SARS-CoV-2 Research in Dogs (Canis lupus familiaris) and Felines (Felis silvestris catus) Domiciled in an International Border Region (Paraguay and Brazil).

Introduction: COVID-19 is an infectious disease caused by SARS-CoV-2 that has become a serious threat to public health owing to its rapid spread from aerosols from infected people. Despite being considered a strictly human disease, there are reports in the literature about animals with confirmed presence of the virus. Aim: Owing to the scarcity of scientific literature on the potential for infection of animals and their importance for One Health, the objective of this work was to research SARS-CoV-2 RNA in felines (Felis silvestris catus) and dogs (Canis lupus familiaris) domiciled. Materials and Methods: Oropharyngeal swabs were collected from domestic dogs and cats belonging to patients diagnosed with COVID-19 from August to October 2021 and residents of the northwest and west regions of Paraná, Brazil. Results: Of the 34 samples collected, 14 were from dogs and 20 from cats. Three of these samples tested positive in real-time PCR, and two of them were also positive in the immunochromatographic test. After testing positive in real-time PCR, the samples underwent genetic sequencing using the Illumina COVIDSeq test. Of the 34 samples collected, three (9%), all of them female and from the feline species, tested positive in real-time PCR, with two of these (67%) also testing positive in the immunochromatographic test. Regarding sequencing, it was possible to sequence the three samples aligned with the AY.101 lineage, corresponding to the Delta variant. Conclusion: The occurrence of SARS-CoV-2 infection in dogs and cats is seen as an unintended event with significant implications for public health, including its potential transmission to other animal species. Further research is required to enhance our understanding of how this disease spreads among these animals and its broader impact on One Health initiatives.

Identification of lactic acid bacteria with anti-listeria activity. Characterization and application of a bacteriocinogenic strain in the control of Listeria monocytogenes in cheese.

The purpose of the research paper was firstly to identify bacteriocin-producing lactic acid bacteria characterizing strains with anti-listeria activity and, secondly, to characterize bacteriocin evaluating its in vitro efficiency as a natural preservative and, thirdly, to evaluate the anti-listeria effect of the bacteriocinogenic strain of Lactiplantibacillus plantarum in cheeses and produce an edible film with anti-listerial effect. Of 355 lactic acid bacteria strains tested, two were able to produce bacteriocin against Listeria monocytogenes and were identified as Lactiplantibacillus plantarum and Lactiplantibacillus pentosus. A bactericidal effect of strain QS494 (Lactiplantibacillus plantarum) was observed in the first 8 h, with a reduction of 1.7 log, using cell-free supernatant with Listeria monocytogenes, where viable cells were counted on listeria selective agar. Both strains showed good technological characteristics and were without production of virulence factors. Changes in the pH of the cell-free supernatant obtained from Lactiplantibacillus plantarum did not affect its antimicrobial activity, which remained stable after heat treatments for up to 15 min at 121°C. Inhibitory activity was also observed after 12 weeks of storage at -20°C. In the evaluation of the anti-listeria effect in cheeses, a 3 log reduction in the Listeria monocytogenes count was observed in 120 h in cheeses produced with bacteriocinogenic lactic acid bacteria, while in cheeses produced with non-bacteriocinogenic culture, we observed a 2 log increase in the count. Edible films produced with the addition of precipitate from the cell free supernatant showed an antimicrobial effect against Listeria monocytogenes. Thus, the two strains studied have technological and biosafety potential.

Effect of an organic acid blend in Nile tilapia growth performance, immunity, gut microbiota, and resistance to challenge against francisellosis.

Organic acids (OAs) are a class of feed additives that have prophylactic and inhibitory properties against pathogenic bacteria. In this study, we investigated growth performance, innate immune response, gut microbiota, and disease resistance against Francisella orientalis F1 in Nile tilapia (Oreochromis niloticus) fed different doses of Bacti-nil®Aqua, a blend of short- and medium-chain OAs. For 21 days, 680 juvenile tilapias were fed a control diet or diets supplemented with a 0.3% (D3) or 0.5% (D5) OA blend. The feed conversion rate of fish fed the 0.5% enriched diet was considerably lower (p < 0.05) than that of the fish fed the basal diet. Lysozyme and serum bactericidal activities were significantly elevated following OA administration. After infection, no differences in the diversity and composition of gut microbiota were observed between the groups. After the bacterial challenge, the mortality was significantly lower in group D5 (p < 0.01). The diet supplemented with Bacti-nil®Aqua (Adisseo) improved the immune response and resistance of tilapia juveniles against F. orientalis infection. Thus, this OA blend could serve as a feed additive with good activity against F. orientalis.

