Morphological and molecular characterization of a species of Tetratrichomonas present in feces of Brazilian sheep (Ovis aries) and goats (Capra hircus).

The trichomonads form part of the phylum Parabasalia, a complex assemblage of diverse species of flagellated protists, with some members recognized as pathogens of men and/or animals. Associations, probably as commensals, between the species Tetratrichomonas ovis and sheep were reported in North America during the 1960s based on morphological and cultural characteristics. Intriguingly, no subsequent studies of this topic have been published. Feces, collected from sheep (n = 55) and goats (n = 14), reared on small-scale, production facilities in Southeastern Brazil, were examined for parabasalids. Protozoa, demonstrating morphologies and motility characteristic of trichomonads, were detected by direct microscopy in 64% of sheep and 43% of goat samples. In contrast to T. ovis, none of the samples could be cultured in Diamond's medium; however, cultures were obtained for three goat and seventeen sheep samples in peptonized broth. Based on morphological analyses, all isolates were classified as members of the genus Tetratrichomonas. Sequencing of the ITS1-5.8S rRNA gene-ITS2 region revealed three highly similar genotypes that were essentially identical to sequences reported for Tetratrichomonas spp. isolated from the preputial cavity of cattle in the USA and Southern Brazil. The findings of this study extend and enhance our knowledge of parasitism in small ruminants by parabasalids.

Detection of Rickettsia spp. in ticks parasitizing toads (Rhinella marina) in the northern Brazilian Amazon.

This study evaluated rickettsial infection in ticks collected on toads from the northern Brazilian Amazon (Amapá state), where to our knowledge there are neither records of ticks from amphibians nor rickettsial infections in ticks. During 2016-2017, a total of 22 free-living toads were captured and identified as Rhinella marina. Overall, 12 (54.5%) toads were parasitized by a total of 97 ticks (6 males, 39 females, 31 nymphs, 21 larvae), giving a mean intensity of 8.1 ticks per infested toad. Two tick species were morphologically identified: Amblyomma rotundatum Koch (31 females, 14 nymphs), and Amblyomma dissimile Koch (6 males, 8 females, 17 nymphs). The 21 larvae were morphologically denoted as Amblyomma sp. Five toads were co-infested by A. rotundatum and A. dissimile. Morphological identifications were confirmed by nucleotide sequencing of fragments of the mitochondrial genes 16S rDNA, 12S rDNA and/or COX1. A total of 54 ticks were analyzed for the presence of rickettsial DNA. Eleven (9 females and 2 nymphs) out of 14 A. rotundatum ticks contained Rickettsia bellii. None of the 25 specimens of A. dissimile (6 males, 6 females, 13 nymphs) contained amplifiable rickettsial DNA. From 15 Amblyomma sp. larvae, a pool of 10 individuals contained Rickettsia sp. strain Colombianensi. Sequencing of the 16S rDNA amplicon derived from the positive pool yielded a sequence of A. dissimile. We detected Rickettsia sp. strain Colombianensi for the first time in Brazil. Prior records of this agent were restricted to Colombia and Honduras. In addition, we report the presence of A. rotundatum for the first time in the state of Amapá, where the only other record of A. dissimile was registered over 20 years ago.

Morphological, ultrastructural, and molecular characterization of intestinal tetratrichomonads isolated from non-human primates in southeastern Brazil.

Non-human primates are our closest relatives and represent an interesting model for comparative parasitological studies. However, research on this topic particularly in relation to intestinal parasites has been fragmentary and limited mainly to animals held in captivity. Thus, our knowledge of host-parasite relationships in this species-rich group of mammals could be considered rudimentary. The current study combined morphological, ultrastructural, and molecular analyses to characterize isolates of intestinal tetratrichomonads recovered from the feces of three species of South American, non-human primates. Fecal samples were collected from 16 animals, representing 12 distinct species. Parabasalid-like organisms were evident in five samples (31%) of feces: two from Alouatta sara, two from Callithrix penicillata, and one from Sapajus apella. The five samples presented morphologies consistent with the description of Tetratrichomonas sp., with four anterior flagella of unequal length, a well-developed undulating membrane, and a long recurrent flagellum. Sequencing of the ITS1-5.8S rRNA-ITS2 region demonstrated that the isolates from A. sara, and C. penicillata were closely related and highly similar to isolates of Tetratrichomonas brumpti, recovered previously from tortoises (Geochelone sp.). The flagellate recovered from S. apella demonstrated a similar morphology to those of the other isolates, however, sequence analysis showed it to be identical to an isolate of Tetratrichomonas sp. recovered from white-lipped peccaries (Tayassu pecari). The findings of this study extend and enhance our knowledge of parasitism of non-human primates by members of the genus Tetratrichomonas and indicate that the host range of these parasites is broader than previously believed.

