Modeling the population impacts of avian malaria on Hawaiian honeycreepers: Bifurcation analysis and implications for conservation.

Avian malaria is a mosquito-borne parasitic disease of birds caused by protists of the genera Plasmodium, most notably Plasmodium relictum. This disease has been identified as a primary cause of the drastic decline and extinctions of birds, in particular Hawaiian honeycreepers (Drepanidinae), where rates of mortality may exceed 90%. We formulate an epizootiological model of the transmission dynamics of avian malaria between populations of bird hosts and mosquito vectors using a system of compartmental ordinary differential equations. We derive the basic reproduction number as well as criteria for the existence and local stability of disease-free and enzootic equilibria. These results provide useful information for evaluating management strategies. A local sensitivity analysis of certain model invariants to uncertain parameter values is performed to ascertain which biological factors have the largest impact on ecological outcomes and, in particular, long-term bird population densities. We discuss and compare the effectiveness of two disease control and conservation strategies: captive propagation of honeycreepers and larval mosquito habitat reduction. We provide examples of combinations of these strategies that either are predicted to eliminate enzootic avian malaria or to increase predicted bird density above a given ecologically meaningful threshold.

Diversity and distribution of avian malaria and related haemosporidian parasites in captive birds from a Brazilian megalopolis

The role of zoos in conservation programmes has increased significantly in last decades, and the health of captive animals is essential to guarantee success of such programmes. However, zoo birds suffer from parasitic infections, which often are caused by malaria parasites and related haemosporidians. Studies determining the occurrence and diversity of these parasites, aiming better understanding infection influence on fitness of captive birds, are limited. Methods: In 2011–2015, the prevalence and diversity of Plasmodium spp. and Haemoproteus spp. was examined in blood samples of 677 captive birds from the São Paulo Zoo, the largest zoo in Latin America. Molecular and microscopic diagnostic methods were used in parallel to detect and identify these infections. Results: The overall prevalence of haemosporidians was 12.6%. Parasites were mostly detected by the molecular diagnosis, indicating that many birds harbour subclinical or abortive infections. In this project, birds of 17 orders (almost half of all the orders currently accepted in taxonomy of birds), 29 families, and 122 species, were tested, detecting positive individuals in 27% of bird species. Birds from the Anatidae were the most prevalently infected (64.7% of all infected animals)...

Malaria in penguins - current perceptions.

Avian malaria is a mosquito-borne disease caused by protozoans of the genus Plasmodium, and it is considered one of the most important causes of morbidity and mortality in captive penguins, both in zoological gardens and rehabilitation centres. Penguins are known to be highly susceptible to this disease, and outbreaks have been associated with mortality as high as 50-80% of affected captive populations within a few weeks. The disease has also been reported in wild penguin populations, however, its impacts on the health and fitness of penguins in the wild is not clear. This review provides an overview of the aetiology, life cycle and epidemiology of avian malaria, and provides details on the strategies that can be employed for the diagnostic, treatment and prevention of this disease in captive penguins, discussing possible directions for future research.

In vivo transmission blocking activities of artesunate on the avian malaria parasite Plasmodium gallinaceum.

Infection and transmission of the avian malaria parasite Plasmodium gallinaceum in domestic chickens is associated with high economic burden and presents a major challenge to poultry industry in South East Asia. Development of drugs targeting both asexual blood stage parasites and sexual stages of the avian malarias will be beneficial for malaria treatment and eradication. However, current drugs recommended for treatment of the avian malaria parasites target specifically the asexual blood stage parasites, but have little or no impact to the gametocytes, the major target for development of transmission-blocking strategies. In the present work, we established a simple procedure to evaluate gametocytocidal and transmission blocking activities in a P. gallinaceum-avian model. The assays involved administration of seven consecutive daily doses of test compounds into P. gallinaceum-infected chickens with 10% parasitaemia and 1% gametocytaemia. Our studies indicated that intramuscular injection with seven daily low doses (the minimum effective dose of 10mg/kg) of artesunate blocked the gametocyte production and transmission to the mosquito vector Aedes aegypti. This assay can be further applicable for testing new compounds against P. gallinaceum and for other parasitic protozoa infecting birds.

Ecology and conservation biology of avian malaria.

