Molecular detection of the avian malaria parasite Plasmodium gallinaceum in Thailand.

Avian malaria is one of the most common veterinary problems in Southeast Asia. The standard molecular method for detection of the avian malaria parasite involves the phenol-chloroform extraction of parasite genomic (g)DNA followed by the amplification of parasite gDNA using polymerase chain reaction (PCR). However, the phenol-chloroform extraction method is time-consuming and requires large amounts of samples and toxic organic solvents, thereby limiting its applications for parasite detection in the field. This study aimed to compare the performance of chelex-100 resin and phenol/chloroform extraction methods for the extraction of Plasmodium gallinaceum gDNA from whole avian blood that had been dried on filter papers (a common field sampling method). The specificity and sensitivity of PCR assays for P. gallinaceum cytochrome B (cytb) and cytochrome oxidase subunit I (coxI) gene fragments (544 and 588bp, respectively) were determined, and found to be more sensitive with gDNA extracted by the chelex-100 resin method than with the phenol/chloroform method. These PCR assays were also performed to detect P. gallinaceum in 29 blood samples dried on filter papers from domestic chickens in a malaria endemic area, where the reliable identification of seven field isolates of P. gallinaceum was obtained with an accuracy of 100%. The analysis of cytb and coxI gene nucleotide sequences revealed the existence of at least two genetically distinct populations of P. gallinaceum in Thailand, both of which differed from the reference strain 8A of P. gallinaceum. In conclusion, the chelex-100 resin extraction method is a simple and sensitive method for isolating gDNA from whole avian blood dried on filter paper. Genomic DNA extracted by the chelex method could subsequently be applied for the PCR-based detection of P. gallinaceum and DNA sequencing. Our PCR assays provide a reliable diagnostic tool for molecular epidemiological studies of P. gallinaceum infections in domestic chickens and wild birds.

Identification and expression of maebl, an erythrocyte-binding gene, in Plasmodium gallinaceum.

Avian malaria is of significant ecological importance and serves as a model system to study broad patterns of host switching and host specificity. The erythrocyte invasion mechanism of the malaria parasite Plasmodium is mediated, in large part, by proteins of the erythrocyte-binding-like (ebl) family of genes. However, little is known about how these genes are conserved across different species of Plasmodium, especially those that infect birds. Using bioinformatical methods in conjunction with polymerase chain reaction (PCR) and genetic sequencing, we identified and annotated one member of the ebl family, merozoite apical erythrocyte-binding ligand (maebl), from the chicken parasite Plasmodium gallinaceum. We then detected the expression of maebl in P. gallinaceum by PCR analysis of cDNA isolated from the blood of infected chickens. We found that maebl is a conserved orthologous gene in avian, mammalian, and rodent Plasmodium species. The duplicate extracellular binding domains of MAEBL, responsible for erythrocyte binding, are the most conserved regions. Our combined data corroborate the conservation of maebl throughout the Plasmodium genus and may help elucidate the mechanisms of erythrocyte invasion in P. gallinaceum and the host specificity of Plasmodium parasites.

Exoerythrocytic development of Plasmodium ga llinaceum in primary fibroblast culture of chicken embryo / Desenvolvimento exoeritrocítico de Plasmodium gallinaceum em cultura primária de fibroblasto de embrião de galinha

In this study we assessed the susceptibility of primary fibroblast culture of chicken embryo to infection of P. gallinaceum sporozoites as well as the initial development of exoerithrocytic stages. Fibroblasts were obtained from the chest muscles of chicken embryos and sporozoites were obtained from experimentally infected Aedes fluviatilis salivary glands. After 1h, 3h, 24h, 48h and 72h periods pos-infection, cell cultures were fixed and analyzed both by indirect immunofluorescent-antibody test with anti-circumsporozoite protein monoclonal antibodies and by transmission electron microscopy. Circumsporozoite protein was detected in all parasitic forms. The mean percentage of fibroblasts with adhered or penetrated sporozoites did not significantly increase proportionately to the concentration of parasites in the inoculum, and independently if fetal calf or normal chicken sera were used in the culture medium. It was noted that the longer the incubation time, higher the possibility of the sporozoites to adhere and penetrate to fibroblats. Spozoites were observed penetrating in the fibroblast after 3h incubation when 0.68% of the cells had adhered parasites. Differentiation and development of the exoerythrocytic forms was observed after 24h incubation, when an average of 0,14% of the parasites have already invaded the cells. Developing parasites were found until 72h, when only 0.04% of fibroblasts were infected. Fibroblast cell culture seems to be a valuable experimental tool for in vitro investigation of the exoerytrocytic cycle of P. gallinaceum.

