Trichinella Nativa Outbreak With Rare Thrombotic Complications Associated With Meat From a Black Bear Hunted in Northern Ontario.

BACKGROUND: Although trichinellosis is known to cause thrombotic disease, serious thrombotic events are rare and have not been previously associated with Trichinella nativa infection. METHODS: Patient interviews and medical chart reviews were conducted on 10 men who became ill following consumption of a common source of black bear meat. Trichinella serology on patient sera as well as polymerase chain reaction (PCR) and larval identification of the meat samples was conducted. RESULTS: All 10 exposed individuals developed an acute illness clinically compatible with trichinellosis, characterized by fever, abdominal pain, and diarrhea, along with eosinophilia ranging from 0.9 × 109/L to 6.1 × 109/L. Within 2 weeks of the diarrheal illness, systemic symptoms developed in all exposed individuals characterized by fever, myalgia, periorbital edema, and fatigue. ST-elevation myocardial infarction and sinus venous tract thrombosis occurred as a complication of trichinellosis in 2 patients. Acute serology was nonreactive in all patients, though convalescent serology was reactive in 6 of 8 (75%) patients for whom sera was available. Multiplex PCR identified T. nativa from the bear meat, and was corroborated by microscopic larval identification. CONCLUSIONS: We report a 100% attack rate of T. nativa from bear meat among those who were exposed, and demonstrate that this species can cause serious thrombotic complications of trichinellosis in humans. Education of hunters and the public regarding the importance of proper preparation of wild game prior to ingestion is warranted.

Ultrastructural preservation of tissues and their reaction to the infection with trichinella in the El Plomo mummy: Muscle fiber ultrastructure and trichinosis/mummy of the Cerro El Plomo.

The El Plomo mummy was a pre-Columbian Incan child who was found mummified in the Andes Mountains above an altitude of 17,700 feet. In the environment, natural mummification occurred due to low temperatures and strong winds. Dating measurements (relative dating) by experts from the National Museum of Natural History of Chile established that the mummified body corresponds the Inca period (1,450 to 1,500 AD). In 2003, the body was transferred to the University of Chile Medical School for exhaustive medical examination. Tissue samples from the right quadriceps muscle were extracted and fixed in glutaraldehyde and postfixed in osmium tetroxide to obtain ultrathin sections to be observed by transmission electron microscope. Images were recorded on photographic paper, digitalized and analyzed by experts on morphology. Results showed a preservation of cell boundaries in striated muscle cells, but specific subcellular organelles or contractile sarcomeric units (actin and myosin) were unable to be recognized. However, the classical ultrastructural morphology of the polypeptide collagen type I was preserved intact both in primary and secondary organization. Therefore, we concluded that the process of natural mummification by freezing and strong winds is capable of damaging the ultrastructure of muscle cells and preserving collagen type I intact.

Trichinella pseudospiralis from a wild pig in Texas.

In December 2001, the routine inspection of a wild boar intended for human consumption revealed the presence of Trichinella ssp. larvae. Biological, morphological and genetic analyses demonstrated the parasite to be Trichinella pseudospiralis. This is the second report of T. pseudospiralis in the United States and the first report of the parasite in a food animal species in the U.S.

Monoclonal antibodies raised in Btk(xid) mice reveal new antigenic relationships and molecular interactions among gp53 and other Trichinella glycoproteins.

Tyvelose-bearing glycoproteins or Trichinella spiralis Group 1 antigens (TSL-1 antigens) are thought to be key molecules in the immunobiology of Trichinella. In the present study, we investigated the binding characteristics of several mAbs produced in Btk(xid) immunodeficient mice that recognise gp53 and some other minor glycoproteins of this parasite. The data obtained reveal the existence of an O-glycan/peptide epitope (recognised by mAb US8) common to all TSL-1 glycoproteins, as well as a specific interaction between the TSL-1 antigen gp53 and other unknown Trichinella glycoproteins in the 35-40 kDa range (these latter react with mAbs US8 and US9, but not with mAb US5). Some of the epitopes recognised by our mAbs are differentially expressed in Trichinella species: the epitope recognised by mAb US5 on gp53 (another O-glycan/peptide epitope) is present only in T. spiralis, whereas those recognised by mAbs US8 and US9 (peptide epitopes) are present in encapsulated Trichinella species. The data obtained also reveal that gp53 is synthesised and glycosylated in beta-stichocytes only. The possible relevance of these findings is discussed.

