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.     

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.     

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.     

Trichinella spiralis: histamine secretion induced by TSL-1 antigens from unsensitized mast cells

Mast cells' hyperplasia and activation are prominent features in Trichinella spiralis infection. Recently, it was shown that TSL-1 antigens from T. spiralis muscle larvae induce IL-4 and TNF release by unsensitized, normal mast cells (MC) involving an Ig-independent mechanism. In this study, we characterized histamine secretion induced by TSL-1 antigens from normal, unsensitized rat peritoneal MC. Maximum histamine secretion (30+/-5.3% SEM, n=13) was achieved with 30 ng/mL TSL-1 antigens. However, TSL-1 did not induce an increase in beta-hexosaminidase release or NADPH oxidase activity by MC. Interestingly, histamine secretion by TSL-1 was completed at 10s, and was inhibited by both Bordetella pertussis toxin and neuraminidase V, characteristics similar to those involved in substance P-induced histamine secretion. However, in contrast to substance P, TSL-1 induced histamine secretion in the absence of detectable changes in intracellular Ca(2+). We are investigating the molecular pathways involved in MC activation by TSL-1.     

Expressed sequence tags of Trichinella spiralis muscle stage larvae

In order to obtain greater insight into the relevant genomic expression patterns of Trichinella spiralis, 992 expressed sequence tags (ESTs) were collected from a cDNA library of T. spiralis muscle stage larvae and assembled into 60 clusters and 385 singletons. Of them, 445 (44.7%) ESTs were annotated to their homologous genes, and small fractions were matched to known genes of nematodes. The annotated ESTs were classified into 25 eukaryotic orthologous groups (KOG). Cytochrome C oxidase (34 clones) was found to be most frequent species.     

Protease-activated receptor 2 is involved in Th2 responses against Trichinella spiralis infection

In order to get a better understanding of the role of protease-activated receptor 2 (PAR2) in type 2 helper T (Th2) cell responses against Trichinella spiralis infection, we analyzed Th2 responses in T. spiralis-infected PAR2 knockout (KO) mice. The levels of the Th2 cell-secreted cytokines, IL-4, IL-5, and IL-13 were markedly reduced in the PAR2 KO mice as compared to the wild type mice following infection with T. spiralis. The serum levels of parasite-specific IgE increased significantly in the wild type mice as the result of T. spiralis infection, but this level was not significantly increased in PAR2 KO mice. The expression level of thymic stromal lymphopoietin, IL-25, and eotaxin gene (the genes were recently known as Th2 response initiators) of mouse intestinal epithelial cells were increased as the result of treatment with T. spiralis excretory-secretory proteins. However, the expression of these chemokine genes was inhibited by protease inhibitor treatments. In conclusion, PAR2 might involve in Th2 responses against T. spiralis infection.     

Hosts and habitats of Trichinella spiralis and Trichinella britovi in Europe

Trichinella spiralis and Trichinella britovi are the two most common species of Trichinella circulating in Europe. Based on data provided to the International Trichinella Reference Centre over the past 20 years (data referring to 540 isolates of T. spiralis and 776 isolates of T. britovi), we describe the host species and habitat characteristics for these two pathogens in Europe. A Geographical Information System was constructed using administrative boundaries, a Corine Land Cover (CLC) map, and an elevation map. In most countries, T. britovi is more widespread (62.5-100% of the isolates) than T. spiralis (0.0-37.5%), although in Finland, Germany, Poland and Spain, T. spiralis is more prevalent (56.3-84.2% of the isolates). Trichinella britovi is more widespread than T. spiralis in sylvatic carnivores (89% versus 11%), whereas T. spiralis is prevalent in both wild boars (62% versus 38%) and domestic swine (82% versus 18%), as well as in rodents (75% versus 25%). Trichinella spiralis and T. britovi circulate in the same environments: 41.1% and 46.0%, respectively, in agricultural areas, and 45.5% and 46.6% in forested and semi-natural areas. Although both pathogens can be transmitted by domestic and sylvatic cycles, their epidemiology is strongly influenced by the higher adaptability of T. spiralis to swine and of T. britovi to carnivores. These results are important because they include information on the countries at risk for these pathogens, the role played by specific species as reservoirs, the role of the pathogens in domestic and sylvatic cycles, and the role of the habitat in their circulation. The results can also be used to identify the most suitable animal species for the monitoring of these pathogens in Europe.     

