Attenuation of pathogenesis of Eimeria stiedae sporulated oocysts using Egyptian alginate propolis nanoparticles.

BACKGROUND: Coccidiosis is a costly and widespread infectious disease that affects mammals and causes huge losses for the global rabbit meat industry. This study evaluated the potency of Egyptian alginate propolis nanoparticles (NPs) in attenuating the infectivity of Eimeria stiedae sporulated oocysts. The gelification method was used to prepare alginate propolis NPs, which were then characterized using a transmission electron microscope and zeta potential analysis. RESULTS: The results revealed that the zeta potential of the prepared alginate propolis NPs increased from - 60.60 ± 9.10 mV to -72.26 ± 6.04 mV. The sporulated oocysts were treated with 50 mg/mL of the alginate propolis NPs. Thereafter, the treated oocysts were tested for their ability to infect rabbits. The rabbits were divided into three groups: the healthy control (G1) group, the infected control (G2) group, and the treated oocyst-infected (G3) group. The rabbits were sacrificed 43 days post-infection (dpi). The infectivity of the oocysts was assessed. The treated oocyst-infected rabbits exhibited slight abdominal distension and dullness symptoms. The G3 group had no oocyst output, with a 100% reduction from 41 dpi until the end of the experiment. Immunologically, the IgG level of the G2 group gradually increased (p ≤ 0.05) much more than that of the G3 group. The IL-12 level in the G3 group significantly increased from 16 dpi until the end of the experiment, nearly reaching the level in healthy animals. Decreased CD4+ and CD8+ immunolabelling was observed in the liver sections of the group infected with the alginate propolis NP-treated oocysts, and there was a remarkable improvement in the histopathological parameters. CONCLUSIONS: These data indicate that Alg propolis NPs are sufficient to reduce the infectivity of E. stiedae oocysts.

Evaluation of a vaccine candidate isolated from Cryptosporidium parvum oocyst in mice.

Background and Aim: Cryptosporidiosis is a leading cause of diarrheal disease worldwide and is an animal and public health burden. This study aimed to evaluate the protective potential of affinity-purified Cryptosporidium parvum oocyst antigen as a vaccine candidate according to fecal oocyst shedding, humoral and cellular immune responses, histopathological changes, and the number of parasite developmental stages in ileal and hepatic tissues. Materials and Methods: We isolated oocysts from naturally infected buffalo calves and identified them molecularly as C. parvum isolates (GenBank: ON730707 and ON730708) by targeting the Cryptosporidium oocyst wall protein gene. We propagated the C. parvum oocysts in mice. In addition, we prepared crude antigen from the isolated oocysts by purification using cyanogen bromide-activated Sepharose-4B affinity chromatography coupled with rabbit hyperimmune serum. Then, we divided 81 parasite-free mice into three groups: (1) non-vaccinated non-infected mice, (2) mice orally infected with 1 × 105 C. parvum oocysts on week 4 of the experiment, and (3) mice immunized twice with 40 µg/kg of the purified fraction at 2-week intervals. Then, we challenged the vaccinated group with C. parvum oocysts after 2 weeks, and the positive control group was infected at the same time. Results: We observed a prolonged prepatent period and decreased oocyst shedding in the vaccinated infected mice compared with the non-vaccinated infected mice (t < 0.001). The vaccinated mice had significantly higher immunoglobulin G levels than those in the other two groups at all examined weeks. In addition, the production of cytokines interferon-gamma, interleukin (IL)-10, IL-12, and IL-15 was activated post-vaccination. After the challenge, all tested cytokines were significantly increased (p < 0.001) in the two infected groups compared with the non-vaccinated non-infected group, with the highest levels in the vaccinated infected group. Vaccinated infected mice exhibited significantly fewer pathological lesions in the ileum and liver than non-vaccinated infected mice, which showed prominent histopathological lesions. Endogenous developmental stages of C. parvum indicated that the ileum was more parasitized than the liver and that vaccination resulted in a lower number of oocysts in ileal and hepatic tissues (p < 0.05). Conclusion: Our prepared affinity-purified vaccine candidate could be promising in protecting against cryptosporidiosis.

