Improved Method for High-Yield Excystation and Purification of Infective Sporozoites of Eimeria spp.1

This report describes a new, gentle procedure for rapid and efficient excystation of large numbers of infective sporozoites of Eimeria vermiformis and Eimeria stiedai. Excysted sporozoites are purified using modifications of a previously described ion-exchange chromatography method. The procedure avoids physical breakage of oocysts and results in greater than 70% recovery of the sporozoites present as sporulated oocysts (i.e. 5–6 sporozoites per sporulated oocyst). The recovered sporozoites are greater than 95% pure and are infective in vivo. We routinely isolate greater than 2 times 10⁸ sporozoites without the use of specialized or expensive equipment.     

Extraintestinal Development of Caryospora simplex (Apicomplexa: Eimeriidae) in Experimentally Infected Mice,Mus musculus1

Developmental stages of Caryospora simplex were found in connective tissue of the cheek, tongue, and nose of Swiss-Webster and C57 BL/6 mice (Mus musculus) from 8 through 70 days after oral inoculation with 50,000 or 250,000 oocysts, or 60,000 free sporocysts of the same species obtained from an Ottoman viper, Vipera xanthina xanthina. The earliest developmental stages were seen on day 8 post-inoculation (PI) and consisted of two types of meronts and gamonts (undifferentiated sexual stages). Gamonts, microgametocytes, macrogametes, and unsporulated oocysts were found on days 10 and 12 PI. Fully sporulated, thin-walled oocysts containing eight sporozoites surrounded by a thin sporocyst membrane were first seen 12 days PI. Monozoic cysts (caryocysts) were first seen 12 days PI and appeared fully viable throughout the duration of the study, 70 days PI. Four mice injected intra-peritoneally with 150,000 free sporozoites and killed 12 days PI contained unsporulated and sporulated oocysts in connective tissues of the cheek, tongue, and nose, suggesting that sporozoites may be carried to the site of infection via the lymphatic/circulatory system. Four cotton rats, Sigmodon hispidus, inoculated orally with 250,000 oocysts all had unsporulated and sporulated oocysts of C. simplex in connective tissue of the cheek, tongue, and nose when killed on day 12 PI, indicating extraintestinal development in the secondary host is not species specific. This is the first report of a heteroxenous coccidium with both asexual and sexual development in the primary (predator) and secondary (prey) hosts.     

Tissue and Organ Specificity of Eimeria tenella (Railliet and Lucet, 1891) Fantham, 1909 in Cecectomized Chickens*†

SYNOPSIS. The ceca of 2-week-old chicks were surgically removed. One week post-operation each cecectomized bird was given 2 × 10⁶ sporulated Eimeria tenella oocysts per os. Birds from the same hatch, with intact ceca, served as controls and were infected the same time as the cecectomized birds. However, in order to reduce mortality, control birds were each given 1 × 10⁴ sporulated E. tenella oocysts per os. Four cecectomized and 5 control birds were killed 12, 24, 48, 72, 96, 120, and 144 hours after inoculation. Tissues from the small and large intestine of each bird proximal to the cecal junction were removed, processed by the dioxan method, and studied microscopically for developmental stages of the parasite. Developmental stages of the parasite were observed in all sections from the large intestine of cecectomized chickens. Initial sporozoite penetration and subsequent development of the parasite in this location was similar to that observed in the cecal mucosa of non-cecectomized chickens. No parasites were observed in sections of the small intestine of cecectomized birds 12 or 24 hours after inoculation, and findings after 48 and 72 hours were inconsistent. However, numerous parasites were observed in sections 96, 120, and 144 hours post-inoculation. In contrast, endogenous stages of the parasite were not seen in tissue sections of the small and large intestine of birds with intact ceca until 120 hours after inoculation. Numerous young gametocytes were then observed in sections from all birds. Similarly, mature gametocytes were observed in all fixed sections 144 hours after inoculation. No evidence was found that would indicate whether or not infection in the small and large intestine of birds with intact ceca or the small intestine of cecectomized birds was initiated by sporozoites or merozoites, nor was evidence found to suggest that development of any stage of the parasite was suppressed in these organs.     

