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.

Diversity of Ehrlichia spp., Anaplasma spp. and Neorickettsia spp. in vampire bats.

Although bats (Mammalia: Chiroptera) act as natural reservoirs for many zoonotic pathogens around the world, few studies have investigated the occurrence of Anaplasmataceae agents in bats, especially vampire bats. The family Anaplasmataceae (order Rickettsiales) encompasses obligate intracellular bacteria of the genera Anaplasma, Ehrlichia, Neorickettsia, Neoehrlichia, Wolbachia, and Allocryptoplasma. The present study aimed to investigate, using molecular techniques, the presence of species of Anaplasma, Ehrlichia, and Neorickettsia in vampire bats sampled in northern Brazil. Between 2017 and 2019, spleen samples were collected from vampire bats belonging to two species, Desmodus rotundus (n = 228) from the states of Pará (n = 207), Amazonas (n = 1), Roraima (n = 18) and Amapá (n = 3), and Diaemus youngii (n = 1) from Pará. Positivity rates of 5.2% (12/229), 3% (7/229), and 10.9% (25/229) were found in PCR assays for Anaplasma spp. (16S rRNA gene), Ehrlichia spp. (dsb gene) and Neorickettsia spp. (16S rRNA gene), respectively. The present study revealed, for the first time, the occurrence of Anaplasma spp. and different genotypes of Ehrlichia spp. in vampire bats from Brazil. While phylogenetic analyses based on the dsb and ftsZ genes of Ehrlichia and 16S rRNA of Anaplasma spp. revealed phylogenetic proximity of the genotypes detected in vampire bats with Anaplasmataceae agents associated with domestic ruminants, phylogenetic inferences based on the gltA and groEL genes evidenced the occurrence of genotypes apparently exclusive to bats. Neorickettsia sp. phylogenetically associated with N. risticii was also detected in vampire bats sampled in northern Brazil.

Molecular survey of hemoplasmas and Coxiella burnetii in vampire bats from northern Brazil.

In addition to zoonotic viral pathogens, bats can also harbor bacterial pathogens, including hemoplasmas (hemotropic mycoplasmas) and Coxiella burnetii. The present study aimed to investigate, using molecular techniques, the presence of hemoplasmas and C. burnetii in spleen samples from vampire bats in northern Brazil. For this purpose, between 2017 and 2019, spleen samples were collected from Desmodus rotundus (n = 228) and Diaemus youngii (n = 1) captured in the states of Pará (n = 207), Amazonas (n = 1), Roraima (n = 18) and Amapá (n = 3). DNA samples extracted from the bat spleen and positive in PCR for the endogenous gapdh gene were subjected to conventional PCR assays for the 16S rRNA, 23S rRNA and RNAse P genes from hemoplasmas and to qPCR based on the IS1111 gene element for C. burnetii. All spleen samples from vampire bats were negative in the qPCR for C. burnetii. Hemoplasmas were detected in 10 % (23/229) of spleen samples using a PCR based on the 16S rRNA gene. Of these, 21.73 % (5/23) were positive for the 23S rRNA gene and none for the RNAseP gene. The seven hemoplasma 16S rRNA sequences obtained were closely related to sequences previously identified in vampire bats from Belize, Peru and Brazil. The 23S rRNA sequence obtained revealed genetic proximity to hemoplasmas from non-hematophagous bats from Brazil and Belize. The analysis revealed different circulating genotypes among Brazilian vampire bats, in addition to a trend towards genera-specific hemoplasma genotypes. The present study contributes to the knowledge of the wide diversity of hemoplasmas in vampire bats.

Molecular detection of vector-borne agents in wild boars (Sus scrofa) and associated ticks from Brazil, with evidence of putative new genotypes of Ehrlichia, Anaplasma, and haemoplasmas.

