Emerging alternatives to traditional anthelmintics: the in vitro antiparasitic activity of silver and selenium nanoparticles, and pomegranate (Punica granatum) peel extract against Haemonchus contortus.

Haemonchus contortus (H. contortus) is one of the most prevalent gastrointestinal nematodes, causing health problems and economic losses in ruminants. Nanotechnology holds great promise as a field of science, with potential applications in veterinary medicine. This study investigated the in vitro anthelmintic activity of silver nanoparticles (AgNPs), selenium nanoparticles (SeNPs), and pomegranate peel extract (Punica granatum; PPE) on different stages of H. contortus: eggs, larvae, and adults. The in vitro anthelmintic efficacy was evaluated using the egg hatching inhibition assay (EHA), the third larval stage paralysis assay (LPA), and the adult worm motility inhibition assay (WMI). Six dilutions of PPE were utilized for EHA, LPA, and WMI, ranging from 0.25 to 6 mg/ml. AgNPs dilutions ranged from 0.00001 to 1.0 µg/ml for EHA and LPA and 1 to 25 µg/ml for WMI. SeNPs were utilized at dilutions of 1, 5, 10, and 15 µg/ml for EHA, LPA, and WMI. The results showed that the lowest concentration of AgNPs, SeNPs, and PPE significantly inhibited egg hatching. To further assess larvicidal activity, AgNPs at the highest concentration of 1 µg/ml induced a strong larvicidal effect, as did SeNPs at the lowest concentration. On the contrary, PPE displayed a significant larvicidal effect at 1 mg/ml compared to the control. The percentage mortality of adult H. contortus was measured as follows (mortality (%) = the number of dead adult H. contortus/total number of adult H. contortus per test × 100). The death of the adult H. contortus was determined by the absence of motility. Adult H. contortus mortality percentage was also significantly affected by all three agents when compared to the control. The AgNPs, SeNPs, and PPE have effective antiparasitic activity on gastrointestinal parasitic nematodes. These results provide evidence of the excellent antiparasitic properties of AgNPs, SeNPs, and PPE, demonstrating their effectiveness in controlling eggs, larvae, and adult H. contortus in vitro.

Immune Cell Reaction Associated with Coenurus cerebralis Infection in Sheep with Particular Reference to ELISA as a Diagnostic Tool.

Sturdy is a disease caused by Coenurus cerebralis (C. cerebralis) that typically affects the brain and spinal cord of sheep. So, this study aimed to detect the pathological, hematological and immunological changes caused by C. cerebralis in sheep. On examination, a total of 17 sheep out of 30 sheep (56.7%) from various regions in Egypt were found infected with C. cerebralis from May to August 2019. Each cyst was extracted from the sheep brain; in addition, tissue specimens were taken from the brain tissues for histopathological examination. The hematological profile was analyzed. Enzyme-Linked Immunosorbent Assay's (ELISA) specificity and sensitivity were evaluated using cystic fluid and protoscolices antigens (Ag). The cell-mediated immunity against the C. cerebralis cyst was also assessed via quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) to show alterations in mRNA expression of the Tumor Necrosis Factor-alpha (TNF-α) and gamma Interferon (IFN-γ) cytokines qRT-PCR. In histopathological sections, cerebral tissue showed an areolar cyst wall with many protoscolices attached to the tissue. The affected part showed prominent necrosis together with inflammatory cells' aggregation. Hyperplastic proliferation of the ependymal cells was a common finding. The infected sheep exhibited significantly lower total erythrocyte numbers (ER), hemoglobin levels (Hb), packed cell volume (PCV), platelet numbers (PN) and segmented cell numbers compared to apparently healthy sheep. Despite the sensitivity for the indirect ELISA being 100% for both of the Ags (fluid and scolex), the evaluation of ELISA specificity using the two antigen (Ag) preparations showed specificities of 46.2% and 38.5% for fluid and scolex Ag, respectively. Meanwhile accuracy ranged from 76.7% and 73.3% for the fluid and scolex Ags, respectively, that showed the priority was directed to the fluid to be used as an ideal sample type for ELISA. Levels of TNF-α and IFN-γ were significantly elevated in infected sheep compared to non-infected control ones. In conclusion, C. cerebralis is a serious disease infecting sheep in Egypt revealing economic losses. Although this investigation supports preliminary information about the prevalence, pathological and serological characterization of C. cerebralis, further sequencing and phylogenetic analysis is needed to understand better the T. multiceps epidemiology in ruminants and canines in Egypt.

Morphomolecular characterization of Strongylus vulgaris isolated from donkeys with special references to histopathological study on the affected organs.