Identification and characterization of a full-length cDNA encoding paramyosin of Trichinella spiralis

A full-length cDNA encoding Trichinella spiralis paramyosin (Ts-Pmy) was cloned by immunoscreening a cDNA library of the adult T. spiralis worm. Ts-Pmy cDNA consists of 2655 bp that encode 885 amino acids. The recombinant protein (rTs-Pmy) was expressed and purified by Ni-affinity chromatography. Western blot analysis showed that rTs-Pmy could be recognized by sera from T. spiralis-infected humans, swine, rabbits, and mice. Immunolocalization demonstrated that Ts-Pmy was abundant on the surface of T. spiralis larvae. BALB/c mice vaccinated with rTs-Pmy demonstrated 36.2% reduction in muscle larvae burden following T. spiralis larvae challenge. Vaccination of the mice with rTs-Pmy resulted in a high level of specific anti-Ts-Pmy IgG antibodies and generated a Th1/Th2 mixed type of immune response, with Th2 predominant. These studies showed that rTs-Pmy induced protective immunity in mice and could be considered as a potential vaccine candidate for trichinellosis.     

Trichinella spiralis: modulation of experimental autoimmune encephalomyelitis in DA rats

Helminth infection has a potent systemic immunomodulatory effect on the host immune response, which also affects the development of autoimmune diseases. We investigated the dose-dependent influence of Trichinella spiralis infection on experimental autoimmune encephalomyelitis (EAE). Our model of concomitant T. spiralis infection and EAE demonstrates that established infection of Dark Agouti (DA) rats with the parasite causes amelioration of the clinical course of induced EAE in a dose-dependent way. Infection with T. spiralis L1 stage muscle larvae (TSL1) reduced the severity of the autoimmune disease as judged by lower maximal clinical score, cumulative index, duration of illness and degree of mononuclear cell infiltration in T. spiralis infected animals compared to control, EAE-induced group. This study provides a valuable model of worm infection to investigate helminth-induced regulatory mechanisms for optimal benefit to the host.     

Glucose uptake and metabolism in the Trichinella spiralis nurse cell

Isolated Trichinella spiralis nurse cells transport a significantly greater amount of glucose/mg of protein than the normal skeletal muscle cell line (L6). V(max) and K(m) estimations revealed that nurse cells have a much higher saturation point than L6 cells for glucose. The effects of numerous physiological conditions (Na(+) concentration, pH, and temperature) on nurse cell glucose uptake were investigated. It was determined that sodium concentration had no effect on glucose uptake. Low (<6.5) and high (>7.3) pH and low (5 degrees C) temperatures significantly effected glucose uptake. The two hormones, insulin and epinephrine, appeared to have little, if any, influence on the rate of glucose uptake by nurse cells. Glucose uptake was inhibited in the presence of 6-carbon carbohydrates. The H(+)/glucose symport inhibitors, dicyclohexylcarbodiimide (DCCD) and Carbonyl cyanide 4-trifluoromethoxyphenlhydrazone (FCCP), and the facilitated diffusion inhibitor phloretin also inhibited glucose uptake. Oubain, a Na(+)/glucose symport inhibitor, did not inhibit glucose uptake. These data, in conjunction with Western blot analyses, revealed that the transport of glucose occurs via H(+)/glucose symport and facilitated diffusion, perhaps through the glucose transport proteins GLUT 1 and/or 4. It was also demonstrated that nurse cells are capable of synthesising glycogen. It appears that glycogen is in a constant state of flux and physiological conditions, such as glucose concentration, significantly influence the synthesis of this macromolecule. We conclude that these results are consistent with the hypothesis that nurse cells, at least maintained in vitro, are metabolically highly active but show significant divergence from normal muscle cells in several fundamental aspects of sugar metabolism.     

Susceptibility of Laboratory Rodents to Trichinella papuae

Members of the genus Trichinella are small nematodes that can infect a wide range of animal hosts. However, their infectivity varies depending on the parasite and host species combination. In this study, we examined the susceptibility of 4 species of laboratory rodents, i.e., mice, rats, hamsters, and gerbils to Trichinella papuae, an emerging non-encapsulated Trichinella species. Trichinella spiralis and Trichinella pseudospiralis were also included in this study for comparison. Fifteen animals of each rodent species were infected orally with 100 muscle larvae of each Trichinella species. Intestinal worm burden was determined at day 6 and 10 post-inoculation (PI). The numbers of muscle larvae were examined at day 45 PI. The reproductive capacity index (RCI) of the 3 Trichinella species in different rodent hosts was determined. By day 6 PI, 33.2-69.6% of the inoculated larvae of the 3 Trichinella species became adult worms in the small intestines of the host animals. However, in rats, more than 96% of adult worms of all 3 Trichinella species were expelled from the gut by day 10 PI. In gerbils, only 4.8-18.1% of adult worms were expelled by day 10 PI. In accordance with the intestinal worm burden and the persistence of adults, the RCI was the highest in gerbils with values of 241.5±41.0 for T. papuae, 432.6±48 for T. pseudospiralis, and 528.6±20.6 for T. spiralis. Hamsters ranked second and mice ranked third in susceptibility in terms of the RCI, Rats yielded the lowest parasite RCI for all 3 Trichinella species. Gerbils may be an alternative laboratory animal for isolation and maintenance of Trichinella spp.     

