Immune system development in foals

Equine neonates are susceptible to a wide variety of pathogens because of their naïve immune systems. As a result, sepsis in foals is a matter of concern to the equine industry.1 Maria Julia Felippe, Med.Vet., MS, PhD, DACVIM, associate professor of large animal medicine at Cornell University's Equine Immunology Lab, is part of a Cornell group studying the development of the foal's immune system. The following discussion outlines several critical aspects of their work that's expanding our understanding of immunity.

Immunity basics

Foals begin to develop their innate and adaptive immune functions partially during gestation but more significantly after birth when exposed to environmental organisms.1

GETTY IMAGES/RICHARD LOADER

Colostrum is an essential source of immune components for newborn foals, the most important of which is IgG. Inadequate IgG absorption results in a partial or total failure to transfer immunoglobulins to the newborn, causing increased susceptibility to infectious diseases. Even with sufficient IgG, foals are susceptible to certain pathogens that rarely affect adult horses, such as Rhodococcus equi.

Transferring proinflammatory cytokines to the foal

A group of Cornell researchers that included Felippe found that "the transfer of cytokines in colostrum to equine neonates could have immunomodulatory effects and help with protection early in life. Perhaps foals with inadequate colostral ingestion and absorption may be more susceptible to infectious diseases not only because of the critical concentrations of IgG but also because of low proinflammatory cytokine transfer from colostrum—both important to help fight off pathogens before competence of their own immune systems are developed."1

This research determined that "serum TNF-alpha concentrations were high in postsuckle samples, though essentially undetectable in presuckle samples, as the TNF-alpha concentration in postsuckle serum predicted the concentration in colostrum."1 The data suggested that "TNF-alpha is transferred to the foal via colostrum absorption and may play a role in early immunity. It is possible that the transfer of proinflammatory cytokines in colostrum helps with immune alertness and protection before the foal can efficiently elaborate an immune response. TNF-alpha has a key role in sepsis due to its potent proinflammatory properties."1

"It's possible that intake and absorption of proinflammatory cytokines in the colostrum might help," says Felippe. "In the manuscript, we cited previous work done in calves where they found that when you give IL-1 orally, it did have an immunomodulatory effect. If foals are drinking colostrum that contain cytokines, these cytokines are going to be absorbed, either to work systemically or locally in the gastrointestinal tract, and have an immunomodulatory effect. By analogy with what's been done in calves, it's very possible."

Felippe's team specifically looked into TNF-alpha, but other cytokines could be transferred via the colostrum. Some have suggested that IL-6 is one of them, though it has not been studied as systemically. "Maybe other anti-inflammatory cytokines, such as IL-10 could be transferred as well," Felippe says. "But we still need to learn how they are transferred through colostrum and what the effects are."

The potential for the immunomodulatory effects for cytokines is speculation at this point, but it may aid the neonate's immune response. "One answer that we don't know is the magnitude of an effect," Felippe says. "Is it dose-dependent? Do you need a lot of TNF-alpha to get an effect or a little bit? That is really an open question.

"This type of work is interesting, as we're learning that the importance of colostrum may be beyond the IgG, though perhaps IgG is the essential part," continues Felippe. "But it also tells us that there are other 'goodies' in the colostrum that can be very helpful during early life. Perhaps there is a quantitative correlation. We know the greater the amount of IgG, the better protected the foal is. Maybe more cytokines of one kind may be very beneficial for the foal. And maybe too much of another kind may not be beneficial for the foal. We just don't know. Too much of a good thing sometimes is not as good in immunology."

Another important aspect of this research is that the team questioned whether serum cytokine concentrations in neonates could be used to predict the severity or outcome of septicemia, therefore predicting foal survival.

"A lot of equine neonates get sick in early life, even when there are perhaps still high levels of those cytokines absorbed from the colostrum," Felippe says. "If you're trying to correlate the level of TNF-alpha, which is a prime inflammatory cytokine, with the severity of disease and perhaps death outcome, you need to keep in mind that in some foals that have more or less TNF-alpha, despite their inflammatory process, the level may be high or low because of maternal transfer from the colostrum. That is a major confounding value that can change your ability to use that as a prognostic indicator.

"There were some incoherencies in the findings of cytokine levels in septic neonates and how that correlated to survival outcome," continues Felippe. She says part of the problem probably relates to the fact that these foals, independent of their disease, have different backgrounds—some cytokine concentrations are coming via the colostrum, and others may have come from plasma transfusion, which is another way to transfer immunoglobulins and cytokines to foals.

