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Thesis (PhD (Biology))--University of Limpopo, 2010. / Digenean parasites are known to be a large and diverse group of parasites. Some of these parasites are free-living, a few are ecto-parasitic, while the majority are endo-parasitic in most invertebrates and vertebrates. Digenean parasites have always been known to be host specific. However, the degree to which these parasites are host specific, is determined by the number of hosts they are able to utilise and the parasitic stages they would currently be at. Morphologically the link between the cercarial and other parasitic stages such as metacercarial and adult stages, were found to be very difficult to establish, since different developmental stages utilise different types of hosts. For instance, cercariae may use the same or different hosts for their metacercarial stages. An example of this is in the case of freshwater snails where the cercariae re-penetrate the same snail and encyst as metacercariae, and then the snail hosts serve also as second intermediate hosts. Adult digenean parasites on the other hand utilise vertebrate hosts different from those serving as second intermediate hosts, as final or definitive hosts. Digenean trematodes like any other helminth parasites have been well researched for decades due to their widespread health-related diseases that they cause and their economic impact globally, especially in third world countries. Research in this field included aspects of species diversity, morphology, distribution, epidemiology and immunology. Despite all of these aspects, these parasites continue to thrive in the face of numerous strategies aimed at their control. Lately polymerase chain reaction (PCR) techniques have been employed to assist with parasite biology and identification, especially with regards to round- and flatworms. Several genome projects like the Schistosoma Genome Project (SGP) initiated in 1992, was established in an attempt to create gene banks and to allow researchers to utilise technology for genomic analysis in the study of organisms relevant to public health in developing countries. ABSTRACT________________________________________ The methods of gene discovery and their functional discovery have been accelerated significantly and are being progressively applied in numerous organisms of medical and veterinary importance. On the other hand parasitic helminths lag behind parasitic protozoa in the sense that in vitro cultivation systems have not been developed to support the entire life cycle of these helminth parasites, genomic databases are far from being complete and lastly there are no established methods for the highly efficient manipulation of endogenous genes within living worms. The present study was aimed at supplying morphological descriptions and additional information through PCR techniques to enable us later to complete the life cycles of the lesser known parasites experimentally. The study was achieved by collecting materials from six localities, namely Boekenhoutskloof farm dam, Supersand dam, Rietvlei dam, Kiewiet farm dam and Northern farm dam. All these five localities were located in the Gauteng Province, proximal to Tshwane. The sixth locality was Metsi-pepa in the North-West Province that was selected due to the unique eye source that feeds the Mooi River. The collected materials were then studied employing standard light and scanning electron microscopy techniques, as well as applying PCR techniques in order to identify and classify the digenean parasites collected during the study. Life cycle studies were also attempted through experimental infections of potential definitive hosts. Seven different types of snail species were collected during the research study, namely Bulinus africanus, Bulinus tropicus, Lymnaea natalensis, Gyraulus connollyi, Burnupia mooiensis, Biomphalaria pfeifferi and Ferrissia fontinalis. Of these, Lymnaea natalensis was found to be the most abundant snail of the entire snail species collected over a period of four years. Of the above-mentioned snail species only four types were found to be infected with various types of cercariae. ABSTRACT________________________________________ Lymnaea natalensis produced three different cercarial types: a) strigeid cercaria B with its characteristic three pairs of linear penetration glands and a very large sinous intestinal caeca, b) a xiphidio cercaria with three pairs of penetration glands, and c) an avian schistosome cercaria. Bulinus tropicus was found to produce two cercarial types: a) an echinostome cercaria with a collar consisting of 27-spines and b) strigeid cercaria A. The third infected snail species was B. africanus, found to be infected with only one type of cercaria, namely xiphidio cercaria B with its characteristic clustered penetration glands. The fourth infected snail species, G. connollyi, housed two types of monostome cercariae: a) monostome cercaria A possessing 3 pairs of linear penetration glands and b) a clinostomatid cercaria with its characteristic head membrane. Nine metacercarial types were collected from various second intermediate hosts. 