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Curing processes for pork meat in the U.S. currently require individual validation of methods to demonstrate inactivation of Trichinella spiralis, a nematode parasite historically associated with pork. However, for protozoan parasites, no such strictures exist. It has been assumed, with little evidence, that curing processes required to inactivate Trichinella also inactivate Toxoplasma gondii. Currently no model of meat chemistry exists that can be correlated with inactivation of T. gondii. Given the possibility of the presence of T. gondii in pork meat, and the frequent use of pork for ready-to-eat (RTE) products not intended to be cooked, curing methods which inactivate T. gondii early in the curing process would be of great value to producers. In this study, we tested the effect of five variables – salt/brine concentration, water activity (aw), pH, temperature, and time on inactivation of T. gondii bradyzoites in tissue cysts using low and high endpoints for common curing treatments during preparation of dry cured pork sausage. Survival of T. gondii bradyzoites at each stage of preparation was assessed using a mouse bioassay. Results indicated that encysted T. gondii bradyzoites do not survive the early stages of the dry curing process within the endpoint parameters tested here, even at levels of NaCl that are lower than typically used for dry curing (1.3%). Exposure of T. gondii encysted bradyzoites to curing components in the formulated batter resulted in rapid inactivation of bradyzoites. These data suggest that the use of dry curing components may be effective for controlling T. gondii potentially transmitted through RTE meats, rendering them safe from risk with respect to T. gondii transmission to human consumers., Highlights • We have investigated salt/brine concentration, water activity (αw), pH, temperature, and time- for their effect on inactivation of Toxoplasma gondii bradyzoites in tissue cysts in pork meat during the dry cure process used to make pepperoni sausage. • NaCl at 1.3% or above is effective at rapidly inactivating bradyzoites within tissue cysts in ground pork during the fermentation process. • The treatments reliably predict inactivation of Toxoplasma gondii, and can be extrapolated to various curing processes within the high and low endpoints tested to eliminate the need for individual ready-to-eat product validation.

Natural infection with Trichinella has been described in more than 150 mammalian species. However, few reports of Trichinella infection in wild animals have come from Argentina. In this study, muscle tissue was obtained from wild animals in Argentina with the aim of evaluating the presence of Trichinella. A total of 169 muscle samples were collected to determine the presence of Trichinella larvae by artificial digestion. The 169 muscle samples originated from 12 species including 36 opossums (Didelphis albiventris), 19 armadillos (Chaetophractus villosus), 9 capybaras (Hydrocaeris hydrocaeris), 1 puma (Puma concolor), 3 grey fox (Lycalopex gymnocercus), 6 coypus (Myocastor coypus), 6 skunks (Conepatus chinga), 2 ferrets (Galictis cuja), 66 rats (Rattus norvegicus), 6 mice (Mus musculus), 12 wild boars (Sus scrofa), and 3 wild cats (Felis geoffroyi). Trichinella infection was detected in 1 puma [2 larvae per gram (LPG)], 3 wild boars (8-420 LPG), 3 armadillos (0.04-0.08 LPG), and 9 rats (0.1 to 150 LPG). Only 3 Trichinella isolates, of 1 rat and 2 wild boars from Neuquén, were identified as Trichinella spiralis by nested PCR. The presence of Trichinella infection among wild animal populations suggests a sylvatic cycle of transmission in Argentina, which can serve as a reservoir for humans and domestic animals. Further, evidence of high prevalence in rats emphasizes the need to improve pig management, mainly in small individual farms without adequate technology, to enhance the quality of feeds, and to improve veterinary services to avoid exposure of pigs to Trichinella.

Recently, there has been interest in programs that certify pork production practices that minimize the risk of exposure of pigs to Trichinella spiralis. Certification might be useful for reducing the risk of human trichinellosis from pork in Argentina, but more information is needed on pig production practices and sources of Trichinella infection in Argentinian pigs. In this study, 21 pig farms were assessed for Trichinella infection including some farms using total and partial confinement management, and others with pigs raised exclusively outdoors. A total of 3224 muscle samples were collected from pigs raised on these farms and tested to determine the presence of T. spiralis larvae by artificial digestion. Serum samples from the same 3224 pigs were tested for antibodies to T. spiralis by ELISA. For each farm, a questionnaire was completed summarizing information about management factors and this information was used to assess risk factors for exposure of T. spiralis. Based on the results, pigs raised outdoors were more likely to be infected than pigs raised in total or partial confinement (por =0.05). Pigs fed waste products containing meat were 12.5 times more likely to be infected than pigs not fed waste containing meat (p0.01). The role played by rats in transmission of Trichinella is unclear; however, on farms with evidence of wild animals and access of pigs to wildlife carcasses, the prevalence of Trichinella infection was significantly higher. All pigs raised under good hygienic and sanitary conditions were negative for Trichinella infection by both artificial digestion and ELISA.

