The Effect of Eimeria nieschulzi Infection on Leukocyte Levels in the Rat*

SYNOPSIS. Rats inoculated with 10,000 sporulated oocysts of Eimeria nieschulzi had significantly higher total leukocyte counts on postinoculation days (PI) 1, 5, 6 and 7 when compared to control rats. Relative and absolute neutrophil counts increased concomitantly with a decrease in the relative lymphocyte levels in E. nieschulzi-infected rats on PI day 7. Absolute and relative neutrophil counts in infected rats on PI days 7 and 8 were closely correlated with the host's total oocyst discharge. The E. nieschulzi infection had no significant effect on the relative or absolute levels of monocytes or eosinophils. The described changes in leukocyte levels were not paralleled by a significant change in the erythrocyte count.     

Intestinal Disaccharidase and Peroxidase Deficiencies during Eimeria nieschulzi Infections in Rats*

SYNOPSIS Experiments were designed to study intestinal pathophysiologic changes associated with coccidial infections in mammalian hosts. Pairs of male Sprague-Dawley rats were killed at various times postinoculation (PI) with 10⁴ or 10⁶ sporulated occysts of Eimeria nieschulzi. The small intestine from each rat was removed, weighed, measured, and divided into thirds. From the middle 11 cm of each third, one cm was fixed for histologic examination. Mucosa was scraped from the remaining 10 cm and was assayed for protein content and for peroxidase, sucrase and trehalase activities. Infection with E. nieschulzi was associated with increased mass of the small bowel. Histologically, crypt depth throughout the small bowel was significantly greater (P≤ 0.005) in infected rats than in non-infected ones on PI days 8 and 16. Villus height did not change drastically during low-dose infections (10⁴ oocysts) and varied during high-dose infections (10⁶ oocysts). As a result of these morphologic changes in the mucosa, crypt/villus ratios were usually significantly greater (P≤ 0.005) in all infected rats throughout the small bowel. In general, increased gut weight and changes in crypt and villus dimensions became evident by PI day 2, were most pronounced at PI day 8, and began to return to control values by PI day 16. Peroxidase, sucrase, and trehalase levels equaled or were slightly higher than in controls on PI day 2, dropped significantly below controls (P≤ 0.05) by PI day 8, and returned to, or exceeded control levels by PI day 16. The intensity of all changes was directly dose-dependent.     

Eimeria and Klossia spp. (Protozoa: Sporozoa) from Wild Mammals in Borneo*

SYNOPSIS. During a survey of parasites of Sabah mammals, coccidia oocysts were found in 9 of 22 host species examined. Sporulated oocysts of Klossia sp. from Rattus whiteheadi are described. New geographic records are reported for Eimeria callosciuri, Eimeria sabani, and Eimeria tupaiae. New host and geographic records are reported for Eimeria separata from Rattus cremoriventer, for E. tupaiae and Eimeria ferruginea from Tupaia tana, for Eimeria nieschulzi from Rattus muelleri, and for E. sabani and Klossia sp. from R. whiteheadi.     

Eimeria nieschulzi and Trichinella spiralis: Analysis of concurrent infection in the rat

The effects of concurrent primary infection of the rat with Eimeria nieschulzi and Trichinella spiralis on the number of oocysts of E. nieschulzi shed by the host and on the number, distribution, and fecundity of adult T. spiralis were analyzed. When rats were initially infected with E. nieschulzi followed 9 days later by infection with T. spiralis there occurred a significant decrease in the total numbers of adult worms in the small intestine, a significant shift in the position of these worms along the length of the small gut, a decrease in the fecundity of adult female worms, and a decrease in muscle parasitism when compared with rats infected with T. spiralis alone. When rats were initially infected with T. spiralis, followed 9 days later by infection with E. nieschulzi, there occurred a significant decrease in the numbers of oocysts shed over 24 hr on Days 7, 9, and 11 postinfection below that seen with rats infected only with Eimeria. These changes are discussed in terms of the enteropathophysiologic lesions and enteric inflammation known to occur during infections with these two parasites.     

