Important Food-borne Parasite Research Articles

Protective effect of benzaldehyde combined with albendazole against brain injury induced by Angiostrongylus cantonensis infection in mice

Angiostrongylus cantonensis, which is also known as rat lungworm, is an important food-borne zoonotic parasite that causes severe neuropathological damage and symptoms, including eosinophilic meningitis and eosinophilic meningoencephalitis, in humans. At present, the therapeutic strategy for cerebral angiostrongyliasis remains controversial. Benzaldehyde, an important bioactive constituent of Gastrodia elata (Tianma) reduces oxidative stress by inhibiting ROS production. This study aimed to evaluate the therapeutic effect of benzaldehyde in combination with albendazole on angiostrongyliasis in animal models. First, the data from body weight monitoring and behavioural analyses demonstrated that benzaldehyde improve body weight and cognitive function changes after A. cantonensis infection. Next, blood‒brain barrier breakdown and pathological changes were reduced after benzaldehyde and albendazole treatment in A. cantonensis-infected BALB/c mice. Subsequently, we established four RNA-seq datasets from different treatment mouse brains, including the normal, infection, infection+albendazole, and infection+albendazole+3-HBA groups. Ultimately, we found that benzaldehyde regulates cell apoptosis, oxidative stress, and sonic hedgehog signalling in A. cantonensis-infected mouse brains. This study evaluated the therapeutic effect of benzaldehyde on angiostrongyliasis and provided a potential therapeutic strategy for human angiostrongyliasis in the clinic. Moreover, we elucidated the molecular mechanism of benzaldehyde in A. cantonensis-infected mouse brains.

The therapeutic effect of tanshinone IIA in mouse astrocytes after treatment with Angiostrongylus cantonensis fifth-stage larval excretory-secretory products.

Angiostrongylus cantonensis is an important food-borne zoonotic parasite that causes eosinophilic meningitis and meningoencephalitis in humans. Excretory-secretory products (ESPs) are valuable targets for studying host-parasite relationships. ESPs are composed of a variety of molecules that are used to penetrate defensive barriers and avoid immune attack of the host. Tanshinone IIA (TSIIA) is a vasoactive cardioprotective drug that is widely used in studies evaluating potential therapeutic mechanisms. In this study, we will evaluate the therapeutic effects of TSIIA in mouse astrocytes after A. cantonensis fifth-stage larvae (L5) ESPs treatment. Here, we examined the therapeutic effect of TSIIA by real-time qPCR, western blotting, activity assay, and cell viability assays. First, the results showed that TSIIA can elevate cell viability in astrocytes after stimulation with ESPs. On the other hand, TSIIA downregulated the expression of apoptosis-related molecules. However, the expression of molecules related to antioxidant, autophagy, and endoplasmic reticulum stress was significantly increased. The results of antioxidant activation assays showed that the activities of superoxide dismutase (SOD), glutathione S-transferase (GST), and catalase were significantly increased. Finally, we found that cell apoptosis and oxidative stress were reduced in TSIIA-treated astrocytes by immunofluorescence staining. The findings from this study suggest that TSIIA can reduce cellular damage caused by A. cantonensis L5 ESPs in astrocytes and clarify the related molecular mechanisms.

Inactivation of Toxoplasma gondii in dry sausage and processed pork, and quantification of the pathogen in pig tissues prior to production

