Fabiana Miraglia

Molecular characterization, serotyping, and antibiotic susceptibility profile of Leptospira interrogans serovar Copenhageni isolates from Brazil

Leptospira interrogans serogroup Icterohaemorrhagiae is the major serogroup infecting humans worldwide, and rodents and dogs are the most significant transmission sources in urban environments. Knowledge of the prevalent serovars and their maintenance hosts is essential to understand the epidemiology of leptospirosis. In this study, 20 Leptospira isolates were evaluated by pulsed-field gel electrophoresis (PFGE), variable number tandem-repeat analysis (VNTR), serotyping, and determination of antimicrobial resistance profile. Isolates, originated from bovine, canine, human, and rodent sources, were characterized by microscopic agglutination test with polyclonal and monoclonal antibodies and were identified as L. interrogans serogroup Icterohaemorrhagiae serovar Copenhageni. MICs of antimicrobials often used in veterinary medicine were determined by broth microdilution test. Most of tested antibiotics were effective against isolates, including penicillin, ampicillin, and ceftiofur. Higher MIC variability was observed for fluoroquinolones and neomycin; all isolates were resistant to trimethoprim/sulfamethoxazole and sulphadimethoxine. Isolates were genotyped by PFGE and VNTR; both techniques were unable to discriminate between serovars Copenhageni and Icterohaemorrhagiae, as expected. PFGE clustered all isolates in 1 pulsotype, indicating that these serovars can be transmitted between species and that bovine, rodent, and dogs can maintain them in the environment endangering the human population.

Isolation and characterization of Leptospira interrogans from pigs slaughtered in São Paulo State, Brazil

With the aim of isolating Leptospira spp., blood serum, kidney, liver and genital tract of 137 female swine (40 sows and 97 gilts) and also urine samples from 22 sows were collected in a slaughterhouse in the State of Sao Paulo, from April 2003 to August 2004. Four isolates were obtained from animals that presented microagglutination test (MAT) titers > 100 for the serovar Pomona and one was obtained from an animal negative by MAT in which Leptospira was isolated from the liver and reproductive tract. The presence of leptospiral DNA was investigated by PCR, and positive results were found in kidneys of 11 females, liver of two, genital tract of two and urine of one of them. Nephrosis, interstitial multifocal nephritis, moderate to severe changing, hyalines cylinders and hemorrhagic focuses, hepatic and uterine horns congestion were histological lesions observed in higher frequency in animals positive for leptospira. The silver impregnation (Warthin Starry) confirmed the presence of spirochetes in renal tubules of four females with positive leptospira cultures from kidneys. The serogroup of the five isolates was identified as Pomona by cross agglutination with reference polyclonal antibodies. Molecular characterization of the isolates was carried out by variable-number tandem-repeats analysis. All the isolates revealed a pattern distinct from the L. interrogans Pomona type strain, but identical to a previously identified pattern from strains isolated in Argentina belonging to serovar Pomona.

Molecular and serological characterization of Leptospira interrogans serovar Canicola isolated from dogs, swine, and bovine in Brazil

The identification of Leptospira clinical isolates through genotyping and serotyping, besides the recognition of its reservoirs, are important tools for understanding the epidemiology of leptospirosis, and they are also keys for identifying new species and serovars. Fourteen clinical isolates from animals were characterized by means of single enzyme amplified length polymorphism, variable number of tandem repeat analysis, pulsed field gel electrophoresis, and serotyping. All isolates were identified as Leptospira interrogans, serovar Canicola. Infections by this serovar occur in urban regions, where dogs represent the main maintenance hosts, whereas bovine and swine may act as reservoirs of serovar Canicola in rural areas. Both urban and rural aspects of leptospirosis, and the role of domestic animals as maintenance hosts, cannot be neglected in developing and developed countries.