Two enzyme immunoassays (EIA) for trichinellosis in swine (a research version and a commercial kit obtained from LMD Agro-Vet) were used to test pig whole blood, serum, and tissue fluids as sample sources. Five pigs inoculated with 2,500 Trichinella spiralis were bled weekly and then sacrificed after 42 days. Blood and serum samples from each sampling time and tissue fluids collected at slaughter were tested in both EIA formats. Whether samples were undiluted or diluted, blood could be used as effectively as serum for detecting pigs infected with T. spiralis . Further, it was as effective to use tissue fluids as either blood or serum. With both tests, the research and commercial versions, it is possible to detect infected pigs by 28 days following infection. The results of this study suggest that a rapid EIA test such as the LMD Agro-Vet Trichinella Serology Microwell ELISA can be used as an effective tool for testing pigs for trichinellosis on the farm. Additionally, because it can enable the detection of antibodies in tissue fluids, this test could be used effectively to determine trichinellosis in meat samples following slaughter.

The objective of this study was to demonstrate the reliability of current and proposed methods for the inspection of swine and other species for infection with the parasite Trichinella spiralis. Five groups of pigs were infected with doses of 2500, 500, 100, 50, and 20 T. spiralis larvae to establish moderate and low-level infections. Pigs were bled periodically during the study for samples to be tested by enzyme immunoassay (EIA). At the conclusion of the study, pigs were slaughtered and tissues collected for analyses of worm burdens and for comparison of digestion testing methods. Comparisons of pooled sample digestion methods were made using inspection methods prescribed by European Union Directives and the USDA, Code of Federal Regulations. Pooled sample digestion testing using 1-g samples was effective for detecting pigs with larval densities of10 larvae per gram (LPG) of tissue but only partially effective for pigs with infections of3 LPG. Pooled sample digestion testing using 5-g samples detected all pigs with infection levels1 LPG. The EIA detected all T. spiralis-infected pigs, but did not detect infections in some pigs until 49 days after inoculation. These results demonstrate that the pooled sample digestion method using a 5-g sample size is the most effective inspection method for reducing the risk of human exposure to T. spiralis in pork.

This research was carried out to determine the time/temperature exposure of Trichinella spiralis to freezing conditions necessary to destroy the infectivity of the trichinae. Experimentally infected pork was subjected to temperatures of -1 to -193°C for one sec to 182 d and the treated pork samples, which contained about 1000 larvae per gram of tissue, were subjected to rat bioassay to determine infectivity of the larvae. A linear regression equation, log10t = 5.98 + 0.40T where t = required inactivation time in hours and T = temperature in degrees Celsius, described the exposure necessary to destroy the trichinae. The correlation for that relationship was r = 0.942. The predicted thermal death times (+7 min) at -20, -15, and -10°C were 8 min, 64 min, and 4.0 d, respectively. The predicted upper confidence limits (99%) for the thermal death times (+7 min) for exposure at -20, -15, and - 10°C, were 48 min, 63 h, and 266 d, respectively. These data provide a continuum of definitive times and temperatures necessary to destroy T. spiralis by freezing and are of value to the meat industry and the regulatory agencies.

Treatment by hydrodynamic pressure is an attractive alternative for meat tenderization and might also have an effect on foodborne pathogens. Numbers of Trichinella spiralis recovered from infected pork were significantly reduced by treatment with the Hydrodyne process, as compared with untreated, infected pork. However, treatment with the hydrodynamic force described in this paper (55 to 60 MPa) did not eliminate the infectivity of this parasite when the larvae from Hydrodyne-treated meat were inoculated into mice.

