Echinococcus Multilocularis Research Articles

Wild Carnivore Survey of Echinococcus Species in Slovenia.

Simple SummaryWild carnivores can harbor a dangerous tapeworm Echinococcus sp. that causes an important food-borne disease called echinococcosis. This study uses molecular methods to assess the prevalence of the tapeworm Echinococcus multilocularis, E. canadensis, and E. granulosus sensu stricto in the stool of red foxes, wolves, golden jackals, martens, lynxes, badgers, and otter. Red foxes (29.1%) and golden jackals (18%) were positive for E. multilocularis (EM), while all other animals were negative for all Echinococcus species tested in this study. Statistical analysis showed that the prevalence of EM is associated only with the region where the sample originated and not by species, age, or sex of the animal. Central and south regions of Slovenia have a higher EM prevalence and risk of infection. Due to habitat expansion and an increasing population, golden jackal may soon become an important source for human infection with EM.Wild carnivores are definitive hosts and potential reservoirs for the tapeworm Echinococcus sp. which can cause cystic and alveolar echinococcosis. Both are considered neglected and important food-borne pandemics. This study is the first to molecularly test Slovenian wild carnivores for Echinococcus species that can cause disease in humans. Fecal samples from 210 red foxes (Vulpes vulpes), 42 wolves (Canis lupus), 39 golden jackals (Canis aureus), 18 martens (Marten sp.), 2 Eurasian lynx (Lynx lynx), 2 European badger (Meles meles), and 1 Eurasian otter (Lutra lutra) were examined for Echinococcus granulosus sensu lato (EGsl: E. granulosus sensu stricto, E. canadensis) and E. multilocularis (EM) using real-time PCR. Red foxes (29.1%) and golden jackals (18%) were positive for EM. All animals examined were negative for EGsl. Univariate analysis showed no significant differences in EM prevalence with respect to animal species (red fox vs., golden jackal) (p = 0.22), age (p = 0.12), and sex (p = 0.18). Prevalence of EM was associated with the region (p < 0.001), with regions in central and southern Slovenia having higher EM prevalence and risk of infection. Due to the increase in population and expansion of habitat, the golden jackal may soon become as important definitive host for EM as the red fox.

Advances in the pharmacological treatment of hepatic alveolar echinococcosis: From laboratory to clinic.

Hepatic alveolar echinococcosis (HAE) is a zoonotic parasitic disease caused by the larvae of Echinococcus multilocularis. Because of its characteristics of diffuse infiltration and growth similar to tumors, the disability rate and mortality rate are high among patients. Although surgery (including hepatectomy, liver transplantation, and autologous liver transplantation) is the first choice for the treatment of hepatic alveolar echinococcosis in clinic, drug treatment still plays an important and irreplaceable role in patients with end-stage echinococcosis, including patients with multiple organ metastasis, patients with inferior vena cava invasion, or patients with surgical contraindications, etc. However, Albendazole is the only recommended clinical drug which could exhibit a parasitostatic rather than a parasitocidal effect. Novel drugs are needed but few investment was made in the field because the rarity of the cases. Drug repurposing might be a solution. In this review, FDA-approved drugs that have a potential curative effect on hepatic alveolar echinococcosis in animal models are summarized. Further, nano drug delivery systems boosting the therapeutic effect on hepatic alveolar echinococcosis are also reviewed. Taken together, these might contribute to the development of novel strategy for advanced hepatic alveolar echinococcosis.

Dual inhibition of the Echinococcus multilocularis energy metabolism.

