Eimeria ahtanumensis n. sp. from the Northwestern Fence Lizard Sceloporus occidentalis in Central Washington

SYNOPSIS Eimeria ahtanumensis n. sp. is described from the northwestern fence lizard Sceloporus occidentalis. The sporulated oocysts are generally cylindrical with rounded ends, averaging 34.2 by 19.7 μ. No oocyst residuum or micropyle is present. The ellipsoidal sporocysts average 10.5 by 8.8 μ; each has a distinct sporocyst residuum composed of spherical granules located against the sides of the sporocysts. The elongate sporozoites, lying head to tail in the sporocyst, average 16.5 by 2.5 μ when excysted. Endogenous development occurs in the epithelial lining of the gall bladder and bile duct. E. ahtanumensis is compared to other similar lizard coccidia.     

Studies on cryopreservation of Cryptosporidium parvum

Neonatal BALB/c mice received oocysts or sporozoites of Cryptosporidium parvum pretreated by a variety of cryopreservation protocols. Histologic sections of infected and control mice were examined to determine if pretreated organisms established infection in the intestine. Sporozoites were inoculated rectally, oocysts orally. Freshly excysted sporozoites were frozen in Hanks' balanced salt solution (HBSS) containing dimethylsulfoxide (DMSO) or glycerol at concentrations of 5%, 10%, or 15% at cooling rates of -1 C and -10 C per min. Other sporozoites were frozen to -70 C in the absence of cryoprotectant without controlled reduction of temperature, others placed in HBSS with 10% DMSO but not subjected to freezing, whereas others were placed in vitrification media containing 5.5 M propylene glycol, 6.5 M glycerol, or 8 M ethylene glycol for 1 min before resuspension in fresh HBSS and inoculation into mice. Intact oocysts were frozen without controlled reduction of temperature directly to -70 C in HBSS containing no cryoprotectant or in HBSS that contained 10% DMSO. Others were cooled at -0.3 C per min from 4 C to -70 C in HBSS with 5% or 10% DMSO. Still others were cooled at a rate of -1 C per min until reaching -40 C and then cooled at -10 C per min until reaching -70 C in HBSS with 7.5% DMSO. Oocysts and sporozoites not exposed to cryoprotectants were inoculated into mice orally and rectally, respectively, for control purposes. Only unfrozen oocysts and sporozoites not exposed to cryoprotectant, and some of the unfrozen oocysts and sporozoites exposed to 10% DMSO, successfully established infections in mice.     

Two new Eimeria species (Apicomplexa: Eimeriidae) from the yellow-crowned Amazon Amazona ochrocephala (Aves: Psittacidae) in Brazil

In this study, we describe 2 new species of Eimeria associated with the yellow-crowned Amazon Amazona ochrocephala. Eimeria amazonae n. sp. has bilayered, ellipsoidal, and smooth oocysts that measure 48.9 × 36.2 μm; the length/width ratio is 1.35. The micropyle and oocyst residuum are both absent, but the polar granule is present. Ovoidal sporocysts are 22.2 × 11.9 μm. Stieda and sub-Stieda bodies and sporocyst residuum are present. The 2 elongate sporozoites are curved and measure 18.1 × 3.4 μm; both have 2 refractile bodies. Eimeria ochrocephalae n. sp. has bilayered, ellipsoidal, and smooth oocysts that measure 43.8 × 27.7 μm; the length/width ratio is 1.58. The micropyle and oocyst residuum are absent, but the polar granule is present; ovoidal sporocysts are 20.6 × 10.1 μm. Stieda and sub-Stieda bodies and sporocyst residuum are present; 2 elongate and curved sporozoites are 15.8 × 3.4 μm, each of which has 2 refractile bodies.     

Developing an in vitro method for Eimeria tenella attachment to its preferred and non-preferred intestinal sites

A frozen section method utilising chicken intestinal tissue was developed to study the Eimeria tenella attachment ex vivo. In order to examine Eimeria-epithelial cell attachment, 10⁵E. tenella sporozoites were incubated with each caecal frozen section (6, 10 and 14 μm) for 1 h in 5% CO₂ incubator at 41 °C. E. tenella sporozoites attached successfully to enterocytes in 14 μm thick of caecal sections. Sporozoite attachment to caecal sections was shown to be dependent on the number of parasites added. To evaluate the method, E. tenella sporozoites were incubated to its preferred (caecum) and non-preferred (duodenum and jejunum) intestinal sites. The number of sporozoites attached to the caecal enterocytes was significantly greater (P < 0.0001) in comparison with the limited number of sporozoites attached to enterocytes of non-preferred intestinal sites. This method was shown to be able to reveal differences in binding capability and allows for comparison of intestinal site attachment.     

