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1.  Comparative genomic analysis of Brazilian Leptospira kirschneri serogroup Pomona serovar Mozdok 
Memórias do Instituto Oswaldo Cruz  2016;111(8):539-541.
Leptospira kirschneri is one of the pathogenic species of the Leptospira genus. Human and animal infection from L. kirschneri gained further attention over the last few decades. Here we present the isolation and characterisation of Brazilian L. kirschneri serogroup Pomona serovar Mozdok strain M36/05 and the comparative genomic analysis with Brazilian human strain 61H. The M36/05 strain caused pulmonary hemorrhagic lesions in the hamster model, showing high virulence. The studied genomes presented high symmetrical identity and the in silico multilocus sequence typing analysis resulted in a new allelic profile (ST101) that so far has only been associated with the Brazilian L. kirschneri serogroup Pomona serovar Mozdok strains. Considering the environmental conditions and high genomic similarity observed between strains, we suggest the existence of a Brazilian L. kirschneri serogroup Pomona serovar Mozdok lineage that could represent a high public health risk; further studies are necessary to confirm the lineage significance and distribution.
doi:10.1590/0074-02760160174
PMCID: PMC4981118  PMID: 27581124
L. kirschneri; serovar Mozdok; genomics
2.  A multilocus variable number tandem repeat analysis assay provides high discrimination for genotyping Leptospira santarosai strains 
Journal of Medical Microbiology  2015;64(Pt 5):507-512.
Considering the prevalence of Leptospira santarosai infections in the Americas and the scarce information about the species, we aimed to apply a multilocus variable number tandem repeat (VNTR) analysis (MLVA) for the molecular typing of L. santarosai isolates from various sources. Amplification of three VNTR loci selected from L. santarosai genome sequences resulted in a wide range of sizes for the amplified products amongst the 21 L. santarosai strains analysed. This suggested a variation in tandem repeat copy numbers in the VNTR loci. secY sequencing also showed a high nucleotide diversity, confirming the MLVA data. In conclusion, this novel MLVA provided a high level of discrimination between L. santarosai isolates, and this new typing tool could be used to investigate leptospirosis in regions where L. santarosai predominates.
doi:10.1099/jmm.0.000045
PMCID: PMC4857445  PMID: 25721051
3.  Profiling of Leptospira interrogans, L. santarosai, L. meyeri and L. borgpetersenii by SE-AFLP, PFGE and susceptibility testing—a continuous attempt at species and serovar differentiation 
Leptospirosis is a widespread systemic zoonosis, considered as reemerging in certain developing countries. Although the cross agglutinin absorption test is still considered the standard method for Leptospira identification, it presents several disadvantages. The aim of this study was to characterize Leptospira spp. isolated from various hosts by genotyping and broth microdilution susceptibility testing in an attempt to differentiate Leptospira species, serogroups and serovars. Forty-seven isolates were studied. They were previously serotyped, and species confirmation was performed by 16S rRNA sequencing. Single-enzyme amplified fragment length polymorphism (SE-AFLP) and pulsed-field gel electrophoresis (PFGE) analysis enabled the distinction of L. interrogans from L. santarosai, L. meyeri and L. borgpetersenii in two main clusters. Among L. interrogans, it was possible to differentiate into two new clusters the serogroup Icterohaemorrhagiae from the serogroups Canicola and Pomona. L. santarosai isolates presented higher genetic variation than the other species in both techniques. Interestingly, the minimum inhibitory concentration (MIC) cluster analysis also provided Leptospira serogroup differentiation. Further studies are necessary regarding serovar Bananal isolates, as they presented the highest MIC values for most of the antimicrobials tested. All studied techniques successfully distinguished Leptospira species and serogroups. Despite being library-dependent methods, these approaches are less labor intensive and more economically viable, particularly SE-AFLP, and can be implemented in most reference laboratories worldwide to enable faster Leptospira typing.
doi:10.1038/emi.2016.16
PMCID: PMC4820670  PMID: 26956446
leptospira; MIC; PFGE; SE-AFLP; sequencing; serotyping
4.  Draft Genome Sequence of Brazilian Leptospira noguchii Serogroup Panama Strain U73, Isolated from Cattle 
Genome Announcements  2015;3(5):e01179-15.
