Doubletree Tape 4

3/6/02

**Dr. John Kolb (continued)**

\--vaccines in the early 1990\'s, I think they\'ve been around 10 years
Brad? Plus or minus 10 years for mycoplasma vaccines. There was some
work done in EU looking at strategic use of medication to try to help
pigs develop an active immunity. I think you mentioned that this morning
with the concept of high levels of CTC one week a month followed by some
other type of antibiotic for three weeks a month. So using strategic
medications to minimize or reduce the impact of the mycoplasma pneumonia
in the pig yet removing those products to allow continued replication of
that organism within the pig. Okay.

The term that was utilized in a swine health and production paper
published in 2000 was the term metaphylaxis which really talks about
utilizing this concept of allowing a pig to be exposed to the virulent
field organism and then placing a strategic use of a medication prior to
the actual on set of overt clinical disease. Allow the pig to be
exposed, begin to develop some type of immune response either alone or
in combination with the vaccine but then medicate that pig to reduce the
clinical and biologic impact on performance. Okay, next one.

So, maybe the best use would be strategic use of a product, okay? Which
allows that natural exposure and infection of the pig to have a virulent
field organism in their system but immediately following that utilizing
short term bursts or strategic pulse of medication to really either
inhibit or shut down the incubation period to prevent expression of
clinical disease. And again, that customer, the pig doesn\'t want to be
sick, so is the way, is there a way for him to get exposed to the
organism and begin to develop an immune response but yet limit or
prevent entirely that negative biologic and economic consequence. Next.

And again, that exposure may help the pig, either by itself or may even
developing acquired immunity from the vaccine provide a long term
protection against these endemic diseases where we know they will be
re-exposed again such as mycoplasma. Next.

And that, a key point of that is a limited duration of a therapeutic
dose, this has some advantages that limits the cost of the producer,
certainly it limits the use of antibiotics and that\'s something we are
all concerned about, how to appropriately utilize those. It limits the
amount of exposure to the antibiotic that the organism has and a recent
piece in Swine Health and Production looking at *E. coli* and
*Salmonella*, high levels of short use of an antibiotic may actually
reduce the development of resistance or prevent development of
resistance completely. Okay. Go ahead.

It\'s important as we said because we know with things like mycoplasma
we\'re not going to keep the organism out of the pigs. It\'s going be
difficult to exclude that infection. If we treat for a period of time
and then remove that therapeutic pressure and we\'ll see in a case
example where that may have happened. They either get subsequent
exposure to the disease or actual clinical disease outbreaks at a later
point in the production phase. Examples of this might be this SEW
systems where you have what we call the 18 week wall that\'s a
respiratory disease in the growing and finishing phase the pigs are
medicated at one point in time those medications are withdrawn, they may
have subsequent disease rebreaks. Another example might be *Lawsonia*,
where we want to utilize medications to prevent clinical disease, we
know they are going to get exposed to that organism later on. Next
please.

Another place where strategic dosing might be effective is where
vaccines aren\'t available or those vaccines aren\'t very effective
today. Dr. Brad Thacker mentioned APP as one potential example there.
Where there\'s really only one vaccine that has some clinical
effectiveness with it. I have *Streptococcus suis* there really are none
today so pulse dosing might be effective there as well or with a vaccine
actually was not used appropriately might be another opportunity where
strategic dosing of medication may play an important role. Next.

Again with the epidemiology of a disease is changed, you are placing a
vaccine at 10 weeks of age into a pig for mycoplasmal pneumonia and
where there\'s change in PRRS virus circulation or change in the actual
mycoplasma circulation. Now pigs can no longer develop an active immune
response in time to be protected. So, in the interim as you change
vaccination strategy, you could use a strategic medication strategy to
control disease or an early diagnose disease where there is no current
vaccine strategy in place. I have to take one from Dr. Walther\'s papers
multiple disease challenges require kind of a broad spectrum a set of
interventions. Next please.

Now, we\'ll go through this case it has a couple of nice examples with
it. There was a commercial three-site production system located in the
Midwest, it was a very typical production system for today\'s pork
production. PRRSV, influenza, mycoplasma, multiple common endemic
disease agents present where the history of this decreased performance 8
to 12 weeks after they were placed in the finishing barn. That\'s about
18 to 22 weeks of age for the pig, plus or minus 60 kilograms, 120
pounds, somewhere in that weight range. Pigs suffered from typical PRDC,
this respiratory disease complex. As a result the growth rate was
reduced, feed efficiency was increased, feed intake was variable, a
response of the disease to feed intake. Okay.

Diagnostically, Dr. Schwartz would say keep sampling cause there was a
lot of different things found on different occasions and it really was
not a consistent single agent that could be identified as being the
major cause of problems, okay.

And this case it was actually a prospective study where animals were
serially bled every two weeks as an indicator of disease exposure.
Serology was performed at these various times looking for at least some
of the pathogens that were present on this farm, *Mycoplasma*, PRRSV
virus, influenza virus, Transmissable Gastroenteritis virus,
*Salmonella* and *Lawsonia*. It sounds like a lot of stuff but as we
learned earlier today this is very typical for a common pig production
system. Then serology was additionally performed on those pathogens that
were shown to be present in the herd. Next please.

There were three treatment groups in this study. First were
non-medicated controls, pigs that had no medication in their feed at all
and this may be a little bit uncommon for production systems today but I
think it\'s where we are starting to head to at least attempting to get
there. Treatment two was two products which have some respiratory
activity that were placed strategically five different times throughout
the production period, with no medication in between them. There\'s a
graphic that will help explain this. The third treatment was those same
products but then a continuous use of medication in between there as
well. So, if we think through this concept of limited exposure, then a
therapeutic use of the medication, another limited exposure and another
therapeutic use of the medication, we\'ll get to some performance data
here next. Okay, outcomes. The typical 18-week wall, severe clinical or
respiratory disease didn\'t occur in this production system because we
develop models to look at mycoplasmal disease maybe remembering that
clinical productions, majors are important. Next please. Next.

Even though potentially there may not been a great deal of clinical
difference between groups of pigs overt, observed clinical disease,
there was a significantly beneficial biologic result between those
groups, growth rate, potential feed efficiency etcetera. So, as we would
begin to look at models that develop an evaluation for mycoplasma, lung
scores are nice, those things are important, cough scores are important
etcetera but we don\'t sell those as producers, data gained, food
efficiency, economically important parameters for the producer and for
the pig, the final customer should be included as well. Next.