"The plasma is from adult horses that are healthy, but they carry some cytokines along with them," Felippe says. "So we need to be careful about measuring things that are not necessarily inherent to the foal, or endogenous to the foal, but are being absorbed via the colostrum and may affect our ability to use those parameters as prognostic indicators."

Can foals respond to antigens?

Another critical question is how foals respond to antigens and vaccines.

"The equine neonate is considered immunocompetent at the time of birth because the equine fetus is capable of producing antigen-specific antibodies about day 180 to 200 of gestation when vaccinated in utero," says research associate Rebecca Tallmadge, PhD, and her colleagues at Cornell's Equine Immunology Lab.2

It is also known that because the equine placenta prevents transfer of maternal antibodies during gestation and because of the absence of exposure of the fetus to microorganisms, presuckle foals are born hypogammaglobulinemic and naïve to environmental pathogens.2

Humoral protection of the newborn foal depends on the absorption of preformed antibodies from the colostrum immediately after birth. Failure of immunoglobulin transfer via the colostrum increases the susceptibility of the foal to infection from environmental pathogens.2

Data collected by Tallmadge's team suggests that the equine neonatal immune system is naïve but competent, including potential B and T helper cell preparedness for the production of all immunoglobulin isotypes, which can be explored for prophylaxis. "Although the equine neonate humoral response seems competent, B cell activation factors derived from antigen presenting cells and T cells may control critical development regulation and immunoglobulin production during the initial months of life."2

"The major question is how can the foal respond to antigens?" Felippe says. "There are some general concerns that foals do not respond very well to vaccines. Interestingly, foals that received colostrum from mares that were vaccinated during the last month of gestation had a delayed ability in their humoral response to vaccination."

One explanation for this delay is the phenomenon called maternal antibody interference, according to Felippe. "The theory is that the circulating antibodies that came from the colostrum interfere with the ability of the foal to produce new antibodies in response to vaccination. How would that occur? A few mechanisms have been proposed. They would interfere by perhaps neutralizing the vaccine antigens. So if you're trying to put a vaccine antigen to stimulate the foal's immune system, and it is immediately neutralized by an antibody that is circulating against it, it doesn't do what it should. It doesn't stimulate the cells. It doesn't make them develop immunity against it."

Felippe says she questions this theory based on some fundamental principles of "mother nature." "The horse is an animal that has been selected for millions of years. One of the interesting things about horses is that they don't transfer immunoglobulins during gestation," she says. "Instead, the foal needs to get its immunoglobulins from the colostrum intake after birth. This is a major pressure in their selection process. If this foal is not healthy enough to nurse quickly and the mare is not healthy enough to produce good-quality colostrum, that transfer doesn't happen in a timely manner, and the foal will be very susceptible to infection.

"It would be a major selection pressure for colostrum, which essentially gives foals survival during the early weeks of life, to paradoxically prevent their own ability to produce protection for continued survival. The species would possibly be in major trouble if that was the case. Therefore, simplistically, I question it," says Felippe.

What is the basis for this hypothesis? Studies done across species have shown some limitations of neonatal response to some vaccines, whereas other vaccines were good at inducing immunity in the neonate despite circulating maternally derived antibodies. "The first thing that comes to mind is that one of the limiting factors here is not necessarily what is going on in the background, but it is the quality of what you are using to elaborate an immune response," says Felippe. "There is good research showing that depending on what you use to stimulate the immune system of the neonate, you are going to have a good response or not-so-good response."

Dosage has also been studied. "In fact, smaller doses may do a better job than higher doses for some pathogens," Felippe says. "We cannot generalize across antigens, but these are some observations that we know may play a role."

As for maternal antibody interference, there have been some conflicting results in the literature, and it depends on the type of model that has been used. "But some of the literature points to the fact that in the presence of circulating maternal antibodies, the immune response to some vaccine products is not as good," says Felippe. "If you vaccinate a foal that doesn't have that much circulating immunoglobulin specific to that antigen, the immune response could be more robust.

"I question some of the conclusions from some of the studies, because if these studies are basing their results on total serum immunoglobulin levels, I think there may be some limitations in understanding what the true response is," she continues. "Because when you measure serum immunoglobulin levels against an antigen, you're measuring the maternally derived and the foal's at the same time. There is no way to discern whose is whose. You're trying to identify an immune response that is going to be minor because it was from a neonate, a naïve individual, in the mix with a lot of other antibodies that belong to the mare. So to find that very small amount within that large sample can be difficult technically."