Genome Mining Reveals High Biosynthetic Potential of Biocontrol Agent Bacillus velezensis B.BV10.

The present study demonstrates the biocontrol potential of a plant growth-promoting bacterial strain using three different approaches: (i) an in vitro evaluation of antagonistic activity against important phytopathogenic fungi; (ii) an evaluation under greenhouse conditions with strawberry plants to assess the control of gray mold; and (iii) an in silico whole genome sequence mining to assign genetic features such as gene clusters or isolated genes to the strain activity. The in vitro assay showed that the B.BV10 strain presented antagonistic activity, inhibiting the mycelial growth in all the phytopathogenic fungi evaluated. The application of the Bacillus velezensis strain B.BV10 under greenhouse conditions reduced the presence of Botrytis cinerea and increased the mean fruit biomass. The genome of B.BV10 was estimated at 3,917,533 bp, with a GC content of 46.6% and 4088 coding DNA sequences, and was identified as B. velezensis. Biosynthetic gene clusters related to the synthesis of the molecules with antifungal activity were found in its genome. Genes related to the regulation/formation of biofilms, motility, and the important properties for the rhizospheric colonization were also found in the genome. The current study offers a comprehensive understanding of the genomic architecture and control activity of phytopathogenic fungi by the B. velezensis strain B.BV10 that may substantiate the industrialization of this strain in the future.

Insights into the Vibrio Genus: A One Health Perspective from Host Adaptability and Antibiotic Resistance to In Silico Identification of Drug Targets.

The genus Vibrio comprises an important group of ubiquitous bacteria of marine systems with a high infectious capacity for humans and fish, which can lead to death or cause economic losses in aquaculture. However, little is known about the evolutionary process that led to the adaptation and colonization of humans and also about the consequences of the uncontrollable use of antibiotics in aquaculture. Here, comparative genomics analysis and functional gene annotation showed that the species more related to humans presented a significantly higher amount of proteins associated with colonization processes, such as transcriptional factors, signal transduction mechanisms, and iron uptake. In comparison, those aquaculture-associated species possess a much higher amount of resistance-associated genes, as with those of the tetracycline class. Finally, through subtractive genomics, we propose seven new drug targets such as: UMP Kinase, required to catalyze the phosphorylation of UMP into UDP, essential for the survival of bacteria of this genus; and, new natural molecules, which have demonstrated high affinity for the active sites of these targets. These data also suggest that the species most adaptable to fish and humans have a distinct natural evolution and probably undergo changes due to anthropogenic action in aquaculture or indiscriminate/irregular use of antibiotics.

Laboratory-controlled challenges of streptococcosis in Nile tilapia using the oral route (infected-feed) for infection.

The study aimed to evaluate the oral infected-feed, intragastric-gavage, and intraperitoneal routes of the Streptococcus agalactiae infection in Nile tilapia (Oreochromis niloticus). For this purpose, 270 juveniles of Nile tilapia, with an average weight of 2 g, were distributed in 18 experimental units of 90 L, acclimatized, and raised for 55 days, until reaching 50 g (median weight). The experimental design was entirely randomized in six treatments, three of which were composed by bacterial infection routes: intraperitoneal 100 µL fish-1 [108 CFU], intragastric 100 µL fish-1 [108 CFU], feed inoculum 100 µL g feed-1 [109 CFU], and three respective control groups. Clinical signs were observed, and mortalities monitored until reaching 50% in the infected groups. Then, tissue samples from the spleen, liver, intestine, brain, and blood were collected from 20 fish per treatment for histopathological and hemato-immunological analyses. In addition, a related mortality curve was established at the end of the experimental challenge. The intraperitoneal and intragastric routes were more aggressive than the oral inoculum, causing greater brain damage, acute hemato-immunological response, and early mortality. While the orally fed inoculum, fish presented brain lesions with less intensity, and a chronic haemato-immunological response, the mortalities occurred twice as long as the other routes. The present research demonstrated that the S. agalactiae oral (feed inoculum) administration can be an innovative methodology to future experimental challenges in aquaculture research.

