The Presence of a Virulent Clone of Leptospira interrogans Serovar Canicola in Confirmed Cases of Asymptomatic Dog Carriers in Mexico.

Leptospirosis is a neglected zoonotic disease that commonly affects cattle, pigs, horses, and dogs in many countries. Infection in dogs is usually subclinical, but acute cases of leptospirosis may occur along with systemic failure, which may become fatal. After recovery from an acute infection, dogs may become asymptomatic carriers and shed pathogenic leptospires through urine for long periods of time. Here, a study of ten different cases of leptospirosis is presented, showing the relevance of dogs as asymptomatic carriers of pathogenic Leptospira. The diagnosis was confirmed via isolation and further serological and genetic identification. Four Leptospira isolates (LOCaS28, 31, 34, and 46) were obtained from the kidneys and urine samples of 58 dogs destined for destruction (6.89%) at a Canine Control Center in Mexico City. No spirochetes were observed in the urine samples of those Leptospira-positive dogs examined under dark-field microscopy, and no clinical signs of disease were observed either. Six additional isolates were obtained: two came from asymptomatic carrier dogs (CEL60 and UADY22); another isolate came from an asymptomatic dog that was a pack companion of a clinically ill dog with fatal leptospirosis (AGFA24); and finally, three isolates were taken from dogs that died of leptospirosis (LOCaS59, Citlalli, and Nayar1). Nine out of the ten isolates were identified as being from the serogroup Canicola via cross-absorption MAT using reference strains and specific antisera, and their identity was genetically confirmed as Canicola ST34 via multi-locus sequencing typing (MLST). In contrast, the isolate Nayar1 was identified as serovar Copenhageni ST2. Interestingly, the asymptomatic dogs from which Leptospira isolates were recovered consistently showed high antibody titers in the microscopic agglutination test (MAT), revealing values of at least 1:3200 against serogroup Canicola and lower titer values against other serogroups. Isolates showed different virulence levels in the hamster model. Taken as a whole, all these findings confirmed that dogs may act as asymptomatic carriers of pathogenic leptospires and possibly spread them out to the environment, thus representing an active public health risk. The results also showed that the Canicola ST34 clone is the most prevalent Leptospira serovar in dogs in Mexico, and finally that the old-fashioned MAT is a good alternative for the detection of presumptive Leptospira asymptomatic carrier dogs.

GspD, The Type II Secretion System Secretin of Leptospira, Protects Hamsters against Lethal Infection with a Virulent L. interrogans Isolate.

The wide variety of pathogenic Leptospira serovars and the weak protection offered by the available vaccines encourage the search for protective immunogens against leptospirosis. We found that the secretin GspD of the type II secretion system (T2S) of Leptospira interrogans serovar Canicola was highly conserved amongst pathogenic serovars and was expressed in vivo during infection, as shown by immunohistochemistry. Convalescent sera of hamsters, dogs, and cows showed the presence of IgG antibodies, recognizing a recombinant version of this protein expressed in Escherichia coli (rGspDLC) in Western blot assays. In a pilot vaccination study, a group of eight hamsters was immunized on days zero and 14 with 50 µg of rGspDLC mixed with Freund's incomplete adjuvant (FIA). On day 28 of the study, 1,000 LD50 (Lethal Dose 50%) of a virulent strain of Leptospira interrogans serovar Canicola (LOCaS46) were inoculated by an intraoral submucosal route (IOSM). Seventy-five percent protection against disease (p = 0.017573, Fisher's exact test) and 50% protection against infection were observed in this group of vaccinated hamsters. In contrast, 85% of non-vaccinated hamsters died six to nine days after the challenge. These results suggest the potential usefulness of the T2S secretin GspD of Leptospira as a protective recombinant vaccine against leptospirosis.

Culture-attenuated pathogenic Leptospira lose the ability to survive to complement-mediated-killing due to lower expression of factor H binding proteins.