What was interesting that Dr. Walter saw in the study, I\'ll go through
the slide for a few minutes just to explain what you are seeing. Along
the X-axis are the number of weeks that these pigs are in the finishing
barn so if you\'ll add 10 to that, that would be approximately the pigs,
the weeks of age. The little circular spots here, these five represent
the five strategic medication periods when they had a very high level of
respiratory active therapeutic product in the feed. This flap right here
represents when they\'ll be at a low level of a therapeutic product for
controlling respiratory disease. On the Y-axis is the serologic
response, again knowing that this is not indicative of protection by any
means, it just indicated exposure to mycoplasma. This will be the, Tween
20 ELISA from Iowa State. The three groups here we have the continuous
medication, the strategic or pulse medication group and then those
control pigs that didn\'t receive any medication at all. What was seen
with the two groups here that, that follow one another in terms of
mycoplasma, they came in with some proportionate pigs positive to
mycoplasma on the ELISA and on subsequent, with exposure to the organism
in the finish room there was zero conversion to mycoplasma. This
medication protocol economically from a performance standpoint was equal
to the continuous medication but yet it allowed pigs to be exposed to
the mycoplasma organism and develop some natural immunity to the agent.
Continuous medication was removed here, subsequently those pigs then
zero converted to mycoplasma. The continuous medication had some level
of inhibitory effect on zero conversion in response to that organism.
And to take home, as we look at approving feed products produced to
control mycoplasma, there may be a role for these two products to be
used in combination both a feed ray product and a biologic product where
you can use either or both to develop active immunity in pigs while
still providing protection economic losses associated with the disease.
Next please.

Both of those medication strategies significantly improved economic
performance in fact, there was no difference between the two groups.
Next. Next.

And there was some level of natural exposure in naturally acquired
immunity to mycoplasma that developed hence the theory at least those
pigs could have had some protection further on down the line in their
lifetime. Next, please.

As we would think or look through potential questions for a model, I
think we\'ll kind of get into this tomorrow a little bit. Remember, the
customer - the pig first and the producer, growth rate certainly should
be one of those parameters, protecting lungs from the clinical
respiratory disease is important but it shouldn\'t be the only end point
and certainly not just biologics can be used to generate active immunity
to the mycoplasma organisms. Next, please. Next please. That\'s it,
okay. That it? Is that it? And that\'s it. Rather then repeat this we
said it already. Any questions or comments?

Dr. Carlos Pijoan:

John, do you have any comments on, on putting all these control
therapies and vaccinations on the sow versus on the growing line
especially in three site production systems?

**Dr. John Kolb**:

I think there\'s opportunity to explore eradication using those
strategies. I don\'t know if vaccination certainly not by itself would
reduce colonization, at least to the extent that you could eliminate
*Mycoplasma*. Potentially the combination of medication and vaccination
has been successful. Some of the early MMEW projects done again, about
10 years ago were successful at least in a very small herds, eliminating
mycoplasma. Some of the European work using medications on a herd basis
has been successful at least eliminating *Mycoplasma* with the
technologies we had available ten years ago. So, I think they, they
could be applied in the sow herd probably where those herds are more
valuable, genetic herds etcetera or at least attempted in a commercial
system but in those cases where eradication fails you may also look at
control of the production line itself.

Dr. Eileen Thacker:

Well, I\'m, I\'m still confused about using vaccination in sow herds
because we really don\'t see much of an impact of vaccines on the
organism itself, the numbers and on medication perhaps but as far as
boosting up and while you said that maternal antibodies were somewhat
protective, they did nothing to reduce the number of organisms in the
little pigs so while it helped to, to reduce at a very young age the
percentage of pneumonia, Brad\'s study found, it did not reduce the
number of organisms. So, if we\'re talking about using it for control
and eradication and stuff, that\'s kind of a tough one. I think, I think
medication management and things like that, like the Swiss have talked
about, now whether that would work in our large units with multiple pigs
in Iowa, I have no idea. Do you want to say something?

Dr. Brad Thacker:

Yeah, I think the, I guess the, with the one study that you sited on the
maternal immunity, what we\'ve found is a reduction in lesions okay?
Now, the thing that, what we, there was no difference in organisms which
is also somewhat true of vaccine studies, most of our vaccine studies we
don\'t find any difference in organisms, some we do, never eliminating
it, okay?

Dr. Eileen Thacker:

(inaudible)

Dr. Brad Thacker:

Right. The other thing that was kind of intriguing to us and that we
haven\'t followed up on is the pigs with maternal immunity compared to
those that didn\'t have maternal immunity had a different antibody
profile in their lavage fluid indicating that they had less IgG and IgA,
more IgM which suggested to us that maybe it was somehow inhibiting the
development of active immunity. I guess the other thing on the sow
vaccination that, that is a question, it\'s a researchable question that
we haven\'t done the experiment yet. How\'s that sound? And that\'s, if
you look at the study we did that Eileen had reported in American
Journal of Vet Research, you vaccinate a pig, you get high systemic
antibody levels but you get very little local antibody until you
challenge the animal. Okay, now if an animal\'s vaccinated the local
antibody levels rise fairly rapidly and so it\'s kind of like that\'s
why it wins the race, right?

**Dr. John Kolb:**

slow infection (inaudible)

Dr. Brad Thacker:

Yeah. With APP if you don\'t have pre-existing antibody there locally,
you know within three hours you can kill a pig with APP. So, so again to
the sow vaccination and what I have trouble trying to resolve in my mind
is that if I vaccinate the sow I get serum antibody tighters but if I
don\'t have exposure locally which I may not have very much in a sow
because she\'s not carrying very much or that whole kind of host
parasite relationship is evolved in this stable, they\'re living happily
together then the question is am I going to get much local immunity
which may or may not then reduce the number of organisms but if, so
it\'s a, it\'s a real question. The one thing and I know they\'re, one
of the other speakers might talk about sow vaccination but if, if we
have had several situations where we have practitioners call us with
herds that are really having difficulty with mycoplasmal pneumonia in
finishing pigs and they are vaccinating sows pre‑farrowing and they
just, it just doesn\'t seem to work or what they described is something
even beyond what we would normally think so I mean I, it\'s a tough one.

**Dr. John Kolb:**

I think as we visit customers who are using that, the most typical use
of sow vaccination would be in those situation where maybe in late
nursery they are already seeing some, what they feel is clinical disease
in some pigs but attempting to try to get all pigs be they\'re
infectable or susceptible at about the same time.

(male voice)

(inaudible)

**Dr. John Kolb:**

Is there anybody here that\'s got European experience because we get
that question, we get that question from our colleagues that, why, why
they do so much more sow vaccination then we do?