Evaluation of a panel of microsatellite markers to study their applications in Serrapinnus notomelas and to reveal the genetic diversity in Hyphessobrycon eques.

Jewel tetra (Hyphessobrycon eques) is a freshwater fish found in several rivers and basins in South America. The present study is the first study to create a panel of microsatellite markers for detecting genetic diversity in H. eques and evaluating the application of these markers in Serrapinnus notomelas. In total, 44 individuals were genotyped from the natural (WIL, n = 20) and stock in captivity (CAP, n = 24) population. Moreover, 19 microsatellite markers were obtained, of which only 8 loci presented a high degree polymorphism. In total, 45 alleles were detected, ranging from 126 bp (Hype2G2) to 420 bp (Hype2E2). The Hardy-Weinberg equilibrium (p < 0.05) revealed significant difference in one locus in WIL (Hype1G4) and three loci in CAP (Hype1F4, Hype2C3, and Hype2G2). Null alleles (p < 0.05) were present in only one locus (Hype1G4). The WIL and CAP populations revealed high genetic diversity during FST analysis. The cross-amplification test for S. notomelas revealed that only two loci (Hype2C3 and Hype2G2B) presented satisfactory transferability results. The developed microsatellite primers will be useful in studying the genetic diversity and population structure of H. eques in wild populations and fish farms in the Brazilian and other South American basins.

Urban capuchin monkeys Sapajus nigritus (Goldfuss, 1809) (Primates, Cebidae) as environmental bioindicators of leishmaniasis.

The aim of this study was to investigate the presence of Leishmania sp. DNA and anti-Leishmania spp. antibodies in free-ranging Sapajus nigritus from an urban forest located in a city in the North Central region of the state of Paraná. For the indirect diagnosis, the direct agglutination test was used with promastigote forms of Leishmania (V.) braziliensis, where it was possible to detect the agglutination reaction in 53.33% of the S. nigritus blood samples. For direct diagnosis, the samples were submitted to real-time quantitative polymerase chain reaction, which confirmed the presence of Leishmania spp. DNA in 26.66% of the tested samples. It reinforces the importance of considering the concept of One Health in the face of diseases with high prevalence, such as leishmaniasis and the need for health education measures. This result shows that the animals in the present study have a role as environmental bioindicators for leishmaniasis.

Effect of Enterococcus faecium as a Water and/or Feed Additive on the Gut Microbiota, Hematologic and Immunological Parameters, and Resistance Against Francisellosis and Streptococcosis in Nile Tilapia (Oreochromis niloticus).

In the present study, we evaluated the effects of administering Enterococcus faecium in food and/or water on the hematological and immunological parameters, intestinal microbiota, resistance to bacterial diseases (streptococcosis and francisellosis) and growth of Nile tilapia. Before the in vivo experiment, probiotic bacteria isolated from Nile tilapia were selected via inhibition tests. Sequencing, annotation, and assembly of the complete genome of the selected bacteria as well as other tests were performed using bioinformatics tools. Three treatments were implemented: G1 (probiotic feeding), G2 (probiotic in water), and G3 (probiotic in food and water); and a negative control (NC) was also employed. Treatment lasted 38 days, and each group consisted of fish and two repetitions. The fish were divided and infected with Streptococcus agalactiae S13 (serotype Ib) and Francisella orientalis. The G1 group had a higher average final weight gain than the G2, G3, and NC groups. Further, a significant increase in the number of thrombocytes was observed in the groups administered probiotics in the diet (G1 and G3). A statistical difference was observed in the mortality of fish infected with S. agalactiae in the NC compared to the treated groups. Cetobacterium was the 43 most abundant genus in the intestinal microbiota of all groups, including the NC group. E. faecium increased the immunity of fish administered the treatment and decreased the mortality caused by S. agalactiae. As an autochtone probiotic, E. faecium does not interfere with the local ecosystem and thus has a great probiotic potential for Nile tilapia in Brazil.

Leptospira spp. in Naturally Infected Dairy Cow from a Brazilian Border Region.

