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Strategic Use of Diagnostics
Tuesday, April 30, 2013
Industry Showcase
With such a wide choice of diagnostic tests
available, Life Technologies explains how to
choose the best option in any given situation.
Large animal veterinarians have never had such a wide range of diagnostic tools to
help them maintain and improve the health and productivity of their clients’ animals.
The development of new and better tests over the last few decades has made it
possible to get faster, more accurate, and more precise information about the
health status of individuals or groups of animals. The development of commercial
ELISA (enzyme linked immunosorbent assay) and PCR (polymerase chain reaction)
technologies in particular has revolutionised diagnostic services, so that
veterinarians now have access to dozens of different tests covering all the major
production animal species and a wide range of pathogens.
But with such a wide choice of tests available, how do you know which one is the
best option in any one given situation?
Culture, ELISA or PCR?
Unfortunately there is no simple answer, and currently no definitive or standardised
guidance made available on PubMed. Although ELISA was a big step forward from
the classical serology methods, and PCR is a more advanced method than the
culture method, it is not simply a question of each successive development
replacing the previous technology. Each has its role: even culture still has a place
in the diagnostic laboratory, as Dr Gerard Wellenberg from GD Animal Health
Services in The Netherlands explains:
"There will always be a need for culturing. Virology departments have shrunk as
culture of many viruses has been replaced by specific PCR methods in recent
years, because it is faster, cheaper and less laborious. That's why PCR was initially
aimed at viruses and slow or difficult to grow bacteria. But there are still a lot of
bacteria, such as Staph aureus, that can be grown in one day and are easy to grow
– and, most importantly, it can still be done more cheaply than PCR."
Conversely, there are some bacteria which can now be tested for much faster and
therefore more cost-effectively thanks to PCR, such as Mycobacterium avium subspecies paratuberculosis (MAP) the causative agent of Johne'-’s disease, which
used to take up to six to eight weeks to culture but can be identified by PCR in a
day.
"Another advantage of PCR over culture is that it can detect bacterial virulence
(associated) factors and different genotypes, for example with Streptococcus suis or
Haemophilus parasuis, which you can't do with culture. That makes PCR
particularly useful to researchers who are investigating the epidemiology and
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aetiology of different pathogens."
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Basic Differences between ELISA and PCR
ELISA is generally used to detect antibodies and so provides indirect evidence of
infection based on the animal's natural response to infection. An inherent drawback
of this approach is that the presence of antibodies also could be maternally derived,
or the result of a previous infection or vaccination. The latter was the driving force
behind the development of so-called marker/diva vaccines with their accompanying
diagnostic tools, which allow animals that have previous infection to be
differentiated from those that have been vaccinated.
ELISA's for the detection of antibodies are still routinely used for surveillance and
control of diseases. In the case of FMD (foot and mouth disease), the presence of
antibodies to non-structural proteins by ELISA indicates that the animal was
naturally infected by the virus and not positive due to vaccination.
PCR can also be developed to differentiate between field strains and vaccine
strains, which can be of added value if you have a vaccination or eradication
programme. Unlike most ELISA test methods, PCR is designed to detect the
pathogen itself, whether a virus, bacterium, parasite, etc, and in general, it can
detect the presence of a pathogen already during the incubation period, even
before it produces clinical signs. However, many samples that are sent into animal
health testing laboratories come from infected animals that are showing signs of
disease.
There are some exceptions to the antibody/antigen differentiation between ELISA
and PCR: for some pathogens, such as BVDV (Bovine Viral Diarrhoea Virus) in
cattle, ELISA can also be used to detect the virus itself. However, although the
basic science underlying each technology is clearly defined their usage in the field
is less clear cut.
Strategic Use of Diagnostics - Pig Articles from The Pig Site - The Pig Site
ELISA testing
Complementary Technology
In some cases, the laboratories are asked to conduct both types of test, says Dr
Wellenberg. "If the antibody ELISA is negative, customers still want to know if there
is the pathogen present, because samples could have been collected during the
incubation period of an infection and thus ELISA alone may not give the whole
picture."
One inherent characteristic of ELISA antibody tests is that immune response takes
time to develop. In the case of MAP, in some infected animals antibodies may not
be detected until they are two years of age. So, the use of PCR or culture to detect
MAP can be of added diagnostic value.
This synergistic use is more indicative of the way in which these tests are best
viewed, he says.
"They are different systems: with ELISA you look in general for antibodies, with
PCR in general you look for the agent. They are complementary. The key is to
know which one to use for which pathogen."
According to Dr Wellenberg, there are a number of key parameters that need to be
considered before deciding which type of test is most appropriate in a given
situation.
"There is no one way of using diagnostic tools. It all depends on a whole range of
factors, such as what type of programme you have: do you have a monitoring
programme, or an eradication programme, or a programme for certification. All
those programmes dictate how you should use your diagnostic systems.
"If you have an endemic disease or if you are virtually free of disease, then your
use of diagnostic tools may be completely different."
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ELISA: mode of action
Disease Prevalence
The prevalence of disease can be a major factor in deciding the best diagnostic
approach. For example, in Holland about 70 per cent of dairy herds are seropositive for BVDV, so it is not practical to cull all the antibody positive animals. The
strategy is to identify the PI (persistently infected) animals which shed high levels of
virus into the dairy herd, and then cull those individuals. It is simply not practical to
test every individual animal using PCR, so bulk tank samples are tested initially to
see if the herd is positive for BVDV. If it is positive, then pooled blood samples
from groups of 15 animals are PCR tested to narrow down the search. Any groups
that test positive are then given individual tests and the positive animals culled – for
this last step, an ELISA antigen test can be used because it is cheaper than the
equivalent PCR.
