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.

In Vitro: The Effects of the Anticoccidial Activities of Calotropis procera Leaf Extracts on Eimeria stiedae Oocysts Isolated from Rabbits.

Hepatic coccidiosis is an infectious and mortal disease that causes global economic losses in rabbits. The research aimed to assess the efficacy of Calotropis procure leaf extracts on the inhibition of Eimeria stiedae oocysts and to determine the optimal dosage for suppressing the parasite's infective phase. In this experiment, oocyst samples per milliliter were tested, and 6-well plates (2 mL) of 2.5% potassium dichromate solution containing 102 non-sporulated oocysts on Calotropis procera leaf extracts were exposed after 24, 48, 72, and 96 h, and the treatments were as follows: a nontreated control, 25%, 50%, 100%, and 150% of C. procera for oocyst activities. In addition, amprolium was utilized as a reference drug. The Calotropis procera was analyzed by GC-Mass, and results showed that the botanical extract contained 9 chemical components that were able to inhibit the oocysts of E. stiedae at 100% and 150% concentrations by about 78% and 93%, respectively. In general, an increase in the incubation period and a greater dose resulted in a decrease in the inhibition rate. The results showed that C. procera has an effective ability, inhibitory potential, and protective effect on the coccidian oocyst sporulation of E. stiedae. It can be used in the disinfection and sterilization of poultry and rabbit houses to get rid of Eimeria oocysts.

Antitumour metastasis and the antiangiogenic and antitumour effects of a Eimeria stiedae soluble protein.

Some protozoa (Plasmodium falciparum, Toxoplasma gondii, etc) are used to treat cancer because they can improve tumour-induced immunosuppression. This study aims to evaluate the antitumour effect of Eimeria stiedae oocyst soluble protein (ESSP). ESSP was extracted, and mice were injected with 5 × 105 CT26 cells in the right axilla, and then, 50 µg of ESSP was intraperitoneally injected for 5 continuous days. The effect of ESSP on tumour immunity was detected by flow cytometry 25 days after the CT26 inoculation. The results showed that ESSP can inhibit the growth of CT26 subcutaneous tumours; significantly increase the expression of MHC I, MHC II, CD80 and CD86 on the surface of splenic dendritic cells; and enhance the level of IL-12 secretion. ESSP induced an increase in the number of NK cells in the mouse spleen, and the levels of IFN-γ and CD107 were upregulated in the NK cells and CD8+ T cells. The number of metastatic nodules in the lung tumours in the mice was significantly reduced, and the number of tubes, area of the loops and total length of the tubes were significantly reduced. ESSP enhances the antitumour immune response and inhibits tumour growth, metastasis and angiogenesis.

Potential Vaccine Targets against Rabbit Coccidiosis by Immunoproteomic Analysis.

The aim of this study was to identify antigens for a vaccine or drug target to control rabbit coccidiosis. A combination of 2-dimensional electrophoresis, immunoblotting, and mass spectrometric analysis were used to identify novel antigens from the sporozoites of Eimeria stiedae. Protein spots were recognized by the sera of New Zealand rabbits infected artificially with E. stiedae. The proteins were characterized by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF/TOF-MS) analysis in combination with bioinformatics. Approximately 868 protein spots were detected by silver-staining, and a total of 41 immunoreactive protein spots were recognized by anti-E. stiedae sera. Finally, 23 protein spots were successfully identified. The proteins such as heat shock protein 70 and aspartyl protease may have potential as immunodiagnostic or vaccine antigens. The immunoreactive proteins were found to possess a wide range of biological functions. This study is the first to report the proteins recognized by sera of infected rabbits with E. stiedae, which might be helpful in identifying potential targets for vaccine development to control rabbit coccidiosis.

Molecular diagnosis of Eimeria stiedae in hepatic tissue of experimentally infected rabbits.

The early detection of Eimeria stiedae in the hepatic tissue of experimentally infected rabbits was investigated using molecular assay. Forty 6-week-old male New Zealand rabbits were divided into two groups. Group A (30 animals) was infected with 2.5 × 10(4) sporulated oocysts of E. stiedae per animal on Day 0 and Group B (10 animals) was used as the uninfected controls. Three animals from Group A and one from Group B were sacrificed at 0, 3, 6, 9, 12, 15, 18, 21, 24 and 27 days post infection (PI). Gross and microscopic post-mortem findings were recorded. Polymerase chain reaction (PCR) of the E. stiedae internal transcribed spacer 1 genomic region was conducted on blood, liver tissue, and feces from the Group A experimentally infected animals. Macroscopically, the liver showed irregular yellowish white nodules pathognomonic to E. stiedae infection beginning on Day 15 PI. Hepatomegaly and ascites were obvious from Day 21-24 PI. The presence of different E. stiedae schizonts and gametocytes in the histopathological sections of the biliary epithelium were evident on Day 15 PI. The E. stiedae PCR was first positive in liver tissues on Day 12 and in fecal samples on Day 18 PI, but the blood samples were negative. In conclusion, the PCR can be used for early diagnosis and control of E. stiedae schizonts before shedding of the oocysts in feces.

