----start-----
lab med
2/24

feldsburg

his part of the exam will be 
short answer questions, 2-3 sentences. 
we're responsible for what we 
went over in class.
not clinical signs.
know immunologic mechanisms of 
disease, tests, and interpretation of test results. read the handout.


tests of normal immune function:

serum protein electrophoresis - 
discussed this before - used as screening for immunodeficiency, or as 
diagnostic test for one particular disease only. this is multiple 
myeloma, which produces monoclonal gammopathies. this is a tumor of a 
plasma cell. the test is diagnostic because instead of seeing a broad 
based elevation - polyclonal gammopathy - you see a monoclonal spike - a 
very narrowly based homogeneous spike in gamma or beta region of the 
pattern. this is really diagnostic for myeloma. if it's way out in gamma 
region, it's generally IgG. also seen in beta region. these are very 
large spikes. 

B and T cell quantitation - we talked about functional 
tests but there are also tests to quantitate their numbers. this wasn't 
very practical in dogs/cats until a few years ago when more monoclonal 
Abs were developed. quantitating B and T cells may give a clue about 
whether there is a deficiency, but you still have to do functional tests. 


ID and quantitation of leukocyte subpopulations uses antibodies against 
various surface receptors on the B and T cells - like IL2 receptor, TcR, 
adhesion molecules, complement receptors, activation receptors, cytokine 
receptors (which are often activation markers), immunoglobulin receptors. 
you get Abs to react with these things so we can tell apart B and T 
cells.  we now have a lot of monoclonal Abs available to look at B and T 
cells in the dog. the main thing is we have Ab that quantitate mature T 
cells - mature CD3, CD4, CD8 are all available. CD11 and CD18 antibodies 
can test for leukocyte adhesion deficiency. every year, more and more of 
these antibodies are available. there's also an NK marker in the dog now. 
Some of these are also available for cats as well.

how do you quantitate 
the cells, actually? You have these antibodies labeled fluorescently - 
and you use a flow cytometer - a laser type thing that counts 10,000 
cells/second. you can do this on whole blood, even. 

so far, we've 
talked about an easy, inexpensive way to evaluate B cell function, and 
the gold standard of evaluating T cell function,and how to quantitate B 
and T cells.

the Phagocytic System 

we need to assess neutrophil 
function also. we want to know if they can kill bacteria. 

bacteria are 
phagocytosed and killed inside the phagosome in the neutrophil.

standard 
test: obtain patient neutrophils, obtain bacteria, and obtain serum. 
you 
need the serum for opsonins. neutrophils will phagocytize bacteria, but 
they like Ab coated bacteria better. you do the test with plain bacteria, 
Ab coated bacteria, and Ab/complement coated bacteria (bacteria with 
serum). almost all bacteria are killed in two hours when you use Ab and 
complement.

bactericidal assay:
take neutrophils from normal animal and 
patient. mix bacteria with serum from patient and from normal. incubate 
bacteria with neutrophils, in two hours you lyse the neutrophils and see 
how many viable bacteria are left. in normal animal there should be a 2 
log decrease in viable bacteria. if neutrophils aren't able to kill 
bacteria, there is no drop.

this is the gold standard test, remember. 
take patient cells (neuts) and patient serum (this is your test sample). 
also take normal cells and nromal serum (this is your control). then do 
your combinations:

patient cells + serum + bacteria - if patient has 
defect this is abnormal
normal cells and normal serum and bacteria - will 
be normal

now, take patient cells and normal serum - if abnormal, 
problem is in patient cells.

normal cells and patient serum - if 
abnormal, problem is in patient opsonins (antibody)

this is rather time 
consuming. there are other screening tests that are useful, and faster.


test developed by pediatric immunologists in TX because it's hard to get 
blood from little babies - this is also good for small animals. 
phagocytes like to adhere to glass or plastic. they took a fingerprick 
and dripped blood onto coverslip, incubated in moist petri dish - as clot 
retracts,neutrophils adhere to coverslip. then you add bacteria and serum 
onto the slip, incubate, and then look for killing. how do you look for 
killing? there are some stains that can tell apart dead from live 
bacteria - some kind of orange stain - you use fluorescent microscopy to 
look at it.

slide: a cell stained with this special stain - viable 
things are green, nonviable things are orange. we see green nucleus, and 
lots of green debris. 60 min later, we see lots of orange dots inside the 
cell - dead bacteria.

this is a good screening test. 

slide: dog with 
chronic pyoderma - vet suspected neutrophil killing defect - these are 
neutrophils from that dog incubated for 90 min with opsonized bacteria - 
there are very few orange dots - mostly green debris. the dog does have a 
neutrophil killing defect. 

