommii.     

Detection and isolation of tick‐borne bacteria (Anaplasma spp., Rickettsia spp., and Borrelia spp.) in Amblyomma varanense ticks on lizard (Varanus salvator)

Ticks are one of the arthropods that play an important role in the transmission of numerous pathogens to livestock and humans. We investigated the presence of tick‐borne bacteria in 23 Amblyomma varanense that fed on a water monitor (Varanus salvator) in Indonesia. Anaplasmataceae and borreliae were detected by PCR in 17.4% and 95.7% of ticks, respectively. “Candidatus Rickettsia sepangensis”, spotted fever group of Rickettsia, was detected in 21.7% of ticks. The water monitor is a common reptile that is widely encountered in city areas in Asian countries. Our results suggested that Am. varanense on water monitor in Indonesia harbored several kinds of bacteria.     

Amblyomma triste (Koch, 1844) (Acari: Ixodidae): morphological description of the ovary and of vitellogenesis

The present study presents the morphology, histology, and the dynamics of vitellogenesis in females of the tick Amblyomma triste. The ovary in this species is of the panoistic type, therefore it lacks nurse cells. It is composed of a layer of epithelial cells that outwardly form the wall of the ovary, but also originate the pedicel, the structure that attaches the oocytes to its external margin, as well the oocytes themselves. In Amblyomma triste, the oocytes develop in four synchronic stages, which differs from the process in other tick species. The classification of the stages of the oocytes was carried out based on the presence of four morphologic characteristics: cytoplasm appearance; site of the germ vesicle; presence, quantity, and constitution of the yolk granules and presence of chorium.     

Characterization of protective antigens from the midgut of Amblyomma variegatum ticks

Separation of midgut membrane proteins from the tick,Ambylomma variegatum, using a non-ionic detergent (TritonX-114), resulted in two protein fractions, namely DET (detergent) and AQ (aqueous). In immunoblotting analysis with polyclonal antibodies against these fractions, 4 proteins (M_r ∼27,000, 67,000, 86,000 and 95,000,)and 2 proteins (M_r ∼54,000 and 67,000) were detected in the DET and AQ fractions, respectively. Three of the DET fraction proteins M_r∼27,000, 67,000 and 95,000 were glycosylated since they bound to the lectin, concanavalin A. In 2-dimensional gel electrophoresis, the AQ and DET fraction proteins were found to be acidic in nature. In a series of bioassay experiments, rabbits were first immunised with both DET and AQ fractions and then infested with ticks. The egg batch weights of these ticks were reduced by 50% compared to control ticks. Furthermore, there was a significant reduction in the hatchability of eggs laid by ticks fed on rabbits previously immunised with both DET (14%) and AQ (33%) fractions. Based on the egg hatchability, there productive capacity of ticks was reduced by 77 and 48% by DET and AQ fractions, respectively. This revised version was published online in July 2006 with corrections to the Cover Date.     

Amblyomma sculptum: genetic diversity and rickettsias in the Brazilian Cerrado biome

Amblyomma sculptum (Ixodida: Ixodidae) Berlese, 1888 is the most important tick vector in Brazil, transmitting the bioagent of the most severe form of spotted fever (SF) in part of the Cerrado (in the states of Minas Gerais and São Paulo). In another part of the Cerrado (Central‐West region of Brazil), a milder form of SF has been recorded. However, neither the rickettsia nor the vector involved have been characterized. The aim of the current study was to analyse genetic variation and the presence of rickettsia in A. sculptum in Cerrado, from silent areas and with the milder form of SF. Samples were subjected to DNA extraction, amplification and sequencing of 12S rDNA, cytochrome oxidase subunit II and D‐loop mitochondrial genes (for tick population analyses), and gltA, htrA, ompA and gene D (sca4) genes for rickettsia researches. Exclusive haplotypes with low frequencies, high haplotype diversity and low nucleotide diversity, star‐shaped networks and significant results in neutrality tests indicate A. sculptum population expansions in some areas. Rickettsia amblyommatis, Candidatus Rickettsia andeanae and Rickettsia felis were detected. The A. sculptum diversity is not geographically, or biome delimited, pointing to a different potential in vector capacity, possibly associated with differing tick genetic profiles.     