Alveolar echinococcosis is caused by the metacestode stage of the zoonotic parasite Echinococcus multilocularis. Current chemotherapeutic treatment options rely on benzimidazoles, which have limited curative capabilities and can cause severe side effects. Thus, novel treatment options are urgently needed. In search for novel targetable pathways we focused on the mitochondrial energy metabolism of E. multilocularis. The parasite relies hereby on two pathways: The classical oxidative phosphorylation including the electron transfer chain (ETC), and the anaerobic malate dismutation (MD). We screened 13 endochin-like quinolones (ELQs) in vitro for their activities against two isolates of E. multilocularis metacestodes and isolated germinal layer cells by the phosphoglucose isomerase (PGI) assay and the CellTiter Glo assay. For the five most active ELQs (ELQ-121, ELQ-136, ELQ-271, ELQ-400, and ELQ-437), EC50 values against metacestodes were assessed by PGI assay, and IC50 values against mammalian cells were measured by Alamar Blue assay. Further, the gene sequence of the proposed target, the mitochondrial cytochrome b, was analyzed. This allowed for a limited structure activity relationship study of ELQs against E. multilocularis, including analyses of the inhibition of the two functional sites of the cytochrome b. By applying the Seahorse XFp Extracellular Flux Analyzer, oxygen consumption assays showed that ELQ-400 inhibits the E. multilocularis cytochrome bc1 complex under normoxic conditions. When tested under anaerobic conditions, ELQ-400 was hardly active against E. multilocularis metacestodes. These results were confirmed by transmission electron microscopy. ELQ-400 treatment increased levels of parasite-released succinate, the final electron acceptor of the MD. This suggests that the parasite switched to MD for energy generation. Therefore, MD was inhibited with quinazoline, which did not induce damage to metacestodes under anaerobic conditions. However, it reduced the production of succinate compared to control treated parasites (i.e., inhibited the MD). The combination treatment with quinazoline strongly improved the activity of the bc1 inhibitor ELQ-400 against E. multilocularis metacestodes under anaerobic conditions. We conclude that simultaneous targeting of the ETC and the MD of E. multilocularis is a possible novel treatment approach for alveolar echinococcosis, and possibly also other foodborne diseases inflicted by platyhelminths, which cause substantial economic losses in livestock industry.

EmCyclinD-EmCDK4/6 complex is involved in the host EGF-mediated proliferation of Echinococcus multilocularis germinative cells via the EGFR-ERK pathway

The larval stage of the tapeworm Echinococcus multilocularis causes alveolar echinococcosis (AE), one of the most lethal helminthic infections in humans. The tumor-like growth and development of the metacestode larvae within host organs are driven by a population of somatic stem cells, the germinative cells, which represent the only proliferative cells in the parasite. Host-derived factors have been shown to promote germinative cell proliferation. Since cells sense the external signal mainly in G1 phase of the cell cycle, host factors are expected to exert impacts on the machinery regulating G1/S phase of the germinative cells, which still remains largely unknown in E. multilocularis. In this study, we described the characterization of two key members of the G1/S phase cell-cycle regulation, EmCyclinD and EmCDK4/6. Our data show that EmCyclinD and EmCDK4/6 display significant sequence similarity to their respective mammalian homologs, and that EmCyclinD interacts with EmCDK4/6, forming a kinase-active complex to activate its substrate Rb1. EmCyclinD was actively expressed in the germinative cells. Addition of human EGF caused an elevated expression of EmCyclinD while inhibition of the EGFR-ERK signaling pathway in the parasite reduced the expression of EmCyclinD and downstream transcriptional factors. Treatment with Palbociclib, a specific CDK4/6 inhibitor, downregulated the expression of cell cycle-related factors and impeded germinative cell proliferation and vesicle formation from protoscoleces. Our data demonstrated that the EmCyclinD-EmCDK4/6 complex participates in the cell cycle regulation of germinative cells which is mediated by host EGF via the EGFR-ERK-EmCyclinD pathway in E. multilocularis.

High variability in the number of E. multilocularis eggs in cat feces collected in the field.

