Analysis of the effect of a 2-(trifluoromethyl)-1H-benzimidazole derivative on Trichinella spiralis muscle larvae.

Albendazole and mebendazole are widely used in the treatment of trichinellosis; however, chemotherapy failure has been reported. In an effort to develop new anthelminthic compounds, we examined a previously synthesized 2-(trifluoromethyl)-1H-benzimidazole derivative (1) that showed good in vitro activity against Trichinella spiralis muscle larvae but low in vivo efficacy. In order to improve the solubility of compound 1, an inclusion complex with 2-hydroxypropyl-ß-cyclodextrin (1/HP-ßCD) was prepared. When 1/HP-ßCD was tested in vivo, it significantly reduced the ML burden (84%). In addition, a proteomic analysis of T. spiralis ML treated with 1 revealed significant changes in the expression levels of proteins involved in energy metabolism and the cytoskeleton of the parasite. Compound (1) also induced extensive ultrastructural changes in the cuticle, hypodermis and midgut of the parasite.

Efecto de la temperatura sobre la viabilidad de larvas de Trichinella spiralis / Effect of temperature on the viability of Trichinella spiralis larvae

El objetivo de este estudio fue determinar el efecto de diferentes temperaturas sobre la viabilidad de larvas libres y enquistadas de Trichinella spiralis aisladas en el sudoeste de la provincia de Buenos Aires, Argentina. Se trataron larvas libres y enquistadas a diferentes temperaturas (-30 °C, -20 °C, 4 °C, 20 °C, calentamiento gradual entre 0-100 °C). Se determinó el tiempo necesario para matar el 100 % de las larvas. Durante los primeros días, la mortalidad larvaria en todos los tratamientos con frío aumentó signifcativamente en función del tiempo. En todos los casos, las larvas libres sobrevivieron menor cantidad de días que las enquistadas. A -30 °C, -20 °C y 20 °C no se observaron diferencias signifcativas entre las curvas de mortalidad de cada estadio larvario, pero a 4 °C la mortalidad fue menos intensa entre las larvas enquistadas. El calentamiento disminuyó la viabilidad, sin observarse diferencias entre estadios larvarios. La totalidad de las larvas libres y enquistadas había muerto a los 61 y 95 días (-30 °C), a los 160 y 180 días (-20 °C), a los 280 y 330 días (4° C), y a los 460 y 590 días (20 °C), respectivamente. Fue necesaria una cocción durante 15 minutos a 90 °C para matar al 100 % de las larvas libres y a 100 °C para lograr igual mortalidad de las enquistadas. Nuestros resultados indican que la temperatura y los tiempos tradicionalmente utilizados para tratar productos cárnicos con potencial de transmisión de T. spiralis no serían los más efectivos para lograr la inactivación de la totalidad de larvas vivas de este parásito.

The aim of this work was to study the effect of temperature on the viability of free and encysted larvae of Trichinella spiralis from southwest Buenos Aires province, Argentina. Larvae were treated at variable temperatures (-30 °C, -20 °C, 4 °C, 20 °C, gradual heating between 0-100 °C). The time necessary to kill 100 % of larvae was calculated. During the frst days of freezing, larval mortality signifcantly increased as a function of time. Regardless of temperature, encysted larvae survived longer than the free ones. At -30 °C, -20 °C, and 20 °C there were no signifcant differences between the survival curves for each larval stage. At 4 °C, mortality was less severe for encysted larvae. All free and encysted larvae died at 61 days and 95 days (-30 °C), 160 days and 180 days (-20 °C), 280 days and 330 days (4 °C) and 460 days and 590 days (20 °C), respectively. Cooking at 90 °C and 100 °C during 15 minutes killed 100 % of free and encysted larvae, respectively. Our results suggest that temperatures and exposure times traditionally used to treat meat products with a potential to transmit T. spiralis are not entirely effcient.

[Effect of temperature on the viability of Trichinella spiralis larvae]. / Efecto de la temperatura sobre la viabilidad de larvas de Trichinella spiralis.

