Sanguinicola inermis Plehn, 1905, a recently introduced fish pathogen in Britain, has caused considerable damage to the carp industry. Two severely affected fish farms in England were included in the study. The incidence of fish infection for both farms was high, being 74-84% in 0+ and 1+ carp. Studies were made on the infection process and details of route of entry, migration and maturation of the worm are presented. An apharyngeate, furcocercous, lophocercous cercaria which develops in the snail Lymnaea (Radix) peregra (Mü11) was shown experimentally to develop into an adult Sanguinicola inermis. Maximum penetration of cercaria was achieved within 30 minutes and fins appeared to be the preferred site. Large numbers of worms were found to remain in the skin after penetration where they continued to develop to maturity, a previously unreported feature of S. inermis infection. The greatest migration to preferred loci occurred at 60 days post infection immediately prior to egg production. The migratory route used was found to be the loose connective tissue as well as the circulatory system. The distribution of mature worms in major blood vessels changed with season. Light microscopical studies were made on the morphology of specimens collected from the two farms and these were identified as S. inermis. Scanning electron microscopy of the surface topography and transmission electron microscopy revealed details of the tegument and provided evidence for the absence of spines. The major developmental features of the worm are described. A marked increase in size occurred up to 36 hours post penetration. Subsequent growth was slower. Egg production by mature worms began 10 weeks post infection at 15°C. Distribution and development of eggs in tissues is described. Egg production continued throughout the year with peaks during the summer months despite the constant environmental conditions. The growth rate of 0+ fry was studied over a period of 16 weeks and heavily infected fish showed stunted growth and poor Specific Growth Rates, Protein Efficiency Ratios and Food Conversion Ratios. Haematological studies showed that infected fish suffered from hypochromic macrocytic anaemia with leucocytosis and an increased Erkhrocyte Sedimentation Rate. Three different phases of infection were recognized. During phase I, the invasive stage, mortality may occur if infection levels are extremely high, but lightly infected fish present as clinically normal. Phase II was found to be the most critical phase since the majority of fish (over 90%) died at this stage. Histopathology revealed the progress of the infection from cercarial invasion to worm migration, maturation, egg production and miracidial hatching. Pathological changes were observed in the skin in phase I. In phase II, the heart, gills and kidneys were severely affected by both mature worms and developmental stages. The infection reached a chronic stage during phase III evidenced by a granulomatous tissue reaction largely in response to residual eggs in tissues. An attempt was made to integrate the phases of infection, development of the worm and pathogenesis in order to elucidate the host-parasite relationships.

No description available.

<I>Gyrodactylus salaris</I> Malmberg, 1957, a monogenean parasite of salmonid fish, has caused the death of up to 95% of salmon parr in 37 Norwegian rivers. In order to prevent further spread of this parasite, a reliable method of identifying <I>G. salaris</I> and distinguishing it from other closely related species is required. This study, the first investigation of <I>Gyrodactylus</I> genetics, has demonstrated that DNA technology can provide methods of gyrodactylid species identification suitable for routine use. DNA was extracted from <I>G. salaris</I> and two other species common on salmonid fish; <I>G. derjavini</I> and <I>G. truttae</I>. The small subunit ribosomal RNA (srRNA) gene was amplified from this DNA by polymerase chain reaction (PCR). The complete nucleotide sequence of the srRNA gene from <I>G. salaris</I> was determined. This was used to predict a secondary structure for gyrodactylid srRNA and to construct molecular phylogenies of platyhelminths including <I>Gyrodactylus. </I>Fragments of the srRNA gene from each species were compared by Denaturing Gradient Gel Electrophoresis. Mobility differences in <I>G. truttae</I> fragments were found and one fragment showed variation between and within species. The V4 region of srRNA was amplified from single specimens of gyrodactylids using a combined lysis and PCR reaction and sequenced. Examination of these sequences enabled prediction of Restriction Fragment Length Polymorphisms (RFLPs) between species and the design of oligonucleotide probes specific for each species. Digestion of the srRNA gene V4 region with two restriction enzymes produced restriction fragment polymorphisms that can be used to discriminate between <I>G. salaris</I>, <I>G. derjavini</I> and <I>G. truttae</I>. The digoxigenin labelled oligonucleotides GsV4, GdV4 and GdV4 are specific for <I>G. salaris, G. derjavini</I> and <I>G. truttae</I> respectively and can detect PCR amplified DNA from single specimens in dot blots. Both RFLP and probe methods of identifying <I>Gyrodactylus </I>species are suitable for use in diagnostic laboratories.