Echinococcus multilocularis is the causative agent of alveolar echinococcosis that is considered as the most severe parasitic disease in Europe. The contribution of cat to environmental contamination by E. multilocularis is generally considered as extremely low based on results of experimental infections and worm burden estimations from natural infections. However, the recent collection of numerous cat feces from kitchen gardens in high endemic areas and the detection of E. multilocularis DNA in a significant number of these feces raise the question of the risk of human transmission from cats. This study aimed to provide a quantitative estimation of E. multilocularis eggs in feces from naturally infected cats. A field sampling conducted in 192 kitchen gardens during a joint study led to the collection and analysis of 597 cat feces, among them 7 (1.2%) yielded positive results for E. multilocularis real-time PCR. The entire pellets obtained after homogenization, filtration and centrifugation of a 5g-sample for each of these 7 feces were examined under a stereoscopic microscope. After assessing their number, 20 taeniid eggs were individually isolated and specifically identified by real-time PCR. Morphologically mature E. multilocularis eggs were identified in 4 samples and the counting of 4 to 43 E. multilocularis eggs per gram in these samples, i.e. 62 to 2331 eggs per feces when the total mass of the feces is considered. The number of eggs counted in 2 feces suggests a biotic potential of some naturally infected cats that largely exceed the previous experimental estimations.

Dog Ownership and Risk for Alveolar Echinococcosis, Germany.

Human alveolar echinococcosis is caused by the parasite Echinococcus multilocularis, and dog ownership has been identified as a risk factor. We sought to specify the factors of dog ownership underlying this risk by conducting a case–control study among dog owners in Germany. The analysis revealed an increased odds ratio of ≈7-fold for dog owners whose dogs roam unattended in fields, 13-fold for dog owners who feed their dogs organic waste daily, 4-fold for dog owners who take their dog to a veterinarian only in case of illness, and 10-fold for dog owners who have never been informed by a veterinarian about the risk for infection. The results highlight the risk for infection associated with various factors of dog ownership and the value of veterinarians informing owners about prevention.

Effects of Echinococcus multilocularis metacestodes infection and drug treatment on the activities of biotransformation enzymes in mouse liver.

The changes of biotransformation enzymes will substantially affect the host's ability to metabolize drugs and other xenobiotic compounds. In order to further elucidate this process and promote the development in treatment of echinococcosis, we investigated the effects of Echinococcus multilocularis infection and drug treatment on biotransformation enzymes in mouse liver. In microsomal and cytosolic fractions, from the six activities assayed, significant decrease of glutathione S-transferases (GST) activity and significant increase of 7-pentoxyresorufin (PROD) and NADPH-cytochrome P450 reductase (CPR) activity were observed in the mice infected withE. multilocularismetacestodes. In addition, after six weeks treatment of albendazole inE. multilocularisinfected mice, significant decreased GST activity and significant increase of 7- ethoxyresorufin (EROD), PROD, and particularly 3-fold higher 7-methoxyresorufin (MROD) activity were observed. The 3-bromopyruvate treated mice only exhibited significantly lower GST activity. Our results demonstrate thatE. multilocularismetacestodes infection can affect the activities of main hepatic biotransformation enzymes and such alterations of activity may further affect the hepatic biotransformation of xenobiotics. Moreover, albendazole and 3-bromopyruvate, the promising potential drug againstEchinococcus, affected different hepatic biotransformation enzymes and may affect their metabolism. The findings will help to develop rational treatments with less side effects and promote the development of more efficient treatments againstE. multilocularis.

Anthelmintic Baiting of Foxes against Echinococcus multilocularis in Small Public Area, Japan

We distributed anthelmintic baits on a university campus in Japan inhabited by foxes infected with Echinococcus multilocularis to design an effective baiting protocol for small public areas. High-density baiting can reduce the risk for human exposure to the parasite to near zero. However, monthly baiting is recommended to maintain this effect.

