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Pomatoschistus microps at Salthouse Point, Burry Inlet, Wales return to the estuary and spawn during June, the 1-group adults then disappear and presumably die but the developing juveniles become heavily parasitized with overdispersed, innocuous, encysted digenean metacercariae namely Cryptocotyle jejuna and C.lingua in subcutaneous muscle, C.concava (Heterophydae), in the body cavity, Labratrema minimus (Bucephalidae), in the liver, Timoniella imbutiforme and T.praeterita (Acanthostoidae) in subcutaneous muscle. Host weight, condition and other growth parameters do not appear to be affected by these parasites. However, declining rates of heterophyid infections after August suggest that host defence responses or hyperparasitism may kill some parasites. Gobies leave the estuary after November and, as suggested by increasing C.lingua and L.minimus infections, may migrate to overwinter near West Cross, Swansea Bay. Sixteen allozyme loci were resolved in these fish, using gel electrophoresis based on a sample of 575 fish. Five of these loci are polymorphic namely LDH, IDH, PGM-1, PGM-2 and PGI. The mean heterozygosity per locus is 0.06 - 0.004 and individual heterozygosity is not usually correlated with host growth parameters but is usually negatively correlated with parasite burden. The relationship is statistically significant, however, only with C.jejuna in September, the sole parasite species which elicits an intense melanized host defence response namely 'black spot' disease. The possible reasons for this are discussed in detail. In Pomatoschistus minutus at Oldbury, Severn Estuary, very lightly parasitized with the larvae of Ligula intestinalis (Cestoda: Pseudophyllidea) and Hysterothylacium sp. (Nematoda: Anisakidae), five of seventeen loci are polymorphic. The mean heterozygosity per locus is 0.03 - 0.014. The results provide equivocal support for the parasite version of the Red Queen hypothesis which predicts that the most heavily parasitized populations will have the highest mean heterozygosity and, within populations, individuals with least parasites will have highest heterozygosity.

In recent years the bewildering diversity of myosin types and function has become apparent. Over ten different classes have been identified in a wide variety of organisms - ranging from protozoans to vertebrates and even higher plants. The intriguing question is - what do they all do? C. elegans, "the worm", is a relatively simple organism, both anatomically and genetically and has been studied extensively with respect to development, cell lineage and genetics. However only myosins of the conventional type (muscle or "class II" myosins) had been identified prior to the work described here. The first novel myosin from C. elegans has been cloned and fully sequenced. The predicted amino-acid sequence shows that this myosin contains the conserved motor or "head" domain responsible for actin-activated ATPase activity. This is followed by two motifs thought to be capable of interaction with members of the calmodulin class of Ca2+ binding proteins and a tail with a general positive charge. The sequence of the entire protein, along with expression and immunolocalisation pattern, suggests that it may be the nematode homologue of a recently discovered unconventional myosin from the rat, myr4. A multiple alignment of the 86 available conserved motor domain sequences has enabled the construction of an unrooted phylogenetic tree, which indicates that the myosins fall into 13 classes. C. elegans Myosin IA is a member of the class I myosins. The conservation of residues within this alignment has been explored further by scrutinising their position within the available myosin crystal structures. From this important residues involved in myosin function can be identified. Furthermore, the alignment allows the positioning of residues from myosins, whose structure is unknown, within the framework provided by the existing crystal structures - enabling, for example, the interpretation of existing mutations in unconventional myosins.

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.

This study is divided into four sections beginning with a consideration of the effects of an hitherto undescribed coccidian parasite of the ovary of this bivalve on the dynamics of a particular population from Kames Bay, Millport, Isle of Cumbrae, Scotland. The parasite was found to bring about complete or partial castration of female Tellina tenuis but had no effect on males. The general biology of Tellina-tenuis is reviewed and discussed in relation to observations that were carried out over one year on the age structure, growth, density, reproduction and degree of parasitization of this bivalve. The population parameters were found to have changed little over the last fifty years. There was not found to be any significant change in the condition index that could be related to the destruction of the gonad by the parasite. This is the first description of a coccidian parasite of the ovaries of any marine mollusc. The second section describes the life cycle and developmental stages of the coccidian parasite. The parasite is believed to be monoxenous with sporogony and anisogamy orcurring in the ovary of the host. Endogenous stages were observed in the primary germ cells of the gonadal follicles. A diagnosis is presented based on the number of sprozoites per sporocyst and sporocysts per oocyst. The name Mcrocystis tellinovum (sp. nov. ) is proposed for this coccidian. The genus Merocystis belongs to the family Aggregatidae within the sub-order Eimeriorina of the order Eucoccidiorida. An ultrastructural study of both sexual and asexual stages is presented in support of this diagnosis. The third section of this study is an investigation of the observation that a large proportion of the Tellina tenuis examined histologically contained inclusion bodies within the secretory cells of the digestive gland. These inclusions were comprised of dense masses of pleomorphic mycoplasma-like organisms. The first part of this section describes the morphology and ultrastructure of these organisms and the histopathological effects on the host digestive gland. A description of the normal digestive gland iss-given including observations of the cyclic changes in appearance that take place in response to the influence of tidal rhythms. This is believed to be the first description of a mycoplasma-like organism from a marine invertebrate. The fourth section is concerned with a series of experiments to determine the nature of a virus seen in association with the mycoplasma-like organism. The virus was isolated from the host cells by density gradient centrifugation and its morphology was compared with a second virus isolated by Hill (1975) through the medium of a fish cell culture. It was found that these two viruses were quite distinct from each other. Attempts were made to propagate both viruses in an established cell line from the Atlantic salmon and the results are described and discussed.

