Effect of Conditioned Media from Several Cell Types on Invasion by Eimeria adenoeides Sporozoites1

ABSTRACT. The effect of conditioned media (media aspirated from a variety of cell cultures after 4 d of growth) on cellular invasion by sporozoites of the turkey coccidium, Eimeria adenoeides, was examined. Conditioned medium from turkey kidney cells and baby hamster kidney cells failed to alter invasion. However, conditioned medium from turkey cecal cell cultures produced a significant (P ≤ 0.05), two-fold increase in invasion over control medium in a variety of cell types. Retentates of conditioned medium from the turkey cecal cells that were passed through microconcentrators having molecular mass cutoffs of 50, 100, and 300 kDa similarly enhanced invasion over retentates from control medium. However, retentates from microconcentrators with a cutoff of 1,000 kDa failed to enhance invasion. Pretreatment in conditioned medium, followed by washing of sporozoites prior to inoculation into cultures, did not result in enhanced invasion. Moreover, when the interval between inoculation of sporozoites into cells and fixation of cultures was reduced to less than 3 h, no enhancement of invasion occurred. Conditioned medium from turkey cecal cells that were grown in the presence of ³⁵S-translabel had at least two labeled bands at 150 kDa and > 200 kDa that were absent in conditioned media from turkey kidney and baby hamster kidney cells.     

Immunoparasitology in Avian Species

There has been no work on the immunological response of birdsto helminth infections since the late 1960s, an area of investigationthat has been too long ignored. Similarly, studies of arthropod-mediatedresponses in birds are lacking except for a few scattered investigations.Recently, a serum antibody response has been seen against onearthropod, the northern fowl mite. The appearance of antibodiesrecognizing an 8–10 kilodalton mite antigen seems to correlatewith a reduction in the mite population on infested chickens.Most of the studies on parasite immunity in avian species havecentered on the economically important Eimeria species, protozoanparasites that infect the intestine of chickens and turkeys.These investigations encompass wide areas of interest includingthe effect of immunity on parasite invasion, development ofT-cell proliferation assays and T-cell clones, inhibition ofparasite penetration and development by hybridoma antibody treatment,production of genetically engineered Eimeria antigens used inbird immunization studies, and studies using inbred or congeniclines of birds to determine what effect the major histocompatibilitycomplex has on parasite immunity. From these efforts it hasbeen learned that not only is the immunity species-specific,but also depending on where in the intestine the parasite invades,penetration is either not affected or inhibited by as much as50%. The T-cell proliferation assays suggest that this specificitymay be due to a species-specific T-cell response. Immunizationstudies using a genetically engineered antigen have indicatedthat at least partial protection against one species of Eimeriais possible. Studies done with the inbred congenic lines ofbirds have shown that the genetic makeup of the bird is importantin how it responds to either a natural infection or to immunizationwith a genetically engineered antigen. Clearly, these resultsshow not only the complexity of the bird response to parasiteinfection, but also the amount of work still undone.     

Eimeria tenella: Vitamin Requirements for Development in Primary Cultures of Chicken Kidney Cells

SYNOPSIS. Development of Eimeria tenella was studied in primary cultures of chicken kidney cells maintained in Medium 199 lacking each of the following: vitamin A. biotin, p -aminobenzoic acid, folic acid, nicotinamide, Ca pantothenate, pyridoxine, pyridoxal, riboflavin, thiamin, ascorbic acid, calciferol, α-tocopherol, and menadione. Data obtained concerning numbers of mature schizonts or total numbers of parasites or both indicated that all of the vitamins are needed for 1st- and 2nd-generation schizogony, and all except calciferol and folic acid are needed for gametogony.     

Eimeria dispersa and Eimeria gallopavonis: Infectivity, Survival, and Development in Primary Chicken and Turkey Kidney Cell Cultures

SYNOPSIS. Eimeria dispersa (turkey strain) and Eimeria gallopavonis sporozoites were inoculated into primary cultures of chicken kidney (CK) and turkey kidney (TK) cells. Eimeria dispersa sporozoites were more infective in either cell type than those of E. gallopavonis : at 4 hr, the percentage of infection was 67-98 for E. dispersa but only 23-56 for E. gallopavonis . E. dispersa also survived better in culture: at 2 days, losses of E. dispersa in both cell types were only 4-19%, whereas losses of E. gallopavonis were 35-47% in TK cells and 60–95% in CK cells. However, E. gallopavonis developed further than E. dispersa . Location and increase in numbers of intracellular stages at 4 days indicated that E. dispersa proceeded through 2 schizogonic generations before development stopped.     