Toxoplasma gondii is an important zoonotic foodborne parasite. Meat of infected animals appears to be a major source of infection in Europe. Pork is the most consumed meat in France, with dry sausages well represented. The risk of transmission via consumption of processed pork products is largely unknown, mainly since processing will affect viability but may not entirely inactivate all T. gondii parasites.We investigated the presence and concentration of T. gondii DNA in the shoulder, breast, ham, and heart of pigs orally inoculated with 1000 oocysts (n = 3) or tissue cysts (n = 3) and naturally infected pigs (n = 2), by means of magnetic capture qPCR (MC-qPCR). Muscle tissues of experimentally infected pigs were further used to evaluate the impact of manufacturing processes of dry sausages, including different concentrations of nitrates (0, 60, 120, 200 ppm), nitrites (0, 60, 120 ppm), and NaCl (0, 20, 26 g/kg), ripening (2 days at 16–24 °C) and drying (up to 30 days at 13 °C), by a combination of mouse bioassay, qPCR and MC-qPCR.DNA of T. gondii was detected in all eight pigs, including in 41.7% (10/24) of muscle samples (shoulder, breast and ham) and 87.5% (7/8) of hearts by MC-qPCR. The number of parasites per gram of tissue was estimated to be the lowest in the hams (arithmetic mean (M) = 1, standard deviation (SD) = 2) and the highest in the hearts (M = 147, SD = 233). However, the T. gondii burden estimates varied on the individual animal level, the tissue tested and the parasitic stage used for the experimental infection (oocysts or tissue cysts). Of dry sausages and processed pork, 94.4% (51/54) were positive for T. gondii by MC-qPCR or qPCR, with the mean T. gondii burden estimate equivalent to 31 parasites per gram (SD = 93). Only the untreated processed pork sample collected on the day of production was positive by mouse bioassay.The results suggest an uneven distribution of T. gondii in the tissues examined, and possibly an absence or a concentration below the detection limit in some of them. Moreover, the processing of dry sausages and processed pork with NaCl, nitrates, and nitrites has an impact on the viability of T. gondii from the first day of production. Results are valuable input for future risk assessments aiming to estimate the relative contribution of different sources of T. gondii human infections.

Prevalence and Haplotypes of Toxoplasma gondii in Native Village Chickens and Pigs in Peninsular Malaysia.

Toxoplasma gondii is an important zoonotic foodborne parasite capable of infecting almost all warm-blooded animal species worldwide. Toxoplasmosis is usually acquired via ingestion of undercooked infected animal tissues resulting in life-threatening consequences for unborn foetus and immunocompromised individuals. A cross-sectional study was carried out to determine the prevalence of T. gondii infection, its associated risk factors in farms, and haplotypes isolated from the native village chicken and pig populations in Peninsular Malaysia. The seroprevalence of T. gondii in village chickens at the animal level was low at 7.6% (95% CI: 4.60-11.60), while at the farm level, it was 52.0% (95% CI: 31.30-72.20). For pigs, the animal-level seroprevalence of T. gondii was 3.0% (95% CI: 1.60-5.10), while the farm-level, it was 31.6% (95% CI: 12.60-56.60). The PCR-based DNA detection on meat samples from chickens (n = 250) and pork (n = 121) detected 14.0% (95% CI: 9.95-18.9) and 5.8% (95% CI: 2.4-11.6) positive, respectively. Six unique T. gondii haplotypes were isolated from the tissue samples. Multivariable logistic regression analysis showed that feeding the chickens farm-produced feeds and allowing wild animals access to pig farms were significant determinants for farm-level seropositivity. Providing hygienic and good quality feeds to chickens and increasing biosecurity in pig farms through prevention of access by wildlife may reduce the risk of transmission of T. gondii infection in the local chickens and pig farms.

Clonorchis sinensis aggravates biliary fibrosis through promoting IL-6 production via toll-like receptor 2-mediated AKT and p38 signal pathways.

Clonorchis sinensis is an important food-borne zoonotic parasite which has been linked to biliary fibrosis and cholangiocarcinoma. However, the details of the pathogenesis of C. sinensis were unclear. To explore the role and regulatory mechanism of toll-like receptor 2 (TLR2) in C. sinensis-induced biliary fibrosis, we established the C. sinensis-infected C57BL/6 mouse model with TLR2-/- and wild type (WT) mice. The mortality rate, liver lesions, TLR2 and TGF-β1 expression, phosphorylation of Smad2/3, AKT, p38, ERK and p65, and cytokine productions were analyzed. Furthermore, similar parameters were examined in mouse biliary epithelial cells (BECs) co-cultured with C. sinensis excretory/secretory proteins (ESPs). The results showed that TLR2 expression was enhanced significantly in C. sinensis-infected WT mice and mouse BECs. C. sinensis-infected TLR2-/- mice exhibited an increased weight and a decreased mortality rate; significantly alleviated liver lesions and biliary fibrosis, reduced numbers of myofibroblasts; decreased expression of TGF-β1 and phosphorylation level of AKT, p38 and Smad2/3; significantly decreased production of IL-6, TNF-α and IL-4, while increased production of IFN-γ compared with C. sinensis-infected WT mice. Furthermore, C. sinensis ESPs could activate TLR2-mediated AKT and p38 pathways to increase the production of IL-6 in mouse BECs. In conclusion, these data indicate that C. sinensis infection activated TGF-β1-Smad2/3 through TLR2-mediated AKT and p38 pathways to promote IL-6 production, which resulted in myofibroblast activation and aggravating biliary fibrosis in mice.

