PCR vs. EM Adenovirus testing

Hello Everyone,

I have been hesitant to post on this subject. The controversy surrounding this embattled issue is upsetting but I do feel there are a few Adenovirus testing questions relating to the differences between PCR and EM testing that I can answer.

There have been some questions about the differences between blood vs fecal swabs as a specimen type for PCR testing. In theory blood can be used in place of a Cloacal swab only if the dragon has viremia (means the virus is found in the blood stream). If the virus is in the bloodstream it means basically that the host’s immune system has not been able to keep the virus in check. As a scientist you want to develop a method that has the best chance of picking up the virus so you look for the virus in the system that it affects first or lays dormant in. Adenovirus like particles have been found in the Liver which is part of the digestive system. Feces are excreted from the digestive system so this is the best place to find the virus. So the Fecal swab would be much more likely to find the virus if it is present in the animal. So if I was to develop a PCR Adenovirus test my sample type would be Feces.

Here are some of the differences between Polymerase Chain Reaction (PCR) and Electron Microscopy (EM). One of the big differences between PCR and EM is that EM is looking for whole adenovirus like particles and is also unable to distinguish between most types of adenovirus. In the PCR test you are looking for a specific sequence of DNA. Not to mention that DNA is inherently more stable than most viruses. When developing a PCR test of this nature your whole goal is to construct a primer that is specific for a species or strain that is habitually isolated from the particular animal of interest. So what I am saying is that you want to find a DNA sequence that is specific or only found in the animal of interest. In doing this you significantly narrow down the likelihood of picking up other types of Adenovirus. Another advantage of PCR is that in theory your PCR should be able to pick up DNA from animals that are not actively shedding whole virions (virus progeny). Here is the reason why, Infected cells like normal cells will eventually die and the cells will release DNA fragments into the feces even know the animal is not actively shedding large numbers of whole virus. The PCR method should be able to pick up some of this DNA from just the natural death of infected cells. This is another advantage of the PCR method.

Here is a great question asked on another forum:

When a beardie dies and is suspected to have AV, a necropsy is done and the liver is biopsied and looked at through an electron microscope, would it be safe to say that a DNA test at that point would also show the virus?

Yes, If you extract the DNA from the tissue and the animal has Adenovirus it will be picked up by PCR. In actuality one could also identify the Genus, species and hopefully someday the serotype or strain by PCR.

Kind Regards,

Randy

Thanks for the great information! That did clear up some questions I have had while preparing to get mine all tested, and trying to get everything worked out with my local vet.

Any work done on the sensitivity and specificity of the tests?

No problem Cat!! My pleasure.

Hey Joe good question!! As far as specificity goes Jacobson's Lab has isolated and partially sequenced four proposed new species of AdV including a species(AAdV-1) found in Bearded Dragons. They compared the AAdV - 1 to known genetic sequences from other adenovirus and did not find a match. The closest match had less than 90% homology. "These lizard AdV sequences are adequately different (less than 90% sequence identity) to be considered distinct adenovirus species."(Wellahan et. al J Virol. 2004 Dec;78(23):13366-9.). So for now this is the most specific test available.

As far as sensitivity goes you would have to ask Dr. Jacobson's lab about that. In the process of validating their technique they probably did a titration curve or a spiking curve to figure out how sensitive their test is. Sorry I can't help you with that one, but I am sure Jacobson's lab can.

Kind Regards,

Randy

Thanks for the info Randy. I'm more curious than anything, I'm more or less retired from the reptile industry and haven't kept a dragon in 15 years. I do have some limited epidemiology background, and I'm curious to see how this situation plays out. It seems to me some folks are throwing out a lot of absolutes when there isn't enough known about the situation. If I were managing a colony of bearded dragons, I'd probably want to run both tests, than sacrifice a whole lot of animals and have histo work done and compare the results. It seems like people are being asked to make some hard choices with little more than hearsay, anecdotal evidence and clinical cases to base things on. Again, I have no vested interest in this, and haven't done a lit search, I'm just curious to see how this plays out.

Joe

Thank you very much for that information.

Hello,

Thanks for that bit of information, it was very helpful!! Very generous of you.

Tracie

Randy, please correct me if I'm wrong, but what your saying then,is that at the present time the EM/fecal would be the best way to determine if your dragon has adeno even though the test can't determine the specific type.
Right? or wrong?
Thanks,

Randy, Thanks for the information. I've been trying to find exactly what you are talking about in literature. I'm trying to decide if I'll take the frozen poop I have and send it in to Illinois in the kit they sent for the EM test or if I'll call Jacobsen's lab to see about sending it here. My vet told me we could do a liver biopsy because tissue is required for the test at the lab in Mississippi that he uses, but I don't want to do that.

I lost my female, Etouffee, after she laid her first clutch. It was septicemia due to egg stasis. I had her sent to Texas A&M Vet School clinic. They did a lot of special mounts and I spoke with the DVM that actually looked at the slides. I asked her why I didn't see the words "adenovirus negative" anywhere on the report but everyone that interpreted it - said "no adenovirus". She told me she was positive that the samples she processed did not have any signs of adenovirus. She amended her conclusion and sent a new copy to the Vet and they faxed it to me.

So, if I could get something close to a definitive answer about the status of the other three, that could mean that my babies should be negative. If it one or more of them are positive, I have a closed situation here were they are not exposed to other BD's and it might help with anecdotal notes for research as three were kept together in a 175 gal enclosure and the big male is in an enclosure by himself. I would think at least all three of the ones kept together would be positive. If not, maybe something in their system keeps them from getting it.

So I'm trying to determine the best way to test.

Hello Karen,

Good to see you!! Actually what I am saying is that the nested PCR that University of Gainesville, Florida does is much better than the EM test. It is more pricey but the fact that they are using two sequential primers and sequencing the viral DNA is one of the big reasons why this test is more specific and more costly.