Echinococcus multilocularis protoscoleces enhance glycolysis to promote M2 Macrophages through PI3K/Akt/mTOR Signaling Pathway

ABSTRACT Alveolar Echinococcosis (AE) is a zoonotic parasitic disease caused by Echinococcus multilocularis, but its pathogenesis remains unclear. The primary objective of this study is to explore whether Echinococcus multilocularis protoscoleces (PSCs) regulate macrophage polarization and glucose metabolism by PI3K/Akt/mTOR signaling pathway. We found that large numbers of CD68+ macrophages gathered in close liver issue from the lesion in AE patients. PSCs preferentially differentiated into M2 macrophages and the expressions of HK1, PFKL, PKM2, PI3K, Akt, p-Akt, mTOR and p-mTOR increased. The above results show that Echinococcus multilocularis protoscoleces enhance glycolysis to promote M2 macrophages through PI3K/Akt/mTOR signaling pathway.

HIV protease inhibitor nelfinavir is a potent drug candidate against echinococcosis by targeting Ddi1-like protein.

SummaryBackgroundAlveolar echinococcosis (AE), which is caused by larval Echinococcus multilocularis, is one of the world's most dangerous neglected diseases. Currently, no fully effective treatments are available to cure this disease.MethodsIn vitro protoscolicidal assay along with in vivo murine models was applied in repurposing drugs against AE. Genome-wide identification and homology-based modeling were used for predicting drug targets. RNAi, enzyme assay, and RNA-Seq analyses were utilized for investigating the roles in parasite survival and validations for the drug target.FindingsWe identified nelfinavir as the most effective HIV protease inhibitor against larval E. multilocularis. Once-daily oral administration of nelfinavir for 28 days resulted in a remarkable reduction in parasite infection in either immune-competent or immunocompromised mice. E. multilocularis DNA damage-inducible 1 protein (EmuDdi1) is predicted as a target candidate for nelfinavir. We proved that EmuDdi1 is essential for parasite survival and protein excretion and acts as a functionally active protease for this helminth. We found nelfinavir is able to inhibit the proteolytic activity of recombinant EmuDdi1 and block the EmuDdi1-related pathways for protein export. With other evidence of drug efficacy comparison, our results suggest that inhibition of EmuDdi1 is a mechanism by which this HIV proteinase inhibitor mediates its antiparasitic action on echinococcosis.InterpretationThis study demonstrates that nelfinavir is a promising candidate for treating echinococcosis. This drug repurposing study proves that the widely prescribed drug for AIDS treatment is potent in combating E. multilocularis infection and thus provides valuable insights into the development of single-drug therapy for highly prevalent co-infection between HIV and helminth diseases.FundingThis work was supported by the National Natural Science Foundation of China (31802179), the Natural Science Foundation of Gansu Province, China (No. 21JR7RA027), and the State Key Laboratory of Veterinary Etiological Biology (No. SKLVEB2021YQRC01).

