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In vitro development of Strongylus edentatus to the fourth larval stage with notes on Strongylus vulgaris and Strongylus equinus.

Strongylus edentatus was successfully cultured in vitro to the fourth larval stage (L4). Some growth continued for periods of 40-50 days at which time reductions in viability were observed in some of the culture systems tested. Various combinations of media, sera, buffers and organ explant cultures were tested. All cultures were incubated at 37 C in an atmosphere of 95% air and 5% CO2. Larvae underwent growth and differentiation to the L4 in all medium-serum combinations with and without organ explant cultures. Development and growth did occur but viability was reduced to insignificant levels in media without serum or cells. Optimal growth, differentiation, and longevity were observed in bicarbonate buffered RPMI-1640 containing 10% fetal calf serum and gerbil (Meriones unguiculatus) cecum explant cultures. Observations indicated that Strongylus vulgaris and Strongylus equinus also developed to the L4 stage using similar techniques. However, viability of S. vulgaris L4 was markedly limited. Specific morphological changes marked phases of development of S. edentatus, categorized as early, middle and late third stage, third molt and early fourth stage. Strongylus equinus appeared to follow the same developmental pattern in vitro as S. edentatus. Distinct differences in morphological features during differentiation were observed between S. edentatus and S. vulgaris.

Animals↗

Production and characterization of monospecific adult worm infections of Strongylus vulgaris and Strongylus edentatus in ponies.

Since 1978, 20 surgical implantations of either Strongylus vulgaris or Strongylus edentatus have been performed in our laboratory for the purpose of obtaining single species cultures of these parasites. Following surgical implantation peak EPG values of 13-327 (S. vulgaris) and 363-1284 (S. edentatus) generally occurred during the first 3 weeks post-implantation. Duration of infections was as long as 5 years. Successful outcome of such surgeries appears to be related to the total number of parasites used (> or = 38) and the ratio of female to male worms implanted (1:1 or 2:1).

Animals↗

Strongylus asini (Nematoda, Strongyloidea): genetic relationships with other Strongylus species determined by ribosomal DNA.

Genomic DNA was isolated from adult Strongylus asini collected from zebra. The second ribosomal transcribed spacer (ITS-2) was amplified and sequenced using polymerase chain reaction (PCR) based techniques. The DNA sequence was compared with previously published data for 3 related Strongylus species. A PCR-linked restriction fragment length polymorphism method allowed the 4 species to be differentiated unequivocally. The ITS-2 sequence of S. asini was found to be more similar to those of S. edentatus (87.1%) and S. equinus (95.3%) than to that of S vulgaris (73.9%). This result confirms that S. Asini and S vulgaris represent separate species and supports the retention of the 4 species within 1 genus.

Animals↗

Necropsies of eight horses infected with Strongylus equinus and Strongylus edentatus.

Ponies (n = 8) approximately 18 months old, were infected with 20,000 to 30,000 infective larvae of Strongylus equinus with less than 10% contamination with Strongylus edentatus larvae and necropsied 7 months post-infection. Lesions were present in the omentum, liver, pancreas, ventral colon, caecum and occasionally in the lungs. There were numerous intraabdominal adhesions and severe multiple granulomatous omentitis. Pancreatic damage, which characterises S. equinus, was exceptionally mild and was manifested mainly by slight periductular infiltration of eosinophils. Granulomas associated with larvae were found in the connective tissue associated with the pancreas. Larvae were recovered from the flanks, the peritoneum, the caecum and the kidney. Larval recovery was low, with a high percentage of the total number of larvae recovered in some of the carcasses being S. edentatus.

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Comparison of the activity of selamectin, imidacloprid and fipronil for the treatment of cats infested experimentally with Ctenocephalides felis felis and Ctenocephalides felis strongylus.

