An intronless gene encoding a protein of 674 amino acid residues with a molecular mass of 73,403 Da showing homology to the cytoplasmic form of the 70 kDa heat shock proteins has been cloned and sequenced from the intestinal pathogen Cryptosporidium parvum. Monospecific polyclonal antibodies obtained to recombinant protein recognized a single band with an approximate molecular mass of 70 kDa on a Western blot of C. parvum proteins, as well as the 70 kDa heat shock protein from bovine brain. Southern blot analysis suggested the gene was single copy in the C. parvum genome. Eleven perfect repeats of the sequence GGMP were found in the predicted protein near the carboxyl terminus.

We report improved separation of chromosome-sized DNA molecules of the coccidian parasite Cryptosporidium parvum with contour-clamped homogeneous electric fields (CHEF). We used scanning densitometry to determine that the most likely number of chromosomes is eight. Molecular probes consisting of cloned genes were used to distinguish each of five bands visible on CHEF gels. We have also identified a low-molecular-size DNA molecule possibly related to the 35-kb circular DNAs found in other Apicomplexa.

We determined the nucleotide (nt) sequence of the putative gene encoding acetyl-coenzyme A synthetase (ACS) from the parasitic protozoan Cryptosporidium parvum. The gene is single copy, located on a chromosome of approximately 1.08 mb, and has no introns. The gene is characterized by low codon usage bias and encodes a 694-amino acid (aa) protein with a predicted molecular size of 78 kDa, similar to other ACSs from different prokaryotic and eukaryotic species. Comparison of multiple protein alignments of ACSs revealed a new conserved sequence motif PKT(R/V/L)SGK(I/V/T)(T/M/V/K)R(R/N) near the C-terminus, which may be a signature for ACSs. This motif shares significant homology with sequences from other members of the AMP-binding family, has secondary structure similar to the purine-binding motif of ATP- and GTP-ases, and may play a role in the enzymatic activity of proteins from the AMP-binding family.

Methods for killing Echinococcus multilocularis eggs within stool or intestinal samples, without damaging the diagnostic value of the sample, would significantly reduce the risk of animal health providers acquiring alveolar hydatid disease. The first objective of this study was to determine whether E. multilocularis eggs located in fox intestines can survive storage at -70 C for at least 4 days. Results showed that none of 72,000 E. multilocularis eggs remained infectious to defined strains of mice under these conditions, yet, similar eggs recovered from nonfrozen carcasses stored at 4 C for the same time period were viable. The structural identities of adult worms and eggs were not significantly altered by the freezing and thawing processes. These results indicate that ultracold temperatures can be used to kill or inactivate E. multilocularis eggs, making them safe to handle when diagnosing this parasite in definitive hosts. The second objective of this study was to determine whether E. multilocularis eggs could survive freezing to -70 C if commonly used cryopreservation protocols were used. The use of the cryoprotectant solution, 5% dimethyl sulfoxide-35% saline-60% lamb serum, with a -1 C/min freezing rate was unable to prevent the eggs from being killed by freezing to -70 C. Rapid cooling by plunge freezing into liquid nitrogen was also lethal to E. multilocularis eggs. Only a few of the many potential cryopreservation protocols were tested in this study, so it is not yet possible to completely rule out the possibility of preserving these eggs at ultralow temperatures, but it does indicate that temperatures below -70 C are lethal to eggs even under favorable storage conditions.

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