Not Mareks?!?!?!?! What could it have been?

[Leihamarie]

Hi all,

So this is an odd result I received yesterday on some blood tests I had done on my flock. This is not an emergency, I have no sick birds but I want to get your opinion/experience in what this could have been.

I had what I thought was Marek's disease take my whole flock over the period of a few months From October 2016- Feb 2017. I thought it was Marek's because the symptoms matched and the 2 Necropsies I had done by the CA Animal Health & Food Safety Office confirmed it was Marek's. Just this week, 5 blood tests I sent to a lab in Texas that performed Real-Time Quantitative PCR DNA testing for Marek's came back completely negative. WHAT THE HECK?!?!?!?! I'm SOOOOO confused. Here's the timeline and events, with necropsy documents and lab test results:

In October, 2016 2 of my 3 pullets died over the course of 15 days. The vet suspected Marek's and I sent one of the pullets (Fleur) off for a necropsy. It came back as death due to Marek's. None of my 3 birds were vaccinated. I've attached the necropsy for Fleur at the bottom of post.

A week later, when my remaining pullet (Mabel) showed no symptoms, I bought 3 new pullets from a local farm. The 3 pullets were vaccinated and the same age as Mabel, who had just lost her flock. I had hoped Mabel was immune. She wasn't. In February of 2017 (4 months later), she also died. Her symptoms started suddenly and she declined quickly. I had the vet euthanize her and sent her for a necropsy. It came back Marek's again. I've attached Mabel's necropsy

So at this point, I had active Marek's and 3 vaccinated pullets who had been exposed to Mabel and the environment.

In June of 2017 (4 months after Mabel died) I picked up 5 baby chicks. I brooded them in the house for about 10 weeks and had SUPER strict PITA bio-security in the house so that their Marek's Vaccine had time to develop resistance.

Since Mabel died, I haven't had any chicken losses due to disease or otherwise. I did raise some meat birds at the end of Summer in 2017 and I remember thinking it was strange that their organs were perfect, being exposed to Marek's and all. I processed them at around 12 weeks old which would have been time enough to develop visible tumors.

So, last week I sent off blood samples from 5 of my 7 hens to a lab in Texas that does DNA gender identification and real-time quantitative PCR testing for many diseases. I had them test for Marek's and my expectation was for them to tell me how virulent the Marek's I have here is, and how big of a virus load my birds have.

ALL FIVE BLOOD TESTS CAME BACK NEGATIVE FOR MAREK'S!!!!!!! WHAT THE HELL?!? I was absolutely floored! This makes ZERO sense! I live in an arid climate, so... no ground freezing and no moisture saturation to eliminate Marek's but even if that weren't the case, I have had CONSTANT chickens on site through the suspected Marek's outbreak! I've thought about it from every direction and can't for the life of me figure out how my flock is Marek's- free. Even if the 3 pullets I got to keep Mabel company were immune, there were only 6 months that passed between Mabel dying and my baby chicks being allowed outside which is not enough time for Marek's to go away. I've attached the lab results at the bottom of post.

Can someone help me make sense of this?!? I have a call into CAHFS and the lab is re-running the blood tests for Lymphoid Leukosis, although I highly doubt that was what was going on, because again... I've had no more losses. This is the most baffling thing!

 

[Kiki]

It has been my understand that Marek's can't be detected until death.
Could it be possible this blood testing isn't good/doesn't work for Marek's disease?

I have actually contacted this lab via email before to ask how the Marek's testing is done.
Never got a response.

 

[Kiki]

One thing I notice is that Mabel had what your lab calls rare coccidia!
Interesting (to me) THANKS for sharing your reports.

 

[micstrachan]

Did the lab verify that the DNA sample was good? How exactly was the blood shipped? As a former molecular biologist, I can tell you if the DNA was badly degraded, it could (would likely) produce a false negative. If they are a reputable lab, however, I would think they could tell you if the sample was good. Do you know what controls were performed?