Extracellular vesicles of the liver fluke Opisthorchis felineus stimulate the angiogenesis of human umbilical vein endothelial cells

The liver fluke Opisthorchis felineus is a clinically important food-borne parasite of humans. Infection with O. felineus in mammals is associated with liver morbidities such as periductal fibrosis, bile duct neoplasia, and chronic inflammation. Previously we have shown that excretory-secretory products (ESP) can stimulate the healing of skin wounds in mice, which may be due to stimulated angiogenesis and extracellular matrix remodeling. However, there are no studies analyzing the angiogenic character of O. felineus, and its effects on angiogenesis, vascularity, and vascular endothelium. The aim of this study was to evaluate the capacity of ESP and extracellular vesicles (EVs) of O. felineus to stimulate angiogenesis and the formation of pseudo-capillaries in vitro. We also aimed at the assessment of the angiogenesis during the infection in vivo, and estimation of the endothelial cell type abundances from heterogeneous bulk liver transcriptome between uninfected and infected animals with single-cell information. The study revealed significant alterations in vascularity in the hamster liver and significant involvement of portal endothelial cells at the transcriptome level. We also demonstrated that the ESP and EVs of O. felineus have the capacity to stimulate the formation of pseudo-capillaries in vitro. Both ESP and EVs appeared to have similar effects on all four parameters, increasing node formation and total master segments length, and significantly decreasing total isolated branches length and number of isolated segments of pseudo-capillaries. The liver flukes manipulate the hostʼs angiogenic response, a fact that has been related to the pathogenesis caused by these parasites. Understanding these pathogenic mechanisms may uncover new therapeutic targets to relieve or prevent the most severe complications of opisthorchiasis.

Foodborne Parasites and Their Complex Life Cycles Challenging Food Safety in Different Food Chains

Zoonotic foodborne parasites often represent complex, multi host life cycles with parasite stages in the hosts, but also in the environment. This manuscript aims to provide an overview of important zoonotic foodborne parasites, with a focus on the different food chains in which parasite stages may occur. We have chosen some examples of meat-borne parasites occurring in livestock (Taenia spp., Trichinella spp. and Toxoplasma gondii), as well as Fasciola spp., an example of a zoonotic parasite of livestock, but transmitted to humans via contaminated vegetables or water, covering the 'farm to fork' food chain; and meat-borne parasites occurring in wildlife (Trichinella spp., Toxoplasma gondii), covering the 'forest to fork' food chain. Moreover, fish-borne parasites (Clonorchis spp., Opisthorchis spp. and Anisakidae) covering the 'pond/ocean/freshwater to fork' food chain are reviewed. The increased popularity of consumption of raw and ready-to-eat meat, fish and vegetables may pose a risk for consumers, since most post-harvest processing measures do not always guarantee the complete removal of parasite stages or their effective inactivation. We also highlight the impact of increasing contact between wildlife, livestock and humans on food safety. Risk based approaches, and diagnostics and control/prevention tackled from an integrated, multipathogen and multidisciplinary point of view should be considered as well.

Oral immunization of mice with recombinant Lactobacillus plantarum expressing a Trichinella spiralis galectin induces an immune protection against larval challenge

