WormMail.2015-06-18. Molecular detection of parasites.Depp dogs and rabies.Surprising wild dog foray. Blood type O and cognitive decline
In this issue:
- Molecular detection of parasites – Hunt
- Depp, dogs and rabies – Sparkes
- Surprising wild dog foray – Ballard
- Blood type O and cognitive decline
Molecular detection of parasites for sheep and cattle production
An invited overview by:
Dr Peter Hunt
CSIRO McMaster laboratory,
Armidale, NSW, Australia
Take home points
- Don’t guess, worm test – As we always say..
- Use it or lose it – Testing for parasitic diseases is provided by both government organisations and private laboratories. The continued provision of these services will depend on livestock owners using them. The continued improvement of these services will also depend on demand from livestock owners.
- DNA technology – There is one suite of DNA-based tests available for livestock owners to use now. Much more can be done with DNA-based testing in the future.
- Australian researchers have been at the forefront of developments in DNA-based tests for parasite diseases of livestock since the early 1990s. Will this continue?
The DNA technology age
Excluding some viruses, all living things possess DNA at all stages of their lifecycle. DNA is a polymer made of just four chemical units, the “bases” A, C, G and T. Because of the simplicity and universality of DNA we can use it to detect organisms, detect changes within organisms, predict the function of organisms and also manipulate organisms to change their function.
Since the 1970s we have been living in an age of DNA technology which has transformed medical diagnostics, pharmaceutical manufacturing and biological research. The same technology has had some impact in crop agriculture through enhanced breeding capacity (genetic diagnostics) and to a far lesser extent, transgenic crops.
Livestock agriculture has not seen the same rewards from the age of DNA technology as medicine and crop agriculture, but there has been some adoption of the technology. In animal breeding, especially dairy cattle, pigs and chickens, the adoption of the same genetic diagnostics technology used by crop breeders has had an impact on genetic progress, and this is beginning to occur for sheep and beef cattle as well. The use of DNA technology to detect pathogens and other organisms in livestock animal samples or in the animal’s environment has taken longer to adopt.
Nevertheless, the detection and characterisation of a range of viral, bacterial and protozool diseases has become part of state-organised biosecurity monitoring and disease control. Mostly, the use of DNA-based technologies has been limited to surveillance and research programs, however some use of the technology as part of routine diagnosis occurs. Examples include the detection of ovine herpesvirus (bovine malignant catarrh) by AgWest and the detection of six different disease organisms in cattle by Gribbles (http://www.gribblesvets.com.au/). Also, at NSWDPI’s Elizabeth Macarthur Agriculture Institute, there are many PCR-based diagnostic tests for genetic-, virus- and bacteria-related diseases (http://www.dpi.nsw.gov.au/research/centres/emai).
DNA technology and the detection of parasites – the early years
The use of DNA technology to detect helminth parasites dates from the early 1990s. During those years a number of labs produced tests that can distinguish between species of nematodes. The first tests we used at CSIRO in Armidale were those developed by Robin Gasser at the University of Melbourne. These involved the extraction of DNA from individual larvae or eggs, amplification of a specific gene fragment, called ITS2, and the cleavage of the amplified fragment with enzymes (restriction endonucleases). ITS2 varies in its sequence between, but not within species, so the pattern of DNA pieces produced by enzymatic cleavage can be used to tell which species the larvae belonged to. The Gasser lab developed tests for a barrage of species infecting sheep, goats, pigs, horses, humans and cattle.
Therefore, since the mid-1990s we have had the ability to tell the species composition of worms from faecal eggs, or worms from faecal cultures. The only barrier to adopting this type of test for commercial use is the cost. One hundred or so DNA extractions and subsequent steps per sample is a significant cost, even at today’s more reasonable consumables prices. The labour involved is also prohibitively costly. Although the latter parts of the procedure are easily automated in today’s laboratories, the individual worm or egg picking step remains a significant bottle neck. Despite this approach being unsuitable for routine diagnostic use, it is still used in research, and recent work in New Zealand by Stewart Bisset and colleagues has further improved the method (Bisset et al., 2014).
It was also in the 1990s that the development of genetic markers to distinguish between populations within a species of parasite began. This initial research was in Haemonchus contortus, H. placei and Trichostrongylus colubriformis, and genetic markers were discovered for benzimidazole (‘white drench’) resistance in H. contortus (Marlene Roos and colleagues in the Netherlands – Kwa et al., 1994) and T. colubriformis (Warwick Grant and Lisa Mascord at CSIRO Armidale – Grant and Mascord, 1996). At the same time Leo Le Jambre (CSIRO Armidale) and Marlene Roos were working on developing microsatellite markers for H. contortus and H. placei, and Ken Beh (CSIRO Glebe) was developing markers in T. colubriformis (e.g. Otsen et al., 2000; Le Jambre et al., 1999; Callaghan and Beh, 1994). These markers were to be used to map resistance genes for other drug resistance, the focus at the time being ivermectin.