Antiparasitic and immunomodulating effects of nitazoxanide, ivermectin and selenium on Cryptosporidium infection in diabetic mice.

The present work aims to investigate the antiparasitic and the immunomodulating effects of nitazoxanide (NTZ) and ivermectin (IVC) alone or combined together or combined with selenium (Se), on Cryptosporidium infection in diabetic mice. The results revealed that the combined NTZ and IVC therapy achieved the highest reduction of fecal oocysts (92%), whereas single NTZ showed the lowest reduction (63%). Also, adding Se to either NTZ or IVC resulted in elevation of oocyst reduction from 63% to 71% and from 82% to 84% respectively. All treatment regimens, with the exception of NTZ monotherapy, showed a significant improvement in the intestinal histopathology, the highest score was in combined NTZ and IVC therapy. The unique results of immunohistochemistry in this study showed reversal of the normal CD4/CD8 T cell ratio in the infected untreated mice, however, following therapy it reverts back to a normal balanced ratio. The combined (NTZ+ IVC) treatment demonstrated the highest level of CD4 T cell expression. Taken together, NTZ and IVC combined therapy showed remarkable anti-parasitic and immunostimulatory effects, specifically towards the CD4 population that seem to be promising in controlling cryptosporidiosis in diabetic individuals. Further research is required to explore other effective treatment strategies for those comorbid patients.

Antiparasitic and immunomodulating effects of nitazoxanide, ivermectin and selenium on Cryptosporidium infection in diabetic mice / Efeitos antiparasitários e imunomodulantes da nitazoxanida, ivermectina e selênio sobre a infecção por Cryptosporidium em camundongos diabéticos

Abstract The present work aims to investigate the antiparasitic and the immunomodulating effects of nitazoxanide (NTZ) and ivermectin (IVC) alone or combined together or combined with selenium (Se), on Cryptosporidium infection in diabetic mice. The results revealed that the combined NTZ and IVC therapy achieved the highest reduction of fecal oocysts (92%), whereas single NTZ showed the lowest reduction (63%). Also, adding Se to either NTZ or IVC resulted in elevation of oocyst reduction from 63% to 71% and from 82% to 84% respectively. All treatment regimens, with the exception of NTZ monotherapy, showed a significant improvement in the intestinal histopathology, the highest score was in combined NTZ and IVC therapy. The unique results of immunohistochemistry in this study showed reversal of the normal CD4/CD8 T cell ratio in the infected untreated mice, however, following therapy it reverts back to a normal balanced ratio. The combined (NTZ+ IVC) treatment demonstrated the highest level of CD4 T cell expression. Taken together, NTZ and IVC combined therapy showed remarkable anti-parasitic and immunostimulatory effects, specifically towards the CD4 population that seem to be promising in controlling cryptosporidiosis in diabetic individuals. Further research is required to explore other effective treatment strategies for those comorbid patients.

Resumo O presente trabalho tem como objetivo investigar os efeitos anti-parasitários e imunomodulantes da nitazoxanida (NTZ) e ivermectina (IVC), isoladas ou em associação, e do selênio (SE), associado à NTZ ou à IVC, sobre a infecção por Cryptosporidium em camundongos diabéticos. Os resultados revelaram que a terapia combinada com NTZ e IVC resultou em maior redução de oocistos fecais, enquanto a NTZ isolada mostrou a menor redução de oocistos fecais (63%). Além disso, a associação do SE com a NTZ ou IVC resultou em redução do número de oocistos fecais de 63% para 71% e de 82% para 84%, respectivamente. Todos os tratamentos, com exceção da monoterapia com NTZ, mostraram uma melhora significativa nos índices relacionados à histopatologia intestinal. Os resultados da imuno-histoquímica mostraram reversão da razão celular CD4/CD8 T normal nos camundongos infectados não tratados, no entanto, após a terapia, houve retorno à razão celular CD4/CD8 T normal. O tratamento combinado (NTZ+ IVC) demonstrou o mais alto nível de expressão celular CD4 T. Em conclusão, a terapia combinada com NTZ e IVC mostrou efeitos anti-parasitários e imunoestimuladores notáveis, especificamente para a população CD4, que parecem ser promissores para o controle da criptosporidiose em indivíduos diabéticos.