Equine gastrointestinal tract is infected with Strongylus vulgaris (S. vulgaris) which is highly pathogenic parasite for its harmful effect on cranial mesenteric artery during its migration. So, this study was applied for identification of S. vulgaris in donkeys ultramorphologically and molecularly. In addition to, detection of the pathological effect of larval stage of S. vulgaris on the mesenteric arterial system using histopathology and immunohistochemistry. During the period from September to December; 2019, 60 male and 20 female donkeys at the Giza Zoo was postmortem examined. S. vulgaris adults and larvae were collected from the large intestine and cranial mesenteric arteries (CMAs), respectively. Ultramorphological examination of the collected adults was done using scanning electron microscope (SEM). DNA was extracted from 5 larvae and 6 adults for further conventional PCR studies and sequencing of the internal transcriped spacer 2 (ITS2) gene. The ITS2 gene were amplified and showed bands at 148 base pair (bp). The ITS2 gene nucleotide sequences of all isolates were aligned using Basic Local Alignment Search Tool (BLAST). Histological sections of S. vulgaris affected mesenteric arteries exhibited the presence of the parasite larvae either in the lumen with thrombus formation or attached to the intima. Most of the detected inflammatory cell populations were CD68-positive cells. From these results, it can be concluded that the ribosomal spacers genes could be used as markers for Strongylus species identification in eggs collected from equine feces as a beneficial method of diagnosis. Also, it could be important in disease surveillance, improving preventive measures and developing an effective control strategy.

Evaluation of the cytokines response in buffaloes focused on its milk as a newly emerging indicator tracing for toxocariasis.

This study aimed to evaluate the immune responses and oxidative stress provoked by Toxocara vitulorum infection in buffaloes with special reference to milk parameters as an emerging tool. The use of the milk tool was reported for the first time in tracing T. vitulorum infection in Egyptian buffaloes. Intestine, milk, serum, and liver samples were gathered from flocks in Cairo and Giza districts to evaluate buffalo immune responses provoked by T. vitulorum. The compositional items and somatic cells of milk were monitored. The intestine and milk were evaluated for interleukin IL-1ß, IL-6, and tumor necrosis factor-alpha (TNF-α) by quantitative real-time polymerase chain reaction protocol and the analysis of malondialdehyde (MDA) as an oxidative stress marker. The mean percentages for the total solids, fats, proteins, lactose, salts, pH, and somatic cell count/ml in positive samples were 11.23 ± 0.37, 5.1 ± 0.17, 4.44 ± 0.14, 3.9 ± 0.14, 0.81 ± 0.02, 6.8 ± 0.22, and 4.23 × 106± 1.41 × 105 cells/ml, respectively. A significant difference (p < 0.05) was observed in the mean values of compositional items except for the total protein %, salts %, and pH. For T. vitulorum-contaminated samples, the milk IL-1ß, IL-6, TNF-α, and MDA (nmol/ml) were 7 ± 0.23, 18 ± 0.6, 17 ± 0.56, and 3.7 ± 0.12, respectively (which were less than the values for intestinal cytokines). There is a statistical difference (p < 0.05) between positive and negative samples in the intestinal, milk cytokines, and MDA. This study is an initial investigation of the utilization of intestine and milk cytokines in the evaluation of buffalo toxocariasis.

Evaluation of immune responses and oxidative stress in donkeys: Immunological studies provoked by Parascaris equorum infection.

This study aimed to assess the effects of Parascaris equorum (P. equorum) in infected donkeys through evaluation the oxidative stress and different gene parameters in infected tissues. Fifty donkeys were examined in Giza Zoo abattoir from the period of January to March 2021. Blood and sera samples were collected from each examined donkey. P. equorum were subjected for identification through scanning electron microscope study and the infected tissues were subjected into gene expression analysis using two genes; interleukin 1ß (IL1- ß); and pro-inflammatory cytokines (TNF-α) with assessment of the antioxidant and free radicals released from the animals during the infection. Eighteen donkeys were positive for P. equorum adult or larvae by postmortem examination of the intestine and abdomen with prevalence rate of 36 %. The examined infected donkeys with P. equorum showed significantly higher of Total antioxidant capacity (TAC) levels and the serum malondialdehyde (MDA) 2.45 ± 0.53 than that in non-infected control donkeys. The levels of AST enzyme were 278.54 ± 0.45 while ALT enzyme was 14.97 ± 0.87 which was significantly higher than that of control negative donkeys. The infected donkeys with P. equorum showed significantly upregulation of the TNF-α and IL-1ß which classify according to number of collected worms. The P. equorum infected donkeys exerted at least 100 eggs of parasite in feces. The fecal egg count was marked decreased after treatment with moxidectin. Moxidectin is considered a novel active ingredient that has a marvelous result with high persistency and protection for long time, in addition to, broad spectrum activity and low or no resistance. We recommend the periodical deworming with different molecules as more economic and lifesaving over a single treatment every 12 months parallel with parasitic testing.

