Except for Sicily, where Trichinella spiralis was docu- mented in pigs and humans from 1933 to 1961 , Tri- chinella spp. have never been recorded from the islands of the Mediterranean Basin until 2004, when Trichinellabritovi was detected in free-ranging pigs in a remote mountainous area of Corsica . One year later, a hu- man outbreak of trichinellosis occurred in neighboring Sardinia following the consumption of pork from a free-ranging pig reared in a remote area of the island . In the following years, extensive surveys showed that T. britovi was circulating among free-ranging pigs and wildlife of the two islands and an epidemiological link between the Corsican and Sardinian parasite populations was suspected due the almost simultaneous detection of the parasites on both islands, their geographical proxim- ity, and the illegal animal trade between the two regions [6 – 8]. In 2015, an outbreak of trichinellosis occurred in the region of Nice, the South of France, due to the con- sumption of raw sausages imported from Corsica .
functions. The surface-exposed paramyosin is thought to act as a protective agent during the host inflamma- tory processes by inhibiting the complement activation cascade and membrane attack complex (MAC) forma- tion . However, V-type proton ATPase subunit E, a member of the ATPase protein family, is activated at a wide pH range and possesses interesting properties under certain biochemical conditions. ATPases are in- volved in metabolite movements, purging of toxins and energy generation for metabolic processes; they also take part in the environmental response [49, 50] and hence are thought to be involved in the nematode im- mune response course. Most of the analyzed T. britovi antigens are derived from the muscle stage of the lar- vae. GO analysis of the obtained results showed that some of the proteins participate in various cellular and metabolic processes mostly associated with the synthe- sis and degradation of macromolecules (nucleotides, proteins) which play an important role in the invasion Table 3 Results of LC-MS/MS analysis of Trichinellabritovi muscle larvae (ML) selected spots which reacted with pig sera collected at 10 dpi
Under the experimental conditions used in the present study, a clear pattern of T. spiralis CWE reacting proteins able to distinguish between T. spiralis-, T. britovi- and T. papuae-infected sera was not defined. Encapsulated and non-encapsulated species of the genus Trichinella diverged from their most recent common ancestor about 21 million years ago (mya), with taxon diversifications commencing 7– 10 mya . Biochemical and immunological differences among the species are well documented [20–23]. However, most of these studies focussed on T. spiralis and T. pseudos- piralis showing substantial differences in the inflammatory response and modulation of the muscle cell phenotype [24– 31], in larval and adult size , naked larva kinesis , morphology of the stichocyte granules  and protein con- tent [33–38] and therefore highlighted discrepancies in im- munological properties [39–42]. Differences in the electrophoretic protein patterns of T. spiralis and T. pseu- dospiralis have been reported by several authors. Rodriguez-Perez et al.  showed that the electrophoretic reactivity pattern of T. pseudospiralis CWE with a monoclo- nal antibody displayed fewer protein bands than that of T. spiralis CWE with the same serum. Wu et al.  using homologous murine infected sera revealed 100 and 20 – 30 antigenic peptide spots in T. spiralis CWE and T. pseudos- piralis CWE, respectively, by two-dimensional Wb, despite
Interestingly, both antisera also recognized common proteins, such as those identified from spots 19, 37 and 52 (gp43, serine protease/proteinase). This finding indi- cates that these proteins are more efficiently recognized by the specific antibodies against T. spiralis, since according to 2-D DIGE the corresponding protein spots are more abundant in T. britovi compared to those detected in T. spiralis E-S proteome (Additional file 1: Table S2, Figure 3B). Both of these proteins have been previously shown to be stage-specific ML proteins; the cross-reacting gp43 possess a DNase II-like motif and is suggested to be involved in the formation of nurse cells, while the identified protease is believed to play a crucial role in the development or migration of NBL in host tis- sues . In addition, proteinase can also serve as an immunodominant antigen, stimulating a protective immune response . Previous studies have demon- strated that T. spiralis ML antigens fall into eight groups according to their recognition by monoclonal and/or