L'utilisation de tests serologiques pour le diagnostic de l'infection a Trichinella chez les animaux domestiques et sauvages et chez l'homme n'a pas ete standardisee juqu'a present. Cette revue fournit un ensemble de recommandations pour le developpement et l'utilisation de tests serologiques pour detecter les anticorps circulants dans les echantillons de serum. Les recommandations sont basees sur les meilleures informations scientifiques publiees et sur des donnees non publiees issues des laboratoires qui possedent une grande experience dans ce domaine. Elles representent la position officielle de la Commission internationale sur la trichinellose a propos des methodes acceptables et de l'evaluation de leur specificite et sensibilite. Ces recommandations sont susceptibles d'etre modifiees en fonction de nouvelles donnees scientifiques.

Vertical migration by infective larvae (L3) of 3 species of nematodes was investigated. Upright truncated agarose cones were used to test upward migration, and comparable truncated cone-shaped agarose hollows were used to test downward migration. Flat agarose plates were control surfaces. When placed at the bases of upright cones, 74% of Ancylostoma caninum L3 migrated up, whereas only 16.5% migrated down the indented cones; this latter value was not significantly different from larval migration on flat plates. Strongyloides stercoralis L3 also migrated upward in significant numbers (80%). These larvae also failed to migrate downward under normal conditions. However, when the bottoms of the indented cones were 3-5 degrees C warmer than the tops, 75.5% of S. stercoralis L3 migrated down. In contrast, Haemonchus contortus L3 showed no tendency to crawl up or down cones, when compared with normal crawling behaviour on flat plates. Thus, L3 of A. caninum and S. stercoralis exhibited negative geotaxis, crawling against the pull of gravity, while H. contortus did not. The biology of these parasites may explain these behavioural differences.

As a basis for studies of thermal behavior of infective larvae (L3) of Haemonchus contortus resulting from ablation of amphidial neurons, the locations of the amphidial cell bodies in the hatchling larva (L1) were compared with their locations in the L3. We sought to verify that killing each targeted cell body in L1 destroys the putative corresponding dendrite of the L3. These comparisons confirmed the predicted cell body-to-dendrite connections, as well as similarities in the general amphidial structure of the two stages. We then conducted a series of studies using laser microbeam ablation of amphidial cell bodies in the L1 to determine the role of specific neurons in the thermal behavior of the L3. In a thermal gradient, normal L3 of H. contortus migrate to the temperature at which they were cultured and/or maintained. Larvae grown at 16 degrees or 26 degrees C migrate appropriately to either of these temperatures. Larvae grown to the L3 stage at 16 degrees C and then moved to 26 degrees C become acclimated to this temperature and thereafter migrate to it. However, when the putative thermosensory neurons, the finger cell neurons (AFD), were ablated in hatchling larvae with a laser microbeam, and these were grown to the L3 stage and tested on a radial thermal gradient, they failed to migrate to their culture temperature. Instead, they moved actively and continuously over much of the assay plate surface, with no obviously oriented cryo- or thermotactic movement. Ablation-control larvae, those in which putatively chemosensory neuron classes ASE or AWC were killed, migrated normally to their culture temperature. When the RIA interneurons (identified by positional homology with those of Caenorhabditis elegans) were ablated, the operated larvae moved actively, but circled near the initial placement point; control larvae, in which other nonamphidial neurons were killed, migrated normally. These results indicate that the finger cell neurons (AFD) are the primary thermosensory class in H. contortus. The RIA-class neurons integrate thermal responses in H. contortus, as do their putative structural homologs in C. elegans, but the behavior of H. contortus subsequent to RIA ablation is strikingly different.

When infective larvae of Haemonchus contortus (a highly pathogenic, economically important, gastric parasite of ruminants) are ingested by grazing hosts, they are exposed to environmental changes in the rumen, which stimulate resumption of development. Presumably, resumption is controlled by sensory neurons in sensilla known as amphids. Neuronal function can be determined by ablation of specifically recognized neurons in hatchling larvae (L1) in which neuronal cell bodies are easily visualized using differential interference microscopy. Using three-dimensional reconstructions from electron micrographs of serial transverse sections, amphidial structure of the L1 is described. Each amphid of H. contortus is innervated by 12 neurons. The ciliated dendritic processes of 10 neurons lie in the amphidial channel. Three of these end in double processes, resulting in 13 sensory cilia in the channel. One process, that of the so-called finger cell, ends in a number of digitiform projections. Another specialized dendrite enters the amphidial channel, but leaves it to end within the sheath cell, a hollow, flask-shaped cell that forms the base of the amphidial channel. Although not flattened, this process is otherwise similar to the wing cells in Caenorhabditis elegans; we consider it AWC of this group. Two other neurons, ASA and ADB, appear to be homologs of wing cells AWA and AWB in C. elegans, although they end as ciliated processes in the amphidial channel, rather than as flattened endings seen in C. elegans. Each of the 12 amphidial neurons was traced to its cell body in the lateral ganglion, posterior to the worm's nerve ring. The positions of these bodies were similar to their counterparts in C. elegans; they were named accordingly. A map for identifying the amphidial cell bodies in the living L1 was prepared, so that laser microbeam ablation studies can be conducted. These will determine which neurons are involved in the infective process, as well as others important in establishing the host-parasite relationship.

