The Life Cycle of Eimeria bateri (Protozoa, Eimeriidae) in the Japanese Quail Coturnix coturnix japonicum

SYNOPSIS. Eimeria bateri, a parasite of the Indian quail, was described from laboratory infections in Japanese quail. First generation schizonts developed in the glands of the duodenum and upper intestine. Second, 3rd and 4th generation schizonts and gametocytes occurred in the villous epithelium. There was a gradual spread along the small intestine until the whole organ was affected. The prepatent period was 4 days and the patent period 6 days. Graded doses from 5,000 to 1,280,000 oocysts produced very little pathogenic effect in young quail. E. bateri did not infect young pheasants or chicks.     

Observations on Haemogregarina balli sp. n. from the Common Snapping Turtle,Chelydra serpentina*

SYNOPSIS. Haemogregarina balli sp. n. is described from the blood and organs of the common snapping turtle Chelydra serpentina serpentina and from the gastric and intestinal ceca of the presumed invertebrate hosts, the leeches Placobdella parasitica and Placobdella ornata. In the peripheral blood of the turtle, male and female gametocytes and immature erythrocytic schizonts are found within erythrocytes. The maturation of erythrocytic schizonts containing 6–8 merozoites is recorded from liver imprints. Schizonts with 13–25 merozoites are found in various cells of the liver, lung and spleen. In the gastric ceca of the leeches the host erythrocytes are digested, releasing the gametocytes and immature erythrocytic schizonts. Immature erythrocytic schizonts degenerate. Association of the gametocytes occurs in the intestinal ceca. The microgametocyte apparently gives rise to 4 nonmotile microgametes, one of which fertilizes the macrogamete while the other remain as condensed, residual nuclei on the periphery of the developing oocyst. The oocyst increases in size with maturity. A mature oocyst produces 8 sporozoites from a single germinal center. Sporozoites liberated from the oocyst are found in the tissues of the leech. Transovarial transmission of the parasite does not occur in the turtle. Attempts at experimental transmission failed. Previously unfed (control) leeches were negative for the parasite. Haemogregarina balli is compared with other haemogregarines described from C. serpentina. Features of species of Haemogregarina and Hepatozoon as well as the taxonomy of these genera are discussed.     

Comparative Cultivation of Poultry Coccidia in Mammalian Kidney Cell Cultures

SYNOPSIS. Excysted sporozoites of Eimeria meleagrimitis, E. necatrix, E. acervulina , and E. gallopavonis were inoculated into monolayer cell cultures of bovine, ovine, porcine, and human kidney. E. meleagrimitis developed only in bovine embryonic kidney. Mature schizonts were found in the 11th, 16th, and 20th serial passages, but only immature schizonts were in the 4th and 6th passages. E. necatrix developed to mature schizonts in the 3rd, 4th, 6th, 11th, 16th, and 20th passages of bovine kidney and also to immature schizonts in the 175th and 189th passages of PK-15 (cell line porcine kidney). Schizonts, however, did not develop in the 140th and 145th passages of CCI-33 (cloned PK-15). Neither E. meleagrimitis nor E. necatrix developed in the primary, 1st or 2nd passages of bovine embryonic kidney, primary porcine kidney, 45th and 52nd passages of a human embryonic kidney cell line, or in the primary, 5th and 18th passages of ovine kidney. Eimeria acervulina and E. gallopavonis did not develop in any of the cultures.
E. meleagrimitis and E. necatrix probably completed only one asexual generation in culture. The structure of mature schizonts of both species differed greatly from those in the natural host. Schizonts of E. meleagrimitis present at 48 hours were small (13–18 by 12–14 μ) and contained only 12–28 merozoites that were 3.2–3.8 μ long. At 48 hours, E. necatrix schizonts were 15–18 μ in diameter or less and contained only 15–20 merozoites (2.0–3.5 μ long); at 96 hours they were 50–70 by 10–35μ and contained either hundreds of small merozoites (2.0–3.5 μ long) or a lesser number of larger merozoites (9–11 μ).     