New host records of Haemaphysalis leporispalustris (Acari: Ixodidae) on birds in Brazil.

Birds are an important component of the life histories and bioecology of a number of tick species and of some tick associated pathogens. An examination of the data concerning bird/tick associations in the Neotropics, showed that the tick Haemaphysalis leporispalustrisis (Packard, 1869) was rarely recorded infesting birds. The current study reports parasitism by H. leporispalustris in wild birds collected from Atlantic rain forest environments in the states of Rio de Janeiro (4LL) and Minas Gerais (17LL, 1NN), Brazil. All ticks were identified morphologically to the genus level; total DNA was extracted from each Haemaphysalis tick and examined by PCR and nucleotide sequencing of fragments of the eukaryotic genes encoding 16S rRNA and 12S rRNA. The bird species Arremon semitorquatus, Corythopis delalandi, Fluvicola nengeta, Troglodytes musculus, and Volatinia jacarina were recorded as hosts for H. leporispalustris for the first time in South America, and Turdus rufiventris represented a new record for Brazil.

The Use of Sucroferric Oxyhydroxide Prior to Sigmoidoscopy in Patients With End-Stage Kidney Disease: A Case Report.

Rationale: Sucroferric oxyhydroxide is an iron-based phosphate-binding medication that has been approved for the treatment of hyperphosphatemia in patients with end-stage kidney disease. Given the low overall iron release from the polynuclear iron(III)-oxyhydroxide molecule, recommendations regarding its use prior to colonoscopy/sigmoidoscopy have not been developed. Presenting concerns of the patient: A 51-year-old male with a known history of end-stage renal disease treated with hemodialysis was referred to Gastroenterology for consideration of colonoscopy to rule out malignancy because of a history of rectal bleeding. This was to be completed prior to proceeding with a living-donor kidney transplant. Diagnoses: Flexible sigmoidoscopy done after non-diagnostic colonoscopy demonstrated diffuse "charcoal-like" material that prevented adequate visualization of the bowel despite standard bowel preparation. The findings were believed to be secondary to the use of sucroferric oxyhydroxide prescribed for hyperphosphatemia. Interventions: The patient was subsequently instructed to discontinue sucroferric oxyhydroxide for 2 weeks prior to his repeat sigmoidoscopy procedure. Outcomes: The patient's repeat sigmoidoscopy after discontinuing sucroferric oxyhydroxide allowed for adequate bowel visualization that revealed only a benign lipoma. Teaching Points: This case demonstrates the potential for sucroferric oxyhydroxide use to result in poor bowel preparation and resulting inadequate visualization on lower gastrointestinal endoscopy. It serves to highlight the clinical implications leading to the need for repeated procedures, which contributes to resource waste and unnecessary costs to the healthcare system, as well as delays in diagnostic evaluation required for transplantation; patient frustration was evident.