Comparative Development of Eimeria tenella from Sporozoites to Oocysts in Primary Kidney Cell Cultures from Gallinaceous Birds

Eimeria tenella completed its endogenous life cycle in primary cultures of kidney cells from 2- to 3-week-old-chickens, guinea fowl, partridges, pheasants, quail, and turkeys. Similarity in percentage of infection at 4 hr suggested that sporozoites entered cells from all birds in equal numbers. Development was better, however, in chicken cells in that the percentage of survival and of developmental stages during the first 2 days were greater, developmental stages occurring after 2 days usually were found earlier, mature 2nd-generation schizonts and oocysts were larger, and oocyst production was far greater than in nonhost cells. Multinucleate macrogametes, which sometimes reached sizes 3–4 times greater than normal oocysts, are reported for the first time.     

Effects of Cationized Ferritin and Neuraminidase on Invasion of Cultured Cells by Eimeria meleagrimitis Sporozoites

Primary turkey kidney cells and Eimeria meleagrimitis sporozoites were treated with cationized ferritin (CF) or neuraminidase (NANase), and the effects on the invasion of the cells by the sporozoites were measured. Cultures of host cells pretreated with either compound contained significantly fewer intracellular sporozoites than did control cultures. There was little additive effect if cultures were first treated with NANase and then with CF. In contrast, pretreatment of sporozoites with CF or low concentrations of NANase had no effect on invasion. The inhibition of invasion was apparently due to an interaction between treatment substances and host cell surface rather than to direct effect on the sporozoites. The CF bound to the randomly distributed anionic sites on the surfaces of both host cells and sporozoites and then rapidly aggregated. Sporozoites, probably in the process of invading cells, were invariably found with the conoid in close association with aggregates of CF on the host cell membrane. The CF on the sporozoites was apparently shed before or during invasion because all intracellular sporozoites were completely devoid of the label.     

Development of Eimeria meleagrimitis Tyzzer from Sporozoites and Merozoites in Turkey Kidney Cell Cultures*

SYNOPSIS. Sporozoites and 1st-, 2nd-, and 3rd-generation merozoites of Eimeria meleagrimitis were inoculated into primary cultures of turkey kidney cells. In vitro-excysted sporozoites developed into mature macrogamonts in 8 days; in vivo-excysted sporozoites developed into 2nd- or 3rd-generation schizonts within 5 to 7 days. First-generation merozoites obtained from infected turkeys produced mature 2nd-generation schizonts within 24 h. Second-generation merozoites from turkeys produced mature macrogamonts and oocysts within 72 h, whereas 3rd-generation merozoites produced these stages within 48 h. The oocysts that developed from 3rd-generation merozoites sporulated at 25 C and were infective for turkeys. The timing of the early stages and the intervals between schizogonic generations in cultures were comparable with those in turkeys. Morphologic parameters, however, indicated that some differences existed between in vitro and in vivo development. Second- and 3rd-generation schizonts and gamonts that developed after inoculation of cultures with merozoites were similar to stages in turkeys. Oocysts, however, were significantly smaller (P < 0.05) in cultures. All stages that developed after inoculation of cultures with sporozoites were smaller (P < 0.05) than their in vivo counter parts.     

CULTURE AND VOLUNTARY INFORMED CONSENT IN AFRICAN HEALTH CARE SYSTEMS

This paper discusses how to apply a collective decision model of the principle of voluntary informed consent in African communitarian culture, in a culturally sensitive way, in order to protect research candidates from potential exploitations and abuses. Dismissing cultural and ethical skepticism surrounding the global application of the principle of voluntary informed consent, the paper ultimately concludes that international collaboration on diagnostic and therapeutic medical research in Africa, especially HIV vaccine trials, is a moral imperative.     

Histomonas meleagridis after One Thousand in vitro Passages

SYNOPSIS. During a 7-year period Histomonas meleagridis survived 1000 passages in Medium 199 fortified with serum and antibiotic-killed bacteria. The histomonads were originally isolated from a chicken's cecal dropping, and the bacteria were cultivated from the cecal contents of a normal turkey. In many respects, the histomonads remained unchanged during cultivation, but in some other respects they changed considerably, perhaps irreversibly.
Morphologically, the histomonads propagated in vitro showed only slight changes. When returned to birds, they resumed the structure characteristic of lumen-dwelling individuals of this species. However, they had long since lost their ability to produce disease in either chickens or turkeys, and organisms of the tissue-dwelling type were rarely seen. The long-cultivated histomonads are actually as nonpathogenic as Histomonas wenrichi , but they have none of the distinguishing morphologic characteristics of the latter.
As has been reported elsewhere, H. meleagridis long maintained in the tissue culture medium has also lost much of its ability to immunize either chickens or turkeys against infection with virulent strains of this parasite. Also lost in the process of adaptation to its restricted medium has been the ability to multiply satisfactorily in vitro with the complement of bacteria normal to the ceca of the birds. However, in some instances, histomonads which have become adapted to in vitro cultivation are still able to live in birds with this diversified flora.
Activity of the histomonads cultivated in vitro differed but little from that of H. meleagridis freshly isolated from birds, when both were viewed under the same conditions. Histomonads from each of the above sources multiplied most satisfactorily in their accustomed habitat, probably because of a difference in nutritional requirements.