Avian Malaria Species Research Articles

Gene regulation of the avian malaria parasite Plasmodium relictum, during the different stages within the mosquito vector

The malaria parasite Plasmodium relictum is one of the most widespread species of avian malaria. As in the case of its human counterparts, bird Plasmodium undergoes a complex life cycle infecting two hosts: the arthropod vector and the vertebrate host. In this study, we examined transcriptomes of P. relictum (SGS1) during crucial timepoints within its vector, Culex pipiens quinquefasciatus. Differential gene-expression analyses identified genes linked to the parasites life-stages at: i) a few minutes after the blood meal is ingested, ii) during peak oocyst production phase, iii) during peak sporozoite phase and iv) during the late-stages of the infection. A large amount of genes coding for functions linked to host-immune invasion and multifunctional genes was active throughout the infection cycle. One gene associated with a conserved Plasmodium membrane protein with unknown function was upregulated throughout the parasite development in the vector, suggesting an important role in the successful completion of the sporogonic cycle. Gene expression analysis further identified genes, with unknown functions to be significantly differentially expressed during the infection in the vector as well as upregulation of reticulocyte-binding proteins, which raises the possibility of the multifunctionality of these RBPs. We establish the existence of highly stage-specific pathways being overexpressed during the infection. This first study of gene-expression of a non-human Plasmodium species in its vector provides a comprehensive insight into the molecular mechanisms of the common avian malaria parasite P. relictum and provides essential information on the evolutionary diversity in gene regulation of the Plasmodium's vector stages.

Preliminary results on species composition of the biting midges-fauna (Culicoides) in a wetland on Lower Danube Flow, Bulgaria

Biting midges of the genus Culicoides Latreille, 1809 (Diptera: Ceratopogonidae) are competent vectors of various pathogens in Europe with veterinary importance such as: Bluetongue virus, Schmallenberg virus, Epizootic hemorrhagic disease virus and avian haemosporidians. In order to reveal the potential role that they may play in spreading diseases in wildlife and thus the impact on ecosystems which they render, a reliable estimation of their faunistic diversity is required. Previous surveys conducted in the region of Kalimok Biological Station – a wetland on Lower Danube flow – were focused mainly on the ornithophilic species acting as vectors of avian haemosporidians, and thus leaving out the whole Culicoides diversity in the area underestimated (Bobeva et al. 2013, Bobeva et al. 2014, Bobeva et al. 2015). In the present study, we combined morphological and molecular approaches for species identification of the biting midges collected in the region of the field station in 2011, 2012, 2013 and 2018 years. Out of the 37 Culicoides species reported previously as national fauna (Nedelchev 2013, Pudar et al. 2018), we recorded 13 species in the studied area. In addition, three species namely, Culicoides duddingstoni, C. griseidorsum and C. кibunensis were newly recorded for the Bulgarian fauna, which raises the number of species of national fauna to 40. Interesting specimens affiliated to C. pseudopallidus have been collected in Kalimok. This latter species has not been reported in Central and Eastern Europe so far and therefore further attention is required prior being considered as a new record. Regarding the potential vectors of avian haemosporidians in the studied area, the present investigation raised the list of species suspected for spreading this disease in wildlife from three to six, namely: C. alazanicus, C. circumscriptus, C. duddingstoni, C. griseidorsum, C. kibunensis and C. pictipennis. The current study of the biting midges’ biodiversity improved the knowledge about the Bulgarian Culicoides fauna including the list of the potential vector species of avian malaria. However, faunistic studies on Culicoides in Europe remain sparse and such data serves as a basic knowledge for a better understanding of the Culicoides-borne diseases.

Complete avian malaria parasite genomes reveal features associated with lineage-specific evolution in birds and mammals.

Avian malaria parasites are prevalent around the world and infect a wide diversity of bird species. Here, we report the sequencing and analysis of high-quality draft genome sequences for two avian malaria species, Plasmodium relictum and Plasmodium gallinaceum. We identify 50 genes that are specific to avian malaria, located in an otherwise conserved core of the genome that shares gene synteny with all other sequenced malaria genomes. Phylogenetic analysis suggests that the avian malaria species form an outgroup to the mammalian Plasmodium species, and using amino acid divergence between species, we estimate the avian- and mammalian-infective lineages diverged in the order of 10 million years ago. Consistent with their phylogenetic position, we identify orthologs of genes that had previously appeared to be restricted to the clades of parasites containing Plasmodium falciparum and Plasmodium vivax, the species with the greatest impact on human health. From these orthologs, we explore differential diversifying selection across the genus and show that the avian lineage is remarkable in the extent to which invasion-related genes are evolving. The subtelomeres of the P. relictum and P. gallinaceum genomes contain several novel gene families, including an expanded surf multigene family. We also identify an expansion of reticulocyte binding protein homologs in P. relictum, and within these proteins, we detect distinct regions that are specific to nonhuman primate, humans, rodent, and avian hosts. For the first time in the Plasmodium lineage, we find evidence of transposable elements, including several hundred fragments of LTR-retrotransposons in both species and an apparently complete LTR-retrotransposon in the genome of P. gallinaceum.