DNA technology and the detection of parasites – a new hope
In the late 1990s two new types of DNA detection technology were developed. The first of these was microarrays or “chips” (Schena et al., 1995; Gupta et al., 1999). Microarrays are capable of detecting many different pieces of DNA simultaneously, and they found uses in detecting organisms from environmental and pathology samples. To my knowledge, this use has never been applied to samples of DNA from livestock animals for detecting parasites, but it could be. The more common uses of microarrays became the analysis of gene expression in research and, as a genetic diagnostic test in medicine, plant and animal breeding where many variants of genes can be assayed simultaneously. Despite some changes to the technology, the second use remains important today, through the application of genomic selection in agriculture. The use of arrays for gene expression analysis has been superseded by newer technologies, but was a core part of biological research for slightly more than a decade. Microarrays were used by UNE, CSIRO and others in the SheepGenomics program, for both gene expression and genetic diagnostic work, and the use of gene chips for “genotyping” animals was subsequently developed and consequently is now in use for sheep breeding.
The polymerase chain reaction (PCR) is the method used to amplify specific gene fragments, a crucial step in most diagnostic tests except for microarrays. The second innovation of the late 1990s which is relevant to our discussion was real time PCR (Heid et al., 1996). This enables the reaction’s progress to be monitored over time as it occurs. PCR reactions typically take 1-2 hours and by the end the reactions become less efficient as reagents are exhausted. This is why real time PCR was important as it can be made quantitative by monitoring the reactions during the earlier linear phases of the reaction. Over the years refinements to real time procedures have allowed the development of reliable reactions which allow quantification of the target, qPCR. Target quantification meant that a sample which was derived from a mixture of individuals could be used and the relative content of each type to be calculated.
When I returned to Armidale in 2002 I had some experience with qPCR and Steve Walkden-Brown (UNE) had just purchased a qPCR machine for poultry virus detection work in collaboration with CSIRO. Within a year we had embarked on a program of research to develop qPCR tests to detect parasites, realising that the costs of the original Gasser lab tests could now be reduced by up to 100 fold because mixed samples containing hundreds of larvae or eggs could be used. The first work we did used pasture samples in collaboration with Brown Besier (DAFWA) and Ian Carmichael (SARDI) (Sweeny et al., 2012). The tests we developed were based on the ITS2 gene and used SYBR green technology, which allows the detection of only one type of DNA in each tube. So, we needed a separate reaction tube for each species to be detected; for example to detect H. contortus, Teladorsagia circumcincta and T. colubriformis three tubes are needed, and six if you want to do the reactions in duplicate for better quality control. We had begun to make progress on these tests when a change of funding meant we had to adapt them for use on faecal samples, rather than pasture washings. Although this slowed technical progress, it did allow a new collaborative agreement with Robin Gasser at the University of Melbourne, and this helped us produce two options of tests for future development.
In the first of these (Bott et al., 2009), we used nematode eggs purified from faeces as the target material, avoiding the problems of extracting DNA from whole faeces which contains inhibitory chemical substances. This suite of SYBR green tests was used to demonstrate the potential of the technology by multiple students from the University of Melbourne, including Florian Roeber (Roeber et al., 2011). Although this technology was only intended as a prototype, its use attracted commercial interest and the E-DNA tests which are now available are these same tests. For each sample, nematode eggs are separated from the faecal sample, and for each species assayed, a separate reaction is prepared. In the currently used format, no attempt is made to use the tests to quantify the total number of nematode parasite eggs in the sample. Rather, the sample is subject to a standard microscopy-based faecal worm egg count (FWEC) and the qPCR is used to quantify the relative proportions of each species assayed which are present. There is nothing wrong with this approach, but it is not the only way in which the tests could be used.
The second test option was to use a different chemistry, fluorescent hydrolysis probes, to simultaneously assay more than one species of parasite, in what are termed multiplex tests. In this option, the labour of setting up the reactions was reduced as there were fewer reaction tubes necessary per sample. At the same time, we set out to use intact faecal samples to extract DNA rather than purified eggs. This was because we could further reduce labour, using qPCR to do the FWEC at the same time as the species composition, and also to allow the flexibility of detecting non nematode parasites and other pathogens using the same sample. We reported the use of these whole faeces multiplex tests (McNally et al., 2013 Hunt et al., 2014) some time ago, and have also demonstrated that Fasciola hepatica can be detected specifically using the same type of samples (McNally 2013). Unfortunately a pathway to commercialisation of these tests has not yet been found. We attempted to validate the tests using the SCAHLS committee criteria, but refining the method in response to the SCAHLS evaluation was abandoned due to funding issues for both CSIRO and SCAHLS.
