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Alveolar echinococcosis caused by Echinococcus multilocularis is an important zoonotic disease, a great threat to human health due to limited interventions. microRNAs are a type of small non-coding RNA that plays a key role in many diseases and is considered as a potential therapeutic target for control of parasitic diseases. However, naked miRNAs are difficult to enter into cells and are easily degraded in both external and internal environments. Chitosan (CS) has recently been used as a promising vehicle for delivery of nucleic acids. Therefore, we prepared miRNA-bearing CS nanoparticles and investigated the physicochemical properties as well as the delivery efficiency. We found that CS nanoparticles was relatively stable, offered miRNA strong protection from degradation and had low cytotoxicity with no significant effects on cell proliferation and apoptosis. CS nanoparticles were shown to be easily absorbed by cells and have remarkable liver tropism. Furthermore, CS nanoparticles were used to efficiently deliver E. multilocularis miR-4989 in vitro and in vivo and caused a significant reduction in the expression of UBE2N in the liver, a potential target of emu-miR-4989, at both mRNA and protein levels. Our data demonstrate that CS nanoparticles can act as a vehicle for efficient liver-targeted delivery of miRNAs and for development of miRNA-based therapeutics against E. multilocularis infection.

Cystic echinococcosis (CE) is of increasing public health and socio-economic concern because of the large morbidity rates and produced high economic losses in the livestock industry. The objective of the current research was to study the reliability of indirect ELISA in detecting CE, based on two dif-ferent types of crude antigens of camel origin; protoscolex and germinal layer antigens from hydatid cyst. Blood samples were collected from 284 (125 slaughtered and 159 live camels). Out of 125 slaughtered camels examined visually, 55 (44%) were found to have hydatid cysts. Of them, 52/125 (41.6%) and 3/125 (2.4%) harboured hydatid cysts in lungs and livers respectively. Fertile lung cysts were 32.8%; 26.9% were sterile, while 40.3% of lung and liver cysts were calcified. The sensitivity of ELISA was 83% and 46.5% when protoscolex and germinal layer antigens were used, respectively. The respective specificity of antigens of protoscolex and germinal layer was 70.3% and 41.7%. The protoscolex antigen showed higher accuracy (73.6%) compared to the germinal layer antigen (52.8%). The cross reactivity of these antigens were evaluated with antigens and hyperimmune sera of CE and Fasciola spp. and Haemonchus contortus using ELISA. The results showed also weak immunogenic potency of each antigen with Fasciola spp. hyperimmune sera at dilution 1:50 while hyperimmune sera of Haemonchus contortus did not bind any antigen.

The current study was carried out to assess in vitro and in vivo effects of Moringa oleifera seed methanolic extract on Fasciola hepatica to develop an alternative source of treatment. The in vitro ovicidal effect of M. oleifera seed extract on immature F. hepatica eggs has provided evidence of inhibitory activity on the vitality and hatchability of F. hepatica eggs. This inhibitory activity was concentration-dependent and also correlated strongly with the exposure time. In the in vivo trial, the oral administration of F. hepatica experimentally infected rabbits with doses of 150 mg/kg BW prepared extract per day for 3 consecutive days on the 63rd day post infection confirmed potent fasciolicide activity of the extract. A gradual decrease in fecal egg count (FEC) was detected from the 1st day post treatment until reaching 100% FEC reduction by the 7th day post treatment. No flukes could be found at post mortem examinations. Significant increments of serum total protein, globulin, the activities of ALT and AST, total cholesterol, triglycerides and urea were recorded during the period of infection, which were improved by treatment. Remarkable histopathological alterations were observed in the infected liver and gallbladder tissues which decreased clearly in the treated rabbits. This study proposes that the used extract has promising and potent fasciolicide activity.

