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Serum samples from 296 horses slaughtered for food in the United States were tested for antibodies to Neospora caninum by the Neospora-agglutination test (NAT). Antibodies were found in 69 (23.3%) horses with titers of 1:40 (19 horses), 1:80 (19 horses), 1:100 (3 horses), 1:200 (7 horses), 1:400 (4 horses), and 1:800 (17 horses). This is the first serologic survey for N. caninum antibodies in horses.

Serum samples from 143 hunter-killed black bears were collected during the 1996 and 1997 black bear hunting seasons in eastern North Carolina. All samples were tested for antibodies to Toxoplasma gondii by the modified agglutination test. Antibodies to T. gondii were present in 120 of 143 (84%) bears. Females had significantly higher titers than males (Wilcoxon rank sums test, P = 0.045), and titers increased with age (Jonckheere test, P = 0.01). Samples collected during 1996 (n = 79) were tested for antibodies to Trichinella spiralis by enzyme-linked immunosorbent assay. No samples were positive for antibodies to T. spiralis.

Parasitic third-stage larvae of the sheep abomasal nematode Haemonchus contortus develop and molt in vitro to the fourth stage (L4) in 48-72 hr, at which time they begin feeding. Coincident with the third molt, larvae begin to secrete significant amounts of protein into culture fluids, including a zinc metalloproteinase. This culture-derived zinc metalloproteinase differs from a previously described metalloproteinase from infective third-stage larvae (L3[2M]), which mediates the ecdysis process. These differences include time of expression, molecular mass, and substrate specificity. The purified proteinase, from cultures of L4, has a molecular weight of approximately 46 kDa, functions as an endopeptidase, and digests several native proteins of host origin including fibrinogen and fibronectin.

A nematode from the genus Trichinella was observed in histological sections of breast and tracheal muscles from a black vulture Coragyps atratus from Alabama. Larvae obtained from breast muscle tissue that had been refrigerated for 8 days were infectious for laboratory mice. No nurse cell was observed around larvae in the black vulture or in experimentally infected mice examined 7 or 9 wk postinoculation. The identity of the parasite as Trichinella pseudospiralis was confirmed by DNA hybridization using the species-specific probe, pTsp 5.32. Infectivity trials showed that this isolate was also infective for pigs and chickens. This is the first report of isolation and transmission of T. pseudospiralis from an animal from North America.

Phosphorus resonances consistent with phosphoarginine and ATP were observed in the in vivo 31P-nuclear magnetic resonance (NMR) spectra of infective larvae of Haemonchus contortus. The level of phosphoarginine quickly declined when nematode suspensions were purged with nitrogen and was restored upon return to aerobic conditions. Saturation transfer NMR demonstrated forward and reverse exchange of phosphorus between phosphoarginine and ATP.

Scanning electron microscopy was used to describe the infective and parasitic third-stage larvae of Haemonchus contortus, the large stomach worm of ruminants. Infective larvae are ensheathed in the cuticle of the second stage, so the descriptions are of the second- and third-stage cuticles. Both larval stages had an inner circle of 6 labial papillae, an outer circle of 6 labial papillae and 4 somatic papillae, and lateral amphidial pits. Infective larvae (cuticle of the second stage) had the 6 internal labial papillae on prominent bluntly rectangular lappets in a star-shaped arrangement around a large triradiate mouth, small triangular or round amphidial pits, flattened ribbonlike lateral alae, and phasmidial apertures opening on the ventral surface of the lateral alae. Parasitic third-stage larvae had the 6 internal labial papillae on small elevations without lappets around a small mouth; large, oval amphidial pits; ribbonlike lateral alae for most of their length, but with the anterior 30-40 microns of the alae cordlike; and phasmidial apertures on the body cuticle ventral to the lateral alae.

Parasitic third-stage larvae of the sheep abomasal nematode Haemonchus contortus develop and molt in vitro to the fourth stage (L4) in 48-72 hr, at which time they begin feeding. Coincident with the third molt, larvae begin to secrete significant amounts of protein into culture fluids, including a zinc metalloproteinase. This culture-derived zinc metalloproteinase differs from a previously described metalloproteinase from infective third-stage larvae (L3[2M]), which mediates the ecdysis process. These differences include time of expression, molecular mass, and substrate specificity. The purified proteinase, from cultures of L4, has a molecular weight of approximately 46 kDa, functions as an endopeptidase, and digests several native proteins of host origin including fibrinogen and fibronectin.

