Isospora as an Extraintestinal Parasite of Passerine Birds1

Toxoplasma-like avian parasites inhabiting mononuclear phagocytes have been called Haemogregarina, Toxoplasma. avian Toxoplasma, Atoxoplasma, and Lankesterella by various authors. My attempts to transmit the parasites by bloodsucking mites or by transfer of blood and tissues of infected sparrows and canaries were unsuccessful. However, it was noted that the infection was exacerbated under conditions that favored transmission of coccidiosis: crowding and lack of cleanliness. Oral inoculation of sporulated oocysts of Isospora resulted in death from overwhelming macrophage infection with Toxoplasma-like organisms. Experiments using sparrows and canaries showed that the Isospora species involved was not cross infectious. Further investigations using canaries demonstrated that after oral oocyst inoculation, infection of macrophages spread from the submucosa of the duodenum to the liver. spleen, and lungs. After several generations in the internal organs, asexual multiplication, occured in the intestinal epithelium of the small intestinc. Fecal oocysts first appeared at the end of 9–10 days. Oocysts continued to be passed in the feces for months after infection. This chronicity may be explained by the relatively long life of the macrophages that serve as host cells for the asexual stages as compared to the intestinal epithelium which is the cell type parasitized by conventional coccidia.     

Eimeria cicaki sp. n. and Isospora thavari sp. n. from the House Lizard Gehyra mutilata Boulenger in Malaysia*

SYNOPSIS. Oocysts and endogenous stages of new species of Eimeria and Isospora from the house lizard, Gehyra mutilata, are described. The ellipsoid to subspherical 2-layered oocysts of E. cicaki averaged 24.0 × 21.0 μm. Polar granules are present. Micropyle and oocyst residuum are absent. Ellipsoid sporocysts average 12.2 × 9.0 μm. A sporocyst residuum is present, but the Stieda body is absent. Endogenous stages are in epithelial cells of the small intestine. The subspherical single-layered oocysts of I. thavari average 23.8 × 22.8 μm. The polar granule is present; micropyle and oocyst residuum are absent. Ellipsoid sporocysts average 12.8 × 9.4 μm. Stieda body and sporocyst residuum are present. There are endogenous stages in epithelial cells of the small intestine.     

Fuchsin fluorescence and autofluorescence in Cryptosporidium, Isospora and Cyclospora oocysts

Cryptosporidium parvum and Isospora belli oocysts stained with carbol–fuchsin, as in a modified Ziehl–Neelsen technique, fluoresce bright red under green light (546 nm). Cryptosporidium oocysts tend to fluoresce more brightly the less intensely stained they appear under transmitted light; this is not the case with Isospora. Fuchsin-stained Cyclospora cayetanensis oocysts fluoresce rather dimly, but those not taking the dye retain their typical autofluorescence. Cryptosporidium and Isospora oocysts are also autofluorescent, appearing violet under u.v. light (365 nm), and green under violet (405 nm) and blue–violet light (436 nm). Their autofluorescence does not survive the staining procedure.     

Isospora Species of the Cat and Attempted Transmission of I. felis Wenyon, 1923 from the Cat to the Dog*

SYNOPSIS Fecal samples from 130 domestic cats from Illinois were examined for coccidia. Three species of Isospora were found: (1) I. felis Wenyon, 1923, with oocysts 38-51 by 27-39 μ with a mean of 41.6 by 30.5 μ and sporocysts 20-26 by 17-22 μ with a mean of 22.6 by 18.4 μ; it was found in 13% of the cats; (2) I. rivolta (Grassi, 1879) Wenyon, 1923, with oocysts 21-28 by 18-23 μ with a mean of 25.0 by 21.1 μ, and sporocysts 14-16 by 10-13 μ with a mean of 15.2 by 11.6 μ; it was found in 3% of the cats; and (3) I. bigemina (Stiles, 1891) Lühe, 1906, with oocysts 12-15 by 10-13 μ with a mean of 13.2 by 11.8 μ. and sporocysts 8-10 by 6-8 μ with a mean of 8.8 by 6.5 μ it was found in 1.5% of the cats. Four coccidia-free puppies 1.5 months old were inoculated with 100,000 oocysts each of I. felis from the cat, but patent infections did not occur. Partial development of I. felis was not seen in tissue sections of the small intestine of a 5th pup killed 96 hours after inoculation with 150,000 I. felis oocysts. This coccidium is therefore presumably host-specific.     