27-spined echinostomatid metacercaria A and 43-spined metacercaria B were found encysted on the gills of the following fish hosts: Pseudocrenilabrus philander and Tilapia sparrmanii, as well as in the mantle of the snail hosts, L. natalensis and B. africanus. Two strigeid metacercarial types, a) strigeid metacercaria A encysted within a green cyst and b) strigeid metacercaria B with distinct fore- and hindbodies, were collected from the fish hosts, P. philander. Strigeid metacercaria A was sporadically also found in T. sparrmanii. Two diplostomatid metacercariae were collected from their fish hosts, a) diplostomatid metacercaria A from the cranial cavity of Clarias gariepinus and b) diplostomatid metacercaria B from the vitreous chamber of T. sparrmanii and P. philander. Two metacercarial types of the family Clinostomatidae, a) a clinostomatid metacercaria and b) an Euclinostomatid metacercaria, were collected from the buccal cavity and the muscle tissue of T. sparrmanii, respectively. The last metacercarial stages, namely xiphidio metacercariae, were collected from various hosts such as freshwater shrimps (branchial region), T. sparrmanii (gill filaments) and L. natalensis (mantle). This parasite was found to have developed sexually more in freshwater shrimps compared to the same stage in other infected second intermediate hosts. ABSTRACT________________________________________ Three adult parasites were also identified and described. They include an amphistome fluke of the genus Cotylophoron and the liver fluke, Fasciola gigantica. These two parasites were collected from the rumen and the hepatic ducts of a heifer at the Northern farm, respectively. The third fluke, Echinoparyphium elegans, was obtained from experimentally infected kittens. It was, however, difficult to link the different stages within the same family using only morphological characteristics. The morphological characterization of digenean parasites, especially the adult stages, has been well-documented over the past few decades worldwide. This has, however, not been the case with larval stages. Recent studies have shown that there were many attempts by researchers pertaining to molecular studies using PCR techniques. In most cases the studies were achieved by using matured (adult) stages of digenean parasites. These include the studies done on digenean parasites at species level, family level, superfamily level, suborder level and on general digeneans. Most of these phylogenetic studies were only conducted on medically and veterinary important digeneans. The present study focused more on the amplification of parasites at family level. The universal primers were used to target ITS-1, ITS-2 and LSU regions. Not all the specimens yielded desired amplicons. Only certain stages of the following four families; a) Clinostomatidae, b) Schistosomatidae, c) Echinostomatidae and d) Strigeidae were able to be amplified and sequenced. From this study, it is evident that in future, specific primers for specific digenean parasites need to be designed and used in order for us to achieve our desired goals (i.e. being able to amplify as many digenean specimens as possible including the lesser known trematodes). ABSTRACT________________________________________ The recent study also demonstrated that much more work needs to be done in order for us to understand parasite-host relationships in the localities studied. Experimental life cycle studies are therefore imperative in order to solve most of our cercaria, metacercaria and adult trematode questions raised during the present study.

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The widespread use and subsequent release of pesticides into aquatic environments have sparked concerns about how organisms within these aquatic systems are affected by pesticide pollution. While many studies have examined the effects of pesticides on individual organisms, in a series of experiments, I investigated the effects of a pesticide mixture on members of a complex host-parasite system and on host susceptibility to infection. In my first experiment in the laboratory, I examined changes in survivorship when trematode parasites (Echinostoma trivolvis) and their first intermediate host, Planorbella trivolvis snails, were exposed to a low concentration (10 ppb: 15 ppb) and high concentration (85 ppb: 100 ppb) mixture of atrazine and metolachlor, respectively. There was a significant decline in parasite survivorship in the high concentration treatment at 14 hours, while snail survivorship was unaffected across all treatments. In my second experiment, prior to infection, I exposed the parasites and/or second intermediate hosts, Rana clamitans and Rana sylvatica tadpoles, to the pesticide mixtures and examined subsequent infection levels in the tadpoles. The atrazine and metolachlor mixtures had no significant effects on parasite load in the laboratory. Newly shed parasites were more likely than 10 hours old parasites to infect tadpoles, regardless of pesticide exposure. In my final experiment, I utilized outdoor mesocosms to expose parasites, snail hosts, and Rana sylvatica tadpoles to the pesticide mixture, and I examined differences in parasite load within the tadpoles after two weeks. The pesticides had no significant effect on parasite loads in the field. Overall, my findings suggest the atrazine and metolachlor mixtures used in this study had no significant effects on disease dynamics in a system involving Echinostome parasites, snails, and tadpoles. / Master of Science