The present study investigated aspects of the biology of the cestode, Proteocephalus filicollis from the three-spined stickleback Gasterosteus aculeatus from Airthrey Loch, Scotland. The population biology study demonstrated that the parasite has an annual cycle of recruitment, which occur mostly in late summer and early autumn. The cestode did not show preference for any sex of the host. Maturation of the cestode also showed a seasonal cycle with the majority of worms maturing in late spring and early summer, but this period may be extended in different generations. Proteocephalus filicollis was overdispersed throughout the year in all sizes of fish, moreover variance to mean ratio always exceeded unity. No severe pathology was observed due to attachment of the worm to the intestine of the fish. The worm population in different sections of the intestine varied according to season and maturity stage. The P. filicollis migrate from the rectum to the anterior intestine as they mature and it is suggested that growth and maturation of the worm is a major stimulus for this migration. Proteocephalus filicollis has a high fecundity as indicated by the higher number of eggs per mm of gravid portion of the strobila and high fertility. Infrapopulation size did not show any relationship with length of worm, percentage gravid portion, number of gravid segments or mean length of gravid segments. Numbers of eggs are correlated to length of the worm, but not to infrapopulation size. Numbers of eggs per mm of the gravid portion are not correlated to length of worm or infrapopulation size. Acanthocyclops robustus was used as an experimental intermediate host. 15-16°C was the optimum experimental temperature for growth and a fully developed larva was formed in 23-27 days at this temperature. No growth was observed at 4°C, growth was slow at 10°C, but rapid at 21-22°C. The eggs are infective for 25 days at 4°C, 10°C and 15-16°C, but for only 15 days at 21-22°C. Prevalence and mortality of copepods are significantly correlated to their exposure time to parasite eggs, but mean intensity of infection did not show any relationship to the exposure time to the eggs. Ultrastructural studies demonstrated that a mature egg is surrounded by at least four embryonic envelopes, the capsule, the outer envelope, the inner envelope, and the oncospheral membrane. All these envelopes originate differently and undergo definite changes during their development.

This research study is divided into 4 major sections. Section A deals with the world-wide distribution of the haematoprotozoan parasites of marine fishes. These are tentatively divided into 3 major groups: the Haemogregarines, the Trypanosomes and the Trypanoplasms and one group of ill defined and controversal organisms including Haemohormidium sp. and Dactylosoma sp. The results of extensive surveys of the coastal waters of France, Scotland and Wales with added examples from the Mediterranean Sea substantiate the zoogeographical distribution of these parasites. Two species of haematoprotozoan parasites Haemogregarina simondi and Trypanosoma soleae are re-described, earlier accounts dating from the beginning of the century being considered incomplete. A new species of haemogregarine is described from the farmed turbot (Scophthalmus maximus) as Haemogregarina sachai n. sp. and an unidentified species of Haemohormidium found occasionally in turbot and Dover sole (Solea soles) is also described. In addition Haemogregarina sp. were encountered in certain wild marine fish from the Atlantic coast of France e. g. Zeus faber, Sebastes sp., Trisopterus luscus, Pagellus bogaraveo and Raja sp. and from the coastal waters of Malta e. g. Peristedion cataphractum and Oblade melanura. However, they were not found in sufficient numbers to allow a definite description. The value of surveys of wild fish populations is discussed in the light of zoogeographical distribution, the apparent periodicity of these parasites and a seasonal variation of parasitism. Section B attempts to summarize the knowledge of the mode of transmission of marine haematoprotozoan parasites by piscicolid leeches as intermediate hosts and vectors. The developmental stages of a trypanosome, probably Trypanosoma murmanensis from the Atlantic cod Gadus morhua are described in the marine leech Calliobdella nodulifera. Stages of a haemogregaririe were observed in the same leech. The development of the turbot haemogregarine Haemogregarina sachai n. sp. in artificially infected leeches is also described up to the 20th day post infection. Transmission of this haemogregarine to apparently uninfected turbot via this leech was not successful. Various stages of development of Haemogregarina simondi are described in its apparently natural vector, the marine leech Hemibdella soleae, and transmission with infected leeches to apparently healthy hatchery reared soles was achieved. Thus it was shown for the first time that marine leeches can serve also as vectors for haemogregarines. Stages of this haemogregarine are also described in the blood-sucking ectoparasitic copepod Lernaeocera sp. parasitizing the haemogregarine infected soles. These results are discussed in relation to the feeding behaviour and migration patterns of the fish hosts, the periodicity of the parasites and possible other vectors or other ways of acquiring an infection with these haematoprotozoan parasites. In Section C the pathogenicity of the haematoprotozoan parasites of marine fishes is summarized from previous accounts among wild fish populations and compared with the situation in aquaculture. The pathogenicity of the two haemogregarines, Haemogregarina simondi and Haemogregarina sachai n. sp., accidentally introduced into several fish farming establishments connected with the effluent cooling waters of a nuclear power station is described with special reference to the possible source of the infection. The results of therapy trials and control programs are discussed in the light of the periodic reappearance of the parasites, the possibility of carrier fish existing, the immune status of the host and the possible role of an intermediate host or vector in maintaining the infection. Possible means of controlling the pathogenicity of marine haemogregarines and perhaps other haematoprotozoan parasites when they occur in farmed marine fishes are also discussed. In Section D are described for the first time, the ultrastructural characteristics of various stages of the haematoprotozoan parasites of marine fishes in fish hosts and vectors. The electronmicroscopical studies are limited to Haemogregarina sachai n. sp. and Haemogregarina simondi, for which the ultrastructure of schizonts from the spleen and blood, intracellular merozoites and free gametocytes were contrasted. In addition stages of H. simondi were demonstrated in Hemibdella soleae and Lernaeocera sp. The fine structure of the various organelles encountered was compared with that of related organisms from other cold-and warm-blooded vertebrates. In conclusion attention is drawn to the need for more investigations in this field of host-parasite relationship of marine haematoprotozoan parasites and their vectors and their pathogenic-action as seen in a confined and artificial environment such as the marine aquaculture.

