Acroeimeria lineri (McAllister, Upton, Freed, 1988) Paperna and Landsberg, 1989 in Mediterranean Geckos (Hemidactylus turcicus): Oocyst Morphometrics, Endogenous Developmental Stages, and Molecular Sequencing Support its Placement into Acroeimeria.

Between June 2016 and June 2019, we surveyed 62 Mediterranean geckos, Hemidactylus turcicus, from Abu Rawash, Giza, Egypt, for the presence of endoparasites. In June 2016, we found 3 individuals to be infected with Eimeria lineri. We studied the morphology and inner structures of its sporulated oocysts, and the locations of its intestinal endogenous stages. We also extracted genomic DNA from these sporulated oocysts and successfully sequenced a 632-bp fragment of the 18S rRNA gene. Phylogenetic analyses using this partial sequence allowed us to support previous studies that assigned E. lineri to the genus Acroeimeria. Our consensus sequence was used to query similar 18S rDNA sequences from GenBank, and 14 sequences were selected. The phylogenetic analysis inferred by maximum likelihood and Bayesian inference methods gave similar results, as both separated the sequences into 2 clades: (1) a monophyletic group of Goussia species (from fish); and (2) a strongly supported clade that separated 4 Choleoeimeria species from a polyphyletic group of species that clustered A. lineri with 3 other Acroeimeria species and 3 Eimeria species from lizards, including Eimeria tiliquae from Tiliqua rugosa (Gray, 1825), Eimeria tokayae from Gecko gecko (L., 1758), and Eimeria eutropidis from Eutropis macularia (Blyth, 1853). Our study supports the placement of E. lineri into the Acroeimeria and contributes additional life history information toward understanding the evolutionary origin of the Eimeria-like species that have sporocysts without Stieda bodies in their oocysts and that infect saurian reptiles. We also support the concept that several traits (morphological, endogenous, and gene sequences) are both necessary and important for authors to include when making generic reassignments within the eimeriid coccidia.

Preliminary studies on in vitro methods for the evaluation of anticoccidial efficacy/resistance in ruminants.

Ovine Eimeria spp. infections cause increased mortality, reduced welfare and substantial economic losses, and anticocccidials are important for their control. Recent reports of anticoccidial resistance against ovine Eimeria spp. necessitate the development of in vitro methods for the detection of reduced anticoccidial efficacy, especially since the in vivo methods are both expensive, time consuming and requires the use of otherwise healthy animals. The aim of the present study was therefore to approach a preliminary standardization of in vitro assays for evaluation of the efficacy of the most commonly used anticoccidials in ruminants. For this purpose, apart from the evaluation of inhibition of oocyst sporulation, most effort was concentrated on assessment of the capacity of the different anticoccidials to inhibit both the invasion and further development (up to the first schizogony) of E. ninakohlyakimovae sporozoites in bovine colonic epithelial cells (BCEC). For this purpose, infected cultures were monitored 1, 8 and 15 days post infection to determine the infection rate, number of immature schizonts and number, size and appearance of mature schizonts, respectively. No clear inhibitory effect was found with any of the anticoccidial formulations tested, and we could not identify why there were no measurable effects from the different anticoccidials. Despite the lack of positive results, further investigations should be encouraged, as this could decrease the need for animal experiments and could be used in the initial assessment of anticoccidial efficacy of new drugs.

Distribution and Processing of Eimeria nieschulzi OWP13, a New Protein of the COWP Family.

