Chemo-prophylaxis of turkey coccidiosis: Activity of clopidol with methylbenzoquate and amprolium with ethopabate against a mixed infection of Eimeria meleagrimitis and Eimeria adenoeides

A comparison has been made between the use of clopidol-methyl benzoquate and amprolium-ethopabate mixtures for the prophylactic control of Eimeria adenoeides and Eimeria meleagrimitis in turkeys. Twenty four poults were randomly allocated to each of the following treatments: non contaminated non treated; contaminated non treated; contaminated treated with 100 ppm + 8.35 ppm clopidol methyl benzoquate; contaminated treated with 200 ppm + 16.70 ppm clopidol methyl benzoquate; contaminated treated with 125 ppm + 8 ppm amprolium ethopabate. The amprolium-ethopabate mixture only partially controlled an infection with 50,000 sporulated oocysts of E. adenoeides and 200,000 oocysts of E. meleagrimitis. The clopidol-methyl benzoquate mixture completely controlled such an infection at the higher concentration (200 ppm + 16.70 ppm) and almost completely controlled it at the lower concentration (100 ppm+ 8.35 ppm). Drug withdrawal studies showed that the clopidol-methyl benzoquate mixture is coccidiostatic for the two species studied.     

Selection of a precocious line of the rabbit coccidium Eimeria flavescens Marotel and Guilhon (1941) and characterisation of its endogenous cycle

The SPF rabbits were inoculated with oocysts of Eimeria flavescens and the first newly developed oocysts were recovered. They were used for inoculation of other rabbits which conseqeuntly excreted oocysts sooner than in the previous passage. By repeated use of this method, the prepatent period was shortened after 18 passages by more than 60 h. The endogenous development of this precocious line (PL) differed from that of the original strain (OS). Compared to OS, two asexual generations, second (or third) and fourth, were absent in PL. The first merogony took place in the jejunum and ileum in OS and, in contrast, in the large intestine in PL. Like in other rabbit coccidia, two types of meronts (A and B) were seen in each generation. However, the ratio of B: A meronts in the last (fifth) asexual generation as well as ratio of microgamonts:macrogamonts differs in OS and PL.     

Selection and characterization of a precocious line ofEimeria intestinalis,an intestinal rabbit coccidium

A precocious line ofEimeria intestinalis was obtained by selection for early development of oocysts in rabbits and after six consecutive passages in animals. This line (EiP) was derived from a wild strain (EiO) isolated in 1975 from the caecal content of a rabbit with coccidiosis. The prepatent period of the EiP strain was reduced from 215 h to<144 h, the result being that the oocyst sporulation time was the same for both lines. The excreted and unsporulated oocysts had exactly the same shape, but microscopical examination of the sporulated oocysts showed a marked difference between EiP and EiO strains. A huge refractile globule was located in each of two sporocysts of the precocious line, whereas no refractile globule was seen in the other two. The EiP line had a reproductive potential much lower (1000 times) than that of its parent strain EiO and, as judged by the weight gain, mortality and lesions that also occurred in the jejunum and above all in the ileum, its pathogenicity was substantially reduced.     

Electron microscopic study on the endogenous development of Eimeria mulardi, Chauve, Reynaud and Gounel, 1994: a coccidium from the mule duck

An electron microscopic study of the endogenous development of Eimeria mulardi Chauve, Reynaud and Gounel, 1994 was carried out in mule ducks which are hybrids of the domestic duck (Anas platyrhynchos) and the muscovy duck (Cairina moschata). All of the endogenous stages were seen within the nucleus of the host cell. Merozoites arose from ectomerogony and three mutually similar merogonies were noted. The asexual stages were found in leukocyte-like cells in the lamina propria of the jejunum, ileum and caecum, while the gamonts developed in glandular epithelial cells in the same part of the intestine.     

Characterization and immunoprotective properties of a monoclonal antibody against the major oocyst wall protein of Eimeria tenella.