The literature relating to the development of the technique of arthropod tissue culture, its application to biological research, and the cultivation of trypanosomes is reviewed. Cultivation of tissues and organs from Glossina morsitans morsitans was investigated using several culture media. Of the media tested, modified Trager's medium was found to be the best for the growth and maintenance of tissues. Successful cultures of developing adult tissues from ticks, Rhipicephalus appendiculatus, R.bursa and Amblyomma hebraeum and from partially engorged adult Ixodes ricinus were obtained in VP4 medium. The greatest success in the cultivation of Trypanosoma brucei and T.congolense in association with Glossina tissues was achieved in the presence of complete alimentary tract of pupae older than 21 days in 15 -20a1 hanging -drops of modified Trager's medium incubated at 28 ?C. Growth of the trypanosomes also occurred in cultures in which the flagellates were separated from the insect tissues by a semi -permeable membrane. No multiplication of the parasites was obseived in (a) culture medium alone, (b) medium containing extracts of alimentary tract and (c) medium in which alimentary canal had been cultured for 3 or 4 days. In all the cultures in which multiplication of the trypanosomes occurred, it was accompanied by morphological transformation. Bloodstream trypomastigotes in the inoculum changed into long slender forms similar to those seen in the midgut of the tsetse fly. Infective stages were not observed. Complete digestive tract from G.morsitans submorsitans, G.austeni and G.palpalis was as suitable as that from G.m.morsitans for the cultivation of the trypanosomes and the same applied to the alimentary tract of the non- haematophagous fly Sarcophaga, a dipteran closely related to Glossina. On the other hand, cultures of comparable tissues from mosquitoes, Aedes aegypti and ticks, R.appendiculatus failed to support the growth of trypanosomes. Fly transmissible as well as laboratory adapted strains of T.brucei and T.congolense could be cultivated in tsetse organ cultures. Negative results were obtained, however, with laboratory adapted strains of T.gambiense and T.vivax and with the monomorphic T.evansi which is not cyclically transmitted in nature by tsetse flies. Stercorarian species, Trypanosoma musculi, T.lewisi, T.mzilophagium and T.theileri were also grown in the presence of tsetse alimentary tract. All these species transformed into and multiplied in forms resembling those formed in the hindgut and rectum of their arthropod vectors. Unlike the cultures of the salivarian trypanosomes, those of T.musculi and T.lewisi remained infective to laboratory rodents for many days. Amino acid analyses of haemolymph of pupae, pre- emerged and adult G.morsitans revealed that of the 21 amino acids, proline was present in highest concentrations in all the stages examined. There were also considerable differences in the concentrations of the individual amino acids in these three stages. The results of the amino acid analyses formed bases for the design of culture media which were capable of supporting growth of T.brucei and T.congolense.

In part one, epidemiology of fasciolosis in eastern Nepal, a 19 months field survey on the epidemiology of fasciolosis is described. Four Lymnaea spp; L. auricularia race rufescens, L. auricularia sensu stricto, L. viridis and L. luteola were identified. L. auricularia race rufescens was the predominant species. The snail main habitats were spring or stream fed rice-fields, irrigation channels, ponds and road-side pools. The monsoon rains and rice cultivation practices contributed to the creation and expansion of the habitats. The snail population density was high during the dry period and declined with the onset of the monsoon. Snail egg masses and young snails were observed throughout the year. Mature Fasciola spp, infections were found in the hills from May to February and throughout the year in the Terai. In part two, experimental studies on pathogenesis of fasciolosis with special reference to its effects on productivity of ruminants is described. Monitoring included clinical, parasitological, haematological, biochemical and pathological observations. Pilot comparative studies in Scottish Blackface and Suffolk cross sheep conducted in Edinburgh indicated that F. gigantica was more pathogenic than F. hepatica. In another pilot experiment in Nepal using local Baruwal sheep, it was also found that very low infections with F. gigantica caused measurable production losses. Pathogenesis in goats was investigated using Nepalese hill goats. Infection caused production losses including weight loss. Burdens of more than 1.3 flukes/kg of initial liveweight produced clinical chronic fasciolosis. In part three, the relative merits of the current methodologies for speciation and differentiation of Fasciola spp. are reviewed. The development of species-specific (MHFh and MHFg) and cross-reactive (MHFx1 and MHFx2) DNA probes for the identification of Fasciola spp. are described. If used in conjunction these probes clearly differentiate F. hepatica and F. gigantica.

This work is a study of both active and passively acquired immunity to Taenia taeniaeformis infection. The role of humoral components in serum as demonstrated by passive transfer of immunity and both active immunity following infection or immunisation were investigated. The antigens derived from larval T. taeniaeformis for immunisation studies were also used to raise immune sera, to monitor experimental infections, and to absorb out protective antibodies. Netacestodes retrieved from mice were maintained in a defined medium for between 10 and 23 days. The tegumental integrity of the metacestodes during maintenance was assessed by dye uptake and transmission electron microscopy (TENT). The medium in which the larvae had been held was used as a source of excretory-secretory antigen (ESA). Somatic antigen (SA) was derived from a saline extract of homogenised larval somata. Anti sera raised against both ESA or SA preparations, were used to detect antigens in the used medium by the enzyme linked immunosorbent assay (ELISA). After verifying that the ELISA technique could quantitate antigens, and that the assay was not affected by the presence of serum in the medium, the effect-of several factors on antigenic activity was investigated. The antigenic activity of the used medium did not appear to be unduly affected by the age of the larvae, by sealing the maintenance vessels, by variations in the integrity of the larval tegument or by the absence or presence of serum during maintenance. However, the beneficial effect of including serum in the maintenance was reflected in the dye uptake and MM results. The aim of studying these factors was to determine the optimal conditions under which the larvae would release 7nax4ma1 amounts of ESA. ELISA was also used to monitor the humoral response of mice and rats experimentally infected with TT taeniaeformis against either ESA or SA as the detecting antigens in the assay. It was found that ESA was a more sensitive indicator of the infection than SA. The ability of ESA and SA to protect rats against infection was investigated. Both these crude antigenic complexes seemed to possess similar immunising capacities. An association was found between the reactivity of these antigens in vitro by ELISA and their immunising or protective activity in vivo, which suggests that such antigens may provide a useful measure of the levels of protective antibody in the serum of the host. Homologous and heterologous immune sera were obtained by immunising rats and sheep respectively with ESA or SA. The immune sera obtained in this manner was protective in passive transfer experiments. This appears to be the first time that serum from animals immunised with ESA has been demonstrated to confer protection against a helminth infection. Immune serum from experimental infections of six weeks duration conferred almost absolute protection, confirming previous published reports. The highly protective immune globulins from active infections was absorbed with ESA, SA or live larvae. The absorption had a measure of success when assessed by in vitro estimation. However, when used in passive transfer, the absorbed globulins retained their protective ability. Several possible reasons were adduced for this apparent failure. Thus there may have been insufficient concentration of antigen to remove all the protective antibodies from the highly active serum. Alternatively the protective antibodies produced in an active infection may not be induced exclusively by the soluble components in ESA or SA, but possibly by both of these and even other antigens. Either of these suppositions would account for the partial efficacy of absorption found in vitro.