Toxoplasma gondii coinfection with diseases and parasites in wild rabbits in Scotland.

In wild rabbits (Oryctolagus cuniculus) on an estate in Perthshire, central Scotland, the seroprevalence of Toxoplasma gondii was 18/548 (3·3%). The wild rabbit could be a T. gondii reservoir and it has potential value as a sentinel of T. gondii in environmental substrates. Toxoplasma gondii was associated with female sex (P < 0·001) and with relatively heavy infections by Eimeria stiedae (P = 0·036). It was not associated with the intensity of coccidial oocysts, the severity of myxomatosis caused by the virus Myxomatosis cuniculi, the intensity of roundworm eggs, the year or season, rabbit age or distance from farm buildings. Coinfections could have been affected by gestational down regulation of type 1 T helper cells. A sudden influx or release of T. gondii oocysts might have occurred. This is the first report of T. gondii in any wild herbivore in Scotland and also the first report of lapine T. gondii as a coinfection with E. stiedae, M. cuniculi and helminths.

Occurrence, Histopathological Findings, and Molecular Identification of Pathogenic Eimeria Infections in Rabbits (Mammalia: Lagomorpha) in Japan.

PURPOSE: Eimeria spp. are commonly found among rabbits (Mammalia: Lagomorpha) worldwide. Among the 11 Eimeria species, several are highly virulent, including E. intestinalis and E. flavescens, which cause intestinal coccidiosis, and E. stiedae, which causes hepatic coccidiosis. Unlike other countries, the occurrence of Eimeria infections in rabbits in Japan remains unknown, except for one reported case of natural infection. METHODS: We surveyed Eimeria infections in clinically affected rabbits over the past approximately 10 years at Livestock Hygiene Centers in 42 prefectures. A total of 16 tissue samples (14 liver, 1 ileum, and 1 cecum) were collected from 15 rabbits in 6 prefectures. RESULTS: Characteristic histopathologic findings were observed, especially around the bile ducts, depending on the developmental stages of the parasites. Eimeria stiedae and E. flavescens were successfully identified by PCR and sequencing analyses in 5 liver samples and 1 cecum sample, respectively. CONCLUSION: Our results could enhance understanding of infection with Eimeria spp. in rabbits in Japan and contribute to pathological or molecular diagnoses.

Hepatic Coccidiosis in Wild Rabbits in Greece: Parasite Detection on Liver Imprints and the Associated Biochemical Profile.

(1) Background: Rabbit hepatic coccidiosis, caused by Eimeria stiedae, is a devastating disease with high morbidity and mortality rates. The disease is well described in rabbits, but little is known about E. stiedae infection in wild rabbits. In this study, we investigated the presence of E. stiedae infection in wild rabbits from the island of Lemnos, Greece, where this species is overpopulated, and the effects of infection on common hepatic biomarkers. (2) Methods: We used liver impression smears to detect the coccidian oocysts, and we defined the liver biochemical profile of the infected individuals. (3) Results: Overall, 13.3% of the liver imprints examined were positive for the presence of coccidial oocysts. The activities of liver enzymes, that is, alanine aminotransferase (ALT), aspartate aminotransferase (AST) and glutamyltransferase (GGT), as well as globulins (GLOB), were increased while the concentrations of albumins (ALB), total proteins (TP) and the albumin to globulin (A/G) ratio were decreased in the infected individuals compared to the non-infected ones. (4) Conclusions: This study adds to the current knowledge on the pathogens affecting wild rabbits and those circulating in this population on the island of Lemnos, Greece. Moreover, we showed that E. stiedae infection exerts pathological effects on the hepatocyte integrity and liver function of wild rabbits, as reflected by the abnormal values of liver injury and dysfunction biomarkers.

Camellia sinensis Aqueous Extract: A Promising Candidate for Hepatic Eimeriosis Treatment in Rabbits.