The importance of foodborne parasitic zoonoses remains high in many regions of the world. Although control efforts have been exerted for quite some time, overall progress has not been satisfactory, even in many well developed countries. An important drawback in control programmes for parasites such as Trichinella, Toxoplasma and Taenia has been the absence of rapid, accurate and sensitive diagnostic tests for these meatborne parasites. However, the rapid advances in the molecular biology of these organisms has yielded concomitant gains in precision of detection. This review highlights these advances and their impact or potential application to the control of foodborne parasites.

Studies in mice infected with the gastrointestinal nematode parasite Nippostrongylus brasiliensis demonstrated that IL-4/IL-13 activation of Stat6 suppresses development of intestinal mastocytosis and does not contribute to IL-4/IL-13 production, but is still essential for parasite expulsion. Because expulsion of another gastrointestinal nematode, Trichinella spiralis, unlike N. brasiliensis expulsion, is mast cell dependent, these observations suggested that T. spiralis expulsion would be Stat6 independent. Instead, we find that Stat6 activation by IL-4/IL-13 is required in T. spiralis-infected mice for the mast cell responses that induce worm expulsion and for the cytokine responses that induce intestinal mastocytosis. Furthermore, although IL-4 induces N. brasiliensis expulsion in the absence of B cells, T cells, and mast cells, mast cells and T cells are required for IL-4 induction of T. spiralis expulsion. Thus, Stat6 signaling is required for host protection against N. brasiliensis and T. spiralis but contributes to expulsion of these two worms by different mechanisms. The induction of multiple effector mechanisms by Stat6 signaling provides a way for a cytokine response induced by most gastrointestinal nematode parasites to protect against most of these parasites, even though different effector mechanisms are required for protection against different nematodes.

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.

Single muscle larvae (ML) of Trichinella belonging to 5 species and 2 genotypes collected from 126 experimentally infected pigs and preserved in 75% ethyl alcohol at room temperature were identified by random amplified polymorphic DNA (RAPD) analysis to evaluate the specificity and sensitivity of this assay. A double-blind protocol was used to ensure that the interpretation of electrophoretic patterns after RAPD amplification was not influenced by an expected result. RAPD analysis of one larva from each sample showed a sensitivity of 100% when only samples with undamaged DNA were considered, and the specificity resulted in an 88% match with the species or genotype that had been used to infect pigs, whereas the identification reached 100% specificity when an additional one to four ML were examined.

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.

Forty-seven pigs were infected with varying doses of Trichinella spiralis and tested for evidence of infection by serology, using an enzyme immunoassay (EIA), and by artificial digestion methods. Using a 1-g sample, as prescribed in accordance with European Union (EU) directives, the sensitivity of the pooled-sample artificial digestion method was between three and five larvae per gram (LPG) of tissue. Using a 5-g sample size, in accordance with methods described in the U.S. Code for Federal Regulations, and as required for the inspection of horses exported to the EU, the sensitivity of the test was increased to approximately 1 LPG. Serological testing by EIA detected pigs with as few as 0.02 LPG, but detection times varied from 4 to 8 weeks after infection. Mean postinoculation times for detection by serology were 32 to 42 days. On the basis of these results, it is clear that digestion testing using a 5-g sample size is the only method of those tested here that is completely reliable for detection of trichinae infection at a level that will protect public health. Both digestion testing using a 1-g sample and EIA have drawbacks. However, EIA remains a highly effective tool for epidemiological purposes and for monitoring trichinae infection on the farm.

