High-resolution electrophoretic procedures for the identification of fiveEimeria species from chickens, and detection of population variation

Woods, Wayne G.; Richards, Grant; Whithear, Kevin G.; Anderson, Glenn R.; Jorgensen, W.K.; Gasser, Robin B.

doi:10.1002/1522-2683(200011)21:17<3558::aid-elps3558>3.0.co;2-2

Cited by 29 publications

(24 citation statements)

References 17 publications

(23 reference statements)

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“…The smallest amount of C. parvum genomic DNA detectable by amplification with primer set 18SiF-18SiR (pSSU) or YA56F-YA54R (pITS-2) and subsequent detection on agarose gels was estimated at 1 to 2 pg, which is comparable to that in a previous study of Eimeria species (Protozoa: family Eimeriidae) (35).…”

Section: Resultssupporting

confidence: 72%

“…While the subgenotypic classification of the type 2 samples by DPGE was the same as that for SSCP analysis, ϳ30% less profile variation (9 instead 13 profiles) was detectable among the type 1 samples, which is attributable to a lack of length (rather than sequence) variation among some of the samples. These results reinforced the superior ability of SSCP to detect subtle nucleotide variation among molecules of the same length within an amplicon (13,35) and reflected the finding of increased sequence and length variability in pITS-2 within and among type 2 samples compared with type 1.…”

Section: Vol 69 2003supporting

confidence: 63%

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Electrophoretic Analysis of Genetic Variability within Cryptosporidium parvum from Imported and Autochthonous Cases of Human Cryptosporidiosis in the United Kingdom

Gasser

EL-Osta

Chalmers

2003

Appl Environ Microbiol

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Cryptosporidium parvum oocyst DNA samples (n ‫؍‬ 184) from humans with cryptosporidiosis contracted during foreign travel or during outbreaks in the United Kingdom were characterized genetically and categorized by single-strand conformation polymorphism (SSCP)-based analysis of the small-subunit gene (pSSU) (ϳ300 bp) and second internal transcribed spacer (pITS-2) (ϳ230 bp) of nuclear ribosomal DNA. The two recognized genotypes (types 1 and 2) of C. parvum could be readily differentiated by a distinct electrophoretic shift in the pSSU SSCP profile, associated with a nucleotide difference of ϳ1.3 to 1.7%. Of the 102 samples from cases contracted during foreign travel, 88 (86.3%) were identified as C. parvum type 1 and 14 (13.7%) were identified as type 2. For outbreak samples, unequivocal differentiation between type 1 (n ‫؍‬ 20; one child nursery outbreak) and type 2 (n ‫؍‬ 62; two waterborne outbreaks) was also achieved. Nucleotide variation in pITS-2 (both within and among samples representing each genotype) was substantially greater (10 to 13 different profiles for each genotype, relating to sequence differences of ϳ1 to 42%) than that in pSSU. SSCP analysis of pITS-2 for all samples revealed that some profiles had a broad geographical distribution whereas others were restricted to particular locations, suggesting a link between some subgenotypes and the geographical origin or source. Comparative denaturing polyacrylamide gel electrophoretic analysis revealed the same genotypic identification and a similar subgenotypic classification of samples as SSCP analysis. The findings of this study, particularly the detection of intragenotypic variation by SSCP, should have significant diagnostic implications for investigating transmission patterns and the monitoring of outbreaks.Species of Cryptosporidium are oocyst-forming apicomplexan parasites which infect a wide range of vertebrate hosts. Intestinal disease caused by these parasites is called cryptosporidiosis and is transmitted via the fecal-oral route (e.g., in child day care centers) and can be associated with waterborne outbreaks (27). The clinical signs of cryptosporidiosis in humans are mainly diarrhea, dehydration, malabsorption, weight loss, and/or wasting (27). In many cases, infection is self-limiting and resolves spontaneously, although chronic infections may develop in high-risk patient groups, particularly neonates and immunocompromised individuals (those with congenital or acquired immunodeficiencies or those undergoing immunosuppressive chemotherapy). The accurate identification of Cryptosporidium to the species or "strain" (i.e., genotypic) level is important for studying transmission patterns and genetic structures and is central to the control of cryptosporidiosis (9, 26, 34). Currently, 11 species of Cryptosporidium are recognized (9, 10), of which C. parvum is of major significance in humans. However, these species cannot be distinguished from one another simply on the basis of host occurrence and/or parasite morphology (9, 27). Consequently, a...

