Publications by authors named "Odir Antônio Dellagostin"

81 Publications

Polymerase chain reaction and loop-mediated isothermal amplification targeting lic13162, lic20239, and lipL32 genes for leptospirosis diagnosis.

Braz J Microbiol 2022 Feb 5. Epub 2022 Feb 5.

Laboratório de Vacinologia, Núcleo de Biotecnologia, Centro de Desenvolvimento Tecnológico, Universidade Federal de Pelotas, Pelotas, RS, 96010-900, Brazil.

Leptospirosis is a zoonotic disease caused by pathogenic species of Leptospira. Due to the similarity with clinical signs of other febrile diseases, early diagnosis remains challenging. Real-time PCR has been used for direct detection of Leptospira, but it requires thermocyclers and highly trained personnel. Loop-mediated isothermal amplification (LAMP) is a simple and rapid DNA-based assay. Therefore, here we have developed PCR and LAMP assays targeting two novel genes, lic13162 and lic20239, and also lipL32 gene to detect pathogenic Leptospira. Analytical and diagnostic performances were compared with bacterial isolates (including different Leptospira species and serovars) and clinical samples. The results demonstrated that PCR assays targeting lic13162 and lic20239 were successful to amplify Leptospira, but LAMP not. However, both PCR and LAMP targeting lipL32 could detect pathogenic Leptospira. LAMP lipL32 could be performed in 30 min with a detection limit of 156 cells/mL. Diagnostic performance of lipL32-LAMP presented 84.2% sensitivity and 93.2% specificity. In conclusion, lipL32 PCR and LAMP are effective methods to detect pathogenic Leptospira directly from clinical samples.
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http://dx.doi.org/10.1007/s42770-022-00698-1DOI Listing
February 2022

Development of ELISA Using Recombinant Proteins for the Diagnosis of Infection.

Indian J Microbiol 2022 Mar 16;62(1):88-95. Epub 2021 Sep 16.

Núcleo de Biotecnologia, Centro de Desenvolvimento Tecnológico, Universidade Federal de Pelotas, Pelotas, RS Brazil.

In order to develop a more sensitive and reliable method for detection of serum antibodies against infection in pigs, six recombinant proteins of (P102, P95, P46, P97 like, Lppt, and hypothetical P987) were used for the standardization of an indirect enzyme-linked immunosorbent assay (ELISA). The proteins were evaluated against 50 sera of the specific pathogen-free and 50 sera of pigs with lesions suggestive of infection. The sensitivity was 88%, 86%, 78%, 74%, 66%, and 60% for the proteins P102, P95, P46, P97 like, Lppt, and hypothetical protein P987, respectively. Moreover, the proteins were used to establish the seroprevalence in two different commercial herds (254 sera pigs from farm considered free of and 246 from farm with clinical signs of enzootic pneumonia and positive serology for ) and the positive rate was 65.2% for P95, 54.6% for P102, 40.2% for P46, 37.2% for P97 like, 17.4% for the hypothetical P987, and 14% for Lppt protein. In addition, the ELISA with six recombinant proteins was compared to commercial HerdCheck kit using 118 random pig sera samples and the results showed that ELISA with recombinant proteins were more sensitive than the commercial test. These data show that the recombinant proteins P95 and P102 are potential targets to be used in diagnostic tests to detect antibodies against . Although more studies are necessary, this study provides insights that these recombinant proteins can be useful in epidemiological investigations and as potential biomarkers in differentiating infected animals from those vaccinated.
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http://dx.doi.org/10.1007/s12088-021-00981-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8758847PMC
March 2022

Development of Human Recombinant Leptospirosis Vaccines.

Methods Mol Biol 2022 ;2410:325-344

Universidade Federal de Pelotas, Centro de Desenvolvimento Tecnológico, Campus Universitário s/n, Pelotas, RS, Brazil.

Leptospirosis is a bacterial zoonotic disease with significant impact on health all over the world. Currently, bacterins are the only vaccines available for prevention of this disease, despite several drawbacks. In an effort to develop a more effective vaccine against leptospirosis, reverse and structural vaccinology have been applied to design recombinant constructions composed of leptospiral surface-exposed antigens. Herein, we describe a protocol for design and development of Leptospirosis recombinant vaccines using immunoinformatic approaches.
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http://dx.doi.org/10.1007/978-1-0716-1884-4_16DOI Listing
January 2022

Protective efficacy of whole-cell inactivated Leptospira vaccines made using virulent or avirulent strains in a hamster model.

Vaccine 2021 09 13;39(39):5626-5634. Epub 2021 Aug 13.

Centro de Desenvolvimento Tecnológico, Núcleo de Biotecnologia, Universidade Federal de Pelotas, Pelotas, RS, Brazil.

Whole-cell inactivated vaccines remain the only licensed vaccines used to control human and animal leptospirosis worldwide. Although they are protective against lethal infections, the efficacy of these vaccines has been divergent. The manufacturing process often involves the use of standard bacterial strains subjected to serial in vitro passages, with a risk of loss of virulence, and may affect the immunogenicity and consequently decrease protection. Thus, the objective of this study was to perform a comparative analysis of the efficacy of in-house bacterins produced with standard (avirulent) and virulent strains. Hamsters were immunized with killed bacteria produced using avirulent and virulent strains of L. interrogans serovars Copenhageni and Canicola. Vaccine efficacy was determined in terms of protection against lethal homologous or heterologous challenges. The results showed that immunization with both avirulent and virulent Canicola strains resulted in 100% protection against homologous challenge. Conversely, Copenhageni bacterins produced using an avirulent strain conferred only 25-37.5% protection against homologous challenge (P > 0.05), while virulent Copenhageni bacterin conferred 100% protection (P < 0.001). A single vaccine dose was sufficient to induce protection, and administration of a prime boost significantly reduced the bacterial load in the kidneys and improved the humoral immune response to the virulent Copenhageni strain. These findings suggest that the maintenance of virulent strains in bacterin formulations is essential for improving the immunogenicity and efficacy of leptospirosis vaccines.
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http://dx.doi.org/10.1016/j.vaccine.2021.08.014DOI Listing
September 2021

SARS-CoV-2 mutations in Brazil: from genomics to putative clinical conditions.

