Diego G. Ramirez

Immature argasid ticks: diagnosis and keys for Neotropical region

Many argasid tick species are known only through their larval descriptions, in which the chaetotaxy, together with other external morphological characteristics, has been used to separate genera and species. However, the illustrations of these features are based on optical microscopy alone and many of these features are not clearly defined. Because of the difficulties in determining the larval and nymph stages of some genera, we have prepared illustrated keys for the immature stages of argasids, including an up-to-date list of the known species of the Neotropical region. We have also included an illustrated key for larvae of the Ornithodoros species from Brazil, based on scanning electron microscopy.

Experimental infection with Rickettsia rickettsii in an Amblyomma dubitatum tick colony, naturally infected by Rickettsia bellii

Amblyomma dubitatum engorged females, naturally infected by Rickettsia bellii, were used to establish a laboratory colony. Larvae, nymphs, and adults were exposed to two strains of Rickettsia rickettsii by feeding on needle-inoculated guinea pigs, and thereafter reared on uninfected guinea pigs. After acquisition feeding, engorged larvae and nymphs molted to nymphs and adults, respectively, which were shown to be infected (confirming transstadial perpetuation), and were able to transmit both strains of R. rickettsii to uninfected animals, as demonstrated by clinical, serological, and molecular analyses. However, the larval, nymphal, and adult stages of A. dubitatum showed to be only partially susceptible to R. rickettsii infection, since in all cases, only part of the ticks became infected by this agent, after being exposed to rickettsemic animals. While transovarial transmission of R. rickettsii was inefficient in the A. dubitatum engorged females of the present study, 100% of these females passed R. bellii transovarially. Because it has been reported that a primary infection by a Rickettsia species would preclude transovarial transmission of a second Rickettsia species, it is likely that the ineffectiveness of A. dubitatum to perpetuate R. rickettsii by transovarial transmission was related to its primary infection by R. bellii; however, it could also be related to unknown factors inherent to A. dubitatum. The relevance of A. dubitatum as a natural vector of R. rickettsii to humans or animals is discussed.

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.

Isolation of the Pathogen Rickettsia sp. Strain Atlantic Rainforest From Its Presumed Tick Vector, Amblyomma ovale (Acari: Ixodidae), From Two Areas of Brazil

Abstract In this study, Amblyomma ovale Koch ticks were collected from domestic dogs in two localities of the Atlantic rainforest biome of Brazil: 1) the Paty Valley of the Chapada Diamantina National Park, Bahia state (northeastern Brazil), and 2) Adrianópolis, Paraná state (southern Brazil). Ticks were screened for the presence of Rickettsia-like structures by the hemolymph test with Giménez staining, and then processed for isolation of rickettsiae in Vero cell culture by the shell-vial technique. Rickettsiae were isolated from one A. ovale tick of each of the two localities. The two isolates were successfully established in the laboratory with several passages, each one reaching >90% infection of the cells. The two isolates were identified as the spotted fever group (SFG) agent Rickettsia sp. strain Atlantic rainforest, as their gltA (350 bp), ompB (781 bp), and ompA (567 bp) gene fragments were 100% equal to GenBank corresponding sequences of the original strain Atlantic rainforest, reported to be infecting a human in southeastern Brazil, and also 100% equal to the available ompA sequence of strain Bahia, reported to be infecting a human in Paty Valley, the same area of the present study in Bahia state. Ten dogs from Paty Valley were serologically tested against rickettsial antigens by the indirect immunofluorescence antibody test. At least 60% of them were seroreactive to SFG rickettsiae. The role of A. ovale as vector of Rickettsia sp. strain Atlantic rainforest in the Paty Valley area, as well as in other parts of Latin America, is discussed.

Toxoplasma gondii in domestic and wild animals from forest fragments of the municipality of Natal, northeastern Brazil.

Toxoplasmosis stands out as a global disease that has felines as definitive hosts. In the municipality of Natal, Rio Grande do Norte State, Brazil, two parks are notable for their ecological and social importance. This study aimed to investigate the presence of Toxoplasma gondii in short hair cats, bats and small non-volant mammals in these two ecological reserves. Altogether, biological samples were obtained from 154 mammals, 92 wild animals from both areas and 62 domestic cats of the Parque da Cidade. In total, 22 (53.7%) non-volant wild mammals, 11 (21.5%) bats and 28 (52.8%) cats were positive for IgG anti-T. gondii antibodies using the Modified Agglutination Test (≥ 25). It was possible to detect the presence of T. gondii DNA, by means of a molecular amplification of a B1 gene fragment (155bp), in 92 tissue samples from wild animals, including Didelphis albiventris, Monodelphis domestica, Artibeus lituratus, Carollia perspicillata and Glossophaga soricina. Of the 62 cats examined by the same molecular method, T. gondii DNA could be detected in 4 cats. In this study, it was observed the circulation of T. gondii in wild species and domestic cats, demonstrating the involvement of wild and domestic animals in the cycle of T. gondii.