Leishmaniasis is a worldwide disease prevalent in many tropical and sub tropical countries. Treatment of Leishmaniasis by chemotherapy is not wholly effective and is usually accompanied by unpleasant side effects. The development of an effective and inexpensive vaccine represents a practical way to control the disease, however at present no safe and effective vaccine is available. In the first part of the present study, the immunity induced by four different L. mexicana potential vaccines, including killed leishmania vaccine, Soluble L. mexicana Antigen (SLA), L. mexicana gp63 cDNA and CT26 tumour cells transfected with L. mexicana gp63, were compared. It was shown that DNA immunisation using L. mexicana gp63 generated the highest immunity to the parasite among the four tested vaccines where the killed leishmania vaccine and L. mexicana gp63 transfected CT26 tumour cells did not generate significant immunity. The efficacy of DNA immunisation by intramuscular injection or using gene gun, in generating immunity to leishmania was compared. Gene gun immunisation induced more immunity to the parasite and high levels of Th1 immune response, which were detected, one week after immunisation through determination of the IgG2a levels in blood serum. Gene gun immunisation also induced long-lasting CTL activity, which was detectable before and during the course of infection for up to 6 months. Immunogenicity of MHC class I restricted peptides derived from L. mexicana gp63 have been investigated. Using 'SYFPEITHI' software, four peptides with high affinity to human HLA-A2 and four peptides with high affinity to mouse H2-Ld were predicted, synthesized and tested in HHD II and BALB/c mice respectively. Only three of the peptides predicted with high affinity to HLA-A2 were immunogenic but only two of them were likely to be naturally processed, however, none were protective in HHD II mice against leishmania infection. Purification and application of OX40L, a ligand for T-cell co-stimulatory receptor, was investigated in L. mexicana BALB/c model. In addition to purification by protein A sepharose, the murine OX40L-IgG fusion protein produced by B9B8E2 cells (cells transfected with OX40L and IgG) was successfully purified by two novel resins, MBI & MEP. The biological activity of the OX40L-IgG purified by MBI resin was significantly higher than that of MEP or protein A sepharose resins. Application of OX40L-IgG resulted in healing of leishmania lesions or delaying in development of the lesions in leishmania-infected mice.

Diphyllobothrium dendriticum and D. ditremum have a wide distribution in the trout of Scottish lochs, although no specific trends in their overall distribution have been observed. The seasonal recruitment trends and development of infections with Diphyllobothrium spp. pleroceroids in wild rainbow trout, in one particular Scottish loch, were monitored regularly for the period 1986 to 1989. Infection varied between these years, but in 1987 intensities from March to November reached their highest levels in November when sampling ended. These two species reached infection intensities, in individual fish, higher than any previous reports and trout stocks appeared to be affected. Plerocercoids, in host-derived cysts, were located on the peritoneal stomach surface, the gross pathology of which is described. The cellular components of the host-derived cyst were examined in detail by light and electron microscopy. A number of leucocyte types, found within a collagenous matrix with associated fibroblasts, were observed. Leucocyte types included neutrophils, eosinophilic granular leucocytes, macrophages and occasional plasma cells. Antibody production, in response to natural infection, was examined by indirect immunofluorescence using serum on cryostat sections of plerocercoids obtained from wild-caught rainbow trout. The tegument of these larvae showed a positive fluorescence, indicating the presence of serum antibody in these trout. Semi-quantitative estimates of antibody titres were estimated by an optimised enzyme-linked immunosorbent assay (ELISA). To facilitate further examination of the events associated with the establishment of plerocercoids and associated host-derived cyst, under controlled conditions, a routinely maintained laboratory life cycle of D. dendriticum was established. This laboratory life cycle utilised Herring gulls (Larus agentatus) and the copepod, Cyclops abyssorum, as the definitive and first intermediate hosts respectively, and provided plerocercoid infections in trout which were examined at various times post infection. Tegumental extracts from the plerocercoids of D. dendriticum, obtained by freezing and thawing specimens, and conditioned medium obtained after in vitro maintenance of live plerocercoids, were prepared. These extracts were tested in respiratory burst assays and were found to stimulate the production of both hydrogen peroxide and superoxide anion by rainbow trout macrophages. The migratory responses of trout macrophages and neutrophils to these agents were also investigated, by an optimised Boyden technique using a 48 Well Micro Chemotaxis Chamber. Leucocytes were found to have an increased chemokinetic motility following stimulation/contact with these antigen preparations. Finally, to investigate if antigens on live parasites were attractive/stimulatory, an in vitro adherence assay was carried out. Procercoid stages, which share common antigens with the plerocercoid stage, were maintained in vitro and incubated with rainbow trout leucocyte suspensions, in the absence and presence of normal or immune serum from infected fish. Leucocytes adherence was considerably increased by the presence of immune serum, indicating the possible interaction of the non-specific and specific immune response in the host inflammatory reaction.