Eimeria species are important veterinary coccidian parasites and are transmitted between hosts via oocysts. The infectious sporozoites are protected by the oocyst and sporocyst wall. Tyrosine-rich proteins are well-known components of the Eimeria oocyst wall. Recently, cysteine motif containing proteins (COWP family), as described in Toxoplasma gondii and Cryptosporidium spp., have also been characterized in Eimeria. Here, we identified a novel COWP-related protein, EnOWP13, and tracked it via transfection technology in Eimeria nieschulzi. The subsequent analysis suggests that the mCherry-tagged EnOWP13 localizes to the wall-forming bodies I and the outer wall. Immunohistochemical analysis confirmed the distribution of wall-forming bodies similar to avian Eimeria species and revealed that the wall-forming bodies I show peroxidase activity. The EnOWP13 amino acid composition and FITC-cadaverine-positive wall-forming bodies I suggest a participation of an enzyme with transglutaminase activity. This is the first description and characterization of this novel outer oocyst wall protein, which is also orthologous to other Eimeria species and Toxoplasma gondii, suggesting a new potential cross-linking mechanism of wall-forming proteins via isopeptide bonds.

Profiling of jejunum inflammatory gene expression during murine eimeriosis.

To understand the host-parasite relationship during coccidiosis it is necessary to identify the transcriptional profile of the local host. In this study, gene profiling in the mouse jejunum due to infection with Eimeria papillata was investigated using Agilent microarray technology. On day 5 post-infection, the characterization of infected and non-infected mice jejunum transcriptional response was compared. There was an increase in the level of tumour necrosis factor-α, nitrite/nitrate and nitric oxide synthase activity was observed following infection. Also, the activity of glutathione peroxidase was reduced from 86.5 to 38.2 mU/g. In addition, E. papillata infection was associated with an increase in the activities of both the mice alkaline phosphatase and lactate dehydrogenase. Moreover, experimental E. papillata infection in mice induced a significant elevation in protein carbonyl content, by about 70%. Agilent genome microarray detected 11 genes whose expression was up-regulated by more than 10-fold, and 30 genes whose expression was down-regulated by a similar amount five days after infection with E. papillata. The expression profiles of the Fas apoptotic inhibitory molecule 3(FAIM3), chemokine (C-X-C motif) receptor 5 (Cxcr5), succinate receptor 1 (SUCNR1), hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 3 (Hsd3b3) and cytochrome P450, family 2, subfamily b, polypeptide 9 (Cyp2b9) genes, arbitrarily selected from the microarray analysis, closely resemble the expressions determined by quantitative PCR. The data indicate that, E. papillata is associated with the induction of inflammatory response and with gene regulation in mice jejunum.

The endogenous development and pathogenicity of Eimeria anseris (Kotlan, 1932) in domestic goslings.

Twenty-one, 25-day-old, artificially reared, coccidia-free goslings (Anser cygnoides var. domestica) were inoculated orally with 0.5 × 104, 1 × 104, or 100 × 104 sporulated oocysts of Eimeria anseris and sacrificed at intervals from 24 to 216 h post-inoculation (HPI). Nine uninfected goslings served as negative controls. Parts of the visceral organs from goslings, including the intestines, kidneys, and liver, were fixed, sectioned, and observed microscopically. The results revealed that two generations of meronts occurred in the life cycle of E. anseris. The first generation of meronts developed at 24-96 HPI and the second generation at 90-128 HPI. Each meront contained 4-10 merozoites. Development of gamonts began at 128 HPI and mature oocysts appeared at 168 HPI. Developmental stages presented mainly in the epithelial cells of crypts and lamina propria in the posterior parts of the jejunum and ileum. Parasites localized mostly in the cytoplasm and occasionally in the nuclei of host cells. Histological lesions were pronounced in the jejunum and ileum. Desquamation and necrosis of the epithelium of intestine and crypts, infiltration of inflammatory cells, and hemorrhage and mucosal edema were associated with aggregates of endogenous stages. The infected goslings mainly showed severe diarrhea, depression, anorexia, and emaciation, suggesting that E. anseris is highly pathogenic in goslings.

Anticoccidial activities of Chitosan on Eimeria papillata-infected mice.