Candidatus Rickettsia andeanae,a spotted fever group agent infecting Amblyomma parvum ticks in two Brazilian biomes

Adult ticks of the species Amblyomma parvum were collected from the vegetation in the Pantanal biome (state of Mato Grosso do Sul) and from horses in the Cerrado biome (state of Piauí) in Brazil. The ticks were individually tested for rickettsial infection via polymerase chain reaction (PCR) targeting three rickettsial genes, gltA, ompA and ompB. Overall, 63.5% (40/63) and 66.7% (2/3) of A. parvum ticks from Pantanal and Cerrado, respectively, contained rickettsial DNA, which were all confirmed by DNA sequencing to be 100% identical to the corresponding fragments of the gltA, ompA and ompB genes of Candidatus Rickettsia andeanae. This report is the first to describe Ca. R. andeanae in Brazil.     

Seasonal dynamics of the Cayenne tick, Amblyomma cajennense on horses in Brazil

The population dynamics of all stages of the Cayenne tick, Amblyomma cajennense (Fabricius) (Acari: Ixodidae) on horses was evaluated over a period of 2 years in the district of Pedro Leopoldo, State of Minas Gerais, Brazil. Every 14 days, the left side of 20 horses was brushed for collection of immature stages; counts of adults were also undertaken. Infestation by larvae was detected from April to August, whereas nymphs were observed from June to October. Infestation by adults was detected throughout the year, and the highest population density occurred from September to March. The number of males was always higher than the number of females, but with considerable reduction in the male : female ratio between April and July. It was observed that 25% of the horses carried 41% of the infesting ticks, and 20% carried only 10% of the ticks during the entire period of the study.     

A prevalent alpha-proteobacterium Paracoccus sp. in a population of the Cayenne ticks (Amblyomma cajennense) from Rio de Janeiro,Brazil

As Rocky Mountain Spotted Fever is the most common tick-borne disease in South America, the presence of Rickettsia sp. in Amblyomma ticks is a possible indication of its endemicity in certain geographic regions. In the present work, bacterial DNA sequences related to Rickettsia amblyommii genes in A. dubitatum ticks, collected in the Brazilian state of Mato Grosso, were discovered. Simultaneously, Paracoccus sp. was detected in aproximately 77% of A. cajennense specimens collected in Rio de Janeiro, Brazil. This is the first report of Paracoccus sp. infection in a specific tick population, and raises the possibility of these bacteria being maintained and/or transmitted by ticks. Whether Paracoccus sp. represents another group of pathogenic Rhodobacteraceae or simply plays a role in A. cajennense physiology, is unknown. The data also demonstrate that the rickettsial 16S rRNA specific primers used forRickettsia spp. screening can also detect Paracoccus alpha-proteobacteria infection in biological samples. Hence, a PCR-RFLP strategy is presented to distinguish between these two groups of bacteria.     

Distributions of the vectors of heartwater, Amblyomma hebraeum and Amblyomma variegatum (Acari: Ixodidae), in Zimbabwe

The tick vectors of heartwater (Cowdria ruminantium infection) in Zimbabwe, Amblyomma hebraeum and Amblyomma variegatum, historically were believed to be confined to the low-lying regions of the south and north-west of the country. However, country-wide surveys performed in 19751980 and 19881991 demonstrated that both species were also established in western parts of the highveld plateau and had started to encroach on the predominantly heartwater-free central and eastern highveld regions. To determine the current distributions of both the vectors and evaluate the potential threat of heartwater to animals in the highveld, a survey of ticks infesting cattle was performed in 1996 at 2994 locations in small-holder and large-scale commercial farming areas throughout Zimbabwe. Amblyomma hebraeum was collected at 1329 locations, A. variegatum at 72 locations and both A. hebraeum and A. variegatum at 13 locations. The results demonstrated that A. hebraeum was present, as previously recorded, throughout the southern half of the country and appeared to have undergone further limited spread into the central and eastern highveld regions. Only the northern-most region of the country appeared to be free of this species. Amblyomma variegatum was collected mainly in the north-west, as previously recorded, but was also found at isolated locations across the central highveld region and along the eastern border with Mozambique. This species was, however, still absent from the southern half and the northern-most regions of the country. An overlap of the distributions of the two species existed within a zone along the southern and eastern regions of the distribution of A. variegatum. These results suggest that the vectors of heartwater are spreading and threaten to introduce heartwater into intensive livestock-producing regions of the country.Exp Appl Acarol 22: 725740 © 1998 Kluwer Academic Publishers     