Trichinella zimbabwensis n.sp. (Nematoda), a new non-encapsulated species from crocodiles (Crocodylus niloticus) in Zimbabwe also infecting mammals.

Since 1995, Trichinella larvae have been detected in 39.5% of farmed crocodiles (Crocodylus niloticus) in Zimbabwe. Morphological, biological, biochemical and molecular studies carried out on one isolate from a farmed crocodile in 2001 support the conclusion that this parasite belongs to a new species, which has been named Trichinella zimbabwensis n.sp. This species, whose larvae are non-encapsulated in host muscles, infects both reptiles and mammals. The morphology of adults and larvae is similar to that of Trichinella papuae. Adults of T. zimbabwensis cross in both directions with adults of T. papuae (i.e. male of T. zimbabwensis per female of T. papuae and male of T. papuae per female of T. zimbabwensis), producing F1 offspring which produce very few and less viable F2 larvae. Muscle larvae of T. zimbabwensis, like those of T. papuae, do not infect birds. Three allozymes (of a total of 10) are diagnostic between T. zimbabwensis and T. papuae, and five are diagnostic between T. zimbabwensis and Trichinella pseudospiralis, the third non-encapsulated species. The percentage of the pairwise alignment identity between T. zimbabwensis and the other Trichinella species for the cytochrome oxidase subunit I gene, the large subunit ribosomal-DNA (mt-lsrDNA) gene and the expansion segment five, shows that T. zimbabwensis is more similar to the two non-encapsulated species T. papuae (91% for cytochrome oxidase I; 96% for mt-lsrDNA; and 88% for expansion segment five) and T. pseudospiralis (88% for cytochrome oxidase I; 90% for mt-lsrDNA; and 66-73% for expansion segment five) than to any of the encapsulated species (85-86% for cytochrome oxidase I; 88-89% for mt-lsrDNA; and 71-79% for expansion segment five). This is the first non-encapsulated species discovered in Africa. The finding of a new Trichinella species that infects both reptiles and mammals suggests that the origin of Trichinella parasites dates back further than previously believed and can contribute to understanding the phylogeny and the epidemiology of the genus Trichinella.

A freeze-fracture study of the surface of the infective-stage larva of the nematode Trichinella.

The surface layers of the cuticle of the infective, first-stage larva of the nematodes Trichinella spiralis and T. spiralis var. pseudospiralis have been studied by means of the freeze-fracturing technique. No obvious differences between the two nematodes were found. A double-layered structure covers the cuticle. Its outermost layer consists of particles embedded in an amorphous matrix; its inner layer is composed of a sheet of fine filaments which may be composed of globular subunits. This unique double layered structure is not like a normal cell membrane in structure. The surface of the cuticle beneath it is relatively smooth except for impressions from the inner surface of the double-layered structure. The cuticle surface did not fracture in the manner of a cell membrane.

A freeze-fracture study of muscle fibres infected with Trichinella spiralis.

The muscle fibres of mice containing the infective-stage larvae of the nematode Trichinella spiralis have been studied by means of the freeze-fracturing technique. The larva lies in what appears to be a fluid-filled cavity within the cytoplasm of an altered muscle fibre. There is no membrane separating the cytoplasm of the nurse cell from the cavity surrounding the larva which is therefore truly intracellular, unlike many parasites that reside within a membrane-lined parasitophorous vacuole within the host cell. This altered muscle fibre, known as a nurse cell, lacks myofilaments but does contain extensive cisternae of endoplasmic reticulum; membrane-bound vesicles are budded off from the endoplasmic reticulum and traverse the cytoplasm towards the cavity containing the nematode where they apparently pass into the cavity. It is suggested that the contents of these vesicles are used to sustain the nematode. Attention is drawn to the similarity to giant cells that have been induced by the plant-parasitic nematode Meloidogyne in the roots of host plants and which sustain the nematode. The conversion of the muscle fibre into a nurse cell is probably brought about by the presence of a metabolic sink, the larval nematode, within the cell. This take-over of the control of a metazoan cell by another metazoan organism is most unusual and warrants further study.

