Orofacial Deformities in 3 Related Rhesus Monkeys (Macaca mulatta) Resembling Human Binder's Syndrome.

Binder's syndrome is a rare congenital deformity characterized by midface hypoplasia, particularly around the nasomaxillary area. Genetic etiology or developmental failure caused by prenatal exposure to teratological agents has been considered. In this article, we present 3 related rhesus monkeys born with orofacial deformities similar to those found in infants with the Binder phenotype. For the first time, a primate biomodel for this condition is presented. The clinical description and association with management and environmental factors are discussed. These findings reinforce the knowledge about the relationship between possible vitamin K metabolism interference and Binder's syndrome.

Ciliate Diversity From Aquatic Environments in the Brazilian Atlantic Forest as Revealed by High-Throughput DNA Sequencing.

Rainforest aquatic ecosystems include complex habitats with scarce information on their unicellular eukaryote diversity and community structure. We have investigated the diversity of ciliates in freshwater and brackish environments along the Brazilian Atlantic Forest, based on the hypervariable V4 region of the 18S-rDNA obtained by high-throughput DNA sequencing. Our analyses detected 409 ciliate taxonomic units (OTUs), mostly attributed to the classes Oligohymenophorea and Spirotrichea. A total of 11 classes, 12 subclasses, 112 genera, and 144 species were reported. We found the following: (a) the ciliate communities are more diverse in freshwater- than in Atlantic Forest-associated brackish environments; (b) the ciliate communities are composed by a small amount of highly abundant OTUs, but a high number of low-abundant or rare OTUs; (c) nearly one-third of the ciliate OTUs share less than 97% sequence identity to reference sequences and (d) phylogenetic inference supports the hypothesis that the V4 region of the Ciliophora 18S-rDNA is a suitable marker for accurate evolutionary inferences at class level. Our results showed that a considerable fraction of the HTS-detected diversity of ciliates from Brazilian Atlantic Forest is not represented in the currently available molecular databases.

The Hidden World of Rickettsiales Symbionts: "Candidatus Spectririckettsia obscura," a Novel Bacterium Found in Brazilian and Indian Paramecium caudatum.

Symbioses between bacteria and eukaryotes are widespread and may have significant impact on the evolutionary history of symbiotic partners. The order Rickettsiales is a lineage of intracellular Alphaproteobacteria characterized by an obligate association with a wide range of eukaryotic hosts, including several unicellular organisms, such as ciliates and amoebas. In this work, we characterized the Rickettsiales symbionts associated with two different genotypes of the freshwater ciliate Paramecium caudatum originated from freshwater environments in distant geographical areas. Phylogenetic analyses based on 16S rRNA gene showed that the two symbionts are closely related to each other (99.4% identity), belong to the family Rickettsiaceae, but are far-related with respect to previously characterized Rickettsiales. Consequently, they were assigned to a new species of a novel genus, namely "Candidatus Spectririckettsia obscura." Screening on a database of short reads from 16S rRNA gene amplicon-based profiling studies confirmed that bacterial sequences related to the new symbiont are preferentially retrieved from freshwater environments, apparently with extremely scarce occurrence (< 0.1% positive samples). The present work provides new information on the still under-explored biodiversity of Rickettsiales, in particular those associated to ciliate host cells.

Molecular phylogeny and comparative morphology indicate that odontostomatids (Alveolata, Ciliophora) form a distinct class-level taxon related to Armophorea.

The odontostomatids are among the least studied ciliates, possibly due to their small sizes, restriction to anaerobic environments and difficulty in culturing. Consequently, their phylogenetic affinities to other ciliate taxa are still poorly understood. In the present study, we analyzed newly obtained ribosomal gene sequences of the odontostomatids Discomorphella pedroeneasi and Saprodinium dentatum, together with sequences from the literature, including Epalxella antiquorum and a large assemblage of ciliate sequences representing the major recognized classes. The results show that D. pedroeneasi and S. dentatum form a deep-diverging branch related to metopid and clevelandellid armophoreans, corroborating the old literature. However E. antiquorum clustered with the morphologically discrepant plagiopylids, indicating that either the complex odontostomatid body architecture evolved convergently, or the positioning of E. antiquorum as a plagiopylid is artifactual. A new ciliate class, Odontostomatea n. cl., is proposed based on molecular analyses and comparative morphology of odontostomatids with related taxa.

Understanding the Influence of Flow Velocity, Wall Motion Filter, Pulse Repetition Frequency, and Aliasing on Power Doppler Image Quantification.

