Molecular detection of Bartonella spp. in bat-associated macronyssid mites (Acari: Macronyssidae) from Southern and Southeastern Brazil.

Despite the worldwide occurrence and high genetic diversity of Bartonella spp. in bats, few studies investigate their occurrence in bat-associated mites. To date, 26 species of Macronyssidae mite species have been reported from Brazil, and 15 of which were found parasitizing bats. The present study aimed to investigate the presence of Bartonella DNA in bat-associated macronyssid mites from Brazil. For this purpose, 393 macronyssid specimens were selected by convenience from the tissue bank of the Acari Collection of the Instituto Butantan (IBSP). These mites were collected from 14 different bat species in three different Brazilian States (Minas Gerais, Paraná, and Rio de Janeiro). Out of 165 mites positive in the PCR for the endogenous 18S rRNA gene, only eight were positive in the qPCR for Bartonella spp. based on the nuoG gene, and we were able to obtain two sequences base in this same gene, and one sequence based on the 16S rRNA gene. The phylogenetic inference based on the nuoG gene grouped the obtained sequences with Bartonella genotypes previously detected in bats and associated bat flies, while the phylogeny based on the 16S rRNA grouped the obtained sequence in the same clade of Bartonella genotypes previously detected in Dermanyssus gallinae. These findings suggest that macronyssid mites might be associated with the maintenance of bartonellae among bats.

Eutrichophilus cordiceps Mjöberg, 1910 (Ischnocera: Trichodectidae) in Spiny Tree Porcupines (Coendou villosus): New locality records and the first molecular evidence of association with Bartonella sp.

The chewing louse genus Eutrichophilus Mjöberg has 19 species only associated with porcupines (Rodentia: Erethizontidae). Of these species, E. cercolabes, E. cordiceps, E. emersoni, E. minor, E. moojeni, and E. paraguayensis have been recorded in Brazil. In the present study, we report E. cordiceps for the first time in the São Paulo State (Bauru Municipality) and for the second time in the Santa Catarina State (Lages Municipality), providing scanning electron images and light microscopy for the eggs, as well as the first molecular data (18S rRNA) for the genus. Additionally, Bartonella sp. was detected for the first time in this chewing lice species.

The genus Perissopalla Brennan and White, 1960 (Trombidiformes: Trombiculidae) in Brazil: Redescriptions, new localities, and host records with the first molecular data for the genus.

The genus Perissopalla Brennan and White (Trombidiformes: Trombiculidae) is currently represented by ten species with three from Brazil: Perissopalla barticonycteris Brennan, Perissopalla ipeani Brennan, and Perissopalla tanycera Brennan. In the present study, these three species are redescribed and illustrated based on the types and additional non-type specimens. New host and locality records for P. ipeani and P. tanycera are included. Additionally, a partial sequence for the 18S rRNA gene for P. ipeani was provided.

Co-infestation caused by Goniodes pavonis (L., 1758) (Ischnocera: Goniodidae) and Goniocotes rectangulatus Nitzsch [In Giebel], 1866 on white Indian peafowl, Pavo cristatus L., 1758 (Galliformes: Phasianidaede) in Brazil.

The genus Goniodes Nitzsch and Goniocotes Burmeister (Ischnocera: Goniogodidae) are distributed worldwide, and exclusively parasitizing avian hosts of the Galliformes. In Brazil, there are only four species recorded: Goniodes dissimilis Denny, Goniodes gigas (Taschenberg), Goniodes pavonis (L.), and Goniocotes gallinae (DeGeer). In the present study, we are reporting the co-parasitism of G. pavonis and G. rectangulatus Nitzsch [In Giebel] on specimens of the white Pavo cristatus, popularly known as 'white Indian peafowl' for the first time. Furthermore, a new Brazilian locality for G. pavonis species has been reported, as well as the first time that G. rectangulatus is reported to Brazil. Additionally, we provide the first molecular information for G. pavonis.

