Development of a diagnostic PCR assay for the detection and discrimination of four pathogenic<i>.Eimeria</i>species of the chicken

Schnitzler, Beate E.; Thebo, P.; Mattsson, Jens G.; Tomley, Fiona M.; Shirley, M. W.

doi:10.1080/03079459808419373

Cited by 112 publications

(72 citation statements)

References 25 publications

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“…The development of molecular techniques has allowed precise diagnosis of Eimeria species, investigation of the genetic variability of these pathogens, and a search for molecular characteristics associated with phenotypical characteristics that may constitute the use of molecular markers (Schnitzler et al, 1998;Costa et al, 2001). Molecular techniques may also contribute to the development of new vaccines and selection of anticoccidial drugs to be used in control programs (Morris and Gasser, 2006;Sun et al, 2009;Lee et al, 2010).…”

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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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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show abstract

“…This test was found to be highly sensitive and could be used to differentiate between the seven species of Eimeria. [40][41][42] However, the method required a different primer pair for each species, resulting in seven separate PCR reactions to test a given sample, and was therefore deemed labor-intensive and time-consuming. 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.…”

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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“…Supplementary evidence can be gathered through microscopic detection of the environmentally resistant oocyst lifecycle stage in faecal or litter samples, although overlapping morphology can confound all but the expert 6,7 . Molecular alternatives using polymerase chain reaction (PCR), random amplification of polymorphic DNA PCR (RAPD-PCR) and quantitative PCR technologies have been available for up to 20 years [8][9][10] , but to date they have failed to become popular. Relative expense and the requirement for specialist laboratory equipment or processing have limited their uptake, despite the often subjective and technically demanding nature of the older pathology-and microscopybased approaches 10,11 .…”

Section: Introductionmentioning

confidence: 99%

Loop-mediated Isothermal Amplification (LAMP) Assays for the Species-specific Detection of <em>Eimeria</em> that Infect Chickens

Barkway

Pocock

Vrba

et al. 2015

JoVE

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Eimeria species parasites, protozoa which cause the enteric disease coccidiosis, pose a serious threat to the production and welfare of chickens. In the absence of effective control clinical coccidiosis can be devastating. Resistance to the chemoprophylactics frequently used to control Eimeria is common and sub-clinical infection is widespread, influencing feed conversion ratios and susceptibility to other pathogens such as Clostridium perfringens. Despite the availability of polymerase chain reaction (PCR)-based tools, diagnosis of Eimeria infection still relies almost entirely on traditional approaches such as lesion scoring and oocyst morphology, but neither is straightforward. Limitations of the existing molecular tools include the requirement for specialist equipment and difficulties accessing DNA as template. In response a simple field DNA preparation protocol and a panel of species-specific loop-mediated isothermal amplification (LAMP) assays have been developed for the seven Eimeria recognised to infect the chicken. We now provide a detailed protocol describing the preparation of genomic DNA from intestinal tissue collected post-mortem, followed by setup and readout of the LAMP assays. Eimeria species-specific LAMP can be used to monitor parasite occurrence, assessing the efficacy of a farm's anticoccidial strategy, and to diagnose sub-clinical infection or clinical disease with particular value when expert surveillance is unavailable.

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Development of a diagnostic PCR assay for the detection and discrimination of four pathogenic.Eimeriaspecies of the chicken

Cited by 112 publications

References 25 publications

Molecular diagnosis of Eimeria species affecting naturally infected Gallus gallus

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

Loop-mediated Isothermal Amplification (LAMP) Assays for the Species-specific Detection of <em>Eimeria</em> that Infect Chickens

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