Haemonchus contortus is one of the most pathogenic and economically important parasites of sheep. Different H. contortus antigens; crude somatic antigen (CSA), excretory/secretory antigen (ESA), crude larval antigen (CLA), glutathione-S-transferase antigen (GST) and recombinant protein (rhcp 26/23) were prepared and characterized using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot. The antigens were immunologically evaluated through indirect enzyme linked immunosorbent assay (ELISA) for diagnosis of haemonchosis in experimentally and naturally infected sheep. Analysis of the resultant bands of SDS-PAGE demonstrated that 13, 6, 11, 2 and 1 protein bands from CSA, ESA, CLA, GST and rhcp 26/23, respectively and analysis of the resultant bands of western blot showed that 13, 6, 4 and 1 reactive bands detected from CSA, ESA, CLA and GST, respectively. The results of ELISA of different antigens revealed that sero-prevelance of CSA, ESA, CLA, GST and rhcp 26/23 were 78.51, 82.34, 85.319, 45.319 and 90.8% respectively, sensitivity were 100, 90, 100, 96.66 and 90%, respectively and specificity were 0, 70, 10, 70 and 6.66%, respectively with diagnostic potency were 50, 80, 55, 83.33 and 48.33%, respectively. Statistical analysis using Chi square test found that GST is the best one that can be used. The cross reactivity of GST antigen, crude Fasciola antigen and crude Moniezia antigen tested versus their homologous hyper immune sera at different dilutions using ELISA. The current study reported that GST antigen could be considered as a promising antigen for diagnosis of haemonchosis.

Additional file 4: Figure S3. Secondary structure prediction of B. caballi SBP4 ( http://minnou.cchmc.org/ ).

Additional file 3: Figure S2. a Sequence alignment among the deduced SBP-4 amino acid sequences from Egypt and USA B. caballi isolates (1, 2) and SBP4 from Babesia bovis (3). Consensus sequences are shown in the bottom row. bIn silico prediction of trans-membrane domains and signal peptides in Egyptian B. caballi SBP-4 using TMHMM2.

Additional file 5: Figure S4. a Anti-histidin antibodies recognize a 37 kDa antigen in a cell lysate of recombinant E. coli transformed with TOPO® TA plasmid containing the full-length Bcsbp4 gene. Lane 1: (+ ve) cultures induced with L-Arabinose; Lane 2: (- ve) non-Arabinose induced cultures. b Specific recognition of rBcSBP4 protein by B. caballi positive serum in ELISA (a 1:100 dilution of serum was serially diluted by two-fold) using: serum from an equid derived from a horse experimentally infected with B. caballi (blue columns); field horse positive for B. caballi infection (orange columns); and horse that tested negative to infection with B. caballi (grey columns).

Additional file 2: Figure S1. Features of the BcSBP4 protein: the BcSBP4 was predicted to be secreted. The location of the predicted signal peptide and the regions containing the two peptides selected for antibody production are indicated by a green rectangle. The predictions were performed using online software at http://wlab.ethz.ch/protter .

Additional file 6: Figure S5. Specificity of the anti BcSBP4 peptide rabbit antibodies: western blot analysis against (i) normal horse RBC lysate, (ii) B. caballi-infected RBC lysate, (iii) recombinant B. caballi Spb4 and (iv) recombinant B. bovis Hap2 using serum indicated above. Abbreviation: M, size markers.

Additional file 1: Dataset S1. Amino acid sequence of BcSPB4.

Alveolar echinococcosis caused by Echinococcus multilocularis is an important zoonotic disease. In the infected mice, emu-miR-4989-3p is present in sera, but its role remains unknown. Using high-throughput sequencing and qPCR, emu-miR-4989-3p was herein confirmed to be encapsulated into E. multilocularis extracellular vesicles. In the transfected macrophages, emu-miR-4989-3p was demonstrated to significantly inhibit NO production compared to the control (p < 0.05). Moreover, transfection of emu-miR-4989-3p also gave rise to the increased expression of TNF-α (p < 0.01). Furthermore, emu-miR-4989-3p induced the dysregulation of several key components in the LPS/TLR4 signaling pathway compared with the control, especially TLR4 and NF-κB that both were upregulated. Conversely, the NO production and the expression of TNF-α, TLR4 and NF-κB tended to be increased and decreased in the mimics-transfected cells upon emu-miR-4989-3p low expression, respectively. These results suggest that emu-miR-4989-3p is one of ‘virulence’ factors encapsulated into the extracellular vesicles, potentially playing a role in the pathogenesis of E. multilocularis.

