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.     

Cell: sporozoite interactions and invasion by apicomplexan parasites of the genus Eimeria

The site specificity that avian Eimeria sporozoites and, to a more limited degree, other apicomplexan parasites exhibit for invasion in vivo suggests that specific interactions between the sporozoites and the target host cells may mediate the invasion process. Although sporozoite motility and structural and secreted antigens appear to provide the mechanisms for propelling the sporozoite into the host cell,there is a growing body of evidence that the host cell provides characteristics by which the sporozoites recognise and interact with the host cell as a prelude to invasion. Molecules on the surface of cells in the intestinal epithelium, that act as receptor or recognition sites for sporozoite invasion, may be included among these characteristics. The existence of receptor molecules for invasion by apicomplexan parasites was suggested by in vitro studies in which parasite invasion was inhibited in cultured cells that were treated with a variety of substances designed to selectively alter the host cell membrane. These substance included cationic compounds or molecules, enzymes that cleave specific linkages, protease inhibitors, monoclonal antibodies, etc. More specific evidence for the presence of receptors was provided by the binding of parasite antigens to specific host cell surface molecules.Analyses of host cells have implicated 22, 31, and 37 kDa antigens, surface membrane glycoconjugates,conserved epitopes of host cells and sporozoites, etc., but no treatment that perturbs these putative receptors has completely inhibited invasion of the cells by parasites. Regardless of the mechanism,sporozoites of the avian Eimeria also invade the same specific sites in foreign host birds that they invade in the natural host. Thus, site specificity for invasion may be a response to characteristics of the intestine that are shared by a number of hosts rather than to a unique trait of the natural host. Protective immunity elicited against avian Eimeria species is not manifested in a total blockade of parasite invasion. In fact, the effect of immunity on invasion differs according to the eliciting species and depends upon the area of the intestine that is invaded. Immunity produced against caecal species of avian Eimeria, for example Eimeria tenella and Eimeria adenoeides, inhibits subsequent invasion by homologous or heterologous challenge species, regardless of the area of the intestine that the challenge species invade. Conversely, in birds immunised with upper intestinal species, Eimeria acervulina and Eimeria meleagrimitis, invasion by challenge species is not decreased and often is significantly increased.     

Malaria's deadly secret: a skin stage

The role skin plays in malaria infection has long been overlooked. Recent analysis, however, suggests skin-infecting sporozoites initiate rapid suppression of immunity, establishing early tolerance to subsequent lifecycle stages. This explains susceptibility to reinfection by mosquito bite, independent of blood stage-induced immunosuppression or semi-immunity. Vaccine trials corroborate skin-initiated immunosubversion due to skin-infecting forms, tightly correlating bite pre-exposure, live parasites in the skin and endemic vaccine failure. Rapidly advancing skin immunobiology and recently described parasite development in host skin further substantiate the proposed model, consolidating a new concept in parasite biology, exemplified by malaria: natural infection has a defined, potently immunosubversive skin stage, crucially affecting vaccine function and vitally relevant to eradication.     

Reproducible infection of intact Aotus lemurinus griseimembra monkeys by Plasmodium falciparum sporozoite inoculation

Aotus lemurinus griseimembra is considered one of the best nonhuman primate species for malarial studies because of its susceptibility to infection by Plasmodium falciparum asexual blood stages. However, reproducible transmission of infective P. falciparum sporozoites by mosquito inoculation has been difficult to achieve even in splenectomized monkeys. Characterization of an Aotus-P. falciparum cyclical transmission model has become a top priority as a result of the significant progress toward the development of preerythrocytic malaria vaccines. Herein, we describe a reproducible model developed using intact A. lemurinus griseimembra monkeys intravenously inoculated with sporozoites from a monkey-adapted P. falciparum (Santa Lucia) strain and a wild Falciparum-Cali-Colombia-4 (FCC-4) strain. Sporozoites were obtained by salivary gland dissection of laboratory-reared Anopheles albimanus mosquitoes. Parasitemia was monitored by thick-smear microscopy, parasite lactate dehydrogenase (pLDH) determination, and mosquito xenodiagnosis. The last method proved to be the most sensitive method for monitoring parasitemias. Infection with the Santa Lucia strain showed a mean prepatent period of 16 days (range 6-21 days), whereas infection with the wild FCC-4 strain resulted in a 24-day prepatent period. Mean peak parasite density was approximately 900 parasites/microliter for both parasite strains. The prepatent period, the peak of parasitemia, and the duration of patency were independent of the size of the sporozoite inoculum and the presence of spleen in the host. This model is being successfully used to test the protective efficacy of P. falciparum preerythrocytic vaccine candidates.     

