The insidious invasive verminous antigens of the horse

THE S E C O N D A N N U A L I M M U N O L O G Y S E M I N A R AND R.D. TURK MEMORIAL LECTURE Texas A & M University March 9, 1985

It was a p p r o p r i a t e t h a t Dr. T o m Bello give this T u r k M e m o r i a l Lecture since he was Dr. T u r k ' s first g r a d u a t e student.

THE I N S I D I O U S I N V A S I V E V E R M I N O U S

ANTIGENS

OF T H E H O R S E

T h o m a s R. Bello, D V M , P h D

Within our scope of equine parasitology, we include the viruses, bacteria, fungi, protozoa, helminths and arthropods. These parasites are antigenic, perhaps immunogenic, and if the horse reacts to them, he may vastly improve his health or he may die. Usual reality is a compromise between these extremes. The horse is a logistical challenge as a research model. Verminous parasites of the horse present a number of difficulties for the study of host reaction. Some of these problems are (1) maintenance of parasites in the laboratory experimental a n i m a l ; (2) a n t i g e n i c c o m p l e x i t y o f v a r i o u s developmental stages: (3) and characterization and measurement of the immunity in the horse to its infection. J The foal is born worm-free as there is no prenatal infection. The foal may become infected by Strongloides westeri through the mare's milk starting at 4 days of life, but not through the colostrum. The foal may become infected through the skin as it lies on contaminated soil. Patent infections occur in the first 6 to 10 days in foals, Author's address: Sandhill Equine Center, Southern Pines, North Carolina. Volume 5, Number 3

increasing to high numbers of eggs in the feces by 4 to 6 weeks, then falling rapidly to low numbers or zero by 12 to 15 weeks without anthelmintic treatment. 2 This infection may produce moderate inflammation of the anterior one-third of the small intestine with atrophy of intestinal villi and increased lymphocyte concentration in the lamina propria, particularly surrounding adult worms. After initial infection, foals become refractory to s u b s e q u e n t S. w e s t e r i challenge. In e x p e r i m e n t a l infection in initially worm-free pony yearlings, there was an increase in 13-globulin corresponding to loss of infection, if a foal is dewormed as the infection approaches its peak at 4 weeks of age, the anthelmintic effect is impressive, but if the foal is treated early (at 2 and 4 weeks, then at 4 week intervals) there apparently is r e d u c e d a n t i g e n i c mass to s t i m u l a t e a d e q u a t e inflammatory response and the infection persists. Although the foal does not appear to react to skin penetration by S. westeri larvae, such penetration of human skin rapidly produces an intense inflammatory process in the dermis, with infiltration of lymphocytes and eosinophils and by microscopic necrosis around the parasite? In the hypersensitive human, edema and 163

erythema are prominent features of the area. If the parasite is not arrested by this reaction, it may travel in the dermis, producing streaky red channels of creeping eruption. This infection would be assumed to stimulate lgE and histamines in this abnormal host. Parascaris e q u o r u m is a highly a n t i g e n i c and immunogenic parasite of the young horse. Previously the life cycle of P. equorum was thought to mimic that of Ascaris sumrn in that the infective 2nd-stage larva hatched from the egg within the lumen of the foal's small intestine, with subsequent parasitic molts in the liver and small intestine, but not in the lung. However, based upon experimental infection of P. equorum in foals, Clayton and Duncan 4 stated that they were not able to determine if molts had occurred in the intestinal submucosa, liver or lungs by day 14 of infection as the larvae all appeared similar morphologically, except for size. Between days 14 and 23 ascarids in the small intestine of foals molted into the L5 (early adult) stage. By day 14 there began a division into 2 populations related to size; the smaller larvae were in the respiratory tract, the larger larvae had reached the small intestine. By day 23 there was a population of small larvae within the lung parenchyma and of larger larvae in the small intestine, but none within the respiratory tract. It was supposed that the smaller larvae were the target of an immunological response leading to their destruction. Within the small intestine, the Parascaris grew rapidly with the largest numbers of intestinal parasites present by day 37. From day 45 onward there was a steady expulsion of the worms in feces. This was attributed to sensitization of foals to molting fluid produced during the last two parasitic molts. The separation of a single challenge experimental infection into different larval populations is of practical significance for explaining differences in effects of anthelmintic treatment. We conducted a controlled trial of ivermectin injectable or paste vs. nontreated controls in pony foals each experimentally infected with 10,000 P. equorum embryonated eggs. There was a distinct difference in drug effect against 28-day-old infections by the two formulations of ivermectin. The injectable formulation was 99.9 percent and 47.7 percent effective against larger and smaller immature larvae, respectively. The ivermectin oral paste formulation was 99.9 percent and 97.3 percent effective against larger and smaller larvae, respectively. We c o n d u c t e d a s t u d y of response to n a t u r a l acquisition of Parascaris infections in Quarter Horse foals for the first 52 weeks of life. 5 Infections in foals started becoming patent at l 0 weeks, peaking at 19 and 23 weeks, then decreasing steadily by 32 weeks, fluctuating moderately thereafter but continuing to decrease. The mares did not have ascarid eggs in the feces. Parascarid whole-worm antigen was used to react against sera from mares and foals. In foals, average precipitin titers increased gradually from a nonspecific reaction in the sample collected at birth before nursing to a titer of 24 by 5 to 8 weeks, maintaining these levels to the 20th week, thereafter rising sharply to week 28. The titers continued to climb as the yearlings developed an active protective immunity. The mares, resistant to the daily challenge by 164