Eimeria spp. multiply within the intestinal tract causing severe inflammatory responses. Chitosan (CS), meanwhile, has been shown to exhibit anti-inflammatory activities in different experimental models. Here, we investigated the effect of CS on the outcome of inflammation caused by Eimeria papillata in the mouse intestine. Investigations were undertaken into the oocyst output in feces and developmental stages and goblet cells in intestinal tissue. Assays for lipid peroxidation, nitric oxide (NO), and myeloperoxidase (MPO) were also performed. T cells in intestinal tissue were counted using immunohistochemistry while total IgA in serum or intestinal wash was assayed using ELISA. In addition, mRNA expression of tumor necrosis factor alpha (TNF-α), transforming growth factor ß (TGF-ß), interleukin (IL)-10, and IL-4 were detected using real-time PCR. The data indicated a reduction in both oocyst output and in the number of parasite developmental stages following CS treatment, while the goblet cell hypoplasia in infected mice was also inhibited. CS decreased lipid peroxidation, NO, and MPO but did not alter the T cell count or IgA levels in comparison to the infected group. The expression of TNF-α and TGF-ß decreased but IL-10 and IL-4 increased after CS treatment in comparison to the non-treated infected group. In conclusion, CS showed anti-inflammatory and protective effects against E. papillata infection.

Targeting essential Eimeria ninakohlyakimovae sporozoite ligands for caprine host endothelial cell invasion with a phage display peptide library.

Eimeria ninakohlyakimovae is an important coccidian parasite of goats which causes severe diarrhoea in young animals. Specific molecules that mediate E. ninakohlyakimovae host interactions and molecular mechanisms involved in the pathogenesis are still unknown. Although strong circumstantial evidence indicates that E. ninakohlyakimovae sporozoite interactions with caprine endothelial host cells (ECs) are specific, hardly any information is available about the interacting molecules that confer host cell specificity. In this study, we describe a novel method to identify surface proteins of caprine umbilical vein endothelial cells (CUVEC) using a phage display library. After several panning rounds, we identified a number of peptides that specifically bind to the surface of CUVEC. Importantly, caprine endothelial cell peptide 2 (PCEC2) and PCEC5 selectively reduced the infection rate by E. ninakohlyakimovae sporozoites. These preliminary data give new insight for the molecular identification of ligands involved in the interaction between E. ninakohlyakimovae sporozoites and host ECs. Further studies using this phage approach might be useful to identify new potential target molecules for the development of anti-coccidial drugs or even new vaccine strategies.

Antioxidant and anti-inflammatory activities of pomegranate (Punica granatum) on Eimeria papillata-induced infection in mice.

Coccidiosis is the most prevalent disease causing widespread economic loss, especially in poultry farms. Here, we investigated the effects of pomegranate peel extract (PPE) on the outcome of coccidiosis caused by Eimeria papillata in mice. The data showed that mice infected with E. papillata and treated with PPE revealed a significant decrease in the output of oocysts in their faeces by day 5 p.i. Infection also induced inflammation and injury of the jejunum. This was evidenced (i) as increases in reactive oxygen species, (ii), as increased neutrophils and decreased lymphocytes in blood (ii) as increased mRNA levels of inducible nitric oxide synthase (iNOS), Bcl-2 gene, and of the cytokines interferon gamma (IFN-γ), tumour necrosis factor-α (TNF-α), and interleukin-1ß (IL-1ß), and (iv) as downregulation of mucin gene MUC2 mRNA. All these infection-induced parameters were significantly altered during PPE treatment. In particular, PPE counteracted the E. papillata-induced loss of the total antioxidant capacity. Our data indicated that PPE treatment significantly attenuated inflammation and injury of the jejunum induced by E. papillata infections.

Control of eight predominant Eimeria spp. involved in economic coccidiosis of broiler chicken by a chemically characterized essential oil.

