First Report of the Isolation and Molecular Characterization of Rickettsia amblyommii and Rickettsia felis in Central America

During 2010, 15 adult ticks, identified as Amblyomma cajennense, were collected from horses in Cahuita and Turrialba districts, whereas 7 fleas, identified as Ctenocephalides felis, were collected from a dog in San Jose city, Costa Rica. In the laboratory, three A. cajennense specimens, two from Cahuita and one from Turrialba, were individually processed for rickettsial isolation in cell culture, as was a pool of seven fleas. Rickettsiae were successfully isolated and established in Vero cell culture from the three ticks and from a pool of seven fleas in C6/36 cell culture. The three tick isolates were genotypically identified as Rickettsia amblyommii, and the flea isolate was identified as Rickettsia felis through DNA sequencing of portions of the rickettsial genes gltA, ompA, and ompB of each isolate. In addition, other seven ticks were shown to contain rickettsial DNA. Polymerase chain reaction products of at least two of these ticks were sequenced and also showed to correspond to R. amblyommii. Overall, 66.7% (10/15) of the A. cajennense adult ticks were found to be infected with rickettsiae. This is the first report of a successful isolation in cell culture of R. amblyommii and R. felis from Central America.

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.

ISOLATION OF Rickettsia bellii FROM Amblyomma ovale AND Amblyomma incisum TICKS FROM SOUTHERN BRAZIL

Objective. To isolate and characterize rickettsiae from the ticks Amblyomma ovale and Amblyomma incisum collected in the state of Sao Paulo. Materials and methods. Adult, free-living A. ovale and A. incisum were collected in an Atlantic rainforest area in the state of Sao Paulo, Brazil. Each tick was tested using the hemolymph assay; samples from positive ticks were placed in shell vials in order to isolate rickettsiae and subsequently grown in Vero cells. Amplification of three rickettsial genes ( gltA, htrA and ompA) was attempted using polymerase chain reaction (PCR) for each isolate obtained. Amplicons were subsequently sequenced. Results. A total of 388 A. incisum and 50 A. ovale were collected. Only one A. incisum and one A. ovale were hemolymph-test positive. Rickettsiae were successfully isolated from these ticks; however establishment in Vero cell culture was successful only for the isolate from A. ovale. Bacterial contamination in the first cell passage of the A. incisum isolate precluded successful isolation of the organism. PCR products were obtained with the gltA and htrA primers for the two isolates, but no product was obtained with the ompA primers. By BLAST analysis, partial gltA and htrA sequences of isolates from A. ovale and A. incisum were similar to the corresponding sequences of R. bellii. Conclusions. This is the first report of R. bellii infecting A. incisum and the first successful isolation from A. ovale.

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.

Experimental Infection of Opossums Didelphis aurita by Rickettsia rickettsii and Evaluation of the Transmission of the Infection to Ticks Amblyomma cajennense

The present study evaluated the infection of opossums (Didelphis aurita) by Rickettsia rickettsii and their role as amplifier hosts for horizontal transmission of R. rickettsii to Amblyomma cajennense ticks. Three groups of opossums were evaluated: on day 0, group 1 (G1) was inoculated intraperitoneally with R. rickettsii; group 2 (G2) was infested by R. rickettsii-infected ticks; and group 3 (G3) was the uninfected control group. Opossum rectal temperature was measured daily. Blood samples were collected every 2 to 4 days during 30 days, and used to (1) inoculate guinea pigs intraperitoneally; (2) extract DNA followed by real-time polymerase chain reaction (PCR) targeting the rickettsial gene gltA; (3) study hematology; (4) detect R. rickettsii-reactive antibodies by indirect direct immunofluorescence assay (IFA). Blood was also collected every 10 days from days 30 to 180, to be tested by serology. Opossums were infested by uninfected A. cajennense larvae and nymphs from days 3 to 15. Engorged ticks were collected and allowed to molt in an incubator. Thereafter, the subsequent flat ticks were allowed to feed on uninfected rabbits, which were tested for seroconversion by IFA. Samples of flat ticks were also tested by real-time PCR. All G1 and G2 opossums became infected by R. rickettsii, as demonstrated by real-time PCR or/and guinea pig inoculation, but they showed no clinical abnormality. Rickettsemia was first detected at days 2 to 8, lasting intermittently till days 1 to 30. Approximately 18% and 5% of the flat ticks previously fed on G1 and G2 opossums, respectively, became infected by R. rickettsii, but only the rabbits infested with G1-derived ticks seroconverted. The study demonstrated that R. rickettsii was capable of infecting opossums without causing illness and developing rickettsemia capable of causing infection in guinea pigs and ticks, although the infection rate in ticks was low.