Co-infections and environmental conditions drive the distributions of blood parasites in wild birds.

Experimental work increasingly suggests that non-random pathogen associations can affect the spread or severity of disease. Yet due to difficulties distinguishing and interpreting co-infections, evidence for the presence and directionality of pathogen co-occurrences in wildlife is rudimentary. We provide empirical evidence for pathogen co-occurrences by analysing infection matrices for avian malaria (Haemoproteus and Plasmodium spp.) and parasitic filarial nematodes (microfilariae) in wild birds (New Caledonian Zosterops spp.). Using visual and genus-specific molecular parasite screening, we identified high levels of co-infections that would have been missed using PCR alone. Avian malaria lineages were assigned to species level using morphological descriptions. We estimated parasite co-occurrence probabilities, while accounting for environmental predictors, in a hierarchical multivariate logistic regression. Co-infections occurred in 36% of infected birds. We identified both positively and negatively correlated parasite co-occurrence probabilities when accounting for host, habitat and island effects. Two of three pairwise avian malaria co-occurrences were strongly negative, despite each malaria parasite occurring across all islands and habitats. Birds with microfilariae had elevated heterophil to lymphocyte ratios and were all co-infected with avian malaria, consistent with evidence that host immune modulation by parasitic nematodes facilitates malaria co-infections. Importantly, co-occurrence patterns with microfilariae varied in direction among avian malaria species; two malaria parasites correlated positively but a third correlated negatively with microfilariae. We show that wildlife co-infections are frequent, possibly affecting infection rates through competition or facilitation. We argue that combining multiple diagnostic screening methods with multivariate logistic regression offers a platform to disentangle impacts of environmental factors and parasite co-occurrences on wildlife disease.

Avian Plasmodium infection in field-collected mosquitoes during 2012-2013 in Tarlac, Philippines.

Global warming threatens to increase the spread and prevalence of mosquito-transmitted diseases. Certain pathogens may be carried by migratory birds and transmitted to local mosquito populations. Mosquitoes were collected in the northern Philippines during bird migration seasons to detect avian malaria parasites as well as for the identification of potential vector species and the estimation of infections among local mosquito populations. We used the nested PCR to detect the avian malaria species. Culex vishnui (47.6%) was the most abundant species collected and Cx. tritaeniorhynchus (13.8%) was the second most abundant. Avian Plasmodium parasites were found in eight mosquito species, for which the infection rates were between 0.5% and 6.2%. The six Plasmodium genetic lineages found in this study included P. juxtanucleare -GALLUS02, Tacy7 (Donana04), CXBIT01, Plasmodium species LIN2 New Zealand, and two unclassified lineages. The potential mosquito vectors for avian Plasmodium parasites in the Philippines were Cq. crassipes, Cx. fuscocephala, Cx. quinquefasciatus, Cx. sitiens, Cx. vishnui, and Ma. Uniformis; two major genetic lineages, P. juxtanucleare and Tacy7, were identified.

Identification and characterization of the merozoite surface protein 1 (msp1) gene in a host-generalist avian malaria parasite, Plasmodium relictum (lineages SGS1 and GRW4) with the use of blood transcriptome