Parascaris had continuously high titers throughout the year. When we found that young horses developed a strong resistance to natural Parascaris infections, the next step was to attempt to improve on that protection. We gave embyronated Parascaris equorum eggs attenuated with 100 K r of Co 6° to three groups of worm-free pony foals in two immunizing oral doses of 50,000 and 100,000 eggs at 10 day intervals. The challenges of 10,000 normal embryonated eggs were given 10 days later. A fourth group was given irradiated eggs only and a fifth group was given 10,000 normal eggs only as controls. The ages of the. foals at the time of initial immunization was day of birth, day 21 or day 42. Foals were euthanatized and necropsied for a count of adult worms at 80 days from the final dose in each group. The foals in groups given irradiated eggs were protected 67 percent (49 percent - 77 percent) based on numbers of worms in comparison with infected controls which had massive and fatal impactions. Attenuation of the embryonated eggs by Co 6° irradiation was 98 percent effective when compared with controls. We conducted a study on the development in ponies of experimental populations of normal and irradiated Gasterophilus intestinalis larvae. Twenty-five bot-free pony foals and 5 naturally infected mares were given single or multiple experimental exposures of normal and / or Co6~-irradiated Gasterophilus intestinalis first-instar larvae. The recovery percentages and sizes of second instars from the initially bot-free foals were similar to those from the mares, indicating that previous n a t u r a l i n f e c t i o n did not s t i m u l a t e resistance to subsequent experimental infection. However, recovery percentages of bots from a first experimental exposure in 10 bot-free foals exceeded recovery percentages from the 2nd exposure by 2.8 times. In= contrast', recovery percentages of bots from a first exposure by irradiated (128 and 192 Kr) first instars were 0.4 and 0.5 times as great as recovery of normal larvae from the second exposure. Sizes of second instars from second exposure in the several groups were similar, indicating that the radiation-attenuated larvae of the first exposure did not stimulate the hosts to inhibit growth of larvae from the second exposure. However, the high levels of radiation altered the orientation of the larvae so that they migrated into the gingiva of the dorsal premolars. Researcher's anecdotes have indicated that horses injected intravenously with saline-extracted Strongylus vulgaris w h o l e - w o r m a n t i g e n p r o m p t l y died in anaphylactic shock; other horses injected intradermally with Parascaris equorum antigen for skin tests likewise "dropped off the end of the needle". Our controlled research demonstrated that a drug may be highly effective but it can be lethal if the responsiveness of the horse is not considered from all aspects. We conducted a 60 animal trial to demonstrate effectiveness of ivermectin injectable or oral paste against migrating Gasterophilus intestinalis. 6 The manufacturer requested that the drug be given to one group by intravenous injection to compare with intramuscular injection. There were 2 ponies in this trial that had immediate EQUINE VETERINARY SCIENCE

