Applicability of LAMP as a field diagnostic test for haemonchus contortus and fasciola hepatica infection
- Authors: Bari, Tanjina
- Date: 2021
- Type: Text , Thesis , PhD
- Full Text:
- Description: Gastrointestinal parasites Haemonchus contortus and Fasciola hepatica are major impediments to livestock production worldwide. Faecal egg counts remain the most commonly used and widely accepted diagnostic tool for these parasites; however, there is a need for improved, field-applicable diagnostics. In this study, loop-mediated isothermal amplification (LAMP) was optimised and evaluated for the detection of H. contortus (in sheep) and F. hepatica (in cattle) infection. LAMP assays were optimised to enable visual detection using calcein dye. DNA extraction techniques were developed that have the potential for on-farm application. Faeces suspended in water, heated, then centrifuged, with two cheap and stable chemicals, enabled detection of H. contortus at clinically relevant infection burdens. For F. hepatica, a faeces-water suspension was sieved to remove particulate matter, then physical disruption (bead-beating) was applied. LAMP was conducted under laboratory conditions and in the field; compared to FEC (the most commonly used diagnostic for the target parasites) and PCR. LAMP was conducted using three incubation methods: a commercially available thermocycler; a field-friendly low-cost portable styrofoam esky; and a dedicated field applicable LAMP incubator. The general trend was for LAMP to have high sensitivity but only moderate specificity when compared to FEC. However, the use of PCR (both pathogens) and a highly sensitive amended FEC (F. hepatica only) suggested that the apparent low specificity was the result of LAMP being able to detect low-level parasite infection when conventional FEC could not. A LAMP assay paired with a potentially field-applicable DNA extraction was able to adequately detect haemonchosis at ‘clinically relevant’ parasite burdens in a laboratory study. A field study for the detection of F. hepatica demonstrated the potential utility of LAMP on-farm. The studies conducted in this thesis demonstrate the potential of LAMP for parasitic disease diagnosis in agriculture.
- Description: Doctor of Philosophy
- Authors: Bari, Tanjina
- Date: 2021
- Type: Text , Thesis , PhD
- Full Text:
- Description: Gastrointestinal parasites Haemonchus contortus and Fasciola hepatica are major impediments to livestock production worldwide. Faecal egg counts remain the most commonly used and widely accepted diagnostic tool for these parasites; however, there is a need for improved, field-applicable diagnostics. In this study, loop-mediated isothermal amplification (LAMP) was optimised and evaluated for the detection of H. contortus (in sheep) and F. hepatica (in cattle) infection. LAMP assays were optimised to enable visual detection using calcein dye. DNA extraction techniques were developed that have the potential for on-farm application. Faeces suspended in water, heated, then centrifuged, with two cheap and stable chemicals, enabled detection of H. contortus at clinically relevant infection burdens. For F. hepatica, a faeces-water suspension was sieved to remove particulate matter, then physical disruption (bead-beating) was applied. LAMP was conducted under laboratory conditions and in the field; compared to FEC (the most commonly used diagnostic for the target parasites) and PCR. LAMP was conducted using three incubation methods: a commercially available thermocycler; a field-friendly low-cost portable styrofoam esky; and a dedicated field applicable LAMP incubator. The general trend was for LAMP to have high sensitivity but only moderate specificity when compared to FEC. However, the use of PCR (both pathogens) and a highly sensitive amended FEC (F. hepatica only) suggested that the apparent low specificity was the result of LAMP being able to detect low-level parasite infection when conventional FEC could not. A LAMP assay paired with a potentially field-applicable DNA extraction was able to adequately detect haemonchosis at ‘clinically relevant’ parasite burdens in a laboratory study. A field study for the detection of F. hepatica demonstrated the potential utility of LAMP on-farm. The studies conducted in this thesis demonstrate the potential of LAMP for parasitic disease diagnosis in agriculture.