Dr. Carlos Pijoan:

Well, I guess, I guess like we can agree to disagree. The first thing is
that there is a lot of very salient producers that do use sow
vaccination and these are not a bunch of dummies that they seem to be
having reasonable success with this. I would just mention for example
the Saint Peter clinic in Minnesota, and the other thing is we just
finished a piece of work on sow vaccination, looking at prevalence of
infection in weaned pigs using PCR and what that finds statistically
reduction in piglet colonization. This is, going to present that DVS and
it\'s already being submitted to, to a publication so you know, you can
get different results cause that\--

(female voice)

(inaudible)

Dr. Carlos Pijoan:

So, the point is a lot of people are using it, we\'re talking obviously
high (inaudible) herd, three sites, specific solution for a specific
production system. I would never vaccinate sows in a continuous flow
system or even in a all in, all out, one site system, I wouldn\'t do
that but in a three-site there are some, there are some reasonings that
may support that and if you add on strategic medications to the sows you
may come to, I\'m not going, I\'m not, I\'m not talking about
eradicating *Mycoplasma* but I\'m talking about bringing the prevalence
of weaning to levels are so low that you may actually not have any
problems in the line.

(male voice)

(inaudible)

Dr. Carlos Pijoan:

I think, I think the difference is that, the difference in their system,
their so called Swiss system of eliminating sows that are less then 10
months of age and then giving treatment to the other sows and stopping
farrowing what it is however is adult sows have very robust immunity.
They are generally coming from their one-site farms. They probably got
early on infection, they have been infected for many months and so by
the time that they are 10 months presumably they have developed a
relatively strong immunity. In our systems where we have delayed
infection or maybe even we're bringing in gilts from negative farms that
were trying to acclimatize either by vaccine or exposure or something.
It's going, I think it's going to be tough for a 10 month protocol to
work but it needs to be tried and in fact there are already a lot of
people that are trying but the essence is that, that the production
system over here is quite different and I think the immunity of the sow
in there is going to be much higher then here.

**Dr. John Kolb**:

Other questions, hold on.

(male voice)

I'll yield to the experts but some of the discussion in the field as why
you vaccinate a sow herd is to really come up with a uniform decline of
maternal antibody, decline within the herd so you're pigs all decline
the antibody at the same time. It's probably not going to help with the
18 week wall but it will have a more uniform decline of antibody in, in
the early part but as Brad said you get that with antibiotics.

Dr. Brad Thacker:

I guess I would, in my experience world wide---

(laughter)

Dr. Brad Thacker:

To me, and again I, it depends on the herd situation but I guess I've
had fairly good success with strategic medication of the sow herd. Now,
that sometimes is a little bit of a sticker shock because if you go 10
milligrams per pound of CTC or OTC per pound, you're looking at 1600 to
2000 grams per ton and I was in a vet clinic here about three weeks ago
with a fairly well known practitioner who had a client call in and gave
a level that was about half of that and I said well, you know, I got to
thinking gosh, maybe I've been calculating this wrong and so we went
through the calculations and he was wrong and so there's, and I see that
every once in a while---

(female voice)

(inaudible)

Dr. Brad Thacker:

Yeah, I mean people, it's so far out of the realm of what they think
they do before so, but in those herds we seem to be able to keep the sow
herds seronegative, now we vaccinate the gilts again before they come
into the breeding herd, they stay seronegative. The pigs are
seronegative enables me, I can vaccinate those pigs anytime I want
usually we don't do it till weaning time so I think, you know, there's
different ways to do it. But those are typically operations that are one
site or not strict multi site operations. The issue you get into for me
is that if you can, if you're sure you can prevent lateral transmission
down the line then maybe, technically, vaccination is not necessary but
in some areas of Iowa like central Iowa I think that's a, that an
impossibility.

**Dr. John Kolb:**

Any other questions? Okay.

(applause)

Dr. Gillian Comyn:

Okay, we're going to take a very quick break here before Dr. Pijoan's
talk due to some computer problems on my end I was not able to download
Dr. Pijoan's talk even though he did get it into us on time so I
apologize to you and because he's such a good speaker, thank goodness
you know, so we rely on your, on your extreme talking talents and again
I apologize for the computer glitch. Anyway, \--

(male voice)

(inaudible)

Dr. Gillian Comyn:

Okay, all right and Nabil is going to go ahead and introduce Dr. Pijoan
who needs no introduction but we have a little, just a little brief bio.

(a lot of conversation)

Dr. Gillian Comyn:

All right we'll just, we'll take a quick five minute break before our
next talk and then we'll go ahead and finish up.

(a lot of conversation)

(The tape goes blank near the end of side A, starts again on side B)

**End of side A/Start of side B:**

(a lot of conversation)

Dr. Nabil Anis:

Our last speaker for the day is Dr. Carlos Pijoan. Dr. Pijoan get his
DVM degree from the National University of Mexico, his Ph.D. from
University of Surrey in England. He work with the Wade Bridge Center,
(inaudible) and he worked with the Mexico Nation and University then he
moved to Minnesota, he became the Director of Swine Disease Eradication
Center at the University. Dr. Pijoan's talk will be diagnostic tools for
*Mycoplasma hyopneumoniae* today.