This thesis sets out to understand interactions between T cells and T. annulata infected cells, both in vitro and in vivo and the consequences for the generation of immunity. In vitro stimulation of peripheral blood T cells from naive animals by IC caused the cells to proliferate, peaking 5 days post stimulation. Phenotypic analysis showed that CD25 and MHC class II were expressed upon the surface of all T cells (CD4, CD8 and γδT cells) within 24hrs of stimulation, reaching a peak at 48hrs and remaining stably expressed for up to 7 days post activation. The parasite infected cells could activate both "memory" and "naive" CD4 T cells, with little change in the CD45RB isoforms during activation. Activation of T cells was contact dependent. T annulata infected cells can therefore cause the activation of the majority of T cells from naive animals irrespective of memory status and , presumably, antigen specificity. The cytokines produced by IC stimulated T cells 1-7 days post stimulation were assessed by reverse transcription polymerase chain reaction (RT-PCR) using primers for IL2, IL2 receptor (IL2R), IL4 and interferon gamma (IFNγ). None of these cytokines were found to be expressed by IC. T cells within PBM expressed mRNA for IL2, IL2R, IL4 and IFNγ 24-48 hours post IC stimulation. IL2 and its receptor were still expressed at day 5 (peak proliferation), and waned by day 7. IFNγ was expressed by all tested animals' cells at all timepoints, while IL4 was intermittently found at day 5 and was always absent at day 7. IL4 was only expressed by CD4 T cells, while IL2/IL2R/IFNγ was expressed by all T cell types. The presence of CD4 cells was required for IL2 and IL2R expression by non CD4 T cells. In summary, this thesis has shown that T. annulata infected cells possess an innate ability to activate naive T cells. Although all T cell types are activated in PBM, this is dependent upon cytokine release by CD4 cells, subsequently leading to a type 1 response. In vivo, a similar mechanism leads to activation of DLN T cells primarily by IC. Such interactions do not lead to the induction of an antigen specific immune response, but to the loss of GC and suppression of further T cell activation.

The work described in this thesis examined 44 stocks of T.evansi, 41 of which were isolated from camels in various areas of Kenya. These trypanosomes had a mean length of 25.5 ± 2.8 μm a mean pre-patent period of 4.7 ± 3.4 days and fulfilled the classical criteria of this species in regard to these two parameters. Initially, the sensitivity of these trypanosome stocks was determined to four trypanocidal drugs active against T.evansi. The 44 stocks exhibited resistance to Berenil (57%), Samorin (7%), suramin (22%) and Trypacide (18%). It was noted that although Berenil is not recommended for use in camels due to its toxicity, more than half of the stocks were resistant to this drug. The malic enzyme patterns of these stocks were determined to investigate possible correlation with resistance to the trypanocides used. Most of the stocks (66%) possessed malic isoenzyme pattern II (66%), although patterns X (23%), IV (7%) and VII (5&37) were also observed. Malic enzyme patterns IV and X had not been identified previously in this trypanosome species. No linkage was found between any of these patterns and resistance to the four drugs. Further work concentrated on three trypanosome stocks which were highly resistant, of intermediate resistance or sensitive to the action of suramin. Investigations on the effect of trypanosome inoculum and timing of suramin administration in obtaining cures in T.evansi infected mice were carried out. The time of administration after infection was shown to be an important factor that influenced the outcome of suramin treatment. Mice which were treated immediately after infection had higher cure rates than those treated at the onset of parasitaemia. This was observed particularly in a trypanosome stock normally resistant to suramin at a dose rate of 160 mg/kg; if treatment was administered immediately after infection, most of the infected mice were cured. Except in the highly resistant stock, the role of trypanosome inoculum was not as important as timing of treatment in determining cures.

A study was made of Nematodirus battus and Trichostrongylus colubriformis infections in New Zealand White rabbits. Experimental studies with N. battus were not continued because of the unsuitability of the parasite in the host. The distribution and development of T. colubriformis in the intestinal environment of N.Z.W. rabbits was studied at various dose levels. The clinical symptoms and pathology of infected animals was studied. The nature of the changes in gut histology and the architecture of villi was established at two sites in the small intestine by examining paraffin sections and studying the surface architecture of villi. The structure of epithelial cells was examined with the electron microscope and the cellular changes were correlated with observations made on enzyme activity using histochemical techniques. The general picture during infection by large doses of T. colubriformis was that of the intestinal malabsorption syndrome. The development of challenge infections given on days 12 and 20 of a primary infection was studied on day 8 post challenge infection. The dose level at which no marked clinical, pathological or immunological changes occurred was used as the dose for the anthelmintic selection study. Rabbits were dosed with a rat strain T. colubriformis and the anthelmintic thiabendazole was used as a selection pressure at 45 mg/body weight against the adult parasite population. After three generations of selection pressure the anthelmintic selected and non-anthelmintic selected povalations were compared in an anthelmintic titration. Following this experiment it was recognised that an anthelmintic resistant population was present. The criterion of resistance was established by studying uterine e-9 counts and the cell development stages in the two populations in anthelmintic treated and untreated rabbits. It was also noted that the response of sheep and rabbit strain T. colubriformis to the anthelmintic was different, the latter was less responsive to the drug. The role of the host in the modification of the parasite phenotype was considered.