BackgroundTrichinella spiralis is an important foodborne parasite that presents a severe threat to food safety. The development of an anti-Trichinella vaccine is an important step towards controlling Trichinella infection in food animals and thus ensure meat safety. Trichinella spiralis galectin (Tsgal) is a novel protein that has been identified on the surface of this nematode. Recombinant Tsgal (rTsgal) was found to participate in larval invasion of intestinal epithelium cells (IECs), whereas anti-rTsgal antibodies impeded the invasion.MethodsThe rTsgal/pSIP409- pgsA′ plasmid was constructed and transferred into Lactobacillus plantarum strain NC8, following which the in vitro biological properties of rTsgal/NC8 were determined. Five groups of mice were orally immunized three times, with a 2-week interval between immunizations, with recombinant NC8-Tsgal, recombinant NC8-Tsgal + α-lactose, empty NC8, α-lactose only or phosphate-buffered saline (PBS), respectively. The vaccinated mice were infected orally with T. spiralis larvae 2 weeks following the last vaccination. Systemic and intestinal local mucosal immune responses and protection were also assessed, as were pathological changes in murine intestine and skeletal muscle.ResultsrTsgal was expressed on the surface of NC8-Tsgal. Oral immunization of mice with rTsgal vaccine induced specific forms of serum immunoglobulin G (IgG), namely IgG1/IgG2a, as well as IgA and gut mucosal secretion IgA (sIgA). The levels of interferon gamma and interleukin-4 secreted by cells of the spleen, mesenteric lymph nodes, Peyer's patches and intestinal lamina propria were significantly elevated at 2–6 weeks after immunization, and continued to rise following challenge. Immunization of mice with the oral rTsgal vaccine produced a significant immune protection against T. spiralis challenge, as demonstrated by a 57.28% reduction in the intestinal adult worm burden and a 53.30% reduction in muscle larval burden, compared to the PBS control group. Immunization with oral rTsgal vaccine also ameliorated intestinal inflammation, as demonstrated by a distinct reduction in the number of gut epithelial goblet cells and mucin 2 expression level in T. spiralis-infected mice. Oral administration of lactose alone also reduced adult worm and larval burdens and relieved partially inflammation of intestine and muscles.ConclusionsImmunization with oral rTsgal vaccine triggered an obvious gut local mucosal sIgA response and specific systemic Th1/Th2 immune response, as well as an evident protective immunity against T. spiralis challenge. Oral rTsgal vaccine provided a prospective approach for control of T. spiralis infection.Graphical

Study on the antagonistic effects of koumiss on Toxoplasma gondii infection in mice.

Toxoplasma gondii is an important food-borne zoonotic parasite, and approximately one-third of people worldwide are positive for T. gondii antibodies. To date, there are no specific drugs or vaccines against T. gondii. Therefore, developing a new safe and effective method has become a new trend in treating toxoplasmosis. Koumiss is rich in probiotics and many components that can alleviate the clinical symptoms of many diseases via the functional characteristics of koumiss and its regulation of intestinal flora. To investigate the antagonistic effect of koumiss on T. gondii infection, the model of acute and chronic T. gondii infection was established in this study. The survival rate, SHIRPA score, serum cytokine levels, brain cyst counts, β-amyloid deposition and intestinal flora changes were measured after koumiss feeding. The results showed that the clinical symptoms of mice were improved at 6 dpi and that the SHIRPA score decreased after koumiss feeding (P < 0.05). At the same time, the levels of IL-4, IFN-γ and TNF-α decreased (P < 0.001, P < 0.001, P < 0.01). There was no significant difference of survival rate between koumiss treatment and the other groups. Surprisingly, the results of chronic infection models showed that koumiss could significantly reduce the number of brain cysts in mice (P < 0.05), improve β-amyloid deposition in the hippocampus (P < 0.01) and decrease the levels of IFN-γ and TNF-α (P < 0.01, P < 0.05). Moreover, koumiss could influence the gut microbiota function in resisting T. gondii infection. In conclusion, koumiss had a significant effect on chronic T. gondii infection in mice and could improve the relevant indicators of acute T. gondii infection in mice. The research provides new evidence for the development of safe and effective anti-T. gondii methods, as well as a theoretical basis and data support for the use of probiotics against T. gondii infection and broadened thoughts for the development and utilization of koumiss.

Benzaldehyde Attenuates the Fifth Stage Larval Excretory-Secretory Product of Angiostrongylus cantonensis-Induced Injury in Mouse Astrocytes via Regulation of Endoplasmic Reticulum Stress and Oxidative Stress.

