Serological evidence of Rickettsia parkeri as the etiological agent of rickettsiosis in Uruguay

We report three new rickettsiosis human cases in Uruguay. The three clinical cases presented clinical manifestations similar to previous reported cases of Rickettsia parkeri in the United States; that is mild fever (< 40 ºC), malaise, headache, rash, inoculation eschar at the tick bite site, regional lymphadenopathy, and no lethality. Serological antibody-absorption tests with purified antigens of R. parkeri and Rickettsia rickettsii, associated with immunofluorescence assay indicated that the patients in two cases were infected by R. parkeri. Epidemiological and clinical evidences, coupled with our serological analysis, suggest that R. parkeri is the etiological agent of human cases of spotted fever in Uruguay, a disease that has been recognized in that country as cutaneous-ganglionar rickettsiosis.

Experimental Infection of the Opossum Didelphis aurita by Rickettsia felis, Rickettsia bellii, and Rickettsia parkeri and Evaluation of the Transmission of the Infection to Ticks Amblyomma cajennense and Amblyomma dubitatum

This work evaluated the infection of opossums (Didelphis aurita) by Rickettsia felis, Rickettsia bellii, and Rickettsia parkeri and their role as amplifier hosts for horizontal transmission to Amblyomma cajennense and/or Amblyomma dubitatum ticks. Infection in D. aurita was induced by intraperitoneal inoculation with R. felis (n = 4 opossums), R. bellii (n = 4), and R. parkeri (n = 2). Another group of six opossums were inoculated intraperitoneally with Leibovitz-15 sterile culture medium, representing the uninfected groups (n = 2 opossums simultaneously to each infected group). Opossum blood samples collected during the study were used for DNA extraction, followed by real-time polymerase chain reaction targeting the rickettsial gene gltA, hematology, and detection of Rickettsia spp.-reactive antibodies by indirect immunofluorescence assay. Opossums were infested with uninfected A. cajennense and/or A. dubitatum for 30 days postinoculation (DPI). Flat ticks molted from ticks fed on opossums were allowed to feed on uninfected rabbits, which were tested for seroconversion by immunofluorescence assay. Samples of flat ticks were also tested by real-time polymerase chain reaction. Inoculated opossums showed no clinical abnormalities. Antibodies to Rickettsia spp. were first detected at the second to fourth DPI, with detectable titers until the 150th DPI. Rickettsemia was detected only in one opossum inoculated with R. parkeri, at the eighth DPI. Only one A. cajennense tick (2.0%) previously fed on a R. parkeri-inoculated opossum became infected. None of the rabbits infested with opossum-derived ticks seroconverted. The study demonstrated that R. felis, R. bellii, and R. parkeri were capable to produce antibody response in opossums, however, with undetectable rickettsemia for R. felis and R. bellii, and very low rickettsemia for R. parkeri. Further studies must be done with different strains of these rickettsiae, most importantly the strains that have never gone through in vitro passages.

Redescription of the female, description of the male, and several new records of Amblyomma parkeri (Acari: Ixodidae), a South American tick species

The tick Amblyomma parkeri Fonseca and Arago was described in 1952, based on female and immature ticks collected in the states of So Paulo and Santa Catarina, Brazil. Thereafter, there has been no further report of A. parkeri, and the male has remained unknown. Herein, we examined ticks collected on porcupines from a locality in the state of So Paulo. Some of the ticks were identified as Amblyomma longirostre (Koch, 1844), whereas others as A. parkeri, including male specimens, for which we provide the first description. We also provide additional reports of A. parkeri after examining collections of A. longirostre and Amblyomma geayi Neumann, 1899 from different tick collections. Morphological evidence to support the original description of A. parkeri is presented, supported by molecular analyses of portions of the 16S rRNA and 12S rRNA mitochondrial genes. Morphological particularities to separate A. parkeri, A. longirostre, and A. geayi are provided.

Serological evidence of Rickettsia parkeri as the etiological agent of rickettsiosis in Uruguay

We report three new rickettsiosis human cases in Uruguay. The three clinical cases presented clinical manifestations similar to previous reported cases of Rickettsia parkeri in the United States; that is mild fever (< 40 ºC), malaise, headache, rash, inoculation eschar at the tick bite site, regional lymphadenopathy, and no lethality. Serological antibody-absorption tests with purified antigens of R. parkeri and Rickettsia rickettsii, associated with immunofluorescence assay indicated that the patients in two cases were infected by R. parkeri. Epidemiological and clinical evidences, coupled with our serological analysis, suggest that R. parkeri is the etiological agent of human cases of spotted fever in Uruguay, a disease that has been recognized in that country as cutaneous-ganglionar rickettsiosis.