A freeze-fracture study of the digestive tract of the parasitic nematode Trichinella.

Freeze-fracture preparations of the esophagus and intestine of larvae and adults of the nematode Trichinella spiralis illustrate the distribution of intramembranous particles in membranes of a number of cell types, and several specializations were found. Esophageal glands are prominently linked by gap junctions, but gap junctions were not found between intestinal cells. Muscle cells of the esophagus have rectilinear arrays of particles, thought to be points of adherence of the muscles to the esophageal epithelium. Clusters of particles are associated with these arrays and particle-free areas (probably Z bodies) also occur. Intestinal cells have small particles in their microvilli, large particles in the cells' apical membranes, and intermediate size particles, similar to membranes of other cells, in the lateral and basal membranes. Apical smooth septate junctions and tricellular junctions occur between intestinal cells.

A freeze-fracture study of the body wall of adult, in utero larvae and infective-stage larvae of Trichinella (Nematoda).

The surface layers of the cuticle, the hypodermal membranes and the muscle membranes of the adult, the in utero larvae and the infective-stage larvae of the nematode Trichinella spiralis have been studied by means of the freeze-fracturing technique. The surface of the cuticle of both adults and larvae fractures in ways different from membranes of internal cells. The surface coat on top of the epicuticle is probably the layer that changes antigenically. Reticulate ridges, with associated particles, on the E face of the outer hypodermal membrane of the adult are probably sites of attachment of the hypodermis to the cuticle. Longitudinally arranged ridges, with associated particles, of the outer hypodermal membrane are probably points of attachment to the cuticle in the in utero and infective larvae. Rectilinear arrays of particles are present on the P face of the inner hypodermal membrane and the P face of the muscle membrane adjacent to the hypodermis of adults and larvae and probably play a role in adhesion of the muscle membrane to the hypodermis. Particle-free areas of membrane lie external to the Z bundles of the muscle cell and are similar to the sites of attachment of Z lines in insect muscles.

Immunocytolocalization study of the external covering of Trichinella spiralis muscle larva.

The antibody-binding sites of the muscle larva of Trichinella spiralis were investigated by immunogold staining on the ultrathin sections of LR white resin. The antibodies, which were produced in the course of T. spiralis infection in rats, specifically bound to the inner layers of the body cuticle and the cuticle of the hindgut, but not to the cuticle of the esophagus. This is the first report that reveals the antigenic nature of the inner layers of the external coverings of T. spiralis larva.

Trichinella spiralis: an intracellular parasite in the intestinal phase.

Trichinella spiralis has been examined by electron microscopy after fixation in situ in the intestine of mice. The worms lie within the cytoplasm of cells of the intestinal mucosa and may occupy both absorptive and goblet cells. They cause little damage to host cells. A few worms have been seen protruding from tissue by SEM techniques. These unusual observations suggest that the nematodes may be capable of exit and reentry into the epithelium.

The stichosome and its secretion granules in the mature muscle larva of Trichinella spiralis.

The stichosome of the mature muscle larva of Trichinella spiralis consists of a single row of 45 to 55 stichocytes. Each stichocyte is about 25 mum in diameter and possesses a single nucleus. A duct leads from each stichocyte to the lumen of the esophagus. The stichocyte cytoplasm contains mitochondria, structures resembling Golgi-complexes, rough endoplasmic reticulum, and usually 1 of 2 types of secretory granules. The alpha-granule measures about 800 mn in diameter, contains a prominent inclusion, and has a granular matrix. The beta-granule is about 600 mn in diameter and is homogeneous in appearance. Both granule types are surrounded by a single membrane. Ten to thirteen stichocytes containing alpha-granules are confined to the posterior portion of the stichosome. After isopycnic centrifugation in sucrose gradient of large granule fractions obtained from cell-free homogenates, the alpha- and beta-granules show characteristic distribution patterns as revealed by the morphology of the fractions. The median equilibrium density of the alpha-granules is 1.245, while that of the beta-granules is 1.230. There is a correlation between the distribution of the granules and of antigens reacting with hyperimmune antitrichinella seruma. At least 4 unique antigens can be attributed to each of the granule types. Fractions enriched in mitochondria do not contain specific antigens. Antigens from both types of secretory granules cross react totally with those present in the excretion-secretion products of living muscle larvae. Cytoimmunochemical data show that antigens are distributed in a patchy fashion throughout the stichocyte cytoplasm. This finding is consistent with the distribution of the secretory granules in the intact stichocyte.

