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.

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.

Novel Tick-Borne Anaplasmataceae Genotypes in Tropical Birds from the Brazilian Pantanal Wetland.

Despite numerous reports of Anaplasmataceae agents in mammals worldwide, few studies have investigated their occurrence in birds. The present study aimed to investigate the occurrence and molecular identity of Anaplasmataceae agents in birds from the Pantanal wetland, Brazil. Blood samples were collected from 93 different species. After DNA extraction, samples positive for the avian ß-actin gene were subjected to both a multiplex quantitative real-time (q)PCR for Anaplasma and Ehrlichia targeting the groEL gene and to a conventional PCR for Anaplasmataceae agents targeting the 16S rRNA gene. As a result, 37 (7.4%) birds were positive for Anaplasma spp. and 4 (0.8%) for Ehrlichia spp. in the qPCR assay; additionally, 13 (2.6%) were positive for Anaplasmataceae agents in the PCR targeting the 16S rRNA gene. The Ehrlichia 16S rRNA sequences detected in Arundinicola leucocephala, Ramphocelus carbo, and Elaenia albiceps were positioned closely to Ehrlichia sp. Magellanica. Ehrlichia dsb sequences detected in Agelasticus cyanopus and Basileuterus flaveolus grouped with Ehrlichia minasensis. The 16S rRNA genotypes detected in Crax fasciolata, Pitangus sulphuratus and Furnarius leucopus grouped with Candidatus Allocryptoplasma. The 23S-5S genotypes detected in C. fasciolata, Basileuterus flaveolus, and Saltator coerulescens were related to Anaplasma phagocytophilum. In conclusion, novel genotypes of Anaplasma, Ehrlichia, and Candidatus Allocryptoplasma were detected in birds from the Pantanal wetland.

Molecular detection and characterization of Bartonella spp. in small mammals in the Amazonia and Cerrado biomes, midwestern Brazil.

Although Bartonella spp. have been worldwide described in rodents and bats, few studies have reported these agents in marsupials. The present work aimed to investigate the occurrence and genetic diversity of Bartonella in small mammals (rodents, marsupials, and bats) and associated ectoparasites in two ecoregions (Amazonia and Cerrado biomes) in midwestern Brazil. For this purpose, DNA samples from 378 specimens of small mammals (128 rodents, 111 marsupials, and 139 bats) and 41 fleas (Siphonaptera) were screened for the Bartonella genus employing a quantitative real-time PCR assay (qPCR) based on the nuoG (nicotinamide adenine dinucleotide dehydrogenase gamma subunit) gene. Then, positive samples in qPCR were submitted to conventional PCR (cPCR) assays targeting the gltA, ftsZ, and rpoB genes. One (0.78 %) rodent, 23 (16.54 %) bats, and 3 (7.31 %) fleas showed positive results in the qPCR for Bartonella sp. After cPCR amplification and sequencing, 13 partial Bartonella DNA sequences of the following genes were obtained only from bats´ blood samples: 9 gltA (citrate synthase), 3 ftsZ (cell division protein), and 1 rpoB (RNA polymerase beta subunit). The maximum likelihood inference based on the gltA gene positioned the obtained sequences in three different clades, closely related to Bartonella genotypes previously detected in other bat species and bat flies sampled in Brazil and other countries from Latin America. Similarly, the ftsZ sequences clustered in two different clades with sequences described in bats from Brazil, other countries from Latin America, and Georgia (eastern Europe). Finally, the Bartonella rpoB from a specimen of Lophostoma silvicolum clustered with a Bartonella sp. sequence obtained from a Noctilio albiventris (KP715475) from French Guiana. The present study provided valuable insights into the diversity of Bartonella genotypes infecting bats from two ecoregions (Amazonia and Cerrado) in midwestern Brazil and emphasized that further studies should be conducted regarding the description and evaluation of different lineages of Bartonella in wild small mammals and their ectoparasites in different Brazilian biomes.

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.

Characterization of the bacterial microbiome of non-hematophagous bats and associated ectoparasites from Brazil.

