Thomas R. Klei

Prophylactic activity of tetracycline against Brugia pahangi infection in jirds (Meriones unguiculatus)

The ability of oral tetracycline to inhibit the development of third-stage infective larvae (L3) of Brugia pahangi to adult worms in jirds was studied using 2 experimental protocols. Jirds treated with 1.4% tetracycline in drinking water for a period beginning 30 days before inoculation of L3 until 30 days post-inoculation (DPI) had 97% reduction in adult worm recovery compared to untreated controls. Jirds that received 1.2% tetracycline in drinking water beginning 1 day before until either 12 or 26 DPI had adult worm recoveries of 11% and < 1%, respectively. Untreated jirds and those given tetracycline beginning at or later than 13 DPI had similar adult worm recovery (27-29%). Prepatent periods were prolonged, and circulating microfilariae were reduced in jirds given tetracycline from 27 to 54 DPI compared to controls. These data indicate that tetracycline administered to jirds in drinking water inhibits B. pahangi development from L3 to adult worms and suggest that this effect occurs during early larval development. Tetracycline administered to infected jirds prior to and continuing through the onset of patency can also affect development of microfilaremia.

IDENTIFICATION AND CHARACTERIZATION OF A PYRANTEL PAMOATE RESISTANT CYATHOSTOME POPULATION

Three fecal egg count reduction assays (FECR) and one critical trial were performed to determine the efficacy of pyrantel pamoate (PP) at 6.6 mg base kg-1 on a well managed stud farm in Louisiana where a loss of efficacy was suspected. Efficacy of PP based on FECR varied from 25% in mares to 83% in yearlings. Second treatments with PP 2 weeks following an initial treatment failed to reduce eggs per gram (EPG). A critical trial was performed to determine the cyathostome species resistant to PP. Three strongyle-naive ponies which acquired infections on the farm were used for this purpose. Following treatment with PP at the recommended dose, 11 species of cyathostomes remained in the intestine of the tracer ponies. Reduced efficacies (62%-88%) were noted for seven species. Resistance to oxibendazole (OBZ), which was > 90% effective on this farm in 1982, was also evaluated by FECR and found to exist. The results of one experiment indicate that dual resistance of parasites to PP and OBZ also exists.

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.

Role of gamma interferon and interleukin-4 in host defense against the human filarial parasite Brugia malayi.

ABSTRACT We have investigated the roles of gamma interferon (IFN-γ) and interleukin-4 (IL-4) in host defense against Brugia malayi. Our data suggest that the lack of either IFN-γ or IL-4 prolongs the time required to achieve sterile immunity, suggesting that both canonical type 1 and type 2 responses are involved in the clearance of infection.

Evaluation of ivermectin at an elevated dose against encysted equine cyathostome larvae

The efficacy of a high dose of ivermectin (1.0 mg per kg Eqvalan liquid drench) on encysted cyathostomes was tested in a controlled study using 12 adult ponies with naturally acquired cyathostome infections. Six treated ponies and six non-treated controls were held in separate stalls for a period of 5 weeks. Cyathostome burdens, which included lumenal larvae, adults and encysted larvae, were determined at necropsy. The viability of encysted larvae, based on morphologic integrity, was assessed by observation of mural transillumination and by the histologic appearance of 12 larvae per pony. Efficacy against adult cyathostomes was 99.9%. Lumenal cyathostome larval numbers were reduced by 87%. Numbers of encysted cyathostome larvae, identified by transillumination of the large intestine, were reduced by 35%. However, this reduction was not statistically significant (P > 0.05) and differences in viability of encysted larvae were not observed. The data strongly indicated that ivermectin has little demonstrable effect on encysted equine cyathostomes.

New Method for Simultaneous Species-Specific Identification of Equine Strongyles (Nematoda, Strongylida) by Reverse Line Blot Hybridization

ABSTRACT The ability of a reverse line blot (RLB) assay to identify 13 common species of equine small strongyles (cyathostomins) and to discriminate them from three Strongylus spp. (large strongyles) was demonstrated. The assay relied on the specific hybridization of PCR-amplified intergenic spacer DNA fragments of the nuclear ribosomal DNA to membrane-bound species-specific probes. All cyathostomins examined were unequivocally identified and simultaneously discriminated from each other and from three large strongyles (Strongylus edentatus, Strongylus equinus, and Strongylus vulgaris). This assay will enable the accurate and rapid identification of equine cyathostomins irrespective of their life cycle stage, opening important avenues for a better understanding of their biology and epidemiology and of the pathogenesis of cyathostomin-associated disease. In particular, this RLB method promises to be a powerful diagnostic tool to determine the roles of individual species in the pathogenesis of mixed infections and to elucidate some aspects of cyathostominosis. Also, it could represent a basic step toward the development of a rapid and simple molecular test for the early detection of drug-resistant genotypes of horse strongyle species.

