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.

Novel genotypes of Hepatozoon spp. in small mammals, Brazil.

BACKGROUND: Small mammals (rodents and marsupials) have been poorly explored for the occurrence of apicomplexan (genus Hepatozoon and genera of the order Piroplasmorida) and Anaplasmataceae agents in Brazil. Thus, this study investigated the occurrence of Hepatozoon spp., Piroplasmorida, and Anaplasmataceae agents in small mammals in seven forest fragments in Brazil. METHODS: During 2015-2018, small mammals were captured in six forest fragments in the State of São Paulo (Cerrado and Atlantic Forest biomes) and one fragment in the State of Mato Grosso do Sul (Pantanal biome). Mammal blood, liver, spleen, and lung samples were tested molecularly for the presence of DNA of Hepatozoon, Piroplasmorida, and Anaplasmataceae agents. RESULTS: A total of 524 mammals were captured, comprising seven species of marsupials, 14 rodents, two carnivores, and one Cingulata. Four novel haplotypes (1, 2, 3, 4) of Hepatozoon spp. were detected in small mammals from different biomes. In São Paulo state, haplotype 1 was detected in rodents from Cerrado and a transition area of Cerrado and Atlantic Forest biomes, whereas haplotype 2 was detected in rodents from the Atlantic Forest biome. On the other hand, haplotypes 3 and 4 were restricted to rodents and marsupials, respectively, from the Pantanal biome of Mato Grosso do Sul. No host species shared more than one haplotype. Despite these distinct geographical and host associations, our phylogenetic analyses indicated that the four Hepatozoon haplotypes belonged to the same clade that contained nearly all haplotypes previously reported on rodents and marsupials, in addition to several reptile-associated haplotypes from different parts of the world. No mammal samples yielded detectable DNA of Piroplasmorida agents. On the other hand, the Anaplasmataceae-targeted polymerase chain reaction (PCR) assay amplified a sequence 100% identical to the Wolbachia pipientis endosymbiont of the rodent filarid Litomosoides galizai. CONCLUSIONS: We report a variety of Hepatozoon haplotypes associated with small mammals in three Brazilian biomes: Cerrado, Atlantic Forest, and Pantanal. Through phylogenetic analyses, the Hepatozoon agents grouped in the rodent-marsupial-reptile large clade of Hepatozoon spp. from the world. The detection of a W. pipientis associated with the rodent filarid L. galizai indicates that the rodent was infected by filarial nematodes.

A Novel Genospecies of Borrelia burgdorferi Sensu Lato Associated with Cricetid Rodents in Brazil.

Borrelia burgdorferi sensu lato (Bbsl) spirochetes thrive in sylvatic transmission cycles infecting vertebrates and their ticks. Rodents and ticks of the genus Ixodes are important hosts of these spirochetes globally. Although evidence suggests that Borrelia burgdorferi sensu stricto does not exist in South America, genospecies of the group (Bbsl) can be found in this region but have been poorly characterized from a genetic viewpoint, and data on their ecoepidemiology are still incipient. Aiming to detect the natural foci of Borrelia in Brazil, we targeted small mammals inhabiting seven forests fragments during a period of three years (2015-2018). Organs (lung) from two Oligoryzomys rodents over a total of 382 sampled mammals were positive, and we performed a molecular characterization of 10 borrelial genes to achieve a robust analysis. Phylogenetic trees inferred from 16S rRNA, flaB, ospC, and seven MLST loci (clpA, nifS, pepX, pyrG, recG, rlpB, and uvrA) support the characterization of a novel genospecies of Bbsl that we herein name "Candidatus Borrelia paulista" Rp42. Remarkably, "Ca. B. paulista" is phylogenetically related to Borrelia carolinensis, a genospecies that infects Ixodes ticks and cricetid rodents in North America. A previous study performed in the same area identified Ixodes schulzei feeding on Oligoryzomys rodents. Although this tick species could be considered a probable host for this novel Borrelia sp., further research is needed to confirm this hypothesis.

Ticks and rickettsial exposure in lowland tapirs (Tapirus terrestris) of three Brazilian biomes.

