CHARACTERIZATION OF THE FUNGAL MICROBIOTA IN THE NOSTRILS AND RECTUM OF AMAZONIAN MANATEES (TRICHECHUS INUNGUIS) FROM A REHABILITATION PROGRAM IN BRAZIL.

The present study characterized the filamentous and yeast-like fungal microbiota of the nasal cavity and rectum of Amazonian manatees (Trichechus inunguis) undergoing rehabilitation at the Laboratory of Aquatic Mammals, National Institute of Amazonian Research, Manaus, Amazonas, and determined the antifungal susceptibility of these organisms. Nasal and rectal swabs were collected from 22 calves and three juveniles. The samples were seeded in Sabouraud agar supplemented with chloramphenicol 10%, incubated at 26°C, and observed daily for up to 7 d. The growth of different filamentous and yeast-like fungi was observed among the two anatomical sites. Filamentous fungi were categorized by macro- and microscopic characteristics of the colonies. Representatives of each group were selected for molecular identification based on the internal transcribed spacer region. Yeast identification was performed using MALDI-TOF MS and molecular analyses. Thirteen genera of filamentous fungi and six genera of yeasts were isolated and identified. The dominant filamentous species were Fusarium spp., Aspergillus spp., and Cochliobolus lunatus in the nostril samples and Aspergillus melleus in the rectal samples. Candida was the dominant genus among the identified yeasts at both anatomical sites. In the antifungal susceptibility test, 28 isolates showed resistance to fluconazole (78%), itraconazole (39%), and nystatin (42%). The knowledge of fungal microbiota composition of Amazonian manatees provides information that assists in monitoring the health status of individuals maintained in captivity, as these organisms can behave either as opportunists or as primary pathogens. Moreover, the composition and resistance of these organisms may vary among different rehabilitation institutions or different time frames of search, reinforcing the importance of constant in loco surveillance of these microorganisms. This study provides new perspectives on the fungal diversity in the microbiota of manatees and supports future studies concerning the clinical and epidemiological aspects and the impacts of these agents on the health of Amazonian manatees undergoing rehabilitation.

Edwardsiella tarda in Tambaqui (Colossoma macropomum): A Pathogenicity, Antimicrobial Susceptibility, and Genetic Analysis of Brazilian Isolates.

Edwardsiella tarda is a crucial pathogenic bacterium in tropical aquaculture. This bacterium was recently isolated from tambaqui (Colossoma macropomum), a commercially important fish species in Brazil. This study assessed the antimicrobial susceptibility, pathogenicity, and genetic diversity of the tambaqui-derived E. tarda isolates. Fourteen bacterial isolates isolated from tambaqui were identified as E. tarda by using matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry and dnaJ gene sequencing. Antimicrobial susceptibility tests were conducted against seven drugs using the disc diffusion assay. The pathogenicity test conducted by intraperitoneal injection of 2.4 × 107 colony-forming units (CFU) fish-1 of E. tarda (ED38-17) into tambaqui juveniles eventually revealed that neither clinical signs nor death were present. However, splenomegaly and whitish areas in the spleen and kidneys were observed. The histological investigation also revealed granulomatous splenitis, nephritis, and hepatitis occurring internally. Repetitive extragenic palindromic-PCR fingerprinting separated the 14 isolates into three genetic groups. The antibiogram revealed that all E. tarda isolates were wild-type (WT) to florfenicol (FLO), norfloxacin (NOR), neomycin (NEO), erythromycin (ERY), and oxytetracycline (OXY); however, some were non-wild-type to sulfamethoxazole/trimethoprim (7.1%) and amoxicillin (21.4%). Therefore, through experimental infection, E. tarda ED38-17 could induce pathogenic effects in C. macropomum. Additionally, three distinct genetic types were found, and the E. tarda isolates were WT to FLO, NOR, NEO, ERY, and OXY. These findings raise awareness of a bacteria causing unseen lesions, a pathogen that will potentially impact tambaqui aquaculture in the future.

Synergic Effect of Brachyspira hyodysenteriae and Lawsonia intracellularis Coinfection: Anatomopathological and Microbiome Evaluation.