adverse reactions following intravenous injection of vehicle,or of ivermectin. One pony was injected with 3.4 ml of vehicle, and then walked about 3 m toward her accustomed stall. She reared, became unbalanced and fell in a saw-horse stance on her right side. Her gingivae were pale, her breathing was in rapid pants and heart rate was elevated. After 5 minutes, she rolled onto her sternum, stood, and was led back into her stall as though there had been no problem. Another pony was given an intravenous injection of 1.3 ml of ivermectin. After one minute she staggered, whinnied repeatedly, then fell. The gingivae were blanched; she breathed deeply once, and died in 6 min. in spite of attempted resuscitation. Necropsy examination at one hour after death indicated gross lesions of acute anaphylactic shock which was confirmed by histopathologic examination. The intestinal tract was covered with lesions from ruptured capillaries throughout the large colon, and in the mesenteric vessels of the ileum and jejunum, and with congestion of stomach vessels. The spleen surface was covered with petecchial hemorrhages and the liver had a dark mottled purple-tinged cast of congestion. There was a harsh reddish appearance of the medulla of the left kidney. There were petecchial hemorrhages throughout the surface of both lungs. The right ventricle of the heart was dilated, a typical sign of anaphylaxis. The vehicle of i v e r m e c t i n injection for horses contained 120 mg of polysorbate 80 (Tween 80) per ml, according to the manufacturer. All emulsifying agents or stabilizers are likely to be toxic to a certain extent. Meng and Freeman reported that intravenous injections of emulsifying agents oft.he Tweens group in dogs produced dilatation of blood vessels, fall in blood pressure, urticaria, urination, defecation, and vomiting, v These and other workers proposed that the mechanism of the adverse reaction may be due partly to histamine f o r m a t i o n / T h e s e reactions could be prevented in dogs by subcutaneous injection of epinephrine 5 to l0 minutes prior to injection of Tween 20. There were no adverse reactions by the ponies to the ivermectin oral paste nor to the intramuscular injection site in our work and that of others. The association of worm larvae with aneurysm of horses was known to the Romans. The infection of the horse by Strongylus vulgaris may result in arterial and intestinal inflammation for 6 months before the specific patent infection can be confirmed by larval culture. Clinical signs of S. vulgaris infection may be confused with bacterial infection. Experimental infections of infective larvae given to worm-free ponies revealed three phases of normal migration of S. vulgaris larvae: migration to the cranial mesenteric artery, development at the predilection site, and return migration to the intestinal lumen/ According to these experimental results, ingested infective larvae exsheath and penetrate the mucosa and submucosa of the small and large intestine 1 to 3 days after infection and molt to the fourth stage by day 7. These larvae then p e n e t r a t e into the l u m e n s of submucosal arterioles, along which they migrate against the flow of the blood. They reach the cranial mesenteric Volume 5, Number 3

artery by day 21. They are only I to 2 mm long at that time, but increase in size considerably during the next 3 to 4 months, becoming 10-18 mm long and usually molted to the fifth stage. The retained fourth-stage sheath is discarded before the larvae begin their return migration to the intestine within the lumens of the intestinal arteries. When the larvae arrive at the serosal surface of the intestine, still within the arteries, pea-sized nodules form around the worms and then rupture into the lumen of the large intestine, releasing the early adult parasites. These worms require another 6 to 8 weeks to mature and begin reproduction. Thus, the prepatent period is 6 to 7 months long. In 1965 we studied the in vitro response to Strong.vlus vulgaris larvae. The parasitic t h i r d - s t a g e larvae, suspended in nonimmune and hyperimmune rabbit serums (prepared against fifth-stage S. vulgaris wholeworm antigen) for 6 days prior to molting, produced no in vitro precipitin reactions in hanging-drop cultures, although the hyperimmune serum produced a strong tube precipitin reaction with the fifth-stage whole-worm antigen. However, Tyrode's solution-extracted somatic antigen of fourth- and early fifth-stage S. vulgaris recovered from aneurysms of the cranial mesenteric artery, produced strong positive tube precipitin reactions when tested against serums from horses naturally infected by this parasite. This suggested that the fourthstage larvae, which stimulate development of the verminous arteritis, may possess stronger antigenic characteristics than the third stage. Then, S. vulgaris late fourth-stage larvae, removed from aneurysms of the cranial mesenteric artery, were maintained in vitro for 11 weeks, transfers of the Medium 199 supplemented with either calf or horse serum being made weekly. Molting to the fifth stage occurred between the third and fifth weeks. The metabolic antigens containing molting fluids resulted in stronger precipitin reactions with 74 percent of the positive serums from 38 horses than did metabolites of the larvae after molting. To determine if the horse reacted to a specific antigenic stage of S. vulgaris, larvae were maintained in Medium 199 supplemented with serum from a horse with a large palpable aneurysm of the cranial mesenteric artery and with clinical signs of verminous colic. In these cultures, precipitates were produced around the body orifices and on the cuticle of the fourth-stage larvae, but the fifthstage larvae primarily produced a field precipitate in the medium. The cuticular precipitates on the fifth-stage larvae were in locations similar to those of attached fibrin tags when the larvae were removed from the aneurysms. These larvae were washed in sterile Tyrode's solution and the cuticular precipitates removed with a sterile transfer needle before replacing the Medium 199-20 percent calf serum. Larvae maintained in the medium containing calf serum did not produce precipitates, but did so when replaced into the medium containing serum from the colicy horse. These preliminary results suggest a local reaction to s u r f a c e antigens by the host in the development of a verminous aneurysm. We studied the host response to experimentally induced infections of S. vulgaris in worm-free and naturally infected ponies. ~0 The worm-free ponies had 165