Several pathogens including Gram-negative bacteria hijack complement regulators to escape host's innate response. Pathogenic Leptospira species bind Factor H, C4b binding protein and vitronectin from the complement system. We evaluated the ability of low passage (LP) and culture-attenuated (CA) pathogenic strains of Leptospira, to bind Factor H. We used LOCaS46 (Leptospira interrogans sv Canicola), LOVe30 (L. interrogans sv Icterohaemorrhagiae) and MOCA45 (L. santarosai sv Tarassovi), and ten high passage strains of Leptospira [used in the microscopic agglutination test (MAT)]. Afterwards, we assessed their survival in normal human serum (NHS). Interestingly, the ability in binding Factor H was higher for LOCaS46 and LOVe30 LP strains, than for the respective CA strains suggesting that the ability of evading the alternative complement pathway is lost after culture attenuation. Accordingly, the level of mRNA expression of the Factor H binding proteins, LigA, LigB and Lsa23 was higher in these LP strains than in the corresponding CA strains. Unexpectedly, no difference in Factor H binding and surviving was observed between LP and CA MOCA45 strains. The high passage MAT-reference strains showed variation in Factor H binding ability, but, in most cases, the ability for capturing Factor H by Leptospira strains correlated with their survival in NHS.

Kinetics of Leptospira interrogans infection in hamsters after intradermal and subcutaneous challenge.

BACKGROUND: Leptospirosis is a zoonosis caused by highly motile, helically shaped bacteria that penetrate the skin and mucous membranes through lesions or abrasions, and rapidly disseminate throughout the body. Although the intraperitoneal route of infection is widely used to experimentally inoculate hamsters, this challenge route does not represent a natural route of infection. METHODOLOGY/PRINCIPAL FINDINGS: Here we describe the kinetics of disease and infection in hamster model of leptospirosis after subcutaneous and intradermal inoculation of Leptospira interrogans serovar Copenhageni, strain Fiocruz L1-130. Histopathologic changes in and around the kidney, including glomerular and tubular damage and interstitial inflammatory changes, began on day 5, and preceded deterioration in renal function as measured by serum creatinine. Weight loss, hemoconcentration, increased absolute neutrophil counts (ANC) in the blood and hepatic dysfunction were first noted on day 6. Vascular endothelial growth factor, a serum marker of sepsis severity, became elevated during the later stages of infection. The burden of infection, as measured by quantitative PCR, was highest in the kidney and peaked on day 5 after intradermal challenge and on day 6 after subcutaneous challenge. Compared to subcutaneous challenge, intradermal challenge resulted in a lower burden of infection in both the kidney and liver on day 6, lower ANC and less weight loss on day 7. CONCLUSIONS/SIGNIFICANCE: The intradermal and subcutaneous challenge routes result in significant differences in the kinetics of dissemination and disease after challenge with L. interrogans serovar Copenhageni strain Fiocruz L1-130 at an experimental dose of 2×106 leptospires. These results provide new information regarding infection kinetics in the hamster model of leptospirosis.

Leptospira and leptospirosis.

Leptospirosis is the most wide spread zoonosis worldwide; it is present in all continents except Antarctica and evidence for the carriage of Leptospira has been found in virtually all mammalian species examined. Humans most commonly become infected through occupational, recreational, or domestic contact with the urine of carrier animals, either directly or via contaminated water or soil. Leptospires are thin, helical bacteria classified into at least 12 pathogenic and 4 saprophytic species, with more than 250 pathogenic serovars. Immunity following infection is generally, but not exclusively, mediated by antibody against leptospiral LPS and restricted to antigenically related serovars. Vaccines currently available consist of killed whole cell bacterins which are used widely in animals, but less so in humans. Current work with recombinant protein antigens shows promise for the development of vaccines based on defined protective antigens. The cellular and molecular basis for virulence remains poorly understood, but comparative genomics of pathogenic and saprophytic species suggests that Leptospira expresses unique virulence determinants. However, the recent development of defined mutagenesis systems for Leptospira heralds the potential for gaining a much improved understanding of pathogenesis in leptospirosis.

Selection of the internal control gene for real-time quantitative rt-PCR assays in temperature treated Leptospira.