Factors affecting the distributions of the ticks Amblyomma hebraeum and A. variegatum in Zimbabwe: implications of reduced acaricide usage

The ticks Amblyomma hebraeum and A. variegatum are the main vectors of heartwater, a disease of ruminants caused by Cowdria ruminantium, in the agricultural areas of Zimbabwe. At present, A. hebraeum is widely distributed in the dry southern lowveld, and occurs in at least seven foci in the higher rainfall highveld. Amblyomma variegatum occurs in the Zambezi valley and surrounding dry lowveld areas in the northwest. The distribution of A. hebraeum has changed considerably over the past 70 years, while that of A. variegatum appears to have remained fairly static. The distribution patterns of both species in Zimbabwe display anomalous features; the ticks occur in areas of lowest predicted climatic suitability for survival and development and in areas where the densities of cattle, the most important domestic host, are lowest. The only factor favouring the survival of the species in the lowveld habitats in which they occur is the presence of alternative wildlife hosts for the adult stage. Their absence from more climatically favourable highveld habitats appears to have been the result of intensive acaricide treatment of cattle over a long period and a historic absence of significant numbers of wildlife hosts. Eradication of A. hebraeum and A. variegatum by intensive acaricide treatment of cattle can be achieved in the absence of significant numbers of alternative hosts, because of the long attachment and feeding periods of the adults of these tick species. However, eradication becomes impossible when alternative hosts for the adult stage are present, because a pheromone emitted by attached males attracts the unfed nymphal and adult stages to infested hosts. The unfed ticks are not attracted to uninfested hosts, such as acaricide-treated cattle.Regular acaricide treatment of cattle is expensive and so, for economic reasons, the Government of Zimbabwe is no longer enforcing a policy of strict tick control. It is likely that reduced tick control will result in the spread of Amblyomma ticks to previously uninfested areas. Added to this, recent introductions of various wildlife species to highveld commercial farming areas have created conditions in which the ticks could become established in higher rainfall areas. Amblyomma hebraeum is more likely to spread than A. variegatum, because its adults parasitize a wider range of wildlife hosts (warthogs, medium to large-sized antelope, giraffe, buffalo and rhinoceros), whereas adults of A. variegatum appear to be largely restricted to one wildlife species (buffalo) in Zimbabwe, the distribution of which is now confined to very limited areas of the country, as part of foot and mouth disease control measures. A model to predict the rate of spread of A. hebraeum through the highveld is described.Possible control options for dealing with the spread of Amblyomma ticks and heartwater to previous unaffected highveld areas, include (1) continuation of intensive acaricide treatment of cattle to prevent the spread, (2) establishment of a buffer zone of intensive tick control around affected areas to contain the spread and (3) allow the spread to occur and control heartwater by means of immunization. An economic analysis to determine the costs and benefits of the control options, which takes into account the development of Amblyomma-specific tick control technologies and improved heartwater vaccines, is recommended.Deceased.     

Attraction of Amblyomma variegatum (ticks) to the attraction-aggregation-attachment-pheromone with or without carbon dioxide

The responses of adult Amblyomma variegatum ticks released from various distances to different doses of the synthetic attraction-aggregation-attachment pheromone (AAAP) (made up of ortho-nitrophenol, methyl salicylate and nonanoic acid in paraffin oil), dispensed from the center of circular field plots, were studied in the presence or absence of elevated levels of CO₂. Up to 90% of the ticks released were attracted to the pheromone source in the presence of CO₂ within 3h. CO₂ alone was unattractive, similar to previous findings in Zimbabwe, but unlike results from a Caribbean A. variegatum population, which was significantly attracted to this signal. In the absence of CO₂, smaller but significant proportions of the released ticks were attracted to the pheromone, albeit more slowly, suggesting another variation in the responses of this bont tick to inter- and intra-specific signals. Our results are interpreted in the light of a study undertaken elsewhere demonstrating relatively high heterozygosity among tick populations. Possible directions of further research to explore the use of the pheromone in off-host control of the tick are also highlighted.     