Rats that were infected with 10, 25 or 50 ML showed a serological response in the T. spiralis western blot 2 weeks post infection. Other rodents like mice have been experimentally infected with low doses of 50, 10, and 5 ML of Trichinella spiralis per animal [20,21]. Serocon- version was observed in these experiments at 30 dpi with an ES-ELISA and specific antibodies increased until 80 dpi. Measured splenic T-lymphocyte activity increased from day 10 to day 15 pi, even with 10 ML as an infective dose . This implies that for epidemiolo- gical studies, low infection levels can be detected by ser- ology, both in rats and mice and we confirmed that low infection doses of T. spiralis larvae induce the produc- tion of specific antibodies at detectable levels in rats. These low doses reflect the infection level that we can find in the sylvatic cycle. Hurnikova and Dublinskyl  underlined that Trichinella (britovi, pseudospiralis and spiralis) infection in wild foxes is usually below 20 LPG and far less with wild boar. In The Netherlands, Trichi- nella britovi infection in wild foxes is even lower with LPG ranging from 0.04 to 0.71 . For confirmation of rat sera from animals with low Trichinella infection levels or higher infection levels early in the time course of infection that exhibit OD values around the cut-off, we showed that western blots are a suitable instrument.
Trichinellabritovi was the only species detected in Italian wildlife up to few years ago when T. pseudospira- lis began to be documented in northern and central Italy [7,35]. The prevalence of Trichinella spp. infections in the wild boar populations of Italy has been always ex- tremely low. From 1985 to 1999, T. britovi was detected in 9 (0.002%) out of 370,000 hunted wild boars . In the last 13 years, there has been an increase of the wild boar population in Italy and, in parallel, an increase of the number of hunted wild boar and an increase of wild boar tested for Trichinella spp. per year according to the Commission Regulation 2075/2005 . From 2007 to 2012, a total prevalence of 0.017% (95% CI 0.0001- 0.0002; 47 positive/268,200 tested), was detected [8-14], which is significantly higher ( P < 0.001) than the preva- lence detected in the previous period, despite the great differences among the Italian regions (data not shown).
Gynura angulosa DC. is a herbal plant which is most commonly used as an anthelmintic in the folk medicine of several native tribes in northeast India The objective of the present study was to evaluate scientifically the anthelmintic efficacy of G. angulosa, using Trichinella-mice model. The efficacy of G. angulosa extract was tested against three developmental stages of T. spiralis, namely- adults, migrating and encysted larvae employing Trichinella spiralis – mouse model. Plant extract given orally as a single dose for 2 days (day 5 and 6 p.i.) at doses of 200, 400, 800 and 1600 mg/kg body weight eliminated 60.8, 66.61, 76.85 and 86.22% of adult worms. Mebendazole (MBZ), a broad spectrum antinematodal drug, at 25 and 50 mg/kg eliminated 94.75 and 100% of adult worms. The mice subjected to 3 days (day 8 to 10) course of extract treatment during the migrating stage led to 60.26, 64.74, 73.08 and 78.53% decline in the number of muscle larvae at 200, 400, 800 and 1600 mg/kg dose of plant extract. MBZ at 25 and 50 mg/kg reduced the number of larvae by 93.96 and 96.92%. For the encysted parasite stage, the treatment was given for 7 days (beginning day 30 p.i.) and reduction of the larvae was noted to be 57.01, 62.76, 65.34 and 72.36% (at day 48 p.i.). MBZ at 25 and 50 mg/kg dose reduced the larval count by 90.52 and 92.930% respectively. In conclusion, the study suggests that leaf extract of G. angulosa possesses significant anthelmintic efficacy against Trichinella infections in mice and lends support to suggested folkloric use of the plant as anthelmintic.