Anthelmintic activity of pomegranate peel extract (Punica granatum) and synthetic anthelmintics against gastrointestinal nematodes in cattle, sheep, goats, and buffalos: in vivo study.

Parasitic gastroenteritis (PGE) is one of the most important parasitic diseases that causes economic losses and health problems in ruminants. PGE causes a drop in milk, meat, and wool production in addition to decreasing animal fertility and sometimes leading to animal death. Conventional anthelmintics used for animal treatment are expensive, especially for farmers in developing countries. Moreover, the concern of anthelmintic resistance to these synthetic drugs is rising. Therefore, this study aimed to evaluate the anthelmintic activity of plant extract pomegranate (Punica granatum L) peel extract (PPE) against PGE infestations among ruminants. A total of 120 ruminants of different species (20 cattle, 12 buffalos, 68 sheep, and 20 goats) were examined for PGE eggs in their fecal samples. The animals under experiment were divided into four groups: the first group (negative control) was not given any drugs, the second group was given ivermectin (0.5 ml/25 kg bwt) (positive control 1), the third group was given albendazole (2.5 mg active principle/kg bwt) (positive control 2), and the fourth group was given PPE (200 mg/kg bwt). Fecal egg count (FEC) was performed on day 0 prior to the 1st dose of treatment. On day 15, an additional treatment (with the same doses) was administered and FEC was performed on days 7 and 21. Our results showed that on the 7th day of the experiment, there was an increase in FEC in the negative control group by 5%, while in the second, third, and fourth groups, there was a decrease in FEC with 95%, 90%, and 85% respectively. On the 21st day (7 days from the second dose), there was an increase in FEC in the control group by a 10% and 100% reduction in FEC in both the second and third groups. While in the fourth group, there was a decrease in FEC by 97%. In conclusion, PPE could be used as a safe, cheap, and effective natural anthelmintic against PGE.

Genetic Characterization of Hydatid Cysts of Different Intermediate Hosts.

Cystic echinococcosis is an important cosmopolitan parasitic zoonosis that causes public health and economic problems in Egypt. The present study was undertaken to identify genotypes of hydatid cyst (HC) DNA isolated from different animal isolates and to identify the genotype of secondary hydatid cysts (HCs) developed in rabbits experimentally infected with camel HC for detection of any genetic mutation. In the present study, we extracted DNA from the germinal layers of 8 HCs collected from 3 camels, 1 cattle, 1 sheep and 3 donkeys in addition to 3 secondary HCs collected from rabbits experimentally infected with camel HC. PCR amplification of the ITS1 gene of all examined samples showed an amplified DNA band at 1115 bp. The partial nucleotide sequences of the ITS1 gene of all isolates were aligned and compared with the reference sequences of the genotypes G1-G8 in GenBank. The camel and rabbit samples were identified as Echinococcus canadensis genotype 6 (G6), while the cattle and sheep samples belonged to E. granulosus sensu stricto (G1). The donkey isolates belonged to E. equines (G4). Alignment of the ITS1 partial nucleotide sequences of the camel HCs and rabbit secondary HCs isolates with the G6 partial nucleotide sequence in GenBank was performed. Both camel HCs and rabbit secondary HCs isolates exhibited the same sequence identity matrix, which indicated the absence of mutation in the rabbit secondary HCs. It can be concluded that camel and rabbit samples were identified as E. canadensis (G6), the cattle and sheep samples belonged to E. granulosus sensu stricto (G1) and donkey isolates belonged to E. equines (G4). No mutation occurred during HCs transmission from camel to rabbit.

Evaluation of tumor necrosis factor-alpha (TNF-α); gamma interferon (IFN-γ) genes and oxidative stress in sheep: immunological responses induced by Oestrus ovis (Diptera: Oestridae) infestation.

This study aimed to evaluate the cell mediated immune responses against Oestrus ovis (O. ovis) in sheep through measurement of the changes in mRNA expression of the tumor necrosis factor alpha (TNF-α) and gamma interferon (IFN-γ) cytokines using quantitative Real time-PCR (qRt-PCR). Also; to detect the role of Oestrus ovis infestation in the oxidative stress markers in sheep. Fifty sheep head were examined in Cairo abattoir from the period of May to August 2019. Sera were separated and collected for measurement of nitric oxide, zinc and malondialdehyde (MDA). While TNF-α and IFN-γ mRNA were extracted from nasal mucosa. Levels of IFN-γ and TNF-α were significantly higher in infested sheep than that in non-infested one. Also, oxidative stresses were indicated by high level of nitric oxide as one of reactive oxygen species (ROS) and serum MDA as oxidative stress marker and low antioxidant capacity (zinc concentration in serum) in infested sheep. The obtained results indicated that measurements of TNF-α and IFN-γ cytokines using qRT-PCR could be used as an association and reproducible quantitative method for the diagnosis of O. ovis infestation in sheep.