A nematode from the genus Trichinella was observed in histological sections of breast and tracheal muscles from a black vulture Coragyps atratus from Alabama. Larvae obtained from breast muscle tissue that had been refrigerated for 8 days were infectious for laboratory mice. No nurse cell was observed around larvae in the black vulture or in experimentally infected mice examined 7 or 9 wk postinoculation. The identity of the parasite as Trichinella pseudospiralis was confirmed by DNA hybridization using the species-specific probe, pTsp 5.32. Infectivity trials showed that this isolate was also infective for pigs and chickens. This is the first report of isolation and transmission of T. pseudospiralis from an animal from North America.

Scanning electron microscopy was used to describe the infective and parasitic third-stage larvae of Haemonchus contortus, the large stomach worm of ruminants. Infective larvae are ensheathed in the cuticle of the second stage, so the descriptions are of the second- and third-stage cuticles. Both larval stages had an inner circle of 6 labial papillae, an outer circle of 6 labial papillae and 4 somatic papillae, and lateral amphidial pits. Infective larvae (cuticle of the second stage) had the 6 internal labial papillae on prominent bluntly rectangular lappets in a star-shaped arrangement around a large triradiate mouth, small triangular or round amphidial pits, flattened ribbonlike lateral alae, and phasmidial apertures opening on the ventral surface of the lateral alae. Parasitic third-stage larvae had the 6 internal labial papillae on small elevations without lappets around a small mouth; large, oval amphidial pits; ribbonlike lateral alae for most of their length, but with the anterior 30-40 microns of the alae cordlike; and phasmidial apertures on the body cuticle ventral to the lateral alae.

Plasma membrane from the brush border isolated from the tegument of Hymenolepis diminuta contains membrane-bound ribonuclease (RNase) and alkaline phosphatase activities. RNase (yeast RNA substrate), alkaline phosphatase (p-nitrophenyl phosphate substrate), and additional membrane proteins were solubilized by sonication or treatment with the detergents dodecyl trimethylammonium bromide, beta-octyl-D-glucopyranoside, sodium dodecyl sulfate (SDS), or ZwittergentTM 3-12 (N-dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate). At optimal conditions, greater than 90% of both enzymes and total protein were solubilized by the latter two detergents, whereas beta-octyl-D-glucopyranoside, dodecyl trimethylammonium bromide, and sonication were only partially effective. Nonionic detergents did not solubilize the membrane effectively.

Sera collected from patients with suspected or confirmed exposure to Trichinella spiralis were tested for circulating parasite antigens and antiparasite antibodies. Using an immunoradiometric assay, excretory--secretory antigens from muscle-stage larvae of T. spiralis were detected in the sera of 47% of 62 patients with clinical trichinellosis and 13% of 39 patients without clinical signs but suspected of exposure to infected meat. In comparison, antibodies were detected using an indirect immunofluorescent test in the circulation of 100% of the 62 patients with clinical trichinellosis and 46% of the 39 patients with suspected exposure. The presence of antibodies specific to excretory-secretory products of T. spiralis muscle larvae was confirmed in the majority of the samples tested by a monoclonal antibody-based competitive inhibition assay. These results indicate that antibody detection is a more sensitive diagnostic method for human trichinellosis, but that antigen detection might be a useful confirmatory test because it is a direct demonstration of parasite products in the circulation.

The isolated brush border membrane of Hymenolepis diminuta contained ribonuclease (RNase) activity which was demonstrable using yeast RNA or synthetic homopolymers of adenylic, cytidylic, inosinic, or uridylic acids as substrates. Polyguanylic acid was not hydrolyzed by worm RNase. RNase activity was inhibited by EDTA and divalent cations as well as sulfhydryl blocking and reducing agents. Polyguanylic acid and DNA were also inhibitors of RNase activity; these compounds were not hydrolyzed, but inhibited the hydrolysis of other substrates, possibly by nonproductive substrate binding. Data suggested that RNase (endonuclease) was probably the major enzyme activity in the degradation of long chain polyribonucleotides at the work's surface, while phosphodiesterase (exonuclease) activity did not contribute significantly to the hydrolysis of these compounds.