Excretory–secretory products (ESPs) are the main research targets for investigating the hosts and helminths interaction. Parasitic worms can migrate to parasitic sites and avoid the host immune response by secreting this product. Angiostrongylus cantonensis is an important food-borne zoonotic parasite that causes severe neuropathological damage and symptoms, including eosinophilic meningitis or meningoencephalitis in humans. Benzaldehydes are organic compounds composed of a benzene ring and formyl substituents. This compound has anti-inflammatory and antioxidation properties. Previous studies showed that 3-hydroxybenzaldehyde (3-HBA) and 4-hydroxybenzaldehyde (4-HBA) can reduce apoptosis in A. cantonensis ESP-treated astrocytes. These results on the protective effect underlying benzaldehyde have primarily focused on cell survival. The study was designed to investigate the molecular mechanisms of endoplasmic reticulum stress (ER stress) and oxidative stress in astrocytes in A. cantonensis ESP-treated astrocytes and to evaluate the therapeutic consequent of 3-HBA and 4-HBA. First, we initially established the RNA-seq dataset in each group, including normal, ESPs, ESPs + 3-HBA, and ESPs + 4-HBA. We also found that benzaldehyde (3-HBA and 4-HBA) can stimulate astrocytes to express ER stress-related molecules after ESP treatment. The level of oxidative stress could also be decreased in astrocytes by elevating antioxidant activity and reducing ROS generation. These results suggested that benzaldehyde may be a potential therapeutic compound for human angiostrongyliasis to support brain cell survival by inducing the expression levels of ER stress- and oxidative stress-related pathways.

Challenges for Cryptosporidium Population Studies.

Cryptosporidiosis is ranked sixth in the list of the most important food-borne parasites globally, and it is an important contributor to mortality in infants and the immunosuppressed. Recently, the number of genome sequences available for this parasite has increased drastically. The majority of the sequences are derived from population studies of Cryptosporidium parvum and Cryptosporidium hominis, the most important species causing disease in humans. Work with this parasite is challenging since it lacks an optimal, prolonged, in vitro culture system, which accurately reproduces the in vivo life cycle. This obstacle makes the cloning of isolates nearly impossible. Thus, patient isolates that are sequenced represent a population or, at times, mixed infections. Oocysts, the lifecycle stage currently used for sequencing, must be considered a population even if the sequence is derived from single-cell sequencing of a single oocyst because each oocyst contains four haploid meiotic progeny (sporozoites). Additionally, the community does not yet have a set of universal markers for strain typing that are distributed across all chromosomes. These variables pose challenges for population studies and require careful analyses to avoid biased interpretation. This review presents an overview of existing population studies, challenges, and potential solutions to facilitate future population analyses.

High prevalence, intensity, and genetic diversity of Trichinella spp. in wolverine (Gulo gulo) from Yukon, Canada

BackgroundSpecies of Trichinella are globally important foodborne parasites infecting a number of domestic and wild vertebrates, including humans. Free-ranging carnivores can act as sentinel species for detection of Trichinella spp. Knowledge of the epidemiology of these parasites may help prevent Trichinella spp. infections in northern Canadian animals and people. Previous research on Trichinella spp. in wildlife from Yukon did not identify risk factors associated with infection, or the diversity and identity of species of Trichinella in regional circulation, based on geographically extensive sampling with large sample sizes.MethodsIn a cross-sectional study, we determined the prevalence, infection intensity, risk factors, and species or genotypes of Trichinella in wolverine (Gulo gulo) in two regions of Yukon, Canada, from 2013–2017. A double separatory funnel digestion method followed by mutiplex PCR and PCR-RFLP were used to recover and identify species of Trichinella, respectively.ResultsWe found larvae of Trichinella in the tongues of 78% (95% CI 73–82) of 338 wolverine sampled. The odds of adult (≥ 2 years) and yearling (1 year) wolverine being Trichinella spp.-positive were four and two times higher, respectively, compared to juveniles (<1 year). The odds of Trichinella spp. presence were three times higher in wolverine from southeast than northwest Yukon. The mean intensity of infection was 22.6 ± 39 (SD, range 0.1–295) larvae per gram. Trichinella T6 was the predominant genotype (76%), followed by T. nativa (8%); mixed infections with Trichinella T6 and T. nativa (12%) were observed. In addition, T. spiralis was detected in one wolverine. Out of 22 isolates initially identified as T. nativa in multiplex PCR, 14 were analyzed by PCR-RFLP to distinguish them from T. chanchalensis, a recently discovered cryptic species, which cannot be distinguished from the T. nativa on multiplex PCR. Ten isolates were identified either as T. chanchalensis alone (n = 7), or mixed infection with T. chanchalensis and T. nativa (n = 2) or T. chanchalensis and Trichinella T6 (n = 1)].ConclusionsWolverine hosted high prevalence, high larval intensity, and multiple species of Trichinella, likely due to their scavenging habits, apex position in the food chain, and wide home range. Wolverine (especially adult males) should be considered as a sentinel species for surveys for Trichinella spp. across their distributional range.Graphical