OBJECTIVES: Although power Doppler imaging has been used to quantify tissue and organ vascularity, many studies showed that limitations in defining adequate ultrasound machine settings and attenuation make such measurements complex to be achieved. However, most of these studies were conducted by using the output of proprietary software, such as Virtual Organ computer-aided analysis (GE Healthcare, Kretz, Zipf, Austria); therefore, many conclusions may not be generalizable because of unknown settings and parameters used by the software. To overcome this limitation, our goal was to evaluate the impact of the flow velocity, pulse repetition frequency (PRF), and wall motion filter (WMF) on power Doppler image quantification using beam-formed ultrasonic radiofrequency data. METHODS: The setup consisted of a blood-mimicking fluid flowing through a phantom. Radiofrequency signals were collected using PRFs ranging from 0.6 to 10 kHz for 6 different flow velocities (5-40 cm/s). Wall motion filter cutoff frequencies were varied between 50 and 250 Hz. RESULTS: The power Doppler magnitude was deeply influenced by the WMF cutoff frequency. The effect of using different WMF values varied with the PRF; therefore, the power Doppler signal intensity was dependent on the PRF. Finally, we verified that power Doppler quantification can be affected by the aliasing effect, especially when using a PRF lower than 1.3 kHz. CONCLUSIONS: The WMF and PRF greatly influenced power Doppler quantification, mainly when flow velocities lower than 20 cm/s were used. Although the experiments were conducted in a nonclinical environment, the evaluated parameters are equivalent to those used in clinical practice, which makes them valuable for aiding the interpretation of related data in future research.

A House for Two--Double Bacterial Infection in Euplotes woodruffi Sq1 (Ciliophora, Euplotia) Sampled in Southeastern Brazil.

Several ciliated protists form symbiotic associations with a diversity of microorganisms, leading to drastic impact on their ecology and evolution. In this work, two Euplotes spp. sampled in Rio de Janeiro, Brazil, were identified based on morphological and molecular features as Euplotes woodruffi strain Sq1 and E. encysticus strain Sq2 and investigated for the presence of endosymbionts. While E. woodruffi Sq1 stably hosts two bacterial populations, namely Polynucleobacter necessarius (Betaproteobacteria) and a new member of the family "Candidatus Midichloriaceae" (Alphaproteobacteria, Rickettsiales), here described as "Candidatus Bandiella woodruffii," branching with a broad host range bacterial group found in association with cnidarians, sponges, euglenoids, and some arthropods; in E. encysticus Sq2 no symbiotic bacterium could be detected. The dispersion ability of this novel bacterium was tested by co-incubating E. woodruffi Sq1 with three different ciliate species. Among the tested strains "Ca. B. woodruffii" could only be detected in association with E. encysticus Sq2 with a prevalence of 20 % after 1 week and 40 % after 2 weeks, maintaining this level for up to 6 months. Nevertheless, this apparent in vitro association was abolished when E. woodruffi Sq1 donor was removed from the microcosm, suggesting that this bacterium has the capacity for at least a short-term survival outside its natural host and the aptitude to ephemerally interact with other organisms. Together, these findings strongly suggest the need for more detailed investigations to evaluate the host range for "Ca. B. woodruffii" and any possible pathogenic effect of this bacterium on other organisms including humans.

Boronated tartrolon antibiotic produced by symbiotic cellulose-degrading bacteria in shipworm gills.

Shipworms are marine wood-boring bivalve mollusks (family Teredinidae) that harbor a community of closely related Gammaproteobacteria as intracellular endosymbionts in their gills. These symbionts have been proposed to assist the shipworm host in cellulose digestion and have been shown to play a role in nitrogen fixation. The genome of one strain of Teredinibacter turnerae, the first shipworm symbiont to be cultivated, was sequenced, revealing potential as a rich source of polyketides and nonribosomal peptides. Bioassay-guided fractionation led to the isolation and identification of two macrodioloide polyketides belonging to the tartrolon class. Both compounds were found to possess antibacterial properties, and the major compound was found to inhibit other shipworm symbiont strains and various pathogenic bacteria. The gene cluster responsible for the synthesis of these compounds was identified and characterized, and the ketosynthase domains were analyzed phylogenetically. Reverse-transcription PCR in addition to liquid chromatography and high-resolution mass spectrometry and tandem mass spectrometry revealed the transcription of these genes and the presence of the compounds in the shipworm, suggesting that the gene cluster is expressed in vivo and that the compounds may fulfill a specific function for the shipworm host. This study reports tartrolon polyketides from a shipworm symbiont and unveils the biosynthetic gene cluster of a member of this class of compounds, which might reveal the mechanism by which these bioactive metabolites are biosynthesized.

Coxiella symbionts are widespread into hard ticks.

