Trichinella spiralis: in vitro cytotoxicity of peritoneal cells against synchronous newborn larvae of different age

Two different immune sera obtained from mice infected with muscle larvae (ML) of Trichinella spiralis (oral infection) or injected with 20-h-old newborn larvae (NBL) via retro-orbital venous plexus (intraocular injection) were compared in antibody-dependent cellular cytotoxicity test. Four synchronous stages of NBL, 0–2 h old, 22–24 h old, 46–48 h old and 6 days old (sNBL) were used to study susceptibility of larvae to the cytotoxic reaction of peritoneal cells. Peritoneal cells adhered to and destroyed sNBL of T. spiralis only in the presence of both immune sera. Living sNBL, living sNBL covered by peritoneal cells and dead sNBL were observed in all experimental groups. The lowest percentage of dead sNBL was detected in the 22- to 24-h-old groups. A dramatic increase in mortality was observed in older groups. A greater susceptibility of sNBL to serum obtained from mice infected per os with ML was observed. Serum obtained after oral infection showed higher levels of IgG1 isotype specific to ML than serum obtained after ocular injection. Received: 16 April 1998  / Accepted: 25 June 1998     

The antiphosphorylcholine plaque-forming cell responses induced by the nematode Trichinella in BWF1 mice

We have tested the ability of the nematode Trichinella to infect young and old (NZB × NZW) F₁ (BWF1) mice. We report the capacity of these mice to respond to the parasite antigens containing the epitope phosphorylcholine. The values obtained in adult worm intestinal retention rates and in the number of encysted larvae on the 35th day postinfection showed that the old BWF1 mice are more susceptible than young BWF1 and control (BALB/c × CBA/j)F₁ (BCF1) mice to Trichinella infection. However, unlike the BCF1 mice, young BWF1 mice were unable to produce a good anti-phosphorylcholine plaque-forming cell response after the killing of Trichinella larvae by the anthelminthic mebendazole. Old BWF1 mice presented a discrete response which is discussed. Finally, our results seem to indicate that the lack of anti-phosphorylcholine response in young BWF1 mice after mebendazole treatment may be related to the high susceptibility of these mice to the suppressive properties of encysted Trichinella larvae against their own antigens.     

Epidemiology, histpathology, and muscle distribution of Trichinella T9 in feral raccoons (Procyon lotor) and wildlife of Japan

The prevalences of Trichinella T9 in trapped raccoons (Procyon lotor) and several other potential mammalian reservoirs in Hokkaido, Wakayama, and Nagasaki Prefectures were investigated. Muscle samples were collected from 2003 to 2006 from 1,080 raccoons, 113 raccoon dogs including 2 species (Nyctereutes procyonoides albus and N. p. viverrinus), 41 wild boars (Sus scrofa leucomystax), 14 martens (Martes melampus), 10 badgers (Meles meles), 5 Siberian weasels (Martes sibirica coreana), 7 mink (Mustela vison), and 1 red fox (Vulpes vulpes japonica). The samples were digested, and the prevalence and mean intensity of infection with the Trichinella muscle larvae were determined. The prevalence and intensity of the muscle larvae were 0.9% and 93.3 larvae/g (range 0.4–201.8) in raccoons, and 1.6% and 61.6 larvae/g in raccoon dogs, respectively. The infected animals were captured in different areas in Hokkaido Prefecture. These results confirmed that raccoons, which have been introduced from North America since the 1970s, are involved in the sylvatic cycle of Trichinella in Japan. In raccoons, the muscle density of Trichinella T9 larvae was highest in the tongue, and larvae were not found in the heart muscle or diaphragm. This is the first report of Trichinella T9 infection of feral raccoons in Japan.     

