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
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
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