Limits...
Development of a multiplex fluorescence immunological assay for the simultaneous detection of antibodies against Cooperia oncophora, Dictyocaulus viviparus and Fasciola hepatica in cattle.

Karanikola SN, Krücken J, Ramünke S, de Waal T, Höglund J, Charlier J, Weber C, Müller E, Kowalczyk SJ, Kaba J, von Samson-Himmelstjerna G, Demeler J - Parasit Vectors (2015)

Bottom Line: The capability of a multiplex platform for the simultaneous detection of three pasture associated parasite species was evaluated and compared to existing ELISAs.The results between the assays were compared and kappa tests revealed an overall good agreement.This platform provides rapid, high-throughput results and is highly sensitive and specific in comparison to existing serological as well as coproscopical diagnostic techniques.

View Article: PubMed Central - PubMed

Affiliation: Institute for Parasitology and Tropical Veterinary Medicine, Freie Universität Berlin, Berlin, Germany. Sofia.Karanikola@fu-berlin.de.

ABSTRACT

Background: A major constraint for the effective control and management of helminth parasites is the lack of rapid, high-throughput, routine diagnostic tests to assess the health status of individual animals and herds and to identify the parasite species responsible for these helminthoses. The capability of a multiplex platform for the simultaneous detection of three pasture associated parasite species was evaluated and compared to existing ELISAs.

Methods: The recombinant antigens 14.2 kDa ES protein for Cooperia oncophora, major sperm protein for Dictyocaulus viviparus and Cathepsin L1 for Fasciola hepatica were recombinantly expressed either in Escherichia coli or Pichia pastoris. Antigens were covalently coupled onto magnetic beads. Optimal concentrations for coupling were determined following the examination of serum samples collected from experimentally mono-infected animals, before and after their infection with the target species. Absence of cross-reactivity was further determined with sera from calves mono-infected with Haemonchus contortus, Ostertagia ostertagi and Trichostrongylus colubriformis. Examination of negative serum samples was characterised by low median fluorescence intensity (MFI).

Results: Establishment of the optimal serum dilution of 1:200 was achieved for all three bead sets. Receiver Operating Characteristic analyses were performed to obtain cut-off MFI values for each parasite separately. Sensitivity and specificity at the chosen cut-off values were close to, or 100% for all bead sets. Examination of serum samples collected on different days post infection from different animals showed a high reproducibility of the assays. Serum samples were additionally examined with two already established ELISAs, an in-house ELISA using the recombinant MSP as an antigen and a DRG ELISA using Cathepsin L1 for liver fluke. The results between the assays were compared and kappa tests revealed an overall good agreement.

Conclusions: A versatile bead-based assay using fluorescence detection (xMAP technology) was developed to simultaneously detect antibodies against C. oncophora, D. viviparus and F. hepatica in cattle serum samples. This platform provides rapid, high-throughput results and is highly sensitive and specific in comparison to existing serological as well as coproscopical diagnostic techniques.

No MeSH data available.


Related in: MedlinePlus

Cross reactivity analysis using sera from target and non-target species. Results are presented as box-plots showing the median fluorescence intensity (MFI) values obtained from multiple testing of sera from negative and mono-infected animals. Bead set were coupled with recombinant antigen for the detection of Cooperia oncophora (a), Dictyocaulus viviparus (b) and Fasciola hepatica (c). Whiskers represent 5 % and 95 % percentage quantiles and the mean is indicated by a +. Outliers are shown as individual dots
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4492007&req=5

Fig3: Cross reactivity analysis using sera from target and non-target species. Results are presented as box-plots showing the median fluorescence intensity (MFI) values obtained from multiple testing of sera from negative and mono-infected animals. Bead set were coupled with recombinant antigen for the detection of Cooperia oncophora (a), Dictyocaulus viviparus (b) and Fasciola hepatica (c). Whiskers represent 5 % and 95 % percentage quantiles and the mean is indicated by a +. Outliers are shown as individual dots

Mentions: All bead sets were examined using positive and negative control sera as well as sera from non-target species infections. Since no obvious differences were observed between singleplex and multiplex assays, results were combined and are shown in Fig. 3. Box plots indicated clear differentiation between positive and negative control serum samples for all three target species. Regarding cross-reactivity antigens used for the detection of C. oncophora and F. hepatica could clearly distinguish between infections with target and non-target species (H. contortus, T. colubriformis, O. ostertagi, D. viviparus and F. hepatica or C. oncophora, respectively). This was different for the recombinant MSP antigen, where cross-reactivity was more pronounced for sera from C. oncophora and F. hepatica infected animals; particularly for a few C. oncophora positive sera differences to the lowest observed MFI value for D. viviparus were only minimal. Determination of the cut-off MFI values, sensitivity and specificity was achieved by ROC analysis separately for each bead set. Since serum samples were derived from experimentally infected animals and either clearly negative (parasite naïve prior to infection) or positive, two cut-off values were defined, one discriminating negative and one positive, leaving grey zone in between. For D. viviparus the situation was slightly different with some cross-reactivity present particularly for the C. oncophora coupled beads, so that for this assay only one cut-off value was determined. The cut-off values with sensitivity and specificity including the 95 % confidence intervals are presented in Table 1.Fig. 3


Development of a multiplex fluorescence immunological assay for the simultaneous detection of antibodies against Cooperia oncophora, Dictyocaulus viviparus and Fasciola hepatica in cattle.