Puerperal Septic Fever and Helminthosis in a 5-Year-Old German Shepherd Bitch

Puerperium is a period post-partum characterized by physiological changes whereby the upper and lower reproductive tracts of the female genital system progressively tries to histologically and endocrinologically return to its pre-gravid state and the period is accompanied by adequate uterine tone for lochia elimination and epithelial regeneration (Voorwald and Tiosso, 2015). Normally, during puerperium the bitch exhibit initially greenish discharge (due to marginal haematoma), then serosanguinous vaginal discharge for 1–6 weeks being the time considered, as the time of normal uterine involution (Feldman and Nelson, 1986). In the first three weeks brownish, non-odorous lochia will be discharged from the vulva, gradually decreasing in volume after the first week (Von Heimendahl et al., 2009). In bitches, the normal uterine involution requires more and adequate time compared to other species due to its nature of the endothelial placentation and deep uterine wounds created at placentation sites after detachment (Al-Bassam et al., 1981). The duration of the puerperium/periparturient period in the bitch has been defined to be 12 weeks (Al-Bassam et al., 1981, Orfanou et al., 2009) or 15 weeks (Yeager et al., 1990). The Bitch, being a monoestrous, polytococcus, non-seasonal animal has a physiological anestrous stage that lasts between 15 to 30 weeks following either parturition (whelping) or diestrous which is characterized by normal quiescence of the gonads and the tubular reproductive tracts. The peripaturient period that occurs during the anaestrous stage of the reproductive/oestrous cycle of the bitch is very delicate and animals are usually susceptible to varying pathological conditions owing to stress and increased demands from the environment and the newborns. For this reason, follow-up of uterine involution is essential to ensure future reproductive capacity/fertility, since failure to complete reproductive organs regression can lead to endometritis, metritis and placental retention (Hirt et al., 2000; Magata et al., 2013). Many assumptions were raised due to lots of biological activities such as nursing of the puppies, uterine involution, milk production and hormonal changes that take place during postpartum period. Thus, bitches are supposed to be more susceptible to pathologic conditions and therefore, it is important to monitor vital functions, appetite and behaviour of postpartum bitches on a daily basis to enable early detection of illness (Grundy and Davidson, 2004). Increase demand in lactation/milk production may predispose the bitch to hypocalcaemia (puerperal tetany) during this period and also a few days post whelping the cervix is physiologically open, paving ways for ascending uterine infection by pyogenic microbes which may lead to septicemia. Thus, a good health surveillance by both the owners and veterinarians is of paramount significance.Infestation of the gastro-intestinal tract and other organs of the animal body by both the adult and immature stages of endo-parasitic helminths is well documented in dogs, especially in newly whelped or neonates and it constitute one of the main causes of pathologies of the intestinal tract in dogs (Blagburn et al., 1996). The life cycle of the many occurring canine helminth species usually involved a vertical transmission from the dam to her offspring. The route of vertical transmission can be: prenatal (e.g Toxocara canis), lactogenic (e.g Ancylostoma caninum, Strongyloides stercoralis) or via coprophagy (Echinococcus multilocularis, Filaroides hirthi, Oslems osleri). Hypobiosis or developmental arrest allows for certain and specific life-cycle stages of the helminthic parasites (mostly L3) to be temporary in-activated in the somatic tissues of the host until the condition for survival is conducive. Such occurs mostly during pregnancy or when the immunity of the host is severely compromised (Shoop, 1999). The L3 re-activates during gestation and is transmitted to the offspring or neonates via milk during puerperal period, the parasites then infest the intestinal tract of the neonates and cause pathologies such as protein loss, diarrhea, obstruction of lumen, iron-deficiency anaemia (especially hook worms) and mortality in unattended condition (Cromptom, 1989).

Prediction and identification of epitopes in the Echinococcus multilocularis thrombospondin 3 antigen.

BACKGROUND: Alveolar echinococcosis is an epidemic disease caused by the parasitism of Echinococcus multilocularis (Em) larvae in the intermediate or final host.OBJECTIVE: To identify and analyze B-cell and T-cell (Th1, Th2, and Th17) epitopes of the Em antigen protein thrombospondin 3 (TSP3).METHODS: The amino acid sequence of TSP3 was obtained, and the secondary structural characteristics of TSP3 were predicted using bioinformatics software to further predict its potential T-cell and B-cell epitopes. The spleen lymphocytes of BALB/c mice, which were immunized with the TSP3 protein, were collected for co-culture with B-cell and T-cell antigen small peptides. The B-cell epitopes and T-cell epitope subtypes Th1, Th2, and Th17 were identified as having good immunogenicity.RESULTS: After identification, it was found that the predominant epitopes of B cells existing in TSP3 were T18-33, T45-55, and T110-122. Furthermore, the predominant epitopes of T cells existing in TSP3 were T33-42, T45-55, T80-90, and T110-122 in the T1 subtype, T45-55, T68-77, and T92-104 in the Th2 subtype, and T53-63 and T80-90 in the Th17 subtype.CONCLUSIONS: Six T-cell and eight B-cell dominant epitopes of the TSP3 antigen were revealed; these results may be applied in the development of a dominant epitope vaccine.

Global analysis of neuropeptides in cestodes identifies Attachin, a SIFamide homolog, as a stimulant of parasite motility and attachment.