Sci Rep 2021 06 7;11(1):11998. Epub 2021 Jun 7.

Department of Pharmaceutical and Medicinal Chemistry, Institute of Pharmacy, University of Tübingen, Tübingen, Germany.

Due to the high rate of transmissibility, Brazil became the new COVID-19 outbreak epicenter and, since then, is being monitored to understand how SARS-CoV-2 mutates and spreads. We combined genomic and structural analysis to evaluate genomes isolated from different regions of Brazil and show that the most prevalent mutations were located in the S, N, ORF3a and ORF6 genes, which are involved in different stages of viral life cycle and its interaction with the host cells. Structural analysis brought to light the positions of these mutations on protein structures, contributing towards studies of selective structure-based drug discovery and vaccine development.
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http://dx.doi.org/10.1038/s41598-021-91585-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8184806PMC
June 2021

Biofilm formation of from milk and expression of the adhesion genes and at different temperatures.

Can J Microbiol 2021 Sep 4;67(9):677-685. Epub 2021 May 4.

Departamento de Ciência e Tecnologia Agroindustrial, Faculdade de Agronomia Eliseu Maciel, Campus Universitário Capão do Leão s/nº, Universidade Federal de Pelotas (UFPel), Capão do Leão, Rio Grande do Sul, Caixa Postal 354, 96160-000 Brazil.

This study investigated the ability of isolates from milk to form biofilm, through detection of adhesion genes, investigating exopolysaccharide (EPS) production and biofilm formation on polystyrene (PS) and stainless steel (SS) surfaces, and by quantifying the expression of and genes under different temperatures and culture media. Among the 31 isolates, the adhesion genes and were found in 81% and 61% of the isolates, respectively. The screening tests for phenotype revealed that 58% of the isolates were EPS producers, and 45% showed the ability to produce biofilm on PS. Nine of the 31 isolates were selected to verify their ability to form biofilm on SS, of which 3 were non-biofilm producers, 3 were poor biofilm producers, and 3 were moderate biofilm producers. However, all nine isolates produced biofilm on SS, regardless of their phenotypic profile on PS. Reverse-transcriptase quantitative PCR (RT-qPCR) revealed no variation in the expression levels of and genes at different temperatures, except for isolate S24 at 10 °C, for both genes tested. Moreover, RT-qPCR assays revealed that the expression levels of the adhesion genes and are isolate- and temperature-dependent; however, they are independent of the phenotypic biofilm-formation profile.
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http://dx.doi.org/10.1139/cjm-2021-0065DOI Listing
September 2021

Three challenging cases of infections by multidrug-resistant Serratia marcescens in patients admitted to intensive care units.

Braz J Microbiol 2021 Sep 8;52(3):1341-1345. Epub 2021 Apr 8.

Laboratório de Pesquisa em Ciências da Saúde, Universidade Federal da Grande Dourados - UFGD, Dourados, Mato Grosso do Sul, Brazil.

The occurrence of multidrug-resistant Serratia marcescens strains represents a serious public health threat. The purpose here is to report three cases of carbapenem-resistant S. marcescens infections with unfavorable clinical outcomes and provide a molecular description of the antibiotic resistance determinants at a genomic level. We performed bacterial identification by VITEK 2 and MALDI-TOF. The minimal inhibitory concentrations of antimicrobials were determined according to the Clinical and Laboratory Standards Institute guidelines, except for tigecycline, for which they were determined using Etest strips. Preliminary screening for the presence of carbapenemases was performed by ertapenem hydrolysis using MALDI-TOF MS. Whole-genome sequencing was provided to identify genes responsible for virulence and antimicrobial resistance. Here we report three challenging cases of S. marcescens that were resistant to the most commonly used antibiotics. Otherwise, we performed a genome description, which includes several genes involved in the resistance and virulence. These cases illustrate serious infection due to multidrug-resistant organisms and the complexity of treatment. Our results highlight the need to evaluate isolates regularly during long-term hospital stay to achieve optimal quality of clinical care and thus improve patient outcomes.
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http://dx.doi.org/10.1007/s42770-021-00477-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8324748PMC
September 2021

The impact of gender on scientific writing: An observational study of grant proposals.

J Clin Epidemiol 2021 08 2;136:37-43. Epub 2021 Feb 2.

Centre for Journalology, Clinical Epidemiology Program, Ottawa Hospital Research Institute, The Ottawa Hospital, General Campus, 501 Smyth Rd, Ottawa, K1H8L6 Canada. Electronic address:

Objectives: This study aimed to determine whether there are differences in the language used in grant applications submitted to a Southern Brazil Research Support Foundation (FAPERGS) according to the gender, career stage, and the number of publications of applicants.

Study Design And Setting: This observational study also evaluated the relationship between gender, career stage, curriculum, and writing characteristics. Summaries of all research proposals in the biomedical field of FAPERGS during the years of 2013 and 2014 were evaluated according to six language patterns (Positive emotions, Negative emotions, Analytic thinking, Clout, Authenticity, and Emotional tone) defined by the LIWC software. Applicant's gender, career stage, and the number of publications were also collected.

Results: Three hundred and forty-four (344) grant proposals met the inclusion criteria and were included in the analysis. No statistical differences were observed in the language pattern used by different gender applicants. In the language used by successful and unsuccessful applicants, we only found a small difference for clout (score 54.5 for not funded and 56.5 for funded grants). However, the principal investigators of successful applications had a significantly higher number of papers published (mean number of papers: 104 versus 58.5).

