Melanie R. Chapman

Immunity in equine cyathostome infections

Defining the characteristics of immunity and immune responses to equine cyathostome infections is clearly important to advancing our understanding of the development of these nematodes within the host, the clinical conditions attributed to them, and in developing more rational and novel strategies for their control. Nonetheless, little is currently known on this topic. Current data based on field observations, worm burdens and fecal egg counts suggest that horses acquire a resistance to cyathostome infection with age. This response is slow to develop and incomplete in that most horses regardless of age harbor significant populations of these nematodes. More convincing evidence has been obtained from experimental infections which indicate that mature horses previously exposed to infection are resistant to re-infection and this resistance is directed at all stages of the parasite life cycle. Further, some immunity against the developing stages within the mucosa appears to require less exposure and occurs in younger animals. Some non-specific events which induce expulsion of all species of lumenal dwelling nematodes also appear to take place post-infection with L3. Antibodies have been detected in limited studies against somatic extracts of adult worms. Not surprisingly, titers of these antibodies do not correlate resistance to re-infection. Serendipitous observations have, however, associated a greater expression of the gene for IL-4 with the spontaneous expulsion of lumenal parasites. The development of a usable model is required to further advance our knowledge in this area.

A SURVEY IN LOUISIANA OF INTESTINAL HELMINTHS OF PONIES WITH LITTLE EXPOSURE TO ANTHELMINTICS

Ponies reared with minimal or no exposure to anthelmintics were surveyed for intestinal helminths in order to estimate prevalence and intensity of parasite populations unaltered by frequent exposure to anthelmintics. Thirty-seven mixed breed ponies of varying ages were examined. Thirty-four species of nematodes and 2 species of cestodes were found. Twenty-four of the nematode species (including 1 new species) were in the subfamily Cyathostominae (small strongyles). Eighty-seven percent of the total burden of adult small strongyles in the large intestine was composed of 10 species. By comparing the results of the present survey with those of recent surveys of horses from herds which had been subjected to treatments with anthelmintics, the effect of prolonged usage of anthelmintic treatment on the prevalence of individual species possibly can be estimated. The general ranking of the 10 most common cyathostome species was similar to those described in recent surveys of horses, suggesting that anthelmintic pressure does not affect the prevalence of most cyathostome species. The lack of anthelmintic treatment appeared not to affect prevalence rates for Anoplocephala perfoliata and Anoplocephala magna when compared to other studies. Conversely, prevalence rates for Strongylus spp., Triodontophorus spp., Craterostomum acuticaudatum, Oxyuris equi, and Parascaris equorum were higher than those reported for these species in recent studies of horses.

GASTROINTESTINAL HELMINTHS OF PONIES IN LOUISIANA: A COMPARISON OF SPECIES CURRENTLY PREVALENT WITH THOSE PRESENT 20 YEARS AGO

A survey in Louisiana of gastrointestinal helminths recovered at necropsy from 117 ponies with minimal exposure to anthelmintics between 1989 and 2000 is compared with a survey conducted 20 yr earlier in the same region. An objective of this study was to determine whether species diversity has been affected by the advent and use of the macrocyclic lactone (ML) parasiticides and by the increased anthelmintic pressure on the helminth species infecting the general equine population. Twenty-six cyathostome species and 8 strongyle species were recovered. Two cyathostome species that were not found before, Cylicostephanus asymetricus and C. bidentatus, and 1 species of large strongyle, Oesophagodontus robustus, were added to the list of species found in Louisiana. All cyathostome and large strongyle species found previously were still present. But prevalences and intensities were significantly reduced for almost all large and small strongyle species. Prevalences and intensities of Oxyuris equi adults and larvae were reduced, whereas the prevalence of Parascaris equorum remained constant. The tapeworm Paranoplocephala mamillana was added to the list of parasite species found in Louisiana. Anoplocephala perfoliata remained the most common cestode. This species was found at the same level of intensity but increased slightly in prevalence. Anoplocephala magna was found less frequently than previously. The overall diversity of species remained relatively unchanged. The reasons for the differences in intensity and prevalence of strongyles between these 2 periods are unknown but might be related to the development and use of the broad-spectrum ML anthelmintics in the intervening period, a difference in the population of equids surveyed, different techniques used to identify the parasites, or differences in numbers of parasites identified (or to all).