No presente estudo, avaliamos a susceptibilidade de cultura primária de fibroblastos de embrião de galinha à infecção por esporozoítas de P. gallinaceum, assim como o desenvolvimento de estágios do ciclo exoeritrocítico. Fibroblastos foram obtidos a partir da musculatura do peito de embriões de galinha e esporozoítas foram obtidos de glândulas salivares de Aedes fluviatilis experimentalmente infectados. Após períodos de 1h, 3h, 24h, 48h e 72h após a infecção, culturas de células foram fixadas e analisadas através de imunofluorescência indireta empregando-se anticorpos monoclonais contra a proteína circum-esporozoíta e microscopia eletrônica de transmissão. Proteína circum-esporozoíta foi detectada em todas as formas parasitárias. O percentual médio de fibroblastos com esporozoítas aderidos ou já penetrados não aumentou proporcionalmente com a concentração de parasitos no inóculo e independeu se o soro utilizado no cultivo celular era soro bovino fetal ou soro de galinha normal. Foi observado que, quando maior é o período de incubação, maior é a possibilidade dos esporozoítas aderirem e penetrarem nos fibroblastos. Esporozoítas foram observados penetrando em fibroblastos depois de 3h de incubação, quando 0,68% das células tinham parasitos aderidos. A diferenciação e o desenvolvimento das formas exoeritrocíticas foram observados após 24h de incubação, quando somente 0.04% dos fibroblastos achavam-se infectados. A cultura primária de fibroblastos de galinha parece ser um valioso modelo experimental para a investigação in vitro do ciclo exoeritrocítico do P. gallinaceum.

Malarial infection in Aedes aegypti : effects on feeding, fecundity and metabolic rate.

We have examined metabolic rate, lipid and carbohydrate of female Aedes aegypti during 10 days following a malaria-infected bloodmeal. In parallel, we determined bloodmeal size, portions retained and diuresed, and subsequent fecundity. We found that mosquitoes obtained identical masses of blood when feeding on an infected or control host. However, infected mosquitoes lost more mass during diuresis and retained a smaller mass. Infection led to a significant reduction in fecundity, the extent of which could not be explained by the difference in post-diuresis bloodmeal mass alone. We found no differences in lipid or carbohydrate content between infected and control mosquitoes during the 10 days post-infection, although infected mosquitoes had a lower body mass than controls. Metabolic rates were not different between groups, except during blood digestion, where the metabolic rate was lower in infected mosquitoes. These results suggest that infection by malaria does not lead to an increase in metabolic rate during the phases of midgut invasion and sporogony. However, infection does have a measurable effect on fecundity and subsequent body mass of the infected females.

Prevalência e variação dos estádios eritrocíticos do plasmodium (Novyella) juxtanucleare em Gallus gallus sob condições naturais, no período de um ano / Prevalence and variation of the erytrocytic forms of plasmodium (novyella) juxtanucleare in Gallus gallus about natural conditions, in the period of one year

The avian malaria caused by Plasmodium juxtanucleare in Gallus gallus, is a tipical plasmodiose from Brazilian gallinaceous. This disease can causes morbidy and mortality in its vertebrate hosts. This work was conducted at Boa Vista farm, Municipality of Santa Bárbara do Tugúrio, Minas Gerais, Brazil and the ours objectives were to evaluate the hight prevalence found in previous studies and to accompany the variation of the erytrocytic forms during one year. The bloods smears, dyed with Giemsa were examined in microscopy immersion. Twenty five half-breed fowls were accompany duting one year, monthly (from November/00 to May/01) and biweekly (from June/01 to October/01). The erytrocytic forms were registered and quantified by the observation of 100 microscopic fields. Was verified a prevalence of 100 por ciento by P. juxtanuclerare, but there wasn't statistics correlation between the increase of the erytrocytic forms during the year. The trophozoites were the more abundant form found in this studie.