This study evaluated ticks and rickettsial exposure in 220 free-ranging lowland tapirs, Tapirus terrestris, from 2006 to 2018 in selected areas of three major biomes of Brazil - Atlantic Forest, Pantanal, and Cerrado. Overall, a total of 5970 tick specimens representing the following nine species were collected from tapirs: Amblyomma brasiliense, Amblyomma coelebs, Amblyomma dubitatum, Amblyomma ovale, Amblyomma parvum, Amblyomma sculptum, Amblyomma triste, Haemaphysalis juxtakochi, and Rhipicephalus microplus. Amblyomma sculptum was the most prevalent and abundant tick species in all three biomes; however, mean intensity values for A. sculptum were significantly lower in Atlantic Forest than in the Pantanal or Cerrado, and at the same time, statistically similar among tapirs from Pantanal and Cerrado. Contrastingly, mean intensity values for A. coelebs were significantly higher in the Atlantic Forest than in the other biomes. The remaining tick species were collected in lower numbers, or were exclusive for one biome, e.g., A. brasiliense and H. juxtakochi only in the Atlantic Forest. A total of 177 blood sera (123 individuals plus 54 recaptures) were collected from tapirs and tested for the presence of reactive antibodies to six Rickettsia species by immunofluorescence assay. Overall, 69% (9/13), 49% (62/126), and 66% (25/38) tapir sera from Atlantic Forest, Pantanal and Cerrado, respectively, were seroreactive to at least one Rickettsia species, with no significant difference between the three areas. Although many tapir sera reacted simultaneously to ≥2 Rickettsia species, Rickettsia parkeri elicited highest % seroprevalence and endpoint titers, and was incriminated as the possible agent involved in a homologous reaction in tapirs from the three biomes, where A. ovale was previously found infected by R. parkeri. In fact, seroconversion to R. parkeri was demonstrated in five tapirs that were captured at least twice during the study. This study demonstrated that tapirs were found to be constantly infested by several tick species in the Atlantic Forest, Pantanal and Cerrado biomes; however, the richness of tick infestations was concordant to the tick species known to be established in each biome. Under natural conditions, lowland tapirs were shown to be exposed to tick-borne spotted fever group rickettsiae.

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.

Epidemiological aspects of Rickettsia parkeri in the Atlantic forest biome of Espírito Santo state, Brazil.

Since 2010, a new rickettsiosis caused by Rickettsia parkeri, a species included in the spotted fever group (SFG) and transmitted by the tick Amblyomma ovale, has been described in Brazil. Considering that A. ovale does occur in the Espírito Santo state (ES), the present study aimed at preliminarily describing the epidemiology of R. parkeri in the Atlantic forest of this state. Between June 2016 and September 2018, 33 villages from nine municipalities of the ES were included in a study for the molecular detection and isolation of SFG rickettsiae from adult Amblyomma ticks collected on dogs with free access to the forest. Serologic screenings against SFG rickettsiae in these animals and their owners (humans) were performed through immunofluorescence assay (IFA) using antigens of Rickettsia rickettsii and R. parkeri. Additionally, local health secretariats were informed on clinical manifestations of R. parkeri infection and told to communicate any suspected case. A total of 280 adult ticks were collected and taxonomically classified as A. ovale (n = 152), Amblyomma aureolatum (n = 127) and Amblyomma sculptum (n = 1). Overall, Rickettsia DNA was detected in 12/266 ticks. The sequencing of PCR products revealed that 0.7% (1/144) and 0.8% (1/121) of the analyzed A. ovale and A. aureolatum ticks were infected by R. parkeri strain Atlantic rainforest, respectively, and 8.3% (10/121) of the A. aureolatum ticks infected by Rickettsia bellii. Among the above PCR-positive ticks, only one isolate from one A. aureolatum tick was successfully established in the laboratory. DNA extracted from the third passage of this isolate was designated as strain M9A and molecularly characterized using primers targeting the Rickettsia gltA gene, whose sequence matched 100% the corresponding sequences of R. bellii. Seroprevalence against SFG rickettsiae in sampled dogs (n = 83) was 41% or 57%, depending on the rickettsial antigen (R. rickettsii strain Taiaçu or R. parkeri strain Atlantic rainforest, respectively). A total of 37 (45%) canine sera showed titers to R. parkeri at least fourfold higher than to R. rickettsii antigen. Among humans, 10% (4/41) of the samples reacted to at least one rickettsial antigen, with the highest endpoint titer varying from 64 to 128 for R. rickettsii and R. parkeri; no human serum showed ≥4-fold difference between the highest endpoint titers. Finally, during the study period, suspicions on cases of R. parkeri-rickettsiosis were not informed by the health secretariats. Our results confirm the presence and exposure to R. parkeri strain Atlantic rainforest, associated with two anthropophilic tick species (A. ovale and A. aureolatum) parasitizing domestic dogs with unrestrained access to forest areas. Consequently, the occurrence of R. parkeri infection in humans inhabiting the Atlantic forests of ES should not be discarded.