True Intermediate Hosts for Eimeria funduli (Apicomplexa) from Estuarine Fishes1

Grass shrimp (Palaemonetes pugio) fed liver containing sporulated oocysts of Eimeria funduli permitted development of sporozoites that became infective to a variety of killifishes. The shrimp's gastric mill mechanically ruptured the oocysts. Sporozoites then excysted through an opening in the sporocyst, and by 12 and 13 h postinfection (p.i.) numerous empty sporocysts and free sporozoites occurred extracellularly in the intestine of the grass shrimp. Even at 5, 7, 8, 11, 46, 79, and 83 days p.i., and presumably for many months, numerous sporozoites still occurred free in the alimentary tract or between intestinal cells. The coccidium did not infect killifish at either 2 or 4 days p.i., but did at 5 days; after release from the sporocyst, it became more elongate with a distinct nucleus and two relatively large refractile bodies. Infections of E. funduli resulted in about one half of the fish that were fed either entire hepatopancreas or tips of hepatopancreas from experimentally infected shrimp. Feeding either the entire alimentary tract proximal to the first abdominal segment or any portion of that section from experimentally infected shrimp produced infections in nearly all tested fish. Feeding portions of the cephalothorax without any attached hepatopancreas or alimentary tract failed to produce an infection. Feeding killifish with wild grass shrimp from an enzootic area produced infections in only a fourth of the fish sample; however, feeding experimentally infected wild, laboratory-reared, and juvenile grass shrimp produced infections in nearly all fish. Palaemonid shrimps other than P. pugio also can serve as intermediate hosts for E. funduli, and these shrimps include Palaemonetes vulgaris, P. paludosus, P. kadiakensis, and Macrobrachium ohione. In contrast, a penaeid shrimp, mysidacean, amphipod, and crab fed liver with sporulated oocysts did not produce infections when fed to killifish.     

Effect of dietary zinc level on serum carotenoid levels,body and shank pigmentation of chickens after experimental infection with coccidia

Abstract

Two experiments were conducted to test the effects of a dietary zinc amino acid complex (Zn-AA) and an anticoccidial drug on Eimeria acervulina or Eimeria tenella infections. In each experiment, 288 day-old Three-Yellow-Chickens were used in a 2 × 3 factorial experimental design. Six groups were arranged randomly to receive three levels of Zn-AA (0, 40, or 80 mg/kg) alone or with salinomycin (60 mg/kg). Additionally an uninfected group was set as negative control. At the age of 21 days birds in Exp. 1 were inoculated with 3 · 10⁴ sporulated E. acervulina oocysts, while birds in Exp. 2 were inoculated with 1.5 · 10⁴ sporulated E. tenella oocysts. In Exp. 1, E. acervulina did not suppress growth performance significantly, but in groups without salinomycin it significantly reduced serum carotenoid levels on day 7 after inoculation and body and shank pigmentation on day 42. Salinomycin medication maintained serum carotenoids and visual colour of inoculated birds, but Zn-AA did not influence these parameters. In Exp. 2, growth performances of infected and uninfected chickens were similar. Infection decreased to only serum carotenoid levels on day 14 after infection, and colour scores on day 42 in the inoculated group without salinomycin and Zn-AA supplementation. The birds that received Zn-AA had significantly higher serum carotenoid levels and colour scores than those that did not. Although supplementation of Zn-AA cannot avoid coccidial damage of caecum, it prevents the reduction of serum carotenoids and pigmentation of Three-Yellow-Chicken infected with E. tenella, but not after infection with E. avervulina. The interactive effects between Zn-AA and salinomycin on growth performance and pigmentation were not significant.     

Development of Eimeria meleagrimitis Tyzzer from Sporozoites and Merozoites in Turkey Kidney Cell Cultures*

SYNOPSIS. Sporozoites and 1st-, 2nd-, and 3rd-generation merozoites of Eimeria meleagrimitis were inoculated into primary cultures of turkey kidney cells. In vitro-excysted sporozoites developed into mature macrogamonts in 8 days; in vivo-excysted sporozoites developed into 2nd- or 3rd-generation schizonts within 5 to 7 days. First-generation merozoites obtained from infected turkeys produced mature 2nd-generation schizonts within 24 h. Second-generation merozoites from turkeys produced mature macrogamonts and oocysts within 72 h, whereas 3rd-generation merozoites produced these stages within 48 h. The oocysts that developed from 3rd-generation merozoites sporulated at 25 C and were infective for turkeys. The timing of the early stages and the intervals between schizogonic generations in cultures were comparable with those in turkeys. Morphologic parameters, however, indicated that some differences existed between in vitro and in vivo development. Second- and 3rd-generation schizonts and gamonts that developed after inoculation of cultures with merozoites were similar to stages in turkeys. Oocysts, however, were significantly smaller (P < 0.05) in cultures. All stages that developed after inoculation of cultures with sporozoites were smaller (P < 0.05) than their in vivo counter parts.     