Leptospira noguchii is a current zoonotic pathogen in Brazil. Here, we report the draft genome sequence of the Brazilian L. noguchii serogroup Panama strain U73, isolated from asymptomatic cattle urine.
doi:10.1128/genomeA.01179-15
PMCID: PMC4611683  PMID: 26472831
5.  Draft Genome Sequences of Leptospira santarosai Strains U160, U164, and U233, Isolated from Asymptomatic Cattle 
Genome Announcements  2015;3(4):e00910-15.
In the present work, we announce the draft genomes for three new strains (U160, U164, and U233) of Leptospira santarosai, isolated from urine samples from asymptomatic cattle in Rio de Janeiro, Brazil.
doi:10.1128/genomeA.00910-15
PMCID: PMC4536688  PMID: 26272577
6.  Molecular characterization of the first leptospires isolated from goats in Brazil 
Brazilian Journal of Microbiology  2015;45(4):1527-1530.
Two Leptospira sp. isolates were obtained by the first time from goats in Brazil and characterized by sequencing rrs, rpoB and secY genes, PFGE and typing with monoclonal antibodies. Both isolates are identical and belong to Leptospira santarosai. Analysis of the rrs and the rpoB genes sequences revealed 100% identity between the goat isolates and the Bananal reference strain. When secY sequences of the two isolates were compared to each other, it was observed that they had identical sequences. However, when compared to that of the Bananal reference strain, there were 15 mismatches along the 549 bp secY sequence. In conclusion, molecular methods are increasingly useful for the characterization of leptospires and allowed to identify those isolates of caprine origin as closely related but not identical to serovar Bananal, and constitute a new type named Carioca.
PMCID: PMC4323332  PMID: 25763063
Leptospira; serogrouping; sequencing; PFGE; rrs
8.  Identification of immunodominant antigens in canine leptospirosis by Multi-Antigen Print ImmunoAssay (MAPIA) 
BMC Veterinary Research  2014;10:288.
Background
The microscopic agglutination test (MAT), the standard method for serological diagnosis of leptospirosis, may present limitations regarding its sensitivity. Current studies suggest that Leptospira immunoglobulin-like (Lig) proteins and LipL32 are of particular interest as serodiagnostic markers since they are present only in pathogenic species of the Leptospira genus. The purpose of this study was to identify leptospiral immunodominant proteins that are recognized by canine sera from diseased dogs.
Results
A total of 109 dogs were studied, including seroreactive dogs (MAT ≥800) and dogs with no seroreactivity detectable by MAT. Eight recombinant fragments (31–70 kDa) of pathogenic Leptospira were tested for their use as diagnostic markers for canine leptospirosis using the Multi-antigen Print Immunoassay (MAPIA) platform: LigB [582-947aa] from L. interrogans serovar Pomona, L. interrogans serovar Copenhageni and L. kirschneri serovar Gryppotyphosa, LigB [131-649aa] from L. interrogans serovar Copenhageni, L. interrogans serovar Canicola and L. kirschneri serovar Gryppotyphosa, LigA [625-1224aa] L. interrogans serovar Copenhageni and LipL32 from L. interrogans serovar Copenhageni. The data were analyzed and ROC curves were generated. Altogether, LigB [131-649aa] L. interrogans Canicola, LigB [131-649aa] L. kirschneri Gryppotyphosa and LipL32 L. interrogans Copenhageni showed best accuracy (AUC = 0.826 to 0.869), with 70% specificity and sensitivity ranging from 89% to 95%.