This strategic use of diagnostics provides a cost-effective means of identifying
individual PI animals in dairy herds in a high prevalence country or area, Dr
Wellenberg says.
However, in other countries which have a very low prevalence of BVDV or in
countries that are BVDV-free, a totally different approach is most appropriate:
"If your country is almost BVDV free, like Sweden for example, you can aim to cull
all antibody positive animals. In that case, it makes more sense to use ELISA for
the initial screening because you know most animals will be antibody negative. If
you get a positive result for a test, then you can use PCR to identify the individual
infected animal and cull it."
Disease prevalence programmes, such as BVDV eradication in Switzerland and
Germany, have been started to test all animals in order to identify all persistently
infected animals via real time (RT)-PCR or ELISA antigen tests.
For monitoring programmes, for example for classical swine fever (CSF) or
Salmonella in swine, ELISA tests are widely used in Holland because they are less
expensive than PCR and because many laboratories have fully automated systems
that can handle the large numbers of samples that are required for monitoring
Strategic Use of Diagnostics - Pig Articles from The Pig Site - The Pig Site
programmes. The ability for mass implementation and specificity always needs to
be well balanced: Germany changed its testing paradigm during the last big CSF
outbreaks in 2006 in pigs from antibody testing to antigen detection via RT-PCR.
The same applies at the farm level as well. A specific pathogen-free herd would
take a different approach to diagnostic testing to a herd which was endemically
infected with the same target pathogen. For example, a swine herd that is free of
PRRS (Porcine Reproductive and Respiratory Syndrome) would take a different
approach to one that has infection and sero-positive animals.
PCR: mode of action
Time Window
The natural history of a disease is probably the factor that comes closest to defining
the place of ELISA and PCR diagnostics. In diseased animals it generally takes
seven to 14 days after infection before antibodies can be detected, so the use of
ELISA is generally not appropriate during this time. Conversely, with PCR, you can
get a positive result after one or two days of infection. So if you have diseased
animals, many people will use PCR for identification of the causative agent.
As with most rules, there are exceptions. Schmallenberg virus for example, is only
detectable in the blood of infected animals for a relatively short time, but antibody
titres remain high for a relatively long time. So the use of PCR is more limited.
Influenza is another example: virus particles are only present in lung and nasal
swabs between day 2 to approximately day 9 – after that time, a PCR can be
negative because by that stage antibodies may neutralise the virus. Again, in this
situation, PCR is of limited use and ELISA may be a better option. Conversely, in
PRRS infection the virus can be detected in blood up to 28 days or even longer
after infection. Every animal and every agent has its own time window, and this can
fundamentally affect the choice of diagnostic system used.
Sample Type
The suspected pathogen also influences the type of sample to be collected. The
type of sample needed for detection is related to the tissue or cell tropism of the
Strategic Use of Diagnostics - Pig Articles from The Pig Site - The Pig Site
organism and where the organism is shed in the body during its lifecycle of
infection. Swine influenza viruses, for example, cannot be detected in blood or
aborted foetuses, even when the abortion is a consequence of the influenza
infection. Brachyspira can be detected by faecal samples but cannot be detected in
blood or lung samples.
The range of sample types that can be tested with PCR was initially quite
restrictive, but more sample types are being validated for existing and new tests.
ELISA, of course, can only be used on samples that will carry the antibodies or
antigens that are being tested for – in most cases, that means blood.
Finances
In the current economic climate cost will always be a factor for any service linked to
production animals. Diagnostic costs are a product of several factors including the
resources of individual stockholders, mandatory programmes and government
support, as well as the cost of individual tests or test programmes.
All these factors, and others, influence the choice of diagnostic tools and their
strategic use. But, as Dr Wellenberg points out, the most costly test of all is the one
that gives a misleading result because it has been applied in the wrong
circumstances. Pooling, for example, is a very effective way of reducing diagnostic
costs, but if your test is not sensitive enough, then pooling may provide misleading
results.
There are quite a few antigen capture ELISA (ACE) which are often used as the
alternative to PCR for cost reasons. Their ability to effectively replace PCR may
depend on the actual viral load in the infected animals. In the case of two look-alike
diseases, African Swine Fever (ASFV) and Classical Swine Fever (CSF), the titre of
virus in CSF may be below the threshold of detection by ACE and thus PCR will
pick up cases at the farm level that may be missed by ELISA. On the other hand,
the load of viral antigens to ASFV in an infected animal are at a sufficient level that
veterinary laboratories in poor countries could adequately detect ASFV by ACE if
that is all that their limited resources would allow.
Many laboratories have automated ELISA systems that can handle many samples
at once in an attempt to keep the cost per test down. However, PCR has become
less expensive as more and more products are developed and so the cost issue is
becoming less of a factor.
Talk to Your Laboratory
The large number of variables shows that it is clearly impossible to give a set of
guidelines on the most appropriate diagnostic approach for each and every
situation that a veterinarian might encounter in the field. Dr Wellenberg’s advice is
to use the laboratories and specialized vets as a source of advice:
"Talk to your laboratory and specialized vets. Make sure you know their capabilities:
what kind of tests they offer. And don’t be afraid to ask their advice – after all, they
should be the diagnostic experts. They know all the latest ideas and can help you
formulate the most effective test programme for your area or for individual herds."
The future is likely to see the number of diagnostic tools continue to grow: not only
in the form of more PCR and ELISA systems but also the development of
multiplexing and micro-chips. That means knowing which ones to use will become
even more important.
May 2013
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