Short-term and long-term effects of food supply on parasite burdens in Tawny Owls, Strix aluco

1. The relationships among food supply (Field Voles, Microtus agrestis ), reproduction and blood parasites was investigated in Tawny Owls, Strix aluco , in Kielder Forest, Northumberland, in 1994 and 1995. Vole populations were significantly lower in 1995 than in 1994.
2. Birds did not lose parasites after initial infection, and the level at which infections were maintained was characteristic of individual birds.
3. In 1994, the number and intensity of parasites was higher in adult owls that had experienced low food supply when they themselves were reared. This indicated that food supplied to chicks in the nest has a long-term effect on the parasite burden of adults.
4. In addition, there was evidence that parasite burdens of adults were influenced by their current food supply. Birds that suffered a decline in food abundance on their territories between 1994 and 1995 showed an increase in parasite load over the same period. In 1995, there was also a significant negative correlation between the parasite loads of owls and vole abundance on their territories.
5. The best predictor of parasite number of chicks reared in 1995 was the parasite load of their fathers. The parasites chicks developed were not the parasites with which their fathers were heavily infested. This result could be due to inherited immunity.
6. Our results indicated that food resources should be measured when investigating interactions between parasites and their hosts, and that offspring quality as well as quantity might suffer when food abundance is low.     

Quantitative imaging of Plasmodium transmission from mosquito to mammal

Plasmodium, the parasite that causes malaria, is transmitted by a mosquito into the dermis and must reach the liver before infecting erythrocytes and causing disease. We present here a quantitative, real-time analysis of the fate of parasites transmitted in a rodent system. We show that only a proportion of the parasites enter blood capillaries, whereas others are drained by lymphatics. Lymph sporozoites stop at the proximal lymph node, where most are degraded inside dendritic leucocytes, but some can partially differentiate into exoerythrocytic stages. This previously unrecognized step of the parasite life cycle could influence the immune response of the host, and may have implications for vaccination strategies against the preerythrocytic stages of the parasite.     

Effects of Cyclophosphamide on Visceral Leishmaniasis in the Mouse*

SYNOPSIS Cyclophosphamide (Cy), 125 or 200 mg/kg body weight, injected intraperitoneally (i.p.) into BALB/c mice one day before infection with amastigotes of Leishmania donovani, by the 8th day postinfection caused a significant decrease in the mean numbers of parasites in spleens and livers when compared to mice injected with phosphate buffered saline (PBS). When 125 mg/kg was injected into chronically infected mice (on day 34 of infection), however, within 2 days (day 36) mean parasite levels in both the spleens and livers were statistically greater than in PBS-treated controls. Similarly, when a series of 6 Cy injections, 50 mg/kg each, was injected over a period of 8 days during the chronic stage of infection, the mean parasite levels in both spleens and livers were significantly increased over those of PBS-treated controls. Druing the chronic stage of infection, Cy injections suppressed the immunity to superinfection. Neither plasma nor parasitized peritoneal macrophages obtained from Cy-treated mice, when compared to PBS-treated mice, differed in their respective capacities to influence the growth of intracellular amastigote of L. donovani in vitro. Passive transfer of hyperimmune mouse serum could not reverse the immunosuppressive effects of Cy upon chronic leishmaniasis in the mouse. It is suggested that neither readily demonstrable anti-leishmanial humoral factors nor “immune” macrophages per se, plays a major role in acquired immunity to leishmaniasis in the mouse.     

