Comparative efficacy of flubendazole and mebendazole on encysted larvae of Trichinella spiralis (USA strain) in the diaphragm of mice and rats

Comparative efficacy of mebendazole and flubendazole, a p-fluor analogue of mebendazole against the encysted larvae of Trichinella spiralis (USA strain) in the diaphragm of mice and rats were studied in order to provide a better understanding of the structure-activity relationship within the benzimidazole series. Drugs given 10-100 mg/kg/day for 3 consecutive days (35-37 days post-infection) or at 300 mg/kg, 35 days post infection were significantly effective in decreasing early encysted larvae in mice. No significant differences in effectiveness against the early encysted larvae could be observed between the drugs under the present experimental conditions. Mebendazole was found to be more effective that flubendazole in decreasing old encysted larvae in mice treated 70-72 days post-infection based on a comparative study of their ED50 values. When rats were given the drugs at the dose of 10 mg/kg/day for 3 consecutive days, mebendazole was significantly effective against both early and old encysted larvae while flubendazole was not.     

Antigen production by encysted muscle larvae of Trichinella spiralis

The production of excretory-secretory antigens by encysted muscle larvae of Trichinella spiralis has been investigated immuno-histochemically using an antiserum raised by infection in rabbits and purified both before and after conjugation by ion-exchange chromatography. The specificity of the antibody for excretory-secretory products was demonstrated by the pattern of staining of live worms in vitro and the failure of the labelled antibody to stain dead, non-metabolizing worms. Using this labelled antibody, and unlabelled antibody in the immunoperoxidase system, the presence of parasite antigen-bearing cells in close proximity to encysted muscle larvae has been demonstrated. This is believed to be the first demonstration of antigen production by encysted muscle larvae in vivo. The implications of this observation to current concepts of immunity to Trichinella spiralis are discussed.     

Therapeutic Potential of Myrrh and Ivermectin against Experimental Trichinella spiralis Infection in Mice

Trichinosis is a parasitic zoonosis caused by the nematode Trichinella spiralis. Anthelmintics are used to eliminate intestinal adults as well as tissue-migrating and encysted larvae. This study aimed to investigate the effects of ivermectin and myrrh obtained from the aloe-gum resin of Commiphora molmol on experimental trichinosis. Ninety albino mice were orally infected with 300 T. spiralis larvae. Drugs were tested against adult worms at day 0 and day 5 and against encysted larvae on day 15 and day 35 post-infection (PI). Mature worms and encysted larvae were counted in addition to histopathological examination of muscle specimens. Serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), total protein, albumin, globulin, urea, and creatinine values were estimated. Significant reductions in mean worm numbers were detected in ivermectin treated mice at day 0 and day 5 PI achieving efficacies of 98.5% and 80.0%, while efficacies of myrrh in treated mice were 80.7% and 51.5%, respectively. At days 15 and 35 post-infection, ivermectin induced significant reduction in encysted larval counts achieving efficacies of 76.5% and 54.0%, respectively, while myrrh efficacies were 76.6% and 35.0%, respectively. AST, ALT, urea, and creatinine levels were reduced, while total proteins were increased in response to both treatments compared to their values in the infected non-treated mice. Ivermectin use for controlling T. spiralis could be continued. Myrrh was effective and could be a promising drug against the Egyptian strains of T. spiralis with results nearly comparable to ivermectin.     

Immunity to Trichinella spiralis VI. The specificity of the immune response stimulated by the intestinal stage.

Mice were immunized to the intestinal stage of T. spiralis by using infections terminated with methyridine before production of newborn larvae had commenced. The muscle larvae which encysted following a normal complete challenge infection were reduced by 87 and 95% in immunized mice. No statistically significant reduction in a challenge infection of intravenously injected parenteral larvae was produced (8% and 15% actual reduction). Previous work has shown that adult worms in a challenge infection are stunted and expelled earlier as well as having a reduced fecundity; it is concluded that the immunity generated by the intestinal stage is largely specific in its action to that phase in a challenge infection.     

