Oral inoculation of ultraviolet-irradiated Eimeria species oocysts protects chickens against coccidiosis.

Prevention of coccidiosis is one of the best ways of controlling disease. Therefore, the present study was carried out to evaluate the protective effect of ultraviolet (UV)-irradiated sporulated oocysts of Eimeria species against coccidiosis in layer chickens. One hundred forty-four one-day-old layer chicks were randomly divided into 4 groups (n = 36), including non-immunized/non-challenged negative control group (NC group), non-immunized/challenged control group (NIC group), non-irradiated sporulated oocyst/challenged group (CA group), and UV-irradiated sporulated oocyst/challenged (UV group). At the age of 4 days, chickens in groups UV and CA were both orally inoculated with 1.0 × 104 UV-irradiated and non-irradiated sporulated oocysts of Eimeria species, respectively. Chickens in groups NIC and NC were served as positive and negative controls, respectively. Chickens in all groups were orally challenged with 7.5 × 104 sporulated oocysts of Eimeria species except the NC group at the age of 21 days. The results revealed that chicks receiving UV-irradiated sporulated oocysts had no signs of illness with minimal or no changes in the cecal integrity and a significantly lower oocyst shedding (OPG) than in the NIC group. Additionally, the cytokine gene expression profiles were evaluated. Expression levels of IL-2, IL-12, and IFN-γ were significantly higher in the spleen of chicks in the UV and CA groups than in the NC group post-challenge. As expected, treatment with irradiated oocysts resulted in a significant reduction in oocyst shedding and maintenance of cecal mucosal integrity. Furthermore, the body weight was higher in chickens inoculated with UV-irradiated oocysts than their non-irradiated counterparts. In conclusion, our results demonstrate that inoculation with UV-irradiated sporulated oocysts of Eimeria species can produce a substantial reduction in infection symptoms.

Evaluation of an experimental irradiated oocyst vaccine to protect broiler chicks against avian coccidiosis.

The current study investigates the use of irradiated oocysts to protect broiler chicks, raised on litter, from infection with multiple species of Eimeria. In order to determine the optimum radiation dose for each Eimeria species, 1-day-old chicks were immunized with oocysts of Eimeria maxima, Eimeria acervulina, or Eimeria tenella exposed to gamma radiation ranging from 0-500 Gy. The litter oocyst counts at 7 days postimmunization, and the effect on weight gain following a challenge infection, decreased with an optimum dose between 150-200 Gy. Based on this finding, broiler chicks were immunized with a mixture of E. maxima, E. acervulina, and E tenella that had been exposed to 150 or 200 Gy. This resulted in more than a 100-fold reduction in litter oocyst counts and significant protection from a challenge infection, as measured by improved weight gain and feed conversion ratio (FCR). Immunization of birds with oocysts receiving 200 Gy was less effective in providing protection from a challenge infection. An additional formulation of vaccines containing two different oocyst doses of the three species that had been irradiated with 150 Gy were evaluated in their ability to attenuate oocyst output and convey protection to challenge. Results were similar with both high and low numbers of irradiated oocysts. Immunized chicks shed less oocysts at 7 days postimmunization and were protected from negative effects of challenge infection as measured by FCR, changes in weight gain, lesion scores, and measurement of body composition. However, the level of protection was somewhat less than that achieved by immunization with nonirradiated oocysts. The overall conclusion is that an irradiated oocyst vaccine developed in this study can effectively protect chicks that are raised on litter from challenge infection with multiple species of Eimeria, comparable to vaccines with virulent or precocious strains.

High hydrostatic pressure and UV light treatment of produce contaminated with Eimeria acervulina as a Cyclospora cayetanensis surrogate.

The prevalence, size, genome, and life cycle of Eimeria acervulina make this organism a good surrogate for Cyclospora cayetanensis, a protozoan that causes gastroenteritis in humans, including recent outbreaks in the United States and Canada associated with contaminated raspberries and basil. Laboratory studies of C. cayetanensis are difficult because of the lack of readily available oocysts and of infection models and assays. UV radiation and high-hydrostatic-pressure processing (HPP) are both safe technologies with potential for use on fresh produce. Raspberries and basil were inoculated with sporulated E. acervulina oocysts at high (10(6) oocysts) and low (10(4) oocysts) levels, and inoculated and control produce were treated with UV (up to 261 mW/cm2) or HPP (550 MPa at 40 degrees C for 2 min). Oocysts recovered from produce were fed to 3-week-old broiler chickens, which were scored for weight gain, oocyst shedding, and lesions at 6 days postinoculation. Oocysts exhibited enhanced excystation on raspberries but not on basil. Birds fed oocysts from UV-treated raspberries had reduced infection rates, which varied with oocyst inoculum level and UV intensity. Birds fed oocysts from UV-treated raspberries (10(4) oocysts) were asymptomatic but shed oocysts, and birds fed oocysts from UV-treated basil (10(4) oocysts) were asymptomatic and did not shed oocysts. Birds fed oocysts from HPP-treated raspberries and basil were asymptomatic and did not shed oocysts. These results suggest that UV radiation and HPP may be used to reduce the risk for cyclosporiasis infection associated with produce. Both treatments yielded healthy animals; however, HPP was more effective, as indicated by results for produce with higher contamination levels.