Serum and liver zinc,copper, and iron in chicks infected withEimeria acervulina orEimeria tenella

Two-wk-old broiler chicks were inoculated via crop intubation withEimeria acervulina at two doses: 10⁵ or 10⁶ sporulated oocysts/bird or withEimeria tenella at a dose of 10⁵ sporulated oocysts/bird. Serum and liver samples were collected on days 3 and 6 post-inoculation (PI). There were no significant changes in serum or liver zinc, copper, and iron concentrations in any of the infected groups by 3 d PI. However, on d 6, PI serum protein was significantly reduced in all of the infected groups compared to their pair-fed controls. The chicks infected withE. tennella had significantly reduced serum zinc (1.20 vs 1.77 μg/mL) and iron (0.44 vs 1.28 μg/mL) concentrations and significantly elevated serum copper (0.28 vs 0.17 μg/mL) and ceruloplasmin levels (20.33 vs 11.11 μg/mL) compared to their pair-fed counterparts. Those chicks infected withE. acervulina (10⁶ oocysts/bird) exhibited significantly reduced serum iron concentration by 6 days PI (0.90 vs 1.14 μg/mL). Liver zinc was significantly increased in the chicks infected withE. tenella (349 vs 113 μg/g dry liver wt), as was copper (24 vs 19 μg/g), whereas liver iron concentration was significantly reduced (172 vs 243 μg/g) compared to pair-fed controls. At both dose levels, the chicks infected withE. acervulina exhibited a significant reduction in liver iron by 6 d PI. Hepatic cytosol metals generally reflected whole tissue levels. Metallothionein (MT)-bound zinc was significantly elevated in the chicks infected withE. tenella. Iron bound to a high molecular weight, heat-stable protein fraction (presumably cytoplasmic ferritin) was significantly reduced in chicks infected withE. acervulina, as well as those infected withE. tenella. Collectively, the changes in serum zinc, copper, and iron concentrations, as well as the changes in hepatic zinc and MT-zinc concentrations in the chicks infected withE. tenella were similar to changes evoked during an acute phase response to infection. It is possible that a secondary bacterial infection or inflammation stemming from erosion of the lining of the cecum may play a role in the response of trace element metabolism to theE. tenella infection. Mentions of a trademarkr, proprietary product or specific equipment does not consitute a guarantee or warranty by the US Department of Agriculture and does not imply its approval to the exclusion of other products.     

Development of Caryospora bigenetica n. sp. (Apicomplexa,Eimeriidae) in Rattlesnakes and Laboratory Mice1

During a survey of the coccidian parasites of reptiles from Iowa, three specimens of Crotalus horridus L., the Timber Rattlesnake, and one of Sistrurus catenatus (Rafinesque), the Massasauga Rattlesnake, were found to be passing oocysts of a Caryospora, here described as C. bigenetica n. sp. Since these snakes (family Crotalidae) are known to subsist mainly on small mammals, oocysts from one of the Timber Rattlesnakes were fed to laboratory white mice (Mus musculus L.) to determine if mammals might be involved as alternate hosts in the life cycle. At necropsy, tissues of the tongue and dermis of the mice revealed a sequence of stages which included mature male and female gamonts, fully sporulated sporocysts, “excysted” sporozoites, and “resting” sporozoites that lay individually in solitary, cyst-like host cells termed “caryocysts.” A coccidia-free Massasauga that was fed an infected mouse, at a time when caryocysts in the mouse would have been present, later passed oocysts similar to those of the original inoculum. These results, along with the discovery of endogenous stages (asexual and sexual) in the intestine of the Timber Rattlesnake and the experimentally infected Massasauga, suggest that this parasite has a heteroxenous life cycle pattern, with sexual stages occurring both in the ophidian and the mammalian hosts.     

