Failure to differentiate Cryptosporidium parvum from C. meleagridis based on PCR amplification of eight DNA sequences

Abstract

In order to determine the specificities of PCR-based assays used for detecting Cryptosporidium parvum DNA, eight pairs of previously described PCR primers targeting six distinct regions of the Cryptosporidium genome were evaluated for the detection of C. parvum, the agent of human cryptosporidiosis, and C. muris, C. baileyi, and C. meleagridis, three Cryptosporidium species that infect birds or mammals but are not considered to be human pathogens. The four Cryptosporidium species were divided into two groups: C. parvum and C. meleagridis, which gave the same-sized fragments with all the reactions, and C. muris and C. baileyi, which gave positive results with primer pairs targeting the 18S rRNA gene only. In addition to being genetically similar at each of the eight loci analyzed by DNA amplification, C. parvum and C. meleagridis couldn't be differentiated even after restriction enzyme digestion of the PCR products obtained from three of the target genes. This study indicates that caution should be exercised in the interpretation of data from water sample analysis performed by these methods, since a positive result does not necessarily reflect a contamination by the human pathogen C. parvum.

FIG. 1

Ethidium bromide-stained agarose gels of PCR products amplified from C. parvum, C. muris, C. baileyi, and C. meleagridis DNA. Size markers are HaeIII-digested ΦX174; arrowheads in all panels point to the 603-bp fragment. (A) Amplification of a 435-bp product of the 18S rRNA gene (method of Johnson [13]). Lanes: 1, C. parvum; 2, C. meleagridis; 3, C. muris; 4, C. baileyi isolate B1; 5, negative control. (B) Amplification of a 556-bp product of the 18S rRNA gene (Awad-El-Kariem [1]). Lanes: 1, C. parvum; 2, negative control; 3, C. meleagridis; 4, C. baileyi isolate B1; 5, C. baileyi isolate O.96.2; 6, C. muris. (C) Electrophoresis of PCR products obtained in the experiment shown in panel B, prior to and after MaeI treatment. As expected (1), incomplete digestion occurred. Lanes: 1, C. parvum; 2, same sample as in lane 1 but after restriction with MaeI; 3, C. meleagridis; 4, same sample as in lane 3 but after restriction with MaeI. (D) Amplification of a 452-bp product of an undefined gene (Laxer [15]). Lanes: 1, C. parvum; 2, C. meleagridis; 3 and 4, C. muris; 5 and 6, C. baileyi isolate B1; 7, negative control. In lanes 4 and 6, C. parvum DNA was added to the reaction mixtures prior to amplification. (E) Amplification of a 898-bp fragment from the CpR1 gene (Wagner-Wiening [28]). Lanes: 1, C. parvum; 2, negative control; 3, C. meleagridis; 4 and 5, C. baileyi isolate B1; 6 and 7, C. baileyi isolate O.96.2; 8 and 9, C. muris. For lanes 5, 7, and 9, C. parvum DNA was added to the reaction mixtures prior to amplification. (F) Electrophoresis of PCR products obtained in the experiment represented in panel E prior to and after treatment with BamHI. Lanes: 1, C. parvum; 2, same sample as in lane 1 but after restriction with BamHI; 3, C. meleagridis; 4, same sample as in lane 3 but after restriction with BamHI. (G) Amplification of a 358-bp fragment of the CpR1 gene (Laberge [14]). Lanes: 1, C. parvum; 2, C. meleagridis; 3 and 4, C. muris; 5 and 6, C. baileyi isolate B1; 7, negative control. For lanes 4 and 6, C. parvum DNA was added to the reaction mixtures prior to amplification. (H) Amplification of a 1,500-bp fragment from an undefined DNA region (Bonnin [3]). Lanes: 1, C. parvum; 2, C. meleagridis; 3 and 4, C. muris; 5 and 6, C. baileyi isolate B1; 7, negative control. For lanes 4 and 6, C. parvum DNA was added to the reaction mixtures prior to amplification. (I) Electrophoresis of PCR products obtained from the 1,500-bp fragment shown in panel H prior to and after treatment with HinfI and RsaI. Lanes: 1, C. parvum; 2 and 3, same product as in lane 1 but after digestion with HinfI and RsaI, respectively; 4, C. meleagridis; 5 and 6, same product as in lane 4 after digestion with HinfI and RsaI, respectively. (J) Amplification of a 680-bp fragment from an undefined gene (Morgan [18]). Lanes: 1, C. parvum; 2, C. meleagridis; 3 and 4, C. muris; 5 and 6, C. baileyi isolate B1; 7 and 8, C. baileyi isolate O.96.2; 9, negative control. For lanes 4, 6, and 8, C. parvum DNA was added to the reaction mixtures prior to amplification. (K) Amplification of a 361-bp fragment of the Hsp70 gene (Rochelle [24]). Lanes: 1, C. parvum; 2, C. meleagridis; 3 and 4, C. muris; 5 and 6, C. baileyi isolate B1; 7 and 8, C. baileyi isolate O.96.2; 9, negative control. For lanes 4, 6, and 8, C. parvum DNA was added to the reaction mixtures prior to amplification.