The aim of this work was to study the effect of temperature on the viability of free and encysted larvae of Trichinella spiralis from southwest Buenos Aires province, Argentina. Larvae were treated at variable temperatures (-30 °C, -20 °C, 4 °C, 20 °C, gradual heating between 0-100 °C). The time necessary to kill 100 % of larvae was calculated. During the first days of freezing, larval mortality significantly increased as a function of time. Regardless of temperature, encysted larvae survived longer than the free ones. At -30 °C, -20 °C, and 20 °C there were no significant differences between the survival curves for each larval stage. At 4 °C, mortality was less severe for encysted larvae. All free and encysted larvae died at 61 days and 95 days (-30 °C), 160 days and 180 days (-20 °C), 280 days and 330 days (4 °C) and 460 days and 590 days (20 °C), respectively. Cooking at 90 °C and 100 °C during 15 minutes killed 100 % of free and encysted larvae, respectively. Our results suggest that temperatures and exposure times traditionally used to treat meat products with a potential to transmit T. spiralis are not entirely efficient.

[Lifetime diagnosis of trichinosis in experimentally infected laboratory animals].

The authors propose a new method for lifetime diagnosis (LTD) of experimental invasion of Trichinella spiralis in migratory and muscular phases, which is based on thin-layer microscopy of minor samples of skeletal muscles (weighing 1-1.5 mg) on gradually increased impulse manual compression. The LTD method permits the monitoring of the most important follow-up qualitative and quantitative invasion parameters in each infected animal in normalcy and under the action of specific therapeutic and preventive agents (the onset and termination of a migratory phrase, the elimination time of intestinal Trichinella after infection, the number, sizes, morphological features, viability, and motor activity of nonincapsulated and incapsulated muscular Trichinella larvae of different age). The most important feature of LTD is its possibility of detecting and determining the viability of neonatal T. spiralis larvae. LTD enhances the informative value, reduces the costs of and makes experimental studies of the basic and applied problems of trichinosis shorter.

Characterisation of novel protein families secreted by muscle stage larvae of Trichinella spiralis.

Proteins secreted by Trichinella spiralis have a potential role in remodelling host skeletal muscle. However, whilst many parasite-secreted proteins have been identified, it has rarely been demonstrated that these are secreted into the nurse cell. Using an informatics-based analysis, we have searched the T. spiralis expressed sequence tag (EST) datasets for cDNAs encoding potential secreted proteins. Here we describe the characterisation of three of the top candidates isolated from our analysis, termed secreted from muscle stage larvae (SML)-1, -2 and -3. All three proteins were demonstrated to be secreted by muscle stage larvae, and immunohistochemical analysis established that SML-1 and -2 are secreted into developing nurse cells. We also show that SML-2 is processed from a precursor into smaller peptides by a metalloprotease contained within T. spiralis-secreted products. With the identification of these and other secreted proteins, we now have molecules to test in functional assays designed to dissect molecular features of the developing nurse cell.

Immunization against trichinellosis using microwaved larvae of Trichinela spiralis.

The present study evaluated the effect of microwave irradiation on infective larvae of Trichinella spiralis (T. spiralis) by the ultrastructure changes of the microwaved larvae (ML) using scanning electron microscope (SEM). The ML tested the ability to immunize mice against a challenge infection with T. spiralis. For the optimal dose and the best route of immunization inducing protection against challenge infection, two doses were used; 300 & 600 ML as one or two-dose regimen, each dose was given orally and intraperitoneally (IP). SEM revealed tegumental damage of the ML in the form of distortion, loss of normal fold pattern and depressions or papillae protruded from their outer surface. After administration of the ML (orally or IP) to the non-infected control mice, neither adults nor larvae were detected in the intestines or muscles respectively. This indicated loss of larvae infectivity after exposure to the microwave irradiation. Also, a significant protection against challenge infection with T. spiralis was demonstrated in experimental mice immunized by ML, orally or IP. This was assessed by a statistically significant decrease in adult and muscle larval count, compared with the non-immunized infected control. Complete protection against both adults and larvae (100%) was achieved by IP injection of two doses of 600 ML, two weeks apart. The results suggested the feasible application of the microwave irradiation on meat for its decontamination from T. spiralis larvae. Such a method might be a promising a prophylaxis vaccine against trichinellosis in animals and/or humans.

