Histological Alterations in the Body Wall of the Tropical Earthworm Eudrilus eugeniae Exposed to Hexavalent Chromium.

The tropical earthworm Eudrilus eugeniae was chronically exposed to hexavalent chromium (Cr) in its substrate over a concentration range from 0.24 to 893 mg kg(-1). Histological alterations in the body wall epithelium included cell fusion, reduction in thickness of the epithelial layer, a marked increase in pyknotic nuclei and epithelial sloughing. Similar changes were noted in the circular and longitudinal muscles with damage being indicated by the prominent inter-muscular cell spaces and disintegration. Many of these noted alterations intensified with increasing levels of exposure. It is significant that some of the changes recorded here were evident even at the lowest concentration of 0.24 mg kg(-1), an environmentally relevant concentration. Hence, the observed trends could be taken as an early warning to the imminent threats of heavy metal pollution to epigeic earthworm species.

Association of high plasma TNF-alpha levels and TNF-alpha/IL-10 ratios with TNF2 allele in severe P. falciparum malaria patients in Sri Lanka.

Plasma levels of pro- and anti-inflammatory cytokines of Plasmodium falciparum-infected patients with severe malaria (SM; n = 62) and uncomplicated malaria (UM; n = 69) from Sri Lanka were assessed. SM patients had significantly higher levels of TNF-alpha (P < 0·01), IL-6 (P < 0·01), and IL-10 (P < 0·05) compared to the UM patients. Plasma IL-2 levels of these patients were undetectable. TNF-alpha levels of a third group of patients with uncomplicated P. falciparum malaria, who were recruited during their fever episodes (UMF; n = 14) were significantly higher than those of the UM patients (P < 0·001) and comparable to SM patients. Plasma IFN-gamma levels of SM patients were higher compared to UM patients, but was not statistically significant. Body temperature in both SM and UMF groups were significantly higher compared to UM group, whereas percentages of parasitemia in all three groups were comparable. Analysis of plasma TNF-alpha levels and the ratio of TNF-alpha/IL-10 in UM (n = 34) and SM (n = 34) patients carrying TNF1 and TNF2 allelic types showed that SM patients carrying TNF2 had significantly higher TNF-alpha levels as well as TNF-alpha/IL-10 ratio compared to UM patients carrying TNF1, UM patients carrying TNF2 and SM patients carrying TNF1 (P < 0·05). These results suggest that the high circulating TNF-alpha levels and the inadequate IL-10 response in the SM patients carrying TNF2 allele could have contributed to the development of severe falciparum malarial disease.

Seroprevalence of Toxoplasma gondii in cats from Colombo, Sri Lanka.

Cats are essential in the life cycle of Toxoplasma gondii because they are the only hosts that can excrete the environmentally resistant oocysts in nature. Nothing is known of the prevalence of Toxoplasma gondii in cats from Sri Lanka. Serum samples from 86 cats from Colombo, Sri Lanka, were tested for antibodies to T. gondii using the modified agglutination test; antibodies were found in 26 (30.2%) cats with titers of 1:25 in 4, 1:50 in 4, 1:100 in 3, 1:400 in 2, 1:800 in 3, 1:1,600 in 4, and 1:3,200 or higher in 6 cats. Seropositivity increased with age and was higher in stray cats versus pet cats. This is the first report of seroprevalence of T. gondii in cats from Sri Lanka.

TNFalpha*2 marks high risk of severe disease during Plasmodium falciparum malaria and other infections in Sri Lankans.

We have investigated the association between alleles of the genes for tumour necrosis factor-alpha (TNF-alpha) and TNF-beta and severity of disease during malarial (Plasmodium falciparum) and other infections in the Sri Lankan population. Patients were categorized as having either (i) uncomplicated malaria, (ii) severe and complicated malaria, or (iii) severe and complicated infection in which a diagnosis of malaria had been excluded. For all the patients, as well as for a group of matched healthy controls, TNF-alpha and TNF-beta allelic types were identified using the polymerase chain reaction (PCR) and allele-specific oligonucleotide probes and restriction enzyme digestion. The odds in favour of carrying the TNFalpha*2 allele, mainly of the heterozygous genotype (TNFalpha*1,*2), were two to three times greater among individuals with severe disease, of either malarial or other infectious origin, relative to healthy controls or to those with uncomplicated malarial infections. No significant risk was associated with either of the alleles of TNF-beta.

