Research Focuses on the Nature of Protective Immune Responses in Horses

Thomas R. Klei, PhD
Boyd Professor, Department of Veterinary Microbiology & Parasitology
and Department of Veterinary Science

Vaccination remains the most cost effective strategy for preventing infection. Since the immune system will respond to both live and killed pathogens, it is possible to stimulate immunity without causing infection. Indeed, this is the basis of attempts to vaccinate individuals. While this appears to be a rather obvious approach, it does not always yield the expected result. Why some vaccines work and others fail is a complex issue. Since the stimulation of an appropriate immune response is critical, current research at LSU has focused on identifying the nature of the protective immune responses in ponies to the intestinal parasite Strongylus vulgaris and equine influenza virus. These studies are being conducted in the laboratories of Drs. David W. Horohov and Thomas R. Klei. This work is being supported through grants from the Equine Veterinary Research Program, Grayson Jockey Club Research Foundation, Inc., several pharmaceutical companies, and a USDA National Research Initiative grant awarded to Drs. Horohov, Klei, Wayne Taylor and Rustin Moore.

Strongylus vulgaris

Strongylus vulgaris is considered the most pathogenic nematode parasite of horses due to the severe damage it causes in the cranial mesenteric artery during larval migration. Infective third-stage larvae (L3) ingested from contaminated pasture penetrate the mucosa of the large intestine, molt to fourth-stage larvae (L4) in the submucosa, and then proceed along arterioles and arteries that supply the intestine to the root of the cranial mesenteric artery. Once there, larvae molt to become immature adults causing severe arteritis before returning, again via the vasculature, to complete their life cycle in the large intestine. Arterial lesions include the formation of tortuous subintimal tracts, thrombi, and in severe cases verminous aneurysms that can compromise perfusion of intestinal vascular beds. This syndrome, known as verminous arteritis or thromboembolic disease, is characterized by ischemic infarctions of the bowel which result in toxemia, abdominal pain, and death in severe cases.

Previous studies in Dr. Klei's laboratory have demonstrated that oral vaccination with irradiated S. vulgaris L3 induces resistance to challenge infection and prevents classical lesions of verminous arteritis. This protection observed correlated to both prechallenge anti-S. vulgaris antibody titers specific for surface antigens of late L3 stages and to the induction of a post challenge anamnestic-like eosinophilia. Infections by S. vulgaris have been shown to activate eosinophils and neutrophils in vitro. Eosinophils from S. vulgaris primed but not unprimed ponies kill S. vulgaris L3 in vitro in an antibody-dependent manner. These findings indicate that an antibody-dependent phenomenon involving eosinophils may contribute to the resistance seen in immune ponies.

While ponies receiving a parenteral vaccination containing killed worm L3 in adjuvant also generated strong antibody responses, the vaccine failed to stimulate a protective immune response and instead caused increased pathology. Though both vaccines induced immune responses in the ponies, it was clear that there were differences in the type of immune response being generated. To determine what these differences are, Dr. Horohov and his colleagues have utilized state-of-the-art molecular biology techniques to evaluate gene expression in these ponies. Lymphocytes (white blood cells) were collected from the ponies before and after vaccination and challenge with S. vulgaris, and some of the genetic material (messenger RNA) was isolated from these cells. Messenger RNA (mRNA) contains the information necessary for the cells to make various proteins. Using a technique called reverse transcription-polymerase chain reaction (RT-PCR), it is possible to determine what proteins the mRNA is specifying. By comparing the mRNA of ponies receiving the two different vaccines, Dr. Horohov and his colleagues can identify any differences between them. Lymphocytes collected from ponies receiving the irradiated vaccine contained mRNA for proteins called interleukin-4 (IL-4) and IL-5. These proteins, also called cytokines, play a central role in regulating immune responses. In particular, IL-4 induces the production of a certain type of antibody called IgE. These IgE antibodies are thought to play an important role in immunity to intestinal parasites. The other protein, IL-5, plays an important role in regulating eosinophil production. Lymphocytes collected from ponies receiving the killed L3 vaccine produced mRNA for a different protein, gamma-interferon. Unlike IL-4, gamma- interferon prevents the production of IgE antibodies and stimulates instead the production of IgG antibodies. While these IgG antibodies play an important role in immunity to viruses and other pathogens, they fail to provide protection against parasites. These results indicate that protective immunity to S. vulgaris is associated with lymphocyte production of IL-4 and IL-5 and pathology is associated with the production of gamma-interferon. While similar results have been seen in parasitic diseases of other animals, this was the first demonstration of this type of response in the horse.

Equine Influenza Virus

While the production of gamma-interferon was not associated with protection against S. vulgaris, this was not the case with equine influenza virus. Equine influenza virus remains a persistent management problem for the horse owner. This is due, in part, to the short-lived protection offered by the commercially available vaccines. Previous work has shown that horses require frequent boosting with the vaccine to maintain levels of protection. Interestingly, horses that recover from an influenza infection are protected against re-infection for much longer periods of time. This suggests that the immune response to the vaccine is different from the immune response generated following an infection. To address this question, Dr. Horohov's laboratory has used the same RT-PCR procedure to compare the immune responses of influenza-vaccinated versus infected ponies. Here their attention was focused on gamma-interferon since its production is known to be important for immunity to this virus. As expected, lymphocytes collected from ponies infected with equine influenza virus had high levels of gamma-interferon mRNA whereas those lymphocytes collected from vaccinated ponies did not contain mRNA for gamma-interferon and instead had elevated levels of mRNA for IL-4.

Both of these studies indicate that protective immunity depended upon the production of certain cytokines, either IL-4 and IL-5 or gamma-interferon. Furthermore, the role each cytokine played was dependent upon the disease. This apparent dichotomy in cytokine responses also extends to non-infectious diseases. Human asthma is associated with the production of IgE antibodies and the presence of IL-4 mRNA in the lymphocytes from asthmatic individuals. Horses suffering from chronic obstructive pulmonary disease (COPD) share certain characteristics with human asthmatics. Could IL-4 be playing a similar role in COPD? Ongoing research at the School of Veterinary Medicine involving Dr. Horohov and Dr. Ralph Beadle is trying to answer this question. Dr. Beadle has identified a COPD-like condition in Louisiana horses called summer pasture-associated obstructive pulmonary disease (SPAOPD). Dr. Horohov is currently using the RT-PCR procedure to determine if horses affected with SPAOPD have elevated levels of mRNA for IL-4. Information gained from these studies would be useful in identifying the causative agent for SPAOPD and could lead to better treatments in the future.