Clay G. Huff

Changes in Infectiousness of Malarial Gametocytes. II. Analysis of the possible Causative Factors.

The former paper (Huff and Marchbank, 1955) clearly indicated a general pattern among seven host-parasite combinations involving avian malarial parasites; this pattern being a rapid and continuing fall in the infectivity of gametocytes for mosquitoes during the course of infection in the bird. An analysis has been made in the present paper of the factors which might contribute to this change in infectivity of gametocytes. The aim has been the testing of two hypotheses to account for the decrease in this infectivity, namely (1) that it was due to a deficiency in the host which developed as the infection progressed, or (2) that an immune mechanism was responsible for the decrease. The following substances were administered to birds during the course of their infections: uninfected whole blood, coenzyme A, ferrous sulfate, sodium glutathione, calcium pantothenate, and sucrose. These substances did not enhance the infectivity of the gametocytes. In two experiments the hosts were bled daily during the course of their infections. No appreciable differences in oocysts numbers or infectivity values (oocyst-gametocyte ratios) were observed between the bled birds and the controls. Two experiments were performed testing whether mixed infections would yield evidence of a greater depleting effect. The results were not clearly conclusive but were suggestive that at least the principal cause of decrease in infectivity of the gametocytes was not a depleting effect. In one experiment in which blood infected with P. gallinaceum was transfused to a naturally immune host (duck) there was an appreciable increase in infectivity values of the mosquitoes. Two attempts to demonstrate effects on the gametocytes by the passive transfer of serum from hyperimmunized birds gave negative results. Two transfusion experiments were carried out in which infected blood was transmitted to birds with acquired immunity. No detectable change was observed in infectivity of gametocytes in one of these, while there were indications of a rapid fall in their infectivity in the second. Four experiments (three with P. gallinaceum in chickens and one with P. fallax in turkeys) testing the effect of immunization with killed parasites prior to the infecting inoculation uniformly indicated an early, more rapid fall in infectivity of gametocytes in the immunized than in the control animals. The results of all experiments can better be explained on the hypothesis that active immunity is the cause of decreasing infectivity of gametocytes than one in which this effect is assumed to result from a depletion effect of infection on the host. As in the former paper the lowering in mean numbers of merozoites produced by segmenters observed by other investigators during the crisis was not observed. However, significant differences were observed between the mean numbers of merozoites of infections in birds on adequate diets and those deficient in pantothenic acid.

Procedures for maximum production of exoerythrocytic stages of Plasmodium fallax in tissue culture.

Abstract For the first time a method is described for the cultivation of sufficient numbers of exoerythrocytic stages of a malarial parasite to facilitate the study of fine structure, the action of chemotherapeutic agents, the completion of timelapse cinemicrographic studies, and the more precise determination of growth rates and longevity. The method will be useful also for physiological studies, chemical analyses, and immunological studies on the extracellular, exoerythrocytic parasites. The use and modifications of commercially available media are described as well as alterations of procedures and techniques which meet the need for the production of exoerythrocytic stages in quantities not attained by other methods.

Changes in infectiousness of malarial gametocytes. I. Patterns of oocyst production in seven host-parasite combinations

Abstract The infectiousness of gametocytes for mosquitoes during the course of infection in the vertebrate was determined in seven combinations of the following parasites, avian hosts, and mosquitoes: Plasmodium gallinaceum, P. fallax, and P. cathemerium; chicks, pigeons, guinea fowl, turkeys, and canaries; Aedes aegypti, A. albopictus, Culex pipiens, and C. tarsalis. In 22 infections the peak of oocyst production consistently preceded the peak of parasitemia by 1 to 4 days (average 2 days) with the result that there was a precipitate fall in oocyst numbers during a period when the total numbers of parasites—as well as gametocytes—was still increasing. The patterns in change of oocyst numbers during the course of infection was similar in infections with the three species of parasites, except that in P. cathemerium infections in canaries there was a recovery in the ability to produce oocysts three days after the beginning of the decline. In the only instance where the experiment was continued long enough to determine the subsequent happenings, a second peak of oocyst production occurred. In the one experiment where comparisons could be made between the results in two different species of mosquitoes, simultaneous feedings of both C. pipiens and C. tarsalis were made daily during the course of infection of P. cathemerium in a canary. The oocyst counts in C. tarsalis were at all times higher than in C. pipiens and there was a very close parallelism between the counts in the two species of mosquitoes. Infections of P. fallax were studied in pigeons, chicks, guinea fowl and turkeys. Patterns of change in oocyst numbers were comparatively alike in turkeys but very variable in pigeons. The chick was a poor host and the guinea fowl a very good host in respect to the relation between degree of parasitemia and number of oocysts produced. Fairly good infections resulted in mosquitoes fed on guinea fowl even though gametocytes were not seen in the blood smears on 5 of the 8 days they were fed. Oocyst size did not vary with fluctuations in degree of parasitemia or number of oocysts per mosquito. There was, however, some evidence that the size of oocysts was affected by differences in individual avian hosts. No changes in rate of reproduction of asexual stages paralleling the changes in infectiousness of gametocytes were reflected in the mean numbers of merozoites formed during the course of infection.