Rickettsia amblyommatis infecting ticks and exposure of domestic dogs to Rickettsia spp. in an Amazon-Cerrado transition region of northeastern Brazil

This study was performed in Maranhão state, a transition area two Brazilian biomes, Amazon and Cerrado. During 2011–2013, 1,560 domestic dogs were sampled for collection of serum blood samples and ticks in eight counties (3 within the Amazon and 5 within the Cerrado). A total of 959 ticks were collected on 150 dogs (9.6%). Rhipicephalus sanguineus sensu lato (s.l.) was the most abundant tick (68% of all collected specimens), followed by Amblyomma cajennense sensu lato (s.l.) (12.9%), Amblyomma parvum (9.2%), and Amblyomma ovale (5.2%). Other less abundant species (<1%) were Amblyomma oblongoguttatum, Rhipicephalus microplus, Haemaphysalis juxtakochi, and Amblyomma rotundatum. Females of A. cajennense s.l. ticks were morphologically identified as A. cajennense sensu stricto (s.s.) or A. sculptum. Molecular analyses of 779 canine ticks revealed three Rickettsia species: Rickettsia amblyommatis in 1% (1/100) A. cajennense s.l., ‘Candidatus Rickettsia andeanae’ in 20.7% (12/58) A. parvum, Rickettsia bellii in 6.8% (3/44) A. ovale and 100% (1/1) A. rotundatum ticks. An additional collection of A. sculptum from horses in a Cerrado area, and A. cajennense s.s. from pigs in an Amazon area revealed R. amblyommatis infecting only the A. cajennense s.s. ticks. Serological analysis of the 1,560 canine blood samples revealed 12.6% canine seroreactivity to Rickettsia spp., with the highest specific seroreactivity rate (10.2%) for R. amblyommatis. Endpoint titers to R. amblyommatis were significantly higher than those for the other Rickettsia antigens, suggesting that most of the seroreactive dogs were exposed to R. amblyommatis-infected ticks. Highest canine seroreactivity rates per locality (13.1–30.8%) were found in Amazon biome, where A. cajennense s.s. predominated. Lowest seroreactivity rates (1.9–6.5%) were found in Cerrado localities that were further from the Amazon, where A. sculptum predominated. Multivariate analyses revealed that canine seroreactivity to Rickettsia spp. or R. amblyommatis was statistically associated with rural dogs, exposed to Amblyomma ticks.

Survey of Leishmania Infantum Chagasi in Wild and Domestic Animals in Urban Area and Atlantic Rainforest Fragment in Northeast, Brazil

Objective: Survey of Leishmania infantum chagasi in domestic and wild mammals in urban area and a Biological Reserve in Natal, Rio Grande do Norte, Brazil. Methods: Domestic and wild mammals were captured in Brazilian northeast Atlantic Rainforest, Rio Grande do Norte state. Serological and parasitological studies were conducted; Leishmania isolates were positioned in phylogeny based on small subunit rDNA (SSU rDNA) and glycosomal-3-phosphate dehydrogenase (gGAPDH) gene sequences. Results: Blood samples were collected from 138 wild and domestic mammals, comprising 66 dogs, 52 cats and 20 marsupials. Antibodies were found in 27 dogs (40.91%), two cats (3.85%) and one Didelphis albiventris (5%). The cultures of popliteal lymph node aspirates were positive in nine dogs (13.64%) and from seven (10.61%) the parasite was isolated and cryopreserved. All isolates were positioned in phylogeny based on SSU rDNA and gGAPDH in the same branch with L. infantum chagasi. Conclusions: The proximity of the forest fragment with humans and their domestic animals provide interference in the health of wild animals. Measures to control the population of feral cats and environmental conservation should be implemented due the importance of visceral leishmaniasis

New Trypanosoma species, Trypanosoma gennarii sp. nov., from South American marsupial in Brazilian Cerrado