Life Cycle of Eimeria christenseni Levine,Ivens & Fritz, 1962 from the Domestic Goat,Capra hircus L.1

The endogenous development of Eimeria christenseni was studied in 10 two- to four-week-old kids inoculated with 10⁶-10⁷ sporulated oocysts. They were killed at intervals from two to 26 days after inoculation, and their tissues were examined for endogenous stages of the coccidian by light microscopy. Such stages were found in the small intestine and mesenteric lymph nodes. In the sexual cycle, two generations of meronts were found. The first generation developed in endothelial cells of lacteals in the jejunun and ileum and mesenteric lymph nodes, and mature meronts were first seen 14 days after inoculation. The second generation developed in epithelial cells of the glands of Lieberkuehn in the jejunum and ileum and in mesenteric lymph nodes, and its mature meronts were first seen by 16 days. Sexual stages were present mostly in epithelial cells of the tips and sides of the villi and less frequently in crypt cells of the jejunum and ileum. Mature macrogametes and microgamonts and oocysts were also first seen by 16 days. The prepatent period was 17 (14-23) days; the patent ranged from 8 to more than 30 days. Sporulation time was 3-4 days at 30°C. E. christenseni was found to be pathogenic, kids inoculated with 1-5 × 10⁵ sporulated oocysts exhibited the following signs: severe diarrhea, anorexia, polydipsia, poor hair coat, and extreme weakness. They recovered about a month later, but their growth rates appeared to be lower than those of uninoculated animals kept under the same conditions. One kid died 20 days after inoculation with 10⁷ oocysts.     

Cultivation of Eimeria tenella in Japanese quail embryos (Coturnix coturnix japonica).

Complete development of Eimeria tenella in Japanese quail embryos was observed. Sporozoites were inoculated into the allantoic cavity of 7-day-old Japanese quail embryos (Coturnix coturnix japonica), after which the infected embryos were incubated at 41 C. In the chorioallantoic membrane mature first generation schizonts, mature second generation schizonts, and gametes were detected at 48 hr postinoculation of sporozoites (PI), 84 hr PI, and 126 hr PI, respectively. Mature gametes and zygotes were found at 132 hr PI, and oocysts were detected at 138 hr PI. Mortality of embryos increased with increment of inoculum size of sporozoites. LD50 was 1.7 x 10(2) sporozoites. Oocyst production was also dependent on inoculum size. Oocysts harvested from embryos sporulated. The oocysts were inoculated into 13-day-old chickens, and oocysts, capable of sporulating normally, were recovered from ceca 7 days after inoculation.     

Extraintestinal development of Caryospora simplex (Apicomplexa: Eimeriidae) in experimentally infected mice, Mus musculus

Developmental stages of Caryospora simplex were found in connective tissue of the cheek, tongue, and nose of Swiss-Webster and C57 BL/6 mice (Mus musculus) from 8 through 70 days after oral inoculation with 50,000 or 250,000 oocysts, or 60,000 free sporocysts of the same species obtained from an Ottoman viper, Vipera xanthina xanthina. The earliest developmental stages were seen on day 8 post-inoculation (PI) and consisted of two types of meronts and gamonts (undifferentiated sexual stages). Gamonts, microgametocytes, macrogametes, and unsporulated oocysts were found on days 10 and 12 PI. Fully sporulated, thin-walled oocysts containing eight sporozoites surrounded by a thin sporocyst membrane were first seen 12 days PI. Monozoic cysts (caryocysts) were first seen 12 days PI and appeared fully viable throughout the duration of the study, 70 days PI. Four mice injected intra-peritoneally with 150,000 free sporozoites and killed 12 days PI contained unsporulated and sporulated oocysts in connective tissues of the cheek, tongue, and nose, suggesting that sporozoites may be carried to the site of infection via the lymphatic/circulatory system. Four cotton rats, Sigmodon hispidus, inoculated orally with 250,000 oocysts all had unsporulated and sporulated oocysts of C. simplex in connective tissue of the cheek, tongue, and nose when killed on day 12 PI, indicating extraintestinal development in the secondary host is not species specific. This is the first report of a heteroxenous coccidium with both asexual and sexual development in the primary (predator) and secondary (prey) hosts.     

