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The Immunology of Large Animals
David J. Hurley, PhD
Associate Professor of Population Health and Large Animal Medicine, and
Molecular Microbiologist, Food Animal Health and Management Program
College of Veterinary Medicine
University of Georgia
Athens, GA 30602
Abstract
Immunology is generally taught as if all animals shared a uniform set of
mechanisms to effect the protection of the body and regulate those processes. Most of
current immunology has been “established” by studies conducted with mice. Mice have a
unique set of environmental and evolutionary challenges not shared by all other animals.
We know that cattle, swine and horses are not just “big mice” in other ways, and it turns
out that they are not immunologically identical with mice (or each other) either. This
essay will summarize the differences between the immunology generally taught to
veterinary students and how cattle, swine and horses mount and regulate inflammatory
and adaptive immune responses.
Introduction
Thus far in your training, you have learned immunology based primarily on the
lessons taught to us by mice and as applied to human conditions. To really appreciate the
complexity of the infectious diseases of large animals, their diagnosis and their treatment;
it is important for you to see the outstanding features that guide the response these
animals make to dangerous and damaging insults, such as the tissue damage caused by
colonization and infection, metabolic and structural products of invaders, and how the
ecology of each host has directed the process of response on the innate, intermediate and
adaptive levels.
Understanding the relationship between immunology and the diagnosis and
treatment of disease is a critical factor in being an informed physician. As a veterinary
physician, you must have a feel for the factors that drive and control the responses of
each group of animals to the dangerous and damaging events in their internal
environment. Large animals are a particularly interesting group. They represent, in my
opinion, the greatest diversity in the control of innate immunity and range in dependence
on adaptive immunity to resolve problems that one has the opportunity to study in all of
medicine. The contrast between cattle and horses in the control of inflammatory function
and use of intermediate response (between direct pathogen associated molecular pattern
(PAMP) recognition and major histocompatibility complex (MHC) restricted) mechanisms is
the most clearly defined in all of medicine. Understanding these differences will instruct
you in ways to approach the treatment and care of these animals.
Most of the immunology we teach in introductory classes, either in undergraduate
school or to veterinary and medical students, is based on studies conducted with mice.
This is for good reason. We have the largest body of data from studies with mice. They
are small and inexpensive to use, we have generated a tremendous number of genetically
defined and knockout variants for use in reductive study of specific pathways, and there
are many research tools available for purchase or exchange to assist us in doing our
studies. In presenting the complex field of immunology to students for the first time, we
attempt to teach a family of “general rules and principals”. This helps the student to
organize and understand the processes and responses more easily, and to connect the
protective and damaging aspects of the same families of responses more clearly in their
general framework. (For example, understanding the relationship between antibodycomplement immune complexes in bacterial clearance and the development of type III
hypersensitivity responses as two sides of one coin). One caveat for the student is that all
animals (including humans) are not just big mice.
Why should we learn about the immunology of large animals? Large animals
represent several families that have evolved under different sets of ecological pressures.
Most obvious are the ruminants. Cattle and their relatives carry a large, shedding
bioreactor within their body cavity that presents huge quantities of PAMPs on a regular
basis to indicate regular danger. Cattle have adapted the most exquisite inflammatory
control of any animal it has been my honor to study. Horses faced different pressures.
Much better contained fermentation systems for extracting nutrition from cellulose than
cattle, but highly responsive inflammatory activity on sensing danger. The most difficult
and feared diseases of the horse, colic, chronic obstructive pulmonary disease (COPD)
and laminitis, all appear to have significant systemic inflammatory components in their
pathogenesis. Swine were domesticated to live on garbage. They are low to the ground
and eat a highly varied diet and raise a lot of dust finding it. These are all factors that
should instruct our study of their diseases.