AIM: To control eight most predominant Eimeria spp. involved in the economic disease of coccidiosis in broiler chicken, by a chemically characterized essential oil of eucalyptus and peppermint. METHODS AND RESULTS: The experimental design consisted of 160 day-old-broiler chicks, divided into four equal groups (G1 , G2 , G3 and G4 ), with 40 birds per group. Each group was divided into four equal subgroups. Birds in G1 were deprived of essential oil treatment and of Eimeria challenge. Birds in G2 were unchallenged, and administered the essential oil in drinking water at 0.69 ml kg(-1) body weight. Birds in G3 were untreated with essential oil, and each of its four subgroups was challenged at a different age (14, 21, 28 and 35 days). Birds in G4 were treated with essential oil, and challenged in the same manner as for G3 . Equal number of birds from all subgroups (n = 10) were sacrificed at the sixth day after the time allocated for each challenge. The 6 day incubation period post challenge resulted in respective mean per cent weight increase in G2 and G1 birds equivalent to 57.8 and 53.1% (P < 0.05). In addition, the essential oil improved the per cent weight increase in challenged birds (54.6%) compared to the challenged-untreated birds (18.6%) (P < 0.05). The mean feed conversion, mortality, intestinal lesion scores and oocyst counts were significantly reduced in the challenged-treated birds compared to the challenged-untreated birds (P < 0.05). CONCLUSIONS: The results support the hypothesis of using the essential oils of eucalyptus and peppermint to control the most prevalent Eimeria spp. involved in coccidiosis of broiler chicken, helping in improvement of their production, alleviation of lesions and reduction in intestinal oocyst counts. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides information about the possibility of using this blend of essential oil as a coccidiostat for the protection of broiler chickens against the prevalent eight Eimeria spp. of coccidiosis.

Re-description of a genetically typed, single oocyst line of the turkey coccidium, Eimeria adenoeides Moore and Brown, 1951.

The Guelph strain of Eimeria adenoeides was obtained from a commercial turkey flock in Ontario, Canada, in 1985. Single oocyst derived lines of E. adenoeides were propagated, and one of them used to re-describe biological and morphological features of E. adenoeides in the turkey. Oocysts of this strain are within the lower size ranges in the original species description reported by Moore and Brown (1951); oocysts of the Guelph strain averaged 18.7 ± 1.4 µm (16.7-22.5) by 14.3 ± 0.9 µm (13-16.2, n = 30) with a shape index (SI) of 1.3 ± 0.1. It is possible that the original species description was based, at least in part, on a mixed culture of two or more Eimeria species. Immature first-generation meronts of E. adenoeides Guelph strain were observed histologically at 32 h post-infection in the ileum and cecal neck. Early studies reported only two asexual generations suggested that first asexual cycle observed at 32 h post-infection was overlooked. In the present study, three asexual generations were observed before the start of gametogony. The Guelph strain is also characterized by a prepatent period of 112 h. The Guelph strain of E. adenoeides is a highly pathogenic coccidium that forms classic cecal lesions, including prominent caseous cecal cores, during moderate to severe infections. The maximum output of oocysts (1.77 × 10(7) per bird) was obtained from birds inoculated with 1 × 10(3) oocysts; maximum fecundity (1.55 × 10(5) oocyst shed per oocyst inoculated) was obtained with an inoculation of 1 × 10(2) oocysts, but fecundity dropped dramatically as the inoculation dose increased. To promote stability of the E. adenoeides species concept, neotype specimens (a parahapantotype slides series and phototype) have been designated and deposited for future reference.

Evidence for a structural role for acid-fast lipids in oocyst walls of Cryptosporidium, Toxoplasma, and Eimeria.