Proteomic Insights into the Biology of the Most Important Foodborne Parasites in Europe.

Foodborne parasitoses compared with bacterial and viral-caused diseases seem to be neglected, and their unrecognition is a serious issue. Parasitic diseases transmitted by food are currently becoming more common. Constantly changing eating habits, new culinary trends, and easier access to food make foodborne parasites’ transmission effortless, and the increase in the diagnosis of foodborne parasitic diseases in noted worldwide. This work presents the applications of numerous proteomic methods into the studies on foodborne parasites and their possible use in targeted diagnostics. Potential directions for the future are also provided.

Toxoplasma gondii in Chickens (Gallus domesticus) from North India.

Toxoplasma gondii, an important food-borne parasite has been reported in a wide range of food animals globally. Poultry meat production is on rise in several developing nations including India. Several studies on status of T. gondii conducted globally had shown substantial risk of this parasite through poultry meat. Such information is lacking in the north India, where poultry meat is a preferred non-vegetarian food over other meats; therefore, this study determined the seroprevalence (based on detection of antibodies) and tissue prevalence (based on detection of DNA) of T. gondii in poultry meat intended for human consumption in north India. Samples (skeletal muscle and/heart as well as blood) were collected from slaughter poultry intended for human consumption reared in Punjab state (n = 366), Himachal Pradesh (n = 44) state, and Chandigarh union territory (n = 112) in North India. Serum samples were tested using commercially available Chicken T. gondii IgG ELISA kit to determine the serostatus. The DNA of T. gondii was detected by nested PCR. To determine the agreement between ELISA and PCR, Kappa value was estimated. Overall, study reports 2.3% (95% CI 1.3%-4.1%) tissue prevalence and 6.5% (95% CI 4.6%-9.1%) seroprevalence from chickens reared in north India. Backyard poultry had significantly higher proportion of T. gondii (24.4%, 10/41; p < 0.001) than caged (0%, 0/284) and deep litter poultry (16.2%, 32/197). The sequence alignment score of three sequences obtained in this study showed 97-100% nucleotide identity with Type I RH strain and VEG type III strain. The agreement between ELISA and PCR was poor (Kappa = 0.145; 95% CI 0.07-0.22). Thus, a combination of serological and molecular tests can improve the diagnosis of T. gondii. A low prevalence indicates that T. gondii in poultry presents a lower food safety risk for public health in north India; however, this study recommend cooking of poultry meat before consumption. Further studies are required to check the viability of T. gondii in poultry meat intended for human consumption, and to determine the risk factors associated and the genotypes circulating in poultry in north India.

Excretory/secretory products of Angiostrongylus cantonensis fifth-stage larvae induce endoplasmic reticulum stress via the Sonic hedgehog pathway in mouse astrocytes