Leptospirosis is an important infectious disease, which can generate large economic losses, especially in the dairy herd. The pathogen that causes this disease may have its entry in Brazilian herds facilitated by the existence of a large extension of land borders. Therefore, the objective of this work was to investigate the presence of DNA and antibodies against Leptospira spp. in samples of vaginal mucus and serum from naturally infected bovine females from small rural dairy farms in a border region. Blood and vaginal mucus samples were collected from 70 Holstein cows, from small rural dairy farms between October 2017 and June 2018. The inclusion criteria for dairy cattle of any breed were aged over 2 years, not vaccinated against leptospirosis, and presenting a history of any reproductive problem such as abortion, stillbirth, repetition of heat, absence of heat, and lack of conception. Blood was collected by puncturing the coccygeal vein; for the collection of vaginal mucus, it was necessary to use a tampon with an applicator. For the detection of anti-Leptospira spp. antibodies, the sera were submitted to microscopic agglutination test (MAT) and, for DNA detection, the vaginal mucus was submitted to the PCR technique. Among the 70 cows, 42.86% had reagents in MAT and the most likely serovar was Wolffi (43.47%). In 74.28% of the vaginal mucus samples, it was possible to amplify the Leptospira spp. DNA. The results of this work show the presence of Leptospira spp. antibodies and DNA in samples of serum and vaginal mucus from naturally infected bovine females from small rural dairy farms in a border region (Brazil × Paraguay). These results demonstrate the importance of considering bovine females as potential vaginal carriers of Leptospira spp. Thus, it highlights the importance of further studies to better understanding of this issue, in addition to carrying out molecular and serological tests, to monitor the infection and further characterize epidemiological studies of leptospirosis in herds from regions that face this international frontier challenge.

Molecular biology as a diagnostic tool for detection of Leptospira spp. in cows of a border region ­ case report

The reproductive efficiency of livestock is the basis for the success of livestock, dairy or beef, and having high reproductive performance depends on several factors within the production system and the presence of infectious diseases of the reproductive sphere in the herd is one of the factors that can compromise that efficiency. The aim of this study was to use molecular biology as a diagnostic tool for the detection of Leptospira spp. DNA in cows with reproductive disorders on a rural property in the municipality of Boca do Acre, Amazonas, Brazil. Vaginal mucus was collected from nine Nelore breeding cows with a history of abortion and birth of weak calves submitted to DNA extraction and nested-PCR technique for 16S gene amplification at the bacterial genus level. Of the nine samples analyzed, five (55.55%) amplified a product of 331bp. The municipality of Boca do Acre is bordered by Peru and Bolivia, and knowledge of the prevalence of the disease, serovars, and circulating Leptospira species is essential for the adoption of measures related to animal husbandry, as well as health education for ranchers and their workers to avoid a possible occupational infection since this disease is considered an important zoonosis. New molecular studies using primers that allow the identification of the Leptospira species and mainly pathogenic species should be conducted in this region in order to elucidate the possible species of this etiological agent and the possible reservoirs of the disease to begin the understanding of the epidemiology of this disease in cattle in this region of border.

In silico Prediction of New Drug Candidates Against the Multidrug-Resistant and Potentially Zoonotic Fish Pathogen Serotype III Streptococcus agalactiae.

Streptococcus agalactiae is an invasive multi-host pathogen that causes invasive diseases mainly in newborns, elderly, and individuals with underlying health complications. In fish, S. agalactiae causes streptococcosis, which is characterized by septicemia and neurological signs, and leads to great economic losses to the fish farming industry worldwide. These bacteria can be classified into different serotypes based on capsular antigens, and into different sequence types (ST) based on multilocus sequence typing (MLST). In 2015, serotype III ST283 was identified to be associated with a foodborne invasive disease in non-pregnant immunocompetent humans in Singapore, and the infection was related to raw fish consumption. In addition, a serotype III strain isolated from tilapia in Brazil has been reported to be resistant to five antibiotic classes. This specific serotype can serve as a reservoir of resistance genes and pose a serious threat to public health. Thus, new approaches for the control and treatment of S. agalactiae infections are needed. In the present study, 24 S. agalactiae serotype III complete genomes, isolated from human and fish hosts, were compared. The core genome was identified, and, using bioinformatics tools and subtractive criteria, five proteins were identified as potential drug targets. Furthermore, 5,008 drug-like natural compounds were virtually screened against the identified targets. The ligands with the best binding properties are suggested for further in vitro and in vivo analysis.