--end of testing. 

now, we've evaluated b 
cell, t cell, and neutrophil function.

to do this screening test, you 
can clip a toenail and use the blood from there, or you can use a small 
clot tube to collect a sample and send it to lab.

how do you use these 
tests?

to diagnose immunodeficiency disease:
the key thing is that you 
have increased susceptibility to infection.

increased frequency of 
infection, increased severity of infection, chronic or prolonged course 
of infection, incomplete or lack of response to treatment,or infection 
with usually nonpathogenic organisms may all point to immunodeficiency. 


say your patient has a bacterial infection and you treat with the right 
antibiotic and it gets worse - this is a sign of immunodeficiency.

infections with usually nonpathogenic organisms may produce clinical 
disease or fatal disease in immunocompromised hosts. 

canine primary 
immunodeficiency diseases:

Xlinked SCID
selective IgA deficiency
LAD

transient hypogammaglobulinemia of infancy
these four were all documented 
first here at penn.

GUS: our first patient documented to have genetic 
immune deficiency.
Gus was an 8 wk old Basset Hound who had a 3 wk 
history of chronic infxn. a littermate had died one week before. he 
looked sad when he came in, but all bassets look sad. he had a big skin 
infection - started as small area of pustules - tx'd with topical abx - 
got worse - txd with systemic abx - got worse - now the dog is a 
festering mess. many pustules, alopecia, - first thing you'd think of 
would be demodex. but this is a staphylococcal infection, you can see the 
pustules. the alopecia is from self trauma. Gus also had severe otitis 
unresponsive to antibiotic therapy. 

One remarkable thing was you 
couldn't palpate any LNs in this puppy. a dog with bacterial infections 
should have enlarged LNs. lymphocyte count was WNL but near the bottom - 
about 1000. 

this was around 1983. clinical immunology in vetmed was 
just starting. they decided to quantitate immunoglobulins, look for B 
cell deficiency. there were no normal puppy values out there, so they 
also evaluated normal littermates. 
Gus's antibody levels were far below 
his littermates'
They then used a polyclonal T cell activator and 
compared Gus' response to littermates - Gus didn't respond. his T cells 
couldn't recognize the stimulus to proliferate. SO Gus had a combined 
immune deficiency. So, the medical  people vaccinated Gus with distemper 
vaccine because he was in the hospital
and they felt he needed it. 10 
days later he died from vaccine induced distemper. 

remember, they 
couldn't palpate any peripheral LNs. Well,they couldn't find any 
mesenteric LNs on necropsy. the spleen histo was a sheet of immature 
lymphocytes - no follicles. the tonsillar crypts didn't have any 
lymphocytes. the small intestine didn't have any peyer's patches. he just 
didn't have any lymphoid tissue. his thymus was small and had no normal 
architecture, and very few lymphocytes. 

the problem is, CDV (distemper) 
can cause lymphoid depletion. This dog died of distemper. At first, they 
weren't sure it was vaccine induced distemper -took a year to get that 
info.

so they took a family history to see what was going on. the mom 
was 5 yrs old, this was her first litter.they asked the breeder to redo 
the breeding. she had 13 puppies that Penn had to buy. this was to see if 
they could reproduce the problem. 

litter 2: at about 3 wks of age, 3 
males got skin lesions. they became full blown by around 5 wks of age. 
the affected puppies were very stunted. they had "failure to thrive", a 
hallmark of combined immune deficiency and other T cell deficiencies. is 
the most typical clinical feature of SCID. 