Carbon Dioxide as the Initial Stimulus for Excystation of Eimeria tenella Oocysts*

SYNOPSIS. The stimulus necessary to initiate in vitro excystation of the chicken coccidium Eimeria tenella was provided by exposure of intact sporulated oocysts to an atmosphere of carbon dioxide. This stimulus produced a thinning and indentation at the micropylar region and oocysts became permeable to trypsin and bile. Sporozoites became active and began to escape from sporocysts into the oocyst cavity and then to the outside thru the altered micropyle after incubation in the enzyme-bile mixture. Activation of sporozoites when CO₂-pretreated oocysts were incubated in trypsin and bile, was used as the criterion to determine the number of oocysts responding to the initial stimulus. Thus, activation of sporozoites within intact oocysts was an indirect measurement of the number of oocysts stimulated during CO₂-pretreatment. Approximately 90% of the oocysts contained active sporozoites after 18 hr of pretreatment with carbon dioxide and 8 hr incubation in trypsin and bile at 38 or 41 C, respectively. Pretreatment of oocysts with air, N₂, O₂, or He resulted in 8% or less activation during incubation in trypsin and bile. Approximately 83% of the oocysts responded to the stimulus during 8 hr CO₂-pretreatment at 41 C, whereas at 38 C, 16 hr of pretreatment were required for a similar response. The stimulus did not elicit a response from oocysts held at 23 C during the pretreatment gasphase. No significant difference occurred in number of oocysts containing active sporozoites after sufficient CO₂-pretreatment for maximum stimulation of oocysts and incubation in trypsin and bile at 38 or 41 C.     

The Oocysts of Eimeria vermiformis sp. n. and E. papillata sp. n. (Protozoa: Eimeriidae) from the Mouse Mus musclus

SYNOPSIS. Eimeria vermiformis sp. n. and E. papillata sp. n. are described from the mouse Mus musculus. The sporulated oocysts of E. vermiformis are 18–26 by 15–21 μ (mean 23.1 by 18.4 μ); its sporocysts are 11–14 by 6–10 μ (mean 12.8 by 7.9 p). The sporulated oocysts of E. papillata are 18–26 by 16–24 μ (mean 22.4 by 19.2 μ); its sporocysts are 10–13 by 6–9 μ (mean 11.2 by 8.0 μ). A substiedal body is present in E. papillata sporocysts. Patent infections were produced in white laboratory mice with both species. Fourteen species of Eimeria have now been described from the genus Mus.     

柔嫩艾美耳球虫孢子发育阶段虫体抑制性消减文库的构建

为了分离鉴定柔嫩艾美耳球虫(Eimeria tenella)孢子发育阶段虫体的差异表达基因,分别以柔嫩艾美耳球虫未孢子化卵囊和孢子化卵囊为驱动组、子孢子为实验组,或未孢子化卵囊为驱动组、孢子化卵囊为实验组,利用抑制性消减杂交(SSH)技术,构建了2个子孢子cDNA消减文库和1个孢子化卵囊cDNA消减文库。随机从3个cDNA消减文库中分别挑取50个克隆,经PCR鉴定2个子孢子cDNA消减文库的重组率都为96%,孢子化卵囊cDNA消减文库的重组率为98%。从每个文库中随机挑取50个克隆测序,并进行同源性比较分析,结果显示:从孢子化卵囊cDNA消减文库中获得了13个单一有效序列,其中8个EST与已知蛋白同源性很高;从2个子孢子cDNA消减文库中共获得了40个单一有效序列,其中9个EST与已知蛋白同源,其余可能为柔嫩艾美耳球虫的新基因。这些结果为分离柔嫩艾美耳球虫新功能基因和进一步探索防治球虫病的方法提供了理论基础。     

Intestinal Transit Time During Infection with Eimeria nieschulzi in Rats*

SYNOPSIS. Experiments were designed to test whether or not intestinal transit time increases significantly during severe coccidiosis in the rat. Intraduodenal catheters were surgically implanted into 25 rats. Six to 12 days after surgery 11 rats were inoculated orally with 10⁴ sporulated oocysts of Eimeria nieschulzi Dieben, and 11 were inoculated with 10⁶ oocysts; 3 rats were retained as uninfected controls. At 2, 4, 8, 9, and 16 days postinoculation (PI) Na₂⁵¹CrO₄ was injected through the catheter into the duodenum of fasted rats and allowed to progress through the small bowel for 15 min, at which time the rats were killed. The distribution of ⁵¹Cr in the gut was plotted as a function of gut length. The leading edge of radioactivity traversed 70% of the gut length in controls, and ～ 50–60% in parasitized rats on days 2, 4, 8, and 9 PI. Also, a reflux of gut contents, as evidenced by radioactivity in the stomach, occurred early (PI days 2 & 4) in rats infected with 10⁴ oocysts and throughout patency in rats infected with 10⁶ oocysts. A 2nd study was undertaken to determine if chemically induced suppression of gut transit time during early infection would enhance infectivity as measured by increased parasite fecundity. Nine rats were injected subcutaneously with an antidiarrheal agent, Loperamide®, known to slow small bowel motility significantly. Another group of 9 control rats was injected with the ethanol-propylene glycol solvent. Ten min after injection, all rats were inoculated per os with 10⁴E. nieschulzi oocysts. The daily number of oocysts discharged/rat was followed from PI days 5–11. Patency began for all rats on PI day 7. The total number of oocysts discharged by the drugged rats as compared with controls was not significantly different.     

