The significance of infection dose,body weight and age of the host on the pathogenic effect of Eimeria acervulina in the fowl

Summary The pathogenic effect of E. acervulina was enhanced by increasing the initial infection doses. However the relation between infection dose and the pathogenic effect was disproportionate; generally it was enhanced less than the infection dose was increased.It appeared that the moderate doses caused the greatest oocyst productions and are therefore of great epidemiological importance. However also the smaller infection doses caused oocyst productions at comparable levels.The oocyst output did not coincide with the increasing pathogenic effect and there was no correlation between individual body weight, weight loss and oocyst output within a group of chickens of the same age infected with the same number of oocysts. It is concluded that the oocyst output is not a reliable guide to the severity of the individual infection.The significance of the moderate doses was also evident from a marked reduction of the intestinal pH and the serum protein concentration compared with only a slightly stronger reduction of these values in birds infected with higher doses.The most important factor in the pathogenic effect appeared to be the age of the host. The reaction was progressively more severe in birds of increasing age infected with an uniform dose.     

Induction of protective immunity against Eimeria tenella, Eimeria maxima, and Eimeria acervulina infections using dendritic cell-derived exosomes

This study describes a novel immunization strategy against avian coccidiosis using exosomes derived from Eimeria parasite antigen (Ag)-loaded dendritic cells (DCs). Chicken intestinal DCs were isolated and pulsed in vitro with a mixture of sporozoite-extracted Ags from Eimeria tenella, E. maxima, and E. acervulina, and the cell-derived exosomes were isolated. Chickens were nonimmunized or immunized intramuscularly with exosomes and subsequently noninfected or coinfected with E. tenella, E. maxima, and E. acervulina oocysts. Immune parameters compared among the nonimmunized/noninfected, nonimmunized/infected, and immunized/infected groups were the numbers of cells secreting T(h)1 cytokines, T(h)2 cytokines, interleukin-16 (IL-16), and Ag-reactive antibodies in vitro and in vivo readouts of protective immunity against Eimeria infection. Cecal tonsils, Peyer's patches, and spleens of immunized and infected chickens had increased numbers of cells secreting the IL-16 and the T(h)1 cytokines IL-2 and gamma interferon, greater Ag-stimulated proliferative responses, and higher numbers of Ag-reactive IgG- and IgA-producing cells following in vitro stimulation with the sporozoite Ags compared with the nonimmunized/noninfected and nonimmunized/infected controls. In contrast, the numbers of cells secreting the T(h)2 cytokines IL-4 and IL-10 were diminished in immunized and infected chickens compared with the nonimmunized/noninfected and the nonimmunized/infected controls. Chickens immunized with Ag-loaded exosomes and infected in vivo with Eimeria oocysts had increased body weight gains, reduced feed conversion ratios, diminished fecal oocyst shedding, lessened intestinal lesion scores, and reduced mortality compared with the nonimmunized/infected controls. These results suggest that successful field vaccination against avian coccidiosis using exosomes derived from DCs incubated with Ags isolated from Eimeria species may be possible.     

Protective immunity against Eimeria acervulina following in ovo immunization with a recombinant subunit vaccine and cytokine genes

A purified recombinant protein from Eimeria acervulina (3-1E) was used to vaccinate chickens in ovo against coccidiosis both alone and in combination with expression plasmids encoding the interleukin 1 (IL-1), IL-2, IL-6, IL-8, IL-15, IL-16, IL-17, IL-18, or gamma interferon (IFN-gamma) gene. When used alone, vaccination with 100 or 500 mug of 3-1E resulted in significantly decreased oocyst shedding compared with that in nonvaccinated chickens. Simultaneous vaccination of the 3-1E protein with the IL-1, -15, -16, or -17 gene induced higher serum antibody responses than 3-1E alone. To evaluate protective intestinal immunity, vaccinated birds were challenged with live E. acervulina oocysts 14 days posthatch, and fecal-oocyst shedding and body weight gain were determined as parameters of coccidiosis. Chickens vaccinated with 3-1E protein showed significantly lower oocyst shedding and normal body weight gain than nonvaccinated and infected controls. Simultaneous immunization with 3-1E and the IL-2, -15, -17, or -18 or IFN-gamma gene further reduced oocyst shedding compared with that achieved with 3-1E alone. These results provide the first evidence that in ovo vaccination with the recombinant 3-1E Eimeria protein induces protective intestinal immunity against coccidiosis, and this effect was enhanced by coadministration of genes encoding immunity-related cytokines.     