Co-infestation caused by Goniodes pavonis (L., 1758) (Ischnocera: Goniodidae) and Goniocotes rectangulatus Nitzsch [In Giebel], 1866 on white Indian peafowl, Pavo cristatus L., 1758 (Galliformes: Phasianidaede) in Brazil

The genus Goniodes Nitzsch and Goniocotes Burmeister (Ischnocera: Goniogodidae) are distributed worldwide, and exclusively parasitizing avian hosts of the Galliformes. In Brazil, there are only four species recorded: Goniodes dissimilis Denny, Goniodes gigas (Taschenberg), Goniodes pavonis (L.), and Goniocotes gallinae (DeGeer). In the present study, we are reporting the co-parasitism of G. pavonis and G. rectangulatus Nitzsch [In Giebel] on specimens of the white Pavo cristatus, popularly known as ‘white Indian peafowl’ for the first time. Furthermore, a new Brazilian locality for G. pavonis species has been reported, as well as the first time that G. rectangulatus is reported to Brazil. Additionally, we provide the first molecular information for G. pavonis.

Noteworthy records of the ticks Ornithodoros rostratus and Amblyomma sculptum parasitizing Pteronura brasiliensis in the central-western region of Brazil, with pathogen investigation notes.

A male of Pteronura brasiliensis (Carnivora: Mustelidae) was found dead on the banks of the Rio Negro, in the Pantanal wetlands of Mato Grosso do Sul state, Aquidauana municipality. Two ticks found attached to its skin were morphologically identified as a second-instar nymph of Ornithodoros rostratus (Argasidae) and a male of Amblyomma sculptum (Ixodidae). In order to complement the morphological identification, these tick specimens were subjected to DNA extraction, and tested using PCR assays to confirm the molecular identity the specimens. Also, the tick DNA samples were tested and were negative in the PCR assays for all the pathogens tested. We also examined 30 batches, consisting of 174 individuals of O. rostratus deposited in the Acari Collection of the Butantan Institute, and we found material from four Brazilian states, including one batch containing 2 males and 2 females from Aquidauana, of Mato Grosso do Sul state, collected from the soil. This was therefore the first record of O. rostratus parasitizing P. brasiliensis and the first locality record (Aquidauana). Likewise, A. sculptum is commonly found in the Pantanal and is reported here for the second time parasitizing the giant otter, which is a host little studied regarding the ectoparasites.

Characterization of the bacterial microbiome of non-hematophagous bats and associated ectoparasites from Brazil.

Introduction: Bats, along with their ectoparasites, harbor a wide diversity of symbiotic and potential pathogenic bacteria. Despite the enormous diversity of bats (181 species), few studies aimed to investigate the bacterial microbiome of Brazilian chiropterans and associated ectoparasites. This study aimed to characterize the bacterial microbiome of non-hematophagous bats and associated Streblidae flies and Macronyssidae and Spinturnicidae mites in the state of Mato Grosso do Sul, midwestern Brazil. Methods: Oral and rectal swabs were collected from 30 bats (Artibeus lituratus [n = 13], Artibeus planirostris [n = 9], Eptesicus furinalis [n = 5], Carollia perspicillata [n = 2], and Platyrrhinus lineatus [n = 1]). In addition, a total of 58 mites (15 Macronyssidae and 43 Spinturnicidae) and 48 Streblidae bat flies were collected from the captured bats. After DNA extraction and purification, each sample's bacterial composition was analyzed with metagenomic sequencing. Results: The microbiome composition of both oral and rectal bat swab samples showed that Gammaproteobacteria was the most abundant bacterial class. Spiroplasma, Wolbachia and Bartonella represented the most abundant genera in Streblidae flies. While Wolbachia (Alphaproteobacteria) was the most abundant genus found in Spinturnicidae, Arsenophonus (Gammaproteobacteria) was found in high abundance in Macronyssidae mites. In addition to characterizing the microbiome of each sample at the class and genus taxonomic levels, we identified medically significant bacteria able to infect both animals and humans in oral (Streptococcus and Anaplasma) and rectal swabs (Enterobacter, Klebsiella, Escherichia, Enterococcus, Streptococcus), Macronyssidae (Anaplasma, Bartonella, Ehrlichia) and Spinturnicidae (Anaplasma, Bartonella) mites as well as Streblidae flies (Spiroplasma, Bartonella). Discussion and conclusion: Besides expanding the knowledge on the bacterial microbiome of non-hematophagous bats and Streblidae flies from Brazil, the present work showed, for the first time, the bacterial community of bat-associated Macronyssidae and Spinturnicidae mites.