Eimeria stiedae (E. stiedae) is a common coccidian species that infects the liver and causes economic losses for the rabbit industry. This study aimed to determine the efficiency of green tea aqueous extract (GTE) as a natural treatment for eimeriosis caused by E. stiedae. Male rabbits Cuniculus L. (Oryctolagus) of the New Zealand White rabbit strain (4-4.5 months) were used, as they are suitable for research and conducting experiments. Thirty rabbits were allocated into six groups, with five rabbits in each group; the G1 group (non-infected untreated) served as a negative control group; the G2 group was not infected and treated with 250 mg GTE; the G3 group was not infected and treated with 500 mg GTE; the G4 group was untreated and was infected with 3 × 104 Sporulated E. stiedae oocysts, which served as a positive control group; the G5 group was infected and treated with 250 mg GTE; and the G6 group was infected and treated with 500 mg GTE. The hematological and biochemical analyses of each group of rabbit sera were carried out. Phytochemical analysis was performed to evaluate the active components in GTE leaves using the following methods: IR spectroscopy, liquid chromatography-mass spectrometry (LC-MS), energy-dispersive X-ray spectroscopy (EDX), and scanning electron microscopy. The infected rabbit groups treated with GTE at both doses of 250 and 500 mg/kg exhibited a significant decrease in the extent of E. stiedae oocyst shedding compared with the infected untreated group at 14, 21, and 28 days post-infection. Also, treatment with green tea showed improvement in liver weight compared with the enlarged livers of infected, untreated rabbits. The disturbance in serum liver enzymes' gamma-glutamyl transferase (GGT) and aspartate aminotransferase (AST/GOT) levels, as well as serum glucose, potassium, uric acid, cholesterol, and urea levels, were improved after the treatment of infected rabbit groups with green tea compared with the infected untreated group. Moreover, in this study, the images of the egg stages of the parasite were taken using a fluorescence microscope at 25 µm and 26 µm magnifications. This study provides promising results for the effective cell absorption of the aqueous extract of green tea, which was confirmed in the analyzed images using a scanning electron microscope at 5 µm and 20 µm magnifications.

Eimeria stiedae causes most of the white-spotted liver lesions in wild European rabbits in Cambridgeshire, United Kingdom.

In rabbits, a white-spotted liver can be indicative of one of several disease processes, frequently caused by parasites. To date, the prevalence of white-spotted liver in wild rabbits, Oryctolagus cuniculus, in the United Kingdom is undetermined. We evaluated the prevalence and main parasitic etiologies of this entity in a U.K. population of wild rabbits. Wild rabbits (n = 87) were shot in Cambridgeshire for population control, and cadavers were donated for research. Postmortem examination was undertaken, including gross and histologic hepatic examination. Macroscopic lesions consistent with white-spotted liver were found in 46 of 87 (53%) rabbits examined; most of these lesions were considered to be mild. For 28 of 46 (59%) rabbits with gross hepatic lesions, an etiologic agent was apparent histologically. Eimeria stiedae was detected in 21 of 87 (24%) rabbits, and Calodium hepaticum (syn. Capillaria hepatica) was detected in 7 of 87 (8%). In the subset of rabbits killed in the summer, there was a significant association between white-spotted liver and juvenile age class. There was also an association between white-spotted liver caused by E. stiedae and juvenile age class. When restricting analysis to rabbits with white-spotted liver caused by E. stiedae and submitted in the summer, both juvenile age class and female had significant effects. E. stiedae and C. hepaticum can be transmitted to pet lagomorphs via contaminated vegetation, and to humans in the case of the latter, which demonstrates the importance of monitoring the prevalence of these parasitic diseases in wild rabbits.

Protective Effect of Litchi chinensis Peel Extract-Prepared Nanoparticles on Rabbits Experimentally Infected with Eimeria stiedae.

The present study used Litchi chinensis peel extract to synthesize silver nanoparticles (AgNPs). This technique is eco-friendly and can be performed in a single step; thus, it has attracted great attention for NPs biosynthesis. Herein, we biosynthesized AgNPs with L. chinensis peel extract and examined their anticoccidial activity in rabbit hepatic coccidiosis induced by E. stiedae infection. Thirty-five rabbits were allocated into seven groups: a healthy group (G1), an infected control group (G2), four groups infected before treatment with 10 mg/kg L. chinensis peel extract-biosynthesized AgNPs (G3, G5) or 50 mg/kg amprolium (G4, G6), and rabbits infected after two weeks of pretreatment with 10 mg/kg L. chinensis eel extract-biosynthesized AgNPs (G7). In this study, both pre-and post-treatment with AgNPs produced a substantial reduction in fecal oocyst output, liver enzyme levels, and histopathological hepatic lesions relative to the infected group. In conclusion, L. chinensis peel extract-prepared AgNPs should be considered harmless and efficient in the cure of hepatic coccidiosis in rabbits.

