Trichinella Spiralis Muscle Larvae Research Articles

Benzimidazoles Containing Piperazine Skeleton at C-2 Position as Promising Tubulin Modulators with Anthelmintic and Antineoplastic Activity.

Benzimidazole anthelmintic drugs hold promise for repurposing as cancer treatments due to their interference with tubulin polymerization and depolymerization, manifesting anticancer properties. We explored the potential of benzimidazole compounds with a piperazine fragment at C-2 as tubulin-targeting agents. In particular, we assessed their anthelmintic activity against isolated Trichinella spiralis muscle larvae and their effects on glioblastoma (U-87 MG) and breast cancer (MDA-MB-231) cell lines. Compound 7c demonstrated exceptional anthelmintic efficacy, achieving a 92.7% reduction in parasite activity at 100 μg/mL after 48 hours. In vitro cytotoxicity analysis of MDA-MB 231 and U87 MG cell lines showed that derivatives 7b, 7d, and 7c displayed lower IC50 values compared to albendazole (ABZ), the control. These piperazine benzimidazoles effectively reduced cell migration in both cell lines, with compound 7c exhibiting the most significant reduction, making it a promising candidate for further study. The binding mode of the most promising compound 7c, was determined using the induced fit docking-molecular dynamics (IFD-MD) approach. Regular docking and IFD were also employed for comparison. The IFD-MD analysis revealed that 7c binds to tubulin in a unique binding cavity near that of ABZ, but the benzimidazole ring was fitted much deeper into the binding pocket. Finally, the absolute free energy of perturbation technique was applied to evaluate the 7c binding affinity, further confirming the observed binding mode.

Evaluation of indirect-ELISA using eluted antigens from Trichinella spiralis muscle larvae for diagnosis of swine trichinellosis

Trichinellosis is caused by Trichinella spiralis muscle larvae (TsML), which is transmitted to human when they eat infected raw or undercooked meat. T. spiralis infection is detected by an enzyme-linked immunosorbent assay (ELISA) using excretory–secretory antigens (ESAg); however, the preparation of ESAg is challenging, and yields are low, which hampers screening efforts. In this study, crude somatic antigens (CSAg) of TsML with molecular weights (MWs) of 43, 79 and 101 kDa have been identified in swine trichinellosis sera with less cross-reaction with uninfected sera and other parasitic infected sera. After that, the CSAg at MWs of 43, 79 and 101 kDa (TsCSAg-43, TsCSAg-79, and TsCSAg-101, respectively) were isolated from sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The eluted antigens were analyzed by IgG-ELISA for sensitivity and specificity, and specific antigens from the three regions were identified by two-dimensional polyacrylamide gel electrophoresis (2-DE) and liquid chromatography-tandem mass spectrometry (LC-MS-MS). The sensitivity of IgG-ELISA using the three eluted antigens was 100% with specificities of 97.77%, 95.54%, 90.63% and for TsCSAg-43, TsCSAg-79, and TsCSAg-101, respectively. The LC-MS-MS results of immunomics showed that 18/20 spots of the antigens with MWs of 43, 79, and 101 kDa represent 11 different proteins identified. TsCSAg-43 showed the highest specificity, indicating that the specific proteins identified, including 45 kDa antigen–trichina [fragment], DNA topoisomerase 2-alpha antigen targeted by protective antibodies, and a conserved hypothetical protein (gi339234223), should be developed and produced in large volumes for further immunodiagnostic studies.

First Serbian external quality assessment to detect Trichinella larvae in meat by the Magnetic Stirrer Method

Introduction. Inter-laboratory comparative testing must be conducted under the direction of the National Reference Laboratory for Trichinella in each Member State of the EU with the aim to ensure the quality of the approved digestion method and to evaluate the competence of laboratories in Trichinella detection. For the first time in Serbia, the National Reference Laboratory for Trichinellosis (NRLT INEP) organized such external quality assessment (EQA) for the detection of Trichinella larvae in meat by the Magnetic Stirrer Method (MSM). The aim of this study was to enable laboratories interested to take part in the EQA and to assess the quality of their MSM performance. Materials and Methods. The EQA was organized by NRLT INEP according to ISO/IEC 17043:2010. The EQA test panel, which was sent to eight laboratories within Veterinary Institutes, consisted of three 100?5 g minced pork meat balls with identical numbers of larvae. Two meat balls were spiked with five viable Trichinella spiralis muscle larvae (L1), and one meat ball was not spiked. Results and Conclusions. Evaluation made on the basis of qualitative results showed that 100% of participants successfully completed the testing. Quantitative evaluation showed that, on average, only 60% of Trichinella larvae were detected among all participants. The main importance of this study is that it enabled laboratories across the country to be compared. This should be efficient stimulus for improvement of analytical performance over time. Therefore, it will be of value if the participants and other official laboratories in Serbia take part annually in EQAs organized at national level. The results obtained in this study could provide useful information to the Ministry of Agriculture, Forestry and Water Management, Veterinary Directorate and be of importance for promoting the one health concept in the country as a whole.