Emprego de estirpes Leptospira spp. isoladas no Brasil na microtécnica de soroaglutinação microscópica aplicada ao diagnóstico da leptospirose em rebanhos bovinos de oito estados brasileiros

The aim of this study was to investigate the adequacy of the use of autochthonous strains of leptospires isolated in Brazil, added to antigen collection of the microscopic agglutination test (MAT) applied to the diagnosis of bovine leptospirosis. By means of non-probability sampling, 109 farms and 9,820 cattle, females at reproductive age were chosen from 85 municipalities in the states of Goias, Mato Grosso, Mato Grosso do Sul, Minas Gerais, Parana, Rio Grande do Sul, Santa Catarina and Sao Paulo. Among the 9,820 examined animals, 5,806 (59.12%) were reactants at MAT for at least one serovar using the 23 reference serovars. Employing the collection of reference serovars and the ten autochthonous strains, 6,400 (65.24%) reactants and significant difference (p=0.001) was found. The most probable serovars identified by the collection of reference antigens were Hardjo (43.03%), Shermani (20%), Wolfi (9.96%), Grippothyphosa (5.42%) and Pomona (4.28%). With the collection amplified with the ten strains isolated in Brazil, the most probable serovars were Hardjo (31%), Guaricura-M4/84 (22.50%), Shermani (15.43%), Wolffi (4.76%), Grippothyphosa (3.71%) and Autumnalis (3.24%). The serovar Guaricura, strain M4/84, isolated from bovines and buffaloes in the State of Sao Paulo, was ranked as one of the three most probable serovars in the states of Goias, Mato Grosso, Mato Grosso do Sul, Minas Gerais and Sao Paulo. The addition of autochthonous strains to the MAT antigen collection provided the confirmation of the diagnosis of leptospirosis in 594 cattle (6%) which have been classified as non-reactants by the reference collection (p=0.001).

Comparison of four antibiotics for inactivating leptospires in bull semen diluted in egg yolk extender and experimentally inoculated with Leptospira santarosai serovar guaricura

Inactivation of leptospires in pools of semen from three Holstein Friesian bulls, collected in an artificial vagina, was investigated. Spermatic concentration was adjusted in egg yolk citrate extender, submitted to the following treatments: A (control; without antibiotics); B (penicillin, 1,000 UI/mL - streptomycin, 1,000 µg/mL); C (amoxicillin, 1,000 µg/mL); D (ceptiofur sodium, 1,000 µg/mL); E (amoxicillin 1,000 µg/mL - ceptiofur sodium 1,000 µg/mL). Leptospires (2.0 x 106 leptospires/mL) were added into the diluted semen. Recovery of leptospires was obtained in modified EMJH semi-solid medium with and without antibiotics. The antibiotics in the concentrations used did not affect means of percentage of progressive motility and individual progressive motility of spermatozoids. Penicillin-streptomycin presented the best results in leptospire inactivation (97.1%). Amoxicillin, ceptiofur sodium and their combination at the concentrations studied presented poor results: 59.29%; 32.5% and 60.36% of inactivation, being less effective in leptospire inactivation than penicillin-streptomycin.

Cross-protection between experimental anti-leptospirosis bacterins

We investigated the existence of cross-protection between two anti-leptospirosis monovalent experimental bacterins produced with two strains of Leptospira serogroup Pomona: Fromm strain of serovar Kennewicky, isolated from pigs in the United States, and strain GR6 of serovar Pomona isolated from pigs in Brazil. Both were added of aluminum hydroxide as an adjuvant. Experimental bacterins were tested with the hamster potency test in order to assess protection provided against the disease and against the establishment of kidney infection. Controls were polyvalent commercial vaccine produced with Leptospira strains isolated outside Brazil, which included a representative of Pomona serovar, or Sorensen solution added of aluminum hydroxide adjuvant. The challenge was performed with cross-strains of serogroup Pomona tested in accordance with international standards established for the potency test. After 21 days of the challenge, survivors were killed to evaluate the condition of Leptospira renal carrier. Experimental bacterins protected hamsters against homologous and heterologous strains, demonstrating the existence of cross-protection. The commercial vaccine protected the hamsters challenged with both strains, but there was a high proportion of animals diagnosed as renal carriers when the challenge was performed with strain GR6, isolated from pigs in Brazil.

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.

