Muscle Larvae Research Articles

A Large-Scale Survey of Trichinella spp. Infection in Japanese Wild Boars.

This study presents the results of a large-scale, one-year survey of Trichinella spp. in Japanese wild boars (Sus scrofa). We analyzed the tongues of 1,168 wild boars captured by hunters in 30 prefectures of Japan, most of which were boar habitats, from October 2014 to January 2015. The samples were digested, and the prevalence of Trichinella spp. muscle larvae was examined. Examination of pooled samples from 10 individuals (15 g per head) or 117 randomly selected samples (10% of the total number of samples) that were individually processed showed no larval infection. Thus, our data suggests that Japanese wild boars do not play a major role in the sylvatic cycle of Trichinella parasites.

Practical methods for collecting Trichinella parasites and their excretory-secretory products

Trichinella is a unique nematode. Its developmental stages include adult worms, newborn larvae, and muscle larvae. Besides humans, the parasite also infects many kinds of animals, including mice. Mice are widely used as an animal model in the research fields of immunology, cell biology, and host-parasite relationships of trichinellosis. The different developmental stages of Trichinella share similar, but unique characteristics. Therefore, it is important to collect different sources of Trichinella-derived materials for research with appropriate methods. In the present study, we introduce methods to collect Trichinella at different stages as well as their ES products. By optimizing the concentration of artificial gastric juice, volume of medium, and time of incubation for ES collection in vitro, muscle larvae, adult worms, and newborn larvae were collected with less contamination by host materials, and the ES products collected were confirmed to be originally antigenic and biologically active. The DNA, RNA, and proteins isolated from the parasites collected were confirmed to be applicable to analyses, including PCR, real-time PCR, Western blotting, and stimulators of cell cultures (macrophages, splenocytes, and tumor cells). The present study compiled protocols to collect materials from Trichinella and provides a reference for research on Trichinella.

The roles of supernatant of macrophage treated by excretory-secretory products from muscle larvae of Trichinella spiralis on the differentiation of C2C12 myoblasts

The excretory-secretory products (ESPs) released by the muscle-larvae (ML) stage of Trichinella spiralis have been suggested to be involved in nurse cell formation. However, the molecular mechanisms by which ML-ESPs modulate nurse cell formation remain unclear. Macrophages exert either beneficial or deleterious effects on tissue repair, depending on their activation/polarization state. They are crucial for skeletal muscle repair, notably, via their actions on myogenic precursor cells. However, these interactions during T. spiralis infection have not been characterized. In the present study, the ability of conditioned medium (CM) from J774A.1 macrophages treated with ML-ESPs to influence the differentiation of murine myoblasts, and the mechanisms of this influence, were investigated in vitro. The results showed that the expression of Myogenic Regulatory Factors (MRFs) MyoD and myogenin, myosin heavy chain (MyHC), and the p21 cyclin-dependent kinase inhibitor were reduced in CM treated cells compared to their expression in the control group. These findings indicated that CM inhibited myoblast differentiation. Conversely, CM promoted myoblast proliferation and increased cyclin D1 levels. Taken together, results of our study suggested that CM can indirectly influence myoblast differentiation and proliferation, which provides a new method for the elucidation of the complex mechanisms involved in cell-parasite and cell–cell interactions during T. spiralis infection.

Developmental profile of select immune cells in mice infected with Trichinella spiralis during the intestinal phase

Trichinella spiralis can cause immunosuppression during the intestinal phase of early infection. However, changes in the peripheral blood during T. spiralis early infection remain unclear. Here, select immune cells in mice infected with 500 muscle larvae (ML) of T. spiralis during the intestinal phase of infection were studied. First, the recovery rates of the intestinal worms and female fecundity were determined, and the results showed that the intestinal worms were completely eliminated at 17 days post-infection (dpi) and that large numbers of new-born larvae (NBL) were generated from 5 to 9dpi. Using flow cytometry, it was shown that the number of CD4+ T cells and CD8+ T cells increased over the entire intestinal phase, except on 7dpi when CD4+ T cells decreased significantly compared to the control groups. Although both CD4+ and CD8+ T cells increased, CD8+ T cells increased more than CD4+ T cells, leading to a lower CD4+/CD8+ ratio compared to the control group. Subsequently, a depression of the proliferative response of T cells to concanavalin A (Con A) was noticed at 7 and 11dpi. Although the proliferative response of B cells to LPS was enhanced, the number of B cells from mouse peripheral blood stimulated by T. spiralis antigens showed no differences with the control group prior to 11dpi. The expression of CD14 on monocyte-macrophages decreased during the same period, which meant that the antigen-presenting response was reduced in the immune system of the infected mice. Moreover, the alternatively activated macrophages were induced in T. spiralis early infection. These data provide a better understanding of the development of the intestinal immune response in mice infected with T. spiralis.