BackgroundThe merozoite surface protein 1 (msp1) is one of the most studied vaccine candidate genes in mammalian Plasmodium spp. to have been used for investigations of epidemiology, population structures, and immunity to infections. However methodological difficulties have impeded the use of nuclear markers such as msp1 in Plasmodium parasites causing avian malaria. Data from an infection transcriptome of the host generalist avian malaria parasite Plasmodium relictum was used to identify and characterize the msp1 gene from two different isolates (mtDNA lineages SGS1 and GRW4). The aim was to investigate whether the msp1 gene in avian malaria species shares the properties of the msp1 gene in Plasmodium falciparum in terms of block variability, conserved anchor points and repeat motifs, and further to investigate the degree to which the gene might be informative in avian malaria parasites for population and epidemiological studies.MethodsReads from 454 sequencing of birds infected with avian malaria was used to develop Sanger sequencing protocols for the msp1 gene of P. relictum. Genetic variability between variable and conserved blocks of the gene was compared within and between avian malaria parasite species, including P. falciparum. Genetic variability of the msp1 gene in P. relictum was compared with six other nuclear genes and the mtDNA gene cytochrome b.ResultsThe msp1 gene of P. relictum shares the same general pattern of variable and conserved blocks as found in P. falciparum, although the variable blocks exhibited less variability than P. falciparum. The variation across the gene blocks in P. falciparum spanned from being as conserved as within species variation in P. relictum to being as variable as between the two avian malaria species (P. relictum and Plasmodium gallinaceum) in the variable blocks. In P. relictum the highly conserved p19 region of the peptide was identified, which included two epidermal growth factor-like domains and a fully conserved GPI anchor point.ConclusionThis study provides protocols for evaluation of the msp1 gene in the avian malaria generalist parasite P. relictum. The msp1 gene in avian Plasmodium shares the genetic properties seen in P. falciparum, indicating evolutionary conserved functions for the gene. The data on the variable blocks of the gene show that the msp1 gene in P. relictum might serve as a good candidate gene for future population and epidemiological studies of the parasite.

FINE-SCALE GENETIC STRUCTURE IN A WILD BIRD POPULATION: THE ROLE OF LIMITED DISPERSAL AND ENVIRONMENTALLY BASED SELECTION AS CAUSAL FACTORS

Individuals are typically not randomly distributed in space; consequently ecological and evolutionary theory depends heavily on understanding the spatial structure of populations. The central challenge of landscape genetics is therefore to link spatial heterogeneity of environments to population genetic structure. Here, we employ multivariate spatial analyses to identify environmentally induced genetic structures in a single breeding population of 1174 great tits Parus major genotyped at 4701 single-nucleotide polymorphism (SNP) loci. Despite the small spatial scale of the study relative to natal dispersal, we found multiple axes of genetic structure. We built distance-based Moran's eigenvector maps to identify axes of pure spatial variation, which we used for spatial correction of regressions between SNPs and various external traits known to be related to fitness components (avian malaria infection risk, local density of conspecifics, oak tree density, and altitude). We found clear evidence of fine-scale genetic structure, with 21, seven, and nine significant SNPs, respectively, associated with infection risk by two species of avian malaria (Plasmodium circumflexum and P.relictum) and local conspecific density. Such fine-scale genetic structure relative to dispersal capabilities suggests ecological and evolutionary mechanisms maintain within-population genetic diversity in this population with the potential to drive microevolutionary change.

Urbanization, Trace Metal Pollution, and Malaria Prevalence in the House Sparrow

Anthropogenic pollution poses a threat for the environment and wildlife. Trace metals (TMs) are known to have negative effects on haematological status, oxidative balance, and reproductive success in birds. These pollutants particularly increase in concentration in industrialized, urbanized and intensive agricultural areas. Pollutants can also interfere with the normal functioning of the immune system and, as such, alter the dynamics of host-parasite interactions. Nevertheless, the impact of pollution on infectious diseases has been largely neglected in natural populations of vertebrates. Here, we used a large spatial scale monitoring of 16 house sparrow (Passer domesticus) populations to identify environmental variables likely to explain variation in TMs (lead, cadmium, zinc) concentrations in the feathers. In five of these populations, we also studied the potential link between TMs, prevalence of infection with one species of avian malaria, Plasmodium relictum, and body condition. Our results show that lead concentration is associated with heavily urbanized habitats and that areas with large woodland coverage have higher cadmium and zinc feather concentrations. Our results suggest that lead concentration in the feathers positively correlates with P. relictum prevalence, and that a complex relationship links TM concentrations, infection status, and body condition. This is one of the first studies showing that environmental pollutants are associated with prevalence of an infectious disease in wildlife. The mechanisms underlying this effect are still unknown even though it is tempting to suggest that lead could interfere with the normal functioning of the immune system, as shown in other species. We suggest that more effort should be devoted to elucidate the link between pollution and the dynamics of infectious diseases.