Resveratrol inhibits the development of obesity-related osteoarthritis via the TLR4 and PI3K/Akt signaling pathways

ABSTRACT

Aim of the study: Obesity leads to mild, chronic inflammation which is a primary risk factor for osteoarthritis (OA). Resveratrol exerts a protective effect on OA through its anti-inflammatory properties, but the precise mechanism remains unknown. The present study aimed to investigate the mechanism by which resveratrol alleviates obesity-related OA, and whether it is linked to the TLR4 and PI3K/Akt signaling pathways.

Materials and methods: C57BL/6J male mice were fed a high-fat diet (HFD) with or without resveratrol treatment and knee joints were collected for analysis. In addition, IL-1β-induced SW1353 cells were used to study in vitro the reciprocal effects of TLR4 and PI3K/Akt pathways.

Results: Resveratrol inhibited the development of OA in mice fed a HFD. TLR4 and PI3K/Akt signaling pathways were both activated in the articular cartilage; resveratrol treatment down-regulated TLR4 but up-regulated PI3K/Akt signaling. Further in vitro results showed that the effect of resveratrol alone on activation of PI3K/Akt was attenuated but not abolished by the TLR4 inhibitor CLI-095, and resveratrol failed to reduce TLR4 protein expression in IL-1β stimulated cells pretreated with the PI3K inhibitor LY294002.

Conclusions: Resveratrol may exert an anti-osteoarthritic effect by inhibiting TLR4 via the activation of PI3K/Akt signaling pathways. Resveratrol has potential as a drug for OA prevention.     

Specificity and sensitivity of random amplified polymorphic DNA analysis for the identification of single larvae of Trichinella after experimental infection of pigs

Single muscle larvae (ML) of Trichinella belonging to 5 species and 2 genotypes collected from 126 experimentally infected pigs and preserved in 75% ethyl alcohol at room temperature were identified by random amplified polymorphic DNA (RAPD) analysis to evaluate the specificity and sensitivity of this assay. A double-blind protocol was used to ensure that the interpretation of electrophoretic patterns after RAPD amplification was not influenced by an expected result. RAPD analysis of one larva from each sample showed a sensitivity of 100% when only samples with undamaged DNA were considered, and the specificity resulted in an 88% match with the species or genotype that had been used to infect pigs, whereas the identification reached 100% specificity when an additional one to four ML were examined. Received: 30 September 1998 / Accepted: 21 December 1998     

Molecular epidemiology of <Emphasis Type="Italic">Trichinella</Emphasis> spp. in three Baltic countries: Lithuania,Latvia, and Estonia

Meat of domestic pigs and wild boars has been the significant source of emerged human trichinellosis in Lithuania, Latvia, and Estonia over the past two decades. However, there is very little known on the occurrence of Trichinella spp. in main wildlife reservoirs and its transmission in domestic and sylvatic cycles in these countries. The present study demonstrated considerably higher endemicity of Trichinella spp. in main sylvatic reservoirs (28.9–42% in foxes (Vulpes vulpes) and raccoon dogs (Nyctereutes procyonoides) in all three countries than previously reported. Molecular identification of Trichinella larvae from more than 500 sylvatic and domestic animals revealed four Trichinella species (Trichinella spiralis, Trichinella britovi, Trichinella nativa, and Trichinella pseudospiralis) sympatric in a relatively small area and several as the first records for the respective countries. The nonencapsulating T. pseudospiralis is found for the first time in the Eastern Europe. Sylvatic T. britovi was found in domestic pigs in Lithuania and Latvia (16 and 57.1%, respectively) and only in these countries, domestic T. spiralis was detected in sylvatic animals in areas where domestic trichinellosis was registered. The study suggests that transmission of Trichinella between domestic and sylvatic cycles in Lithuania and Latvia is favored by improper human behavior, e.g., pig and slaughter waste management.     