Validation studies of the latex agglutination test for the detection of Trichinella larvae in meat products

Human trichinellosis is a foodborne disease caused by ingestion of meat infected with Trichinella muscle larvae. To control Trichinella spp. infection in the European Union, all slaughtered pigs from holdings that are not officially recognized as applying controlled housing conditions and other animals susceptible to Trichinella infection and intended for human consumption should be examined by one of the approved digestion methods described in Regulation (EU) No. 2015/1375.In the past, Trichinella outbreaks due to the consumption of cured wild boar or pork products have been described in several European countries, making the identification of the larvae from these products relevant for Trichinella control.Therefore, this study aimed to validate the newly approved latex agglutination test (Trichin-L) for routine testing of cured meat products. The test was validated based on the OIE Guidelines using pork products spiked with Trichinella larvae. The sensitivity of the method varied greatly depending on the investigated meat product and was usually lower than for the gold standard, the magnetic stirrer method. The detection rate reached 80% for three larvae and 60% for one larva in cured pork sausages. A detection rate of 100% for three larvae and 50% for one larva was found in bacon. For frozen samples (−20°C) the Trichin-L kit is similarly sensitive as for cured samples. Further, to determine the performance of the test under field conditions, pork products from regions with known high Trichinella prevalences confiscated by customs authorities at two German international airports were analyzed. Problems associated with the Trichin-L test were incomplete digestion due to fatty ingredients, spices and very dry meat products, resulting in data which could not be evaluated. Therefore, the test is currently not suitable for the detection of Trichinella larvae in cured meat products and needs further adaptation steps to increase both usability and sensitivity.

Effect of probiotic bacteria on phagocytosis and respiratory burst activity of blood polymorphonuclear leukocytes (PMNL) in mice infected with Trichinella spiralis

This study focusses on the effect of probiotic (bacteriocinogenic) strains on parasite infection and innate immunity – phagocytosis and oxidative burst of blood monocytes and polymorphonuclear leukocytes (PMNL) in mice infected with Trichinella spiralis. Bacteriocinogenic and probiotic strains of different origin (Enterococcus faecium AL41=CCM8558, Enterococcus durans ED26E/7, Lactobacillus fermentum AD1=CCM7421, Lactobacillus plantarum 17L/1) were administered daily in dose of 109CFU/ml in 100μl and mice were infected with 400 larvae of T. spiralis on 7th day of treatment. Phagocytic activity of blood leukocytes was inhibited at week 3 and 4 post infection (p.i.), i.e. in the time of massive muscle invasion with larvae T. spiralis. Administration of bacterial strains to mice prior to T. spiralis infection elevated and prolonged phagocytic activity of blood leukocytes and their ingestion capability from week 1 to 3 of the infection and the phagocytosis was inhibited only at week 4 p.i. The highest stimulative effect on phagocytosis was induced by strains E. durans ED26E/7, L. fermentum AD1=CCM7421, and L. plantarum 17L/1. The percentage of cells with respiratory burst and their enzymatic activity was increased after T. spiralis infection with the exception of week 3 p.i. In contrast, in all mice treated with bacterial strains the enzymatic stimulation was observed after the infection, with the highest intensity caused by strains E. durans ED26E/7, L. fermentum AD1=CCM7421 and L. plantarum 17L/1. The administration of probiotic strains stimulated phagocytosis and respiratory burst of blood PMNL that could contribute to a decreased larval migration and a destruction of muscle larvae and then reduced parasite burden in the host. The protective effect against T. spiralis infection was induced by all strains, but the highest reduction was recorded by E. faecium AL41=CCM8558.