Coccidian Parasites of Intertidal Fishes from Wales: Systematics, Development, and Cytochemistry

SYNOPSIS. Examination of littoral fish Blennius pholis and Cottus bubalis caught at Aberystwyth and Porth Cwyfan, Wales. U.K., revealed 2 species of coccidia.
Eimeria dingleyi sp. n. Oocysts spherical (16.1–19.2) to subspherical (13.9–14.2 × 18.8–20.0) μm, with thin walls; sporulation outside the host to produce ellipsoid sporocysts; endogenous phases in epithelial cells throughout intestine; 26 of 58 B. pholis infected.
Eimeria variabilis (Thélohan) Reichenow. Oocysts spherical (11.9–14.6) to subspherical (9.2–10.9 × 13.9–14.3) μm; sporulation in lining of pyloric ceca and rectum; previously unrecorded schizonts and gametocytes present; 21 of 25 C. bubalis infected.
Electron microscopy revealed that the oocyst wall of E. variabilis consists of a thin membrane whereas the sporocyst wall is thick and 3-layered. Typical oocyst wall-forming bodies were absent from the macrogamete. Cytochemical tests on the endogenous stages of E. dingleyi and E. variabilis indicated that in general they resembled other coccidia in their chemical constitution.     

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.     

Simultaneous purification of merozoites and schizonts of Eimeria tenella (Apicomplexa) by Percoll flotation and assessment of cell viability with a double fluorescent dye assay.

The asynchronous development of Eimeria tenella in orally infected chickens makes it possible to purify second generation merozoites (meros) and shizonts from a single mucosal homogenate. After centrifugation in 30% Percoll in phosphate-buffered saline (Percoll-PBS), debris, villi, and schizonts float, whereas meros and erythrocytes are pelleted. Erythrocytes are lysed by a mild hypotonic shock; meros are filtered through a cotton wool plug and collected by centrifugation. The 30% Percoll-PBS supernatant fraction is diluted to 25% Percoll-PBS and centrifuged to sediment mature schizonts. By repeated slow-speed centrifugation, schizonts are separated from nuclei and small-sized debris. In less than 3 hr, 8.8 +/- 2.3 x 10(8) meros and 7.2 +/- 3.9 x 10(6) schizonts are collected from 10 infected chickens. Contamination with host material is 2% for meros but variable for schizonts. For the assessment of cell viability, ethidium bromide (EB) and acridine orange (AO) have been used as markers for dead and living cells, respectively, in a single step method. More than 95% of the schizonts and meros accumulate AO and no EB, whereas lysed erythrocytes and all cells hosting a schizont are permeable to EB. After incubation of meros and schizonts in synthetic media with [5,6- 3H]uracil, label accumulates in the perchloric acid-soluble and -insoluble fractions, indicating transport, salvage, and incorporation of the pyrimidine precursor in nucleic acids. If stored on ice, meros and schizonts retain metabolic activity for at least 5 hr, but metabolism declines rapidly during incubation at 41 C.     

Redescriptions of Eimeria irresidua Kessel & Jankiewicz, 1931 and E. flavescens Marotel & Guilhon, 1941 from the domestic rabbit

Eimeria flavescens and E. irresidua from the domestic rabbit are redescribed. The relatively smaller ovoidal oocysts of E. flavescens which measure on average 31.7 X 21.4 micrometer, possess a wide micropyle at the broad end. First-generation schizonts of this species develop deep in the glands of the lower small intestine. Merozoites migrate to the caecum and colon where second, third and fourth-generation schizonts develop in the superficial epithelium followed by the fifth-generation schizonts and gametocytes which form in the glands. In young Dutch rabbits E. flavescens is very pathogenic; low doses of oocysts produce a severe enteritis with high mortality and morbidity. The larger, broadly ellipsoidal oocysts of E. irresidua measure on average 38.4 X 23.2 micrometer and often possess a very small cryptic oocyst residuum. The endogenous stages develop in the small intestine only; first-generation schizonts in the glands and second-generation schizonts in the lamina propria whilst third and fourth-generation schizonts and gametocytes develop in the villous epithelium. E. irresidua is not pathogenic in young Dutch rabbits; even heavy infections produce only a transient pause in weight gain.     