Coccidial contamination of raspberries: mock contamination with Eimeria acervulina as a model for decontamination treatment studies.

Numerous outbreaks have been reported since 1995 in the United States and Canada that were linked to the consumption of imported fresh raspberries contaminated with Cyclospora. Because Cyclospora has no laboratory animal hosts, Eimeria acervulina, a common chicken coccidium similar in characteristics to Cyclospora, was used as a surrogate to test decontamination treatments. Raspberries were mock contaminated with E. acervulina-sporulated oocysts in a water suspension, then exposed to washing, freezing, heat, or irradiation before they were fed to chicks. The presence of oocysts in the contaminated raspberries was confirmed either by duodenal lesions or oocysts in cecal contents 5 days postinoculation (PI) or in fecal contents 6 days PI, after 24 h of fecal collection. Washing of raspberries was generally not adequate in removing coccidial contamination, but freezing and heat treatment appeared effective. Gamma irradiation of E. acervulina-sporulated oocysts at a dose of 0.5 kGy was partially effective, but it was completely effective at 1.0 kGy and higher. We suggest that E. acervulina, for mock contamination of raspberries and subsequent decontamination treatments, is easy to handle, safe, and economical to study.

Protection against coccidiosis in outbred chickens elicited by gamma-irradiated Eimeria maxima.

In an effort to develop an attenuated coccidiosis vaccine against coccidiosis, we exposed Eimeria maxima oocysts to an optimum dose of gamma irradiation (17 kRad) that does not affect sporozoite invasion of the intestinal mucosa but does prevent asexual parasite development. Irradiated E. maxima oocysts were suspended in gelatin slabs and placed in battery cages for ingestion by 1-day-old chickens. Separate groups of chickens were given gelatin slabs containing nonirradiated E. maxima oocysts or were inoculated per os with either irradiated or nonirradiated E. maxima oocysts. Chickens infected with irradiated or nonirradiated oocysts by either oral inoculation or gel delivery showed a dose-dependent protection against weight loss associated with E. maxima challenge compared with unimmunized controls. In general, nonirradiated oocysts elicited protective immunity at lower immunization doses compared with irradiated oocysts. These experiments were extended to a floor pen study wherein 1-day-old male and female broiler chickens were given irradiated or nonirradiated E. maxima oocysts in gelatin slabs in hatching boxes and challenged at 4 wk of age. A significant reduction (P < 0.05) in lesion scores was observed for chickens immunized with either irradiated or nonirradiated oocysts compared with unimmunized controls. Although no significant difference (P > 0.05) was observed in weight gain between these groups, both male and female chickens inoculated with irradiated E. maxima oocysts showed about a 10% greater weight gain than unimmunized controls. For both male and female chickens, average weights at challenge were greater in groups that were immunized with 17-kRad-irradiated E. maxima oocysts compared with those animals immunized with nonirradiated oocysts.

A new coccidian (Apicomplexa: eimeriidae) in the northern pocket gopher (Thomomys talpoides) and a comparison of oocyst survival in hosts from radon-rich and radon-poor soils.

Forty (93%) of 43 northern pocket gophers (Thomomys talpoides) from the Jemez Mountains, Sandoval County, New Mexico (USA), had coccidian oocysts in their feces when examined. We describe this parasite, Eimeria jemezi, n. sp. Sporulated oocysts were subspheroidal, 13.3 by 12.2 (10 to 17 by 9 to 15) microns, with sporocysts ellipsoidal, 7.1 by 4.4 (5 to 9 by 4 to 5) microns; micropyle and oocyst residuum were absent, but polar bodies, Stieda bodies and sporocyst residua were present. All gophers were collected from two sites of similar habitat 7 km apart. One site (R+) had a high soil radon content (> or = 50 to 70 picocuries (pCi) per liter of air) whereas the other site (R-) had soils that were near average natural levels (1.2 to 1.6 pCi/g uranium nucleotides per gram of soil; < 1.9 pCi/g thorium nucleotides). Twenty-one (88%) of 24 gophers from the R+ site had coccidian oocysts in their intestines when examined, but none of these oocysts ever sporulated, whereas all 19 (100%) gophers from the R- site had coccidian oocysts in their intestines and 16 (84%) of these samples sporulated normally under laboratory conditions. The elevated radon content of the soil may have had an adverse effect on the sporulation of this coccidian while it still was intracellular within its host.