DNA technology and the detection of parasites – the future
Labour is the biggest cost in diagnostic laboratories, and any tests developed that reduce labour input per sample would be desirable. An economic reality however is the need for high throughput to make the most of any labour input, irrespective of the efficiency per sample. Parasite detection in the laboratories will continue to improve if the uptake of testing by livestock, because of efficiencies of scale. This will reduce costs for the laboratories which could be passed on to the customer, irrespective of the type of test employed (e.g. microscopy or qPCR). However, even my basic knowledge of economics leads me to the obvious caveat: there has to be competition between labs to create downward pressure on prices.
The role of extension in this process cannot be overstated. Better communication to livestock owners of the benefits of testing is to my mind a key future requirement. Research to provide better, more persuasive evidence for consultants will help.
Another factor which might assist is a change in the amount of attention given to livestock kept as a side-line or even as pets. Even though these animals are not part of a highly efficient production system, their welfare and health is just as important to their owners, the disease organisms they harbour can affect other livestock enterprises and some of the operations are able to utilise parasite control strategies which would be uneconomic on larger scales.
I believe it is these questions of scale of operation for both diagnostics labs and livestock owners, and the perceived need for parasite testing from livestock owners which will dominate the near future of these technologies. Having said that, let’s look at some of the technological possibilities which might also arise.
First, as indicated above, there are already improvements to the qPCR tests in the pipeline. Some of the technical issues around faecal DNA extraction could be solved with some more empirical work, and a more thorough evaluation of the technology should be able to demonstrate its use in place of a microscopy-based count in time.
Second, the use of ITS2 is somewhat of a historical artefact. With mitochondrial genome sequence now available for many nematode parasites of livestock and whole genome sequence now available for H. contortus and T. circumcincta, there are many more genome fragment options which might prove to be more reliable than ITS2. One issue with ITS2 and mitochondrial DNA is an unknown copy number. If gene segments which were highly species specific and unique within the genome were used, there may be an increase in the reliability of the relationship between worm numbers (as eggs or larvae) and qPCR outcomes. This might come at the cost of decreased sensitivity, but optimising PCR reaction conditions can minimise this effect. Such improvements would not change the type of information given to livestock owners, or the way in which samples were submitted, but would increase the quality of the information.
Third, the use of DNA to merely count worms is not really making the most of this technology. DNA-based tests should, and could, be used to tell livestock owners so much more.
For some time I have advocated the use of genetic diagnostics for parasites. The monitoring of genetic change over time in parasite populations would add a dimension which is not available using any currently existing methods. The effects of stock introductions, failed quarantine drenching, movement of parasites through flood water or the selection of drug resistant genotypes might all be monitored through genetic diagnostics for parasites. Such technology would rely on microarray platforms, and be used a small number of times on bulk, flock or herd-level samples to track population change. Properly engineered, the microarrays would be able to detect species composition change in conjunction with intra-species genetic change.
Adopting a lesson from livestock breeding would make the best use of the technology. Accumulating data for a series of properties over time including some basic information about stock and management procedures and weather data, could generate self-improving algorithms which predict animal health and parasite control outcomes. This is similar to the ways in which the breeding values of individual animals for given traits are estimated to a higher degree of accuracy as more data from relatives is combined into the database. In this case we are interested in individual properties, rather than animals, and individualised farm management rather than genetic progress.
Bisset, S. A.; Knight, J. S.; Boucher, C. L. G. (2014) A multiplex PCR-based method to identify strongylid parasite larvae recovered from ovine faecal cultures and/or pasture samples. Veterinary Parasitology 200 (1/2): 117-127.
Nathan J. Bott, Bronwyn E. Campbell, Ian Beveridge, Neil B. Chilton, Dianne Rees, Peter W. Hunt, Robin B. Gasser (2009) “A combined microscopic-molecular method for the diagnosis of strongylid infections in sheep” International Journal for Parasitology 39(11): 1277-1287.
Callaghan, M. J. ; Beh, K. J. (1994) A middle-repetitive DNA sequence element in the sheep parasitic nematode, Trichostrongylus colubriformis. Parasitology 109(3): 345-350.
Grant, W. N.; Mascord, L. J. (1996) Beta-tubulin gene polymorphism and benzimidazole resistance in Trichostrongylus colubriformis. International Journal for Parasitology 26(1): 71-77.
Gupta, P. K.; Roy, J. K. ; Manoj Prasad (1999) DNA chips, microarrays and genomics. Science 77(7): 875-884.
Heid, CA; Stevens, J; Livak, KJ; Williams, PM. (1996) Real time quantitative PCR. Genome Research 6(10): 986-994.