Optimized excystation protocol for ruminant Eimeria bovis- and Eimeria arloingi-sporulated oocysts and first 3D holotomographic microscopy analysis of differing sporozoite egress

Successful excystation of sporulated Eimeria spp. oocysts is an important step to acquire large numbers of viable sporozoites for molecular, biochemical, immunological and in vitro experiments for detailed studies on complex host cell-parasite interactions. An improved method for excystation of sporulated oocysts and collection of infective E. bovis- and E. arloingi-sporozoites is here described. Eimeria spp. oocysts were treated for at least 20 h with sterile 0.02 M _L-cysteine HCl/0.2 M NaHCO₃ solution at 37 °C in 100% CO₂ atmosphere. The last oocyst treatment was performed with a 0.4% trypsin 8% sterile bovine bile excystation solution, which disrupted oocyst walls with consequent activation of sporozoites within oocyst circumplasm, thereby releasing up to 90% of sporozoites in approximately 2 h of incubation (37 °C) with a 1:3 (oocysts:sporozoites) ratio. Free-released sporozoites were filtered in order to remove rests of oocysts, sporocysts and non-sporulated oocysts. Furthermore, live cell imaging 3D holotomographic microscopy (Nanolive®) analysis allowed visualization of differing sporozoite egress strategies. Sporozoites of both species were up to 99% viable, highly motile, capable of active host cell invasion and further development into trophozoite- as well as macroment-development in primary bovine umbilical vein endothelial cells (BUVEC). Sporozoites obtained by this new excystation protocol were cleaner at the time point of exposure of BUVEC monolayers and thus benefiting from the non-activation status of these highly immunocompetent cells through debris. Alongside, this protocol improved former described methods by being is less expensive, faster, accessible for all labs with minimum equipment, and without requirement of neither expensive buffer solutions nor sophisticated instruments such as ultracentrifuges.     

Excystation of the Sporozoites of Eimeria tenella in Apparently Unbroken Oocysts in the Chicken

SYNOPSIS. Chickens experimentally fed sporulated oocysts of Eimeria tenella were necropsied 5–300 minutes later to study excystation, especially in apparently unbroken oocysts. In birds killed 75–125 minutes after inoculation, there was evidence of excystation in some oocysts recovered from the small and large intestines. Altho not visibly broken, the shells of about 10% of the ones studied were wrinkled or otherwise deformed; many contained active sporozoites, some inside and some outside the sporocysts. During examinations, numerous sporozoites emerged from the sporocysts. Altho evidence was not obtained that excystation from unbroken oocysts was occurring inside the birds, the fact that it occurred in some oocysts during examinations may indicate that it could be taking place. Evidence of excystation was not seen in oocysts with shells that were not visibly altered.     

Parental Development of Eimerian Coccidia in Sandhill and Whopping Cranes1

In contrast with isosporoid species of coccidia that have established extraintestinal phases of development, the eimeriids, except for a few species, generally have been considered inhabitants of the intestinal tract. Eimeria infection in sandhill cranes (Grus canadensis) and whooping cranes (G. americana) may result in disseminated visceral coccidiosis. Nodules were observed in the oral cavity of 33% (n = 95) of the G. canadensis at the Patuxent Wildlife Research Center (PWRC) in Laurel, MD. Necropsy of six of the afflicted cranes revealed granulomatous nodules in many tissues and organs. Histologic studies disclosed protozoan organisms morphologically resembling schizonts in the granulomas, and endogenous stages of coccidia were present in the intestines of four birds. Fecalysis of three of four sandhill cranes yielded oocysts of E. reichenowi and E. gruis. Only E. reichenowi-type oocysts were recovered from a dead whooping crane sample. Domestic broiler chicks each intubated with about 1 times 10⁶ pooled sporulated oocysts of E. reichenowi and E. gruis were not infected. Exposure of six incubator-hatched and hand-reared sandhill crane chicks to oocysts artificially (two chicks) and naturally (four chicks) resulted in typical infection of intestinal epithelium with invasion of subepithelial tissues extending to the muscular layer and widespread extraintestinal development. Asexual and sexual stages occurred primarily in macrophages in the liver, spleen, heart, and lung. In the lung, oocysts were found in bronchial exudate and epithelial lining cells. Six of ten G. canadensis chicks, one adult G. americana, and three of five G. americana chicks that died naturally at PWRC had disseminated visceral coccidiosis.     

