Isolation and molecular characterization of a novel relapsing fever group Borrelia from the white-eared opossum Didelphis albiventris in Brazil.

This study aimed to detect, isolate and to characterize by molecular methods a relapsing fever group (RFG) Borrelia in white-eared opossums (Didelphis albiventris) from Brazil. During 2015-2018, when opossums (Didelphis spp.) were captured in six municipalities of the state of São Paulo, Brazil, molecular analyses revealed the presence of a novel RFG Borrelia sp. in the blood of seven opossums (Didelphis albiventris), out of 142 sampled opossums (4.9% infection rate). All seven infected opossums were from a single location (Ribeirão Preto municipality). In a subsequent field study in Ribeirão Preto during 2021, two new opossums (D. albiventris) were captured, of which one contained borrelial DNA in its blood. Macerated tissues from this infected opossum were inoculated into laboratory animals (rodents and rabbits) and two big-eared opossums (Didelphis aurita), which had blood samples examined daily via dark-field microscopy. No spirochetes were visualized in the blood of the laboratory animals. Contrastingly, spirochetes were visualized in the blood of the two D. aurita opossums between 12 and 25 days after inoculation. Blood samples from these opossums were used for a multi-locus sequencing typing (MLST) based on six borrelial loci. Phylogenies inferred from MLST genes positioned the sequenced Borrelia genotype into the RFG borreliae clade basally to borreliae of the Asian-African group, forming a monophyletic group with another Brazilian isolate, "Candidatus B. caatinga". Based on this concatenated phylogenetic analysis, which supports that the new borrelial isolate corresponds to a putative new species, we propose the name "Candidatus Borrelia mimona".

New records of soft ticks (Acari: Argasidae) from caves in Brazil, with a morphological study of Ornithodoros fonsecai and an analysis of the taxonomic status of Antricola inexpectata.

In this study, we report soft ticks from bat-inhabiting caves in different areas of Brazil. From 2010 to 2019, we collected 807 tick specimens from nine caves located in four Brazilian states among two biomes. Ticks were morphologically identified as Antricola guglielmonei (282 specimens), Ornithodoros cavernicolous (260 specimens), and Ornithodoros fonsecai (265 specimens). Whereas A. guglielmonei was collected on bat guano in hot caves, O. cavernicolous and O. fonsecai were collected in cracks and crevices on the walls of cold caves, sometimes in the same chamber. Morphological identifications were corroborated by molecular and phylogenetic analyses inferred from tick mitochondrial 16S rRNA gene partial sequences. The sequences of A. guglielmonei, O. cavernicolous and O. fonsecai collected in this study clustered with conspecific GenBank sequences from different localities of Brazil. Remarkably, a clade containing 12 sequences of O. fonsecai was clearly bifurcated, denoting a degree of genetic divergence (up to 5 %) of specimens from Cerrado/Atlantic Forest biomes with the specimens from the Caatinga biome. To further evaluate this divergence, we performed morphometric analysis of the larval stage of different O. fonsencai populations by principal component analysis, which indicated that the larvae from Caatinga populations were generally smaller than the larvae from other biomes. Some of the present A. guglielmonei specimens were collected from the type locality of Antricola inexpectata. Comparisons of these specimens with the type specimens of A. inexpectata and A. guglielmonei indicated that they could not be separated by their external morphology. Hence, we are relegating A. inexpectata to a synonym of A. guglielmonei. This proposal is corroborated by our phylogenetic analysis.

A Novel Relapsing Fever Group Borrelia Isolated from Ornithodoros Ticks of the Brazilian Caatinga.

Tick-borne relapsing fever group (RFG) borreliosis remains neglected as a human disease and little is known on its maintenance in ticks and vertebrates, especially in South America. Therefore, this study investigated borrelial infection in Ornithodoros ticks collected in rodent-inhabited rock formations in the Brazilian semiarid region, within the Caatinga biome. Collected ticks (Ornithodoros rietcorreai and Ornithodoros cf. tabajara) were allowed to feed under laboratory conditions on guinea pigs, which had blood samples examined daily by dark-field microscopy. No spirochetes were visualized in the blood of any of four O. rietcorreai-infested guinea pigs. Contrastingly, spirochetes were visualized between 9 and 39 days after tick feeding in the blood of three guinea pigs, each infested with O. cf. tabajara ticks from a different locality. Guinea pig infection was confirmed by passages into experimental animals and by generating DNA sequences of Borrelia spp. from the blood of spirochetemic guinea pigs. Three O. cf. tabajara populations were infected by the same borrelial organism, which was characterized as a novel RFG agent (named as 'Candidatus Borrelia caatinga') based on 10 Borrelia loci (rrs, flaB, glpQ, gyrB, clpX, pepX, pyrG, recG, rplB and uvrA). We demonstrated that O. cf. tabajara is a competent vector of the novel Borrelia sp. isolates, although none of the infected rodents developed clinical illness.