To assess performance of a synthetic carbohydrate antigen for use in an enzyme immunoassay for diagnosis of trichinellosis in swine. ANIMALS AND SAMPLE POPULATION: 47 York X Duroc pigs and field sera from 265 farm pigs raised under various management systems.Each of 47 pigs was inoculated with 25 to 500 Trichinella spiralis larvae, and blood samples were obtained weekly. At postinoculation week 12, pigs were euthanatized, and tissues recovered from the tongue and diaphragm were digested to determine worm burden. Serum samples from experimentally infected pigs and sera obtained from pigs on a farm were tested by enzyme immunoassay, using standard excretory-secretory and synthetic glycan antigens.Of the 47 pigs, 46 (97.8%), with worm burden ranging from 0.02 to 248.8 larvae/g of tissue in the tongue and diaphragm, tested seropositive using both antigens. Time of seroconversion varied among pigs and was negatively correlated with intensity of infection. Minor differences were observed in time of seroconversion between the 2 antigens in 11 of 46 pigs, suggesting some differences in the antibody response. One pig with a worm burden of 0.01 larva/g was not detected by enzyme immunoassay using either antigen. Background values obtained using the 2 antigens did not differ among confinement-raised pigs, but background values for pigs raised outdoors on dirt lots were significantly lower using the glycan antigen Low-level, naturally acquired T spiralis infections in pigs were detected in most instances by use of both antigens, although there were some differences in antibody responses of infected pigs.The synthetic glycan antigen has potential for replacing excretory-secretory antigens as a standardized reagent for diagnosis of trichinellosis in pigs.

Three parasites pose a public health risk from the ingestion of raw or undercooked pork, namely: Trichinella spiralis, Taenia solium and Toxoplasma gondii. Inspection procedures, when practised according to prescribed methods, are effective in eliminating the majority of risks from T. spiralis and T. solium. No suitable methods for the post-slaughter detection of T. gondii are available. All three parasites are inactivated by various methods of cooking, freezing and curing; some information is also available on inactivation by irradiation. Good production practices, including a high level of sanitation, rodent and cat control on farms, can prevent opportunities for exposure of pigs to these parasites. Alternatively, meat inspection, proper commercial processing and adherence to guidelines for in-home preparation of meat are effective methods for reduction of risks for human exposure.

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.

Eighteen female lambs with prior exposure to Haemonchus contortus infections were ovariectomized and assigned to 1 of 3 replacement regimens: 0, 25, or 250 mg of progesterone/d delivered IM. After 3 weeks of hormonal treatment, all lambs were inoculated with 100,000 infective larvae of H contortus. After 8 weeks of hormonal treatment, a blastogenic assay was performed on blood lymphocyte populations, and the abomasum from each lamb was obtained for larval and adult worm recoveries of H contortus. Lambs of the 25 mg of progesterone group had significantly (P0.05) reduced blastogenic response to concanavalin A and greater adult and larval populations, compared with controls. Lambs of the 250 mg of progesterone group had worm burdens and lymphocyte blastogenesis values intermediate between those of the other treatment groups.

First-stage larvae of Protostrongylus rufescens were detected in the feces of a ram with diarrhea, anorexia, weight loss, and mucopurulent nasal discharge. Subsequently, a survey was initiated in the experimental sheep flocks maintained at the Agricultural Research Service's Helminthic Diseases Laboratory in Beltsville, Md to determine the extent and effect of infection with the parasite. Five of 31 ewes and 4 of 16 rams were found to be infected with the lungworm. Clinical signs of infection, which ranged from mild to severe, included diarrhea, weight loss, and respiratory distress. Natural transmission of the parasite to parasite-naive lambs grazing on pasture occurred during the course of this study. Three lambs born to infected ewes during this study were not infected, suggesting that there is no vertical transmission. Additionally, cattle grazing the same pastures as infected sheep were not infected with the parasite. Results of this study indicated that Protostrongylus rufescens can cause serious disease in domestic sheep in the United States and should be considered as a differential diagnosis in sheep with nonspecific respiratory tract signs.

1. By transmission electron microscopy, the eggshell of Haemonchus contortus was seen to be similar to previously studied nematodes, with an outer vitelline layer bounded by a trilaminate membrane, a broad medial region, containing chitin, and an electron dense basal region, containing lipid and protein. 2. Exposure of Haemonchus contortus eggs to proteases resulted in disruption of the shell with removal of components of the outer, medial and basal regions. Exposure to chitinase depleted fibrillar components of the medial region of the shell, while collagenase had no effect. 3. Chloroform/methanol extraction of fresh eggshells caused a minor condensation of the outer, vitelline layer and some depletion of the basal layer. 4. After normal hatching, shells appeared similar to those treated with protease and chitinase, but also lacked the basal, lipid layer. 5. Extracts of isolated unhatched eggshells and hatched eggshells, and extracts of biotin-labelled whole fresh eggs showed three major protein bands when run on sodium dodecyl sulphate-polyacrylamide gels indicating that these three proteins are most likely structural in nature and do not participate in the release of the larva from the eggshell. 6. Biotin-labelled protein bands were degraded by proteases and chitinase, but not collagenase or lipase.