Disturbed intestinal absorption of vitamin A and carotenes and the effect of a low pH during Eimeria acervulina infection in the domestic fowl (gallus domesticus)

Summary It could be shown that in E. acervulina infected birds the absorption both of vitamin A and carotenes is strongly disturbed, chiefly in the proximal parts of the intestine. When the infection is stronger, more vitamin A is absorbed in the distal part of the intestine.At day 5 after infection a caseous substance is found over the mucosa of the affected parts of the intestine.It appeared that a similar caseous substance is formed after experimental exposure of the intestinal wall to a pH of about 4.5 for 4 hours.The supposition is given that in infected birds the mucosal protein in the affected parts of the intestine is denaturated thanks to the low pH caused by the infection.As a consequence of this denaturation the absorption of vitamin A and of carotene is strongly disturbed and the carotene in the blood decreases. This can be observed already at day 2 after infection.Finally it has been shown that it seems very unlikely that administration of extra vitamin A to normally fed, infected chickens will decrease the influence of the infection on the chickens.     

Biochemical, genetic and applied aspects of drug resistance in Eimeria parasites of the fowl

Anticoccidial drugs are widely used for the control of coccidiosis in the fowl which has inevitably led to the development of drug resistance. Resistance has developed to all of the compounds that have been introduced and if chemotherapy is to remain the principal method of control of coccidiosis, it will be important to continue the search for new anticoccidial agents. Knowledge of biochemical pathways present in the Eimeria parasite and how they differ from those of the host might help identify novel targets for inhibition. Studies of the mode of action of drugs are required if the biochemical mechanisms of resistance are to be understood. Information on the genetic origins of resistance, the stability of resistance and the factors involved in the spread of resistance throughout parasite populations is required. Since there are no methods at present to prevent resistance, more attention should be given to developing strategies for preserving the efficacy of anticoccidial drugs.     

Histological Observations with Respect to the Immune Mechanism in Eimeria acervulina Infection in the Domestic Fowl

Summary A strong repulsion of epithelial tissue into the intestinal lumen associated with a lowered intestinal pH, thin liquid intestinal contents and leakage of serumprotein into the lumen was observed in E. acervulina immunized birds, some hours following oral reinfection. The extruded epithelial tissue degenerated and died in the lumen; massive numbers of dying cells could also be observed in tissue sections of the intestinal contents. Most likely the sporozoites are pushed off together with the cells. The repulsion was always associated with a prominent swelling of the muscles in the intestinal villi. In the not immune birds however, some cell repulsion and a little swelling of the villous muscles was observed but only at the tips of the villi. In the normal not infected individual contraction of the villous muscles supports the physiological repulsion of epithelial cells at the so called extrusion zone.In the immunized birds the reaction of the tissue after reinfection could be completely suppressed by cortisone treatment.Incubation experiments of intestinal pieces both from immunized and not immunized birds with oocyst fluid, sporocysts and glucose in vivo as well as in vitro showed reactions in the same way as mentioned above. The reaction was much stronger in the immunized than in the not immunized birds. These results indicate a local tissue immunity.The hypothesis is given that after a primary infection compounds stay behind in the intestinal wall enhancing the reaction of oocyst fluid and sporocysts with glucose to such an extent that much lactic acid is formed, which in turn might affect the intestinal wall, resulting in repulsion of the epithelial tissue.     