Trichinella spiralis: migration of larvae in the rat

Counts were made of Trichinella spiralis “migratory” larvae recovered from blood, abdominal cavity, lungs, liver, kidneys, and thoracic duct lymph of male albino rats from 4–15 days postinoculation. From these data, the pathways the larvae utilized to travel from the small intestine to skeletal muscle were determined. Approximately 70% of the encysted muscle larvae were accounted for by the lymph-blood circulatory system pathway. The data indicate that the other 30% probably migrated by way of both (a) the hepatic portal circulatory system to the heart and then to the general circulation and skeletal muscle; and (b) the abdominal cavity and/or abdominal fluids to skeletal muscle.     

CCR3 is required for tissue eosinophilia and larval cytotoxicity after infection with Trichinella spiralis

The CCR3 binds at least seven different CC chemokines and is expressed on eosinophils, mast cells (MC), and a subset of Th cells (Th2) that generate cytokines implicated in mucosal immune responses. Using mice with a targeted disruption of CCR3 (CCR3(-/-)) and their +/+ littermates, we investigated the role of CCR3 in the amplification of tissue eosinophilia and MC hyperplasia in the mouse after infection with Trichinella spiralis. In CCR3(-/-) mice, eosinophils are not recruited to the jejunal mucosa after infection and are not present in the skeletal muscle adjacent to encysting larvae. In addition, the number of cysts in the skeletal muscle is increased and the frequency of encysted larvae exhibiting necrosis is reduced. The CCR3(-/-) mice exhibit the expected MC hyperplasia in the jejunum and caecum and reject the adult worms from the small intestine at a normal rate. This study is consistent with distinct functions for MC (adult worm expulsion) and eosinophils (toxicity to larvae) in immunity to a helminth, T. spiralis, and defines the essential requirement for CCR3 in eosinophil, but not MC recruitment to tissues.     

Trichinella spiralis: major histocompatibility complex-associated elimination of encysted muscle larvae in swine

A heretofore undescribed host-mediated reactivity against encapsulated muscle larvae (ML) of the nematode Trichinella spiralis is reported. Inbred miniature swine (NIH minipigs) of three independent SLA phenotypes, which received a primary oral dose of 300 T. spiralis ML, successfully resisted a secondary infection of 10,000 ML; however, only pigs of the SLAa/a phenotype exhibited an unusual and highly significant reduction in the numbers of encysted ML from the primary infection (P less than 0.0003). This initial anti-encysted ML reactivity was confirmed in subsequent trials by comparing the prechallenge ML burdens with the reduced ML numbers in primary-infected aa pigs after challenge. Analyses of inbred strains of mice, selected for major histocompatibility type and for resistance or susceptibility to infection with T. spiralis, showed no such anti-encysted ML response. Because elimination of encysted T. spiralis ML had been accomplished previously only through selected drug regimens, our demonstration of a nonpharmacological, host-mediated reactivity against this stage of the parasite in swine highlights the importance of MHC genes in regulating disease resistance in a livestock species.     

Trichinella spiralis: specificity of ES antigens from pre-encysted larvae.

Excretory/secretory (ES) antigens were obtained by culturing pre-encysted Trichinella spiralis larvae which were recovered from muscles of experimentally infected mice 14-15 days postinfection. Analyses of these antigens (PEL ES) with immunoblotting, SDS-PAGE and Triple Antibody ELISA showed that they yielded a low sensitivity and specificity when tested with antisera against the common nematodes of Chinese pigs. As compared to ES antigens from encysted larvae, PEL ES also contained more low molecular mass proteins.     

Developmentally regulated gene expression in Artemia: Histone gene expression in newly hatched larvae

The synthesis of histones and presence of histone mRNA sequences in embryos and larvae of the brine shrimp, Artemia, were investigated. Radiolabeling of proteins synthesized in vivo followed by electrophoretic and fluorographic analysis confirmed the prediction that histone synthesis is coordinated with the wave of DNA replication in newly hatched larvae. No histone synthesis occurs during development of encysted embryos. Hybridization of cloned Artemia histone gene DNA to total cell RNA indicated that dormant encysted embryos do not contain “masked” messenger RNA.     

