Development of resistance to nymphs of Amblyomma cajennense ticks (Acari : Ixodidae) in dogs

Ticks have long been regarded as constraints to humans and domestic animals, but hosts often develop resistance to ticks after repeated infestations. The purpose of this investigation was to study the possible acquisition of immunity in domestic dogs to nymphs of A. cajennense by determining the tick alimentary performance after successive controlled infestations. Mean engorged weight of nymphs was not significantly different among the three infestations; molting rate from nymph to adult ticks, and the percentage of nymph recovery were also very close in all infestations. These results are similar to those obtained in studies of the dog-adult Rhipicephalus sanguineus interface. It is concluded that domestic dogs do not develop resistance against nymphs of A. cajennense ticks.

GUS expression in sweet oranges (Citrus sinensis L. Osbeck) driven by three different phloem-specific promoters

Huanglongbing (HLB) is associated with Candidatus Liberibacter spp., endogenous, sieve tube-restricted bacteria that are transmitted by citrus psyllid insect vectors. Transgenic expression in the phloem of specific genes that might affect Ca. Liberibacter spp. growth and development may be an adequate strategy to improve citrus resistance to HLB. To study specific phloem gene expression in citrus, we developed three different binary vector constructs with expression cassettes bearing the beta-glucuronidase (GUS) reporter gene (uidA) under the control of one of the three different promoters: Citrus phloem protein 2 (CsPP2), Arabidopsis thaliana phloem protein 2 (AtPP2), and Arabidopsis thaliana sucrose transporter 2 (AtSUC2). Transgenic lines of 'Hamlin', 'Pera', and 'Valencia' sweet oranges [Citrus sinensis (L.) Osbeck] were produced via Agrobacterium tumefaciens transformation. The epicotyl segments collected from in vitro germinated seedlings were used as explants. The gene nptII, which confers resistance to the antibiotic kanamycin, was used for selection. The transformation efficiency was expressed as the number of GUS-positive shoots over the total number of explants and varied from 1.54 to 6.08 % among the three cultivars and three constructs studied. Several lines of the three sweet orange cultivars analyzed using PCR and Southern blot analysis were genetically transformed with the three constructs evaluated. The histological GUS activity in the leaves indicates that the uidA gene was preferentially expressed in the phloem, which suggests that the use of the three promoters might be adequate for producing HLB-resistant transgenic sweet oranges. The results reported here conclusively demonstrate the preferential expression of GUS in the phloem driven by two heterologous and one homologous gene promoters. Key message The results reported here conclusively demonstrate the preferential expression of GUS in the phloem driven by two heterologous and one homologous gene promoters.

Weedy Hosts and Prevalence of Potential Leafhopper Vectors (Hemiptera: Cicadellidae) of a Phytoplasma (16SrIX group) Associated With Huanglongbing Symptoms in Citrus Groves

Huanglongbing (HLB) is a severe citrus (Citrus spp.) disease associated with the bacteria genus Candidatus Liberibacter, detected in Brazil in 2004. Another bacterium was found in association with HLB symptoms and characterized as a phytoplasma belonging to the 16SrIX group. The objectives of this study were to identify potential leafhopper vectors of the HLB-associated phytoplasma and their host plants. Leafhoppers were sampled every other week for 12 mo with sticky yellow cards placed at two heights (0.3 and 1.5 m) in the citrus tree canopy and by using a sweep net in the ground vegetation of two sweet orange, Citrus sinensis (L.) Osbeck, groves infected by the HLB-phytoplasma in Sao Paulo state. Faunistic analyses indicated one Agalliinae (Agallia albidula Uhler) and three Deltocephalinae [Balclutha hebe (Kirkaldy), Planicephalus flavicosta (Stal), and Scaphytopius (Convelinus) marginelineatus (Stal)] species, as the most abundant and frequent leafhoppers (Hemiptera: Cicadellidae). Visual observations indicated an association of leafhopper species with some weeds and the influence of weed species composition on leafhopper abundance in low-lying vegetation. S. marginelineatus and P. flavicosta were more frequent on Sida rhombifolia L. and Althernantera tenella Colla, respectively, whereas A. albidula was observed more often on Conyza bonariensis (L.) Cronq. and B. hebe only occurred on grasses. DNA samples of field-collected S. marginelineatus were positive by polymerase chain reaction and sequencing tests for the presence of the HLB-phytoplasma group, indicating it as a potential vector. The association of leafhoppers with their hosts may be used in deciding which management strategies to adopt against weeds and diseases in citrus orchards.