Attractivity of cattle infested by Amblyomma variegatum (Acari: Ixodidae) for conspecific adult ticks from the field in Guadeloupe

Four field experiments were conducted with creole cattle in Guadeloupe to study the attractivity for adult ticks of animals previously infested with males of Amblyomma variegatum producing aggregation attachment pheromones (AAPs). In each experiment, two groups of cattle were used; the first group was infested with adult A. variegatum and the second group was kept free of ticks and pheromones by acaricidal treatment and by washing the hair and skin of previously infested sites. Both groups were then exposed to natural infestation by ticks present in the environment or by ticks bred in the laboratory and released on the pasture. The number of additional ticks infesting animals were compared between the treated and control groups. There were no significant differences between both groups in the numbers of new male ticks infesting the animals during the first days following the beginning of the experiment. Thereafter, comparison of the slopes of infestation indicated that infested cattle were no more attractive for male ticks than uninfested hosts. Uninfested cattle were spontaneously and rapidly infested by male ticks. It is concluded that carbon dioxide or another host component was a major attractant for A. variegatum males. Female ticks attached on predilection sites exclusively in the presence of AAP-producing males, i.e. 5–10 days after the attachment of the first males. Based on these results we postulate that slow-release devices impregnated with pheromones have no additional attractive effect on A. variegatum males under Caribbean conditions. However, they may be useful in inducing the attachment of females, but only in the first days preceding the arrival of pioneer AAP-producing males. © Rapid Science Ltd. 1998     

Experimental infection of the tick Amblyomma cajennense, Cayenne tick, with Rickettsia rickettsii, the agent of Rocky Mountain spotted fever

In the laboratory, Amblyomma cajennense (Acari: Ixodidae) (Fabricius) larvae, nymphs and adults were exposed to Rickettsia rickettsii by feeding on needle-inoculated animals, and thereafter reared on uninfected guinea pigs or rabbits. Regardless of the tick stage that acquired the infection, subsequent tick stages were shown to be infected (confirming transstadial and transovarial transmissions) and were able to transmit R. rickettsii to uninfected animals, as demonstrated by serological and molecular analyses. However, the larval, nymphal and adult stages of A. cajennense were shown to be partially refractory to R. rickettsii infection, as in all cases, only part of the ticks became infected by this agent, after being exposed to rickettsemic animals. In addition, less than 50% of the infected engorged females transmitted rickettsiae transovarially, and when they did so, only part of the offspring became infected, indicating that vertical transmission alone is not enough to maintain R. rickettsii in A. cajennense for multiple generations. Finally, the R. rickettsii-infected tick groups had lower reproductive performance than the uninfected control group. Our results indicate that A. cajennense have a low efficiency to maintain R. rickettsii for successive generations, as R. rickettsii-infection rates should decline drastically throughout the successive tick generations.     

Rickettsial infections of dogs, horses and ticks in Juiz de Fora, southeastern Brazil, and isolation of Rickettsia rickettsii from Rhipicephalus sanguineus ticks