Multi-locus phylogenetic analysis groups the New World bacterium Rickettsia sp. strain ApPR with the Old World species R. africae; proposal of "Candidatus Rickettsia paranaensis".

Rickettsia parkeri sensu stricto (s.s.) is an emerging human pathogen in the Americas. Comprehension of the etiology of R. parkeri infections in South America is complicated by the existence of genetic variants (Atlantic rainforest, NOD and Parvitarsum) of this species that are associated with specific groups of Amblyomma ticks. The rickettsial bacterium strain ApPR was first reported in Amblyomma parkeri ticks in Southern Brazil in 2012 and was considered, based on sequencing of fragments of the gltA, htrA, ompA and ompB genes, to represent yet another genetic variant of R. parkeri. In the current work, a multi-locus phylogenetic analysis employing additional genes and intragenic regions was performed using DNA extracted from (a) larvae of A. parkeri and Amblyomma species haplotype Nazaré ticks collected from wild birds, (b) a nymph of Amblyomma sp. haplotype Nazaré recovered from a monkey (Callicebus nigrifons), representing the first report of that tick parasitizing a non-human primate and (c) from a cultured isolate of ApPR, isolated from colony-reared adults of Amblyomma geayi. Phylogenetic inference performed using Maximum-likelihood (ML), Maximum Parsimony (MP) and Bayesian (B) methods, consistently placed strain ApPR outside the New World R. parkeri complex and instead grouped it in proximity to the Old World species Rickettsia africae and Rickettsia sibirica. Estimates of evolutionary divergence provided additional support for the inferred phylogenetic relationship. Given the clear evolutionary distance between strain ApPR and R. parkeri we propose the recognition of "Candidatus Rickettsia paranaensis".

Novel Ehrlichia sp. detected in Magellanic penguins (Sphenicus magellanicus) and in the seabird tick Ixodes uriae from Magdalena Island, southern Chile.

Ehrlichia spp. are obligatory intracellular microorganisms that infect hematopoietic, endothelial or blood cells of mammals. Ticks are the only vectors of these agents in nature. To date, the role of birds and their associated ticks as reservoirs of ehrlichiae remains almost unexplored. In this study, we performed a molecular screening for bacteria of Anaplasmataceae family in samples of spleen (n = 72) and lung (n = 17), recovered from 72 carcasses of Magellanic penguins (Spheniscus magellanicus) in Brazil and Chile. One apparently unengorged tick (Ixodes uriae) was also collected while wandering upon one of the carcasses and submitted to molecular analyses as well. Through conventional and nested PCR protocols three genes (16S rRNA, dsb and groEL) of a new Ehrlichia sp. were partially characterized upon organs of three penguins and in the tick coming from Magdalena Island (Chile). First matches after BLASTn comparisons showed that our sequences share 99.4% (16S rRNA), 94.6% (groEL) and 79.3% (dsb) of identity with "Candidatus Ehrlichia ornithorhynchi", Ehrlichia sp. NS101 and Ehrlichia canis CCZ, respectively. Matrixes of genetic distance including other representatives of the Ehrlichia genus point a 99.4%, 94.0%, and 80.0% of identity with 16S rRNA, groEL and dsb genes from Ehrlichia sp. It25, Ehrlichia sp. NS101, and Ehrlichia chaffeensis San Louis, respectively. A Bayesian phylogenetic analysis of Anaplasmataceae 16S rRNA gene places the detected Ehrlichia sp. into a group with Ehrlichia sp. BAT and Ehrlichia sp. Natal. Although depicting different topologies, Bayesian unrooted phylogenetic trees constructed for groEL and dsb genes position this Ehrlichia sp. into well-supported branches, which reinforces the finding of a new taxon. For the moment, any pathogenic effect of this new Ehrlichia sp. on penguins is still unknown. However, this fact becomes important to assess from a conservation point of view since populations of Magellanic penguins are currently threatened and in an ongoing decrease.