Differentiation between Human and Animal Isolates of Cryptosporidium parvum Using rDNA Sequencing and Direct PCR Analysis

During 1996 and 1997, a new and damaging disease of leek (Allium porrum) was observed on greenhouse-produced transplants and field-grown plants in California. Symptoms were water-soaked lesions at leaf tips, which eventually expanded down the length of the leaf and resulted in brown, elongated, stripe-like lesions with yellow margins. Diseased leaves eventually wilted. A blue fluorescent pseudomonad was consistently recovered from lesions, and biochemical and physiological tests indicated that it was Pseudomonas syringae. Pathogenicity tests established that representative strains of this P. syringae induced disease symptoms in leek that were similar to those observed on leek plants in the greenhouse and field, and that this bacterium caused similar symptoms in onion, chives, and garlic plants. Representative strains were further characterized by fatty acid analysis, repetitive bacterial sequence-polymerase chain reaction (rep-PCR), and rDNA sequencing. Fatty acid analysis confirmed that these isolates were P. syringae, but did not provide a clear pathovar designation. Rep-PCR analysis revealed that all the California leek P. syringae strains had identical DNA fingerprints and that these strains were indistinguishable from those of known strains of P. syringae pv. porri. In addition, the rDNA sequence of the spacer region between 16S and 23S rDNA genes was identical among the California leek P. syringae strains and P. syringae pv. porri. Together, these results established that the new leek disease in California is caused by P. syringae pv. porri. P. syringae pv. porri was recovered from a commercial leek seed lot imported into California, which suggests that the pathogen was introduced in association with seed. Commercial leek production in California is favorable for development of this disease because transplants are produced in greenhouses with high plant densities, overhead irrigation, and mowing of plants.

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Role of CpSUB1, a Subtilisin-Like Protease, in Cryptosporidium parvum Infection In Vitro

Eukaryotic Cell ◽

10.1128/ec.00306-08 ◽

2009 ◽

Vol 8 (4) ◽

pp. 470-477 ◽

Cited By ~ 27

Author(s):

Jane W. Wanyiri ◽

Patsharaporn Techasintana ◽

Roberta M. O'Connor ◽

Michael J. Blackman ◽

Kami Kim ◽

...

Keyword(s):

Serine Protease ◽

Cryptosporidium Parvum ◽

Protease Activity ◽

Diarrheal Disease ◽

Genomic Sequence ◽

Host Cells ◽

Pcr Analysis ◽

Apical Region ◽

Gene Encoding

ABSTRACTThe apicomplexan parasiteCryptosporidiumis a significant cause of diarrheal disease worldwide. Previously, we reported that aCryptosporidium parvumsubtilisin-like serine protease activity with furin-type specificity cleaves gp40/15, a glycoprotein that is proteolytically processed into gp40 and gp15, which are implicated in mediating infection of host cells. Neither the enzyme(s) responsible for the protease activity inC. parvumlysates nor those that process gp40/15 are known. There are no furin or other proprotein convertase genes in theC. parvumgenome. However, a gene encoding CpSUB1, a subtilisin-like serine protease, is present. In this study, we cloned the CpSUB1 genomic sequence and expressed and purified the recombinant prodomain. Reverse transcriptase PCR analysis of RNA fromC. parvum-infected HCT-8 cells revealed that CpSUB1 is expressed throughout infection in vitro. In immunoblots, antiserum to the recombinant CpSUB1 prodomain revealed two major bands, of ∼64 kDa and ∼48 kDa, forC. parvumlysates and proteins “shed” during excystation. In immunofluorescence assays, the antiserum reacted with the apical region of sporozoites and merozoites. The recombinant prodomain inhibited protease activity and processing of recombinant gp40/15 byC. parvumlysates but not by furin. Since prodomains are often selective inhibitors of their cognate enzymes, these results suggest that CpSUB1 may be a likely candidate for the protease activity inC. parvumand for processing of gp40/15. Importantly, the recombinant prodomain inhibitedC. parvuminfection of HCT-8 cells. These studies indicate that CpSUB1 plays a significant role in infection of host cells by the parasite and suggest that this enzyme may serve as a target for intervention.

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Reverse Transcriptase Activity in Mature Spermatozoa of Mouse

The Journal of Cell Biology ◽

10.1083/jcb.148.6.1107 ◽

2000 ◽

Vol 148 (6) ◽

pp. 1107-1114 ◽

Cited By ~ 70

Author(s):

Roberto Giordano ◽

Anna Rosa Magnano ◽

Germana Zaccagnini ◽

Carmine Pittoggi ◽

Nicola Moscufo ◽

...

Keyword(s):

Reverse Transcriptase ◽

Pcr Amplification ◽

Reverse Transcriptase Activity ◽

Pcr Analysis ◽

Immunogold Electron Microscopy ◽

Direct Pcr ◽

Vitro Fertilization ◽

Rna Molecules ◽

Exogenous Rna

We show here that a reverse transcriptase (RT) activity is present in murine epididymal spermatozoa. Sperm cells incubated with human poliovirus RNA can take up exogenous RNA molecules and internalize them in nuclei. Direct PCR amplification of DNA extracted from RNA-incubated spermatozoa indicate that poliovirus RNA is reverse-transcribed in cDNA fragments. PCR analysis of two-cell embryos shows that poliovirus RNA-challenged spermatozoa transfer retrotranscribed cDNA molecules into eggs during in vitro fertilization. Finally, RT molecules can be visualized on sperm nuclear scaffolds by immunogold electron microscopy. These results, therefore, reveal a novel metabolic function in spermatozoa, which may play a role during early embryonic development.

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Differentiating Human from Animal Isolates of Cryptosporidium parvum

Emerging Infectious Diseases ◽

10.3201/eid0404.980424 ◽

1998 ◽

Vol 4 (4) ◽

pp. 681-685 ◽

Cited By ~ 108

Author(s):

Irshad Sulaiman

Keyword(s):

Cryptosporidium Parvum ◽

Animal Isolates

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Method Detection Limits of PCR and Immunofluorescence Assay for Cryptosporidium parvum in Soil

Applied and Environmental Microbiology ◽

10.1128/aem.64.6.2281-2283.1998 ◽

1998 ◽

Vol 64 (6) ◽

pp. 2281-2283 ◽

Cited By ~ 14

Author(s):

Mark J. Walker ◽

Carlo Montemagno ◽

Juliet C. Bryant ◽

William C. Ghiorse

Keyword(s):

Cryptosporidium Parvum ◽

Detection Limits ◽

Immunofluorescence Assay ◽

Pcr Analysis ◽

Quantitative Nature

We determined and compared the method detection limits (MDLα) of a PCR and an immunofluorescence assay (IFA) for detection of Cryptosporidium parvum oocysts in soils. Based on the MDLα and the quantitative nature and stability of the IFA, PCR analysis is not a useful screening step for soil studies of oocyst transport.

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