Brachylaima spp. (Digenea: Brachylaimidae) are terrestrial trematodes of mammals and birds and have land snails as their first and second intermediate hosts. This thesis describes a new species of Brachylaima and investigates infection in both snail intermediate hosts and definitive host animals. A laboratory life-cycle was established using brachylaimid eggs recovered from the faeces of an infected human. Five species of introduced European helicid and hygromiid snails, Theba pisana, Cernuella virgata, Cochlicella acuta, Cochlicella barbara and Microxeromagna armillata were susceptible first intermediate hosts. These same snails and introduced Helix aspersa as well as the native snails Succinea australis and Strangesta gawleri were suitable second intermediate hosts. Field and laboratory studies revealed that in addition to humans and mice, various species of birds and reptiles were also definitive hosts. On the basis of its unique morphological and lifecycle features, a new species, Brachylaima cribbi was described. The scanning electron microscopical appearances of the various life-cycle stages were detailed. Studies of Swiss albino outbred mice and 8 strains of inbred mice revealed that C57BL/6J mice were most susceptible to B. cribbi infection. The peak infection occurred 4 weeks after inoculation with metacercariae following which worms were expelled over the next few weeks. Exposure to a second infection in C57BL/6J mice did not result in accelerated expulsion of adult worms but did significantly inhibit their fecundity. In contrast, when immunodeficient NOD SCID mice were infected with B. cribbi metacercariae the adult worms persisted for the life span of the host mice. 6,432 land snails were collected over a distance of 3,000 km across southern Australia. Sporocyst-infected snails were found in all districts of South Australia and Victoria with the percentages of infected T. pisana, C. virgata, C. acuta and C. barbara ranging from 1.7 to 4.7%. These 4 species together with M. armillata, S. australis and S. gawleri were infected with metacercariae being found in 18-63% of snails and the mean number of metacercariae per infected snail ranged from 2.1 to 6.1. Laboratory studies revealed that eggs may remain viable for almost 12 months in mouse faeces. The prepatent period for a sporocyst infection is 7-10 weeks after egg ingestion. Metacercariae 7 weeks of age are capable of developing into adult worms. Detailed studies of seasonal variations in sporocyst and metacercarial infection rates were studied at 4 ecologically diverse sites on the Yorke Peninsula of South Australia. The clinical features and epidemiological circumstances of B. cribbi infections of 12 humans are detailed, as is their satisfactory response to treatment with praziquantel. / Thesis (Ph.D.)--School of Molecular and Biomedical Science, 2003.

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Digenetic trematodes are a common pest problem in aquaculture where their unappetizing appearance often reduces the marketability of food fish. Aquatic snails are intermediate hosts in the trematode lifecycle and are commonly targeted with control measures to prevent the crop fish from becoming infected. I evaluated several chemical and biological snail control strategies as alternatives to the potentially invasive black carp. Copper sulfate, hydrated lime slurry, and several fish and decapod species were tested for effectiveness against physid (Physa spp.) and planorbid (Helisoma spp.) snails in laboratory aquaria trials. Hydrated lime demonstrated effectiveness with the least potential to be toxic to cultured fish in regional application. Hybrid sunfish (redear × green sunfish) consumed large quantities of both snails in ad libitum feedings. The most effective biological (redear × green sunfish) and chemical (hydrated lime) control methods identified in the laboratory were evaluated further in research ponds. Hydrated lime applications of 9.07 kg over 9.14 m2 were found to be effective against Helisoma spp. confined to enclosures along the pond shoreline; average survival was 2%. When stocked in aquaculture ponds, hybrid redear sunfish did not significantly influence snail capture rates; however ponds stocked with redear sunfish experienced a gradual decrease in snail populations throughout the 2008 growing season. Hydrated lime and a combination of redear sunfish and hybrid redear sunfish were evaluated separately and in tandem as a combined chemical/biological treatment in the 2009 growing season. Evaluation occurred under mock production conditions in which hybrid striped bass were raised in the research ponds to determine snail treatment effects on trematode abundance. Ponds stocked with sunfish at 494 fish/ha had snail densities significantly (P ≤ 0.05) lower than control ponds after two months. Ponds treated with hydrated lime at 31.7 kg/31.5 m of shoreline in a 1 m swath experienced 99% estimated reductions in snail densities following application, but snail populations rebounded to previous levels within two months. The mean snail density in ponds treated with both hydrated lime and sunfish was significantly lower than control one month post treatment; this mean rebounded slightly by the conclusion of the trial, but not as much as in the chemical treatment group. Hybrid striped bass examined thoroughly for trematodes revealed a positive relationship between trematode abundance in fish and increasing Helisoma densities. This relationship was most apparent when estimates of snail density from only the beginning of the trial were used. Based on these results, it appears that a nearly complete reduction of Helisoma, particularly at the time of stocking fingerlings, is necessary to avoid a high abundance of trematodes in cultured fish. To this end, an early-season application of molluscicides followed closely by stocking of predator sunfish has potential to achieve a uniformly low density of snails throughout the growing season.