Isolation, characterization, and ELISA applications of alkaline phosphatase and acetylcholinesterase from Moniezia expansa

Moniezia expansa worms are a significant source of alkaline phosphatase (ALP) and acetylcholinesterase (AChE) enzymes. The current study presents a simple and reproducible ALP and AChE purification method from Moniezia expansa helminthes by precipitating the proteins with ammonium sulfate and chromatography on the Sephacryl S-300 column. The M. expansa ALP purified at 1070.8 U/mg, displaying 6.0 purification folds and 53.6% yield, while M. expansa AChE is at 5250 U/mg, displaying 2.0 purification folds and 43% yield. The M. expansa ALP isoenzyme displayed its optimum activity at pH 9.6, while the M. expansa AChE isoenzyme displayed its optimum activity at pH 8.0. The affinity of M. expansa ALP for several substrates revealed that p-nitrophenyl phosphate preferentially cleaved with a Km value of 4.4 mM. M. expansa AChE preferentially cleaved acetylthiocholine iodide with a Km value of 0.9 mM. M. expansa ALP is strongly stimulated with Co2+, Mn2+, Ni2+, and Mg2+and reduced with Zn2+, Cu2+, Ca2+, EDTA and DTT. On the other hand, M. expansa AChE is significantly induced with Co2+, Zn2+, and Ni2+and inhibited with Mg2+, Ca2+, EDTA, 1,10-phenanthroline and eserine. The antisera of the purified M. expansa ALP and AChE found effective for determining the two enzymes in different unknown sera from different animal species, including humans, sheep and fish. These results may provide a possible future application of such enzymes in producing ALP and AChE-coated ELISA plates for research purposes

A Novel Designed Sandwich ELISA for the Detection of <i>Echinococcus granulosus</i> Antigen in Camels for Diagnosis of Cystic Echinococcosis

Echinococcus spp. are important cosmopolitan zoonotic parasitic tapeworms that cause a disease called hydatidosis or cystic echinococcosis (CE), which has remarkable economic losses. The objective of our study was to develop a specific IgG polyclonal antigen-based ELISA (Sandwich ELISA; capture ELISA) method for the detection of circulating Echinococcus granulosus (E. granulosus) antigens in camels infected with hydatid cysts before slaughtering and its application in serodiagnosis of CE in animals to assess the positive rate of hydatidosis in camels slaughtered in Giza governorate abattoirs in Egypt. In this study, molecular identification of Echinococcus sp. isolate was performed based on the NADH dehydrogenase subunit 1 (NAD1) gene, revealing the isolate (GenBank: OQ443068.1), which is identical to the G6 E. granulosus sensu lato genotype. The positive rate of hydatid cysts was determined in slaughtered camels’ organs (n = 587). The results revealed that hydatid cysts were found in 46.5% (273/587) of the examined camels. Pulmonary echinococcosis was significantly more prevalent in the slaughtered camels (60%, 164/273) than hepatic echinococcosis (39.9%, 109/273), (p = 0.001, Chi Square = 11.081). Cyst fertility rates were higher in hepatic (90.8%, 99/109) than in pulmonary cysts (83.5%, 137/164) and the most viable protoscoleces were recorded from fertile the hepatic cysts (67.85 ± 12.78). In this study, hydatid cyst germinal layer antigen (GlAg) was isolated and used for the immunization of rabbits to raise IgG polyclonal antibodies (anti-Echinococcus GlAb IgG). These IgG polyclonal antibodies were purified by affinity chromatography using a protein A column, then labeled with horseradish peroxidase. Electrophoretic analysis of IgG polyclonal antibodies and crude GlAg was performed in 10% polyacrylamide gels. The SDS-PAGE revealed four bands at molecular weights of 77 kDa, 65 kDa, 55 kDa, and 25 kDa. The Sandwich ELISA was performed to evaluate the sensitivity and specificity and cross-reactivity of the prepared IgG polyclonal antibodies. The circulating hydatid antigen was found in 270 out of the 273 samples with hydatidosis, with a sensitivity of 98.9% (270/273), a specificity of 94.9% (296/312) and a diagnostic efficacy of 96.8%. Regarding the cross reactivity, anti-Echinococcus GlAb IgG showed a low cross-reactivity with Fasciola gigantica infected camel sera (3/8), and Myiasis (Cephalopina titillator larvae; 3/20). No cross-reactivity was recorded with uninfected camel sera (negative sera for E. granulosus), and no cross-reactivity was found with antigens of Eimeria spp., Toxoplasma gondii, Cryptosporidium sp., and Hyalomma dromedarii (ticks’ infestation). Then, Sandwich ELISA was conducted again to detect E. granulosus antigen in all the collected camel sera, which resulted in a 48.7% (286/587) positive rate of CE compared to 46.5% (273/587) using a postmortem inspection (PM diagnosis) (p = 0.5, Chi Square = 0.302). In conclusion, the Sandwich ELISA technique introduced in this study appears to be a sufficiently sensitive diagnostic assay for the detection of camels’ echinococcosis using anti-Echinococcus GlAb IgG. In addition, it might offer a significant medical and veterinary importance in helping the early detection of hydatidosis, as well as its early treatment

