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.

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 and serological detection of Anaplasma spp. in small ruminants in an area of Cerrado Biome in northeastern Brazil.

Anaplasmosis, caused by bacteria of the genus Anaplasma, is an important tick-borne disease that causes economic losses to livestock farms in many countries. Even though Anaplasma spp. have been detected in goats and sheep worldwide, few studies investigate the occurrence and genetic identity of these agents in small ruminants from Brazil. Thus, this work aimed to detect and determine the genetic identity of Anaplasma spp. in small ruminants from the Baixo Parnaíba region, state of Maranhão, northeastern Brazil. For this purpose, blood samples were collected from 161 animals (91 goats; 70 sheep) from 4 municipalities in the Baixo Parnaíba region. Sheep and goat serum samples were subjected to recombinant membrane surface protein (MSP5)-based iELISA. Whole blood samples were subject to DNA extraction and molecular diagnosis using PCR assays for Anaplasma spp. targeting msp1ß, msp1α, 16S rRNA and msp4 genes. Positive samples were sequenced and then subjected to Anaplasma marginale msp1α genetic diversity analysis and phylogenetic inferences based on the 16S rRNA and msp4 genes. The serological survey detected the presence of anti-A. marginale IgG antibodies in 18 animals (11.1%): 2.9% (2/70) sheep and 17.4% (16/91) goats. Anaplasma marginale DNA was detected in 2 goats (1.2%) using qPCR based on the msp1ß gene. Two distinct A. marginale msp1α strains, namely α ß and α ß ΓγΓγΓγΓγ were found in the infected goats, each one found in a different animal, both belonging to the H genotype. Phylogenetic analysis based on the 16S rRNA gene showed the sequences positioned in three different clades and grouped with sequences from 'Candidatus Anaplasma boleense', A. platys and A. marginale. Phylogenetic inferences based on the msp4 gene positioned the sequence variants in the A. marginale clade. The present work represents the first molecular detection of sequence variants phylogenetic associated to 'Candidatus Anaplasma boleense' and A. platys and α ß and α ß ΓγΓγΓγΓγ in goats from Brazil.

Multi-locus Sequencing Typing of Bartonella henselae isolates reveals coinfection with different variants in domestic cats from Midwestern Brazil.

Bartonella henselae is a zoonotic pathogen responsible for causing Cat Scratch Disease (CSD) and other clinical manifestations in humans. Domestic cats are the main reservoirs of this Bartonella species. Previous studies have suggested that certain genotypes of B. henselae seem to be more associated with human infections. The present study aimed to genotype B. henselae isolates from domestic cats' blood samples in the state of Goiás, midwestern Brazil. The association of quantitative real-time PCR (qPCR) based on the nuoG gene from Bartonella spp. of blood samples, before and after incubation in pre-enrichment liquid medium (BAPGM) and isolation on chocolate agar, showed a positivity frequency of 42% (42/100) for Bartonella spp. Twelve B. henselae isolates obtained on agar chocolate from six cats' blood samples (two isolates from each animal) were characterized by Multi-locus Sequencing Typing (MLST) and revealed to belong to Sequence Types ST1 and ST5. One of the cats (1/6) presented both STs, demonstrating that domestic cats can be coinfected with different variants of B. henselae. The STs detected in this study are distributed worldwide and have already been detected in humans with clinical manifestations of bartonellosis. This is the first report of the zoonotic variants ST1 and ST5 of B. henselae in domestic cats from Brazil.

Integrative taxonomy of Metastrongylus spp. in wild boars from Brazil.