Conclusions: Gender bias appears to be a more complex problem than just the type of language used; the way society is organized causes several gender biases that may be reflected throughout the women's career.
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http://dx.doi.org/10.1016/j.jclinepi.2021.01.018DOI Listing
August 2021

Protection against leptospirosis conferred by Mycobacterium bovis BCG expressing antigens from Leptospira interrogans.

Vaccine 2020 12 8;38(51):8136-8144. Epub 2020 Nov 8.

Programa de Pós-Graduação em Biotecnologia, Centro de Desenvolvimento Tecnológico, Universidade Federal de Pelotas, Pelotas, RS, Brazil. Electronic address:

Leptospirosis is a zoonotic disease worldwide and caused by the pathogenic spirochetes of the genus Leptospira. Bacterins make up the vaccines used against leptospirosis, but they only succeed in providing short-term and serovar-specific protection. The use of Mycobacterium bovis BCG as a live vaccine vector expressing leptospiral antigens is a promising alternative, particularly due to its adjuvant properties. Four distinct portions P (lipL32), P (ligAni), P (lemA:ligAni) and P (lipL32:lemA) of a recombinant chimera composed of the lipL32, lemA and ligANI genes from Leptospira interrogans were cloned individually according to the BioBricks® strategy in the plasmid pUP500/P. These constructs were individually transformed into a BCG Pasteur strain, and protein expression was detected by Western blot. For vaccination, 5 groups of 10 Golden Syrian hamsters were used, aged 4-6 weeks - group 1, rBCG (LipL32); group 2, rBCG (LigAni); group 3, rBCG (LemA:LigAni); group 4, (LipL32:LemA); group 5, wild-type BCG Pasteur (negative control). Two doses containing 10 CFU of rBCG were administered subcutaneously, the challenge was performed with 5 × LD of Leptospira interrogans serovar Copenhageni L1-130, and the animals were observed for a 30-day period until the endpoint was reached. Humoral immunity was assessed via indirect ELISA, while renal colonisation was assessed by culture and quantitative real-time PCR. All vaccinated groups were protected against lethal challenge and renal colonisation, in comparison with negative control group (P < 0.05). Recombinant vaccines were not effective at inducing significant humoral immunity, which suggests the induction of cellular immunity - a characteristic of M. bovis BCG. In conclusion, all formulations provide 100% significant protection against leptospirosis in hamsters with no renal colonisation. The use of rBCG as a vaccine vector represents a promising alternative for the control of animal leptospirosis, allowing for protection against clinical signs of leptospirosis and renal colonisation.
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http://dx.doi.org/10.1016/j.vaccine.2020.10.086DOI Listing
December 2020

Seroepidemiological investigation of animal leptospirosis and molecular characterization of the first Leptospira strain isolated from Fernando de Noronha archipelago, Brazil.

Transbound Emerg Dis 2021 Jul 27;68(4):2477-2488. Epub 2020 Nov 27.

Universidade Santo Amaro, São Paulo, Brazil.

Leptospirosis has been widely reported in insular environments worldwide, characterizing a major public health threat. Although low-genetic biodiversity is expected in these regions, the introduction of domestic and synanthropic mammals may contribute to the wider diversity of leptospiral strains in insular settings. This study proposes a large-scale seroepidemiological investigation of Leptospira infection in animals from Fernando de Noronha archipelago and describes the characterization of the first leptospiral strain ever isolated from an insular setting in Brazil. A total of 1,265 blood samples from domestic (n = 682), synanthropic (n = 133) and wild (n = 450) animals were collected between 2007 and 2014, totalling 12 species. The presence of anti-Leptospira spp. antibodies was investigated by the microscopic agglutination test (MAT), and kidney samples from 20 synanthropic rodents were collected for the isolation of Leptospira spp. The leptospires recovered were further characterized by serogrouping with polyclonal antibodies, whole-genome sequencing and multilocus sequence typing (MLST). The MAT results revealed the presence of agglutinins in 90 samples (7.1%) and the most frequently found serogroup was Icterohaemorrhagiae (n = 57) in practically all species included. Viable leptospires were recovered from one brown rat, and characterization revealed that the isolate belongs to L. interrogans serogroup Pyrogenes. The results suggest that synanthropic rodents might play an important role in leptospiral infection among wildlife and domestic species in the archipelago.
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http://dx.doi.org/10.1111/tbed.13915DOI Listing
July 2021

EPICOVID19 protocol: repeated serological surveys on SARS-CoV-2 antibodies in Brazil.

Cien Saude Colet 2020 Sep 28;25(9):3573-3578. Epub 2020 Aug 28.

Universidade Federal de Pelotas, Pelotas, RS, Brazil,

The first case of COVID-19 was reported in China in December 2019, and, as the virus has spread worldwide, the World Health Organization declared it a pandemic. Estimates on the number of COVID-19 cases do not reflect it real magnitude as testing is limited. Population based data on the proportion of the population with antibodies is relevant for planning public health policies. We aim to assess the prevalence of SARS-CoV-2 antibodies, presence of signs and symptoms of COVID-19, and adherence to isolation measures. A random sample comprising 133 sentinel cities from all states of the country will be selected. Three serological surveys, three weeks apart, will be conducted. The most populous municipality in each intermediate region of the country, defined by the Brazilian Institute of Geography and Statistics, was chosen as sentinel city. In each city, 25 census tracts will be selected, and 10 households will be systematically sampled in each tract, totaling 33,250 participants. In each household, one inhabitant will be randomly selected to be interviewed and tested for antibodies against SARS-CoV-2, using WONDFO SARS-CoV-2 Antibody Test. By evaluating a representative sample of Brazilian sentinel sites, this study will provide essential information for the design of health policies.
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http://dx.doi.org/10.1590/1413-81232020259.25532020DOI Listing
September 2020

Genetic diversity, antimicrobial resistance, and virulence genes of thermophilic Campylobacter isolated from broiler production chain.