so they evaluated 
immunoglobulins over time. IgM levels developed in all puppies equally. 
IgG levels were similar in all puppies early on, then dropped off in 
affected animals as maternal Ab levels declined. same with IgA. all 
similar to Gus (except IgM). no T cell proliferation in affected pups. so 
this was definitely genetic. a primary immune deficiency. but, is it 
combined?
IgM was normal. IgA and IgG abnormal. T cells abnormal. is this 
combined? well, in humans, they started documenting immune deficiency in 
kids in the 50s - if they lacked Ab, it was B cell defic, if they didn't 
have T cell response, it was cell mediated defic, if both,combined. there 
are probably no true combined deficiencies in humans - probably, it's a T 
cell problem and you don't have the help to make some Ab. Same thing 
here.that's why IgM is normal. doesn't require T cell help. we still call 
it combined though.

so - pedigree - when bitch was bred to a different 
male, there were no affected puppies. all the affected puppies turned out 
to be male. this is an  X linked SCID - most common type in children. 
this means the female is a carrier. she's clinically normal, but 
shouldn't be bred. half of the male offspring are affected and die.the 
other half will be normal. half the females are carriers, and half are 
normal. this is "bubble boy" disease. David the bubbleboy was played by 
John Travolta in a movie - he lived in a gnotobiotic environment for 12 
yrs and was healthy. his mom had had a previous son with this disease. 
when she got pregnant again they decided raise him this way.what finally 
happened was, he was 12, they were doing bone marrow transplants with 
60-70% efficacy, so he opted for a transplant, and died. 

untreated 
kids/animals die very young.
there are also bubble bassetts that do fine 
in the gnotobiotic environment.

one thing that dogs were valuable for 
was that there was no animal model for this disease - the horse is a good 
model for mouse, and mouse is good model for human, but this dog model is 
the only good model for studying human disease (huh? he just said the 
mouse! whatever). 

for 30 yrs, all we knew was patients had low levels 
of Ig, had few T cells, and so forth. in the dog, we showed that -we know 
the T cell growth factor is IL2
- we wanted to know did it work in these 
dogs? no, it didn't. so there might be defect in IL2 receptor - and we 
showed that XSCIDdogs lacked IL2 receptors. a genetecist at NIH found the 
mutation in David's IL2 receptor - in the gamma chain. this is one of 
three parts of this receptor, and is part of all major lymphocyte growth 
factor receptors. so a mutation in this shared receptor explains why it 
is such a devastating defect. the defect in the bassets is a 4bp deletion 
producing a trunkated protein.

so, knowing what the disease is,what the 
mutation is - it's very easy to quickly show yes your patient has it and 
what's the patient's defect? this is good because it may not help your 
patient but on a population level can help detect carriers.with this 
disease, knowing the mutation - it's different in every patient unless 
it's in the same family. there are 50 different known mutations in the 
human- these are spontaneous mutations. but with dogs, people line bree,d 
and it is more consistent. so you can make a PCR based assay to find 
carriers and try to eliminate the disease by spaying the female carriers.


mutation is in exon 1 - they made primers such that in a normal dog they 
should get a 169 bp segment, and affected would be 165 - missing four bp. 
an affected male has a 165 bp segment. carrier female has one of each. 
you can do this test on a simple drop of blood. easy to find carriers. 


stunted growth is really a nonspecific sign. there is always a runt in a 
litter. how many of those runts have immune deficiency? it would be nice 
to document these diseases and try to get rid of them. there are 60 known 
primary immunodeficiencies in people. they probably all exist in dogs, at 
higher incidence due to inbreeding. we just have to find them. 

cure: 
bone marrow transplant is the only way. we've had a 90% success rate with 
our colony dogs although it's probably not feasable for pets for 
financial reasons.


---break---


principles of immunization

we're 
going to go over some examples that aren't in the handout and are 
important so...

one major advance in vet and human medicine over the 
past 50yrs is the generation of vaccines to help prevent infxs dz in 
animals and people. in the 40s, infectious dz was a huge problem, but now 
it's really not as much of a big deal. however, they're making a 
comeback. today, we're going to talk immunization. this is administration 
of an antigen to an individual to create immunity. you give the antigen, 
and host makes antibodies. this occurs naturally all the time. the issue 
with vaccination is to try not to make the animal sick, and to promote 
antibody responses.