This study sought to identify antigens involved in protective immune responses to Cowdria by Western blotting using immune sera and surface labelling of elementary body (EB) proteins using biotin to identify which of these are outer membrane proteins. Antigens which could be considered as potential vaccine candidates were identified. Immunisation of goats with live Cowdria or with inactivated elementary bodies (IEBs) leads to development of antibodies to at least six antigenic components of the EB of 24kDa, 27kDa, 31kDa, 58kDa and 66kDa. In contrast immunisation of goats with detergent extracted soluble antigens stimulated production of antibodies to only four antigens of 24kDa, 27kDa, 28kDa and 31kDa. Six surface exposed antigens of the Cowdria elementary body were identified by biotin labelling, with molecular masses of 21kDa, 28kDa, 31kDa, 62kDa, 74kDa and 115kDa and are therefore considered outer membrane proteins. These proteins reacted with antibodies in sera raised by immunisation of goats with live or inactivated EBs. The 58kDa heat shock protein (GroEL) of C. ruminantium is an immunodominant antigen. The immune responses to 58kDa antigen expressed as a recombinant in E. coli were investigated by immunisation of mice and sheep. The immunised animals stimulated specific antibody which reacted with the native homologue on the EB 58kDa. Immunisation with recombinant 58kDa heat shock protein of C. ruminantium led to development a of specific antibody response of the IgG1 isotype. Challenge of immunised sheep showed a highly significant reduction (p

Tropical theileriosis, caused by the haemoprotozoan parasite Theileria annulata, is one of the major threats to cattle health in Tunisia. The aim of the study described in this thesis was to assess the extent of diversity in T. annulata parasite population from within a single country in order to provide a better understanding of the epidemiology of the disease and biology of the parasite. The work also provided useful background information to vaccine development studies in Tunisia. Different T. annulata stocks, isolated from different bioclimatic zones in Tunisia were characterised using monoclonal antibodies (MAbs), isoenzyme and DNA analyses and compared with each other and with a number of recognised laboratory stocks from other theileriosis endemic areas of the world. The study comprises seven chapters. In the first, an introduction to the literature describing the parasite T. annulata and the disease it causes are presented and the epidemiology of tropical theileriosis is discussed with particular regard to the situation in Tunisia. Finally an overview on diversity in protozoan parasites, including Theileria, is given to emphasise the rationale behind the present study. The next chapter details how the biological material, including 51 field isolates, was generated for the three parasite life-cycle stages, sporozoites, piroplasms and schizonts. In the third chapter the production of antischizont MAbs and their use as immunological makers to reveal differences between stocks of T. annulata is described. The fourth chapter demonstrates the use of a biochemical marker, glucose phosphate isomerase isoenzyme to study diversity in T. annulata isolated in Tunisia.

TheUeria an ,plat, infection is a tick -borne protozoal disease of cattle. The high mortality and lack of any effective treatment make this disease one of great economic importance. The success achieved in adaptation of the parasite to culture in vitro facilitated investigation of the disease and the consequent attenuation of the parasite led the production of experimental vaccines. The literature relevant to the aspects studied was reviewed. Methods of isolation and adaptation of strains of the parasite in tissue culture were compared. A simple and new method of isolation from whole blood was introduced. The first isolation of a very virulent strain in tissue culture was achieved. Growth rates of five strains of T. annulate in vitro showed an inverse relationship between rate of growth and virulence of strain. Several growth media and supplements were compared and the most satisfactory were shown to be Eagle's MEM with calf serum, lactalbumin hydrolysate and yeast extract. Growth of T. annulata- infected cells in various conditions was measured and factors affecting it were investigated. Of these, pH of the medium and percentage of viable cells in suspension were very important. For morphological studies, from several methods of preparation of smears and staining, a modified method of linear smears was adopted. The morphology of the macroschizont and nuclear particles in the host cells was studied. Various factors including developmental changes of the macroschizont were demonstrated. Attempts to infect normal bovine cells with schizonts in vitro using infected cells and freed parasites and to infect calves with separated schizont particles were unsuccessful. Establishment of cell lines from normal bovine leucocytes and lymphoid cells using mitogens failed. The amounts of glucose uptaken and lactate produced in the culture of schizont -infected cells were measured and these correlated with cell growth. A surplus of glucose in growth medium did not affect its utilisation by the infected cells. The methods for cryopreservation of T. annulata- infected cells were investigated. The cryoprotectants used, the methods of freezing and thawing, the duration of viability of the infected cells in low temperature storage and re- establishment of the cells after preservation at low temperatures were studied.

Theileria annulata is an economically important protozoan parasite (Api-complexa) which cycles between bovine and invertebrate tick hosts. Within the bovid, sporozoites invade leucocytes and develop into macroschizonts: macroschizonts divide, initially by binary fission, in synchrony with the newly transformed host cell, but later merogony takes place, producing merozoites which invade erythrocytes. These are subsequently ingested by the tick. The post-macroschizont life cycle stages are poorly defined, and this experimental project was aimed at investigating the basic biology of these stages at the molecular level. The polypeptide complement of the infected erythrocyte membrane has been investigated. The precise location of piroplasm polypeptides in the infected cells was not determined, but the experimental results indicate that several (most notably molecules of 122kDa, 98-100kDa & 77kDa) are associated with the erythrocyte membrane. Monoclonal antibodies have been raised against infected erythrocytes. By immunofluorescence microscopy, several antibodies recognise, mainly, either the outer perimeter of the piroplasm, vesicular structures (dots), or molecules located within the piroplasm. For the purpose of strain differentiation, a panel of these monoclonal antibodies distinguish between un-cloned preparations of Ankara, Hissar and Gharb stocks. The vast majority of the monoclonal antibodies (27 cloned lines) are stage specific and do not recognise slide preparations of macroschizonts or sporozoites. Merogony has been induced in vitro, in recently transformed macroschizont infected cell lines, and four anti-piroplasm monoclonal antibodies recognise preparations of merozoites. Immunoelectron microscopy results have shown that antibody 5E1 recognises the surface of heat induced merozoites. Western blot analysis suggests that the 30kDa and 120kDa polypeptides, recognised by antibody 5E1, also elicit an antibody response in the bovine host. These antigens are preliminary candidates for part of a molecular sub-unit vaccine against the disease (Tropical theileriosis) caused by T.annulata. The T.annulata gene, which encodes the epitope determining antibody 5E1 has been cloned from a lambda gtl 1 genomic expression library. A model system for investigating the molecular mechanisms of stage differentiation has been developed. Macroschizont merogony can be induced in recently infected cell lines by elevating culture temperatures. Similar heat-treatment, of the same cell line after prolonged passage, fails to result in the differentiation into merozoites. The epitope recognised by antibody 5E1 is stage specific to merozoite and piroplasm stages and preliminary Northern slot-blot analysis, with the cloned gene sequence, suggests that expression is regulated at the level of transcription.

