Environmental and Occupational Health Impact of Bauxite Mining in Malaysia: A Review

In the perspective of recent bauxite mining in Malaysia, this review aims to identify the potential environmental and health impacts on miners and surrounding communities. The environmental issues of bauxite mining include, air, water and soil pollution due to bauxite dust; leaching of bauxite into water sources resulting in reduced soil fertility as well as affecting agricultural food products and aquatic life. Bauxite occupational exposure affects the health of miners, and has negative consequences on the health of surrounding communities, such as increased respiratory symptoms, contamination of drinking water, other potential health risks from ingestion of bauxite and heavy metals, including noise-induced hearing loss and mental stress. This review discusses the processes of bauxite mining, its constituents and residual trace elements, and their impact on the environment and health of exposed workers and communities. It also explores the Malaysian legal requirements and standards of occupational exposure to bauxite.

Saccharomyces cerevisiae recombinant Pfs25 adsorbed to alum elicits antibodies that block transmission of Plasmodium falciparum.

Antibodies to Pfs25, a cysteine-rich 25-kDa protein present on the surface of Plasmodium falciparum zygotes, can completely block the transmission of malaria parasites when mixed with infectious blood and fed to mosquitoes through a membrane feeding apparatus. Recently, a polypeptide analog, Pfs25-B, secreted from recombinant Saccharomyces cerevisiae was found to react with conformation-dependent, transmission-blocking monoclonal antibodies and to elicit transmission-blocking antibodies in experimental animals when emulsified in either Freund's or muramyl tripeptide adjuvant. In this study, Pfs25-B adsorbed to alum induced transmission-blocking antibodies in both rodents and primates. Bacterially produced Pfs25, however, did not elicit complete transmission-blocking antibodies in rodents. Furthermore, unlike monoclonal antibodies to Pfs25, which block transmission only after ookinete development, antisera to Pfs25-B adsorbed to alum appeared to block the in vivo development of zygotes to ookinetes as well.

Cloning and expression of the gene coding for the transmission blocking target antigen Pfs48/45 of Plasmodium falciparum.

The gene encoding the gametocyte/gamete-specific membrane protein Pfs48/45 of Plasmodium falciparum has been cloned. The Pfs48/45 gene is a non-interrupted, single copy gene that codes for a hydrophobic, non-repetitive protein of 448 amino acid residues containing a putative signal peptide at the N-terminus, a hydrophobic C-terminus and 7 potential N-glycosylation sites. Antibodies directed against a Pfs48/45-glutathione-S-transferase fusion protein reacted with both the 45-kDa and 48-kDa proteins of gametocytes. When Pfs48/45 is expressed in the baculovirus-insect cell system the recombinant Pfs48/45 protein is targeted and exposed to the insect cell surface in such a configuration that it is recognized by transmission-blocking anti-45/48-kDa monoclonal antibodies.

Pfs2400 can mediate antibody-dependent malaria transmission inhibition and may be the Plasmodium falciparum 11.1 gene product.

Monoclonal antibodies (mAb) have been raised against Plasmodium falciparum gametocyte stage protein extracts, in an effort to identify novel parasite antigens that might mediate malaria transmission-blocking immunity. mAb 1A1 identified Pfs2400, a sexual stage-specific antigen of greater than 2 megadaltons, that is associated with the outer leaflet of the parasitophorous vacuole membrane in mature circulating gametocyte-infected red blood cells. Upon induction of gametogenesis, Pfs2400 partitions between the gamete plasmalemma and the degenerating erythrocyte membrane. The antigen is no longer detectable in the fully emerged gamete. mAb 1A1 dramatically reduces the number of oocysts formed in P. falciparum gametocyte-fed mosquitoes. The cognate antigen is probably the product of the Pf11.1 gene (Scherf et al. 1988. EMBO [Eur. Mol. Biol. Organ.]J. 7:1129) on the basis that a peptide composed of two copies of the degenerate nine amino acid repeat sequence in the Pf11.1 protein, can inhibit binding of mAb1A1 to the native antigen. The mechanism of transmission inhibition mediated by the Pfs2400 is discussed.

Transmission blocking antibody of the Plasmodium falciparum zygote/ookinete surface protein Pfs25 also influences sporozoite development.

The Plasmodium falciparum zygote/ookinete surface protein, Pfs25, persists in the oocyst wall throughout its development. Anti-25 kD transmission blocking antibody, given to infected Anopheles stephensi or A. gambiae mosquitoes in an additional bloodmeal, 3-6 days after being fed gametocyte infected blood, penetrated the oocyst and reacted with the 25 kD protein within it. This reaction caused a significant reduction in the number of developing sporozoites. Mouse serum containing antibodies raised by immunization with a recombinant 25 kD yeast product showed a similar effect.