During the second ecdysis of ruminant trichostrongyles, a region of the second molt cuticle is digested by a 44-kDa Zn-metalloprotease. We have examined this digestion process by light and scanning electron microscopy (SEM). The substrate region of the cuticle appeared, during the ecdysis process, as an indented ring at the 20th cuticular annulus coincident with the anterior terminus of the lateral alae. Continued digestion of the cuticle resulted in holes in the ring region that expanded until they became continuous and separation occurred between the anterior and posterior portions of the cuticle. Mechanical movements of the L3 forced aside the cuticle cap that generally remained attached on one side to the posterior portion as the larva escaped from the sheath. The site of secretion of the 44-kDa ecdysing enzyme causing cuticle digestion was not clear from morphological observations; however, existing evidence strongly points to the release of enzyme from the esophageal (pharyngeal) glands through the mouth.

Crude and immunoaffinity-purified excretory-secretory antigens derived from a domestic pig isolate of Trichinella spiralis were used in an enzyme-linked immunosorbent assay to test serum from mice infected with 25 different pig and wild animal isolates of T. spiralis sspp. All of the sera were found positive by ELISA using either of the antigen preparations, indicating all isolates shared certain antigen epitopes. Excretory-secretory antigens were prepared from 3 distinct isolates of T. spiralis sspp.--Trichinella spiralis spiralis (pig isolate), Trichinella spiralis nativa (polar bear isolate), and Trichinella spiralis pseudospiralis--and compared by electrophoresis and monoclonal antibody binding. While protein profiles varied among the isolates, a monoclonal antibody recognizing a major immunodiagnostic antigen epitope bound all 3 antigen preparations. However, this antigen epitope occurred on different molecular weight excretory-secretory proteins from the different isolates.

Crude and immunoaffinity-purified excretory-secretory antigens derived from a domestic pig isolate of Trichinella spiralis were used in an enzyme-linked immunosorbent assay to test serum from mice infected with 25 different pig and wild animal isolates of T. spiralis sspp. All of the sera were found positive by ELISA using either of the antigen preparations, indicating all isolates shared certain antigen epitopes. Excretory-secretory antigens were prepared from 3 distinct isolates of T. spiralis sspp.- Trichinella spiralis spiralis (pig isolate), Trichinella spiralis nativa (polar bear isolate), and Trichinella spiralis pseudospiralis-and compared by electro- phoresis and monoclonal antibody binding. While protein profiles varied among the isolates, a monoclonal antibody recognizing a major immunodiagnostic antigen epitope bound all 3 antigen preparations. However, this antigen epitope occurred on different molecular weight excretory-secretory proteins from the different isolates. Improved immunodiagnostic tests have been developed for the detection of swine trichinel

ABSTRAcr: During the second ecdysis of ruminant trichostrongyles, a region of the second molt cuticle is digested by a 44-kDa Zn-metalloprotease. We have examined this digestion process by light and scanning electron microscopy (SEM). The substrate region of the cuticle appeared, during the ecdysis process, as an indented ring at the 20th cuticular annulus coincident with the anterior terminus of the lateral alae. Continued digestion of the cuticle resulted in holes in the ring region that expanded until they became continuous and separation occurred between the anterior and posterior portions of the cuticle. Mechanical movements of the L3 forced aside the cuticle cap that generally remained attached on one side to the posterior portion as the larva escaped from the sheath. The site of secretion of the 44-kDa ecdysing enzyme causing cuticle digestion was not clear from morphological observations; however, existing evidence strongly points to the release of enzyme from the esophageal (pharyngeal) glands through the mouth.