BACKGROUND: Wild boars (Sus scrofa) may cause substantial damage to crops and can spread zoonotic parasites to domestic animals, posing a risk to health and animal production. Metastrongylus spp. can negatively affect the wild boar population, increasing piglet mortality. In addition to that, studies with Metastrongylus genetic characterization are still scarce in Brazil. The present study aims to characterize Metastrongylus spp. from wild boars hunted in the states of São Paulo, Paraná, and Rio Grande do Sul, Brazil, using traditional morphological description and DNA sequences in an integrative taxonomic approach. METHODS: After nematode collection from 58 wild boars, the parasites were morphologically identified and genetically characterized by the amplification of 18S ribosomal DNA (rDNA), 28S rDNA, internal transcribed spacer (ITS) region, and cox-1 mitochondrial DNA (mtDNA). Descriptors of infection were determined and Pearson's Chi-square test was applied to compare the prevalence of infections among the identified parasite species, host age group (juveniles and adults), and sex. The Mann-Whitney U test was performed to compare the mean intensity between the age groups and sex. RESULTS: Metastrongylus salmi, Metastrongylus apri, and Metastrongylus pudendotectus were identified in 77.6% (45/58) of the necropsied wild boars. Metastrongylus salmi was the most prevalent and abundant species (70.7%, 11.1), followed by M. pudendotectus (18.9%, 4.3) and M. apri (17.2%, 2.2). Metastrongylus pudendotectus showed the highest mean intensity and range (25.2, 1-93), followed by M. salmi (15.7, 1-58) and M. apri (12.6, 3-27). We found a significantly higher prevalence of Metastrongylus spp. and M. salmi in adult wild boars, probably associated with a more prolonged time of exposure to intermediate host species. The phylogenetic analysis revealed that ITS2 region and cox-1 mtDNA are the most suitable genetic markers for Metastrongylus species characterization. Genetic variability between M. apri and M. salmi isolates was verified. CONCLUSIONS: We expand the knowledge about the Metastrongylus community in the non-captive wild boar population from Brazil as well as the importance of this exotic species in the maintenance of Metastrongylus spp. in its areas of occurrence. The novel genetic sequences obtained may help further studies to understand the genetic diversity in other nematode populations from Brazil and other countries.

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 survey and genetic diversity of Bartonella spp. in domestic cats from Paraguay.

Although Bartonella spp. is described in cats worldwide, little is known about the occurrence and genetic diversity of Bartonella spp. in cats from South America. To date, it has only been detected in cats from Brazil, Chile and Argentina. This study aimed to undertake a molecular survey and explore the genetic diversity of Bartonella spp. in domestic cats from Paraguay. A TaqMan real-time quantitative PCR (qPCR) targeting the nuoG gene (83 bp) for Bartonella spp. was used to screen 125 blood samples from cats in Asuncion, Paraguay. nuoG qPCR-positive samples were further submitted to conventional PCR assays based on the ITS (453- 717 bp), gltA (767 bp), ftsZ (515 bp), rpoB (333 bp), ribC (585-588 bp), and pap-31 (564 bp) loci. Positive samples were sequenced for species identification, phylogenetic, and haplotype analyses. Bartonella D.N.A. was present in 20.8% (26/125) cat blood samples, with low levels of Bartonella nuoG D.N.A. cPCR products targeting gltA, ftsZ, ITS, and rpoB loci from sixteen cats were successfully sequenced. However, all nouG qPCR-positive samples were negative for the ribC and pap-31 genes. Bartonella henselae [62.5% (10/16)] and Bartonella clarridgeiae [37.5% (6/16)] were identified among the sequenced samples. Upon phylogenetic analysis, B. henselae and B. clarridgeiae from Paraguay clustered with sequences detected in domestic and wild cats, dogs, and cat fleas worldwide. Two to four haplotypes of B. henselae and B. clarridgeiae in cats from Paraguay were observed, with some being exclusive and others shared with worldwide distributed haplotypes. Here, we report B. henselae and B. clarridgeiae for the first time in cats from Paraguay. Its circulation in cats suggests the need to consider Bartonellae when testing clinical samples from suspected infectious diseases in humans from Paraguay.

First Report of Bartonella spp. in Marsupials from Brazil, with a Description of Bartonella harrusi sp. nov. and a New Proposal for the Taxonomic Reclassification of Species of the Genus Bartonella.