SOME PROPERTIES OF THE MULTIPLICITY SEQUENCE FOR ARBITRARY IDEALS

In this work we prove that the Achilles-Manaresi multiplicity sequence, like the classical Hilbert-Samuel multiplicity, is additive with respect to the exact sequence of modules. We also prove the associativity formula for his mulitplicity sequence. As a consequence, we give new proofs for two results already known. First, the Achilles-Manaresi multiplicity sequence is an invariant up to reduction, a result first proved by Ciuperca. Second, I subset of J is a reduction of (J,M) if and only if c(0)(I(p), M(p)) = c(0)(J(p), M(p)) for all p is an element of Spec(A), a result first proved by Flenner and Manaresi.

Serum cardiac troponin I concentration in dogs with ehrlichiosis

Background: Ehrlichiosis is a multisystemic disease with the potential to cause cardiomyocyte injury in naturally infected dogs. Hypothesis: Myocardial injury occurs in dogs infected with Ehrlichia canis. Animals: One-hundred and ninety-four dogs from Brazil with clinical and laboratory abnormalities indicative of ehrlichiosis. Sixteen healthy dogs served as controls. Methods: Electrocardiogram, echocardiogram, noninvasive blood pressure measurement, and serum cardiac troponin I (cTnI) concentrations were evaluated. Serologic assays and PCR determined the exposure and infection status for E. canis, Anaplasma spp., Babesia canis vogeli, Bartonella spp., Borrelia burgdorferi, Dirofilaria immitis, Ehrlichia chaffeensis, Ehrlichia ewingii, Leishmania chagasi, and spotted-fever group Rickettsia. Dogs were assigned to groups according to PCR status: E. canis infected, infected with other vector-borne organisms, sick dogs lacking PCR evidence for infection, and healthy controls. Results: E. canis-infected dogs had higher serum cTnI concentrations than controls (median: 0.04 ng/dL; range 0.04-9.12 ng/dL; control median: 0.04 ng/dL; range: 0.04-0.10 ng/dL; P = .012), and acute E. canis infection was associated with myocardial injury (odds ratio [OR]: 2.67, confidence interval [CI] 95%: 1.12-6.40, P = .027). Severity of anemia was correlated with increased risk of cardiomyocyte damage (r = 0.84, P < .001). Dogs with clinical signs of systemic inflammatory response syndrome (SIRS) were at higher risk for myocardial injury than were other sick dogs (OR: 2.55, CI 95%: 1.31-4.95, P = .005). Conclusions and Clinical Importance: Acute infection with E. canis is a risk factor for myocardial injury in naturally infected Brazilian dogs. Severity of anemia and SIRS might contribute to the pathophysiology of myocardial damage.

Experimental infection of capybaras Hydrochoerus hydrochaeris by Rickettsia rickettsii and evaluation of the transmission of the infection to ticks Amblyomma cajennense

The present study evaluated the infection of capybaras (Hydrochoerus hydrochaeris) by Rickettsia rickettsii and their role as amplifier hosts for horizontal transmission of R. rickettsii to Amblyomma cajennense ticks. Two groups of two capybaras each were evaluated: on day 0, group 1 (G1) was infested by R. rickettsii-infected ticks, and group 2 (G2) was inoculated intraperitoneally with R. rickettsii. Two additional groups were control groups, not exposed to R. rickettsii, being CG1 group the control of G1, and CG2 group the control of G2. Capybara rectal temperature was measured daily. Blood samples were collected every 3 days during 30 days, and used to (i) inoculate guinea pigs intraperitoneally; (ii) DNA extraction followed by real-time PCR targeting the rickettsial gene gltA; (iii) hematology; (iv) detection of R. rickettsii-reactive antibodies by indirect immunofluorescence assay (IFA). Blood was also collected from G I capybaras every approximate to 10-30 days till the 146th day, to be tested by serology. Capybaras were infested by uninfected A. cajennense nymphs from the 3rd to the 18th day. Engorged nymphs were collected, allowed to molt to adults in an incubator. Thereafter, the subsequent flat ticks were tested by PCR. All G1 and G2 capybaras became infected by R. rickettsii, as demonstrated by guinea pig inoculation and seroconversion, but they showed no fever. Rickettsemia was continually detected from the 6th (G2 capybaras) or 9th (G1 capybaras) to the 18th day post inoculation or infestation with R. rickettsii-infected ticks. A total of 20-25% and 30-35% of the flat ticks previously fed on G1 and G2 capybaras, respectively, became infected by R. rickettsii. The study demonstrated that R. rickettsii was capable to infect capybaras without causing clinical illness, inducing rickettsemia capable to cause infection in guinea pigs and ticks. Our results indicate that capybaras act as amplifier host of R. rickettsii for A. cajennense ticks in Brazil. (c) 2008 Elsevier B.V. All rights reserved.