A new species of Eutrombicula Ewing, 1938 (Trombidiformes: Trombiculidae) from the Galápagos Islands, with a checklist of Ecuadorian chiggers.

Few studies have been carried out on the Ecuadorian chigger fauna. In this study, we are describing a new species of Eutrombicula from the Galápagos Islands. Also, we provide an updated checklist of Ecuadorian and Galápagos Islands chiggers, including a new host association and locality record for Eutrombicula nerudai Stekolnikov and González-Acuña.

Artificial Feeding of Ornithodoros fonsecai and O. brasiliensis (Acari: Argasidae) and Investigation of the Transstadial Perpetuation of Anaplasma marginale.

Anaplasma marginale is a Gram-negative, obligate intraerythrocytic bacterium that causes bovine anaplasmosis. While hard ticks of the genera Dermacentor and Rhipicephalus can be biological vectors, transmitting this pathogen via saliva during blood meals, blood-sucking insects, and fomites play a role as mechanical vectors. Little is known about the interaction between Anaplasma marginale and Argasidae ticks. Among soft ticks, Ornithodoros fonsecai (Labruna and Venzal) and Ornithodoros brasiliensis Aragão inhabit environments surrounding localities where many cases of bovine anaplasmosis have been reported. Ticks of the species O. fonsecai parasitize bats, while O. brasiliensis can parasitize different vertebrate species. Therefore, the present study aimed to feed third-instar nymphs artificially (N3) of O. fonsecai and O. brasiliensis using blood samples obtained from a calf naturally infected with A. marginale and rabbit blood added to A. marginale-containing bovine erythrocytes, to investigate the ability of these nymphs to acquire, infect and transstadially perpetuate this agent. For the artificial feeding system, adapted chambers and parafilm membranes were used. Nymphs of both tick species were submitted to different replications weighed before and after each feeding. Blood samples and molted ticks were submitted to DNA extraction, quantitative real-time PCR for the msp1ß gene to detect A. marginale DNA, while a semi-nested polymerase chain reaction for the msp1α gene was performed for genotyping. Using calf blood naturally infected with A. marginale, among the three artificial feeding replications performed with O. fonsecai and O. brasiliensis nymphs, the DNA of A. marginale was detected in both nymphs after 30-50 days of molting. For artificial feeding with rabbit blood added to bovine erythrocytes containing A. marginale, the DNA of this pathogen was also detected in both nymph species. As for the assay for the msp1α gene, strains were found Is9; 78 24-2; 25; 23; α; and ß. It was concluded that nymphs (N3) of O. fonsecai and O. brasiliensis could feed artificially through a parafilm membrane using blood from calves and rabbits infected by A. marginale. The DNA of A. marginale was detected in nymphs fed artificially of both tick species studied after molt. However, further studies are needed to confirm transstadial perpetuation in other instars and their host transmission capacity.

Contribution to the knowledge of Neotrichodectes (Nasuicola) pallidus (Piaget, 1880) (Phthiraptera: Trichodectidae).

The species in the genus Neotrichodectes (Phthiraptera: Ischnocera) infest carnivores. Neotrichodectes (Nasuicola) pallidus (Piaget, 1880), which has been primarily found parasitizing Procyonidae mammals, has been recorded in ring-tailed coatis (Nasua nasua) in the Brazilian states of Minas Gerais, Pernambuco, Santa Catarina, Rio Grande do Sul and Pernambuco. We report a new record of N. pallidus in coatis in the state of Mato Grosso do Sul, central-western Brazil, using morphological (Light and Scanning Electronic Microscopy) and molecular approaches (PCR, sequencing and phylogenetic analysis). Coatis were sampled in two peri-urban areas of Campo Grande city, Mato Grosso do Sul state, Brazil, between March 2018 and March 2019, as well as in November 2021. Lice were collected and examined under light and Scanning Electron Microscopy. DNA was also extracted from nymphs and adults and submitted to PCR assays based on the 18S rRNA and cox-1 genes for molecular characterization. One hundred and one coatis were sampled from 2018 to 2019 and 20 coatis in 2021 [when the intensity of infestation (II) was not accessed]. Twenty-six coatis (26/101-25.7%) were infested with at least one louse, with a total of 59 lice collected in 2018-2019. The II ranged from one to seven lice (mean 2.2 ± SD 1.7). The louse species was confirmed based on the following morphological characteristics: female gonapophyses rounded with the setae along anterior region but not in the medial margin; the male genitalia with a parameral arch not extending beyond the endometrial plate. The same ornamentation was observed on the abdomen of the females, males, and nymphs. The nymphs and the eggs were described in detail for the first time. The obtained 18S rRNA and cox1 sequences from N. pallidus clustered in a clade with other sequences of Ischnocera species. In the present study, a new record of the louse N. pallidus in central-western Brazil was provided, along with new insights into the morphological features of this species, with the first morphology contribution of nymphal and eggs stages.