Avian Eimeria: effects of gamma irradiation on development of cross-species immunity in foreign and natural host birds.

Repeated inoculation (immunization) of white leghorn chickens with oocysts of the turkey coccidium, Eimeria adenoeides, resulted in significantly improved weight gain and feed-conversion ratios (feed efficiency) after E. tenella challenge. However, the development E. tenella in the immunized chickens did not differ markedly from that in unimmunized chickens, and intestinal lesions remained severe (greater than 3.0). Apparently improved weight gain and feed efficiency can be maintained in the presence of extensive parasite development. The ability to elicit cross-protective immunity was abrogated when the E. adenoeides oocysts were exposed to 15 kRad of gamma irradiation before inoculation into the chickens. Sporozoites existing from irradiated oocysts of E. acervulina also failed to immunize the chickens against challenge with E. tenella but protected chickens almost completely against homologous challenge with E. acervulina. The results indicate that cross-species immunity is not elicited by all developmentally arrested sporozoites and that the ability to produce cross-species immunity can be destroyed by gamma irradiation of the immunizing species.

Protective immunity against coccidiosis elicited by radiation-attenuated Eimeria maxima sporozoites that are incapable of asexual development.

Eimeria maxima oocysts were exposed to various doses of gamma radiation that did not affect sporozoite invasion of intestinal epithelium but did prevent subsequent merogonic development therein. Although merogony and oocyst formation were inhibited, parasites exposed to 12 kRad radiation induced a level of immunity against E. maxima challenge equivalent to that induced by non-irradiated oocysts. Chickens immunized per os with 20 kRad-treated E. maxima oocysts were not protected against coccidial challenge. Immunization of chickens with a single low dose (five oocysts) of non-irradiated (0 kRad) or irradiated (12 kRad) E. maxima was effective in preventing weight depression after coccidial challenge. Immunofluorescence staining of intestinal tissue from chickens infected with irradiated (12 or 20 kRad) or non-irradiated (0 kRad) E. maxima oocysts with developmental stage-specific monoclonal antibodies showed that sporozoite invasion was similar in all groups. However, merogonic development was not observed at any time postinfection in chickens infected with irradiated oocysts, unlike the case with chickens infected with non-irradiated parasites. These results suggest that sporozoite-infected host cells are capable of eliciting complete protection against E. maxima challenge.

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.

Ultrastructural changes following treatment with a microwave pulse in the oocyst of Eimeria magna Perard, 1925.

The ultrastructure of oocysts of Eimeria magna was studied before and after their exposure to unique pulse of microwaves (2.450 MHZ; 600 W) of different durations (10, 15 and 20 s). Following treatment, the progressive destruction of the three layers of the oocyst wall was observed, the innermost being destroyed first. Internal structures were also affected, resulting in swollen mitochondria, a loss of ribosomes and fragmentation of the rough endoplasmic reticulum; moreover, the wall-forming bodies were no longer identifiable. Further studies using microwave pulses on biological material should be carried out to improve our understanding of the consequences of such treatment and to investigate its utility in the control of transmissible pathogenic organisms.

Eimeria tenella: in vitro development in irradiated bovine kidney cells.

The initial infection and first-generation development of Eimeria tenella was quantified using a cloned MDBK (Madin-Darby Bovine Kidney) cell line, irradiated with gamma radiation prior to infection, as the host cell. Irradiated cell cultures were found to be more susceptible to infection and had a greater capacity to support parasite development than non-irradiated cultures. It was suggested that the larger proportion of cells in the G2 phase of the cell cycle, the larger individual cell size and the inhibition of cell division in the irradiated cultures were all factors contributing to the increased susceptibility to infection and capacity to support parasite growth and development. The application of this technique (host cell irradiation) to the cultivation of other intracellular, protozoan parasites is discussed.

The microwave oven: a novel means of decontaminating parasitological specimens and glassware.

This study was conducted to determine the effects of microwave radiation on developmental and infective stages of Eimeria nieschulzi, Strongyloides ratti, and Taenia taeniaeformis. Fecal samples and laboratory preparations containing these three parasites were subjected to microwave radiation for brief periods in a microwave oven, and then in vitro and/or in vivo assessments of viability were made for each organism and preparation. Our results showed that microwave irradiation is extremely effective in killing or preventing development of helminth and protozoan parasites without unduly distorting eggs or developmental stages. Therefore, microwaves may prove useful for decontaminating diagnostic samples or sterilizing contaminated materials in the laboratory and thus for reducing risk to laboratory personnel from parasites of public health importance.

The immunizing potential of sporulated oocysts of Eimeria nieschulzi exposed to heat and Co-60 gamma-radiation.

