Phylogenetic inferences based on distinct molecular markers reveals a novel Babesia (Babesia pantanalensis nov. sp.) and a Hepatozoon americanum-related genotype in crab-eating foxes (Cerdocyon thous).

Piroplasmids and Hepatozoon spp. Are apicomplexan protozoa that may cause disease in several canid species. The present study aimed to expand the knowledge on the diversity of piroplasmids and Hepatozoon in crab-eating foxes (Cerdocyon thous; n = 12) sampled in the Pantanal of Mato Grosso do Sul State, central-western Brazil. PCR assays based on the 18S rRNA were used as screening. Three (25%) and 11 (91.7%) were positive for piroplasmids and Hepatozoon spp., respectively. Co-infection was found in three C. thous. Phylogenetic analyses based on the near-complete 18S rRNA, cox-1 and hsp70 genes evidenced the occurrence of a novel of Babesia spp. (namely Babesia pantanalensis nov. sp.) closely related to Rangelia vitalii and Babesia sp. 'Coco'. This finding was supported by the genetic divergence analysis which showed (i) high divergence, ranging from 4.17 to 5.62% for 18 S rRNA, 6.16% for hps70 and 4.91-9.25% for cox-1 and (ii) the genotype network (which displayed sequences separated from the previously described Piroplasmida species by median vectors and several mutational events). Also, phylogenetic analysis based on the 18S rRNA gene of Hepatozoon spp. positioned the sequences obtained herein in a clade phylogenetically related to Hepatozoon sp. 'Curupira 2', Hepatozoon sp. detected in domestic and wild canids from Uruguay and Hepatozoon americanum. The present study described Babesia pantanalensis nov sp. and Hepatozoon closely related to H. americanum in crab-eating foxes from Brazil. Moreover, the coinfection by piroplasmids and Hepatozoon sp. for the first time in crab-eating foxes strongly suggesting that this wild canid species potentially acts as a bio-accumulate of hemoprotozoan in wild environment.

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.

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.

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.

Molecular detection of piroplasmids in synanthropic rodents, marsupials, and associated ticks from Brazil, with phylogenetic inference of a putative novel Babesia sp. from white-eared opossum (Didelphis albiventris).

The order Piroplasmida encompasses tick-borne pathogens of veterinary and medical importance positioned in two main families: Babesiidae and Theileriidae. Even though previous studies carried out in Brazil recorded the occurrence of piroplasmid species circulating in small mammals, 18S RNA gene sequences were only partially sequenced, preventing the assessment of their phylogenetic positioning. The current study aimed to detect and characterize, using morphological, molecular, and bioinformatic approaches, piroplasmids from wild mammals and associated ticks sampled in Central-Western Brazil. Out of 67 Didelphis albiventris sampled, 22 (16.4%) were positive for piroplasmids by PCR. In contrast, none of the 48 small rodents and 14 capybaras (Hydrochoerus hydrochaeris) was PCR-positive. Four Amblyomma dubitatum ticks-one from Rattus rattus, one from H. hydrochaeris, and two from D. albiventris-out of 114 Amblyomma spp. DNA samples were positive for piroplasmids by PCR. The phylogenetic inference performed using the near-complete 18S rRNA gene positioned the putative novel piroplasmid species detected in D. albiventris and associated A. dubitatum ticks near to Babesia sensu lato clade (Western group-cluster III) and distant from the Australian marsupial-associated piroplasms. Phylogenetic inferences based on two additional molecular markers, namely hsp-70 and cox-1, supported the near-complete 18S rRNA gene phylogenetic inference. Finally, the partial 18S rRNA gene sequences detected in ticks from rodents (R. rattus and H. hydrochaeris) showed 97.2-99.4% identity with the Piroplasmida previously detected in a capybara from Brazil, raising evidence that a still uncharacterized piroplasmid species has been identified in the capybara, the largest rodent species from South America.

Description of Leptus (Leptus) haitlingeri n. sp. (Trombidiformes: Erythraeidae), parasitising horse flies (Diptera: Tabanidae), and a key to the larvae of Leptus spp. in Brazil.

The genus Leptus Latreille, 1796 (Trombidiformes: Erythraeidae) is represented by more than 250 species worldwide, and the larval stages of these mites are ectoparasites of many arthropod orders. In Brazil, there are 12 species of Leptus, some of which have been reported parasitising representatives of the orders Opiliones, Araneae, Coleoptera, Heteroptera, Hymenoptera and Diptera. This paper describes Leptus (Leptus) haitlingeri n. sp. collected from horse flies (Diptera: Tabanidae) in the state of Sao Paulo, Brazil. The new species is distinguished from L. (L.) adaminae Haitlinger, 2004 and L. (L.) fozicus Haitlinger, 2004 by the presence of 4 branched setae on palptarsus (vs 2 branched setae). A key to the larvae of Leptus spp. in Brazil is provided.

Genetic Diversity of Bartonella spp. in Wild Mammals and Ectoparasites in Brazilian Pantanal.

The present work aimed to investigate the genetic diversity of Bartonella in mammals and ectoparasites in Pantanal wetland, Brazil. For this purpose, 31 Nasua nasua, 78 Cerdocyon thous, 7 Leopardus pardalis, 110 wild rodents, 30 marsupials, and 42 dogs were sampled. DNA samples were submitted to a quantitative real-time PCR assay (qPCR). Positive samples in qPCR were submitted to conventional PCR assays targeting other five protein-coding genes. Thirty-five wild rodents and three Polygenis (P.) bohlsi bohlsi flea pools showed positive results in qPCR for Bartonella spp. Thirty-seven out of 38 positive samples in qPCR were also positive in cPCR assays based on ftsZ gene, nine in nuoG-cPCR, and six in gltA-cPCR. Concatenated phylogenetic analyses showed that two main genotypes circulate in rodents and ectoparasites in the studied region. While one of them was closely related to Bartonella spp. previously detected in Cricetidae rodents from North America and Brazil, the other one was related to Bartonella alsatica, Bartonella pachyuromydis, Bartonella birtlesii, Bartonella acomydis, Bartonella silvatica, and Bartonella callosciuri. These results showed that at least two Bartonella genotypes circulate among wild rodents. Additionally, the present study suggests that Polygenis (P.) bohlsi bohlsi fleas could act as possible Bartonella vectors among rodents in Pantanal wetland, Brazil.