Avian malaria is a worldwide mosquito-borne disease caused by Plasmodium parasites. These parasites occur in many avian species but primarily affect passerine birds that have not evolved with the parasite. Host pathogenicity, fitness, and population impacts are poorly understood. In contrast to continental species, introduced avian malaria poses a substantial threat to naive birds on Hawaii, the Galapagos, and other archipelagoes. In Hawaii, transmission is maintained by susceptible native birds, competence and abundance of mosquitoes, and a disease reservoir of chronically infected native birds. Although vector habitat and avian communities determine the geographic distribution of disease, climate drives transmission patterns ranging from continuous high infection in warm lowland forests, seasonal infection in midelevation forests, and disease-free refugia in cool high-elevation forests. Global warming is expected to increase the occurrence, distribution, and intensity of avian malaria across this elevational gradient and threaten high-elevation refugia, which is the key to survival of many susceptible Hawaiian birds. Increased temperatures may have already increased global avian malaria prevalence and contributed to an emergence of disease in New Zealand.

Apical surface expression of aspartic protease Plasmepsin 4, a potential transmission-blocking target of the plasmodium ookinete.

To invade its definitive host, the mosquito, the malaria parasite must cross the midgut peritrophic matrix that is composed of chitin cross-linked by chitin-binding proteins and then develop into an oocyst on the midgut basal lamina. Previous evidence indicates that Plasmodium ookinete-secreted chitinase is important in midgut invasion. The mechanistic role of other ookinete-secreted enzymes in midgut invasion has not been previously examined. De novo mass spectrometry sequencing of a protein obtained by benzamidine affinity column of Plasmodium gallinaceum ookinete axenic culture supernatant demonstrated the presence of an ookinete-secreted plasmepsin, an aspartic protease previously only known to be present in the digestive vacuole of asexual stage malaria parasites. This plasmepsin, the ortholog of Plasmodium falciparum plasmepsin 4, was designated PgPM4. PgPM4 and PgCHT2 (the P. gallinaceum ortholog of P. falciparum chitinase PfCHT1) are both localized on the ookinete apical surface, and both are present in micronemes. Aspartic protease inhibitors (peptidomimetic and natural product), calpain inhibitors, and anti-PgPM4 monoclonal antibodies significantly reduced parasite infectivity for mosquitoes. These results suggest that plasmepsin 4, previously known only to function in the digestive vacuole of asexual blood stage Plasmodium, plays a role in how the ookinete interacts with the mosquito midgut interactions as it becomes an oocyst. These data are the first to delineate a role for an aspartic protease in mediating Plasmodium invasion of the mosquito and demonstrate the potential for plasmepsin 4 as a malaria transmission-blocking vaccine target.

Expression of a mutated phospholipase A2 in transgenic Aedes fluviatilis mosquitoes impacts Plasmodium gallinaceum development.

The genetic manipulation of mosquito vectors is an alternative strategy in the fight against malaria. It was previously shown that bee venom phospholipase A2 (PLA2) inhibits ookinete invasion of the mosquito midgut although mosquito fitness was reduced. To maintain the PLA2 blocking ability without compromising mosquito biology, we mutated the protein-coding sequence to inactivate the enzyme while maintaining the protein's structure. DNA encoding the mutated PLA2 (mPLA2) was placed downstream of a mosquito midgut-specific promoter (Anopheles gambiae peritrophin protein 1 promoter, AgPer1) and this construct used to transform Aedes fluviatilis mosquitoes. Four different transgenic lines were obtained and characterized and all lines significantly inhibited Plasmodium gallinaceum oocyst development (up to 68% fewer oocysts). No fitness cost was observed when this mosquito species expressed the mPLA2.

Genetic control of malaria parasite transmission: threshold levels for infection in an avian model system.

Genetic strategies for controlling malaria transmission based on engineering pathogen resistance in Anopheles mosquitoes are being tested in a number of animal models. A key component is the effector molecule and the efficiency with which it reduces parasite transmission. Single-chain antibodies (scFvs) that bind the circumsporozoite protein of the avian parasite, Plasmodium gallinaceum, can reduce mean intensities of sporozoite infection of salivary glands by two to four orders of magnitude in transgenic Aedes aegypti. Significantly, mosquitoes with as few as 20 sporozoites in their salivary glands are infectious for a vertebrate host, Gallus gallus. Although scFvs hold promise as effector molecules, they will have to reduce mean intensities of infection to zero to prevent parasite transmission and disease. We conclude that similar endpoints must be reached with human pathogens if we are to expect an effect on disease transmission.