Use of ELISA and Western blot for serological detection of antibodies to E-S antigens of Trichinella spiralis muscle larvae in sera of swine experimentally infected with Trichinella spiralis

The aim of the present study was to evaluate the detection of trichinellosis using ELISA, and to determine the degree of variation of IgG antibodies against excretory-secretory (E-S) antigens of T. spiralis muscle larvae. Ten young Polish Large White native breed pigs were experimentally inoculated with a low dose of 300 invasive Trichinella spiralis (T. spiralis) muscle larvae (ML). Pig sera were collected at 7 and 9 days prior to the experimental infection with T. spiralis and at 9, 14, 20, 23, 25, 27, 30, 33, 37, 41 and 46 days post-infection (d.p.i.). Western blotting was used as a follow-up test to detect anti-T. spiralis IgG and confirm the results provided by ELISA. The molecular weight of the E-S antigenic proteins of T. spiralis muscle larvae reacting with the sera from the infected pigs was determined. Finally, the intensity of T. spiralis infection (lpg) was determined as an average value of 16 muscles taken from each pig. The E-S antigen of T. spiralis muscle larvae in the in-house ELISA and immunoblotting assays did not demonstrate any cross-reaction with non-infected pig sera and sera from pigs naturally infected with Oesophagostomum spp. The ELISA assay did not recognize trichinellosis in pigs until 27 days after the T. spiralis infection. The anti-Trichinella IgG antibodies were first detected on day 30 post-infection. The immunoblotting technique confirmed the presence of anti-Trichinella IgG antibodies in all serum samples evaluated as ELISA-positive. The Western blot detected anti-Trichinella IgG antibodies on the same day they were detected for the first time by ELISA in five pigs, whereas the immunoblot assay detected seroconversion 2, 3 or 4 days earlier than ELISA in the remaining three pigs. The swine sera reacted with a broad range of T. spiralis ML E-S antigens with molecular weights ranging from 30 to 88 kDa and the most frequently recognized proteins were 45, 49 and 60 kDa.

Immunomodulatory potential of particular Trichinella spiralis muscle larvae excretory–secretory components

Excretory–secretory antigens of Trichinella spiralis muscle larvae can induce the semi-matured status of rat dendritic cells. This may at least partly be the consequence of transient activation of extracellular signal-regulated kinases 1/2 (ERK1/2). Here we investigated the potential of several components of excretory–secretory antigens (native fraction containing 45, 49 and 53kDa proteins and recombinant Tsp53, representing one of the constituents of this fraction) to demonstrate previously observed effects of excretory–secretory antigens on dendritic cells in vitro, characterised by establishment of a particular phenotype (very low MHC II expression, moderate CD86 expression and significant ICAM-1 expression) and functional properties (low production of pro-inflammatory cytokine IL-12p70, and high production of IL-10 and TGF-β). Dendritic cells activated by these components were able to provoke proliferation of naïve T cells and their polarisation towards Th2 and anti-inflammatory responses. The investigated antigens had almost the same capacity to induce IL-4 and IL-10 production from T cells as excretory–secretory antigens, but failed to induce significant TGF-β synthesis. It could be concluded that the investigated excretory–secretory antigens components can largely reproduce the immunomodulatory effects of the complete excretory–secretory antigens and therefore may be considered as molecules important for creation of the anti-inflammatory milieu achieved by the parasite.