Coxiella symbiont in the tick Ornithodoros rostratus (Acari: Argasidae)

In the present study, the presence of tick-associated bacteria and protozoa in Ornithodoros rostratus ticks (adults, nymphs, and eggs) from the Pantanal region of Brazil were determined by molecular detection. In these ticks, DNA from protozoa in the genera Babesia and Hepatozoon, and bacteria from the genera Rickettsia, Borrelia, Anaplasma, and Ehrlichia were not detected. Conversely, all tested ticks (100%) yielded PCR products for 3 Coxiella genes (16S rRNA, pyrG, cap). PCR and phylogenetic analysis of 3 amplified genes (16S rRNA, pyrG, cap) demonstrated that the agent infecting O. rostratus ticks was a member of the genus Coxiella. This organism grouped with Coxiella symbionts of other soft tick species (Argasidae), having different isolates of C. burnetii as a sister group, and these 2 groups formed a clade that grouped with another clade containing Coxiella symbionts of hard tick species (Ixodidae). Analysis of tick mitochondrial 16S rRNA gene database composed mostly of tick species previously shown to harbor Coxiella symbionts suggests a phylogenetic congruence of ticks and their Coxiella symbionts. Furthermore, these results suggest a very long period of coevolution between ticks and Coxiella symbionts and indicates that the original infection may have occurred in an ancestor common to the 2 main tick families, Argasidae (soft ticks) and Ixodidae (hard ticks). However, this evolutionary relationship must be confirmed by more extensive testing of additional tick species and expanded populations. (c) 2012 Elsevier GmbH. All rights reserved.

Molecular characterization of a phytoplasma of group 16SrIX related to 'Ca. Phytoplasma phoenicium' in periwinkle in Brazil

Periwinkle (Catharanthus roseus), a tropical perennial plant, was found to be infected by a phytoplasma. Plants exhibiting virescence, phyllody and variegation symptoms were collected in the states of Minas Gerais and Sao Paulo, Brazil. The phytoplasma was transmitted by grafting from an infected periwinkle plant to healthy plants and by dodder to a citrus plant. Phytoplasma isolates from periwinkle plants from Brazil had the 16S rDNA gene sequenced and were classified in the 16SrIX group, subgroup A, belonging to the 'Candidatus P. phoenicium' species.

Nine New Species of Bugula Oken (Bryozoa: Cheilostomata) in Brazilian Shallow Waters

Background: Bugula is a speciose genus of marine bryozoans, represented by both endemic and cosmopolitan species distributed in tropical and temperate waters and important to marine biologists because of the occurrence of many species in harbor and fouling communities, therefore as potential invaders. The southeastern Brazilian coast in the southern Atlantic hosts the highest known diversity of the genus, a status intimately associated with the intensity of collecting efforts. Methodology: Morphological data based on the examination of living specimens, scanning electron and light microscopic images, and morphometric analyses were used to assess the diversity of Bugula along the coastal areas of southern, northeastern, and southeastern Brazil. In this study, morphological species boundaries were based mainly on avicularian characters. For two morphologically very similar species, boundaries are partially supported by 16 S rDNA molecular data. Results: Nine species are newly described from Brazil, as follows: Bugula bowiei n. sp. (= Bugula turrita sensu Marcus, 1937) from the southern, northeastern, and southeastern coasts; Bugula foliolata n. sp. (= Bugula flabellata sensu Marcus, 1938), Bugula guara n. sp., Bugula biota n. sp. and Bugula ingens n. sp from the southeastern coast; Bugula gnoma n. sp. and Bugula alba n. sp. from the northeastern coast; Bugula rochae n. sp. (= Bugula uniserialis sensu Marcus, 1937) from the southern coast; and Bugula migottoi n. sp., from the southeastern and southern coasts. Conclusion: The results contribute to the morphological characterization and the knowledge of the species richness of the genus in the southwestern Atlantic (i.e., Brazil), through the description of new species in poorly sampled areas and also on the southeastern coast of that country. Additionally, the taxonomic status of the Brazilian specimens attributed to B. flabellata, B. turrita and B. uniserialis are clarified by detailed studies on zooidal and avicularia morphology.