Transferability of heterologous primers in Brycon falcatus

The genus Bryconcomprises fish species of significant socioeconomic and biological importance in Brazil. Despite that, the genetic knowledge about these species is scarce, especially regardingBrycon falcatus. Thus, the objective of this study was to evaluate the transferability of heterologous microsatellite primers inB. falcatus for the first time. Heterologous primers obtained from B. opalinus, B. hilarii, B. insignis, B. orbignyanus, B. amazonicus, Prochilodus argenteus, Prochilodus lineatus, Piaractus mesopotamicus, and Colossoma macropomum were evaluated. The primers that showed the best amplification patterns were applied to a sample of 22 individuals and the genetic parameters were calculated. Nine primers displayed satisfactory cross-amplification withB. falcatus: BoM5 (Brycon opalinus); Bh8, Bh13 and Bh16 (B. hilarii); Borg59 (B. orbignyanus); Bag22 (B. amazonicus); Par12 and Par80 (P. argenteus), and Cm1A8 (C. macropomum). The genetic parameters (number of alleles, effective alleles, allele richness, and expected and observed heterozygosity) and the polymorphic information content (PIC) confirmed the viability of these primers for population genetics analyses. Our study demonstrates the potential of transferability of microsatellite markers from related species and even different genera to B. falcatus, providing usefull tools for future population genetic studies in this species. (AU)

Effects of dietary supplementation with a microalga (Schizochytrium sp.) on the hemato-immunological, and intestinal histological parameters and gut microbiota of Nile tilapia in net cages.

Nutritional improvements in intensive aquaculture production systems is necessary for the reduction of stress, maximum utilization of nutritional components, and expression of the genetic potential of fish. The objective of this study was to evaluate the hemato-immunological, and histological parameters and gut microbiota of Nile tilapia fed with the microalga Schizochytrium sp. Males of Nile tilapia were distributed among eight net cages (6 m3), and fed for 105 days with two diets: control (CON), without Schizochytrium sp., and supplemented (SUP), with 1.2% Schizochytrium sp. in the diet. The final weight, mortality, hematocrit, total erythrocyte count (RBC), hemoglobin, hematimetric indices, white blood cell count (WBC), total protein, and serum lysozyme were measured. Alterations in intestinal morphology were evaluated. The gut microbiota was evaluated with next-generation sequencing. No significant differences (p>0.05) were found in the final weight and mortality between diets. Regarding the hematological parameters, a difference (p<0.05) was detected only in RBC, with there being lower values in the SUP, although this group also showed a tendency toward having an increased mean corpuscular hemoglobin level. There were no differences (p>0.05) in total protein and serum lysozyme concentrations or in WBCs between diets, except for lymphocytes, which presented lower values (p<0.05) in the SUP, suggesting immunomodulation by the polyunsaturated fatty acids present in the microalga. There was no difference (p>0.05) in the intestinal morphology between diets. Metagenomic data indicated greater richness (represented by the Chao index) and a higher abundance of the bacterial phylum Firmicutes in the gut microbiota of the tilapia fed with the SUP diet, demonstrating that the digestion and use of the components of the microalga could influence the microbial community. The results indicated that the microalga had modulatory effects on blood cells and the intestinal microbiota, without affecting the structure and integrity of the intestinal villi.

Genomic Insights Into the Antifungal Activity and Plant Growth-Promoting Ability in Bacillus velezensis CMRP 4490.

