Chemiluminescence and phagocytic responses of rat polymorphonuclear neutrophils to leptospires

The interaction of leptospires with polymorphonuclear neutrophils (PMN) was examined by the luminol-dependent chemiluminescence (CL) test. Whole blood CL changed in relation to the stage of leptospiral infection both in susceptible (SUS) and resistant (RES) rats. The intensity of CL grew with an increasing number of leptospires in the blood. CL responses were observed in isolated PMN upon exposure to living leptospires. In contrast, the same bacteria, having been inactivated by formalin, did not stimulate PMN. A variation was found in the CL response by different living strains of Leptospira. The CL intensity was arranged as follows: L. illini greater than L. biflexa greater than L. interrogans avirulent strains greater than L. interrogans virulent strains. The CL response was markedly enhanced by an opsonization of leptospires. Specific opsonization was shown to increase the rate of phagocytosis of leptospires with relation to the CL response.     

The detection of leptospires by a chemiluminescent immunoassay

Rabbit serum hyperimmune to Leptospira interrogans var icterohaemorrhagiae serovar icterohaemorrhagiae, reference strain RGA was conjugated to the chemiluminescent label ABEI (6-[N-(4-aminobutyl)-N-ethyl amino]-2,3-dihydrophthalazine-1,4-dione) in the presence of the coupling agent, EDC (1-ethyl-3(3-dimethyl amino-propyl)-carbodiimide HCl). The luminescent conjugate was incubated with homologous and heterologous antigens. The results indicate that chemiluminescence may provide an accurate and rapid method of detecting leptospires in biological fluids.     

Genetic and antigenic differences of serologically indistinguishable leptospires of serovar hardjo.

Pathogenic leptospires of serovar hardjo isolated from North American cattle were compared genetically and antigenically to reference strain hardjoprajitno of the Sejroe serogroup. Guanine-plus-cytosine (G+C) contents were determined for the genomes, and microscopic agglutination, Western blotting (immunoblotting), and immunoprecipitation were used to characterize antigenic relatedness. Major differences were demonstrated between the isolates and the reference strain. The G+C content of the reference strain was calculated to be approximately 34 +/- 1%, and those of the isolates were calculated at 39 +/- 1%. Antigenic differences between the isolates and the reference strain were identified by using rabbit immune serum raised against a hardjo isolate exhaustively adsorbed with hardjoprajitno whole and sonicated cells. Western blot analysis and immunoprecipitation using this adsorbed serum revealed antigens apparently unique for the hardjo isolates. Microscopic agglutination with the adsorbed rabbit serum did not agglutinate hardjoprajitno when diluted 1:2 but agglutinated bovine isolates to a 1:32 dilution. Bovine antiserum raised against the isolates was also used to identify antigens by immunoprecipitation.     

Survival of leptospires in commercial blood culture systems revisited

AIM: To assess the ability of commercial blood culture systems to maintain leptospires. METHODS: Nine different commercial blood culture bottles were compared for their ability to maintain four leptospiral strains at two temperatures, 30 degrees C and 37 degrees C. Bottles were subcultured at 48 hours, and one, two, three, and four week intervals and examined microscopically for the presence of viable leptospires. RESULTS: The results were comparable with those of an earlier study, which showed that different commercial blood culture systems varied in their ability to maintain leptospires. CONCLUSIONS: No single factor appears to influence the viability of leptospires in blood culture systems. In general, the combination of an aerobic blood culture and an incubation temperature of 30 degrees C enhances the viability of leptospires, and hence would increase the chances of their subsequent isolation from suspected cases of leptospiraemia.     

Lipids and HCV

Chronic hepatitis C virus (HCV) infection is associated with an increase in hepatic steatosis and a decrease in serum levels of total cholesterol, low-density lipoprotein cholesterol (LDL) and apolipoprotein B (apoB), the main protein constituent of LDL and very low-density lipoprotein (VLDL). These changes are more marked in HCV genotype 3 infection, and effective treatment results in their reversal. Low lipid levels in HCV infection correlate not only with steatosis and more advanced liver fibrosis but also with non-response to interferon-based therapy. The clinical relevance of disrupted lipid metabolism reflects the fact that lipids play a crucial role in the life cycle of hepatitis C virus. HCV assembly and maturation in hepatocytes depend on microsomal triglyceride transfer protein and apoB in a manner that parallels the formation of VLDL. VLDL production from the liver occurs throughout the day with an estimated 10¹⁸ particles produced every 24 h whilst the estimated hepatitis C virion production rate is 10¹² virions per day. HCV particles in the serum exist as a mixture of complete low-density infectious lipo-viral particles (LVP) and a vast excess of apoB-associated empty nucleocapsid-free sub-viral particles that are complexed with anti-HCV envelope antibodies. Apolipoprotein E (apoE) is also involved in HCV particle morphogenesis and is an essential apolipoprotein for HCV infectivity. ApoE is a critical ligand for the receptor-mediated removal of triglyceride rich lipoprotein (TRL) remnants by the liver. The dynamics of apoB-associated lipoproteins, including HCV-LVP, change post-prandially with an increase in large TRL remnants and very low density HCV-LVP which are rapidly cleared by the liver (at least three HCV receptors are cellular receptors for uptake of TRL remnants). In summary, HCV utilises triglyceride-rich lipoprotein pathways within the liver and the circulation to its advantage.     