A surrogate life cycle of Amblyomma ovale Koch, 1844

As ticks spend most of their time off-host, the environment is a major selective force of these parasites. In fact, human impact on landscapes has favored a minority of tick species which became well-known pests. However, this is an ongoing process and novel pests may arise. We herein report a surrogate life cycle of a neotropical tick species. Amblyomma ovate, and which may be related to an increased risk of human rickettsiosis. Under natural conditions, adults of this tick species feed on carnivores and exhibit non-nidicolous ambush behavior, whereas larvae and nymphs feed on small rodents and birds. In an anthropized spot within an Atlantic rainforest reserve of Brazil, an A. ovate population exhibited a nidicolous behavior with all 3 tick stages feeding on the dog. This dog's infestation was outstandingly high, and it displayed the highest anti-Rickettsia titers and harbored Rickettsia-infected ticks. (c) 2012 Elsevier GmbH. All rights reserved.

Brazilian spotted fever: Real-time PCR for diagnosis of fatal cases

Suspicion of Brazilian spotted fever (BSF) should occur in endemic regions upon surveillance of the acute febrile icteric hemorrhagic syndrome (AFIHS). However, limitations associated with currently available laboratory tests pose a challenge to early diagnosis, especially in fatal cases. Two real-time PCR (qPCR) protocols were evaluated to diagnose BSF in 110 fatal AFIHS cases, collected in BSF-endemic regions in 2009-2010. Of these, 24 were positive and 86 negative by indirect immunofluorescence (IFA) assay (cutoff IgG and/or IgM >= 128). DNA from these samples was used in the qPCR protocols: one to detect Rickettsia spp. (Citrate synthase gene) and another to determine spotted fever group (SFG) Rickettsia species (OmpA gene). Of the 24 IFA-positive samples, 5 (21%) were positive for OmpA and 9 (38%) for citrate synthase. In the IFA-negative group (n = 86), OmpA and citrate synthase were positive in 23 (27%) and 27 (31%), respectively. These results showed that the 2 qPCR protocols were about twice as sensitive as the IFA test alone (93% concordance). In conclusion, qPCR is a sensitive method for the diagnosis of fatal BSF cases and should be considered for routine surveillance of AFIHS in places like Brazil, where spotted fever-related lethality is high and other endemic diseases like dengue and leptospirosis can mislead diagnosis. (C) 2012 Elsevier GmbH. All rights reserved.

Rickettsial Infection in Ticks (Acari: Ixodidae) Collected on Birds in Southern Brazil