Braz J Microbiol 2020 Dec 9;51(4):2021-2032. Epub 2020 Jun 9.

Departamento de Ciência e Tecnologia Agroindustrial, Universidade Federal de Pelotas, Pelotas, Rio Grande do Sul, Brazil.

The aim of this study was to investigate the prevalence of thermophilic Campylobacter in the broiler production chain of southern Brazil, by evaluating broiler farms and slaughter line samples, and to determine the genetic diversity, antimicrobial resistance, and virulence genes of the isolates. Of the 140 samples investigated in this study, 75 (53.6%) were positive for thermophilic Campylobacter, and all isolates were identified by phenotypic and molecular tests as C. jejuni. The resistance to nalidixic acid was the most common (74%), followed by resistance to enrofloxacin (67.3%) and ciprofloxacin (37.1%). However, there was no resistance to the macrolides tested which are recommended for the treatment of human campylobacteriosis. The PFGE showed that the isolates were grouped in eight macrorestriction patterns (P1 to P8). A representative isolate of each macrorestriction pattern was investigated for the presence of virulence genes and all isolates carried the cadF, ciaB, cdtA, cdtB, cdtC, and flaA genes. The dnaJ gene was detected in 87.5% (7/8) of the isolates. The flhA and racR genes were detected in 75% (6/8), while the pldA gene was present in 62.5% (5/8) and the wlaN gene in 25% (2/8). The presence of C. jejuni in broiler farms and in the slaughterhouse is a hazard to consumer given that this pathogen can be maintained throughout the broiler production chain and contaminates the final product. Moreover, the presence of the major virulence genes in the isolates demonstrates that they have the ability to develop campylobacteriosis in humans.
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http://dx.doi.org/10.1007/s42770-020-00314-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7688733PMC
December 2020

Monoclonal antibodies against LipL32 confer prophylactic protection against lethal leptospirosis challenge in animal model.

Microb Pathog 2020 Apr 11;141:103975. Epub 2020 Jan 11.

Programa de Pós-graduação em Biotecnologia, Centro de Desenvolvimento Tecnológico, Universidade Federal de Pelotas, Pelotas, RS, Brazil; Departamento de Microbiologia e Parasitologia, Instituto de Biologia, Universidade Federal de Pelotas, RS, Brazil.

Leptospirosis is a widespread zoonotic disease caused by pathogenic spirochetes of the genus Leptospira. The commercially available vaccines are bacterins that offer limited protection, short-term effect, and serovar-specific immunity. The development of novel immunization strategies is crucial to control the infection and decrease the chances of new outbreaks. In this study, purified monoclonal antibodies (mAbs) anti-LipL32 (1D9 and mAb3) were evaluated by their capacity to bind and neutralize the pathogen improving host survival. For that, an in vitro growth inhibition assay, and in vivo passive immunization were performed in animal model. Syrian hamsters were passively immunized by three different strategies. Hamsters immunized with mAb3 6 h prior to the lethal challenge showed a significantly higher survival rate of 61.1%, and a significant reduction in tissue damage in the lungs. Cumulatively, our results showed that anti-LipL32 mAbs inhibited the growth of L. interrogans in vitro, and that passive immunization offered significant protection in animal model when administered prior to infection.
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http://dx.doi.org/10.1016/j.micpath.2020.103975DOI Listing
April 2020

Campylobacter jejuni isolated from poultry meat in Brazil: in silico analysis and genomic features of two strains with different phenotypes of antimicrobial susceptibility.

Mol Biol Rep 2020 Jan 20;47(1):671-681. Epub 2019 Nov 20.

Centro de Desenvolvimento Tecnológico, Núcleo de Biotecnologia, Universidade Federal de Pelotas, Pelotas, RS, Brazil.

Campylobacter jejuni is the most common bacterial cause of foodborne diarrheal disease worldwide and is among the antimicrobial resistant "priority pathogens" that pose greatest threat to public health. The genomes of two C. jejuni isolated from poultry meat sold on the retail market in Southern Brazil phenotypically characterized as multidrug-resistant (CJ100) and susceptible (CJ104) were sequenced and analyzed by bioinformatic tools. The isolates CJ100 and CJ104 showed distinct multilocus sequence types (MLST). Comparative genomic analysis revealed a large number of single nucleotide polymorphisms, rearrangements, and inversions in both genomes, in addition to virulence factors, genomic islands, prophage sequences, and insertion sequences. A circular 103-kilobase megaplasmid carrying virulence factors was identified in the genome of CJ100, in addition to resistance mechanisms to aminoglycosides, beta-lactams, macrolides, quinolones, and tetracyclines. The molecular characterization of distinct phenotypes of foodborne C. jejuni and the discovery of a novel virulence megaplasmid provide useful data for pan-genome and large-scale studies to monitor the virulent C. jejuni in poultry meat is warranted.
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http://dx.doi.org/10.1007/s11033-019-05174-yDOI Listing
January 2020

A standardized BioBrick toolbox for the assembly of sequences in mycobacteria.

Tuberculosis (Edinb) 2019 12 19;119:101851. Epub 2019 Jul 19.

Department of Microbial and Cellular Sciences, Faculty of Health and Medical Sciences, University of Surrey, Guildford, Surrey, GU2 7XH, United Kingdom. Electronic address:

For more than 25 years, recombinant Mycobacterium bovis BCG has been genetically engineered for use as a vehicle for antigen expression and immunomodulation, typically through introducing or deleting a gene from BCG genome. However, BCG transformation efficacy is still unpredictable, and cloning and expression of sequences from mycobacteria is difficult to predict due to the lack of standardization. To overcome such limitations, we have employed the BioBrick format to construct a toolbox of several mycobacterial parts, including coding sequences, reporter genes, selective markers, promoters, and other regulatory sequences. Additionally, we have developed and characterized BioBrick-compatible episomal vectors that are able to replicate in M. bovis BCG to enable expression of heterologous antigens. The availability of a BCG Biobrick toolbox will enable any coding sequence to be optimally expressed in BCG. We believe that this mycobacterial toolbox represents a standardized and useful kit to enhance the efficacy and use of recombinant BCG.
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http://dx.doi.org/10.1016/j.tube.2019.07.002DOI Listing
December 2019

Assessment of the immunogenicity of the leptospiral LipL32, LigAni, and LigBrep recombinant proteins in the sheep model.

