Answers to Questions
In the invasive front, host culling is a measure that can be taken. Is the culling contingent on the testing result?
- A key challenge when using culling to eradicate a pathogen (as opposed to reducing host densities to decrease transmission) is the identification and removal of all infected individuals, which is difficult even with effective diagnostic tools. (Langwig et al., 2015) Does the difficulty have to do with the "effective" diagnostic device limitations?
Answer: ( Dr. Penner) No, culling is not a strategy currently promoted for Bd or Bsal. This has several reasons. Partly because diagnostics are never 100% reliable. Partly because this will not stop the spread of the disease because it is also transmitted by other vectors (i.e. other species and other means (e.g. soil, water). Testing all vectors is impossible. For Bd and BSal other researchers test avenues to cure the infected individuals, which looks promising in the case of Bsal. So the current idea is to remove infected individuals, keep them in captivity until free of the pathogen, monitor the environment until pathogen free as well, then release the individuals back into the wild.
On the roles of researchers: In pre-arrival, the protocols we have seen so far involve sample collection and release of animals. Would there be an added advantage to a field use device besides convenience in this case? If researchers were to get the results faster, would they not release? Would they be able to cull infected animals?
Answer: (Dr. Penner) Researchers would be faster and would not release infected animals. These would not be culled but cured and released at a later point in time. This is not possible with current methods. There one has to wait until infections have spread widely, then find living animals which will all be collected (not only infected individuals) and treated in captivity until release.
Literature so far suggests that sample collection and management requires experience. Where does the necessity of proficiency stem from in the current procedures? Does it have to do with complexity in the collection method such as swabbing or perhaps later handling of sample in wet lab?
Answer: (Dr. Penner) In my opinion from two points: Finding and identifying the animals in the wild; second: later handling and analyses of the samples in the laboratory.
Who carries out prevalence assessments in the invasion and epidemic stage? Is prevalence assessment taken out by governments seeking to contain spread or is there involvement by researchers in gathering sample, testing etc, and the government taking on a more managerial role?
Answer: (Dr. Penner) This differs from country to country. Currently the government in Germany only takes an active role with diseases related to birds and mammals. Amphibian and reptile diseases are analysed by researchers.
Are you aware of active frequent testing carried out as part of risk assessment (without any detected cases)?
Answer: (Dr. Penner) Yes, that used to be the case for Bd and Bsal.
One of the barriers to a rigorous measure during the pre-arrival stage is the significant cost and impact on trade. (Langwig et al., 2015) Given the other elements of this stage (testing, quarantine, and banning), how significant is the cost of testing as a limitation for the biosecurity measures?
Answer: (Dr. Penner) In my opinion costs are significant because research is conducted on the basis of money acquired by third party funds. Thus limited and only available for specific projects. Thus there are "waves" of higher rates of testing and lower efforts but not really continuos comparable standardised monitoring. Your machine could enable that.
How many amphibians need to be tested per trip to the field?
Answer: (Colleen Kamoroff) In my research with field based molecular techniques, I was able to detect Bd on every frog I swabbed, although Bb prevalence was known to be high. The swabbs were run in triplicate qPCR reactions (three technical replicates... three wells with DNA extract), and I did not detect Bd in every technical replicate. I cannot speak to how many frogs would need to be swabb in order to determine if Bd was present (especially if it was present at low densities), but I would suggest running the extracted DNA from the swabbs in triplicate qPCR reactions.
In your opinion, what is the most important factor to take into consideration when picking a point-of-care testing device for amphibian testing? Time, accuracy, cost, etc?
Answer: (Colleen Kamoroff) I just did a study where analyzed the effectiveness of using an in-situ DNA extraction method combined with a handheld mobile thermocycler for real-time qPCR analysis from Biomeme Inc. In this study, the limiting factor was accuracy. The probability of detecting Bd DNA in the technical replicates was lower for the Biomeme field protocol compared to samples extracted using the lab protocol, suggesting the field protocol has reduced sensitivity and may not detect low quantities of DNA. Our results suggest the field extraction protocol using a handheld qPCR platform is a promising tool for rapid detection of Bd in susceptible amphibian populations. The field protocol yielded accurate results in less than 60 minutes. However, the applied field protocol may be prone to false negatives when analyzing low-quantity DNA samples (i.e. eDNA).