Immunomodulatory effects of Moringa oleifera leaves aqueous extract in sheep naturally co-infected with Fasciola gigantica and Clostridium novyi.

The current study was designed to evaluate the in vivo fasciolicidal activity of Moringa (M.) oleifera leaf aqueous extract oral administration as well as its antibacterial activity against Clostridium (C.) novyi in sheep naturally co-infected with fascioliasis and C. novyi. Sheep naturally infected with fascioliasis were divided into 3 groups, heavily infected treated group, lightly infected treated group and mixed infection control (non-treated) group. Treatment groups were orally administered M. oleifera leaves aqueous extract at a dose of 150 mg/kg every 48 h for 21 days. Animal body weights, fecal egg count, serum levels of anti-Fasciola IgG, cytokines (IL-2, IL-17, IL-10), and bacterial count of C. novyi were evaluated. The results showed that treatment with M. oleifera improved the body weight gain and decreased fecal egg count in lightly and heavily infected groups compared to the nontreated group with 100% reduction in egg count in lightly infected sheep. Furthermore, the treatment with M. oleifera significantly reduced the serum levels of IgG, IL-2, and IL-17. Interestingly, elevated levels of IL-10 were recorded in both heavily and lightly infected sheep. The treatment with Moringa extract significantly decreased the fecal bacterial count of C. novyi in both heavily and lightly infected groups. In conclusion, this study highlights the potential beneficial effects of M. oleifera leaf against Fasciola (F.) gigantica and C. novyi.

Assessment of Haemonchus contortus larval and adult somatic antigens in sero-diagnosis of haemonchosis in naturally infected sheep and goats.

The current work was carried out to evaluate the potency of larval and adult somatic Haemonchus contortus (H. contortus) antigens in detection of haemonchosis among sheep and goats using ELISA. Two hundred and forty-three fecal and blood samples were randomly collected from small ruminants (107 sheep and 136 goats) in Beni-Suef Governorate, Egypt, during the period from June to August 2018. The fecal analysis exhibited that 26.33% of the small ruminants were infected with gastrointestinal nematodes. The overall prevalence of H. contortus was reached 22.22% whereas it was 27.10% and 18.38% among sheep and goats, respectively. The current study elucidated that the larval antigen has claimed more superior diagnostic results compared to the adult somatic H. contortus antigen. The apparent overall sero-prevalence among small ruminants was reached 51.85%. Separately, it was 64.48% in sheep and 41.91% in goats. The larval antigen had proved 96.55% sensitivity and 47.43% specificity, for sheep serum samples. Meanwhile, sensitivity and specificity for goats' sera were 100% and 71.17%, respectively. Diagnostic efficacy of ELISA was recorded 60.74% in sheep and 76.47% in goats. This study deduced that the larval antigen has proved the priority and the potency for diagnosis of H. contortus infection. Moreover, haemonchosis is a prevalent disease among the examined sheep and goats.

Cellular immune response and scanning electron microscopy in the evaluation of Moringa leaves aqueous extract effect on Cryptosporidium parvum in buffalo intestinal tissue explants.