Comp Immunol Microbiol Infect Dis 2019 Aug 30;65:176-180. Epub 2019 May 30.

Centro de Desenvolvimento Tecnológico, Núcleo de Biotecnologia Universidade Federal de Pelotas, Pelotas, Rio Grande do Sul, Brazil. Electronic address:

Veterinary leptospirosis vaccines are composed of bacterins and present limitations, for example, the need for bacteriological culture and serovar-dependent immunity. Recombinant antigens represent a promising alternative. LigAni, LigBrep, and LipL32 proteins have been shown to promote a protective immune response against the homologous challenge in hamsters. Therefore, the next step is to evaluate the immunological properties of these immunogens in the actual hosts, as ruminants, which has never been performed before. The objective of this study was to evaluate the immunogenicity and potential adverse effects of the recombinant proteins LigAni, LigBrep, and LipL32 in the ovine model. For this, 16 Santa Inês sheep were allocated into three groups: two experimental (Groups A and B) and one control group (Group C). Group A was inoculated with a formulation containing the recombinant proteins in combination with the aluminum hydroxide adjuvant; Group B was inoculated with a formulation containing the recombinant proteins in combination with the Montanide adjuvant; and Group C was inoculated with adjuvants only. The results revealed that formulations containing the recombinant proteins induced total IgG seroconversion and led to a significant increase in antibody titers in the sheep model. Besides, there were no clinical changes or adverse effects. Thus, LigAni, LigBrep, and LipL32 proteins elicited a significant humoral immune response with elevated serum IgG levels, demonstrating that they possess the immunogenic and safety characteristics necessary to sustain their potential use as leptospirosis vaccines in the ruminant model.
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http://dx.doi.org/10.1016/j.cimid.2019.05.012DOI Listing
August 2019

Evaluation of different strategies to promote a protective immune response against leptospirosis using a recombinant LigA and LigB chimera.

Vaccine 2019 03 28;37(13):1844-1852. Epub 2019 Feb 28.

Biotecnologia, Centro de Desenvolvimento Tecnológico, Universidade Federal de Pelotas, Pelotas, Brazil. Electronic address:

Leptospirosis is a zoonosis of worldwide distribution, caused by infection with pathogenic Leptospira species. The vaccines that are currently available are bacterins, with limited human use, that confer short-term, serovar-specific immunity. Lig proteins are considered to be the best vaccine candidates to date. Here, we aimed to construct a recombinant Lig chimera (LC) comprised of LigAni and LigBrep fragments, and to evaluate it as subunit or DNA vaccine using different administration strategies. Vaccines were formulated with 50 µg of recombinant LC associated with different adjuvants or with 100 µg of pTARGET/LC. Four-week-old hamsters received two doses of vaccine with different strategies and were challenged with 5 × DLLeptospira interrogans serovar Copenhageni strain Fiocruz L1-130. The immune response generated by Lig chimera conferred 100% protection to hamsters treated with at least one dose of recombinant LC. Despite the high levels of antibodies that vaccinated animals produced, a sterilizing immunity was not achieved. The lack of a sterilizing immunity could indicate the importance of a mixed humoral and cellular immune response. The present study generated insights that will be useful in the future development of improved subunit vaccines against leptospirosis.
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http://dx.doi.org/10.1016/j.vaccine.2019.02.010DOI Listing
March 2019

Heterologous expression and characterization of a new galactose-binding lectin from Bauhinia forficata with antiproliferative activity.

Int J Biol Macromol 2019 May 2;128:877-884. Epub 2019 Feb 2.

Laboratório de Genômica Estrutural, Programa de Pós-Graduação em Biotecnologia, Centro de Desenvolvimento Tecnológico, Universidade Federal de Pelotas, Pelotas, RS, Brazil.

In this study, we describe the characterization of a new lectin, BfL-II, purified from the seeds of Bauhinia forficata, which is distinct, at sequence-level, from the previously reported lectin from the same specie (BfL). In addition, the gene for this lectin was cloned and expressed in Escherichia coli and its antiproliferative activity was evaluated against human breast and colorectal cancer cells (MCF-7 and HT-29, respectively). The treatment with 100 μg/μL of either native or recombinant BfL (nBfL or rBfL) significantly reduced the proliferation of both cancer cell lines (p < 0.01). The inhibition of HT-29 cell proliferation was as high as 82.5% and 93.6% with 100 μg/μL of rBfL-I and nBfL after 24 h, respectively. Therefore, BfL-II presents a promising antiproliferative activity, which may be applied in the development of new anticancer treatments.
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http://dx.doi.org/10.1016/j.ijbiomac.2019.01.090DOI Listing
May 2019

Recombinant BCG strains expressing chimeric proteins derived from Leptospira protect hamsters against leptospirosis.

Vaccine 2019 02 7;37(6):776-782. Epub 2019 Jan 7.