Molecular Survey of Hemotropic Mycoplasma spp. and Bartonella spp. in Coatis (Nasua nasua) from Central-Western Brazil.

Even though previous works showed molecular evidence of hemotropic Mycoplasma spp. (hemoplasmas) in ring-tailed coatis (Nasua nasua) from Brazil, Bartonella sp. has not been reported in these mammals so far. The present study aimed to detect the above-mentioned agents in coatis' blood and associated ectoparasites, assessing the association between these infections and red blood parameters. Between March 2018 and January 2019, coati (n = 97) blood samples, Amblyomma sp. ticks (2242 individual ticks, resulting in 265 pools), and Neotrichodectes pallidus louse (n = 59) were collected in forested urban areas from midwestern Brazil. DNA extracted from coatis' blood, and ectoparasite samples were submitted to quantitative PCR (qPCR) (16S rRNA) and conventional PCR (cPCR) (16S rRNA and 23S rRNA) for hemoplasmas and qPCR (nuoG gene) and culturing (only blood) for Bartonella spp. Two different hemoplasma genotypes were detected in blood samples: 71% coatis positive for myc1 and 17% positive for myc2. While 10% of ticks were positive for hemoplasmas (myc1), no louse was positive. The estimated bacterial load of hemoplasmas showed no association with anemia indicators. All coatis were negative for Bartonella sp. in qPCR assay and culturing, albeit two Amblyomma sp. larvae pools, and 2 A. dubitatum nymph pools were positive in the qPCR. The present work showed a high occurrence of hemoplasmas, with two distinct hemoplasma genotypes, in coatis from forested urban areas in midwestern Brazil.

Bacterial pathogens' screening in Brazilian chigger mites (Trombidiformes: Trombiculidae), with the first report of 'Candidatus Rickettsia colombianensi'-like in avian-associated chiggers.

Chiggers are larval ectoparasites of the Trombiculidae that can transmit pathogens to their hosts. In this study, chiggers collected from birds in Brazil were morphologically identified as Blankaartia sinnamaryi, Eutrombicula batatas, Eutrombicula daemoni, Eutrombicula goeldii, Eutrombicula tinami, and Parasecia gilbertoi. For these specimens, a beginning attempt at molecular identification were also provided, as well as, were genetically screened to detect bacterial pathogens. The species B. sinnamaryi and E. tinami were positive for Rickettsia felis-like and 'Candidatus Rickettsia colombianensi'-like, respectively. For the other agents (Anaplasmataceae, Borrelia spp. and Orientia tsutsugamushi), the tests were negative. This is the first report of 'Ca. R. colombianensi'-like and the second record of R. felis-like in chigger collected on birds from Brazil.

Multilayer Networks Assisting to Untangle Direct and Indirect Pathogen Transmission in Bats.