Coccidia of Malaysian Mammals: New Host Records and Descriptions of Three New Species of Eimeria*

SYNOPSIS. In a survey of parasites of wild mammals of Malaysia 3 new species of Eimeria were found. Eimeria tupaiae sp. n. is described from the common tree shrew, Tupaia glis. Its ellipsoidal to spherical, 3-layered oocysts average 20 × 19 μm. A micropyle is absent; an oocyst residuum and polar granule are present. Ellipsoidal sporocysts average 11 × 7 μm. A sporocyst residuum and Stieda body are present. Eimeria ptilocerci sp. n. is described from the pen-tail tree shrew, Ptilocercus lowii. The ellipsoidal to spherical, 2-layered oocysts average 23 × 20 μm. A micropyle and oocyst residuum are absent; polar granules are present. The ellipsoidal sporocysts average 13 × 7 μm. A sporocyst residuum and Stieda body are present. Eimeria muuli sp. n. is described from the pencil-tailed tree mouse, Chiropodomys gliroides. The ellipsoidal single-layered oocysts average 25 × 19 μm. A micropyle and oocyst residuum are absent; a polar granule is present. The ellipsoidal sporocysts average 13 × 8 μm. A sporocyst residuum and Stieda body are present. In addition, new host records are reported as follows: E. miyairii Ohira from Whitehead's rat Rattus whiteheadi and the Malaysian wood rat, R. tiomanicus; E. separata Becker & Hall from Mueller's rat, R. muelleri, the chestnut rat, R. fulvescens, and the Malaysian wood rat, R. tiomanicus; E. nieschulzi Dieben from the red spiny rat, R. surifer and the chestnut rat, R. fulvescens; and E. callosciuri Colley from the grey-bellied squirrel, Callosciurus caniceps and the black-banded squirrel, C. nigrovittatus.     

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.     

Role of intraepithelial lymphocytes in mucosal immune responses of mice experimentally infected with Cryptosporidium parvum

In order to investigate the role of intestinal intraepithelial lymphocytes (IELs) in host defense against Cryptosporidium parvum infection, conventionally bred immunocompetent (ImCT) ICR mice and immunosuppressed (ImSP) littermates were infected orally with 10(6) C. parvum oocysts. Then fecal oocyst excretion, the number and location of IELs, and their T lymphocyte subsets were observed on days 4, 7, 10, 13, 16, and 20 postinfection (PI). Uninfected ImCT and ImSP mice were used as controls. The starting point of oocyst excretion was day 4 PI in both ImCT- and ImSP-infected mice. The highest oocyst excretion occurred on day 7 PI in both groups, though the number of oocysts excreted was 3 times greater in ImSP than in ImCT mice. In ImCT mice, IELs greatly increased in number on days 16 and 20 PI (P < 0.05), but the increase was minimal in ImSP mice. IELs changed their location from the basal area to intermediate and apical areas of villous epithelial cells during the early stage of infection. In ImCT-infected mice, IEL phenotypes also changed; whereas CD4+ cells increased temporarily on day 7 PI (P < 0.05), CD8+ cells increased significantly on days 16 and 20 PI (P < 0.05). The results strongly suggest that IELs play a significant role in host defense against C. parvum infection, with helper T cells initiating control of the infection and cytotoxic T cells eliminating the parasites.     

The Life Cycle of Cryptosporidium baileyi n. sp. (Apicomplexa, Cryptosporidiidae) Infecting Chickens

