Real-time PCR-based quantification of<i>Eimeria</i>genomes: a method to outweigh underestimation of genome numbers due to PCR inhibition

Raj, G. Dhinakar; Aarthi, S; Selvabharathi, R; Raman, Muthusamy; Blake, Damer P.; Tomley, Fiona M.

doi:10.1080/03079457.2013.790531

Cited by 15 publications

(14 citation statements)

References 8 publications

(19 reference statements)

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“…This method has the advantage of requiring lower group numbers due to within reduced intra-group variation between chickens given the same oocyst challenge dose compared with fecal oocyst counts (27). DNA extracted directly from feces can be unsuitable due to the presence of inhibitors which can impact qPCR efficiency and therefore quantification accuracy, although an internal qPCR control can be used for standardization (90). Extraction from tissues or oocysts is preferable given lower occurrence of PCR inhibitors.…”

Section: Measures Of Parasite Replicationmentioning

confidence: 99%

Poultry Coccidiosis: Design and Interpretation of Vaccine Studies

et al. 2020

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Eimeria infection impacts upon chicken welfare and economic productivity of the poultry sector. Live coccidiosis vaccines for chickens have been available for almost 70 years, but the requirement to formulate blends of oocysts from multiple Eimeria species makes vaccine production costly and logistically demanding. A multivalent vaccine that does not require chickens for its production and can induce protection against multiple Eimeria species is highly desirable. However, despite the identification and testing of many vaccine candidate antigens, no recombinant coccidiosis vaccine has been developed commercially. Currently, assessment of vaccine efficacy against Eimeria, and the disease coccidiosis, can be done only through in vivo vaccination and challenge experiments but the design of such studies has been highly variable. Lack of a "standard" protocol for assessing vaccine efficacy makes comparative evaluations very difficult, complicating vaccine development, and validation. The formulation and schedule of vaccination, the breed of chicken and choice of husbandry system, the species, strain, magnitude, and timing of delivery of the parasite challenge, and the parameters used to assess vaccine efficacy all influence the outcomes of experimental trials. In natural Eimeria infections, the induction of strong cell mediated immune responses are central to the development of protective immunity against coccidiosis. Antibodies are generally regarded to be of lesser importance. Unfortunately, there are no specific immunological assays that can accurately predict how well a vaccine will protect against coccidiosis (i.e., no "correlates of protection"). Thus, experimental vaccine studies rely on assessing a variety of post-challenge parameters, including assessment of pathognomonic lesions, measurements of parasite replication such as oocyst output or quantification of Eimeria genomes, and/or measurements of productivity such as body weight gain and feed conversion rates. Understanding immune responses to primary and secondary infection can inform on the most appropriate immunological assays. The discovery of new antigens for different Eimeria species and the development of new methods of vaccine antigen delivery necessitates a more considered approach to assessment of novel vaccines with robust, repeatable study design. Careful consideration of performance and welfare factors that are genuinely relevant to chicken producers and vaccine manufacturers is essential.

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Section: Measures Of Parasite Replicationmentioning

confidence: 99%

Poultry Coccidiosis: Design and Interpretation of Vaccine Studies

et al. 2020

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“…This study represents the first validation of an objective, highly sensitive, and efficient published quantitative real-time PCR technique to expedite the process of determining Eimeria parasite replication in tissue for both small and large-scale investigations in laboratory and field-place settings. Previous reports using qPCR methods have focused on developing assays for the specific identification of multiple chicken-infecting Eimeria species [19,45–48] , or for quantification of a single species (i.e., Eimeria acervulina [18,27] and Eimeria maxima [29] ). Here, we directly compared qPCR of tissue samples with FOC and lesion scores in experimentally infected chickens to demonstrate a robust correlation between oocyst dose, qPCR test results, and FOC.…”