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.

Vaccination against animal parasites offers an alternative to chemotherapy for the control of losses due to morbidity and mortality. However, only a few vaccines are currently available, and these are based on controlled infections with living parasites. Further advancement in the development of defined vaccines against parasite infections has been hindered by incomplete knowledge of the immunological relationship between the host and the parasite. The advent of monoclonal antibody technology has provided a powerful new tool for the identification and isolation of parasite antigens. Exploitation of this technique in veterinary parasitology has greatly facilitated progress toward the development of vaccines against several animal parasites.

The passage of non-Toxoplasma antibodies from dam to fetus through damaged placenta was studied in sheep inoculated with Toxoplasma gondii. Six ewes were inoculated with chicken globulins and Leptospira bacterins 2 months before oral inoculation with Toxoplasma gondii oocysts. Ewes were euthanatized between 42 and 62 days after T gondii inoculation. Antibody titers against chicken globulins, Leptospira spp, Haemonchus contortus, Sarcocystis spp, and T gondii were measured in the maternal and fetal sera. All ewes became infected with T gondii and had grossly visible necrotic foci in the placentas, and T gondii antibodies were found in the fetuses and the ewes. Appreciable amounts of antibodies to Haemonchus contortus, Sarcocystis sp, Leptospira spp, and chicken globulins did not cross the placental barrier. Seemingly, serologic examination of the fetus was reliable for the diagnosis of ovine congential toxoplasmosis.

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.

A field evaluation of an enzyme-linked immunosorbent assay (ELISA) for swine trichinosis was done with sera obtained from 5 herds experiencing ongoing transmission of Trichinella spiralis. Epizootiologic studies conducted on these herds offered an opportunity to evaluate the accuracy of an ELISA, using larval T spiralis excretory-secretory antigens. Sera from 162 infected pigs and 143 serum samples from noninfected pigs originating from the same farms were tested. The infection status of the pigs was determined by digestion of diaphragm or tongue muscle samples. Two criteria were established to classify the ELISA optical density (OD) readings: Criterion I stated that an OD greater than or equal to 5 times the mean OD of several normal swine sera pools was positive; criterion II stated that a OD greater than or equal to 4 times the normal sera values was positive. The results obtained did not reveal obvious serologic variations among infected herds located in the 4 states involved. Overall, the test detected 93% (criterion I) and 96% (criterion II) of infected pigs. The majority of false-negative sera was from hogs that had less than 5 larvae/g of muscle; 1 hog had 73.8 larvae/g of diaphragm muscle. The false-positive rates were 8% for criterion I and 9% for criterion II. The actual rate for these false-positive samples may have been overestimated, because generally, only small tissue samples (0.4 to 10 g) were digested; larger sample sizes might have altered the results. The relevance of this qualification is that these pigs originated from herds with prevalence rates greater than 50%. Other factors that may account for occasional false-positive sera or false-negative sera in the swine trichinosis ELISA are discussed.(ABSTRACT TRUNCATED AT 250 WORDS)

SUMMARYAromatic amino acids (phenylalanine, tryptophan and tyrosine) are absorbed by Hymenolepis diminuta through a combination of mediated (non-Na+-sensitive) transport and diffusion. All 3 amino acids are accumulated against an apparent concentration difference during a 30-min incubation of tapeworms in 0·1 mM 3H-labelled amino acid. Inhibitor studies demonstrate that phenylalanine, tryptophan and tyrosine are mutually competitive inhibitors of the uptake of each other, and the uptake of these amino acids is inhibited by aliphatic amino acids but not by basic or dicarboxylic amino acids. The D- and L-isomers of aromatic amino acids are equally effective in inhibiting aromatic amino acid uptake. The data indicate that at least 3 amino acid transport loci are involved in aromatic amino acid transport by H. diminuta.

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.