The importance of species that connect the different types of interactions is becoming increasingly recognized, and this role may be related to specific attributes of these species. Multilayer networks have two or more layers, which represent different types of interactions, for example, between different parasites and hosts that are nonetheless connected. The understanding of the ecological relationship between bats, ectoparasites, and vector-borne bacteria could shed some light on the complex transmission cycles of these pathogens. In this study, we investigated a multilayer network in Brazil formed by interactions between bat-bacteria, bat-ectoparasite, and ectoparasite-bacteria, and asked how these interactions overlap considering different groups and transmission modes. The multilayer network was composed of 31 nodes (12 bat species, 14 ectoparasite species, and five bacteria genera) and 334 links, distributed over three layers. The multilayer network has low modularity and shows a core-periphery organization, that is, composed of a few generalist species with many interactions and many specialist species participating in few interactions in the multilayer network. The three layers were needed to accurately describe the multilayer structure, while aggregation leads to loss of information. Our findings also demonstrated that the multilayer network is influenced by a specific set of species that can easily be connected to the behavior, life cycle, and type of existing interactions of these species. Four bat species (Artibeus lituratus, A. planirostris, Phyllostomus discolor, and Platyrrhinus lineatus), one ectoparasite species (Steatonyssus) and three bacteria genera (Ehrlichia, hemotropic Mycoplasma and Neorickettsia) are the most important species for the multilayer network structure. Finally, our study brings an ecological perspective under a multilayer network approach on the interactions between bats, ectoparasites, and pathogens. By using a multilayer approach (different types of interactions), it was possible to better understand these different ecological interactions and how they affect each other, advancing our knowledge on the role of bats and ectoparasites as potential pathogen vectors and reservoirs, as well as the modes of transmission of these pathogens.

Artificial feeding of Ornithodoros fonsecai and O. brasiliensis (Acari: Argasidae) and investigation of the Transstadial perpetuation of Anaplasma marginale

Anaplasma marginale is a Gram-negative, obligate intraerythrocytic bacterium that causes bovine anaplasmosis. While hard ticks of the genera Dermacentor and Rhipicephalus can be biological vectors, transmitting this pathogen via saliva during blood meals, blood-sucking insects, and fomites play a role as mechanical vectors. Little is known about the interaction between Anaplasma marginale and Argasidae ticks. Among soft ticks, Ornithodoros fonsecai (Labruna and Venzal) and Ornithodoros brasiliensis Aragão inhabit environments surrounding localities where many cases of bovine anaplasmosis have been reported. Ticks of the species O. fonsecai parasitize bats, while O. brasiliensis can parasitize different vertebrate species. Therefore, the present study aimed to feed third-instar nymphs artificially (N3) of O. fonsecai and O. brasiliensis using blood samples obtained from a calf naturally infected with A. marginale and rabbit blood added to A. marginale-containing bovine erythrocytes, to investigate the ability of these nymphs to acquire, infect and transstadially perpetuate this agent. For the artificial feeding system, adapted chambers and parafilm membranes were used. Nymphs of both tick species were submitted to different replications weighed before and after each feeding. Blood samples and molted ticks were submitted to DNA extraction, quantitative real-time PCR for the msp1β gene to detect A. marginale DNA, while a semi-nested polymerase chain reaction for the msp1α gene was performed for genotyping. Using calf blood naturally infected with A. marginale, among the three artificial feeding replications performed with O. fonsecai and O. brasiliensis nymphs, the DNA of A. marginale was detected in both nymphs after 30–50 days of molting. For artificial feeding with rabbit blood added to bovine erythrocytes containing A. marginale, the DNA of this pathogen was also detected in both nymph species. As for the assay for the msp1α gene, strains were found Is9; 78 24-2; 25; 23; α; and β. It was concluded that nymphs (N3) of O. fonsecai and O. brasiliensis could feed artificially through a parafilm membrane using blood from calves and rabbits infected by A. marginale. The DNA of A. marginale was detected in nymphs fed artificially of both tick species studied after molt. However, further studies are needed to confirm transstadial perpetuation in other instars and their host transmission capacity.

Contribution to the knowledge of Neotrichodectes (Nasuicola) pallidus (Piaget, 1880) (Phthiraptera: Trichodectidae)