Gene disruption of Plasmodium falciparum p52 results in attenuation of malaria liver stage development in cultured primary human hepatocytes

Difficulties with inducing sterile and long lasting protective immunity against malaria with subunit vaccines has renewed interest in vaccinations with attenuated Plasmodium parasites. Immunizations with sporozoites that are attenuated by radiation (RAS) can induce strong protective immunity both in humans and rodent models of malaria. Recently, in rodent parasites it has been shown that through the deletion of a single gene, sporozoites can also become attenuated in liver stage development and, importantly, immunization with these sporozoites results in immune responses identical to RAS. The promise of vaccination using these genetically attenuated sporozoites (GAS) depends on translating the results in rodent malaria models to human malaria. In this study, we perform the first essential step in this transition by disrupting, p52, in P. falciparum an ortholog of the rodent parasite gene, p36p, which we had previously shown can confer long lasting protective immunity in mice. These P. falciparum P52 deficient sporozoites demonstrate gliding motility, cell traversal and an invasion rate into primary human hepatocytes in vitro that is comparable to wild type sporozoites. However, inside the host hepatocyte development is arrested very soon after invasion. This study reveals, for the first time, that disrupting the equivalent gene in both P. falciparum and rodent malaria Plasmodium species generates parasites that become similarly arrested during liver stage development and these results pave the way for further development of GAS for human use.     

Intraseasonal variation in immune defence,body mass and hematocrit in adult house martins Delichon urbica

The intensity of parasite infections often increases during the reproductive season of the host as a result of parasite reproduction, increased parasite transmission and increased host susceptibility. We report within‐individual variation in immune parameters, hematocrit and body mass in adult house martins Delichon urbica rearing nestlings in nests experimentally infested with house martin bugs Oeciacus hirundinis and birds rearing nestlings in initially parasite‐free nests. From first to second broods body mass and hematocrit of breeding adult house martins decreased. In contrast leucocytes and immunoglobulins became more abundant. When their nests were infested with ectoparasites adults lost more weight compared with birds raising nestlings in nests treated with pyrethrin, whereas the decrease in hematocrit was more pronounced during infection with blood parasites. Neither experimental infestation with house martin bugs nor blood parasites had a significant effect on the amount of immune defences.     

Plasmodium protease ROM1 is important for proper formation of the parasitophorous vacuole

Apicomplexans are obligate intracellular parasites that invade host cells by an active process leading to the formation of a non-fusogenic parasitophorous vacuole (PV) where the parasite replicates within the host cell. The rhomboid family of proteases cleaves substrates within their transmembrane domains and has been implicated in the invasion process. Although its exact function is unknown, Plasmodium ROM1 is hypothesized to play a role during invasion based on its microneme localization and its ability to cleave essential invasion adhesins. Using the rodent malaria model, Plasmodium yoelii, we carried out detailed quantitative analysis of pyrom1 deficient parasites during the Plasmodium lifecycle. Pyrom1(-) parasites are attenuated during erythrocytic and hepatic stages but progress normally through the mosquito vector with normal counts of oocyst and salivary gland sporozoites. Pyrom1 steady state mRNA levels are upregulated 20-fold in salivary gland sporozoites compared to blood stages. We show that pyrom1(-) sporozoites are capable of gliding motility and traversing host cells normally. Wildtype and pyrom1(-) sporozoites do not differ in the rate of entry into Hepa1-6 hepatocytes. Within the first twelve hours of hepatic development, however, only 50% pyrom1(-) parasites have developed into exoerythrocytic forms. Immunofluorescence microscopy using the PVM marker UIS4 and transmission electron microscopy reveal that the PV of a significant fraction of pyrom1(-) parasites are morphologically aberrant shortly after invasion. We propose a novel function for PyROM1 as a protease that promotes proper PV modification to allow parasite development and replication in a suitable environment within the mammalian host.     