Programa de Pós-Graduação em Biotecnologia, Centro de Desenvolvimento Tecnológico, Universidade Federal de Pelotas, Pelotas, RS, Brazil. Electronic address:

Leptospirosis is a zoonosis that is responsible for one million human cases per year. Fusing multiple immunogenic antigens represents a promising approach to delivering an effective vaccine against leptospirosis. Mycobacterium bovis bacillus Calmette-Guérin (BCG) is a potential vaccine vector due to its adjuvant properties and safety. Two chimeric genes based on genic sequences of ligANI, ligBrep, lipL32, and lemA, were individually cloned into five BioBrick vectors with different promoters (pAN, Hsp60, 18 kDa, Ag85B and Ag85B plus signal sequence) for antigen expression in BCG. Groups of ten hamsters were vaccinated with recombinant BCG (rBCG) strains in two doses of 10 CFU and challenged with 5 × LD of L. interrogans serovar Copenhageni. All rBCG vaccines expressing chimera 1, based on antigens LipL32, LigANI, and LemA, under the control of any promoter, protected 80-100% of the hamsters from challenge (P < 0.05) and four of them also protected from renal carrier status; for chimera 2, based on LigANI and LigBrep antigens, the only vaccine that afforded survival rates statistically different from the control was the vaccine that incorporated the pAN promoter (60% of survival). A single vaccine dose was sufficient to induce significant IgG levels by all vaccine compositions evaluated; however, humoral response was not related to protection. These findings suggest that the combination of potential vaccine candidates in chimeric antigens and the use of BCG as a live vector are promising strategies by which it is possible to obtain an effective and sterilizing vaccine against leptospirosis.
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http://dx.doi.org/10.1016/j.vaccine.2018.12.050DOI Listing
February 2019

Presence of genes associated with adhesion, invasion, and toxin production in Campylobacter jejuni isolates and effect of temperature on their expression.

Can J Microbiol 2019 Apr 11;65(4):253-260. Epub 2018 Dec 11.

a Departamento de Ciência e Tecnologia Agroindustrial, Faculdade de Agronomia Eliseu Maciel, Universidade Federal de Pelotas, Pelotas, RS, Brazil.

The aims of this study were to evaluate the presence of genes associated with adhesion (cadF), invasion (ciaB), and cytotoxin production (cdtA, cdtB, and cdtC) among Campylobacter jejuni isolates from a poultry slaughterhouse and to investigate the effect of different temperatures on the expression of these virulence-associated genes. A total of 88 C. jejuni isolates from cecum, liver, chicken carcasses, chilled water, and scalding water were submitted to PCR assay for detection of virulence genes. Representative isolates were selected for gene expression evaluation at 37 and 42 °C, according to their virulence gene profile and genotypic typing. All C. jejuni isolates carried the five virulence-associated genes, which play an important role in the infectious process. Differential gene expression by RT-qPCR was observed among C. jejuni isolates at 37 and 42 °C. The expression levels at 37 °C showed upregulation of the ciaB, cdtA, cdtB, and cdtC genes in five isolates, with the exception of ciaB for isolate 4. At 42 °C, upregulation was observed for ciaB and cdtC, cdtA and cdtB, and cadF in four, three, and two isolates, respectively. The C. jejuni isolates expressed the virulence genes evaluated, and the expression is gene- and isolate-dependent and varied according the temperature.
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http://dx.doi.org/10.1139/cjm-2018-0539DOI Listing
April 2019

Complete genome sequence and in silico analysis of L. interrogans Canicola strain DU114: A virulent Brazilian isolate phylogenetically related to serovar Linhai.

Genomics 2019 12 17;111(6):1651-1656. Epub 2018 Nov 17.

Núcleo de Biotecnologia, Centro de Desenvolvimento Tecnológico (CDTec), Universidade Federal Pelotas, Capão do Leão, Rio Grande do Sul, Brazil.

Canine leptospirosis is often caused by Leptospira interrogans serovar Canicola. Infected dogs may become asymptomatic carriers of the pathogen, which leads to many public health concerns. In this work, we present the complete genome sequencing and in silico analysis from a virulent Brazilian strain of L. interrogans serovar Canicola, previously isolated from a stray dog in Sao Paulo City. Comparative genomic analysis with a reference genome allowed identification of 1031 INDELs and several arrangement variations. Out of 35,361 SNPs identified, 6780 were missense mutations and 16,114 were synonymous mutations. The Gene Ontology terms more affected by mutations were described. Interestingly, phylogenetic analyses indicated a genetic relatedness of the isolate with serovar Linhai strain 56,609. In addition, we found several virulence-related genes and main outer membrane proteins associated with pathogenesis. This genomic information about canine isolates may help to elucidate the molecular diversity and mechanisms of Leptospira spp. pathogenicity.
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http://dx.doi.org/10.1016/j.ygeno.2018.11.015DOI Listing
December 2019

LemA and Erp Y-like recombinant proteins from Leptospira interrogans protect hamsters from challenge using AddaVax™ as adjuvant.

Vaccine 2018 05 3;36(19):2574-2580. Epub 2018 Apr 3.

Programa de Pós-Graduação em Biotecnologia, Centro de Desenvolvimento Tecnológico, Universidade Federal de Pelotas, Pelotas, RS, Brazil; Departamento de Microbiologia e Parasitologia, Instituto de Biologia, Universidade Federal de Pelotas, Pelotas, RS, Brazil. Electronic address:

Background: Recombinant subunit vaccines have been extensively evaluated as promising alternatives against leptospirosis. Here, we evaluated two proteins in formulations containing the adjuvant AddaVax™ as vaccine candidates for prevention and control of leptospirosis.

Methods: Recombinant proteins rErp Y-like and rLemA were characterized by ELISA to assess their ability to bind extracellular matrix (ECM) components and fibrinogen. Groups of eight hamsters were immunized intramuscularly with rErp Y-like or rLemA mixed with a squalene-based adjuvant (AddaVax), and then vaccine efficacy was determined in terms of protection against a lethal challenge. The humoral immune response was determined by ELISA, and the evidence of sub-lethal infection was evaluated by histopathology and kidney culture.

Results: rLemA protein binds laminin, fibrinogen, and collagen type IV, while rErp Y-like interacts with fibrinogen. Significant protection was achieved for rLemA and rErp Y-like vaccines, which showed 87.5% and 62.5% survivals, respectively. On day 28, the humoral immune response was significantly greater in the vaccine groups as compared to that in the control group, and the response was predominantly based on IgG2/3. The surviving animals showed negative results in culture isolation but presented with tissue lesions in the lungs and kidneys.