Scanning Electron Microscopy of Schizogony in Eimeria tenella

SYNOPSIS. Developing 2nd- and 3rd-generation schizonts of Eimeria tenella were found in the ceca of chicks infected orally with sporulated oocysts. Several free 2nd-generation schizonts, which varied in diameter from 11 to 21.6 μm, were found on the epithelial surface of the cecum. Some schizonts appeared to have lost merozoites. Other schizonts were intact, one of which was surrounded by an unbroken membrane that followed the contours of the merozoites. Third-generation schizonts, much smaller than 2nd-generation schizonts and with fewer merozoites, were found only on cut or fractured surfaces of the cecal tissue. Third-generation merozoites appeared shorter and thicker than those of the 2nd-generation and were attached to the schizont residuum. A form with conical protuberances and another with 4 triangular segments were found; they were believed to be developing stages 3rd-generation schizonts.     

Emergence of influenza A virus variants after prolonged shedding from pheasants

We previously demonstrated the susceptibility of pheasants to infection with influenza A viruses of 15 hemagglutinin (HA) subtypes: 13/23 viruses tested were isolated for >or=14 days, all in the presence of serum-neutralizing antibodies; one virus (H10) was shed for 45 days postinfection. Here we confirmed that 20% of pheasants shed low-pathogenic influenza viruses for prolonged periods. We aimed to determine why the antibody response did not clear the virus in the usual 3 to 10 days, because pheasants serve as a long-term source of influenza viruses in poultry markets. We found evidence of virus replication and histological changes in the large intestine, bursa of Fabricius, and cecal tonsil. The virus isolated 41 days postinfection was antigenically distinct from the parental H10 virus, with corresponding changes in the HA and neuraminidase. Ten amino acid differences were found between the parental H10 and the pheasant H10 virus; four were in potential antigenic sites of the HA molecule. Prolonged shedding of virus by pheasants results from a complex interplay between the diversity of virus variants and the host response. It is often argued that vaccination pressure is a mechanism that contributes to the generation of antigenic-drift variants in poultry. This study provided evidence that drift variants can occur naturally in pheasants after prolonged shedding of virus, thus strengthening our argument for the removal of pheasants from live-bird retail markets.     

Life Cycle of Eimeria ferrisi Levine & Ivens, 1965 in the Mouse, Mus musculus

SYNOPSIS. The life cycle of Eimeria ferrisi is described from experimentally infected Mus musculus. The prepatent period was 3 days and the patent period was 3–4 days. The endogenous stages were found only in the cecum and colon. Three generations of schizonts were found. Mature 1st-generation schizonts first seen 24 hr postinoculation (PI) measured 10.9 (7–14) × 10.2 (6–13) μm and had 9.6 (7–14) merozoites. Some 2nd-generation schizonts had uninucleate merozoites and others had multinucleate merozoites. The former were first seen in small numbers 36 hr PI and were most abundant 48 hr PI. They measured 9.6 (5–13) × 7.9 (6–12) μm and had 18 (6–25) merozoites. Schizonts with multinucleate merozoites were seen 72 hr PI. Mature 3rd-generation schizonts were seen 72 hr PI. They measured 14.0 (12–18) × 11-0 (9–13) μm and had 12.5 (5–16) merozoites. Macrogamonts were first seen in 72 hr sections. Each young macrogamont had a large nucleus with a prominent nucleolus. Only one type of cytoplasmic granule appeared to be involved in the formation of the oocyst wall. Mature macrogamonts were 11.0 (5–14) × 10.0 (6–13) μm. Crescent-shaped bodies were observed in the parasitophorous vacuole of trophozoites and young macrogamonts. Early microgamonts were first recognized at 96 hr by the presence of darkly stained and irregularly shaped nuclei. Usually, mature microgametes were arranged in long, narrow whorls at the periphery of the microgamont or in whorls at the surface of 2–5 compartments.     

