Thesis (M.Sc. (Tropical Medicine))--Mahidol University, 1979.

MSc (Applied Mathematics) / Department of Mathematics and Applied Mathematics / In this study, we develop a multiscale model of soil transmitted helminths in humans with a special reference to hookworm infection. Firstly, we develop a single scale model that comprises of five between host scale populations namely; susceptible humans, infected humans, eggs in the physical environment, noninfective worms in the physical environment and infective worms in the physical environment. Secondly, we extend the single scale model to incorporate within-host scales namely; infective larvae within-host, immature worms in small intestine, mature worm population and within-host egg population which resulted to a multiscale model. The models are analysed both numerically and analytically. The models are epidemiologically and mathematically well posed. Numerical simulation results show that there is a bidirectional relationship between the between-host and within-host scales. This is in agreement with the sensitivity analysis results, we noted that the same parameters that reduce reproductive number R0 are the same parameters that reduce the infective worms endemic equilibrium point. From the comparative effectiveness of hookworm interventions analysis results, we notice that any intervention combination that include wearing shoes controls and reduces the spread of the infection. The modelling framework developed in this study is vigorous to be applicable to other soil transmitted helminths infections / NRF

The aim of this study was to investigate the occurrence of the soil transmitted parasitic nematode Necator americanus ("Old World" hookworm) in soils of different texture in KwaZulu-Natal. The key questions being asked were: (i) Is hookworm infection in KwaZulu-Natal confined to the coastal plain? (ii) Is there any association between hookworm prevalence and the different soil types in the province? (iii) Since several examples exist in the province of soil types on which hookworm is transmitted on the coastal plain, occurring inland, what is the status of infection in communities situated in these areas? (iv) What properties of soil are important in the transmission ecology of hookworm larvae? All available hookworm prevalence data of KwaZulu-Natal were mapped on Land Type maps of the province (Land Type Survey Staff, 1986). Several additional surveys were carried out to supplement this database. Faecal egg counts were obtained by the Formal-Ether Concentration Method and positive infections were confirmed as N. americanus by larval morphology after coproculture using the Harada-Mori Technique. Univariate analysis was carried out for significant associations between hookworm prevalence, altitude, climatic variables (rainfall and temperature) and soil type. The results showed that areas ≤ 150m above sea level (i.e. the coastal plain) support high prevalences (x ‾ = 45 %), and are characterised by low-clay textured soils, warm temperatures and relatively high rainfall. Areas > 150 m (i.e. inland) have low hookworm prevalences (x ‾ = 6 %), and are characterised by high-clay textured soils, cool temperatures and moderate rainfall. Hookworm prevalence also decreased southwards as climatic conditions (rainfall and temperature) become unfavourable, and the coastal plain also narrows in this direction. Multivariate analysis was done to determine which environmental factors combine best to provide favourable conditions for hookworm transmission. From the variables used, prevalence of infection was most significantly correlated with the mean daily minimum temperature for January followed by the mean number of rainy days for January. This points to the importance of summer conditions in the transmission of hookworm infection in KwaZulu-Natal. Moderate hookworm prevalences (x ‾ = 17.3 %) were found in the inland sandy areas, dropping to low prevalences (x ‾ = 5.3 %) in the surrounding non-sandy areas. The intensity-related data could not be significantly correlated with the environmental variables used in this study. The Spearman Correlation Coefficient was used to test for relationships between hookworm prevalence and soil variables. In the results, only the fine and medium sand fractions showed positive correlations with hookworm prevalence. Clay showed a significant negative correlation with hookworm prevalence. No significant correlations were found between soil pH or its organic matter content and hookworm prevalence. Age and sex related infection data could not be drawn into the analysis due to the small sample size of study localities. / Thesis (M.Sc.)-University of Natal, Durban, 1998.