Application of Giemsa stain for easy detection of Trichinella spiralis muscle larvae.

The application of Giemsa technique to stain compressed diaphragm samples obtained from rodents experimentally infected with Trichinella spiralis is described. Diaphragm samples from rats heavily infected with 20 muscle larvae per gram of body weight (20 ML/gbw) were cut into several pieces and stained with Giemsa; on the other hand, whole diaphragms from slightly infected mice (1 ML/gbw) were also stained with Giemsa. Besides, muscle samples were also stained with Giemsa. Observation at 10 x magnification revealed that both ML and nurse cells (NC) look as bluish structures clearly contrasting with the pinkish color of the non-infected muscle fibers. NC in the diaphragms of mice could be easily observed at naked eye as blue points contrasting with the pink surrounding areas formed by the non-infected muscle fibers. Among NC observed in the diaphragms of rats infected with 20 ML/gbw, 4.4% was multiple infection. These findings were confirmed in sectioned and hematoxylin-eosin stained specimens. This data could be usefulness for a rapid diagnosis of trichinellosis in post-mortem mammals without magnification procedures.

Contrast staining of Trichinella spiralis larvae in fresh, frozen and formalin preserved muscles.

Fresh, frozen-thawed and formalin-preserved muscle samples heavily infected with Trichinella larvae were cut into several pieces and stained with Giemsa and Leishman and the reference Haematoxylineosin (H & E) stain. Observation under microscope revealed that both muscle larvae and nurse cells in fresh and formalin preserved specimens appeared as purplish blue structures contrasting with the pinkish color of non-infected muscle fibers in both Giemsa and Leishman stains. These findings were confirmed in H & E stained samples. However, frozen samples did not show contrast stain.

Persistence of reactivity against the 45 k Da glycoprotein in late trichinellosis patients.

Over the years, the opinions of clinicians on the existence of the so-called chronic trichinellosis or late sequelae of infection have differed. However, the persistence of a humoral immune response against Trichinella in these late-stage patients has been confirmed using specific tests such as the competitive inhibition assay (CIA). We evaluated sera from late-stage trichinellosis patients (2--8 years from acute infection), for their reactivity against Trichinella spiralis antigens. The following tests were carried out: (i) indirect immunofluorescence assay (IFA), performed on muscle sections from mice, 30 days following synchronous infection by intramuscular injection with T. spiralis newborn larvae (NBL); (ii) enzyme immunoassay, employing a synthetic beta-tyvelose antigen conjugated to bovine serum albumin (BSA-Ag); and (iii) western blot (WB) with both an "in house" kit and a commercial kit. The results of IFA obtained by confocal laser microscopy showed that sera reacted against both surface and internal structures of L(1) larvae but at varying levels. Employing the synthetic antigen, EIA showed that 50% of sera tested were positive for the presence of specific antibodies against beta-tyvelose. By WB, all sera were reactive with the 45 k Da glycoprotein (45 gp). These data suggest that reactivity against the beta-tyvelosylated 45 gp persists even in very late stages of human trichinellosis.

Are bacillary bands responsible for expulsion of Trichinella spiralis?

