Detection of Cryptosporidium and Giardia in molluscan shellfish by multiplexed nested-PCR.
A multiplexed nested-PCR procedure (ABC-PCR) previously developed to detect Cryptosporidium spp. and Giardia duodenalis assemblages A and B in whole human faeces was applied to DNA extracted from filter-feeding molluscs. Species of Cryptosporidium and G. duodenalis were identified by restriction fragment analysis of the PCR products and by DNA sequencing. The extraction and ABC-PCR procedures were shown to be suitable for application to shellfish by amplification of specific target sequences using DNA from Cryptosporidium parvum genotype 2 and G. duodenalis assemblages A and B which were spiked into DNA extracted from mussels. Using 49 molluscan shellfish specimens (18 clam, 22 mussel and 9 oyster samples) from Spain, cryptosporidial oocysts were detected in 56% by immunofluorescence microscopy, and in 44% by ABC-PCR. For detection of Cryptosporidium, there was a significant association, but not total agreement, between the results of microscopy and PCR. G. duodenalis assemblage B was detected from one oyster sample by PCR. Amongst 38 specimens (20 mussel and 18 cockle samples) collected in the UK and tested by the ABC-PCR, G. duodenalis was not detected, and Cryptosporidium was detected in 11% of the samples. Overall, the 26 samples where Cryptosporidium was detected, C. hominis/C. parvum genotype 1 was detected in 1, C. parvum genotype 2 in 22, and the remaining three samples contained either sequences similar to C. parvum genotype 2 or heterogeneous mixtures of Cryptosporidium species. There was no significant association between the level of Escherichia coli detected by conventional microbiological methods and the presence of Cryptosporidium detected by ABC-PCR.
Gómez-Couso
H
H
Laboratorio de Parasitología, Departamento de Microbiología y Parasitología/Instituto de Acuicultura, Universidad de Santiago de Compostela, 15782 Santiago de Compostela, A Coruña, Spain.
Freire-Santos
F
F
Amar
C F L
CF
Grant
K A
KA
Williamson
K
K
Ares-Mazás
M E
ME
McLauchlin
J
J
D000818:Animals; D003257:Consumer Product Safety; D003458:Cryptosporidium / Q000235:genetics / Q000302:isolation & purification*; D016054:DNA, Protozoan / Q000032:analysis*; D005516:Food Microbiology; D005784:Gene Amplification; D005838:Genotype; D005872:Giardia / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D008974:Mollusca / Q000382:microbiology*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012150:Polymorphism, Restriction Fragment Length; D012680:Sensitivity and Specificity; D012758:Shellfish / Q000382:microbiology
D016054:DNA, Protozoan