Cryptosporidium is an apicomplexan parasite of human and animals and is considered as an important co-factor in neonatal diarrhea. In this study, an explant culture was used as an in vitro model of buffalo intestine to evaluate the effect of Moringa leaves extract on Cryptosporidium parvum (C. parvum) oocysts using light and scanning electron microscopy and measuring IFN-γ, IL-12 and IL-14 in the culture supernatants. C. parvum oocysts were collected from naturally-infected calf feces, isolated, excysted and then co-inoculated with ileal tissue explants culture medium. The prepared Moringa leaves extract was then introduced to the infected tissues in the concentrations of 100 mg/ml and 300 mg/ml. After 24 h, tissues were collected and processed for light and scanning electron microscopy. Also, culture supernatants were collected for cytokines measurement. C. parvum parasitophorous vacuoles were found attached to the surface of tissue in Cryptosporidium-infected ileal tissue explants. High magnification imaging of ileal tissue explants using scanning electron microscopy showed that Moringa leaves extracts had a great effect on Cryptosporidium-infected ileal tissue explants. There was a high significant (P < 0.001) increase in IFN-γ, IL-12 and IL-14 (375, 275 and 90 pg/ml, respectively) in the supernatants of infected non-treated ileal tissue explant culture plate wells compared to the control non-infected ones (74.66, 75 and 50 pg/ml, respectively). A concentration of 100 mg/ml Moringa extract exhibited the highest anticryptosporidial effect causing a significant decrease in IFN-γ, IL-12 and IL-14 levels (225, 150 and 65 pg/ml, respectively) compared with supernatants of infected non-treated ileal explant culture plate wells. In this study, explant culturing of buffalo ileal tissues allowed investigating the pathogenesis of cryptosporidiosis using light and scanning electron microscopy and studying changes in cytokine levels in tissues with and without Moringa leaves extract treatment. This model could help to understand the regulation of intestinal secretory and inflammatory responses, and could be useful for the screening of potential anticryptosporidial candidate compounds.

Toxocara vitulorum cuticle glycoproteins in the diagnosis of calves' toxocariasis.

AIM: The current study was designed to isolate and characterize Toxocara vitulorum glycoprotein antigens and then to evaluate its potency in accurate diagnosis of toxocariasis. MATERIALS AND METHODS: T. vitulorum glycoprotein fractions were isolated using Con-A affinity chromatography. The fractions characterized using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and immunoblot assay. Mass spectrometric analysis was used for identification of proposed structure of the N-acetylglucosamine (GlcNAc) fraction. Enzyme-linked immunosorbent assay (ELISA) was used to assess the diagnostic potential of the isolated fractions. RESULTS: Surface of T. vitulorum adult worm revealed two glycoprotein fractions rich in glucose (Glc) and GlcNAc. Three bands of molecular weight 212kDa, 107 kDa, and 93 kDa were detected in Glc fraction by SDS-PAGE. These bands were also detected in GlcNAc fraction with an additional band of 49 kDa. GlcNAc fraction showed more diagnostic potency of calves' toxocariasis; 79% than Glc fraction; 46.9% by indirect ELISA. The additional band of 49 kDa in GlcNAc fraction is probably responsible for its higher diagnostic potentials. Western blotting verified the immunoreactivity of the Glc and GlcNAc isolated fraction as they reacted with calves sera infected with toxocariasis. The proposed structure of GlcNAc fraction was Ser-Meth-Arg-O-methylated GlcNAc. CONCLUSION: GlcNAc-rich fraction of T. vitulorum can be successfully utilized in the diagnosis of calves' toxocariasis.

Medico-legal aspects of disabilities due to orthopedic injuries and compensations in Egypt.