In vitro association of leptospires with host cells.

Interactions of Leptospira interrogans with cultured endothelial and kidney epithelial cells were assayed by examining (i) cytoadherence of intrinsically radiolabeled leptospires to eucaryotic cell monolayers and (ii) penetration of leptospires through cell monolayers grown on polycarbonate filters in chemotaxis chambers. L. interrogans serovars attached to cultured cells in a dose- and time-dependent manner. Adherence was diminished following pretreatment of organisms with proteases, rabbit immune serum, or heat. When observed by scanning electron microscopy, most leptospires attached by both ends, rather than just one tip like Treponema pallidum. In penetration assays, 9.7% of added L. interrogans migrated through the monolayer-filter barrier, while only 0.3% of L. biflexa penetrated in the same time interval. Transmission electron microscopy revealed that organisms entered the host cell cytoplasm. These in vitro results indicate that leptospires have an invasive capacity that may be related to pathogenicity in vivo and suggest that further investigation of interactions with host cells may enhance knowledge of leptospiral virulence.     

Screening urinalysis in dogs with urinary shedding of leptospires

The aim of this study was to determine the utility of urinalysis and dipstick results in predicting leptospiruria in dogs. Ninety dogs, regardless of health status, were included in this study since January to August 2008. All the dogs went through a complete physical examination, and urine samples were collected by catheter or by cystocentesis. Polymerase chain reaction (PCR) testing was performed on urine samples to detect leptospiral DNA. Urinalysis was done at the same time. Of the 90 dogs included in the study, 28 (31%) were diagnosed as shedding leptospires in their urine. In terms of urinalysis test results, no significant difference was found between dogs with or without leptospiruria. Indeed, most of the dogs with positive PCR assay had normal urinalysis findings. The present study suggests that using urinalysis or dipstick is not recommended for screening the dogs that are actively shedding leptospires in their urine.     

Keratinophilic and saprophytic fungi isolated from students' nails in Egypt

In order to determine the presence of dermatophytes and saprophytes in healthy toe and finger nails, 120 students (60 male and 60 female) from preparatory schools at Sohag Governorate (Upper Egypt) were studied. 54 species in addition to 3 varieties belonging to 17 genera were isolated. Six species of true dermatophytes were collected: Microsporum audouinii var. rivalieri, M. cookei, Trichophyton mentagrophytes, T. simii, T. terrestre and T. verrucosum. Chrysosporium, a well-known keratinophilic genus, was prevalent and represented by 7 species (C. asperatum, C. indicum, C. keratinophilum, C. luteum, C. pannorum, C. tropicum and Chrysosporium state of Thielavia sepedonium). The commonest saprophytes in order of frequency were members of the genera Aspergillus, Penicillium, Alternaria, Scopulariopsis, Fusarium, Paecilomyces, Chaetomium, Syncephalastrum, Mucor, Rhizopus and Acremonium.     

Detection of leptospires in urine by polymerase chain reaction.

Primers for polymerase chain reaction (PCR) were synthesized from clones derived from a Leptospira hardjo (type hardjobovis) library. One pair of synthetic oligonucleotide primers was selected for further analysis. Under experimental conditions an amplification was obtained with DNA of Leptospira interrogans of some serovars belonging to serogroup sejroe. However, very little or no amplification was observed with DNA from other serovars of this group. No amplification was observed with DNA from other serogroups, other bacteria, or eucaryotic organisms. Cattle urine, seeded with hardjobovis, was processed in several ways and subsequently subjected to PCR. Boiling of the samples or treatment with detergents appeared to be most effective. Urine samples containing fewer than 10 leptospires gave a positive result in the PCR assay. Twenty urine samples obtained from a slaughterhouse or farm cows were investigated using the PCR assay, culture isolation, dot and quick blot hybridization, and serological tests. This comparative study suggests that amplification by PCR may be a valuable method for the detection of leptospires in cattle urine.     

Comparative analysis of the genomes of mycobacteriophages infecting saprophytic and pathogenic mycobacteria

Summary The genomes of a series of mycobacteriophages have been analyzed to disclose possible relationships between genetic characteristics and host range. The percent guanine-plus-cytosine in the DNA of 14 phages was found to be 34.4 to 47.5, as determined by a double-labelling procedure, which is unaffected by the presence of modified bases. The DNA of few mycobacteriophages yielded discordant values when the G+C content was estimated by buoyant density determination and by the double labelling procedure. This observation suggests the possible presence of modified bases in these genomes. The reduced susceptibility of viral DNAs to several restriction endonucleases is suggestive of the occurrence of both methyladenine and methylcytosine in the genome of all the mycobacteriophages studied. Heterologous annealing among the 14 DNAs analyzed yielded 6 hybridization groups. Within one group, the homology level among viral genomes was estimated by comparing the electrophoretic mobilities of restriction fragments: values of 0.8 to 1.3% base substitution have thus been found. A comparison of the genomic characteristics and host range of the mycobacteriophages analyzed suggests a possible relationship between restriction pattern, G+C content, crosshybridization level and host range.