ABSTRACT. The life cycle and morphology of a previously undescribed species of Cryptosporidium isolated from commercial broiler chickens is described. The prepatent period for Cryptosporidium baileyi n. sp. was three days post oral inoculation (PI) of oocysts, and the patent period was days 4–24 PI for chickens inoculated at two days of age and days 4–14 for chickens inoculated at one and six months of age. During the first three days PI, most developmental stages of C. baileyi were found in the microvillous region of enterocytes of the ileum and large intestine. By day 4 PI, most parasites occurred in enterocytes of the cloaca and bursa of Fabricius (BF). Mature Type I meronts with eight merozoites first appeared 12 h PI and measured 5.0 × 4.9 μm. Mature Type II meronts with four merozoites and a large granular residuum first appeared 48 h PI and measured 5.1 × 5.1 μm. Type I meronts with eight short merozoites and a large homogeneous residuum first appeared 72 h PI and measured 5.2 × 5.1 μm. Microgamonts (4.0 × 4.0 μm) produced 16 micro-gametes that penetrated into macrogametes (4.7 × 4.7 μm). Macrogametes gave rise to two types of oocysts that sporulated within the host cells. Most were thick-walled oocysts (6.3 × 5.2 μm), the resistant forms that passed unaltered in the feces. Some were thin-walled oocysts whose wall (membrane) readily ruptured upon release from the host cell. Sporozoites from thin-walled oocysts were observed penetrating enterocytes in mucosal smears. The presence of thin-walled, autoinfective oocysts and the recycling of Type I meronts may explain why chickens develop heavy intestinal infections lasting up to 21 days. Oocysts of C. baileyi were inoculated orally into several animals to determine its host specificity. Cryptosporidium baileyi did not produce infections in suckling mice and goats or in two-dayold or two-week-old quail. One of six 10-day-old turkeys had small numbers of asexual stages only in the BF. Four of six one-day-old turkeys developed mild infections only in the BF, and sexual stages of the parasite were observed in only one of the four. All seven one-day-old ducks and seven two-day-old geese developed heavy infections only in the BF with all known developmental stages present.     

Development of Caryospora simplex (Apicomplexa: Eimeriidae) from Sporozoites to Oocysts in Human Embryonic Lung Cell Culture1

Leighton tubes containing monolayers of human embryonic lung cells were inoculated with 70,000 or 30,000 sporozoites of the viperid coccidium Caryospora simplex and examined at 1, 2, 4, 6, 8, 10, 12, 14, 16, and 18 days post-inoculation (PI). By day 1 PI, sporozoites had penetrated cells and were within parasitophorous vacuoles. Most sporozoites became spherical and then underwent karyokinesis several times between days 2 and 6 PI. Mature Type I meronts were found on days 6–16 PI and contained 8 to 22 short, stout merozoites. Mature Type II meronts were present on days 10–18 PI and contained 8 to 22 long, slender merozoites. Developing gamonts (undifferentiated sexual stages) were observed on days 14 and 16 PI. Mature micro- and macrogametes and thin-walled unsporulated oocysts were present on days 16 and 18 PI. Attempts to sporulate oocysts in tissue culture medium or in a 2.5% (w/v) aqueous solution of K₂Cr₂O₇ at 25/°C and 37°C were unsuccessful; only a few oocysts developed to the contracted sporont stage. Four Swiss-Webster mice injected intraperitoneally with merozoites obtained from Leighton tubes on day 10 PI did not acquire infections. This is the second coccidium reported to complete its entire development, from sporozoite to oocyst, in cell culture.     

Fine Structure of Zygotes and Oocysts of Eimeria nieschulzi*

SYNOPSIS. Mature macrogamonts were present in the small intestine of rats 5.5 to 7.5 days postinoculation with Eimeria nieschulzi oocysts; oocysts were present at 6 to 7.5 days. Types I and II wall-forming bodies in macrogamonts began to undergo ultrastructural changes within zygotes to form the outer and inner layers of the oocyst wall. Before and during oocyst wall formation a total of 5 membranes (M1–5) were formed at or near the surface of the zygote. The outer and inner oocyst wall layers formed between M2 and M3, and M4 and M5, respectively. The mature oocyst was loosely surrounded by M1 and M2, had an electron-dense outer layer, 100–275 nm thick, and an electron-lucent inner layer, 160–180 nm thick. It also contained an electron-lucent line consisting of M3 and M4 interposed between the outer and inner layers of the oocyst wall. The micropyle, measuring 935 × 47 nm, was located in the outer layer of the oocyst wall and consisted of 10–14 alternating layers of electron-dense and lucent material. The sporont of mature oocysts was covered by M5, immediately beneath which were M6 and M7. The sporont contained a nucleus and nucleolus, lipid and amylopectin bodies, mitochondria, ribosomes, as well as smooth and rough endoplasmic reticulum. Canaliculi, Golgi complexes, and types I and II wall-forming bodies were absent.     