UNLABELLED: Coccidia are protozoan parasites that cause significant human disease and are of major agricultural importance. Cryptosporidium spp. cause diarrhea in humans and animals, while Toxoplasma causes disseminated infections in fetuses and untreated AIDS patients. Eimeria is a major pathogen of commercial chickens. Oocysts, which are the infectious form of Cryptosporidium and Eimeria and one of two infectious forms of Toxoplasma (the other is tissue cysts in undercooked meat), have a multilayered wall. Recently we showed that the inner layer of the oocyst walls of Toxoplasma and Eimeria is a porous scaffold of fibers of ß-1,3-glucan, which are also present in fungal walls but are absent from Cryptosporidium oocyst walls. Here we present evidence for a structural role for lipids in the oocyst walls of Cryptosporidium, Toxoplasma, and Eimeria. Briefly, oocyst walls of each organism label with acid-fast stains that bind to lipids in the walls of mycobacteria. Polyketide synthases similar to those that make mycobacterial wall lipids are abundant in oocysts of Toxoplasma and Eimeria and are predicted in Cryptosporidium. The outer layer of oocyst wall of Eimeria and the entire oocyst wall of Cryptosporidium are dissolved by organic solvents. Oocyst wall lipids are complex mixtures of triglycerides, some of which contain polyhydroxy fatty acyl chains like those present in plant cutin or elongated fatty acyl chains like mycolic acids. We propose a two-layered model of the oocyst wall (glucan and acid-fast lipids) that resembles the two-layered walls of mycobacteria (peptidoglycan and acid-fast lipids) and plants (cellulose and cutin). IMPORTANCE: Oocysts, which are essential for the fecal-oral spread of coccidia, have a wall that is thought responsible for their survival in the environment and for their transit through the stomach and small intestine. While oocyst walls of Toxoplasma and Eimeria are strengthened by a porous scaffold of fibrils of ß-1,3-glucan and by proteins cross-linked by dityrosines, both are absent from walls of Cryptosporidium. We show here that all oocyst walls are acid fast, have a rigid bilayer, dissolve in organic solvents, and contain a complex set of triglycerides rich in polyhydroxy and long fatty acyl chains that might be synthesized by an abundant polyketide synthase. These results suggest the possibility that coccidia build a waxy coat of acid-fast lipids in the oocyst wall that makes them resistant to environmental stress.

Eimeria bovis-induced modulation of the host cell proteome at the meront I stage.

The proteome of Eimeria bovis meront I-carrying host cells was analyzed by two-dimensional gel electrophoresis (2DE) at 14 days p.i. and compared to non-infected control cells. A total of 221 protein spots were modulated in their abundance in E. bovis-infected host cells and were subsequently analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectometry (MALDI-TOF-MS). These analyses identified 104 proteins in total with 25 host cell proteins being up-regulated and 79 proteins being down-regulated in E. bovis-infected host cells. Moreover, 20 newly expressed proteins were identified exclusively in E. bovis-infected host cells and were most likely of parasite origin. Parasite-induced differences in protein abundance concerned distinct functional categories, with most proteins being involved in host cell metabolism, cell structure, protein fate and gene transcription. Some of the modulated molecules also indicated regulatory processes on the level of host cell stress response (HSP70, HSP90), host cell apoptosis (caspase 8) and actin elongation/depolymerization (α-actinin-1, gelsonin, tropomodulin-3, transgelin). Since merozoites I were already released shortly after cell sampling, the current data reflect the situation at the end of first merogony. This is the first proteomic approach on E. bovis-infected host cells that was undertaken to gain a rather broad insight into Eimeria-induced host cell modulation. The data processed in this investigation should provide a useful basis for more detailed analyses concerning Eimeria-host cell interactions.

Recombinant Eimeria protozoan protein elicits resistance to acute phlebovirus infection in mice but not hamsters.