Fine structural changes associated with microgametogenesis of Eimeria acervulina in chickens

Summary The fine structure of the changes occurring during microgametogenesis and of the microgametes of Eimeria acervulina was studied in experimentally infected chickens. Early microgametocytes had relatively electron-transparent nuclei with few, scattered, dense masses. Micropores were seen at the surface of these microgametocytes. Intranuclear microtubules extended inwards from a nuclear protuberance (centrocone) oriented towards the cell membrane. One to several fissures appeared in the cytoplasm of the microgametocytes and the nuclei oriented themselves along either the outer membrane or a fissure. One or 2 centrioles were seen between the nuclei and the cell membrane at this stage. Later, flagella and their basal bodies were seen in this position. A wide, dense band appeared in the nucleus perpendicular to the surface nearest the flagella. Later, the pale portion of the nucleus was pinched off and remained within the residual body. Mature microgametes had an anterior perforatorium, 3 basal bodies, 3 flagella, 4 microtubules running adjacent to the anterior mitochondrion, and a dense, elongated nucleus mostly posterior to this, with a narrow portion extending anteriorly nearly as far as the anterior end of the mitochondrion.Supported by an Ontario Health Research Grant No. PR 297 and by the Ontario Ministry of Agriculture and Food.     

Intestinal absorption following challenge of immune chicks with Eimeria acervulina

The absorption of glucose or L-methionine was measured in vitro in the intestine of broilers challenged with 1 x 10(6) sporulated oocysts of Eimeria acervulina. These broilers had various degrees of immunity resulting from previous inoculation with several dosages of oocysts given one to six times. Unimmunised broilers were also given the challenge inoculation of oocysts. The level of immunity had a marked effect on nutrient absorption post challenge (p.c.). Partially immune birds showed an enhanced absorption at 3 hours p.c. compared with absorption in unchallenged birds. With high levels of immunity, the enhanced absorption at 3 hours p.c. was not seen. A significant malabsorption was seen 6 hours p.c., but absorption returned to normal by 24 hours p.c. The absorption pattern during the first 24 hours in unimmunised control broilers was similar to that in partially immune birds. The degree of immunity was directly related to the protection against malabsorption at 6 days p.c. In solidly immune birds, absorption at 6 days p.c. was significantly greater than in immunised, unchallenged birds and equal to that in the unimmunised, unchallenged controls. The diffusion and mediated components of methionine absorption were, in general, both affected during malabsorption or stimulation of absorption. There was no evidence histologic-ally of epithelial loss in the 24 hours p.c. The pH in the duodenum was significantly decreased at 8 and 24 hours p.c. in immunised birds but was not affected by a primary inoculation given to unimmunised birds.     

Vaccination of broilers with HVT expressing an Eimeria acervulina antigen improves performance after challenge with Eimeria

The sequence encoding part of the 100 kDa refractile body protein (Ea1A) from Eimeria acervulina was cloned into the US10 locus of herpesvirus of turkeys (HVT) downstream of LTR promoter. Expression of the fusion protein was shown in vitro. Recombinant HVT showed a delayed and slightly reduced level of viremia compared to the parent strain in SPF chickens as well as in broilers. Effect on the performance of broilers vaccinated with recombinant or parent HVT was measured by challenge at day 24 with a high dose of E. acervulina and E. maxima oocysts. A significant improvement in weight of animals vaccinated with the recombinant HVT was detected at the end of the challenge period.     

Eimeria adenoeides sporozoites: enhanced invasion of cultured cells previously inoculated with Eimeria acervulina sporozoites

The effects of prior or concurrent administration of Eimeria acervulina on invasion of cultured cells by Eimeria adenoeides sporozoites and possible mechanisms of action were examined. Baby hamster kidney (BHK) cell cultures that were inoculated with E. acervulina sporozoites were significantly more permissive for invasion by E. adenoeides sporozoites than uninoculated cultures. Enhancement of invasion by E. adenoeides did not occur when the two species were inoculated into cultures concurrently, or within 30 h of each other. However, 48 and 72 h after inoculation of BHK cells with E. acervulina, invasion by E. adenoeides sporozoites was significantly greater than invasion in uninoculated cultures. At 96 h postinoculation with E. acervulina, the enhancing effect on invasion was variable. Culture media collected from E. acervulina-inoculated cultures also significantly enhanced invasion by E. adenoeides. Slight changes in proteins of E. acervulina-inoculated versus uninoculated cell cultures were detected by Western blots of biotinylated and nonbiotinylated cells. Biotinylated bands between 10 and 25 kDa increased in the inoculated cultures. In addition, when chicken anti-E. acervulina sporozoite serum was used as a probe, labeling of a 10 kDa antigen increased in the inoculated cultures.     

