Biomedical subjects

P C Augustine

Publications and source records attributed to P C Augustine.

At least 19 recordsLinked to original sources

PubMed2003

The effect of dietary betaine on intestinal and plasma levels of betaine in uninfected and coccidia-infected broiler chicks.

Chicks fed betaine supplemented diets and infected with Eimeria acervulina and Eimeria maxima had markedly higher levels of betaine in the duodenum and mid-gut than unsupplemented, infected chicks. Uninfected chicks fed betaine exhibited almost twice the levels of betaine in the gut as infected chicks. Plasma betaine levels were lower in E. maxima-infected chicks than in E. acervulina-or Eimeria tenella-infected chicks. Betaine supplementation reversed the decrease in weight gain in E. maxima- infected chicks but had no effect on the decrease in weight gains in E acervulina- and E. tenella-infected chicks. Coccidia-infected birds on normal diets regularly exhibit increases in plasma NO(2)(+)NO(3). This increase was abolished in E.tenella-infected birds on betaine supplement. Betaine feeding did not alter this effect in E. acervulina- and E. maxima-infected birds. Results indicate that betaine supplementation has a positive effect on gut betaine levels in birds infected with E. acervulina and E. maxima. In all treatment groups, infection lowered the levels of betaine.

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PubMed2001

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.

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PubMed2001

Cell: sporozoite interactions and invasion by apicomplexan parasites of the genus Eimeria.

The site specificity that avian Eimeria sporozoites and, to a more limited degree, other apicomplexan parasites exhibit for invasion in vivo suggests that specific interactions between the sporozoites and the target host cells may mediate the invasion process. Although sporozoite motility and structural and secreted antigens appear to provide the mechanisms for propelling the sporozoite into the host cell,there is a growing body of evidence that the host cell provides characteristics by which the sporozoites recognise and interact with the host cell as a prelude to invasion. Molecules on the surface of cells in the intestinal epithelium, that act as receptor or recognition sites for sporozoite invasion, may be included among these characteristics. The existence of receptor molecules for invasion by apicomplexan parasites was suggested by in vitro studies in which parasite invasion was inhibited in cultured cells that were treated with a variety of substances designed to selectively alter the host cell membrane. These substance included cationic compounds or molecules, enzymes that cleave specific linkages, protease inhibitors, monoclonal antibodies, etc. More specific evidence for the presence of receptors was provided by the binding of parasite antigens to specific host cell surface molecules. Analyses of host cells have implicated 22, 31, and 37 kDa antigens, surface membrane glycoconjugates,conserved epitopes of host cells and sporozoites, etc., but no treatment that perturbs these putative receptors has completely inhibited invasion of the cells by parasites. Regardless of the mechanism,sporozoites of the avian Eimeria also invade the same specific sites in foreign host birds that they invade in the natural host. Thus, site specificity for invasion may be a response to characteristics of the intestine that are shared by a number of hosts rather than to a unique trait of the natural host. Protective immunity elicited against avian Eimeria species is not manifested in a total blockade of parasite invasion. In fact, the effect of immunity on invasion differs according to the eliciting species and depends upon the area of the intestine that is invaded. Immunity produced against caecal species of avian Eimeria, for example Eimeria tenella and Eimeria adenoeides, inhibits subsequent invasion by homologous or heterologous challenge species, regardless of the area of the intestine that the challenge species invade. Conversely, in birds immunised with upper intestinal species, Eimeria acervulina and Eimeria meleagrimitis, invasion by challenge species is not decreased and often is significantly increased.

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PubMed2001

Chasing coccidia--new tools enter the race.

The 8th International Coccidiosis Conference, held on 9--13 July 2001 in Palm Cove, Australia, was a showcase of the latest studies on widely known coccidia, including Eimeria and Toxoplasma in addition to the emerging or re-emerging parasites such as Neospora, Cryptosporidium and Cyclospora. This meeting was staged in conjunction with the Annual Scientific Meeting of the Australian Society for Parasitology.

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PubMed1999

Effect of polyclonal antisera developed against dense granule-associated Neospora caninum proteins on cell invasion and development in vitro by N. caninum tachyzoites.