more adverse reaction to 5,000 infective larvae than did the naturally sensitized ponies given similar doses, based on several clinical parameters examined. The B-globulin changes may have been formed in response to parasitic metabolic or somatic antigens or to both. Changes in 13globulin have been reported from infections of S. vulgaris. Strongyloides westeri, and small strongyles. The naturally infected ponies may have had prolonged sensitization, when given an additional experimental infection resulting in lack of 13-globulin increase. Damage to the equine arterial system resulting from S. vulgaris infections has been shown to lead to conditions resembling arteriosclerosis in man. If initial damage to the equine arterial system by S. vulgaris larvae causes a l t e r a t i o n of the a r t e r i a l i n t i m a , t h e n s o l u b l e glycoproteins could be readily released into the blood. The primary damage to the endothelium is an arteritis. The increased concentrations of 13-glycoprotein occurred at the time the fourth-stage larvae were growing in size, metabolizing, and becoming embedded in host-response in the cranial mesenteric artery and branches. Several host-parasite models have been used to determine the effects of immunosuppressants on altering the host resistance to helminth or bacterial infections. Methotrexate, a folic acid antagonist which suppresses both antibody production and delayed hypersensitivity reactions, has been used in guinea pigs given diphtheria toxoid or Mycobacterium tuberculosis, in cattle infected with M. paratuberculosis or Dermatophilus congolensis, in Swiss albino mice injected with Schistosoma mansoni eggs, in Chinese hamsters injected with Trichinella spiralis and in dogs infected with Toxocara canis. ~j Various degrees of methotrexate toxicosis in the several host species have been reported. We had no information on how this drug would affect the Equidae. Both wormfree and naturally infected ponies were given 5,000 Strongylus vulgaris infective larvae. Members of each group were either given daily injections of methotrexate or not treated. By day 2 methotrexate-treated ponies became anorectic with constipation or diarrhea. On day 3, there was a steady reduction in total leukocytes, mainly due to sharply decreasing numbers of neutrophils. By day 9, the entire leukocyte population was lymphocytes. Time of death ranged from 9 to 11 days. Gross lesions produced by methotrexate toxicosis were seen in gastrointestinal tracts, mesenteric lymph nodes, adrenal glands and livers. There was an absence of intestinal mucosa (thus eliminating the protective barrier of the intestinal epithelium); lymph nodes had a loss of germinal centers (explaining immunocellular depressant effects), with medium-sized lymphocytes filling the sinusoids; there was atrophy of the zona fasciculata of the adrenal gland and marked vacuolation of hepatocytes. P o n i e s that were given S. vulgaris had extensive gastrointestinal hemorrhages. The profound immunosuppressant effects of methotrexate appeared to potentiate the early invasion of Strongylus vulgaris. Many horses have recurrent colic despite rigorous anthelmintic therapy and good management. In most cases, colic is most obvious about 3 to 4 weeks after deworming with synergistic compounds. In addition, some horses have been given larvicidal doses of 166