**Dr. Carlos Pijoan:**

Okay, thank you very much. Well, we have a little bit of a slight
problem and I guess it's going to be a presentation like the old style
presentations where we use, where we talk and maybe a little bit of an
overhead and stuff like and I'm sorry, I'm sure it was my fault cause
I'm a total bone head with computers so I'm sure that I did not send it
to her or something. When they invited me to do this presentation I
tried to, to look at mycoplasma diagnostics in the perspective of what
this symposium or summary is all about which is about designing trials
that deal with measures of antibiotics or in general of drug treatments
and, and I think that, so what I'm going to discuss then, first I'm
going to discuss diagnostics and I guess I'm going to discuss the
limitations of a lot of our diagnostics based on how can we use these
diagnostics for, for more efficiently, set up trials and more
efficiently be able to measure the, the benefits or non-benefits of
antibiotic treatments. Okay. A lot of the stuff I was going to say has
already been very, very well covered today so I will just be briefly
report it. First of all the microscopic lesions, as has been said are,
are you know, very suggestive but are not (inaudible) there's a number
of organisms that produce it. We were able to produce mycoplasmal
pneumonia that looks exactly like mycoplasmal pneumonia by using
Pseudorabies with *Pasteurella* with no mycoplasma what so ever and we
have also reproduced lesions that are exactly identical to mycoplasmal
pneumonia with *Haemophilus parasuis* infections in pigs that are
susceptible. So, I guess we're all in agreement this, that by in large a
microscopic picture of the, of the mycoplasma lesion is not enough to
warrant a diagnostic on the disease and so you are using these lesions,
if you are using it in a very controlled exposure situation say you have
pigs that are in a controlled environment and you're exposing them to
mycoplasma and you are really convinced that there is no other exposure
then, maybe, lesions can be used but if it's on, it's a setting that is
on a farm setting, if it's on a field setting then lesions are much more
difficult to use because there is a number of other things that can be
involved in producing these lesions, okay? So, I think we'll deal with
that. Then the other thing that I wanted to present very briefly was
immunofluorescence and immunohistochemistry. We have not been very lucky
with these technologies in Minnesota. It may be again, we're the dummies
here but in our hands it has not been very sensitive and we have found a
lot of false negatives that of animals that were inoculated to
mycoplasma that, that yielded mycoplasma culture, that were
PCR-positive, that had mycoplasma and electromicroscopy, but were
immunofluorescent negative. And so to us, fluorescence, we haven't done
immunohistochemistry, I imagine the same thing, because low sensitivity
and tends to detect microorganisms specifically at certain periods of
the disease so it's not a test that you can rely on if you are going to
use it to be assessing the impact of the disease. So, that to me is also
one of the tests that we don't have a lot of good handle. I just want to
briefly look at this microscopic lesions thing and Brad Thacker talked
about this briefly. This (inaudible) a very long time ago and what we
did here, we repeated this once more in, in Spain. I'm trying to look
for a pointer. All right, thank you. So what we did here was this is an
unfound deal and we didn't actually do any intervention to these
animals, all we did was follow them and this is a big part. It's a long
trial and this was actually done actually in the (inaudible) the
continuous whole farm. They don't even exist anymore, so it's kind of
historical but in 1988, in this farm, we followed these pigs to
slaughter and we took radiographs on the farm, of thoraxic reography for
all these pigs every 15 days and then once they got to slaughter we
looked at their slaughter lesions and each of these little, little
mountains here represents a particular pig and you can see the weight at
180 days. This pig was only 158 pounds so, but anyway, the point I'm
making here is each of these measures here in gray represents the
pneumonia at the score by radiography and the lighter band represents
the pneumonia score at slaughter and you can see for example that this
pig here had a tremendous amount of pneumonia practically all life it
had pneumonia scores, very high but by the time it went to slaughter it
was kind of resolving pneumonia, stuff came up at, at as far as 35%
lesions score was very high but it did not really represent what had
been going on because it was already in resolution. This pig is much
more worrisome and this particular animal here, they had, this animal
had pneumonia scores although they picked up and then it started coming
down by the time it went to slaughter which is this tiny little blip
here, essentially had no lesions. So, when you look at the slaughter
lesions of this pig it had no lesion yet it had a lot of pneumonia. In
contrast for example, this other guy here had no pneumonia at all
throughout most of his life, started developing pneumonia at the end of
the life and by the time it got to slaughter it had a huge lesion but
yet it, but yet the actual pneumonia score, average pneumonia lifetime
score was low. So, what I'm trying to show here in this slide here is
and, and we've done more trials like this and it's kind of a pain to go
and pick and radiograph some farms but what we tried to show with this
is that yes, this is a resolving process and what you detect at
slaughter is more a measure of the time of infection then the actual
severity of infection and so if you use pneumonia scores in a lot of, a
lot of traditional old trials for drugs and even some for vaccines use
lung scores at slaughter as their measuring or end point measure. If you
do that again, if it's a control situation and you did, you challenge
the pigs and you took them slaughter and what not and they've been in
isolation this could work and they were all challenged the same time but
if it's on a farm situation in which you are not controlling for the
time of infection then by the time they reach slaughter you're, the
predictability of looking at lesions at slaughter is not very good. And
so I would say that this is probably not the best measure to use from
these kinds of trials even though it's being very, very commonly used.
So, we are not going to use lesions because they have these problems.
What can we use? Well, one of the things that has been used quite alot
and we've already seen a lot of it today is serology. There's a trial we
did in Spain some years ago and again in this trial with the pneumonic
lung score based on radiographs exactly the same thing, this is again an
unfound trial. This is a two site farm but here what we also did is that
we added on some serology into the system and, okay and anyway, very
briefly these animals had some antibodies to Pseudorabies which came
down, then actually did not reappear, and then they also had a little
bit of antibodies to APP, this were all maternal stuff. They also came
down and did not reappear and then these antibodies here are mycoplasma
to us. And this particular group of pigs and we have several groups,
this is the average pneumonic lung score for the group and you can see
that the pneumonic lung score starts going up in peaks and this, in
particular farm the pigs usually about 12 weeks of age which is the time
they were being moved into the finishing then it starts coming down. One
of the interesting things here is that yes, serology was a good
predictor, it was not ELISA by the way, the serology in this trial was a
good a predictor of the lesion as seen in radiography but the time it
took between the peak of pneumonia which is actually not even done with
infection is the peak of pneumonia for, for this group which was at 12
weeks of age. They didn't start seroconverting until they were between
16 and 18 weeks of age so there was very delayed seroconversion and this
has, and this has been one of the big problems with ELISA tests in
serology for *Mycoplasma* is that serum conversion is a delayed event.
It's not a predictable two-week event after infection like it is for
most other disease and the, the most other problem that we have is that
this delaying serum version is variable. If it was always six weeks or
it was always seven weeks it'd be okay, it would correct for that and it
would (inaudible), but if actually in, in experimental trials, the kind
of stuff that they lead us, she gets her conversion very quickly because
challenge doses are very high and so in those situations two to three
weeks after infection you do get serum version but under field
conditions serum version can, can run from three to six to even eight
weeks and three weeks looks like it depends on how much challenge the
pigs are actually getting. And so when you are looking at this and you
are trying to figure zero conversion to define for example, I was
listening with, to Jim who was telling me a second ago about pulse
medication and what do you call it? Something meta-therapeutics, what
was it? Well, it's all very good if you know when to give them but to
predict when to give them you need to have a serology so you can do a
serum profile and the serum profile in mycoplasma is very difficult to
interpret under farm conditions. You are really always guessing because
you can't really pinpoint what kind of serum conversion delay do you
have in your particular group. We have a number of ELISAs in this
country the Tween ELISA where I will call the "in house" Tween ELISA
which essentially is the Iowa State ELISA as the gold standard. That's
what we all use. Other ELISAs like the commercial Idexx ELISA is coming
out right now and the (inaudible) are used as confirmatory tests. We
have a lot of fun with the Idexx ELISA and I said that some of the
people may not have and that will be okay because do a lot of money
doing the clean herds and these clean herds where\'s there\'s a lot
false negatives now. Eileen is telling us that they are not false
negatives and they are true infector and that may be. I don\'t know.

Dr. Eileen Thacker:

No, they are false negatives not false positives.

**Dr. Pijoan:**

Sorry, false positives but, I know\--

(female voice)

Doesn\'t make a difference.

