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 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.

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.

Ultrastructural characteristics of nurse cell-larva complex of four species of Trichinella in several hosts.

The nurse cell-larva complex of nematodes of the genus Trichinella plays an important role in the survival of the larva in decaying muscles, frequently favouring the transmission of the parasite in extreme environmental conditions. The ultrastructure of the nurse cell-larva complex in muscles from different hosts infected with T. nativa (a walrus and a polar bear), T. spiralis (horses and humans), T. pseudospiralis (a laboratory mouse) and T. papuae (a laboratory mouse) were examined. Analysis with transmission electron microscope showed that the typical nurse cell structure was present in all examined samples, irrespective of the species of larva, of the presence of a collagen capsule, of the age of infection and of the host species, suggesting that there exists a molecular mechanism that in the first stage of larva invasion is similar for encapsulated and non-encapsulated species.

The effect of changes in the gastric pH value on experimental trichinosis.

The effect of increasing or decreasing the gastric pH of mice on the course of experimental infection with Trichinella spiralis was studied, by administration of 1% NaHCO3 in PBS (pH 9.0) or 1% HCl in PBS (pH 5.0) to mice half an hour prior to infection respectively. The results revealed that raising the gastric pH led to a significant increase in the adult worm count with all increase in their fecundity both in vivo and in vitro. On on the other hand, the more acidic gastric pH induced prior to infection led to its amelioration. This was obvious by the significant reduction in the adult worm count and their inability to give birth to newborn larvae. Several factors may be incriminated, among which are decreased larval infectivity and affection of the maturation of the reproductive organs, mainly the uterus and the testis. Changes in their morphology were observed by both light and transmission electron microscopic studies, which could account for the impairment in their functions, namely embryogenesis and spermatogenesis.

Immunofluorescent localization of intermediate filaments (IFs) in helminths using anti-mammalian IFs monoclonal antibody.

Intermediate filaments (IFs) make up the cytoskeleton of most eukaryotic cells. In vertebrates, a number of IF proteins have been identified, showing distributions unique to tissue or cell type. Information on helminth IFs is limited to some nematode species. To observe immunofluorescent localization of IFs in helminth tissues, we selected a murine hybridoma clone producing IgM antibody to multiple types of mammalian IF proteins and examined cross-reactivity to helminth proteins. The selected monoclonal antibody (HUSM-9) cross-reacted well with IFs from nematode species such as Toxocara canis, Dirofilaria immitis, Anisakis simplex, and Trichinella britovi; strong immunofluorescence on cryostat sections was detected in the hypodermis, cords, body muscle, smooth muscle of the uterus, and other epithelial structures. In platyhelminths, i.e., adult Schistosoma mansoni, larval Taenia taeniaeformis, adult Taenia crassiceps, and Echinococcus multilocularis protoscolex, the reactivity was weaker than in nematodes, and localized in the body wall muscle and subtegumental tissue. Western blotting of 8 M urea extracts of parasites with the antibody detected a pair of clear bands in nematodes but not in S. mansoni or the cestodes. These results might be explained by sparse distribution of IFs in platyhelminths, or low affinity of the used antibody to platyhelminth IF proteins, or both.

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.

Polymyositis caused by a new genus of nematode.

We report two patients who presented with increasing malaise and myalgia, and had biopsy-proven polymyositis. Their conditions deteriorated after corticosteroid treatment, and repeat muscle biopsies showed adult and larval nematodes. Anthelminthic treatment was completely successful in both cases. The infecting nematode appears to belong to a new genus and is, to our knowledge, the first known muspiceoid nematode to infect humans. Its life cycle and the route of infection are unknown.

Differences and similarities between Trichinella spiralis and T. pseudospiralis in morphology of stichocyte granules, peptide maps of excretory and secretory (E-S) products and messenger RNA of stichosomal glycoproteins.

The ultrastructure of muscle larvae of Trichinella pseudospiralis was studied by electron microscopy. The overall structure of muscle larvae of T. pseudospiralis resembled that of T. spiralis except for the stichocyte granules. T. pseudospiralis had at least 3 kinds of stichocyte granules distinguishable from each other by their shape, size and inclusions. The granules had some resemblance to alpha granules or beta granules of T. spiralis, but no resemblance to gamma granules. In favour of these morphological differences and similarities among T. spiralis and T. pseudospiralis, excretory and secretory (E-S) products (originating from stichocyte granules) of the 2 species differed to some degree. In an analysis by 2-dimensional electrophoresis, some peptide spots migrating at 45 kDa were shared by the 2 species but the other spots were unique to each of the 2 species. Messenger RNA encoding the 43 kDa glycoprotein of stichocyte granules was detected in the muscle larvae of both species but mRNA encoding the 53 kDa glycoprotein was detected only in muscle larvae of T. spiralis.

[The comparative morphology of Trichinella spiralis and Trichinella pseudospiralis]. / Sravnitel'naia morfologiia Trichinella spiralis i Trichinella pseudospiralis.

The comparative study of the morphology of invasive larvae and mature males and females T. spiralis and T. pseudospiralis at the head end of the parasites has revealed 14 papillae which are located in 3 circles and 2 which are placed bilaterally, 2 amphidae, and a lanceolar stiletto with a spicular cutting edge. There are no differences in the structure of the heads of these two Trichinella species. The larval cuticle is decorated only with cross folds while longitudinal small-sized folds which are arranged in regular rows appeared in mature species. The vulva in females is located at the levels of a fifth of the body from the head, it is slit-like and oval, that in T. pseudospiralis is situated at the cuticular projection. The anus in T. pseudospiralis larvae is as a cross oval slit, that in adult females is as a hemisphere, and in adult males it is coincident with the copulation pore. As for the structure of the pseudobursa, differences are found in the size and shape of copulation processes, in the arrangement of pre- and postanal papillae against the cloaca, in the size of the formed cuticular projections in T. spiralis, in the shape of the cloacal hole. In the unscrewed copulatory bell, it opens transversely in T. spiralis and vertically in T. pseudospiralis.