Twenty adult, domestic short hair cats were randomly allocated into four groups of five cats and housed in separated cages. Each cat was infested with 25 fleas Ctenocephalides felis felis and 25 Ctenocephalides felis strongylus and 2 days later (day 0) the cats in group 1, 2 and 3 received a spot on application of selamectin, imidacloprid or fipronil, respectively, while the cats in group four were not treated. The cats were combed 48 h later, the fleas were removed, counted and their subspecies were determined. All the cats were reinfested with the same number of the two subspecies of fleas on days 7, 14, 21, 29 and 35. The efficacy of each treatment was calculated 48 h after each infestation. The mean number of fleas on the control cats was 16.4 C. f. felis and 13.4 C. f. strongylus. The three treatments were effective for the first 31 days for C. f. felis and for the full 37 days for C. f. strongylus. Over the first 31 days, the efficacy of selamectin ranged from 89 to 100% and 85 to 100% against C. f. felis and C. f. strongylus, respectively, the efficacy of imidacloprid ranged from 76 to 100% and 92 to 100% and the efficacy of fipronil ranged from 98 to 100% and 97 to 100% against C. f. felis and C. f. strongylus. There were no significant differences between the control of C. f. felis and C. f. strongylus by the three products.

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[Some ecological parameters of Ctenocephalides felis strongylus (Jordan, 1925) (Siphonaptera: Pulicidae)].

Among the fleas of medico-veterinary interest, Ctenocephalides felis (Bouché, 1 835) is the one most studied. This taxon includes two subspecies: Ctenocephalides f. felis, and Ctenocephalides f. strongylus (Jordan, 1925); only C. f. felis has been the subject of almost all the studies available. We were, thus, interested in C. f. strongylus which can be regarded as the species of substitution of C. f. felis on the African continent. The purpose of our work was to establish some biological parameters such as: hatching of eggs, cycle of development and emergence of adults. These data were compared with those available on C. f. felis. With temperatures ranging between 19 degrees C and 29 degrees C and a relative humidity (HR) of 75 % +/- 5, the hatching rates of eggs observed from the two subspecies of C. felis, are higher than 88 %. The optimal temperature of eggs hatching for C. felis is 29 degrees C, with more than 70 % of hatching obtained in 1-2 days after the laying. The larval developments of the two subspecies are almost identical and function of the temperature 18-9 days with 27 degrees C). Only differs the minimal duration of the progressive cycle. For C. f. strongylus, it lasts in 16-17 days at 29 degrees C, 20-21 days at 27 degrees C and 38 days at 19 degrees C. For C. f. felis, published values give report of 15 days at 27 degrees C and 17 days at 24 degrees C. The emergence of adults of C. f. strongylus takes eight to ten days between 19 degrees C and 29 degrees C, while data published on C. f. felis are about 26 days at 19 degrees C and 15 days at 27 degrees C.

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Scanning electron microscopy of Strongylus spp. in zebra.

The external ultrastructure of the anterior and posterior extremities of the nematodes, Strongylus asini , Strongylus vulgaris, Strongylus equinus and Strongylus edentatus, was studied with scanning electron microscopy (SEM). Fresh specimens of S. asini were collected from the caecum, ventral colon and vena portae of Equus burchelli and Equus zebra hartmannae ; S. vulgaris from the caecum, colon and arteria ileocolica of E. burchelli ; S. equinus from the ventral colon of E. z. hartmannae and S. edentatus from the caecum and ventral colon of both zebras , during surveys of parasites in zebras in the Etosha Game Reserve, South West Africa/Namibia, and the Kruger National Park, Republic of South Africa. The worms were cleaned, fixed and mounted by standard methods and photographed in a JEOL JSM - 35C scanning electron microscope (SEM) operating at 12kV . The SEM showed the following differences: the tips of the external leaf-crowns varied and were fine and delicate in S. asini , coarse and broad in S. vulgaris and, in S. equinus and S. edentatus, closely adherent, separating into single elements for half their length. The excretory pores showed only slight variation, and the morphology of the copulatory bursae did not differ from those seen with light microscopy. The genital cones differed markedly: S. asini had a ventral triangular projection and laterally 2 finger-like projections: in S. vulgaris there were numerous bosses on the lateral and ventral aspects of the cone; in S. equinus 2 finger-like processes projected laterocaudally ; and in S. edentatus 2 pairs of papilla-like processes projected laterally on the ventral aspects, and a pair of rounded projections and a pair of hair-like structures adorned the dorsal aspects.(ABSTRACT TRUNCATED AT 250 WORDS)

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Role of lipids in the transmission of the infective stage (L3) of Strongylus vulgaris (Nematoda: Strongylida).