The excretions, secretions and surface components of a parasite are by their nature centrally involved in the host/parasite interaction. Rats, like cattle, are capable of developing resistance to fasciolosis after primary infection and are therefore considered a suitable laboratory model for cattle. The objective of this study was to characterise the excretory/secretory (ES) and surface components of Fasciola hepatica as it develops in the rat, and to identify those components involved in the host/parasite interaction that may have diagnostic and/or protective value. Three trials were conducted during the study in order to produce supplies of rat antiserum which was protective against F. hepatica, Rats were infected with either 10 (first trial) or 20 (second and third trials) F. hepatica metacercariae as information from the literature indicated that these doses were adequate to stimulate the production of protective antibody levels. The rat sera from the three trials were checked for the presence of protective antibodies by passive protection studies. Only in the latter two trials was the level of protection conferred on recipient statistically significant. The probable causes for the lack of significant protection in trial 1 are discussed. The silver stained protein profiles of ES from newly excysted (D0) flukes and one-day old (D1) flukes were characteristic and were similar to each other. The ES of parenchymal 14-day old (D14) and adult (D56) flukes were markedly different from D0 and D1 flukes but similar to each other. The silver stained total ES protein profiles of the developing flukes were very different from the total biosynthetically (35S-methionine) radio-labelled ES protein profiles. Possible reasons for this are discussed. However, as with the total silver stained ES protein profiles there were clear changes in the profiles of the biosynthetically radio-labelled ES as the flukes developed. It is suggested that these differences in the ES products may reflect the changing environment and activities of the flukes. The possible functions of the changing ES products are discussed.

Onchocerciasis or river blindness is a major cause of infectious blindness in the world. In severely affected areas, half of the adult population may be blinded by the disease. The human parasite, O.volvulus, is very closely related to the cattle parasite Onchocerca gibsoni which provides an abundant source of biological material for analysis. Thus, O.gibsoni offers a model system for biochemical analysis and drug screening. In these organisms, as in other eukaryotes, microtubules are essential, multifunctional, subcellular components. They are involved in chromosome segregation, cell architecture, motility, intracellular transport, and secretion. The major protein of microtubules is tubulin, a heterodimer of two distinct polypeptides designated α and β. Each subunit has a molecular weight of about 50kD and is encoded by a distinct set of genes. Tubulins are highly conserved proteins and the number of genes coding for both α and β-tubulin vary dramatically between different species. This major structural protein of eukaryotic cells has particular importance in helminthic parasites as it is a target for anthelmintic benzimidazoles, which directly bind to tubulin and inhibit the assembly of microtubules. The effectiveness of such drugs depends on differences in the structure of the tubulin molecules from the parasite and its host. In this context, a genomic library from O.gibsoni has been constructed, screened with a β-tubulin gene from Plasmodium falciparum and the gene isolated. The chromosomal copy of the gene has been completely cloned and sequenced. This has revealed the gene to possess a very complex structure compared to the organisation of all the known β-tubulin genes as the coding region is interrupted by 11 introns. The deduced polypeptide is 444 amino acids long, and its sequence is highly conserved. The position of some introns appear to demarcate functional domains within the protein. The results that have emerged from this thesis are a step forward not only in the rational design of drugs, but also offer an insight into the biology and evolution of an Onchocerca gene and its product.

The relationship between the feeding of Amblyomma variegatum ticks and Dermatophilus congolensis infections has been studied under laboratory conditions. The local effects of hypersensitive or inflammatory reactions to larval and nymphal A.variegatum on subsequent D.congolensis infections were investigated using rabbits and sheep respectively. Multiple or single infestations of ticks were used to produce hypersensitivity or inflammatory reactions respectively. These reactions were confirmed by histological assessment of the tick attachment sites. Identical titrated doses of D.congolensis were applied to the tick attachment sites after the ticks had detached, a control titration was set up on skin with no previous exposure to ticks. The progression of the resulting lesions was assessed using a non-parametric ranking system. There was no significant difference (P > 0.05) between the severity or duration of the three groups of dermatophilosis lesions, either on the sheep or the rabbits. Therefore it was concluded that the local effects of the feeding of immature instars of this tick do not affect the pathogenesis of subsequent D.congolensis infections. The local effect of hypersensitive or inflammatory reactions to A.variegatum nymphs on simultaneous D.congolensis infections on rabbits was also studied. There was an increase in the initial severity of the dermatophilosis lesions and a positive correlation between inflammatory tick attachment sites and dermatophilosis foci. However, the local effects of the feed of nymphal A.variegatum did not result in the development of chronic dermatophilosis lesions. The systemic effect of adult and nymphal A.variegatum on simultaneous dermatophilosis lesions was compared, using sheep as the experimental hosts. Dermatophilosis congolensis infections on sheep infested with adult A.variegatum developed into chronic lesions which persisted for several months. Serological and skin tests revealed significantly (P < 0.01) reduced humoral and cellular immune responses in sheep infested with adult A.variegatum compared with sheep infested with nymphs or control sheep not exposed to ticks.

Pasteurella haemolytica serotypes A1 and A2 and Pasteurella trehalosi serotype T10 were cultured in "in-vivo" fluids (ruminant tracheobronchial washings and serum), under defined iron-restrictive conditions, and compared for changes in cellular composition. In analyses by SDS PAGE and Western blotting, envelope and cell contents of the bacteria showed differences in protein content and changes in antigens recognised by convalescent antiserum. Capsule size was not influenced by growth medium. Lipopolysaccharide was detected by PAGE in all the fluids except bovine tracheobronchial washings. However, a Limulus amoebocyte lysate assay did detect trace amounts of endotoxin. Leukotoxin activity was only detected in broth and iron-restricted cultures and not in any "in vivo" fluids. Neutralisation of the toxin with homologous and heterologous convalescent antiserum showed little cross-reactivity in the neutralisation capacity against A1 leukotoxin but heterologous antiserum was effective with leukotoxin of the other serotypes. Using the same fluids to monitor long term culture, all serotypes showed the capacity for extended survival and resuscitation with the addition of nutrients. All three strains survived better in ruminant than in other species' fluids. In non-ruminant fluids and natural water viability was detected only at low temperatures. Morphological changes in both colonial and microscopic appearances were apparent during long term survival. These results imply that this organism possesses some starvation survival mechanisms. Immunomagnetic beads with bound antibody specific for the three serotypes proved a useful reagent for the isolation of target organisms from host tissues and fluids. Differing protein profiles from those seen in bacteria grown in-vitro were demonstrated. Western blotting unexpectedly failed to identify many antigens when recovered whole cells were compared to those grown in laboratory culture, indicating that perhaps mechanisms were present which helped to evade an immune response. Superoxide dismutases were detected in all three strains. Level of enzyme activity, electrophoretic mobility on native PAGE, and metal-active sites were shown to differ.