The effects of polyclonal antisera to 2 recombinant dense granule (DG) antigens of Neospora caninum on invasion and development by N. caninum tachyzoites in baby hamster kidney cell cultures were examined. In immunofluorescent antibody tests, the anti-DG sera, at dilutions of 1:100 to 1:500, reacted intensely with individual intracellular tachyzoites and groups of tachyzoites enclosed in parasitophorous vacuoles at 2 and 52 h p.i., respectively. Tachyzoites suspended in diluted anti-DG sera and inoculated immediately into the cell cultures invaded cells in significantly fewer numbers (53-68% fewer by 2 h p.i.) than tachyzoites suspended in similarly diluted normal rabbit serum. In contrast, tachyzoites suspended in anti-Eimeria tenella sporozoite serum invaded cells as efficiently as those suspended in normal rabbit serum. Addition of anti-DG sera at the time of inoculation of tachyzoites, or to the cell cultures after the parasites had entered cells, had little effect on either the percentage or rate of their subsequent development.

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PubMed1998

Dietary modulation of avian coccidiosis.

During the past several years, our laboratory has been investigating the anticoccidial activities of various natural products that have potential use as dietary supplements for coccidiosis control. Sources of fats containing high concentrations of n-3 fatty acids such as menhaden oil and flaxseed oil and flaxseed, when added to starter rations and fed to chicks from one day of age, effectively reduce lesions caused by the caecal parasite Eimeria tenella, but not lesions caused by Eimeria maxima. Our results are consistent with reports of effects of diets high in n-3 fatty acids on other protozoan parasites which suggest that the state of oxidative stress induced by these diets in the cells of both host and parasites is responsible for their parasitic actions. Artemisinin, a naturally occurring (Artemisia annua) endoperoxide and effective antimalarial significantly lowers lesions from E. tenella when given at low levels as a feed additive. The mechanism of its action is also considered to involve induction of oxidative stress. Diets supplemented with 8 p.p.m. gamma-tocopherol (abundant in flaxseeds) or with 1% of the spice tumeric, reduce mid-small intestinal lesion scores and improve weight gains during E. maxima infections. These compounds may exert their anticoccidial activity because they are effective antioxidants. Betaine, a choline analogue found in high concentrations in sugar beets, improves nutrient utilisation by animals under stress. When provided as a dietary supplement at a level of 0.15% it has enhanced the anticoccidial activity of the ionophore, salinomycin. Betaine may act as an osmoprotectant whereby it improves the integrity and function of the infected intestinal mucosa. In in vivo studies, betaine plus salinomycin significantly inhibit invasion of both E. tenella and E. acervulina. However, subsequent development of E. acervulina is inhibited more effectively with this combination treatment than development of E. tenella.

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PubMed1998

Use of monoclonal antibodies against chicken coccidia to study invasion and early development of Eimeria gruis in the Florida sandhill crane (Grus canadensis).

Eimeria gruis and E. reichenowi are common coccidial parasites of a number of crane species. In the present study, monoclonal antibodies (McAbs), elicited against Eimeria spp. of chickens and turkeys, cross-reacted with sporozoites and developmental stages of E. gruis in the tissues of Florida sandhill cranes. These McAbs were used to define the area of the intestine that was invaded by sporozoites of E. gruis and to demonstrate the feasibility of using McAbs to study the early development of E. gruis in the intestines and visceral organs of cranes. At 6 hr postinoculation (PI), E. gruis sporozoites were found primarily from just proximal to Meckle's diverticulum in the jejunum to the ileocecal juncture. Fewer sporozoites were found in the ceca and rectum, and none were found in the duodenum. Most of the sporozoites were in the middle third of the villi and within the lamina propria. At 14 days PI, developmental stages were detected in the ceca, jejunum, liver, and lungs but not in the heart, kidney, or brain. In the ceca and jejunum, the number, location, and maturity of the stages differed markedly.

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PubMed1997

Effect of betaine on the growth performance of chicks inoculated with mixed cultures of avian Eimeria species and on invasion and development of Eimeria tenella and Eimeria acervulina in vitro and in vivo.

At 7 d postinoculation (DPI) with a mixed culture of avian Eimeria species, 21-d-old chicks maintained in batteries and floor pens on a diet containing 0.15% (3 lb/ton) betaine plus 66 ppm (60 g/ton) salinomycin were significantly heavier and had significantly lower feed conversion ratios and mortality than chicks fed diets containing 0.15% betaine or 66 ppm salinomycin alone, or the control diet. At 31 DPI, when the chicks were 45 d old, the differences between the diet groups were not as great as at 7 DPI. In vitro, except at high concentrations, betaine was nontoxic to sporozoites of Eimeria tenella or Eimeria acervulina and had little effect on their invasion and development in cultured cells. In vivo, invasion by E. tenella and E. acervulina sporozoites was significantly reduced in all chicks fed diets containing betaine or salinomycin compared with that in control chicks. There was a significant interaction between betaine and salinomycin that impacted on invasion by both species. Overall development of E. tenella did not appear to be adversely affected by addition of betaine to diets containing salinomycin. Conversely, development of E. acervulina was reduced in chicks fed diets containing 0.075% (1.5 lb/ton) betaine plus 66 ppm salinomycin as compared with that in chicks fed salinomycin alone.