Analysis of gene expression requires sensitive, precise, and reproducible measurements for specific mRNA sequences. To avoid bias, real-time RT-PCR is referred to one or several internal control genes. Here, we sought to identify a gene to be used as normalizer by analyzing three functional distinct housekeeping genes (lipL41, flaB, and 16S rRNA) for their expression level and stability in temperature treated Leptospira cultures. Leptospira interrogans serovar Hardjo subtype Hardjoprajitno was cultured at 30 degrees C for 7 days until a density of 10(6) cells/ml was reached and then shifted to physiological temperatures (37 degrees C and 42 degrees C) and to environmental temperatures (25 degrees C and 30 degrees C) during a 24 h period. cDNA was amplified by quantitative PCR using SYBR Green I technology and gene-specific primers for lipL41, flaB, and 16S rRNA. Expression stability (M) was assessed by geNorm and Normfinder v.18. 16S rRNA registered an average expression stability of M = 1.1816, followed by flaB (M = 1.682) and lipL41 (M = 1.763). 16S rRNA was identified as the most stable gene and can be used as a normalizer, as it showed greater expression stability than lipL41 and flaB in the four temperature treatments. Hence, comparisons of gene expression will have a higher sensitivity and specificity.

[Evaluation of the transcription of locus rfb for the biosynthesis of LPS in Leptospira interrogans subtype hardjoprajitno]. / Evaluacion de la transcripción del locus rfb para la biosíntesis de LPS en Leptospira interrogans subtipo hardjoprajitno.

Plasmid pGL4W15-96 was constructed from the pGL4W74 vector without promoter for kanamycin and a sequence of 480pb containing the supposed J15 promoter with the objective of confirming the role of J15 sequence as a promoter for Leptospira, which restored the transcription of the gene of resistance to kanamycin in Escherichia coli and Leptospira biflexa, corroborating this way the function of the insertion as a promoter for both organisms.

[Expression in Escherichia coli of the gspd(l) gene of the type II secretion system in Leptospira borgpetersenii serovariety hardjo]. / Expresión en Escherichia coli del gen gspd(l) del sistema de secreción tipo II de Leptospira borgpetersenii serovariedad hardjo.

A fragment of 1.539 pb of the gene homologous to gspD of Hardjobovis was clonated in the pET28a vector and it was transformed into E. coli C43 and Rosetta strains. The sequence of GspD(L) showed 46 % of similitude with E. coli GspD secretin. The expression of recombinant GspD(L) was obtained in Rosetta strain.

Seroprevalencia de leptospirosis en perros callejeros del norte de la ciudad de México / Leptospirosis seroprevalence in stray dogs from northern Mexico City

Se colectó el suero de 135 perros capturados en las calles del norte de la ciudad de México y que fueron alojados en el Centro de Control Canino Luis Pasteur. Los sueros fueron probados por aglutinación microscópica para detectar anticuerpos antileptospira. Cincuenta y dos sueros (38.51 por ciento) fueron positivos a una o más serovariedades. Las serovariedades más comúnmente detectadas fueron: L. castellonis (50 por ciento), L. pyrogenes (38.46 por ciento) y L. canicola (26.92 por ciento). L. icteroheamorrhagiae fue detectada en 21.15 por ciento de los sueros positivos. Los títulos de anticuerpos más altos fueron contra las serovariedades: L. castellonis, L. canicola, L. icterohaemiorrhagiae y L. pyrogenes con 1:1600

Detección de anticuerpos contra Leptospira interrogans en equinos dedicados a la producción de sueros hiperinmunes / Detection of Leptospira interrogans antibody in horses used for production of hyperimmune sera

Sueros de 106 equinos de 1 a 21 años (89 machos y 17 hembras), procedentes del Instituto Nacional de Higiene y dedicados a producir sueros hiperinmunes, fueron analizados por aglutinación microscópica (AM) contra 19 serovariedades de L. interrogans. Considerando el estado inmunológico de los equinos, se examinaron sueros con títulos 1:400 mediante inmunodifusión doble en agar (IDD) y contrainmunoelectroforesis (CIEF), para descartar reacciones inespecíficas. Con el mismo fin, se inocularon conejos con los antígenos utilizados en los equinos (virus rábico, toxoides tetánico y diftérico y venenos de alacrán y víbora) y se probaron por AM. Con reacciones 1:100, 83 por ciento de los equinos (88 de 106) fueron positivos a una o más serovariedades con títulos de hasta 1:6500. Las serovariedades con más reacciones positivas fueron: autumnalis, australis y pomona. Las serovariedades con títulos más altos fueron: autumnalis, cynopteri y pyrogenes (1:6400) y australis, celledoni, szwajizak e icterohaemorrhagiae (1:3200). La IDD detectó un caso de identidad inmunológica total entre virus rábico y L. pomona (suero 545). La CIEF no detectó líneas de precipitación. Ninguno de los conejos inmunizados mostró títulos contra leptospira