Anticoccidial effect of Origanum majoranum aqueous extract on Eimeria tenella-infected chicken

@#Avian coccidiosis, an important protozoal disease of chicken triggered by coccidian protozoa of genus Eimeria, causes considerable economic losses to broiler producers. The study was designed to assess the efficiency of Origanum majoranum aqueous extract (OMAE) on E. tenella-infected broiler chicken. Birds were divided into four groups including: positive control (PC, challenged with 5×104 sporulated oocysts of E. tenella at the 12th day of age), PC+OMAE (challenged with E. tenella oocysts at the 12th day of age and received OMAE (125 mg/kg BW) orally, started at the 7th day of age, and continued for 14 consecutive days), OMAE (received OMAE (125 mg/kg BW) orally, at the 7th day of age, for 14 consecutive days), and negative control (received basal diet only). Anticoccidial efficacy of OMAE was evaluated by complete blood picture, serum chemistry, serum protein electrophoresis, antioxidants markers, cecal oocysts count, and cecal lesions score. Briefly, collected data indicated that supplementation of OMAE could increase antioxidants concentrations and improve changes in hematobiochemical parameters and serum protein fractions, as well as decrease cecal oocysts count and reduce cecal lesion scores in E. tenella-infected birds. In conclusion, OMAE restores oxidant-antioxidant balance, and its supplementation in broiler chicken can alleviate E. tenella-infection and reduce its severity.

Immunological and histopathological evaluation of Eimeria tenella oocysts Egyptian local isolate vaccine and its comparative efficacy with a commercial live vaccine

@#Coccidiosis is the most important protozoan disease in broilers all over the world. Controlling of broilers coccidiosis via vaccination rather than chemicals is a new trend with promising results. Thus, the present work describes an evaluation of Eimeria tenella Lab-made vaccine of local Egyptian strain and its comparative efficacy with a commercial live vaccine “Fortegra®”. Eighty broiler chickens one day old were used; they were divided in to 4 equal groups; 20 chicks each. Group 1 (G1) kept as control negative, G2 administrated orally with lab-made sporulated oocysts vaccine at 5 days old, the birds of G3 vaccinated orally with Fortegra® at day 6 of age, and G4 served as control positive. All birds were challenge by 50,000 sporulated oocysts of E. tenella at day 21. For testing the efficacy and comparison; OPG (oocyst per gram), serum Interleukin4 (IL4) levels, Immunoglobulin A (IgA) levels in both serum and ceca, cecal lesion score, as well as histopathological changes in ceca of tested groups were evaluated. The results demonstrated significantly elevated IL4 level in serum and IgA level in serum and cecum of G2 than G3. IgA in cecum significantly elevated in G2 than G3. OPG significantly decreased in both vaccinated groups (G2 and G3), and have lower lesion score than nonimmunized group. Cecal tissues of vaccinated groups had mild pathological changes. Conclusively, good immunization by the currently tested vaccine, against experimental E. tenella infection was observed.

Identification and Characterization of the ATG8, a Marker of Eimeria tenella Autophagy / Identificação e Caracterização do ATG8, um marcador de autofagia em Eimeria tenella

Abstract Autophagy plays an important role in maintaining cell homeostasis through degradation of denatured proteins and other biological macromolecules. In recent years, many researchers focus on mechanism of autophagy in apicomplexan parasites, but little was known about this process in avian coccidia. In our present study. The cloning, sequencing and characterization of autophagy-related gene (Etatg8) were investigated by quantitative real-time PCR (RT-qPCR), western blotting (WB), indirect immunofluorescence assays (IFAs) and transmission electron microscopy (TEM), respectively. The results have shown 375-bp ORF of Etatg8, encoding a protein of 124 amino acids in E. tenella, the protein structure and properties are similar to other apicomplexan parasites. RT-qPCR revealed Etatg8 gene expression during four developmental stages in E. tenella, but their transcriptional levels were significantly higher at the unsporulated oocysts stage. WB and IFA showed that EtATG8 was lipidated to bind the autophagosome membrane under starvation or rapamycin conditions, and aggregated in the cytoplasm of sporozoites and merozoites, however, the process of autophagosome membrane production can be inhibited by 3-methyladenine. In conclusion, we found that E. tenella has a conserved autophagy mechanism like other apicomplexan parasites, and EtATG8 can be used as a marker for future research on autophagy targeting avian coccidia.