A protein antigen from an Eimeria protozoan has recently been reported to induce antitumor activity in mice. This activity most likely results from the strong induction of interkeukin-12 (IL-12) and gamma interferon (IFN-gamma), which are also essential factors in the establishment of protective immunity against viral infection. We evaluated recombinant Eimeria antigen (rEA) as a potential immunotherapeutic agent in mouse and hamster models of acute phleboviral disease. Punta Toro virus (PTV) was highly sensitive to a single dose of nanogram quantities of rEA in the mouse infection model. Intraperitoneal treatment with rEA also reduced virus load and liver damage associated with PTV infection. IL-12 was elicited following exposure of uninfected mice to quantities of rEA of 10 ng or greater, and the levels peaked at between 3 and 8 h postexposure. IFN-gamma release was induced more slowly and required less rEA (1 ng) to produce a significant rise in systemic levels. The induction of IL-12 and IFN-gamma involved in the coordination of innate and adaptive immune responses to microbial pathogens required myeloid differentiation factor 88, a signaling adaptor shared by most members of the Toll-like receptor (TLR) family. Despite encouraging results in the murine system, rEA failed to protect hamsters challenged with PTV. Our findings suggest that hamsters may lack functional TLR11, which has recently been shown to recognize a profilin-like protein homologous to rEA from the protozoan Toxoplasma gondii. Further investigation into the immunostimulatory capacity of rEA in other mammalian systems is necessary.

[The effect of betain on biology and morphology of developmental stages of Eimeria acervulina in broiler chicks experimentally infected]. / Efeito do uso da betaína na biologia e morfologia dos estádios evolutivos de Eimeria acervulina em frangos de corte infectados experimentalmente com oocistos esporulados.

Purposing to investigate the betaine effect on biology and morphology of developmental stages of Eimeria acervulina, 420 broiler chicks Cobb were experimentally inoculated with 2 x 10(5) sporulated oocysts and housed in battery cages in a block design with five treatments and six replicates each, including a positive control, a group treated with salinomycin and growth promoter plus three levels of betaine as additive in the feed at 0.05, 0.10 and 0.15%. Measurements of oocysts, sporocysts and endogenous stages were performed as morphological parameters, while pre patent and patent periods and sporulation time were taken as biological parameters. Morphology was also associated with the mathematical constant Phi (1.618) to evaluate possible relationship. Betaine was able to cause modifications in both biology and morphology of oocysts and sporocysts, whereas it was weakly able to affect developmental stages based on trophozoites and macrogamonts measurements. According to the measures of sporocysts E. acervulina development was closely related to Phi.

Pathology and pathogenesis of disseminated visceral coccidiosis in cranes.

Disseminated visceral coccidiosis (DVC) caused by Eimeria spp. was recognized as a disease entity in captive sandhill cranes (Grus canadensis) and whooping cranes (Grus americana) in the late 1970s. While most avian species of Eimeria inhabit the intestinal tract of its host, the crane eimerians, Eimeria reichenowi and Eimeria gruis, invade and multiply systemically and complete their development in both digestive and respiratory tracts. In DVC, cranes, especially chicks, may succumb to acute infections resulting in hepatitis, bronchopneumonia, myocarditis, splenitis, and enteritis. Cranes may also develop chronic, subclinical infections characterized by granulomatous nodules in various organs and tissues. This paper reviews the pathology and pathogenicity of natural and experimental DVC in sandhill and whooping cranes. Naturally infected birds appeared clinically normal, but progressive weakness, emaciation, greenish diarrhea, and recumbency before death were observed in birds administered doses > or = 10 x 10(3) sporulated oocysts per os. At necropsy, naturally infected birds had nodules in the mucosa of the oral cavity and the esophagus, and in thoracic and abdominal viscera. Experimentally infected birds necropsied less than 7 days after infection (a.i.) had no gross lesions. Birds examined later had hepatosplenomegaly, liver mottling, lung congestion and consolidation with frothy fluid in airways, and turgid intestinal tracts with hyperemic mucosa. From 28 days a.i., grossly visible granulomatous nodules were seen in the esophagus, heart, liver, cloaca, and eyelids. By light microscopy, the basic host response was a granulomatous inflammation with non-suppurative vasculitis affecting many organs and tissues. With time, multifocal aggregates of mononuclear cells, many laden with asexual coccidial stages, increased in size and number. Widespread merogony resulted in morbidity and death, particularly in birds administered 20 x 10(3) sporulated oocysts. Ultrastructural examination revealed developing asexual coccidian stages in the cytoplasm of large lymphocytes or monocytes within a parasitophorous vacuole, often indenting the nucleus. Oocysts and gametocytes were found in the intestines by 12 days a.i., and in the esophagus, trachea, bronchi, and lung by 14 days a.i., indicating that crane eimerians can complete their life cycle at these sites. Thus, DVC in cranes could be a useful animal model for the study of eimerian extra-intestinal stages and the evaluation of potential systemic anticoccidial drugs.