Trichinella spiralis infection and transplacental passage in human pregnancy

Infection by Trichinella spp. during pregnancy still arises many questions. Worldwide an important number of trichinellosis outbreaks have occurred over time where pregnant women were or might have been involved.Parasitological and immunoserological parameters, clinical background and transplacental passage of Trichinella spiralis of six pregnant women, from different outbreaks, were studied.Serum samples were collected from patients and their children: at pregnancy, at delivery, newborn and/or infant (≥1 year-old). Total immunoglobulins (Igs) and isotypes against excretion-secretion products from muscle larvae (ESP-ML) and newborn larvae (NBL) of T. spiralis were detected by immunoserological techniques. Sera helminthocytotoxic activity against NBL was evaluated by antibodies dependent cell cytotoxicity assay.Patients presented similar clinical symptoms. Mother's sera showed differences in the presence and titres of Igs, IgE, IgG1 and IgG4 against NBL surface. All of them showed helminthocytotoxic activity against NBL, four in progesterone dependent manner and the other two by antibodies. Igs anti ESP-ML were present in mother's sera, with differences in the isotypes detected. Anti-ESP-ML IgE, IgA and/or IgM in newborn sera were observed. Three out of four infants showed specific Igs; one even at 8 years-old.The distinct immune response between mothers at pregnancy evidenced by sera helminthocytotoxic activity and immunoserological parameters may be associated with a different parasite burden exposition. Specific Igs observed in newborns would be a suggestion of transplacental passage of this Igs and/or their production by the fetus. Specific Igs found in infants sera suggests that newborn larvae transplacental passage is possible.

Proteomic analysis and immunodetection of antigens from early developmental stages of Trichinella spiralis

Trichinella spiralis is an ubiquitous parasitic nematode that lives in muscle tissue of many hosts and causes trichinellosis in humans. Numerous efforts have been directed at specific detection of this infection and strategies for its control. TSL-1 and other antigens, mainly from muscle larvae (ML), have been used to induce partial protection in rodents. An improvement in protective immunity may be achieved by using antigens from other parasite stages. Further, identification of other parasite antigens may provide insights into their role in the host-parasite interaction. In this study, T. spiralis antigens from early developmental parasite stages, namely ML and pre-adult (PA) obtained at 6h, 18h and 30h post-infection, were identified by proteomic and mass spectrometry analyses. Our findings showed a differential expression of several proteins with molecular weights in the range of 13–224kDa and pI range of 4.54–9.89. Bioinformatic analyses revealed a wide diversity of functions in the identified proteins, which include structural, antioxidant, actin binding, peptidyl prolyl cis-trans isomerase, motor, hydrolase, ATP binding, magnesium and calcium binding, isomerase and translation elongation factor. This, together with the differential recognition of antigens from these parasite stages by antibodies present in intestinal fluid, in supernatants from intestinal explants, and in serum samples from mice infected with T. spiralis or re-infected with this parasite, provides information that may lead to alternatives in the design of vaccines against this parasite or for modulation of immune responses.

Partially protective immunity induced by the 14-3-3 protein from Trichinella spiralis

Trichinellosis, a widespread zoonosis, is considered to be an emerging or re-emerging infectious parasitic disease. The development of vaccines to prevent Trichinella infection in domestic animals and humans is important for disease control. In a previous study, we identified Ts14-3-3 as an immunodominant protein from Trichinella spiralis (T. spiralis) adult worms recognized by early infection sera from pigs and mice. In this study, we further confirmed that Ts14-3-3 mRNA is expressed in both adult worms and in the larval stages of T. spiralis. Immunostaining with anti-Ts14-3-3 mouse sera further confirmed that native Ts14-3-3 is highly expressed on the surface of T. spiralis muscle larvae. The immune recognition by infected sera, its expression in both adult and larval stages and its exposure on the surface of the parasite led us to explore Ts14-3-3 as a vaccine antigen. Recombinant Ts14-3-3 formulated with an ISA50v2 adjuvant produced strong total IgG and balanced IgG1 and IgG2a responses in vaccinated mice and stimulated mouse splenocytes to produce high levels of Th1 (INF-γ, IL-2) and Th2 (IL4, IL5) cytokines. These results indicate that Ts14-3-3 is highly immunogenic and is able to induce balanced Th1/Th2 immune responses. These vaccine-induced immune responses resulted in a reduction in muscle larvae of up to 46.2% in vaccinated mice upon subsequent larval challenge relative to the number of larvae in mice received PBS control. The significant reduction in muscle larvae in vaccinated mice suggests that Ts14-3-3 is a promising vaccine target for potential use in domestic pigs to prevent trichinellosis transmission.