Section: Discussionmentioning

confidence: 99%

“…Biological and technical factors can influence gDNA-based qPCR results and must be considered carefully before this tool can be used widely as a realistic alternative to FOC. In previous qPCR studies with Eimeria carried out by Morgan et al [48] and Raj et al [45] , gDNA was extracted directly from faeces employing (singly or in combination) a QIAamp® DNA Stool Mini Kit (Qiagen), DNeasy® Tissue Kit (Qiagen), and/or a standard cetyl trimethylammonium bromide (CTAB) [50] extraction protocol. These methods are at least partially ineffective at removing faecal components inhibitory to PCR, as demonstrated using an internal positive control (IPC) qPCR in the latter study.…”

Section: Discussionmentioning

confidence: 99%

“…These methods are at least partially ineffective at removing faecal components inhibitory to PCR, as demonstrated using an internal positive control (IPC) qPCR in the latter study. Using tissue samples in place of caecal contents or faeces can reduce the risk of inhibition and could be confirmed by inclusion of an IPC assay [45] . Quantification of Eimeria genome numbers in tissue, rather than faecal or litter samples, offers the additional benefit of removing sporulation as a variable.…”

Section: Discussionmentioning

confidence: 99%

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Quantitative real-time PCR (qPCR) for Eimeria tenella replication — Implications for experimental refinement and animal welfare

Nolan¹,

Tomley²,

Kaiser³

et al. 2015

Parasitology International

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The Eimeria species are highly pathogenic parasites of chickens. Research aimed at reducing their impact is hindered by a lack of non-subjective, quantitative, tools to measure parasite replication in the host. The time-consuming, and often time-sensitive, nature of existing approaches precludes their use in large-scale genetic, epidemiological, and evolutionary analyses. We have used quantitative real-time PCR (qPCR) to accurately quantify Eimeria tenella in chicken tissue and shown this to be more efficient and sensitive than traditional methodologies. We tested four chicken-specific reference qPCR assays and found beta-actin (actb) to be optimal for sample normalisation. In an experimental setting, chickens were inoculated with 500, 1500, or 4500 E. tenella oocysts and parasite replication and the impact of infection measured by i) qPCR analysis of DNA extracted from caecal tissues collected at five and eight days post-infection (dpi), ii) faecal oocyst counts (FOCs) on samples taken from six to eight dpi, and iii) lesion scoring on caeca collected post-mortem at five and eight dpi. Quantitative real-time PCR test results indicated a significant dose-dependent increase in parasite numbers among study groups for samples collected five dpi (i.e., prior to gametogony) (R2 = 0.994) (p < 0.002) but not in those from day eight (after most oocyst shedding) (R2 = 0.006) (p > 0.379). A strong dose-dependent increase in parasite replication and severity of infection was also revealed by FOC (R2 = 0.997) and lesion scoring. Importantly, qPCR offers substantial improvements for animal welfare via improved statistical power and reduced group sizes in experimental studies. The described qPCR method overcomes subjective limitations of coproscopic quantification, allows reproducible medium- to high-throughput examination of tissues, faeces, and oocysts, and is a valuable tool for determining the impact of Eimeria infections in both experimental and field settings.

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“…The oocyst is the most readily accessible phase of the eimerian lifecycle, but routine DNA extraction requires laboratory facilities 22 . Other, more labile intestinal lifecycle stages require purification prior to DNA preparation to prevent PCR inhibition and a consequent loss of sensitivity 23,24 . The ability to extract eimerian DNA of a quality suitable for LAMP using equipment no more specialised than a microcentrifuge and a water bath, supplemented by inhibitor adsorption using chelex resin, now promotes the wider use of molecular biology in eimerian diagnostics.…”

Section: Discussionmentioning

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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Real-time PCR-based quantification ofEimeriagenomes: a method to outweigh underestimation of genome numbers due to PCR inhibition

Cited by 15 publications

References 8 publications

Poultry Coccidiosis: Design and Interpretation of Vaccine Studies

Poultry Coccidiosis: Design and Interpretation of Vaccine Studies

Quantitative real-time PCR (qPCR) for Eimeria tenella replication — Implications for experimental refinement and animal welfare

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

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