Survival of released pheasants, Phasianus colchicus, in Ireland

The survival of 250 hand-reared pheasants was assessed by resightings of tagged individuals following their release into an open-roofed pen on an estate in Ireland. The death rates per 10 days after release were approximated as 5.5% during the first 30 days, 11.6% between 31–70 days, 6.0% between 70–240 days and 2.3% between 241–365 days. The birds suffered their highest rate of loss (48.2%) during their first 10 days after leaving the release pen. This emergence-related mortality was the major factor influencing changes in the observed death rate following release. Predators, mainly foxes, were initially attracted to the area by the presence of inaccessible prey. Males suffered a higher rate of loss following their emergence from the release pen than did the females, possibly associated with the greater proportion of time thereafter spent outside the pen and hence at risk. The factors affecting the survival of hand-reared pheasants are discussed. It is suggested that reducing densities of birds within pens may increase subsequent survival without resorting to predator control.     

Survival and Development of Eimeria meleagrimitis Tyzzer, 1929 in Bovine Kidney and Turkey Intestine Cell Cultures

SYNOPSIS. Monolayer cell cultures of embryonic turkey intestine (primary) and bovine kidney (cell line, 20th passage), maintained at 40.6 and 43 C for alternating intervals of approximately 12 hours in Basal Medium Eagle and fetal calf serum at pH 7.0–7.4, were observed for 144 hours after inoculation of Eimeria meleagrimitis sporozoites.
In turkey intestine cultures, which consisted of fibroblast-like cells and patches of epitheliul-like cells, there were decreases of 80 and 81% in the numbers of parasites between 5 and 48 hrs; in bovine cultures, 21–41% decreases. Decreases in the turkey cultures, however, were due to the nonsurvival of sporozoites in fibroblast-like cells; in epitheliul-like cells there was a 42% dcrease between 5 and 48 hrs and only 27% between 48 and 144 hours.
Trophozoites were present in bovine cells at 5 hrs. Small, mature schizonts containing only 12-28 merozoites were present in the bovine cultures and in the epitheliul-like cells within turkey intestine cultures from 48-144 hrs. Larger schizonts (50-115 by 20-70 μ) were present in bovine but not in turkey cultures from 72–144 hrs. Many of these schizonts contained far more merozoites than schizonts of any of the 3 generations described from the host.
In bovine cultures, there was an abundance of liberated merozoites at 50, 52, 74, and 76 hrs; many had reinvaded cells, sometimes as many as 50–60 per cell. In turkey cultures, liberated merozoites were found once at 144 hrs and none were intracellular. At 120 and 144 hrs in bovine cultures, abnormally developed and degenerate forms appeared; in turkey cultures, all were normal.     

Effects of Calcium-Loading on Egg Production in Ring-Necked Pheasants

Abstract: Ring-necked pheasants (Phasianus colchicus) are able to store dietary calcium as medullary bone, which they may mobilize for future eggshell synthesis. We define this mechanism as calcium-loading. Previous experiments on pheasants conducted to document the importance of calcium in limiting distribution did not account for calcium-loading. We hypothesized that calcium-loading could override experimental calcium treatments of the diet. We measured egg production, egg characteristics, and femoral mineral content for pheasants that were not calcium-loaded on 7 diets differing in calcium from 0.2% to 4.5% and compared these results to a similar study on calcium-loaded pheasants. We predicted that calcium-loaded pheasants would produce more eggs than those that were not calcium-loaded. We also predicted that there would be no significant difference between femur ash fractions in non-calcium-loaded pheasants, but that the ash fraction in calcium-loaded pheasants would differ significantly between the beginning and end of the experiment. Egg production was higher in calcium-loaded pheasants above 2% dietary calcium. Femur ash fraction was not different in non–calcium-loaded pheasants but differed significantly before and after the experiment and between high (>2%) and low (<2%) dietary levels in calcium-loaded pheasants. Calcium-loading may account for short-term persistence of captive pheasants introduced on calcium-poor soils, followed by their eventual population failure. Managers may improve survival of captive pheasants before introduction by surveying habitat for adequate calcium and by calcium-loading.     

