Anemia in Ducks Infected with Leucocytozoon simondi*†

SYNOPSIS. Thirty-four white Pekin ducklings were used to study the anemia associated with infection by Leucocytozoon simondi. The first appearance of gametocytes in the peripheral blood, as detected by thin smears, marked the onset of anemia. This anemia lasted for the duration of the initial parasitemia, usually reaching a low point early in the infection (1 to 5 days post patency) and returning slowly to normal as the parasitemia decreased. Greatest gametocyte density occurred 5 to 8 days post patency. In a number of cases recovery from anemia began simultaneously or even prior to the highest level in the gametocyte density. In low level parasitemias a fluctuation occurred in erythrocyte numbers which corresponded with the peaks of gametocyte density. In none of the infections was a sufficient number of parasite observed to account for the existing anemia. Haemopoietic activity was observed for a brief period at the time of maximum erythrocyte loss in only a few birds. The overcompensation for erythrocyte loss at the end of the primary parasitermia favors the view that increased erythrocyte production may account for the short duration of haemopoiesis.     

Experimental Studies of the Life Cycle of Leucocytozoon simondi in Ducks in Norway*

SYNOPSIS. Pekin ducks were infected naturally and experimentally. The life cycle of Leucocytozoon simondi was followed in the ducks and the vector, a new species of Simulium, close to Simulium dogieli. The prepatent period in the ducks was 4–5 days and developing megaloschizonts appeared in 6–7 days. Schizonts were found in liver, spleen, brain, and kidney. In the kidneys they were located in the glomeruli. Sporogony in some individuals was completed in 7 days at 13–14 C, other individuals developed more slowly, as ookinetes were found in flies 8 days after feeding. The rapid asexual cycle combined with a sporogonic cycle, in which some ookinetes develop rapidly and others more slowly, favors the maintenance of the parasite in an environment with relatively low daily temperatures. This and the size of the megaloschizonts indicate differences between this strain of the parasite and the one occurring in North America.     

Hemagglutination and Erythrophagocytosis Associated with the Anemia of Plasmodium berghei Infections of Rats*

SYNOPSIS. The presence of anemia that often seems excessive for the amount of parasitemia in Plasmodium berghei infections had led to the suggestion that autoimmunity might be in part responsible for the anemia. In another erythrocytic infection, Anaplasma marginale of cattle, the association of erythrophagocytosis, autohemagglutination and anemia with infection has led to the suggestion that autoimmunization may occur in anaplasmosis. The possibility that similar findings might be present in P. berghei infections of rats has been investigated. Groups of rats infected with P. berghei were examined at 2–3 day intervals during the course of infection. Red blood cell counts, hematocrit values and percentages of parasitized erythrocytes were determined. The rats were bled at intervals and the sera tested for agglutinins for trypsinized rat erythrocytes. Other infected rats were killed, and their spleens and bone marrow were examined for evidence of erythrophagocytosis. Parasitemia reached a peak on the 9th day of infection and became subpatent by the 14th. The greatest depression in erythrocyte numbers occurred on the 11th day, and the counts remained below normal until the 23rd day. Phagocytized erythrocytes, predominantly uninfected, were found in the phagocytes of the spleen and bone marrow from the 5th through the 21st day. Agglutinins for trypsinized normal rat erythrocytes were present in the sera of the rats in titers as high as 1:64 from the 5th through 14th day of infection. Lower agglutinin titers (1:8) were found from time to time in sera of rats made anemic by repeated bleedings. It is not clear whether these agglutinins are responsible for erythrophagocytosis; however, the fact that predominantly uninfected erythrocytes were phagocytized suggests that the erythrocytes might have been opsonized by an autoantibody associated with the P. berghei infections.     

Zoonotic Echinostome Infections in Free-Grazing Ducks in Thailand

Free-grazing ducks play a major role in the rural economy of Eastern Asia in the form of egg and meat production. In Thailand, the geographical location, tropical climate conditions and wetland areas of the country are suitable for their husbandry. These environmental factors also favor growth, multiplication, development, survival, and spread of duck parasites. In this study, a total of 90 free-grazing ducks from northern, central, and northeastern regions of Thailand were examined for intestinal helminth parasites, with special emphasis on zoonotic echinostomes. Of these, 51 (56.7%) were infected by one or more species of zoonotic echinostomes, Echinostoma revolutum, Echinoparyphium recurvatum, and Hypoderaeum conoideum. Echinostomes found were identified using morphological criteria when possible. ITS2 sequences were used to identify juvenile and incomplete worms. The prevalence of infection was relatively high in each region, namely, north, central, and northeast region was 63.2%, 54.5%, and 55.3%, respectively. The intensity of infection ranged up to 49 worms/infected duck. Free-grazing ducks clearly play an important role in the life cycle maintenance, spread, and transmission of these medically important echinostomes in Thailand.     

