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.

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.

A NEW SPECIES OF CHIGGER MITE AND THE RECORD OF A GASTROINTESTINAL NEMATODE IN SCELOPORUS CAUTUS (SQUAMATA: PHRYNOSOMATIDAE) FROM THE CHIHUAHUAN DESERT PROVINCE IN COAHUILA, MEXICO.

The chigger species Acomatacarus coahuilensis n. sp. (Acariformes: Trombidiformes: Leeuwenhoekiidae) inhabiting the neck scales of the wary spiny lizard Sceloporus cautus Smith, 1938 (Squamata: Iguania: Phrynosomatidae), from Chihuahuan Desert Province, Coahuila, Mexico, is described herein. The new mite species is assigned to the genus AcomatacarusEwing, 1942, based on a palptarsus with 6 branched setae plus the tarsala (ω). This new species can be separated from the other species in this genus mainly based on the combination of the following characters: presence of mastisetae (MTa) on tarsus III and tibiala (φ) on tibia III, subterminala (ζ1), parasubterminala (z), and pretarsala (ζ2) on tarsus I, 2 genualae (σ1-2) on genu I, onychotriches and claw-like empodium of legs I-III, and absence of mastisetae on tibia III. In addition to the new species, the gastrointestinal nematode Skrjabinoptera phrynosoma (Ortlepp, 1922) Schulz, 1927 (Physalopteridae), was also found, which represents a new host record in the wary spiny lizard. This study reports the third species of the genus Acomatacarus recorded from Mexico and the first record of chigger mites and helminth parasites associated with Sc. cautus.

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 detection and characterization of vector-borne agents in common opossums (Didelphis marsupialis) from northeastern Brazil.

Opossums are synanthropic marsupials able to interchange among wild, peri­urban and urban environments, playing an epidemiologically important role as hosts for emerging pathogens and ectoparasites of relevance in public health. The present study aimed to detect and molecularly characterize vector-borne agents in a population of common opossums (Didelphis marsupialis) from the Island of São Luís do Maranhão, northeastern Brazil. Of the 45 animals analyzed, one (2.22%) was positive in the nested PCR assay based on the 18S rRNA gene of piroplasmids. The obtained sequence was phylogenetically positioned in a clade containing sequences of Babesia sp. previously detected in Didelphis aurita, Didelphis albiventris and associated ticks from Brazil. Eight (17.77%) samples were positive in PCR for Ehrlichia spp. based on the dsb gene; four samples were sequenced and positioned into a new clade, sister to E. minasensis and Ehrlichia sp. clade detected in Superorder Xenarthra mammals. No samples tested positive in the screening PCR assays based on the 16S rRNA gene of Anaplasma spp. Two samples were positive in the qPCR for Bartonella spp. based on the nuoG gene. Seven animals (15.56%) were positive in the nPCR based on the 16S rRNA gene of hemoplasmas. Of these, three were positive in a PCR based on the 23S rRNA gene. The phylogenies based on both 16S rRNA and 23S rRNA genes corroborated to each other and positioned the sequences in the same clade of hemoplasmas previously detected in D. aurita and D. albiventris sampled in Brazil. Finally, three (6.66%) animals were positive in the PCR for Hepatozoon spp.; the obtained 18S rRNA sequence was positioned into the H. felis clade.The present study showed, for the first time, the circulation of piroplasmids, Hepatozoon spp., Ehrlichia spp., hemoplasmas and Bartonella spp. in D. marsupialis sampled in northeastern Brazil, with description of putative novel genotypes of Ehrlichia and Hepatozoon and copositivity by different vector-borne agents. The present work consolidates the "South American Marsupialia" piroplasmid clade, adding one more genotype of Babesia sp. to this clade.

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.

Contribution to the knowledge of Parichoronyssus bakeri Morales-Malacara and Guerrero, 2007 (Mesostigmata: Macronyssidae): new locality and host-association records with additional molecular data

The mite genus Parichoronyssus Radovsky (Mesostigmata: Macronyssidae) comprises twelve known species found parasitizing bats in the New World. This study aimed to identify morphologically and molecularly macronyssid mites collected from bats and kept as backlog in the tissue bank (-80C) at the Acarological Collection of the Butantan Institute (IBSP). After the examination, the material was listed and identified as Parichoronyssus bakeri Morales-Malacara and Guerrero. Here, we were able to provide diagnostic microscopic images for the females of this species, as well as molecular information (18S rRNA and 16S rRNA) that were deposited in Genbank for reference. New locality records were also provided for P. bakeri in Rio de Janeiro and Rondônia States, Brazil.

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 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.

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.

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.