No description available.

A thesis submitted to the Faculty of Health Science, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Doctor of Philosophy Johannesburg, 2014 / Malaria is the most serious tropical infectious disease in humans, caused by parasites of the Plasmodium genus and transmitted by anopheline mosquitoes. The interaction between the parasites and vectors has become a focus for malaria research as it may present an alternative disease control method by enhancing anti-plasmodial factors within the mosquito to impede parasite development and transmission. Anopheles gambiae is the best studied African malaria vector and is often used with the murine malaria parasite, Plasmodium berghei, for investigating parasite-vector interactions in the laboratory setting. Anopheles funestus has not been studied and its interactions with Plasmodium were unknown, until now. Although the Vector Control Reference Laboratory routinely maintains An. funestus and a number of An. gambiae colonies, none have been infected with Plasmodium since their establishment. This study aimed to use P. berghei to determine the vectorial capacity of these colonies and to examine the involvement of the 2La paracentric chromosomal inversion and antimicrobial peptides during Plasmodium infection in An. gambiae and An. funestus, respectively. Most of the An. gambiae complex colonies were susceptible to P. berghei, but the range of feeding and infection rates varied considerably. The infection rates for some of the older colonies were lower than previously documented. Anopheles funestus colonies were all viable vectors and there was an inverse correlation between the insecticide resistance profile and parasite susceptibility. Increased detoxification enzyme activities may have been contributing to a greater degree of parasite elimination. In An. gambiae, molecular karyotyping of the 2La inversion using PCR was validated against traditional cytogenetic techniques. The PCR was shown to be a reliable substitute for identifying the inversion. Using molecular karyotyping on 2La polymorphic colonies infected with P. berghei, it was found that infected females were more likely to carry the 2La inversion, indicating possible correlation between the inversion and susceptibility to parasites. In An. funestus, the expression of antimicrobial pepetide genes during P. berghei infection was examined using real-time PCR. Although all three genes showed increased activity at certain points of the infection, none displayed significant anti-plasmodial properties. However, in the less parasite susceptible strain, expression of two genes was higher towards the end of the infection, which was not observed in the other strains. It is possible that the co-expression of both peptides has led to a decrease in parasite load in late infection, but given the multi-factorial nature of the parasite-vector interaction, further investigation is required.

Leishmania are protozoan parasites responsible for the range of infections collectively known as leishmaniasis. Currently, drugs represent the only course of treatment. The nitroaromatic prodrugs are a class of agents that are used clinically used to treat trypanosomatid diseases; and in the parasites Trypanosoma brucei and Trypanosoma cruzi these compounds undergo reduction activation by enzymes homologous with bacterial type I nitroreductases (NTRs). From the Leishmania major genome database, we have identified a protein (LmNTR) that could catalyse this reaction. Based on co-factor, oxygen-insensitive activity and substrate range, we demonstrate that the LmNTR displays many characteristics of its bacterial counterparts. Gene deletion studies revealed that LmNTR is essential to the insect and mammalian stages of L. major. Null mutant parasites could not be generated while loss of a single LmNTR allele in the insect form conferred resistance to a range of nitroaromatic compounds without affecting their growth rate or ability to differentiate into infectious forms. Heterozygous lines could not establish an infection in vitro using a tissue culture model, or in vivo, in mice. As NTRs are absent from most eukaryotes, with trypanosomatids being a major exception, we exploit this difference to evaluate a library of nitroaromatic compounds against L. major parasites. Biochemical screens against the purified enzyme revealed that several compounds were effective substrates for LmNTR, generating higher activities than nifurtimox, the clinically used nitro-based agent that targets trypanosomes. Using phenotypic screens, we demonstrated that growth inhibition mirrored enzyme activity, with the most potent compounds generating IC50’s <100 nM whilst having little effect on mammalian cells. L. major NTR was shown to play a key role in parasite killing as heterozygous lines displayed resistance to the compounds. In conclusion, we have shown that LmNTR is essential to the parasite and by exploiting its prodrug activating properties, identified several novel agents that provide new lead structures to treat leishmanial infections.

No description available.