The main objective of this study was to evaluate Bacillus velezensis strain CMRP 4490 regarding its ability to inhibit soil-borne plant pathogens and to increase plant growth. The study included evaluation of in vitro antifungal control, sequencing the bacterial genome, mining genes responsible for the synthesis of secondary metabolites, root colonization ability, and greenhouse studies for the assessment of plant growth-promoting ability. The strain was obtained from soil samples in the north of Paraná in Brazil and was classified as a B. velezensis, which is considered a promising biological control agent. In vitro assay showed that B. velezensis CMRP 4490 presented antagonistic activity against Sclerotinia sclerotiorum, Macrophomina phaseolina, Botrytis cinerea, and Rhizoctonia solani with a mycelial growth inhibition of approximately 60%, without any significant difference among them. To well understand this strain and to validate its effect on growth-promoting rhizobacteria, it was decided to explore its genetic content through genome sequencing, in vitro, and greenhouse studies. The genome of CMRP 4490 was estimated at 3,996,396 bp with a GC content of 46.4% and presents 4,042 coding DNA sequences. Biosynthetic gene clusters related to the synthesis of molecules with antifungal activity were found in the genome. Genes linked to the regulation/formation of biofilms, motility, and important properties for rhizospheric colonization were also found in the genome. Application of CMRP 4490 as a coating film on soybean increased from 55.5 to 64% on germination rates when compared to the control; no differences were observed among treatments for the maize germination. The results indicated that B. velezensis CMRP 4490 could be a potential biocontrol agent with plant growth-promoting ability.

Complete Genome Sequence of Bacillus velezensis LABIM40, an Effective Antagonist of Fungal Plant Pathogens.

Bacillus velezensis strain LABIM40 holds high potential for biological control of plant pathogens. Its complete genome contains one chromosome of 3,972,310 bp with 3,777 DNA coding sequences and displays 33 gene clusters potentially involved in the suppression of fungal pathogens.

Skin antisepsis protocols for the collection of blood from donor dogs / Protocolos de antissepsia de pele para colheita de sangue de cães doadores

ABSTRACT: The objective of this study was to evaluate and compare the bactericidal efficacy of2% chlorhexidine surfactant solution + 70% alcohol and 2% chlorhexidine surfactant solution + 0.5% chlorhexidine-alcohol, and standardize skin antisepsis for blood collection from donor dogs. One hundred and twenty skin swabsof the jugular regions of 20 dogs were evaluated. Swabs were distributed into six treatment(T) groups according to the disinfectant used and removal or retention of local hair: T1involved neither antisepsisnorhair removal; T2comprised 2% chlorhexidine + 0.5% chlorhexidine-alcoholwithout hair removal;T3 comprised 2% chlorhexidine + 70% alcohol without hair removal; T4comprised hair removal but no antisepsis;T5comprised 2% chlorhexidine + 0.5% chlorhexidine-alcohol withhair removal; and T6comprised 2% chlorhexidine + 70% alcohol with hair removal. Antiseptic agents were continuously applied in a single direction for a total of 3 min. Use of antiseptics was effective with or without hair removal, resulting in the absence of bacterial growth. Complete efficacy of the technique used in this study may have been due to the increased antiseptic application time. In conclusion,the antisepsis protocols tested in this study can be safely used for the collection of blood from dogs; although,removal of hair prior to antisepsis is still recommended.

RESUMO: O objetivo deste estudo foi avaliar e comparar o potencial de redução bacteriana proporcionado peloclorexidinadegermante 2% + álcool 70% eclorexidinadegermante 2% + clorexidinaalcoolica 0,5% e padronizar a antissepsia de pele para colheita de sangue de cães doadores. Foram avaliados 120 zaragatoas de pele da região da jugular de 20 cães, que foram distribuídos em seis tratamentos (T) de acordo com oagente usado para desinfecção, associado, ou não, a tricotomia local: T1 - Tratamento sem tricotomia e sem antissepsia, T2 - Tratamento clorexidinadegermante 2% + clorexidinaalcoolica 0,5% sem tricotomia, T3 - Tratamento clorexidina 2% + álcool 70% sem tricotomia, T4 - Tratamento com tricotomia e sem antissepsia, T5 - Tratamento clorexidina 2% + clorexidina alcoólica 0,5% com tricotomia, T6 - Tratamento clorexidina 2% + álcool 70% com tricotomia. A antissepsia foi feita de forma contínua em um único sentido, totalizando 3 minutos. O uso dos antissépticos se mostraram eficazes nos tratamentos com e sem tricotomia não apresentando crescimento bacteriano. A eficácia de 100% da técnica utilizada no presente trabalho pode ser decorrente do maior tempo de antissepsia. Conclui-se que os protocolos de antissepsia realizados neste estudo podem ser utilizados com segurança para a colheita de sangue de cães, embora ainda o recomendado seja a tricotomia antes da antissepsia.

