Electrophoretic analysis of ITS from Piscirickettsia salmonis Chilean isolates.

Piscirickettsia salmonis is the most important pathogen in salmonid mariculture in Chile. Since it was reported numerous piscirickettsiosis outbreaks have occurred differing in virulence and mortality. Genetic variability of P. salmonis isolates has been suggested as one factor to explain this. However until now isolates obtained from outbreaks have not been analyzed. Knowledge of genetic variability of P. salmonis is very limited and also a useful screening method for genetic variations in isolates without sequencing is not available. Here we report an electrophoretic analysis of internal transcribed spacer region (ITS) of eleven P. salmonis isolates obtained from different salmon species and places in southern Chile. When PCR products were submitted to polyacrylamide gel electrophoresis (PAGE) a characteristic electrophoretic pattern was observed, distinguishable from ITS of other bacteria, including fish pathogens. Even though this pattern is conserved in all isolates, a difference in ITS electrophoretic mobility was observed, determining clearly two groups: ITS with higher or with lower electrophoretic mobility, including LF-89 and EM-90 isolates, respectively. A higher ITS sequence homology inside each group was shown by heteroduplex mobility assay (HMA). Our results show that genetic variability between Chilean P. salmonis isolates allows the differentiation of two groups with similar behavior observed previously when six P. salmonis isolates from three geographic origins were analyzed by 16S, 23S and ITS sequencing. PAGE analysis of ITS and HMA could be a basis to develop an assay for screening genetic variability between P. salmonis isolates.

tRNA genes were found in Piscirickettsia salmonis 16S-23S rDNA spacer region (ITS).

Piscirickettsia salmonis is the etiological agent of Salmonid Rickettsial Septicemia, a disease affecting salmon aquaculture industry. We analyzed the 16S-23S rDNA spacer region (internal transcribed spacer, ITS) of Chilean P. salmonis isolates LF-89 and EM-90. Two main ITS amplification products were obtained by PCR using L1 and G1 primers, differing from that described where only one ITS region was found. By Southern blot, it was established that these two amplification products contained sequences related to P. salmonis ITS. Sequence analysis confirmed that P. salmonis had two ITS regions: ITS A and ITS B. In both isolates, the smaller (ITS B) corresponded to ITS sequences previously described for each one, and the larger (ITS A) were almost the same as their respective ITS B sequences interrupted by an insert which contained two tRNAs genes: tRNA-Ile and tRNA-Ala.

Inhibitors of infectious pancreatic necrosis virus (IPNV) replication.

In attempts to detect inhibitors of infectious pancreatic necrosis virus (IPNV) replication, we have evaluated, by an IPNV plaque inhibition assay, a group of compounds that have broad spectrum antiviral activity for both single- and double-stranded RNA viruses. The inosine monophosphate dehydrogenase (IMP dehydrogenase) inhibitors 1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide (ribavirin) and 5-ethynyl-1-beta-D-ribofuranosylimidazole-4-carboxamide (EICAR), and the orotidine monophosphate decarboxylase (OMP decarboxylase) inhibitor 4-hydroxy-3-beta-D-ribofuranosylpyrazole-5-carboxamide (pyrazofurin), were found to inhibit IPNV replication. For EICAR and pyrazofurin the concentrations that inhibited the IPNV plaque formation by 50% (EC50) were 0.01 micrograms/ml and 0.5 micrograms/ml, respectively. The cytotoxic concentrations required to reduce cell viability by 50% (CC50) were 50 micrograms/ml and 100 micrograms/ml, respectively, and the concentrations that reduced [methyl-3H] thymidine incorporation by 50% (IC50) were 0.5-1 and 50 micrograms/ml. Thus, for both compounds the IPNV-inhibitory concentration was 50-100 times lower than the concentration that affected DNA synthesis in growing cells. EICAR and pyrazofurin seem to be good candidates for further evaluation in an in vivo model of IPNV infection.

[Evaluation of an immunoenzyme technique (ELISA) to diagnose typhoid fever]. / Evaluación de un ensayo inmunoenzimático (ELISA) para el diagnóstico de la fiebre tifoídea.

The efficiency of an ELISA method, designed to detect polyvalent IgG and IgM antibodies to Salmonella typhi polysaccharide was evaluated in patients admitted or convalescing from typhoid fever and in control subjects. Polyvalent antibodies to S typhi were demonstrated in 28/30 (93%) typhoid patients, 0/11 bacteremic patients (E coli or S paratyphi A) and 0/15 asymptomatic individuals. Widal test showed significant anti-0 agglutinin values (> = 1: 160) in only 12/30 (40%), 1/11 (9%) and 0/15 subjects from each group respectively. Typhoid patients tested on admission or at discharge showed similar high reactivity rates to ELISA. On the contrary, the Widal test detected only 2/15 (13%) patients on admission (p < 0.02) and 10/15 (67%) at discharge. These results and additional immunoblotting tests suggest that ELISA developed to detect polyvalent anti-LPS antibodies could represent a highly specific diagnostic tool to confirm typhoid fever in endemic areas.

[The IgG antibody response in patients colonized by Helicobacter pylori]. / Respuesta de anticuerpos IgG en pacientes colonizados por Helicobacter pylori.

The IgG antibody response specific to Helicobacter pylori was evaluated through ELISA in a group of 92 gastric patients colonized by this bacteria. 74 had gastritis and 19 gastroduodenal ulcer. Three control groups were studied in a similar way: normal adult volunteers (n = 17), adults with E coli or S typhi bacteremia (n = 30) and normal infants (n = 30). IgG antibody response to H pylori was demonstrated in 98% of colonized patients and 0% of infants. Asymptomatic individuals and those with bacteremia had high rates of antibody response (76 and 90% respectively), although this rate and also the titers of antibody response were significantly lower than that of colonized patients (p < 0.05). ELISA reactive sera from colonized patients and asymptomatic individuals evidenced a similar antibody pattern when tested by blotting. This profile was absent in non reactive sera, including those with high antibody titers to C jejuni. The presence of specific IgG antibodies to H pylori in the majority of colonized gastric patients and asymptomatic adults suggests that this infection is very common in our population.

Respuesta de anticuerpos IgG en pacientes colonizados por helicobacter pylori / Antibodies to helicobacter pylori in colonized patients with gastric symptoms and in asymptomatic groups

The IgG antibody response specific to Helicobacter pylori was evaluated through ELISA in a group of 92 gastric patients colonized by this bacteria. 74 had gastritis and 19 gastroduodenal ulcer. Three control groups were studied in a similary way: normal adult volunteers (n=17), adults with E coli or S typhi bacteremia (n=30) and normal infants (n = 30). IgG antibody response to H pylori was demonstrated in 98% of colonized patients and 0% of infants. Asymptomatic individuals and those with bacteremia had high rates of antibody response (76 and 90% respectively), although this rate and also the titers of antibody response were significantly lower than that of colonized patients (p < 0.05). ELISA reactive sera from colonized patients and asymptomatic individuals evidenced a similar antibody pattern when tested by blotting. This profile was absent in non reactive sera, including those with high antibody titers to C jejuni. The presence of specific IgG antibodies to H pylory in the majority of colonized gastric patients and asymptomatic adults suggest that this infection is very common in our population