Prior infection of rodents or pigs with Trichinella spiralis results in nearly complete immunity to challenge infection (Culbertson, 1942, Journal of Parasitology 28: 197-202; Murrell, 1985, Experimental Parasitology 59: 347-354). The expression of this immunity is characterized by accelerated expulsion of adult or preadult parasites, stunting of the growth of enteral stages, a reduction in female worm fecundity, and failure of newborn larvae to survive and establish in muscle tissue (Despommier et al., 1977, Parasitology 74: 109-119; Wakelin and Wilson, 1980, International Journal of Parasitology 10: 37-41). These different expressions of immunity against challenge can be induced by abbreviated primary infections. For example, James and Denham (1975, Journal of Helminthology 49: 43-47) demonstrated that exposure of mice to only the intestinal stage of the parasite produced high levels of resistance to oral challenge, but failed to protect against intravenous challenge with newborn larvae. Conversely, exposure to the parenteral stages resulted in good protection against intravenous challenge but was less effective against oral challenge (James et al., 1977, Journal of Parasitology 63: 720-723). Antigens, derived from various stages of T. spiralis, likewise have been demonstrated to induce protective immunity. With one exception (Chipman, 1957, Journal of Parasitology 43: 593598), these antigens have been derived from the muscle larvae (L1), probably due to the ready availability of large numbers of worms (see Silberstein, 1983. In Trichinella and trichinellosis, W. C. Campbell (ed.). Plenum Press, New York, pp. 309-334). While most antigen fractions derived from the muscle larvae induce protective immunity, this protection is only partial, resulting in a 60-80% reduction in muscle larvae accumulating from a challenge infection and has not resulted in the nearly complete immunity characteristically induced by primary infections with living parasites. With a renewed interest in control methods for swine trichinosis, recent studies have tested the efficacy of antigen fractions derived from muscle larvae as vaccines for pigs (Murrell and Despommier, 1984, Veterinary Parasitology 15: 263-270). These experiments have shown significant, but again partial, levels of protection against challenge infection. Because of the limited levels of protection induced using antigens derived from muscle larvae and the potential for additive effects of multiple stage antigens due to stage-specific immunity (Bell et al., 1979, Experimental Parasitology 47: 140-157), we sought to examine the immunization efficacy in mice of crude antigen preparations from muscle larvae and adult worms and the relationship between immunization with these antigens and adult worm expulsion, female worm fecundity, and muscle larvae burdens. The parasite used in this study was the Beltsville pig strain of T. spiralis maintained by serial passage in female Sprague-Dawley rats. Larval excretory-secretory (ES) products were recovered from the muscle larvae stage of T. spiralis by maintenance of worms in Dulbecco's minimum essential medium for periods of 18 to 20 hr as described by Gamble et al. (1983, Veterinary Parasitology 13: 349-361). A crude saline extract of 3-day-old adult worms was prepared by homogenization in a Potter-Elvejin tissue grinder at 4 C followed by centrifugation at 20,000 g (Gamble and Graham, 1984, American Journal of Veterinary Research 45: 67-73). Protein concentration of antigens was determined spectrophotometrically by absorbance at 280/260 nm; antigen doses were expressed as total protein. Outbred female Swiss-Webster mice 2 to 3 mo old were immunized 3 times at biweekly intervals by intraperitoneal injection of antigen or saline emulsified in complete Freund's adjuvant (CFA). One week after the final immunization all treatment groups were challenged with 150 infective T. spiralis muscle larvae. To assess accelerated expulsion of adult worms and female worm fecundity mice were killed and parasites

The isolated, brush-border membrane of Hymenolepis diminuta contained an enzyme which hydrolyzed phosphodiester bonds. This enzyme appeared to be a Type I phosphodiesterase (E. C. 3.1.4.1) (produces nucleoside 5'-phosphates) and had no activity against synthetic, Type II phosphodiesterase substrates (mononucleotides substituted at the 3' position). The effects of various potential inhibitors of enzymatic activity, and cation requirements of this enzyme, demonstrated a distinct difference between the phosphodiesterase and alkaline phosphatase activities of the isolated, brush-border membrane. SDS-polyacrylamide gel electrophoresis of the isolated membrane preparation, followed by localization of phosphodiesterase activity in the gels, indicated the enzyme had a molecular weight of approximately 87,000. Thus, the phosphodiesterase activity represents a previously undescribed, membrane-bound enzyme of the brush-border of Hymenolepis diminuta.

Plasma membrane from the brush border isolated from the tegument of Hymenolepis dimi- nuta contains membrane-bound ribonuclease (RNase) and alkaline phosphatase activities. RNase (yeast RNA substrate), alkaline phosphatase (p-nitrophenyl phosphate substrate), and additional membrane pro- teins were solubilized by sonication or treatment with the detergents dodecyl trimethylammonium bro- mide, /3-octyl-D-glucopyranoside, sodium dodecyl sulfate (SDS), or ZwittergentTM 3-12 (N-dodecyl-N,N- dimethyl-3-ammonio-l-propanesulfonate). At optimal conditions, greater than 90% of both enzymes and total protein were solubilized by the latter two detergents, whereas f3-octyl-D-glucopyranoside, dodecyl trimethylammonium bromide, and sonication were only partially effective. Nonionic detergents did not solubilize the membrane effectively.