Rickettsia parkeri: a Rickettsial pathogen transmitted by ticks in endemic areas for spotted fever rickettsiosis in southern Uruguay

At first Rickettsia conorii was implicated as the causative agent of spotted fever in Uruguay diagnosed by serological assays. Later Rickettsia parkeri was detected in human-biting Amblyomma triste ticks using molecular tests. The natural vector of R. conorii, Rhipicephalus sanguineus, has not been studied for the presence of rickettsial organisms in Uruguay. To address this question, 180 R. sanguineus from dogs and 245 A. triste from vegetation (flagging) collected in three endemic localities were screened for spotted fever group (SFG) rickettsiosis in southern Uruguay. Tick extracted DNA pools were subjected to PCR using primers which amplify a fragment of the rickettsial gltA gene. Positive tick DNA pools with these primers were subjected to a second PCR round with primers targeting a fragment of the ompA gene, which is only present in SFG rickettsiae. No rickettsial DNA was detected in R. sanguineus. However, DNA pools of A. triste were found to be positive for a rickettsial organism in two of the three localities, with prevalences of 11.8% to 37.5% positive pools. DNA sequences generated from these PCR-positive ticks corresponded to R. parkeri. These findings, joint with the aggressiveness shown by A. triste towards humans, support previous data on the involvement of A. triste as vector of human infections caused by R. parkeri in Uruguay.

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.

Morphological description of the nymphal stage of Amblyomma geayi and new nymphal records of Amblyomma parkeri

The external morphology of the nymph of Amblyomma geayi Neumann is described by optical and scanning electron microscopy. Unfed nymphs were obtained from an engorged A. geayi female, which had been collected on a sloth (Bradypus variegatus) from Belém municipality, State of Pará, northern Brazil, and was kept under laboratory conditions. With the present description, we propose a modification of a taxonomic key published in 2010 for the Amblyomma nymphs that occur in Brazil, through the inclusion of A. geayi. The nymph of A. geayi is morphologically very similar to the nymph of Amblyomma parkeri Fonseca and Aragão, with only slight morphological differences related to scutal surface and punctuations (more shagreened and less punctuated in A. geayi). These 2 nymphs differ from all other known Amblyomma nymphs from Brazil by the combination of auriculae present as small posterolateral rounded projections, eyes located at the level of the scutal midlength, and a rounded hypostome. These nymphal similarities as well the morphology of the adult stage corroborate previous studies that showed that A. geayi and A. parkeri are genetically closely related. Unpublished host records of the nymphs of both A. geayi and A. parkeri are provided. Established populations of A. geayi and A. parkeri seem to be geographically separated, since all confirmed records of A. geayi are from the northern half of South America (mainly the Amazonian region) and Central America, whereas all known records of A. parkeri are from the Atlantic rainforest biome in northeastern, southeastern, and southern Brazil. © 2013 Elsevier GmbH.

Taxonomic Status and Redescription of the Gillbacker Sea Catfish (Siluriformes: Ariidae: Sciades parkeri)

The Gillbacker Sea Catfish is a valid species of ariid catfish from the northeastern coast of South America. There are many synonyms In the literature for the Gillbacker Sea Catfish and even recent classifications have used different scientific names. Examination of a wide range of sizes of Individuals from different localities and examination of types and original species descriptions of Silurus parkeri, Bagrus flavescens, B. emphysetus, Arius physacanthus, A. bonneti, A. clavispinosus, and A. despaxi has lead us to the conclusion that all these names refer to the Gillbacker Sea Catfish and the valid name for the species is Sciades parkeri. The species is distinguished from all other ariid species by the following combination of features: body coloration yellow; swim bladder divided Into three chambers, posterior chamber moderately sized; nuchal plate shield-shaped, usually larger than supraocciptal process; anterior notch of nuchal plate absent; head shield exposed and granulated In orbital and postorbital regions; lateral edge of accessory patches not emarginated or shallowly notched; fleshy furrow connecting posterior nares absent; and mesial gill rakers absent from first two gill arches. Striking intraspecific and/or ontogenetic variation In eye size, maxillary-barbel length, supraoccipital-process size, nuchal-plate size and shape, and dorsal-spine thickness contributed to the numerous synonyms and misidentifications for Sciades parkeri. Bagrus albicans, described from French Guiana, has at times been listed as a synonym of Sciades parkeri. Our examination of the holotype of B. albicans, however, led us to conclude that It is a synonym of Sciades proops.