Oral administration with attenuated Salmonella encoding a Trichinella cystatin-like protein elicited host immunity

Trichinellosis is a public health problem and is regarded as an emergent/re-emergent disease in various countries. The cDNA encoding a cystatin-like protein (Ts-cystatin) was identified by immunoscreening intestinal muscle larvae cDNA libraries with serum from pigs experimentally infected with 20,000 Trichinella spiralis muscle larvae. To study its impact on host immunity, we chose a eukaryotic expression system based on several comparisons of immunogenicity between the two Salmonella typhimurium administration schemes, which indicated that the eukaryotic expression system was superior. Humoral IgG and mucosal IgA were measured to determine the antibody response. To explore whether Th1 and Th2 responses were responsible for the induced protection, Th1- and Th2-specific cellular transcription factors and the cytokine profile were examined. Changes in the T lymphocyte and macrophage populations were detected by flow cytometry. Lastly, parasitological examination was examined. The results showed that Ts-cystatin induced a Th1/Th2-mixed type of immune response and decreased STAT6 transcription. The intestinal adult recovery increased by 10.9% in the Ts-cystatin group, the Ts-cystatin group fecundity rate was decreased by 91%. Furthermore, the number of muscle larvae did not change compared with the control group. In conclusion, our results suggest that Ts-cystatin plays an important role in Trichinella resistance to rapid expulsion by the host and is worth further study.

Influence of adjuvant formulation on the induced protection of mice immunized with total soluble antigen of Trichinella spiralis

Vaccination of pigs against the helminth nematode Trichinella could be a good alternative to prevent the risk of human infection. In order to develop an efficient and safe vaccine, the choice of the adjuvant is an important issue. In this study, two adjuvants were selected to prepare vaccines based on total soluble Trichinella spiralis muscle larvae (ML) antigen: Montanide ® ISA70 water in oil emulsion and Montanide ® IMS nanoparticles. Aluminium hydroxide was used as a reference adjuvant. The immune response was checked by ELISA of parasite antigen specific IgG1 and IgE. Finally, protection induced in vaccinated mice was measured after a T. spiralis challenge by counting ML burdens. The results clearly showed an impact of adjuvants on the specific IgG1 and IgE antibody responses against T. spiralis. Differences were observed between the rates of protection induced according to the type of formulation, although the three adjuvants tested were able to enhance the humoral immune response. This work demonstrated the need to use an adjuvant to obtain a specific IgG1 and IgE responses directed against the total soluble extract of T. spiralis.

Murine model study of the practical implication of trypanosome-induced immunosuppression in vaccine-based disease control programmes

The relevance of trypanosome-induced immunosuppression in relation to the efficacy of vaccine-induced immunity was studied in mice. Mice were immunised with crude Trichinella spiralis muscle larvae homogenate vaccine and infected with T. spiralis and/or Trypanosoma brucei. Vaccination significantly decreased adult worm burden ( p<0.05) and accelerated worm expulsion in mice infected with T. spiralis only. T. brucei superinfection resulted in monocytosis, suppressed eosinophilia, significant decrease in PCV ( p<0.001), higher numbers of adult worms ( p<0.001) and failure to expel all adult worms by Day 12 post infection (p.i.). Regardless, they produced anti- Trichinella IgG 1 responses similar to those of the vaccinated non- T. brucei-infected group. T. brucei also suppressed the proliferative responses of spleen cells to stimulation with Con A and T. spiralis antigen, and induced strong production of interferon-gamma (IFN-γ) in culture supernatants of antigen stimulated spleen and mesenteric lymph node cells. Interleukin-5 (IL-5) production was suppressed by T. brucei in supernatants of Con A- and antigen-stimulated spleen cells. It was concluded that trypanosome infections and the associated immunosuppression are of great practical significance in trypanosome endemic areas, especially with regards to disease control programmes involving vaccine-induced herd immunity.

Detection of Trichinella infection in slaughter horses by ELISA and western blot analysis

In order to determine the presence of Trichinella infections in horses slaughtered at an abattoir in Mexico, 147 serum samples were examined by two immunoenzymatic methods. Specific antibodies were detected by ELISA in 7% of the serum samples at a dilution 1:400 and in 10% at lower dilutions (1:20, 1:40) using Trichinella spiralis muscle larvae (ML) excretory/secretory (E/S) products. Serum samples from four naturally infected horses (confirmed by direct methods) gave negative O.D. values in an ELISA at a 1:400 dilution and only two of them were positive at a 1:20 and 1:40 dilutions. Serum samples from experimentally infected horses reacted by Western blotting with ML components with molecular weights of 47, 52, 59, 67, 72 and 105 kDa which correspond to the TSL-1 antigens. Serum samples from the four naturally infected horses and from the abattoir horses that were positive in ELISA using E/S antigens recognized several ML components, some of them reacted with all the TSL-1 antigens mentioned above and others recognized preferentially two or three of these molecules. Since the serologic assays may not offer the sensitivity required in the diagnosis of horses trichinellosis and the direct methods had not always been useful in the detection of larvae in horsemeat related to trichinellosis outbreaks in Europe, it is proposed that additional assays are performed to determine Trichinella infection in horses. These can include detection of parasite antigens by ELISA and Dot ELISA or PCR, which in turn may also help to determine the presence of the parasite in early and late infections of horses.