Justification: L'oxyhydroxyde sucro-ferrique, un médicament à base de fer liant le phosphate, a été approuvé pour le traitement de l'hyperphosphatémie chez les patients atteints d'insuffisance rénale terminale. La molécule polynucléaire fer (lll) ­ oxyhydroxyde ne libérant qu'une faible quantité globale de fer, aucune recommandation n'a été développée concernant son utilisation avant une coloscopie/sigmoïdoscopie. Présentation du cas: Un homme de 51 ans connu pour insuffisance rénale terminale et traité par hémodialyse a été orienté en gastroentérologie pour subir une coloscopie afin d'exclure une tumeur maligne en raison d'antécédents de saignement rectal. L'examen devait être complété avant de procéder à la greffe de rein par donneur vivant. Diagnostic: Une sigmoïdoscopie souple réalisée après une coloscopie non diagnostique a révélé une matière diffuse de type « charbon de bois ¼ qui empêchait de bien voir l'intestin malgré une préparation intestinale adéquate. Ce résultat a été jugé secondaire à l'utilisation d'oxyhydroxyde sucro-ferrique prescrit pour traiter l'hyperphosphatémie. Intervention: On a demandé au patient d'interrompre le traitement par oxyhydroxyde sucro-ferrique pendant deux semaines avant de répéter la procédure de sigmoïdoscopie. Résultats: La sigmoïdoscopie répétée après l'arrêt de l'oxyhydroxyde sucroferrique a permis une visualisation adéquate de l'intestin qui n'a révélé qu'un lipome bénin. Enseignements tirés: Ce cas démontre que l'utilisation d'oxyhydroxyde sucro-ferrique peut entraîner une mauvaise préparation intestinale et entraver la visualisation lors d'une endoscopie gastro-intestinale basse. Il met en évidence les répercussions cliniques qui justifient des procédures répétées, lesquelles contribuent au gaspillage des ressources et entraînent des coûts inutiles pour le système de santé, ainsi que les retards dans l'évaluation diagnostique requise pour la transplantation; dans ce cas, la frustration du patient était évidente.

Extended genetic analysis of Brazilian isolates of Bacillus cereus and Bacillus thuringiensis.

Multiple locus sequence typing (MLST) was undertaken to extend the genetic characterization of 29 isolates of Bacillus cereus and Bacillus thuringiensis previously characterized in terms of presence/absence of sequences encoding virulence factors and via variable number tandem repeat (VNTR). Additional analysis involved polymerase chain reaction for the presence of sequences (be, cytK, inA, pag, lef, cya and cap), encoding putative virulence factors, not investigated in the earlier study. MLST analysis ascribed novel and unique sequence types to each of the isolates. A phylogenetic tree was constructed from a single sequence of 2,838 bp of concatenated loci sequences. The strains were not monophyletic by analysis of any specific housekeeping gene or virulence characteristic. No clear association in relation to source of isolation or to genotypic profile based on the presence or absence of putative virulence genes could be identified. Comparison of VNTR profiling with MLST data suggested a correlation between these two methods of genetic analysis. In common with the majority of previous studies, MLST was unable to provide clarification of the basis for pathogenicity among members of the B. cereus complex. Nevertheless, our application of MLST served to reinforce the notion that B. cereus and B. thuringiensis should be considered as the same species.

Isolation and characterization of Cyniclomyces guttulatus (Robin) Van Der Walt and Scott, 1971 in dogs in Brazil.

Vegetative cells of an ascomycetous yeast, morphologically consistent with published descriptions of Cyniclomyces guttulatus, were observed in large numbers in the feces and stomach washes of three dogs with a recurrent medical history characterized by vomiting and diarrhea. Nucleotide sequence analysis of an approximately 600 base pair fragment of the variable D1/D2 domain of large subunit (26S) ribosomal DNA of a pure culture, isolated from a Siberian Husky, revealed 98-99 % homology to sequences deposited in the GenBank as C. guttulatus. These data represent the first observation of C. guttulatus in association with canine gastrointestinal illness in the southern hemisphere and add weight to the hypothesis that this yeast may act as an opportunistic pathogen of dogs. An extended examination of wet mounts and smears prepared from feces collected from 63 dogs with no clinical symptoms of gastrointestinal illness, identified C. gluttulatus in 14 (22.2 %) of the animals, albeit at lower numbers than in diseased dogs, indicating that this yeast species is widely distributed as a component of the normal microflora of the canine gastrointestinal tract.

Identification of Pentatrichomonas hominis in preputial washes of bulls in Brazil.

The parabasalid Pentatrichomonas hominis is generally considered to represent a symbiotic component of the gastrointestinal microbiota in a wide variety of vertebrate hosts including humans. Nevertheless, a limited number of studies have raised the possibility that it may act as a pathogen of humans, dogs, and pigs and that some human infections may have a zoonotic origin. Data from North America revealed an association between P. hominis and the bovine urogenital tract, principally in bulls and rarely in cows. The importance of this observation is linked to possible interference in the accurate diagnosis of the economically important venereal pathogen Tritrichomonas foetus. The current study employed culture-based and molecular methods to examine the preputial cavities of four breeding bulls, raised in open pasture in southeastern Brazil, for the presence of trichomonads. Motile protozoa were isolated from three of the bulls and were definitively identified as P. hominis based on nucleotide sequencing of polymerase chain reaction (PCR) amplicons derived from the ribosomal RNA operon (ITS1-5.8S rDNA-ITS2) of the parasite. The potential implications of these findings for bovine and human health are discussed.