Of mice and not men – diversity in immunity
There was a great review of the comparative immune activity of mice and men
published by Metas and Hughes in the Journal of Immunology (J. Immunol. vol 172, pp
2731-2738, 2004). The authors compiled a list of differential features of the mouse and
human immune system for those of us who are really into immunology. If you chose to
read it, and I do recommend it to you, you will find it assumes you are highly conversant
with modern immunology. The vocabulary and intensity of the paper are at a quite high
level. Yet, the major points will be clear to you as you review it. (The basic areas they
address were covered in your immunology courses). What is their point? Well, they are
trying to show us that we must be careful in making assumptions about immunological
assessments and treatments developed in mice, as they may not translate directly into the
same outcomes in humans (or in our case large animals). They point out significant
differences in every aspect of immune system function and regulation between mice and
humans including: hemopoesis, the use of toll-like receptors (TLR) in PAMP recognition,
recognition of antigen, the intermediate immune response, chemokine usage in staging
immune function, and how grafts are tolerated and rejected. The table in their paper is
packed with great information. Probably too dense for your everyday purposes, but what
you may eventually need to find someday is likely at least hinted at there.
Mice and humans have big differences in their approach to inflammation. Mice
have only about 10% neutrophils in circulation, but humans have about 45%. Mice tend
to deal with dangerous invaders using tissue macrophages and recruiting monocytes to
the tissues. Humans recruit neutrophils, which in turn recruit monocytes to the site of
danger or damage. The use of specific members of the TLR family of receptors is also
different in mice and humans, with the greatest differences observed in the distribution of
TLR 2, 3 and 9. Defensins are also ecologically different between mice and humans.
Mice store pro-enzymes, humans store active proteins, and mice do not produce defensins
in leukocytes.
Mice and humans also have big differences in control of Th1/Th2 polarization.
Mice rapidly and permanently differentiate lymphocyte responses into Th1 and Th2
contexts. This polarization is maintained in memory cells and provides a fixed
reactivation context. This is not observed in humans. While, Th1 and Th2 response
contexts are measured in active responses, the context is more plastic and memory
reactivation can have a Th1, Th2 or Th0 bias. The role of IL-10, an anti-inflammatory
cytokine, is also different between mice and humans in that IL-10 is produced in humans
as part of both Th1 and Th2 response, but only in Th2 responses by rodents.
Differences in antigen presentation are also observed between mice and humans.
Antigen presentation by epithelial cells is observed in humans, but only presentation by
dendritic cells, macrophages and B cells have been observed in mice. It also appears that
activated T cells can participate in antigen presentation through MHC class II in humans,
but never in mice. These enhanced antigen presentation capacities in humans also appear
to be related to differential presentation of CD40 on epithelial cells and T cells not
observed in rodents.
Table 1. Comparison of the Immunity of Mice and Men
Feature
Percent neutrophils in blood
Percent lymphocytes in blood
Percent monocytes in blood
Percent gamma-delta in blood
CD4:CD8 ratio in adult
Polarization of Th1/Th2
NO production to LPS
ROS production to LPS
MHC class II expressed on T cells
epithelial cells present AG
Predominant panT cells CD
Transplasental immune transfer
Immunoglobulin classes
Light Chain usage
Secretory Ig
lymph
lymph nodes
Mouse
Human
10
75
10
2
2:1
very strong
very strong
weak
never
no
3
major
IgG1,2a,2b,3
M, E, A
kappa
A
cellular
normal
45
40
10
2
3:1
moderate
moderate-weak
moderate
activated
yes
2,3
moderate
IgG1,2,3,4
A1,A2, M, E, D
Both used
A
cellular
normal
Humans and rodents each produce a unique set of chemokines. Chemokines
appear to act as the “vote” each local environment offers to inform the secondary
lymphoid tissue about the state of danger and damage it is experiencing to inform the
overall response to danger in the regional and systemic response. Humans produce CXC
receptor 1 and the ligands CXCL8 (IL-8), CXCL7, CXCL11, CCL13 (MCP-4), CCL15,
CCL18, CCL23, and CCL24/26 (eotaxins 2/3). These have all been identified in humans
and play critical roles in disease states, but have not been identified in mice. In contrast,
CCL6, CCL9, CXCL15 (lungkine), and CCL12 (MCP-5) play a role in mouse infectious
diseases, but have not been identified in humans.