**Dr. Pijoan:**

I know. (laughter) But the point, the point is that, that when, when
those are usually confirmed with DAKO tests and the DAKO will usually
come negative on those things. So, ELISA, they are quite good. They\'re
pretty good but they are not perfect and so and, and it\'s a little bit
difficult, difficult to equate what we do in this country to what is
done in other countries because most other countries do not have that
in-house Tween 20 ELISA, they have some commercial Tween 20 ELISAs which
are not very good and so, so then when you are looking at the, when you
are trying to compare what the results that we get here to what they may
be getting in Latin America or they buy commercial Tween 20, it\'s
difficult to equate. There\'s some difficulties there but DAKO is a very
good test and in Europe of course it is the gold standard but it is
expensive and so that is something that one has to take into account
when one is looking at ELISA s. The other point about serology and I
think in general mycoplasma infection is how mycoplasma infection,
that\'s on the face of diagnostics really changes in view of the
production system. Both Brad and Monte talked about this and I think we
are all kind of in agreement, I don\'t see any disagreement in this, as
we have changed production system the disease has changed. But this has
a big impact when you start thinking therapeutics and it has the big
impact when you start thinking about licensing drugs and whatnot.

Let\'s look at what happens, this is the, if you like the old style
continuous flow system where they used to have these, I remember one
that\'s the European style stuff where you get pigs coughing in the
nurse, or late nursery or this kind of thing. In this models you get
infection very quickly usually at about four to five weeks of age, your
pigs, most of your pigs are ready, I\'m putting a 50% group infection
and I\'ll tell you in a little while why I\'m using that rate but let\'s
say that about four to five weeks about 50% of the pigs in the group are
already infected and usually from six to eight weeks of age, very short
time serum conversion, you start to get positive conversions in the
group. So, so, so this is the classical model of mycoplasma that we were
all taught in school, this still the classical model that is used for
vaccine recommendation so when you, when I buy a vaccine and they tell
me well, you have to vaccinate at one week and three weeks of age which
is quite ridiculous, well the reason they say that is because they
expected pigs will get infected at four weeks of age and of course if
you\'re expecting them to be infected at four weeks of age you have to
have put both vaccines before they\'re four weeks. That however, is a
scenario that in this country is nowadays is very rare. It\'s going to
be very rare that you find a farm that actually still has that kind of
disease. Most times in this country except in the Midwest, have gone to
a one-site, all-in-all-out, or three cycle all-in-all-out and when you
go in the all-in-all-out and now you have minimized the pressure, the
infection pressure between the older animals and the younger animals in
the growing line then all you are doing is actually delaying infection.
So, now in this kind of model generally pigs will get infected or at
least the 50 percentile will be infected at late nursery and they will
probably start showing serology at about middle of the finishing barn.
So, that, that you will see still others along this path come in the
Midwest in which there are still family farms, they\'re still on one
site but they are quite willing to all-in-all-out them. They have
delayed effects and I think it\'s this, this is very clearly a model
that is happening but one of the things that you have to see here that
if you contrast these two different farms or these two farm systems, in
the first one if you are looking at the time it takes for the 50
percentile to get infected it was only, let\'s say four weeks. So,
during those four-week windows practically everyone got infected. So,
you are looking treatment, you know that if you, you got a prophylactic
antibiotic you look, you know that if you give it during those four
weeks your change of success are quite good but here now, it\'s taking
ten weeks for this 50 percentile to get infected and they probably
started when they put them in the nursery. So, now infection has taken
seven weeks to occur. Of course, it\'s a much lower level. There\'s a
lot less microbial presence, that\'s why it\'s taking so long but now it
is, it is also much more difficult to say well, when am I going to treat
these guys. I mean this is full seven weeks, am I going to give them
seven weeks antibiotics. The other time I just have to give two weeks
treatment now here I\'m looking at seven weeks and in order to get some
effect and even worse then that, that\'s not even the worst scenario,
the worst scenario is a three site, multi site system. Because in the
three site, multi site system we still have a much more delayed deal. In
fact, some groups what may actually go all the way started with being
zero negative. We very commonly find in three-site system, groups of
pigs that are seronegative at slaughter and yet have lesions, that\'s a
very, very common finding and what all that is saying is that these guys
got infected, they developed lesion, they don\'t even have time to
convert, very late infection, okay? So, now in these models maybe it\'s
taking 14 to 16 weeks for the 50 percentile to get infected but they
started getting infected in the nursery at four weeks, so now infection
has been going on in the group for ten weeks. When are you going to give
the antibiotic during this whole ten weeks of pressures obviously again,
it\'s a lot less micro pressure but the pigs have become very, very
strung out and so, so to me when I started looking at, at getting
antibiotics as treatment, I\'m a great believer actually but I\'m
thinking well, yes but when? I need to figure out the way to, to in
this, in this model which is now becoming the common model of
production, in this model and in this disease which is now becoming the
common disease I need to figure a way to treat this pig successfully and
I cannot use the traditional tested methods because they\'re not going
to work because the disease has changed in the last five years so we
need to be able to start you know, figuring out what, what I\'m going to
do for this new model that has changed so much. Okay, so that is as far
as serology and I think serology does have a, quite a bit of a role to
play, the interpretation has (inaudible) as I said.