Trichostrongylus vitrinus is one of the principal causative nematodes of ovine parasitic gastro-enteritis within Scotland and infests the proximal small intestine of the sheep. At present, control is achieved mainly by the administration of anthelmintic drugs, but with the increasing emergence of anthelmintic resistance, much research is now centred on vaccine development. Recent evidence has suggested that the excretory-secretory components (ES) from parasitic nematodes may be an important source of host-protective antigens. The overall aim of the present study was to characterise the nature and properties of T. vitrinus ES components. The initial part of the work involved the partial characterisation of two of the enzymes, acetylcholinesterase (AChE) and proteinases, excreted and secreted during the in vitro culturing of adult T. vitrinus. These enzymes were defined on the basis of their substrate specificity, molecular size, pH optima and inhibitor sensitivity. Attempts were also made to isolate cDNA fragments encoding AChE from an adult T. vitrinus cDNA pool, using the polymerase chain reaction (PCR) and degenerate oligonucleotide primers directed towards highly conserved regions of AChEs that had previously been identified by comparison of known polypeptide sequences from a number of higher eukaryotic organisms. No adult T. vitrinus cDNA fragments encoding AChE were amplified, suggesting that T. vitrinus AChE(s) is/are distinctly different to AChEs from higher eukaryotes, at least at the level of nucleic acid sequence. Subsequent research focused on the molecular characterisation of adult T. vitrinus ES. Immunoscreening of an adult T. vitrinus cDNA lambda gt11 library with antiserum raised against adult T. vitrinus ES, resulted in the isolation of ten immunopositive clones. Their inserts were sequenced and the results were analysed using computer databases. Three of the clones were identified as harbouring inserts that encoded proteins that shared significant homology to myosin heavy chain, vitellogenin and serine proteinase inhibitor (serpin) respectively. The other seven clones contained inserts that showed no significant homology to any of the sequences present in the computer databases.

Two T. evansi Ag-ELISAs based on different monoclonal antibodies (2G6 Ag-ELISA and Tr7 Ag-ELISA) were evaluated using buffaloes in Southeast Asia, where T. evansi is endemic. The repeatability and robustness of the two Ag-ELISAs were shown to be high. Profiles of antigenaemia varied between individual buffaloes and between the two Ag-ELISAs. Antigen and antibody responses were first detected 7 to 42 days after infection, but in some buffaloes responses fluctuated below cut-off values during infection, whilst in other buffaloes antigen and antibody responses persisted after trypanocidal drug treatment. With the naturally-infected buffaloes, the diagnostic sensitivity estimate of the Tr7 Ag-ELISA (81%) was significantly higher than that of the 2G6 Ag-ELISA (71%), and the IgG ELISA sensitivity (89%) was significantly higher than either the IgM ELISA or CATT sensitivities (78%). In Central Java, 2387 buffaloes were blood sampled in 59 villages, and estimates of test prevalence were 4% with the MHCT, 9% with MI, 58% with the 2G6 Ag-ELISA and 70% with the Tr7 Ag-ELISA, but prevalence values differed between districts and between villages. True incidence rates per animal-year at risk were 0.44 with the Tr7 Ag-ELISA and 0.22 with the 2G6 Ag-ELISA. of 239 market buffaloes sampled, 10% were parasitaemic, 39% antigenaemic, 56% positive by IgG ELISA and 47% positive by CATT, representing an important source of T. evansi. The T. evansi Ag-ELISAs and antibody-detection tests used in this study have many advantages as screening tests over commonly used parasitological tests, in terms of their diagnostic sensitivity and ability to rapidly test large numbers of samples. The two T. evansi Ag-ELISAs could be applied in high prevalence areas, whilst antibody-detection tests (in particular, the IgG ELISA or CATT) would be more appropriate to test buffaloes in low prevalence areas or to confirm the negative-status of buffaloes prior to movement within Indonesia or export. Future work should aim to improve the specificities of the Ag-ELISAs, which were low in this study in contrast to previous reports.

The objective of this work has been to investigate the frequency of cross-fertilisation between gametes of genetically distinct clones of the human malaria parasite Plasmodium falciparum. Previous genetic experiments involving both rodent malaria parasites in vivo and human malaria parasites in vitro have demonstrated higher than expected numbers of recombinants among the progeny of crosses. It has been suggested that this could be due to a favouring of cross-fertilisation over self-fertilisation in the mosquito phase of the life-cycle. The work has involved examining the genotypes of individual oocysts (derived from individual zygotes) resulting from mixed infection of two clones in mosquitoes. In preliminary work using the mouse malaria parasite P. yoelii nigeriensis attempts were made to examine the chromosomes of single oocysts using pulsed field gradient gel electrophoresis. However there was insufficient DNA in an oocyst to allow chromosomes to be visualised using this technique. The bulk of the work has been concerned with P. falciparum. In the first stage oligonucleotide primers suitable for use in the polymerase chain reaction (PCR) were designed to allow amplification of repetitive regions of two polymorphic antigen genes, denoted MSP1 and MSP2. The two clones of P. falciparum used in the crossing experiments possessed a different allele of each gene. These alleles were found to be recognisable as size differences of the PCR-amplified fragments on agarose gels. Gametocytes of the two clones were grown in vitro. Mixtures of gametocytes of each clone were made and fed to Anopheles stephensi or A. gambiae mosquitoes through membrane feeders. 9 to 10 days later the mosquitoes were dissected and their midguts were examined for the presence of oocysts. Individual oocysts were dissected from the midguts and the DNA extracted from them.