Proteomic analysis of differentially expressed proteins in the three developmental stages of Trichinella spiralis

Trichinella spiralis, an intracellular parasitic nematode, can cause severe foodborne zoonosis, trichinellosis. The life cycle of T. spiralis consists of adult (Ad), muscle larvae (ML) and newborn larvae (NBL). The protein profiles in different developmental stages of the parasite remain unknown. In the present study, proteins from lysates of Ad, ML and NBL were identified by isobaric tags for relative and absolute quantitation (iTRAQ). A total of 4691 proteins were identified in all the developmental stages, of which 1067 proteins were differentially expressed. The number of up-regulated proteins in NBL was higher than that of the other two groups. The protein profiles from Ad, ML and NBL were compared in pairs. The identified proteins were involved in various functions of T. spiralis life cycle, including sexual maturity, metabolism, utilization of carbohydrates, lipids and nucleotides, and other crucial developmental processes that occur at distinct stages. Further investigation of the transcriptional levels of major sperm protein, serine protease, zinc finger protein, etc. from the different protein profiles using quantitative RT-PCR showed identical results to the iTRAQ analysis. The differentially expressed proteins that are involved in developmental regulation and host-parasite interactions should be further studied.

An invasive species as an additional parasite reservoir: Trichinella in introduced American mink (Neovison vison)

The American mink (Neovison vison Schreber, 1777) is one of the most invasive alien mammal species which has been introduced to many countries in Europe, Asia and South America. As a predator which feeds on various prey, American mink carry many endoparasites; however, data about infection rate and prevalence of different parasites in mink are limited, especially concerning Trichinella spp. The aim of our study was to evaluate the role of American mink in the circulation of Trichinella species in its introduced range. In total, 812 muscle samples of mink from six feral populations and three farms in Poland were examined for presence of Trichinella muscle larvae using the artificial HCl–pepsin digestion method. Muscle larvae were found in 27 out of 812 individuals (3.3%) from six localities in western and north-eastern Poland. The intensity of infection varied from 0.1 to 274.8 LPG. The majority of Trichinella-positive individuals – 15 (55.6%) were infected with T. britovi, one with T. spiralis, and one with T. pseudospiralis. Two animals harboured T. britovi and T. spiralis in co-infection and this data confirmed the first report of a mixed Trichinella infection in American mink.

Cloning and characterization of thioredoxin peroxidases from Trichinella spiralis

The intracellular parasitic nematode, Trichinella spiralis, can initiate a high level of oxidative stress, especially during rapid growth and generative propagation phases. Thioredoxin peroxidases (TPXs) protect helminths against oxidative stress, but none has been identified in T. spiralis. Here, 3 members of the TPX family were cloned from T. spiralis muscle larvae (ML). The lengths of TsTPX ORFs were 747bp, 588bp and 594bp, respectively, and the deduced proteins predicted to contain AhpC-TSA and 1-cys Prx_C domains. Interestingly, qRT-PCR data showed that TsTPX genes were expressed in all three developmental stages of T. spiralis. The TsTPX2 and TsTPX3 genes were up-regulated in day 3 adults (Ad3) compared with newborn larvae (NBL) and ML (P<0.05); expression levels of the TsTPX1 gene in ML were higher compared with Ad3 and NBL amounts (P<0.05). After prokaryotic expression, the reactivity of rTsTPX proteins was assessed by Western-blotting: only rTsTPX1 was specifically recognized by T. spiralis infection sera from pigs. Enzyme catalytic experiments showed that rTsTPX proteins could deoxidize H2O2 in the presence of DTT, with the catalytic ability increasing with protein concentration and time.