BackgroundAngiostrongylus cantonensis is an important food-borne zoonotic parasite. Humans are non-permissive hosts, and this parasite develops into fifth-stage larvae (L5) in the brain and subarachnoid cavity and then induces eosinophilic meningitis and eosinophilic meningoencephalitis. Excretory/secretory products (ESPs) are valuable targets for the investigation of host-parasite interactions. These products contain a wide range of molecules for penetrating defensive barriers and avoiding the immune response of the host. Endoplasmic reticulum (ER) stress has been found to be associated with a wide range of parasitic infections and inflammation. ER stress can increase cell survival via the activation of downstream signalling. However, the mechanisms of ER stress in A. cantonensis infection have not yet been clarified. This study was designed to investigate the molecular mechanisms of ER stress in astrocytes after treatment with the ESPs of A. cantonensis L5.ResultsThe results demonstrated that A. cantonensis infection activated astrocytes in the mouse hippocampus and induced the expression of ER stress-related molecules. Next, the data showed that the expression of ER stress-related molecules and the Ca2+ concentration were significantly increased in activated astrocytes after treatment with the ESPs of L5 of A. cantonensis. Ultimately, we found that ESPs induced GRP78 expression via the Sonic hedgehog (Shh) signalling pathway.ConclusionsThese findings suggest that in astrocytes, the ESPs of A. cantonensis L5 induce ER stress and that the Shh signalling pathway plays an important role in this process.

Opinions and knowledge on globally important foodborne parasites among healthcare professionals at a tertiary teaching hospital in Nigeria

Foodborne diseases are important everywhere in the world, but the level of attention they receive varies by region. We surveyed the current opinions and knowledge regarding the globally most important foodborne parasites (FBP) among healthcare professionals in Abakaliki, Ebonyi State, Nigeria, by conducting a questionnaire survey among healthcare professionals in a tertiary hospital. We focused on the FBP ranked as the top five globally: Taenia solium, Echinococcus granulosus, Echinococcus multilocularis, Toxoplasma gondii, and Cryptosporidium spp., and gathered local expert opinions regarding their importance in Nigeria. Moreover, we surveyed the extent of healthcare community knowledge on transmission, manifestations and pathologies, and prevention of infections with these five FBP. Among the 205 healthcare professionals completing the survey, T. solium was selected as important in Nigeria by 143 (70%), E. granulosus by 91 (44%), E. multilocularis by 62 (30%), T. gondii by 132 (64%), and Cryptosporidium spp. by 123 (60%). Only 44 (21%) of the participants selected at least 15 of the 25 answers to knowledge questions that we considered as correct to select. The proportion selecting at least 15 of the correct answers was not statistically significantly associated with gender nor with experience level. Our results suggest that further education about FBP should target healthcare professionals at all levels.

A novel protocol to isolate, detect and differentiate taeniid eggs in leafy greens and berries using real-time PCR with melting curve analysis

BackgroundZoonotic taeniid cestodes are amongst the most important food-borne parasites affecting human health worldwide. Contamination of fresh produce with the eggs of Echinococcus granulosus (s.l.), Echinococcus multilocularis, and some Taenia species pose a potential food safety risk. However, very few studies have attempted to investigate the potential contamination of fresh produce with taeniid eggs and the available methods are not standardized for this purpose. Established protocols do exist for testing leafy greens and berries for contamination with protozoan parasites and are used in national surveillance programmes. This methodology could be suitable for the detection of taeniids. The objective of this project was to develop and standardize a sensitive and reliable method to detect contamination of leafy greens and berries with eggs of zoonotic taeniids and to differentiate between E. multilocularis, E. granulosus (s.l.) and Taenia spp.MethodsWe compared the efficacy of different wash solutions to remove Taenia spp. eggs from spiked produce, assessed two DNA extraction kits for their performance on Taenia spp. eggs, and adapted a published conventional multiplex PCR into a real-time PCR with fluorescence melting curve analysis (MCA) that was optimized for use on produce washes. Analytical specificity of this protocol was assessed using non-spiked produce washes as well as a variety of other potentially contaminating parasites.ResultsThe protocol as established in this study had an analytical sensitivity of detecting five eggs per spiked sample for both romaine lettuce and strawberries. Unequivocal identification of E. multilocularis, E. granulosus (s.l.) and Taenia spp. was possible through MCA. Amplicon sequencing allowed identification of Taenia to the species level. The real-time PCR also amplified DNA from Dicrocoelium sp., but with a clearly discernable melting curve profile.ConclusionThe new protocol for screening produce for taeniid contamination was highly sensitive. Melting curve analysis and the possibility of amplicon sequencing made this assay very specific. Once further validated, this method could be employed for surveillance of produce for contamination with taeniid parasites to assess potential risks for consumers.

Characterization of the complete mitochondrial genome of the echinostome Echinostoma miyagawai and phylogenetic implications.