Fine Structure of Oocyst Transformation and the Sporozoites of Leucocytozoon dubreuili*

SYNOPSIS. The sporogonic stages of Leucocytozoon dubreuili in the midgut and salivary glands of the simuliid vectors was studied by electron microscopy. Young uninucleate oocysts have a pellicle that initially resembles that of the ookinetc. Numerous electron-dense bodies and microtubules in the peripheral cytoplasm may be involved in the formation of the cyst wall. The dense bodies appear to give rise to the amorphous material of the wall. The tubules which run circumferentially beneath the oocyst's boundary probably serve as a skeletal support for the cell surface during deposition of the wall material. A subcapsular “space” which provides area for expansion of the developing sporozoites is formed in early multinucleate oocysts. The subcapsular “space” appears to be formed through a condensation of the peripheral cytoplasm, resulting in an osmotic gradient across the oocyst's limiting membrane. Consequently water diffuses out, creating a fluid-filled space. Sporozoite formation begins with localized thickenings on the oocyst's limiting membrane. Subsequent extension of the thickened regions into the subcapsular “space” marks the onset of sporozoite budding. The process is highly synchronized, and culminates with the production of up to 150 sporozoites about the sporoblastoid body. The structure of sporozoites from mature oocysts and of the salivary glands of the vector is basically similar, although salivary gland sporozoites are more elongate and have numerous electron-dense micronemes. The paired rhoptries in the latter sporozoites are more elongate and uniformly electron-dense than in oocyst sporozoites.     

A new species of Eimeria (Apicomplexa: Eimeriidae) from the eastern pipistrelle, Perimyotis subflavus (Chiroptera: Vespertilionidae), in Arkansas

During November 2009 and March 2010, 20 adult eastern pipistrelles, Perimyotis (= Pipistrellus) subflavus, were collected from Polk County, Arkansas, and their feces were examined for coccidian parasites. Two (10%) of the bats were found to be passing oocysts of an undescribed species of Eimeria. Oocysts of Eimeria heidti n. sp. were ovoidal to ellipsoidal, 26.1 × 20.5 μm (23-31 × 18-23 μm), with a bilayered wall, externally rough, internally smooth, and with a shape index of 1.3. Micropyle and oocyst residuum were absent, but a subspherical polar granule was often present. Sporocysts were ovoidal, 13.0 × 8.8 μm (11-15 × 7-13 μm), the shape index was 1.6, a Stieda body was present and sub-Stieda and para-Stieda bodies were absent. A sporocyst residuum consisting of multiple globules dispersed along the perimeter of the sporocyst and between the sporozoites were present, sporozoites were elongate, with a subspherical anterior refractile body and elongate posterior refractile body; a nucleus not discernible. This is the second coccidian reported from this host and the fourth instance of a coccidian species reported from an Arkansas bat.     

Cryopreservation of Leucocytozoon caulleryi Sporozoites

Leucocytozoon caulleryi sporozoites that had been stored at - 196° C or -80° C for 6 or 12 months in Eagle's minimum essential medium or Medium 199 supplemented with 5% glycerol and 10% chicken serum showed infectivity to chickens. Glycerol at a concentration of 10% and dimethyl sulfoxide at 10% and 5% were found to be ineffective cryoprotective agents for the low temperature preservation of sporozoites. Sporozoites isolated from the intact females of Culicoides arakawae , which had been stored at -80° C for 6 or 12 months without cryoprotective agents, retained their infectivity. No differences were observed in the prepatent period, duration of parasitemia, and presence of serum-soluble antigens between chickens infected with frozen sporozoites and those infected with fresh sporozoites.     

Cryopreservation of Leucocytozoon caulleryi sporozoites

Leucocytozoon caulleryi sporozoites that had been stored at -196 degrees C or -80 degrees C for 6 or 12 months in Eagle's minimum essential medium or Medium 199 supplemented with 5% glycerol and 10% chicken serum showed infectivity to chickens. Glycerol at a concentration of 10% and dimethyl sulfoxide at 10% and 5% were found to be ineffective cryoprotective agents for the low temperature preservation of sporozoites. Sporozoites isolated from the intact females of Culicoides arakawae, which had been stored at -80 degrees C for 6 or 12 months without cryoprotective agents, retained their infectivity. No differences were observed in the prepatent period, duration of parasitemia, and presence of serum-soluble antigens between chickens infected with frozen sporozoites and those infected with fresh sporozoites.     