A simple and efficient purification method of native immunoreactive antigen for diagnosis of camel hydatidosis

Background: Cystic echinococcosis (CE), a zoonotic disease that affects animal and human health, is of increasing economic importance due to high morbidity rates and high economic losses in the livestock industry. Aim: The present study was conducted to purify the antigen from hydatid cyst fluid (HCF) with high diagnostic efficacy of camel hydatidosis using indirect enzyme-linked immunosorbent assay (ELISA). Materials and Methods: The HCF antigen was purified using Sephacryl S-300 column chromatography. Characterization of fractions was performed using reducing and non-reducing sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis. Further, antibodies against Echinococcus granulosus cysts in camel serum were detected using indirect ELISA. Results: The purification process resulted in three fractions of antigens: FI, FII, and FIII. Indirect ELISA showed that higher diagnostic efficacy was observed in FI than in FII and FIII. Indirect ELISA, in which FI was utilized, showed 88% sensitivity and 91.7% specificity. Non-reducing SDS-PAGE showed that FI had two bands of molecular weights 120 and 60 kDa. Western blot analysis of FI demonstrated that 60, 38, and 22 kDa were antigenic bands when reacted with naturally infected camel sera with E. granulosus cysts. Using indirect ELISA, F1 recorded an infection percentage of 81.7% in randomly collected camel serum samples. Conclusion: FI is a promising antigen for accurate diagnosis of camel CE using indirect ELISA

A Cryptosporidium parvum vaccine candidate effect on immunohistochemical profiling of CD4+, CD8+, Caspase-3 and NF-κB in mice

Abstract Background Cryptosporidium parvum is a protozoan parasite of medical and veterinary importance that causes neonatal diarrhea in many vertebrate hosts. In this study, we evaluated the efficacy of an affinity-purified antigen as a C. parvum vaccine candidate using ileal and liver tissues of experimentally infected neonatal mice by immunohistochemical profiling and immune scoring of CD4+, CD8+, Caspase-3, and nuclear factor kappa B (NF-κB). This vaccine was prepared from the C. parvum oocysts antigen using immune affinity chromatography with cyanogen bromide-activated Sepharose-4B beads. Methods Thirty neonatal mice were divided into three groups (10 mice/group): (1) non-immunized non-infected, (2) non-immunized infected (using gastric tubes with a single dose of 1 × 105 of C. parvum oocysts in 250 µl PBS solution 1 h before a meal) and (3) immunized (twice with 40 µg/kg of purified C. parvum antigen at 2-week intervals and then infected with 1 × 105 C. parvum oocysts simultaneously with the second group). After euthanizing the animals on the 10th day, post-infection, their ileal and liver tissues were collected and prepared for immunohistochemistry (IHC) staining to detect CD4+, CD8+, Caspase-3, and NF-κB levels, which are indicators for T helper cells, cytotoxic T cells, apoptosis, and inflammation, respectively. Results The IHC results showed that CD4+, CD8+, Caspase-3, and NF-κB expression varied significantly (P < 0.001) in both organs in all the groups. We also recorded high CD4+ levels and low CD8+ expression in the non-immunized non-infected mice tissues, while the opposite was observed in the non-immunized infected mice tissues. In the immunized infected mice, the CD4+ level was higher than CD8 + in both organs. While the Caspase-3 levels were higher in the ileal tissue of non-immunized infected than immunized infected mice ileal tissues, the reverse was seen in the liver tissues of both groups. Furthermore, NF-κB expression was higher in the liver tissues of non-immunized infected mice than in immunized infected mice tissues. Therefore, the IHC results and immune-scoring program revealed a significant difference (P < 0.001) in the CD4+, CD8+, Caspase-3, and NF-κB expression levels in both ileal and liver tissues of all mice groups, which might be necessary for immunomodulation in these tissues. Conclusions The improvement observed in the immunized infected mice suggests that this vaccine candidate might protect against cryptosporidiosis

Preparation and Characterization of Alginate Nanocarriers as Mucoadhesive Intranasal Delivery Systems for Ameliorating Antibacterial Effect of Rutin Against Pasteurella Multocida Infection in Mice