Immunology of cattle (including goats, sheep and water buffalo)
The rumen is an ecological factor in cattle. The rumen produces huge quantities
of microbes in the digestion of grasses, and products that represent PAMPs escape the
rumen environment and insult cattle on a regular basis. Therefore, cattle have evolved
very tight regulation of innate inflammatory responses. This can be readily observed
after field surgery for hardware disease. The operation is often done in the mud and the
surgical wound closed, then the animals released to the field. The next day or the day
after, the wound is in the process of healing and the cow eating happily. This is a truly
remarkable sight.
Cattle have a balance between neutrophils and mononuclear cells in circulation.
The inflammatory cells produce both nitric oxide (NO) and radical oxygen species (ROS)
in moderate concentrations after stimulation in vitro with LPS (particularly from
Salmonella). It appears that both NO and oxygen radicals play important roles in direct
inflammatory control of dangerous invaders in cattle. Cattle also appear to rely on
intermediate immune responses (regulated by lipid and phospho- or lipo- protein) for
control of infection to a much greater extent than mice or humans. Cattle have a
relatively high number of circulating gamma-delta cells. These cells appear to recognize
antigens in the context of CD1 antigens, similar to men. Three different CD1 antigens
have been identified in cattle, CD1d, a probable CD1b and a possible CD1c that can
provide the context for intermediate responses. This dependence on immune responses
that are not PAMP dependent, but directly recognize structures produced by dangerous
invaders may be part of the method cattle use to regulate inflammatory responses and
minimize damage caused by regular exposure to microbial products.
Another example of the highly regulated inflammatory response of cattle is their
tolerance of a wide variety of strong adjuvants. Many different formulations of adjuvant,
including those that cause significant side-effects in other species, have been successfully
used in commercial products for cattle. These include very potent oil in water and water
in oil formulations containing multiple components that lead to a very broad and lasting
immune response in cattle. Other evidence can also be seen in the generally welltolerated vaccines produced as crude bacterins used in cattle. While the level of
lipopolysaccharide in many of these products has been documented to cause problems in
some individual animals, large numbers of cattle have been successfully vaccinated with
these products to induce short-term immunity to specific bacterial diseases, like blackleg.
Cattle are more like humans than mice in the differentiation of Th1/Th2 responses
to antigen. Cattle can be shown to mount specific responses in the context of Th1 or Th2,
but it does not appear that memory responses are locked into these response patterns.
Adaptive, classically MHC restricted, responses also appear to play a “completing”,
rather than central role in the management of infections.
Immunoglobulins of mice and humans are different (see table 1). Cattle have
features similar to both mice and humans, but some unique features as well. Cattle, like
mice have a very high representation of a single light chain gene used in antibody (about
95%). However, unlike mice that use primarily kappa, cattle use lambda. In addition,
cattle utilize IgG1 as a secreted immunoglobulin in addition to IgA. Unlike other
animals, they make a secretory chain for monomeric IgG1 that is related in structure to
that for IgA. Cattle also have a unique FcR that recognizes only IgG2b and is important
to control of bacterial infections.
Antigen presentation presentation in cattle appears to be a complex process. The
tightly regulated inflammatory responses in cattle, significant level of MHC class II
expression on epithelial cells, T cells and B cells, and expression of several CD1 epitopes
by bovine mononuclear cells suggests that multiple pathways of antigen presentation are
occurring in concert. Development of novel and complex adjuvants for cattle vaccines
were driven by the empirical recognition of this complex set of processes. Further, cells
that appear to recognize virally infected autologous cells, but are not dependent on MHC
class I antigen presentation for killing, have been documented in vaccinated cattle.