Further Studies on Host-Cell Preferences by Exoerythrocytic Stages of Avian Malaria.

Abstract Two experiments in which chickens were given first infections different from the second infection tested whether the malarial infection itself has some nonspecific depleting action which alters the natural occurrence and distribution of the exoerythrocytic stages. P. fallax and P. lophurae were used respectively as first infections and P. gallinaceum was used as the second infection in each case. There was no evidence of any effect of the first infections on the course of the exoerythrocytic infection of P. gallinaceum in chickens. Two attempts to alter the known course of exoerythrocytic infection of P. lophurae in turkeys by the administration of hyperimmune chicken serum failed to produce any alteration of exoerythrocytic stages from that which occurs in birds not previously immunized. In one of them the serum was given daily between the day following the infecting inoculation and the day the bird was sacrificed. In the other one successive daily injections of the immune serum were given prior to the inoculation of infective parasites. There was essentially no effect of this passive transfer upon the distribution of exoerythrocytic stages either temporally or anatomically. Sporozoites of P. gallinaceum were incubated for one hour in hyperimmune serum and then inoculated into chickens. There was no indication that this treatment altered the pattern of exoerythrocytic development of the parasites resulting from these sporozoites. Turkeys were given seven successive daily inoculations of killed erythrocytic stages of P. fallax previous to the inoculation of live parasites. Examination of the principal tissues of the birds killed, one each day, from the 3rd to 10th post-inoculation day revealed no evidence that the active immunization by killed parasites had altered the pattern of development. Inoculation of exoerythrocytic stages of P. fallax from primary tissue cultures of chick embryo liver was followed by infections more like those produced by sporozoite inoculations than those following inoculations of erythrocytic stages. These experiments are not conclusive in determining what factors are responsible for the changes which occur in the distribution of the parasites among the various types of host cells during the course of primary infection in the host. Changes in host-cell selectivity by the parasite tend to persist until some other factor intervenes whether the change is from preference for erythrocytes to endothelial cells or vice versa. These changes, whatever their cause, resemble transformations or “Dauermodifikationen”.

The Growth of Exoerythrocytic Stages of Avian Malaria within Diffusion Chambers in Different Hosts.

Abstract Exoerythrocytic stages of Plasmodium gallinaceum and P. fallax from infected chick embryos were placed in chambers made of lucite rings and porous membranes which were inserted intraperitoneally into birds and mice, respectively. The chambers containing P. gallinaceum were made of membranes with pores of 0.45 ± 0.02 μ. Those placed into chickens were allowed to remain from 1 to 14 days then removed. Infections resulted in 11 of 66 chickens which served as recipients of the chambers. Similar chambers were also placed in turkeys and ducks. In the former nine of 11 turkeys either became infected or were proved to have latent infections by subinoculations into chicks, whereas no infections resulted in seven ducks in which the chambers were allowed to remain from 2–14 days. The ability of the parasites (probably the merozoites) to penetrate membranes with pore size of 0.45 ± 0.02 μ is clearly proved by these experiments. Greater foreign-body response around the chambers was observed on the part of chickens than of turkeys. Twenty chambers of the preceding type were placed in chick embryos. In two of these—removed on the 2nd and 5th days—parasites were observed microscopically, the latter one being more heavily infected than any tissue yet observed. The chambers containing chick embryo cells infected with P. fallax were placed into mice and allowed to remain for 3 days to 3 weeks. Membranes of three different pore-sizes were used — HA similar to those used in the experiments with P. gallinaceum , PH (pore-size: 0.3 ± 0.02 μ) and VM (pore-size: 50 ± 3 mμ). At 3-, 7-, 14-, and 21-day intervals the chambers were removed and the contents were subinoculated into turkey poults. The parasites in six of 20 chambers were shown to be viable by these subinoculations—three after 7 days, two after 14 days, and one after 21 days. No infections were observed in the mice. No evidence of survival of the parasites of P. fallax was obtained in a parallel series in which exoerythrocytic stages of this parasite were inoculated directly into mice. These experiments give evidence that the use of porous chambers has promising possibilities for the study of many problems in parasitology.