 

[Eggcessive]

Everything I have read about Mareks says the best way to diagnose Mareks is to test feather shafts and tumor tissue after death. I do think that some labs offer this sort of thing, and it may not be accurate. Someone recently posted about a lab where they could send in a feather, and get it tested for Mareks. Hers came back negative on all of her chickens, but it sure sounded as though she had Mareks in her sick chicken.

Just because a lab offers a test, such as the PCR test for mycoplasma for example, they may have a lot of false negative readings. A good friend is supervisor of a lab microbiology unit, and he said that they had so many false negatives, they stopped doing those tests. They now do a dna test for mycoplasma.

So, I would trust the poultry labs which do necropsies and testing. There may even be some false negatives there as well, but they look at the whole body for evidence of Mareks tumors.

 

[Leihamarie]

It has been my understanding that Marek's can't be detected until death.
Could it be possible this blood testing isn't good/doesn't work for Marek's disease?

I have actually contacted this lab via email before to ask how the Marek's testing is done.
Never got a response.

I do know that standard PCR is a good way to test for Marek's in a live bird, but it's not good at differentiating between types/ virulence and has been cost prohibitive when done through veterinary offices. That's why real-time quantitative PCR is so exciting. Here's an article about testing on live birds: http://www.fwi.co.uk/poultry/new-faster-test-for- marek-s-disease.htm

also, here's an article on using q-PCR to identify Rispens vaccine in feather tips. If they're able to differentiate between Rispens vaccine and MDV, it's an accurate method of testing. https://www.ncbi.nlm.nih.gov/pubmed/17058503

I called the lab and had about an hour long conversation prior to sending samples in. I also had them DNA sex my chicks that hatched last week from their shells. They seem to work with a lot of vet offices and chicken people. I'm surprised that you didn't get a response from them. They've responded to me usually very quickly and always answer the phone (I literally called them every morning for like...5 days with new questions, lol!).

 

[micstrachan]

@Leihamarie, you’re my kind of girl! Just sayin’.

 

[Leihamarie]

Did the lab verify that the DNA sample was good? How exactly was the blood shipped? As a former molecular biologist, I can tell you if the DNA was badly degraded, it could (would likely) produce a false negative. If they are a reputable lab, however, I would think they could tell you if the sample was good. Do you know what controls were performed?

This is their "Marek's sample guideline" sheet (attached). I bought sterile, rayon-tipped swabs, prepared the sample myself by wiping each chicken's clean toenail with alcohol, clipping it just to the quick, saturating the swab, securing the swab in a sterile tube and then tending to the chicken's toenail (styptic powder, mealies for their trouble, etc...). I sent the samples by overnight mail and they arrived the next morning, along with my hatching shells to gender ID my new chicks.

They didn't outrightly confirm that the sample was good, but I will double check their procedure for verifying samples.

 

[micstrachan]

Thanks for sharing that. I am stumped. qPCR should be a VERY reliable assay. Apparently there is much more to Marek’s than meets the eye.

 

[Leihamarie]

Everything I have read about Mareks says the best way to diagnose Mareks is to test feather shafts and tumor tissue after death. I do think that some labs offer this sort of thing, and it may not be accurate. Someone recently posted about a lab where they could send in a feather, and get it tested for Mareks. Hers came back negative on all of her chickens, but it sure sounded as though she had Mareks in her sick chicken.

Just because a lab offers a test, such as the PCR test for mycoplasma for example, they may have a lot of false negative readings. A good friend is supervisor of a lab microbiology unit, and he said that they had so many false negatives, they stopped doing those tests. They now do a dna test for mycoplasma.

So, I would trust the poultry labs which do necropsies and testing. There may even be some false negatives there as well, but they look at the whole body for evidence of Mareks tumors.

That's a really good point! I'll call the lab again on Monday and ask more questions about accuracy and false negatives, etc... The guy I spoke with said that blood was more reliable/accurate than feathers, which is why I did blood... much to the dismay of my unsuspecting ladies, lol!