The ParaSight-F dipstick test as a routine diagnostic tool for malaria in Sri Lanka.

Blood from 1053 persons who presented for treatment at outpatient clinics of government health institutions in Sri Lanka, and 250 who took part in a blood survey for malaria, was examined by thick blood film microscopy under routine field conditions, and by the ParaSight-F dipstick method. All the samples were also examined microscopically under laboratory conditions when 4 times the number of microscope fields were examined. Compared with this reference standard, the sensitivity and specificity of the ParaSight-F test were 90.2% and 99.1%, and those of microscopy in the field were 92.4% and 98.4% respectively, there being no statistically significant difference between the 2 methods. The ParaSight-F test reading correlated significantly and positively with the intensity of clinical disease of patients but not with their peripheral parasitaemia, indicating that it may be a more accurate measure of the true parasite load than microscopy, which detects only parasites which are in the peripheral blood and not those which are sequestered in deep organs. The ParaSight-F test, however, failed to detect Plasmodium falciparum infections with only gametocytes in the blood (19.6% of the infected blood samples in this study). The time taken for a patient to revert to negativity by the ParaSight-F test was also significantly longer, up to 14 d. This would make the test unsuitable for checking the response to antimalarial treatment within 14 d. In an endemic area it would therefore fail to detect drug resistant populations of parasites.

Transmission blocking immunity in Plasmodium vivax malaria: antibodies raised against a peptide block parasite development in the mosquito vector.

One approach towards the development of a vaccine against malaria is to immunize against the parasite sexual stages that mediate transmission of the parasite from man to mosquito. Antibodies against these stages, ingested with the blood meal, inhibit the parasite development in the mosquito vector, constituting "transmission blocking immunity." Most epitopes involved in transmission-blocking immunity depend on the tertiary conformational structure of surface antigens. However, one of the transmission-blocking monoclonal antibodies we have raised against Plasmodium vivax reacts with a linear epitope on both asexual stages and gametes. This monoclonal antibody (A12) is capable of totally blocking development of the parasite in the mosquito host when tested in membrane feeding assays with gametocytes from P. vivax-infected patients. Immune screening of a P. vivax lambda gt11 genomic expression library with A12 led to the isolation of a clone to which was mapped the six-amino acid epitope recognized by A12. Antisera raised in mice against a 12-mer synthetic peptide containing this epitope coupled to bovine serum albumin not only had high titers of antipeptide antibodies as measured by enzyme-linked immunosorbent assay, but in addition recognized the same 24- and 57-kD parasite components as A12 on Western blots and reacted with the parasite by immunofluorescence. When tested in membrane feeding assays, these antibodies have significant suppressive effects on parasite development in the mosquito.

Plasmodium falciparum malaria transmission-blocking immunity under conditions of low endemicity as in Sri Lanka.

Sera from acute primary Plasmodium falciparum patients in Sri Lanka were tested for the presence of antibodies against gamete antigens and for their functional effects of transmission blocking activity. Comparisons were made with corresponding data from a previous study from sera of patients from Papua New Guinea where malaria is more highly endemic. Although the prevalence of anti-gamete antibodies in the two groups were broadly similar, the prevalence of infectivity suppressive effects in the Sri Lankan sera (56%) was less than in Papua New Guinea sera (75%), suggesting that the generation of functionally effective transmission blocking antibodies requires prolonged exposure to multiple inoculations of malaria. In Papua New Guinea sera there was a good correlation between transmission blocking effects and antibody responses to Pfs 230, a known target of transmission blocking antibodies. Among the Sri Lankan sera no strong correlation was found between transmission blocking effects and the presence of antibodies to gamete surface antigens Pfs 230 nor Pfs 48/45 as detected by immunoprecipitation of radio-iodinated gamete proteins; a strong correlation was however, found between the intensity of response to gamete surface antigens by IFA and transmission blocking effects of these sera. It is possible therefore, that the antigens identified by IFA include non-protein moieties and that these may be the targets of transmission blocking antibodies in sera from acute primary infections of P. falciparum.

Plasmodium vivax:recombination between potential allelic types of the merozoite surface protein MSP1 in parasites isolated from patients.