Use of a DNA probe to measure the neutralization of Plasmodium berghei sporozoites by a monoclonal antibody

A specific DNA probe has been used to quantify the neutralizing effects of monoclonal antibodies (3D11) against the circumsporozoite protein of Plasmodium berghei sporozoites. The amount of parasite DNA was measured in the livers of Norway Brown rats at the peak of proliferation of the exoerythrocytic forms (EEF). In vitro treatment of 1.5 X 10(5) sporozoites with 0.36 microgram/0.5 ml of whole 3D11 IgG neutralized about 90% of the sporozoite infectivity. When the dose was 3.6 micrograms no signal was detected, indicating that less than ten sporozoites developed into EEF in the liver. In contrast, 3.6 micrograms of Fab obtained from 3D11 neutralized sporozoite infectivity by only 60%. Although the neutralizing effect of 3D11 was very marked, the infected rats developed parasitemias after a prolonged delay in patency, suggesting that a small proportion of sporozoites was resistant to the effects of 3D11. The sporozoites were subjected to four cycles of 3D11-mediated selection, each one involving treatment of sporozoites with the antibodies, injection of the mixture into rats, infection of hamsters with blood stage parasites obtained from the rats, feeding of Anopheles stephensi on these hamsters, and obtaining sporozoites from the salivary glands of the infected mosquitoes. After four cycles of selection, the susceptibility of the resulting sporozoites to different concentrations of 3D11 was compared with that of nonselected sporozoites. No differences were detected, indicating that the capacity of a few sporozoites to escape the neutralizing effect of 3D11 antibodies is not inherited.     

Transport of Eimeria necatrix sporozoites in the chicken: effects of irritants injected intraperitoneally

Light and electron microscopic observations confirmed that Eimeria necatrix sporozoites first enter villous epithelial cells of the chicken small intestine and are transported to the crypts by mononuclear cells. Ultrastructurally, these cells resemble granulated intraepithelial lymphocytes (IEL) rather than macrophages, as suggested previously. The injection of chickens intraperitoneally (i.p.) with a variety of irritants, including proteose peptone, at the time of oocyst inoculation or up to 12 hr postinoculation (PI) resulted in a delay in the arrival of sporozoites at the crypt. Significantly fewer sporozoites had arrived at the crypt by 24 hr PI in i.p.-injected birds as compared to controls. This delay in the arrival of sporozoites at the crypts was reflected by a delay in the development of intestinal lesions and in peak oocyst production. However, there was no significant decrease in the total numbers of oocysts produced by these birds as compared to controls, indicating that no significant loss of sporozoites occurs during the possible rerouting of the parasites. The presence of infective stages in extraintestinal sites was detected by transferring various tissues to coccidia-free recipients. Infection was transferable by gut, liver, and spleen from irritant-injected and control birds at all time intervals studied (12, 24, 36, and 48 hr PI). Infection was also transferable with blood and kidney, but not consistently. A small number of oocysts was passed by the recipients of peritoneal wash from irritant-injected birds at 12 hr PI. In all transfers, the prepatent period was normal, suggesting that the migrant stages are sporozoites.     

Hepatozoon species of the timber rattlesnake in northern Florida: description of a new species, evidence of salivary gland oocysts, and a natural cross-familial transmission of an Hepatozoon species

Two species of Hepatozoon, i.e., H. sauritus and H. horridus n. sp., were present in 1 of 8 timber rattlesnakes, Crotalus horridus. The narrow gamonts of H. sauritus are 15.0-19.0 x 3.5-5.0 microm, with LW 58-86 microm2 and L/W 3.2-4.7, with a narrow, rounded anterior end. The spherical to slightly ovoid oocysts produce ovoid to elongate sporocysts, 21-43 x 12-24 microm, L/W 1.20-2.7, containing on average 22.1 (10-34) sporozoites. This is the first report of a natural cross-familial transfer of a Hepatozoon species. Gamonts of H. horridus n. sp. are 13.0-17.0 x 4.0-6.0 microm, with LW 63-102 microm2 and L/W 2.6-4.0, and have broadly rounded ends. The gamont cytoplasm is vacuolated. The spherical to ovoid oocysts form spherical to elongate sporocysts 14-45 x 11-25 microm, L/W 1.0-2.3, producing an average of 13.0 (8-21) sporozoites. The salivary gland in 1 of 5 mosquitoes dissected contained 1 mature oocyst.     