Brachyspira hyodysenteriae and Lawsonia intracellularis coinfection has been observed in the diagnostic routine; however, no studies have evaluated their interaction. This study aimed to characterize lesions and possible synergisms in experimentally infected pigs. Four groups of piglets, coinfection (CO), B. hyodysenteriae (BRA), L. intracellularis (LAW), and negative control (NEG), were used. Clinical signals were evaluated, and fecal samples were collected for qPCR. At 21 days post infection (dpi), all animals were euthanized. Gross lesions, bacterial isolation, histopathology, immunohistochemistry, and fecal microbiome analyses were performed. Diarrhea started at 12 dpi, affecting 11/12 pigs in the CO group and 5/11 pigs in the BRA group. Histopathological lesions were significantly more severe in the CO than the other groups. B. hyodysenteriae was isolated from 11/12 pigs in CO and 5/11 BRA groups. Pigs started shedding L. intracellularis at 3 dpi, and all inoculated pigs tested positive on day 21. A total of 10/12 CO and 7/11 BRA animals tested positive for B. hyodysenteriae by qPCR. A relatively low abundance of microbiota was observed in the CO group. Clinical signs and macroscopic and microscopic lesions were significantly more severe in the CO group compared to the other groups. The presence of L. intracellularis in the CO group increased the severity of swine dysentery.

Comparative polyphasic characterization of Weissella strains isolated from beaked whale and rainbow trout (Oncorhynchus mykiss): confirmation of Weissella ceti sp. nov. and description of the novel Weissella tructae sp. nov. isolated from farmed rainbow trout.

The weissellosis agent bacterium (WS08T = CBMAI 2730) was isolated from diseased rainbow trout (Oncorhynchus mykiss) in Brazil. The whole genome sequence of this strain was compared with the Mexican W-1 strain, also isolated from diseased rainbow trout, and with the Weissella ceti type strain CECT 7719 T (= 1119-1A-09 T = CCUG 59653 T), recovered from the beaked whale. Digital DNA-DNA hybridization pairwise analyses scored 98.7% between the Mexican W-1 and Brazilian WS08T but just 24.4% for both fish isolates compared to the W. ceti type strain CECT 7719 T. The 16S rRNA gene sequence comparisons with isolates of W. ceti, available at GenBank, were conducted. All rainbow trout-pathogenic isolates grouped close (97% bootstrap confirmation), but when this group was compared to the W. ceti type strain CECT 7719 T the similarity varied from 78.9 to 79.1%. Phenotypic assays were also conducted, and the W. ceti type strain diverged from WS08T and W-1 in the hydrolysis of aesculin, D-mannose, and potassium gluconate and in the hydrolysis of hippurate. Moreover, WS08T and W-1 showed weak growth at 5 °C whereas no growth was observed for W. ceti CECT 7719 T. The major fatty acids (> 10% total fatty acids) presented by WS08T and W-1 were summed feature 8 (C18:1 ω7c/C18:1 ω6c), summed feature 3 (C16:1 ω6c/C16:1ω7c), and C16:0. The results of phylogenetic and phenotypic analyses clearly differentiated the W. ceti CECT 7719 T type strain from the assessed pathogenic strains obtained from rainbow trout. Therefore, Weissella strains isolated from rainbow trout, here represented by strain WS08T (= CBMAI 2730), should be known as members of a novel species for which the name Weissella tructae sp. nov. is proposed.

First report of infectious spleen and kidney necrosis virus in Nile tilapia in Brazil.

In June 2020, an atypical fatal outbreak in a Brazilian Nile tilapia farm was investigated. Twenty-three animals were collected and different tissues were used for bacterial isolation, histopathological and electron microscopic examination and viral detection using molecular methods. A large number of megalocytes were observed in the histopathological analysis of several tissues. Icosahedral virions, with a diameter of approximately 160 nm, were visualized inside the megalocytes through transmission electron microscopy of the spleen tissue. The virions were confirmed to be infectious spleen and kidney necrosis virus (ISKNV) through PCR and sequencing analyses of the fish samples. Phylogenetic analysis indicated that the virus belongs to the Clade 1 of ISKNV. This viral pathogen is associated with high mortality in the early stages of cultured Nile tilapia in the United States, Thailand and Ghana; however, until now, there have been no reports from ISKNV affecting cultured fish in Brazil. Additionally, in 14 out of 23 sampled fish, Streptococcus agalactiae, Edwardsiella tarda or Aeromonas hydrophila infections were also detected. This is the first report of fatal ISKNV infections in the Brazilian Nile tilapia fish farms and represents a new challenge to the aquaculture sector in the country.