Spatial determinants of infection risk in a multi‐species avian malaria system

Spatially‐variable processes can be an important element of host–parasite interactions, but their longer term demographic and evolutionary effects depend on the magnitude of variation in space, the scale at which variation occurs and the degree to which such processes are temporally stable. Here, we use multiple years of data from a study of two closely related tit species (Paridae), infected with two congeneric species of avian malaria (Plasmodium), to evaluate the roles of extrinsic and intrinsic factors in driving spatial heterogeneity in infection risk, and to address questions of scale and temporal stability in these vector‐driven host–parasite interactions. We show that the two malaria parasite species exhibit markedly different spatial epidemiology:P. relictuminfections are effectively randomly distributed in space, with no temporal consistency, whereasP. circumflexuminfections exhibit pronounced spatial structuring that is stable over the six years of this study and similar in both host species. We show that both conspecific and heterospecific host density contribute to elevated infection risk, but that the main determinants of elevated risk ofP. circumflexuminfection risk are habitat features probably associated with vector distribution and abundance. We discuss the implications of these findings, both for our understanding of the epidemiology of malaria in the wild, but also in terms of the longer‐term evolutionary and demographic consequences that spatially variable parasite‐mediated selection may have on host populations.

A Cautionary Note on the Use of Nested PCR for Parasite Screening—An Example From Avian Blood Parasites

The use of new powerful nested polymerase chain reaction (PCR) techniques to identify and screen for prevalence of parasites has a huge potential. It allows for the detection and identification of low-intensity infections, but its high sensitivity and technical setup may also induce problems. Here, we report a cautionary note regarding misleading amplification of avian malaria species (Haemoproteus and Plasmodium) during Leucocytozoon spp. detection. We used a previously described nested PCR method for the molecular detection of avian malaria and Leucocytozoon spp. In the first step of the PCR protocol, these parasites are detected simultaneously; in the second PCR, Haemoproteus and Plasmodium spp. are separated from Leucocytozoon spp. However, in certain cases when a bird was infected with avian malaria, we obtained a slightly longer PCR product during the detection of Leucocytozoon spp. Our data imply that these "false" Leucocytozoon fragments are the consequences of strong amplification of certain malaria lineages in the first PCR, which can also be detected after the second PCR amplification that is specific to Leucocytozoon spp. parasites. Because these "false" Leucocytozoon fragments are slightly longer than the normal Leucocytozoon fragments, we suggest the use of well-separating agarose gels, several positive controls, and molecular standards to facilitate their separation. If one obtains a fragment that differs in length from the one expected for Leucocytozoon spp., sequencing is essential. More generally, in order to limit this type of problem with nested PCR protocols, we suggest that the first and the second primer pair be chosen so that they have different annealing temperatures.

Not just for the birds

Many people are surprised when they discover that there is malaria in birds or that it is found in all the different climates throughout the world, yet Avian Malaria Parasites and Other Haemosporidia fills 932 pages on this topic. Not since Garnham's classic Malaria Parasites and Other Haemosporidia (1966) has such a complete work about the description of malaria species been written and illustrated. Avian malaria species provided much of the basis of the understanding and treatment of malaria, from Sir Ronald Ross' demonstration of parasite transmission to antimalarial discovery from testing compounds against the model Plasmodium gallinaceum.

The Blood Protozoa of Seventeen Species of Sparrows and Other Fringillidae

SUMMARY. A total of 1037 birds, belonging to 17 species of Fringillidae (order Passeres) and nearly all collected in or near Syracuse, N. Y., were examined for blood parasites. Of the species of avian malaria, Plasmodium cathemerium occurred most frequently, followed by P. hexamerium, P. relictum, P. elongatum, and P. circumflexum. Other species were not seen, although some (e.g. P. nucleophilum, P. polare, P. vaughani) are known to occur in Central New York.A number of song, chipping, and field sparrows were given experimental inoculations of Plasmodium vaughani‐infected blood and liberated. A few were recaught at varying intervals, but none were infected, suggesting that these species are insusceptible to this type of avian malaria.Malaria in song sparrows, which were caught in large numbers, is common and probably endemic, not varying greatly in frequency from year to year. But the incidence shows a sharp peak in the spring, most cases apparently being low‐grade relapses, which probably serve to reestablish the infection in the new crop of mosquitoes. There are indications that Haemoproteus and Leucocytozoon behave similarly.Infection with these last two parasites occurred in most of the species examined, but the incidence varied greatly, being especially high in white‐throated and white‐crowned sparrows. Reproductive stages of Haemoproteus were observed in the lungs of song and chipping sparrows.