It does take time for an immune system to expand the population that will respond to an antigen, according to Felippe. With a more trained adult immune system, you can anticipate that fewer doses would be necessary for that population expansion. But in an untrained immune system, such as the foal, you would probably need more boosters to achieve the same level of population expansion that you would have with an adult horse. "We go back to principles that are not new, but the use of boosters, and perhaps more than one or two, are necessary for some antigenic stimulation to accomplish what we consider protective levels, or standard adult horse levels," Felippe says.

All of this leaves Felippe with two questions: Are we using the correct tools to measure this? And are we limiting our interpretation to our ability of measuring the foal's response to vaccination?

"What our lab developed with Rebecca Tallmadge, the creator of the technique, was the ability to measure the foal immune response to antigens without the confounding factor of maternally derived antibodies," Felippe says. "Tallmadge's technique measures the immunoglobulin expressed on B cells that is being generated by the foal against that antigen."

Within the immunoglobulin is a "variable" region. This region has specificity to the antigen that is being produced against it. Without the confounding element of the maternal antibodies, Tallmadge can measure the true production of immunoglobulins by the foal when they are still expressed on the surface of the foal's B cells. Not only that, she can measure how well these immunoglobulins are evolving with the boosters.

"With each round of boosters you are stimulating either the same cells or creating new cells that can bind to those antigens, and you are creating what we call diversity in the repertoire of immunoglobulins," Felippe says. "In the end, you are going to have immunoglobulins that can bind to any single small area on that antigen, and in a large amount of them. The efficiency in binding and neutralizing antigens increases with the number of exposures you have to that specific antigen. So we get better immunity as we go along with the boosters."

Tallmadge can measure initially and quantitatively how much of a response a neonate can elaborate, and she can measure the dynamics of the response over time with the boosters, without the technically confounding element of the maternal antibodies.

The development of memory cells also has been quite limited, even in the human literature. Several studies in humans discuss the inability of the infant to respond to vaccines and the maternal antibody interference. "But, very few of these studies address the elaboration of memory cells, which is important," says Felippe. "In some neonatal models of study, neonates are more interested at first in producing memory than they are necessarily in producing a significant amount of immunoglobulin, which is a differentiation of these cells into memory cells instead of plasma cells, respectively. We don't know the result in the foal, but we are studying it. Perhaps there is a bias in the neonate in producing memory, before they can necessarily devote the capacity to producing a high quantity of immunoglobulins. That's why they tend to increase their antibody levels kind of gently in the beginning, and then all of a sudden they spike their antibody production. There is an interesting aspect in the dynamics."

Next steps in research

To summarize, Felippe says her team has plenty of evidence that the horse fetus is well-prepared to face the world and respond to antigens with antibody production. Foals have all the "machinery" to produce antibodies, including elaborated antibodies, those antibodies that have diversity. "It's hit and go," says Felippe. "The foal will be exposed to antibodies when it hits the ground, and it is ready to respond to them."

Now Felippe's team is trying to evaluate the maternal antibody interference phenomenon by using sequences of the variable region of the immunoglobulins expressed on the surface of B cells as a means of, first, initiating a response to vaccination, and, second, determining their ability to elaborate that immune response efficiently.

"So we hope to have some evidence—not confounded by maternally derived antibody levels—that the neonate can indeed respond to vaccinations," Felippe says. "The foal might not respond to everything because some vaccines induce better immunity than others, but it seems that it is our job to find out what makes the 'trick' and take the opportunity that the foals can elaborate an immune response and then vaccinate accordingly."

The American Association of Equine Practitioners offers guidelines for neonatal vaccinations (aaep.org/vaccination_guidelines.htm) that state that it is important to note if a mare has been vaccinated during the last month of gestation. With the information Felippe and her colleagues are gathering, those guidelines could be revised.

In Part 2, the Cornell Equine research team explains two more important factors of the development of the foal's immune system—opsonization and the foal's defenses against R. equi.

Ed Kane, PhD, is a researcher and consultant in animal nutrition. He is an author and editor on nutrition, physiology and veterinary medicine with a background in horses, pets and livestock. Kane is based in Seattle.

References

1. Secor EJ, Matychak MB, Felippe MJB. Transfer of tumour necrosis factor-alpha via colostrum to foals. Vet Rec 2012;170(2):51.

2. Tallmadge RL, McLaughlin K, Secor E, et al. Expression of essential B cell genes and immunoglobulin isotypes suggests active development and gene recombination during equine gestation. Dev Comp Immunol 2009;33(9):1027-1038.