Newborn larvae, muscle larvae, intestinal larvae, and adults of Trichinella spiralis were examined by light, scanning and transmission electron microscopy for the presence of bacillary bands and pores of the gland cells. Vital staining of living adults disclosed that the bacillary bands, approximately 5 microm in width, begin at the cephalic end of the worms, on each side of the worm, as a row of single cells. The bands extend, gradually increasing in width, to the posterior end of the worms; and in the mid-body and posterior end of the worms they measure approximately 17 microm wide and accommodate at least two cells side by side. Each gland cell communicates with the outside of the worms through a 1.5--2 microm pore, containing dense material, in the cuticle. The pores constitute anatomic weak points of Trichinella since they can be can be targeted by the host's humoral immune responses and enzymatic attacks. The appearance of patent pores after the first intestinal molt correlates in time with the early expulsion of Trichinella from the intestine, suggesting that the bands may be the source of secreted metabolic or antigenic irritants that initiate, or contribute, to the expulsion mechanisms. Further studies to elucidate the functions of these organs would greatly increase our understanding of the biology of Trichinella and the mechanisms of expulsion.

[Ultrastructural analysis of capsule and nurse-cell morphology examined seven months after Trichinella spiralis mouse infection]. / Badania ultrastrukturalne torebki i "nurse-cell" Trichinella spiralis w siedem miesiecy od zarazenia myszy.

Ultrastructural changes in muscles cells of mice infected with T. spiralis larvae in 220 day of infection were evaluated. The object of study was in the region of the "nurse-cell" being in direct contact with the larva wall. Electron microscopic observations revealed the continuity of the muscle cell membrane adjacent to larva surface.

Bacterial-parasite interaction between Salmonella and each of Fasciola gigantica and Trichinella spiralis.

The possibility of bacterial-parasite interaction between Salmonella typhimurium and the surface of each of Fasciola gigantica metacercariae and Trichinella spiralis larvae was investigated in vitro. Two studies were carried out. In the first, S. typhimurium were incubated in vitro with the metacercariae of F. gigantica. In the second, S. typhimurium was incubated with larvae of T. spiralis. The interactions of S. typhimurium with each of F. gigantica metacercariae and T. spiralis larvae were studied by scanning electron microscope (SEM). In the first study, numerous bacilli were found adhered to the metacercariae surface. In the second study, no S. typhimurium was observed on the wall of T. spiralis larvae. The results indicated that only F. gigantica metacercariae act as a carrier for S. typhimurium with the possibility of occurrence of mixed infections with both organisms. So, both fascioliasis and salmonellosis must be treated concomitantly.

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.

Five types of nuclei present in the capsule of Trichinella spiralis.

This study describes morphological changes during capsule formation of Trichinella spiralis with emphasis on nuclei within the capsule. The nuclei of the pre-existing muscle cell may become hypertrophied or degrade soon after the entrance of newborn larvae. Cytoplasm associated with these hypertrophied nuclei was basophilic in H&E staining and composed of mitochondria and rough endoplasmic reticulum. Satellite cells underwent cell division and joined to the nurse cell. These nuclei may also become hypertrophied or degrade. Cytoplasm associated with nuclei of satellite cell origin was eosinophilic in H&E staining and composed of mitochondria, polysomes, and smooth and rough endoplasmic reticulum. Eosinophilic cytoplasm (of satellite cell origin) became prevalent in the capsule as capsule formation proceeded. Infiltrated cells were observed within the capsule. Thus this study showed diversity of nuclei in the capsule of Trichinella.

A reversible protein phosphorylation system is present at the surface of infective larvae of the parasitic nematode Trichinella spiralis.

Trichinella spiralis infective larvae have externally oriented enzymes catalysing reversible protein phosphorylation on their surface. Incubation of larvae with exogenous ATP resulted in phosphorylation of surface bound and released proteins. Exposure of the parasites to bile, a treatment which renders them infective for intestinal epithelia, resulted in increased release of protein and an altered profile of phosphorylation. Both serine/threonine and tyrosine phosphorylation and dephosphorylation reactions took place at the parasite surface. Examination of the structural characteristics of the larvae following exposure to bile showed that the non-bilayer surface coat was not shed but was structurally reorganised.

Synergistic action of a cyclic depsipeptide and piperazine on nematodes.