The merozoite surface protein MSP1, which is one of the most promising candidates for a malaria vaccine directed against erythrocytic stages, has been shown to be polymorphic in different malarial species. Characterization of the Plasmodium vivax MSP1 gene (Pv200) in two strains (Belem and Salvador-1) revealed the existence of several polymorphic regions. One of these regions has been examined here in primary parasite isolates obtained from patients in Sri Lanka. Oligonucleotide primers hybridizing to conserved parts of the gene on either side of a polymorphic region were used to amplify DNA from 22 isolates. Sequence analysis of the amplified portion of the MSP1 gene in five patients showed the existence of three types of polymorphic regions. Two were almost identical either to that of the Belem or to that of the Salvador-1 strain. The third polymorphic type appeared to have resulted from recombination between the two others. This recombination event took place inside a repeated part of the sequence.

A stage specific gene expressed at the onset of gametocytogenesis in Plasmodium falciparum.

The gene encoding the gametocyte specific cytoplasmic protein Pfg27/25 of the human malaria parasite Plasmodium falciparum has been cloned. The gene encodes a highly hydrophilic non-repetitive protein which does not share obvious homologies with other polypeptides. The stage specificity of Pfg27/25 is controlled at the stage of the production of stable mRNA, which is detectable only in the sexual stages of the parasite, and contains long additional sequences outside the Pfg27/25 coding region. As the activation of Pfg27/25 gene expression occurs at an early stage of gametocytogenesis, the study of its regulation might provide information on the molecular events occurring after the parasite commitment to sexual differentiation and at the beginning of gametocyte formation.

Target antigens of transmission blocking immunity of Plasmodium vivax malaria. Characterization and polymorphism in natural parasite isolates.

A panel of 20 anti-Plasmodium vivax female gamete mAb has been established and was characterized with respect to their transmission-blocking properties in membrane-feeding experiments and their target Ag identified. Seven mAb suppressed the infectivity of P. vivax parasites to Anopheles tesselatus mosquitoes. The m.w. of the Ag recognized by these mAb were ascertained by SDS-PAGE and Western blots. Three sets of polypeptides of low Mr--20, 24, and a doublet of 37/42 kDa--have been defined as target Ag of transmission-blocking antibodies of P. vivax. All epitopes of these target Ag were found to be dependent on the tertiary conformational structure of the Ag. Polymorphism of target Ag of transmission-blocking immunity was investigated in over 30 natural isolates of P. vivax in Sri Lanka based on the reactivity of a mAb with an isolate as assessed by the indirect immunofluorescent test with the use of live extracellular female gametes, and in Western blots with the use of extracted gametes. The functional consequences of antigenic polymorphism on immunity was investigated in transmission-blocking assays by using membrane-feeding experiments. A majority of target Ag of transmission-blocking immunity were found to be polymorphic, exhibiting size as well as epitope polymorphism. Results indicate that failure of a mAb to affect the infectivity of a parasite isolate of P. vivax to mosquitoes can be caused by polymorphism of the target Ag among isolates.

Monoclonal and polyclonal antibodies both block and enhance transmission of human Plasmodium vivax malaria.

Antibodies against gametes of the malarial parasite inhibit the development of the parasite in the mosquito and curtail the transmission of malaria. We now report that a monoclonal antibody against gametes of the human malaria pathogen Plasmodium vivax and antibodies induced during natural infections of P. vivax in humans which suppress infectivity of the parasites to the vector at high concentrations can, at lower concentrations, have the opposite effect and enhance the level of malaria infection in the mosquitoes. Infectivity enhancing effects of up to 12-fold were demonstrated when a transmission blocking monoclonal antibody and immune human sera were diluted, in some undiluted immune human sera, and in the sera of vivax malaria patients during convalescence after drug cure.

A survey of glucose-6-phosphate-dehydrogenase deficiency in the North Central Province of Sri Lanka (formerly Ceylon).

A high frequency of the G6PD deficient gene was detected in the North Central Province of Sri Lanka. The frequency in the ancient villages is much higher than that of the recently colonised areas. The Sinhalese and Ceylon Moors have a significantly higher frequency as opposed to the Ceylon Tamils. The distribution appears to be related to a history of exposure to malarial endemicity.