So, let\'s talk about PCR, somebody made a little fun of me saying that
I never see pigs and I always do these PCR\'s. I do see a couple of pigs
now and then and we do do some other things that are not only PCR\'s
but, but PCR has become a very powerful tool and you cannot deny it.
It\'s there and it\'s not going to go away and we are already I mean in
our biological diagnostics by in large it\'s very rapidly substituting
bioculture because it\'s much easier to do, it\'s much more sensitive,
much faster, gives it a diagnosis. I mean nowadays you have boor
(inaudible) and you don't know if it\'s infected with PRRS you are not
going to wait two weeks to find out the culture of the virus you want it
tomorrow the answer, you want it today and the only way you are going to
get it, with PCR. So, PCRs now they do have a lot of drawbacks but they
are undoubtedly the most powerful diagnostic tools that are coming up in
the arsenal and we have just start assuming them. PCRs for mycoplasma
could be in this fight for a long time. There is a number of them, I
will just simplify in three groups. I will call the one-step PCRs or
one-reaction PCRs then the nested‑PCRs which are two steps or two
reactions and now there is already real‑time PCR available which is one
time but it\'s on the fluorescent PCR asset. Okay, so those three types
are a little bit different. They, all of them have their benefits and
all of them have their problems, okay? Let\'s start with the one step
PCR, one step PCR that we use or our (inaudible) PCR is based on the
described one step PCR and, and depending on the needs of our
(inaudible) sometimes we\'re run it just one step and sometimes we run
it as two steps. Now, the one step was described by Matheson over in
England, in the UK that\'s, that PCR is the standard diagnostic test. A
tremendous amount people use it and nobody questions it, it\'s used
extensively and we have, as I said we use that as our first primary
application is exactly the match in the (inaudible). What it does it\'s,
it\'s in our hands it\'s very good because very, very few false
positives because one step PCRs do not get a lot of contamination in the
laboratory so you have a very clean reaction. You don\'t have a lot of
false positives for contamination of the lab, which are the bane of the
mass PCRs but they have lower sensitivity. They are not as sensitive as
domestic PCRs and depending where you want to detect your bug they may
or may not work. If you are going to, for example, our fluorescent PCR
that we have, real time PCR, Tagmon PCR that is offered by the
Veterinary Diagnostic Laboratory at the University of Minnesota right
now. It's essentially a one step in a fluorescent (inaudible). It\'s
more sensitive then the traditional (inaudible) because techments are
more sensitive then looking in a gel but the actual reactions the same.
Now, what they do in the diagnostic lab is that they use that routinely
with a lot of success for all the submissions that are either lung
submissions or dead pig submissions or what not and in those types of
scenarios all they do is they swab, tracheal bronchial swab. They submit
that to PCR, once that PCR gets (inaudible) in three hours and it worked
fantastically well. So, if you can kill the pig, nested PCRs, one steps,
sorry, one step PCR or fluorescent PCRs are the way to go. They have
very few cross contamination. They work very well. The fluorescent has
no cross contamination it\'s totally automated assets but there, there
is sensitivity is to the level that it will pick up the mycoplasma in an
infected pig from a tracheal bronchial washing or swab but it will not
pick it up from the nose. Now, that may not be an important issue
depending on how you are going to set up your trial and if your trial,
if you are setting up a trial and say my trial contemplates killing all
the pigs well, then fine use this PCR because it\'s by far the best one
there is but if you are contemplating not killing the pigs or you are
contemplating some kind of monitoring that you have to monitor the pigs,
the pigs for a while while they\'re alive and maybe at the end you can
kill them then you run into trouble because antigen protection in live
pigs is very difficult and that\'s where we use a nested PCR which has
gone through a lot of scrutiny but the advantage that it has is that it
is sensitive and the nasal PCR will pick up mycoplasmas from nasal swabs
in certain particular conditions. It does have false positives. It does
have some problems. It really is, it really is an experimental tool for,
for experimental license. It has not been successfully run at the
diagnostic laboratory level for example. It has not been successful, we
tried it for a while and we would do 1000 samples each week. It\'s not
very good for a 1000 samples each week. You\'ve got a lot of
contaminations. You run into trouble. It\'s good for playing around in
the lab and a few samples and a little control trial, those kinds of
things it works very well. You want to set it up as a traditional
diagnostic then I don\'t know. Okay?

So, I\'m going to talk a little bit more about the, the detection of
mycoplasma in the nose because I\'m sure you all are worried that
that\'s not a normal site for mycoplasma to be in and so once you start
figuring out well, how come you\'re detecting it there and what does
this all mean? It has taken us about three years to play around with the
PCR to have a handle on what all it means to be detecting PCR, detecting
getting false, getting positive signals of PCR from the nose. I want to
show you just one thing here. This is, nested PCR, nasal PCR from
different parity sows in commercial herds. Now, you are all aware and we
are all aware that there\'s a tradition literature mycoplasma that
suggests that the older sow and by older sow we are talking about three
parities or more, does not affect the litter. Now, that does not mean
that it\'s a mycoplasma negative animal but does not affect the litter.
There\'s a very, very well supported literature in this, in this comes
from the 60\'s in England and Kirk Clark did some work on that and I
think we all agree on it. The older sow or the medium to older sow
generally does not infect the litter but when we go out there on that
and then I will say that these are three-site farms and not one-site
farms. When you go to a three-site farm you start swabbing different
parities of the sows and we\'ve done this a number of times. You get a
picture that is very confusing. You start getting a lot of positive
signals, practically about the same level across parities, up to six or
seven parities. When you go to real old sows they\'re negative but
across parities you get something that you really don\'t have a parity
distribution as far as nasal prevalence and so at first you know we
started with the questions I said well, maybe it's not working, maybe
these are not true positives, there\'s something wrong here. We went to
another farm, different technician, same result. Another farm, we\'ve
done it so many times that we just have to accept that this is true. So,
then we did another trial and said okay, let\'s see if these sows here
actually infect the baby pigs. We would recognize if they were infected
but let\'s see if they infect the baby pigs. So, now we are doing nasal
PCRs from the pigs, not on the sows. And when we do the nasal PCR on the
pigs at weaning then we do find that these old gals or semi old gals,
they\'re medium age gals, they don\'t infect the pigs. They are infected
but the baby pigs by and large are not. So, when you actually do a do a
(inaudible) of, of infection at weaning, baby pigs. Most of the pigs are
infected, there are very few pigs infected at weaning anyway. You only
get about 10 to 15% best case scenario of actually infected pigs at
weaning but most of them come from litters of young sows and not of old
sows. So, this supports what we\'ve always heard but then there is
something here that is funky. This old sow is infected but not infecting
the pigs. The young one, is infected and it is infecting this pigs.
Since PCR\'s are not quantitative, the different maybe in the level of
nasal secretion. It may be that these ones actually had very few
organisms. They are giving you a positive signal but there\'s very, very
few mycoplasmas there and they can\'t even infect the baby pigs and this
ones may have a lot. It\'s quite difficult to quantitate (inaudible) if
the PCR does not do that. The other alternative scenario is that these
old gals have a very robust immunity and that that immunity is
protecting the baby pigs from infection throughout that three-week
period (inaudible) for three weeks here. (inaudible) Can you protect the
pigs through immunity from (inaudible) for months, no? But the question,
can you protect it for three weeks? Can you protect these pigs for three
weeks from a presumably very low level of shedding and I would say yes
and I would say immunity can protect the pig. Now, which part of the
disease antibodies, I don\'t know. Is this transfer of immune cells from
the sow to the baby pig I have no idea but what I will say is that the,
the older sows even though they are infected by and large generally
don\'t infect the baby pigs and, and in our mind it has to be,
associated to transfer of passive immunity or, or to the fact that the
immunity of the sow decreases its shedding level, one or the other.