The aims of this study were an establishment of the domestic rabbit as an intermediate host for cystic echinococcosis (CE) and to evaluate the potency of the crude germinal layer and the protoscoleces antigens to protect against the CE. Firstly; Two groups of white Newzeland rabbits were infected orally either by 5000 active oncospheres or viable protoscoleces separately. After 20 weeks, the slaughtered rabbits showed the presence of hydatid cysts at different internal organs. Molecular detection of the resulted cysts was conducted. Secondly; 27 rabbits were divided into nine groups (n = 3). Groups 1 and 2 were immunized with the crude germinal layer antigen while the groups 3 and 4 were immunized with the crude protoscoleces antigen. Groups 5 and 6 received the adjuvant mineral oil. Groups 7 and 8 were used as positive control. The last 9 group was kept as a negative control. The obtained results showed a significant high protection percentage of 83.4% and high antibody titer was recorded in groups that received the crude germinal layer antigen comparing with the groups that immunized with the crude protoscoleces antigen as their protection percentage was 66.7% with lower IgG response. In conclusion, the domestic rabbits could be used as a laboratory model for CE. Developing of the germinal layer antigen is more immunogenic than the protoscoleces one and could be used as a promising vaccine. Attention should be directed towards the existing rabbit in the environment adjacent to infected dogs as it could be a part of Echinococcus life cycle.

Haemonchus contortus (H. contortus) remains important nematode that infecting sheep all over the world. Truthful diagnosis of haemonchosis needs reliable Enzyme linked immune sorbent assay test as well as the immuno-reactive protein profile of different prepared H. contortus antigens; larval (L), excretory secretory product (ESP) and adult somatic H. contortus (AS). The current study fulfilled that L antigen is the talented antigen for such serological diagnosis. Immunodominant band at molecular weight 57 kDa were answerable for highest specificity and accuracy of positive predictive value of this antigen. Moreover, the highest apparent prevalence value was 92 and 75% obtained by L and ESP antigens, respectively. The results of cross reactivity among AS, Monezia expansa (M. expansa) and Fasciola spp. revealed that AS antigen appeared major cross reactivity with other cestode and trematode. Best dilution of serum was (1:800) to rise above this phenomenon.

Extracellular vesicles (EVs) are heterogeneous populations of different membrane-wrapped vesicles in size and encapsulated cargo and have recently emerged as a crucial carrier with the functions in intercellular communication, being involved in host-parasite interactions. However, Echinococcus granulosus EVs are not fully described. To separate EVs with a different size, the culture supernatant of E. granulosus protoscoleces (PSCs) was sequentially centrifuged at 2,000g, 10,000g and 110,000g, and the resulting precipitates were accordingly named as 2K, 10K and 110K EVs, respectively. The size and morphology of three different EVs were identified using ZETASIZER NANO and transmission electron microscopy (TEM), respectively. Then mass spectrometry was applied to define protein cargo of EVs and EV internalization was assessed using fluorescent microscopy and flow cytometry. The results showed that 2K EVs mainly ranged from 450 to 950 nm in diameter, 10K EVs ranged from 220 to 390 nm and 110K EVs from 60 to 150 nm. A total of 901 EV proteins were identified, 328 of which were commonly found in the three types of EVs. GO analysis revealed that these proteins were mainly involved in binding (44%) and catalytic activity (44%). Three types of EVs were different in biomarkers (Enolase and 14-3-3) and in reactivity with anti-echinococcosis positive serum. Moreover, 110K EVs were more easily internalized by hepatic cells than 10K EVs as well as 2K EVs (p < 0.0001). These results reveal the physical and biological discrepancy among 2K, 10K and 110K EVs, suggesting a distinct role in host-parasite interactions.

In this study, the essential oils of camphor and lavender were tested in vitro against the third instar larvae of Lucilia sericata for the first time, following dipping toxicity technique. The toxicity results revealed that L. sericata larvae were susceptible to the applied essential oils. Lavender oil was more effective than camphor in killing of L. sericata larvae. With 32 % concentration, the mortality percentages of larvae were 100 and 93.3 %, respectively. Light and scanning electron microscopic examinations were done to determine the cuticular changes of L. sericata larvae following exposure to the applied essential oils. Larvae showed cuticular swelling and distortion after oil treatment, but its level was greater with lavender oil. The current study suggested that an alternative, effective and natural product can be developed as larvicides against L. sericata using camphor and lavender oils.