The aim of the present study was to develop a farm-to-fork quanti- tative microbial risk assessment (QMRA) model that simulates occur- rence of the parasite in wildlife, its transmission dynamics through the food chain from meat inspection to consumption of pork or wild boar meat, and consequent human trichinellosis risks. We focus on meat from shoulder, belly and loin, since these meat cuts are purchased raw and cooked by consumers at home. Using the model, we estimate the number of human trichinellosis cases from consuming pork reared in different husbandry systems and from consuming wild boar meat. For this purpose, we evaluated the meat production system in a Trichinella endemic country in Europe (Poland), identi ﬁed critical points at which Trichinella ML may escape detection or inactivation, collected and criti- cally appraised relevant datasets to estimate model parameters and de- veloped a stochastic model representing variability due to systematic and random effects. Reported incidence rates of human trichinellosis over a period of six years in Poland are used to evaluate the outcomes of our model. Finally, we discussed uncertainty of model outcome for different parts of our QMRA model.
Background: We have previously reported that Trichinella spiralis Nudix hydrolase (TsNd) bound to intestinal epithelial cells (IECs), and vaccination of mice with recombinant TsNd protein (rTsNd) produced a partial protective immunity. The aim of this study was to investigate the immune protection induced by TsNd DNA vaccine. Methods: The full-length cDNA sequence of TsNd gene was cloned into pcDNA3.1 and used to immunize BALB/c mice by intramuscular injection. Transcription and expression of TsNd were detected by RT-PCR and IFT. The levels of specific IgA, IgG, IgG1 and IgG2a, and cytokines were assayed by ELISA at weeks 0, 6 and 8 post-immunization. The immune protection of TsNd DNA vaccine against challenge infection was investigated.
Schematic illustration of the involvement of death receptor pathway (right half) and mitochondrial pathway mediated (left half) apoptosis in nurse cell formation. Upon binding with TNF-D, TNF-RI recruits TRADD which functions as a platform adapter that recruits several signaling molecules. The recruitment of TRADD and FADD results in autocata- lytic activation of procaspase 8. Activated caspase 8 cleaves effector procaspase 3 which plays a role in apoptosis in the basophilic cytoplasm of Trichinella infected muscle cells. On the other hand, the binding of TNF-D and TNF-RI induces the sequential recruitment of TRADD, TRAF2 and RIP, which leads to the activation of NF-kB. The activated NF-kB acts for anti-apoptosis in the basophilic cytoplasm. In mito- chondrial pathway, Bax induces apoptosis by forming the membrane pore in mitochondria from which cytochrome c is released. Cytochrome c activates caspase 9 which in turn activates caspase 3 to induce apoptosis in infected muscle cells. As a co-factor, Apaf-1 plays a role with caspase 9 in apoptosis in the basophilic cytoplasm. On the other hand, Akt plays an anti-apoptosis role in the eosinophilic cytoplasm by inactivating proapoptotic proteins such as Bad and caspase 9. This figure referred the review by Gupta .
Background: The nurse cell (NC) constitutes in mammalian skeletal muscles a confined intracellular niche to support the metabolic needs of muscle larvae of Trichinella spp. encapsulating species. The main biological functions of NC were identified as hypermitogenic growth arrest and pro-inflammatory phenotype, both inferred to depend on AP-1 (activator protein 1) transcription factor. Since those functions, as well as AP-1 activity, are known to be regulated among other pathways, also by Wnt (Wingless-Type of Mouse Mammary Tumor Virus Integration Site) signaling, transcription profiling of molecules participating in Wnt signaling cascades in NC, was performed.