The present study was performed in an area endemic for Brazilian spotted fever (BSF) in Juiz de Fora, state of Minas Gerais, Brazil, during the years 2007 and 2008, when fatal cases of BSF (caused by Rickettsia rickettsii) were reported. Adult ticks (Acari: Ixodidae) identified as Rhipicephalus sanguineus (Latreille) and Amblyomma cajennense (Fabricius) were collected from dogs and horses, respectively, and tested by polymerase chain reaction (PCR). Overall, 13.1% of the Rh. sanguineus ticks and none of the A. cajennense were found to be infected with R. rickettsii. Two isolates of R. rickettsii were successfully established in Vero cell culture from two Rh. sanguineus ticks. An indirect immunofluorescence assay (IFA) using R. rickettsii antigens detected blood serological reaction to R. rickettsii in 67.9% (53/78) of dogs and 41.0% (16/39) of horses living in the study area. Larval offspring from two Rh. sanguineus engorged females, naturally infected by R. rickettsii, were reared to adult stage in the laboratory. All active stages (larvae, nymphs, adults) remained 100% infected by R. rickettsii, which was efficiently transmitted to naïve rabbits. Overall, the results of the present study indicate a potential risk for transmission of R. rickettsii to humans by Rh. sanguineus, an occurrence yet to be documented in Brazil.     

Detection of Rickettsia and Anaplasma from hard ticks in Thailand

We collected a total of 169 adult hard ticks and 120 nymphs from under the leaves of plants located along tourist nature trails in ten localities. The results present data examining the vector competence of ticks of different genera and the presence of Rickettsia and Anaplasma species. The ticks belonged to three genera, Amblyomma, Dermacentor, and Haemaphysalis, comprising 11 species. Rickettsia bacteria were detected at three collection sites, while Anaplasma bacteria were detected at only one site. Phylogenetic analysis revealed new rickettsia genotypes from Thailand that were closely related to Rickettsia tamurae, Rickettsia monacensis, and Rickettsia montana. This study was also the first to show that Anaplasma bacteria are found in Haemaphysalis shimoga ticks and are closely related evolutionarily to Anaplasma bovis. These results provide additional information for the geographical distribution of tick species and tick‐borne bacteria in Thailand and can therefore be applied for ecotourism management.     

Ecology and genetic variation of Amblyomma tonelliae in Argentina

The ecology of Amblyomma tonelliae (Ixodida: Ixodidae), including its seasonal distribution and the development periods of each stage, was investigated during a study carried out over two consecutive years in northwestern Argentina. In addition, the genetic variation of this tick was studied through analyses of 16S rDNA sequences. Amblyomma tonelliae has a 1‐year lifecycle characterized by a long pre‐moult period in larvae with no development of morphogenetic diapause. Larvae peak in abundance during late autumn and early winter; nymphs peak in abundance in spring, and adults do so from late spring to early summer. Amblyomma tonelliae shows a marked ecological preference for the driest areas of the Chaco ecoregion. In analyses of 16S rDNA sequences in genes from different populations of A. tonelliae, values for nucleotide diversity and the average number of nucleotide differences showed genetic diversity within this species to be low. No significant differences were found in comparisons among populations.     

Ecdysteroids in females and eggs of the Ixodid tick Amblyomma hebraeum

Summary

A mated Amblyomma hebraeum female will engorge on a host for about 8 days before detaching and beginning the maturation of its single egg batch which is laid during a period of about 30 days. The feeding period is characterized by an important synthesis of endocuticular material occurring before the rapid feeding phase. This latter phase, correlated with an enormous weight uptake, shows an increase of ecdysteroid levels measured in the whole animal by RIA. However, the hemolymphatic levels of ecdysteroids remain very low (12 pg 20-hydroxyecdysone equivalent (20-OH-E eq.) per μ1. Within 4 days after detachment, the salivary glands degenerate. Ecdysteroid levels in the whole animal continue to increase, reaching high values (about 500 ng 20-OH-E eq./tick) at the moment of oviposition which begins 10–14 days after dropping. During the same period, hemolymphatic ecdysteroid levels increase, rising to a peak (600 pg 20-OH-E eq./μ1) 1 day prior to the beginning of oviposition. Then, the levels decrease and stabilize around 250 pg 20-OH-E eq./μl during egg-laying. Freshly laid eggs contain large amounts of ecdysteroids (2744 pg 20-OH-E eq./mg).

20-Hydroxyecdysone and ecdysone have been found to be the major free ecdysteroids in hemolymph, ovaries and eggs (verified by the HPLC-RIA technique and GC-MF of silylated HPLC fractions). Helix juice (or esterase) labile ecdysteroid conjugates do not seem to be present to any noticeable extent in hemolymph, ovaries and eggs.