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.     

Goussia girellae N. Sp. (Apicomplexa: Eimeriorina) in the Opaleye,Girella nigricans1

ABSTRACT Goussia girellae n. sp. is described from the opaleye fish, Girella nigricans. Merogonic stages were observed in the apices of intestinal epithelial cells, in the lamina propria, and in extra-intestinal sites including liver, gills, and spleen. Gamonts were observed in the intestinal epithelial cells. Only unsporulated oocysts were detected in the intestine, and sporulation occurred when feces containing oocysts were incubated for 48 h in seawater at 21°C. Oocysts are elongated (24.8 × 14.7 μm) with a wall about 200 nm thick and have no residuum, micropyle, or polar granule. Sporocysts are ellipsoid (8.5 × 4.5 μm), have a thin two-layered wall approximately 30 nm thick, and consist of two valves joined by a suture. Although moribund opaleye were also infected with Gyrodactylus sp., Cryptobia sp., Cardicola sp., and epitheliocystis organisms (chlamydia), all fish were heavily infected with G. girellae and morbidity was thus attributed to the coccidium.     

Eimeria acervulina: The influence of inoculation dose on transmission between broiler chickens

The course and clinical appearance of an Eimeria species infection in chicken flocks depend on the response of an individual bird to infection and on population-dynamics of the infection in the flock. Differences in ingested numbers of oocysts may affect oocyst load in the flock and the subsequent infectious dose for not yet infected birds. To study the link between numbers of oocysts excreted by infected birds and transmission of Eimeria acervulina, experiments were carried out with 42 pairs of broiler chickens using inoculation doses with 5, 50, 500 or 50,000 sporulated oocysts. In each pair one bird was inoculated and the other bird was contact-exposed. All contact birds became infected, which occurred on average within 34 h after exposure to an inoculated bird. Although a higher inoculation dose resulted in higher oocyst excretion in inoculated and contact-infected birds, only small non-significant differences in transmission rates between groups were found.     

Evaluation of water treatment plant UV reactor efficiency against Cryptosporidium parvum oocyst infectivity in immunocompetent suckling mice

Aim: To assess the efficiency of a medium‐pressure UV reactor under full‐scale water treatment plant (WTP) conditions on the infectivity of Cryptosporidium parvum oocysts in an Naval Medical Research Institute (NMRI) suckling mice infectivity model. Methods and Results: Six/seven‐day‐old mice were administered orally 2–10 × 10⁴Cryptosporidium parvum oocysts. Compared with nonirradiated oocysts, 40 mJ cm^?2 UV irradiation of ingested oocysts resulted 7 days later in a 3·4–4·0 log₁₀ reduction in the counts of small intestine oocysts, using a fluorescent flow cytometry assay. Conclusion: Present data extend to industrial conditions previous observations of the efficiency of UV irradiation against Cryptosporidium parvum oocyst in vivo development. Significance and Impact of the study: Present results suggest that in WTP conditions, a medium‐pressure UV reactor is efficient in reducing the infectivity of Cryptosporidium parvum oocysts, one of the most resistant micro‐organisms present in environmental waters.     

Development of Toxoplasma gondii Chinese I genotype Wh6 Strain in Cat Intestinal Epithelial Cells