Resumo A autofagia desempenha um papel importante na manutenção da homeostase celular através da degradação de proteínas desnaturadas e outras macromoléculas biológicas. Nos últimos anos, muitos pesquisadores se concentraram no mecanismo da autofagia em parasitas apicomplexos, mas pouco se sabe sobre esse processo na coccidia aviária. No presente estudo, a clonagem, sequenciamento e caracterização de gene relacionado à autofagia Etatg8 foram investigados pela PCR quantitativa em tempo real (RT-qPCR), mancha ocidental (WB), ensaios indiretos de imunofluorescência (IFAs) e microscopia eletrônica de transmissão (TEM), respectivamente. Os resultados mostraram que o gene Etatg8 de E. tenella possui uma ORF de 375 bp, codificando uma proteína de 124 aminoácidos com estrutura e propriedades semelhantes à de outros apicomplexos. RT-qPCR revelou que Etatg8 é expresso durante os quatro estágios de desenvolvimento de E. tenella. Entretanto, seus níveis transcricionais foram significativamente mais elevados na fase de oocisto não esporulados. Os ensaios de manchas ocidental (WB) e de imunofluorescência (IFA) mostraram que a proteína EtATG8 foi lipidada para ligar-se à membrana do autofagossomo sob condições de deficiência nutritiva (em presença de rapamicina) e se agregar no citoplasma de esporozoítas e merozoítas. No entanto, o processo de produção de membrana do autofagossomo pode ser inibido por um inibidor de autofagia (3-meetiladeninatiladenina, 3-MA). Em conclusão, foi demonstrado que E. tenella tem um mecanismo de autofagia conservado, semelhante ao de outros parasitas apicomplexos, e que EtATG8 pode ser usado como um marcador para futuras pesquisas sobre autofagia direcionada à coccidiose aviária.

Anticoccidial effects of the Plantago asiatica extract on experimental Eimeria tenella infection / 한국실험동물학회지

Anticoccidial effects of the Plantago asiatica extract (PAE) were evaluated in chickens following oral infection with Eimeria (E.) tenella. This study was conducted on the 3-day-old chickens (n=30). Those animals were divided with 3 groups; PAE 0.1% treated/infected (n=10), PAE untreated/infected (n=10) and non-infected control (n=10). Chickens were fed a standard diet supplemented with or without PAE for 1 week prior to infection with E. tenella (10,000 sporulated oocysts per chicken). The effects of PAE on E. tenella infection were assessed by two parameters; fecal oocysts shedding and body weights gain. The PAE-fed chickens produced significantly reduced fecal oocysts (P<0.05) when compared to the E. tenella-infected group fed standard diet. Also, PAE-based diet, improved body weight loss caused by E. tenella infection. Our data demonstrated that PAE had remarkable anticoccidial activities against E. tenella. This finding might have implications for the development of anticoccidial drug. This study is the first to demonstrate anticoccidial effect of PAE on Eimeria parasites.

Effect of Diclazuril on the Bursa of Fabricius Morphology and SIgA Expression in Chickens Infected with Eimeria tenella

The effects of diclazuril on the bursa of Fabricius (BF) structure and secretory IgA (SIgA) expression in chickens infected with Eimeria tenella were examined. The morphology of the BF was observed by hematoxylin and eosin staining, while ultrastructural changes were monitored by transmission electron microscopy. E. tenella infection caused the BF cell volumes to decrease, irregularly arranged, as well as, enlargement of the intercellular space. Diclazuril treatment alleviated the physical signs of damages associated with E. tenella infection. The SIgA expression in BF was analyzed by immunohistochemistry technique. The SIgA expression increased significantly by 350.4% (P<0.01) after E. tenella infection compared to the normal control group. With the treatment of diclazuril, the SIgA was relatively fewer in the cortex, and the expression level was significantly decreased by 46.7% (P<0.01) compared with the infected and untreated group. In conclusion, E. tenella infection in chickens induced obvious harmful changes in BF morphological structure and stimulated the expression of SIgA in the BF. Diclazuril treatment effectively alleviated the morphological changes. This result demonstrates a method to develop an immunological strategy in coccidiosis control.