Comparative analysis of mobility and ultrastructure of intramuscular larvae of Trichinella spiralis and Trichinella pseudospiralis.

The muscle phase of Trichinella spiralis and Trichinella pseudospiralis was studied by using scanning electron microscope techniques, closed circuit television, and video tape recording. The complete absence of any cyst structure including the pseudocapsule allows T. pseudospiralis to move freely between the muscle layers. Its rate of activity, measured as distance moved between two points, was 2.83 mm/min compared to that of the encysted T. spiralis larvae which was 0.237 mm/min. There was an absence of cellular reaction to T. pseudospiralis infection which may have been the result of either an absence of active antigen or specific suppression of the cellular response by the metabolites released by by the parasite. THe absence of a capsule around the muscle larvae of T pseudospiralis suggest a different host-parasite relationship than T spiralis.

Ultrastructure, antigenicity, and histochemistry of stichocyte granules of adult Trichinella spiralis.

The stichosome of adult Trichinella spiralis was studied to determine its ultrastructural, antigenic, and histochemical characteristics. Stichocytes of adult worms had 2 types of granules, type I and type II, the ultrastructure of which was different from those of muscle larvae. Both types of granules consisted of a membrane surrounding a homogeneous matrix, and type I granules were rounder than type II granules. Sera from C3H mice immunized against excretory-secretory products of muscle larvae produced positive immunostaining of type I but not type II granules. Differences in antigenicity were observed between larval and adult stichocyte granules; monoclonal antibodies against alpha-granules of muscle larvae failed to label the adult granules. Azan staining revealed a histochemical difference between larval and adult stichocytes; adult stichocytes stained yellow, whereas larval stichocytes are known to stain red or blue. Thus, the present contribution revealed the existence of 2 distinct types of stichocyte granules in adult T. spiralis and showed them to differ profoundly from those characterized previously in muscle larvae.

Comparison of three subspecies of Trichinella spiralis by scanning electron microscopy.

The surface morphology of three subspecies of Trichinella spiralis was examined by SEM in an attempt to find characteristics useful for distinguishing the subspecies. The subspecies studied were T. spiralis spiralis, which had been maintained in swine and laboratory animals for about 50 yr; T. spiralis nativa collected from Ursus maritimus at 58 degrees N latitude and 95 degrees W longitude in 1976; and, T. spiralis pseudospiralis, which was derived from the original isolation of this subspecies from Procyon lotor at 43 degrees N latitude and 47 degrees 30'E longitude in 1972. All three subspecies were passed in CFW mice and adult worms were collected from the small intestine at 4, 5, 6, 7, 8, and 11 days PI. Characteristics examined included labial and cephalic papillae, cuticular ridges and folds, hypodermal gland cell pores, pseudobursal lobes, genital papillae, cloacal aperture, copulatory bell and vulval morphology. Previous reports of subspecies differences within Trichinella spiralis in the number and distribution of hypodermal gland cell pores, position of genital papillae, shape of the cloacal aperture and shape of pseudobursal lobes were not confirmed and are believed to have been in error resulting from artifacts of fixation and a lack of knowledge of variations within the subspecies caused by low numbers of samples. Differences in surface morphology were not found among the three subspecies. The available names of the recognized biological populations of Trichinella were used at the subspecies level rather than species level because this more clearly represents the state of our knowledge of the nematodes. The question of whether the epidemiology of trichinosis is complicated by the presence of more than one species has not been answered, and it is important that our nomenclature reflect this.

Freeze tolerance, morphology, and RAPD-PCR identification of Trichinella nativa in naturally infected arctic foxes.