Trichinella britovi biomass in naturally infected pine martens (Martes martes) of Latvia

Parasites of the genus Trichinella are cosmopolitan nematodes infecting primarily wild animals, which represent the main reservoirs of these zoonotic pathogens. To investigate the transmission patterns of Trichinella spp. from wild to domestic animals and to humans and for the risk assessment of these parasites in a geographical area, it is important to know the number of possible transmission events deriving from carcasses of infected hosts. For this purpose, the evaluation of the larval biomass in reservoir hosts is needed. No data is available on how to estimate the biomass of Trichinella spp. larvae in muscles of naturally infected animals. The aim of this study was to evaluate the larval biomass in naturally infected pine martens (Martes martes) of Latvia, in which the prevalence of Trichinella britovi infection was over 50%. Single muscles or group of muscles (abdomen, back, diaphragm, intercostal muscles, muscles from the head, left and right shoulders, lower and upper parts of the forelimbs and hind limbs, neck, rump with tail, and base and tip of the tongue) were collected from five skinned and eviscerated carcasses of T. britovi infected pine martens. Muscles were entirely removed from the bones and weighted. Each muscle or group of muscles was separately digested to detect the larvae per gram (LPG). Using linear regression, the larval burden in each muscle or group of muscles was evaluated to measure the possible prediction of the total animal larval burden (both as total number of larvae and as average LPG). All muscles were significantly predictive of the total burden with high “goodness of fit” (all adjusted R2>0.80; P≤0.01), and the left shoulder provided the highest adjusted R2 (0.999). Then, to estimate the Trichinella britovi biomass in the pine marten population of Latvia, recent literature data on prevalence (56.2%, 95% CI: 47.8–64.3) and geometric mean LPG (1.26, 95% CI: 0.89–1.79) in the limb muscles of a sample representative of the whole Latvian pine marten population, were used. Using the predictive estimated relationship between LPG in the limb and that in the entire animals and by the estimated animal population and their mean total muscle weight, the Trichinella britovi biomass in the pine marten population of Latvia was estimated to be of 6,647,092 (95% CI: 3,840,030–11,100,000) larvae. The assessment of the biomass in nature can help to understand the epidemiological pattern of these pathogens, to implement actions aimed at controlling the infection in target animal species, and to acquire basic information on the complex biology of this group of zoonotic nematodes.

The occurrence of nematodes of the genus Trichinella in wolves (Canis lupus) from the Bieszczady Mountains and Augustowska Forest in Poland

The aim of the present study was to investigate the prevalence of Trichinella infection in wolves (Canis lupus) in two regions in Poland. Muscle samples were collected from 21 wolves between 1999 and 2015 and processed by artificial digestion. In two cases, the muscle larvae (ML) were obtained and stored in alcohol. ML were detected in 12 wolves and genotyped by multiplex PCR. Trichinella britovi was confirmed in 12 wolves (54.5%). The larval burdens in infected animals ranged from 0.009 to 27 larvae per gram. The high prevalence of Trichinella infection in wolves might suggest that this predator is a significant reservoir of Trichinella species in the sylvatic cycle in Poland.

Carbonic anhydrase enzyme as a potential therapeutic target for experimental trichinellosis.

Trichinellosis is a globally distributed helminthic infection. There is a considerable interest in developing new anti-helminthic drugs affecting all the developmental stages of Trichinella. Acetazolamide (carbonic anhydrase (CA) inhibitor) involves a novel mechanism of action by inhibiting such an essential enzyme for parasite metabolism. This work aimed to study the effect of acetazolamide against different stages of T. spiralis in experimental animals. Mice were divided into three groups: group I: infected and treated with acetazolamide on day 2 post infection (P.I.), group II: infected and treated with acetazolamide on day 12 P.I., and group III: infected non-treated. From each group, small intestine and muscles were removed for histopathological and immunohistochemical studies. Also, total adult and muscle larval count were estimated. We found that acetazolamide was effective in reduction of both adult and muscle larval counts. When given early, the effect was more pronounced on the adults (62.7%). However, the efficacy of the drug against muscle larvae was increased when given late (63%). Improvement of the intestinal histopathological changes was observed in all the treated groups. Degeneration of encysted larvae with minimal pathologic changes of infected skeletal muscle was observed in the treated groups. Expression of matrix metalloproteinase-9 showed a statistically significant decrease in the intestinal and muscle tissues in all treated groups as compared to the control group. In conclusion, the present study revealed that acetazolamide, carbonic anhydrase inhibitor, could be a promising drug against both adults and larvae of T. spiralis.