Recently a dramatic development of the cancer drug discovery has been shown in the field of targeted cancer therapy. Checkpoint kinase 2 (Chk2) inhibitors offer a promising approach to enhance the effectiveness of cancer chemotherapy. Accordingly, in this study many pyrimidine-benzimidazole conjugates were designed and twelve feasible derivatives were selected to be synthesized to investigate their activity against Chk2 and subjected to study their antitumor activity alone and in combination with the genotoxic anticancer drugs cisplatin and doxorubicin on breast carcinoma, (ER+) cell line (MCF-7). The results indicated that the studied compounds inhibited Chk2 activity with high potency (IC50 = 5.56 nM - 46.20 nM). The studied candidates exhibited remarkable antitumor activity against MCF-7 (IG50 = 6.6 μM - 24.9 μM). Compounds 10a-c, 14 and 15 significantly potentiated the activity of the studied genotoxic drugs, whereas, compounds 9b and 20–23 antagonized their activity. Moreover, the combination of compound 10b with cisplatin revealed the best apoptotic effect as well as combination of compound 10b with doxorubicin led to complete arrest of the cell cycle at S phase where more than 40% of cells are in the S phase with no cells at G2/M. Structure-activity relationship was discussed on the basis of molecular modeling study using Molecular modeling Environment program (MOE).

Infection of Cysticercus tenuicollis, the larval stage of Taenia hydatigena, is extensively found in sheep and pigs and jeopardizes the breeding and meat industry. miRNAs are a subclass of small noncoding regulatory RNAs and closely associated with the pathogenesis and biology of parasites. Here, using HiSeq sequencing we identified 49 known and 2 potential novel miRNAs in C. tenuicollis, of which both thy-miR-71 and -87 were predominant. Using RT-qPCR, 6 selected miRNAs were validated, and thy-miR-71 and -miR-87 were confirmed to be highly expressed, with the copy number of approximately 82,340 ± 2079 and 19,580 ± 609 per 1 ng total RNA, respectively. Similar to other cestodes, T. hydatigena was predicted to have two conserved miRNA clusters thy-miR-71/2c/2b and thy-miR-4989/277, and three members of the former were confirmed to reside sequentially within the genomic region of 253 bp by PCR. The current data provide us a valuable resource for further studies of a role of miRNAs in T. hydatigena biology and infection.

Haemonchus contortusis a major parasite of small ruminants and its blood-feeding behaviour causes effects ranging from mild anaemia to death. Knowledge of the genetic variation within and amongH. contortuspopulations can provide the foundation for understanding transmission patterns and aid in the control of haemonchosis. Adult maleH. contortuswere collected from three geographical regions in Egypt. The second internal transcribed spacer (ITS2) of nuclear ribosomal DNA was amplified using the polymerase chain reaction (PCR) and sequenced directly. The population genetic diversity and sequence variations were determined. Nucleotide sequence analyses revealed one genotype (ITS2) in all worms, without genetic differentiation. The similarity in population genetic diversity and genetic patterns observed among the three geographical regions could be attributed to possible movement between the sites. This is the first study of genetic variation inH. contortusin Egypt. The present results could have implications for the rapid characterization ofH. contortusand other trichostrongyloid nematodes, and evaluation of the epidemiology ofH. contortusin Egypt.

In this study, the effect of the methanolic extract of Balanites aegyptiaca fruits (BAE) on adult Toxocara vitulorum was evaluated after incubating the parasites in Ringer solution containing 10, 30, 60, 120 and 240 μg/ml of the methanolic extract, for 24h using light and scanning electron microscopic observations. Differences in response to BAE action were concentration dependent. These changes occurred in definite sequences in response to BAE concentration and were consisted of slightly swelling which became pronounced and so severe, with lips showed wrinkled cuticular surface and deformed sensory papillae on increasing the BAE concentration. The strongest effects were reached with the highest BAE concentration, where disorganization of the cuticle and body musculature was observed. Additionally, the ovicidal effect of BAE, at the previous concentrations, on the development of T. vitulorum eggs was examined after 12h exposure. The inhibitory activity of BAE on egg development was concentration dependent and the highest value reached to 100% with the concentration of 240 μg/ml. These results were compared with those observed in the worm cuticle and eggs following incubation in albendazole, as it was a broad-spectrum nematodicidal compound with well-known ovicidal activity.