Eimeria saudiensis n. sp. (Apicomplexa: Eimeriidae) from the Arabian oryx (Oryx leucoryx) in Saudi Arabia

Oocysts of Eimeria saudiensis n. sp. (Apicomplexa: Eimeriidae) are described from the feces of the Arabian oryx, Oryx leucoryx, from the Riyadh Zoo, Saudi Arabia. The oocysts were ellipsoidal or slightly ovoid, 31.2 x 24.5 (24.3-36.5 x 20.0-27.6) micron with a bilayered wall about 1.7 micron thick. The micropyle was covered by a dome-shaped cap. The oocyst residuum was absent, but tiny polar granules were present. The sporocysts were elongate ovoid, 14.3 x 7.2 (11.5-18.5 x 6.0-9.0) micron, had a Stieda body, but lacked a substiedal body. The sporocyst residuum was present, composed of numerous small granules. The sporozoites were elongate club-shaped, and contained two prominent refractile bodies.     

Wenyonella baghdadensis sp. n. from the Bandicoot Rat Nesokia indica in the Baghdad Area

SYNOPSIS. Oocysts of Wenyonella baghdadensis sp. n. were found in the feces of 2 of 12 bandicoot rats Nesokia indica Gray & Hardwicke from the Baghdad area. Sporulated oocysts were subspherical to broadly ellipsoidal, 25.4 (18–22) × 20.8 (15–28) μm, with a 2-layered wall, the outer wall being mammillated, without micropyle, micropylar cap, residuum, or polar granule. Sporozoites were ovoid, 11.8 (9–15) × 8.4 (6–10) μm, with Stieda body and residuum. Two sporozoites in each sporocyst were elongate, with granular cytoplasm and a refractile globule, the other 2 were bean-shaped, without granules or refractile globule. Sporulation time equalled 3 days at 22–24 C. Entamoeba coli and Giardia sp. cysts were also found in 3 rats each.     

The entry of sporozoites of Theileria parva into bovine lymphocytes in vitro. Immunoelectron microscopic observations

In an electron microscopic investigation of the entry of sporozoites of Theileria parva into bovine lymphocytes, the fate of the surface coat of the parasite was traced by immunocytochemical methods. A monoclonal antibody (MAbD1) raised in mice and directed against a surface antigen of sporozoites, was applied to ultrathin frozen sections of bovine lymphocytes infected in vitro. Sites of binding of MAbD1 were localized using a protein A-colloidal gold conjugate as an electron-dense label. The surface of all free sporozoites was labelled. Sporozoites in the process of entering were labelled only on that portion of the membrane not yet tightly bound to the lymphocyte membrane. No label was detected on sporozoites that had completed entry. After fixation with formaldehyde, but not with glutaraldehyde, local areas of labelling were found on lymphocytes in contact with sporozoites and on cells already invaded. The sporozoite organelles, called micronemes, occasionally appeared to contain labelled antigen. No label was found on sporozoites or lymphocytes in control preparations previously exposed to non-specific antibody or treated with protein A-colloidal gold alone. The findings support the conclusion that the sporozoite surface coat, containing the antigen recognized by MAbD1, is shed as the sporozoite enters the host cell.     

Exit of Plasmodium sporozoites from oocysts is an active process that involves the circumsporozoite protein

Plasmodium sporozoites develop within oocysts residing in the mosquito midgut. Mature sporozoites exit the oocysts, enter the hemolymph, and invade the salivary glands. The circumsporozoite (CS) protein is the major surface protein of salivary gland and oocyst sporozoites. It is also found on the oocyst plasma membrane and on the inner surface of the oocyst capsule. CS protein contains a conserved motif of positively charged amino acids: region II-plus, which has been implicated in the initial stages of sporozoite invasion of hepatocytes. We investigated the function of region II-plus by generating mutant parasites in which the region had been substituted with alanines. Mutant parasites produced normal numbers of sporozoites in the oocysts, but the sporozoites were unable to exit the oocysts. In in vitro as well, there was a profound delay, upon trypsin treatment, in the release of mutant sporozoites from oocysts. We conclude that the exit of sporozoites from oocysts is an active process that involves the region II-plus of CS protein. In addition, the mutant sporozoites were not infective to young rats. These findings provide a new target for developing reagents that interfere with the transmission of malaria.     

Eimeria saudiensis N. Sp. (Apicomplexa: Eimeriidae) from the Arabian Oryx (Oryx leucoryx) in Saudi Arabia

Oocysts of Eimeria saudiensis n. sp. (Apicomplexa: Eimeriidae) are described from the feces of the Arabian oryx, Oryx leucoryx , from the Riyadh Zoo, Saudi Arabia. The oocysts were ellipsoidal or slightly ovoid, 31.2 times 24.5 (24.3–36.5 times 20.0–27.6) μm with a bilayered wall about 1.7 μm thick. The micropyle was covered by a dome-shaped cap. The oocyst residuum was absent, but tiny polar granules were present. The sporocysts were elongate ovoid, 14.3 times 7.2 (11.5–18.5 times 6.0–9.0) μm, had a Stieda body, but lacked a substiedal body. The sporocyst residuum was present, composed of numerous small granules. The sporozoites were elongate club-shaped, and contained two prominent refractile bodies.     