First Record of the Genus Trochiliphagus Carriker 1960 (Amblycera: Ricinidae) Infesting a Hummingbird from Brazil.

Four genera and at least fifty species of chewing lice have been recorded infesting hummingbirds (Trochilidae). Here, we record for the first time, the genus Trochiliphagus in Brazil, as well as, a new host record for Trochiliphagus mellivorus Carriker, the fork-tailed woodnymph, Thalurania furcata.

First report of unusual case of parasitism by Amblyomma nodosum (Neumann, 1889) in a yellow cururu toad (Rhinella icterica) in the Northeastern Brazilian Caatinga.

The Amblyomma genus (Arachnida: Ixodidae) is widely distributed in South America, with 34 species occurring in Brazil. Amblyomma nodosum Neumann 1889 is a species that predominantly feeds on Passeriformes during immature stages (larvae and nymphs) and anteaters (Myrmecophagidae) during adult stages. The aim of the present study is to report, for the first time, an unusual case of parasitism by adults of A. nodosum on a yellow cururu toad (Rhinella icterica) captured in the city of Nossa Senhora da Glória, Sergipe state (Northeastern Brazil) in the Caatinga biome, and also investigate the presence of DNA of Rickettsia in the collected material. DNA was extracted from all specimens collected (N=8) and subjected to PCR assays based on the tick 16S rRNA endogenous gene and gltA gene for Rickettsia sp. All samples (8/8; 100%) were positive for the 16S rRNA endogenous gene and two amplicons (obtained from one male and one female) were purified and sequenced. The BLASTn analysis of the sequences revealed a high degree of similarity (95-100%) with A. nodosum sequences previously deposited on GenBank, while the phylogenetic analysis clustered the sequences obtained in the same clade as A. nodosum sequences from Brazil.

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.

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.

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.

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.

Life cycle of Ornithodoros rostratus (Acari: Argasidae) under experimental conditions and comments on the host-parasite relationship in the Pantanal wetland region, Brazil.

The genus Ornithodoros is represented by 15 species in Brazil, on which no detailed life cycle studies have been published, except for O. talaje and O. mimon. The aim of the present study was to evaluate life cycle parameters of O. rostratus based on ticks collected in the Pantanal wetland region of the state of Mato Grosso do Sul, Brazil, using domestic rabbits as experimental hosts. The periods of pre-attachment and feeding of the larvae lasted an average of 39 min (range 15-76 min). Five or six nymphal instars were found. The emergence of adults started with N3 in the following sequence: N3-two males; N4-13 males; N5-three males and 16 females; and N6-two females. Mean weight of N4 that molted to males was 31.7 ± 13.6 mg, whereas mean weight of N5 that molted to females was 100.1 ± 36.2 mg. The overall sex ratio was 1:1. Oviposition lasted 14 days, with a sharp decline beginning with the 7th day. The overall duration of the life cycle of O. rostratus ranged from approximately 66 to 136 days. Comments on the tick-host relationship in the Pantanal region are offered.

Life cycle and behavior of Amblyomma rotundatum (Acari: Ixodidae) under laboratory conditions and remarks on parasitism of toads in Brazil.

The life cycle and behavior of Amblyomma rotundatum were evaluated under laboratory conditions. The experiment started with four engorged females collected from toads (Rhinella schneideri) naturally infested at the Pirapitinga Ecological Station in the state of Minas Gerais, Brazil. Developmental periods of free-living stages were assessed in an incubator at 27 ± 1 °C, >80 % RH and darkness. The complete life cycle, including pre-attachment periods for each parasitic stage, ranged from 126 to 228 days. The pre-attachment, feeding and molting periods increased as the life cycle progressed from larva to adult female. Oviposition lasted about 20 days, with the peak occurring on days 4 and 5. Longevity of nymphs and adult females was quite similar (approximately 250 and 240 days, respectively) and slightly longer than that of larvae. Lesions caused by tick feeding are discussed and a list of known hosts, including new host records for A. rotundatum, is offered.

Description of nymphal instars of Ornithodoros mimon Kohls, Clifford & Jones, 1969 (Acari: Argasidae).

Ornithodoros mimon is an argasid tick common on Chiroptera in the Neotropical region, where it also bites humans aggressively. Here we describe for the first time all nymphal instars (N1, N2 and N3) of O. mimon based on optical and scanning electron microscopy. Although the nymphal instars of O. mimon resemble each other closely, there are characters that differentiate them: the N3 are taller that N1 and N2; the genital primordium occurs in some N2 and all N3; the spiracular plate in N1 and N2 is cone-like, but in N3 it is semicircular; and the submarginal dorsal groove is less distinct in N1 but more evident in N3. Nymphs of 0. mimon closely resemble the bat-associated species of the genus Ornithodoros included in the Alectorobius group. We review prior descriptions of nymphs of the Alectorobius group and make comparisons with nymphs of O. minon, highlighting characters with diagnostic information, such as the idiosomal shape, presence of discs and hood and absence subapical protuberance of tarsus I. The description of nymphal instars of O. mimon herein presented, improves the taxonomy of the family Argasidae, performing a work more detailed about the immature stage of this species.

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.