Conservation of a gliding motility and cell invasion machinery in Apicomplexan parasites

Most Apicomplexan parasites, including the human pathogens Plasmodium, Toxoplasma, and Cryptosporidium, actively invade host cells and display gliding motility, both actions powered by parasite microfilaments. In Plasmodium sporozoites, thrombospondin-related anonymous protein (TRAP), a member of a group of Apicomplexan transmembrane proteins that have common adhesion domains, is necessary for gliding motility and infection of the vertebrate host. Here, we provide genetic evidence that TRAP is directly involved in a capping process that drives both sporozoite gliding and cell invasion. We also demonstrate that TRAP-related proteins in other Apicomplexa fulfill the same function and that their cytoplasmic tails interact with homologous partners in the respective parasite. Therefore, a mechanism of surface redistribution of TRAP-related proteins driving gliding locomotion and cell invasion is conserved among Apicomplexan parasites.     

T cells as mediators of protective immunity against liver stages of Plasmodium

T cells from different subsets play a major role in protective immunity against pre-erythrocytic stages of malaria parasites. Exposure of humans and animals to malaria sporozoites induces (alphabeta CD8(+) and CD4(+) T cells specific for antigens expressed in pre-erythrocytic stages of Plasmodium. These T cells inhibit parasite development in the liver, and immunization with subunit vaccines expressing the respective antigenic moieties confers protection against sporozoite challenge. gammadelta and natural killer T cells can also play a role in protective immunity. Recent studies with mice transgenic for the alphabeta T-cell receptor have revealed the existence of complex mechanisms regulating the induction and development of these responses.     

Immunity to malaria.

Malaria remains prevalent throughout tropical and subtropical regions and almost a third of the World's population is exposed to the risk of infection. There is currently a serious resurgence of the disease in Asia and Central America. The failure of global eradication measures based upon the use of insecticides and chemotherapy has resulted from difficulties of practical implementation compounded by the spread of insecticide and drug resistance. Repeated natural infection does not produce detectable resistance to the exo-erythrocytic cycle of malaria in man. Irradiated sporzoite vaccines do, however, induce stage specific immunity in murine malaria and in a proportion of human subjects. Vaccinated individuals remain susceptible to blood stage infection which causes clinical malaria. In addition the vaccine is unstable and must be administered by intravenous inoculation. Since neither sporogonic nor exo-erythrocytic parasite development is cyclical in human malarias, there is little prospect for vaccine production through cultivation of these stages. The inhabitants of hyperendaemic areas become increasingly resistant to malaria during childhood and adolescence, through the slow development of specific, acquired immunity to asexual blood stage parasites. Immunity is mediated by antibody, which blocks merozoite invasion of red cells, as well as by cell mediated mechanisms and non-specific cytotoxic agents. Vaccination with merozoites induces long lasting immunity of broad serological specificity active against the blood-stage of the parasite. Merozoite vaccines can be preserved by freeze drying and harvested from continuous cultures of blood stage parasites. The major problem in development of a human merozoite vaccine concerns the requirement for Freund's complete adjuvant which is not acceptable for man. The effective immunity induced by vaccination contrasts with the slow development of incomplete resistance which follows repeated natural infection. The latter is associated with the generation of immune suppressor cells, lymphoid cell mitogens and soluble antigens, and in some species by the occurrence of antigenic variation--all of which may favour parasite survival. It is probable that vaccination with non-viable antigen of appropriate composition, induces immune effector processes without activating mechanisms which allow parasites to escape the consequences of immunity. Many effective vaccines such as those against measles, poliomyelitis, tetanus and rabies are commercially available but barely used in the developing world. The affected nations cannot afford their purchase, nor do the means exist for their distribution. It follows that if a safe and effective malaria vaccine were to be developed, its bulk manufacture and administration would require massive international support and cooperation.     