Elongate and Round Gametocytes of Leucocytozoon simondi (Mathis & Léger) in Ducks Inoculated with Megaloschizonts

SYNOPSIS. Single megaloschizonts give rise to elongate and round gametocytes, the former outnumbering the latter. Male and female elongate gametocytes develop from merozoites of a single megaloschizont. Elongate gametocytes were seen 2–7 days and round gametocytes 6–11 days after megaloschizonts had been inoculated into ducklings. Experimental evidence indicates that merozoites of megaloschizonts invade blood cells and develop into elongate gametocytes. Other merozoites infect tissue cells and develop into secondary exoerythrocytic schizonts which give rise to round gametocytes. Relapse in Leucocytozoon simondi infections is discussed in relation to megaloschizont-induced exoerythrocytic schizogony.     

Three Cases of Hemolytic Anemia with Erythrocyte Pyruvate Kinase Deficiency in Alberta

Erythrocyte pyruvate kinase (PK) activity was determined by the lactate dehydrogenase-coupled spectrophotometric assay. The effects of modifications in the buffer and in substrate concentrations were studied. Three patients with congenital non-spherocytic hemolytic anemia were deficient in erythrocyte PK, and were evidently homozygous for this deficiency. The daughter of one patient and the parents of another had intermediate PK levels and were probably heterozygous. The erythrocyte adenosine triphosphate (ATP) level was low in one patient, high in another. Adenosine triphosphatase activity of the erythrocyte membranes of one patient was normal.     

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.     

Further Studies in the Chemotherapy of Plasmodium hexamerium Infections in Ducks

Five standard antimalarial drugs have been tested, in the present study, against trophozoite-induced Plasmodium hexamerium infections in ducks; namely, chloroquine, proguanil (paludrine), pyrimethamine (daraprim), pentaquine and isopentaquine. (Four others were used previously.) Two doses daily were given for a treatment period of 7 days. The effectiveness of treatment was measured in terms of its ability to suppress parasitemia and to completely prevent relapse ( i.e. to achieve sterilization). Of the five drugs, the two pentaquines were about equally effective. They rapidly cleared the blood of parasites, and completely aborted the infection in nearly all cases. The other three drugs failed to sterilize any of the cases treated, and were not even effective as suppressants.
The pentaquines, however, proved quite toxic to the host and, even in the dosages used, retarded the normal rate of weight gain. Pyrimethamine had a similar effect, and in addition interfered with normal feathering. Neither of these effects, whether due to pentaquines or pyrimethamine, was recovered from after treatment stopped.     

Schizogony and Gametogony of Leucocytozoon simondi and Associated Reactions in the Avian Host*

SYNOPSIS. Stages of development of Leucocytozoon simondi in White Pekin ducklings and their reactions to the parasite were studied on successive days after infecting them artificially with sporozoites from Simulium rugglesi. The minimum prepatent period was 5 days. The first asexual cycle occurred exclusively in the parenchymal cells of the liver. Progeny of these hepatic schizonts followed one of 3 courses: (a) invaded parenchymal liver cells to give rise to another hepatic cycle, (b) penetrated blood cells to form round gametocytes, and (c) were phagocytized by macrophages and grew into megaloschizonts thruout the body. The appearance of elongating gametocytes coincided with the period of maturation and release of merozoites from the megaloschizonts. Experimental evidence supports the hypothesis that the round gametocytes arise from the hepatic schizonts and the elongate forms from the megaloschizonts. Mature megaloschizonts released millions of merozoites, but a high 2nd peak in parasitemia did not develop because of retention of developing gametocytes in the deep circulation, particularly the liver and spleen, and a pronounced host reaction.     

Crystalloid Inclusions in Species of Leucocytozoon,Parahaemoproteus, and Plasmodium*†

Cytoplasmic vacuoles seen in methanol-fixed, Giemsa's-stained ookinetes of Leucocytozoon simondi, Parahaemoproteus fringillae and Plasmodium gallinaceum, when studied with the electron microscope, were found to correspond with crystalloid inclusions of similar structure, particle size, and arrangement. Cytochemical examination of these “crystalloids” revealed their lipid-protein nature. Morphologically similar inclusions were found also in ookinetes of Leucocytozoon ziemanni and Parahaemoproteus velans. In L. simondi, crystalloid is formed rapidly after fertilization, from amorphous electron dense material seen in mature macrogametocytes. The arrangement and distribution of crystalloids in the zygote, ookinete, oocyst, and sporozoite are described. On the basis of differences in structure and particle size, it is proposed that the crystalloid inclusions in Haemosporina be divided into 2 types. Type I—lipid-protein in nature, characterized by electron dense irregularly spherical particles, 25–40 nm in diameter, with individual particles not invested by membrane. Type II—probably virus, characterized by electron dense, irregularly spherical, membrane-bounded particles, with a diameter usually greater than 40 nm.