Conclusion: Cumulatively, our findings suggest that LemA and Erp Y-like proteins act as adhesins and are able to protect against mortality, but not against tissue lesions. Moreover, AddaVax is a novel adjuvant with potential for improving the immunogenicity of leptospiral vaccines.
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http://dx.doi.org/10.1016/j.vaccine.2018.03.078DOI Listing
May 2018

Genome of Leptospira borgpetersenii strain 4E, a highly virulent isolate obtained from Mus musculus in southern Brazil.

Mem Inst Oswaldo Cruz 2018 Feb;113(2):137-141

Núcleo de Biotecnologia, Universidade Federal de Pelotas, Capão do Leão, RS, Brasil.

A previous study by our group reported the isolation and characterisation of Leptospira borgpetersenii serogroup Ballum strain 4E. This strain is of particular interest because it is highly virulent in the hamster model. In this study, we performed whole-genome shotgun genome sequencing of the strain using the SOLiD sequencing platform. By assembling and analysing the new genome, we were able to identify novel features that have been previously overlooked in genome annotations of other strains belonging to the same species.
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http://dx.doi.org/10.1590/0074-02760170111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5722270PMC
February 2018

Whole-genome sequencing of Leptospira interrogans from southern Brazil: genetic features of a highly virulent strain.

Mem Inst Oswaldo Cruz 2018 Feb;113(2):80-86

Centro de Desenvolvimento Tecnológico, Universidade Federal de Pelotas, Pelotas, RS, Brasil.

BACKGROUND Leptospirosis is the most widespread zoonotic disease. It is caused by infection with pathogenic Leptospira species, of which over 300 serovars have been described. The accurate identification of the causative Leptospira spp. is required to ascertain the pathogenic status of the local isolates. OBJECTIVES This study aimed to obtain the complete genome sequence of a virulent Leptospira interrogans strain isolated from southern Brazil and to describe its genetic features. METHODS The whole genome was sequenced by next-generation sequencing (Ion Torrent). The genome was assembled, scaffolded, annotated, and manually reviewed. Mutations were identified based on a variant calling analysis using the genome of L. interrogans strain Fiocruz L1-130 as a reference. FINDINGS The entire genome had an average GC content of 35%. The variant calling analysis identified 119 single nucleotide polymorphisms (SNPs), from which 30 led to a missense mutation. The structural analyses identified potential evidence of genomic inversions, translocations, and deletions in both the chromosomes. MAIN CONCLUSIONS The genome properties provide comprehensive information about the local isolates of Leptospira spp., and thereby, could facilitate the identification of new targets for the development of diagnostic kits and vaccines.
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http://dx.doi.org/10.1590/0074-02760170130DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5722262PMC
February 2018

Complete genome sequence of native strains isolated from intestinal tract of the crab sp.

Data Brief 2018 Feb 20;16:381-385. Epub 2017 Nov 20.

Federal University of Rio Grande, Institute of Biological Sciences, Rio Grande, RS, Brazil.

is a gram positive bacterium with sporulation capacity. Here, we report the complete genome sequence of two native strains (#25 and #29) isolated from intestinal tract of the crab sp. from Pacoti River in the State of Ceará, Brazil. The findings of this study might increase the molecular information for strains. The data can be used in comparative analyses, origin and distribution, as well support for genetic engineering.
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http://dx.doi.org/10.1016/j.dib.2017.11.049DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5723261PMC
February 2018

Parenteral adjuvant potential of recombinant B subunit of Escherichia coli heat-labile enterotoxin.

Mem Inst Oswaldo Cruz 2017 Dec;112(12):812-816

Universidade Federal de Pelotas, Centro de Desenvolvimento Tecnológico, Biotecnologia, Pelotas, RS, Brasil.

Background: The B subunit of Escherichia coli heat-labile enterotoxin (LTB) is a potent mucosal immune adjuvant. However, there is little information about LTB's potential as a parenteral adjuvant.

Objectives: We aimed at evaluating and better understanding rLTB's potential as a parenteral adjuvant using the fused R1 repeat of Mycoplasma hyopneumoniae P97 adhesin as an antigen to characterise the humoral immune response induced by this construct and comparing it to that generated when aluminium hydroxide is used as adjuvant instead.

Methods: BALB/c mice were immunised intraperitoneally with either rLTBR1 or recombinant R1 adsorbed onto aluminium hydroxide. The levels of systemic anti-rR1 antibodies (total Ig, IgG1, IgG2a, and IgA) were assessed by enzyme-linked immunosorbent assay (ELISA). The ratio of IgG1 and IgG2a was used to characterise a Th1, Th2, or mixed Th1/Th2 immune response.

Findings: Western blot confirmed rR1, either alone or fused to LTB, remained antigenic; anti-cholera toxin ELISA confirmed that LTB retained its activity when expressed in a heterologous system. Mice immunised with the rLTBR1 fusion protein produced approximately twice as much anti-rR1 immunoglobulins as mice vaccinated with rR1 adsorbed onto aluminium hydroxide. Animals vaccinated with either rLTBR1 or rR1 adsorbed onto aluminium hydroxide presented a mixed Th1/Th2 immune response. We speculate this might be a result of rR1 immune modulation rather than adjuvant modulation. Mice immunised with rLTBR1 produced approximately 1.5-fold more serum IgA than animals immunised with rR1 and aluminium hydroxide.

Main Conclusions: The results suggest that rLTB is a more powerful parenteral adjuvant than aluminium hydroxide when administered intraperitoneally as it induced higher antibody titres. Therefore, we recommend that rLTB be considered an alternative adjuvant, even if different administration routes are employed.
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http://dx.doi.org/10.1590/0074-02760170133DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5719549PMC
December 2017

DNA vaccines against leptospirosis: A literature review.