Exoerythrocytic Stages of Plasmodium durae

In July, 1941, Herman reported the discovery of a new species of avian malaria, pathogenic to turkeys, and named it Plasmodium durae. In November, 1941, a duck infected with this species was secured and biological studies were started. It proved impossible to secure turkey poults at the time the infected duck was obtained, therefore two 13-week-old turkeys were bought from a local breeder and were inoculated intravenously with 3 cc and 5cc, respectively, of blood, obtained by heart puncture, from the infected duck. It was felt that the rather large doses were necessary because of the age of the recipient animals. Nothing is known about the previous parasite history of these two turkeys, but blood examination at the time of their inoculation with P. durae was negative for parasites. Both turkeys were inoculated on November 14, 1941, and, in each case, parasites were first seen in blood samples eleven days later. Both birds continued to show parasites until their deaths. Turkey No. 2 (Table 1) died after 26 days. This animal seemed to be partially paralyzed for several hours before its death, and although it was able to flap its wings, it could not raise its head or stand on its legs. An autopsy was performed and tissue smears made from the spleen, liver, lungs, and brain. These were stained with Giemsa. In smears from all of these organs, numerous bodies were found which were thought to be exoerythrocytic stages of Plasmodium dutrae. They were especially numerous in the lungs and brain, being present in the latter organ in such numbers as to occlude completely many of the capillaries. Some of these bodies were in a comparatively early stage of development, with only one or two nuclei, while many others were in later developmental stages, some apparently approaching segmentation. Other investigators in this laboratory who had had experience with exoerythrocytic stages, including Dr. Robert W. Hegner, Dr. Fruma Wolfson, and Miss Evaline West, were unanimous in the opinion that these bodies were unquestionably the exoerythrocytic stages of Plasmodium durae. Turkey No. 1 (Table 1) was killed 78 days after inoculation; tissue smears of spleen, liver, lungs and brain were made and carefully examined, but no typical exoerthrocytic stages could be found in any of these organs. Subsequently, as shown in the table, out of a total of 8 young (5-10 days old) turkeys which were inoculated intravenously with P. durae, two have been found to have exoerythrocytic stages in the spleen, liver, lungs and brain. Bird No. 4, which was 8 days old, was injected with 0.3 cc of inoculum from bird No. 1. The infected poult died 15 days later and tissue smears demonstrated the presence of exoerythrocytic stages in the usual sites. Bird No. 6, which was 10 days old, was injected with 0.8 cc of inoculum from a duck previously infected. This poult first showed parasites the next day, was patent for 10 days, remained latent for 5 days, then had a relapse and died 23 days after the initial infection. Tissue smears showed the presence of exoerythrocytic stages in the spleen, liver, lungs and brain.

Comparative Pathology of Infections of Plasmodium Rouxi in Canaries with Other Malarias of Birds, Monkeys, and Man 1

Summary and Conclusions 1. The object of this work was to follow the course of infections and pathological effects of a relatively non-virulent species of avian malaria caused by P. rouxi. These observations were used to make a comparison with other types of malaria in birds, monkey, and man. Twenty-four adult female canaries were used in this work. Over 1000 slides of tissue sections and blood smears form the basis of the parasitological and pathological studies. 2. The spleen responded rapidly to infections with P. rouxi. The resultant splenomegaly was due mainly to an increase of the mononuclear cells in the red pulp. The mononuclear cells were phagocytic, Deposition of pigment in the spleen was heavy and in a band-like pattern. 3. Very active phagocytosis occurred in the Kupffer cells of the liver. One case of vacuolation of the hepatic cells was seen during the peak of the infection but as no additional ones were seen it was concluded that this response was not a constant accompaniment of this infection. In general, the infection caused no disorientation nor necrosis of the liver parenchyma. 4. The bone-marrow became hyperplastic during the acute stage losing some but not all of the fat cells. 5. Both the bone-marrow and spleen showed a hyperplasia even in old chronic stages. The spleen remained enlarged and soft, and microscopically showed a continued hyperplasia of the red pulp. Much pigment was present in the spleen even in a six-months old infection. 6. The Kupffer cells of the liver remained large and actively phagocytic in the chronic patent cases. 7. A comparison of the pathology of infections caused by P. rouxi, P. cathemerium (birds), P. brasilianum (monkeys), and the plasmodia of man was made. In general, the human plasmodia had the most widespread and severe effects and P. rouxi the least. 8. P. rouxi exerted its greatest effects on the spleen, bone-marrow, and liver. There was less initial activation and hyperplasia of these organs than that found in P. cathemerium. 9. Activation of the spleen, bone-marrow, and liver persisted at a high rate and for a period of time (at least six months) much longer than that found in P. cathemerium (one to two months). 10. The immunological responses of infections with P. rouxi and P. cathemerium were reviewed. The type of host reaction elicited by P. rouxi (low) correlated with its immunity to superinfections (relatively inefficient) suggests that the severity of the primary attack of avian malaria is an important factor in determining the immune response; the more intense the attack, the greater the immunity conferred.

