A spectrum of antibody response with time after Trichinella spiralis infection in rats

A chronology of class-specific antibody response against the Trichinella spiralis infection in Fischer rats was investigated. G-class antibodies against the cuticle inner layer(s), hypodermis, hemolymph, glycogen aggregates, discrete areas in genital primordial cells, intestinal gland cell granules, and cytoplasmic granules in the cords were detectable 2 wk after infection (the rapid-responding group), whereas G-class antibodies against the cuticle surface, stichocyte granules, and the esophagus-occupying substance were detected 6 wk after infection (the slow-responding group). M-class antibodies recognized a narrower spectrum of antigens than did G-class antibodies; M-class antibodies against hemolymph, cord granules, and intestinal gland cell granules were not detectable. M-class antibodies tended to decrease in titer with time after infection. This tendency was more striking with antibodies against the rapid-responding group than with those against the slow-responding group. This information sheds light upon antibody response against many antigenic components of T. spiralis muscle larvae.     

Direct evidence that the cuticle surface of Trichinella spiralis muscle larvae shares antigenicity with stichocyte alpha-granules and the esophagus-occupying substance.

Antibodies against the cuticle surface of Trichinella spiralis muscle larvae were purified by means of immunoaffinity chromatography and incubated with ultrathin sections of muscle larvae. Major constituents of the parasite reactive with the purified antibodies included the cuticle surface, stichocyte alpha-granules, and the esophagus occupying substance of the muscle larvae. Thus the present data suggest that the cuticle surface is an antigenically different entity from the cuticle inner layers and its origin is likely stichocyte alpha-granules.     

Stichosome ultrastructure of the fish nematode Capillaria pterophylli Heinze, 1933

The stichosome (posterior glandular esophagus) of Capillaria pterophylli Heinze, 1933 consists of large gland cells (stichocytes) and lumenal epithelium with cuticular lining. Both structures are enclosed in a reticulum of muscle cells. The stichocyte cytoplasm contains small cisternae of rough endoplasmic reticulum, Golgi complexes, one kind of electron dense secretory granules, mitochondria and a branching system of intracellular collecting ducts without filament bundles around them.     

Prolactin-like hormone in the nematode Trichinella spiralis larvae

Expression of prolactin (PRL) or prolactin-like hormone has been reported in invertebrates. We investigated the larval phase of Trichinella spiralis: (a) to express 23 kDa PRL, (b) to define its localization and (c) to test its possible biological activity. Immunostaining in isolated larvae demonstrated positive material to 23 kDa PRL by all along the stichosome, specifically in the stichocytes. Homogenized immunoblot larvae showed a 23 kDa protein band. To assess PRL release and its biological activity, larvae were incubated in culture medium and the excretory/secretory products were analyzed by the Nb2 cells bioassay. A cellular growth equivalent until 10 nM PRL and using antibody against 23 kDa PRL, the growth was blocked. In conclusion our result provides evidence that PRL-like hormone is expressed and secreted by the larvae of T. spiralis.     

Ontogeny of the endocrine pancreas in sea bass (Dicentrarchus labrax): an ultrastructural study. II. The big and secondary islets

The big and secondary islets of sea bass larvae were characterized ultrastructurally from, 25 to 60 days after hatching. From the 25th day, big islets consisted of inner type II and III, external type I and peripheral type IV cells. From the 55th day, type V cells appeared in limited peripheral areas. Secondary islets, first found in 32-day-old larvae, were made up of inner type II and III, external type I, and peripheral either type IV and V cells (type I islets), or only type V cells (type II islets). Type I cells contained secretory granules with a fine granular, low-medium electron-dense material, whereas the secretory granules of type II cells were smaller and had a high electron-dense core with diffused limits; needle and rod-like crystalloid contents were occasionally found. Type III secretory granules posessed a homogeneous, high or medium electron-dense material with or without a clear halo. Type IV cells had secretory granules with a polygonal dense core embedded in a granular matrix and granules containing a high or medium electron-dense material. Type V cells had secretory granules with a fine granular, high or medium electron-dense content. These cell-types correlated with cells previously identified immuno-cytochemically, as regards to their distribution in the islets, and related to those characterized ultrastructurally in adult specimens. Thus, types I, II, III, IV and V correspond to D₁, B, D₂, A and PP cells, respectively. From the 32nd day onwards, endocrine cells of all the different types were found grouped, type V cells also being observed in isolation close to pancreatic ducts and/or blood vessels. Small groups consisting of type I and II cells were found in 40-day-old larvae. A mitotic centroacinar ductular cell containing some secretory granules similar to those of type I cells, was seen adjacent to a type I cell. As the larvae grew older, the endoplasmic reticulum developed, the number of free ribosomes decreased, and the number and size of the secretory granules increased. Dark type I, II, III, IV and V cells were found in the islets and cell clusters from the 55th day onwards.     