A preliminary investigation on the infectivity of<Emphasis Type="Italic"> Trichinella</Emphasis> larvae in traditional preparations of walrus meat

This study evaluated the infectivity of Trichinella nativa in freshly frozen walrus meat and traditionally aged walrus meat (igunaq) associated with two human outbreaks of trichinellosis in the Canadian Arctic. Trichinella larvae recovered from walrus meat stored at –20°C for up to 20 months remained infective for guinea pigs inoculated with 135 or 716 larval doses. However, none of the 4–5 and 10-month-old igunaq preparations contained infective T. nativa larvae as measured by bioassays using mice and guinea pigs at inoculation doses ranging from 6 to 500 larvae. This indicates that the degradation process that occurred in the field can be sufficient to either kill Trichinella larvae or render them non-infective for mice and guinea pigs. Further research is needed to evaluate the food safety risk of traditional walrus igunaq aged under different field conditions and storage times.     

Distribution of muscle larvae and antibody dynamics in goats experimentally infected with Trichinella spiralis

Herbivorous animals can play a very important role in spreading trichinellosis. In the study presented here, the susceptibility and distribution of Trichinella spiralis infection was examined in 16 goat kids. The goats were inoculated with 10,000 T. spiralis larvae isolated by artificial digestion methods. The animals were necropsied per two animals in weekly intervals, and the larval burdens in different muscle tissue and anti-Trichinella antibodies measured with the indirect enzyme-linked immunosorbent assay (ELISA) serological method using excretory–secretory (E/S) antigen for detecting anti-Trichinella antibodies were assessed during the experiment. T. spiralis larval burden was maximal at 6 weeks postinoculation (480–5,057 larvae/g according to locality), and the larvae were also found in the myocardium (0.77 larvae/g). In this paper, our next step was to compare the specificity and the time of seroconversion by means of ELISA based on E/S antigen prepared from T. spiralis. Antibody response was detected in all 16 goats. The ELISA test carried out showed the first increments in optical density 2 weeks postinfection (p.i.), reached their peak 4 weeks p.i., and remained elevated from that day until the end of the experiment (10 weeks p.i.). These results indicated that specific anti-Trichinella antibodies in goats persist for a relatively long time.     

旋毛虫新生幼虫T668重组抗原对小鼠的保护性免疫

目的研究旋毛虫新生幼虫T668重组抗原的免疫原性,制备基因工程疫苗。方法以旋毛虫新生幼虫期特异性基因T668在大肠杆菌中的表达蛋白为抗原免疫小鼠,每间隔10d免疫1次,共免疫3次。末次免疫后10d,每只小鼠攻击感染200条旋毛虫感染性肌幼虫,感染后5周用消化法检查肌幼虫(ML)负荷。结果T668重组抗原免疫组肌幼虫减虫率明显高于佐剂组和对照组。结论T668重组抗原能诱导小鼠产生一定程度的保护性免疫,且可激发特异性体液免疫。     

Efficacy of Lasia spinosa leaf extract in treating mice infected with Trichinella spiralis

Trichinellosis is a widespread zoonoses for which no effective drug treatment is available at this time. Though anthelmintics such as mebendazole and albendazole are commonly used to treat human trichinellosis, none of these drugs are fully effective against the encysted or new-born larvae of Trichinella spiralis. In recent years, there has been a growing interest in developing newer anthelminthics from medicinal plants, particularly the ones used in traditional medicines in many parts of the world, due to the increasing spread of anthelminthic resistance and/or decreasing activity against encapsulated larval stages of parasites. The aim of the present study was to investigate the efficacy of leaf extract of Lasia spinosa (Araceae) against different life cycle stages of T. spiralis, i.e. adult (days 3 and 4 post-infection), migrating larvae (days 8, 9 and 10 post-infection) and encysted muscle larvae (days 31–37 post-infection). The study showed that L. spinosa leaf extract is effective against all the three life cycle stages of parasite. Against the adult stage, an oral administration of plant extract at 800 mg/kg dose revealed a 75.30% reduction in the number of adult worms, as compared to untreated controls at day 10 post-infection. Whereas against migrating larvae, the same dose of plant extract given for 3 days, reduced the number of larvae recovered from musculature of treated animals by 72.23%. However, in comparison of preceding two stages, the extract showed comparatively less efficacy against the encysted larvae of parasite. In this case, the 800 mg/kg dose of extract given for 7 days (after 30 day of post-infection) revealed only 64.84% reduction in the number of encysted larvae, as was evident from larval count on day 49 post-infection. Therefore, the results of this study indicate that leaf extract of L. spinosa possesses significant anthelminthic efficacy against the adult stages and migrating larvae of T. spiralis. On the other hand, the encysted muscle larvae of parasite are comparatively less sensitive to L. spinosa leaf extract treatment.     