- Description: Doctor of Philosophy
Application of molecular diagnostic methods for the detection and quantification of Fasciola hepatica infection in cattle faecal samples
- Authors: Thakur, Sameer
- Date: 2020
- Type: Text , Thesis , Masters
- Full Text:
- Description: Fasciola hepatica, commonly known as liver fluke, is a globally distributed trematode causing significant production losses in ruminant livestock. Due to reduced drug efficacy, there is a need for appropriate diagnostic tools, which would allow alternative management practices to be developed and minimize economic losses. The traditional ‘gold standard’ method for diagnosis, faecal egg count (FEC), is associated with low sensitivity when diagnosing F. hepatica infection in livestock using faecal samples. The present study aimed to compare the sensitivity and specificity of the molecular diagnostic methods [conventional PCR (cPCR), Loop Mediated Isothermal Amplification (LAMP) and quantitative real time PCR (qPCR)] with the conventional diagnostic method FEC, for detecting F. hepatica infection using cattle faecal samples. Faecal samples were collected from 94 experimentally-infected cattle 12 weeks post infection and 40 faecal samples were collected from cattle with no previous history of F. hepatica infection, as a comparative control. The sensitivity of conventional PCR, LAMP and qPCR was 86.2%, 87.2% and 96.8% respectively, which was similar to the faecal egg count (97.9%). While the specificity of all the molecular methods were 97.5%, and for FEC the specificity was 100%. The potential advantage of these molecular diagnostic tests, with further development, suggest they may be a viable alternative diagnostic test when compared to FEC. In addition, a pilot study was conducted to evaluate the potential use of a commercial snail trap in catching and detecting the intermediate host of F. hepatica in irrigated farmland, as an alternative management strategy. However, under the parameters tested in these experiments, the use of commercial snail traps to catch the intermediate host of F. hepatica from farm irrigation channels was shown to be ineffective.
- Description: Masters by Research
- Authors: Thakur, Sameer
- Date: 2020
- Type: Text , Thesis , Masters
- Full Text:
- Description: Fasciola hepatica, commonly known as liver fluke, is a globally distributed trematode causing significant production losses in ruminant livestock. Due to reduced drug efficacy, there is a need for appropriate diagnostic tools, which would allow alternative management practices to be developed and minimize economic losses. The traditional ‘gold standard’ method for diagnosis, faecal egg count (FEC), is associated with low sensitivity when diagnosing F. hepatica infection in livestock using faecal samples. The present study aimed to compare the sensitivity and specificity of the molecular diagnostic methods [conventional PCR (cPCR), Loop Mediated Isothermal Amplification (LAMP) and quantitative real time PCR (qPCR)] with the conventional diagnostic method FEC, for detecting F. hepatica infection using cattle faecal samples. Faecal samples were collected from 94 experimentally-infected cattle 12 weeks post infection and 40 faecal samples were collected from cattle with no previous history of F. hepatica infection, as a comparative control. The sensitivity of conventional PCR, LAMP and qPCR was 86.2%, 87.2% and 96.8% respectively, which was similar to the faecal egg count (97.9%). While the specificity of all the molecular methods were 97.5%, and for FEC the specificity was 100%. The potential advantage of these molecular diagnostic tests, with further development, suggest they may be a viable alternative diagnostic test when compared to FEC. In addition, a pilot study was conducted to evaluate the potential use of a commercial snail trap in catching and detecting the intermediate host of F. hepatica in irrigated farmland, as an alternative management strategy. However, under the parameters tested in these experiments, the use of commercial snail traps to catch the intermediate host of F. hepatica from farm irrigation channels was shown to be ineffective.