Many anthelmintics target the neuromuscular system, in particular by interfering with signaling mediated by classical neurotransmitters. Although peptidergic signaling has been proposed as a novel target for anthelmintics, current knowledge of the neuropeptide complement of many helminth groups is still limited, especially for parasitic flatworms (cestodes, trematodes, and monogeneans). In this work, we have characterized the neuropeptide complement of the model cestode Hymenolepis microstoma. Peptidomic characterization of adults of H. microstoma validated many of the neuropeptide precursor (npp) genes previously predicted in silico, and identified novel neuropeptides that are conserved in parasitic flatworms. Most neuropeptides from parasitic flatworms lack significant similarity to those from other animals, confirming the uniqueness of their peptidergic signaling. Analysis of gene expression of ten npp genes by in situ hybridization confirmed that all of them are expressed in the nervous system and identified cryptic features, including the first evidence of dorsoventral asymmetry, as well as a new population of peripheral peptidergic cells that appears to be conserved in the trematode Schistosoma mansoni. Finally, we characterized in greater detail Attachin, an SIFamide homolog. Although its expression is largely restricted to the longitudinal nerve cords and cerebral commissure in H. microstoma, it shows widespread localization in the larval nervous system of Echinococcus multilocularis and Mesocestoides corti. Exogenous addition of a peptide corresponding to the highly conserved C-terminus of Attachin stimulated motility and attachment of M. corti larvae. Altogether, this work provides a robust experimental foothold for the characterization of peptidergic signaling in parasitic flatworms. Cover Image for this issue:https://doi.org/10.1111/jnc.15418.

Alveolar echinococcosis in nutria (Myocastor coypus), invasive species in Slovenia

The present report describes a case of Echinococcus multilocularis infection in nutria (Myocastor coypus) culled in the central area of Slovenia. Post-mortem exam showed multiple cystic lesions in the liver. Gross examination, as well as parasitological and histopathological examinations, revealed numerous cysts of various sizes, filled with yellow clear fluid and displacing most of the liver parenchyma. The cyst lumina contained numerous protoscolices approximately 100 μm in diameter and calcareous corpuscles. The protoscolices had two visible suckers and a rostellum with birefringent hooks. The lesions were consistent with an E. multilocularis cyst. Molecular analysis confirmed that the nutria was infected with E. multilocularis. To our knowledge, this is the first report of echinococcosis in nutria in Slovenia that presents gross, parasitological, and histological lesions and the result of molecular analysis. Nutrias in Slovenia are dangerous invaders but can also be a relevant bioindicator of the presence of the parasite in the environment.

Tim-3/Galectin-9 signaling pathway is involved in the cytokine changes in mice with alveolar echinococcosis.

Tim-3/Galectin-9 is involved in the immune escape of many pathogens. However, the role of Tim-3/Galectin-9 in persistent infection of Echinococcus multilocularis (Em), which is related to immune escape, is still unclear. To investigate the role of Tim-3/Galectin-9 and related cytokines in mice with persistent infection of Em. Em infection model was established by injecting the protoscoleces. Serum was collected at days 2, 8, 30, 60, 90, 180 and 270 after infection. Lymphocytes were isolated from liver tissue samples with Ficoll. Tim-3 + CD4 + T percentage was analyzed by flow cytometry. CD4 + T cells were isolated from liver tissues of Em infected mice and cultured in vitro. The mRNA levels of Tim-3, Galectin-9, IFN-γ and IL-4 were detected by qRT-PCR. Cytokine levels in serum and culture supernatant (IFN-γ and IL-4) were analyzed by cytometric bead array. The expression of Tim-3 and Galectin-9 mRNA significantly increased after 30 days of infection, reached peak on day 90, and then decreased slightly on days 180-270. The expression of IFN-γ mRNA, increased on day 2 and 8 after infection, slightly decreased on days 30-60, and obvious decreased on days 90-270, but were still higher than those of the control group. The expression of IL-4 mRNA gradually increased along with the time of infection. In serum of Em infected mice, level of IFN-γ peaked at day 30 and then gradually decreased; whereas IL-4 level peaked at day 90 and then gradually decreased. In vitro experiment found that Tim-3/Galectin-9 directly caused the changes in the levels of IFN-γ and IL-4. Tim-3/Galectin-9 signaling pathway may be involved in the development of persistent infection of Em by regulating the production of Th1 and Th2 cytokines.