Electrophoretic identification of larvae and adults of Anisakis (Ascaridida:Anisakidae)

The relationships between larvae and adults of Anisakis from the Mediterranean Sea and North-East Atlantic Ocean were analysed by multilocus electrophoresis. The correspondence of type I larvae with the A. simplex complex, including the sibling species A. simplex A and B, and of type II larvae with A. physeteris is confirmed. 19 of the 22 loci studied discriminated between the two larval types. Biochemical keys are given for the electrophoretic identification of A. simplex A, A. simplex B and A. physeteris, at both the larval and adult stages.     

Fine structure and immunocytochemistry of cells within the endocrine pancreas of larval and adult sea lampreys, Petromyzon marinus L

Immunocytochemistry with protein A-gold and routine electron microscopy were used to identify cell types within the endocrine pancreas of larvae, juvenile adults, and upstream-migrant adults of the sea lamprey, Petromyzon marinus. The larval pancreatic islets are composed only of insulin-immunoreactive B-cells, which are uniform in their fine structure. The cranial and caudal pancreatic tissue in both adult periods contains three cell types: B-cells, somatostatin-immunoreactive D-cells, and a third cell type of unknown content. No glucagon-immunoreactive cells are present in lampreys, but B- and D-cells exist in equal numbers in the pancreatic tissue of adults. The B-cells of adults have a fine structure similar to those in larvae. D-cells have secretory granules that are distinctly different from those both in B-cells and in the third cell type. Although B- and D-cells in lamprey pancreatic tissues have a basic morphological similarity to these cells in other vertebrates, their granules are generally of smaller dimensions. The inclusion of granules within large pleomorphic bodies in many D-cells indicates that granule turnover is common. Immunocytochemistry will be a useful tool for showing the relationship between the cells in the degenerating bile ducts and those of the developing adult pancreas.     

Alterations of the eyes of Carinaria lamarcki (Gastropoda, Heteropoda) during the long pelagic cycle

 The eyes of different larval stages of Carinaria lamarcki were examined ultrastructurally. In all larval stages the eyes consist of a cornea, a lens and an everse retina. The photoreceptors in young larvae are exclusively of the ciliary type. In old larvae, however, two types of photoreceptors are present and the retina is composed of two segments: a posterior segment with altered ciliary photoreceptors (=type I sensory cells) and an anterior segment with what are presumably rhabdomeric photoreceptors (=type II sensory cells). The anterior retina is interpreted here as an accelerted character. Furthermore, the arrangement of the pigment granules changes during the long larval development being cup shaped in young larvae versus ribbon shaped in old larvae. The findings allow for the conclusions that: (a) the ciliary photoreceptors are correlated with the long larval period of Heteropoda and that (b) the eyes are altered continuously during the larval cycle. Accepted: 6 July 1998     