Occurrence of ticks (Acari: Ixodidae) in the municipality of Goiatins, Tocantins

Due to a suspected human case of Brazilian Lyme-like disease in the city of Goiatins, Tocantins State, an epidemiological survey was carried out in eight counties in this region during September 2007 and February 2008, where 1,890 ticks were collected from domestic animals and from the environment. A total of eight tick species were identified: Rhipicephalus sanguineus, Rhipicephalus (Boophilus) microplus, Dermacentor nitens, Amblyomma cajennense, Amblyomma oblongoguttatum, Amblyomma ovale, Amblyomma parvum and Amblyomma tigrinum. The last four species were described for the first time in this region. Although human parasitism by ticks is frequently described in Goiatins, no ticks collected from humans were analyzed. The Study of ixodids in this region contributes with the survey of Brazilian ticks, as well as the elucidation of the possible transmission of the agent that caused the Brazilian Lyme-like disease case in Goiatins.

Surveillance of Rickettsia sp infection in capybaras (Hydrochoerus hydrochaeris) a potential model of epidemiological alert in endemic areas

Introduction. Capybaras (Hydrochoerus hydrochaeris) are considered amplifying hosts of Rickettsia sp. These rodents are usually parasitized by the tick vector, Amblyomma cajennense, the main vector of rickettsioses in humans and animals in South America. Capybaras can be used as sentinels in detection of circulation of rickettsiae. Objective. Antibodies to rickettsiae of spotted fever group were detected in capybaras in a rural area of Cordoba Province, northern Colombia. Materials and methods. Sera were analyzed from 36 capybaras in a rural area of Monteria (village of San Jeronimo) in Cordoba. For the detection of IgG antibodies, indirect immunofluorescence was performed. The antigens were derived from R. rickettsia strain Taiacu isolated in Brazil. Capybara sera were diluted 1:64 for IFA analysis. Ticks were collected from each capybara (also known as chiguiro) and identified to species. Results. The seroprevalence of spotted fever group Rickettsia was 22% (8 capybaras). Four sera had a titer of 1:64, 3 had a titer of 1:128 and one serum had a titer of 1:512. All ticks removed from the capybaras (n=933) were taxonomically identified as Amblyomma cajennense. Conclusion. Colombia has areas endemic for rickettsioses, as indicated by confirmed annual outbreaks. The current study reports the first evidence of natural rickettsial infection of the spotted fever group in capybaras from Colombia. The findings suggest that capybaras can be used as sentinels for the circulation of rickettsiae and can identify endemic areas for the transmission of rickettsial diseases.

Canine infection by Rickettsiae and Ehrlichiae in southern Brazil

This study evaluated the infection caused by Rickettsia and Ehrlichia agents among dogs in southern Brazil. A total of 389 dogs were tested by the indirect immunofluorescence assay (IFA) for Rickettsia rickettsii, Rickettsia parkeri, Rickettsia amblyommii, Rickettsia rhipicephali, Rickettsia bellii, and Ehrlichia canis. Overall, 42.4% (165/389) of the dogs were seroreactive to at least one Rickettsia species, but only 11 canine sera reacted with another Rickettsia species without reacting with R. parkeri. A total of 100 (25.7%) canine sera showed titers to R. parkeri at least 4-fold higher than those to any of the other rickettsial antigens, allowing us to consider that these dogs were infected by R. parkeri. Dogs that had direct contact with pasture or forest areas were > 2 times more likely to be seroreactive to Rickettsia than dogs with no such direct contact. Only 19 (4.8%) of the 389 dogs were seroreactive to E. canis.

Ticks (Acari : ixodidae) infesting wild birds in an Atlantic forest area in the state of Sao Paulo, Brazil, with isolation of Rickettsia from the tick Amblyomma longirostre

During field work in Nazare Paulista, state of Sao Paulo, Brazil, we found 13 (56.5%) of 23 birds (mostly Passeriformes) to be infested by 28 larvae and I nymph of Amblyomma spp. Two larvae were reared to the adult stage, being taxonomically identified as Amblyomma parkeri Fonseca and Aragao, whereas five larvae and one nymph were identified as Amblyomma longirostre Koch. All six A. longirostre specimens were shown to be infected by rickettsia, as demonstrated by polymerase chain reaction (PCR) targeting two rickettsial genes (gltA and ompA) or isolation of rickettsia in cell culture from one of the ticks. This isolate was designated as strain AL, which was established in Vero cell culture and was molecularly characterized by DNA sequencing fragments of the rickettsial genes gltA, htrA, ompA, and ompB. Phylogenetic analyses inferred from ompA and ompB partial sequences showed a high degree of similarity of strain AL with Rickettsia sp. strain ARANHA, previously detected by PCR in A. longirostre ticks from Rondonia, northern Brazil. We conclude that strain AL is a new rickettsia genotype belonging to the same species of strain ARANHA, which are closely related to Candidatus `R. amblyomniii`. Further studies should elucidate if strains AL and ARANHA are different strains of Candidatus `R. amblyommii` or are a new species.