Changes in host muscles induced by excretory/secretory products of larval Trichinella spiralis and Trichinella pseudospiralis.

Excretory/secretory (ES) products obtained by in vitro culture of infective-stage larvae of Trichinella spiralis and T. pseudospiralis were injected intramuscularly at various intervals into mice. Mini-osmotic pumps containing T. spiralis ES products were also implanted subcutaneously and intraperitoneally into rats. The introduction of ES materials into muscles elicited extensive lesions which included dissolution of myofibres, mobilization of mononuclear and polymorphonuclear leucocytes, angiogenesis, hypertrophy of myonuclei, myotube formation, mitosis, muscle bundles becoming rounded and separated from each other, disappearance of Z, I and A bands of sarcomeres, increase in endoplasmic reticulum and Golgi complexes, decrease in glycogen and relocation of mitochondria. These are considered as degenerative/regenerative changes of muscles to injury. Immunodominant epitopes of specific 45-53 kDa glycoproteins in ES antigens of T. spiralis could not be detected in hypertrophic nuclei of injected muscles by using polyclonal and monoclonal antibodies and immunocytochemical methods. ES products of T. spiralis failed to stimulate unsensitized lymphocytes in the lymphocyte transformation test. Infective-stage larvae of T. spiralis released from muscles were found capable of forming nurse cells after injection subcutaneously into rats. It is postulated that the invasion of muscles by trichinellids elicits two independent events, i.e. a general degenerative/regenerative response of muscles and a specific change in genomic expression of myonuclei. The two events are probably mediated by different effector molecules.

Scanning electron microscopic study of Trichinella pseudospiralis muscle phase after modulation of host response using an immunostimulant drug.

In a previous study, evident collagen deposition around T. pseudospiralis larvae was noticed after administration of Levamisole to infected mice. The present investigation was undertaken to confirm these findings. Scanning electron microscopic examination revealed multiple clear cystic formations on the surface of the diaphragm muscle. The present investigation was undertaken to confirm these findings. Scanning electron microscopic examination revealed multiple clear cystic formations on the surface of the diaphragm muscle. The presence of such structures was seen to be more frequent when the drug was given as early as the 6th day post infection. Membrane like structure was observed around T. pseudospiralis larvae, an interesting finding which was by far not reported before.

Studies on T. spiralis and T pseudospiralis larvae recovered from mice immunized with heterologous Trichinella antigen.

In this study, S3 antigen of T. spiralis and T. pseudospiralis was given to the heterologous infection in albino mice. The recovered larvae were studied as regards: their number, length and steroscan examination. It was found that vaccination of mice with T. spiralis (S3) antigen induced reduction in T. pseudospiralis larval count by 66.43% and significant reduction in length. Using T. pseudospiralis (S3) antigen reduction of 74.77% in T. spiralis larval count was detected as well as significant reduction in length. Scanning electron microscopic (SEM) examination revealed more or less similar finding in both species: loss of integrity of the cuticle, some of the larvae were attacked by granulocytes mainly eosinophils and few larvae appeared completely destructed with loss of normal architecture. A peculiar finding was noticed in case of T. pseudospiralis larvae in form of fine electron dense deposits giving the cuticular surface a motling appearance.

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.

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.

An immunocytochemical analysis of a class-specific antibody response against Trichinella spiralis in humans.

The response of different classes of antibodies against antigens of the muscle larvae of Trichinella spiralis was tested using an immunocytochemical approach. Ultrathin sections of resin-embedded larvae were treated with sera from patients with trichinosis, then exposed to a biotinated second antibody and stained with avidin-gold complex. Antibody of the M-class was a major component in the response against a slow-responding group of antigens that included stichocyte granules, the cuticle surface, and the esophagus-occupying substance; a minor component in the response against antigens of the rapid-responding group that included cuticle inner layers, hypodermis, hemolymph, and intestinal gland granules. The response of G-class antibody against the rapid-responding group of antigens was detected in all patients tested, while against the slow-responding group of antigens it was detected in only half of the patients, suggesting that an antibody shift from the M to the G class occurred in some patients. The results, obtained in humans, were similar to those we obtained previously in rats (J. Parasitology, 76,230-239, 1990), suggesting that the rat immune system can serve as an experimental model of human trichinosis.

Interferon-gamma treatment in mice experimentally infected with Trichinella spiralis.

Interferon-tau (IFN-tau) treatment of Trichinella spiralis-infected BALB/c mice was investigated. The therapeutic regimen consisted of daily intraperitoneal injection of 10(4) U murine IFN-tau for 7 days, starting at 2 weeks post-infection. Striated muscle samples (diaphragm, thigh) were collected at 4, 8 and 12 weeks after infection. The muscle larval burden, the degree of encystation and the digestion of T. spiralis larvae were investigated. Furthermore, immunohistochemical studies of the inflammatory cell infiltrate around encysted larvae were performed. The results demonstrated an influence of IFN-tau treatment on the CD4+ and CD8+ subset distribution during the immune response but revealed no difference in the degree of encystation or digestion of encapsulated larvae as compared with control values.