Studies on the Exoerythrocytic Stages of Plasmodium gallinaceum during the " Transitional Phase ".

Abstract Studies made during the transitional stage infection of P. gallinaceum in chicks (i.e., the period of transition from tissue parasitism to parasitemia) have yielded the following results: 1. 1. The relative frequency of exoerythrocytic stages expressed as number of parasites per square millimeter of section is fairly consistent for the two birds examined except for the kidney and spleen. The brain ranked low in relation to kidney, lung, heart, and liver as the organ of localization. 2. 2. At the time when the preponderance of parasites in the fixed tissue consisted of large schizonts and segmenters the preponderance of eryth-rocytic parasites consisted of young trophozoites. The larger schizonts and segmenters were fairly equally distributed among the different organs whereas in one of the birds 32% of the exoerythrocytic stages of the lung were small trophozoites. It would appear that not only were large numbers of exoerythrocytic merozoites changing to erythrocytic stages but that relatively few of them were continuing as exoerythrocytic stages. 3. 3. Degenerative changes in the exoerythrocytic stages, possibly due to the development of antibodies by the host, were seen as early as 53 hours after the first observed parasitemia. The type of degenerative change in the parasite was similar to that observed previously as the result of the action of natural immunity and of antimalarial drugs. 4. 4. So-called macromerozoites and micromerozoites were found to be only the extremes in size of a population of merozoites with normal variation in size. Thus, no dimorphism exists in the schizonts of this species. 5. 5. It is believed that, although no dimorphism of the schizonts exists, the average size of the merozoite decreases during the transitions from a condition in which the host cells are predominantly of the lymphoid-macrophage system to the condition in which they are predominantly endothelial in type, and then to the stage where the erythrocytes are the cells which are predominantly inhabited. 6. 6. The prevalent view that the smaller merozoites are destined to produce erythrocytic stages while the larger merozoites are destined to continue the exoerythrocytic cycle has not been adequately proved.

Merozoite Size in Exoerythrocytic Infections of Plasmodium gallinaceum, P. fallax, P. lophurae, and P. cathemerium.

Abstract The diameter of merozoites was chosen as the quantitative measure for comparison of exoerythrocytic stages of avian malaria, since it was possible to obtain this measurement with the greatest accuracy. Statistical studies, using this measure, on the exoerythrocytic stages of P. gallinaceum , P. lophurae, P. fallax , and P. cathemerium revealed the following information: 1. 1. The mean diameter of P. gallinaceum was significantly smaller than the diameters of P. fallax and P. lophurae ; the diameters of the latter two species were not significantly different. The morphology of the merozoites of P. cathemerium is such as to prevent the use of the diameter as a measure of comparison of merozoite size with the other three species. 2. 2. Significant differences were found between the merozoites in certain individual hosts of the same species and between the merozoites of P. gallinaceum in the duck and the quail. These differences between the parasites in different individuals of the same species and in different species of host were of the same order of magnitude. 3. 3. Significant differences among merozoites from different organs of the host were found only between those of P. gallinaceum in the brain of the chick as compared with those in spleen, lung, and kidney. A corresponding difference was not found between the merozoites of P. fallax in the brain and lung of the turkey. 4. 4. The merozoites of P. gallinaceum in blood-induced infections were significantly smaller in early (4-day) as compared with late (10, 16, 17 days) infections. This smaller size of the merozoites in early, blood-induced infections is interpreted as a persistence of a characteristic of the parasites following a conversion of its host cell preferences from erythrocytes to cells other than erythrocytes. The differences between 5-day and 10-day merozoites from sporozoite-induced infections were not significant. 5. 5. Although statistically significant differences were found between exoerythrocytic merozoites (of P. gallinaceum and P. fallax) in blood-induced as compared with those from sporozoite-induced infections, it is believed that the bias in the sampling of the merozoites either invalidates these differences completely or reduces them in degree. If differences do exist between the size of exoerythrocytic merozoites in blood-induced and sporozoite-induced infections these differences must be very small. 6. 6. The absence of any evidence of bimodality in the distributions of all sets of the data collected indicates that the absence of two categories of exoerythrocytic merozoites is probably a general phenomenon in avian malaria.