Assessment of probiotics supplementation via feed or water on the growth performance,intestinal morphology and microflora of chickens after experimental infection with Eimeria acervulina,Eimeria maxima and Eimeria tenella

In this study, the effect of probiotic supplementation via drinking water or feed on the performance of broiler chickens experimentally infected with sporulated oocysts of Eimeria acervulina (5 × 10⁴), Eimeria maxima and Eimeria tenella (2 × 10⁴ each one) at 14 days of age was evaluated. Two hundred and forty 1-day-old Ross 308 male chicks were separated into eight equal groups with three replicates. Two of the groups, one infected with mixed Eimeria oocysts and the other not, were given a basal diet and served as controls. The remaining groups were also challenged with mixed Eimeria species and received the basal diet and either water supplemented with probiotic (three groups) or probiotic via feed (two groups); the probiotic used consisted of Enterococcus faecium #589, Bifidobacterium animalis #503 and Lactobacillus salivarius #505 at a ratio of 6:3:1. Probiotic supplementation was applied either via drinking water in different inclusion rates (groups W1, W2 and W3) or via feed using uncoated (group FN) or coated strains (group FC). The last group was given the basal diet supplemented with the anticoccidial lasalocid at 75 mg/kg. Each experimental group was given the corresponding diet or drinking water from day 1 to day 42 of age. Throughout the experimental period of 42 days, body weight and feed intake were recorded weekly and feed conversion ratios were calculated. Seven days after infection, the infected control group presented the lowest weight gain values, while probiotics supplied via feed supported growth to a comparable level with that of the lasalocid group. Probiotic groups presented lesion score values and oocyst numbers that were lower than in control infected birds but higher than in the lasalocid group. In the duodenum, jejunum and ileum, the highest villous height values were presented by probiotic groups. In conclusion, a mixture of probiotic substances gave considerable improvement in both growth performance and intestinal health in comparison with infected control birds and fairly similar improvement to an approved anticoccidial during a mixed Eimeria infection.     

Amino-acid malabsorption and intestinal leakage of plasma-proteins in young chicks infected with Eimeria acervulina

The in vivo absorption of 14C-labelled amino-acids and the leakage of radioactive plasma proteins into the gut of infected birds were measured together with their food-intake and growth. The results suggested that anorexia and protein leakage from the gut are major factors in the pathogenesis of E. acervulina infection.     

Changes in the generation cycle of duodenal crypt cells in chickens infected with Eimeria acervulina

Summary Changes in the duration of the progenitor cycle and its four phases were determined for duodenal crypt cells in chickens infected with Eimeria acervulina. Metaphase curves were constructed using percent labelled metaphase nuclei in duodenal crypt cells at short intervals after the injection of [³H]thymidine. The duration of the progenitor cycle and its four phases were calculated using the synthetic index and data obtained from the metaphase curves. The cycle time was reduced from 14 h in control birds to 10.2 h at 2 days and 10.6 h at 4 days postinfection. The change was attributable entirely to a reduction in G₁ or the presynthetic phase. In addition, the population of dividing cells within each duodenal crypt was almost doubled in infected birds. These increases in cell production precedes all the histological changes observed earlier in the intestines of E. acervulina infected chickens. At least in this instance, changes in crypt morphology seems, therefore, to result from an induced change in the functional activity of the crypt.     