Infectivity, predilection sites, and freeze tolerance of Trichinella spp. in experimentally infected sheep

A total of 36 sheep in groups of 4 were inoculated with 9 isolates of Trichinella and euthanized after 10 weeks. Thereafter, numbers of muscle larvae were determined in 13 different muscles/muscle groups. Muscle larvae were found in high numbers in all four sheep inoculated with T. spiralis, in lower numbers in two sheep inoculated with T. pseudospiralis (USA isolate), and in very low numbers in one sheep inoculated with T. pseudospiralis (USSR isolate) and one inoculated with T. britovi. In infections of high and moderate larval intensity, predilection sites of T. spiralis were the masseter muscles, the tongue, and the diaphragm and those of T. pseudospiralis were the masseter muscle and the neck. In low-intensity infections, muscle larvae were detected only in the diaphragm or in pooled muscle samples. For evaluation of the freeze tolerance of the different Trichinella species in sheep-muscle tissue, samples taken from the filet were stored at +5°, −5°, and −18 °C, respectively. After exposure for 1 and 4 weeks the tissue was digested and the released larvae were inoculated into mice for determination of the reproductive capacity index (RCI). Larvae of both T. spiralis and T. pseudospiralis survived freezing at −5° and −18 °C for 4 weeks. Received: 10 September 1999 / Accepted: 22 October 1999     

Effect of resveratrol in experimental osteoarthritis in rabbits

Objective: Resveratrol (trans-3,4,5-trihydroxystilbene) is a phytoalexin found in high concentration in the skins of grapes and red wines which has been shown to have antiinflammatory, anticancerogen and antioxidant properties. Resveratrol is a potent and specific inhibitor of nuclear factor kappa B (NF-B). Resveratrol also inhibits COX-2 gene expression and enzyme activity. We aimed to determine the in vivo effects of intra-articular injections of resveratrol on cartilage and synovium in an experimental osteoarthritis (OA) model in rabbits.Methods: As OA model, rabbits underwent unilateral anterior cruciate ligament transection (ACLT). Five weeks after test group was injected with 10 Mol/kg resveratrol in dimethylsulphoxide (DMSO) in the knees once daily for two weeks and as the control group at the same time DMSO was injected into the knees. All rabbits were killed one week after the last injection. Cartilage tissue and synovium were evaluated with a histological scoring system.Results: Histological evaluation of cartilage tissue by H&E staining revealed a significantly reduced average cartilage tissue destruction score of 1.7 in the resveratrol group versus 2.8 in the control group (p = 0.016). Loss of matrix proteoglycan content in cartilage was also much lower, as determined by safranin O staining. Scores of synovial inflammation didnt show difference between groups (1,3 vs 2,2; p = 0.057).Conclusion: A characteristic parameter in arthritis is the progressive loss of articular cartilage. This study suggests that intraarticular injections of resveratrol starting at the onset of disease may protect cartilage against the development of experimentally induced OA.Received 24 November 2004; returned for revision 29 November 2004; accepted by J. Hamilton 13 December 2004     

Survey of Toxoplasma gondii and Trichinella spp. in hedgehogs living in proximity to urban areas in the Czech Republic