Infective larvae (L3) of Strongylus vulgaris have limited energy stores for host finding and for infection. For transmission to occur, the larvae must have sufficient energy to (a) migrate onto grass, where they are ingested by their equine host (host finding), and (b) penetrate into the host gut. This study is designed to test the hypothesis that L3 larvae of S. vulgaris partition their energy stores between locomotory activity (used in host finding) and infection activity (penetration). Chronic locomotory activity was stimulated by incubating S. vulgaris L3 larvae at a constant temperature (38 C). After 8 days of treatment, locomotory activity ceased (exhaustion). Exhausted L3 larvae had significantly decreased total lipid when compared to controls (P < 0.05), but there was no decrease in levels of protein of carbohydrate. Lipids of S. vulgaris L3 larvae are comprised of 9 fatty acids, some of which are depleted in exhausted worms (14:0, 14:1, 16:0, 16:1, 18:1, 18:2), whereas others (18:0, 20:4, 24:0) remain unchanged. These data suggest that specific fatty acids provide the energy source for locomotory activity in S. vulgaris. Exhausted L3 larvae were also less able to penetrate host cecal tissue in in vitro penetration assays when compared to controls (P < 0.05), suggesting that the depletion of individual fatty acids during locomotory activity also reduced infectivity. These data do not support the hypothesis that S. vulgaris L3 larvae partition their energy stores between host-finding and infection activities. A comparison of lipid storage profiles in the L3 larvae of 4 nematode species with similar transmission strategies (S. vulgaris, Strongylus edentatus, Strongylus equinus, and Haemonchus contortus) revealed similarities in the fatty acid composition of these species. These data suggest a relationship between transmission patterns and energy storage strategies in the L3 larvae of nematode parasites of vertebrates.

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Differences in a ribosomal DNA sequence of Strongylus species allows identification of single eggs.

In the current study, molecular techniques were evaluated for the species identification of individual strongyle eggs. Adult worms of Strongylus edentatus, S. equinus and S. vulgaris were collected at necropsy from horses from Australia and the U.S.A. Genomic DNA was isolated and a ribosomal transcribed spacer (ITS-2) amplified and sequenced using polymerase chain reaction (PCR) techniques. The length of the ITS-2 sequence of S. edentatus, S. equinus and S. vulgaris ranged between 217 and 235 nucleotides. Extensive sequence analysis demonstrated a low degree (0-0.9%) of intraspecific variation in the ITS-2 for the Strongylus species examined, whereas the levels of interspecific differences (13-29%) were significantly greater. Interspecific differences in the ITS-2 sequences allowed unequivocal species identification of single worms and eggs using PCR-linked restriction fragment length polymorphism. These results demonstrate the potential of the ribosomal spacers as genetic markers for species identification of single strongyle eggs from horse faeces.

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Generation and partial characterization of an eosinophil chemotactic cytokine produced by sensitized equine mononuclear cells stimulated with Strongylus vulgaris antigen.

Supernatants generated by stimulation of peripheral blood mononuclear cells (PBMC) from Strongylus vulgaris sensitized or immunized ponies were assayed in vitro for eosinophil chemotactic activity (ECA) using the filter system in blind well chambers. The supernatants from these cultures were chemotactic for eosinophils, but not for neutrophils. Supernates from cultures of unsensitized PBMC stimulated with S. vulgaris antigen were not chemotactic for eosinophils. ECA was first detected in culture supernatants after 1.5 h of incubation and was dependent on both antigen and PBMC concentrations, but independent of serum concentrations. Both female and male S. vulgaris worm antigens stimulated ECA production from sensitized PBMC. ECA was not induced by in vitro stimulation of sensitized S. vulgaris PBMC by female Strongylus edentatus worm antigen. Partial characterization of the eosinophil chemotactic cytokine showed it to be nondialyzable, greater than 8000 molecular weight (MW), and sensitive to heating (56 and 95 degrees C), trypsin, and sodium metaperiodate treatments, suggesting that the cytokine is a protein containing some essential carbohydrate moieties. The cytokine described in this paper could partially contribute to the in vivo blood and tissue eosinophilia in experimental S. vulgaris infection.

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Dose titration of moxidectin oral gel against migrating Strongylus vulgaris and Parascaris equorum larvae in pony foals.