Coccidia of New World passerine birds (Aves: Passeriformes): a review of Eimeria Schneider, 1875 and Isospora Schneider, 1881 (Apicomplexa: Eimeriidae).

In the New World, the avian order Passeriformes comprises 47 families and 2,453 species, yet to date only 21 (45%) of the families and 58 (2%) of the species have been examined for coccidia, and from these only two species of Eimeria Schneider, 1875 and 81 species of Isospora Schneider, 1881 have been described. This review contributes to our understanding of the morphology and systematics of coccidian parasites of passeriforms, providing a scientific basis for the identification of sporulated oöcysts recovered from the faeces of passerine birds from North, Central and South America. To this end, the coccidia were organised and grouped according to the family of the host, following the widely recognised concept of family-specificity and the updated systematics of the class Aves. Details of 83 eimeriid species are presented along with an illustration and tabulated data.

Detection of Rickettsia sp. strain Itinguçú in Ornithodoros faccinii (Acari: Argasidae) parasitizing the toad Rhinella ornata (Anura: Bufonidae) in Brazil.

The pivotal role of amphibians in food webs and their value as indicators of disequilibrium in ecosystem health have long been recognized by wildlife biologists. However, massive pathogen-induced declines in global amphibian populations reported during the last 30 years served to alert the scientific community that knowledge of amphibian disease ecology, including parasitic and vector-borne conditions, was and remains incipient. Herein, we report the detection of a Rickettsia bacterium infecting larvae of the argasid tick Ornithodoros faccinii, collected from the toad Rhinella ornata, in Southeastern Brazil. Fragments of the genes 16S rDNA, gltA, htrA, sca1, sca4, and ompB were amplified by polymerase chain reaction (PCR), but the sequence encoding the ompA antigen was not detected. Nucleotide sequencing and multi-locus (gltA, htrA, sca1, and sca4) phylogenetic analyses characterized the bacterium, designated Rickettsia sp. strain Itinguçú, as a novel member of the spotted fever group (SFG) of the Rickettsia, closely related to the Rickettsia massiliae and to a lesser extent the Rickettsia helvetica subgroups. The apparent absence of the ompA protein together with limited levels of nucleotide (90.5 %) and amino acid (82-83 %) sequence identity, relative to the ompB gene of other species in the R. massiliae subgroup, were unusual features that may reflect adaptation to selective pressures exerted by the tick and/or amphibian immune systems. The ompB sequence was exploited to develop a low-cost method for differential identification of Rickettsia sp. strain Itinguçú, based on restriction fragment length polymorphism analysis of amplicons (PCR-RFLP). The characterization of this novel bacterium provided an unprecedented record of infection by an SFG Rickettsia in a member of the family Argasidae infesting a cold-blooded animal and raised the number of tick-associated Rickettsia reported in Brazil to sixteen. Moreover, it highlighted the value of and the requirement for continued and extended surveillance of wildlife as potential sources of emerging tick-borne pathogens.

Platynosomum illiciens (Trematoda: Dicrocoeliidae) in a hybrid marmoset (Callithrix sp.) in the Municipality of Seropédica, RJ, Brazil - Case report.

Platynosomum illiciens is a liver trematode encountered infecting mainly felids although it has also been reported in birds and in additional mammalian species, including non-human primates. The current study reports a natural P. illiciens infection primate of the genus Callithrix. The diagnosis was made using a combination of copro-parasitological techniques, morphological evaluation of adult specimens recovered from the liver during necropsy, and molecular analyses. Eggs were brown in color, oval, operculated, and contained a miracidium. Adult specimens recovered during necropsy were measured and showed dimensions compatible with P. illiciens. Molecular characterization of the trematode involved amplification by polymerase chain reaction (PCR), in combination with nucleotide sequencing, of an approximately 900 base pairs fragment corresponding to 18S-ITS1-5.8S ribosomal DNA. Sequenced amplicons showed 100% nucleotide identity with sequences deposited in the GenBank database as derived from specimens of P. illiciens recovered from cats in Malaysia and Brazil. It was concluded that the morphological and molecular analyses presented herein, confirmed the identification of the trematode recovered as P. illiciens.