Identification of immunodominant surface antigens of Eimeria acervulina sporozoites and merozoites

Immunodominant surface antigens of Eimeria acervulina sporozoites and merozoites were identified by 125I-labeling and immunoblotting studies. Using these methodologies 60% of the immunodominant sporozoite antigens and 90% of the immunodominant merozoite antigens were observed to be 125I-surface labeled. However, several major 125I-labeled sporozoite and merozoite proteins did not represent prominent antigens as measured by immunoblotting. Immunodominant surface antigens were found over a wide size range for sporozoites (21-110 kDa) and for merozoites (20-250 kDa). In order to relate these findings to a 'natural' infection, two groups of 3-week old chickens were inoculated 5 times over a 2.5 week period with either a low or high dose of E. acervulina oocysts. The serum response to sporozoites and merozoites, indicated by enzyme-linked immunosorbent assay titers, was rapid; less than or equal to 7 days post-infection with 10(4) oocysts and less than or equal to 3 days with 10(5) oocysts. Many of the antigens identified by immunoblotting of sera from sporozoite- and merozoite-immunized animals were recognized by sera from both high dose and low dose E. acervulina-infected chickens. Furthermore, the sporozoite and merozoite antigens could be grouped into those constituents which induced a serum response early or late in the infection.     

The specific characters of the Eimeria, with special reference to the coccidia of the fowl

The differentiation of species of Eimeria is discussed with special reference to those occurring in the domestic fowl. Details of the morphology of the oocysts and other developmental stages of the parasites, their location in the host and the characteristics of the lesions produced, the timing of the patent and prepatent periods, host- and site-specificity and immunological specificity may be used as aids for specific identification. A single criterion is usually insufficient for differentiation and depending upon the species, different characters vary in significance. Quantitative cross-immunity tests appear to be the most satisfactory means available for the differentiation of species of Eimeria affecting chickens.     

Sporozoites of Eimeria acervulina within intestinal macrophages in normal experimental infections

Summary Sporozoites of Eimeria acervulina were observed in macrophages of the intestinal epithelium 5 and 6 days post-infection. These sporozoites lay within a well developed parasitophorous vacuole, were normal in structure and showed no signs of development. Macrophages harbouring sporozoites showed considerable structural changes, most pronounced being an absence of lysosomes, an enlarged nucleolus and extensive proliferation of the Golgi complex and endoplasmic reticulum. Possible mechanisms of survival and transport of sporozoites to preferred sites of development are discussed.Key to Lettering of Figures ER Endoplasmic reticulum - ERM Endoplasmic reticulum of macrophage - GOM Golgi zone of macrophage - INM Invaginations of macrophage cell membrane - LM Lipid globule of macrophage - LYM Lysosomes of macrophage - MI Mitochondria - MIM Mitochondria of macrophage - MN Micronemes - MP Micropore - N Nucleus - NM Nucleus of macrophage - PO Rhoptries (paired organelles) - PV Parasitophorous vacuole - RB Refractile body - V Vacuole This study was carried out in the Department of Poultry Research, Wye College (University of London), Wye, Kent, while the author was a Wellcome Trust Research Fellow.     

In-vitro studies on intestinal malabsorption of amino-acids in young chicks infected with Eimeria acervulina

In vitro uptake of L. histidine by intestinal tissue from chicks infected with E. acervulina was slightly reduced. This was confirmed using radio-actively labelled mixed amino-acid substrates in the form of a Chlorella protein hydrolysate.     

Ultrastructural studies of the effects of amprolium and dinitolmide on Eimeria acervulina macrogametes

Medication of chicks with 125 ppm amprolium or dinitolmide adversely affected oocyst sporulation of Eimeria acervulina (Weybridge strain). Dinitolmide delayed oocyst production and no oocyst wall formation was seen up to 168 h post infection. Both drugs caused large numbers of abnormally small wall-forming bodies to be produced in the macrogametes. In amprolium-fed chicks, abnormal oocyst wall formation was seen. It was concluded that the main drug action was against wall forming bodies of type 2.Abbreviations C canaliculi - CL cytoplasmic limiting membrane - L lipid - M1, M2, M3 membranes - N nucleus - NU nucleolus - OW outer oocyst wall - PG polysaccharide granule - PV parasitophorous vacuole - WFB1 wall forming bodies of type 1 - WFB2 wall forming bodies of type 2