The aim of the study was to evaluate rickettsial infection in ticks from wild birds of the Semidecidual and Atlantic Rainforest remnants of three municipalities of the State of Parana, southern Brazil. Overall, 53 larvae and nymphs collected from birds were checked for the presence of Rickettsia DNA by molecular tests. Five tick species were tested: Amblyomma aureolatum (Pallas), Amblyomma calcaratum Neumann, Amblyomma longirostre (Koch), Amblyomma ovale Koch, and Amblyomma parkeri Fonseca and Aragao. A. longirostre ticks were infected with the spotted fever group agents Rickettsia amblyommii strain AL (32.3% infection rate) and Rickettsia parkeri strain NOD (5.9% infection rate). A new rickettsial genotype was detected in the tick A. parkeri (50% infection rate), which had never been reported to be infected by rickettsiae. Through phylogenetic analysis, this new genotype, here designated as strain ApPR, grouped in a cluster composed by different strains of Rickettsia africae, Rickettsia sibirica, and R. parkeri. We consider strain ApPR to be a new genotype of R. parkeri. This study reports for the first time rickettsial infection in ticks from birds in southern Brazil. The role of migrating birds in the dispersal of these rickettsial strains should be considered in ecological studies of spotted fever group agents in Brazil.

Rickettsial Infection in Ticks Collected from Road-Killed Wild Animals in Rio de Janeiro, Brazil

During 2008D2010, ticks were collected from road-killed wild animals within the Serra dos Orgaos National Park area in the state of Rio de Janeiro, Brazil. In total, 193 tick specimens were collected, including Amblyomma dubitatum Neumann and Amblyomma cajennense (F.) from four Hydrochoerus hydrochaeris (L.), Amblyomma calcaratum Neumann and A. cajennense from four Tamandua tetradactyla (L.), Amblyomma aureolatum (Pallas) and A. cajennense from five Cerdocyon thous L., Amblyomma longirostre (Koch) from one Sphiggurus villosus (Cuvier), Amblyomma varium Koch from three Bradypus variegatus Schinz, and A. cajennense from one Buteogallus meridionalis (Latham). Molecular analyses based on polymerase chain reaction targeting two rickettsial genes (gltA and ompA) on tick DNA extracts showed that 70.6% (12/17) of the A. dubitatum adult ticks, and all Amblyomma sp. nymphal pools collected from capybaras were shown to contain rickettsial DNA, which after DNA sequencing, revealed to be 100% identical to the recently identified Rickettsia sp. strain Pampulha from A. dubitatum ticks collected in the state of Minas Gerais, Brazil. Phylogenetic analysis with concatenated sequences (gltA-ompA) showed that our sequence from A. dubitatum ticks, referred to Rickettsia sp. strain Serra dos Orgaos, segregated under 99% bootstrap support in a same cluster with Old World rickettsiae, namely R. tamurae, R. monacensis, and Rickettsia sp. strain 774e. Because A. dubitatum is known to bite humans, the potential role of Rickettsia sp. strain Serra dos Orgaos as human pathogen must be taken into account, because both R. tamurae and R. monacencis have been reported infecting human beings.

Epidemiology of Brazilian spotted fever in the Atlantic Forest, state of Sao Paulo, Brazil

The tick-borne bacterium Rickettsia rickettsii is the aetiological agent of Brazilian spotted fever (BSF). The present study evaluated tick infestations on wild and domestic animals, and the rickettsial infection in these animals and their ticks in 7 forest areas adjacent to human communities in the Sao Paulo Metropolitan Area (SPMA). The results were compared to ecological traits of each sampled area. Two main tick species, Amblyomma aureolatum and Rhipicephalus sanguineus, were collected from dogs. The major ticks found on small mammals and birds were Ixodes loricatus and Amblyomma longirostre, respectively. Both anti-R. rickettsii antibodies and R. rickettsii-infected ticks were detected on dogs from only 2 areas in the southern part of the SPMA, which were considered to be endemic for BSF; the remaining 5 areas were considered to be non-endemic. Ecologically, the BSF-endemic areas clearly differed from the non-endemic areas by the presence of significantly more degraded forest patches in the former. The present results corroborate historical observations that have indicated that all human cases of BSF in the SPMA were contracted in the southern part of this metropolitan area. However, not all forest patches in the southern part of the SPMA were shown to be associated with BSF endemism.