DsRNA-mediated silencing of Nudix hydrolase in Trichinella spiralis inhibits the larval invasion and survival in mice

The aim of this study was to investigate the functions of Trichinella spiralis Nudix hydrolase (TsNd) during the larval invasion of intestinal epithelial cells (IECs), development and survival in host by RNAi. The TsNd-specific double-stranded RNA (dsRNA) was designed to silence the expression of TsNd in T. spiralis larvae. DsRNA were delivered to the larvae by soaking incubation or electroporation. Silencing effect of TsNd transcription and expression was determined by real-time PCR and Western blotting, respectively. The infectivity of larvae treated with dsRNA was investigated by the in vitro larval invasion of IECs and experimental infection in mice. After being soaked with 40 ng/μl of dsRNA-TsNd, the transcription and expression level of TsNd gene was inhibited 65.8% and 56.4%, respectively. After being electroporated with 40 ng/μl of dsRNA-TsNd, the transcription and expression level of TsNd gene was inhibited 74.2% and 58.2%, respectively. Silencing TsNd expression by both soaking and electroporation inhibited significantly the larval invasion of IECs in a dose-dependent manner (r1 = −0.96798, r2 = −0.98707). Compared with the mice inoculated with untreated larvae, mice inoculated with larvae soaked with TsNd dsRNA displayed a 49.9% reduction in adult worms and 39.9% reduction in muscle larvae, while mice inoculated with larvae electroporated with TsNd dsRNA displayed a 83.4% reduction in adult worms and 69.5% reduction in muscle larvae, indicating that electroporation has a higher efficiency than soaking in inhibiting the larval development and survival in mice. Our results showed that silencing TsNd expression in T. spiralis inhibited significantly the larval invasion and survival in host.

Helminth parasites in black rats (Rattus rattus) and brown rats (Rattus norvegicus) from different environments in the Netherlands

BackgroundRattus norvegicus (brown rat) and Rattus rattus (black rat) are known carriers of bacteria, viruses, and parasites of zoonotic and veterinary importance. Moreover, rats may play a role in the transmission of muscle larvae of the zoonotic nematode Trichinella spiralis to farm animals. We aimed to study the intestinal and intramuscular helminths in wild rats from three different environments to assess the relevance of rats as carrier of zoonotic parasites for public health.Materials and methodsWild brown rats (117 individuals) and black rats (44 individuals) were captured at farms, in suburban and in rural environments in the Netherlands. Intestinal helminths were isolated and identified morphologically. Artificial digestion was used to isolate muscle larvae.Results and discussionMorphological analysis of rat intestinal contents yielded six nematode species (Syphacia muris, Heterakis spumosa, Aonchotheca murissylvatici, Trichuris muris, Nippostrongylus brasiliensis, and Strongyloides sp.), three cestode species (Hymenolepis diminuta, H. nana and Hymenolepis (=Rodentolepis) fraterna), and four trematode species (Plagiorchis muris, Plagiorchis proximus, Echinostoma chloropodis, and Notocotylus imbricatus).Black rats at farms displayed the lowest intestinal helminth species variation (six species) and carried overall on average 0.93 species simultaneously. In comparison, brown rats at farms carried seven helminth species and 1.91 species simultaneously. Brown rats from suburban environments displayed the highest species variation (11 species) at 1.82 simultaneous helminth species. Absence of trematodes from rats at farms may suggest limited exchange of rats between farms and surrounding wet rural environments. We report four species of veterinary (Syphacia muris) or zoonotic relevance (Hymenolepis diminuta, Hymenolepis nana and Plagiorchis muris). We did not find Trichinella muscle larvae, consistent with long-term prevalence in Dutch wild rats.