The oocyst wall of Eimeria spp. consists of a 10-nm-thick outer lipid layer and a 90-mm-thick inner layer of glycoprotein which has been described previously to be composed of a single major protein. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions and (125)I labelling of a oocyst wall fragments and of delipidated intact oocysts revealed a molecule of approximately 12 kDa as the major protein component of the oocyst wall of Eimeria tenella. An immunoglobulin M monoclonal antibody (c11B9F3) was produced against this 12-kDa oocyst wall protein sliced from a preparative SDS-polyacrylamide gel. Its reactivity by immunofluorescence against oocyst wall fragments and sporozoites or by immunoperoxidase assays of infected tissue sections was stage restricted to gametocytes and oocysts but pan-specific against all face of the oocyst wall. In chicks passively immunized with C11B9F3, oocyst output was significantly (P<0.01) reduced by 42 to 54% after homologous E. tenella infection and by 35% after heterologous Eimeria maxima infection compared with that of control groups. The results demonstrate the presence of a highly conserved, low-molecular-weight antigen on the oocyst wall and the gametocytes of Eimeria spp. which is a candidate for inclusion in a pan-specific, transmission-blocking vaccine against avian coccidiosis.     

Single oocyst infection: a simple method for isolation of Eimeria spp. from the mixed field samples

The study described a simple method for single oocyst infection which is usually used to maintain the Eimeria spp. as pure strains in the laboratory and to isolate a single species from the mixed field samples.     

The pathogenicity, immunogenicity and endogenous development of a precocious line of Eimeria brunetti

A precocious line of Eimeria brunetti with a prepatent period of 75 hours was compared with the parent strain (normal prepatent period of 120 hours) in a battery study. The precocious line was less pathogenic than the parent, and caused less weight depression and lower lesion scores in chickens given any dose of oocysts (1,000, 5,000, 25,000 or 125,000). No chickens receiving the precocious line died, whilst 19% and 25% mortality occurred in groups receiving 25,000 and 125,000 oocysts of the parent strain. The precocious line was also highly immunogenic. Chickens given 1,000 or 5,000 oocysts of the precocious line or parent strain developed immunity sufficient to protect against a challenge of the parent strain which caused 83% weight depression and 13% mortality in non-immunised, challenged controls. Histological observations showed that the endogenous development of the parent strain and precocious line was identical up to 48 hours. However, by 72 hours macrogametocytes and microgametes were seen only with the precocious line. The abbreviated asexual cycle of the precocious line would account for its lower reproductive potential.     

A merozoite-specific 22-kDa rhoptry protein of the coccidium Eimeria nieschulzi (Sporozoa,Coccidia) is exocytosed in the parasitophorous vacuole upon host cell invasion

A monoclonal antibody (Mab D12A4) was used to follow the genesis and fate of rhoptries from early first-generation merogony through second-generation merozoites of the rat coccidium Eimeria nieschulzi. The epitope recognized by Mab D12A4 belongs to a 22-kDa protein which can be localized first in developing meronts 25 h post-infection. Early rhoptries appear as distinct granules in the cytoplasm of developing meronts and elongate into mature organelles before merozoite release. The 22-kDa protein is found in the parasitophorous vacuole after host cell invasion. Western blotting and immunofluorescence showed that the 22-kDa rhoptry protein is expressed in schizonts and merozoites but not in sporozoites. Received: 9 August 1997 / Accepted: 14 October 1997     

Characterization of rhoptry proteins of Eimeria tenella sporozoites: antigenic diversity of rhoptry epitopes within species of the genus Eimeria and among three asexual generations of a single species, E. tenella.

Rhoptry organelles from sporozoites of the apicomplexan parasite Eimeria tenella contain at least 60 independent polypeptides that can be resolved by two-dimensional gel electrophoresis. Rhoptries from three species of Eimeria that infect chickens share very few antibody cross-reactive epitopes, and there is poor conservation of epitopes among three distinct asexual generations of zoites within the developmental life cycle of a single parasite, E. tenella.     

Activation of murine macrophages and a bovine monocyte cell line by bovine lymphokines to kill the intracellular pathogens Eimeria bovis and Toxoplasma gondii.