Arctic foxes (Alopex lagopus) were collected from Greenland and Svalbard (N = 319). Twenty-four were infected with Trichinella (7.5%). Molecular analysis (random-amplified polymorphic DNA polymerase chain reaction) confirmed that all animals were infected with Trichinella nativa. Motile larvae were found in muscle tissue from all foxes after carcasses had been frozen for 1 yr at -18 C. Infective larvae were found in 2 foxes after a total of 4 yr storage at -18 C, which is longer than any previous observations. Morphological examination of the cysts showed large nurse cells and significant deposition of collagen and connective tissue. It is suggested that, within the geographical distribution of T. nativa, the more freeze-resistant isolates are found at higher latitudes.

Surface morphology of Trichinella spiralis by scanning electron microscopy.

The surface morphology of larval and adult Trichinella spiralis was studied by scanning electron microsocopy (SEM) of fixed, dried, and metal-coated specimens. The results are compared with those found earlier by various investigators using light and transmission electron microscopy. Some morphological features reported here are revealed uniquely by SEM. These include the pores of the cephalic sense organs, the character of secondary cuticular folds, variations of the hypodermal gland cell openings or pores, and the presence of particles on the copulatory bell.

Characterization of the accessory layer of the cuticle of muscle larvae of Trichinella spiralis.

The accessory layer of the cuticle of infective larvae of Trichinella spiralis has been studied with electron microscopy using cytochemical techniques and chemical extractions. The accessory layer lacks negative charges and carbohydrates demonstrable in vivo. Staining with ruthenium red and tannic acid is interpreted as being consistent with their reactions with phospholipids. Freeze fractures demonstrate an external layer of granules that can be partially released by means of detergents (CTAB and SDS). The granules are considered to be proteins. Their removal makes the worms acid sensitive and prevents them from infecting mice. Extraction of whole worms with ethanol, acetone and methanol (via reaction with 2,2-DMP), or chloroform and methanol destroys an internal layer of filaments. Thin-layer chromatography of chloroform/methanol extracts showed principally ethanolamine phospholipids from the surface of the worms. A model is presented for the molecular organization of the accessory layer. Ethanolamine phospholipids are suggested to occur as tubular micelles. Proteins may attach to these by lipophilic moieties and perhaps by a cryptic sugar group (demonstrated by others) that may penetrate into the hydrophilic core of the lipid micelles.

Modification of the ultrastructure of the muscle larva of Trichinella pseudospiralis following exposure to acidified pepsin solution.

Examination of the cuticle of Trichinella pseudospiralis by transmission electron microscopy revealed an epicuticle, exocuticle, and mesocuticle, each divided into several layers. The epicuticle consisted of an outermost thin plasmalemmalike infracuticular material covering an inner trilaminar membrane. The exocuticle was granular and could be divided into 2 regions on the basis of density. The mesocuticle was fibrillar and 3 regions could be distinguished based on the orientation of fibrils. The cuticle appears attached to the hypodermis by hemidesmosomes. The infracticular structure was altered following isolation of larvae by pepsin-HCl digestion of host muscle.

Cell injury caused by Trichinella spiralis in the mucosal epithelium of B10A mice.

Most of the mucosal epithelium in the anterior small intestine of B10A mice infected with Trichinella spiralis showed no cytopathology. However, isolated foci of damaged cells or dense masses of multinucleate cytoplasm were seen in the crypt-villus junction, or the base of the villi. Cells occupied by the nematode ranged from a nearly normal appearance, showing only compressed nuclei and organelles, to progressive inflation and vesiculation of endoplasmic reticulum, loss of terminal web and hence disoriented and reduced microvilli, and pycnosis of nuclei. Damaged cells and multinucleate cytoplasmic masses may be derived from the cells previously occupied by the nematode that were linked together by fusion of their lateral cell membranes. Damaged cells and multinucleate masses are apparently sloughed from the epithelium at the villus base without migrating up the villi. Eosinophils were seen in the lamina propria, in the mucosal epithelium (usually associated with damaged cells) and in the intestinal lumen (also with damaged cells). As no eosinophils were seen in contact with the nematode, their activities may be related more to the cells killed by the worm than to the worm itself.