Novel Borrelia Genotypes from Brazil Indicate a New Group of Borrelia spp. Associated with South American Bats.

The bacterial genus Borrelia comprises vector-borne spirochetes that have been classified into three major groups: the relapsing fever group (RFG), the Borrelia burgdorferi Johnson, Schmid, Hyde, Steigerwalt & Brenner sensu lato group (Bbsl), and the reptile-monotreme group (RMG). All three groups have been associated mainly with ticks and wild animals, especially rodents, birds, and reptiles. Here, we searched for Borrelia infection among 99 vampire bats [Desmodus rotundus (É. Geoffroy)] (Chiroptera: Phyllostomidae) from the Brazilian semiarid region. Through molecular investigation of bat internal organs, haplotypes of a potentially novel Borrelia organism were detected in 5% (5/99) of the bats. Borrelia DNA was detected in the liver, blood, spleen, kidney and brain, suggesting a systemic infection. Phylogenetic analyses inferred from partial sequences of the borrelial rrs and flaB genes indicated that the vampire bat-associated Borrelia sp. of this study form a monophyletic group with a newly reported Borrelia associated with a Colombia bat, distinct from the three main currently recognized groups of Borrelia spp., Bbsl, RFG, and RMG. These novel bat-associated Borrelia spp. from South America might have arisen through an independent event along the borrelial evolutionary history, since previous molecular reports of Borrelia organisms in bats or bat-associated ticks from Africa, Europe, and North America were all classified in the RFG.

New records of soft ticks (Acari: Argasidae) in the Caatinga biome of Brazil, with a phylogenetic analysis of argasids using the nuclear Histone 3 (H3) gene.

In Brazil, 19 species of the genus Ornithodoros (Acari: Argasidae) have been reported. The medical and veterinary importance of Ornithodoros ticks has increased substantially in recent decades, with the discovery of various relapsing fever Borrelia infecting Ornithodoros ticks. Herein, argasid ticks were collected during 2019-2020 from caves, abandoned nests and homes in various regions of Ceará State, Brazilian semiarid-Caatinga biome. In total, 289 ticks were collected and identified into five species: Ornithodoros cavernicolous (176 specimens), Ornithodoros fonsecai (81), Ornithodoros mimon (12), Ornithodoros rietcorreai (4), and a fifth species provisionally retained as Ornithodoros sp. Ubajara. Tick identifications were corroborated by a phylogenetic analysis inferred using the 16S rRNA gene. To extend the molecular characterization, DNA samples were tested by an additional PCR assay targeting the nuclear Histone 3 (H3) gene. Because there were no H3 sequences of argasids in GenBank, we extended this PCR assay for additional Ornithodoros species, available in our laboratory. In total, 15 partial sequences of the H3 gene were generated for 10 Ornithodoros species, showing 0% intraspecific polymorphism, and 1.5-11.6% interspecific polymorphism. Phylogenetic analyses inferred segregated Ornithodoros sp. Ubajara as a potential novel species. Our results also highlight the potential of the H3 gene for deeper phylogenetic analyses of argasids. The present study provides new data for argasid ticks of the genus Ornithodoros in the Caatinga biome. Because some of these tick species are human-biting ticks, active surveillance for the incidence of human infection due to Ornithodoros-borne agents is imperative in the Caatinga biome.

Anthelmintic resistance of gastrointestinal nematodes in sheep grazing in irrigated and dry areas in the semiarid region of northeastern Brazil.

This study aimed to determine the AR of gastrointestinal nematodes (GIN) to commercial drugs in sheep flocks naturally infected, grazing in irrigated (IA) and dry (DA) areas of the semiarid region in northeastern Brazil. Fecal egg count reduction tests (FECRT) were performed at 10 farms. From each flock, 36 adult sheep were selected and divided into five groups (G1 (0.08% ivermectin), G2 (10% albendazole), G3 (5% levamisole), G4 (1% moxidectin), G5 (10% closantel) and one control group, G6). All the commercial drugs were found to reduce the number of eggs per gram of feces (EPG). Resistance to ivermectin (37.1%), albendazole (52.1%), and levamisole (52.0%) was detected at all the farms, but nematodes proved to be susceptible to moxidectin (87.9%) and closantel (83.9%). The overall average efficacy of the commercial drugs was significantly higher (P < 0.05) in DA (49.2%), where moxidectin (90.4%) showed high effectiveness. The presence of the parasite Haemonchus contortus predominated at all the farms. The variables irrigated area (P = 0.002), intensive breeding (P = 0.018), uncovered enclosures (P = 0.05), cultivated (P = 0.043) and native/cultivated (P = 0.007) pastures, and rotational grazing (P = 0.013) were significantly associated with GIN infection; irrigated area (P = 0.009), semi-intensive breeding (P = 0.05), rotational grazing (P = 0.045), cultivated (P = 0.021) and native/cultivated (P = 0.04) pastures, and estimated weighing of animals (P = 0.002) were significantly associated with AR. Therefore, improved management practices and strategic deworming must be implemented to prevent the development of AR.