A scanning electron microscopic study of the sporogonic development of Plasmodium falciparum in Anopheles stephensi.

The full development of Plasmodium falciparum in Anopheles stephensi mosquitoes was studied by scanning electron microscopy. Ookinetic development was described from in vitro cultures. Growing oocysts beneath the basal lamina of the midgut wall mechanically stretch this lamina until it is torn and displaced by day 7. In young oocysts the wall appears smooth. In older oocysts wrinkles in the wall are visible after routine fixation. Osmium tetroxide postfixation greatly reduced the occurrence of these wrinkles. Intracapsular development of sporozoites was visualized after mechanical manipulation of the oocysts during sample preparation. In contrast to P. berghei, no ectopic development was seen in P. falciparum in the mosquito midgut. The mechanism of sporozoite escape from the oocyst appears to be similar to that described for rodent malaria. Fracturing of salivary glands provided the first view by scanning electron microscopy of sporozoites located in proximal and distal gland cells and in the draining duct.

Characterization of murine monoclonal antibodies against a repetitive synthetic peptide from the circumsporozoite protein of the human malaria parasite, Plasmodium falciparum.

Monoclonal antibodies (mAbs) were raised in mice against the synthetic peptide (NANP)40, consisting of 40 (NANP) repeats of the circumsporozoite (CS) protein of the human malaria parasite, Plasmodium falciparum, and characterized. (i) Five of these mAbs recognized the P. falciparum CS protein in western blot experiments and in immunofluorescence assays using different preparations of sporozoites. The remaining two mAbs (CT3.2 and CT3.3, both IgG1) gave negative results by both techniques. (ii) When the anti-(NANP)40 peptide mAbs were functionally tested in vitro to assess their ability to inhibit the attachment and penetration of the parasites into cultured human liver cells, six of them exhibited inhibitory activities ranging between 66 and 90%. CT3.2 mAbs, also, inhibited sporozoite attachment and penetration, despite the negative results by immunofluorescence and western blot experiments. However, when immunofluorescence was repeated in the presence of calcium, CT3.2 did reveal a positive recognition of P. falciparum sporozoites, suggesting that this mAb could recognize the (NANP) sequence when calcium was bound to the repetitive peptide. (iii) Furthermore, the binding of an anti-(NANP)40 IgM mAb (CT1) to the solid-phase peptide was not inhibited by preincubation of the peptide with a mAb against the P. falciparum CS protein. (iv) Finally, one anti-(NANP)40 IgG1 mAb (CT3.1) was unable to bind to the shorter (NANP)3 peptide, although it recognized the (NANP)40 peptide and the P. falciparum CS protein. The results presented here suggest that heterogeneous antibody populations are produced upon immunization of mice with (NANP)40 synthetic peptide and that epitopes different from those simply related to the linear (NANP) amino acid sequence are likely to be present in long (NANP)n constructs as well as in the repetitive domain of the P. falciparum CS protein.

Conserved and variant epitopes of target antigens of transmission-blocking antibodies among isolates of Plasmodium falciparum from Malaysia.

Monoclonal antibodies (MAbs) directed against different epitope regions on three sexual stage-specific gamete surface proteins of Plasmodium falciparum, Pfs 25, Pfs 230, and Pfs 48/45, were used to study the genetic diversity of these epitopes among fresh isolates of P. falciparum from Malaysia, using immunofluorescence microscopy (IFA). Among 45 Malaysian isolates, one epitope of Pfs 25, designated region I, showed evidence of variable reactivity with MAbs among different isolates; the Pfs 25 epitope, region II, was universally recognized by MAbs in all isolates. Two apparently distinct epitope regions of Pfs 230 were defined by MAbs, one of which was universally recognized by MAbs among the 45 isolates; the other was conserved in all but three isolates. The epitope regions of gamete-surface protein Pfs 48/45, designated regions I, IIa, IIb, IIc, III, and IV, were examined for reactivity by IFA in 33 isolates. Epitope regions I, IIb, III, and IV were conserved in all isolates; regions IIa and IIc existed in variant forms.

Feeding behaviour and sporozoite ejection by infected Anopheles stephensi.

Anopheles stephensi mosquitoes infected with Plasmodium falciparum sporozoites were allowed to feed individually through fresh whole thickness mouse skin. More sporozoites were ejected into the skin in clusters than into the blood. Deposition of sporozoites in the blood was an infrequent occurrence and always coincided with ejection of these stages into the skin--perhaps a spill-over effect. The number of probes before feeding (median 4.5) was not correlated with the sporozoite inoculum (median 8), nor was the number of sporozoites in the glands (median 14,500). However, the number of sporozoite clusters in the skin (median 1) was positively correlated with the inoculum size. The median value of the sporozoite inoculum was 22, when only those mosquitoes that ejected sporozoites were included. When feeding was interrupted and recommended on a new membrane, sporozoite ejection occurred with equal frequency on both occasions. Sporozoites disappeared from the site of bites in living mice within 2 h of feeding. The epidemiological significance of these observations is discussed.