acquired immunity - we want the dog/cat/person to 
acquire immunity to the disease agent. we can do this either by creating 
an active immune response in the animal, or by passive mechanisms. 
passive - the host immune system isn't participating -> can be natural, 
as in maternal colostrum; or artificial, as in immune globulin 
administration. active can also be natural, as in natural exposure to 
infectious agent that doesn't result in mortality, or artificial, which 
is what we do when we vaccinate a person or animal. 

passive 
immunization is a situation where animal's own immune system isn't 
playing a part - we're giving it preformed antibody. natural passive 
immunity is mainly for the neonate - it gets Ab from the mother. this can 
occur prenatally as Ig crosses the placenta into the fetus, or postnatal, 
via colostrum. whether it's prenatal or postnatal depends on type of 
placenta in that species. see table 2-7 in handout - horse uses colostral 
Ig. primates, rabbits and guinea pigs pass Ig through the placenta and 
none in the colostrum. cats/dogs/rodents use a little transplacental, a 
little more in colostrum.

primate placentas have few layers separating 
fetus from mom. the more layers, the harder it is for something to pass 
through.
in human placenta, trophoblasts have Fc receptors for IgG,and 
they transfer it into the placental circulation so it gets into the 
fetus. but our pets get most of their maternal Ab from their colostrum. 
that's why it's so important for them to get colostrum. a lot of this is 
in the handout.

when we talk about transferring Ab through colostrum, 
keep in mind - it's important for neonate to suckle w/in first 12-24 hrs. 
the gut will only transport the IgG across into the circulation during 
this window of opportunity. after 24 hrs, this won't work any more. this 
is called closure - the GI tract closes,nothing more can be absorbed. 
what causes closure? why can't IgG be absorbed later? for first 24 hrs, 
intestinal lining cells are very immature and they let things through. 
they mature quickly and then are incapable of transferring things across. 
so this is one thing. the other thing is that during first 24 hrs, pH of 
intestine of neonate is neutral. by 24 hrs, it becomes acidic. 
proteolytic enzymes act, and break down any further IgG delivered to gut. 
so it's really important for horse, dog, cat, sheep, cow babies to suckle 
within first day.

important disease in vet med esp horses- immune 
deficiency, not primary, but failure to receive colostrum - occurs in 
about 25% of foals. foals that do not get colostrum are at high risk for 
disease until their own immune systems mature and make Ab, which may take 
14-21 days. this is an important dz in the horse. not so much in 
dogs/cats. how do you dx this? when do you test? how do you document it? 
IgG quantitation of blood. when do you do this? you have to wait til 
after closure. so, if you find out the animal didn't get it, what do you 
do? giving colostrum won't work. you could give IV gamma globulins. 
that's the best thing to do. to diagnose, you have to wait til after 
closure, and if you wait to after closure, you can't give the colostrum - 
that's his point.

keep in mind- the newborn has maternal Ab to whatever 
mom was exposed to. you don't want to move pregnant animal to a new place 
because neonate won't have Ab to the things in the new place. also, 
passive Ab may interfere with active immune response following 
immunization. we'll get back to that.

artificial ways of giving passive 
immunity- giving Ab to the patient. this was common before vaccines. 
before polio vaccine was available, every child got a shot of gamma 
globulin once  a year.  use of gamma globulin almost disappeared with 
advent of vaccines. but now, we use it with autoimmune dz, to tx foals, 
and so forth. 
what happens when you give a patient gamma globulin IV or 
IM. if you give a person antibodies made in other people, which is what 
they do these days (they take plasma from expired blood from blood banks, 
pool it, and fractionate it to make pure gamma globulins), it declines 
over time - half life about 20 days in man, 16-18 days in dog. since host 
isn't making Ab, as Ab is metabolized it disappears. so it's a shortlived 
immunity. immunodeficient kids are treated once a month. they do fine. if 
you give humans gamma globulins made in horses, which they used to do, it 
goes away much more rapidly - this is because not only is normal 
catabolism occuring, but there is an immune response mounted against it. 
the other problem with this is that you develop antigen antibody complex 
formation and you can end up with immune complex disease. 