Felids are the unique definitive host of Toxoplasma gondii. The intestine of felid is the only site for initiating Toxoplasma gondii sexual reproduction. T. gondii excretes millions of infectious oocysts from the intestine, which are the primary source of infection. There are many difficulties in developing vaccines and drugs to control oocyst excretion due to the lack of an appropriate experimental model. Here, we established an in vitro feline intestinal epithelial cell (IEC) infection system and an efficient animal model of T. gondii Chinese 1 genotype, Wh6 strain (TgCtwh6). The Kunming mice brain tissues containing TgCtwh6 cysts were harvested 42-day post-infection. The bradyzoites were co-cultured with cat IECs in vitro at a ratio of 1:10. Five 3-month-old domestic cats were orally inoculated with 600 cysts each. The oocysts were detected by daily observation of cat feces by microscopy and polymerase chain reaction. We found that the parasite adhered and invaded cat IECs in vitro, transformed into tachyzoites, and then divided to form rose-like structures. These parasites eventually destroyed host cells, escaped, and finished the asexual reproduction process. Schizonts associated with sexual reproduction have not been observed during development in vitro cultured cells. However, schizonts were detected in all infected cat intestinal epithelial cells, and oocysts were presented in all cat feces. Our study provides a feasible cell model and an efficient infection system for the following studies of T. gondii sexual reproduction, and also lays a foundation to develop drugs and vaccines for blocking excretion and transmission of oocysts.     

Isospora papionis n. sp. (Eimeriidae) of the Chacma Baboon Papio ursinus (Kerr, 1792)

SYNOPSIS. Isospora papionis n. sp. is described from the chacma baboon Papio ursinis (Kerr, 1792). Histopathologic examination revealed 3 of 20 baboons to be infected, but no organisms were found on examination of the feces. Macrogametes and sporulated oocysts were observed in the small intestine, mainly in the jejunum and ileum, invariably in an epithelial or subepithelial location at the proximal portions of the crypts of Lieberkühn. Intact sporulated oocysts averaged 17 by 11 μ and had very thin walls which were often partially collapsed between the sporocysts. The sporocysts were somewhat flattened on one side and averaged 11 by 8.5 μ. A large sporocyst residuum was present.     

Fat stores in a migratory bird: a reservoir of carotenoid pigments for times of need?

Carotenoids are well known for their immune-stimulant function in birds and other vertebrates. Moreover, they have potential antioxidant capacity, scavenging free radicals and protecting cell compartments from oxidation. Most essential carotenoids are fat soluble and could be stored for times of need especially in adipose tissues, built up by migratory birds as the main source of energy on long-distance flights. In an exclusive diet experiment, garden warblers (Sylvia borin) were fed ad libitum with an experimental diet, enriched with two different dose rates of carotenoids, or with control food, during the period of their first autumn migration. Plasma carotenoid content was measured via HPLC and chroma of plasma and fat examined with a spectrophotometer. Birds were infected with Isospora spp. and intensity of infection determined by oocyst counts 3 days post infection. Plasma lutein levels and chroma of subcutaneous fat stores were positively correlated and chroma values of these fat stores increased in the birds that got the higher dose rate, whereas they decreased significantly in the control group after infection with Isospora spp. Chroma of subcutaneous fat deposits in vivo and intensity of Isospora infection were negatively correlated. By measuring the chroma of fat deposits in vivo, we show that fat can be a reservoir for carotenoids. These colorful antioxidants are stored in the fat and taken from there in times of a higher demand, e.g. when mounting an immune response to parasites.     

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.     

Eimeria indianensis sp. n. and an Isospora sp. from the Opossum Didelphis virginiana (Kerr)*

Two of 15 road-killed opossums examined for coccidia were found to be infected with a hitherto undescribed species of Eimeria, herein named Eimeria indianensis . The oocysts were spherical (63%) or slightly subspherical (37%) with a double-layered wall. The outer layer was ～1.5 μm thick, yellowish, striated, and appeared rough and pitted on the surface. A micropyle was absent. The spherical oocysts were 16.3 (13–18) μm in diameter; the subspherical ones, 17.6 (15–18) × 16.4 (14–17) μm. The sporocysts measured 9.1 (8–10) × 6.2 (6–7) μm and contained a granular residuum. The sporozoites were elongate, measuring 13.4 (13–15) × 1.8 (1.6-2.0) μm; no refractile globules were seen. The prepatent period was 10 days and the patent period ranged from 9–15 days. A few oocysts of an Isospora sp. were present in one opossum. It was not possible to confirm whether they were specifically of the opossum or of spurious origin.     

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.     