Effect of vitamin A deficiency on host intestinal immune response to Eimeria acervulina in broiler chickens.

The effects of vitamin A (VitA) deficiency on the host intestinal immune response and disease susceptibility to coccidiosis were investigated in broiler chickens following oral infection with Eimeria acervulina (EA). Day-old male broilers were fed milo-soybean meal diets either with 8,000 IU VitA/kg feed (CONT) or without added VitA (A-DEF). At 25 d, a group of randomly selected birds from each treatment was inoculated orally with EA-sporulated oocysts. Intestinal immune response was assessed by the changes in the duodenum intraepithelial lymphocyte (IEL) subpopulations using flow cytometry at 35 d in in fected and noninfected birds. Concanavalin A (ConA)-induced spleen lymphocyte proliferation was tested using dimethylthiazol diphenyltetrazolium bromide colorimetric assay. Whether challenged or not with EA, A-DEF birds had fewer IEL expressing the surface markers CD3, CD4, CD8, alphabetaTCR, and gammabetaTCR. Without EA challenge, A-DEF birds had more surface IgA-expressing cells than CONT birds. Upon challenge, A-DEF chickens showed lower CD4+ IEL than CONT chickens. Following EA infection, CD8+ IEL increased in the CONT group, whereas no change was found in CD8+ IEL of A-DEF birds. A higher number of EA oocysts was recovered from A-DEF birds than from CONT birds (9.2 x 10(8) vs 5.4 x 10(8), respectively; P < or = 0.05). Serum samples taken 10 d post challenge showed higher antibody level against a recombinant coccidial antigen in A-DEF birds than in CONT birds. The A-DEF birds showed depressed ConA-induced lymphoproliferation response and produced lower serum interferon-gamma than CONT birds. These data show that VitA deficiency compromised local immune defenses of challenged birds, as reflected in lymphocyte profiles, oocyst shedding, and interferon-gamma levels in A-DEF birds.

Eimeria organisms develop in the epithelial cells of equine small intestine.

Histopathologic and immunohistochemical examinations were performed to determine the origin of host cells parasitized by Eimeria in the small intestines collected from five foals. Eimeria organisms at various stages (mainly microgametes and macrogametes) were frequently found in the cytoplasm of hypertrophied host cells in the lamina propria at the tips of villi of the jejunum and ileum. The cytoplasm of the host cell was immunohistochemically positive for cytokeratin AE1/AE3 and cytokeratin 13 and was negative for vimentin, desmin, alpha-smooth muscle actin, chromogranin A, neuron-specific enolase, and factor VIII. The host cells parasitized by Eimeria species had the immunostaining characteristics of epithelial cells but not of mesenchymal cells, endothelial cells of lacteals or capillaries, smooth muscle cells or neuroendocrine cells. These results suggest that the host cell of Eimeria species is possibly derived from intestinal epithelial cells and then displaced into the lamina propria of the small intestine.

Avian anticoccidial activity of a novel membrane-interactive peptide selected from phage display libraries.