The genus Bartonella (Rhizobiales: Bartonellaceae) encompasses facultative intracellular Gram-negative alphaproteobacteria that parasitize mainly erythrocytes and endothelial cells, as well as macrophages, monocytes and dendritic cells. Although they can infect numerous mammal species and arthropod vectors worldwide, reports of Bartonella infections in marsupials are scarce. In fact, such agents have only been detected in marsupials and/or associated ectoparasites in Australia and the United States of America until the present moment. The present study aimed to isolate and characterize molecularly, morphologically and phenotypically Bartonella infecting free-living marsupials sampled in the Brazilian Pantanal, the largest wetland in South America. Two marsupials were captured in December 2018 and six marsupials in February 2019, totaling eight small mammals sampled: five (62.5%) Thylamys macrurus and three (37.5%) Monodelphis domestica. All blood samples were submitted to qPCR for Bartonella spp. based on the nuoG gene, a pre-enrichment liquid culture and a chocolate agar solid culture. Bartonella sp. was isolated from 3 T. macrurus and one M. domestica. One Bartonella isolate obtained from a T. macrurus blood sample (strain 117A) that showed to be closely related to the Bartonella vinsonii complex and Bartonella machadoae was selected for whole genome sequencing using a hybrid approach based on Illumina NovaSeq and Nanopore sequencing platforms. This strain showed a genome of 2.35 Mbp, with an average C + G content of 38.8%, coding for 2013 genes, and a 29 kb plasmid with an average C + G content of 34.5%. In addition, this strain exhibited an average nucleotide identity (ANI) of 85% with Bartonella species belonging to the B. vinsonii group and 91% with B. machadoae. Phylogenomic analysis based on 291 protein coding genes shared by the genomes of 53 Bartonella species positioned this strain closely to B. machadoae. This new isolated species was named Bartonella harrusi sp. nov., which was characterized as having small capnophilic, microaerophilic and aerobic rods with an absence of pili and flagella. In conclusion, the present work describes the biochemical, phenotypic and genomic characteristics of Bartonella harrusi, a new species isolated from the T. macrurus blood samples of the Brazilian Pantanal. Finally, a review of the taxonomic classification of members of the genus Bartonella is proposed, based on the ANI values accessed by whole genome sequencing analyses.

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.

Bartonella machadoae sp. nov. isolated from wild rodents in the Pantanal wetland.

It has been estimated that 75% of emerging infectious diseases comprise zoonoses, whose majority have free-living animals as reservoirs and are mainly transmitted by arthropod vectors. Although rodents represent important Bartonella reservoirs, there are few studies on the genotypic characterization of Bartonella species commonly found in this taxon and from different Brazilian biomes. Therefore, the present study aimed to investigate the occurrence, isolate and molecularly, morphologically and phenotypically characterize a new Bartonella species infecting free-living rodents sampled in the Brazilian Pantanal, the largest wetland in South America. For this purpose, 129 free-living rodents (79 Thrichomys fosteri, 4 Clyomys laticeps, and Oecomys mamorae) were captured. While blood samples were collected from 57 T. fosteri, 4 C. laticeps and 32 O. mamorae; spleen samples were collected from 22 T. fosteri and 14 O. mamorae. Blood and spleen samples were submitted to a qPCR for Bartonella spp. targeting the nuoG gene, using DNA samples extracted directly from blood/spleen, after passage in pre-enrichment liquid culture, and from colonies obtained from solid culture on chocolate agar. Combining all techniques, occurrence of 24.8% for Bartonella sp. was found among the sampled rodents. One Bartonella isolate (strain 56A) obtained from a T. fosteri's blood sample was closely related to the Bartonella vinsonii complex and selected for Whole Genome Sequencing (WGS) hybrid approach using Illumina NovaSeq and Nanopore sequencing platforms. This strain exhibits a circular 2.7 Mbp genome with an average C+G content of 39% and encoding to 2239 genes. In the phylogenomics based on 291 shared protein-coding genes, this strain was positioned in a unique clade, closely related to Bartonella vinsonii subsp. vinsonii, B. vinsonii subsp. berkhoffii and B. visonii subsp. arupensis. An Average Nucleotide Identity of 85% was found between the obtained isolate and Bartonella species belonging to B. vinsonii complex. These findings supported the separation of this strain, now formally named as Bartonella machadoae sp. nov., from the Bartonella vinsonii complex. In addition, Bartonella machadoae sp. nov. was characterized by capnophilic, microaerophilic and aerobic small rods with absence of pili and flagella. Phylogenetic and distance analyses based on five concatenated molecular markers suggest that Bartonella machadoae may parasite rodents from different Brazilian biomes. In conclusion, we described biochemical, phenotypic and genomic characteristics of Bartonella machadoae nov. sp. isolated from blood samples of T. fosteri rodents from the Brazilian Pantanal.