Rutin is a natural product has various biological activities. Pasteurellosis is crucial bacterial infection of respiratory system caused by Pasteurella multocida. This study aimed to investigate the improved antibacterial effect of Rutin nanocarriers as mucoadhesive intranasal delivery against Pasteurella multocida. Different formulations of Rutin niosomes and nanostructure lipid carriers (NLCs) were formulated and well characterized. The in vivo antibacterial performance of the developed formulations against Pasteurella multocida in infected mice was conducted. Further, cytokines levels of Interferon Gamma (INF-γ) and Interlukin-12 (IL -12) in mice sera were assessed. The results revealed that developed Rutin nanocarriers were in nanosized range and exhibited high drug encapsulation. However, Rutin NLCs showed smaller particle size (240.34 ± 5.5 nm), higher encapsulation% (97.34 ± 0.15%), and higher drug release of 94.5% within 12 h comparing with Rutin niosomes. Further, Rutin NLCs presented the highest antibacterial effect against P. multocida infection compared with other treated groups. The bacterial count in lungs and livers was reduced in treated groups compared to the infected non treated one. Our results indicate that mucoadhesive Rutin nanocarriers introduce a new promising antibacterial agent for intranasal delivery against P. multocida and open vision for veterinary applications to utilize advanced nanocarriers in the management of several infections

Comparative ovicidal activity of Moringa oleifera leaf extracts on Fasciola gigantica eggs

Background: Fasciolosis is an important zoonotic disease affecting the productive performance of farm animals in Egypt. Aim: The aim of the present study was comparing the ovicidal effect of different extracts as an alcoholic (Methanolic and Ethanolic) and aqueous Moringa oleifera leaf extracts on Fasciola gigantica non-embryonated and developed eggs. Materials and Methods: Tested concentrations of extracts ranged from 12.5 to 800 mg/ml. Nitroxynil was used as reference drug with a dose of 100 mg/ml. Results: M. oleifera alcoholic and aqueous extracts showed a concentration-dependent ovicidal effect on F. gigantica non-embryonated and developed eggs. Based on LC50 values, water extract showed the highest ovicidal activity since it registered the lowest values of 2.6 mg/ml on non-embryonated eggs. Non-embryonated eggs were more susceptible to aqueous extract than developed eggs. On the other hand, the developed eggs were more susceptible to ethanolic extract than non-embryonated eggs even the lowest LC50 (12.38 mg/ml). Conclusion: M. oleifera leaf extracts especially aqueous extract could be a promising step in the field of controlling fascioliasis. Further, in vivo studies are needed to enlighten the therapeutic potential of M. oleifera extracts in treating F. gigantica infection

Table_3_Microscopy detection and molecular characterisation of Giardia duodenalis infection in outpatients seeking medical care in Egypt.XLSX

IntroductionGiardiosis remains one of the most prevalent enteric parasitic infections globally. Earlier molecular-based studies conducted in Egypt have primarily focused on paediatric clinical populations and most were based on single genotyping markers. As a result, there is limited information on the frequency and genetic diversity of G. duodenalis infections in individuals of all age groups.MethodsIndividual stool samples (n = 460) from outpatients seeking medical care were collected during January–December 2021 in Kafr El-Sheikh governorate, northern Egypt. Initial screening for the presence of G. duodenalis was conducted by coprological examination. Microscopy-positive samples were further confirmed by real-time PCR. A multilocus sequence typing approach targeted amplification of the glutamate dehydrogenase (gdh), beta-giardin (bg), and triose phosphate isomerase (tpi) genes was used for genotyping purposes. A standardised epidemiological questionnaire was used to gather basic sociodemographic and clinical features of the recruited patients.ResultsGiardia duodenalis cysts were observed in 5.4% (25/460, 95% CI: 3.6–7.9) of the stool samples examined by conventional microscopy. The infection was more frequent in children under the age of 10 years and in individuals presenting with diarrhoea but without reaching statistical significance. Stool samples collected during the winter period were more likely to harbour G. duodenalis. All 25 microscopy-positive samples were confirmed by real-time PCR, but genotyping data was only available for 56.0% (14/25) of the isolates. Sequence analyses revealed the presence of assemblages A (78.6%, 11/14) and B (21.4%, 3/14). All assemblage A isolates were identified as sub-assemblage AII, whereas the three assemblage B sequences belonged to the sub-assemblage BIII. Patients with giardiosis presenting with diarrhoea were more frequently infected by the assemblage A of the parasite.ConclusionThis is one of the largest epidemiological studies evaluating G. duodenalis infection in individuals of all age groups in Egypt. Our molecular data suggest that G. duodenalis infections in the surveyed population are primarily of anthropic origin. However, because assemblages A and B are zoonotic, some of the infections identified can have an animal origin. Additional investigations targeting animal (domestic and free-living) and environmental (water) samples are warranted to better understand the epidemiology of giardiosis in Egypt.</p