Disability is a complex problem that results in detrimental effects on the victim. The number of disabled has increased considerably. The development constitutes a good reason for paying more specific attention to society's growing number of disabled. The physician's duties are not only to describe the anatomic and physiological defects present in the injured individual, but also to answer questions regarding the patient ability to perform specific economic or social function. This study aimed to clear up some of the problems or difficulties that may face the medico-legal physician or any personal that may be concerned with the process of evaluation. In the other words the aim of work is to design a new strategy for evaluating the degree of permanent infirmities and their compensation. The study investigates the pattern of disability in cases referred for medico-legal examination in department of Cairo and the chief's office of medico-legal administration in ministry of justice. The study included 200 cases of disabled. Male gender was more predominate than female gender (were male 78% - female 22%) and the working group (18-60) were the most frequent age group identified as victims of traumatic injuries. It was recommended the overall development plans of the country should have a positive role in prevention and decrease this problem.

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.

Studies on monieziasis of sheep I. Prevalence and antihelminthic effects of some plant extracts, a light and electron microscopic study.

Out of 2,343 examined sheep throughout the whole year, 271 were found to be infected with tapeworms (11.5%). The highest infection rate (22.1%) was recorded in autumn, while the lowest (6%) occurred in summer. Six species of cestodes were identified according to their prevalence: Moniezia expansa (74%), Moniezia denticulata (8.5%), Moniezia benedeni (4.8%), Moniezia trigonophora (2.7%), and Thysaniezia giardi (2.7%), T. giardi was recorded in Egypt for the first time. M. expansa was found throughout the year with two peaks in January (92.3%) and June (88.5%). SEM examination revealed that M. expansa scolex has four triangular suckers guarded by an X-shaped fibrous band that makes them open permanently. Also, there is a Y-shaped apical part at the scolex center which may be an embryonically non-developed rostellum. The whole worm body surface is covered with unidirectional microtriches. TEM showed that the tegument is underlaid by a thick fibrous interstitial layer below which an outer circular and an inner longitudinal muscular layer are arranged. Beneath these structures the subtegumental cells are found which have spherical electron-dense inclusions and various other cell organelles. Moreover, M. expansa has interproglottidal glands which contain secretory vesicles and secretions. For biological control of this parasite, different concentrations of crude plant extract of Artemisia cina were used in vitro and in vivo. In vitro, the results indicated that the plant extract is efficacious at all concentrations tested. Electron microscopic examination showed that many structures of the treated worms were affected. The most affected sites were the scolex and the microtriches of the outer tegumental surface. In vivo, treatment of heavily infected animals showed an antihelminthic effect, since the complete absence of eggs was recorded 9 days after treatment when fecal investigations were done.

Botanical study of Meryta denhamii Seem. and its antihelminthic activity against Fasciola gigantica.

The anthelmintic activity of the ethanolic extracts of stems and leaves of Meryta denhamii Seem. against adult liver flukes "Fasciola gigantica" was studied in vitro. Although leaves extract was inactive, stems extract exhibited anthelmintic activity and recorded LC50 and LC90 values, 16 and 26 gm/l respectively. The mode of action of the ethanolic extract of the stems on the adult flukes was evaluated by scanning electron microscopy (SEM). Tegumental sloughing, loss of spines and deformity of suckers were observed. These damages are responsible for the vermicidal effect of the ethanolic extract of the stems. The saponin contents of the stems (4.25%) and leaves (2.45%) were determined using haemolytic index. The plant was identified based on the macro and micromorphological features of the stem and leaf.

Molluscicidal saponins from leaves of Hedera canariensis.

Activity-guided fractionation of the methanol extract of Hedera canariensis (var. Gloire de Maringo) Wild leaves afforded five saponins 1-5. Chemical and spectral methods (MS, 1HNMR, 13CNMR) showed that they are glycosides of hederagenin and oleanolic acid. The results showed that 4,5 exhibited molluscicidal properties, compound 1 was inactive. Mortality rate of exposed snails increased by increasing plant extract concentration. Lymnaea cailliaudi was more sensitive to plant extract than Biomphalaria alexandrina. The histopathological study revealed distinct damage in the structure of the stomach and ovotestis of treated L. cailliaudi snails. Saponin content was determined in term of haemolytic index.

