A staining technique was developed for locating the sporozoites of Eimeria spp. in both mammalian and avian hosts. Bouin-fixed tissues embedded in Paraffin wax and cut at a thickness of 3 μg were stained by a controlled sequence of Ehrlich's haematoxylin, acid fuchsin and aniline blue–orange G–acetic acid which took only 30 min to perform. The refractile globule of the sporozoite stained a brilliant crimson whilst the tissue had an overall blue colour, thus enabling rapid scanning of material under low-power magnification. The granules of eosinophils and heterophils also stained a brilliant crimson and their significance in immunological responses indicates a wider application for this technique.

The interaction between mouse peritoneal macrophages and ‘Y’ strain Trypanosoma cruzi bloodstream forms was studied at optical and electron microscopical levels. The method of marking lysosomes with Thorotrast, either before or after infection of cell monolayers with parasites, revealed that secondary lysosomes fused with phagosomes shortly after trypanosome interiorization. In spite of this, 24 h later most parasites were no longer in a vacuole but lay free within the host cell cytoplasm, multiplying actively. At this time, and up to shortly before 96 h when parasites escaped to the external milieu, most parasitized cells were not lethally injured, as revealed by the Trypan blue dye-exclusion test. Only when parasites were released into the external medium was this situation reversed and infected macrophages took up the dye.

LLC-MK2 cell monolayers infected with Trypanosoma cruzi were shown by immunofluorescence to present parasite antigens on the surface of both parasitized and non-parasitized cells after completion of the first intracellular cycle and rupture of infected cells. The cell-culture supernatant fluid at this stage, as well as the supernatant fluid of parasites left overnight in culture medium were concentrated and contained antigen capable of binding to uninfected cell monolayers. The origin of this antigen, as well as its eventual role in the pathogenesis of Chagas' disease, are discussed.

Clones of animal-infective bloodstream forms of Trypanosoma brucei (stocks S.427 and LUMP 227) were made by transferring a single organism from bloodstream-form cultures into each well of Microtest II Tissue Culture Plates containing bovine fibroblast-like feeder cells. When the number of trypanosomes increased to 102–103/well on days 4–16, they were transferred into plastic T-25 culture flasks also containing feeder cells and fresh medium. Cultures were thereafter maintained by partially replacing the trypanosome suspension with the same volume of fresh medium (diluting the density to 2–5 × 105 trypanosomes/ml) every 24 h. Sub-cultivations could be made by transferring 1–2 ml of the trypanosome suspension to a new culture flask at 4–5 day intervals. A total of 42 clones in the 3 series TC221, TC52 and TC227, carrying variable antigen types (VATs) 221, 052 and ILTat 1·4, respectively, have been established. Average population doubling times for clones of TC221, TC52 and TC227 were 8·7, 14·5 and 15·5 h respectively. Of 35 populations examined, 34 clones retained the original specificity of their VATs for at least 8–32 days from cloning. One cloned population of TC52 consisted of 99·8% VAT 052 and 0·2% VAT 221 at the time when the first VAT test was made on day 18.

The in vitro absorption of glucose and l-methionine in the intestine of broiler chickens was measured 7, 14 and 21 days post-inoculation (p.i.) with sporulated oocysts of Eimeria acervulina, E. mivati, E. maxima or E. brunetti. The small intestine of each bird was divided into 8 regions of equal length and absorption was measured on 3 tissue disks of equal size from each region. The absorption rate of each substrate with each coccidial species was measured based on (1) an equal area from each region, (2) an equal weight from each region, (3) the total absorption in each region and (4) the total potential absorption in the intestine. Comparisons of absorption rate of equal areas in each intestinal region demonstrated that infected birds at 7 days p.i. absorbed significantly less substrate per unit area in the regions of maximum infection than uninfected controls. Malabsorption was less apparent when the weight of the region was used as the unit of measurement. Compensatory absorption was seen in some uninfected regions with E. acervulina. The total potential intestinal absorption at 7 days p.i. was reduced with E. mivati, E. maxima and E. brunetti but not with E. acervulina. At 14 days p.i., total l-methionine and glucose absorption in some regions of the intestine was significantly increased with E. acervulina but not with E. mivati, E. maxima or E. brunetti. No absorption differences were seen at 21 days p.i. with any species.