Life cycle of Stephanoprora uruguayense (Digenea: Echinostomatidae) in Argentina

The life cycle of Stephanoprora uruguayense Holcman et Olagüe, 1989, was experimentally resolved. In an artificial pond in the Zoological Garden in Buenos Aires City, Argentina, Heleobia parchappei (Hydrobiidae) was found to be releasing large-tailed cercariae with a prepharyngeal body, but lacking collar spines and corpuscles in the excretory system. Metacercariae, which encysted on the gills of naturally and experimentally infected Cnesterodon decemmaculatus (Poecilidae), developed collar spines and corpuscles in the excretory system in 7 days. Sexually mature adults were recovered from chicks and immature adults from mice fed metacercariae from C. decemmaculatus. Eggs shed in chick feces developed to miracidia within 10 days; sporocysts were found on the gills of snails. Stephanoprora uruguayense and S. denticulata from Europe are similar in adult morphology, but can be distinguished by morphological and behavioral features of larvae. Likewise, although S. denticulata and S. paradenticulata from Venezuela are similar to S. uruguayense in adult morphology, they differ considerably in larval morphology and intermediate hosts.     

The first human case of Trichinella spiralis infection in Korea

Three cases of human infection by Trichinella spiralis were first confirmed by detecting encysted larvae in the biopsied muscle in December 1997, in Korea. The patients were one 35- and two 39-year-old males residing in Kochang-gun, Kyongsangnam-do. They had a common past history of eating raw liver, spleen, blood and muscle of a badger, Meles meles melanogenys, and complained of high fever, facial and periorbital edema, and myalgia. Hematologic and biochemical examinations revealed leukocytosis and eosinophilia, and highly elevated levels of GOT, GPT, LDH and CPK. In the gastrocnemius muscle of a patient, roundly coiled nematode larvae were detected. The larvae measured 0.775-1.050 (av. 0.908) mm in length, and 0.026-0.042 (av. 0.035) mm in maximum width. The specific IgG antibody levels in three patients' sera were significantly higher when compared with those of normal controls. The patients were treated with flubendazole and albendazole for 15-30 days, and discharged at 13-34 days post-admission. From the above findings, it was confirmed that T. spiralis is present in Korea, and the badger plays a role of as the natural host.     

Persistence of Gnathostoma hispidum in chronically infected rats

Gnathostomiasis by Gnathostoma hispidum has occurred mainly in southern and western areas of Japan since 1980. In all cases, patients had eaten live loaches, freshwater fish imported from southeast Asia. Chief symptoms were creeping linear skin eruptions and eosinophilia, both of which disappeared in most patients within three months after the onset, and never recurred thereafter. In order to study how gnathostome larvae may survive and behave in humans, rats were infected with gnathostome larvae. Loaches were purchased in city markets in Fukuoka. Gnathostome larvae (G. hispidum) were recovered from the fish and fed to rats. They were killed at intervals of one and six months and one, two and three years postinoculation. Encysted larvae were found in the muscles of all rats and recovered larvae were all viable. A migrating larva was observed in muscle tissue from one rat that was killed three years postinoculation. In histological sections, walls around encysted larva were much thinner in a rat killed three years postinoculation than in a rat killed one month postinoculation.     

Survival of Trichinella spiralis larvae in sewage sludge anaerobic digesters

The survival of some bacteria, viruses, protozoans, and helminths through the sewage digestion process has been a question of considerable concern among researchers throughout the world. Among the most resistant organisms are some of the pathogenic roundworms and tapeworms. Encysted larvae of Trichinella spiralis are sometimes present in animal tissues discarded as waste from slaughterhouses, restaurants or other sources. In experimental anaerobic sewage digesters, encysted larvae of T. spiralis, in rat muscle, were able to survive a maximum of 96 hr in a "batch" digester. In a digester "fed" daily with small numbers of encysted larvae, "draw-off" remained infective for white rats throughout a 16-day experimental period. Potentially infective material could be present when there is continuous "draw-off" from the anaerobic digesters.     