The present study describes the synergistic effects of the cyclic depsipeptide BAY 44-4400 and piperazine in the treatment against the nematodes Trichinella spiralis, Heligmosomoides polygyrus, and Heterakis spumosa. The in vitro anthelmintic activity of a combination of the two compounds (1.7 motility units) against T. spiralis larvae was significantly higher than the sum of the individual drug effects (1.3 motility units). With regard to the rate of expulsion of H. polygyrus worms from the intestine of infected mice, an additive effect was observed; piperazine alone exerted an efficacy of 54.4% and BAY 44-4400 alone, one of 44.4%, whereas the combination of these compounds had an efficacy of 97.5%. With regard to the expulsion of H. spumosa worms, the effect of the combination was more than 5 orders of magnitude greater than the sum of the effects of the single compounds, i.e., there was a considerable potentiation of the actions of BAY 44-4400 and piperazine. Moreover, the combination exerted a significantly higher degree of degenerative effects on the intestine and on the nerve chords of H. spumosa as compared with the single compounds.

Predilection sites of Trichinella spiralis larvae in naturally infected horses.

A total of 120 muscle tissues from three horses naturally infected with Trichinella spiralis were examined. The head was the most infected site. In particular, the muscles harbouring the highest number of larvae were: musculus buccinator (12, 411 and 1183 larvae g-1), the tongue (11, 615 and 1749 larvae g-1), m. levator labii maxillaris (17,582 and 1676 larvae g-1), and the masseter (4.9, 289 and 821 larvae g-1). Compared with the diaphragm, the number of larvae per gram was from 3.5 to 6.8 times higher in the tongue, from 3.5 to 6.5 higher in m. levator labii maxillaris, and from 2.5 to 4.6 higher in m. buccinator. Of the examined muscles, the diaphragm had from the 6th to the 15th highest level of infection (3.1, 166 and 256 larvae g-1). Published data from experimentally infected horses confirm these results, suggesting that efforts to detect predilection sites should focus on the head muscles.

The surface coat of infective larvae of Trichinella spiralis.

The surface coat of the infective larvae of the parasitic nematode Trichinella spiralis was characterized with respect to its biophysical properties, morphology and composition. Labelling of larvae with the fluorescent surface probe PKH26 was lost after activation (by incubation in mammalian medium containing trypsin and bile), or following pronase treatment. Electron microscopical examination revealed that pronase treatment resulted in the loss of an amorphous surface layer only, further demonstrating the specificity of PKH26 for the larval surface coat. Surface coat shedding was inhibited by sodium azide and carbonyl cyanide, or by incubation of larvae at 4 degrees C, suggesting the shedding process required metabolic energy. Pre-labelled, unactivated larvae demonstrated continuous slow surface coat shedding and could be re-labelled with PKH26, indicating that the shed coat is replaced in these parasites. However, pre-labelled larvae which were activated failed to re-label with the probe, suggesting that activation provides an irreversible trigger for surface changes. PKH26, therefore, is a useful marker for larval activation. Examination of the shed coat material by scanning electron microscopy revealed 2 types of morphologies; one comprising thin multilaminate sheets and the other of amorphous material with ridges producing a fingerprint-like motif. Western- and lectin-blotting of the shed coat material demonstrated 2 prominent entities; a 90 kDa glycoprotein, which bound Datura stramonium agglutinin and was resistant to N- and O-glycanase treatment and a 47-60 kDa set of protein(s). Analysis of the surface lipids by electrospray mass spectometry revealed the presence of lysophosphatidic acid (lysoPA, C14:2) and an unidentifiable component of 339.4 Da. These two lipids constituted 36.9% and 36% by mass of surface coat lipids respectively. The presence of lysoPA was confirmed by thin layer chromatography, which also detected phosphatidic acid (PA). The polar lipids detected in solvent rinses of intact parasites by electrospray mass spectrometry were PI (C48:4), PE (C40:4 and C38:4), PS (C40:4), lysoPC (C20:2 and C18:2) and lysoPA (C14:2). These observations are discussed with respect to the role of the surface coat and its shedding in the T. spiralis host-parasite relationship.

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.