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Section: Resultssupporting

confidence: 72%

Section: Vol 69 2003supporting

confidence: 63%

Electrophoretic Analysis of Genetic Variability within Cryptosporidium parvum from Imported and Autochthonous Cases of Human Cryptosporidiosis in the United Kingdom

Gasser

EL-Osta

Chalmers

2003

Appl Environ Microbiol

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“…Analysis of internal transcribed spacers (ITS-1 and ITS-2) permits discrimination of all species of the genus Eimeria, using polymerase chain reaction (PCR) or real-time PCR (Tsuji et al, 1997;Woods et al, 2000;Fernandez et al, 2003a;Morris and Gasser, 2006;Kawahara et al, 2008). Another marker that has also been used is the sequence-characterized amplified region, which was proposed by Fernandez et al (2003b); it allows distinguishing Eimeria species in individual or in multiplex PCR.…”

Section: Introductionmentioning

confidence: 99%

Molecular diagnosis of Eimeria species affecting naturally infected Gallus gallus

Fs¹,

Aa²,

Teixeira³

et al. 2011

Genet. Mol. Res.

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ABSTRACT. We used PCR to test various protocols and define a technique for DNA extraction directly from chicken-shed stool samples for the identification of Eimeria species that parasitize birds. It was possible to extract and amplify DNA of seven Eimeria species from field stool samples, using both protocols tested; extractions made with phenol/chloroform protocols gave the best results. The primers were specific and sensitive, allowing amplification of samples containing as few as 20 oocysts, both in individual and in a multiplex PCR. Individualized PCR with the phenol/chloroform DNA extraction protocol detected a larger number of Eimeria species. Molecular diagnosis was found to be practical and precise, and can be used for monitoring and epidemiological studies of Eimeria.

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“…Additionally, because this approach gave limited information about strain variation, further work was carried out to develop a test based on the use of the second ITS region. 25,27,[43][44][45] Universal probes for family (Eimeriidae) and genus (Eimeria) 28S rRNA (at the 3′ end) and 5.8S rRNA (at the 5′ end) sequences respectively flanking the second ITS region enabled identification of and discrimination between all seven species. It also made it possible to detect variation within a single Eimeria species.…”

Section: Targeted Pcr On Individual Speciesmentioning

confidence: 99%

“…It also made it possible to detect variation within a single Eimeria species. 44,45 Therefore, in addition to abolishing the need for isotopes, electrophoretic gels, and monospecific reference controls, the major advantage of this novel test was the ability to detect all seven Eimeria species and their genetic variants in a single PCR reaction. These universal primers were validated in testing two Eimeria species concurrently as well as field samples, and it was concluded by the authors that these tests are accurate, less laborious than previous tests, and cheap enough to be used for testing field samples in routine diagnostic laboratories.…”

Section: Targeted Pcr On Individual Speciesmentioning

confidence: 99%

Designing strategies for the control of coccidiosis in chickens on poultry farms using modern diagnostic tools

Frölich¹,

Farhat²,

Wallach³

2013

RIP

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Abstract:Coccidiosis is caused by the intracellular protozoan parasite Eimeria and is a major worldwide problem in the poultry industry today. The current strategies to control the disease still rely heavily on anticoccidial drugs and on the use of shuttle programs to periodically introduce attenuated live parasites onto poultry farms. Recently, because of the improved performance of broiler chickens, farmers are able to raise chickens for market within a period of only 5-6 weeks. This short growth period enables the development of strategies for coccidiosis control either by ensuring an even exposure of chickens to parasites in the litter, inducing immunity against reinfection, or by keeping parasite numbers below a threshold level wherein only subclinical infections will occur. Therefore, the development and use of new diagnostic tools to precisely identify the Eimeria species and strain and to measure the number of oocysts in the feces and litter can be very helpful in predicting exposure and designing strategies for the control of coccidiosis in the field. The purpose of this study was to describe recently developed methods of diagnosis in terms of their sensitivity and usefulness, describe ways in which litter oocyst numbers can be quantitated, and start to develop an analytical tool that can be used for designing strategies for the control of coccidiosis. A multiplex polymerase chain reaction-based assay has been developed that enables the identification of all seven Eimeria species in a single reaction tube. Previous reports have shown it is possible to detect one oocyst using a polymerase chain reaction-based assay; however, other reports have found that under both laboratory and field conditions the minimal level of sensitivity is only 20 oocysts. In addition, the present authors' studies have found that when using spiked mixed samples, the level of detection of a contaminating Eimeria species is only 5%. Therefore, it is imperative that a new method be developed with a tenfold higher level of sensitivity for field-testing. Once this test is developed and validated, it can be used to monitor the litter and assess the parameters (climate, hygiene, breed of chicken, etc) that govern coccidial outbreaks on farms.

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High-resolution electrophoretic procedures for the identification of fiveEimeria species from chickens, and detection of population variation

Cited by 29 publications

References 17 publications

Electrophoretic Analysis of Genetic Variability within Cryptosporidium parvum from Imported and Autochthonous Cases of Human Cryptosporidiosis in the United Kingdom

Electrophoretic Analysis of Genetic Variability within Cryptosporidium parvum from Imported and Autochthonous Cases of Human Cryptosporidiosis in the United Kingdom

Molecular diagnosis of Eimeria species affecting naturally infected Gallus gallus

Designing strategies for the control of coccidiosis in chickens on poultry farms using modern diagnostic tools

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