Background Shepherd and stray dogs are thought to represent the primary definitive hosts of Coenurosis by Taenia multiceps, due to their feeding habits which translate into high chances of coming into contact with infected intermediate hosts. Nonetheless, little attention has been paid to the role of the red fox (Vulpes vulpes) in the epidemiology of coenurosis. In fact a knowledge gap exists on the role played by red foxes in the epidemiology of Taenia multiceps and the capability of this parasite to produce fertile and viable eggs in this wild canid, i.e. on the occurrence of a sylvatic cycle. This study investigates the role of the red fox (Vulpes vulpes) in the epidemiology of T. multiceps and related metacestodoses. Methods The small intestine of 63 red foxes was macroscopically examined for the presence of cestodes. Adult parasites were identified morphologically as being T. multiceps. Tapeworm eggs were counted and stored at 4 °C in physiological saline solution prior to experimental infection of four sheep and one goat. Sheep were inoculated orally on Day 0 with 3000 (sheep 1), 5000 (sheep 2 and 3) or 7000 eggs (sheep 4), while the goat was infected with 5000 eggs of T. multiceps. The animals were followed-up regularly by MRI and underwent surgical treatment between days 180 to day 240 post infection. Collected coenuri were identified using morphological and molecular methods. Results A total of 6.3 % of red foxes were found infected with T. multiceps and the eggs obtained from the worms were determined to have a viability of 45.4 %. Two of the challenged sheep and the goat developed disease compatible with T. multiceps. Morphometrical features of the cysts were consistent with those of T. multiceps; nucleotide amplification and sequencing of mitochondrial genes (i.e., cox1 and Nd1) from the metacestode material confirmed the identification. Conclusions The present study is the first to provide evidence of the role of the red fox as a competent definitive host for T. multiceps, thus changing the epidemiological scenarios of infections by this cestode.

Thirty clinically healthy dogs were divided into five equal groups; Gs 1 and 2 were vaccinated with subunit and somatic antigens (Ags) respectively in combination with vitamin E and selenium (Vit E/Se) supplement, Gs 3 and 4 were vaccinated with subunit and somatic Ags respectively and group 5 was kept unvaccinated as control positive. Dogs in the vaccinated-Vit E/Se supplemented groups had a significantly greater (P0.05) serum Se and alpha-tocopherol than un-supplemented Gs. Best immune response against T. hydatigena was observed in the Vit E/Se supplemented groups, as evidenced by increase production of antibody titer and IgG concentration in comparison with either vaccinated un-supplemented or control groups. Moreover, the highest protection level against T. hydatigena infection was observed in groups 1 and 2 (83.3%), while was 66.7% and 50% in groups 3 and 4 respectively. Subunit Ag was more efficient than somatic Ag in improving the immune status of dogs. Vit E and Se proved to be immuno-potentiating to dogs vaccinated with subunit and somatic Ags and increased the possibility for the protection against T. hydatigena infection.

Objective To evaluate the synergetic effect of an ethanolic extract of Egyptian propolis in immunization of BALB/c mice with Taenia saginata ( T. saginata ) crude antigen against bovine cysticercosis, with reference to its effects on liver and kidney functions. Methods Sixty female mice BALB/c strain weighing 20 to 25 g and 6-8 weeks old were randomly allocated into six groups of ten mice each. Mice in groups 1 and 2 (G1 and G2) were immunized intraperitoneally with 100 µg of T. saginata crude antigen in 100 µL phosphate buffer saline emulsified in Freund's adjuvant. Besides, the mice in G2 were administered with propolis extract simultaneously with immunization. Control mice were either administered with propolis extract (G3) or injected with the same volume of phosphate buffer saline emulsified in Freund's adjuvant (G4). The mice in G5 were non-immunized infected control while, those in G6 were non-immunized non-infected control. Two weeks after the last immunization, each mouse was challenged intraperitoneally with 5 000 oncospheres except those of G6. Ethanolic extract of propolis was prepared at a dose 50 mg/kg body weight. Results After 24 weeks of challenge, the mice in G2 showed the highest level of protection (100%), with no cyst being detected rather than mice in G1 (33.3% protection). Additionally, the ELISA results, in this study, showed higher antibody titer in G2 with reduction the alteration in liver and kidney functions compared to G1. Conclusions Egyptian propolis could increase the level of protection against experimental challenge infection with T. saginata eggs when administered simultaneously with immunization. Furthermore, it could enhance the production of antibodies to immunized antigen and decrease the alteration in liver and kidney functions.