Investigation of Ehrlichia spp., Anaplasma spp. and Rickettsia spp. in ectoparasites collected from domestic animals, Rio de Janeiro State, Brazil

A previous study in Paulicéia Municipality, south-eastern Brazil, reported 9.7% of the Amblyomma triste ticks to be infected by Rickettsia parkeri, a bacterial pathogen that causes spotted fever in humans. These A. triste ticks were shown to be associated with marsh areas, where the marsh deer Blastocerus dichotomus is a primary host for this tick species. During 2008-2009, blood serum samples were collected from 140 horses, 41 dogs, 5 opossums (Didelphis albiventris) and 26 humans in farms from Pauliceia Municipality. Ticks were collected from these animals, from vegetation and from additional wildlife in these farms. Overall, 25% (35/140) of the horses, 7.3% (3/41) of the dogs, 3.8% (1/26) of the humans and 100% (5/5) of the opossums were seroreactive (titre ≥64) to spotted fever group (SFG) Rickettsia spp. Multivariate statistical analysis indicated that horses that were allowed to forage in the marsh were 4.8 times more likely to be seroreactive to spotted fever group (SFG) Rickettsia spp than horses that did not forage in the marsh. In addition, horses that had been living in the farm for more than 8.5 years were 2.8 times more likely to be seroreactive to SFG Rickettsia spp than horses that were living for ≤8.5 years. Ticks collected from domestic animals or from vegetation included Amblyomma cajennense, Amblyomma coelebs, Amblyomma dubitatum, Dermacentor nitens and Rhipicephalus microplus. By PCR analyses, only one pool of A. coelebs ticks from the vegetation was shown to be infected by rickettsiae, for which DNA sequencing revealed to be Rickettsia amblyommii. Ticks (not tested by PCR) collected from wildlife encompassed A. cajennense and Amblyomma rotundatum on lizards (Tupinambis sp), and A. cajennense and A. triste on the bird Laterallus viridis. Our results indicate that the marsh area of Paulicéia offers risks of infection by SFG rickettsiae.

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 Paraná, 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 Aragão. 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.

-The previously unknown tadpole of H. giesleri is described and illustrated based on two populations from Southeastern Brazil, one from Rio de Janeiro State and the other from Espirito Santo State. The tadpole of H. microps is redescribed and illustrated from Rio de Janeiro State and compared with that of H. giesleri. The mean length of tadpoles of H. giesleri in stage 33 varies from 17.9 (Rio de Janeiro State) to 22.5 mm (Espirito Santo State). The mean length in H. microps is 16.9 mm. Tadpoles of H. giesleri have the terminal portion of tail orange (red in H. microps). These species can also be distinguished by other details of their color patterns. Remarkable differences of size and color were found between the two studied populations of H. giesleri. The Hyla parviceps group was defined by Duellman and Crump (1974) and modified slightly by Duellman and Trueb (1989). Wild (1992), following the conventional grouping, provided detailed larval characters for this group. The small hylid frogs of the Hyla parviceps group occur mainly in the Amazon Basin and northern South America (Duellman and Crump, 1974). The only exceptions are Hyla microps Peters, 1872 and H. giesleri Mertens, 1950, which occur in southeastern and southern Brazil. These species were originally described from Rio de Janeiro State. Weygoldt and Peixoto (1987) described H. ruschii from Espirito Santo State, suggesting that it could also be a member of the Hyla parviceps group. Hyla microps and H. giesleri were long considered as synonymous due to the great morphological similarity of their adults (Cochran, 1955; Bokermann, 1966; Lutz, 1973; Duellman and Crump, 1974). Heyer (1980) revalidated H. giesleri based on differences found in the mating call and color pattern of subocular region. Bokermann (1963) briefly described the tadpole of H. microps from material collected in Sao Paulo State. Heyer et al. (1990) also described the tadpole of H. microps from Sao Paulo State. Herein, we describe and compare the tadpoles of H. giesleri and H. microps. The description of the former species was based on two distinct populations. MATERIAL AND METHODS All specimens examined in this study belong to the Collection of Departamento de Zoologia, Instituto de Biologia, Universidade Federal do Rio de Janeiro (ZUFRJ). We collected adults and tadpoles of H. giesleri (ZUFRJ 2324, 928) from two populations (Fig. 1), one in Rio de Janeiro State (Municipio de Mage, 22?39'S; 43002'W) and the other in Espirito Santo State (Municipio de Linhares, 19?23'S; 40?04'W). The tadpoles from Rio de Janeiro were obtained from eggs collected in March 1984 and raised in captivity. Those tadpoles from Espirito Santo State were collected in January 1980. The tadpoles of H. microps (ZUFRJ 4145, 4390, 4391) were collected from a single population in Rio de Janeiro State (Municipio de Teresopolis, 22?24'S; 42?57'W) (Fig. 1). The tadpoles ZUFRJ 4145 were obtained from eggs (ZUFRJ 4011) collected in September 1989, the others were collected in February and March 1991. Tadpoles were anesthetized in 0.1% chloretone and stored in 5% formalin. Adults were anesthetized in 0.25% chloretone, preserved in 10% formalin, and stored in 70% alcohol. Some couples collected in amplexus were kept alive to obtain eggs. Tadpoles that emerged from these eggs were reared until the stages desired for study. Tadpoles collected in the field were identified by means of comparisons with those obtained from eggs or reared until metamorphosis and then compared with determined adults. Tadpoles were reared in captivity in aquariums (measurements: 60 x 30 x 30 cm) with slightly acid water and a dense layer of leaves on the bottom. About 20 tadpoles were maintained per aquarium. Fish food was provided regularly. The water temperature was not controlled. Tadpoles in stage 33 (Gosner, 1960) were used in the descriptions and measurements. Measurements were taken according to the methods of Altig (1970) and Duellman (1970), except for interorbital distance, which was taken between This content downloaded from 157.55.39.149 on Sat, 09 Jul 2016 04:57:02 UTC All use subject to http://about.jstor.org/terms