Conclusions
These results reinforce their potential as diagnostic candidates for the development of new methods for the serological diagnosis of canine leptospirosis.
doi:10.1186/s12917-014-0288-2
PMCID: PMC4269070  PMID: 25466383
Leptospirosis; Dogs; MAPIA; LipL32; Lig proteins
9.  Letter to the Editor 
doi:10.1111/jvim.12454
PMCID: PMC4895617  PMID: 25410950
10.  Comparison of cefoxitin disk diffusion test and mecA gene PCR results for methicillin resistance detection in Staphylococcus intermedius group isolates from canine origin in Brazil 
Brazilian Journal of Microbiology  2014;45(1):235-237.
The study evaluated cefoxitin disk diffusion tests breakpoints and their correlation to mecA gene PCR results for detecting Methicillin-resistant Staphylococcus intermedius Group (MRSP) isolates from dogs in Brazil. Agreement using proposed breakpoint (resistant ≤ 30 mm) was encouraging. The current study reinforces that an epidemiological breakpoint can be established to predict presence of MRSP.
PMCID: PMC4059303  PMID: 24948938
methicillin-resistant Staphylococcus intermedius group; cefoxitin; disk diffusion test
11.  The panorama of animal leptospirosis in Rio de Janeiro, Brazil, regarding the seroepidemiology of the infection in tropical regions 
Background
Leptospirosis is an important disease caused by various serovars of Leptospira sp. It can affect humans as well as domestic and wild animals; therefore, it has importance for public health, animal production, and wild species. The aim of this paper is to discuss the epidemiology of animal leptospirosis in Rio de Janeiro, Brazil, as a possible model for other tropical regions. In several studies conducted in the last 20 years, a total of 47 rats, 120 dogs, 875 cows, 695 horses, 1,343 goats, 308 sheep and 351 pigs from all regions of the state, in addition to 107 wild mammals and 73 golden-lion tamarins were tested (MAT) for anti-Leptospira antibodies.
Results
Seroreactivity was frequent in all studied species, confirming that the infection is endemic in Rio de Janeiro. Serogroups Icterohaemorrhagiae and Sejroe were the most prevalent in urban and rural scenarios, respectively. This paper reviews the current knowledge on animal leptospirosis in Rio de Janeiro and describes important differences between urban versus rural cycles of the infection in various species.
Conclusion
Identification of the prevailing serogroups and their reservoirs is essential for understanding agent-host-environment interactions under tropical conditions.
doi:10.1186/1746-6148-9-237
PMCID: PMC4220826  PMID: 24289165
Animal leptospirosis; Tropical scenario; Infection; Brazil
12.  Subunit Approach to Evaluation of the Immune Protective Potential of Leptospiral Antigens ▿ 
Clinical and Vaccine Immunology : CVI  2011;18(12):2026-2030.
Leptospirosis is the most widespread zoonosis in the world. Current vaccines are based on whole-cell preparations that cause severe side effects and do not induce satisfactory immunity. In light of the leptospiral genome sequences recently made available, several studies aimed at identification of protective recombinant immunogens have been performed; however, few such immunogens have been identified. The aim of this study was to evaluate 27 recombinant antigens to determine their potential to induce an immune response protective against leptospirosis in the hamster model. Experiments were conducted with groups of female hamsters immunized with individual antigen preparations. Hamsters were then challenged with a lethal dose of Leptospira interrogans. Thirteen antigens induced protective immune responses; however, only recombinant proteins LIC10325 and LIC13059 induced significant protection against mortality. These results have important implications for the development of an efficacious recombinant subunit vaccine against leptospirosis.
doi:10.1128/CVI.05297-11
PMCID: PMC3232701  PMID: 22030369
13.  In vitro antimicrobial resistance of staphylococci isolated from canine urinary tract infection 
The Canadian Veterinary Journal  2010;51(7):738-742.