Tick-borne rickettsioses in Brazil: what lessons can be learned from the COVID-19 pandemic.

In this commentary, the authors highlight the importance of basic research in the field of public health regarding the recent pandemic Covid-19, using tick-borne rickettsioses as an example. In addition, they alert politicians, government officials and managers of research funding agencies to increase the allocated financial resources to enhance basic research on public health in Brazil.

Serological and molecular detection of infection with Mycobacterium leprae in Brazilian six banded armadillos (Euphractus sexcinctus).

Leprosy was recognized as a zoonotic disease, associated with nine-banded armadillos (Dasypus novemcinctus) in the Southern United States of America in 2011. In addition, there is growing evidence to support a role for armadillos in zoonotic leprosy in South America. The current study evaluated twenty specimens of the six-banded armadillo (Euphractus sexcinctus), collected from rural locations in the state of Rio Grande do Norte (RN), Brazil for evidence of infection with Mycobacterium leprae. Serum was examined using two "in-house" enzyme-linked immunosorbent assays (ELISAs) and via two commercially available (ML flow and NDO-LID®) immunochromatographic lateral flow (LF) tests, for detection of the PGL-I and/or LID-1 antigens of the bacterium. The presence of M. leprae DNA in liver tissue was examined using the multi-copy, M. leprae-specific repetitive element (RLEP), as target in conventional and nested PCR assays. Molecular and anti-PGL-I-ELISA data indicated that 20/20 (100 %) of the armadillos were infected with M. leprae. The corresponding detection levels recorded with the LF tests were 17/20 (85 %) and 16/20 (85 %), for the NDO-LID® and ML flow tests, respectively. Our results indicate that, in common with D. novemcinctus, six banded armadillos (a species hunted and reared as a food-source in some regions of Brazil, including RN), represent a potential reservoir of M. leprae and as such, their role in a possible zoonotic cycle of leprosy within Brazil warrants further investigation.

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".

Two for the price of one: Co-infection with Rickettsia bellii and spotted fever group Rickettsia in Amblyomma (Acari: Ixodidae) ticks recovered from wild birds in Brazil.

The bacterium Rickettsia bellii has been detected in 25 species of ticks in the American continents, but its pathogenic potential is considered as undetermined. A possible role for this species in the phenomenon of transovarial exclusion of pathogenic members of the spotted fever group (SFG) of Rickettsia has been suggested and co-infections with pathogenic species have been reported infrequently in both North and South America. Traditional methods for the molecular detection of rickettsial agents in ticks focus largely on the identification of sequences found in SFG Rickettsia, an approach that may overlook the presence of co-infections with R. bellii. Two novel, species-specific polymerase chain reaction (PCR) assays, targeting the genes encoding the surface cell antigen (Sca), autotransporter proteins sca9 and sca14, were developed and validated for the detection of R. bellii using 150 Amblyomma ticks collected from wild birds in Brazil. Co-infection of R. bellii infected ticks was evaluated using a novel PCR assay targeting the ompA sequence characteristic of SFG Rickettsia. Preliminary species-level identification was achieved by restriction fragment length polymorphism (RFLP) analysis and subsequently confirmed by sequencing of amplicons. Nine out of seventy-three Amblyomma longirostre and one of two Amblyomma calcaratum ticks were shown to be co-infected with R. bellii and Rickettsia amblyommatis, while two out of sixty-seven Amblyomma sp. haplotype Nazaré ticks were recorded as co-infected with R. bellii and the Rickettsia parkeri-like bacterium, strain ApPR. Interestingly, our data represent the first records of R. bellii in association with A. calcaratum and Amblyomma sp. haplotype Nazaré. The novel PCR-RFLP systems reported herein, provide an alternative, rapid and cost-efficient (relative to strategies based on sequencing or real-time PCR), approach to evaluate rickettsial co-infection of ticks, a potentially significant phenomenon that has most likely been underestimated to date.

A new species of Amblyomma (Acari: Ixodidae) associated with monkeys and passerines of the Atlantic rainforest biome, Southeastern Brazil.