Malarial Parasites of the "Jesu Cristo" Lizard Basiliscus basiliscus (Iguanidae) in Panama

SYNOPSIS. The iguanid lizard Basiliscus basiliscus in Panama is parasitized by Plasmodium basilisci and P. achiotense sp. nov. P. basilisci in this host is characterized by schizonts containing 4–14 merozoites, with schizonts parasitizing proerythrocytes containing more merozoites than those in erythrocytes. Asexual parasites lack cytoplasmic projections, while mature gametocytes are round or oval with regular margins.
P. achiotense is characterized by the combination of prominently pigmented, large schizonts containing 36–56 merozoites and oval or round gametocytes which are about 1/3 larger than those of P. basilisci.
EE-schizonts of P. basilisci were observed commonly in thrombocytes and occasionally in lymphocytes, and appeared early in experimental infections induced by blood inoculation.     

Influence of keeping pheasants in captivity vs. nature on the biological value of meat and its use in human nutrition

The life of game birds (pheasants) in nature is coupled with a number of difficulties in all seasons of the year. This refers to finding food, breeding, laying eggs, raising the young, fleeing from their natural enemies and lack of protection from unfavorable climatic conditions. The pheasants that live in captivity--aviaries for pheasants--do not have such difficulties--they are fed regularly by quality feed for pheasants, they are protected from bad weather and natural enemies. Our research was aimed at determining the biological value of meat of pheasants grown in the two different settings--in captivity and in nature. The highest weight achieved wild pheasant males (1232.4 +/- 147.36 g). The differences between tested pheasant groups were statistically very high significant (P < 0.001). The differences between groups related to breast weight and tights with drumsticks weight were statistically very high significant (P < 0.001). Between breast parts (%) and legs parts (%) were notified very high (P < 0.001) i.e. high (P = 0.002) differences. The highest weight breast muscles and tights with drumsticks had wild pheasants (282.6 +/- 63.53 g i.e. 206.2 +/- 37.88g). Wilde pheasants had lower part (%) and lighter (g) skin with subcutaneous fatty tissue on breasts. Female pheasants cultivated on both ways had higher skin part (%) and subcutaneous fatty tissue in tights with drumsticks. Related to chemical composition of breast muscles is established statistically significant differences (P < 0.001 i.s. P = 0.040)) in part of Ca (%) and P (%). In wild pheasant tights with drumsticks muscles established statistically very significant (P < 0.001) higher part of moisture, protein and Ca, i.e. statistically very high significant (P < 0.001) lower part of fat and energetic value. Research results indicate that the quality of meat of pheasants grown in nature has higher biological value than the meat of pheasants kept in aviaries, which means it has advantages in human nutrition.     

Haemoproteus palumbis sp. nov. (Sporozoa, Haemosporina) of the English Wood-Pigeon Columba p. palumbus

SYNOPSIS. Haemoproteus palumbis sp. nov. is described from the English wood-pigeon, Columba p. palumbus. Its pigmented gametocytes inhabit erythrocytes and resemble those of H. columbae (in C. livia ) but may be slightly longer and narrower. It is characterized by having oval or slightly lobed, not elongate, schizonts in endothelial cells of lung and heart, a prepatent period of 14 days, and a sporogonic cycle in the hippoboscid fly Ornithomya avicularia lasting 6 1/2–7 days. H. palumbis sp. nov. cannot infect C. livia but can infect a small proportion of individuals of Pseudolynchia canariensis , the vector of H. columbae.     

