Rhodococcus equi infection: Treatment and immunity in foals

Foals are often exposed to Rhodococcus equi, a common cause of bacterial pneumonia occurring from a few weeks after birth up until 4 to 6 months of age.

"R. equi pneumonia rarely develops in older foals unless immunodeficient or immunocompromised," states a recent study by Dominic R. Dawson, DVM, DACVIM, CCRT, and other researchers from Cornell University's Equine Immunology Lab, which is headed by Maria Julia Felippe, Med. Vet., MS, PhD, DACVIM.1 The group is studying opsonization effect on the outcome of pathogen viability and the development of the immune system to help the foal in its fight. Though foals are affected at an early age, with their innate immunity and the help of a variety of equine practitioners' tools, they have a fighting chance to fend off the disease.

GETTY IMAGESAccording to the study, passive immunoglobulin transfer via colostrum or plasma transfusion could help provide initial protection in the airways before R. equi reaches the intracellular environment and until a foal's own cellular and humoral immunity develops. Neutrophils are important cells of the innate immune system and inhabitants of the upper airways in horses. They're also the first cells recruited to the site of infection and have bactericidal qualities against opsonized R. equi, the group says.1

Phagocytic function in neonatal foals is thought to be important in controlling pathogen expansion and disease development until more complete acquired immunity develops. According to Dawson's study, "foal blood phagocytes are competent with phagocytic capacity and the expression of large amounts of inflammatory cytokines following R. equi infection." But the mechanisms underlying this response are poorly understood by researchers.1

The study investigated the effect of opsonization of R. equi and R. equi-specific antibodies in plasma on bacterial viability and phagocytic activation in a cell culture model of infection. "Results of the study confirmed that opsonization of R. equi with specific antibodies in a commercially available plasma product enhances phagocytic function of neutrophils and macrophages, including increases in oxidative burst activity and TNF-alpha production, and a decrease in bacterial viability," researchers state.1

In the context of the airways, it is possible that enriched plasma containing high concentrations of R. equi-specific antibody may limit the viability of bacteria on the mucosal surface and may increase the intracellular killing by airway phagocytes.

Although neonatal foals are born competent for the production of antibodies, endogenous serum immunoglobulins may take a long time to achieve protective concentrations, according to a study by Rachel B. Gardner, DVM, DACVIM, and other researchers, also at Cornell's Equine Immunology Lab.2 "Therefore, passively acquired maternal antibodies from colostrum play an important role in preventing infection in the initial two to three months of life."2

Opsonization

Phagocytosis is facilitated by the presence of immunoglobulins and complement known as opsonins, which function synergistically. "Adequate and partial colostrum ingestion promotes a positive effect on serum opsonization," according to the Gardner and colleagues study.2 "Complete lack of colostrum ingestion affects opsonization capacity, which was observed in the presuckle serum of control foals in comparison with post-suckle samples. Because sepsis and bacterial infection may result in consumption of opsonins, intravenous plasma transfusion becomes an important component in the treatment of neonatal foals with severe or generalized bacterial infection."2

What is opsonization? There are two major opsonins. One is complement, which is a part of the innate immune system and produced by the liver. Foals are born with low levels of complement, but these levels increase with age. The other critical opsonin is immunoglobulin. Immunoglobulin is produced by the specialized, "acquired" immune system and is either absorbed from the colostrum or produced by the foal with time.

"For a bacterium to be removed by phagocytes, the optimal condition is when it is bound either by complement or immunoglobulin, more efficiently even by both," Felippe says. "That is because phagocytes (i.e., neutrophils, macrophages, monocytes) have receptors for complement and for immunoglobulins. If a bug has an immunoglobulin associated with it, a phagocyte can bind to the immunoglobulin that is bound to it. That helps not only with internalizing the bug but also activates that cell to produce the nasty elements that will then kill the bug."

Neutrophils are very good at doing that. That's their job—to remove all these pathogens. And foal phagocytes have the benefit of functioning optimally from birth. They're also more sensitive to infection than adult phagocytes, as they possess more receptors.

But a conundrum is that the phagocytes are more efficient—and probably most efficient—when opsonins are present. "Without opsonins they are not very good," Felippe says. She goes on to say that the foal's innate immune system can function, but it's really dependent on (acquired) immunoglobulins, which are transferred through the colostrum. And if that transfer doesn't go well, sepsis is likely to occur. However, if there's only a slight environmental challenge, foals can overcome infection and do well, she says.