Recent studies have reported several larvae of an unidentified Amblyomma species on passerine birds in Atlantic rainforest fragments in southeastern Brazil. These larvae yielded a unique 16S rRNA haplotype designated as Amblyomma sp. haplotype Nazaré, which showed nucleotide identity levels of 91% to Amblyomma parkeri Fonseca & Aragão, 1952 and 88% to Amblyomma longirostre (Koch, 1844). Herein, we describe Amblyomma sp. haplotype Nazaré as a new species, Amblyomma romarioi n. sp. Martins, Luz & Labruna, through a formal description of the male and female adult stages. Amblyomma romarioi is morphologically and genetically most closely related to A. parkeri, A. longirostre and Amblyomma geayi Neumann, 1899. Among males, the rectangular basis capituli and rounded coxa I spurs separates A. romarioi from A. parkeri, A. longirostre, and A. geayi, which have basis capituli triangular or slightly hexagonal, and pointed coxa I spurs. Among females, the V-shaped genital aperture and coxa I rounded spurs of A. romarioi contrasts to the U-shaped genital aperture and coxa I pointed spurs in A. parkeri, A. longirostre, and A. geayi. Larvae of A. romarioi have been collected on 24 species of passerines. The few records of nymphs and adults were on the black-fronted titi monkey Callicebus nigrifrons (Spix, 1823). The current distribution of A. romarioi is restricted to the Brazilian Atlantic rainforest, southeastern Brazil, in areas with altitude between 363 and 1600 m, within the distribution of C. nigrifrons. We discuss ecological features of Amblyomma romarioi, comparatively to A. parkeri, A. longirostre and A. geayi. The present study increases the Brazilian tick fauna to 74 species.

Transferable, multiple antibiotic and mercury resistance in Atlantic Canadian isolates of Aeromonas salmonicida subsp. salmonicida is associated with carriage of an IncA/C plasmid similar to the Salmonella enterica plasmid pSN254.

OBJECTIVES: The aim of this study was to examine the molecular basis for multiple antibiotic and mercury resistance in Canadian isolates of Aeromonas salmonicida subsp. salmonicida. METHODS: Phenotypic and genotypic methods were employed to identify plasmid-associated antibiotic and mercury resistance genes and to determine the organization of those genes in multidrug-resistant (MDR) A. salmonicida isolates. RESULTS: The MDR phenotype was transferable via conjugation using Escherichia coli, Aeromonas hydrophila and Edwardseilla tarda as recipients. Antibiotic and mercury resistance genes were carried by a conjugative IncA/C plasmid. Three distinct antibiotic resistance cassettes were characterized; first a class I integron containing an aadA7 gene encoding for an aminoglycoside-3'-adenyltransferase, the second cassette showed 99.9% nucleotide sequence homology to a cassette previously identified in the Salmonella enterica IncA/C plasmid pSN254, containing floR, tetA, sulII and strA/strB sequences. The third cassette showed 100% nucleotide sequence similarity to a transposon-like element, containing a bla(CMY-2) beta-lactamase in association with sugE and blc sequences. This element is known to be widely distributed among clinical and food-borne Salmonella and other Enterobacteriaceae throughout Asia and the United States. Mercury resistance was linked to the presence of a mer operon that showed 100% nucleotide sequence homology to the mer operon carried by plasmid pSN254. CONCLUSIONS: Each MDR A. salmonicida isolate carried the same plasmid, which was related to plasmid pSN254. This is the first report of plasmid-mediated florfenicol-resistant A. salmonicida in North America. In addition, it is the first report of a plasmid-associated AmpC beta-lactamase sequence in a member of the Aeromonadaceae.

Application of 16S rDNA-DGGE and plate culture to characterization of bacterial communities associated with the sawfly, Acantholyda erythrocephala (Hymenoptera, Pamphiliidae).