The species in the genus Neotrichodectes (Phthiraptera: Ischnocera) infest carnivores. Neotrichodectes (Nasuicola) pallidus (Piaget, 1880), which has been primarily found parasitizing Procyonidae mammals, has been recorded in ring-tailed coatis (Nasua nasua) in the Brazilian states of Minas Gerais, Pernambuco, Santa Catarina, Rio Grande do Sul and Pernambuco. We report a new record of N. pallidus in coatis in the state of Mato Grosso do Sul, central-western Brazil, using morphological (Light and Scanning Electronic Microscopy) and molecular approaches (PCR, sequencing and phylogenetic analysis). Coatis were sampled in two peri-urban areas of Campo Grande city, Mato Grosso do Sul state, Brazil, between March 2018 and March 2019, as well as in November 2021. Lice were collected and examined under light and Scanning Electron Microscopy. DNA was also extracted from nymphs and adults and submitted to PCR assays based on the 18S rRNA and cox-1 genes for molecular characterization. One hundred and one coatis were sampled from 2018 to 2019 and 20 coatis in 2021 [when the intensity of infestation (II) was not accessed]. Twenty-six coatis (26/101–25.7%) were infested with at least one louse, with a total of 59 lice collected in 2018–2019. The II ranged from one to seven lice (mean 2.2 ± SD 1.7). The louse species was confirmed based on the following morphological characteristics: female gonapophyses rounded with the setae along anterior region but not in the medial margin; the male genitalia with a parameral arch not extending beyond the endometrial plate. The same ornamentation was observed on the abdomen of the females, males, and nymphs. The nymphs and the eggs were described in detail for the first time. The obtained 18S rRNA and cox1 sequences from N. pallidus clustered in a clade with other sequences of Ischnocera species. In the present study, a new record of the louse N. pallidus in central-western Brazil was provided, along with new insights into the morphological features of this species, with the first morphology contribution of nymphal and eggs stages.

Eutrombicula cochinocaensis n. sp.: a chigger (Trombidiformes: trombiculidae) parasite of camelids (Cetartiodactyla: camelidae) in Argentina

The known chigger fauna of Argentina comprises 17 species organized in the families Trombiculidae (10) and Leeuwenhoekiidae (7). The present study describes a new species of Eutrombicula with drawings, phase contrast microscopy images, and SEM images. This chigger was collected parasitizing a vicuna in Abra Pampa City, Jujuy Province. It is the first time a chigger from Argentina has been collected from the family Camelidae.

Novel Ehrlichia and Hepatozoon genotypes in white-eared opossums (Didelphis albiventris) and associated ticks from Brazil.

White-eared opossums (Didelphis albiventris) are well adapted to anthropized areas. The increased contact with domestic animals and humans mediates the transmission of arthropod-borne pathogens. Despite the worldwide occurrence of tick-borne Anaplasmataceae and Hepatozoidae species in a variety of vertebrates, few studies reported serological evidence or molecular detection of theses agentes in marsupials. Up to now, while Ehrlichia/Anaplasma spp. have only been detected in marsupials from Brazil, Hepatozoon spp. have been reported in marsupials from Chile, Australia and Brazil. The present work aimed to investigate, using molecular techniques and blood smear analysis, the presence of Ehrlichia spp., Anaplasma spp., and Hepatozoon sp. in the blood and ticks collected from D. albiventris in urban forest fragments from midwestern Brazil. Between May and December 2017, 43 D. albiventris (27 males and 16 females) were captured for blood and tick collection in the city of Campo Grande, state of Mato Grosso do Sul, midwestern Brazil. Ticks (46 Amblyomma dubitatum nymphs and 24 Amblyomma spp. larvae) were collected from 14 out 43 (32.5%) of the white-eared opossums. Panoptic-stained blood smears were performed using peripheral blood (tail tip) of the captured opossums. DNA extracted from blood and tick samples were subjected to PCR/qPCR assays for Anaplasmataceae agents (rrs, gltA, groEL, sodB, and dsb genes, and 23S-5S intergenic region) and Hepatozoon spp. (18S rRNA gene), followed by Sanger sequencing, BLASTn and phylogenetic analyses. An inclusion resembling Ehrlichia morulae was found in a white-eared opossum's monocyte from a blood smear stained with Panoptic. Five (11.63% [5/43]) white-eared opossums' blood samples and 7 (25% [7/28]) tick samples (2 pools of Amblyomma spp. larvae and 5 pools of A. dubitatum nymphs) were positive for Anaplasmataceae via a PCR assay targeting the conserved rrs gene. Phylogenetic analysis based on the rrs gene positioned three sequences obtained from opossums and ticks together as a subclade within the Ehrlichia canis clade. However, all samples were negative in a qPCR assay specific for E. canis based on the dsb gene. Phylogenetic analyses positioned the gltA and 23S-5S ITS sequences obtained from opossums' blood samples in a separate clade from the other validated Ehrlichia species. One (2.3% [1/43]) opossum blood sample was positive for the 18S rRNA gene of Hepatozoon sp. The phylogenetic analysis positioned the Hepatozoon sp. sequence obtained from a D. albiventris specimen in a clade with a sequence previously detected in a black storm petrel (Oceanodroma melania) from Mexico. All the other sequences of Hepatozoon sp. previously detected in marsupials from Brazil were positioned in a separated clade. The present work showed the occurrence of putative novel genotypes of Ehrlichia sp. and Hepatozoon sp. in white-eared opossums and associated A. dubitatum ticks from midwestern Brazil.