The present study aimed to investigate, by molecular techniques, the occurrence of Anaplasmataceae, Bartonellaceae, Rickettsiaceae, Mycoplasmataceae, Coxiellaceae, and Babesiidae/Theileriidae agents in blood samples of free-living wild boars (Sus scrofa) and associated ticks in south-eastern Brazil. For this purpose, 67 blood samples and 265 ticks (264 Amblyomma sculptum and one Amblyomma ovale) were analysed. In the screening for Anaplasmataceae agents by a PCR assay based on the 16S rRNA gene, 5.97% blood samples and 50.54% ticks were positive. In the PCR assay for Ehrlichia spp. based on the dsb gene, 9.24% of ticks were positive. Despite the low occurrence, a possible new 16S rRNA genotype of Anaplasma sp. was detected in a wild boar's blood sample. According to phylogenetic analyses based on the 16S rRNA, gltA, and sodB genes and ITS (23S-5S rRNA) intergenic region, it was found that A. sculptum and A. ovale ticks collected from wild boars carry Ehrlichia genotypes phylogenetically associated with Ehrlichia ewingii, Ehrlichia ruminantium, and new Ehrlichia genotypes previously detected in horses, peccaries, and ticks collected from jaguars. In the screening for haemoplasmas by a qPCR based on the 16S rRNA gene, 88.06% of blood samples and 8.69% of ticks were positive. Mycoplasma suis, Mycoplasma parvum, and a possible new haemoplasma genotype were detected in wild boars in south-eastern Brazil. In the screening for Bartonella spp. using a nuoG-based qPCR assay, 3.8% of tick samples were positive. Phylogenetic inferences positioned four nuoG and one r gltA Bartonella sequences into the same clade as Bartonella machadoae. No blood or tick samples from wild boars showed to be positive in the qPCR for Coxiella burnetii based on the IS1111 gene. On the other hand, only 1.6% of ticks were positive in the nested PCR assay for piroplasmids based on the 18S rRNA gene. A 18S rRNA sequence detected in a pool of A. sculptum nymphs was phylogenetically close to Cytauxzoon felis sequences previously detected in cats from the United States. Rickettsia sp. closely related to Rickettsia bellii was detected in a pool of A. sculptum nymphs. This is the first report of haemoplasmas, B. machadoae, and Cytauxzoon spp. in A. sculptum. Wild boars and associated ticks do not seem to participate in the epidemiological cycle of C. burnetii in the region studied. This invasive mammal species may act as a potential disperser of ticks infected with Ehrlichia spp., Bartonella spp., haemotropic mycoplasmas, and Cytauxzoon, and may bring important epidemiological implications in the transmission of bartonelosis, ehrlichiosis, haemoplasmosis, and cytauxzoonosis to humans and animals, more specifically to horses, rodents, pigs, and cats.

Molecular investigation of haemotropic mycoplasmas and Coxiella burnetii in free-living Xenarthra mammals from Brazil, with evidence of new haemoplasma species.

Although mammals of the superorder Xenarthra are considered hosts of a wide range of zoonotic agents, works aiming at investigating the role of these animals as hosts for bacteria with zoonotic potential are rare. The present study aimed to investigate the occurrence and molecularly characterize Coxiella burnetii and haemoplasma (haemotropic mycoplasmas) DNA in blood and spleen samples from 397 free-living Xenarthra mammals (233 sloths, 107 anteaters and 57 armadillos) in five Brazilian states (Mato Grosso do Sul, São Paulo, Pará, Rondônia and Rio Grande do Sul). All biological samples from Xenarthra were negative in the qPCR for Coxiella burnetii based on the IS1111 gene. The absence of C. burnetii DNA in blood and spleen samples from Xenarthra suggests that these mammals may not act as possible hosts for this agent in the locations studied. When performed conventional PCR assays for the endogenous (gapdh) mammalian gene, 386 samples were positive. When screened by molecular assays based on the 16S rRNA gene of haemoplasmas, 81 samples were positive, of which 15.54% (60/386) were positive by conventional PCR and 5.44% (21/386) were positive by real-time PCR; three samples were positive in both assays. Of these, 39.74% (31/78) were also positive for the 23S rRNA gene and 7.69% (6/78) for the haemoplasma RNAse P gene. Among the samples positive for haemoplasmas, 25.64% (20/78) were obtained from anteaters (Tamandua tetradactyla and Myrmecophaga tridactyla), 39.74% (31/78) from sloths (Bradypus tridactylus, Bradypus sp. and Choloepus sp.) 34.61% (27/78) from armadillos (Priodontes maximus, Euphractus sexcinctus and Dasypus novemcinctus). A haemoplasma 16S rRNA sequence closely related and showing high identity (99.7%) to Mycoplasma wenyonii was detected, for the first time, in B. tridactylus. Based on the low identity and phylogenetic positioning of 16S rRNA and 23S rRNA sequences of haemoplasmas detected in anteaters and armadillos, the present study showed, for the first time, the occurrence of putative new Candidatus haemotropic Mycoplasma spp. ("Candidatus Mycoplasma haematotetradactyla" and "Candidatus Mycoplasma haematomaximus") in Xenarthra mammals from Brazil.