Vaccine 2017 10 4;35(42):5559-5567. Epub 2017 Sep 4.

Programa de Pós-Graduação em Biotecnologia, Centro de Desenvolvimento Tecnológico, Universidade Federal de Pelotas, Pelotas, RS, Brazil; Departamento de Microbiologia e Parasitologia, Instituto de Biologia, Universidade Federal de Pelotas, RS, Brazil. Electronic address:

Leptospirosis is an infectious disease caused by pathogenic Leptospira species. The vaccines that are currently available for leptospirosis are composed of whole-cell preparations and suffer from limitations such as low efficacy, multiple side-effects, poor immunological memory and lack of cross-protection against different serovars of Leptospira spp. In light of the global prevalence of this disease, the development of a more effective vaccine against leptospirosis is of paramount importance. Genetic immunization is a promising alternative to conventional vaccine development. In the last 25years, several novel strategies have been developed for increasing the efficacy of DNA vaccines. Examples of such strategies include the introduction of novel plasmid vectors, adjuvants, alternate delivery routes, and prime-boost regimens. Herein we discuss the latest and most promising advances that have been made in developing DNA vaccines against leptospirosis. We also deliberate over the future directions that must be undertaken in order to improve results in this field.
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http://dx.doi.org/10.1016/j.vaccine.2017.08.067DOI Listing
October 2017

Recombinant BCG vaccines: molecular features and their influence in the expression of foreign genes.

Appl Microbiol Biotechnol 2017 Sep 4;101(18):6865-6877. Epub 2017 Aug 4.

Programa de Pós-Graduação em Biotecnologia, Centro de Desenvolvimento Tecnológico, Universidade Federal de Pelotas, Pelotas, RS, Brazil.

Recombinant Mycobacterium bovis BCG vaccines (rBCG) were first developed in the 1990s as a means of expressing antigens from multiple pathogens. This review examines the key structural factors of recombinant M. bovis that influence the expression of the heterologous antigens and the generation of genetic and functional stability in rBCG, which are crucial for inducing strong and lasting immune responses. The fundamental aim of this paper is to provide an overview of factors that affect the expression of recombinant proteins in BCG and the generation of the immune response against the target antigens, including mycobacterial promoters, location of foreign antigens, and stability of the vectors. The reporter systems that have been employed for evaluation of these molecular features in BCG are also reviewed here.
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http://dx.doi.org/10.1007/s00253-017-8439-6DOI Listing
September 2017

Human and animal leptospirosis in Southern Brazil: A five-year retrospective study.

Travel Med Infect Dis 2017 Jul - Aug;18:46-52. Epub 2017 Jul 22.

Centro de Desenvolvimento Tecnológico, Universidade Federal de Pelotas, Pelotas, Brazil.

Background: Leptospirosis is an emerging zoonosis attributed to multiple reservoirs. Climatic conditions influence the transmission of pathogenic leptospires, which require warm and humid conditions for survival. The influence of seasonality in human and animal leptospirosis in the subtropical region of Brazil remains poorly understood.

Methods: We performed a retrospective study to describe the patterns of human and animal exposure to leptospirosis and their association with precipitation events in Southern Brazil. Rainfall data were obtained from satellite images. Serum samples were tested using the microscopic agglutination test (MAT); samples with titer ≥ 100 were defined as seroreactive. Linear regression and Pearson's correlation were performed to assess whether there is a relationship between these variables.

Results: We found that precipitation events were not significantly associated with the exposure to leptospirosis in humans or animal species, except for dogs. The interspecies analysis revealed an association between canine and human exposure to leptospirosis. Leptospira kirschneri serovar Butembo (serogroup Autumnalis) presented the highest seroreactivity in humans.

Conclusion: This study provides valuable insights in human and animal leptospirosis in Southern Brazil. These insights will be essential to design intervention measures directed to reduce disease dissemination.
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http://dx.doi.org/10.1016/j.tmaid.2017.07.010DOI Listing
October 2017

Flow cytometric sex sorting affects CD4 membrane distribution and binding of exogenous DNA on bovine sperm cells.

Zygote 2017 Aug 13;25(4):519-528. Epub 2017 Jul 13.

Laboratório de Genômica Estrutural,Centro de Desenvolvimento Tecnológico,Campus Universitário Capão do Leão s/n°,CEP 96160-000,Capão do Leão,RS,Brasil.

Bovine sex-sorted sperm have been commercialized and successfully used for the production of transgenic embryos of the desired sex through the sperm-mediated gene transfer (SMGT) technique. However, sex-sorted sperm show a reduced ability to internalize exogenous DNA. The interaction between sperm cells and the exogenous DNA has been reported in other species to be a CD4-like molecule-dependent process. The flow cytometry-based sex-sorting process subjects the spermatozoa to different stresses causing changes in the cell membrane. The aim of this study was to elucidate the relationship between the redistribution of CD4-like molecules and binding of exogenous DNA to sex-sorted bovine sperm. In the first set of experiments, the membrane phospholipid disorder and the redistribution of the CD4 were evaluated. The second set of experiments was conducted to investigate the effect of CD4 redistribution on the mechanism of binding of exogenous DNA to sperm cells and the efficiency of lipofection in sex-sorted bovine sperm. Sex-sorting procedure increased the membrane phospholipid disorder and induced the redistribution of CD4-like molecules. Both X-sorted and Y-sorted sperm had decreased DNA bound to membrane in comparison with the unsorted sperm; however, the binding of the exogenous DNA was significantly increased with the addition of liposomes. Moreover, we demonstrated that the number of sperm-bound exogenous DNA was decreased when these cells were preincubated with anti-bovine CD4 monoclonal antibody, supporting our hypothesis that CD4-like molecules indeed play a crucial role in the process of exogenous DNA/bovine sperm cells interaction.
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http://dx.doi.org/10.1017/S0967199417000375DOI Listing
August 2017
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