Ticks are blood-feeding arthropods and can harbor several bacteria, including the worldwide zoonotic disease Q-fever agent Coxiella burnetii. Recent studies have reported a distinct group of Coxiella mostly associated with Ixodidae ticks, including the primary endosymbionts of Amblyomma americanum. In the present work, a screening for Coxiella infection was performed by 16S ribosomal DNA (rDNA) gene analyses in 293 tick samples of 15 different species sampled worldwide, including Brazil, Colombia, Kenya, and China. Different Coxiella phylotypes were identified, and these putative symbiotic bacteria were detected in ten different Amblyomma tick species. Approximately 61 % of Rhipicephalus sanguineus and ∼37 % of Rhipicephalus microplus DNA samples were positive for Coxiella. Sequence analysis and phylogenetic reconstruction grouped all the detected Coxiella with Coxiella-like symbionts from different Ixodidae ticks. This well-defined clade clearly excludes known phylotypes of C. burnetii pathogens and other Coxiella spp. detected in different environmental samples and other invertebrate hosts.

Molecular Phylogeny of the Family Ophryscolecidae (Ciliophora, Litostomatea) Inferred from 18S rDNA Sequences.

The 18S rDNA was used to infer oral ciliature patterns of evolution within the family Ophryoscolecidae, with the addition of five new sequences of ciliates from the genus Ostracodinium. Our data confirmed the monophyly of the subfamilies Entodiniinae and Ophryoscolecinae, but more analysis would be required for the definition of the status of the subfamily Diplodiniinae. The oral infraciliature patterns reflect evolutionary divergence in the family Ophryscolecidae, observing monophyly on Entodinium-type, Diplodinium-type, Ostracodinium-type, Epidinium-type, and Ophryoscolex-type. The ancestral infraciliature of Entodinium-type cannot be proven, however, the position of Entodinium-type showed closer of Diplodinium-type than Ophryoscolex-type, corroborating previous studies using morphological characters. The observed inconsistencies reflect the need to increase the number of 18S rDNA sequences to family Ophryoscolecidae and investigate the evolution of this group using other molecular markers.

Importance of Pulse Repetition Frequency Adjustment for 3- and 4-Dimensional Power Doppler Quantification.

OBJECTIVES: To determine the influence of the pulse repetition frequency (PRF) and wall motion filter on the 3-dimensional (3D) power Doppler vascularization-flow index (VFI) and volumetric pulsatility index (PI) obtained from spatiotemporal image correlation (STIC) data sets acquired from a common carotid artery of a healthy participant. METHODS: We acquired 11 STIC data sets, 1 for each PRF value ranging from 0.6 to 9.0 kHz. Vascularization-flow index and volumetric PI values were determined from the 440 static 3D data sets contained in these STIC data sets. Additionally, 3 sets of radio-frequency data were acquired for offline processing of different wall motion filter values for PRF values of 0.6, 3.3, and 10 kHz. RESULTS: We constructed VFI curves and observed 2 patterns: a flattened pattern with a low PRF and a triphasic pattern with a high PRF, correlating with the known pulsed wave Doppler profile of this vessel. Volumetric PI values were around 0 for low PRF settings and increased with increasing PRF. Analysis of the radiofrequency data showed that increasing wall motion filter values gradually filtered out the low-velocity power Doppler signals while retaining the higher-velocity ones, allowing the distinction of integrated power Doppler signal velocity throughout the cardiac cycle. CONCLUSIONS: We conclude that the PRF and wall motion filter dramatically influence 3D power Doppler indices and the volumetric PI, and the use of PRF values in which minimum VFI values are measured during the diastolic phase in the spectral Doppler wave may validate the use of the volumetric PI.

Transgene expression in tick cells using Agrobacterium tumefaciens.

Ticks transmit infectious agents to humans and other animals. Genetic manipulation of vectors like ticks could enhance the development of alternative disease control strategies. Transgene expression using the phytopathogen Agrobacterium tumefaciens has been shown to promote the genetic modification of non-plant cells. In the present work we developed T-DNA constructs for A. tumefaciens to mediate transgene expression in HeLa cells as well as Rhipicephalus microplus tick cells. Translational fusions eGfp:eGfp or Salp15:eGfp, including the enhanced-green fluorescent protein and the Ixodes scapularis salivary factor SALP15 genes, were constructed using the CaMV 35S (cauliflower mosaic virus) promoter, "PBm" tick promoter (R. microplus pyrethroid metabolizing esterase gene) or the Simian Virus SV40 promoter. Confocal microscopy, RT-PCR and Western-blot assays demonstrated transgene(s) expression in both cell lines. Transgene expression was also achieved in vivo, in both R. microplus and I. scapularis larvae utilizing a soaking method including the A. tumefaciens donor cells and confirmed by nested-RT-PCR showing eGfp or Salp15 poly-A-mRNA(s). This strategy opens up a new avenue to express exogenous genes in ticks and represents a potential breakthrough for the study of tick-host pathophysiology.