An anti-Chitinase malaria transmission-blocking single-chain antibody as an effector molecule for creating a Plasmodium falciparum-refractory mosquito.

Indirect evidence has suggested the existence of a second chitinase gene, PgCHT2, in the avian malaria parasite Plasmodium gallinaceum. We have now identified PgCHT2 as the orthologue of the P. falciparum chitinase gene PfCHT1, a malaria transmission-blocking target. Computational phylogenetic evidence and biochemical and cell biological functional data support the hypothesis that an avian-related Plasmodium species was the ancestor of both P. falciparum and P. reichenowi, and this single lineage gave rise to another lineage of malaria parasites, including P. vivax, P. knowlesi, P. berghei, P. yoelii, and P. chabaudi. A recombinant PfCHT1/PgCHT2-neutralizing single-chain antibody significantly reduced P. falciparum and P. gallinaceum parasite transmission to mosquitoes. This single-chain antibody is the first anti-P. falciparum effector molecule to be validated for making a malaria transmission-refractory transgenic Anopheles species mosquito. P. gallinaceum is a relevant animal model that facilitates a mechanistic understanding of P. falciparum invasion of the mosquito midgut.

Preliminary results of an anticircumsporozoite DNA vaccine trial for protection against avian malaria in captive African black-footed penguins (Spheniscus demersus).

Captive juvenile African black-footed penguins (Spheniscus demersus) housed in an outdoor enclosure at the Baltimore Zoo have an average 50% mortality from avian malarial (Plasmodium sp.) infection each year without intense monitoring for disease and chemotherapeutic intervention. During the 1996 malaria transmission season, the safety and efficacy of an anti-circumsporozoite (CSP) DNA vaccine encoding the Plasmodium gallinaceum CSP protein against P. relictum were studied. The goal was to reduce clinical disease and death without initiating sterile immunity after release into an area with stable, endemic avian malaria. The birds were monitored for adverse clinical signs associated with vaccination, the stimulation of an anti-CSP antibody response, and protection afforded by the vaccine. The presence of P. relictum in trapped culicine mosquitoes within the penguin enclosure was monitored to assess parasite pressure. Among the vaccinated penguins, the parasitemia rate dropped from approximately 50% to approximately 17% despite intense parasite pressure, as determined by mosquito infection rate. During the year of the vaccine trial, no mortalities due to malaria occurred and no undesirable vaccination side effects occurred. This is the first trial of an antimalarial vaccine in a captive penguin colony.

Measuring the effects of an ever-changing environment on malaria control.

The effectiveness of malaria control measures depends not only on the potency of the control measures themselves but also upon the influence of variables associated with the environment. Environmental variables have the capacity either to enhance or to impair the desired outcome. An optimal outcome in the field, which is ultimately the real goal of vaccine research, will result from prior knowledge of both the potency of the control measures and the role of environmental variables. Here we describe both the potential effectiveness of control measures and the problems associated with testing in an area of endemicity. We placed canaries with different immunologic backgrounds (e.g., naïve to malaria infection, vaccinated naïve, and immune) directly into an area where avian malaria, Plasmodium relictum, is endemic. In our study setting, canaries that are naïve to malaria infection routinely suffer approximately 50% mortality during their first period of exposure to the disease. In comparison, birds vaccinated and boosted with a DNA vaccine plasmid encoding the circumsporozoite protein of P. relictum exhibited a moderate degree of protection against natural infection (P < 0.01). In the second year we followed the fate of all surviving birds with no further manipulation. The vaccinated birds from the first year were no longer statistically distinguishable for protection against malaria from cages of naïve birds. During this period, 36% of vaccinated birds died of malaria. We postulate that the vaccine-induced protective immune responses prevented the acquisition of natural immunity similar to that concurrently acquired by birds in a neighboring cage. These results indicate that dominant environmental parameters associated with malaria deaths can be addressed before their application to a less malleable human system.