Sleeping with the enemy: case reports of Ornithonyssus bursa (Berlese, 1888) (Mesostigmata: Macronyssidae) causing human dermatitis in Brazil.

Ornithonyssus bursa, known as the "tropical fowl mite," is a hematophagous mite of domestic and wild birds, which occasionally bites humans. Accidental bites on humans occur mainly when abandoned bird nests are close to homes or when people are handling parasitized birds. In the present study, we describe five case reports of bites on humans and new records of localities for this species. Based on the material examined, we provide morphological and molecular characterizations for this species herein.

Reinstatement of Eutrombicula butantanensis (Fonseca, 1932) (Trombidiformes: Trombiculidae).

Eutrombicula Ewing is currently represented with more than 80 species worldwide parasitizing amphibians, reptiles, birds, and mammals. In the present study, we examined the type series and additional material of two Neotropical chigger's species: Eutrombicula butantanensis (Fonseca) and E. ophidica (Fonseca). Eutrombicula butantanensis (Fonseca) was previously synonymized with E. alfreddugesi (Oudemans). However, we are proposing the reinstatement of E. butantanensis as a valid species. Besides that, we are proposing that E. ophidica is a junior synonym of E. butantanensis. Beyond the morphological similarities, E. butantanensis and E. ophidica were initially described to the state of São Paulo, Brazil, and were accidentally collected parasitizing the skin of a human. Besides that, we are increasing the knowledge about E. butantanensis on Brazil's national territory, recording new locality and host associations.

Establishment and multiapproach characterization of Amblyomma sculptum (Acari: Ixodidae) cell line (ASE-14) from Brazil.

The establishment and characterization of the ASE-14 cell line derived from embryos of Amblyomma sculptum is described here. Primary cultures were started, and after 60 days of culturing a confluent monolayer was formed and the first subculture was then carried out. After this, new subcultures were carried out every 4 weeks. Cryopreservation of cells was successful only after the 14th subculture. We compared the chromosomes of the ASE-14 cell line with those of parental ticks. Cytogenetic analysis revealed occurrences of variable and increased diploid numbers in the ASE-14 cell line in comparison with adult ticks, probably through polyploidization events, chromosome fusions and translocations, which allowed generation of cells with distinct diploid numbers. Confirmation of the origin of the A. sculptum cell line was obtained through conventional PCR and sequencing of a fragment of the mitochondrial 16S rRNA gene. In addition, no DNA from Anaplasma marginale, Anaplasma spp., Babesia/Theileria spp., Bartonella spp., Coxiella spp., Ehrlichia canis, Mycoplasma spp. or Rickettsia spp. was detected in the cells through PCR assays. Cytological analyses were performed using live phase contrast microscopy and cytocentrifuge smears stained with Giemsa, while periodic acid-Schiff and bromophenol blue staining techniques were used to detect polysaccharides and protein, respectively. In conclusion, a new cell line derived from embryos of A. sculptum was generated and characterized in this study. The ASE-14 cell line was deposited in the Tick Cell Biobank at the University of Liverpool, and in the Tick Cell Biobank South America Outpost at the Oswaldo Cruz Foundation (FIOCRUZ). The ASE-14 cell line is an important addition to the existing panel of tick cell lines and can be used as a tool for advancing research in various areas of the virology, bacteriology, biology and control of this tick.