Sporulated oocysts of Eimeria nieschulzi Dieben 1924, a rat coccidium, were exposed to radiation, heat, or both in an effort to attenuate the parasite. Moderate levels of each treatment or combination thereof attenuated the parasite, reduced pathogenesis (as judged by oocyst discharge during primary infection), and produced immunity to challenge when the oocysts were subsequently inoculated into rats. Thus, heat- and/or radiation-treated E. nieschulzi oocysts fed to rats could reduce pathogenesis during a primary infection and yet give good homologous protection.

The effects of heat and cobalt-60 gamma-radiation on excystation of the rat coccidium, Eimeria nieschulzi Dieben, 1924.

Sporulated oocysts of Eimeria nieschulzi Dieben, a rat coccidium, were exposed for 1 hr to Cobalt-60 gamma-radiation (15, 30, or 60k-rads), to heat (35, 40, or 45 C) , or to both concurrently (15, 30, OR 60 K-RADS AT 35 C) to compared the excystation capabilities of treated vs nontreated parasites. Intact treated oocysts appeared structurally unaltered when viewed with the light microscope. Excystation of sporozoites occured in all treated groups when their sporocysts were exposed to a trypsin-sodium taurocholate (TST) fluid, but after 150 min in TST the excystation rate was significantly lower than in nontreated sporocysts. Sporozoites which excysted from treated sporocysts were abnormal both in the excystation process and in their form and movement once outside the sporocysts.

Effect of irradiation (gamma rays) on the biology of Eimeria tenella oocysts.

Effect of gamma rays on the biology of the progeny of the irradiated Eimeria tenella oocysts was investigated. The parent inoculum of sporulated oocysts was exposed to 5 to 60 kR (gamma rays). These oocysts were fed to chicks. The oocysts voided by the chicks were collected and sporulated. The sporulation rate, pathogenicity, immunogenicity and reproduction potential of these oocysts--the progeny of the irradiated oocysts--were compared with those of the unirradiated oocysts. It was observed that increase of irradiation dose caused progressive decrease in the pathogenicity of the oocyst suspension. The oocysts exposed to 30 and 40 kR produced only mild infections whereas those exposed to 50 kR and above, were noninfective. No difference in pathogenicity, immunogenicity and reproduction potential of unirradiated oocysts and the oocysts progeny of the irradiated oocysts, was seen. It was concluded, therefore, that the effect of irradiation was limited to the inoculum exposed to it, and was not transmissible to the progeny of the irradiated oocysts.

[The destruction of parasitic resistant stages in sludge by irradiation with low accelerating voltage electrons (author's transl)]. / Die Vernichtung parasitärer Dauerformen im Klärschlamm durch Bestrahlung mit niederenergetischen Elektronen

The destroying effect of ionizing radiation on parasitic resistant stages in sludge has been tested. Suitable for that process is an electron beam accelerator which will be provided with energy from the electric power supply network which can be switched on and off according to the requirements. Such modern utilities have an enormous beam capacity and a high operating safety. The process is working according to the continuous flow principle and at room temperature. In a series of 13 experiments the effect of different doses has been tested. A dose of 480 kRad (accelerating voltage; 400 kV, beam current; 10 mA, irradiation time: 24 sec.) can easely obtained in practical work and is economically acceptable. By these means approximately 97% of the following parasitic stages have been destroyed: undeveloped eggs of Ascaris suum, Trichuris suis, Fasciola hepatica and gastrointestinal strongylids of pigs, embryonated eggs of Capillaria obsignata and probably of Taenia spec. A few third-stage larvae of Oesophagostomum (Strongylidae) of pigs survived even 108 sec of irradiation; however, they did not develop to maturity in the definitive host. Approximately 25% of the sporulated oocysts of Eimeria renella were still infective after 108 sec of irradiation.

Effect of gamma-irradiation on oocysts of Eimeria necatrix.

Effect of gamma radiation on oocysts of Eimeria necatrix was investigated. It was observed that oocysts exposed to 200 kR or above did not sporulate. Irratiation at 10-150 kR caused a progressive decrease in sporulation. Irradiation affected normal development of unsporulated oocysts as the zygote protoplasm divided into unequal masses or was shattered into granules. Increase in the intensity of irradiation of sporulated oocysts resulted in the progressive decrease in severity of the resultant infections in chicks and their effects - mortality, type of lesions developed, total oocyst production and immunity produced - were comparable with infections induced by decreasing the number of unirradiated oocysts. Infection produced by 1000 unirradiated oocysts was comparable with that resulting from 50 000 oocysts irradiated at 25 kR. Infection obtained with 20 000 unexposed oocysts approximated to that produced by 50 000 oocysts irradiated at 2-5 kR. It was concluded that irradiation abolished infectivity of the oocysts/sporozoites rather than bringing about attenuation of the parasite.