Serosurvey of antibodies against spotted fever group Rickettsia spp. in horse farms in Northern Paraná, Brazil

Brazilian spotted fever (BSF) is an emerging disease most likely caused by Rickettsia rickettsii. The objective of the present study was to estimate the seroprevalence of BSF rickettsia infections in equines from six horse farms located in Londrina County, Paraná, Southern Brazil. Six owners of horse farms situated in Cambé, Santa Fé, Guaraci and Londrina municipalities participated in the study. All farms were located in areas where BSF has not been reported. A total of 273 horses were sampled and their sera were tested by indirect Immunofluorescence assay (IFA) using R. rickettsii and R. parkeri antigens. Titers equal to and greater than 64 were considered positive. Of 273 sera tested, 15 (5.5%) reacted to R. rickettsii and 5 (1.8%) to R. parkeri. Five out of the six farms studied revealed seropositive animals and seropositivity rate ranged from 0 to 13%. The titers ranged from 64 to 512, and four samples had a titer of 512. Nine animals reacted to R. rickettsii with titers four-fold higher than those for R. parkeri. These results suggest that horses in Northern Paraná may have been exposed to rickettsiae identical or closely related to R. rickettsii.

Seroprevalence of Rickettsia bellii and Rickettsia felis in dogs, São José dos Pinhais, State of Paraná, Brazil

Brazilian spotted fever (BSF) is a vector-borne zoonosis caused by Rickettsia rickettsii bacteria. Dogs can be host sentinels for this bacterium. The aim of the study was to determine the presence of antibodies against Rickettsia spp. in dogs from the city of São José dos Pinhais, State of Paraná, Southern Brazil, where a human case of BSF was first reported in the state. Between February 2006 and July 2007, serum samples from 364 dogs were collected and tested at 1:64 dilutions by indirect immunofluorescence assay (IFA) against R. rickettsii and R. parkeri. All sera that reacted at least to one of Rickettsia species were tested against the six main Rickettsia species identified in Brazil: R. rickettsii, R. parkeri, R. bellii, R. rhipicephali, R. amblyommii and R. felis. Sixteen samples (4.4%) reacted to at least one Rickettsia species. Among positive animals, two dogs (15.5%) showed suggestive titers for R. bellii exposure. One sample had a homologous reaction to R. felis, a confirmed human pathogen. Although Rickettsia spp. circulation in dogs in the area studied may be considered at low prevalence, suggesting low risk of human infection, the present data demonstrate for the first time the exposure of dogs to R. bellii and R. felis in Southern Brazil.

Serological survey of Rickettsia sp. in horses and dogs in an non-endemic area in Brazil

Brazilian Spotted Fever (BSF) is a lethal rickettsiosis in humans caused by the bacteria Rickettsia rickettsii, and is endemic in some areas of Brazil. Horses and dogs are part of the disease's life cycle and they may also serve as sentinel animals in epidemiological studies. The first human BSF case in the State of Paraná was reported in 2005. The present study was conducted in the municipality of Almirante Tamandaré, where no previous case of BSF was reported. Serum samples were collected from 71 horses and 20 dogs from nine properties in the area. Ticks were also collected from these animals. All farmers completed a questionnaire about their knowledge of BSF and animal health management. Serum samples were analyzed by indirect immunofluorescent-antibody assay (IFA) using R. rickettsii and R. parkeri as antigens. Ticks were analyzed by PCR for Rickettsia sp., and all of them were PCR-negative. Six horses (8.45%) and 4 dogs (20%) were identified as seropositive. Farmers were not aware of the correlation between the presence of ticks and risk of BSF. Although a non-endemic area, Almirante Tamandaré is a vulnerable environment for BSF and effective tick control measures are required.

Study of infection by Rickettsiae of the spotted fever group in humans and ticks in an urban park located in the City of Londrina, State of Paraná, Brazil

INTRODUCTION: Spotted fevers are emerging zoonoses caused by Rickettsia species in the spotted fever group (SFG). Rickettsia rickettsii is the main etiologic agent of Brazilian spotted fever (BSF) and it is transmitted by Amblyomma spp. ticks. METHODS: The study aimed to investigate SFG rickettsiae in the Arthur Thomas Municipal Park in Londrina, PR, by collecting free-living ticks and ticks from capybaras and blood samples from personnel working in these areas. Samples from A. dubitatum and A. cajennense were submitted for PCR in pools to analyze the Rickettsia spp. gltA (citrate synthase gene). RESULTS: All the pools analyzed were negative. Human sera were tested by indirect immunofluorescence assay with R. rickettsii and R. parkeri as antigens. Among the 34 sera analyzed, seven (20.6%) were reactive for R. rickettsii: four of these had endpoint titers equal to 64, 2 titers were 128 and 1 titer was 256. None of the samples were reactive for R. parkeri. An epidemiological questionnaire was applied to the park staff, but no statistically significant associations were identified. CONCLUSIONS: The serological studies suggest the presence of Rickettsiae related to SFG that could be infecting the human population studied; however, analysis of the ticks collected was unable to determine which species may be involved in transmission to humans.