Table 2. Comparison of the immunological features of cattle, swine and horses
Feature
Percent neutrophils in blood
Percent lymphocytes in blood
Percent monocytes in blood
Percent gamma-delta in blood
CD4:CD8 in adult
Polarization of Th1/Th2
NO production to LPS
ROS production to LPS
MHC class II expressed on T cells
epithelial cells present AG
Predominant panT cells CD
Transplasental immune transfer
Immunoglobulin classes
Light chain use
Secretory Ig
lymph
lymph nodes
# not universally accepted
*also called IgG1,2,3,4,5 &6 in horse
Cattle
Swine
50
30
5
15
3:1
weak
moderate
moderate
activated
probably
2,6
none
IgG1, 2a,2b
M, E, A
lambda
G1 and A
cellular
normal
Horse
40
35
5
20
0.7:1
weak-moderate
Moderate
moderate
always
probably
80
15
5
~1
2:1
moderate
weak
very strong
Activated#
unknown
2
2
none
IgG1, 2a,2b, 3, 4
M, A,E
Both
A
acellular
inverted
none
IgGa,b,c,(B), (T), Tb*
M, A, E
both
A
cellular
normal
Finally, because of the structure and thickness of the placenta in cattle, no maternal
immune components appear to be transferred during development to the fetus. The
placenta is an absolute barrier, excluding proteins and cells. Therefore, colostrum
becomes the critical link between maternal immune protection against local
environmental antigens and the neonate. Failure of passive transfer is a critical problem
in cattle, and an important management issue in rearing both beef and dairy calves.
Maternal cells also appear to play a role in the transfer of immunity to the calf. These
cells can traffic across the gut of the calf during the first 8-24 hours after birth and have
been shown to travel in the neonatal circulation. Maternal cells (based on expression of
maternal MHC class II antigens) have been shown to home to the gut and secondary
immune tissues of the calf beginning about 12 hours after birth. Finally, it has been
shown that enhanced antigen presentation, response to alloantigens (foreign MHC) and
viral antigens are observed in the circulation of the neonatal calf concurrent with the
circulation of maternal cells between 12 and 30 hours after receiving colostrum.
Immunology of the pig
One of the most striking features of the immune system of the pig is the low ratio
of CD4:CD8 T cells, generally less than 1:1 in adult. Mature pigs also have a large
population of CD4, CD8 double positive T cells in circulation. These represent
“experienced” T cells and may represent the circulating activated and memory
populations. The double positive population of T cells in the pig increases after infection
or vaccination. In addition, MHC class II antigens are always expressed on porcine T
cells, unlike most other species. The level of expression increases on activated cells in
the pig and is a good indicator of an active immune response. Pigs show moderate
polariztion of adaptive immunity into Th1 and Th2 responses, with considerable Th3 type
responses reported.
Pigs have what has been called an “inverted” lymph node structure. That is most
immune cells enter and leave the lymph nodes in through the blood. Thus, pigs have
nearly acellular lymph. This makes pigs an interesting biomedical model, as they
represent a unique class of animals in which the mucosal leukocytes circulate in the blood
and can be monitored (against the population of central circulatory compartment
leukocytes) in peripheral blood samples. For this reason, the number of circulating
leukocytes in pigs is generally higher than most other animals. It also means that there is
a high fraction of gamma-delta cells circulating in the blood of pigs. When they are
young, as many as 35-45% of circulating leukocytes can bear gamma-delta TCR.
Further, adults have about 20% gamma-delta cells circulating in the blood. So far, only a
CD1a like antigen gene has been identified in swine, unlike cattle where three different
CD1 genes were found. This CD1 isoform is consistent with presentation to gammadelta cells in a fashion similar to that described for humans.
The pig also has a balanced inflammatory response that is very well regulated.