Exposure of small mammals to ticks and rickettsiae in Atlantic Forest patches in the metropolitan area of Recife, North-eastern Brazil

Between December 2007 and March 2009, small mammals were captured in 6 Atlantic Forest patches in Brazil. We assessed tick-host associations and whether they differ among forest strata, sites, seasons, and host age classes or between sexes. Moreover, we assessed the exposure of animals to Rickettsia spp. In total, 432 animals were captured and 808 ticks were found on 32-9% of them. Significant differences were found among host species, collection sites, and forest strata; microhabitat preference was a strong risk factor for tick infestation. The highest tick density rates were recorded in forest fragments settled in rural areas; 91.3% of the ticks were collected from animals trapped in these forest fragments. A high prevalence (68.8%) of antibodies to Rickettsia spp. was detected among animals. This study suggests that disturbed Atlantic Forest fragments provide an environment for ticks and small mammals, which are highly exposed to rickettsiae. It also indicates that forest patches settled in rural areas are usually associated with higher small mammal diversity as well as with higher tick density rates.

Rickettsial infection in Amblyomma cajennense ticks and capybaras (Hydrochoerus hydrochaeris) in a Brazilian spotted fever-endemic area

Brazilian spotted fever (BSF), caused by the bacterium Rickettsia rickettsii, is the deadliest spotted fever of the world. In most of the BSF-endemic areas, capybaras (Hydrochoerus hydrochaeris) are the principal host for the tick Amblyomma cajennense, which is the main vector of BSF. In 2012, a BSF case was confirmed in a child that was bitten by ticks in a residential park area inhabited by A. cajennense-infested capybaras in Itú municipality, southeastern Brazil. Host questing A. cajennense adult ticks were collected in the residential park and brought alive to the laboratory, where they were macerated and intraperitoneally inoculated into guinea pigs. A tick-inoculated guinea pig that presented high fever was euthanized and its internal organs were macerated and inoculated into additional guinea pigs (guinea pig passage). Tissue samples from guinea pig passages were also used to inoculate Vero cells through the shell vial technique. Infected cells were used for molecular characterization of the rickettsial isolate through PCR and DNA sequencing of fragments of three rickettsial genes (gltA, ompA, and ompB). Blood serum samples were collected from 172 capybaras that inhabited the residential park. Sera were tested through the immunofluorescence assay using R. rickettsii antigen. A tick-inoculated guinea pig presented high fever accompanied by scrotal reactions (edema and marked redness). These signs were reproduced by consecutive guinea pig passages. Rickettsia was successfully isolated in Vero cells that were inoculated with brain homogenate derived from a 3rd passage-febrile guinea pig. Molecular characterization of this rickettsial isolate (designated as strain ITU) yielded DNA sequences that were all 100% identical to corresponding sequences of R. rickettsii in Genbank. A total of 83 (48.3%) out of 172 capybaras were seroreactive to R. rickettsii, with endpoint titers ranging from 64 to 8192. A viable isolate of R. rickettsii was obtained from the tick A. cajennense, comprising the first viable R. rickettsi isolate from this tick species during the last 60 years. Nearly half of the capybara population of the residential park was seroreactive to R. rickettsii, corroborating the findings that the local A. cajennense population was infected by R. rickettsii.

New records and human parasitism by Ornithodoros mimon (Acari: Argasidae) in Brazil

The bat tick Ornithodoros mimon Kohls, Clifford & Jones is currently known by only few reports in Bolivia, Uruguay, Argentina, and the state of São Paulo in southeastern Brazil. Here, we expand the distribution of O. mimon in Brazil to the states of Minas Gerais (southeastern region), Goiás (central-western), Pernambuco, and Rio Grande do Norte (northeastern). Ticks were collected on human dwellings, where there had been repeated complains of tick bites on persons during the night. Tick bites were generally followed by intense inflammatory reactions that lasted for several weeks at the bite site. Bats and opossums were reported to inhabit the attic of the infested houses. In addition, a free-ranging opossum (Didelphis albiventris Lund) trapped in Rio Grande do Norte was found infested by argasid larvae. Based on morphological and/or molecular analysis, all ticks were identified as O. mimon. From one of the sites (Tiradentes, state of Minas Gerais), 20 field-collected nymphs were tested by a battery of polymerase chain reaction protocols targeting tick-borne microorganisms of the genera Babesia, Hepatozoon, Rickettsia, Borrelia, Anaplasma, Ehrlichia, and Coxiella; no tick specimen was found infected by any of these microorganism genera. The current study expands northwards the distribution of O. mimon, which has been shown to be very harmful to humans because of the intense inflammatory response that usually occurs after tick bites.