Anaplasma marginale in goats from a multispecies grazing system in northeastern Brazil.

Anaplasma marginale, a tick-borne α-proteobacterium that causes significant economic losses for the cattle industry worldwide, has been increasingly detected in other animal species. This agent has been previously detected in buffaloes and goats co-grazed with cattle in Brazil. This study aimed to investigate the occurrence of A. marginale in a multispecies (goats, sheep and cattle) grazing farm in the State of Paraíba, northeastern Brazil. A total of 119 goats, 71 sheep, and five cattle were evaluated. An epidemiological questionnaire was applied to the farm owner addressing age, gender, and presence of ticks. Serum samples from goat, sheep and cattle were tested for anti-Anaplasma marginale antibodies by a commercial MSP5-based on indirect enzyme-linked immunosorbent assay (iELISA). EDTA-blood samples were screened for A. marginale- and A. ovis-infection by PCR using primers targeting Anaplasma spp. msp4 gene. Sequencing of the repeat region of the msp1α gene was used for genotyping A. marginale strains found in the present study. A total of 47/119 (39.5 %, 95 % CI: 31.1-48.4 %) goats and 2/71 (3%, 95 % CI: 0.7-9.7 %) sheep were seroreactive for A. marginale rMSP5 by the commercial iELISA. All cattle were seronegative for A. marginale. Anaplasma spp. msp4 PCR results revealed that two out of 119 (1.7 %; 95 % CI: 0.4-5.9 %) goats tested positive and all sheep and cattle samples were negative. It was not possible to sequence one sample. Therefore, the other sequencing sample found tandem repeats of A. marginale msp1α gene demonstrating that goat was infected with the genotype F/91. Rhipicephalus microplus ticks were found parasitizing goats but not on sheep or cattle. Considering that in Brazil A. marginale genotype F/91 and the MSP1a tandem repeat F has only been detected in goats so far, we hypothesized that this genotype may be related to goats.

Multi-locus sequencing reveals a novel Bartonella in mammals from the Superorder Xenarthra.

The superorder Xenarthra consists of sloths, anteaters and armadillos, mammals originated from South America and currently distributed from the south of North America to the south of South America. The present study aimed to investigate the occurrence and genetic diversity of Bartonella spp. in blood and spleen samples from free-living Xenarthra mammals in the states of São Paulo (SP), Mato Grosso do Sul (MS), Rondônia (RO) and Pará (PA). Based on a quantitative real-time PCR (qPCR) assay, a Bartonella spp. nuoG gene fragment was detected in 1.51% (5/330) of the samples: 4 six-banded armadillos (Euphractus sexcinctus) sampled in the MS and 1 southern tamandua (Tamandua tetradactyla) sampled in the PA. Eight sequences (5 ftsZ, 2 gltA and 1 rpoB) were obtained in the conventional PCR assays. In both phylogenetic analyses based on Bayesian and distance (SplitsTree) methods, the obtained ftsZ, gltA and rpoB sequences were positioned in a distinct clade, but related to B. washoensis. The analysis of SplitsTree and genotype networks based on B. washoensis sequences from several hosts from various localities of the world showed that the sequences of the present study were allocated in a group separated from the other sequences, indicating that they probably originated from median vectors and large numbers of mutational events. Additionally, the analyses performed by BLAST showed low percentages of identities of the sequences obtained in the present study when compared to those previously deposited in GenBank. Therefore, we propose a new Candidatus to Bartonella occurring in Xenarthra in Brazil. The present study was the first to report the occurrence of Bartonella sp. in mammals of the superorder Xenarthra in the world, and it was the first to describe a new Candidatus related to B. washoensis in Brazil.