Hundreds of trypanosome species have been described in all mammalian orders, on every continent, including with mixed infections. Trypanosomes circulate in the form of sylvatic enzootic infections transmitted by blood-sucking insects that are associated with the host mammals. Small wild mammals were caught in a fragment of Cerrado terrain on an island in the hydroelectric reservoir of Três Marias, in the central region of the state of Minas Gerais, using pitfall and Sherman traps with different means of attraction. DNA samples from these mammals were subjected to the conventional polymerase chain reaction (PCR) for the full-length genes SSU rDNA and gGAPDH. A total of 232 animals of the orders Didelphimorphia, Rodentia, Chiroptera and Cingulata were caught (total of 17 species). There were also four species of marsupials: Monodelphis domestica, Didelphis albiventris, Gralicinanus agilis and Micoureus paraguaianus. Among these, there were eight positive individuals of Monodelphis domestica. However, nine cultures were established, because one of them was parasitized by two species of trypanosomes: Trypanosoma cruzi and a new trypanosome species. The new species have a large epimastigote forms, and with a well-developed undulating membrane in trypomastigote forms. The new species Trypanosoma gennarii was described in Monodelphis domestica.

Laboratory life cycle of Ornithodoros brasiliensis (Acari: Argasidae): An endemic tick from southern Brazil

ORNITHODOROS BRASILIENSIS Aragão is an endemic tick restricted to the highlands of the state of Rio Grande do Sul, Brazil. This species is very aggressive toward humans, causing fever, great pain and intense inflammatory response at the bite site. It is also very aggressive toward dogs, and tick toxicosis syndrome has been reported in this host. In order to elucidate the biology of this tick, the present study describes its life cycle under laboratory conditions, using guinea pigs as hosts for two generations. In the nonparasitic phase, the ticks were maintained in sand, in an incubator under controlled conditions. The larvae molted to the nymphal stage without feeding, and five nymphal stages (N1, N2, N3, N4 and N5) were observed in both generations. In both generations emergence of adults started from N3 when sex ratio was 0.85:1 (23 males and 27 females) in F1 and 0.63:1 (34 males and 54 females) in F2. For both generations, N4 generated more females, while N5 only produced females. The pre-ecdysis period of the nymphs ranged from 31.1 to 38.6 days. Two gonotrophic cycles were observed, and the first one presented a higher average number of eggs deposited (N=139) than the second (N=73.8). The mean duration of the life cycle (egg to egg) of O. brasiliensis was 215.4 days for the first generation and 195 days for the second.

Ticks, rickettsial and erlichial infection in small mammals from Atlantic forest remnants in northeastern Brazil

We evaluated infection by Rickettsia spp. and Ehrlichia spp in small mammals and their ticks from two Atlantic forest conservation areas in the state of Rio Grande do Norte, northeastern Brazil. A total of 39 small mammals were captured during 2012–2013, encompassing 33 marsupials (29 Didelphis albiventris, four Monodelphis domestica), three Cricetidae rodents (two Necromys lasiurus, one Rattus rattus), one Caviomorpha rodent (Thrichomys apereoides) and two armadillos (Euphractus sexcinctus). The ticks Amblyomma auricularium, Ixodes loricatus, and Ornithodoros mimon were collected from D. albiventris, whereas only A. auricularium was collected from armadillos. Through immunofluorescence assay with Rickettsia spp. antigens, 6/28 (21%) D. albiventris and the single R. rattus specimen reacted to at least one rickettsial antigen, with highest seroprevalence and endpoint titers to Rickettsia amblyommatis. A total of 150 ticks (126 A. auricularium, nine I. loricatus, 15 O. mimon) was tested for rickettsial infection by PCR, which detected only R. amblyommatis in most of the A. auricularium ticks. Lung and spleen samples were collected from small mammals (two N. lasiurus, six D. albiventris, three M. domestica, one T. apereoides, one R. rattus) and were tested by PCR for Anaplasmataceae agents. The spleen from one D. albiventris contained a new ehrlichial agent, here named as Ehrlichia sp. strain Natal. Phylogenetic analysis inferred from the dsb gene of Ehrlichia spp. indicates that this novel agent is potentially a new species. Future studies should monitor the possible role of rickettsial and/or ehrlichial microorganisms as agents of emerging diseases in these degraded areas of Atlantic forest, just as has occurred with other agents in degraded areas of this biome in southeastern Brazil.