4D-Dynamic Representation of DNA/RNA Sequences: Studies on Genetic Diversity of Echinococcus multilocularis in Red Foxes in Poland.

The 4D-Dynamic Representation of DNA/RNA Sequences, an alignment-free bioinformatics method recently developed by us, has been used to study the genetic diversity of Echinococcus multilocularis in red foxes in Poland. Sequences of three mitochondrial genes, i.e., NADH dehydrogenase subunit 2 (nad2), cytochrome b (cob), and cytochrome c oxidase subunit 1 (cox1), are analyzed. The sequences are represented by sets of material points in a 4D space, i.e., 4D-dynamic graphs. As a visualization of the sequences, projections of the graphs into 3D space are shown. The differences between 3D graphs corresponding to European, Asian, and American haplotypes are small. Numerical characteristics (sequence descriptors) applied in the studies can recognize the differences. The concept of creating descriptors of 4D-dynamic graphs has been borrowed from classical dynamics; these are coordinates of the centers or mass and moments of inertia of 4D-dynamic graphs. Based on these descriptors, classification maps are constructed. The concentrations of points in the maps indicate one Polish haplotype (EmPL9) of Asian origin.

Alveolar echinococcosis in a dog in Missouri, USA.

A 10-y-old, castrated male Boxer dog that was born and had lived in Missouri without any travel history to other states, except for a few trips to Kansas, was presented with a distended abdomen and declined health. Ultrasonographic examination revealed a large hepatic mass, and the dog was euthanized. A postmortem examination revealed that the left liver lobes were largely replaced by a white-to-tan multilobular mass with a cobblestone surface. The lesion also involved the diaphragm. Histologically, hepatic architecture was effaced by large areas of necrosis with numerous, ≤0.2-cm, cystic structures that stained positively with periodic acid-Schiff stain and contained calcareous corpuscles. Gross and microscopic hepatic lesions were compatible with alveolar echinococcosis (AE) caused by Echinococcus multilocularis. PCR examination confirmed E. multilocularis, and results from genotyping were consistent with the E4 haplotype. To our knowledge, this is only the second canine AE case and the third pet dog that has been confirmed to be infected by E. multilocularis in the contiguous United States. E. multilocularis is a serious health risk for both pet dogs and humans.

Production and Evaluation of a Novel Multi-Epitope Bivalent Vaccine Against Echinococcus multilocaularis Metacestode

Alveolar Echinococcosis is a globally widespread zoonotic disease caused by the larval stage of Echinococcus multilocularis (Em) and is seriously harmful to human health. In our previous studies, we found that Em-EMY162 had good prophylactic and therapeutic effects against Em infection and the dominant epitopes of Em-EMY162 and Em-TSP3 were also identified. In this study, a multi-epitope vaccine LTB-ETBM targeting both Em-EMY162 and Em-TSP3 was designed and constructed, and then the immunogenicity and protective efficacy were evaluated in Em infected mice. The results showed that LTB-ETBM could induce high levels of specific IgG against Em-EMY162 and Em-TSP3 and a mixed Th1/Th2 lymphocyte response to LTB-ETBM. Moreover, the LTB-ETBM significantly inhibited the formation of cysts in mice challenged with 1000 Em protoscoleces. In a therapeutic mouse model injected intraperitoneally with 1000 protoscoleces, vaccination with LTB-ETBM using either Freund's or CpG as an adjuvant significantly decreased the growth of protoscoleces and the formation of cysts. LTB-ETBM may be efficacious for use as a prophylactic or therapeutic agent against Em infection.