Hunt, PW (2014) MLA project B.AHE.0028 final report – Refinement and validation of a PCR test to replace WEC and FCLD, including commercial feasibility. ISBN: 9781740361859. ( http://www.mla.com.au/Research-and-development/Search-RD-reports/RD-report-details/Animal-Health-and-Biosecurity/Refinement-and-validation-of-a-PCR-test-to-replace-WEC-and-FCLD-including-commercial-feasibility/146 )
Kwa, M. S. G. ; Veenstra, J. G. ; Roos, M. H. (1994) Benzimidazole resistance in Haemonchus contortus is correlated with a conserved mutation at amino acid 200 in beta-tubulin isotype 1. Molecular and Biochemical Parasitology 63(2): 299-303.
Le Jambre, L. F.; Lenane, I. J. ; Wardrop, A. J. (1999) A hybridisation technique to identify anthelmintic resistance genes in Haemonchus. International Journal for Parasitology 29(12): 1979-1985.
McNally, J., Callan, D., Andronicos, N.A., Bott, N. and Hunt, P.W. (2013) DNA-based methodology for the quantification of gastrointestinal nematode eggs in sheep faeces. Veterinary Parasitology 198: p325-335.
McNally, J. (2013) MLA/DAFF Science and Innovation Awards for Young People in Agriculture, Fisheries and Forestry final report. (available on request)
Roeber, F. ; Jex, A. R. ; Campbell, A. J. D. ; Campbell, B. E. ; Anderson, G. A. ; Gasser, R. B. (2011) Evaluation and application of a molecular method to assess the composition of strongylid nematode populations in sheep with naturally acquired infections.
Schena, M. ; Shalon, D. ; Davis, R. W. ; Brown, P. O. (1995) Quantitative monitoring of gene expression patterns with a complementary DNA microarray. Science 270(5235): 467-470.
Sweeny, J.P., Ryan, U.M., Robertson, I.D., Niemeyer, D., Hunt, P.W. (2012) Development of a modified molecular diagnostic procedure for the identification and quantification of naturally occurring strongylid larvae on pastures. Veterinary Parasitology 190(3-4): 467-481.
Depp, dogs and rabies
The following was first published by Jessica Sparkes on 29 May, 2015 under the title, “Little dogs, big issue: Biosecurity scientists review rabies” in DPI Active, an internal NSW DPI blog.
“Johnny Depp’s two terriers, Pistol and Boo, have received a great deal of media attention, locally and internationally. Although this provides comical relief and grist for the Twitter mill, it also opens up myriad questions relating to a disease that could potentially cost Australia billions of dollars and human lives.
Of greatest concern for the illegal importation of dogs into Australia is that of rabies, a devastating disease that kills more than 55,000 people annually on a global basis. Due to our current quarantine protocols, this deadly disease has not established in Australia to-date.
To assist in the preparedness for such an outbreak, Jessica Sparkes, Guy Ballard and Peter Fleming of DPI’s Vertebrate Pest Research Unit, in collaboration with the University of New England and Sydney University are researching how rabies could spread through Australia’s dog populations. Two recent publications summarise this work, see Canine rabies in Australia: A review of preparedness and research needs and Social, conservation and economic implications of rabies in Australia.
These papers review the implications of rabies and our preparedness, emphasising that the illegal importation of infected dogs (e.g. via boats or jets), could spark an epidemic that would be difficult to stop”
Republished here with permission.
Camera traps reveal surprising wild dog foray
Long-term monitoring stations established by DPI staff and local landholders have revealed images of the same wild dog moving almost 40km between livestock production areas before then moving back again. The wild dog’s travel route took him through the rugged Oxley Wild Rivers National Park over a 2 week period.
Both the extent of the wild dog’s movement and how the data was pieced together, i.e. from DPI and landholder’s camera traps, reinforces a fundamental need for wild dog management programs to operate across tenure via effective partnerships between public and private land managers.
Wild dogs, including dingoes, are a declared pest in NSW. All landholders are obliged to control them, to alleviate the burden of wild dog predation and disease on livestock and other domestic animals.”
This story first appeared in ‘DPI Active’ and is republished here with permission.
“Type O protects against cognitive decline via gray (sic) matter”
Like most of you, I take most health/science reports on the web/in the media with a grain of salt, especially now that salt (and dietary fat) have been at least partly rehabilitated following reviews of the evidence. So, scientists are human after all.
Researchers have apparently found that having blood type O was associated with more grey matter….(but this association or correlation somehow, in this article, got transformed into causality: the article heading says ” type O protects against cognitive decline via gray (sic) matter”.
But, I am happy to go with this because …yep, my blood type is O. But that should have been obvious, a no-brainer.
I will, of course, ignore the bits in the article I don’t like.
In fact, I am generally sceptical. But then everything in (good) science is provisional.