anthelmintics without complete amelioration of the syndrome. Satisfactory management of these cases has been with diethylcarbamazine citrate given daily in the feed at 6.6 m g / k g of body weight. 12 Treatment is usually continuous for 6 months. Some horses may have colic 10 to 14 days after treatment ceases; these resume daily medicine. Some horses have been treated daily in feed for 5 to 7 years. This treatment may provide continuous exposure of strongyle larvae in the intestinal wall to the DEC, thus specifically blocl~ing the production of leukotrienes (SRS-A) to parasitic molting fluids and metabolites. The natural sequence to measuring host response is to improve on that response as a means of protection. In a recent study, Klei, et al. vaccinated 9-to-12 months old worm-free ponies with third-stage Strong.vlus vulgaris larvae that had been irradiated with 70, 100 or 130 Kr of g a m m a radiation, t-~ Ponies given an oral inoculation of larvae irradiated with 70 or 100 Kr were protected from the clinical disease and lesions associated with challenge infections of 4,300 third-stage larvae when compared to nonvaccinated controls. The numbers of worms from the challenging population recovered from successfully vaccinated animals were significantly lower than from nonvaccinated controls. The authors believed that the d e g r e e of p r o t e c t i o n t h a t d e v e l o p e d c o u l d be semiquantitated, based on clinical and pathological response. Klei, et al. used an indirect fluorescent antibody assay (IFA) with Strongylus vulgaris parasitic third-stage larvae (L0 as antigens, j4 These tests indicated that a species-specific antibody response to S. vulgaris L 3 develops in S. vulgaris-infected ponies and that some surface LI antigens are shared by adult worms. Sequential antibody levels against S. vulgaris were measured in strongyle-free and in immune ponies following initial and challenge infections using the IFA and an indirect h e m a g g l u t i n a t i o n a s s a y ( I H A ) . A n t i b o d y levels measured by IFA increased faster following initial infections than did levels measured by IHA. Antibody levels appear to increase with IFA only following challenge infections of immune ponies. There were no real differences in antibody titers between colicy ponies and those that did not develop colic after challenge infections. Antibodies were not produced in initially worm-free ponies that had S. vulgaris adults surgically implanted into the cecum. The spirurid worms Draschia, Habronema live in the glandular portion of the stomach. Draschia lesions in the stomach result in excessive granulation and abscess. Embryonated eggs are passed in feces and are eaten by fly maggots. Larvae reach the infective stage within the house or stable flies which are intermediate hosts. Larvae may invade eyes, causing persistent conjunctivitis. Pulmonary abscesses result when they invade the lungs. These parenteral invasions of larvae are considered aberrant because only the larvae gaining direct access to the stomach complete their development. " S u m m e r Sores" are the clinical entities of cutaneous habronemiasis, resulting from excessive host reaction to skin invasion by the infective larvae. Generally only a few EQUINE VETERINARY SCIENCE

larvae are present in the average lesion, so their absence in a p a r t i c u l a r b i o p s y s a m p l e does n o t rule o u t habronemiasis. A toxic action has been p o s t u l a t e d for the necrosis a n d marked hypervascular g r a n u l a t i o n , but it is likely that a local hypersensitivity reaction from reinfection is the basic cause. 15 Local eosinophilia a n d regression of the lesions d u r i n g winter when the i n t e r m e d i a t e hosts cease their activity support this idea. It has been suggested also that the presence of mature H a b r o n e m a in the s t o m a c h may induce a state of general hypersensitivity. Certain horses seem to be more susceptible a n d have lesions year a f t e r year. Horses so affected are almost always heavily parasitized by the adult worms. Ivermectin oral paste is the c u r r e n t t r e a t m e n t o f c h o i c e a g a i n s t c l i n i c a l habronemiasis. The most c o m m o n cause of pruritic alopecia is a n allergic dermatitis caused by hypersensitivity to the bite of any of several species of C u l i c o i d e s biting gnats. 15This is called " Q u e e n s l a n d itch" in Australia, "sweet itch" in England and Ireland, "summer itch" and other u n m e n t i o n a b l e names in the United States, especially when c o n c e r n i n g t h i n - s k i n n e d horses at t h e peak of s u m m e r show season. So me species of gnats may feed on the ventral aspect of the horse; others may c o n c e n t r a t e on the m a n e a n d tailhead. The bite of the C u l i c o i d e s produces a n i m m e d i a t e sharp pain a n d irritation, followed by a red wheal. The intense irritation reportedly lasts for I to 3 weeks. The reaction is cumulative. It is suspected that in the horse, the insect allergen (lgE) reaction causes mast cell d e g r a n u l a t i o n with subsequent release of histamine,SRS-A (leukotriene), bradykinin and proteolytic enzymes. These substances produce the clinical skin disease. We have been treating horses with this c o n d i t i o n o n a repeated a n n u a l basis by the in-feed daily dosage of d i e t h y l c a r b a m z i n e citrate at 6.6 m g . / k g . Some show horses h a v e required c o n t i n u o u s medication from midMarch to m i d - N o v e m b e r . Other horses were refractory to t h i s t r e a t m e n t , b u t r e s p o n d e d t o l o w - l e v e l d e x a m e t h a z o n e or prednisone. All horses required a d d i t i o n a l stabling d u r i n g hours of intense gnat feeding. A n excellent up-dated discussion of C u l i c o i d e s bite hypersensitivity is presented in E q u i n e M e d i c i n e a n d Surgery.