Severe outbreak of Centrocestus formosanus (Trematoda: Heterophyidae) in farm-raised ornamental platies Xiphophorus maculatus.

This report describes a severe outbreak of the gill fluke Centrocestus formosanus in farm-raised platies Xiphophorus maculatus in Brazil, with mortality rate approaching 95%. Typical clinical signs of infection were observed, with microscopic examinations of fresh gills revealing multiple cysts containing a once-folded metacercaria with an X-shaped excretory bladder. The 18S subunit of the metacercariae (BR1) was amplified by PCR, sequenced and analyzed by BLAST. Subsequent phylogenetic analyses revealed that the BR1 isolate was closely related to C. formosanus from Thailand. This is the first report of C. formosanus in ornamental fish in Brazil. Our observations suggest that platies are highly sensitive to this digenetic parasite. Controlling population densities of the parasite's intermediate host, the snail Melanoides tuberculata, would help to reduce outbreaks.

First genome sequencing and comparative analyses of Corynebacterium pseudotuberculosis strains from Mexico.

Corynebacterium pseudotuberculosis is a pathogenic bacterium which has been rapidly spreading all over the world, causing economic losses in the agricultural sector and sporadically infecting humans. Six C. pseudotuberculosis strains were isolated from goats, sheep, and horses with distinct abscess locations. For the first time, Mexican genomes of this bacterium were sequenced and studied in silico. All strains were sequenced using Ion Personal Genome Machine sequencer, assembled using Newbler and SPAdes software. The automatic genome annotation was done using the software RAST and in-house scripts for transference, followed by manual curation using Artemis software and BLAST against NCBI and UniProt databases. The six genomes are publicly available in NCBI database. The analysis of nucleotide sequence similarity and the generated phylogenetic tree led to the observation that the Mexican strains are more similar between strains from the same host, but the genetic structure is probably more influenced by transportation of animals between farms than host preference. Also, a putative drug target was predicted and in silico analysis of 46 strains showed two gene clusters capable of differentiating the biovars equi and ovis: Restriction Modification system and CRISPR-Cas cluster.

Gut microbiome modulation during treatment of mucositis with the dairy bacterium Lactococcus lactis and recombinant strain secreting human antimicrobial PAP.

Mucositis is an inflammatory condition of the gut, caused by an adverse effect of chemotherapy drugs, such as 5-fluorouracil (5-FU). In an attempt to develop alternative treatments for the disease, several research groups have proposed the use of probiotics, in particular, Lactic Acid Bacteria (LAB). In this context, the use of recombinant LAB, for delivering anti-inflammatory compounds has also been explored. In previous work, we demonstrated that either Lactococcus lactis NZ9000 or a recombinant strain expressing an antimicrobial peptide involved in human gut homeostasis, the Pancreatitis-associated Protein (PAP), could ameliorate 5-FU-induced mucositis in mice. However, the impact of these strains on the gut microbiota still needs to be elucidated. Therefore, in the present study, we aimed to characterize the effects of both Lactococci strains in the gut microbiome of mice through a 16 S rRNA gene sequencing metagenomic approach. Our data show 5-FU caused a significant decrease in protective bacteria and increase of several bacteria associated with pro-inflammatory traits. The Lactococci strains were shown to reduce several potential opportunistic microbes, while PAP delivery was able to suppress the growth of Enterobacteriaceae during inflammation. We conclude the strain secreting antimicrobial PAP was more effective in the control of 5-FU-dysbiosis.

Complete genome sequencing of sixteen Francisella noatunensis subsp. orientalis isolates: A genomic approach for molecular characterization and spread dynamics of this clonal population.