Trichinella spiralis: immunization of pigs with newborn larval antigens

The potential of crude Trichinella spiralis newborn larval antigens for pig immunization was investigated. A preparation of whole newborn larvae killed by freezing and thawing, and combined with Freund's complete adjuvant, induced a high level of protection against challenge (78%), compared to a 40% resistance level in pigs immunized with excretory secretory antigens of muscle larvae. Sera from pigs immunized with newborn larvae contained antibodies which bound to the surface of the newborn larvae, as determined by immunofluorescence. In a second trial, the freeze thawed newborn larvae preparation was compared with a soluble and insoluble fraction prepared by sonication of whole newborn larvae. Pigs receiving whole newborn larvae or the insoluble fraction developed strong immunity to challenge (88.2 and 85.5%, respectively); the soluble fraction was ineffective. Immunization with all preparations induced antibody to newborn larval antigens, but not to adult or muscle larvae excretory secretory antigens. Polyacrylamide gel electrophoresis of the soluble and insoluble fractions indicated that sonication was ineffective in solubilizing the larger molecular weight components. These results demonstrate that newborn larval antigens are highly protective in pigs, but that their further development as a vaccine will require more efficient procedures for antigen solubilization and large-scale production.     

Identification and characterisation of a cDNA sequence encoding a glutamic acid-rich protein specifically transcribed in Trichinella spiralis newborn larvae and recognised by infected swine serum

Presently, little is known of the mechanism by which Trichinella penetrates and modulates reprogramming of muscle cells. In light of evidence demonstrating strong protective characteristics of antigens derived from this stage, understanding this process may shed light on potential targets for effective abatement of infection. To this end, a PCR-derived cDNA expression library was constructed using 0.5 micro g of total RNA from Trichinella spiralis newborn larvae. The library consisted of >125000 insert-containing clones. Approximately 40-50 x 10(3) clones were screened immunologically using sera from pigs experimentally infected with 7000 Trichinella L1. Multiple clones reacting positively with the swine infection serum and encoding portions of a glutamic acid-rich protein were identified. Northern and Southern blots indicated at least two distinct genes that encoded the glutamic acid-rich proteins and that these genes were transcribed specifically in the newborn larvae stage. cDNA sequence data predicted open reading frames of 1497 and 1,716 bp generating proteins of 498 amino acids and 571 amino acids, respectively. Both sequences consisted of approximately 39% glutamic acid and 16% serine residues, and differed by the presence of a 219 bp fragment present in the 1716 bp sequence that was absent from the 1497 bp sequence. PCR data indicated that additional isoforms exist within this gene family that are different in length from those described above. In addition, it was found that more than one isoform can exist within a single worm and that this pattern can vary between individual worms within a population. Mouse antibodies to recombinant antigen localised the glutamic acid-rich proteins to the periphery of the developing stichocyte cells within the newborn larvae consistent with the hypothesis that the newborn larval antigens are secreted.     

Multiple actins in drosophila melanogaster

The tissue and developmental specificities of the three Drosophila isoactins, originally identified in primary myogenic cultures and in the permanent Schneider L-2 cell line, have been investigated. Of these three isoactins (I, II, and III), actins I and II are stable and actin III is unstable. Two-dimensional polyacrylamide gel electrophoretic analyses of total cellular extracts after 1-h [(35)S]methionine pulses were performed on a large variety of embryonic, larval, and adult muscle and nonmuscle tissues. The results suggest that isoactins II and III are generalized cellular actins found in all drosophila cell types. Actin I, on the other hand, is muscle-associated and is found exclusively in supercontractile muscle (such as larval body wall and larval and adult viscera) including primary myogenic cell cultures. Although actin I synthesis is not detectable during very early embryogenesis, it is detectable by 25 h and actin I is a major stable actin in all larval muscle tissues. Actin I is synthesized in reduced amounts relative to the other actins in late third instar larvae but is again a major product of actin synthesis in the adult abdomen. A stable actin species with the same pI as actin III has been identified in the adult thorax and appears to be unique to flight muscle tissue. This new stable form of thoracic actin may be the result of a stabilization of the actin III found in other tissues or may be an entirely separate gene product.     