Kappaphycus alvarezii which is endemic to the Indo-Pacific region is the main raw material for kappa carrageenan production. A seedling that was cultivated in Japan (originally from the Philippines) was introduced in a trial, for aquaculture purpose, in 1995, in Sao Paulo State, southeastern region of Brazil. It was later introduced in Santa Catarina State, in Southern Brazil. In 1998, another seedling that was brought from Venezuela, also originally from the Philippines, was commercially introduced at Ilha Grande Bay and later on at Sepetiba Bay, both in Rio de Janeiro State. The aim of this study was to characterize 21 samples from different farms (Rio de Janeiro and Santa Catarina States) and verify if they are K. alvarezii or other species. Based on the intergenic spacer cox2-3 sequences, phylogenetic relationships were inferred through neighbor joining, maximum likelihood, and Bayesian analyses. The topology of the trees suggests that all samples from the different farms form a monophyletic group of K. alvarezii. Statistical analysis of the cox2-3 marker calculated with median-joining network showed 38 variable positions defining 15 haplotypes for the studied samples of Kappaphycus. The most frequent K. alvarezii haplotype grouped the samples cultivated worldwide with the Brazilian samples. These results are important for better productivity and are environmentally desirable for introduction purposes since the species introduced is known and will help focusing the research on this species. This knowledge can be of assistance to the government in setting up environmental and cultivation protocols to achieve sustainability in macroalgae aquaculture.

This study evaluated rickettsial infection in ticks collected on toads from the northern Brazilian Amazon (Amapá state), where to our knowledge there are neither records of ticks from amphibians nor rickettsial infections in ticks. During 2016-2017, a total of 22 free-living toads were captured and identified as Rhinella marina. Overall, 12 (54.5%) toads were parasitized by a total of 97 ticks (6 males, 39 females, 31 nymphs, 21 larvae), giving a mean intensity of 8.1 ticks per infested toad. Two tick species were morphologically identified: Amblyomma rotundatum Koch (31 females, 14 nymphs), and Amblyomma dissimile Koch (6 males, 8 females, 17 nymphs). The 21 larvae were morphologically denoted as Amblyomma sp. Five toads were co-infested by A. rotundatum and A. dissimile. Morphological identifications were confirmed by nucleotide sequencing of fragments of the mitochondrial genes 16S rDNA, 12S rDNA and/or COX1. A total of 54 ticks were analyzed for the presence of rickettsial DNA. Eleven (9 females and 2 nymphs) out of 14 A. rotundatum ticks contained Rickettsia bellii. None of the 25 specimens of A. dissimile (6 males, 6 females, 13 nymphs) contained amplifiable rickettsial DNA. From 15 Amblyomma sp. larvae, a pool of 10 individuals contained Rickettsia sp. strain Colombianensi. Sequencing of the 16S rDNA amplicon derived from the positive pool yielded a sequence of A. dissimile. We detected Rickettsia sp. strain Colombianensi for the first time in Brazil. Prior records of this agent were restricted to Colombia and Honduras. In addition, we report the presence of A. rotundatum for the first time in the state of Amapá, where the only other record of A. dissimile was registered over 20years ago.