Previous studies have shown that, when suspended in a semisolid medium and inoculated onto an IEC cell monolayer cultured in vitro, T. spiralis infective larvae invade and migrate through the cell monolayer, leaving serpentine trails of dead and damaged cells [13,25]. In this study, when anti-rTspst serum was added to the medium, the invasion rate (25.2%) of the infective larvae into the cell monolayer was significantly lower than the 79% of normal serum control groups, indicating that the anti-rTspst serum partially prevented the larval invasion of the IECs. This results demonstrated that antibodies against rTspst could reduce intestinal adult worms and protect IECs in vitro . Anti-Trichinella antibodies could protect epithelia without the assistance of inflam- matory cells, soluble cofactors, or mucus . The mech- anism of the blockage of the larval invasion of IECs by specific antibodies may be related to the formation of lar- val cephalic immune complexes that may physically block invasion or may interfere with sensory reception . Furthermore, after the challenge infection with T. spiralis infective larvae, the mice immunized with the rTspst dis- played a 47.5% reduction of adult worm burden in intes- tines. The results showed the rTspst induced a partial intestinal protective immunity in mice. Tspst might be a larval invasion-related protein, and could be considered as a potential vaccine candidate against intestinal T. spiralis infection.
T he parasitic nematode Trichinella spiralis induces potent Th2 responses that both clear intestinal infections and regulate im- munity in extraintestinal sites (1–5). The innate cellular and mo- lecular drivers of Th2 immunity appear to vary among nematode infections, and the identification of mechanisms that initiate Th2 responses continues to be an area of active research (reviewed in reference 6). Key roles for pattern recognition receptors, including Toll-like receptors (TLRs), Nod-like receptors (NLR), and C-type lectins, have been described for Th2 responses (7–13), although a central paradigm of Th2 activation that parallels that of TLR/in- terleukin-12 (IL-12) and Th1 immunity has yet to be defined. TLR4 has been implicated in Th2 immunity in several different systems, including the response to parasitic worms, ovalbumin (OVA)-lipopolysaccharide (LPS), and house dust mite allergen (8, 14–17). In an indirect mechanism, eosinophil-derived neuro- toxin promotes Th2 immunity by binding to TLR2 (18). MyD88 is a signaling adaptor molecule that propagates the response to most TLRs, as well as responses to IL-1 family members, including IL-18 and IL-33 (19–21). MyD88 binds to the intracellular Toll/ IL-1 receptor (TIR) domain of these receptors (directly or via a bridging adaptor such as MyD88-adaptor-like [MAL]), where it initiates a signaling cascade that ultimately leads to the acti- vation of proinflammatory genes by the transcription factor NF-B (22, 23).
To identify Trichinella antigens suitable for high-specificity and high-sensitivity serodiagnosis of human trichinellosis, we evaluated assays using four antigens: (i) crude first-stage larval extract (CLE), (ii) O- deglycosylated CLE, (iii) tyvelose-bearing antigens (Trichinella spiralis larva group 1 [TSL-1] antigens) puri- fied by US4 affinity chromatography and coupled directly to enzyme-linked immunosorbent assay (ELISA) plates (pTSL-1 antigens), and (iv) TSL-1 antigens immobilized on ELISA plates with the monoclonal antibody (MAb) US4 (cTSL-1 antigens). Assays using these antigens were compared by analysis of sera from healthy individuals (n ⴝ 224) (group 1), individuals with noninfectious intestinal pathologies (n ⴝ 114) (group 2), individuals with other parasitic infections (n ⴝ 107) (group 3), and individuals with confirmed trichinellosis (n ⴝ 42) (group 4). Our results indicate that capture ELISA using cTSL-1 antigens is the most effective method for serodiagnosis of human trichinellosis; this was the only method showing 100% specificity and 100% sensitivity at the patent stage of the infection, and it was also the most sensitive for sera obtained prior to patency in indirect immunofluorescence (IIF). Indirect ELISA with pTSL-1 antigens was also 100% specific but was slightly less sensitive, particularly with sera obtained before IIF patency. Inhibition ELISA with MAb US4 indicated (i) that in Trichinella-infected patients the immune response to TSL-1 antigens is directed mostly against tyvelose-containing epitopes (mean of 84.2% of total anti-TSL-1 immunoglobulin G1 [IgG1] antibody response [range, 51.3 to 97.6%]) and (ii) that in most individuals a large proportion of anti-CLE IgG1 antibodies (mean, 49.5%; range, 7.3 to 92.6%) are directed against tyvelose epitopes.