Expressed sequence tags (ESTs) from the salivary glands of the tick Amblyomma cajennense (Acari : Ixodidae)

The neotropical tick Amblyomma cajennense is a significant pest to domestic animals, the most frequently human-biting tick in South America and the main vector of Brazilian spotted fever (caused by Rickettsia rickettsii), a deadly human disease. The purpose of this study is to characterize the adult A. cajennense salivary gland transcriptome by expressed sequence tags (ESTs). We report the analysis of 1754 clones obtained from a cDNA library, which reveal mainly transcripts related to proteins involved in the hemostatic processes, especially proteases and their inhibitors. Remarkably, five types of possible serine protease inhibitors were found, including a molecule with a distinguished structure that contains repeats of the active motif of hirudin inhibitors. Besides, other components that may be active over the host immune system or acting as defensins against infecting microorganisms were also described, including a molecule similar to insect venom allergens. The conjunction of components from this transcriptome suggests a diverse strategy of A. cajennense tick during feeding, but emphasized in the coagulation system. (c) 2008 Published by Elsevier Ltd.

An integrated database on ticks and tick-borne zoonoses in the tropics and subtropics with special reference to developing and emerging countries

Tick-borne zoonoses (TBZ) are emerging diseases worldwide. A large amount of information (e.g. case reports, results of epidemiological surveillance, etc.) is dispersed through various reference sources (ISI and non-ISI journals, conference proceedings, technical reports, etc.). An integrated database-derived from the ICTTD-3 project (http://www.icttd.nl)-was developed in order to gather TBZ records in the (sub-)tropics, collected both by the authors and collaborators worldwide. A dedicated website (http://www.tickbornezoonoses.org) was created to promote collaboration and circulate information. Data collected are made freely available to researchers for analysis by spatial methods, integrating mapped ecological factors for predicting TBZ risk. The authors present the assembly process of the TBZ database: the compilation of an updated list of TBZ relevant for (sub-)tropics, the database design and its structure, the method of bibliographic search, the assessment of spatial precision of geo-referenced records. At the time of writing, 725 records extracted from 337 publications related to 59 countries in the (sub-)tropics, have been entered in the database. TBZ distribution maps were also produced. Imported cases have been also accounted for. The most important datasets with geo-referenced records were those on Spotted Fever Group rickettsiosis in Latin-America and Crimean-Congo Haemorrhagic Fever in Africa. The authors stress the need for international collaboration in data collection to update and improve the database. Supervision of data entered remains always necessary. Means to foster collaboration are discussed. The paper is also intended to describe the challenges encountered to assemble spatial data from various sources and to help develop similar data collections.

Survey of Ticks (Acari: Ixodidae) and Their Rickettsia in an Atlantic Rain Forest Reserve in the State of Sao Paulo, Brazil

The current study investigated the occurrence of ticks and their rickettsiae in the Serra do Mar State Park, which encompasses one of the largest Atlantic rain forest reserves of Brazil. From July 2008 to June 2009, a total of 2,439 ticks (2,196 free living and 243 collected on hosts) was collected, encompassing the following 13 species: Amblyomma aureolatum (Pallas), Amblyomma brasiliense Aragao, Amblyomma dubitatum Neumann, Amblyomma fuscum Neumann, Amblyomma incisum Neumann, Amblyomma longirostre (Koch), Amblyomma naponense (Packard), Amblyomma nodosum Neumann, Amblyomma ovale Koch, Haemaphysalis juxtakochi Cooley, Ixodes aragaoi Fonseca, Lodes loricatus Neumann, and Rhipicephalus sanguineus (Latreille). Ticks were submitted to polymerase chain reaction assays targeting portions of the rickettsial genes gltA and ompA. Polymerase chain reaction products were DNA sequenced and compared with corresponding sequences available in GenBank. Rickettsia bellii, a rickettsia of unknown pathogenicity, was detected in one A. aureolatum, one A. ovate, and three A. incisum specimens. At least 8.8% (3/34) of the free-living A. ovale ticks, 13.6% (8/59) of the A. ovale ticks collected from dogs, and 1.9% (1/54) of the R. sanguineus (Latreille) ticks were found to be infected by Rickettsia sp strain Atlantic rain forest, a novel strain that has been shown to cause an eschar-associated spotted fever in the state of Sao Paulo. Our results suggest that A. ovale is the vector of Rickettsia sp strain Atlantic rain forest in the state of Sao Paulo.