immunization 
- creating response without exposing host to harmful agent in natural 
environement. this is what happens - the first time an animal sees an 
antigen, nothign happens for about a week to ten days. then you see the 
IgM response, then class switch to IgG occurs. if you give a booster, the 
lag phase is shorter,and response is faster, greater, and almost all IgG. 
what causes all this? well, the antigen, say virus, stimulates the one or 
two B cells in the host which can respond to that antigen. that cell then 
undergoes clonal expansion. it proliferates, (this is the inductive 
phase)  and it starts making IgM - and then there's class switching to 
IgG. also, some memory cells are made. then at second immunization, the 
memory cells are ready. they respond more rapidly.

so why immunize? we 
want to impart memory onto the immune system, so that when the host 
encounters wild Ag, it responds rapidly and aggressively. this is the 
main point of it. we also want the primary Ab response, but that's less 
important. the memory is more important. 

what should a vaccine do? the 
main thing is it should protect the host from disease. that is why we 
give it. we want to prevvent clinical disease. you don't have to prevent 
infection. you want to prevent the signs of infection, the clinical 
disease. of course, ideally, we'd like to prevent infection, but from the 
individual's standpoint, we want to prevent clinical disease.

how do you 
evaluate the vaccine? look at antibody titers? 
the first bordetella 
bronchiseptica vaccine - bordetella hangs out in respiratory epithelium, 
irritates it, and causes coughing. drug reps like to show you serum Ab 
response that the vaccine creates, and whether vaccine prevents clinical 
dz. the first bb vaccine was given IM or SQ- now, a natural infection 
produces very little serum Ab, because the bacteria sits in the 
respiratory epithelium, it doesn't really stimulate the immune system. 
but if you tried to challenge dogs that had had natural exposure and no 
serum Ab present, the dogs didn't get clinical dz, and you couldn't 
isolate the organism from the trachea - because of secretory IgA. it's 
not affected by serum Ab - the IgA prevents infection and disease. now, 
the vaccine created a great serum antibody response! way more than 
natural infection ever produced. they were challenged at a time when they 
had very high serum antibody - and they all got clinical disease. why? 
because they didn't have secretory IgA! so you have to think about these 
things. you have to know what the agent is, where it replicates - you 
can't just look at serum antibody titers. you have to know about response 
to challenge. the point is, you evaluate vaccines by knowing if they 
prevent clinical disease.

two types: killed and live vaccines.
killed: 
advantage is that they're safe. it's been killed. it can't replicate. 

disadvantage (all in handout) won't stimulate local secretory IgA 
response, duration of immunity is shorter, but can be given to all 
animals b/c of safety. hard to make - need certain antigenic mass to 
stimulate immune response, need more for great dane than poodle. 


live/modified life - these are "attenuated" in that they are 
nonpathogenic, but still can replicate in the host. advantage - don't 
worry about antigenic mass, b/c it creates its own as it replicates. 
major problem is you have to be careful - less safe. can spread b/w 
animals. might revert to virulence! 

one important thing - in addition 
to creating local immune response, live vaccine can for some diseases 
create an almost immediate response. study showed people immunizing dogs 
w/modified live distemper, then challenging them, and the design called 
for challenge 4 days post vaccination - and dogs were protected. they had 
no Ab, but the vaccine stimulated the production of interferon. primarily 
RNA viruses and some others are protected against by high levels of 
interferon. the IFN is produced rapidly in the host post vaccination, and 
then drops off as antibody is produced. fibroblasts are making this IFN. 
so this confers near immediate protection, andkilled vaccines don't do 
this.

another important concept - what route of vaccination do you use? 
you can use intranasal, SQ, IM..which is best? you have to know about the 
agent of infection. if it replicates at site of entry, you want local 
immune response - you want to give it by local route. consider k9 
parainfluenza (CPI) - it gains entry through respiratory tract and 
remains local. you'd like an intranasal vaccine. distemper gains entry 
the same way, but then goes to LN, replicates there - so you can give 
vaccine IM or SQ and systemic immune response will prevent replicaton. 
you have to know where the replication occurs. 

in handout is a table. 
go through table on board b/c this is really important. on page 6 - 
										
challenge
parenteral route 	vaccine shed	virus shed	clinical disease
(IM, 
SQ)
CPI					no				yes			mild


the vaccine doesn't prevent infection, but 
it does prevent clinical disease. the virus infects respiratory tract and 
replicates but serum antibody prevents bad disease - if you gave the 
vaccine intranasally, you could prevent infection.

