Acompanhamento sorológico e molecular do sorovar hardjo no diagnóstico de leptospirose em um rebanho bovino leiteiro

The cattle are considered hosts of the Hardjo serovar, causing economic damages due to the reproductive failures like abortions and infertility. The serovar Hardjo usually remains in the reproductive tract and also in the renal tubules where it is eliminated intermittently in the urine for months. Placental remnants, the aborted fetus and contaminated urine promote the permanence of this bacterium within the herd for years. Thus, the objective of this study was to monitor for prolonged period, cows naturally infected with Leptospira ssp. through microbiological culture, serological examination and DNA detection of the pathogen in the urine. The dairy herd was composed of 50 breeding cows with a history of abortion and infertility, without leptospirosis vaccine and located in the northern region of Paraná. Blood and urine samples were collected and laboratorial diagnosis were performed five times at intervals of four months. Blood samples were collected from the all 50 animals and the serum was submitted to the microscopic agglutination test (MAT) for the detection of anti-leptospira antibodies. Of the total cows, 20 showed antibody titres ? 1: 100 in MAT and urine samples were collected from only those animals with higher titers to perform nested-PCR (n-PCR) and bacterial isolation per culture. In addition, two urine samples from five animals with antibody titers < 1: 100 were collected in MAT for n-PCR. Serovar Hardjo was considered the most frequent during the serological monitoring of the animals evaluated. The n-PCR technique was able to detect leptospiral DNA in the urine of animals with MAT ? 1: 100 antibody titers and urine from animals whose titers were < 1: 100. Sequencing of the leptospiral amplicons shared 100% nucleotide sequence identity with the Leptospira interrogans species. Positive n-PCR results from animals with titers of < 1: 100 suggest that the cut-off of MAT is could be not sufficient to detect renal carriers, so it is also important to use n-PCR as an additional diagnostic tool for identify infected animals with Hardjo serovar and whose serology was negative.Os bovinos são considerados hospedeiros do sorovar Hardjo, causando prejuízos econômicos devido aos problemas reprodutivos como abortos e infertilidade. O sorovar Hardjo costuma permanecer no trato reprodutivo e também nos túbulos renais onde é eliminado de forma intermitente na urina por meses. Restos placentários, o feto abortado e a urina contaminada favorecem a permanência dessa bactéria dentro do rebanho por anos. Assim, o objetivo deste estudo foi monitorar por período prolongado, vacas naturalmente infectadas por Leptospira ssp. através de cultura microbiológica, exame sorológico e detecção de DNA do patógeno na urina. O rebanho leiteiro estudado era constituído por 50 vacas reprodutoras com histórico de abortos e infertilidade, sem uso de vacina contra a leptospirose e localizada na região Norte do Paraná. As amostras de sangue e urina foram coletadas e submetidas a análises laboratoriais em cinco vezes em intervalos espaçados de quatro meses. As amostras de sangue foram coletadas dos 50 animais e o soro foi submetido ao teste de soroaglutinação microscópica (SAM) para detecção de anticorpos anti-leptospira. Do total vacas, 20 demonstraram títulos de anticorpos ? 1: 100 na SAM e foram coletadas amostras de urina apenas destes animais com maiores título para realizar a nested-PCR (n-PCR) e isolamento bacteriano por cultura. Adicionalmente, foram coletadas duas amostras de urina de cinco animais com títulos de anticorpos < 1: 100 na SAM para n-PCR. O Sorovar Hardjo foi considerado como sendo o mais frequente durante o monitoramento sorológico dos animais avaliados. A técnica de n-PCR foi capaz de detectar DNA leptospiral na urina de animais com títulos de anticorpos ? 1: 100 e na urina de animais cujos títulos eram < 1:100. O sequenciamento dos amplicons leptospíricos compartilhou 100% de identidade da sequência de nucleotídeos com a espécie Leptospira interrogans. Os resultados positivos da n-PCR dos animais com títulos < 1: 100 sugerem que a diminuição do ponto de corte da SAM pode não ser suficiente para detectar os portadores renais, por isso também é importante usar a n-PCR como uma ferramenta diagnóstica adicional para identificar os animais infectados com serovar Hardjo e cuja sorologia foi negativa

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

Skin antisepsis protocols for the collection of blood from donor dogs

<div><p>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.</p></div

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

Antibiotic Resistance in Enterobacteriaceae Family Members Isolated from Horses Used for Animal Traction

Lately, bacterial drug resistance has become an important worldwide problem in one health, where bacteria have undergone mutation becoming increasingly resistant. The major problem of bacterial drug resistance is the difficulty with eliminating microorganisms from different wounds and infected patients, and the therapeutic option is most often ineffective as a result of the repeated and inappropriate use of antimicrobials. The objective of this work was to detect and identify enterobacteria, to evaluate their resistance profile and the production of extended-spectrum b-lactamases in draft horse isolates from the municipality of Umuarama, Parana, Brazil. A nasal, oral, and ear cavity swab was collected from 38 horses (used for animal traction) for isolation and bacterial identification, phenotypic antibiotic susceptibility testing, and the phenotypic test for the detection of extended-spectrum b-lactamasesproducing strains. In 12 swabs bacterial isolation was possible. Strains of Escherichia coli, Serratia rubidaea, Citrobacterdiversus, Kluyvera species, and Providenciaalcalifaciens were isolated. One hundred percent multidrug resistance was detected, and the antimicrobials that encountered the highest resistance were ertapenem (100%), cefotaxime (100%), cefoxitin (100%), ampicillin (100%), amoxicillin (100%), chloramphenicol (100%), and aztreonam (91.67%) and no extended-spectrum b-lactamases -producing strain was detected. The results of this work reveal the presence of strains of the Enterobacteriaceae family associated with high bacterial resistance in horses used for animal traction in the municipality of Umuarama, State of Parana, Brazil, and these results confirm that these horses can be considered reservoirs of multidrug-resistant microorganisms. This situation can be considered an important problem of one health