Propionsäureabbau in NawaRo-Biogasanlagen

Die Energiewende ist begleitet von dem Ausbau erneuerbarer Energien. Dabei spielt die Energiegewinnung aus Biomasse eine wichtige Rolle. Der optimale Betrieb einer Biogasanlage erfordert eine stabile Methanproduktion, welche jedoch durch die Akkumulation von Propionsäure nachhaltig gestört werden kann. Aus diesem Grund ist der mikrobielle Abbau dieser Substanz von besonderem Interesse. Die Thermodynamik des anaeroben bakteriellen Abbaus von Propionsäure erfordert die syntrophe Verwertung des entstehenden Wasserstoffs durch Wasserstoff-verbrauchende Mikroorganismen, beispielsweise methanogene Archaea.rnMit dem Ziel, die Erkenntnislage der Propionat-Verwertung in NawaRo-Biogasanlagen zu erweitern, sollten Propionat-verwertende Anreicherungskulturen aus NawaRo-Biogasanlagen etabliert, charakterisiert und molekularbiologisch analysiert werden.rnAus landwirtschaftlichen Biogasanlagen wurden reproduzierbar Propionat-verwertende Anreicherungskulturen mittels anaerober Kultivierungstechniken etabliert. Die anaerob Propionat-verwertende Aktivität der Kulturen blieb über Jahre erhalten und konnte unter verschiedenen Bedingungen charakterisiert werden. Die Analyse der sukzessiven Diversität von vier Anreicherungskulturen ermöglichte einen Einblick in die sich während der Propionat-Verwertung sukzessiv verändernde mikrobielle Diversität. Dabei wurden die aus der 16S rDNA-Analyse resultierenden Sequenzcluster MP-1 (Cryptanaerobacter sp./ Pelotomaculum sp.), MP-6 und MP-15 (beide ''Candidatus Cloacamonas sp. ''), sowie MP-9 (Syntrophobacter sulfatireducens) als potentiell Propionat-verwertende Schlüsselspezies identifiziert. Mit S. sulfatireducens wurde eine bekannte syntroph Propionat-verwertende Spezies gefunden. Die Sequenzen von MP-1 waren nahe verwandt mit Pelotomaculum schinkii, ebenfalls eine beschriebene syntroph Propionat-verwertende Spezies. Bei dem nächsten Verwandten der Cluster MP-6 und MP-15 handelte es sich um ''Candidatus Cloacamonas acidaminovorans'', eine bisher unkultivierbare Spezies, dessen Genom für den gesamten Abbauweg der syntrophen Propionat-Oxidation codiert. Syntrophobacter sulfatireducens kam zusammen mit Vertretern der methanogenen Gattungen Methanoculleus, Methanosaeta und Methanomethylovorans vor. Als methanogener Partner von Cryptanaerobacter sp./ Pelotomaculum sp. dominierte die Gattung Methanosarcina. Aufgrund der starken Präsenz der syntroph Acetat oxidierenden Spezies Tepidanaerobacter acetatoxydans (Sequenz-Cluster MP-3), sowie potentiell homoacetogener Arten, wurde zudem ein theoretischer Zusammenhang der Propionat-Verwertung mit der syntrophen Acetat-Oxidation und der autotrophen Homoacetogenese vorgeschlagen.rn