Effects of Toxoplasma gondii (Gleadle strain) on the host-parasite relationship in trichinosis.

The effects of concurrent infection with Toxoplasma gondii on the host-parasite relationship in trichinosis were studied. Infected mice showed a delay in expulsion of Trichinella spiralis adults from the gut. Persisting adult female worms were fecund but the numbers of larvae recovered from the muscles were not increased. Increased resistance to the systemic phase of trichinosis was shown by reduced numbers of muscle larvae after intravenous injection of newborn larvae in animals with toxoplasmosis as compared with control mice. There were no differences in small bowel pathology of trichinous mice with and without toxoplasmosis but inflammation around muscle cysts of T. spiralis was reduced in mice with toxoplasmosis. The eosinophilia which normally develops in mice with trichinosis was suppressed by concurrent toxoplasmosis. Trichinella infection did not alter the numbers of T. gondii cysts recovered from the brain 4 weeks after infection. It is suggested that the delay in expulsion of adult worms, decrease in muscle inflammation around T. spiralis cysts, and inhibition of eosinophilia result from immune suppression, while the reduction in numbers of muscle larvae after intravenous injection of newborn larvae reflects enhanced nonspecific resistance to infection in toxoplasmosis.     

Parasitic freshwater pearl mussel larvae (Margaritifera margaritifera L.) reduce the drift-feeding rate of juvenile brown trout (Salmo trutta L.)

In this paper we describe, for the first time, the effects of freshwater pearl mussel (Margaritifera margaritifera L.) encystment on the drift-feeding behavior of juvenile brown trout (Salmo trutta L.). Because both mussel and salmonid populations are often threatened, this study not only adds knowledge to the understanding of host-parasite systems, but it is also of conservation value. Individual trout, mussel-encysted (25.1?±?5.7 larvae?·?g^?1 body weight, n?=?5) or non-encysted (n?=?5), were fed with chironomid larvae in a flow-through stream aquarium. Feeding trials were filmed and analyzed by counting the numbers of chironomid larvae each individual ate, and by estimating the prey-capture distance. Non-encysted trout had a significantly higher drift-foraging rate than did encysted trout, and they captured significantly more prey further away from their focal point. The reduced foraging success of encysted trout was mainly due to their failure to catch prey relatively further from their focal point. This suggests that reduced foraging success of encysted trout may be due to poorer energetic status, but the physical effects of mussel larvae on prey handling time cannot be ruled out. Encysted trout caught approximately 20 % fewer prey, which would result in a reduction in growth potential during the period of mussel encystment. Reduced energetic status might also result in reduced competitive ability or in increased exposure to predation risk.     

Trichinella spiralis: nonspecific resistance and immunity to newborn larvae in inbred mice

The implantation and development of intravenously injected Trichinella spiralis newborn larvae were examined in different strains of inbred mice by determining muscle larvae burden. This was compared to the numbers of muscle larvae that established after a natural infection during which a quantitative assessment of intestinal newborn larvae production was made. In most inbred strains of mice, newborn larvae do not all successfully implant in muscle. Mice of the DBA/1 strain are the most resistant to successful implantation, and C3H mice are the most permissive. This pattern is evident in the strains studied whether newborn larvae are injected intravenously or are produced by intestinal adults. Thus, after a natural infection, 100% of intestinally produced newborn larvae implanted in C3H mice, whereas in NFR 68% and DBA/1 mice 62% successfully matured in muscle. Immunity to newborn larvae could be demonstrated as early as 10 days after exposure to this stage of the life cycle. This immunity was protective against a complete challenge infection given 9 days after newborn larvae had been injected intravenously. Protection against newborn larvae was identical in male and female mice or in mice from 1 to 9 months of age. We conclude that there are two mechanisms by which mice impair newborn larvae establishment or development in muscle. The first appears to be nonimmunological (non-specific resistance), and the second is immunological. Genetically determined variation in strain-specific expression is apparent with both mechanisms. In strains displaying high intrinsic "resistance" (DBA/1), this process is likely to account for most of the 38% reduction in newborn larvae establishment in a primary infection. However, immunity against newborn larvae develops quickly enough to have a significant effect on migratory larvae in primary infections where adults persist in the intestine (e.g., the B10 congenic mice), or when high adult worm burdens delay adult worm rejection. Muscle larvae burden, therefore, reflects systemic nonspecific resistance to newborn larvae as well as immunological processes that occur in the intestine and systemically.     