Macrophage (M phi)-activating lymphokines present in concanavalin A-stimulated bovine T-lymphocyte cultures (ConAS) were studied by assessing their effects on Eimeria bovis and Toxoplasma gondii growth in cultured bovine monocytes (BM) and mouse M phi. The in vitro development of both parasites was assessed by incorporation of [3H]uracil and by microscopic examination of parallel cultures. Incorporation of [3H]uracil into infected cultures was an accurate indicator of growth of both E. bovis and T. gondii in BM and mouse M phi. Sporozoites of E. bovis underwent merogony in untreated BM but not in mouse M phi, whereas T. gondii developed in both cell types. Inhibition of T. gondii growth was greatest in ConAS-treated BM, whereas preincubation of mouse M phi with ConAS resulted in about 80% growth inhibition. There was no significant difference between the inhibition of either T. gondii sporozoite- or tachyzoite-induced growth in ConAS-treated cells, showing that activation pathways are equally effective against both stages. Treatment of ConAS with glycine-hydrochloride buffer (pH 2) resulted in a total loss of antiviral activity mediated by gamma interferon (IFN-gamma). When pH 2 dialyzed ConAS was used to activate BM, inhibition of T. gondii growth was only partially affected. Because bovine IFN-gamma does not activate mouse M phi and due to the partial effects of pH 2 on ConAS-induced growth inhibition, the major component(s) of ConAS responsible for T. gondii growth inhibition is distinct from IFN-gamma. Furthermore, IFN-gamma may act synergistically rather than being part of a priming sequence for M phi responsiveness to other lymphokines. Murine recombinant granulocyte-macrophage colony-stimulating factor (rGM-CSF) was tested for any microbistatic activity against T. gondii sporozoites and tachyzoites. There was no significant difference in either colony formation or [3H]uracil incorporation between rGM-CSF-treated and control cultures, regardless of host cell type. Thus, rGM-CSF does not induce adequate M phi activation to kill T. gondii and is not a major microbistatic component of ConAS. rGM-CSF also had no effect on T. gondii infection in vivo.     

First record of Eimeria furonis infection in a ferret, Japan, with notes on the usefulness of partial small subunit ribosomal RNA gene sequencing analysis for discriminating among Eimeria species

Eimeria is a genus of apicomplexan parasites found in a variety of vertebrates including the weasel. At present, three species have been reported in members of the weasel family, but the presence of weasel Eimeria in Japan have been quite unclear. The identification of Eimeria species has been performed based on oocyst morphology, host species, and habitat in the host, but sometimes discriminating among morphologically similar species under light microscopy is impossible. The present study detected for the first time the weasel coccidium, E. furonis, in a ferret in Japan. Since molecular information for E. furonis has been quite unclear, this species was compared molecularly with other Eimeria species, and also the usefulness of sequencing analysis of the small subunit ribosomal ribonucleic acid gene (SSUrDNA) for discriminating among Eimeria species was examined. About the 350-bp sequence of SSUrDNA of all species including E. furonis compared differed from each other, and its differences found in Eimeria species were also reflected in the phylogram constructed using the partial nucleotide sequences. The sequencing analysis of partial SSUrDNA examined in the present study thus appears useful for discriminating among morphologically similar Eimeria species.     

Genetic dissection of primary and secondary responses to a widespread natural pathogen of the gut, Eimeria vermiformis

Because most pathogens initially challenge the body at epithelial surfaces, it is important to dissect the mechanisms that underlie T-cell responses to infected epithelial cells in vivo. The coccidian parasites of the genus Eimeria are protozoan gut pathogens that elicit a potent, protective immune response in a wide range of host species. CD4+ alpha beta T cells and gamma interferon (IFN-gamma) are centrally implicated in the primary immunoprotective response. To define any additional requirements for the primary response and to develop a comparison between the primary and the secondary response, we have studied Eimeria infections of a broad range of genetically altered mice. We find that a full-strength primary response depends on beta(2)-microglobulin (class I major histocompatibility complex [MHC] and class II MHC and on IFN-gamma and interleukin-6 (IL-6) but not on TAP1, perforin, IL-4, Fas ligand, or inducible nitric oxide synthetase. Indeed, MHC class II-deficient and IFN-gamma-deficient mice are as susceptible to primary infection as mice deficient in all alpha beta T cells. Strikingly, the requirements for a highly effective alpha beta-T-cell-driven memory response are less stringent, requiring neither IFN-gamma nor IL-6 nor class I MHC. The class II MHC dependence was also reduced, with adoptively transferable immunity developing in MHC class II(-/-) mice. Besides the improved depiction of an immune response to a natural gut pathogen, the finding that effective memory can be elicited in the absence of primary effector responses appears to create latitude in the design of vaccine strategies.     