Immune Correlates of Resistance to Trichinella spiralis Reinfection in Mice

The immune correlate of host resistance induced by reinfection of Trichinella spiralis remains unclear. In this study, we investigated immune correlates between the resistance and serum IgG antibody level, CD23⁺ IgM⁺ B cells, and eosinophil responses induced by T. spiralis reinfection. Mice were primarily infected with 10 or 100 T. spiralis larvae (10 TS, 100 TS), respectively, and after 4 weeks, they were challenge infected with 100 T. spiralis larvae (10–100 TS, 100-100 TS). Upon challenge infections, 10–100 TS mice induced significantly higher levels of T. spiralis-specific total IgG antibody responses in sera and antibody secreting cell responses in spleens compared to 100-100 TS mice, resulting in significantly reduced worm burdens in 10–100 TS mice (60% and 70% reductions for adult and larvae, respectively). Higher levels of eosinophils were found in mice primarily infected with 10 TS compared to those of 100 TS at week 8 upon challenge. CD23⁺ IgM⁺ B cells were found to be increased significantly in mice primarily infected with 10 TS. These results indicate that primary infection of 10 larvae of T. spiralis, rather than 100 larvae, induces significant resistance against reinfection which closely correlated with T. spiralis-specific IgG, eosinophil, and CD23⁺ IgM⁺ B cell responses.     

Enzyme-linked immunosorbent assay for detection of Trichinella spiralis antibodies and the surveillance of selected pig breeding farms in the Republic of Korea

Trichinellosis is a parasitic zoonosis of public health importance. It is caused by Trichinella spiralis which has a wide host range including humans. In the present communication, the ELISA technique was employed on a total of 803 blood samples from 7 selected pig breeding farms in 1996 for diagnosis and surveillance of trichinellosis. Out of the entire 803 samples, nine were found to be suspected while one was positive by ELISA. But western blot analyses employed for further confirmation have shown that all of 10 samples did not react to larval excretory-secretory product antigens. These results indicate that pig breeding farms included in the present study are free from trichinellosis. However, it does not mean Korea is free from trichinellosis since human trichinellosis has recently been reported. The necessity of continued surveillance for trichinellosis in both pigs and wild animals was discussed.     

An Outbreak of Trichinosis with Molecular Identification of Trichinella sp. in Vietnam

The 5th outbreak of trichinosis occurred in a mountainous area of North Vietnam in 2012, involving 24 patients among 27 people who consumed raw pork together. Six of these patients visited several hospitals in Hanoi for treatment. Similar clinical symptoms appeared in these patients within 5-8 days after eating infected raw pork, which consisted of fever, muscle pain, difficult moving, edema, difficult swallowing, and difficult breathing. ELISA revealed all (6/6) positive reactions against Trichinella spiralis antigen and all cases showed positive biopsy results for Trichinella sp. larvae in the muscle. The larvae detected in the patients were identified as T. spiralis (Vietnamese strain) by the molecular analysis of the mitochondrial cytochrome c oxidase subunit III (cox3) gene.     

Activation and Recruitment of Regulatory T Cells via Chemokine Receptor Activation in Trichinella spiralis-Infected Mice

As most infections by the helminth parasite elicit the recruitment of CD4⁺CD25⁺Foxp3⁺ T (T_reg) cells, many scientists have suggested that these cells could be used for the treatment of immune-mediated inflammation and associated diseases. In order to investigate the distribution and alteration of activated T_reg cells, we compared the expression levels of T_reg cell activation markers in the ileum and gastrocnemius tissues 1, 2, and 4 weeks after infection. The number of T_reg cells was monitored using GFP-coded Foxp3 transgenic mice. In mice at 1 week after Trichinella spiralis infection, the number of activated T_reg cells was higher than in the control group. In mice at 2 weeks after infection, there was a significant increase in the number of cells expressing Foxp3 and CTLA-4 when compared to the control group and mice at 1 week after infection. At 4 weeks after infection, T. spiralis was easily identifiable in nurse cells in mouse muscles. In the intestine, the expression of Gzmb and Klrg1 decreased over time and that of Capg remained unchanged for the first and second week, then decreased in the 4th week. However, in the muscles, the expression of most chemokine genes was increased due to T. spiralis infection, in particular the expression levels of Gzmb, OX40, and CTLA-4 increased until week 4. In addition, increased gene expression of all chemokine receptors in muscle, CXCR3, CCR4, CCR5, CCR9, and CCR10, was observed up until the 4th week. In conclusion, various chemokine receptors showed increased expressions combined with recruitment of T_reg cells in the muscle tissue.     