Three experiments were conducted to investigate experimentally the occurrence of periparturient nematode egg rise in ewes and the hormonal modulation of Haemonchus contortus infections. In the first experiment, fall-bred and winter-bred pregnant (n = 4 and 14, respectively) and nonpregnant (n = 5 and 29, respectively) ewes were treated with anthelmintic and were pastured together on fields that were contaminated with H contortus. Three weeks before lambing, all ewes were placed in concrete pens; fecal egg counts for the winter-bred group were obtained on alternate days. Pregnant and lactating ewes had significantly larger numbers (P less than 0.01) of H contortus eggs than did the nonpregnant controls 1 week before and after lambing. Lactating, fall-bred ewes had significantly (P less than 0.01) more adult worms in their abomasum through natural acquisition than the nonpregnant controls. In the second experiment, fall-bred and winter-bred, helminth-free, pregnant (n = 4 and 8, respectively) and nonpregnant (n = 3 and 15, respectively) ewes were inoculated on 5 alternate days, beginning 70 days after breeding with 20,000 infective H contortus larvae. The ewes were maintained on concrete pens throughout pregnancy. Fecal egg counts were significantly (P less than 0.05) greater in pregnant ewes, beginning 1 week before lambing until 1 week after lambing. Abomasums of lactating ewes from both lambing seasons yielded significantly (P less than 0.01) more adult worms at necropsy than nonpregnant ewes. In the third experiment, ewes were ovariectomized (n = 15) or sham-operated (n = 9); half of the control ewes were bred.(ABSTRACT TRUNCATED AT 250 WORDS)

Pigs were inoculated with Trichinella spiralis excretory-secretory products derived from short-term in vitro maintenance of infective muscle larvae. Intraperitoneal administration of excretory-secretory products in Freund's complete adjuvant or aluminum hydroxide induced moderate, but variable, degrees of immunity to challenge exposure in a nondose-dependent manner; IM administration of products in Freund's incomplete adjuvant was less successful. Inoculated pigs harbored fewer adult worms, and the fecundity of female worms (numbers of newborn larvae shed in vitro) recovered after challenge exposure was significantly lower (alpha = 0.05) than the fecundity of females recovered from control pigs. The degree of resistance in inoculated pigs was directly related to serotiter against excretory-secretory antigens, as determined in an enzyme-linked immunosorbent assay.

Monoclonal antibodies generated from spleen cells of Balb/cJ mice infected with Trichinella spiralis reacted with antigenic determinants present in some other common swine parasites, especially Trichuris suis. An antigenic determinant unique to T spiralis was recognized by one immunoglobulin M monoclonal antibody (7C2C5); this determinant was found on larval T spiralis proteins with molecular weights of 53,000, 49,000, and 45,000. These proteins were components of in vitro-derived worm excretory-secretory products. Immunoperoxidase staining techniques and reactions of the monoclonal antibody with living parasites indicated that the source of the antigen was the stichocyte. Two of the proteins (53,000 and 49,000 molecular weight) possessing this determinant were isolated by monoclonal antibody-affinity chromatography, using hybridoma 7C2C5. When used in a serologic enzyme-linked immunosorbent assay, the affinity-isolated antigen was able to completely eliminate false-positive reactions with serum from healthy swine and to detect all pigs infected with T spiralis.

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.

The development of methods for the production of monoclonal antibodies has provided a powerful tool for virtually all areas of the biological sciences. Monoclonal antibodies allow the dissection of complex mixtures mixtures of antigenic molecules; by applying screening procedures to a library of monoclonals, selection for properties such as viral neutralization or enzyme inactivation is possible, thereby identifying proteins with specific biological activities. Similarly, monoclonal antibodies can be used to map the surface of a protein, define cell surface antigens, and localize molecules within cells or tissues. The property of unique specificity associated with clonally-derived antibodies has served to revolutionize the field of diagnostics where the use of polyclonal antisera once dominated. The use of monoclonal antibodies for antigen identification has greatly benefited the study of the immune response to infectious agents and the development of vaccines. With the advent of better methods for the generation of hybridomas such as electrofusion and oncogene transfection, modifications allowing the exploitation of non-immunogenic molecules, and the development of techniques for producing interspecific hybrids, the contribution of this technology as a tool for research in the agricultural sciences will undoubtedly increase.

Nematodes of the genus Trichinella are the causative agents of trichinellosis, a potentially severe disease in humans. Raw or undercooked pork, horse and game meat (predominantly wild boar and bear) poses a health risk to consumers.

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.