IL-18 regulates intestinal mastocytosis and Th2 cytokine production independently of IFN-gamma during Trichinella spiralis infection

Expulsion of the gastrointestinal nematode Trichinella spiralis is associated with pronounced mastocytosis mediated by a Th2-type response involving IL-4, IL-10, and IL-13. Here we demonstrate that IL-18 is a key negative regulator of protective immune responses against T. spiralis in vivo. IL-18 knockout mice are highly resistant to T. spiralis infection, expel the worms rapidly and subsequently develop low levels of encysted muscle larvae. The increased speed of expulsion is correlated with high numbers of mucosal mast cells and an increase in IL-13 and IL-10 secretion. When normal mice were treated with rIL-18 in vivo, worm expulsion was notably delayed, and the development of mastocytosis and Th2 cytokine production was significantly reduced. The treatment had no effect on intestinal eosinophilia or goblet cell hyperplasia but specifically inhibited the development of mastocytosis. Addition of rIL-18 to in vitro cultures of bone marrow-derived mast cells resulted in a significant reduction in cell yields as well as in the number of IL-4-secreting mast cells. In vivo treatment of T. spiralis-infected IFN-gamma knockout mice with rIL-18 demonstrated that the inhibitory effect of IL-18 on mastocytosis and Th2 cytokine secretion is independent of IFN-gamma. Hence, IL-18 plays a significant biological role as a negative regulator of intestinal mast cell responses and may promote the survival of intestinal parasites in vivo.     

The location of parasites within their hosts: Factors affecting longitudinal distribution of Trichinella spiralis in the small intestine of mice

Inocula of encysted larvae, excysted larvae or infected meat all resulted in aggregated distributions of adults in the anterior small intestine. However, implantation studies indicated that the larvae could establish anywhere between the duodenum and the colon. The site of larval establishment determines the subsequent location of adult T. spiralis; the site of establishment was due largely to influences such as intestinal motility, size of inoculum and the size of the vehicle of infection. Predominantly female inocula were dispersed similarly to heterosexual inocula while male populations were dispersed more widely.     

Neutral lipids in cercariae, encysted metacercariae, and rediae of Zygocotyle lunata

High-performance thin-layer chromatography was used to analyze the neutral lipids in the rediae, cercariae, and encysted metacercariae of the paramphistomid trematode Zygocotyle lunata. Visual observations of the chromatograms showed that the most abundant lipid fractions were free sterols and free fatty acids in all larval stages and triacylglycerols in the metacercariae and rediae. The weight of free sterols (x +/- SE) was 120+/-20 ng/cercaria, 56+/-3.8 ng/redia, and 5.9+/-1.5 ng/encysted metacercaria; the weight of triacylglycerols was 13+/-0.88 ng/encysted metacercaria, 6.3+/-0.063 ng/redia, and was not detectable in the cercaria; the weight of free fatty acids was 160+/-17 ng/ cercaria, 76+/-9.1 ng/redia, and 4.2+/-0.46 ng/encysted metacercaria. Oil red O staining of whole larvae showed the presence of neutral lipids in the rediae but not in the cercariae or encysted metacercariae. A dramatic reduction was seen in the quantity of free sterols and free fatty acids in the encysted metacercariae as compared with the cercariae, suggesting that these neutral lipids are used in some way during the transformation from cercaria to metacercaria.