Ciliate diversity in rodrigo de freitas lagoon (Rio de Janeiro, Brazil) from an integrative standpoint.

The Rodrigo de Freitas Lagoon is a highly eutrophic lacustrine system and has one of the longest histories of exploration and anthropic alteration in Brazil. Despite its relevance, limited studies explored the diversity of micro-eukaryotes in the lagoon. Ciliates (Alveolata, Ciliophora) are overlooked in environmental microbiology, especially in tropical and subtropical ecosystems, resulting in limited knowledge about their diversity and functional relevance in South American habitats, particularly in coastal lagoons. To fill this gap, here we investigated the diversity of ciliates in a brackish coastal lagoon in an urban area of Rio de Janeiro, Brazil, applying and comparing the performance of morphological and metabarcoding approaches. The metabarcoding analysis, based on high-throughput sequencing of the hipervariable region V4 of the 18S rRNA genes detected 37 molecular operational taxonomic units (MOTUs) assigned to Ciliophora, representing only about a half (56.9%) of the diversity detected by microscopy, which counted 65 ciliate morphotypes. The most representative classes in both approaches were Spirotrichea and Oligohymenophorea. The metabarcoding analysis revealed that 35.3% of the ciliate MOTUs had less than 97% similarity to available sequences in the NCBI database, indicating that more than one-third of these MOTUs potentially represents still not represented or undescribed ciliate species in current databases. Our findings indicate that metabarcoding techniques can significantly enhance the comprehension of ciliate diversity in tropical environments, but the scarcity of reference sequences of brackish ciliates in molecular databases represents a challenge to the taxonomic assignment of the MOTUs. This study provides new insights into the diversity of ciliates in a threatened coastal lagoon, revealing a vast array of still unknown and rare ciliate taxonomic units in tropical environments.

Identification of bacterial infection in neotropical primates.

Emerging infectious diseases usually arise from wild animal populations. In the present work, we performed a screening for bacterial infection in natural populations of New World primates. The blood cell bulk DNAs from 181 individuals of four Platyrrhini genera were PCR screened for eubacterial 16S rRNA genes. Bacteria were detected and identified in 13 distinct individuals of Alouatta belzebul, Alouatta caraya, and Cebus apella monkeys from geographically distant regions in the states of Mato Grosso and Pará, Brazil. Sequence analyses showed that these Platyrrhini bacteria are closely related not only to human pathogens Pseudomonas spp. but also to Pseudomonas simiae and sheep-Acari infecting Pseudomonas spp. The identified Pseudomonas possibly represents a group of bacteria circulating in natural monkey populations.

A prevalent alpha-proteobacterium Paracoccus sp. in a population of the Cayenne ticks (Amblyomma cajennense) from Rio de Janeiro, Brazil.

As Rocky Mountain Spotted Fever is the most common tick-borne disease in South America, the presence of Rickettsia sp. in Amblyomma ticks is a possible indication of its endemicity in certain geographic regions. In the present work, bacterial DNA sequences related to Rickettsia amblyommii genes in A. dubitatum ticks, collected in the Brazilian state of Mato Grosso, were discovered. Simultaneously, Paracoccus sp. was detected in aproximately 77% of A. cajennense specimens collected in Rio de Janeiro, Brazil. This is the first report of Paracoccus sp. infection in a specific tick population, and raises the possibility of these bacteria being maintained and/or transmitted by ticks. Whether Paracoccus sp. represents another group of pathogenic Rhodobacteraceae or simply plays a role in A. cajennense physiology, is unknown. The data also demonstrate that the rickettsial 16S rRNA specific primers used forRickettsia spp. screening can also detect Paracoccus alpha-proteobacteria infection in biological samples. Hence, a PCR-RFLP strategy is presented to distinguish between these two groups of bacteria.

Morphological and phylogenetic investigations shed light on evolutionary relationships of the enigmatic genus Copemetopus (Ciliophora, Alveolata), with the proposal of Copemetopus verae sp. nov.