T cell epitopes of the circumsporozoite protein of Plasmodium vivax. Recognition by lymphocytes of a sporozoite-immunized chimpanzee.

The humoral and cellular antisporozoite immune responses of a laboratory-born chimpanzee were measured following multiple exposures to the bites of Plasmodium vivax-infected mosquitoes. T cell lines and clones derived from the chimpanzee's PBL were used to identify T cell epitopes of the P. vivax circumsporozoite (CS) protein. Two independently obtained cell lines, established by culturing the PBL with either a recombinant P. vivax circumsporozoite (rPvCS) protein or a pool of synthetic peptides spanning the rPvCS sequence, recognized a 20-mer peptide from a nonpolymorphic region of the carboxyl terminus of the CS protein. This peptide overlaps a sequence homologous to region II of the Plasmodium falciparum CS protein. A third T cell line recognized an epitope within the central repeat domain, which has recently been found to be a polymorphic region of the P. vivax CS protein. The CD4+ clones derived from this third T cell line secreted IFN-gamma and IL-2 when stimulated with either the P. vivax repeat peptide (DRAAGQPAG)2 or the rPvCS protein.

Identification of plasma membrane proteins involved in the hepatocyte invasion of Plasmodium falciparum sporozoites.

To determine whether surface proteins of hepatocytes might be involved in the sporozoite invasion, plasma membrane proteins were prepared from human livers with CHAPS (3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulphonate) and radiolabelled with 125I (Iodogen; 1,3,4,6-tetrachloro-3 alpha,6 alpha-diphenylglycoluril). The labelled proteins were incubated with Plasmodium falciparum sporozoites and cross-linked with DSP (dithio-bis-succinimidylpropionate). Radiolabelled proteins released by reduction after repeated washing of the sporozoite-complex were separated by SDS-PAGE and autoradiographed. Two human hepatocyte membrane proteins of 20 and 55 kDa were found to be involved in the initial binding of P. falciparum sporozoites. The electrophoretically purified 20- and 55-kDa proteins both inhibited the binding of the corresponding radiolabelled proteins to P. falciparum sporozoites and reduced the invasion of sporozoites in an in vitro assay. We propose that these 20-kDa and 55-kDa proteins represent putative human hepatocyte receptors for P. falciparum sporozoite invasion.

Large-scale production of Plasmodium vivax sporozoites.

Mass-scale production of Plasmodium vivax sporozoites in Anopheles stephensi was achieved using the chimpanzee (Pan troglodytes) as a source of infective blood. Membrane feeding was as successful as feeding mosquitoes directly on the animal so long as the time between drawing the blood and feeding was restricted to 45 min. Longer delays such as 2-3 h resulted in loss of infectivity in terms of oocyst production. The selected strain of A. stephensi was highly susceptible to P. vivax (Chesson strain). A strain of A. stephensi relatively refractory to P. falciparum showed no cross-refractoriness to P. vivax. Mixed infections of P. falciparum and P. vivax did not interfere with each other in their development in A. stephensi. A second normal blood meal to mosquitoes infected with P. vivax increased the yield of salivary gland sporozoites.

Biosynthesis of the 25-kDa protein in the macrogametes/zygotes of Plasmodium falciparum.

Synthesis of the 25-kDa protein in the early midgut stages of Plasmodium falciparum was studied, using metabolic inhibitors (colchicine and actinomycin D) and pulse-labeling experiments. Experiments with colchicine showed that, immediately after induction of macrogametogenesis, 25-kDa protein synthesis occurs in both fertilized and nonfertilized macrogametes. The amount of 25-kDa protein synthesized increased slowly during time. Experiments with actinomycin D revealed that the slow increase of synthesis may be dependent on de novo messenger RNA synthesis.

Plasmodium falciparum: studies on mature exoerythrocytic forms in the liver of the chimpanzee, Pan troglodytes.

Mature exoerythrocytic forms (EEF) of Plasmodium falciparum from the chimpanzee were examined by light- and transmission electron microscopy from a liver biopsy taken on Day 6 after sporozoite inoculation. Infectivity of the sporozoites obtained from whole mosquitoes which were membrane fed on cultured gametocytes was about 4-6%. In comparison, salivary gland sporozoites added to human hepatocytes in vitro had only a developmental percentage of 0.02 to 0.05% at Day 5. The EEF found in the liver biopsy were not all at the same stage of development. Immature compact parasites were seen simultaneously with stages with fully formed merozoites, indicating a rapid final maturation or asynchrony. At Day 7.5, large numbers of rings were already seen in the peripheral blood, indicating a duration of the liver development of P. falciparum in the chimpanzee of about 5.5-6 days. The process of merogony at the fine structural level was comparable to that described for rodent and other primate parasites in vivo. Compared to the fine structure of EEF in vitro in cultured human hepatocytes, the parasites described here were much more advanced in development. There appeared to be some cell infiltration with collagen deposition around the intracellular parasite; however, no marked degeneration of EEF was observed.