Brugia pahangi: Effects of duration of infection and parasite burden on lymphatic lesion severity, granulomatous hypersensitivity, and immune responses in jirds (Meriones unguiculatus)

The effects of Brugia pahangi infection duration and parasite burden on parasite-associated inflammatory and immune responses were determined over a 181-day period in jirds receiving from one to eight inoculations of infective larvae. Multiple infections did not produce a protective resistance to reinfection as determined by adult worm recovery at necropsy. Intralymphatic granulomatous lesions, lymph thrombi, were first seen at 48 days post initial inoculation (DPI). The numbers of lymph thrombi reached peak levels in singly inoculated jirds at 90 DPI and significantly decreased to low levels by 160 DPI. The ratio of lymph thrombi to adult worms recovered from the spermatic cord lymphatics followed a similar pattern. Sizes of renal lymph nodes, which drain lymphatics containing parasites, followed a temporal pattern of increase and decrease similar to that of lymph thrombi numbers. Peak granuloma areas around antigen-coated beads embolized in lungs were seen at 27 DPI. Granuloma areas around antigen-coated beads began to decrease after 69 DPI and reached sizes not significantly different from uninfected controls by 118 DPI. Multiple inoculations of infective larvae and increasing worm burdens did not affect the pattern of granulomatous response to antigen-coated beads. Eosinophilia of singly and multiply infected jirds peaked at 26 DPI. Eosinophilia of singly infected jirds returned to normal levels by 103 DPI but those of multiply infected jirds remained elevated until 160 DPI. Lymph node cell blastogenic responses to antigen were greater than those of splenocytes at all time intervals measured. However, significant differences in stimulation indexes between groups with different infection durations were not seen with either cell type. Antibody responses to somatic adult worm antigen as measured by ELISA reached near peak levels by 48 DPI and remained elevated for the course of the study in all infected jirds. The decrease in lymphatic lesion severity seen in chronically infected jirds temporally corresponds to the decrease in granulomatous reactivity measured around antigen-coated beads embolized in the lungs. This observation suggests that host and/or parasite factors associated with these two phenomena may be similar. Although these decreases may be the result of down-regulated immune responses, corresponding decreases in antibody levels and blastogenesis of lymphocytes stimulated by crude worm extracts were not observed in chronic infections.

Comparison of moxidectin oral gel and ivermectin oral paste against a spectrum of internal parasites of ponies with special attention to encysted cyathostome larvae.

Two dosages of moxidectin oral gel were evaluated and compared to a therapeutic dose of ivermectin oral paste in the control of a spectrum of gastrointestinal parasites of ponies naturally infected in southern Louisiana or Mississippi. Thirty-two mixed-breed ponies ranging in age from one to 21 years were used in this controlled test. Eight weeks prior to the experiment, ponies grazing on contaminated pasture were moved to a paddock and fed a pelleted ration, thus reducing or eliminating the potential for additional infection and ensuring the existence of a population of encysted larvae. Ponies were then allocated to replicates of four animals based on values of fecal strongyle egg counts and percent strongyle larvae composition determined from Baermann sedimentations of fecal cultures. Members of replicates were allocated to one of four treatment groups: moxidectin oral gel administered at 300 micrograms kg-1 body weight, moxidectin oral gel at 400 micrograms kg-1, the oral gel vehicle as negative control, and ivermectin oral paste at 200 micrograms kg-1. Prior to treatment, ponies were confined in pairs to covered concrete runs by treatment group. Two weeks following treatment, necropsy examinations of all animals were performed. Parasites were recovered from the lumen of the stomach, the intestinal tract, the cranial mesenteric artery and its major branches, the peritoneal body wall and from pepsin digests of mucosal scrapings taken from the cecum and large colon. Encysted cyathostome larval burdens were also compared using mural transillumination of segments of the large colon for visualization of the encysted forms. Control ponies were not uniformly infected with the spectrum of parasites; however, moxidectin, at either dosage, compared favorably with ivermectin in the control of the adults of Strongylus vulgaris, Strongylus edentatus, Triodontophorus spp., Oesophagodontus robustus, Trichostrongylus axei, Oxyuris equi, Parascaris equorum, Habronema muscae, as well as both the adult and larval Cyathostominae recovered from the lumen. Moxidectin also appears as efficacious as ivermectin against migrating large strongyle larvae at the two weeks post-treatment evaluation. Moxidectin demonstrated a trend towards greater efficacy against encysted cyathostome larvae than a therapeutic dosage of ivermectin, but this difference was not statistically significant. Moxidectin was less effective than ivermectin against Gasterophilus intestinalis and was equally ineffective as ivermectin against Anoplocephala perfoliata.

Identification and localization of glutathione S-transferase as a potential target enzyme in Brugia species.

Brugia filarial nematodes are pathogenic lymphatic-dwelling parasites that, like other helminths, may modify the hosts defense mechanisms by a major detoxification process involving glutathione-binding proteins such as glutathione S-transferases (GSTs). In the present study, soluble extracts of third-stage larvae, adult male and female worms, microfilariae of either B. pahangi or B. malayi or the adult worm excretory–secretory products of B. malayi were used to determine GST activity. These extracts and affinity-purified fractions of B. pahangi adult worms had a specific enzymatic activity when 1-chloro-2,4-dinitrobenzene was used as a substrate. The observance of this enzyme in all life cycle stages of Brugia spp. demonstrates its ubiquitous nature. Lavage of intraperitoneally infected jirds, but not that of uninfected jirds, also showed increased enzymatic activity, suggesting that GST is secreted in vivo. Soluble proteins of both Brugia spp. were strongly recognized by antibodies in sera from rabbits immunized with affinity-purified native GST of Onchocerca volvulus. Immunohistochemical studies localized these proteins in adult worms, demonstrating cross-reactivity between the GST of these two filarial nematodes. The effect of this enzyme on the motility and viability of adult worms, microfilariae, and larvae was tested in vitro using a battery of known GST inhibitors. Of all those tested, ethacrynic acid, N-ethylmalemide, 4-nitropyridine-oxide, or 1-chloro-2,4-dinitrobenzene at micromolar concentrations reduced the viability and motility of microfilariae, third-stage larvae, and adult worms. These results suggest that Brugia GSTs are major metabolic enzymes and may play an important role in the parasites survival.

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.