Molecular identification of Korean Trichinella isolates

Muscle larvae of Trichinella isolates from two outbreaks in Korea were analyzed by a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and multiple-PCR. All of the muscle larvae showed a band similar to that of T. spiralis larvae of the reference strain. The two Korean Trichinella isolates (isolate code ISS623 and ISS1078) might be classifiable to Trichinella spiralis.     

Sleep Deprivation Induces Changes in Immunity in Trichinella spiralis-Infected Rats

Sleep is considered an important predictor of immunity. A lack of sleep may reduce immunity, which increases susceptibility to any type of infection. Moreover, sleep deprivation in humans produces changes in both, the percent of circulating immune cells (T cells and NK cells) and cytokine levels (IL-1, IFNγ, TNΦ-αα, IL-6 and IL-17). The aim of our study was to investigate whether sleep deprivation produces deregulation on immune variables during the immune response generated against the helminth parasite Trichinella spiralis. Because sleep deprivation is stressful per se, we designed another experiments to compared stress alone (consisting in movement restriction and single housing) with sleep deprivation, in both control (uninfected) and experimental (infected) rats. Our results demonstrate that the sleep deprivation and stress have a differential effect in mesenteric lymph nodes (MLN) and spleen. In uninfected rats sleep deprivation alone produces an increase in natural killer cells (NK+) and B cells (CD45+), accompanied by a decrease in cytotoxic T cells (CD3+CD8+) in spleen; while, in MLN, produces only an increase in natural killer cells (NK+). Both, SD and stress, produce an increased percentage of total T cells (CD3+) in spleen. In the MLN both are also associated to an increase in cytotoxic T cells (CD3+CD8+) and B cells (CD45+). In the spleens of parasitized rats, cell populations did not change. In spleens of both, sleep-deprived and stressed infected rats, we observed an increase in B cells (CD45+). In infected rats, sleep deprivation alone produced an increase in NK cells (NK+). In mesenteric node cell populations of parasitized rats, we observed a decrease in NK cells and an increase in T helper (CD4+) cells in both SD and stressed rats. Rats that were only subjected to stress showed a decrease in B cells (CD45+). These findings suggest that the immune response generated against infection caused by T. spiralis is affected when the sleep pattern is disrupted. These results support the notion that sleep is a fundamental process for an adequate and strong immune response generated against this parasite.     

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.     

Trichinella spiralis Infection Suppressed Gut Inflammation with CD4+CD25+Foxp3+ T Cell Recruitment

In order to know the effect of pre-existing Trichinella spiralis infection on experimentally induced intestinal inflammation and immune responses, we induced colitis in T. spiralis-infected mice and observed the severity of colitis and the levels of Th1, Th2, and regulatory cytokines and recruitment of CD4⁺CD25⁺Foxp3⁺ T (regulatory T; T_reg) cells. Female C57BL/6 mice were infected with 250 muscle larvae; after 4 weeks, induction of experimental colitis was performed using 3% dextran sulfate sodium (DSS). During the induction period, we observed severity of colitis, including weight loss and status of stool, and evaluated the disease activity index (DAI). A significantly low DAI and degree of weight loss were observed in infected mice, compared with uninfected mice. In addition, colon length in infected mice was not contracted, compared with uninfected mice. We also observed a significant increase in production of pro-inflammatory cytokines, IL-6 and IFN-γ, in spleen lymphocytes treated with DSS; however, such an increase was not observed in infected mice treated with DSS. Of particular interest, production of regulatory cytokines, IL-10 and transforming growth factor (TGF)-β, in spleen lymphocytes showed a significant increase in mice infected with T. spiralis. A similar result was observed in mesenteric lymph nodes (MLN). Subsets of the population of T_reg cells in MLN and spleen showed significant increases in mice infected with T. spiralis. In conclusion, T. spiralis infection can inhibit the DSS-induced colitis in mice by enhancing the regulatory cytokine and T_reg cells recruitment.