Relapsing Fever Group Borreliae in Human-Biting Soft Ticks, Brazil.

We conducted a molecular survey for Borrelia spp. in Ornithodoros ticks previously reported as biting humans. We collected specimens in natural ecosystems and inside human dwellings in 6 states in Brazil. Phylogenetic analyses unveiled the occurrence of 4 putatively new species of relapsing fever group borreliae.

Ectoparasites of small mammals in a fragmented area of the southern Amazonia: interaction networks and correlations with seasonality and host sex.

The present work aimed to analyze the ectoparasite-host interaction network and possible differences of this interaction related to two seasonal periods and host sex. During November 2016 and July 2017, non-flying small mammals were captured in 17 forest fragments located in the southern portion of the Amazon biome. We captured 96 individuals belonging to 10 host species that were parasitized with a total of 3668 ectoparasites. Overall, we identified 24 ectoparasite taxa belonging to the mite and insect groups Ixodida (ticks), Mesostigmata, Sarcoptiformes, Trombidiformes (mites), Phthiraptera (lice), and Siphonaptera (fleas). The interaction network between all ectoparasites and hosts showed significant deviation from random, with moderately high specialization index (H2' = 0.80). There was seasonal difference in prevalence for Amblyomma cajennense (Fabricius) sensu stricto (s.s), Amblyomma coelebs Neumann and larvae of Amblyomma. This difference was also found in the mean intensity of infestation of Amblyomma larvae and the mite Tur aragaoi (Fonseca). Only mean intensity of infestation differed in relation to host sex for the species Marmosa constantiae Thomas. Our results demonstrate that specificity between ectoparasites and small mammals in this region is moderately high and that the pattern of aggregation of some ectoparasite taxa differed between two seasons, as well as between sexes in M. constantiae.

A third species of Nothoaspis Keirans & Clifford, 1975 (Acari: Argasidae): Nothoaspis setosus (Kohls, Clifford & Jones, 1969) n. comb.

In South America, early descriptions of soft tick species were based on examination of the external morphology of the larval stages. In many cases, specimens were collected only once as it is the case of some bat-associated Ornithodoros spp. If we are to understand the systematic scenario of South American soft ticks, these species become axial questions to be re-studied from a morphological and molecular point of view. The objective of this study was to assess the taxonomic identity of soft tick larvae collected on bats inhabiting crevices of a large rock in the Rondônia State (RO), Brazilian Amazon. After a detailed morphological analysis using light microscopy, three large engorged larvae sharing the same phenotype were identified as Ornithodoros setosus Kohls, Clifford & Jones, 1969, a species collected in 1964 on bats in RO. Remarkably, maximum parsimony and Bayesian phylogenetic analyses based on tick 16S rRNA mitochondrial gene sequences obtained from two of these specimens showed that O. setosus indeed corresponds to a species of Nothoaspis Keirans & Clifford, 1975. Therefore, a new combination, Nothoaspis setosus (Kohls, Clifford & Jones, 1969), is herein proposed. While an elongated triangular dorsal plate with a curvy-notched posterior margin, and bulges in the lateral margins of basis capitulum correspond to common characters in larvae of the genus Nothoaspis, polymorphic traits are represented by minute cornua in the basis of the capitulum, the dentition of the hypostomal tip, triangular spurs on coxae I, and the number of dorsal and circumanal setae.

Rickettsia bellii in Amblyomma rotundatum ticks parasitizing Rhinella jimi from northeastern Brazil.

This study evaluated rickettsial infection in Amblyomma rotundatum ticks collected from toads (Rhinella jimi) in the Brazilian Caatinga biome, an unique semiarid region of South America. Tick infestations were observed in 57.8% toads (26/45); mean infestation: 1.6 ticks/toad. DNA extraction from 42 ticks (6 larvae, 22 nymphs and 11 female adults) was tested by polymerase chain reaction (PCR) targeting Rickettsia organisms, which were detected in 100% of the ticks. Amplicons' DNA sequences were identical to each other and 99% identical to Rickettsia bellii from GenBank. DNA samples extracted from the blood of the 45 toads were negative by rickettsia-PCR protocols.