Eimeria stiedae: weight, oocyst output, and hepatic function of rabbits with graded infections

Groups of 5 (38–40 days old) Eimeria stiedae-naive rabbits were infected with 0 (mock infection), 10², 10³, 10⁴, and 10⁵ sporulated oocysts of Eimeria stiedae (groups A, B, C, D, and E, respectively) and body weight, oocyst output, serum glutamic pyruvic and serum oxalacetic transaminases, bilirubinemia. lipemia, glycemia, and proteinemia were measured on diverse occasions for 50 days. Mortality and carcass and liver weights at the end of the experiment were also recorded. Mortality was 80% in group E, 40% in group D, and 0% in the other groups. Reduction of weight gain was observed from the 8th day of infection and actual loss from the 15th day in infected animals. On Day 50, the average body and carcass weights of all infected rabbits were 71.2 and 63.2%, respectively, of group A. Only groups D and E had absolute hepatomegaly and group C had relative liver enlargement. Patency and rate of increase of oocyst output were not related to dose but peak and declination of oocysts production were delayed in proportion to the infective dose.Serum glutamic pyruvic and glutamic oxalacetic transaminases were increased from Day 8 to Day 36, bilirubinemia and lipemia augmented from Day 22, and glycemia and total serum proteins decreased from Days 22 and 29, respectively. Bilirubinemia tended to recover sooner (toward Day 50) in rabbits with lighter infection and lipemia recovered from Day 36 in proportion to the size of the infective dose. Modifications of glycemia and total proteinemia were consistent but reached statistical significance only occasionally. The asexual reproduction of the parasites caused transient damage to the hepatocytes during the second week of infection, and later sexual stages altered the ductal epithelium from the fourth week.     

Life cycle and cytochemical study of the endogenous developmental stages of Eimeria akeriana (coccidiida, Eimeriidae) from the Asia Minor gerbil

Three generations of schisonts in the life cycle of Eimeria akeriana, the intestinal parasite of Meriones blackleri, were determined. Gametogony begins in 94 hours, the first oocysts discharge in 5.5--6 days and lasts 14.5 to 15 days after the oocyst administration. A cytochemical study of the distribution of the nucleic acids, proteins and amylopectin at the stages of endogenous development of E. akeriana has revealed a considerable similarity among the parasites of Eimeria though each type is characterized by some cytochemical peculiarities.     

Eimeria lancasterensis Joseph, 1969 and E. confusa Joseph, 1969 from the Grey Squirrel Sciurus carolinensis*

SYNOPSIS. Forty grey squirrels Sciurus carolinensis were examined for coccidia during a 2-year period. Eimeria lancasterensis was found in all of them. The ellipsoidal oocysts of this species averaged 24.6 by 14.6 μ. They had no micropyle or oocyst residuum. A polar body was present. The sporocysts averaged 14.1 by 8.4 μ. The endogenous phases of the parasite were found in the epithelial cells of the villi thru the entire length of the small intestine. E. confusa was found in one of 40 squirrels. The oocysts of this species were subspherical, occasionally ellipsoidal or rarely spherical; they averaged 33.2 by 26.7 μ. Oocyst residuum and micropyle were absent. Polar granules ranged in number from 0–5. The sporocysts averaged 19.6 by 12.1 μ. The prepatent period for this species was 7–8 days and the patent period 6–13 days. E. ontarioensis was found in 3 of the 40 squirrels.     

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.     