Fresh specimens were collected from natural infections and from experimentally infected animals. Collections were made in the United Kingdom and in Nigeria and a few specimens were also obtained from other countries abroad. Materials from the specimens was compared by staining, serological techniques and enzyme electrophoresis. Staining ova by the Ziehl-Neeleen method was not successful in differentiating ova of any of the species including T. saginatue and T. solium. Serological techniques that were tried included oval precipitation, agar gel diffusion precipitation, fluorescent antibody, immunoeleotrophoresis and the enzyme-linked immunosorbent assay (ELISA). Strobilate, oval and, in some instances, larval cyst antigens, were used in tests against antisera raised in rabbits and other species. With antisera that had been absorbed by one or two heterologoue antigens, some success was achieved in agar gel diffusion precipitation tests and with ELISA in differentiating strobilate tissue, but serological techniques were not generally adequate for this purpose. Success in differentiating taeniid material was achieved by enzyme electrophoresis on thin layer starch gels. Of the many enzymes tested, five were found to display inter-species zymogram differences in the pattern and mobility of their multiple forms: adenylate kinase, glutamate dehydrogenase, malate dehydrogenase, hexokinase and glucose phosphate isomerase, of which the latter two were the most useful for differentiation. No differences were seen between larval and adult material except for the presence of host enzymes in the former which were easily identified by comparison with host tissue extracts. No differences were seen between immature, mature and gravid proglottides and no individual variation was seen between worm specimens. Anthelmintic treatment did not affect worm enzyme patterns. Taeniid species were compared with one another and also with other tapeworm species. The mobility and pattern of the enzyme forms in zymograms differed more between unrelated than between related species. With glucose phosphate isomerase it was possible to identify most of the taeniid species. When this enzyme was preserved in a solution containing enzyme stabilisers, it stored well over a long period especially in liquid nitrogen or, freeze dried and kept at -20 °C. Dilution of samples did not distort zymogram patterns and crude extracts could be identified using the mobility of the slowest moving band as the criterion. This method was used successfully for a survey in Nigeria. Identification was by comparison with a known species. The other enzymes were useful for confirming the diagnosis. Strain variation between E. granulosus of horse and sheep origin was seen in specimens collected from Belgium and different parts of the United Kingdom. Cysts from oxen were identical to those from sheep, but indirect comparison with cysts from Nigerian camels showed these to be different from both the equine and ovine strains.

Allergens from somatic extracts of adult Nippostrongylus brasilinesis worms were characteriszed by SDS-PAGE and Western blotting. Seven allergens, with molecular weight ranging from 14,000 - 69,000 were identified after blotting with hyperimmune serum (HIS) from rat immunized by several infections with N. brasiliensis. Monoclonal mouse anti rat - IgE was used as a specific probe. The major 14,000-17,000 MW allergens, partially purified by electrolution, retained activity by passive cutaneous anaphylaxis (PCA). Other immunoglobulin isotypes, notably IgC1 also bound to allergens on Western blot. The specificities of other immunoglobulin isotypes and IgE were compared by sequential incubation of blotted allergens with IgE - depleted HIS and with affinity-purified serum IgE. Despite retaining biological activity by PCA, the affinity-purified IgE proved to be insufficiently specific for use on Western blots. The range of allergens detected and the kinetics of appearance of parasite-specific IgE differed between LOU. Hooded Lister, August and F334 undergoing primary infection with N. brasiliensis. Whereas August rats responded uniformly, there were variations in the timing and specificity of IgE responses between individual rats within the other strains. Nevertheless, it was possible to classify LOU and Wister rats as early and F334 rats as late responders. Internalization of IgE within the cytoplasm of intestinal mucosal mast cells (MMC) of rodents infected with intestinal nematodes, and the absence of this isotype from the cytoplasm of connective tissue mast cells (CTMC) suggested that the MMC granule protease, rat mast cell protease II (RMCP II), might fail to catabolize IgE. However, when compared with the granule protease RMCP I from CTMC. RMCP II was more efficient in catabolizing the heavy chains of both IgE and IGG2a. The light chains were apparently resistant to digestion. The presence of IgE-bearing cell populations in bone marrow, peripheral blood, and peritoneal cavity, was monitored by flow cytometry in normal Wister rats and during the course of infection with N. brasiliensis. The proportions of IgE-bearing cells in the peritoneum and bone marrow increased on day 10 and, in peripheral blood on day 15. The IgE-bearing cells in each compartment were of mixed phenotype.

A description of the isolation of new Cowdria isolates by different methods and from different vectors and geographical locations in Kenya is given. These included Amblyomma variegatum, A. gemma and A. lepidum. Isolates from the later two species were also by feeding adults moulted from nymphs collected in the field and is the first report on transtadial transmission by A. gemma ticks. A spectrum of virulence ranging from highly virulent to mildly virulent for sheep was found among the Cowdria isolates. The majority of isolates were highly virulent. There was a range of mouse infectivity among the isolates from inapparent to lethal. The Asembo and Baragoi isolates were pathogenic and lethal, the Kiswani, was infective and non pathogenic for Balb-C mice while the other 8 were avirulent or refractile to mice inducing only antibody production in various proportions of mice. There was a difference in the infectivity for neutrophils both in the frequency of infected cultures and in their level of infection. The different isolates were classified as of low infectivity where even the few positive cultures rarely reached 1% infection rate, medium infectivity if a good number of cultures regularly attained 1% infected neutrophils, or high infectivity if a large proportion of the cultures which became positive regularly attained 1% infected neutrophils and at least some of then attained more than 10% infected neutrophils. The isolates also had a range of infectivity for the brain endothelial cells, from no detectable colonies to greater than 16% infected endothelial cells in individual animals. The author concludes that the agent of heartwater is endemically widespread in many districts in Kenya and poses a potential threat of outbreaks to areas newly invaded by vector ticks and also to areas where immunity due to the local agent may not protect against an invading one. The author recommends that the Baragoi or Marigat isolate should be adopted for possible vaccine development in Kenya.