Okay. So, as you look at PCR\'s you find all kinds of funny things as I
said. So, let\'s look at this one. This is a very traditional PCR. This
stuff that was done by Gary Bosch when he was trying be a Master\'s
student from University of Minnesota, now he\'s decided he\'s going to
be a big honcho in Pharmacia, now he no longer wants to be a master\'s
student but he was puttering around with being a master\'s student he
did a tremendous amount of fun. He ran around alot of farms and he would
do the serology and we would do the PCR and I\'d put it all, everything
together and look at the ending of all these different profiles of the
association, on farm association became nasal PCRs and, and serology and
this is a very traditional one, very clear one. You look at this
diagnostic here, the light gray is the serology and the dark gray over
the black would be the PCR and you can see that in this particular group
of pigs here on this farm the pigs here convert practically the day they
are going to slaughter, at 24 weeks of age. Before that they were
essentially negative to serology, okay? This is the traditional picture
of the delayed mycoplasma or (inaudible) or PRDC whatever you want to
call it. I don\'t care, you can give it any name you want. But this
delayed mycoplasma thing in which we get some groups of pigs that
actually you can, do the only pig serology the very last month of age or
the very last two weeks of age and then they go to slaughter and have
lesions. Okay, so these guys they didn\'t really circumvent them till
the end but their PCRs were positive by in large since 14 weeks of age.
So, again we\'re looking at this delayed conversion here and so in this
particular type of scenario the nasal PCR is a much better predictor of
infection down in serology in this scenario because if this is a very
clean herd, there\'s not a lot of other stuff going on, you only have
delayed mycoplasma infection you start picking up the antigen very
quick. It takes a while for serology and the protection of antigen under
this conditions is, is quite a good way of doing it.

There are however, other farms. They are not all like this and in some,
like this one which is another pattern that this one doesn't show very
well but anyway this is the black lines are here and here and here and
here, sorry about that. So, that will be the serology there and these
are the PCRs, the PCRs being the light ones and you can see that this is
a lot more confusing, a lot more confusing and Gary did a lot of his
work and we tried to figure it out. What is happening here? I mean this
is not just one farm. He went to farms all over the country and we would
find this pattern over and over again. So, he essentially ended up with
four or five different patterns and this was one of them and the with
this pattern you are getting very early infection by PCR from six weeks
of age, you really at, certainly at, 11 weeks of age you have more then
50% of the animals are positive and then you know, they stay positive,
they start coming down as far as having the organism and you\'re
detecting serology really as early as three weeks of age and some were
positive at eleven weeks of age, so what we have done, what we have
concluded from a lot of these observations on farms is that with PCR you
really have to have a large proportion the group PCR positive before
they actually go into seroconversion, now this may sound crazy and I
don\'t fully understand it myself but if you have 20% of the group
infected and it stays and 20% for x or y reason, that group doesn\'t
seroconvert. It just keeps on going, keeps on going, keeps on going.
You\'re sub clinical and sub serological and I don\'t know how that
happens but once you get to a relatively high level, let\'s say half of
the group or more then yes, from there you can start counting your six
weeks or your four weeks and you\'re going to get seroconversion and we
have, this, we have looked at this on farm over farm over farm in which
you have to, it\'s not enough to have a few pigs infected you have to
have a percentage of the group infected before you actually develop
lesions and develops conversion and all of this happens and this has
been very troubling and very confusing because you know, we always been
accused that all of these are false positives and we\'re a bunch of
dummies and we don\'t know what we\'re doing and it\'s not, and maybe
it\'s true but see, we can repeat it over and over and over again, we
have to come to grips and try to interpret it. So, look at this pattern
here which starts conversion from a lot of, a lot of this is an on-farm
pattern but I will show you an experimental infection pattern. And in
this pattern here, you see that the pigs at five weeks of age have a few
positive signals (inaudible) pigs and then at eight weeks of age you
don\'t get any positive signals and then at 14 weeks of age practically
everybody\'s positive so you have early positives then a few negatives.
This is all nasal PCR no tracheobronchial stuff, this is all in the
nose. And so, and then go positive again and you say well, that\'s
weird. I mean, how do they come positive and negative and positive so
we\'ve done some experimental infection in groups of pigs and when
you\'re doing experimental infection, these are actually in house trying
to infect the pigs and follow with PCR\'s and in this kind of scenarios
and I won\'t go with the (inaudible) on that but essentially you get the
same data. You get, you inoculate them and the first week after, the
first, second week after inoculation practically everybody tests
positive then boom, they suddenly go negative on you or at least buy
some joy to you and then a while later, you know maybe two or three
weeks later they come, come up positive again in the nose. And you say,
well, I mean how did this happen? How did I, why did they come, why were
they positive first and the negative second and to be true and honest we
don\'t really know but we have made some hypotheses here of why that
happens and unfortunately I don\'t have the slides so I\'ll just have to
tell you about them. But what we think happens is that when the pigs
originally get infected especially in on farm views not necessarily, and
you start picking up this mycoplasmas the most. Now, we all agree that
(inaudible) the most (inaudible) place for *Mycoplasma* to be so if they
are there it\'s because they are being shed from one way or the other
from the lower bronchial tubes. So, other wise it wouldn\'t be there.
So, what would be the scenarios in which you can pick up a, a mycoplasma
actually in the nose when it\'s being shed and to me the obvious one is
there is active shedding, there is active replication, the pig is
coughing, and shedding mycoplasma (inaudible) but there is another
alternative, scenario and that is the pig that is actually just getting
infected or maybe not even, or getting infected but not getting a true
colonization and the mycoplasma as a percentage of the, of the cells
that you, that (inaudible) do not attach either because they\'re dead or
they don\'t have that D92 whatever protein or whatever you want. They
don\'t attach and so there\'s a percentage of the population of
mycoplasmas that actually abort, they don\'t attach. So, in the early
stages of the infection a lot of this non-attaching bad cells or
whatever you want to call them are being washed by the tracheobronchial
mucus and you are picking them up in the nose. After a while all this
washing has been done and all these dead cells can be taken out of the
system. Now, we only have attaching cells which are stuck to those cilia
and then for a while you don\'t pick them up because these ones are
truly attached and they are not getting washed out and so now you are
getting essentially false negatives. These are truly infected pigs but
when you are sticking nasal swabs in there, the nasal swabs you are not
getting positive signals. After a while those microorganisms
proliferate, they form microcolonies, they destroy the cilia, all these
events that we all know and now you have a true shedding, an infectious
shedding if you like and so after this feeling of negativity now you
have again you\'re positive but now it is true positive shedding. This
is what we think has happened. We need to do a lot more work to be able
to interpret all this stuff to make sure that that\'s where it is but at
least it makes sense in the context of the experimental infections that
we can learn and farm observations that we have done and it makes sense
if you start thinking about it and thinking about how the infection
actually takes place and so then you think about, you go back to your
nurseries here and when we go to nurseries in three-site systems, we get
a lot of "n" (nested ?) PCR positives early on in the nursery a lot of
it but then they become negative and then they come up to become
positive again and what we think is happening that those early on are
just, you know, there are obviously some infected pigs in the group and
those infected pigs may be are touching some other pigs and they are
picking up from the noses. They may even just be transiently, they may
just be conflicting sections from other pigs that hasn\'t even developed
into infection in this pig but you are getting these positives then
after a while you get practically nothing because the pigs have really,
you know that\'s when the mycoplasmas go in the trenches and they stick
themselves to the cilia and so now you\'re negative and then you become
late positive again. Okay.