Effects of antihelminthic treatment on cell-mediated immunity in Gentoo penguin chicks

Intestinal parasites suppose a cost to hosts as they compete directly for nutritional resources. Therefore, hosts must defend themselves against intestinal parasites by mounting an immune response. Many penguin species acquire parasites through their diet and transfer these parasites to their chicks when feeding them. High parasite loads in penguin chicks could have effects on their growth and body condition, and ultimately on their survival. Here, we evaluated the effect of parasites on the cell-mediated immune system in Gentoo penguin (Pygoscelis papua) chicks at Stranger Point (25 de Mayo/King George Island, South Shetland Islands). To this end, 12 chicks were experimentally deparasitized with a mixture of anthelminthic drugs (albendazole and praziquantel), whereas 10 others were kept as control. We measured cutaneous cell-mediated immunity in response to immunization with phytohemagglutinin (PHA). We also analyzed the leukocyte profile in both treated and control groups before and after the treatment. After the treatment, deparasitized birds showed larger foot-web swelling in response to PHA injection than control birds. Deparasitized penguins also showed lower eosinophil and monocyte counts than controls, whereas heterophils, lymphocytes, and total white blood cell counts did not differ between groups. Our results suggest that Gentoo penguin chicks parasitized with intestinal parasites suffer a cost in terms of reduced cell-mediated immune responses that could ultimately affect their survival.     

IL-12 and NK cells are required for antigen-specific adaptive immunity against malaria initiated by CD8+ T cells in the Plasmodium yoelii model.

CD8+ T cells have been implicated as critical effector cells in protection against preerythrocytic stage malaria, including the potent protective immunity of mice and humans induced by immunization with radiation-attenuated Plasmodium spp. sporozoites. This immunity is directed against the Plasmodium spp. parasite developing within the host hepatocyte and for a number of years has been presumed to be mediated directly by CD8+ CTL or indirectly by IFN-gamma released from CD8+ T cells. In this paper, in BALB/c mice, we establish that after immunization with irradiated sporozoites or DNA vaccines parasite-specific CD8+ T cells trigger a novel mechanism of adaptive immunity that is dependent on T cell- and non-T cell-derived cytokines, in particular IFN-gamma and IL-12, and requires NK cells but not CD4+ T cells. The absolute requirement for CD8+ T cells to initiate such an effector mechanism, and the requirement for IL-12 and NK cells in such vaccine-induced protective immunity, are unique and underscore the complexity of the immune responses that protect against malaria and other intracellular pathogens.     

Fine structure of Plasmodium gallinaceum in embryonic and neonate chicks.

The erythrocytic stages of Plasmdoium gallinaceum in chicken embryos injected with parasited blood either from a syringe-passaged infection in chickens or from a chicken infected with sporozoites were characterized by abnormal structure. Particularly evident were large, unstained vacuoles within the cytoplasm; these occurred with greatest frequency in schizonts. The presence of myelin bodies within these vacuoles was revealed by transmission electron microscopy; abnormal cytokinesis and aberrant merozoites provided additional evidence of the parasite's inability to develop naturally within the milieu of the embryonic erythrocytes. Fifty-five passages were necessary to restore normal structure of the parasites in embryos, while only 5 passages were required for such restoration in neonate chicks. The probable adaptation of the parasite to the proportions of hemoglobin of the adult chicken may be responsible for the abnormal growth in the immature host.     

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.     

In vivo imaging of malaria parasites--recent advances and future directions

A new view into the life of malaria parasites is now possible owing to recent advances in imaging techniques and to the generation of tagged parasites. Insights into how parasites interact with their insect vectors and mammalian hosts have been gained by the study of various parasitic forms in their natural environment. Quantitative analysis of Plasmodium ookinete motility has revealed different modes of motility in parasite invasion of the mosquito gut and the extrusion of invaded gut cells from the epithelium. Similar analysis with Plasmodium sporozoites has revealed the importance of parasite motility in transmission from the mosquito vector to the mammalian host.