New vaccine strategies are needed for the prevention of leptospirosis, a widespread human and animal disease caused by pathogenic leptospires. Our previous work determined that a protein leptospiral extract conferred cross-protection in a gerbil model of leptospirosis. The 31-to 34-kDa protein fraction of Leptospira interrogans serovar autumnalis was shown sufficient for this purpose. In the present study, N-terminal sequencing of a 32-kDa fraction and Southern blotting of genomic DNA with corresponding degenerated oligonucleotide probes identified two of its constituents: hemolysis-associated protein 1 (Hap1) and the outer membrane Leptospira protein 1 (OmpL1). Adenovirus-mediated Hap1 vaccination induces significant protection against a virulent heterologous Leptospira challenge in gerbils, whereas a similar OmpL1 construct failed to protect the animals. These data indicate that Hap1 could be a good candidate for developing a new generation of vaccines able to induce broad protection against leptospirosis disease.

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The use of DNA constructs encoding leptospiral proteins is a promising new approach for vaccination against leptospirosis. In previous work we determined that immunization with hemolysis-associated protein 1 (Hap1) (LipL32) expressed by adenovirus induced significant protection against a virulent Leptospira challenge in gerbils. To avoid the use of the adenovirus vector, we checked for clinical protection against lethal challenge by DNA vaccination. A DNA vaccine expressing Hap1 was designed to enhance the direct gene transfer of this protein into gerbils. A challenge was performed 3 weeks after the last immunization with a virulent strain of serovar canicola. Our results show that the cross-protective effect with pathogenic strains of Leptospira, shared by Hap1, could be mediated by the DNA plasmid vector. This finding should facilitate the design and development of a new generation of vaccines against bacteria, particularly Leptospira interrogans sensu lato.

In this study, we used Southern hybridization of genomic DNA with the integral hap1 gene as a probe to show that this gene is only present in pathogenic Leptospira strains. We then selected PCR primers based on the hap1 gene, and tested them on several Leptospira strains and biological samples. Specific amplification was obtained for all pathogenic strains tested. Negative PCR results were observed with all saprophytic leptospire strains used as well as with other spirochetes and bacteria commonly found in biological samples. The results of direct PCR performed on biological samples, such as blood, urine or kidneys correlated with the results obtained with the classical Leptospira tests (culture and MAT). A PCR assay based on this gene would be a very useful tool for the rapid, sensitive and specific identification of pathogenic leptospires in samples for diagnosis or epidemiological survey.

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Twenty‐four specific pathogen‐free beagles were randomly allocated into four groups (three vaccinated groups and one control group) and inoculated at nine and 12 weeks of age with one of three commercial inactivated Leptospira vaccines: A (Vanguard 7; Pfizer Santé Animale), B (Dohyvac 7L; Fort Dodge), and C (Nobivac DHPPi + Lepto; Intervet Intemational); the control group received Nobivac DHPPi (Intervet International). Seven weeks after the second vaccination all the dogs were challenged with Leptospira interrogans serogroup canicola. All the vaccinated dogs developed a mild serological response (microscopic agglutination titres) after the booster vaccination. A significant serological response after the challenge was observed, particularly in the controls. The challenge induced fever and clinical disorders in the control group, whereas in the vaccinated groups the clinical signs were mild. Blood cultures became positive in all control dogs, and in one of six dogs vaccinated with vaccine A and two of four dogs vaccinated with vaccine B; none of the six dogs vaccinated with vaccine C was leptospiraemic at any stage of the experiment. Urine cultures were positive in all the control dogs two weeks after the challenge. One of six dogs vaccinated with vaccine A and two of four dogs vaccinated with vaccine B shed bacteria in their urine after the challenge, but none of the dogs vaccinated with vaccine C shed bacteria in their urine at any time during the experiment.

Nocardia cyriacigeorgica is a recently characterized species within the genus of Nocardia. We report a brain abscess, following a primary pulmonary colonization, due to this species in a human immunodeficiency virus-infected patient. This case confirms that isolation of Nocardia in sputum is associated with a high risk of disseminated infection in immunocompromised patients. CASE REPORTA sub-Saharan 33-year-old human immunodeficiency virus (HIV)-infected woman was admitted for a generalized seizure in April 2004. Six months prior to admission, HIV infection had been diagnosed when she presented with weight loss, fever, cough, dyspnea, and chest pain. The CD4 lymphocyte count was 20 cells/mm 3 . Diagnosis of cavitary pneumonia due to Actinomyces sp. was established by culture of a bronchoscopic sample. She received oral treatment with amoxicillin at 150 mg/kg of body weight per day and minocycline at 100 mg twice a day. She also received primary prophylaxis for pneumocystosis with co-trimoxazole (one double-strength tablet per day) and highly active antiretroviral therapy with didanosine, lamivudine, and lopinavir-ritonavir. The patient was compliant with this regimen, which was associated with a global improvement of her clinical status, and the pneumonia was cured. Five months later, a new sputum sample grew Nocardia cyriacigeorgica. At that time she was asymptomatic, with no evidence of active pneumonia. The immune condition of the patient had also improved with a CD4 lymphocyte count at 80 cells/mm 3 . No change in therapy was made, based on a presumption of respiratory tract colonization by Nocardia. The follow-up sputum cultures did not grow the pathogen.On the day of her admission, she suddenly presented a grand mal seizure. A brain computed tomography scan showed a single ring enhancing nodular hyperdensity, surrounded by an edema, in the right frontal lobe which was confirmed by magnetic resonance imaging. A biopsy was first considered hazardous to perform. The diagnosis of toxoplasmosis abscess was unlikely because of primary prophylaxis with co-trimoxazole with good compliance, negativity of toxoplasmosis serology, and negativity of toxoplasmosis cerebrospinal fluid PCR assay. Because of a presumptive diagnosis of Actinomyces brain abscess, amoxicillin and minocycline therapy was continued, but oral amoxicillin was switched to intravenous administration at a dose of 200 mg/kg per day. However, the patient presented a progressive right arm paresis and new episodes of generalized seizure despite anticonvulsant therapy. Two weeks later, the follow-up brain magnetic resonance imaging showed a progression of the right frontal abscess and three new abscesses in the left cerebral hemisphere. A stereotaxial brain biopsy of the frontal abscess was performed. Direct sample examination by microscopy showed many polymorphonuclear leukocytes without any bacteria, but the culture grew a branched gram-positive rod organism, which was further identified as N. cyriacigeorgica. Antimicrobial therapy was changed to imipe...

We evaluated the ability of Yersinia pestis antigens HmuR, Psn and modified forms of LcrV delivered by live attenuated Salmonella strains to stimulate a protective immune response against subcutaneous or intranasal challenge with Y. pestis CO92. LcrV196 is a previously described truncated protein that includes aa 131–326 of LcrV and LcrV5214 has been modified to replace five key amino acids required for interaction with the TLR2 receptor. Psn is the outer membrane receptor for the siderophore, yersiniabactin, and the bacteriocin, pesticin. Mice immunized with Salmonella synthesizing Psn, LcrV196 or LcrV5214 developed serum IgG responses to the respective Yersinia antigen and were protected against pneumonic challenge with Y. pestis. Immunization with Salmonella synthesizing Psn or LcrV196 was sufficient to afford nearly full protection against bubonic challenge, while immunization with the strain synthesizing LcrV5214 was not protective. Immunization with Salmonella synthesizing HmuR, an outer membrane protein involved in heme acquisition in Y. pestis, was poorly immunogenic and did not elicit a protective response against either challenge route. These findings indicate that both Psn and LcrV196 delivered by Salmonella provide protection against both bubonic and pneumonic plague.