Characterization of Three cDNAs Obtained by Spliced Leader-PCR Screening of a Taenia solium cDNA Library

Cysticerci or metacestodes of Taenia solium cause cysticercosis, being the neurocysticercosis (NCC) the main pathology. The characterization of genes is essential for the knowledge of parasite biology, the understanding of the parasite-host relationship, and the identification of possible targets for diagnosis, treatment, and protection. The objective of this work was the molecular and bioinformatic characterization of three news cDNAs (TsTF10, TsAAP8, and TsrGAP8), obtained by spliced leader-PCR screening of a Taenia solium cDNA library. The sequences of the three cDNA were compared with the sequences in the nucleic acid and protein databases (GenBank, EMBL) and analyzed by bioinformatic programs (CDD-Search of the National Center for Biotechnology Information, Interpro of the European Bioinformatics Institute, Motif scan and Expert Protein Analysis System of the Proteomics Server from Swiss Institute of Bioinformatics). Considering the high identities with similar molecules of related helminths (Taenia asiatica, Echinococcus granulosus, Echinococcus multilocularis, and Hymenolepis diminuta) and the functional domains found, the TsTF10, TsAAP8, and TsrGAP8 genes of Taenia could act as a nuclear transcription factor gamma, a putative vacuolar ATPase membrane sector associated protein and a Rho GTPase activating protein, respectively. Although few B epitopes could be predicted in the sequences, it could be relevant to evaluate them as possible candidates for diagnosis and protection in cysticercosis. The characterization and analysis of these sequences and the prediction of their possible usefulness as antigens, vaccines, or therapeutic targets contribute to the designing and planning of future studies.

Genetic diversity of Echinococcus spp. in wild carnivorous animals in Kazakhstan.

Background and Aim:The study of Echinococcus infection among farm animals in Kazakhstan was carried out to monitor the invasion among livestock and map the data obtained. Unfortunately, there are only partial data on the study of echinococcosis among wild carnivores in Kazakhstan, which makes it difficult to conduct a comparative analysis of the epidemiological situation among wild animals. The present study aimed to estimate the genetic diversity of Echinococcus spp. (Leuckart, 1863) in Kazakhstan based on sequence analysis of cytochrome c oxidase subunit 1 (cox1) and dehydrogenase subunit 1 (nad1) of worms isolated from wild carnivorous animals wolf (Canis lupus), red fox (Vulpes vulpes) and corsac (Vulpes corsac).Materials and Methods:DNA from parasite tissue was used as a template for the amplification of the two mitochondrial genes cox1 and nad1. Sequencing was performed according to the manual for the Seq Studio Genetic Analyzer. The multiple alignments of obtained sequences were performed using the ClustalW algorithm in Mega (v.11) software. Alignments were exported as a Nexus extension and used as input for TCS v1.21 for the identification of haplotypes. The phylogenetic analysis was constructed according to the neighbor-joining method using Mega (v.11) software.Results:Analysis of the extensiveness of echinococcosis invasion showed that 6.3% were wolves, 18.2% were corsacs, and 85% were foxes. In total, 159 adults of Echinococcus spp. from the three species of animals in different parts of Kazakhstan were analyzed, and 17 individual biological samples were successfully sequenced. Sequence analysis of cox1 and nad1 genes revealed two types of echinococcosis – Echinococcus granulosus in red foxes and wolves, and Echinococcus multilocularis in corsacs. Sequencing of a portion of the mitochondrial genome made it possible to determine seven haplotypes of the pathogen in the studied samples of E. granulosus. Molecular analysis of cox1 and nad1 genes of E. multilocularis revealed three new haplotypes, which have significant variability compared with other studied Asian haplotypes.Conclusion:This study made it possible to fill the gaps in understanding the localization of the foci of the spread of the echinococcosis pathogen among the main wild carnivores and to determine the species reservoir of the pathogen in the greater territory of Kazakhstan.