Moxidectin was tested for efficacy in ponies against experimental infections of 56 day Strongylus vulgaris larvae and 11 day Parascaris equorum larvae. Three dosages of moxidectin were tested: 300 micrograms per kg live body weight, 400 micrograms per kg, and 500 micrograms per kg, and the vehicle served as control. Ponies were first infected with 600 S. vulgaris third-stage larvae (L3) on Experiment Day 0 and then with 3000 embryonated P. equorum eggs on Day 45. Moxidectin treatments were administered on Day 56 and necropsy examinations were performed on Day 91. Strongylus vulgaris fourth-stage (L4) and fifth-stage (L5) larvae were recovered at necropsy from the control ponies, in dissections of the cranial mesenteric artery and its branches (L4 and L5), and recovered from nodules in the wall of the cecum and ventral colon (L5). Parascaris equorum larvae were recovered from the small intestine of control ponies. Moxidectin was highly efficacious against S. vulgaris L4 and L5 at all three doses tested (99.6-100%), and appeared to be equally efficacious against P. equorum larvae (100%); however, control ponies had low levels of P. equorum infections compared to previous experimental infections performed using identical methods. This suggests that the prior S. vulgaris infection on Day 0 may have influenced the subsequent experimental P. equorum infection on Day 45 and contributed to the lower recovery.

Administration, Topical↗

Role of the eosinophil in serum-mediated adherence of equine leukocytes to infective larvae of Strongylus vulgaris.

The adherence of equine leukocytes to Strongylus vulgaris infective larvae (L3) in the presence of normal and immune sera was examined in vitro. Immune sera promoted adherence of buffy coat cells from ponies with S. vulgaris-induced eosinophilia (eosinophilic ponies) to S. vulgaris L3. However, eosinophils in the buffy coat cells were the predominant adherent cell type. Studies using leukocyte populations enriched for eosinophils, neutrophils, and mononuclear cells from eosinophilic ponies support the observations using buffy coat cells that eosinophils were the main effector cells. Adherent eosinophils from eosinophilic ponies immobilized L3. Neutrophils were less adherent and did not immobilize L3. Mononuclear cells failed to adhere. Normal eosinophils from strongly-naive ponies did not immobilize S. vulgaris L3 in the presence of immune serum, suggesting the in vivo activation of eosinophils in eosinophilic animals. Immune serum promoted less adherence of buffy coat cells to Strongylus edentatus or mixed species of Cyathostominae L3, suggesting that the serum-mediated cellular adherence phenomenon was species-specific. Normal serum promoted less cellular adherence to S. vulgaris L3 than immune serum. The adherence mediated by normal serum was removed by heat inactivation, suggesting that this nonspecific phenomenon was a complement-mediated reaction. Immune globulins promoted reactions similar to that seen using heat-inactivated immune serum, whereas normal globulins did not promote adherence. Immune globulins absorbed with pieces of S. vulgaris adult worms did not promote the adherence of buffy coat cells to S. vulgaris L3, suggesting that adult and L3 stages share antigens important in this phenomenon that resulted in the removal of specific adherence antibody during absorption.

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Effects of repeated Strongylus vulgaris inoculations and concurrent ivermectin treatments on mesenteric arterial lesions in pony foals.

Eight of 10 pony foals reared under helminth-free conditions were inoculated PO with 50 Strongylus vulgaris infective larvae/week for 4 weeks, at which time 1 foal died of acute verminous arteritis. Inoculation of 7 remaining foals continued at 2-week intervals for 20 weeks. Of the 7 foals, 3 were treated with ivermectin (0.2 mg/kg of body weight) in an oral paste formulation at experiment weeks 8, 16, 24; 4 foals were not treated. Two foals were not inoculated or treated and served as controls. After the first ivermectin treatment, ivermectin-treated foals had fewer days (12 +/- 2.9) with rectal temperatures greater than 38.6 C than did nontreated foals (23.3 +/- 3.8). Mean baseline rectal temperatures were 38 +/- 0.2 C. Adverse clinical reactions to ivermectin treatment were not observed in foals. Foals were euthanatized and necropsied 3 weeks after the last ivermectin treatment (week 24). Ivermectin was effective in reducing S vulgaris arterial larval and intestinal adult parasite numbers by 100% in 3 treated foals. Strongylus vulgaris arterial larvae and/or adults were recovered from all 4 nontreated inoculated foals. One nontreated inoculated foal lacked arterial larvae or active arterial lesions, indicating that protective resistance had developed in this individual. Marked gross and histopathologic lesions typical of chronic S vulgaris infection were observed in the 3 nontreated inoculated foals with arterial larvae. Repeated killing of intra-arterial S vulgaris fourth-stage larvae in ivermectin-treated foals did not exacerbate lesions associated with verminous arteritis or induce unique lesions associated with repeated destruction of arterial larvae.(ABSTRACT TRUNCATED AT 250 WORDS)

Administration, Oral↗

Strongylus equinus: development and pathological effects in the equine host.