Evaluation of the Anticoccidial Activity of Sheep Bile against Eimeria stiedae Oocysts and Sporozoites of Rabbits: An In Vitro Study.

Coccidiosis is one of the most common infectious diseases that causes digestive problems in rabbits, leading to global economic losses. This study was conducted to evaluate the effects of bile obtained from sheep gallbladder on the sporulation and morphology of Eimeria stiedae oocysts and sporozoites affecting rabbit liver cells and to determine the best concentration for sporulation inhibition. Sporulation inhibition per milliliter was measured in samples exposed to five concentrations of sheep bile (SB) in a 2.5% potassium dichromate solution: 12.5%, 25%, 50%, 75%, and 100% concentrations for oocysticidal activity and 125, 250, 500, 750, and 1000 µg/mL concentrations for antisporozoidal activity. A bioassay was performed to assess the in vitro anticoccidial activity of sheep bile against E. stiedae oocysts and sporozoite sporulation. In this assay, six-well plates with 5 mL of bile containing 1000 oocysts showed unsporulated oocysticidal activity after 48, 72, and 96 h and antisporozoidal activity after 12 and 24 h. A chemical assay was performed via infrared spectroscopy to investigate the presence of several anticipated active chemical compounds in sheep bile. Sheep bile was able to inhibit E. stiedae oocysts at 100% and 75% concentrations by about 91% and 81%, respectively. In addition, SB had the highest inhibition of E. stiedae sporozoite viability (92%) at a concentration of 1000 µg/mL and had the lowest inhibition of 8% at a concentration of 125 µg/mL. An increase in the incubation time and a higher dose generally increased the inhibition rate. The results showed that sheep gallbladder bile is effective due to its inhibitory potential and effect on the coccidian oocyst sporulation of E. stiedae. Further studies are needed to determine the precise active chemicals present in SB and their modes of action and application in vivo.

Preliminary evaluation of the protective effects of recombinant AMA1 and IMP1 against Eimeria stiedae infection in rabbits.

BACKGROUND: Eimeria stiedae parasitizes the bile duct, causing hepatic coccidiosis in rabbits. Coccidiosis control using anticoccidials led to drug resistance and residues; therefore, vaccines are required as an alternative control strategy. Apical membrane antigen 1 (AMA1) and immune mapped protein 1 (IMP1) are surface-located proteins that might contribute to host cell invasion, having potential as candidate vaccine antigens. METHODS: Herein, we cloned and expressed the E. stiedae EsAMA1 and EsIMP1 genes. The reactogenicity of recombinant AMA1 (rEsAMA1) and IMP1 (rEsIMP1) proteins were investigated using immunoblotting. For the vaccination-infection trial, rabbits were vaccinated with rEsAMA1 and rEsIMP1 (both 100 µg/rabbit) twice at 2-week intervals. After vaccination, various serum cytokines were measured. The protective effects of rEsAMA1 and rEsIMP1 against E. stiedae infection were assessed using several indicators. Sera were collected weekly to detect the specific antibody levels. RESULTS: Both rEsAMA1 and rEsIMP1 showed strong reactogenicity. Rabbits vaccinated with rEsAMA1 and rEsIMP1 displayed significantly increased serum IL-2 (F (4, 25) = 9.53, P = 0.000), IL-4 (F (4, 25) = 7.81, P = 0.000), IL-17 (F (4, 25) = 8.55, P = 0.000), and IFN-γ (F (4, 25) = 6.89, P = 0.001) levels; in the rEsIMP1 group, serum TGF-ß1 level was also elevated (F (4, 25) = 3.01, P = 0.037). After vaccination, the specific antibody levels increased and were maintained at a high level. The vaccination-infection trial showed that compared with the positive control groups, rabbits vaccinated with the recombinant proteins showed significantly reduced oocyst output (F (5, 54) = 187.87, P = 0.000), liver index (F (5, 54) = 37.52, P = 0.000), and feed conversion ratio; body weight gain was significantly improved (F (5, 54) = 28.82, P = 0.000). CONCLUSIONS: rEsAMA1 and rEsIMP1 could induce cellular and humoral immunity, protecting against E. stiedae infection. Thus, rEsAMA1 and rEsIMP1 are potential vaccine candidates against E. stiedae.