Introduction: Bats, along with their ectoparasites, harbor a wide diversity of symbiotic and potential pathogenic bacteria. Despite the enormous diversity of bats (181 species), few studies aimed to investigate the bacterial microbiome of Brazilian chiropterans and associated ectoparasites. This study aimed to characterize the bacterial microbiome of non-hematophagous bats and associated Streblidae flies and Macronyssidae and Spinturnicidae mites in the state of Mato Grosso do Sul, midwestern Brazil. Methods: Oral and rectal swabs were collected from 30 bats (Artibeus lituratus [n = 13], Artibeus planirostris [n = 9], Eptesicus furinalis [n = 5], Carollia perspicillata [n = 2], and Platyrrhinus lineatus [n = 1]). In addition, a total of 58 mites (15 Macronyssidae and 43 Spinturnicidae) and 48 Streblidae bat flies were collected from the captured bats. After DNA extraction and purification, each sample's bacterial composition was analyzed with metagenomic sequencing. Results: The microbiome composition of both oral and rectal bat swab samples showed that Gammaproteobacteria was the most abundant bacterial class. Spiroplasma, Wolbachia and Bartonella represented the most abundant genera in Streblidae flies. While Wolbachia (Alphaproteobacteria) was the most abundant genus found in Spinturnicidae, Arsenophonus (Gammaproteobacteria) was found in high abundance in Macronyssidae mites. In addition to characterizing the microbiome of each sample at the class and genus taxonomic levels, we identified medically significant bacteria able to infect both animals and humans in oral (Streptococcus and Anaplasma) and rectal swabs (Enterobacter, Klebsiella, Escherichia, Enterococcus, Streptococcus), Macronyssidae (Anaplasma, Bartonella, Ehrlichia) and Spinturnicidae (Anaplasma, Bartonella) mites as well as Streblidae flies (Spiroplasma, Bartonella). Discussion and conclusion: Besides expanding the knowledge on the bacterial microbiome of non-hematophagous bats and Streblidae flies from Brazil, the present work showed, for the first time, the bacterial community of bat-associated Macronyssidae and Spinturnicidae mites.

Molecular detection of blood-borne agents in vampire bats from Brazil, with the first molecular evidence of Neorickettsia sp. in Desmodus rotundus and Diphylla ecaudata.

Bats (Mammalia, Chiroptera) represent the second largest group of mammals. Due to their ability to fly and adapt and colonize different niches, bats act as reservoirs of several potentially zoonotic pathogens. In this context, the present work aimed to investigate, using molecular techniques, the occurrence of blood-borne agents (Anaplasmataceae, Coxiella burnetii, hemoplasmas, hemosporidians and piroplasmids) in 198 vampire bats sampled in different regions of Brazil and belonging to the species Desmodus rotundus (n = 159), Diphylla ecaudata (n = 31) and Diaemus youngii (n = 8). All vampire bats liver samples were negative in PCR assays for Ehrlichia spp., Anaplasma spp., piroplasmids, hemosporidians and Coxiella burnetii. However, Neorickettsia sp. was detected in liver samples of 1.51% (3/198) through nested PCR based on the 16S rRNA gene in D. rotundus and D. ecaudata. This is the first study to report Neorickettsia sp. in vampire bats. Hemoplasmas were detected in 6.06% (12/198) of the liver samples using a PCR based on the 16S rRNA gene. The two 16S rRNA sequences obtained from hemoplasmas were closely related to sequences previously identified in vampire and non-hematophagous bats from Belize, Peru and Brazil. The genotypic analysis identified a high diversity of bat-associated hemoplasma genotypes from different regions of the world, emphasizing the need for studies on this subject, in order to better understand the mechanisms of co-evolution between this group of bacteria and their vertebrate hosts. The role of neotropical bat-associated Neorickettsia sp. and bats from Brazil in the biological cycle of such agent warrant further investigation.

Potentially zoonotic parasites in the soil of public squares in the city of Aracaju (Sergipe, Northeastern Brazil).

This study evaluated the soil contamination of public squares in the city of Aracaju, Sergipe, by potentially zoonotic parasites and correlated their occurrence with climatic variables (temperature, humidity and precipitation). Samples were collected over a 18-month period, from 20 different public squares, and submitted to three different parasitological techniques: Faust's, Hoffman's and Rugai's methods, adapted to soil samples. Results indicated the presence of several potentially zoonotic parasitic species in eighteen of the 20 squares analyzed (90%). The parasites identified included Ancylostoma spp., Strongyloides stercoralis, Toxocara spp., Dipylidium caninum, Trichuris sp., Capillaria sp. and Giardia sp. They were identified during all months of the year and no influence of temperature, humidity or precipitation on the occurrence of those parasites was observed. Such results demonstrate that public squares in the city of Aracaju pose a parasitic contamination risk for people and pets that visit those places as a leisure activity.