The development and pathological effects of Strongylus equinus were studied in 17 pony foals and one horse foal raised in isolation and examined at necropsy from seven days to 40 wk postinfection (PI). Following inoculation of 15000 +/- 6% or 16000 +/- 6% infective larvae by stomach tube foals were monitored for clinical signs and selected blood changes. Larvae penetrated the wall of the ileum, cecum and colon. The molt to the fourth stage occurred mostly in the wall of the ventral colon before 2 wk PI and larvae attained the liver mainly via the peritoneal cavity as early as eight days PI and persisted in the liver until 17 wk PI. Following active migration within the liver, invasion of the pancreas was accomplished at least by 7 wk PI with maximum numbers at 17 wk. The fourth molt occurred about 15 wk PI and preadults were present in the wall of the ventral colon at 30 wk PI and in the lumen of the colon at 40 wk. Strongylus equinus tends to wander retroperitoneally to the flanks, perirenal fat, diaphragm, omentum and occasionally to the lungs. Between 1 and 4 wk PI small raised hemorrhagic areas were present on the serosa of the ileum and colon. Small white foci on the surface of the liver at 1 wk PI were followed by tortuous tracks 3 wk later. Pathological changes in the pancreas were evident at three months PI and more severe by four months. Granulomas containing larvae were common in the flanks, diaphragm, omentum and occasionally beneath the pleura of the lungs. Clinical signs were correlated with invasion of the pancreas, the fourth molt, maximum globulin values and high eosinophil counts.

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[Arterial repair after mechanical injury by migrating fourth-stage larvae of Strongylus vulgaris in the horse (a light and electron microscopic study) (author's transl)].

INTRODUCTION: Migrating fourth-stage larvae of Strongylus vulgaris, a parasite of equines, damage the intima of the anterior mesenteric artery and its larger branches and induce thrombus formation on the injured sites. As the time of larval passage through each of these branches has been exactly determined in earlier experiments, the aim of the present studies is to contribute to a more complete understanding of repair mechanisms in the process of time after thrombotic vascular injuries. MATERIAL AND METHODS: five foals were separated individually to specially cleaned stables and given anthelmintic treatment till the age of one year. One foal was infected per os with 350, the second with 500 and the remaining three with 1,000 third-stage larvae of Strongylus vulgaris...

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Antigenic analyses of tissues and excretory and secretory products from Strongylus vulgaris.

Rabbit antisera were prepared against veronal buffered saline extracts of L4 and L5 Strongylus vulgaris, adult S. vulgaris and adult Strongylus equinus retrieved from naturally infected horses. In agar gel diffusion with these antisera, adult S vulgaris and S. equinus each appeared to have at least one unique antigen; larval S. vulgaris appeared to have two species-specific and two stage-specific antigens. There were several common antigens. Excretory and secretory products were collected also from L4 and L5 an maintained over several days in tissue culture fluid. In agar gel diffusion against the above rabbit antisera, a stage-specific antigen was found also in excretory and secretory products. In addition, excretory and secretory products had three antigens in common with adult and larval S. vulgaris, but only one of these was common to adult S. equinus. The excretory and secretory products appear, therefore, to have two species-specific and one stage-specific antigens.

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Controlled tests of ivermectin against migrating Strongylus vulgaris in ponies.

Twelve pony foals were reared worm-free and inoculated with Strongylus vulgaris. On day 7 after inoculation, 6 ponies were given ivermectin IM at a dose of 200 micrograms/kg of body weight and on day 28 were necropsied. Ivermectin was effective in eliminating early 4th-stage S vulgaris larvae and reducing clinical signs associated with acute arteritis. After administrative ivermectin was effective against early 4th-stage Strongylus vulgaris larvae in ponies when administered at 100, 300, or 800 micrograms/kg of body weight. The purpose of the present study was to report on a more extensive trial, using a single dosage of ivermectin.

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