To summarize all this and I\'m sorry that I didn\'t have my nice color
slide presentation to show but in summarize of this and to look at it in
the prospect of designing trials which is what we are talking about. I
think that if you do an in-house trial, by in-house trial, what I think
of in-house trial is, a trial in which you have a group of pigs that you
have in a separate isolated place. It can be a very large group of pigs
and that you challenge them and you do it and they all challenge the one
point infection or maybe you challenge a percentage and you leave some
context, type of deals that we've all done over and over again. If you
do those things in those kind of, in that kind of scenario practically
any diagnostic will work. The lesions will work because you will be,
have a very controlled situation. You will know when to kill them and
get maximum lesions expression. Serology will work because you need keep
a heavy duty challenge of zero compared, they're also compared to the
challenge at the same time and the PCRs will work, everything will work.
So, in a design like that monitoring the group and monitoring the
benefits of the treatment if you like control versus treated should be a
relatively straight forward scenario. When you go on farms and, me, I'm
a great believer of on-farm trials and so I, I always like to hold
myself on farms. When on-farms then it becomes a lot more complex
because on farms pigs don't all get infected at the same time, they
don't all get infected with the same high doses of microorganisms and so
when you start monitoring a group, it's very difficult to know when to
monitor, when to treat and when to, and so that's when you get this
delayed seroconversion and now the serology becomes a lot more, if
there's nothing wrong with the serology it's just now you have a
population dynamics playing a fact. If you are looking at lesions well,
it depends on when they go infected. If it's an early infection, lesions
are not going to be very informative, if it's a late infection they will
be a lot more informative I think so when tomorrow, we meet in these
little discussion groups and this is really what I wanted to talk about
really, more then, then anything else but start thinking about in the
scenario of a field trial how are we going to monitor these groups so
that we can maximize differences between treated versus untreated and
then we can interpret those differences in a true significant way
because to me I find a lot, a very problematic. I find it very easy to
go on an in-house deal, very difficult to go on a field trial deal. I
guess that was the message I wanted get you. Thanks. (applause) Okay no
questions from the Thackers. (laughter)

Dr. Brad Thacker:

Well, I agreed with everything you said. (laughter)

Dr. Eileen Thacker:

But now you can't say it. (laughter)

Dr. Brad Thacker:

I think one thing that I'd like to clarify though with our experimental
infections with the Tween 20 ELISA the group average of let's say
non-infected or I'm sorry infected non-vaccinated controls reaches the
positive negative cut off typically when we necropsy the pigs at four
weeks so that means that half of the pigs are seropositive, half are
negative. Now, statistically higher then non-infected, non-vaccinated
controls which stay very low. So, we, we see that delayed response even
when we know exactly and we have some pigs that stay very low. The
second question is kind of a question/comment and, and I've given you
know, a lot thought to what some of your data, and the question I have,
Carlos, is, is to add maybe a little extra wrinkle to the, your, kind of
your second theory about the nasal, positive nasal samples and that's to
kind of extrapolate from that Swiss paper I think that, where they,
where they looked at the air sampling and they found the, the variation
in the amount of mycoplasmas in the organisms based on kind of the
severity of disease in the herd and in kind of a general sense so my
question is then is the nose simply representative of kind of the
general amount of mycoplasmas that are floating through the air which
are also then maybe related to the severity of disease in the pigs and,
and so your second theory kind of gets at that but to me it's almost
like a, is it measuring kind of the general environmental contamination.
I just thought I'd seek your comment on that because that's what that
paper told, kind of said to me.

**Dr. Pijoan**:

Yeah, I don't know if you've all read this paper, it's a, it's an old
one, that's not old, five year old paper and they, they use nested PCR
on air samples on the farms in Switzerland and they, a very interesting
paper and they did actually get some positive and some negative samples
and, and it was very interesting in the fact, for example we have been
very, I think their method was very narrow because we did not get those
positive air samples, but the point is that they got positive air
samples in farms where the pigs were coughing. They didn't get positive
air samples in the farms in which the pigs were not coughing even if
they were infected and so what the conclusion of that paper and I think
it's a valid conclusion is that when you have a very large group of pigs
coughing and I mean in a barn that the microbial content of the air is
going to be high and now you can pick it up in the PCR from an air
sample and I agree with that. The following question here is if you had
that scenario, where you have a large group of pigs coughing and you
have a big microbial contamination in that barn are you picking up false
PCR's from pigs that are not infected that are in the barn, that's
essentially what you were saying that just transitorial picking up the
antigen from the air and it may be, and it may be I will contend however
that if you have a barn that is under that clinical scenario that barn,
those pigs are at very high risk anyway. Generally, in the US systems we
don't tend to get a lot of barns that have a lot of coughing like that
nowadays at least most of the place I work. I think part of the problem
is the work that we do is all on three site systems, very high health
kind of farms and those farms don't really cough that much and so for us
again, it's a little bit difficult to be trying to replicate the
European work in here because of the system, production system, like I
cannot think, I don't think we're going to be able to eradicate
mycoplasma by taking away the (inaudible) cells, I don't think so, but
in Europe it works undoubtedly, they've done thousands of farms and we
cannot deny that fact that it has worked but it's a little farm, it's a
one-site farm, this house is a very solidly infected. It's very solid
immunity. There's a scenario that is not our scenario and so even though
I agree with you and so what I'm trying to do, what's I'm trying to say
here is that I'm not saying that all the PCRs that you test, if you pick
PCR and you want to do a single animal decision you're going to be in
trouble, for sure. I have no doubt about that. It, it has to be done on
a, on a group base and population basis and you do have to understand
the dynamics of this early infection which made it meaningless and then
you have to wait to see the late infection occurs or doesn't occur. So,
I think the PCR is a very, it's a valuable tool, I say it has to be used
with caution. It has to be interpreted. Monte?

Dr. Monte McCaw:

Proceeding down those lines, have you in your field studies when you
find the nasal positive animals have you ever had the opportunity and
realize this is major funding at times, to sacrifice the pig and see
when are they nasal positive and then the rate of lung positive and
remember many of, this will be a two fold question. Farm C here when we
had PCR positives for a little bit and they, they went negative but you
had some antibody, it may be more compelling that the page right after
that where you get a few PCR positives at five weeks, nothing at eight
and then 14 and 18 to 24 weeks you got positives again. Could there be
another possible explanation where the early say colonization and, and
even from studies we challenge but if it's an intranasal, there's only a
few takers.

**Dr. Pijoan:**

We, we (inaudible) otherwise we don't get the (inaudible). (laughter)
Okay, so no, I see where you are coming from and we've done that in
challenge experiments but the, and the transference have worked we have
had nice association with, through PCR infection as detected by electron
microcopy. The problem of course we don't have a big---

**(End of side B)**