Immunogold determination of Plasmodium falciparum circumsporozoite protein in Anopheles stephensi salivary gland cells.

The distribution of circumsporozoite (CS) proteins of Plasmodium falciparum sporozoites was observed during the passage of mature sporozoites in the hemocoel of Anopheles stephensi and during their entrance and sojourn in the salivary gland cells (SGC). The CS protein was visualized using a monoclonal antibody (3SP2) and immunogold labeling on ultrathin cryosections. In the hemocoel the sporozoites cease synthesizing CS protein, and some of it is shedded resulting in a patchy labeling pattern on the outer pellicular membrane. No internal labeling was observed. The sporozoites enter the SGC by puncturing the basal or lateral membrane. Inside the SGC, CS protein synthesis is turned on again; the Golgi system, nuclear envelope and all 3 pellicular membranes contain CS immunoreactivity. In the last phase of maturation, micronemes display abundant CS immunoreactivity. Rhoptries also show some immunogold labeling, but not as much as the micronemes.

Infectivity of cultured Plasmodium falciparum gametocytes to mosquitoes.

Various factors that may influence routine and high levels of mosquito infection with cultured Plasmodium falciparum gametocytes are considered in this paper. One of the most important is the choice of an appropriate isolate, with facilities for cryopreservation and a good technique for initiation of cultures. The use of automated culture systems with strict adherence to detail and routine has eliminated much of the variability. The quality of the serum used for the culture of gametocytes and inclusion in the feed material for mosquitoes is of the highest importance. Blood collection for culture purposes must preferably involve alcohol as an antiseptic for cleaning donor skin or suitable receptacles. Mosquito blood meals should not include plasma with citrate phosphate dextrose or sera collected in microtainer tubes or from volunteers on proguanil-chloroquine prophylaxis. Sera of individuals on chloroquine alone do not influence transmission. Haematocrits of from 5 to 10% permit the culture of equally infective gametocytes. It was impossible to predict the outcome of an infection in mosquitoes based on the number of female gametocytes or gametes. Within any experiment, the oocyst load initially increased, followed by a decline with progressively lower numbers of gametocytes accompanied by a progressive increase in the efficiency of transmission. Some of the variability of mosquito infection within an experiment was due to individual differences in the speed of blood digestion of the mosquitoes. A new membrane feeder is described with three different sizes to accommodate a variety of goals.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of epitopes on the 25 kD protein of the macrogametes/zygotes of Plasmodium falciparum.

A sexual stage-specific protein of Plasmodium falciparum with a Mr of 25,000 is one of the target antigens of transmission-blocking antibodies. The contributions of tertiary structure and post-translational modifications (glycosylation and acylation) to the structure of the epitopes on this protein were the subject of detailed investigations. After modification of the three-dimensional structure and modification or cleavage of carbohydrate groups and linked fatty acids, the immunological reactivity was investigated by three different techniques: (i) immunoprecipitation of radiolabelled proteins, (ii) enzyme-linked immunosorbent assay (ELISA), and (iii) Western blotting. The results of the experiments indicate that the immunological reactivity of the major epitopes on the 25 kD protein, including the epitope involved in transmission-blocking immunity, are dependent on the tertiary structure of the protein and on the presence of linked fatty acids, but not on the presence or absence of carbohydrate groups.

Selection of Anopheles stephensi for refractoriness and susceptibility to Plasmodium falciparum.

Variation in susceptibility of the vector Anopheles stephensi Liston to the human malaria parasite Plasmodium falciparum (Welch) was demonstrated using twelve strains of mosquitoes and one strain of parasites cultured in vitro. The Beech strain of An. stephensi exhibited greatest natural refractoriness, but with high intrapopulation variability. By selection for the required characteristic, two refractory lines of the Punjab strain and one highly susceptible line of the Sind strain were obtained. The median number of oocysts in the two refractory lines was less than 4% of that in the unselected line, whilst the highly susceptible line yielded about twice as many oocysts as the unselected line. Selection progressed more by keeping the descendants of individual females separate and selecting between them (individual selection) rather than pooling the progeny of all selected mosquitoes (mass selection). Using the former procedure many lines were lost due to inbreeding depression, but the outcome was more successful.