Occurrence of Bartonella genotypes in bats and associated Streblidae flies from Maranhão state, northeastern Brazil.

Bartonella is a genus of emerging zoonotic bacteria that are mainly associated with mammalian erythrocytes and endothelial cells. Bats are natural reservoirs for a variety of important pathogens that impact human and animal health. Recent reports have highlighted the role of bats and bat flies in the maintenance of Bartonella. Here, we showed that none of the 29 bat DNA blood samples obtained from five bat species in São Luís Island, state of Maranhão, northeastern Brazil, were positive for Bartonella in qPCR assays targeting nuoG. On the other hand, three out of 15 DNA samples (20%) from flies in the family Streblidae were positive for Bartonella. The BLASTn results showed that the gltA and rpoB sequences shared identities ranging from 97.2% to 100%, with Bartonella sequences amplified from bats or bat flies from Costa Rica and Brazil. These findings were supported by phylogenetic analyses based on Bayesian inferences. The present study showed that Bartonella genotypes are present in bat flies, thus shedding some light on the distribution of bat fly-related Bartonella genotypes in South America.

Genetic diversity of Bartonella spp. in vampire bats from Brazil.

Recently, an increasing number of Bartonella species have been emerged to cause human diseases. Among animal reservoirs for Bartonella spp., bats stand out due to their high mobility, wide distribution, social behaviour and long-life span. Although studies on the role of vampire bats in the epidemiology of rabies have been extensively investigated in Latin America, information on the circulation and genetic diversity of Bartonella species in these bat species is scarce. In the present work, 208 vampire bats, namely Desmodus rotundus (the common vampire bat; n = 167), Diphylla ecaudata (the hairy-legged vampire bat; n = 32) and Diaemus youngii (the white-winged vampire bat; n = 9) from 15 different states in Brazil were sampled. DNA was extracted from liver tissue samples and submitted to real-time PCR (qPCR) and conventional PCR (cPCR) assays for Bartonella spp. targeting five genetic loci, followed by phylogenetic and genotype network analyses. Fifty-one out of 208 liver samples (24.51%) were positive for Bartonella DNA in the ITS real-time PCR assay [40 (78.43%) of them were from D. rotundus from 11 states, and 11 (21.57%) samples from D. ecaudata from three states. Eleven genotypes were found for each gltA and rpoB genes. Several ITS sequences detected in the present study clustered within the lineage that includes B. bacilliformis and B. ancachensis. The Bayesian phylogenetic inference based on the gltA gene positioned the obtained sequences in six different clades, closely related to Bartonella genotypes previously detected in D. rotundus and associated ectoparasites sampled in Latin America. On the other hand, the Bartonella rpoB genotypes clustered together with the ruminant species, B. schoenbuchensis and B. chomelii. The present study describes for the first time the molecular detection of Bartonella spp. in D. ecaudata bats. It also indicates that Bartonella spp. of vampire bats are genetically diverse and geographically widespread in Brazil.

Molecular detection of Mycoplasma suis in captive white-lipped peccaries (Tayassu pecari) and wild boars (Sus scrofa) in Brazil.

Mycoplasma suis, the etiological agent of swine hemoplasmosis, is an epicellular bacterium that adheres to the surface of pig erythrocytes leading to deformations of the target cells. Little is known about the occurrence of M. suis in wild swine populations around the world, its economic impact on swine herds, and the risk of human infection. The aim of this study was to investigate, by quantitative real-time PCR (qPCR) based on the 16S rRNA gene, the occurrence of M. suis in a captive population of white-lipped peccaries (100 Tayassu pecari) and in free-living wild boars (14 Sus scrofa) in Brazil. None of the white-lipped peccaries were positive for M. suis, whereas seven (50%) wild boars were positive in qPCR assays. The quantification of M. suis-16S rRNA copies/µL ranged from 1.42 × 10° to 3.906 × 101 in positive animals, indicating a low bacteremia and a chronic carrier status in free-living wild boars. In conclusion, M. suis might be a non-frequent pathogen in wild suids maintained in captivity. Despite the low bacteremia, the prevalence of M. suis in wild boar population in Brazil seems to be high.