Parasitismo de leucocitos y trombocitos de gallus gallus l. por plasmodium (novyella) juxtanucleare (apicomplexa: plasmodiidae) / Parasitism of leukocytes and thrombocytes of gallus gallus L. by plasmodium (novyella) juxtanucleare (apicomplexa: plasmodiidae)

Se realizó un investigación del parasitismo de plasmodium juxtanucleare en gallinas sin razas definida, provenientes de criaderos rústicos en el municipio de Seropédica, estado de Río de Janeiro, de Brasil. Se realizaron frotis sanguíneo periféricos, los cuales fuero coloreados con giemsa diluido en tampón sorensen pH6,8. En el examen hemoscópico se puede observar en aves con alto índice de (< 10 por ciento ) formas parasitarias de trofozoítas y esquizontes en el citoplasma de células de la línea leucocitica y trofozoítas en células de la línea trombocítica. Las observaciones en el presente estudio hacen inferir que la cepa de P. juxtanuclear que ocurre en Seropédica realiza esquizogonia fanerozóica. Este trabajo constituye el primer hallazgo de formas de P. juxtanucleares en leucocitos

Vesicular ATPase-overexpressing cells determine the distribution of malaria parasite oocysts on the midguts of mosquitoes.

In Plasmodium-infected mosquitoes, oocysts are preferentially located at the posterior half of the posterior midgut. Because mosquitoes rest vertically after feeding, the effect of gravity on the ingested blood has been proposed as the cause of such a biased distribution. In this paper, we examined the oocyst distribution on the midguts of mosquitoes that were continuously rotated to nullify the effect of gravity and found that the typical pattern of oocyst distribution did not change. Invasion of the midgut epithelium by ookinetes was similarly found to be biased toward the posterior part of the posterior midgut. We examined whether the distribution of oocysts depends on the distribution of vesicular ATPase (V-ATPase)-overexpressing cells that Plasmodium ookinetes preferentially use to cross the midgut epithelium. An antiserum raised against recombinant Aedes aegypti V-ATPase B subunit indicated that the majority of V-ATPase-overexpressing cells in Ae. aegypti and Anopheles gambiae are localized at the posterior part of the posterior midgut. We propose that the typical distribution of oocysts on the mosquito midgut is attributable to the presence and the spatial distribution of the V-ATPase-overexpressing cells in the midgut epithelium.

Plasmodium gallinaceum: fluorescent staining of zygotes and ookinetes to study malaria parasites in mosquito.

We have developed a fluorescent labeling procedure for staining the mosquito stages of Plasmodium gallinaceum. PKH26, a lipophilic dye, is efficiently and permanently incorporated into the membranes of zygotes and ookinetes. Stained zygotes undergo normal development into ookinetes; the stain does not interfere with ookinete mobility or ability to adhere to the mosquito midgut lumen. Stained zygotes and ookinetes are comparable to untreated parasites in their ability to give rise to oocysts when fed to mosquitoes. This technique can be used to study the development of Plasmodium parasites in the complex cellular environment of the mosquito midgut after a blood meal. It may also be adapted to study other parasite-vector interactions.

Plasmodium: a quantitative molecular assay for detection of sporogonic-stage malaria parasites.

We present a molecular assay to detect malaria parasites during sporogonic development in the mosquito host. Specific primers for Plasmodium-specific small-subunit ribosomal RNA sequences not present in mosquito RNA were used in a reverse transcriptase-polymerase chain reaction (RT-PCR) assay. A synthetic RNA quantitative competitor was made which included targets for two primers and a target sequence for a hybridization probe which is also present in the natural parasite ribosome. The heterobifunctional Tth polymerase was used to carry out both reverse transcription and DNA-dependent polymerase chain reaction in a single reaction tube. Ookinetes and sporozoites, the stages from the beginning and end of sporogonic development, respectively, were both recognized in the assay. The assay was calibrated for quantitation of sporozoites by making a standard curve with counted sporozoites. The linear range of the calibrated assay allowed accurate quantitation of parasite number over at least two orders of magnitude, from 10 to 1000 sporozoites, in each RT-PCR reaction.

A PCR test for avian malaria in Hawaiian birds.

The decline of native Hawaiian forest birds since European contact is attributed to factors ranging from habitat destruction to interactions with introduced species. Remaining populations of Hawaiian honeycreepers (Fringillidae: Drepanidinae) are most abundant and diverse in high elevation refuges above the normal range of disease-carrying mosquitoes. Challenge experiments suggest that honeycreepers are highly susceptible to avian malaria (Plasmodium sp.) but resistance exists in some species. In order to detect low levels of malarial infection and quantify prevalence of Plasmodium in high elevation natural populations of Hawaiian birds, a polymerase chain reaction (PCR) based diagnostic test was developed that identifies rRNA genes of Plasmodium in avian blood samples. Quantitative competitive PCR (QC-PCR) experiments indicate that the detection limit of our test is an order of magnitude greater than that reported for human malaria DNA blot tests. Compared with standard histological methods, the PCR test detected a higher prevalence of diseased birds at mid-elevations. Malaria was detected in three species of native birds living in a high elevation wildlife refuge on the island of Hawaii and in four species from Maui. Our results show that avian malaria is more widespread in Hawaiian forests than previously thought, a finding that has important conservation implications for these threatened species.