Francisella noatunensis subsp. orientalis (FNO) is an important emerging pathogen associated with disease outbreaks in farm-raised Nile tilapia. FNO genetic diversity using PCR-based typing, no intra-species discrimination was achieved among isolates/strains from different countries, thus demonstrating a clonal behaviour pattern. In this study, we aimed to evaluate the population structure of FNO isolates by comparing whole-genome sequencing data. The analysis of recombination showed that Brazilian isolates group formed a clonal population; whereas other lineages are also supported by this analysis for isolates from foreign countries. The whole-genome multilocus sequence typing (wgMLST) analysis showed varying numbers of dissimilar alleles, suggesting that the Brazilian clonal population are in expansion. Each Brazilian isolate could be identified as a single node by high-resolution gene-by-gene approach, presenting slight genetic differences associated to mutational events. The common ancestry node suggests a single entry into the country before 2012, and the rapid dissemination of this infectious agent may be linked to market sales of infected fingerlings.

Transcriptome analysis of Corynebacterium pseudotuberculosis biovar Equi in two conditions of the environmental stress.

Corynebacterium pseudotuberculosis has been widely studied in an effort to understand its biological evolution. Transcriptomics has revealed possible candidates for virulence and pathogenicity factors of strain 1002 (biovar Ovis). Because C. pseudotuberculosis is classified into two biovars, Ovis and Equi, it was interesting to assess the transcriptional profile of biovar Equi strain 258, the causative agent of ulcerative lymphangitis. The genome of this strain was re-sequenced; the reassembly was completed using optical mapping technology, and the sequence was subsequently re-annotated. Two growth conditions that occur during the host infection process were simulated for the transcriptome: the osmotic and acid medium. Genes that may be associated with the microorganism's resilience under unfavorable conditions were identified through RNAseq, including genes present in pathogenicity islands. The RT-qPCR was performed to confirm the results in biological triplicate for each condition for some genes. The results extend our knowledge of the factors associated with the spread and persistence of C. pseudotuberculosis during the infection process and suggest possible avenues for studies related to the development of vaccines, diagnosis, and therapies that might help minimize damage to agribusinesses.

Large-scale genomic analyses reveal the population structure and evolutionary trends of Streptococcus agalactiae strains in Brazilian fish farms.

Streptococcus agalactiae is a major pathogen and a hindrance on tilapia farming worldwide. The aims of this work were to analyze the genomic evolution of Brazilian strains of S. agalactiae and to establish spatial and temporal relations between strains isolated from different outbreaks of streptococcosis. A total of 39 strains were obtained from outbreaks and their whole genomes were sequenced and annotated for comparative analysis of multilocus sequence typing, genomic similarity and whole genome multilocus sequence typing (wgMLST). The Brazilian strains presented two sequence types, including a newly described ST, and a non-typeable lineage. The use of wgMLST could differentiate each strain in a single clone and was used to establish temporal and geographical correlations among strains. Bayesian phylogenomic analysis suggests that the studied Brazilian population was co-introduced in the country with their host, approximately 60 years ago. Brazilian strains of S. agalactiae were shown to be heterogeneous in their genome sequences and were distributed in different regions of the country according to their genotype, which allowed the use of wgMLST analysis to track each outbreak event individually.

Identification of bluetongue virus serotypes 1, 4, and 17 co-infections in sheep flocks during outbreaks in Brazil.

Bluetongue (BT) is a vector-borne viral disease caused by the Bluetongue virus (BTV), an Orbivirus from the Reoviridae family, affecting domestic and wild ruminants. BTV circulation in Brazil was first reported in 1978, and several serological surveys indicate that the virus is widespread, although with varied prevalence. In 2014, BT outbreaks affected sheep flocks in Rio Grande do Sul state, causing significant mortality (18.4%; 91/495) in BTV-infected sheep. In total, seven farms were monitored, and one or two sheep from each farm that died due to clinical signs of BT were necropsied. Apathy, pyrexia, anorexia, tachycardia, respiratory, and digestive disorders were noted. Additionally, an abortion was recorded in one of the monitored farms. The main gross lesions observed were pulmonary edema, anterior-ventral pulmonary consolidation, muscular necrosis in the esophagus and in the ventral serratus muscle, and hemorrhagic lesions in the heart. The blood and tissue samples were tested for BTV RNA detection by RT-qPCR targeting the segment 10. Positive samples were used for viral isolation. The isolated BTVs were typed by conventional RT-PCR targeting the segment 2 of the 26 BTV serotypes, followed by sequencing analysis. BTV-1, BTV-4 and BTV-17 were identified in the analyzed samples. Double or triple BTV co-infections with these serotypes were detected. We report the occurrence of BT outbreaks related to BTV-1, BTV-4 and BTV-17 infections and co-infections causing clinical signs in sheep flocks in Southern Brazil, with significant mortality and lethality rates.