Studies on the Motility of Plasmodium Sporozoites*

Albumin was found to have a striking stimulatory effect on motility of Plasmodium sporozoites, while serum globulins had an inhibitory effect. Albumin also preserved viability of sporozoites in vitro at 4 C for several days. P. berghei, P. cynomolgi, and P. falciparum sporozoites each had a distinct and characteristic type of motility. P. berghei sporozoites from oocysts had a different type of motility from that of salivary gland sporozoites, each type presumably associated with different invasive capacities at different times during the life cycle of the parasite. This change in sporozoite motility during development was also associated with other physiologic developmental changes in the sporozoite. The degree of motility of a given pool of sporozoites was to some degree associated with other parameters of metabolic activity of these sporozoites, i.e. infectivity, immunogenicity, and secretory activity. Secretions of the rhoptry-microneme complex may play a role in sporozoite motility.     

Coccidian Parasites (Apicomplexa: Eimeriidae) of Nerodia spp. (Serpentes: Colubridae), with a Description of a New Species of Eimeria

Eimeria conanli n. sp. (Apicomplexa: Eimeriidae) is described from intestinal contents and feces of Nerodia erythrogaster transversa and N harteri harteri from northcentral Texas. Oocysts of the new species are ellipsoid in shape. 17.9 × 13.0(15–21 × 12–15) μm, with a smooth, thin, single-layered wall; shape index 1.4 (1.2–1.5). One to several (usually 2) polar granule(s) and an oocyst residuum are present, but a micropyie is absent. Sporocysts are elongate, 12.9 × 5.2 (13–15 × 5–6) -m, apparently without a true Stieda body structure. Each sporoeyst contains an ellipsoid residuum, 3.9 × 3.2 (3–6 × 2–4) μm, and elongate sporozoites, 11.4 × 2.5 (10–14 × 2–3) μm in situ, each with a spherical or subspherical anterior refractile body and spherical to ellipsoid posterior refractile body. In addition to the new species, oocysts of 4 previously described eimerians from colubrid snakes were found in these hosts.     

Three New Species of Eimeria (Apicomplexa: Eimeriidae) from Nerodia rhombifem (Serpentes: Colubridae) from Texas1

Three new species of Eimeria (Apicomplexa: Eimeriidae) are described from the intestinal contents of Nerodia rhombifera (Serpentes: Colubridae) from Texas. Oocysts of Eimeria infirmus are irregular in shape, 11.6 (8.8–14.4) μm in diameter, with a smooth, thin wall that ruptures easily, releasing free sporocysts. A small polar granule is usually present, but a micropyle and oocyst residuum are absent. Sporocysts are bean-shaped with one side flattened slightly, 9.1 times 5.0 (7.2–10.4 times 4.4–5.8) μm, with what may be a Stieda body consisting of a slight thickening of one end of the sporocyst. Each sporocyst contains a spherical or ellipsoid residuum and sporozoites, each with a single, posterior refractile body. Oocysts of Eimeria rhombifera are spherical or subspherical, 13.1 times 12.6 (12.0–14.4 times 11.2–14.4) μm, with a smooth, frail wall. A polar granule is present, but a micropyle and oocyst residuum are absent. Sporocysts are ovoid, 8.8 times 5.5 (8.0–9.6 times 5.0–6.0) μm, each with a Stieda body. Each sporocyst contains a spherical or subspherical residuum and sporozoites, each with a single posterior refractile body. Oocysts of Eimeria tenuis are ellipsoidal in shape, 17.2 times 10.8 (15.2–20.8 times 9.6–12.0) μm, with a smooth, thin wall. A polar granule and oocyst residuum are present, but a micropyle is absent. Sporocysts are elongate, 13.2 times 4.9(11.2–15.2 times 4.4–5.6) μm, each with a Stieda body consisting of a thickening of the sporocyst wall. Each sporocyst contains a spherical or subspherical residuum and sporozoites with anterior and posterior refractile bodies.     

Observations on the sporozoite transmission of Plasmodium vivax to monkeys

Saimiri boliviensis monkeys were infected via sporozoites with the Salvador I strain of Plasmodium vivax that had been stored frozen for periods ranging from 12 to 5,312 days. Prepatent periods ranged from 16 to 53 days.