The clinical signs of onchocerciasis are similar to that of C u l i c o i d e s bite allergy. Since C u l i c o i d e s is the vector of the filarial parasite O n c h o c e r c a c e r v i c a l i s the pruritis may be due to both the C u l i c o i d e s a n d to metabolic antigens of O n c h o c e r c a microfilarie within the dermis. The disease can be cumulative in that a pruritic horse m a y scratch, killing microfilariae which release more antigen. lvermectin oral paste at 200 m c g / k g is 99.9 percent effective against O n c h o c e r c a microfilariae. There is some circumstantial evidence that the drug may be effective

Volume 5, Number 3

also against the a d u l t parasites in the ligaments of the neck. The horse h a v i n g clinical signs at time of treatment is supported by low doses of d e x a m e t h a z o n e (10, 10, 5, 5, r a g / d a y for 4 days). For several years d i e t h y l c a r b a m azine citrate given in feed at 6.6 m g / k g for 21 days or longer has been o u r t r e a t m e n t of choice. Rarely, we have found that a horse would not respond to the D E C treatment, based on biopsy e x a m i n a t i o n . The horse then would be given levamisole at 3.5 m g / k g for 3 consecutive days, then not treated for 4 days, and then this schedule repeated for 4 to 6 weeks. If the infection had not been removed, the horse would be given D E C again, usually resulting in p r o m p t clearance of microfilariae from the tissues, in these rare cases, we assumed that the horse was i m m u n o s u p p r e s s e d , allowing the infection to persist. Then the levamisole acted as an i m m u n o e n h a n c e m e n t medium, resulting in effective c h e m o t h e r a p y in a horse now responsive to DEC.

REFERENCES 1. Wakelin D: Immunity to Parasites. How Animals Control Parasite Injections. 165 pp. Edward Arnold, Baltimore MD 1984. 2. Greet GJ, Bello TR, Amborski GF: Experimental Infection of Strongyloides westeri in Parasite-Free Ponies. J Paras# 60:466-472,

1974. 3. Roeckel IE, Lyons ET: Cutaneous Larva Migrans, an Occupational Disease. An Clin and Lab Sci 7:405-410, 1977. 4. Clayton HM, Duncan JL: The Migration and Development of Parascaris equorum in the Horse. Inter J Paras# 9:285-292, 1979. 5. Bello TR, Amborski, GF, Torbert BJ: Practical Equine Parasitology Based Upon Recent Research. Proc Am Assoc Equine Practnr 97, 1974. 6. Bello TR: Controlled Critical Evaluation of lvermectin Given by Intravenous or Intramuscular Injection or as an Oral Paste Against Migrating First-lnstar Gasterophilus intestinalis Resulting From Experimental Infection in Ponies. Am J Vet Res submitted from publication. 7. Meng HC, Freeman S: Experimental Studies on the Intravenous Injection of a Fat Emulsion in Dogs. J Lab and Clin Med33:689-707, 1948. 8. Krantz Jr JC, Culver PJ, Carr CJ, Jones CM: Sugar Alcohols XX VIII. Toxicologic, Pharmacodynamic and Clinical Observations on Tween 80. Bull Seh Med U MD 36:48-56, 1951. 9. Duncan J L, Pirie H M: The LifeCycleof Strongylus ~ulgaris in the Horse. Res Vet S~q 13:374-379, 1972. 10. Amborski GF, Bello TR, Torbert BJ: Host Response to Experimentally Induced Infections of Strongylus vulgaris in ParasiteFree and Naturally infected Ponies. A m J Vet Res 35: I 181-1188, 1974. II. Bello TR, Amborski GF, Torl~ert BJ: Effects of the lmmunosuppressant Methotrexate in Ponies. Am J Vet Res 34: 12911297, 1973. 12. Bello TR: Endoparasitism, in Equine Medicine and Surgery, Third Ed, 67-85 Am Vet Pub Santa Barbara CA, 1982. 13. KleiTR, Torbert BJ, Chapman MR, Ochoa R: Irradiated Larval Vaccination of Ponies Against Strongylus vulgaris. J Parasit 68: 561569, 1982. 14. Klei TR, Chapman MR, Torbert BJ, McClure JR: Antibody Responses of Ponies to Initial and Challenge Infections of Strongvlus vulgaris. Vet Paras# 12:187-198, 1983. 15. McMullan WC: The Skin, in Equine Medicineand Surgery Third Ed 789-843 Am Vet Pub Santa Barbara CA, 1982. -

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