Results: Approximately 300 spots were separated by 2-DE, with molecular weights ranging from 10 to 130 kDa, and pI values ranging from pH 4 to 10. The sera from swine and mice infected with T. spiralis for 7 days recognized 64 proteins. MALDI-TOF/TOF-MS analysis identified 55 proteins, some with different isoforms. Finally, 40 individual immunoreactive proteins were obtained with a wide range of biological functions. Several proteins, such as heat shock protein 70, 14-3-3 protein, and cysteine protease could be used as immunodiagnostic or vaccine antigens. Among these identified proteins, the highly immunodominant Ts14-3-3 was chosen for expression in E. coli and purified recombinant Ts14-3-3 was able to be strongly recognized by the same T. spiralis infected sera used for identifying these antigens, therefore the most promising antigen for early immunodiagnosis of Trichinella infection. Conclusions: A total of 64 proteins from the adult worm were recognized by early infection sera from swine and mice infected with T. spiralis for 7 days. Several proteins, are of particular interest as immunodiagnostic or vaccine antigens, especially with Ts14-3-3 as most promising due to its highly immunogenicity during early infection, expressed protein can be recognized by Trichinella early infection sera and the native Ts14-3-3 expression in both adult and larval stages.
world. Human trichinellosis has been reported in 55 (27.8%) countries (Pozio 2007) and is usually caused by consumption of raw or undercooked meat, especially pork and pork products. There are two cycles of distribution of Trichinella in the natural hosts: one is the domestic cycle in which the transmission involves swine herds that are fed un- cooked pork scraps, carrion, garbage, or carcasses that are not promptly removed from the farm; the second is the sylvatic cycle, in which transmission occurs among wildlife hosts, including mammals, birds and reptiles (Pozio 2005). The domestic cycle and the sylvatic cycle can function either indepen- dently from each other or can also interact (Pozio 2007). With regard to the geographical distribu- tion of the domestic cycle of trichinellosis, there have been no reports of infections on industrialised farms in Canada, the United States, and Western Europe. However, the domestic cycle still occurs on industrial farms in several countries of East-Central Europe and East Asia (Pozio 2007). In the coun- tries with the domestic cycle, domestic pork and pork products remain the most important source of Trichinella infection in humans, especially when pigs are raised under free-ranging or backyard pro- duction conditions (Gottstein et al. 2009).
Trichinella spiralis (T. spiralis), which is a cosmopolitan nematode that infects humans among other species, presents a complex host-parasite relationship that hinders the development of tools to eradicate the parasitosis. The aim of this research was to analyze the host response during a pri- mary infection with T. spiralis in five genetically different mouse lines of the CBi-IGE stock. Adult males from the CBi+, CBi−, CBi, CBi/L and CBi/C lines were infected with 1, 2 or 4 L1 larvae per g of body weight. In the chronic stage, the number of parasites per g of tissue (relative larval load, rLL) showed a significant host genotype-dose interaction, since it did not increase in the same way in the five genotypes. At the lowest dose, both CBi− and CBi/L mice were resistant while CBi+, CBi/C, and CBi were susceptible. At the highest dose, only CBi/L remained resistant, and CBi+ was the most susceptible. The reproductive capacity index of adult worms (RCI = rLL/infective dose) evinced only a genotype effect, allowing rating each line as resistant or susceptible regardless of dose. Animals receiving 2 L1 larvae were also sacrificed in the intestinal phase (6 and 13 days p-i) to determine the number of adult parasites (nAP) recovered in a small intestine segment, and fe- male fecundity (Ff). No differences in nAP were observed among genotypes on day 6 p-i. nAP de- creased between days 6 and 13 p-i, this reduction being different among genotypes and significant only in CBi/L and CBi/C. Ff decreased in CBi/L and CBi/C on day 13 p-i. At the time of infection, se- rum cytokine baseline values showed a Th1 orientation for genotype CBi/L (high IFN-γ and IL-2)