Hedgehogs (Mammalia: Erinaceidae) are omnivorous nocturnal animals typically living in anthropogenic areas. They may be suitable as sentinels for a wide range of zoonotic infections. Only a few studies have investigated hedgehogs (and then as representative wildlife species) to establish their role in the life cycle of such tissue parasites with zoonotic potential as Toxoplasma gondii or Trichinella spp. Working with frozen hedgehog cadavers, we tested for these parasites using T. gondii DNA-specific magnetic capture isolation plus polymerase chain reaction and Trichinella spp. digestion assay. All of 50 examined hedgehogs were negative for Trichinella spp. larvae in their muscles, but brain tissue from 5 out of 26 Erinaceus europaeus (19.2%) and 4 out of 24 E. roumanicus (16.6%) tested positive for T. gondii DNA. Frequency of T. gondii for both hedgehog species was equal, as was distribution between males and females and across age categories. Although a few studies have suggested the possibility of Trichinella spp. infection in hedgehogs, the zero prevalence in the tested hedgehogs is not surprising in view of the generally low prevalence of Trichinella spp. in Central Europe. Our results show that hedgehogs are susceptible to infection by T. gondii and can be used as indicator wildlife animal species in anthropogenic ecosystems.

     

Validation of a Western Blot for the detection of anti-<Emphasis Type="Italic">Trichinella</Emphasis> spp. antibodies in domestic pigs

Trichinellosis is a zoonotic disease in humans caused by Trichinella spp. According to international regulations and guidelines, serological surveillance can be used to demonstrate the absence of Trichinella spp. in a defined domestic pig population. Most enzyme-linked immunosorbent assay (ELISA) tests presently available do not yield 100% specificity, and therefore, a complementary test is needed to confirm the diagnosis of any initial ELISA seropositivity. The goal of the present study was to evaluate the sensitivity and specificity of a Western Blot assay based on somatic Trichinella spiralis muscle stage (L1) antigen using Bayesian modeling techniques. A total of 295 meat juice and serum samples from pigs negative for Trichinella larvae by artificial digestion, including 74 potentially cross-reactive sera of pigs with other nematode infections, and 93 meat juice samples from pigs infected with Trichinella larvae were included in the study. The diagnostic sensitivity and specificity of the Western Blot were ranged from 95.8% to 96.0% and from 99.5% to 99.6%, respectively. A sensitivity analysis showed that the model outcomes were hardly influenced by changes in the prior distributions, providing a high confidence in the outcomes of the models. This validation study demonstrated that the Western Blot is a suitable method to confirm samples that reacted positively in an initial ELISA. Frey and Schuppers contributed equally to this work. This work was funded by the Swiss Federal Veterinary Office, grant number 1.06.03.     

Thermally induced and developmentally regulated expression of a small heat shock protein in <Emphasis Type="Italic">Trichinella spiralis</Emphasis>

A cDNA encoding a small heat shock protein of Trichinella spiralis, Ts-sHsp, was cloned and expressed and is herein characterized. This cDNA encoded a predicted protein of 165 amino acids, which had a high sequence identity in α crystallin domain with various small heat shock proteins of other organisms. A Western blot analysis indicated that anti-Ts-sHsp recombinant antibody recognized the protein of adults and larvae migrating at about 19 kDa. An in situ localization study showed the protein to be abundantly present in the body wall muscle cells, hypodermis, stichocytes, and esophagus of muscle larvae. The Ts-sHsp recombinant protein possessed chaperone activity to suppress the thermally-induced aggregation of citrate synthase. This sHsp was expressed at various developmental stages of T. spiralis, but at different levels. A high level was observed in mature muscle larvae (infective larvae), which was much higher than the levels seen in adults, newborn larvae, or immature muscle larvae. The expression of the sHsp gene was thermal inducible, thus responding to both cold (0°C) and heat shock (43°C) stress; however, at different patterns. The expression of Ts-sHsp increased gradually from 3 to 72 h after cold stress, while the expression was elevated to its highest after 3 h heat stress and then decreased. These results suggest that this small heat shock protein likely plays a role in the tolerance to both chemical and physical stresses, thereby enhancing the survival ability of Trichinella muscle larvae.     