A Novel Recombinant BCG Vaccine Encoding Eimeria tenella Rhomboid and Chicken IL-2 Induces Protective Immunity Against Coccidiosis

A novel recombinant Bacille Calmette-Guerin (rBCG) vaccine co-expressed Eimeria tenella rhomboid and cytokine chicken IL-2 (chIL-2) was constructed, and its efficacy against E. tenella challenge was observed. The rhomboid gene of E. tenella and chIL-2 gene were subcloned into integrative expression vector pMV361, producing vaccines rBCG pMV361-rho and pMV361-rho-IL2. Animal experiment via intranasal and subcutaneous route in chickens was carried out to evaluate the immune efficacy of the vaccines. The results indicated that these rBCG vaccines could obviously alleviate cacal lesions and oocyst output. Intranasal immunization with pMV361-rho and pMV361-rho-IL2 elicited better protective immunity against E. tenella than subcutaneous immunization. Splenocytes from chickens immunized with either rBCG pMV361-rho and pMV361-rho-IL2 had increased CD4+ and CD8+ cell production. Our data indicate recombinant BCG is able to impart partial protection against E. tenella challenge and co-expression of cytokine with antigen was an effective strategy to improve vaccine immunity.

Effects of Different Sizes of Glass Beads on the Release of Sporocysts from Eimeria tenella Oocysts

The oocyst wall is severed by means of mechanical injury or chemical agents. This study reports the percentage of in vitro sporocyst release following mechanical shaking in the presence of varying sizes of glass beads. Glass beads measured 0.5, 1, and 3 mm in diameter and were shaken with the oocysts for different times ranging from 5 sec to 5 min. Approximately 80% of sporocysts were released with 5 min of shaking in the presence of 3 mm glass beads, as well as 30 sec with 0.5 mm beads and 1 mm glass beads. The release of sporocysts of E. tenella was most efficient using 1 mm glass beads and treatment times of 30 sec to 1 min. Therefore, the use of 1 mm glass beads with 30 sec to 1 min of agitation is recommended in order to maximize sporocyst release and recovery and to improve the yield of viable sporozoites for use in biochemical, tissue culture, and immunological applications of coccidia.

Suppression of Eimeria tenella Sporulation by Disinfectants

The disinfectant effects (DEs) of 10 types of chemicals, defined by their ability to destroy or inhibit oocysts and consequently prevent sporulation of Eimeria tenella field isolate, were evaluated in vitro. Correct species assignments and sample purities were confirmed by the singular internal transcribed spacer (ITS)-PCR analysis. A total of 18 treatments were performed, and the disinfection suppression levels were 75.9% for 39% benzene + 22% xylene (1:10 dilution), 85.5% for 30% cresol soup (1:1 dilution), and 91.7% for 99.9% acetic acid (1:2 dilution) group. The results indicate that acetic acid, cresol soup, and benzene+xylene are good candidates for suppression of E. tenella oocyst sporulation.

Anticoccidial effects of Galla rhois extract on Eimeria tenella-infected chicken / 한국실험동물학회지

Anticoccidial effects of Galla rhois (GR) extract were evaluated in chickens after oral infection with Eimeria tenella. This study was performed using 3-day-old chickens (n=30). The animals were divided into 3 groups as follows: GR 0.5%/infected (n=10), untreated/infected (n=10), and non-infected control (n=10). The chickens were fed a standard diet supplemented with or without GR for 1 week before infection with E. tenella (10,000 sporulated oocysts per chicken). The effects of GR on E. tenella infection were assessed by 2 parameters, number of fecal oocysts and body weight gain, and the results of the polymerase chain reaction (PCR). The GR-fed chickens produced significantly lower number of fecal oocysts (P<0.05) than the E. tenella-infected chickens who were fed the standard diet. In addition, GR-based diet improved the loss of body weight caused by E. tenella infection. Positive findings of PCR were identified by distinct bands in the samples of E. tenella-inoculated chickens. However, PCR analysis revealed no E. tenella oocysts in the feces of GR-fed chickens. Our data showed that GR extracts had remarkable anticoccidial activities against E. tenella. This finding might have implications for the development of novel anticoccidial drugs.