Culture-based analysis was employed in parallel with PCR amplification of 16S rDNA, coupled with denaturing gradient gel electrophoresis (DGGE), to profile bacterial species associated with different developmental stages of the pine false webworm (PFW), Acantholyda erythrocephala, a sawfly pest responsible for incidents of severe defoliation in commercially important tree plantations in North America. Culture-based analysis revealed that Pseudomonas spp. along with Bacillus sphaericus and Arthrobacter sp. were the predominant components of the microflora of the internal organs and identified life-stage-specific associations including Photorhabdus temperata with egg and larval samples and a Janthinobacterium sp. with eonymphs. PCR-DGGE confirmed the predominance of Pseudomonas spp. and B. sphaericus in the majority of samples but did not detect Arthrobacter sp., P. temperate, or Janthinobacterium sp. In contrast, DGGE revealed the presence of a Chryseobacterium sp. as the predominant component of the PFW micoflora at all life stages, with the exception of adults. This species had been infrequently cultured, at low levels, from a limited number of samples and the existence of a possible relationship between this bacterium and the PFW had gone unnoticed using the culture-based approach. Our findings highlight the advantages of applying a dual approach to the study of microbe-insect associations and demonstrate that the benefits of one system can be used to overcome some of the limitations of the other.

Morphological and phylogenetic characterization of a novel Unicauda species, infecting the kidney of Astyanax altiparanae (Teleostei: Characidae) in Brazil.

During a parasitological survey of Astyanax altiparanae captured in the Mogi Guaçú river, Pirassununga, state of São Paulo, Brazil, a new species of myxozoa was observed infecting the kidney of 30 of 42 specimens. Plasmodia of the new myxozoa, referred to herein as Unicauda whippsi n. sp. were spherical and varied from 450 to 1000 µm in length. The mature spore was elongated and measured 11 ± 1.2 (10-12) µm long by 5.4 ± 0.6 (4.7-7.2) µm wide. Polar capsules of unequal length with the filament coiled 7-9 times and a unique caudal process characteristic of the genus Unicauda, were recorded. Phylogenetic analysis demonstrated that U. whippsi n. sp. clustered with members of the genus Unicauda and Myxobolus, confirming it's placement within the family Myxobolidae. This is the first record of a species of Unicauda infecting characiform fish, as well as the first registered occurrence of species of this genus in South America.

Molecular analyses reveal an abundant diversity of ticks and rickettsial agents associated with wild birds in two regions of primary Brazilian Atlantic Rainforest.

Brazilian wild birds are recognized as frequent and important hosts for immature stages of more than half of the 32 recognized species of Amblyomma ticks recorded in that country. Several species of Amblyomma harbor rickettsial agents, including members of the spotted fever group (SFG). Most studies on this topic relied primarily on morphological characterization and reported large portions of the collected ticks at the genus rather than species level. Clearly, this factor may have contributed to an underestimation of tick diversity and distribution and makes comparisons between studies difficult. The current investigation combined morphological and molecular analyses to assess the diversity of ticks and rickettsial agents associated with wild birds, captured in two regions of native Atlantic rainforest, in the state of Rio de Janeiro, Brazil. A total of 910 birds were captured, representing two orders, 34 families and 106 species, among which 93 specimens (10.2%), were parasitized by 138 immature ticks (60 larvae and 78 nymphs), representing 10 recognized species of the genus Amblyomma; together with two reasonably well classified haplotypes (Amblyomma sp. haplotype Nazaré and Amblyomma sp. strain USNTC 6792). Amplification by PCR and sequencing of rickettsial genes (htrA, gltA, ompA and ompB), demonstrated the presence of Rickettsia DNA in 48 (34%) of the ticks. Specifically, Rickettsia bellii was detected in a single larva and a single nymph of A. aureolatum; R. amblyomatis was found in 16 of 37 A. longirostre and was recorded for the first time in three nymphs of A. calcaratum; R. rhipicephali was detected in 9 (47%) of 19 Amblyomma sp. haplotype Nazaré ticks. The remaining ticks were infected with genetic variants of R. parkeri, namely strain ApPR in 12 A. parkeri and seven Amblyomma sp. haplotype Nazaré ticks, with the strain NOD found in two specimens of A. nodosum. Interestingly, a single larvae of A. ovale was shown to be infected with the emerging human pathogen Rickettsia sp. strain Atlantic rainforest (ARF), suggesting a possible role for birds in the dispersal of ticks infected with this variant of R. parkeri. The diversity of ticks and Rickettsia recorded in this study is, to our knowledge, the most abundant recorded to date in Brazil and highlighted the value of employing methods capable of providing species level identification of the ixodofauna of wild birds.