This study determined the diversity of species and antimicrobial resistance of staphylococci isolated from dogs with a presumptive diagnosis of urinary tract infection (UTI). Urine samples from 348 dogs with clinical signs of UTI, according to clinical examination and urinalysis, were processed for isolation of Staphylococcus. Colonies in pure culture were identified by biochemical reactions and tested for susceptibility to 15 antimicrobials. Seventy isolates of staphylococci were obtained (20.1%). Staphylococcus pseudintermedius was the most frequent species (32.8%), followed by S. epidermidis (18.6%), S. simulans (15.7%), S. schleiferi schleiferi (11.4%), S. aureus (11.4%), S. schleiferi coagulans (7.2%) and S. saprophyticus (2.9%). All the isolates were resistant to at least 1 drug and 77.1% were multiresistant. The study reports the alarming antimicrobial resistance of members of the Staphylococcus genus isolated from canine UTI and highlights the importance of coagulase-negative staphylococci in its etiology.
PMCID: PMC2885114  PMID: 20885826
14.  Potential Application Of New Diagnostic Methods For Controlling Bovine Tuberculosis In Brazil 
Brazilian Journal of Microbiology  2010;41(3):531-541.
Bovine tuberculosis, a chronic infection in cattle caused by Mycobacterium bovis, remains an economic and public health problem for several countries. Due to its economic impact on international trade, contagious nature, and implications for human health, global programs to eradicate the disease were implemented worldwide. Those programs are based on slaughtering PPD-reactive animals. Despite the National Programs in Brazil, complete eradication has not been achieved, and the disease remains, albeit at a lower prevalence.
The central purpose of this review is to address diagnostic tests for tuberculosis. Considering the course of the infection in cattle, at least two tests, ideally complementary to one another, may be necessary for an adequate diagnosis: the first based on the cellular response, and the second capable of identifying anergic animals by detection of specific anti-M.bovis antibodies.
doi:10.1590/S1517-83822010005000002
PMCID: PMC3768653  PMID: 24031527
Bovine Tuberculosis; Mycobacterium bovis; Diagnosis
15.  Detection of Mycobacterium bovis DNA in nasal swabs from tuberculous cattle by a multiplex PCR 
Brazilian Journal of Microbiology  2010;41(2):386-390.
Detection of tuberculosis in cattle relies on the intradermal tuberculin test (ITT), but a definitive diagnosis requires identification of the pathogen after the animal is slaughtered. DNA in nasal swabs from 50 cows was analyzed by m-PCR, targeting for the RvD1-Rv2031c and IS6110 sequences. M. bovis was identified in two of 34 tuberculous cows (5.9%). The use of mPCR of nasal swabs as an in vivo diagnostic tool for bovine tuberculosis is suggested.
doi:10.1590/S1517-838220100002000020
PMCID: PMC3768681  PMID: 24031509
Mycobacterium bovis; multiplex-PCR; nasal swab; bovine tuberculosis
16.  Identification of Mycobacterium bovis Isolates by a multiplex PCR 
Brazilian Journal of Microbiology  2009;40(2):231-233.
Isolates from suggestive bovine tuberculosis lesions were tested by a multiplex polymerase chain reaction (m-PCR) targeting for RvD1Rv2031c and IS6110 sequences, specific for M. bovis and Mycobacterium tuberculosis complex respectively. The m-PCR successfully identified as M. bovis 88.24% of the isolates.
doi:10.1590/S1517-83822009000200004
PMCID: PMC3769727  PMID: 24031349
Mycobacterium bovis; multiplex-PCR; bovine tuberculosis
17.  An alternative for the preadsorption step in the paratuberculosis serodiagnosis: Mycobacterium fortuitum 
Brazilian Journal of Microbiology  2008;39(3):511-513.
ELISAs for paratuberculosis employ a preadsorption step with Mycobacterium phlei to diminish unspecific reactions As M. fortuitum is one of the most frequent environmental mycobacteria, the purpose of this pilot study was to evaluate its use as an alternative for the preadsorption in ELISAs for paratuberculosis. Results suggest that M. fortuitum can be an alternative instead of or associated to M. phlei with comparable results (κ > 0.8) to conventional ELISAs using M. phlei as a preadsorption antigen.
doi:10.1590/S1517-838220080003000019
PMCID: PMC3768432  PMID: 24031256
Mycobacteria; ELISA; M. fortuitum; M. phlei

Results 1-17 (17)