The Gardner and colleagues study looked at the opsonization capacity and phagocytic capacity of foals admitted to the hospital.2 The foals were divided into groups—those that had a true clinical septicemia and those that did not (sick but not systemically infected foals). There was also a control group of normal foals that were not sick.

The researchers found that phagocytic function of the foals was adequate but low when the foals arrived at the hospital, at the most critical phase of their disease, before antibiotic treatment could help them overcome the infection. The group speculated that the foals' immune systems were trying to keep up with demand and possibly sending out premature infection-fighting cells. However, once an infection is stabilized, even though the demand for an immune response is present, there is time for the cells to become mature and mount a stronger response, Felippe says.

In that transition, when they were really fighting the bugs, their phagocytic function was present but slightly lower than normal, and so was their opsonization capacity. Interestingly, their opsonization capacity was not necessarily correlated with their IgG concentrations. There seems more to it than just IgG concentrations.

"You would expect that there would be a direct association," Felippe says. "But in some foals in which there was enough IgG, opsonization capacity was still reduced in the very beginning of their hospitalization period. Once the plasma therapy and antibiotics were in place, their recovery was quite rapid."

Although IgG is the most important opsonin, other factors in serum seem to be playing a role in opsonization capacity, Felippe says. Complement has an innate dependent development. "Immediately after birth, probably with antigenic challenge, the foal makes the liver produce complement," Felippe says. And since colostrum is not a good source of complement, the foal needs to work at it. In septic foals, the rate of complement level is lower through time than in normal foals, which could be a factor of consumption.

"I would expect that complement is highly used in sepsis, so that there isn't much available circulating as there is in a normal foal," Felippe says. "Complement in concert with IgG work effectively for a robust opsonization capacity. It has opened our eyes to the fact that IgG is important; it plays a major role, but there are other things that are also important to fight certain types of organisms."

Although opsonization is important, the timing of opsonization is probably more important, Felippe says. "That should be well understood when developing strategies for protection. There have been different attempts for the use of vaccines for R. equi and protecting against it, including vaccinating the dam for a better transfer of immunoglobulins," she says. Felippe admits it's a legitimate approach but maintains that it doesn't guarantee 100 percent protection because of the factors discussed above and potential failures in adequate transfer of opsonins to the foal via colostrum. She also points out that innate immunity is dependent on elements received through passive transfer from an acquired immune system, and that both elements must be present at the right time.

A hard look at R. equi

What makes a foal susceptible to R. equi versus resistant to infection? "What we think is that the foal has a window of susceptibility for the organism to penetrate, expand and cause disease," Felippe says.

Felippe says that one key factor in a foal's susceptibility is that R. equi has developed a mechanism that allows it to replicate itself inside macrophages—it's protected from the immune system inside of an immune cell. R. equi also uses macrophages as a means of transportation within lung tissue.

Neutrophils are killers of R. equi if they have the opsonin, particularly immunoglobulin, that is essential for R. equi killing in neutrophils, Felippe says. How can we boost immunoglobulin? "We can give the foal hyperimmune plasma with a lot of immunoglobulin that is against R. equi," Felippe says.

"Others have done that experimentally," she continues. "If you first give the immunoglobulin—high levels of immunoglobulin against R. equi—to foals and then infect them, the foals control infection pretty well. They get infection. They get a little bit of pneumonia, but they do a good job eliminating the organism and resolving disease."

But if you try to apply this in the field, the results of the studies are not as clear, according to Felippe. If specific immunoglobulins are made available before infection, they can bind to the pathogen (opsonize it) before it enters the cell and facilitate active phagocytosis and killing, Felippe says. And that is why some practices perform R. equi-antibody enriched plasma transfusions soon (or immediately) after birth.

Everything in this process is very dynamic. How much a mare transfers R. equi-specific immunoglobulin to a foal can be variable. "If you look in a population of horses, mare A and mare B may have a very different antibody production, colostrum quality and transfer capability to the foal," Felippe says. "And if you would like to even out the efficiency of immunoglobulin transfer across the foals by giving plasma transfusion, you really have to give the plasma early, as soon as the foal is born. You have to give it before the organism gets to the airways, especially in an environment that offers significant exposure to the organism."