Anti-Trichinella antibodies killed T. spiralis larvae through an ADCC mode [42, 43]. Our results from the ADCC test indicate that anti-rTsSP antibodies partici- pated in the killing of T. spiralis NBL and ML. Perito- neal macrophages adhered to and damaged the larvae with the aid of anti-rTsSP serum, and the killing was also antibody dose-dependent. When the mice were inoculated orally with ML treated by ADCC, the reduc- tion of intestinal adult worms reached up to 89.40%. The results indicate that anti-rTsSP antibodies signifi- cantly killed the ML, decreased larval infectivity, and Figure 10 T. spiralis NBL and ML at different times after ADCC assay. In the assay, the NBL (A–F) and ML (G–L) were cultured with anti-rTsSP serum and 2 × 10 5 mouse peritoneal macrophages (PM) at 37 °C for different times, A, G 6 h; B, H 12 h; C, I 24 h; D, J: 48 h; E, K: 72 h; F (NBL) and I
In the present study, the AW ES antigens of T. spiralis were used as diagnostic antigens for the detection of anti- Trichinella IgG in the sera of experimentally infected mice. The sensitivity and specificity of the ELISA with the AW ES antigens were 100 % (35/35) and 100 % (106/106) in the infected mice, respectively. Moreover, there was no sig- nificant difference in the antibody detection rates in the sera of mice infected with T. nativa, T. britovi, and T. nelsoni by ELISA with the AW ES antigens and ML ES antigens (P > 0.05). The results suggested that there were common AW ES antigens among the encapsulated Trichinella species (i.e., T. spiralis, T. nativa, T. britovi, and T. nelsoni), but not in the non-encapsulated Trichinella species (T. pseudospiralis) [35, 36]. Moreover, most of the epitopes of the AW ES antigens recognized by sera from infected mice were common to the encapsulated Trichinella species . Our results indicated that the AW ES antigens of T. spiralis could also be used for the serodiagnosis of trichinellosis caused by other en- capsulated Trichinella species.
T. murrelli, T. spiralis, T. nativa; the genotype Trichinella T6 ( Masuoka et al., 2009 ); and the non-encapsulated species T. pseudospiralis, which is the only Trichinella species pre- viously documented in feral swine in the USA ( Gamble et al., 2005 ). The sylvatic genotypes are minimally infec- tive to domestic pigs as opposed to the domestic genotype (T. spiralis), and show poor persistence in pig tissues ( Kapel and Gamble, 2000 ). On the other hand, even though T. spi- ralis reproduces well in most carnivore hosts, it is found in the USA almost exclusively in pigs and peridomestic car- nivores. Most feral swine in the USA are descendants of escaped or deliberately released domestic pigs, with inter- breeding with imported and released European wild boar. T. spiralis is rarely detected in wildlife species that do not associate with pigs; T. murrelli is the predominant genotype identiﬁed in sylvatic carnivores in North America ( Zarlenga et al., 1991; Pozio and La Rosa, 2000; Hill et al., 2008 ). The seroprevalence of Trichinella spp. in feral pigs varies world- wide, 0.11% in Slovakia ( Hurníková and Dubinsk ´y, 2009 ), 0.2% in Switzerland ( Frey et al., 2009a ), 0.77% in Spain ( García Sánchez et al., 2009 ), and 19.9% in Vietnam ( Vu Thi et al., 2010 ). The Trichinella spp. seroprevalence of 3.0% in feral swine in the USA seen in the current 2006–2010 sur- vey was four times higher than in feral swine of Europe. The reason for the higher Trichinella spp. seroprevalence in the USA compared to Europe may be related to the fact that in contrast to Europe, there has never been a Trichinella spp. control program in domestic pigs in the USA.