Eosinophils Regulate Type 2 Immune Responses Following Infection with the NematodeTrichinella spiralis

Eosinophils are multifunctional leukocytes implicated in protection against helminth infections. Although eosinophils comprise between 1~5% of peripheral blood leukocytes, they primarily reside in the gastrointestinal tract under homeostatic conditions, and rapidly proliferate upon parasitic infection. Intestinal infection with Trichinella spiralis (T. spiralis) induces eosinophilia when the parasite enters the larval stages and larvae finally migrate to the skeletal muscle. Eosinophils are known to mediate parasite death through antibody-dependent cellular cytotoxicity. In this study, we aimed to address the functional significance of eosinophils in the intestinal phase of T. spiralis infection by analysis of immune responses in the Peyer’s patch (PP) of infected BALB/c and eosinophil-ablated ΔdblGATA mice. Trafficking of eosinophils to the PP was significantly increased, with upregulation of interleukin-5 at 14 days post infection. Eosinophil deficiency led to a significant augmentation of serum immunoglobulin (Ig) M and IgG1 antibody levels. In accordance with this, IgG1 + B cells in the PP were substantially increased in ΔdblGATA mice compared to that in BALB/c mice. Transforming growth factor-β expression in the PP of infected ΔdblGATA mice was significantly decreased compared to that in BALB/c mice, whereas the number of T. spiralis larvae in the diaphragm was increased. Taken together, these findings indicate that eosinophils contribute to the regulation of Th2 immune responses, and protect the host from T. spiralis attempting to establish larvae in the skeletal muscle.

Quality Control of Trichinella Testing at the Slaughterhouse Laboratory: Evaluation of the Use of a 400-Micrometer-Mesh-Size Sieve in the Magnetic Stirrer Method

The performance of a 400-μm-mesh-size sieve (sieve400) has not previously been compared with that of a 180-μm-mesh-size sieve (sieve180). Using pork samples spiked with 0 to 10 Trichinella muscle larvae and an artificial digestion method, sieve performance was evaluated for control of Trichinella in meat-producing animals. The use of a sieve400 resulted in 12% lower larval counts, 147% more debris, and 28% longer counting times compared with the use of a sieve180. Although no false-negative results were obtained, prolonged counting times with the sieve400 may have an impact on performance in a high-throughput environment such as a slaughterhouse laboratory. Based on our results, the sieve180 remains the sieve of choice for Trichinella control in meat in slaughterhouse laboratories, according to the European Union reference method (European Commission regulation 2075/2005). Furthermore, the results of the present study contribute to the discussion of harmonization of meat inspection requirements among countries.

Eosinophils and IL-4 Support Nematode Growth Coincident with an Innate Response to Tissue Injury.

It has become increasingly clear that the functions of eosinophils extend beyond host defense and allergy to metabolism and tissue regeneration. These influences have strong potential to be relevant in worm infections in which eosinophils are prominent and parasites rely on the host for nutrients to support growth or reproduction. The aim of this study was to investigate the mechanism underlying the observation that eosinophils promote growth of Trichinella spiralis larvae in skeletal muscle. Our results indicate that IL-4 and eosinophils are necessary for normal larval growth and that eosinophils from IL-4 competent mice are sufficient to support growth. The eosinophil-mediated effect operates in the absence of adaptive immunity. Following invasion by newborn larvae, host gene expression in skeletal muscle was compatible with a regenerative response and a shift in the source of energy in infected tissue. The presence of eosinophils suppressed local inflammation while also influencing nutrient homeostasis in muscle. Redistribution of glucose transporter 4 (GLUT4) and phosphorylation of Akt were observed in nurse cells, consistent with enhancement of glucose uptake and glycogen storage by larvae that is known to occur. The data are consistent with a mechanism in which eosinophils promote larval growth by an IL-4 dependent mechanism that limits local interferon-driven responses that otherwise alter nutrient metabolism in infected muscle. Our findings document a novel interaction between parasite and host in which worms have evolved a strategy to co-opt an innate host cell response in a way that facilitates their growth.