But how does the Cornell team's research contribute to the understanding of the role of R. equi-specific immunoglobulins in the protection against this pathogen? Felippe says that the group's paper didn't bring any new information about the importance of IgG.

"Studies in the 1980s showed beautifully the importance of IgG for neutrophil phagocytosis and killing of R. equi," she says. "We repeated some of those experiments to show that our model was detecting the same principles, but what our paper additionally showed is that antibodies were containing bacterial growth extracellularly and intracellularly, suggesting a direct interference with the bacterium. And we also showed that at least one mechanism of macrophage activation was dependent on immunoglobulin specific against R. equi. Our study supports that immunoglobulin specific to R. equi would be beneficial, especially during the early stages of infection, in the very beginning, before the organism has established disease, when it reaches the airways. If you focus the treatment there, there's a good chance not to develop clinical disease."

Macrophages are not good killers of R. equi, so additional immunity is needed to kill cells that are infected, i.e., cellular immunity, Felippe explains. "There have been some studies suggesting that foals have limiting factors for their ability to kill intracellular Rhodococcus," Felippe says. "One of them is a delayed production of interferon-gamma, a cytokine produced by lymphocytes. It has been well-documented that there is a delay in their production by the foal's immune system. However, if they were stimulated, foals could produce interferon-gamma."

Why is interferon-gamma so important? For at least two reasons: "One is that it activates cellular immunity against intracellular pathogens," Felippe says. "Secondly, it activates a more efficient killing system in the macrophages that not only uses oxygen reactive elements, but also nitrogen reactive elements. The two together produce peroxynitrite, which is effective in killing Rhodococcus. But without both, Rhodococcus is not as effectively eliminated. This activation is primarily interferon-gamma induced, and at some level TNF-alpha induced."

In early life, a foal may not have the ability to prevent the disease and fight the infection—that creates the window of susceptibility and allows the disease. After foals are stimulated with infection, they create this immune response, but it still takes time for them to fight the infection and control disease.

"In fact, we now realize, clinically, that a lot of foals acquire Rhodococcus infection and they manage their disease without antibiotic therapy," Felippe says. "With age, they are capable of eliminating the infection without antibiotics. In the field, it is becoming common practice to monitor the disease, monitor the foals and only give antibiotics when the disease becomes overwhelming. It is an interesting understanding about the development of the immune response in the foal."

So how is this stimulation occurring in the airways? "Ute Schwab in our lab created an in vitro system with respiratory epithelial cells of the horse, and she studied the dynamics between the signaling of the epithelial cells and phagocytes in the presence of R. equi," Felippe says.3 "It is beautiful how activation can control infection and how there is a difference between foal macrophages and adult macrophages in this process. So we believe that the airway epithelium signals the presence of R. equi in the environment, attracts and activates resident macrophages, and subsequently neutrophils. It is very possible that, when neutrophils arrive to the airways with appropriate concentration of R. equi-specific immunoglobulin, Rhodococcus removal is efficient and disease can be prevented. Using this system, we also want to learn more about how the adult horse airway epithelium activates macrophages—if that capacity differs from the foal airway epithelium and how that affects R. equi survival."

Conclusion

Many factors are at play in a foal's immunity—from the efficiency of transfer of immunoglobulins and proinflammatory cytokines to the foal via colostrum, the physiologic developmental phases of the immune system, and a foal's ability to respond to antigens, as covered in part 1 of this series, to the timing of presence of essential opsonization to fight R. equi infections in the airways. Further research will continue to illuminate ways practitioners can better foster the health of foals.

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. Dawson DR, Nydam DV, Price CT, et al. Effects of opsonization of Rhodococcus equi on bacterial viability and phagocyte activation. Am J Vet Res 2011;72(11):1465-1475.

2. Gardner RB, Nydam DV, Luna JA, et al. Serum opsonization capacity, phagocytosis, and oxidative burst activity in neonatal foals in the intensive care unit. J Vet Intern Med 2007;21(4):797-805.

3. Schwab U, Caldwell S, Matychak MB, et al. A 3-D airway epithelial cell and macrophage co-culture system to study Rhodococcus equi infection. J Vet Immunol Immunopathol 2013; in press (ePub available).