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PubMed1995

Enrichment of cecal species of avian Eimeria in a mixed culture by transfer of sporozoites from foreign host to natural host birds.

An effective method for the enrichment of two cecal species of the avian Eimeria in mixed cultures has been developed. The method involves the transfer of sporozoites of Eimeria tenella and Eimeria adenoeides from their specific site of invasion in a foreign host bird to the natural host, and markedly increases the numbers of the cecal species in relation to the other species in the mixture. Donor turkeys and chickens were inoculated with mixed cultures containing equal numbers of oocysts of the chicken coccidia, E. tenella + Eimeria acervulina, or the turkey coccidia, E. adenoeides + Eimeria meleagrimitis, respectively. After 8 to 24 h, the cecal tissues of the donor birds were scraped into phosphate-buffered saline and transferred, per os, to the natural host. On Day 6 to 7 posttransfer, numerous oocysts of E. tenella and E. adenoeides were found in the ceca of the recipient chickens and turkeys, respectively. In contrast, only occasional oocysts were found in a few birds in other areas of the intestine.

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PubMed1994

Ultrastructural observations of development of Eimeria tenella in a novel established avian-derived cell line.

Transmission electron microscopy was used to study the in vitro development of Eimeria tenella in a novel established avian-derived cell line (designated CEV-1/F7) used for antigen production in chicken immunization studies. Sporozoites of E. tenella were inoculated onto cell monolayers and the cells were fixed at 24-h intervals. Large numbers of intracellular sporozoites were seen at 24 h postinoculation (p.i.), and trophozoites were identified at 24-48 h p.i. Immature schizonts, some with budding merozoites, were seen by 48 h p.i. At 72-96 h p.i., immature and mature schizonts and extracellular merozoites were observed. No merozoite invasion occurred, but immature second-generation schizogony was seen in parasitophorous vacuoles of first-generation schizonts. No further development occurred and degeneration of most schizonts was seen by 120-144 h p.i. The results confirmed synchronous development of E. tenella until 48 h p.i., followed by asynchronous development and ultrastructural degeneration with increased incubation time.

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PubMed1994

In vitro and in vivo immunolabeling of sporozoites, schizonts, and sexual stages of Eimeria acervulina and E. tenella by a species- and stage-cross-reactive monoclonal antibody.

A cross-reactive monoclonal antibody (mAb), designated 1205, was used to study redistribution, parasitophorous vacuole (PV) incorporation, and in situ antigen production during the intracellular parasite development of Eimeria acervulina and E. tenella. Western-blot analysis of sporozoite preparations showed that the mAb recognized antigenic bands at 55 and 80 kDa. Indirect immunofluorescent antibody (IFA) labeling of sporozoites produced an internal dot pattern. Immunogold electron microscopy (IM) showed labeling of dense granules within sporozoites. The IFA pattern changed to a general-internal label in immature schizonts followed by a surface-tip pattern in mature merozoites both in vitro and in vivo. IM of the asexual stages revealed the same labeling pattern for the in vivo development of both species, and labeling of rhoptries was seen. In vitro, the PV membrane together with amorphous material within the PV was labeled by IFA during schizont development for E. tenella. No IM labeling of either the PV membrane or material within the PV was observed. Sexual stages seen in vivo for both species had the general-internal IFA pattern.

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PubMed1994

Establishment of a turkey cecal cell line and development of turkey coccidia within the cells.

Cells were dispersed from cecal tissues of 1- to 2-day-old turkeys using a mixture of collagenase and dispase to enrich for epithelial cells. The initial culture produced from these cells (TCC) appeared to be heterogeneous, but, as the cells were cultured through 25 passages, they assumed a more fibroblastic appearance. Cellular invasion of the TCC by two species of turkey coccidia, Eimeria adenoeides and Eimeria meleagrimitis, was not enhanced, as compared with invasion in turkey kidney cells (TKC), the cell culture system standardly used to study the avian coccidia in vitro. However, early development by one of the species, E. meleagrimitis, was markedly increased in the TCC (Passages 6 through 19) over that in TKC. Thirty to 42% of the parasites that invaded the TCC developed beyond the sporozoite stage, as compared with 5% development in TKC. Mature first-generation schizonts were observed within 24 hr postinoculation in the TCC, but not until 48 hr in the kidney cells. There was no evidence that development of the second generation was initiated in either the TCC or kidney cells.