Quantitative Proteomic Analysis Reveals Changes in the Benchmark Corynebacterium pseudotuberculosis Biovar Equi Exoproteome after Passage in a Murine Host.

Corynebacterium pseudotuberculosis biovar equi is the etiologic agent of ulcerative lymphangitis. To investigate proteins that could be related to the virulence of this pathogen, we combined an experimental passage process using a murine model and high-throughput proteomics with a mass spectrometry, data-independent acquisition (LC-MSE) approach to identify and quantify the proteins released into the supernatants of strain 258_equi. To our knowledge, this approach allowed characterization of the exoproteome of a C. pseudotuberculosis equi strain for the first time. Interestingly, the recovery of this strain from infected mouse spleens induced a change in its virulence potential, and it became more virulent in a second infection challenge. Proteomic screening performed from culture supernatant of the control and recovered conditions revealed 104 proteins that were differentially expressed between the two conditions. In this context, proteomic analysis of the recovered condition detected the induction of proteins involved in bacterial pathogenesis, mainly related to iron uptake. In addition, KEGG enrichment analysis showed that ABC transporters, bacterial secretion systems and protein export pathways were significantly altered in the recovered condition. These findings show that secretion and secreted proteins are key elements in the virulence and adaptation of C. pseudotuberculosis. Collectively, bacterial pathogenesis-related proteins were identified that contribute to the processes of adherence, intracellular growth and evasion of the immune system. Moreover, this study enhances our understanding of the factors that may influence the pathogenesis of C. pseudotuberculosis.

Clostridium perfringens and C. difficile in parvovirus-positive dogs.

The aim of this study was to investigate Clostridium difficile and Clostridium perfringens in 82 diarrheic dogs positive for canine parvovirus type 2 (CPV). Enterotoxigenic C. perfringens type A was isolated from three (3.6%) dogs. One (1.2%) strain was also positive for NetE- and NetF-encoding genes, which are commonly associated with diarrhea in dogs. Toxigenic C. difficile was isolated from one animal (1.2%), which was also positive for A/B toxins. The present study identified C. difficile and C. perfringens infection in CPV-positive dogs. Further studies are necessary to clarify if clostridial infections may predispose or potentiate CPV-infection in dogs or vice versa.

Comparative genomic analysis between Corynebacterium pseudotuberculosis strains isolated from buffalo.

Corynebacterium pseudotuberculosis is a Gram-positive, pleomorphic, facultative intracellular pathogen that causes Oedematous Skin Disease (OSD) in buffalo. To better understand the pathogenic mechanisms of OSD, we performed a comparative genomic analysis of 11 strains of C. pseudotuberculosis isolated from different buffalo found to be infected in Egypt during an outbreak that occurred in 2008. Sixteen previously described pathogenicity islands (PiCp) were present in all of the new buffalo strains, but one of them, PiCp12, had an insertion that contained both a corynephage and a diphtheria toxin gene, both of which may play a role in the adaptation of C. pseudotuberculosis to this new host. Synteny analysis showed variations in the site of insertion of the corynephage during the same outbreak. A gene functional comparison showed the presence of a nitrate reductase operon that included genes involved in molybdenum cofactor biosynthesis, which is necessary for a positive nitrate reductase phenotype and is a possible adaptation for intracellular survival. Genomes from the buffalo strains also had fusions in minor pilin genes in the spaA and spaD gene cluster (spaCX and spaYEF), which could suggest either an adaptation to this particular host, or mutation events in the immediate ancestor before this particular epidemic. A phylogenomic analysis confirmed a clear separation between the Ovis and Equi biovars, but also showed what appears to be a clustering by host species within the Equi strains.