In the present work, we describe the discovery of PW2, a novel peptide presenting in vitro activity against Eimeria acervulina and E. tenella sporozoites. PW2 was selected from phage display (Ph.D.) peptide libraries by an alternative method of panning using living purified E. acervulina sporozoites as targets. Our results showed that the peptide disrupts the sporozoite pellicle, resembling the effect caused by most natural antimicrobial peptides. PW2 peptide was also effective against fungi and showed low activity against Toxoplasma gondii tachyzoites, but no activity against Trypanosoma cruzi, Crithidia fasciculata epimastigotes, and bacteria. Additionally, the parasiticidal concentrations of PW2 produced a very low lytic effect on mammalian and avian cells. The effectiveness against Eimeria sporozoites and the absence of adverse effects to host cells indicates that PW2 may be used as a model to generate new drugs for the control of avian coccidiosis.

Invasion of different cell types by sporozoites of Eimeria species and effects of monoclonal antibody 1209-C2 on invasion of cells by sporozoites of several apicomplexan parasites.

Sporozoites of avian Eimeria species differed markedly in their ability to invade cells in vitro. Invasion by E. tenella and E. adenoeides was significantly greater in baby hamster kidney (BHK) and chicken cecal cell (CC) cultures than in primary chicken (PCK) or turkey kidney (PTK) cell cultures. Moreover, invasion of BHK cell cultures by E. adenoeides was significantly greater than that of other Eimeria species, and invasion by E. acervulina sporozoites was significantly lower. Monoclonal antibody 1209-C2 (MAb 1209-C2) reacted by immunofluorescent labeling (IFA) with refractile bodies of sporozoites of 5 species of Eimeria and Caryospora bigenetica, but not with sporozoites of Toxoplasma gondii, Hammondia hammondi, or Cryptosporidium parvum, which have no refractile bodies. The MAb also cross-reacted with formalin-fixed BHK, CC, turkey cecal (TC) cells, and PTK. Pretreatment of BHK cells with MAb 1209-C2 significantly reduced invasion of the cells by sporozoites of E. tenella, E. acervulina, E. meleagrimitis, and C. bigenetica, but did not alter invasion by T. gondii, C. parvum, or H. hammondia. Apparently, reactivity of MAB 1209-C2 with the sporozoites was required for inhibition of invasion despite the fact that the inhibition resulted from pre-treatment of the host cell. Conversely, although MAb 1209-C2 also reacted moderately with PTK and TC cells, pre-treatment of these cell cultures with the MAb did not inhibit invasion by either MAB 1209-C2-reactive or -nonreactive parasites. Collectively, the data indicated that refractile body antigens of sporozoites of Eimeria and Caryospora, which are recognized by MAb 1209-C2, may function in cellular invasion, but also suggest that cellular invasion is probably not mediated by interactions between the conserved epitopes in sporozoites and cultured host cells that are recognized by the MAb.

Endogenous development of Eimeria minasensis in experimentally infected goats.

The endogenous development of Eimeria minasensis was studied in 9 coccidia-free goat kids inoculated with 10(5) sporulated oocysts/kg body weight. Kids were killed 4, 7 (2 animals), 10, 13, 16, 18, 19, and 22 days after inoculation (DAI). In tissue sections of the intestines stained with hematoxylin and eosin and examined by light microscopy, 2 generations of meronts, gamonts, gametes, and oocysts were found. The first generation of meronts developed in cells deep in the lamina propria of the jejunum and ileum. Mature giant meronts (299.4x243.8 microm) found 16 DAI were visible to the naked eye and contained a large number of crescent-shaped merozoites. The second generation of meronts developed in the epithelial cells of crypts of the ileum and above the host cell nuclei. Mature meronts (11.5x10.1 microm) with 18-28 comma-shaped merozoites were first seen 16 DAI. Gametogenesis took place in epithelial cells of the crypts and villi of the terminal part of the ileum, cecum, and colon. Macrogametes (27.8x17.6 microm), mature microgamonts (21.3x17.0 microm), microgametes, and oocysts (30.5x19.4 microm) were found 19 DAI. Sexual stages were below the host cell nucleus.