Immunity to Cross-Infection in Avian Malaria Due to Plasmodium Vaughani

For many years it was believed that infection with one species of avian malaria conferred protection against subsequent infection with that species only, and one of the tests by which a given species could be differentiated from another was to inoculate a subpatent or chronic infection of the former with the suspected new species. Recently it has been shown by Gingrich and others that this is not always the case, but this method is still an important test of specificity. The species used in the present work was originally described by Novy and MacNeal from the common robin in 1904, and the strain employed was isolated from a catbird caught in Syracuse in April, 1934. The other species with which P. vaughani was crossed were secured from sources previously mentioned by the author., Female canaries were used throughout. Before inoculating a bird with a latent infection the continued presence of an infection was checked either by microscopic examination or (since this is usually negative) by subinoculation into fresh birds. Inoculations were generally intravenous, and large doses of parasites were usually used. Ten series of 3 birds each (in addition to one or more controls in each series) were used, 5 being chronic cases of P. vaughani and the others consisting of one series each of P. cathemerium, praecox (relictum), circumflexum, elongatum, and rouxi. Each series of chronic vaughani infections was inoculated with one of the other species named, and the experiment was then reversed, using chronic infections of the latter. The results show that a subpatent (chronic) infection with P. vaughani confers little if any protection against subsequent infection with the 5 other species named, and the reverse of this relationship appears to hold true also, except that there is some indication that a pre-existing infection with P. praecox (relictum) gives a partial immunity to subsequent infection with P. vaughani.

Quinine and Plasmochin Therapy in Infections with Plasmodium Circumflexum

Summary and Conclusions The study here reported had for its object the investigation of the effects of quinine and plasmochin on infections with Plasmodium circumflexum, and comparison of the results with those already obtained by similar treatment of infections with the other four species of avian malaria (Plasmodium capistrani excepted). A total of fifty-five female canaries were used in the treated series. The results may be summarized as follows: 1. Basing the list which is given below partly on the data reported in the present paper and partly on data previously reported, the susceptibility of the five species of avian plasmodia here considered to treatment with quinine and plasmochin appears to be as follows, the most susceptible being given first: Quinine Plasmochin 1. Plasmodium rouxi 1. Plasmodium elongatum 2. Plasmodium praecox Plasmodium rouxi 3. Plasmodium cathemerium 2. Plasmodium praecox 4. Plasmodium circumfiexum 3. Plasmodium circumfiexum 5. Plasmodium elongatum 4. Plasmodium cathemerium It should be noted that in the first list (quinine) the differences between Praecox, cathemerium and circumflexum are very small, and that this is also true of praecox and circumflexum in the second list. Elongatum and rouxi are placed at the top of the second list, and rouxi heads the first list, chiefly because infections with this species could be readily sterilized with the drug in question. Cathemerium and circumflexum occupy the positions they do (even though plasmochin is effective in removing parasites from the peripheral blood in a relatively short time) largely for the contrary reason; in other cases the percentage of parasite-free days was the chief factor governing the place of the species in the scale. 2. As with the other species, Plasmodium circumflexum is more amenable to plasmochin than to quinine. When sterilization is not brought about relapse within a month generally occurs, but does so less frequently than in praecox infections, and may be deferred for two months or even longer. This is correlated with the fact that the parasite-level in chronic infections of circumflexum is generally established at a much lower level than in the case of praecox, and is to be compared in this respect only to cathemerium. 3. Plasmochin in combination with quinine is apparently no better than when used separately, and may even be less satisfactory—due possibly to quinine intoxication. 4. As with the other species, treatment during the incubation period is much more effective than when postponed until parasites appear. A considerable proportion of cases may be completely aborted in the former case, as demonstrated by continued freedom from relapse and by subinoculation, with subsequent reinfection, but so far no case in which parasites had already appeared has been sterilized. 5. There is little evidence that gametocytes are either more or less susceptible to plasmochin and quinine than the asexual stages. 6. Study of the rates of reproduction of the different species, and other peculiarities of the life-cycle, suggests that the degree of susceptibility to drug therapy may depend in large part on these factors—particularly in the case of plasmochin.