BackgroundMatrix Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) has been shown to be an effective tool for the rapid identification of arthropods, including tick vectors of human diseases.Methodology/Principal FindingsThe objective of the present study was to evaluate the use of MALDI-TOF MS to identify tick species, and to determine the presence of rickettsia pathogens in the infected Ticks. Rhipicephalus sanguineus and Dermacentor marginatus Ticks infected or not by R. conorii conorii or R. slovaca, respectively, were used as experimental models. The MS profiles generated from protein extracts prepared from tick legs exhibited mass peaks that distinguished the infected and uninfected Ticks, and successfully discriminated the Rickettsia spp. A blind test was performed using Ticks that were laboratory-reared, collected in the field or removed from patients and infected or not by Rickettsia spp. A query against our in-lab arthropod MS reference database revealed that the species and infection status of all Ticks were correctly identified at the species and infection status levels.Conclusions/SignificanceTaken together, the present work demonstrates the utility of MALDI-TOF MS for a dual identification of tick species and intracellular bacteria. Therefore, MALDI-TOF MS is a relevant tool for the accurate detection of Rickettsia spp in Ticks for both field monitoring and entomological diagnosis. The present work offers new perspectives for the monitoring of other vector borne diseases that present public health concerns.

tional Center for Biotechnology Information basic local alignment sequence tool, BLAST (www.ncbi.nlm.nih.gov/blast/Blast.cgi).To determine whether R. felis occurs on another Caribbean island, we tested 32 C. felis fl eas from Dominica and found 1 (3%) to be positive by PCR when primers targeting ompA were used.The sequence obtained was also identical to that of R. felis URRxCal2.Our study provides further evidence that cats can be sentinels for the presence of rickettsiae (1).However, although rickettsemia can develop in cats experimentally infected with R. felis (3), no compelling evidence shows that cats help maintain the organism or transmit it to humans (8,9).Rather, it appears that C. felis fl eas, which are also commonly found on dogs and to a lesser extent other mammals, are the major reservoir hosts and vectors of infection, although the exact mechanisms are unknown (10).Our study also expands the known distribution of R. felis and should

The eco-epidemiological scenario of spotted fever (SF), a tick-borne disease that affects humans and other animals in several countries around the world, was analyzed in Rio de Janeiro (RJ) State, Brazil. During the last 34 years, 990 SF cases were reported in RJ (the Brazilian state with the highest population density), including 116 cases confirmed by serology (RIFI) or PCR, among 42.39% of the municipalities with reported cases of SF. The epidemiologic dynamics of SF in RJ State are very heterogeneous in time and space, with outbreaks, high mortality rates and periods of epidemiological silence (no SF cases reported). Furthermore, it exhibited a changing epidemiological profile from being rural to becoming an urban disease. This study identified arthropods infected with Rickettsia felis, R. bellii and R. rickettsii, and found that the abundance of ectoparasites was associated with specific hosts. The R. rickettsii-vector-host relationship was most evident in species-specific parasitism. This suggests that the association between dogs, cattle, horses, capybaras and their main ectoparasites, Rhipicephalus sanguineus and Ctenocephalides felis, Rhipicephalus microplus, Dermacentor nitens, and Amblyomma dubitatum, respectively, has a key role in the dynamics of R. rickettsii transmission in enzootic cycles and the maintenance of carrier ectoparasites, thus facilitating the existence of endemic areas with the ability to produce epidemic outbreaks of SF in RJ. This study found confirmed human infections for only the R. rickettsii carrier Amblyomma sculptum, which reinforces the importance of this species as a vector of the pathogen in Brazil. This study can be adapted to different eco-epidemiological scenarios of spotted fever throughout the Americas.

Brazilian spotted fever (BSF), caused by Rickettsia rickettsii, is endemic in the municipality of Americana, southeastern Brazil, where the disease is transmitted by the tick Amblyomma cajennense. This study evaluated the tick fauna and rickettsial infection in free-living ticks that were captured monthly using dry ice traps in areas endemic for BSF in Americana, from July 2009 to June 2010. Two tick species were captured: A. cajennense (6,122 larvae; 4,265 nymphs; 2,355 adults) and Amblyomma dubitatum (7,814 larvae; 3,364 nymphs; 1,193 adults). The immature stages of A. cajennense and A. dubitatum had similar distribution through the 12-month period, with larvae of both species collected in highest numbers between April and July, and nymphs between June and October. The highest numbers of A. cajennense adults were collected between October and December, whereas A. dubitatum adults were collected in relatively similar numbers throughout the 12-month period. Rickettsial infection was evaluated by means of PCR in 1,157 A. cajennense and 1,040 A. dubitatum ticks; only 41 (3.9%) A. dubitatum were found to be infected by Rickettsia bellii. The present study showed that the areas of Americana that are endemic for BSF are characterized by high environmental burdens of A. cajennense and A. dubitatum.