Eimeria callosciuri n. sp. (Protozoa: Eimeriidae) from Prevost's Squirrel Callosciurus prevostii Demarest, 1822 in Malaysia*

SYNOPSIS. Eimeria callosciuri n. sp. is described from Prevost's squirrel Callosciurus prevostii in Malaysia. Its oocysts are 24–31 by 20–2μ with a mean of 21.9 by 28.4μ. Schizogonic and gametogonic stages develop in the tips of the villi of the small intestine, above the nuclei of the host epithelial cells. It is the first species of Eimeria described from the rodent tribe Callosciurini.     

Cyst-Induced Toxoplasmosis in Cats*

SYNOPSIS. The life cycle of Toxoplasma gondii is described from cats orally inoculated with Toxoplasma cysts. Five new structural stages of Toxoplasma designated as “types” A-E were found in the epithelial cells of the small and large intestine. Type A is the smallest of all 5 intestinal Toxoplasma types. It occurs as collections of 2-3 organisms in the jejunum 12–18 hr after infection. Type B organisms are characterized by a centrally located nucleus, a prominent nucleolus and dark blue cytoplasm giving rise to the appearance of bipolar staining with Giemsa. Type B occurs 12–54 hr after infection and appears to divide by simple endodyogeny and by multiple endodyogeny (endopolygeny). Type C organisms are elongate with subterminal nuclei and strongly PAS-positive cytoplasm. They occur at 24–54 hr and divide by schizogony. Type D organisms are smaller than type C and contain only a few PAS-positive granules. They occur from 32 hr to 15 days after inoculation and account for over 90% of all parasites in the small intestine during this time. Three subtypes divide by endodyogeny, schizogony and by splitting of their merozoites from the main nucleated mass without leaving a residual body. Type E organisms resemble one of the subtype D which divide by schizogony, but they leave a residual body. They occur from 3–15 days after inoculation. Gametocytes occur thruout the small intestine but more commonly in the ileum 3-15 days after infection. Male gametocytes contain on an average of 12 microgametes and comprise 2–4% of the gametocyte population. The prepatent period after cystinduced infection is 3–5 days with the peak oocyst production between 5–8 days and a patent period varying from 7–20 days. Variable numbers of trophozoites are present in the lamina propria of the small intestine and in the extra-intestinal tissues within a few hr after inoculation. After 9–10 days cysts were seen in the heart and later in the brain. The lesions of toxoplasmosis are compared in newborn and weanling kittens and in adult cats after oral and subcutaneous inoculation with cysts. After the ingestion of cysts, newborn kittens developed enteritis, hepatitis, myocarditis, myositis, pneumonitis and encephalitis and were moribund by the 9th day. Kittens aged 2 weeks and older developed enteritis, myocarditis, encephalitis and myositis but often survived; adult cats usually remained asymptomatic. After subcutaneous inoculation of cysts, newborn and weanling kittens died of acute toxoplasmosis with severe pneumonia, myocarditis, encephalitis and hepatitis.     

Plasmodium octamerium n. sp., an Avian Malaria Parasite from the Pintail Whydah Bird Vidua macroura*

SYNOPSIS. A new species of avian malaria parasite is described from the pintail whydah Vidua macroura, a very small African finch of the weaver bird family (Ploceidae). Its structure has been studied chiefly in the canary, to which it is easily transmissible by blood inoculation. Since the segmenters most often produce 8 merozoites, the name Plasmodium octamerium n. sp. is proposed. Other characteristics include sexual stages which are usually elongate, often slender, and do not displace the host cell nucleus, and gametocytes indistinguishable from those of many species of Haemoproteus. Erythrocytes are the only blood cells parasitized. The new species resembles Plasmodium fallax in many respects, but gives rise to fewer merozoites and the asexual forms are smaller. Blood-induced infections are also of strikingly different type in some host species. Among susceptible host species are several kinds of finches, pigeons, quail, young chicks, chukars, tree and song sparrows. In most of these hosts infections are mild, but some tree sparrows die as the result of blood infection, and chukars usually die because of massive invasion of the capillary endothelium of the brain by exoerythrocytic forms. These are of the gallinaceum type and may be quite large, producing hundreds of merozoites. Exoerythrocytic stages were sought but not found in other host species.     

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.     

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.