Re-evaluation of ivermectin efficacy against equine gastrointestinal parasites

Two trials were conducted to confirm the efficacy of ivermectin paste against endoparasites of horses. In these trials, 20 ponies were treated with ivermectin oral paste at 200 mcg x kg body weight once on Day 0, and 20 ponies served as unmedicated controls. The animals carried naturally acquired parasite infections as confirmed by pretrial fecal examination. The animals were necropsied for worm recovery on Days 14, 15 or 16. Parasites recovered were identified to species. Horses treated with ivermectin had significantly (P<0.05) fewer (>99.0% reduction) adult small strongyles (Coronocyclus spp including C. coronatus, C. labiatus, C. labratus; Cyathostomum spp including C. catinatum, C. pateratum; Cylicocyclus spp including C. ashworthi, C. elongatus, C. insigne, C. leptostomum, C. nassatus, C. radiatus; Cylicodontophorus bicoronatus; Cylicostephanus spp including C. asymetricus, C. bidentatus, C. calicatus, C. goldi, C. longibursatus, C. minutus; Gyalocephalus capitatus; Parapoteriostomum spp including P. euproctus, P. mettami; Petrovinema poculatum; Poteriostomum spp including P. imparidentatum, P. ratzii) and adult large strongyles (Strongylus edentatus, S. vulgaris; Triodontophorus spp including T. brevicauda, T. serratus; Craterostomum acuticaudatum) than the controls. Ivermectin was also highly effective (94% to >99%, P<0.05-0.01) against Gasterophilus intestinalis larvae, Habronema spp., Oxyuris equi, Parascaris equorum. The data from these two trials confirm that ivermectin paste administered to horses orally at 200mcg x kg(-1) continues to be highly effective for treatment and control of a broad range of small and large strongyle species as well as other species of gastrointestinal parasites.

Efficacy of the nematophagous fungus Duddingtonia flagrans in reducing equine cyathostome larvae on pasture in south Louisiana.

The effectiveness of Duddingtonia flagrans in reducing the free living third stage larvae (L(3)) of equine cyathostomes on pasture when fed to horses has been demonstrated in cold temperate climates. The objective of this experiment was to assess the efficacy of D. flagrans against equine cyathostomes in the subtropical environment of southern Louisiana. Fecal pats were prepared by mixing feces obtained from a parasite-free horse fed D. flagrans at a dose of approximately 2 x 10(6) spores kg(-1), with feces containing cyathostome eggs from a parasitized horse. Control pats contained feces from a parasite-free horse mixed with feces containing cyathostome eggs. The fecal pats were placed on pasture in six replicates at 4-week intervals from March 1997 until January 1998. Comparison of recoveries of L(3) from non-treated control pats in the field with non-treated coprocultures maintained in the laboratory indicated that L(3) survival on pasture was reduced during the months of May, June, July, August and September. The efficacy of the fungus was determined by L(3) recovery from grass surrounding the fecal pats of treated and control groups. D. flagrans significantly reduced L(3) during the months of April, May, and October 1997 to January 1998 (range 66-99% reduction, p=0.0001), and for the year as a whole (p=0.0001).

PREVALENCE OF STRONGYLE NEMATODES IN NATURALLY INFECTED PONIES OF DIFFERENT AGES AND DURING DIFFERENT SEASONS OF THE YEAR IN LOUISIANA

One hundred and seventeen ponies were surveyed for the seasonal prevalence of strongyloid parasites, particularly cyathostomes, and for host- and age-related differences in these infections. For 56 ponies, all stages of the cyathostome life cycle, both mucosal and luminal, were enumerated. Total numbers of cyathostomes and percentage of developing larvae (DL) encysted in the mucosa remained constant in all 4 seasons of the year, whereas a significant increase in the percentage of adults in the cyathostome population occurred in fall. In yearling ponies, encysted early third-stage larvae constituted a significantly lower percentage of the cyathostome population, and DL and adults formed a significantly higher percentage, compared with those stages in older ponies, 2–5 yr of age. More species of cyathostomes were present in yearling ponies than in older ponies. Significant differences occurred in fecal egg counts at different seasons of the year, even though adult cyathostome and large strongyle numbers remained constant. Twenty-four species of cyathostomes were found year-round, and 2 rare species were found in only 2 or 3 seasons of the year. Prevalences for these 24 species were not significantly different during any season, although 5 species had significant differences in intensity levels of infection in certain seasons of the year. Three species of large strongyle (strongylinae) adults had significant seasonal variations in intensities. These were Strongylus edentatus and S. vulgaris, which occurred in significantly higher numbers in summer and fall, and Triodontophorus brevicauda, which was more numerous in spring and summer. Fourth- and fifth-stage larvae of S. vulgaris recovered from the mesenteric vasculature were significantly more numerous in winter and spring than in other seasons.