Do the detection methods depend on the geographical location? If so, what are the factors that contribute to this dependence?
Answer: (Colleen Kamoroff) Aqueous eDNA detection is dependent on collection, extraction, and identification of target DNA particles. eDNA exists in heterogeneous clumps in the environment so collecting samples from multiple locations would increase your chances of collecting target eDNA. Also collecting eDNA samples near the target specimen (near frogs who are hosts of Bd) would increase chances of detection.
What kind of conditions will a point-of-care device for amphibians usually be used in? Are there any extreme conditions such as temperature that should be taken into account when developing a point-of-care device for amphibians?
Answer: (Colleen Kamoroff) This depends on the type of amphibian. I work with high elevation amphibian species... so we experience high UV conditions. DNA degrades more rapidly in high temp, pH, and UV conditions... so those are good conditions to take in account.
When testing for the presence of fungi on amphibians is the required result often qualitative or quantitative?
Answer: (Colleen Kamoroff) Binary detection (i.e. presence/absence) of Bd DNA is an important metric for understanding disease dynamics and host risk. However, DNA quantification of both eDNA and swab samples is critical to the ecological interpretation of the results. Determining when Bd levels and infection intensities rapid/ exponential grown before lethal threshold levels is critical for management to implement conservation strategies. Such determination can only be accomplished through accurate quantification of Bd load on skin swabs and potentially eDNA samples.
From your experience ,on average, what is the cost of a lab PCR?
Answer: (Colleen Kamoroff) Typical costs for lab extraction and analysis of swabs and eDNA samples are ~$10-35 and ~$50-150 respectively depending on type of lab, extraction method, and number of samples processed. Typical qPCR machines used in lab analysis have a 96 well capacity and can multiplex up to five targets per well resulting in a high-volume throughput per run.
What species of amphibians is your research team testing on for Bd and Bsal diagnosis?
Answer: (Dr. Penner) My team and I would like to conduct research with such a device on Grass frogs (Rana temporaria) to test for Bd and on Fire salamanders (Salamandra salamandra) to test for Bsal. However, in general all 3 orders of amphibians (Anura, Gymnophiona and Urodela) have Bd and Bsal has been found on several species of Anura and Urodela. So for my research tests would involve all amphibians encountered in suitable habitats of Rana temporaria and Salamandra salamandra
During your research with the fungus Bd, is it preferred for the point-of-care device to give out quantitative or qualitative results?
Answer: (Dr.Penner) The main aim would be quantitative results for Bd.
What exactly do they calculate, how, and what do they infer from those quantitative measurements? what does low accuracy in quantification mean, how exactly do we determine the accuracy?
Answer: (Research and Colleen Kamoroff) The quantitative measurements allow for the researchers to know the Bd/ Bsal load present in the amphibian this is essential as only once the fungal load reaches a certain level does it become lethal and at another fungal load it becomes contagious. Low accuracy means that when there is a low fungal load the testing results for presence of the fungi will come back negative when in fact there is just a low trace of the fungus. (Steffanie) Accuracy is basically at what quantity of fungi present will the test start to give negative results due to low traces of the fungi.
Why do the researchers use qPCR?
Answer: (Colleen Kamoroff) qPCR is used due to the cost and time efficiency, three test can be carried out at once. Result are also quantitative.
Map of Bd for finding out climate conditions.
Answer: (Source: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3584086/) Prevalence of Bd in an area decreases with increasing temperature, the exact statistic is "decreased by 8.8% with each degree increase in temperature range at a site" although the prevalence begins to decrease once temperatures exceed 28 degrees as Bd does not grow well at temperatures≥28°C. Research found increased infection prevalence and mortality under cooler conditions. The presence of Bd also increases with proximity to a water source. As seen in the maps most sites that are pinned as positive sites are prone to be damp areas. Barely any positive sites are in landlocked areas. Montane, grasslands and shrublands have the highest occurrence of Bd.