Anticorpos anti-rickettsias do grupo da febre maculosa em equídeos e caninos no norte do Estado do Paraná, Brasil

The Brazilian Spotted Fever (BSF) is a zoonosis that can be fatal if not trteated. As there are few studies of the BSF in the Paraná State, the occurrence of BSF was serologically investigated in dogs and horses by indirect immunofluorescence assay (IFA), in the North of that State, in animals that are regarded as sentinels for BSF. A total of 241 samples of sera of equine, four of asinine, and 29 of dogs were collected in nine farms of three municipalities in the North of Paraná: Arapongas, Douradina, and Umuarama. From fifteen samples of equine serum with positive titres for RIFI, eight (53.3 %) had titre of 64 against R. rickettsii, two (13.3 %) 128 against R. rickettsii, and five (33.3 %) were reactants for R. parkeri and R. rickettsii, with titres ranging from 64 to 2048, and 128 to 1024, respectively. The results showed that domestic animals, sentinels for BSF, are under low exposition to ticks infected with spotted fever group Rickettsia, indicating low risk of human infection by these agents in the studied area.

Serologic Survey for Rickettsiosis in Bats from Sao Paulo City, Brazil

Blood serum samples were collected from 451 bats captured within the Sao Paulo city from April 2007 to November 2008, and individually tested by indirect immunofluorescence assay against antigens derived from five Rickettsia species reported to occur in Brazil: the spotted fever group (SFG) species R. rickettsii, R. parkeri, R. amblyommii, R. rhipicephali, and the ancestral group species R. bellii. For this purpose, an anti-bat immunoglobulin G was produced and used in the present study. Overall, 8.6% (39/451), 9.5% (34/358), 7.8% (28/358), 1.1% (4/358), and 0% (0/358) serum samples were reactive to R. rickettsii, R. parkeri, R. amblyommii, R. rhipicephali, and R. bellii, respectively. Endpoint titers of reactive sera ranged from 64 to 256. From 20 bat species of 3 different families (Molossidae, Vespertilionidae, and Phyllostomidae), 46 animals were shown to be reactive to at least one rickettsial antigen. Seropositivity per bat species ranged from 0% to 33.3%. Most of the serologically positive sera reacted with two or more rickettsial antigens. Seropositivity for SFG rickettsial antigens in the absence of reactivity against R. bellii (ancestral group species) suggests that bats from Sao Paulo city can be infected by SFG rickettsiae. The possible role of soft ticks in serving as vectors of SFG rickettsiae to bats within the Sao Paulo city, associated to its public health risks, is discussed.

Tick-Borne Bacteria in Free-Living Jaguars (Panthera onca) in Pantanal, Brazil

Tick-borne bacteria were investigated in 10 free-living jaguars and their ticks in the Pantanal biome, Brazil. Jaguar sera were tested by indirect fluorescent antibody assays using Rickettsia rickettsii, Rickettsia parkeri, Rickettsia amblyommii, Rickettsia rhipicephali, Rickettsia felis, Rickettsia bellii, Ehrlichia canis, and Coxiella burnetii as crude antigens. All 10 jaguar sera reacted (titer >= 64) to at least one Rickettsia species; 4 and 3 sera reacted with E. canis and C. burnetii, respectively. One jaguar presented antibody titer to R. parkeri at least fourfold higher than those to any of the other five Rickettsia antigens, suggesting that this animal was infected by R. parkeri. Ticks collected from jaguars included the species Amblyomma cajennense, Amblyomma triste, and Rhipicephalus (Boophilus) microplus. No Rickettsia DNA was detected in jaguar blood samples, but an A. triste specimen collected on a jaguar was shown by PCR to be infected by R. parkeri. The blood of two jaguars and samples of A. triste, A. cajennense, and Amblyomma sp. yielded Ehrlichia DNA by PCR targeting the ehrlichial genes 16S rRNA and dsb. Partial DNA sequences obtained from PCR products resulted in a new ehrlichial strain, here designated as Ehrlichia sp. strain Jaguar. A partial DNA sequence of the 16S rRNA gene of this novel strain showed to be closest (99.0%) to uncultured strains of Ehrlichia sp. from Japan and Russia and 98.7% identical to different strains of Ehrlichia ruminantium. The ehrlichial dsb partial sequence of strain jaguar showed to be at most 80.7% identical to any Ehrlichia species or genotype available in GenBank. Through phylogenetic analysis, Ehrlichia sp. strain jaguar grouped in a cluster, albeit distantly, with different genotypes of E. ruminantium. Results highlight risks for human and animal health, considering that cattle ranching and ecotourism are major economic activities in the Pantanal region of Brazil.