Culture Strategies for Isolation of Fastidious Leptospira Serovar Hardjo and Molecular Differentiation of Genotypes Hardjobovis and Hardjoprajitno.

The Leptospira serovar Hedjo belongs to the serogroup sejroe and this serovar is the most prevalent in bovine herds worldwide. The sejroe serogroup is the most frequently detected by serology in Brazilian cattle herds suggesting that it is due serovar Hardjo. In the molecular classification, this serovar has two genotypes: Hardjobovis and Hardjoprajitno. This serovar is as considered as fastidious pathogens, and their isolation is one of the bottlenecks in leptospirosis laboratories. In addition, its molecular characterization using genomic approaches is oftentimes not simple and time-consuming. This study describes a method for isolating the two genotypes of serovar Hardjo using culture medium formulations and suggests a get-at-able molecular characterization. Ten cows naturally infected which were seropositive were selected from small dairy farms, and their urine was collected for bacterial isolation. We evaluated three modifications of liquid Leptospira medium culture supplemented with sodium pyruvate, superoxide dismutase enzyme and fetal bovine serum, and the isolates were characterized by molecular techniques. After isolation and adaptation in standard culture medium, the strains were subcultured for 1 week in the three modified culture media for morphologic evaluation using electronic microscopy. Strains were molecularly identified by multilocus variable-number tandem-repeat analysis (MLVA), partial sequencing and phylogenic analyses of gene sec Y. Combining the liquid culture medium formulations allowed growth of the Leptospira serovar Hardjo in three tubes. Two isolates were identified as genotype Hardjobovis, and the other as genotype Hardjoprajitno. Morphologically, compared with control media, cells in the medium supplemented with the superoxide dismutase enzyme were more elongated and showed many cells in division. The cells in the medium supplemented with fetal bovine serum were fewer and lost their spirochete morphology. This indicated that the additional supplementation with fetal bovine serum assisted in the initial growth and maintenance of the viable leptospires and the superoxide dismutase enzyme allowed them to adapt to the medium. These culture strategies allowed for the isolation and convenient molecular characterization of two genotypes of serovar Hardjo, creating new insight into the seroepidemiology of leptospirosis and its specific genotypes. It also provides new information for the immunoprophylaxis of bovine leptospirosis.

New Insights about Antibiotic Production by Pseudomonas aeruginosa: A Gene Expression Analysis.

The bacterial resistance for antibiotics is one of the most important problems in public health and only a small number of new products are in development. Antagonistic microorganisms from soil are a promising source of new candidate molecules. Products of secondary metabolism confer adaptive advantages for their producer, in the competition for nutrients in the microbial community. The biosynthesis process of compounds with antibiotic activity is the key to optimize their production and the transcriptomic study of microorganisms is of great benefit for the discovery of these metabolic pathways. Pseudomonas aeruginosa LV strain growing in the presence of copper chloride produces a bioactive organometallic compound, which has a potent antimicrobial activity against various microorganisms. The objective of this study was to verify overexpressed genes and evaluate their relation to the organometallic biosynthesis in this microorganism. P. aeruginosa LV strain was cultured in presence and absence of copper chloride. Two methods were used for transcriptomic analysis, genome reference-guided assembly and de novo assembly. The genome referenced analysis identified nine upregulated genes when bacteria were exposed to copper chloride, while the De Novo Assembly identified 12 upregulated genes. Nineteen genes can be related to an increased microbial metabolism for the extrusion process of exceeding intracellular copper. Two important genes are related to the biosynthesis of phenazine and tetrapyrroles compounds, which can be involved in the bioremediation of intracellular copper and we suggesting that may involve in the biosynthesis of the organometallic compound. Additional studies are being carried out to further prove the function of the described genes and relate them to the biosynthetic pathway of the organometallic compound.

Whole-Genome Sequence of Streptococcus agalactiae Strain S13, Isolated from a Fish Eye from a Nile Tilapia Farm in Southern Brazil.

Streptococcus agalactiae is an important pathogen to world aquaculture due to its high mortality rates in fish farms and consequent economic losses. Our study presents the complete genome sequence of strain S13, isolated from a tilapia farm outbreak in southern Brazil.