Irradiated larval vaccination of ponies against Strongylus vulgaris

Nonimmune pony foals 9 to 12 mo of age were vaccinated with third-stage Strongylus vulgaris larvae (L3) irradiated with 70, 100, or 130 Kr of gamma radiation. Ponies receiving per os inoculations of L3 irradiated with 70 or 100 Kr were protected from the clinical disease and lesions associated with challenge infections of 4,300 L3, when compared to nonvaccinated controls. Similarly, the numbers of worms from the challenging population recovered from successfully vaccinated animals were significantly lower than from nonvaccinated controls. The degree of resistance that develops in individuals can be semiquantitated based on clinical and pathological responses.

Antibody responses of ponies to initial and challenge infections of Strongylus vulgaris.

An indirect fluorescent antibody assay (IFA) was developed using Strongylus vulgaris third stage larvae (L3) as antigens. Observations using the IFA indicate that a species-specific antibody response to S. vulgaris L3 develops in S. vulgaris-infected ponies and that some surface L3 antigens are shared by adult worms. Sequential antibody levels against S. vulgaris were measured in strongyle-naive and in immune ponies following initial and challenge infections using the IFA and an indirect hemagglutination assay (IHA). Antibody levels measured by IFA increased faster following initial infections than did levels measured by IHA. Antibody levels appear to increase following challenge infections of immune ponies when measured with the IFA, but not with the IHA. Significant differences in antibody titers were not seen between ponies which developed colic following challenge infections and those that did not develop colic. Antibodies were not detectable in ponies unexposed to larval migrations, but which received surgical implantation of S. vulgaris adults into the cecum.

ONE SEASON OF PASTURE EXPOSURE FAILS TO INDUCE A PROTECTIVE RESISTANCE TO CYATHOSTOMES BUT INCREASES NUMBERS OF HYPOBIOTIC THIRD-STAGE LARVAE

The development of acquired resistance to cyathostome challenge after 1 seasons exposure to a cyathostome-contaminated pasture was investigated using 17 parasite-naive ponies, which were 2–3 yr of age. These were divided into 3 groups: 1 to graze a cyathostome-contaminated pasture for 4 mo (exposed ponies), 1 to graze a “clean” pasture not previously grazed by parasitized animals (nonexposed ponies), and 1 group to remain in the barn under helminth-free conditions (parasite-free ponies). After pasture exposure all ponies were housed in stalls in the barn and dewormed with ivermectin (200 μg/kg) and oxibendazole (100 mg/kg), a treatment that eliminated most cyathostomes encysted in the mucosa as well as all luminal parasites, on the basis of necropsies of 5 animals, after 17 days. Remaining ponies were challenged with 100,000 cyathostome-infective third-stage larvae (L3) per os 3 wk after anthelmintic treatment. Necropsies were performed 7 wk after the challenge. Total cyathostome burdens (luminal plus encysted stages) were not significantly different among any of the groups. However, a significantly higher percentage of hypobiotic early L3 (EL3) and a lower percentage of adults were found in exposed ponies. This observation supports the hypothesis that resistance acquired through exposure promotes cyathostome hypobiosis. This increase in EL3 in exposed ponies was associated with a significant increase in weight of cecum and ventral colon biopsies.

EXPERIMENTAL IMMUNIZATION OF PONIES WITH STRONGYLUS VULGARIS RADIATION-ATTENUATED LARVAE OR CRUDE SOLUBLE SOMATIC EXTRACTS FROM LARVAL OR ADULT STAGES

Protection from Strongylus vulgaris infection through immunization with radiation-attenuated third-stage larvae (L3) or crude soluble homogenates from larval or adult stages was examined. Yearling ponies raised parasite-free were divided into 3 immunization groups: radiation-attenuated L3; soluble adult somatic extracts; larval somatic extracts with excretory/secretory products (E/S) from in vitro culture; and 1 medium control group. Ponies were immunized twice; attenuated larvae were administered orally and somatic extracts or controls injected intramuscularly with adjuvant. Approximately 6 wk following the second immunization, all ponies were challenged. Necrospy examinations were performed 6 wk following challenge. Irradiated larvae recipients had the fewest postchallenge clinical signs and lesions and were 91% protected from infection determined by larval recoveries from arterial dissections. Soluble antigen recipients and controls had similar larval recoveries and thus equal susceptibility to challenge. Soluble antigen recipients had more severe clinical signs and lesions than controls, suggesting that parenteral immunization exacerbated postchallenge inflammatory responses. Protection by immunization with irradiated larvae was associated with an anamnestic eosinophilia and postimmunization antibody recognition of S. vulgaris L3 surface antigens. Histologic staining of eosinophils within tissues of this group suggested that this immunization induced a cytophilic antibody response that facilitated degranulation.