Anticoccidial activity of hydrochloric acid (HCl) against Eimeria tenella in broiler chickens

The present study was planned to evaluate the anticoccidial activity of the different concentrations of the HCl against Eimeria tenella infection in broiler chickens in comparison with the amprolium anticoccidial. For this purpose, a total of 198 chicks were placed 11 per pen with three pens per treatment. The different concentrations of HCl (1000ppm, 2000ppm and 3000ppm) and amproilum (at the dose rate of 125ppm) were given to the experimental groups in drinking water from 10 to 19th days of age. One group was kept as infected non medicated control and one as non infected non medicated control. At the 12th day of age, all the groups were inoculated orally with 75,000 sporulated oocysts except non infected non medicated control. Anticoccidial activity was evaluated on the basis of performance (weight gain, feed conversion ratio) and pathogenic (oocyst score, lesion score and mortality percentage) parameters. Among HCl medicated groups, the maximum anticoccidial effect was seen in the group medicated with 1000ppm HCl followed by 2000ppm and 3000ppm HCl medicated groups. Amprolium and 1000ppm HCl were almost equivalent in suppressing the negative performance and pathogenic effects associated with coccidiosis (Eimeria tenella) challenge. In summary, the lower doses of HCl have the potential to be used as alternative to chemotherapeutic drugs for Eimeria tenella control. It is therefore suggested that further studies should be carried out to determine the possible minimum safe levels of HCl with least toxic effects to be used as anticoccidial.

Anticoccidial effects of acetic acid on performance and pathogenic parameters in broiler chickens challenged with Eimeria tenella

The objective of the present study was to evaluate the anticoccidial effect of the different concentrations of the acetic acid in the broiler chickens in comparison with the amprolium anticoccidial. A total of 198 chicks were placed 11 per pen with three pens per treatment. The different concentrations (1 percent, 2 percent and 3 percent) of acetic acid and amproilum (at the dose rate of 125ppm) were given to the experimental groups in drinking water from 10-19th days of age. One group was kept as infected non medicated control and one as non infected non medicated control. All the groups were inoculated orally with 75,000 sporulated oocysts at the 12th day of age except non infected non medicated control. Anticoccidial effect was evaluated on the basis of performance (weight gain, feed conversion ratio) and pathogenic (oocyst score, lesion score and mortality percentage) parameters. Among acetic acid medicated groups, the maximum anticoccidial effect was seen in the group medicated with 3 percent acetic acid followed by 2 percent and 1 percent acetic acid medicated groups. Amprolium and 3 percent acetic acid were almost equivalent in suppressing the negative performance and pathogenic effects associated with coccidiosis (Eimeria tenella) challenge. In summary, acetic acid has the potential to be used as alternative to chemotherapeutic drugs for Eimeria tenella control. Concentration-dependent anticoccidial effect of acetic acid suggests that further studies should be carried out to determine the possible maximum safe levels of acetic acid with least toxic effects to be used as anticoccidial.

Post-challenge hematological evaluation with virulent strain of Eimeria tenella in broilers immunized with attenuated strain or sporozoite proteins from homologous strain / Avaliação hematológica pós-desafio com cepa virulenta de Eimeria tenella em frangos de corte imunizados com cepa atenuada ou proteínas de esporozoítos de cepa homóloga

Hematological parameters were evaluated in broilers immunized and challenged with Eimeria tenella. Broiler chickens of Hubbard strain, females, coccidian-free, were kept in wire cages and inoculated on the third day. The experiment was designed to include five sorts of treatment with three replicates each. T1 was the negative control group, T2 received 500 attenuated sporulated oocysts by gavage, T3 was the positive control, T4 received 50 µg of sporozoite protein + Quil A vaccine, and T5 received Quil A without sporozoite protein + PBS, the last two through nasal route on days 0, 7, and 21. On the 31st day, all treatments were challenged with homologous virulent strain of E. tenella in the dose of 8.0 × 10(4) oocysts, with the exception of T1. One week later, blood sampling, lesion scores, and cecal oocyst count were carried out. The parasitological parameters showed statistical significance (p < 0.05) and there was no damage to the hematological parameters of birds (p > 0.05) by ANOVA test. The correlations suggest that the blood parameters were impaired by effects of the parasite on tissue, showing levels of hemorrhage and/or hydration.