Regulation by thyroid hormones of terminal differentiation in the skeletal dorsal muscle. II. Urodelan amphibians

In the urodelan amphibian Pleurodeles waltlii, spontaneous anatomical metamorphosis was correlated with an increase in the serum level of thyroxine (T4). It was also accompanied by a change in the myofibrillar ATPase profile of the dorsal skeletal muscle; fibers of larval type were gradually replaced by the adult fiber types I, II A, and II B. Likewise, a myosin isoenzymic transition was observed in dorsal muscle, larval isomyosins were replaced by adult isoforms. In a related species, Ambystoma mexicanum, in which no spontaneous external metamorphosis occurs under standard conditions, the serum T4 level was shown to remain low. During further development, the myofibrillar ATPase profile acquired the adult fiber types, but a high percentage of immature fibers of type II C persisted. Myosin isoenzymic transition was also incomplete; larval isoforms were still distinguished in the neotenic adults. In experimental hypothyroidian P. waltlii, no external metamorphosis occurred; the myofibrillar ATPase profile was of the immature type, and the larval isomyosins persisted. Triiodothyronine induced experimental anatomical metamorphosis in A. mexicanum; only limited changes in the myofibrillar ATPase profile resulted from the treatment, but a complete myosin isoenzymic transition was observed. These results tend to indicate that a moderate increase in the level of thyroid hormone is sufficient to induce the differentiation of adult fiber types, together with the production of adult myosin isoforms in the skeletal dorsal muscle of amphibians, while a pronounced increase would be necessary for repressing the initial larval features.     

Infection status of the sea eel (Astroconger myriaster) purchased from the Noryangjin fish market with anisakid larvae]

Although the sea eel (Astroconger myriaster) is suspected as one of the most important fish host for human anisakiasis in Korea, no report has been made on the infection status of the sea eel with anisakid larvae. In the present study, 26 sea eels (Astroconger myriaster) were purchased from the Noryangjin fish market in Seoul, and anisakid larvae were collected from their viscera, muscle, head and skin. The collected larvae were classified by their morphological types. A total of 1,351 anisakid larvae were collected from 15 of 26 fish examined. Among them, 1,269 were recovered from the viscera, 66 from the muscle, and 16 from the head and skin. Morphologically, most of the anisakids were classified into 6 known larval types, Anisakis type I (564 larvae) of Berland (1961), Contracaecum type A (409) and type D (5) of Koyama et al. (1969), Contracaecum type C' (83) and type D' (117) of Chai et al. (1986), and Contracaecum type V (1) of Yamaguti (1935). Remaining 172 specimens were new in the available literature, hence, designated as Contracaecum type A' (new type). The present results revealed that the sea eels caught in the Korean waters are heavily infected with anisakid larvae, not only in their viscera but also in the muscle, and Anisakis type I was the most common among the 7 larval types.     

Larval cells of sponge Halisarca dujardini (Demospongiae, Halisarcida). II. Some cell types marked by polyclonal antibodies

The recent morphological and experimental data concerning the involvement of flagellated cells in sponge larvae are contradictory and testify to or against the germinal layers inversion. A study of morphogenetic processes in sponges, in particular larval metamorphosis, is complicated by difficulties in identification and succession of certain cell types. It is possible to trace the destiny of flagellated and other larval cells by marking them with antibodies (AB) specified for each cell type. We separated larval and adult sponge cells of Halisarca dujardini in percoll density gradient and obtained polyclonal AB for the majority of these cell types. The protein pattern of larval flagellated cells differed significantly from that of other cell types. The major proteins of flagellated, collencyte-like and spherulous cells were used to raise the corresponding AB. Immunoblot showed all AB to be specific for certain proteins and suitable for immunofluorescence. The AB for flagellated cells reacted with the apical cytoplasm, but not with the flagellum, the AB for major protein of collencyte-like cells stained cytoplasm granules. The AB for spherulous cells of the adult sponge reacted with larval spherulous cells supposed to be of maternal origin. So, the method of cell marking with specific polyclonal AB can facilitate analysis of the layers inversion problem, as well as elucidate the degree of cell differentiation in larvae, their conformity to cells of the adult sponge or their provisional destiny.     