Global transcriptome landscape of the rabbit protozoan parasite Eimeria stiedae.

BACKGROUND: Coccidiosis caused by Eimeria stiedae is a widespread and economically significant disease of rabbits. The lack of studies on the life-cycle development and host interactions of E. stiedae at the molecular level has hampered our understanding of its pathogenesis. METHODS: In this study, we present a comprehensive transcriptome landscape of E. stiedae to illustrate its dynamic development from unsporulated oocysts to sporulated oocysts, merozoites, and gametocytes, and to identify genes related to parasite-host interactions during parasitism using combined PacBio single-molecule real-time and Illumina RNA sequencing followed by bioinformatics analysis and qRT-PCR validation. RESULTS: In total, 12,582 non-redundant full-length transcripts were generated with an average length of 1808 bp from the life-cycle stages of E. stiedae. Pairwise comparisons between stages revealed 8775 differentially expressed genes (DEGs) showing highly significant description changes, which compiled a snapshot of the mechanisms underlining asexual and sexual biology of E. stiedae including oocyst sporulation between unsporulated and sporulated oocysts; merozoite replication between sporulated oocysts and merozoites; and gametophyte development and gamete generation between merozoites and gametocytes. Further, 248 DEGs were grouped into nine series clusters and five groups by expression patterns, and showed that parasite-host interaction-related genes predominated in merozoites and gametocytes and were mostly involved in steroid biosynthesis and lipid metabolism and carboxylic acid. Additionally, co-expression analyses identified genes associated with development and host invasion in unsporulated and sporulated oocysts and immune interactions during gametocyte parasitism. CONCLUSIONS: This is the first study, to our knowledge, to use the global transcriptome profiles to decipher molecular changes across the E. stiedae life cycle, and these results not only provide important information for the molecular characterization of E. stiedae, but also offer valuable resources to study other apicomplexan parasites with veterinary and public significance.

Study on impression smears of hepatic coccidiosis in rabbits.

Hepatic coccidiosis is a contagious and lethal disease condition in rabbits. The disease was recorded in six rabbits suffering with watery diarrhoea. Clinically, affected rabbits showed decreased growth rate, anorexia, debilitation, diarrhea and rough hair coat. Examination of the faecal samples revealed the presence of unsporulated oocysts of Eimeria spp. After sporulation Eimeria stiedae oocysts were identified. Postmortem examination revealed hepatomegaly with presence of discrete yellowish-white nodules on the surface of the liver. Impression smears from the liver revealed the presence of numerous developmental stages of E. stiedae corresponding with the stage of the liver lesion and also represent the histological changes of the liver. Rabbits were treated with a combination of sulphaquinoxaline and diaveridine for five days.

Prevalence, pathology and risk factors for coccidiosis in domestic rabbits (Oryctolagus cuniculus) in selected regions in Kenya.

BACKGROUND: The potential for rabbit production in Kenya is high. However, high morbidity and mortality of domestic rabbits were reported. OBJECTIVE: The aim of the study was to determine the pathology, prevalence and the predisposing factors to coccidiosis in domestic rabbits in selected regions in Kenya. ANIMALS AND METHODS: A total of 61 farms keeping rabbits in six different counties were visited in the survey. A total of 2680 live rabbits were examined and 61 rabbits and 302 fecal samples were randomly collected from the farms and examined for coccidian oocysts by ante-mortem and post-mortem methods. The predisposing factors to coccidiosis were assessed through questionnaires and direct observation. Chi square (χ(2)) statistics was used with P values < 0.05 considered statistically significant. RESULTS: Of the 302 fecal samples, 85% (P < 0.001) contained coccidian oocysts and 2% harbored nematode eggs (Passalurus ambiguous). The overall prevalence of Eimeria spp. infestation was 85.1% in the study area and 90.2% in the individual rabbits, while prevalence of intestinal coccidiosis and hepatic coccidiosis was 29.5% and 11.5%, respectively. Higher counts of coccidian oocysts per gram of feces were recovered in weaners than in growers and adults rabbits (P < 0.001), rabbits that were kept in high density group housing (P < 0.05) and housing with more than two tiers. CONCLUSION: This study identified group housing of rabbits of different ages and inadequate control of concurrent infections as the major risk factors associated with coccidiosis in domestic rabbits in Kenya.