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PubMed1992

Maternal immunization with gametocyte antigens as a means of providing protective immunity against Eimeria maxima in chickens.

In the present study, we wished to demonstrate the ability of surface gametocyte antigens to induce protective immunity against Eimeria maxima infections in chickens. In order to accomplish this goal, we employed maternal immunization as a means of providing large amounts of specific antibodies to offspring chicks. Upon challenge with sporulated E. maxima oocysts, chicks from hens immunized with affinity-purified gametocyte antigens showed greatly reduced oocyst production compared with chicks from sham-immunized hens. These results suggest that maternal immunization with gametocyte antigens can be used as a means to provide transmission-blocking immunity against E. maxima infections.

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PubMed1992

Effect of monensin on ultrastructure and cellular invasion by the turkey coccidia Eimeria adenoeides and Eimeria meleagrimitis.

Freshly excysted sporozoites (SZ) of the turkey coccidia Eimeria meleagrimitis and Eimeria adenoeides were incubated at 41 C in concentrations of monensin from .01 to 1.0 microgram/mL, washed free of the drug, and either processed for phase, fluorescence, and transmission electron microscopy or inoculated into cultures of turkey kidney cells. Phase microscopy indicated that after 1.5 h incubation in 1.0 micrograms/mL monensin, about 60% of the SZ of E. meleagrimitis had become notably rounded or displayed localized protrusions. These alterations were accompanied by ultrastructural abnormalities (in 90% of the SZ) including vacuoles in the cytoplasm, bulging and separation of plasma membrane layers, and dense bands in the refractile bodies that extended toward the periphery of the refractile body. Similar morphological and ultrastructural changes were observed in over half of the E. adenoeides SZ after 2 h incubation in 1.0 micrograms/mL monensin. Additionally, some specimens contained a pycnotic nucleus that was usually surrounded by a large vacuole. After 4 h incubation, almost all of the SZ displayed some degree of ultrastructural damage. Indirect fluorescent antibody labeling with parasite-specific monoclonal antibodies demonstrated clouds of antigen surrounding the monensin-treated but not the untreated SZ, suggesting an increase in permeability with incubation in monensin. With both E. meleagrimitis and E. adenoeides, the structural changes were reflected in a significant inhibition of cellular invasion. The inhibitory activity of monensin was concentration- and time-dependent in that the greatest inhibition of invasion was observed in SZ incubated for 4 h in 1.0 micrograms/mL of monensin; shorter incubation times or lower concentrations of monensin having less effect.

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PubMed1992

Localization of a low molecular weight antigen of Eimeria tenella by use of hybridoma antibodies.

Three monoclonal antibodies (Mabs), found by western blot analysis to recognize 10-kDa bands of Eimeria tenella sporozoite preparations, were used with immunoelectron (IE) microscopy, immunogold-silver staining (IGSS), and indirect immunofluorescent antibody (IFA) light microscopy to determine the location and distribution of the antigens in or on extra- and intracellular parasites. All 3 of the Mabs (designated C3, E5, and 1231) were found by IE microscopy to label amylopectin granules of extracellular sporozoites. Additionally, these Mabs extensively gold-labeled the sporocyst wall. In cultured primary chicken kidney cells inoculated with sporozoites of E. tenella, IGSS showed surface labeling of the parasite and intense labeling of the infected host cells by 6 hr postinoculation (PI). At 24 hr PI, host cell vacuoles in infected and uninfected cells were labeled by the 3 Mabs by IFA. The E5 and C3 Mabs also were seen to label the host cell membrane of newly infected cells. The C3 and 1231 Mabs showed little label of the host cells by 48 hr PI, but the parasites still were labeled up to 96 hr PI. The E5 Mab had intense IFA labeling of infected host cells at 48 hr PI. The results of this study indicate that parasites apparently release antigenic material during the early stages of parasite development and that this material is found internally and/or on the surface of the infected host cells.

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PubMed1991

Development of resistance to coccidiosis in the absence of merogonic development using X-irradiated Eimeria acervulina oocysts.