Echinostomes are important intestinal foodborne parasites. Despite their significance as pathogens, characterization of the molecular biology and phylogenetics of these parasites are limited. In the present study, we determined the entire mitochondrial (mt) genome of the echinostome Echinostoma miyagawai (Hunan isolate) and examined the phylogenetic relationship with selected members of the suborder Echinostomata. The complete mt genome of E. miyagawai (Hunan isolate) was 14,468bp in size. This circular mt genome contained 12 protein-coding genes, 22 transfer RNA genes, two ribosomal RNA genes, and one non-coding region. The gene order and genomic content were identical with its congeners. Phylogenetic analyses (maximum parsimony, maximum likelihood, and Bayesian inference) based on the concatenated amino acid sequences of 12 protein-coding genes strongly supported monophyly for the genus Echinostoma; however, they rejected monophyly for the family Echinostomatidae and the genus Fasciola. The mt genomic data described in this study provides useful genetic markers for studying the population genetics, molecular biology, and phylogenetics of these echinostomes.

Identification and molecular characterization of exosome-like vesicles derived from the Taenia asiatica adult worm

Taenia asiatica is an important food-borne parasite that poses a threat to food-safety and animal husbandry hygine, yet little is known about its specific infection and immune escape mechanisms. Exosome-like vesicles have recently emerged as a regulator in the interactions between parasites and hosts, providing a new direction for research on infection of T. asiatica. In this experiment, exosome-like vesicles were collected from the excretory/secretory products of cultured T. asiatica and isolated by differential centrifugation. The purified vesicles, ranging from 30 to 150 nm in size, were identified as exosome-like vesicles by transmission electron microscope and Nanoparticle tracking analysis. Proteomics analysis identified 455 proteins in the exosome-like vesicles. Of these proteins, enzymes involved in metabolic processes were identified, including glyceraldehyde 3 phosphate dehydrogenase, fructose-1, 6-bisphosphate aldolase, cytosolic malate dehydrogenase, and enolase. The two most abundant proteins from proteomic analysis, 14-3-3 and enolase, were shown to be present in the exosome-like vesicles by immunogold labeling. High-throughput RNA sequencing yielded twenty known miRNAs present in exosome-like vesicle sRNA libraries. Nine of the miRNAs, including six known miRNAs (tas-miR-71, tas-miR-1, tas-miR-7, tas-miR-9, tas-miR-10, and tas-let-7) and three newly discovered miRNAs (tas-m0022-3p, tas-m0816-3p, tas-m0082-5p), were confirmed by RT-qPCR as present in T. asiatica adult worm extracts and secreted exosome-like vesicles in T. asiatica. Additionally, we demonstrated that exosome-like vesicles experimentally labeled with PKH67 were internalized by LoVo cells in vitro. These findings provide new insights into the interaction between tapeworms and hosts mediated by exosome-like vesicles.

Molecular Identification and Phylogenetic Analysis of Nuclear rDNA Sequences of Clonorchis sinensis Isolates From Human Fecal Samples in Heilongjiang Province, China.

Studying the genetic diversity of parasite is important for understanding their biogeography and molecular epidemiology, as well as for establishing disease prevention and control strategies. Clonorchis sinensis is an important foodborne parasite worldwide. However, despite its epidemiological significance, the genetic diversity of C. sinensis has not been well studied from human in northeastern China. In this study, a total of 342 fecal specimens were collected from residents living in five villages in Heilongjiang Province and analyzed for the presence of C. sinensis by PCR amplification and sequencing of the internal transcribed spacer 1 (ITS1) and ITS2 regions of nuclear ribosomal DNA. 21.64% (74/342) of fecal samples were found to be positive for C. sinensis by PCR. The sequences of the ITS1 region in 34 of the 74 samples (45.95%) matched that of MK179278, Genetic polymorphisms were observed at six nucleotide sites. The ITS2 gene sequence of 37 of the 74 samples (50%) matched that of MK179281. In conclusion, a low degree of genetic diversity between C. sinensis isolates from China and different geographical regions was found at ITS loci. Despite this conservation, sequencing of the rDNA region has provided important data that will be useful for future studies addressing the molecular evolution, biology, medical implications and ecology of C. sinensis.