Histochemical patterns in normal and splaylegged piglet muscle fibers

Summary The histochemical pattern of muscle fiber types of the longissimus dorsi and biceps femoris muscles was investigated in normal and splaylegged piglets at birth and seven days later. Only slight differences between the muscle fibers at birth were found using histochemical reactions for alkaline adenosine triphosphatase (ATPase), succinate dehydrogenase (SDH), phosphorylase (PH) activities, and for the periodic acid-Schiff (PAS) reaction. With the method for acid-preincubated ATPase activity, high activity was observed in Type I muscle fibers and low activity in Type II muscle fibers in animals of both groups investigated. However, a higher number of Type I fibers was found in muscles of normal piglets, suggesting a faster and more advanced process of transformation of Type II into Type I muscle fibers in unaffected animals. Thus the histochemical conversion appears to be retarded in muscles of splaylegged animals, which have a histochemical pattern similar to that of normal prenatal animals. Cholinesterase activity in motor endplates was well developed; its staining revealed smaller sized and irregularly arranged endplates in muscles of affected piglets. Fiber type differentiation in muscles of animals which recovered from splayleg becomes fully developed and comparable to normal piglets seven days after birth. The number of fibers which became converted from Type II to Type I was increased; the fiber types were differentiated with regard to the PAS reaction and to their ATPase, SDH and PH activities. Morphological features of motor endplates in muscles of normal and surviving splaylegged piglets are similar.Histochemical investigation of the fiber type differentiation thus suggests that full recovery occurs within the first week of postnatal life in muscles affected by pathological changes accompanying splayleg.     

The mouse antibody response to Trichinella spiralis defines a single, immunodominant epitope shared by multiple antigens.

Immunoblot analysis was used to characterize the Trichinella spiralis L1 larval Ag recognized by antisera from T. spiralis-infected AKR/J mice. Antisera were analyzed for reactivity with crude worm extract, excretory/secretory proteins and cuticle proteins from L1 larvae. The response was biphasic; antibodies against one set of Ag were detected 13 days after infection (group I Ag), and antibodies against a different set of Ag were detected 35 days after infection (group II Ag). Excretory/secretory and cuticle proteins were recognized only by antibodies produced late in infection. The predominant isotype in day 42 antiserum was IgG1, and 80% of these IgG1 antibodies reacted with a stage-specific determinant shared by virtually all group II Ag. A mAb reactive with the shared determinant was used to purify the group II Ag from L1 larval extract. Such affinity-purified Ag were protective when used to immunize mice against subsequent infection, and T cells from infected mice proliferated when cultured with these Ag in vitro. Other mouse strains also made a strong serum antibody response to the group II Ag. We hypothesize that immune responses to the shared epitope play an important role in determining the outcome of the host-parasite interaction during infection.     

Ultrastructural and histochemical features of the thymus glands of the adult lungless salamander, Plethodon glutinosus (Caudata: Plethodontidae).

The thymus glands of adult slimy salamanders (Plethodon glutinosus) were examined by light and electron microscopy with the objective of describing the populations of epithelial cells believed to be secretory. The results of various histochemical procedures designed to demonstrate nucleic acids, proteins, lipids, and mucosubstances were evaluated by light microscopy. Each thymus is incompletely subdivided into a variable number of interconnected lobules by trabeculae extending inward from a thin capsule composed of connective tissues. The thymic parenchyma lacks distinct cortical and medullary regions, although developing lymphocytes and plasma cells tend to accumulate in larger numbers in the outermost portions of the glands. Basophils are found regularly in the capsule and trabeculae, but only very rarely within the thymic parenchyma. The epithelial cells of the thymus can be classified into five categories: epithelial reticular cells; three varieties of granulated cells (types I, II, and III), and myoid cells. Epithelial reticular cells form a three-dimensional network which extends throughout all portions of the thymus. Type I and type II granulated cells can be distinguished from one another by various morphological criteria at the ultrastructural level, but only small differences in the composition of their inclusions can be demonstrated histochemically. Both types of granules are composed principally of a proteinaceous material containing an abundance of primary amino and guanidyl groups. In addition, most type I inclusions possess a lipid component that cannot be demonstrated in type II granules. Type III granulated cells possess very small cytoplasmic inclusions resembling those of gastroenteric endocrine cells. Myoid cells contain concentrically arranged myofibrils composed of sarcomeres. In favorably oriented material, small cysts can be identified whose walls are composed of mixtures of type I cells, type II cells, and epithelial reticular cells. Groups of degenerating epithelial cells form lamellated structures corresponding to Hassall's (thymic) corpuscles.