Identification of Differentially Expressed Genes of Trichinella spiralis Larvae after Exposure to Host Intestine Milieu

Although it has been known for many years that T. spiralis muscle larvae (ML) can not invade intestinal epithelial cells unless they are exposed to the intestinal milieu and activated into intestinal infective larvae (IIL), which genes in IIL are involved in the process of invasion is still unknown. In this study, suppression subtractive hybridization (SSH) was performed to identify differentially expressed genes between IIL and ML. SSH library was constructed using cDNA generated from IIL as the ‘tester’. About 110 positive clones were randomly selected from the library and sequenced, of which 33 T. spiralis genes were identified. Thirty encoded proteins were annotated according to Gene Ontology Annotation in terms of molecular function, biological process, and cellular localization. Out of 30 annotated proteins, 16 proteins (53.3%) had binding activity and 12 proteins (40.0%) had catalytic activity. The results of real-time PCR showed that the expression of nine genes (Ts7, Ndr family protein; Ts8, serine/threonine-protein kinase polo; Ts11, proteasome subunit beta type-7; Ts17, nudix hydrolase; Ts19, ovochymase-1; Ts22, fibronectin type III domain protein; Ts23, muscle cell intermediate filament protein OV71; Ts26, neutral and basic amino acid transport protein rBAT and Ts33, FACT complex subunit SPT16) from 33 T. spiralis genes in IIL were up-regulated compared with that of ML. The present study provide a group of the potential invasion-related candidate genes and will be helpful for further studies of mechanisms by which T. spiralis infective larvae recognize and invade the intestinal epithelial cells.     

Trichinella spiralis: Differential effect of host bile on the in vitro invasion of infective larvae into epithelial cells

The differential effect of fox and pig bile and its corresponding low molecular weight fraction (LMW) was investigated on the in vitro invasion of MDCK-AA7 epithelial cell monolayers by Trichinella spiralis muscle larvae. Seven invasion experiments were performed and a total of 274 cell monolayers were examined. Fox and pig raw bile at 1:10 and 1:20 dilution and their LMW fractions at 1:10 dilution activated T. spiralis larvae to invade the cell monolayers. In addition, fox raw bile caused significantly larger cell damage than pig raw bile at both dilutions. The area of cell damage was larger at 1:10 than at 1:20 dilution for both fox and pig raw bile (p < 0.05). On the other hand, there was no significant difference between the areas of cell damage caused by the LMW fractions of fox and pig bile. It is concluded that differences between host bile actions may account for differences in host susceptibility to T. spiralis.     

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.     

Expression of heat shock protein 70a mRNA in Bombyx mori diapause eggs

In an effort to understand whether heat shock protein 70 (Hsp70) participates in the environmental 5 °C signal reception/transduction toward breaking embryonic diapause of the silkworm Bombyx mori, we isolated a cDNA for Hsp70a and examined the expression of Hsp70a mRNA in B. mori diapause and nondiapause eggs by quantitative real-time PCR. Hsp70a mRNA gradually increased in diapause eggs continuously kept at 25 °C after oviposition to maintain diapause. When diapause eggs were exposed to the diapause-terminating condition of 5 °C beginning at 2 days post-oviposition, Hsp70a mRNA increased beginning at 5 days post-cold treatment. Even in nondiapause eggs, Hsp70a mRNA increased slightly with exposure to 5 °C. These results suggest that Hsp70a is involved in reception/transduction of the diapause-terminating (5 °C) signal via gene activation. The expression patterns of Hsp70a mRNA are discussed in relation to those of the cold-response gene Samui.     

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.     

Trichinella spiralis: comparison of stages in host intestine with those of an Arctic Trichinella sp

The intestinal phase of Trichinella spiralis and of Trichinella sp. isolated in the Arctic were compared in experimental animals. Reproductive capacity, pathogenicity, distribution, and persistence of adults in the small intestine, morphological measurements, and release of newborn larvae in vitro were examined. Numerous passages of 40 days each for T. spiralis and the Trichinella sp. isolate in mice did not affect reproductive capacity, distribution of adults in the small intestine, and size of worms. Reproductive capacity index for T. spiralis, I = 151.27 ± 27.30 was significantly higher compared to the Trichinella sp. isolate index, I = 63.46 ± 19.34. The Trichinella sp. isolate was more pathogenic to mice and wild rodents compared to T. spiralis during the intestinal phase. Both parasites were located in the anterior part of the small intestine but the position of T. spiralis adults (P = 17.08) differed from position of the Trichinella sp. isolate adults (P = 23.46) in the small intestine. Intestinal phase of T. spiralis was longer (20 days) compared to the Trichinella sp. isolate (15 days) but sex ratios (:♂) were similar for both parasites. T. spiralis females released significantly higher numbers of larvae in vitro/24 hr compared to the Trichinella sp. isolate. Release of larvae was continuous during the intestinal phase and the average fecundity for T. spiralis was 335 larvae/female and for the Trichinella sp. isolate 114 larvae/female. Adults of T. spiralis and the Trichinella sp. isolate were morphologically indistinguishable and did not differ in size. A comparative index for the intestinal phase is proposed for comparison of any Trichinella spp. isolates and standard T. spiralis.     