Foram avaliados os parâmetros hematológicos em frangos de corte imunizados e desafiados com Eimeria tenella. Pintos de corte fêmeas da linhagem Hubbard, livres de coccídios, foram mantidos em baterias metálicas e inoculados no terceiro dia. O experimento foi delineado por cinco tratamentos com três repetições cada, sendo: T1 controle negativo, T2 recebeu 500 oocistos esporulados atenuados via oral, T3 controle positivo, T4 recebeu vacina contendo 50 µg de proteínas de esporozoítos + Quil A e T5 recebeu Quil A + PBS, sendo os dois últimos por via nasal nos dias 0, 7 e 21. No dia 31, todos os tratamentos foram desafiados com cepa virulenta homóloga de E. tenella na dose de 8,0 × 10(4) oocistos, exceto T1. Uma semana depois, foi realizada amostragem de sangue, escore de lesão e contagem de oocistos cecais. Os parâmetros parasitológicos apresentaram significância estatística (p < 0,05), sem que causassem prejuízos aos parâmetros hematológicos das aves (p > 0,05), pelo teste ANOVA. As correlações sugerem que os parâmetros sanguíneos foram afetados pelos efeitos do parasita no tecido, apresentando níveis de hemorragia e/ou hidratação.

Anticoccidial activity of Curcuma longa L. in broilers

Comparative efficacy of turmeric (Curcuma longa L.) crude powder and salinomycin sodium on the occurrence of coccidiosis and growth performance of broiler was evaluated. A total of 90, day-old chicks were randomly divided into six groups. From first day onward, ration was supplemented with 1, 2 and 3 percent turmeric powder in groups 2, 3 and 4, respectively, group 1 received salinomycin sodium @ 12 g 50 kg-1 feed while groups 5 and 6 were kept as infected un-medicated and uninfected un-medicated controls. First five groups were infected with Eimeria tenella sporulated oocysts @ 1,00,000/chick at the age of 20 days. Body weight gain, feed consumption, feed conversion ratio were investigated throughout the experimental period, and bloody diarrhea and oocysts excretions were investigated at the first and the second week after infection. Maximum coccidiostatic effect was observed with turmeric (3 percent) showing mild bloody diarrhea as compared to other infected groups receiving turmeric containing rations. This effect was comparable with a standard coccidiostat i.e., salinomycin sodium. Similarly, the weight gain in the groups treated with salinomycin sodium (2280g) and 3 percent turmeric (2293g) were also significantly higher (p < 0.05) than that of infected control group (1955g). In the groups treated with ration supplemented with 3 percent turmeric powder and salinomycin sodium, the peak excretion of oocysts was delayed about 1 or 2 days relative to the control infected group. Concentration-dependent coccidiostatic effect of turmeric suggested that further studies should be carried out to determine the possible maximum safe levels of turmeric with least toxic effects to be used as coccidiostat.

Some observations on the adaptation of Eimeria tenella (local isolates) sporozoites on chicken embryos through chorioallantoic membrane

Eimeria (E.) tenella (local isolate) sporozoites were adapted on the chorioallantoic membrane (CAM) of 10-12 days chicken embryos and completed its life cycle in 6~7 days at 39 degrees C and 70 per cent humidity. Only 23 embryos (4.6%) were found dead from 1~4 day post inoculation of sporozoites with mild lesions on CAM with no gametocytes but few sporozoites in chorioallantoic fluid (CAF). On 5~7 day post inoculation, 432 embryos (86.4%) were found dead with severe haemorrhages on CAM and CAF contained uncountable number of gametocytes. After seven days post inoculation, 45 embryos (9%) were found to be alive. Some oocysts were also detected in the CAF on 6~7 days post inoculation. In the histological sections of the CAM, there were abundant small dark colored rounded bodies of gametes; distributed extensively in tissues of CAM on 5~7 days post inoculation of sporozoites. In some cases, cluster of small mature and immature relatively large bodies were seen in increasing numbers on 5~6 days post inoculation.