Identification of novel Plasmodium gallinaceum zygote- and ookinete-expressed proteins as targets for blocking malaria transmission.

The development of transmission-blocking vaccines is one approach to malaria control. To identify novel Plasmodium zygote- and ookinete-secreted proteins as targets of blocking malaria transmission, monoclonal antibodies (MAbs) were produced against parasite-secreted proteins found in Plasmodium gallinaceum ookinete culture supernatants. Four MAbs-1A6, 2A5, 2B5, and 4B6-were identified that bound to P. gallinaceum zygotes and ookinetes in diverse patterns in terms of spatial localization on parasites, time course of antigen expression, and Western immunoblot patterns. MAbs 2A5 and 4B6 recognized more than one protein band as detected by Western immunoblot of P. gallinaceum ookinete supernatants. Beginning at 0 h postfertilization, MAb 2A5 recognized a diverse set of antigens; at 10 h postfertilization, MAb 4B6 recognized several antigens as well. MAb 1A6 recognized a single approximately 17-kDa protein, and 2B5 recognized a single approximately 32-kDa protein at 15 h postfertilization. In membrane feeding assays to assess the effect of these MAbs on P. gallinaceum infectivity for Aedes aegypti mosquitoes, the addition of MAbs 1A6 and 2B5 to infectious blood meals significantly inhibited oocyst development in the mosquito midgut. In contrast, MAb 2A5 seemed to enhance infectivity. These results demonstrate that Plasmodium ookinetes secrete proteins (in addition to previously characterized chitinases) that may be targets for blocking malaria transmission. Future investigation of ookinete-secreted neutralization-sensitive molecules should provide valuable insight into mechanisms by which ookinetes exit the blood meal, penetrate and transverse the peritrophic matrix, and invade the mosquito midgut epithelium.

Immunogenicity of Leucocytozoon caulleryi sporozoites and their reactivity with specific immune sera.

The immunogenicity of Leucocytozoon caulleryi sporozoites for chickens and their reactivity in vitro with specific immune sera were studied. Almost all of the chickens that had been immunized with the sporozoite antigens survived the sporozoite challenge. The degree of parasitemia observed in the immunized chickens was significantly lower than that found in the nonimmunized chickens. Specific antibodies against sporozoites were tested by the circumsporozoite precipitation (CSP) reaction. Antibodies were demonstrated in the sera of chickens that had been immunized with the sporozoite antigens or chickens that had recovered from a primary infection with L. caulleryi sporozoites. When viable mature sporozoites were incubated in vitro with serum from immune chickens, agglutination and a long, thread-like precipitate at one end of the sporozoite could be seen within a few minutes under a phase-contrast microscope. The effects of specific immune serum on the infectivity of sporozoites were examined by the sporozoite neutralization activity (SNA) test. Sporozoites that had been incubated in vitro with serum from immune chickens lost their infectivity to chickens. The CSP reaction and the SNA test in L. caulleryi infection were stage- and species-specific.

Plasmodium gallinaceum: antibodies to circumsporozoite protein prevent sporozoites from invading the salivary glands of Aedes aegypti.

A circumsporozoite protein-specific monoclonal antibody (N2H6D5) was injected into malaria-infected mosquitoes to determine its effect on the sporogonic cycle. After injection of antibody into mosquitoes (100 ng each), positive immunofluorescence (measured on air-dried sporozoites) reactions in hemolymph extracts were observed at a dilution of 1:1000. At 72 hr postinjection the levels dropped to 1:10. Sporozoites coinjected with antibody did not invade the salivary glands. In naturally infected mosquitoes, sporozoites were released over a period of 3 to 4 days. Therefore, mosquitoes were injected twice. The first injection was a day before the beginning of sporozoite release and the second, 2 days later. Sporozoite invasion of the salivary glands was assessed 3 days after the second injection, by microscopic examination of dissected glands. At this stage, all oocysts had completed maturation and released the sporozoites. Salivary gland infections were totally prevented in mosquitoes given two injections of 100 ng N2H6D5. Hence, sustained presence of anti-circumsporozoite antibodies in the hemolymph can render female Aedes aegypti refractory to Plasmodium gallinaceum.