During 2008-2010, ticks were collected from road-killed wild animals within the Serra dos Orgãos 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 Orgãos, 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 Orgãos as human pathogen must be taken into account, because both R. tamurae and R. monacencis have been reported infecting human beings.

The Japanese invasive jumping snail Ovachlamys fulgens is a pest of ornamental plants and an intermediate host of a nematode that causes eosinophilic meningitis. We expand its distribution to eight municipalities from Rio de Janeiro State, and one locality from the Paraná State, and generated for the first time partial sequences of the cytochrome c oxidase subunit I (COI) gene for Brazilian populations. External morphology, reproductive system, shell, radula, and jaw were also analyzed and described. Twenty-one lots were collected from Rio de Janeiro, Niterói, Magé, Miguel Pereira, Petrópolis, Teresópolis, Nova Friburgo, Bom Jardim and Paraty, in Rio de Janeiro State, and from Foz do Iguaçu, Paraná State. External morphology, shell and reproductive system were typical of O. fulgens, with some peculiarities found in the shell and radula. A single haplotype was found, which was 100% similar to sequences of COI available in GenBank for specimens from Japan and Argentina. The species seems to be adapted to many habitats and be rapidly expanding its distribution in Southeastern and Southern Brazil, and other South America countries. We highlight the importance of monitoring O. fulgens, considering its potential to compete with native mollusks, attack several plants, and be a transmitter of diseases.

Pathogens can manipulate the phenotypic traits of their hosts and vectors, maximizing their own fitness. Among the phenotypic traits that can be modified, manipulating vector behavior represents one of the most fascinating facets. How pathogens infection affects behavioral traits of key insect vectors has been extensively investigated. Major examples include Plasmodium, Leishmania and Trypanosoma spp. manipulating the behavior of mosquitoes, sand flies and kissing bugs, respectively. However, research on how pathogens can modify tick behavior is patchy. This review focuses on current knowledge about the behavioral changes triggered by Anaplasma, Borrelia, Babesia, Bartonella, Rickettsia and tick-borne encephalitis virus (TBEV) infection in tick vectors, analyzing their potential adaptive significance. As a general trend, being infected by Borrelia and TBEV boosts tick mobility (both questing and walking activity). Borrelia and Anaplasma infection magnifies Ixodes desiccation resistance, triggering physiological changes (Borrelia: higher fat reserves; Anaplasma: synthesis of heat shock proteins). Anaplasma infection also improves cold resistance in infected ticks through synthesis of an antifreeze glycoprotein. Being infected by Anaplasma, Borrelia and Babesia leads to increased tick survival. Borrelia, Babesia and Bartonella infection facilitates blood engorgement. In the last section, current challenges for future studies are outlined.

Diversity of rickettsiae in a rural community in northern California

Rickettsial infection in ticks (Acari: Ixodidae) from reptiles in the Colombian Caribbean

Spotted fever illness caused by the tick-borne pathogen Rickettsia parkeri has emerged in the Pampa biome in southern Brazil, where the tick Amblyomma tigrinum is implicated as the main vector. Because domestic dogs are commonly parasitized by A. tigrinum, this canid is also a suitable sentinel for R. parkeri-associated spotted fever. Herein, we investigate rickettsial infection in ticks, domestic dogs and small mammals in a natural reserve of the Pampa biome in southern Brazil. The ticks A. tigrinum, Amblyomma aureolatum and Rhipicephalus sanguineus were collected from dogs. Molecular analyses of ticks did not detect R. parkeri; however, at least 34% (21/61) of the A. tigrinum ticks were infected by the non-pathogenic agent 'Candidatus Rickettsia andeanae'. Serological analyses revealed that only 14% and 3% of 36 dogs and 34 small mammals, respectively, were exposed to rickettsial antigens. These results indicate that the study area is not endemic for R. parkeri rickettsiosis. We tabulated 10 studies that reported rickettsial infection in A. tigrinum populations from South America. There was a strong negative correlation between the infection rates by R. parkeri and 'Candidatus R. andeanae' in A. tigrinum populations. We propose that high infection rates by 'Candidatus R. andeanae' might promote the exclusion of R. parkeri from A. tigrinum populations. The mechanisms for such exclusion are yet to be elucidated.