In this paper we report the identification and characterization of a DNA region containing putative nif genes and belonging to a Burkholderia endosymbiont of the arbuscular mycorrhizal fungus Gigaspora margarita. A genomic library of total DNA extracted from the fungal spores was also representative of the bacterial genome and was used to investigate the prokaryotic genome. Screening of the library with Azospirillum brasilense nifHDK genes as the prokaryotic probes led to the identification of a 6,413-bp region. Analysis revealed three open reading frames encoding putative proteins with a very high degree of sequence similarity with the two subunits (NifD and NifK) of the component I and with component II (NifH) of nitrogenase from different diazotrophs. The three genes were arranged in an operon similar to that shown by most archaeal and bacterial diazotrophs. PCR experiments with primers designed on the Burkholderia nifHDK genes and Southern blot analysis demonstrate that they actually belong to the genome of the G. margarita endosymbiont. They offer, therefore, the first sequence for the nif operon described for Burkholderia. Reverse transcriptase PCR experiments with primers designed on the Burkholderia nifH and nifD genes and performed on total RNA extracted from spores demonstrate that the gene expression was limited to the germination phase. A phylogenetic analysis performed on the available nifK sequences placed the endosymbiotic Burkholderia close to A. brasilense.

Wild canids and domestic dogs are the main reservoir of zoonotic visceral leishmaniasis (VL) caused byLeishmania infantum (syn.: Leishmania chagasi). Serological diagnosis of VL is therefore important in both human and dog leishmaniasis from a clinical and epidemiological point of view. Routine diagnosis of VL is traditionally carried out by immunofluorescent antibody test (IFAT), which is laborious and difficult to standardize and to interpret. In the last decade, however, several specific antigens of Leishmania infantum have been characterized, allowing the development of a recombinant-based immunoassay. Among them, the whole open reading frame encoding K9 antigen, the gene fragment encoding the repetitive sequence of K26, and the 3-terminal gene fragment of the kinesin-related protein (K39sub) were previously evaluated as diagnostic markers for canine leishmaniasis and proved to be independent in their antibody reactivity. Since sensitivity of serological test is usually higher in multiple-epitope format, in this study the relevant epitopes of K9, K26, and K39 antigens were joined by PCR strategy to produce the chimeric recombinant protein. The resulting mosaic antigen was found highly expressed in Escherichia coli and efficiently purified by affinity chromatography. Antigenic properties of this recombinant antigen were evaluated by indirect enzyme-linked immunosorbent assay (ELISA) using a panel of human and dog sera previously characterized by parasitological and/or serological techniques. Chimeric ELISA showed 99% specificity in both human (n ‫؍‬ 180) and canine (n ‫؍‬ 343) control groups, while sensitivity was higher in canine VL (96%, n ‫؍‬ 213) than in human VL (82%, n ‫؍‬ 185). Accordingly, concordance between IFAT and canine chimeric ELISA (k ‫؍‬ 0.95, 95% confidence interval ‫؍‬ 0.93 to 0.98) was higher than between IFAT and human chimeric ELISA (k ‫؍‬ 0.81, 95% confidence interval ‫؍‬ 0.76 to 0.87). Results suggest the potential use of this new antigen for routine serodiagnosis of VL in both human and canine hosts. Animal and human leishmaniases are parasitic infections caused by protozoan hemoflagellates belonging to the genus Leishmania.Parasites are transmitted by the bite of phlebotomine sand flies to the mononuclear phagocyte system of the vertebrate host, where the infecting promastigotes differentiate into and replicate as amastigotes. The geographical distribution and the spreading of the infection depend on the presence of sand fly vectors and of animal reservoirs (3).Wild canids and domestic dogs represent the main reservoir hosts of zoonotic visceral leishmaniasis (VL), playing a strategic role for diffusion and maintenance of the infection (25). Zoonotic VL is caused by Leishmania infantum (syn. Leishmania chagasi) (24) and spread in the Mediterranean basin, in the Middle East, and in Latin America.In the past decades, human factors and environmental changes have promoted the diffusion of the disease in areas originally not considered suitable for the spreading of leishmaniasis (9,11,32,...