Characterization of Leptospira santarosai Serogroup Grippotyphosa Serovar Bananal Isolated from Capybara (Hydrochaeris hydrochaeris) in Brazil

Abstract Leptospirosis is a widespread zoonosis caused by bacteria of the genus Leptospira. Rodents appear to be the most important reservoirs of infection. They contaminate the environment and food and can transmit the pathogen when they are consumed by carnivores. Capybara (Hydrochaeris hydrochaeris) are efficient reservoirs of Leptospira, and because they are in close contact with farm animals and are found in semiurban areas, they represent a risk to public health. We isolated five Leptospira strains from capybara kidneys in Sao Paulo State, Brazil, in 2001 and typed them using serologic and molecular techniques. These strains include the Leptospira santarosai serogroup Grippotyphosa serovar Bananal. Pulsed field gel electrophoresis resulted in a unique pattern distinct from the reference strains, and the isolates clustered with greater than 85% similarity. The isolates also presented higher growth rates than other Leptospira serovars, with high minimal inhibitory concentration values for most of the tested antibiotics, with the exception of penicillin and ampicillin. This isolation and characterization of the L. santarosai serogroup Grippotyphosa serovar Bananal from capybara, highlights the importance of wild and sinantropic rodents as carriers of pathogenic leptospires.

Characterization of Leptospira interrogans serovar Pomona isolated from swine in Brazil.

INTRODUCTION Leptospira interrogans swine infection is a cause of serious economic loss and a potential human health hazard. In Brazil, the most common serovars associated with swine infections are Pomona, Icterohaemorrhagie and Tarassovi. Cross-reactions among serovars and the failure of infected animals to seroconvert may complicate the interpretation of serological tests. Molecular methods with better discriminatory powers are useful tools for swine leptospirosis characterization and diagnosis. METHODOLOGY This study evaluated nine L. interrogans isolates from the States of Sao Paulo and Minas Gerais during different time periods. Isolates from diseased and apparently healthy swine were characterized by microscopic agglutination tests with polyclonal antibodies and were genotyped by VNTR, PFGE and MLST techniques. Broth microdilution was used to determine the minimal inhibitory concentration of the antimicrobials of veterinary interest. RESULTS The strains were identified as L. interrogans serogroup Pomona serovar Pomona Genotype A, while MLST grouped all of the isolates in sequence type 37. The PFGE analysis resulted in two pulsotypes with more than 70% similarity, distinguishing serovar Pomona isolates from the serovar Kennewicki reference strain. All of the isolates presented low MIC values to penicillin, ampicillin, ceftiofur and tulathromycin. High MIC values for fluoroquinolones, tiamulin, gentamicin, tetracyclines, neomycin, tilmicosin and sulfas were also observed. CONCLUSIONS All molecular techniques were concordant in L. interrogans serovar Pomona identification. This serovar may have a different antibiotic susceptibility profile than previously reported for Leptospira isolates.

Efficacy of leptospiral commercial vaccines on the protection against an autochtonous strain recovered in Brazil

In swine and bovines, leptospirosis prevention and control is carried out via vaccination of susceptible animals using bacterins. However, the efficiency of leptospirosis vaccines has been questioned. This work aimed to investigate the potency of five leptospirosis vaccines sold commercially in Brazil, challenging the animals with one autochthonous strain of Leptospira, Canicola serovar, denoted LO4, isolated from swine. The standard protocol was followed, and renal carriers of Leptospira were identified among the surviving animals by culture and PCR. Of the five vaccines tested, only two proved effective. None of the surviving animals was positive by culture; however, one animal was positive by PCR. Three of the five vaccines sold commercially in Brazil for the immunization of swine or bovines failed the test of the efficacy to protect the vaccinated animals following challenge with an autochthonous Leptospira strain, Canicola serovar. The two vaccines provided protection against the renal carrier state in the surviving animals. The criteria used to produce leptospirosis bacterins sold commercially in Brazil must be reviewed. The industry should support researches on leptospiral vaccinology to improve the quality of the present vaccines and discover new immunogenic strains, because it is known that vaccination is one of the most important tools to increase the reproduction rates in livestock.