Antisporozoite antibodies with protective and nonprotective activities: in vitro and in vivo correlations using Plasmodium gallinaceum, an avian model.

A correlation was observed between in vivo and in vitro activity of six monoclonal antibodies (mAb) against the major circumsporozoite protein of the avian malaria Plasmodium gallinaceum as follows. (1) Two mAb were protective, totally abrogating sporozoite infectivity to chicks, its natural host, in vivo; they caused 100% inhibition of sporozoite invasion (ISI) in vitro to SL-29 chicken fibroblasts and intense ISI to cultured chicken macrophages, as well as inhibited the exoerythrocytic development of sporozoites taken up by macrophages, the initial cell host of P. gallinaceum sporozoites. (2) Two mAb were partially protective in that they reduced sporozoite infectivity to chicks, caused partial ISI to SL-29 and macrophage cells and partial inhibition to the exoerythrocytic development of sporozoites in macrophages in vitro. (3) Two mAb were totally inactive in vivo although they both bound to the sporozoite antigens as detected by indirect immunofluorescence, western blot, and ELISA; they both failed to induce ISI or inhibit the exoerythrocytic development in macrophages. The possible participation of macrophages as the initial cell type involved in sporozoite destruction in the presence of anti-circumsporozoite antibodies is discussed.

Developmentally regulated infectivity of malaria sporozoites for mosquito salivary glands and the vertebrate host.

Sporozoites are an invasive stage of the malaria parasite in both the mosquito vector and the vertebrate host. We developed an in vivo assay for mosquito salivary gland invasion by preparing Plasmodium gallinaceum sporozoites from infected Aedes aegypti mosquitoes under physiological conditions and inoculating them into uninfected female Ae. aegypti. Sporozoites from mature oocysts were isolated from mosquito abdomens 10 or 11 d after an infective blood meal. Salivary gland sporozoites were isolated 13 or 14 d after an infective blood meal. Purified oocyst sporozoites that were inoculated into uninfected female mosquitoes invaded their salivary glands. Using the same assay system, sporozoites derived from salivary glands did not reinvade the salivary glands after inoculation. Conversely, as few as 10 to 50 salivary gland sporozoites induced infection in chickens, while only 2 of 10 chickens inoculated with 5,000 oocyst sporozoites were infected. Both sporozoite populations were found to express a circumsporozoite protein on the sporozoite surface as determined by immunofluorescence assay and circumsporozoite precipitation test using a circumsporozoite protein-specific monoclonal antibody. We conclude that molecules other than this circumsporozoite protein may be responsible for the differential invasion of mosquito salivary glands or infection of the vertebrate host.

Transmission of Plasmodium gallinaceum by adult Aedes aegypti infected as larvae.

Transmission of Plasmodium gallinaceum to chickens by adult mosquitoes eclosed from larvae that consumed infected adult mosquitoes was investigated. No malarial infections were observed in chicks fed on by mosquitoes that eclosed from larvae that had consumed crushed infected adult mosquitoes. Where cadavers with noncrushed thoraces were used, 3 of 4 chicks fed on by adult mosquitoes developed parasitemias ranging from 6 to 11% infected erythrocytes.

Immunodiagnosis of malaria.

Different approaches to immunodiagnosis of malaria in an endemic country have been described in this paper. Demonstration of circulating malaria antigen may be done by gel-diffusion and counter-immunoelectrophoresis. Parasite associated antigen may be demonstrated by highly sensitive methods like radio-immunoassay or enzyme linked immuno sorbent assay. Malaria antibodies of the IgG type being long lasting do not appear to have any role in immuno-diagnosis. However, determination of malaria specific IgM antibodies by P. gallinaceum haemagglutination test or IgM immunofluorescence test may be simple and useful in immunodiagnosis of malaria. The tests though evaluated in different laboratories may not be applicable in the field for diagnosis of malaria at the present moment. However, it is envisaged that with the availability of different specificities of monoclonal antibodies by way of hybridoma technology and also with the help of recombinant DNA techniques immunodiagnosis of malaria in the field situation may become a reality.