Copemetopus Villeneuve-Brachon, 1940 is a rare, poorly known sapropelic ciliate genus composed of only two valid nominal species. Over time, Copemetopus was taxonomically assigned to Heterotrichea and Armophorea classes, but its phylogenetic affinities remained unknown. Until the present study, there were no molecular data available for Copemetopus representatives. Here, we present the 18S and 28S-rDNA sequences and the phylogenetic position of Copemetopus verae sp. nov., as well as its detailed morphological description based on live observations, protargol impregnation, and scanning electron microscopy. Transmission electron micrographs of the type species C. subsalsus Villeneuve-Brachon, 1940 reveal new morphological traits and a unique somatic ciliature pattern of Copemetopus, composed by short segments of dikinetids with one or two supplementary kinetosomes. The phylogenetic trees recovered Copemetopus as the sister group of the genus Protocruzia, both constituting early-divergent lineages that split first from a common ancestor of Intramacronucleta. Morphological and molecular evidence suggest that Copemetopus is neither a heterotrichean nor an armophorean ciliate, but a distinct clade related to Protocruzia.

Coxiella symbionts in the Cayenne tick Amblyomma cajennense.

Members of the Coxiella genus are intracellular bacteria that can infect a variety of animals including humans. A symbiotic Coxiella was recently described in Amblyomma americanum ticks in the Northern Hemisphere with no further investigations of other Amblyomma species in other geographic regions. These ixodid ticks represent a group of important vectors for human infectious agents. In the present work, we have demonstrated that symbiotic Coxiella (SCox) are widespread, occurring in South America and infecting 100% of all life stages and eggs of the Cayenne ticks Amblyomma cajennense from Brazil and the USA. Using light microscopy, in situ hybridization, and PCR, we demonstrated SCox in salivary glands, ovaries, and the intestines of A. cajennense. These symbionts are vertically and transtadially transmitted in laboratory reared A. cajennense, and quantitative PCR analyses indicate that SCox are more abundant in adult female ticks, reaching values corresponding to an 11×, 38×, and 200× increase in SCox 16S rRNA gene copy number in unfed females, compared to unfed nymphs, larvae, and eggs, respectively. Phylogenetic analyses showed distinct SCox subpopulations in the USA and Brazil and demonstrated that SCox bacteria do not group with pathogenic Coxiella burnetii.

Macronuclear Plasticity in Two South American Populations of Spirostomum (Ciliophora, Heterotrichea) Warns About its Use for Species Classification: Revision and New Insights.

Spirostomum is a widely distributed heterotrichean genus composed of well-known species with described ecology and phylogenetic affinities. The morphological classification of Spirostomum species is mostly based on the body size/shape, number of cortical granule rows and macronuclear characteristics. These features along with molecular phylogenies based on ribosomal genes divide the genus into two phylogroups, one including species with a compact macronucleus, and another including species with a moniliform macronucleus. Here, we present our observations on atypical Spirostomum specimens with unusually two distinct macronuclei and shortened adoral zone of membranelles. These atypical forms appeared in the cultures of S. minus and S. yagiui, sampled at different sites in South America (Chile and Brazil) and associated with unrelated substrate types. Morphological observations of living and stained cells, 18S rRNA gene analyses, and a thorough investigation of the literature suggest that the atypical phenotype may be a result of uncommon pathways during the conjugative process. Thus, we demonstrate that studies of ciliate natural populations and their morphological variations, especially from undersampled biogeographical regions, can reveal the boundaries of widely used morphological characters for Spirostomum taxonomy and species identification.

Parablepharisma (Ciliophora) is not a Heterotrich: A Phylogenetic and Morphological Study with the Proposal of New Taxa.

The genus Parablepharisma Jankowski, 2007 at present includes five species, most of which have not been studied in detail, therefore phylogenetic affinities remained uninvestigated up to now. Parablepharisma is traditionally placed within Heterotrichea based on insufficient existing morphological data, and there are no available Parablepharisma gene sequences in molecular databases to support this placement. This work presents an 18S rDNA-based phylogeny of P. bacteriophora (Kahl, 1932) Jankowski, 2007 and P. brasiliensis sp. nov. We also provide a redescription of P. bacteriophora and P. chlamydophorum (Kahl, 1932) Jankowski, 2007 based on live morphological observations combined with silver impregnation and scanning and transmission electron microscopy. According to characters such as macro- and micronucleus number, the position of the ectosymbiotic bacteria, and the presence/absence of caudal cilia, two new species are described, i.e. P. granulata sp. nov. and P. brasiliensis sp. nov. In addition, we establish Kahlium gen. nov. to include P. chlamydophorum, which has a segmented anterior paroral portion and a twisted posterior paroral section as diagnostic features. To include Parablepharisma and Kahlium gen. nov., we propose Parablepharismidae fam. nov. According to our phylogenetic analyses, Parablepharisma belongs to SAL (Spirotrichea, Armophorea, Litostomatea), being a sister group of Cariacotrichea.