Karanikola SN, Krücken J, Ramünke S, de Waal T, Höglund J, Charlier J, Weber C, Müller E, Kowalczyk SJ, Kaba J, von Samson-Himmelstjerna G, Demeler J - Parasit Vectors (2015)

Cross reactivity analysis using sera from target and non-target species. Results are presented as box-plots showing the median fluorescence intensity (MFI) values obtained from multiple testing of sera from negative and mono-infected animals. Bead set were coupled with recombinant antigen for the detection of Cooperia oncophora (a), Dictyocaulus viviparus (b) and Fasciola hepatica (c). Whiskers represent 5 % and 95 % percentage quantiles and the mean is indicated by a +. Outliers are shown as individual dots
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4492007&req=5

Fig3: Cross reactivity analysis using sera from target and non-target species. Results are presented as box-plots showing the median fluorescence intensity (MFI) values obtained from multiple testing of sera from negative and mono-infected animals. Bead set were coupled with recombinant antigen for the detection of Cooperia oncophora (a), Dictyocaulus viviparus (b) and Fasciola hepatica (c). Whiskers represent 5 % and 95 % percentage quantiles and the mean is indicated by a +. Outliers are shown as individual dots
Mentions: All bead sets were examined using positive and negative control sera as well as sera from non-target species infections. Since no obvious differences were observed between singleplex and multiplex assays, results were combined and are shown in Fig. 3. Box plots indicated clear differentiation between positive and negative control serum samples for all three target species. Regarding cross-reactivity antigens used for the detection of C. oncophora and F. hepatica could clearly distinguish between infections with target and non-target species (H. contortus, T. colubriformis, O. ostertagi, D. viviparus and F. hepatica or C. oncophora, respectively). This was different for the recombinant MSP antigen, where cross-reactivity was more pronounced for sera from C. oncophora and F. hepatica infected animals; particularly for a few C. oncophora positive sera differences to the lowest observed MFI value for D. viviparus were only minimal. Determination of the cut-off MFI values, sensitivity and specificity was achieved by ROC analysis separately for each bead set. Since serum samples were derived from experimentally infected animals and either clearly negative (parasite naïve prior to infection) or positive, two cut-off values were defined, one discriminating negative and one positive, leaving grey zone in between. For D. viviparus the situation was slightly different with some cross-reactivity present particularly for the C. oncophora coupled beads, so that for this assay only one cut-off value was determined. The cut-off values with sensitivity and specificity including the 95 % confidence intervals are presented in Table 1.Fig. 3

Bottom Line: The capability of a multiplex platform for the simultaneous detection of three pasture associated parasite species was evaluated and compared to existing ELISAs.The results between the assays were compared and kappa tests revealed an overall good agreement.This platform provides rapid, high-throughput results and is highly sensitive and specific in comparison to existing serological as well as coproscopical diagnostic techniques.

View Article: PubMed Central - PubMed

Affiliation: Institute for Parasitology and Tropical Veterinary Medicine, Freie Universität Berlin, Berlin, Germany. Sofia.Karanikola@fu-berlin.de.

ABSTRACT

Background: A major constraint for the effective control and management of helminth parasites is the lack of rapid, high-throughput, routine diagnostic tests to assess the health status of individual animals and herds and to identify the parasite species responsible for these helminthoses. The capability of a multiplex platform for the simultaneous detection of three pasture associated parasite species was evaluated and compared to existing ELISAs.

Methods: The recombinant antigens 14.2 kDa ES protein for Cooperia oncophora, major sperm protein for Dictyocaulus viviparus and Cathepsin L1 for Fasciola hepatica were recombinantly expressed either in Escherichia coli or Pichia pastoris. Antigens were covalently coupled onto magnetic beads. Optimal concentrations for coupling were determined following the examination of serum samples collected from experimentally mono-infected animals, before and after their infection with the target species. Absence of cross-reactivity was further determined with sera from calves mono-infected with Haemonchus contortus, Ostertagia ostertagi and Trichostrongylus colubriformis. Examination of negative serum samples was characterised by low median fluorescence intensity (MFI).

Results: Establishment of the optimal serum dilution of 1:200 was achieved for all three bead sets. Receiver Operating Characteristic analyses were performed to obtain cut-off MFI values for each parasite separately. Sensitivity and specificity at the chosen cut-off values were close to, or 100% for all bead sets. Examination of serum samples collected on different days post infection from different animals showed a high reproducibility of the assays. Serum samples were additionally examined with two already established ELISAs, an in-house ELISA using the recombinant MSP as an antigen and a DRG ELISA using Cathepsin L1 for liver fluke. The results between the assays were compared and kappa tests revealed an overall good agreement.

Conclusions: A versatile bead-based assay using fluorescence detection (xMAP technology) was developed to simultaneously detect antibodies against C. oncophora, D. viviparus and F. hepatica in cattle serum samples. This platform provides rapid, high-throughput results and is highly sensitive and specific in comparison to existing serological as well as coproscopical diagnostic techniques.

No MeSH data available.


Related in: MedlinePlus