- Description: Masters by Research
Evaluation of the immune responses induced by four targeted DNA vaccines encoding the juvenile liver fluke antigen, cathepsin B in a mouse model
- Jayaraj, Ramamoorthi, Piedrafita, David, Spithill, Terry, Smooker, Peter
- Authors: Jayaraj, Ramamoorthi , Piedrafita, David , Spithill, Terry , Smooker, Peter
- Date: 2012
- Type: Text , Journal article
- Relation: Genetic Vaccines and Therapy Vol. 10, no. 1 (Art. No:7) (2012), p. 1-9
- Full Text:
- Reviewed:
- Description: Background Liver fluke can infect cattle and sheep, and is also emerging as a human pathogen in developing countries. Cathepsin B (Cat B2) is a major cysteine protease secreted by the juvenile flukes. To enhance the immune responses of Cat B2, the cDNA sequence was fused with four different DNA vaccine vectors. The induced cellular and antibody responses were compared in vaccinated mice. Methods The following recombinant DNA vaccine constructs were constructed: empty vector VR1012 as negative control, cytoplasmic construct pVR1012 Cat B2, secretory construct pVR1020 Cat B2, chemokine-fused construct pMCP3 Cat B2 and lymph node targeting construct pCTLA-4 Cat B2. Plasmids were constructed using standard procedures, and positive constructs screened and selected using restriction digestion analysis followed by sequence analysis. The constructs were then tested in Cos-7 cells for in vitro expression, which was analysed using immunoblotting. Subsequently, female BALB/c mice were immunised with DNA constructs as vaccines. Elicited antibody responses were measured using ELISA. The ratio between IgG1 and IgG2a antibody responses was estimated among different vaccine groups. IgG antibody avidity assay was performed and the relative avidity index was calculated. The induced cytokine production from splenocytes of vaccinated animals was estimated using ELISPOT. Results DNA vaccine constructs carrying Cat B2 were expressed in Cos-7 cell lines and encoded protein was recognised using western blotting using rat anti- cathepsin B antibody. DNA vaccines elicited high Cat B2- specific IgG, IgG1, IgE and also modest IgG2a antibody responses. Cat B2 specific IL-4 T cell responses were also observed in Cat B2 vaccinated mice. The comparison of immunogenic potential in each of these constructs was demonstrated as enhanced antibody responses on the lymph-node targeting vector pCTLA-4 Cat B2, the high antibody avidity of chemo-attractant pMCP3 Cat B2 and stronger T cellular responses of non-secretory DNA vaccine pVR1012 Cat B2 in vaccinated animals. Conclusion This study showed that the targeting DNA vaccine strategies enhanced specific immune responses to juvenile fluke Cat B2. The results of our current study have demonstrated that a gene-based vaccine as an immunotherapeutic approach to combat Fasciola infection may be feasible.
- Authors: Jayaraj, Ramamoorthi , Piedrafita, David , Spithill, Terry , Smooker, Peter
- Date: 2012
- Type: Text , Journal article
- Relation: Genetic Vaccines and Therapy Vol. 10, no. 1 (Art. No:7) (2012), p. 1-9
- Full Text:
- Reviewed:
- Description: Background Liver fluke can infect cattle and sheep, and is also emerging as a human pathogen in developing countries. Cathepsin B (Cat B2) is a major cysteine protease secreted by the juvenile flukes. To enhance the immune responses of Cat B2, the cDNA sequence was fused with four different DNA vaccine vectors. The induced cellular and antibody responses were compared in vaccinated mice. Methods The following recombinant DNA vaccine constructs were constructed: empty vector VR1012 as negative control, cytoplasmic construct pVR1012 Cat B2, secretory construct pVR1020 Cat B2, chemokine-fused construct pMCP3 Cat B2 and lymph node targeting construct pCTLA-4 Cat B2. Plasmids were constructed using standard procedures, and positive constructs screened and selected using restriction digestion analysis followed by sequence analysis. The constructs were then tested in Cos-7 cells for in vitro expression, which was analysed using immunoblotting. Subsequently, female BALB/c mice were immunised with DNA constructs as vaccines. Elicited antibody responses were measured using ELISA. The ratio between IgG1 and IgG2a antibody responses was estimated among different vaccine groups. IgG antibody avidity assay was performed and the relative avidity index was calculated. The induced cytokine production from splenocytes of vaccinated animals was estimated using ELISPOT. Results DNA vaccine constructs carrying Cat B2 were expressed in Cos-7 cell lines and encoded protein was recognised using western blotting using rat anti- cathepsin B antibody. DNA vaccines elicited high Cat B2- specific IgG, IgG1, IgE and also modest IgG2a antibody responses. Cat B2 specific IL-4 T cell responses were also observed in Cat B2 vaccinated mice. The comparison of immunogenic potential in each of these constructs was demonstrated as enhanced antibody responses on the lymph-node targeting vector pCTLA-4 Cat B2, the high antibody avidity of chemo-attractant pMCP3 Cat B2 and stronger T cellular responses of non-secretory DNA vaccine pVR1012 Cat B2 in vaccinated animals. Conclusion This study showed that the targeting DNA vaccine strategies enhanced specific immune responses to juvenile fluke Cat B2. The results of our current study have demonstrated that a gene-based vaccine as an immunotherapeutic approach to combat Fasciola infection may be feasible.
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