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Volume 7, Number 7—June 2001


Waterborne Outbreak of Tularemia Associated with Crayfish Fishing

Pedro Anda*Comments to Author , Javier Segura del Pozo*†, José María Díaz García‡, Raquel Escudero*, F. Javier García Peña§, M. Carmen López Velasco‡, Ricela E. Sellek*, M. Rosario Jiménez Chillarón‡, Luisa P. Sánchez Serrano*, and J. Fernando Martínez Navarro*
Author affiliations: *Instituto de Salud Carlos III, Majadahonda, Madrid, Spain; †Public Health Department, Alcalá de Henares, Madrid, Spain; ‡Cuenca Public Health Department, Cuenca, Spain; §Ministry of Agriculture and Fisheries, Algete, Madrid, Spain

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Table 1

Polymerase chain reaction results of crayfish and water samples tested for Francisella tularensisa

Crayfish, Batch A
Crayfish, Batch B
Water SP
Water R
B S H G W B S H G W I 1 2 3 1-8 LNA
- + + - - - - - - - - + - 3 - +

Batch A = crayfish collected from a patient's house; Batch B = crayfish collected from the river; SP = sewage plant; R = river; LNA = lymph node aspirate; B = branchia; S = stomach; G = gonads; H = hepatopancreas; W = washed pellet; I = intestines.
aSeveral annealing temperatures were tested, from 60°C to 68°C for the 25 cycles of the first round. The second round consisted of 45 cycles with an annealing temperature of 60°C. The rest of the parameters were as described (21). All reagents were from Perkin Elmer (Foster City, CA); the cycling was done in a PCT-100 thermocycler (MJ Research Inc., Watertown, MA). The PCR products, with a size of 1550 bp and 950 bp for the first and second rounds of amplification, respectively, were visualized in 1% low-melt agarose gels (Pronadisa, Alcobendas, Madrid, Spain) stained with ethidium bromide (Sigma-Aldrich). Cross-contamination was avoided by using standard methods. Half the samples studied were kept unprocessed and frozen in a different area. A positive result was confirmed by processing the rest of the sample. To assess the sensitivity of our system, 10 L of serial twofold dilutions of an aqueous suspension of bacteria, ranging from 104 CFU to 102 mL was added to 1-mL volumes of distilled water. These samples were subjected to DNA extraction and PCR as above. Negative controls (autoclaved distilled H2O) were included in all extractions at a ratio of a negative control for each five samples. The specificity was checked against DNA from Salmonella Typhimurium, Escherichia coli, Legionella pneumophila, Yersinia enterocolitica, and Proteus vulgaris.

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