High frequency and molecular characterization of porcine hemotrophic mycoplasmas in Brazil.

Mycoplasma suis and Mycoplasma parvum are the two hemotrophic mycoplasmas species described in pigs. M. suis is involved in infectious anemia, while M parvum infection is commonly subclinical. The objectives of this study were twofold: (i) to investigate the prevalence of porcine hemotrophic mycoplasmas in sows from the southern region of Brazil by quantitative real-time PCR (qPCR) and (ii) to genetically characterize a subset of the samples based on the 16S rRNA gene. A total of 429 blood samples were evaluated from 53 different farm sites. Porcine hemoplasmas was detected at all the 53 tested sites and in 79.72% of the samples (342/429). Two sequences were obtained for Mycoplasma spp. The phylogenetic analysis based on the 16S rRNA gene (900 bp) showed that the Mycoplasma sequences were closely related to the M. suis cluster and that one sequence was positioned in the M. parvum cluster. In conclusion, porcine hemoplasmas have a high rate of prevalence in sows from commercial farms in the southern region of Brazil. This study demonstrated the first molecular detection and characterization of partial 16S rRNA gene of M. parvum in Brazil.

Molecular detection of Mycoplasma suis in extensive pig production systems in the State of Maranhão, northeast Brazil.

Mycoplasma suis is a bacterium that causes hemoplasmosis in pigs. This agent is capable of adhering to the surface of porcine erythrocytes, inducing structural changes on these cells. In Brazil, there are few reports about the disease, its causal agent, and the economic impact of this pathogen on pig production systems and farm sanitation. The present study aimed to investigate the occurrence of M. suis in extensive swine farms located in the counties of Itapecuru Mirim, Santa Rita and Rosario, State of Maranhão, northeast Brazil. For such purpose, 64 blood samples of pigs from these facilities were tested for M. suis using a 16S rRNA gene-based quantitative real-time PCR (qPCR); 82.3%, 65.2% and 25% of blood samples of swine from farms in the cities of Itapecuru Mirim, Santa Rita and Rosario were positive for M. suis by qPCR, respectively. This study shows, for the first time, that M. suis circulates in pig populations from the state of Maranhão, Northeast Brazil.

Genetic diversity of Anaplasma marginale in beef cattle in the Brazilian Pantanal.

There are few studies on the genetic diversity of Anaplasma marginale in Brazilian cattle herds, especially about beef cattle. The objective of this study was to evaluate the genetic diversity of A. marginale, based on the msp1α gene in Bos taurus indicus sampled from the Brazilian Pantanal. Aliquots of blood with and without EDTA were taken from 400 cattle (200 cows and 200 calves) across five extensive farms. The samples were submitted to the indirect immunoenzymatic assay (iELISA), quantitative real-time PCR (qPCR) for the msp1ß gene and to the semi-nested (sn) PCR for the msp1α gene. Positive samples were sequenced by the Sanger method and subjected to diversity analysis using the RepeatAnalyser software. The percentage of positive animals by iELISA, qPCR and (sn) PCR was 72.2% (289/400), 56.7% (227/400) and 23% (52/227), respectively. Cows (154/200) showed to be significantly more seropositive than calves (135/200). In qPCR, the number of calves and average quantification value (138/200; 1.3 × 106) A. marginale msp1α copies per µL proved to be higher when compared to that found for the cows (89/200; 3.9 × 104). The microsatellite analysis of the 26 sequences obtained from the msp1α gene revealed the presence of E (77%), C (15.4%) and B (7.7%) genotypes. Fourteen A. marginale strains were identified in the studied region, with eight that have never before been described in the literature (τ-10-13-13-18; τ-27-18; EV8-EV8-17; α-ß-ß-ß-100; EV7-11-10-15; τ-11-11-27-18; τ-11-10-15; τ-27-13-18). Beef cattle are highly exposed to A. marginale in the Brazilian Pantanal. Moreover, a high genetic diversity of A. marginale, with eight new strains, was found in the studied region. While cows may act as chronic carriers, perpetuating the pathogen within the herd, male beef calves sold to other regions may disperse these strains.