Serological detection of <Emphasis Type="Italic">Trichinella spiralis</Emphasis> in swine by ELISA (enzyme-linked immunosorbent assay) using an excretory–secretory (E/S) antigen

The enzyme-linked immunosorbent assay (ELISA) method is recommended for farm surveillance programs and may be useful for epidemiological studies in wildlife or for establishing Trichinella-free areas. In this study, our interest was to compare the specificity and the time of seroconversion of excretory–secretory (E/S) antigens prepared from Trichinella spiralis. A group of eight pigs was inoculated with 500 T. spiralis larvae per animal, and blood sampling was performed at 3 and 4-day intervals during all experiments. The numbers of muscle larvae were determined in four different muscles groups. The larvae per gram burden shows that the most heavily parasitized muscles were the diaphragm [mean = 43.7 larvae per gram (lpg)] and the tongue (mean = 16.9 lpg). Antibody responses were detected by any of eight infected pigs of T. spiralis. Using the ELISA method with E/S antigen, antibodies to T. spiralis were first found on the day 21st p.i. The initial detection of antibodies varied from 21st to 31st day p.i., and the peak was reported 42nd day p.i. Dynamic of antibodies was stable or increased slightly throughout the experimental period (60 days post-inoculation). Our results represent important data for validation of a serological test, especially if blood samples are taken during early stages of infection.     

Specific IgG4 response directed against the 45-kDa glycoprotein in trichinellosis: a re-evaluation of patients 15 years after infection

The aim of this study was to evaluate the humoral immune response in late human trichinellosis with particular attention to the presence of IgG4 antibodies directed against the Trichinella-45-kDa glycoprotein (gp). This study re-evaluates subjects 15 years after they were involved in a trichinellosis outbreak that occurred in Central Italy following the consumption of raw boar meat infected with Trichinella britovi. The results show that ELISA tests using the E/S antigen identified five IgM- and eight IgG-positive patients and no IgA-positive patients. Tests using immunoblot (IB) with E/S antigens identified three IgM-, five IgA-, seven- IgG1- and three IgG4-positive sera. When the purified 45-kDa gp was used as an antigen, the IB revealed that six of the ten sera tested were positive for IgG4. Sera were also evaluated with a commercial kit, revealing that 11 of 12 patients had a highly sensitive reactivity against Trichinella proteins (64 and 44–43 kDa). In conclusion, humoral immune response against Trichinella is still present in these patients 15 years after the initial infection, including an IgG4 response directed to the 45-kDa gp.     

Resveratrol inhibits dysfunction of dendritic cells from chronic obstructive pulmonary disease patients through promoting miR-34

Background: Resveratrol has demonstrated many beneficial effects against aging, including anti-inflammatory and antioxidant roles. The present study was designed to observe the effects of resveratrol on the dysfunction of dendritic cells (DCs) from COPD patients and its possible mechanism and use in the treatment for COPD. Methods: Flow cytometry analysis was used to examine the expression of costimulatory markers CD80 and CD86 and ELISA was used to examine the secretion of IFN-α. Expression of miR-34 was examined by using real-time PCR. Expression vector of miR-34, LV3-miR-34 was also constructed and transfected into DCs to observe the effects on functions of DCs. Results: The results showed that there was remarkable upregulation of CD80 and CD86 and secretion of cytokines IFN-α in DCs from COPD patients. Resveratrol displayed a dose-dependent cytotoxicity action over 10 µg/mL and pretreatment with resveratrol inhibited upregulation of CD80 and CD86 and secretion of cytokines IFN-α. Further study showed resveratrol upregulated the expression of miR-34, which inhibited the dysfunction of DCs. Conclusion: These proofs suggest that resveratrol inhibited dysfunction of DCs from COPD patients through promoting miR-34.