Abnormal thymus development and impaired function of the immune system in rats after prenatal exposure to aciclovir.

Aciclovir (synonym: acyclovir) causes abnormal thymus development in rats. After treatment on day 10 of gestation a weight reduction of the organ is obvious in 21-day-old fetuses which persists postnatally. Adult male rats exposed in utero to one or three injections of 100 mg aciclovir/kg body wt given to the dam on day 10 of pregnancy showed a reduction of the thymus weight to 333 +/- 158 mg and 276 +/- 61 mg (control: 428 +/- 92 mg; n = 10). Corresponding alterations were detectable in female offspring. Liver weight was also decreased and spleen weight (in relation to body wt) was significantly increased in the offspring after the three exposures. In a host resistance model with Trichinella spiralis the function of the immune system of rats prenatally exposed to aciclovir was examined. Six weeks postnatally 10-12 randomly selected male rat offspring of one control and two treatment groups (1 or 3 injections of 100 mg aciclovir/kg body wt on day 10 of gestation) were infected orally with 500 Trichinella spiralis muscle larvae. Before and several times after the infection blood was taken from a tail vein or obtained by decapitation for examination of the antibody titers (IgM, IgG, IgA, IgE) to antigens of T. spiralis. Six weeks after the infection the weight of relevant organs was determined and tongue preparations were used for T. spiralis muscle larvae counting. Aciclovir exposed animals showed a different immune response than control rats. IgM titers in both treatment groups were higher than in controls two weeks after the infection but not different by the end of the experiment.(ABSTRACT TRUNCATED AT 250 WORDS)

Rapid expulsion of Trichinella spiralis in suckling rats.

Orally administered Trichinella spiralis muscle larvae were rapidly expelled by rat pups suckling an immune dam. The immunity was delivered in the milk; substantial resistance was conferred on normal rat pups suckled for only 24 hours by a Trichinella-immune foster mother. The pups were protected by oral or systemic administration of specific serum antibodies. When infused into a normal lactating dam, these antibodies accumulated in the serum of her suckling pups.

Trichinella spiralis: Selective intestinal immune deviation in the rat

In rats, infections with 100–2000 Trichinella spiralis muscle larvae lead to a prompt immunity that is expressed in parasite expulsion within 14 days. Rats infected with more than 2000 larvae display impaired immunity with rejection delayed by 50% (7 days) or more. Suppression is selective for expulsive immunity as the antifecundity response of rats is directly proportional to dose and is expressed sooner in heavily infected subjects. Suppression of intestinal expulsive immunity was suggested by the fact that, with low doses (2000 larvae or less), worm rejection was inhibited by cortisone, whereas cortisone inhibited antifecundity but had no discernable effect on worm rejection in high-dose infections. Evidence for local immune deviation as opposed to systemic immunosuppression was obtained in experiments using parabiotic rats. When one partner was infected with 6000 worms and the other with 200, the rat infected with 200 parasites showed earlier rejection than was seen in single controls infected with 200 worms. The prolonged survival of high-dose adults was not accompanied by a change in the site of worm residence in the gut. Immunological parameters such as serum antibody levels, the number of activated cells or specific anti- T. spiralis lymphocytes in thoracic duct lymph were all increased in a dose-dependent manner. These experiments therefore demonstrate a novel autoprotective mechanism by which adult T. spiralis selectively reduce the expression of expulsive immunity in the gut.

Abnormal weight gain in mice, induced by Trichinella pseudospiralis

The parasite-induced weight gain in animals is an unusual phenomenon which involves a complex host–parasite relationship. Trichinella pseudospiralis exhibits such a phenomenon in mice. Eighty Swiss mice were orally infected with 600 T. pseudospiralis or Trichinella spiralis muscle larvae. The total body weight of the mice was recorded at the time of infection and at 2 and 10 months after, and compared with an uninfected control group. Trichinella pseudospiralis muscle larvae induced an abnormal weight gain. This weight increase is statistically different from the other two groups of mice after 2 and 10 months postinfection. The results indicate the existence of a different type of host–parasite relationship between the two parasites. Some speculative ideas are advanced to explain the rapid growth effect of T. pseudospiralis on its host.