Molecular detection of Bartonella spp. and Rickettsia spp. in bat ectoparasites in Brazil.

The family Streblidae comprises a monophyletic group of Hippoboscoidea, hematophagous dipterans that parasitize bats. Bartonella spp. and Rickettsia spp. have been reported in bats sampled in Europe, Africa, Asia, North, Central and South America. However, there are few reports on the Bartonella and Rickettsia bacteria infecting Hippoboscoidea flies and mites. While Spinturnicidae mites are ectoparasites found only in bats, those belonging to the family Macronyssidae comprise mites that also parasitize other mammal species. This study investigates the occurrence and assesses the phylogenetic positioning of Bartonella spp. and Rickettsia spp. found in Streblidae flies and Spinturnicidae and Macronyssidae mites collected from bats captured in Brazil. From May 2011 to April 2012 and September 2013 to December 2014, 400 Streblidae flies, 100 Macronyssidaes, and 100 Spinturnicidae mites were collected from bats captured in two sites in northeastern Nova Iguaçu, Rio de Janeiro, southeastern Brazil. Forty (19.8%) out of 202 Streblidae flies were positive for Bartonella spp. in qPCR assays based on the nuoG gene. Among the flies positive for the bacterium, six (18%) were Paratrichobius longicrus, seven (29%) Strebla guajiro, two (40%) Aspidoptera phyllostomatis, five (11%) Aspidoptera falcata, one (10%) Trichobius anducei, one (25%) Megistopoda aranea, and 18 (32%) Trichobius joblingi, and collected from bats of the following species: Artibeus lituratus, Carollia perspicillata, Artibeus planirostris, Sturnira lilium, and Artibeus obscurus. Six sequences were obtained for Bartonella (nuoG [n = 2], gltA [n = 2], rpoB [n = 1], ribC = 1]). The phylogenetic analysis based on gltA (750pb) gene showed that the Bartonella sequences clustered with Bartonella genotypes detected in bats and ectoparasites previously sampled in Latin America, including Brazil. Only one sample (0.49%) of the species Trichobius joblingi collected from a specimen of Carollia perspicillata was positive for Rickettsia sp. in cPCR based on the gltA gene (401bp). This sequence was clustered with a 'Candidatus Rickettsia andaenae" genotype detected in an Amblyomma parvum tick collected from a rodent in the southern region of Brazilian Pantanal. The sampled Macronyssidae and Spinturnicidae mites were negative for Bartonella spp. and Rickettsia spp. This study demonstrated the first occurrence of Bartonella spp. and Rickettsia spp. DNA in Streblidae flies collected from bats in Brazil.

High occurrence of Mycoplasma suis infection in swine herds from non-technified farms in Mossoró, state of Rio Grande do Norte, Northeastern Brazil.

Mycoplasma suis, the etiological agent of swine hemoplasmosis, has been neglected in swine herds around the world. Swine hemoplasmosis is frequently associated with hemolytic anemia, disgalacty, infertility and immunosuppression, and it results in significant economic losses. This study investigates the occurrence of M. suis in non-technified swine herds in the northeastern region of Brazil using quantitative PCR (qPCR) based on the 16S rRNA gene. Between March and August 2013, blood samples from 147 swine were collected during slaughter in the city of Mossoró, state of Rio Grande do Norte, northeastern Brazil. One hundred and twelve samples (76.19%) were positive for M. suis by qPCR assays. The range of Cqs and quantification (copies of a M. suis-16S rRNA gene fragment/µL) was 20.86-37.89 and 1.64×101-6.64×107, respectively. One can conclude that M. suis infection have high occurrence (76,19%) in non-technified swine-rearing systems in Mossoró in the state of Rio Grande do Norte, Brazil.