Experimental Vaccination of Chicks with Plasmodium gallinaceum Sporozoites. I. Circumsporozoite Proteins Are Expressed by Sporozoites Recovered from Both Salivary Glands and Midguts of Mosquitoes1

Immunogenicity of Plasmodium gallinaceum Sporozoites for chicks and their in vitro reactivity with normal and specific immune sera were studied. Two sporozoite populations recovered from experimentally infected Aedes fluviatilis were used: sporozoites from salivary glands and sporozoites from midgut oocysts. Populations seven to nine days old of sporozoites recovered from salivary glands were infective for all chicks until the chicks were three weeks old; however, sporozoites recovered from midguts containing oocysts infected these chicks only if isolated on days 8–9, but not on day 7 after the mosquitoes' infective blood meal. Infectivity of the sporozoites was lost after exposure to ultraviolet (UV) light (30 min) or X-rays (13 krad). Inactivated sporozoites from both sources proved highly immunogenic to chicks that were immunized by several intravenous or intramuscular injections. These parasites elicited a strong humoral immune response in the chicks, as measured by the circumsporozoite precipitation (CSP) reaction. The levels of the CSP antibodies were similar with sporozoites from both sources, there being no detectable differences in the percentage of reactive sporozoites or the intensity of the CSP reaction with sera containing antibodies to either sporozoites from salivary glands or sporozoites from oocysts. These results provide the first evidence that avian malaria sporozoites express the circumsporozoite protein that has been extensively characterized in mammalian malaria (rodent, simian, human sporozoites). Furthermore, we observed that the yields of sporozoites obtained from mosquito midguts, on days 8 and 9 of the P. gallinaceum infection, were at least twice as great as those obtained by salivary gland dissection, even 20 days after a blood meal. This is an advantage since obtaining the midguts is less tedious, as well as more efficient and faster.     

Effects of Carbon Dioxide on Coccidian Oocysts from 6 Host Species*

SYNOPSIS Activation of sporozoites in oocysts of Eimeria acervulina (chicken), E. intricata (sheep), and E. scabra (swine) occurred after pretreatment in aqueous 0.02 M cysteine hydrochloride under an atmosphere of CO₂, followed by incubation in a trypsin-bile mixture. Sporozoites of E. stiedae (rabbit), E. bilamellata (squirrel), and Isospora canis (dog) became activated when incubated in trypsin and bile with or without prior CO₂-pretreatment of oocysts; however, when CO₂-pretreatment was used, activation of these species in trypsin and bile was greatly enhanced. For E. acervulina, 12% of the oocysts were activated after 4 hr CO₂-pretreatment and 10 hr incubation in trypsin and bile at 43 C; higher temperatures or longer pretreatment times did not cause greater activation. Eimeria intricata oocysts became activated after 1 hr pretreatment and 10 hr incubation in trypsin and bile at 37, 39 or 41 C, respectively. The highest activation (31%) occurred after 20 hr pretreatment and 10 hr incubation in trypsin and bile at 41 C. Ninety percent of E. scabra oocysts contained active sporozoites after 1 hr CO₂-pretreatment and 10 hr incubation in trypsin and bile at 37 C. At 39 or 41 C, 100% activation occurred with this species after similar pretreatment and treatment periods. With E. bilamellata, 64% activation occurred in nonpretreated oocysts incubated 10 hr in trypsin and bile at 41 C, whereas 100% activation occurred if oocysts were pretreated with CO₂ for 1 hr before treatment with trypsin and bile. Thirty-one, 35, and 36% of CO₂-pretreated E. stiedae oocysts were activated after 1 hr incubation in trypsin and bile at 37, 39 or 41 C, respectively, whereas 1, 2, and 20% activation occurred in nonpretreated oocysts incubated at the same temperatures. Sporozoites in 99-100% of I. canis oocysts were activated after 10 hr treatment in trypsin and bile with or without 1 hr CO₂-pretreatment at 23, 37, 39 or 41 C.