Endogenous Cycle of the Swine Coccidium Eimeria debliecki Douwes, 1921*

SYNOPSIS. A pure strain of Eimeria debliecki (University of Illinois strain A) established from a single oocyst was used to determine the endogenous cycle. Young parasite-free pigs 2 weeks to 3 months old were used throughout the study. The endogenous cycle was found to take place in the small intestine where the parasites were located in the distal portion of the striated simple columnar epithelial cells of the villi. The first generation schizonts were found in only the jejunum (15% of small intestine). The second generation schizonts and gametes occurred in the jejunum and ileum (70% of small intestine), a slight posterior progression occurring with each stage. The entire cycle required 6.5 days. The schizogonous cycle comprised 2 generations. The first generation schizonts required 2.5 days to reach maturity, measured 8-12 μ, contained 16 merozoites measuring 12-15 μ and had a polar residual mass. The second generation schizonts required 2 days to reach maturity, measured 13-16 μ, contained 32 rotund merozoites measuring 6–8 μ, and had only a few granules of residual material. Gametogony took place in 1.5 days. The macrogametes measured 12-16 μ, and the microgametocytes measured 9-14 μ with microgametes measuring 5–6 μ.     

Histomonas wenrichi n. sp. (Mastigophora: Mastigamoebidae), a Nonpathogenic Parasite of Gallinaceous Birds

SYNOPSIS. A nonpathogenic histomonad, morphologically identical to that found in pheasants by Wenrich in 1943, is described as Histomonas wenrichi n. sp. Its dimensions are about 1.5 × those of H. meleagridis. It has 4 flagella instead of the 1 or 2 that characterize the latter species. It does not multiply in the host tissues nor produce visible responses to its presence there, and it does not exist as the clear "tissue form" that is common in active H. meleagridis infections.
Histomonas wenrichi n. sp. can be transmitted to turkeys, chickens, and pheasants by either rectal inoculation or by feeding embryonated eggs of Heterakis gallinarum grown in birds harboring the nonpathogenic histomonad in the ceca. Usually fewer than half the female heterakids so grown produce eggs capable of transmitting this histomonad, and a total of 200 to several thousand eggs from worms recovered from Histomonas -infected birds may be required to produce one Histomonas infection.
Histomonas wenrichi n. sp. has been propagated in more than 2000 chickens and turkeys over an 8-year period, during which time it has been transmitted by 18 generations of Heterakis. It has frequently been grown in birds in which H. meleagridis was also present, and upon reisolation it maintained all of its original characteristics. It has not yet been cultivated in vitro.     

Schizogonic development of Leucocytozoon smithi.

The schizogonic development of Leucocytozoon smithi in the liver of experimentally infected turkey poults was examined by electron microscopy. Following intraperitoneal injection, sporozoites migrated to the liver and entered hepatic cells to become intracellular trophozoites. Three to four days post inoculation (PI), trophozoites underwent asexual multiple fission known as merogony or schizogony. Two generations of schizonts were observed. The primary or first generation schizonts, abundant on day 4 PI, appeared as interconnected cytoplasmic masses (pseudocytomeres). Each pseudocytomere was enclosed by a membranous vacuole and contained varying numbers of nuclei. As nuclear division and growth of the schizonts continued, larger discrete cytoplasmic masses or cytomeres were formed with rhoptries and multiple nuclei in various stages of division. Synchronous multiple cytoplasmic cleavage of the schizont resulted in the formation of numerous uninucleate merozoites. Second generation schizonts, which developed from hepatic merozoites released from primary schizonts, were abundant in hepatocytes on day 6 PI. Although tissue samples from liver, lung, spleen, kidney, intestine, brain, blood vessels and lymph nodes were examined, schizogonous forms were observed in liver only. No megaloschizonts were detected in any host tissue examined. Schizogonic development was completed by day 7 PI as merozoites developed into gametocytes within mononuclear phagocytes.