Demonstration of Acid Phosphatase in Eimeria spp.: Partial Characterization of the Enzyme in E. vermiformis1,2

Sporozoite extracts of E. vermiformis, E. stiedai, and E. tenella are rich in acid phosphatase activity. They contain specific enzyme activities equal to or greater than those reported for other highly virulent protozoan parasites. The absolute amount of enzyme activity per oocyst dramatically increases during sporulation of E. stiedai and E. vermiformis. Partial characterization of the acid phosphatase activity of E. vermiformis indicates that sporozoites account for greater than 92% of the total activity in sporubted oocysts, that the enzyme is resistant to inhibition by tartrate, and that it can be separated into two forms by anion exchange chroma-tography.     

Pathological Changes and Immunity Associated with Experimental Eimeria vermiformis Infections in Mus musculus1

ABSTRACT. Pathological changes and immunity induced by Eimeria vermiformis (Ernst, Chobotar & Hammond, 1971) were studied in outbred Swiss mice inoculated with 5000, 10,000, 20,000, or 40,000 oocysts. Cross immunity to E. ferrisi was also studied. In the case of E. vermiformis, mortality was dose dependent; most deaths were observed in the intermediate-dose groups. Most deaths also correlated with peak oocyst output. Histopathologic changes consisted of an early neutrophil and mononuclear cell infiltration in the small intestine. Later, villus atrophy and crypt hyperplasia caused a decrease in the villus-crypt ratio. During the acute phase (8-10 days after inoculation), villus tips were eroded and parasites with necrotic debris filled the cryptal and intestinal lumina. Vacuolar changes were observed in epithelial cells of the small intestine. Neither parasites nor significant pathological changes were observed in extra-intestinal organs. Mice were totally immune to reinfection with E. vermiformis 30 and 105 days after inoculation. Cross immunity was not observed between E. vermiformis and E. ferrisi.     

Plasmodium cynomolgi: the comparative infectiousness of individual rhesus monkeys

Five rhesus monkeys were infected with the malaria parasite Plasmodium cynomolgi Mayer. Anopheles stephensi Liston mosquitoes were fed on each monkey over the period of prepremunitive gametocytemia. Individual monkeys did not differ significantly in either mean daily gametocyte count (median = 1300 gametocytes per mosquito blood meal volume per day) or mean daily oocyst production (median = 34 oocysts per mosquito per day). Significant differences among monkeys in daily oocyst/gametocyte conversion ratio were attributable to essentially random correlation effects. The observed range in duration of the period of prepremunitive gametocytemia was 14–43 days. Total oocyst production over this period, as calculated for a unit mosquito biting rate of one per day, ranged from 130 to 2800 oocysts. The overall efficiency of conversion of gametocytes to oocysts in A. stephensi was estimated at 0.02 oocysts per gametocyte.     

Immunity to the protozoan parasite Eimeria nieschulzi in inbred CD-F rats

Immunity to the coccidial parasite, Eimeria nieschulzi, in CD-F rats was assessed by the numbers of oocysts shed in relation to the time after inoculation. Intravenous injections of syngeneic thoracic duct lymphocytes (TDL) from immunized rats elicited various degrees of adoptive immunity against primary infections of E. nieschulzi. Of the 16 rats injected with 10⁹ sensitized TDL, 7 were totally immune to a subsequent challenge by the parasite. This number of injected TDL also raised the serum antibody level to that of immune rats. Contact with immune TDL was deleterious to sporozoites of E. nieschulzi in vitro and produced immunocytoadherence of parasite to cell.     

Chemotherapeutic effect of azithromycin and lasalocid on Cryptosporidium infection in mice.

Prednisolone-immunosuppressed mice (ICR, 7-wk-old female) were each inoculated with 1 x 10(5) oocysts of Cryptosporidium parvum. Medication with azithromycin (400 mg/kg/day) or lasalocid (64, or 128 mg/kg/day) was started 13 h after inoculation and continued for 3 days. The number of oocysts discharged by each mouse was calculated on days 4-12 post-inoculation. Compared with non-medicated controls, oocyst production by the medicated mice was markedly reduced; some mice did not discharge oocysts and the remaining mice discharged less than 1/100 the number of oocysts of the control mice. These results indicate that both azithromycin and lasalocid have prophylactic or therapeutic activity against Cryptosporidium.