Thesis (M.SC. (Aquaculture)) --University of Limpopo, 2011 / The present MSc dissertation emanates from seasonal surveys conducted by the fish parasitological group of the Department of Biodiversity and Aquaculture Research Unit of the University of Limpopo, Turfloop Campus. The first part of the present study was aimed at investigating the metazoan parasites of three cyprinids occurring in the Nwanedi-Luphephe Dams. The main purpose of it was to determine temporal changes in the intensity of infestation in terms of prevalence, mean intensity and abundance of parasite species parasitizing the cyprinids studied over a two year period. Ecological parameters including species host-specificity, seasonality, and gender preference and host size versus species intensity are discussed for each parasite. Altogether 152 specimens were examined for parasites and a total of 2 432 metazoan parasites of ten species were recorded. At the sampling site, all three hosts co-occurred, however, a substantial proportion of Barbus radiatus was collected from the perennial stream feeding one of the twindams. Fish were sampled by means of gill nets and electrofishing or seine netting in accordance with the habitat conditions. Hosts were killed and organs investigated for metazoan parasites. After collection of parasites, standard methods for processing individual parasites were followed. The results obtained revealed the following groups of parasites; monogeneans (ectoparasites) included Dactylogyrus spinicirrus, D. afrolongicornis afrolongicornis, D. afrolongicornis alberti, Afrodiplozoon polycotyleus, Gyrodactylus sp., and Dogielius sp. (all recorded from the gills); Crustacea, Dolops ranarum was found from the mouth cavity, gills and skin of Labeobarbus marequensis. Of these, only two specialists, both monogeneans, were found on Barbus trimaculatus namely, D. afrolongicornis afrolongicornis and D. afrolongicornis alberti. Based on morphology of the haptoral hard parts, these two species were almost similar to each other than to D. spinicirrus. The appreciable difference between D. afrolongicornis afrolongicornis and D. afrolongicornis alberti was mainly in the shape of the marginal bar. Both D. spinicirrus and A. polycotyleus were widely distributed and recorded on the gills of all hosts during all seasons. Both species were recorded for the first time on B. radiatus. Also, D. spinicirrus was recorded for the first time on the gills of B. trimaculatus. Based on comparison with the original material, the species could be identified to species level. These analyses provided sufficient evidence for restoration of Afrodiplozoon polycotyleus as a valid taxon. The existence of two species, Gyrodactylus sp. and Dogielius sp. were recorded for the first time on B. radiatus in South Africa, and this possibly represents new species. The endoparasites included the following groups: digeneans- Diplostomulum metacercariae from the eyes of Lb. marequensis, Ornithodiplostomum sp. and black spot (grubs) were recorded from B. trimaculatus. The latter was also recorded in the muscle of B. radiatus. Unidentified digenean cysts were recovered from the gills and in the body cavity of both Lb. marequensis and B. trimaculatus; nematodes were represented by Contracaecum larvae in the body cavity of both Lb. marequensis and B. trimaculatus; cestodes were represented by gryporynchid larvae from the intestine of B. radiatus. The general high prevalence and intensities of ectoparasites recorded is an indication that the Nwanedi-Luphephe Dams has a biotic mechanism which might have enabled it to sustain the growth rate of ectoparasite intra-population. There was no correlation between either fish length or condition factor and the number of parasites. The study indicated that the abundance of monogeneans is partly influenced by season and that of endoparasites was principally governed by the presence of intermediate hosts and definitive hosts. The second part of this dissertation dealt with the health status of Lb. marequensis. Fish health was assessed using condition-related indices including condition factor and a modified Health Assessment Index (HAI) and the associated Parasite Index (PI). The HAI was performed to determine and examine any macroscopic abnormalities regarding external features and internal organs. The purpose of combining the two indices was to use the infestation of the metazoan parasites found on and/or in Lb. marequensis to determine whether or not the environment they live in was healthy. Both indices together with the condition factor provided relatively simple and rapid indications of how well fish were coping in their environment. The HAI score varied amongst the four sampling seasons. The highest individual mean value was 63 in winter, followed by a score of 50 in autumn, while the lowest were 42 and 33 in summer and spring respectively. To authenticate the HAI and PI data, certain water quality variables were measured and are discussed in detail in this dissertation. The Nwanedi-Luphephe Dams are generally believed to have good water quality. This was supported in this study; conditions assessed in fish using the aforementioned indices did not differ greatly between seasons, nor did the conditions deviate appreciably from normality. The HAI values were low overall which signifies a healthy fish profile for the system. The present investigation showed the existence of differences in the occurrence of individual parasite to be linked to water temperature changes. Thus, seasonal changes do influence parasite developmental stages to a certain degree. Tested heavy and trace metals were within the permissible limits as provided by the Department of Water Affairs and Tourism (DWAF, 1996).