Developmental gene expression of a 230-kilodalton macrogamete-specific protein of the avian coccidial parasite, Eimeria maxima.

We prepared a cDNA library from gametocytes of Eimeria maxima and screened it using antibodies raised against an 82-kDa gametocyte antigen. One cDNA clone designated pEM230 was isolated and characterized. It encodes a portion of a 230-kDa gametocyte protein and its DNA sequence shows the presence of several tandem repeats of 42 bp. In order to determine the stage and sex specificity of the mRNA for the 230-kDa protein, Northern blotting and in situ hybridization studies were performed. The 230-kDa protein is encoded for by a 7 kb mRNA, which is expressed exclusively during the macrogamete stage with no detectable expression seen in any other stage of parasite development.     

LuSIV cells: a reporter cell line for the detection and quantitation of a single cycle of HIV and SIV replication

A single cycle of viral replication is the time required for a virus to enter the host cell, replicate its genome, and produce infectious progeny virions. The primate lentiviruses, human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV), require on average 24 h to complete one cycle of replication. We have now developed and characterized a reporter assay system in CEMx174 cells for the quantitative measurement of HIV/SIV infection within a single replication cycle. The SIV(mac)239 LTR (-225 --> +149) was cloned upstream of the firefly luciferase reporter gene and this reporter plasmid is maintained in CEMx174 cells under stable selection. This cell line, designated LuSIV, is highly sensitive to infection by primary and laboratory strains of HIV/SIV, resulting in Tat-mediated expression of luciferase, which correlates with viral infectivity. Furthermore, manipulation of LuSIV cells for the detection of luciferase activity is easy to perform and requires a minimal amount of time as compared to current HIV/SIV detection systems. The LuSIV system is a powerful tool for the analysis of HIV/SIV infection that provides a unique assay system that can detect virus replication prior to 24 h and does not require virus to spread from cell to cell. Thus these cells can be used for the study of replication-deficient viruses and the high throughput screening of antivirals, or other inhibitors of infection.     

Efficient transposition of a single Minos transposon copy in the genome of the ascidian Ciona intestinalis with a transgenic line expressing transposase in eggs

Transgenesis with transposons is an important technique for studying genetic functions. In the ascidian Ciona intestinalis, methods for germline transformation with the Tc1/mariner transposon Minos have been established. A system to remobilize a single Minos copy in the genome is needed to refine this transgenic technique. In this study, such an experimental system was established with a transgenic line expressing Minos transposase in eggs. In the eggs of a double transgenic animal from a cross between the egg transposase line and a transgenic line having a single Minos insertion, the transposon was transposed into new positions of the Ciona genome, thus creating new insertions. Some of the new insertions caused enhancer detection. The majority of the new insertion sites were mapped on different chromosomes from that of the transposon donor. This characteristic of Minos is in contrast to that of the Sleeping Beauty transposon, which causes frequent intrachromosomal transposition. Developmental Dynamics 239:1076–1088, 2010. © 2010 Wiley‐Liss, Inc.     

Sensitivity of field isolates of Eimeria acervulina to salinomycin, maduramicin, and a mixture of clopidol and methyl benzoquate in the chicken

Lerbek was more effective than salinomycin, and salinomycin more effective than maduramicin, against field isolates of E. acervulina obtained from farms where salinomycin had been used continuously for 30 crops. Salinomycin was the most effective drug against isolates of E. acervulina from farms where various shuttle programmes had been employed. Lerbek and maduramicin were ineffective against these isolates.