A shift in the virulence potential of Corynebacterium pseudotuberculosis biovar ovis after passage in a murine host demonstrated through comparative proteomics.

BACKGROUND: Corynebacterium pseudotuberculosis biovar ovis, a facultative intracellular pathogen, is the etiologic agent of caseous lymphadenitis in small ruminants. During the infection process, C. pseudotuberculosis changes its gene expression to resist different types of stresses and to evade the immune system of the host. However, factors contributing to the infectious process of this pathogen are still poorly documented. To better understand the C. pseudotuberculosis infection process and to identify potential factors which could be involved in its virulence, experimental infection was carried out in a murine model using the strain 1002_ovis and followed by a comparative proteomic analysis of the strain before and after passage. RESULTS: The experimental infection assays revealed that strain 1002_ovis exhibits low virulence potential. However, the strain recovered from the spleen of infected mice and used in a new infection challenge showed a dramatic change in its virulence potential. Label-free proteomic analysis of the culture supernatants of strain 1002_ovis before and after passage in mice revealed that 118 proteins were differentially expressed. The proteome exclusive to the recovered strain contained important virulence factors such as CP40 proteinase and phospholipase D exotoxin, the major virulence factor of C. pseudotuberculosis. Also, the proteome from recovered condition revealed different classes of proteins involved in detoxification processes, pathogenesis and export pathways, indicating the presence of distinct mechanisms that could contribute in the infectious process of this pathogen. CONCLUSIONS: This study shows that C. pseudotuberculosis modifies its proteomic profile in the laboratory versus infection conditions and adapts to the host context during the infection process. The screening proteomic performed us enable identify known virulence factors, as well as potential proteins that could be related to virulence this pathogen. These results enhance our understanding of the factors that might influence in the virulence of C. pseudotuberculosis.

Complete Genome Sequence of the Attenuated Corynebacterium pseudotuberculosis Strain T1.

We present here the genome sequence of the attenuated Corynebacterium pseudotuberculosis strain T1. The sequencing was performed with an Ion Torrent Personal Genome Machine platform. The genome is a circular chromosome of 2,337,201 bp, with a G+C content of 52.85% and a total of 2,125 coding sequences (CDSs), 12 rRNAs, 49 tRNAs, and 24 pseudogenes.

Whole-Genome Sequence of Corynebacterium auriscanis Strain CIP 106629 Isolated from a Dog with Bilateral Otitis from the United Kingdom.

In this work, we describe a set of features of Corynebacterium auriscanis CIP 106629 and details of the draft genome sequence and annotation. The genome comprises a 2.5-Mbp-long single circular genome with 1,797 protein-coding genes, 5 rRNA, 50 tRNA, and 403 pseudogenes, with a G+C content of 58.50%.

Complete genome sequence of Streptococcus agalactiae strain GBS85147 serotype of type Ia isolated from human oropharynx.

Streptococcus agalactiae, also referred to as Group B Streptococcus, is a frequent resident of the rectovaginal tract in humans, and a major cause of neonatal infection. The pathogen can also infect adults with underlying disease, particularly the elderly and immunocompromised ones. In addition, S. agalactiae is a known fish pathogen, which compromises food safety and represents a zoonotic hazard. This study provides valuable structural, functional and evolutionary genomic information of a human S. agalactiae serotype Ia (ST-103) GBS85147 strain isolated from the oropharynx of an adult patient from Rio de Janeiro, thereby representing the first human isolate in Brazil. We used the Ion Torrent PGM platform with the 200 bp fragment library sequencing kit. The sequencing generated 578,082,183 bp, distributed among 2,973,022 reads, resulting in an approximately 246-fold mean coverage depth and was assembled using the Mira Assembler v3.9.18. The S. agalactiae strain GBS85147 comprises of a circular chromosome with a final genome length of 1,996,151 bp containing 1,915 protein-coding genes, 18 rRNA, 63 tRNA, 2 pseudogenes and a G + C content of 35.48 %.