Rapid detection of Trichinella spiralis larvae in muscles by loop-mediated isothermal amplification

Trichinella spiralis is a tissue-dwelling nematode parasite. A loop-mediated isothermal amplification (LAMP) assay was developed and validated for the sensitive and rapid detection of T. spiralis larvae in muscle samples. Sixteen sets of primers were designed to recognise distinct sequences of a conserved gene, a 1.6 kb repetitive element of the Trichinella genome. One set of primers was selected as the most appropriate for rapid detection. The specificity and sensitivity of the primers in LAMP reactions for T. spiralis larvae and muscle samples of mice infected with T. spiralis were determined. Another 10 heterologous parasites were selected for specificity assays. The results showed that target DNA was amplified and visualised by monitoring turbidity and adding calcein detection methods within 70 min at an isothermal temperature of 63 °C. The sensitivity of LAMP with the detection limit of 362 fg/μl was >10 times higher than that for PCR. The designed primers had a good specificity. No cross-reactivity was found with the DNA of any other parasites. The assay was able to detect T. spiralis in all mouse muscle samples infected with 10 T. spiralis larvae on day 20 p.i. We believe this is the first report regarding the application of the LAMP assay for detection of T. spiralis larvae in muscle samples from experimentally infected mice. This method demonstrates a potentially valuable means for the direct detection of T. spiralis larvae in meat inspection.     

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.     

Patterns of p53, p73 and mortalin gene expression associated with haemocyte polyploidy in the soft-shell clam, Mya arenaria

The molecular mechanisms by which haemocytes of clams are transformed in the course of haemic neoplasia remain by far unknown. The aim of this study was to quantify the expression of p53/p73 and mortalin genes, in relation with the ploidy status of clam haemocytes and to correlate the p53 expression with mortalin expression. For this purpose, soft-shell clams, Mya arenaria, were collected from an endemic zone for neoplasia. The ploidy of haemocytes was assessed for each individual clam by flow cytometry using a propidium iodide protocol, while p53/p73 and mortalin gene expressions were quantified by real-time RT-PCR. Results show that haemocytes of some clams with a moderate percentage (15-50%) of tetraploid cells have a significantly high level of p53 and p73 in comparison with clams belonging to categories with low (<15%) or high levels (>50%) of tetraploid cells, where low levels of expression of these genes were observed. Furthermore, mortalin gene expression is strongly correlated (r² = 0.68, p < 0.01) with p53 gene expression level. This reinforces the hypothesis of a cytoplasmic p53 sequestration mechanism in clam haemic neoplasia. Further studies are needed to confirm these preliminary results and further unravel the molecular pathways involved in this process. Our results are believed to provide phenotypic foundation for such studies to be undertaken.     

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.     

Characterisation of a Plancitoxin-1-Like DNase II Gene in Trichinella spiralis

Background

Deoxyribonuclease II (DNase II) is a well-known acidic endonuclease that catalyses the degradation of DNA into oligonucleotides. Only one or a few genes encoding DNase II have been observed in the genomes of many species. 125 DNase II-like protein family genes were predicted in the Trichinella spiralis (T. spiralis) genome; however, none have been confirmed. DNase II is a monomeric nuclease that contains two copies of a variant HKD motif in the N- and C-termini. Of these 125 genes, only plancitoxin-1 (1095 bp, GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"XM_003370715.1","term_id":"339256039","term_text":"XM_003370715.1"}}XM_003370715.1) contains the HKD motif in its C-terminus domain.

Methodology/Principal Findings

In this study, we cloned and characterised the plancitoxin-1 gene. However, the sequences of plancitoxin-1 cloned from T. spiralis were shorter than the predicted sequences in GenBank. Intriguingly, there were two HKD motifs in the N- and C-termini in the cloned sequences. Therefore, the gene with shorter sequences was named after plancitoxin-1-like (Ts-Pt, 885 bp) and has been deposited in GenBank under accession number {"type":"entrez-nucleotide","attrs":{"text":"KF984291","term_id":"594551321","term_text":"KF984291"}}KF984291. The recombinant protein (rTs-Pt) was expressed in a prokaryotic expression system and purified by nickel affinity chromatography. Western blot analysis showed that rTs-Pt was recognised by serum from T. spiralis-infected mice; the anti-rTs-Pt serum recognised crude antigens but not ES antigens. The Ts-Pt gene was examined at all T. spiralis developmental stages by real-time quantitative PCR. Immunolocalisation analysis showed that Ts-Pt was distributed throughout newborn larvae (NBL), the tegument of adults (Ad) and muscle larvae (ML). As demonstrated by DNase zymography, the expressed proteins displayed cation-independent DNase activity. rTs-Pt had a narrow optimum pH range in slightly acidic conditions (pH 4 and pH 5), and its optimum temperature was 25°C, 30°C, and 37°C.

Conclusions

This study indicated that Ts-Pt was classified as a somatic protein in different T. spiralis developmental stages, and demonstrated for the first time that an expressed DNase II protein from T. spiralis had nuclease activity.     

Humoral immune response of Galleria mellonella larvae after infection by Beauveria bassiana under optimal and heat-shock conditions

Natural infection of Galleria mellonella larvae with the entomopathogenic fungus Beauveria bassiana led to antifungal, but not antibacterial host response. This was manifested by induction of gallerimycin and galiomicin gene expression and, consequently, the appearance of antifungal activity in the hemolymph of the infected larvae. The activity of lysozyme increased at the beginning of infection and dropped while infection progressed. Exposure of the naturally infected animals to 43 °C for 15 min extended their life time.Galleria mellonella larvae were injected with 10⁴, 10⁵ and 10⁶ fungal blastospores, resulting in the appearance of strong antifungal activity and a significant increase in lysozyme activity in larval hemolymph after 24 h. Antibacterial activity was detectable only when 10⁵ and increased when 10⁶ blastospores were injected. The number of the injected B. bassiana blastospores also determined the survival rate of animals. We found that exposure of the larvae to 38 °C for 30 min before infection extended their life time when 10³ and 10⁴ spores were injected. The increase in the survival rate of the pre-heat-shocked animals may be explained by higher expression of antimicrobial peptides and higher antifungal and lysozyme activities in their hemolymph in comparison to non-heat-shocked animals.