Treatment of acute schistosomiasis mansoni with praziquantel and an antifibrotic agent in mice. Maintenance of resistance to infection.

This work is a trial to evaluate the effect of the combination of the anthelmintic drug praziquantel (CAS 55268-74-1, PZQ, EMBAY 8440, Biltricide) with the antifibrotic agent beta-aminopropionitrile-monofumarate salt (BAPN, CAS 2079-89-2). It is also a trial to elucidate the repercussions of this drug combination upon worm and tissue egg loads and oogram pattern. Moreover, it aims to study their effects on the hepatic granuloma size and the resistance to reinfection in experimental murine schistosomiasis mansoni. A group of 120 Swiss albino mice was used. This group was further subdivided into six small subgroups. Subgroup I comprised infected untreated control mice. Subgroup II comprised infected untreated challenged control mice. Subgroup III comprised challenged control mice. Subgroup IV comprised infected mice treated with PZQ 500 mg/kg b. w. orally for two successive days. Subgroup V comprised infected mice given BAPN daily as 5 mg powder in 0.5 ml saline for 14 successive days. Subgroup VI comprised mice given both PZQ + BAPN. Animals were sacrificed 12 weeks post primary infection. Mice given the combination regimen PZQ + BAPN, compared to those given each drug solely, revealed absence of worm recovery at perfusion and only dead ova in the oogram (99.2 + 0.6). Inspite of the marked reduction in the hepatic and intestinal tissue egg loads recorded, this drug combination revealed the highest score of percent resistance to reinfection (91.2 + 0.5%). The data were less salient in mice given PZQ or BAPN alone.

Oogram pattern, egg load and hatchability of eggs in mixed infection of Schistosoma mansoni and Schistosoma haematobium in hamster.

In mixed infection with Schistosoma mansoni and S. haematobium in hamsters, either simultaneously or after the full maturation of an initial infection with either schistosome species, a high level of acquired resistance was observed. The degree of immunity was magnified by the significant reduction in the number of eggs per gram tissue and hatchability of mature eggs. Some disturbance was also observed in the oogram pattern.

Incidence of cryptosporidiosis in immunodeficient cancer patients in Egypt.

A coproprotozoal study was carried out on 63 patients suffering from malignancy. The majority had cancer of haemopoietic system. All patients were under chemotherapy and included: Group A (33 children) and Group B (30 adults) of whom 20 immunocompetent diarrhoeic patients of matched age and sex were considered as controls. Stool samples were examined by merthiolate iodine-formaldehyde concentration technique (MIF). Modified Zeihl-Neelsen (ZN) stain was performed for Cryptosporidium oocysts. Detection of Cryptosporidium coproantigen by enzyme-linked immunoassay test (Ridascreen test), was used. Immunoglobulins (IgG, IgM, IgE & IgA), C3, C4 and CD4:CD8 ratio, were measured. According to their levels 25 out of 63 patients had both humoral and cellular immunodeficiency. The incidence of Cryptosporidium in cancer patients was 23.8%, while it was 37.7% and 91% in children and adults immunodeficient patients, respectively. ZN stain was able in diagnosed Cryptosporidium in 13 out of 35 immunodeficient cases while ELISA detected only 11 cases. Cryptosporidium infection in immunodeficient cancer patients had significantly more frequent and prolonged duration of diarrhoea than in negative ones.

Biological and morphological changes in worms in mixed infection of Schistosoma mansoni and S. haematobium in hamsters.

Hamsters infected with Schistosoma mansoni and S. haematobium either simultaneously or after maturation of an initial infection with either schistosome species were examined to estimate the biological and morphological changes under mixed infection conditions. The results indicate a high level of acquired resistance. The degree of immunity was evidenced by the significant reduction in the worm load as well as in the size of mature worms. Some disturbance was observed in the worm distribution. The results are important for epidemiological and immunological uses, as well as in development of a vaccine from one species that might be effective against both species.