ABSTRACT
The long-term storage of Cryptosporidium life-cycle stages is a prerequisite for in vitro culture of the parasite. Cryptosporidium parvum oocysts, sporozoites, and intracellular forms inside infected host cells were stored for 6-12 mo in liquid nitrogen utilizing different cryoprotectants (dimethyl sulfoxide [DMSO], glycerol and fetal calf serum [FCS]), then cultured in vitro. Performance in vitro was quantified by estimating the total Cryptosporidium copy number with quantitative polymerase chain reaction (qPCR) in 3- and 7-day-old cultures. Although few parasites were recovered either from stored oocysts or from infected host cells, sporozoites stored in liquid nitrogen recovered from freezing successfully. More copies of parasite DNA were obtained from culturing those sporozoites than sporozoites excysted from oocysts kept at 4 C for the same period. The best performance was observed for sporozoites stored in Roswell Park Memorial Institute (RPMI) medium with 10% FCS and 5% DMSO, which generated 240% and 330% greater number of parasite DNA copies (on days 3 and 7 post-infection, respectively) compared to controls. Storage of sporozoites in liquid nitrogen is more effective than oocyst storage at 4 C and represents a more consistent approach for storage of viable infective Cryptosporidium aliquots for in vitro culture.
Subject(s)
Cryopreservation/standards , Cryptosporidium parvum/physiology , Animals , Cattle , Cell Line , Cryopreservation/methods , Cryoprotective Agents/standards , Cryptosporidium parvum/genetics , Cryptosporidium parvum/growth & development , Culture Media , DNA, Protozoan/isolation & purification , Dimethyl Sulfoxide/standards , Gene Dosage , Glycerol/standards , Humans , Life Cycle Stages , Nitrogen , Real-Time Polymerase Chain Reaction , Serum , Time FactorsABSTRACT
Viability estimation of the highly resistant oocysts of Cryptosporidium remains a key issue for the monitoring and control of this pathogen. We present here a simple 'one tube' quantitative PCR (qPCR) protocol for viability estimation using a DNA extraction protocol which preferentially solubilizes excysted sporozoites rather than oocysts. Parasite DNA released from excysted sporozoites was quantified by real-time qPCR using a ribosomal DNA marker. The qPCR signal was directly proportional to the number of oocysts excysted, and a power-law relationship was noted between oocyst age and the proportion excysting. Unexcysted oocysts released negligible amounts of DNA making the method suitable for estimating viability of as few as 10 oocysts.
Subject(s)
Cryptosporidiosis/parasitology , Cryptosporidium parvum/physiology , Real-Time Polymerase Chain Reaction/methods , Animals , Biomarkers/analysis , Cryptosporidium parvum/genetics , Cryptosporidium parvum/isolation & purification , DNA, Protozoan/analysis , DNA, Protozoan/genetics , DNA, Ribosomal/analysis , Oocysts , SporozoitesABSTRACT
Heligmosomoides americanus is shown by molecular phylogenetic analysis of 3 nuclear (28S, ITS1, and ITS2) and 2 mitochondrial (cytochrome oxidase 1 and cytochrome b) loci to be a distinct species of heligmosomid nematode with a long-independent evolutionary history, and not a subspecies of Heligmosomoides polygyrus . Rather than being a recent arrival in North America, the species probably originated as a Beringian immigrant with the host vole Phenacomys, approximately 2 million years ago (MYA).
Subject(s)
Arvicolinae/parasitology , Rodent Diseases/parasitology , Trichostrongyloidea/classification , Trichostrongyloidiasis/veterinary , Animals , British Columbia , Cytochromes b/genetics , DNA, Ribosomal Spacer/genetics , Electron Transport Complex IV/genetics , Genetic Markers , Molecular Sequence Data , Montana , Phylogeny , RNA, Ribosomal, 28S/genetics , Sequence Alignment/veterinary , Trichostrongyloidea/anatomy & histology , Trichostrongyloidea/genetics , Trichostrongyloidiasis/parasitologyABSTRACT
PURPOSE: The aims of this study were: (1) to estimate Babesia prevalence in the most common species of tick in Poland, Ixodes ricinus, in two recreational areas (Urwitalt in the Mazury Lake District and Bielanski Forest in Warsaw), and (2) to evaluate the molecular diversity of Babesia isolates in questing I. ricinus in Poland. MATERIAL AND METHODS: Questing ticks were collected from vegetation in forest areas in Urwitalt near Mikolajki and in Bielanski Forest (Warsaw). Purified genomic DNA was used with specific primers to amplify a fragment of the Babesia spp. 18S rRNA gene. RESULTS: Tick-drag indices for I. ricinus were high in both study areas, reaching somewhat higher values in Urwitalt than in Bielanski Forest. The overall prevalence of Babesia spp. in examined ticks was 1.6%. In Urwitalt, two strains of B. microti were identified using rRNA sequences: the enzootic Munich strain and an isolate close to the zoonotic Jena strain. The proportion of infections due to these two strains in questing ticks reversed over a six-year period. During 3 years of study in Bielanski Forest, all Babesia isolates obtained from I. ricinus were identical to Babesia sp. EU1 (B. venatorum), previously recognized as an agent of human babesiosis. CONCLUSIONS: This study has confirmed the presence of enzoonotic and zoonotic Babesia species/strains in the abundant human-biting tick I. ricinus in recreational areas in Poland. It has also shown that the distribution of different genotypes has changed over time, however the reasons for these fluctuations still remain to be investigated.