now, canine distemper 
also enters through the respiratory tract. you give vaccine - vaccine 
isn't shed. you challenge with wild virus, and there's no shedding of the 
challenge virus -this means that this vaccine actually prevents 
infection. this is because the virus is prevented from replicating in the 
LNs by the serum Ab

parvo - if you give vaccine IM Or SQ, there's no 
vaccine shedding, no secretory IgA made in intestine, and when you 
challenge with wild virus there is no shedding - because parvo replicates 
in LNs, and serum Ab prevents infection. so there's no clinical dz. so 
from the standpoint of protecting from infecton, you'd like to give CPI 
vaccine by natural route so you would have antibody in area of 
replication. 
however, when you give CPI via natural route, vaccine virus 
will be shed - because it replicates in respiratory tract. when you give 
vaccines by a parenteral route, there is no vaccine shedding. of course, 
after vaccination there is no clinical disease with parenterally given 
vaccines -but when you give the intranasal vaccine, there are some signs, 
referable to the replicatoin of the vaccine virus. but then there's no 
disease from the wild virus. 

again - route of choice dpends on biology 
of virus, and need to prevent infection. more striking example in cats - 
see handout, he's not discussing it other than to say that for feline 
viral rhinotracheitis you have to use an intranasal vaccine because it's 
a herpes virus, and it becomes latent and you can never get rid of it. 
when you use IM vaccine, the vaccine doesn't prevent infection in 
respiratory tract - it just limits the clinical signs. when you give a 
vaccine against disease that stays local to respiratory tract, it doesn't 
protect against infection. now, these cats, that you've vaccinated, have 
latent herpes infections - and when new cats come in, they get infected 
before vaccine kicks in. you have to give new cats intranasal vaccine to 
prevent infection. 

there's also something here on herd immunity
one 
more example before we go. slide isn't working.
oh, that's ok, it's not 
on the exam anyway. 
the point is that you do not have to vaccinate every 
animal to prevent spread of infection - it may be you only have to 
vaccinate 60-70%. you want to minimize likelihood of infectious animal 
contacting another susceptible animal. herd immunity depends on ease and 
mode of transmission - if it's blood borne it's harder to spread. if it's 
aerosol, it's easier, you need to vaccinate more. population density is 
also important. 

one clinical example before we call it quits:

how to 
evaluate a vaccine:

veterinary vaccines, in contrast to human vaccines, 
aren't licensed through FDA but through USDA. they don't go through the 
rigorous clinical trials of human vaccines. 

companies will give you 
info regarding their vaccines. you have to evaluate it.

first 
parainfluenza vaccine from 1970s -data given to USDA- had to see if 
vaccine produced clinical disease in patients. so they produced data 
showing vaccine was safe, first of all. it didn't cause fever, harsh 
hacking cough. this vaccine is given by IM/SQ route. of inoculated dogs, 
none developed fever, leukocytosis or clinical signs. control animals in 
contact with these dogs didn't get sick either. fine. 

the next data 
were to show if animals produced serum antibody. two groups of dogs were 
vaccinated. none had Ab prior to immunization. animals did develop Ab 
response after SQ or IM vaccination. challenge produced a secondary 
response. 

the important data- they took a group of vaccinated dogs. 
before vacc - no Ab. after vacc- Ab present. after booster - secondary Ab 
response. challenge with wild virus - no clinical signs. control dogs - 
not immunized, challenged. the vaccinated dogs didn't get sick and shed 
the challenge virus for 3-6 days. so they got infected, but didn't get 
sick. the control dogs didn't really get that sick either and shed virus 
for 7 days. the vaccine didn't work. the control dogs weren't getting 
ill. it turned out the company used an attenuated virus for the 
challenge. this vaccine was on the market and it did nothing.

the first 
canine parvo vaccine didn't do much either and provoked many lawsuits. 
before the trial, the warehouse storing the vaccine burned down. hmm.
you 
have to be able to evaluate the data. 

point - serum antibody titer may 
have nothing to do with protection from disease.

----end----