Swine are good responders to environmental antigens and produce high levels of
antibody and cell mediated responses to antigens. Many of the same adjuvants that were
developed for use in cattle have been successfully applied to vaccine for swine. There is
some evidence for the replication of gamma-delta cells in cattle and swine after infection
or vaccination, and that some form of memory based on clonal expansion may be present.
These finding have not yet been reported in other species.
In response to in vitro stimulation with lipopolysaccharide a balanced production
of both NO and ROS is reported in pigs. Analogous to cattle, this may help to explain the
tight regulation of inflammatory responses in the pig, and the capacity of the pig to
tolerate vaccination with strong adjuvants. Pigs also have a moderate number of
neutrophils in circulation, similar to the human. The balance between neutrophil and
mononuclear cells driven inflammatory responses may also be an important factor in the
balance and regulation of inflammation in the pig.
Compared to cattle and horses, pigs utilize a much more restricted selection of Vh
genes to develop their repertoire of antibodies. They have a more diverse set of gamma
heavy chains than cattle, but fewer than horses, with a split into IgG2a and 2b, similar to
rodents, and 4 gamma chain classes like humans have been documented.
Similar to cattle, pigs have no transfer of immunity to the fetus across the
placenta. It is too thick and forms a barrier. Colostrum is again the primary source of
transfer of immunity to neonatal piglets. We will discuss the development of immunity
in neonatal pigs during a future class period in detail.
Immunology of the horse
The immune responses and regulation in the horse make a very interesting study.
I have been studying the inflammatory and immune responses of horses for about five
years now, and have found that everything is more difficult to study in the horse. Horses
mount strong and persistent inflammatory response to signals of danger or damage. Their
circulating leukocytes are loaded with neutrophils, and up to 90% circulating leukocytes
can be neutrophils in the blood, 70% is typical. In addition, unlike many other animals
where monocytes and eosinophils characterize recurring inflammatory responses,
neutrophil recruitment in COPD is the classical mechanism observed, and neutrophil
activation in the circulation and neutrophil trafficking into the laminae are observed in
black walnut induced laminitis. This is consistent with the dominant role of neutrophils
in the horse. Horse leukocytes produce massive quantities of ROS when stimulated with
LPS, but essentially no detectible level of NO by activation of iNOS. This does not
discount the role of monocytes and macrophages in the protection of horses or in
inflammatory disease. Monocyte products, such as tumor necrosis factor and IL-1 play
important roles in colic and the response to endotoxin in horses. One must simply not
discount neutrophil driven mechanisms when considering the pathogenesis of diseases
with an inflammatory component in horses because of their dominating presence.
Another aspect of the strong inflammatory responses of horses is the problems
they encounter after exposure to adjuvants. Many otherwise interesting vaccine
candidates for protection of horses are eliminated before production because of sideeffects encountered in testing. Adjuvants used for other large animals have often proven
too strong for horses. We are working on new classes of adjuvants to better utilize the
inflammatory responses of horses in development of protective immunity at UGA. These
constructs are designed to spare the inflammatory consequences of vaccines. This work is
just beginning, but we have some candidates in development.
The inflammatory responses of horses are strong and prolonged. A role for
intermediate responses has not been well documented in horses. Lymphocytes that lack
CD4 and CD8 receptors have been identified, and a role for gamma-delta lymphocytes
has been postulated for horses. No CD1 antigens have been characterized in horses, so
the mechanism of antigen presentation is not clear for intermediate responses.
In contrast, the classically MHC restricted T cell responses of horses have been
clearly demonstrated and the polarization of Th1 and Th2 responses of horses appear to
be stronger than in humans, but less fixed than in rodents. Studies of MHC restricted
killing by horse lymphocytes and studies of cytokine message production support a
strong role for MHC restricted responses in control of infection and a polarized memory
response. A somewhat limited number of tools to assess equine lymphocyte
subpopulations have impeded the complete description of equine adaptive immunity.