With over a billion people infected world wide, hookworms are considered as important human pathogens, particularly in developing countries which have the highest rates of infections. Hookworms reside in the gastrointestinal tract of the host where they continuously feed on blood, leading to conditions such as chronic irondeficiency anaemia. The majority of blood-feeding parasites rely on proteins found in blood to provide many of their nutritional requirements for growth, reproduction and survival. Of the numerous proteins found in blood, haemoglobin (Hb) is one of the most abundant. In order to acquire amino acids for protein synthesis, it is thought that haematophagous parasites degrade Hb using various classes of endo- and exoproteases, in a manner similar to that which occurs in catabolism of proteins in mammalian cellular lysosomes. This study identified and characterised proteases involved in the Hb degradation process in the human hookworm, Necator americanus, in order to identify potential candidate antigens for a vaccine that interrupts blood-feeding. Red blood cells ingested by hookworms are lysed to release Hb, which is cleaved by various proteases into dipeptides or free amino acids and these are taken up through the gut membrane by amino acid transporters. Proteases expressed in the intestinal tract of hookworms are thought to play a major role in this process and would therefore make good targets for vaccine candidates aimed at interrupting blood-feeding. To identify these proteases, adult hookworms (both N. americanus and Ancylostoma caninum) were sectioned and intestinal tissue was dissected via laser microdissection microscopy. RNA extracted from the dissected tissue was used to generate gut-specific cDNA, which then was used to create plasmid libraries. Each library was subjected to shotgun sequencing, and of the 480 expressed sequence tags (ESTs) sequenced from each species, 268 and 276 contigs were assembled from the N. americanus and A. caninum libraries, respectively. Nine percent of N. americanus and 6.5% of A. caninum contigs were considered novel as no homologues were identified in any published/accessible database. The gene ontology (GO) classification system was used to categorise the contigs to predicted biological functions. Only 17% and 38% of N. americanus and A. caninum contigs, respectively, were assigned GO categories, while the rest were classified as being of unknown function. The most highly represented GO categories were molecular functions such as protein binding and catalytic activity. The most abundant transcripts encoded fatty acid binding proteins, C-type lectins and activation associated secreted proteins, indicative of the diversity of functions that occur in this complex organ. Of particular interest to this study were the contigs that encoded for cysteine and metalloproteases, expanding the list of potential N. americanus haemoglobinases. In the N. americanus cDNA library, four contigs encoding for cathepsin B cysteine proteases were identified. Three contigs from the A. caninum and one contig from the N. americanus cDNA libraries encoded for metalloproteases, including astacin-like and O-sialoglycoprotein endopeptidases, neither of which had previously been reported from adult hookworms. Apart from haemoglobinases, other mRNAs encoding potential vaccine candidate molecules were identified, including anti-clotting factors, defensins and membrane proteins. This study confirmed that the gut of hookworms encodes a diverse range of proteases, some of which are likely to be involved in Hb digestion and have the potential to be hidden (cryptic) vaccine antigens. Four cysteine proteases (Na-CP-2, -3, -4 and -5) were identified from the gut cDNA library of N. americanus. All four proteases belong to the clan CA, family C1, share homology with human cathepsin B and possess a modified occluding loop. Real-time PCR indicated that all transcripts were up-regulated in the adult stage of the hookworm parasite with high levels of mRNA expression detected in gut cDNA. All four proteases were expressed in recombinant form, but only Na-CP-3 was successfully expressed in soluble form in the yeast Pichia pastoris. Proteolytic activity for Na-CP-3 was detected on a gelatin zymogen gel, however no catalytic activity was detected against the class-specific fluorogenic peptides Z-Phe-Arg-AMC and Z-Arg-Arg-AMC. Mass spectrometry analysis of the purified protein suggested that the pro-region had not been processed in trans when the protein was secreted by yeast. Incubation of Na-CP-3 in salt buffers containing dextran sulfate resulted in autoprocessing of the pro-region as detected by Western blot and catalytic activity was detected against Z-Phe-Arg-AMC. Activated Na-CP-3 did not digest intact tetrameric human Hb. The other three cysteine proteases (Na-CP-2, -4, and -5) were expressed in insoluble form in Escherichia coli. Antibodies to all four proteins (Na- CP-2 to 5) immunolocalised to the gut region of the adult worm, supporting mRNA amplification results and strongly indicated that they might play a role in nutrient acquisition. Hb digestion in blood feeding parasites such as schistosomes and Plasmodium spp. occurs via a semi-ordered cascade of proteolysis involving numerous enzymes. In Plasmodium falciparum, at least three distinct mechanistic classes of endopeptidases have been implicated in this process, and at least two classes have been implicated in schistosomes. A similar process is thought to occur in hookworms. An aspartic protease, Na-APR-1, was expressed in P. pastoris and purified protein was shown to cleave the class-specific fluorogenic peptide 7- Methoxycoumarin-4-Acetyl-GKPILFFRLK(DNP)-D-Arg-Amide. Recombinant Na- APR-1 was able to cleave intact human Hb and completely degrade the 16 kDa monomer and 32 kDa dimer within one hour. Recombinant Na-CP-3 was not able to cleave intact Hb, but was able to further digest globin fragments that had previously been digested with Na-APR-1. A clan MA metalloprotease, Na-MEP-1, was identified in gut tissue of N. americanus and was expressed in recombinant form in Hi5 insect cells using the baculovirus expression system. Recombinant Na-MEP-1 displayed proteolytic activity when assessed by gelatin zymography, but was incapable of cleaving intact Hb. However, Na-MEP-1 did cleave globin fragments which had previously been incubated with Na-APR-1 and Na-CP-3. Hb digested with all three proteases was subjected to reverse phase HPLC and peptides were analysed using Liquid Chromatography-Mass Spectrometry (LC-MS). A total of 74 cleavage sites were identified within Hb ƒ¿ and ƒÀ chains. Na-APR-1 was responsible for cleavage of Hb at the hinge region, probably unravelling the molecule so that Na- CP-3 and Na-MEP-1 could gain access to globin peptides. All three proteases were promiscuous in their subsite specificities, but the most common P1-P1Œ residues were hydrophobic and/or bulky in nature, such as Phe, Leu and Ala. Antibodies to all three proteins (Na-APR-1, -CP-3, -MEP-1) immunolocalised to the gut region of the worm, further supporting their roles in Hb degradation. These results suggest that Hb degradation in N. americanus follows a similar pattern to that which has been described in Plasomdium falciparum. Studies conducted in this project have identified a number of potential haemoglobinases and have demonstrated that the gut region of the hookworm contains a multitude of proteases which could be targeted for production of new chemotherapies or as vaccine candidates. Results presented here also suggest that the Hb degradation process occurs in an ordered cascade, similar to those which have been reported in other haematophagous parasites. More importantly, it has been confirmed that Na-APR-1 plays a crucial role in the initiation of the Hb degradation process and therefore targeting this molecule as a vaccine candidate could provide high levels of protection against hookworm infection.