Sporulated oocysts of the protozoan Eimeria acervulina were subjected to 0, 10, 15, 20, or 30 krad of X-irradiation and inoculated into susceptible outbred chickens to determine if radioattenuated coccidia could induce protection against parasite challenge. Irradiation treatment had an appreciable dose-dependent effect on parasite development. Insignificant numbers of oocysts were produced by chickens inoculated with parasites that had been exposed to greater than 10 krad X-irradiation. Sporozoites exposed to 15 or 20 krad irradiation conferred significant protection against the appearance of intestinal lesions after parasite challenge. Sporozoites subjected to the highest dose level (30 krad) did not produce any significant level of protection. To investigate this phenomenon further and assess intracellular parasite development, susceptible outbred strains of chickens were administered either nonirradiated (0 krad) oocysts or oocysts that were exposed to an optimal dose (15 krad) or a high dose (30 krad) of X-irradiation. Immunofluorescence staining of tissue sections from each treatment group at various intervals after the initial administration of irradiated parasites indicated that sporozoites exposed to 15 krad irradiation were as capable of invading the host intestinal epithelium as nonirradiated sporozoites. However, at 48, 60, 72, and 96 hr, there was a marked reduction in merogonic development in groups receiving irradiated sporozoites compared to those inoculated with nonirradiated parasites. The latter parasites underwent profuse merogonic development; in contrast, irradiated parasites demonstrated little (15 krad) or no (30 krad) merogonic development. These results suggest that induction of a protective immune response occurs during a critical period early in intracellular development of E. acervulina.

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PubMed1991

X-irradiation of Eimeria tenella oocysts provides direct evidence that sporozoite invasion and early schizont development induce a protective immune response(s).

Sporulated oocysts of the protozoan parasite Eimeria tenella were attenuated by exposure to various doses of X-radiation to inhibit intracellular replication and thus determine whether sporozoites alone can induce a protective immune response. Exposure to doses greater than 15-kilorads had a significant effect on development, as indicated by the absence of oocyst production in chickens infected with parasites treated with 20 or 30 kilorads of radiation. Infection with nonirradiated or 15-kilorad-exposed parasites led to either normal or reduced oocyst shedding. Equivalent protection was afforded chickens inoculated with a minimum immunizing dose of either nonirradiated or 20-kilorad-irradiated E. tenella oocysts. Immunofluorescence staining of cecal tissue from chickens inoculated with 10(7) nonirradiated or 20- or 30-kilorad-irradiated oocysts with stage-specific monoclonal antibodies showed no significant difference in sporozoite invasion between treatment groups. Normal merogonic development was observed at appropriate times (48, 60, 72, and 96 h) postinfection in chickens inoculated with nonirradiated oocysts. In contrast, irradiated parasites exhibited minimal merogonic development at 48 h postinfection. Furthermore, no merogonic stages were observed at times of otherwise peak merozoite development (60, 72, and 96 h) in cecal tissue from chickens inoculated with irradiated parasites. Infection of chicken cells with irradiated or nonirradiated parasites in vitro corroborated these findings and indicate that events early after sporozoite invasion induce a protective immune response against this parasite.

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PubMed1991

Monoclonal antibody produced against partially purified sporozoite antigen inhibits invasion of cells by sporozoites of avian Eimeria species.

Previous studies showed that molecules of in vitro-cultured primary turkey kidney cells bound to 23-, 40-, and 60- to 65-kDa antigens of sodium dodecyl sulfate-solubilized sporozoites of Eimeria adenoeides. Similar binding to antigens of three other species of avian Eimeria, E. tenella, E. acervulina, and E. meleagrimitis, is now reported. Strips containing the most avidly bound sporozoite antigen (approximately 40 kDa) were excised from the sodium dodecyl sulfate-polyacrylamide gels on which E. adenoeides antigens had been electrophoretically separated. The strips were homogenized and injected into mice to produce hybridoma cell lines. Twelve cell lines secreting monoclonal antibodies (McAb) that reacted with E. adenoeides sporozoites were detected. One of these McAb, H11C3, reacted with structures in the anterior tip of sporozoites of E. adenoeides and five other species of avian Eimeria. When included in the inoculation medium, this McAb significantly inhibited invasion of cultured kidney cells by sporozoites of E. adenoeides and E. tenella. In contrast, when the sporozoites were pretreated with McAb H11C3 and then washed free of the antibody, no inhibition of invasion was observed.

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