Multiple-locus variable-number tandem repeat analysis (MLVA) of Leptospira interrogans serovar Pomona from Argentina reveals four new genotypes

Outbreaks of leptospirosis occur regularly in Argentina, but little is known about their epidemiological relationships. We have analyzed the genetic diversity of a collection of 16 strains of Leptospira interrogans serovar Pomona isolated from animals and humans in Argentina during the past 45 years. Genotyping was performed by multiple-locus variable-number tandem repeat analysis (MLVA) using the loci VNTR4, VNTR7, VNTR9, VNTR10, VNTR19, VNTR23 and VNTR31, as described by Majed et al. [Identification of variable-number tandem-repeat loci in Leptospira interrogans sensu stricto. J Clin Microbiol 2005;43:539-45]. Clustering analysis revealed four new distinct MLVA genotypes, with a dominant one. Strains with this genotype were consistently isolated since 1960 to the present, mainly from cows and pigs, but also from humans, representing 75% of the total strains studied. These strains coexisted temporally and geographically with isolates presenting the other new genotypes. VNTR4 locus, with four different alleles, presented the highest diversity between the VNTR loci analyzed. MLVA patterns obtained will be useful for future diagnostic and epidemiological tracing analysis.     

Detection of a Phytoplasma Associated with Grapevine Flavescence dorée in Clematis vitalba

About 40 different species of wild herbaceous and woody plants were collected in underbrush close to a vineyard where Flavescence dorée (FD) has been reported for several years. Polymerase chain reaction assays were carried out using DNA extracted from leaves of each species for the detection of the presence of phytoplasmas. Only samples of Clematis vitalba were found to be infected with phytoplasmas. Restriction fragment length polymorphism and sequencing data of the 16S ribosomal RNA gene and of a non-ribosomal DNA fragment FD9 revealed that the phytoplasma isolate was identical to that causing FD in the nearby vineyard. The isolate identified in the clematis is the same as the FD-C phytoplasma found in grapevine in north-east Italy.     

Pharmacokinetics and milk penetration of moxifloxacin after intramuscular administration to lactating goats

The pharmacokinetics of moxifloxacin was studied following intramuscular administration of 5mg/kg to healthy lactating goats (n=6). Moxifloxacin concentrations were determined by high performance liquid chromatography assay with fluorescence detection. The moxifloxacin plasma concentration versus time data could best be described by a one-compartment model. The plasma moxifloxacin clearance (Cl) was mean standard deviation (+/-SD) 0.49+/-0.14 L/h kg. The apparent volume of distribution (V(z)) was 0.83+/-0.20 L/kg. The terminal half-life (t(1/2 lambda z)) was 1.31+/-0.64 h. Moxifloxacin penetration from blood to milk was rapid and the high AUC(milk)/AUC(plasma) and C(max-milk)/C(max-plasma) ratios reached indicated a good penetration of moxifloxacin into the milk.     

Babesia bovis contains an abundant parasite-specific protein-free glycerophosphatidylinositol and the genes predicted for its assembly

Autonomous glycosylphosphatidylinositol (GPI) molecules (also protein-free GPIs or free GPIs) have been reported to be particularly abundant in some parasitic protozoa and mediate strong immunomodulatory effects on the host immune system. In the work at hand we have investigated the existence of free GPIs in Babesia bovis. Comparative thin layer chromatographic analysis of the protein-free glycolipid fraction of in vitro cultured B. bovis merozoites and erythrocyte membranes demonstrated the presence of an abundant parasite-specific band. Its chemical analysis revealed a GPI species containing a chain of two mannose residues, N-glucosamine and non-acylated inositol. The lipid moiety linked to inositol was diacylglycerol. The total fatty acid composition showed predominantly long-carbon chain molecules (12% of C_22:0 and 45% of C_24:0). The potential of B. bovis to assemble the presented free GPI species was verified by the existence of seven genes in its genome that putatively encode the following GPI biosynthetic enzymes: PI N-acetyl-GlcN-transferase (PIG-A and GPI-1), N-acetyl-GlcN-PI-de-N-acetylase (PIG-L), acyltransferase (PIG-W), dolichyl-phosphate mannosyl transferase (DPM-1), GPI mannosyltransferase I (PIG-M), and GPI mannosyltransferase II (PIG-V). GPI biosynthesis is vital for the intraerythrocytic parasite stage as mannosamine, an inhibitor of GPI biosynthesis, impaired in vitro growth of B. bovis merozoites. Absence of the vast majority of N-glycan metabolism encoding genes in the B. bovis genome underscores that the growth inhibitory effect of mannosamine is attributable to its interference with GPI biosynthesis and not with assembly of N-linked oligosaccharides, as has been described for higher eukaryotes. Elucidation of the structure and biosynthesis of GPI may allow to facilitate the development of future immune interventions against bovine babesiosis.     

The activity of a selected extract of <Emphasis Type="Italic">Drimys winteri</Emphasis> bark and polygodial on settling and probing behavior of the lettuce aphid <Emphasis Type="Italic">Nasonovia ribisnigri</Emphasis>

An n-hexane extract and the sesquiterpene polygodial isolated from the bark of Drimys winteri J.R. Forster & G. Forster (Winteraceae) were tested to assess their ability to inhibit settling of the aphid Nasonovia ribisnigri Mosley on lettuce leaves; assessments were made using a choice and no-choice leaf-disk assay. The n-hexane extract and the polygodial both significantly inhibited settling in the choice and no-choice tests, but their application on lettuce leaves did not result in any aphid mortality. Probing behavior of N. ribisnigri on polygodial-treated plants was assessed by means of the electrical penetration graph technique. Polygodial interfered with aphid probing behavior by reducing the total probing time, the number of probes, and the number of intracellular punctures. Aphids did not reach the sieve elements of polygodial-treated plants during the 2-h access period. Both tested compounds have the potential to be used in the development of lettuce aphid control agents to reduce the risk of virus transmission.     

Purification and characterization of chymotrypsin from viscera of vermiculated sailfin catfish, Pterygoplichthys disjunctivus, Weber, 1991

Pterygoplichthys disjunctivus viscera chymotrypsin was purified by fractionation with ammonium sulfate (30–70 % saturation), gel filtration, affinity, and ion exchange chromatography. Chymotrypsin molecular weight was approximately 29 kDa according to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), shown a single band in zymogram. Electrofocusing study suggested being an anionic enzyme (pI ≈ 3.9), exhibiting maximal activity at pH 9 and 50 °C, using Suc-Ala-Ala-Pro-Phe-p-nitroanilide (SAAPNA) as substrate. Enzyme was effectively inhibited by phenyl methyl sulfonyl fluoride (PMSF) (99 %), and N-tosyl-l-phenylalanine chloromethyl ketone (TPCK) (94 %). Enzyme activity was affected by the following ions in decreasing order: Hg²⁺, Fe²⁺, Cu²⁺, Li¹⁺, Mg²⁺, K¹⁺, Mn²⁺, while Ca²⁺ had no effect. Chymotrypsin activity decreased continuously as NaCl concentration increased (from 0 to 30 %). K _m and V _max values were 0.72 ± 1.4 mM and 1.15 ± 0.06 μmol/min/mg of protein, respectively (SAAPNA as substrate). Results suggest the enzyme has a potential application where low processing temperatures are needed, such as in fish sauce production.