Horses have a unique distribution of immunoglobulin classes. A family of six
gamma constant region genes, with no pseudogenes identified, has been described. It
appears that horses have six classes of IgG, and renaming them IgG 1-6 has been
proposed. The molecular biologists have also found evidence for at least two classes of
IgA genes in horses. At present, we do not have the tools to test this fully, but it is likely
that a split, similar to that found in humans, has occurred in horses. It is clear that
immunoglobulin plays an important role in the defense of horses and division of that
defense has occurred in the development of the large number of immunoglobulin classes
that they produce.
As with the other large animals reviewed here, horses do not transfer immunity
across the placenta to the fetus. Colostrum is a critical source of immunity and immune
development cues to the neonatal foal. Failure of passive transfer is a critical problem for
horses, and leads to significant problems in the health of the foal. Tests for failure of
passive transfer should be included in a foal workup for neonatal infection. Our group
has recently started to work on other factors, in addition to antibody, that are important in
the transfer of maternal immunity to the foal. We have examined the roles of soluble
CD14 (an LPS transfer molecule and stimulator of immune cell development) and
lactorferrin on the function of leukocytes from presuckle foals. We are also studying the
role of transfer of maternal leukocytes to neonates in our laboratory. It has been observed
that foals are deficient in their ability to produce interferon gamma for about a month
after birth. This suggests that they develop the capacity to mount T cell mediated
responses to intracellular pathogens more slowly than many other species. We hope to
eventually develop a better definition of failure of passive transfer for use in the clinic.
Common immunological features of large animals
Two striking features of the immunology of large animals were presented here.
First, all the large animal families discussed in this essay were absolutely dependent on
colostrum for the passive transfer of immunity and cues from the mother for the
development of the neonatal immune system. This is radically different from mice and
men where very significant transfer of immunity occurs before birth. Mice are born with
much more complete immune protection and a network of responses that look much like
the adult, only on a smaller scale. Humans receive about 30% of the antibody transfer
from their mothers before birth and there is evidence of some cellular transfer during fetal
life.
Second, all these families of large animals have some unique features in the
function of antibody. Cattle have unique Fc receptors directed at IgG2b to protect the
tissues and a secreted form of IgG1. Pigs have a unique system for the generation of
diversity among large animals, using fewer recombinant sites in the building of an
antibody arsenal. Horses have six classes of IgG and probably at least two classes of
IgA. This suggests that the production and regulation of antibody is linked to the ecology
of the threats to each animal and should be considered in development of vaccines and
treatments for disease.
Take home messages
First, this essay represents my viewpoint. It is not a complete and exhaustive
review, but a discussion of points that I believe are important and interesting. Good
reviews of food animal and equine immunology can be found in many places, and I
recommend Vet Clinics in N. Am. Food Animal Practice,Vol. 17.3, 2001, and Vet Clinics
in N. Am., Equine Practice, Vol. 16.1, 2000 to you for further reading. The topic is far
too broad and complex to be covered in a few pages. Second, I intend this essay to serve
as the basis and background for a discussion of the immunology of large animals among
us. I recommend you to address the questions that follow in your preparation for class.
The class will be structured around my interest in comparative immunology and my
“framework” for understanding the ecology, evolutionary pressure and biology of
differential immune responses. The lecture will be very different from this presentation,
much less formal.
1) How does the immunology of cattle (or swine or horses) impact the types of
diseases that affect cattle (or swine or horses)?
2) How does the role of the inflammatory response differ in its impact on the
pathogenesis of disease in cattle, swine and horses?
3) How does an understanding of the immunology of each family of large animals
instruct the development of methods to diagnose disease?
4) How can information about the specific class of antibody response in horses (or
cattle or swine) provide significant prognostic information to the veterinarian?
5) How does the gamm-delta T cell response (and NK and NK-T cell response as
well) act as a transition between innate PAMP driven responses and the classical
MHC restricted response to infection?