Forms of Trypanosoma brucei isolated from lymph nodes of cyclically or intravenously infected rats, at different stages of infection, were morphologically compared with the corresponding bloodstream forms. Unlike the pleomorphic bloodstream trypanosome population, the lymph node trypanosome population was essentially monomorphic and resembled the slender forms of the bloodstream. Values for the morphometric parameters indicated a position between the slender and the stumpy forms. The number of trypanosomes/ml of blood showed the well-known periodic fluctuations while the number of trypanosomes/g of lymph node remained fairly constant. Serology revealed that lymph node trypanosome populations differed antigenically from the trypanosome populations isolated simultaneously from the bloodstream.

Recent microscopic studies of Theilerial parasites in. ticks appear to provide convincing evidence for a sexual cycle for these parasites. This evidence would remove most of the objections to placing piroplasms in the same class as malarial and coccidial parasites. It is suggested, therefore, that the class Piroplasmasida (Piroplasmea) be abolished and that piroplasms be located in a sub-order Piroplasmorina alongside Haemosporina within the class Sporozoa (Teleospora).

If the close relationship of Theilerial and malarial parasites is correct then by analogy meiotic or reduction division of Theileria probably occurs during sporogony in the tick salivary gland acinar cells. This means that all bovine stages are haploid. If these can be cloned and characterized by isoenzyme or drug resistance markers, then genetic recombinant studies, similar to those performed with malarial and coccidial parasites, could be conducted.

The potential of genetic recombination greatly increases the scope of the parasite for developing new variants. This potential is greatest in areas where there is a variety of tick and host species which apply different types of selection pressure to parasites. Atypical strains of Theileria are usually isolated from such areas, and although the characteristics of these strains may not be expressed in the wild, unnatural selection by laboratory passage may reveal their presence. The apparent lability of such strains may simply be cloning of different genetic recombinants.

If different strains can be cloned and characterized it may be possible by genetic recombination techniques to develop new clones which retain desired characters for use as avirulent protective vaccines, but from which undesirable characters have been eliminated.

The use of human diploid cell lines of finite life for the in vitro production of Trypanosoma cruzi is described. Both MRC5 and WI38 cells release trypomastigotes with less than 5% amastigotes. This could form the basis for biochemical and immunological studies, which were previously limited by the problems of separating parasites from blood. By selecting the in vitro passage number of the parasite it is possible to select for either the broad or the slender forms of trypomastigotes, allowing comparative studies of these forms within a single strain of the parasite. It is also possible to isolate amastigotes by disrupting the cells before trypomastigotes appear, and separating them from cell debris with Metrizamide. It is shown that by incorporating [3H]uridine in the cell-culture medium, labelled trypomastigotes are obtained. The release of this label (putative RNA) provides a relatively simple isotopic assay for parasite death. Examples of this assay for testing drug toxicity and in immunological lysis are presented.

The morphology and development of Trypanosoma cobitis during its life-cycle in the blood of 6 species of River Lee fish, and also in its invertebrate vector, the fish leech Hemiclepsis marginata L., are described in detail.

A method has been devised to show that malaria parasites can be detected serologically in infected blood with a high degree of sensitivity. Using a murine malaria model, parasites were demonstrated in a solid-phase radio-immunoassay which measured antibody-binding inhibition. Lysed red blood cells (r.b.c.) were incubated with labelled specific antibody and were then reacted in antigen-coated tubes. The degree of inhibition of antibody binding in the tubes correlated with the level of parasitaemia in the test blood. Using homologous antisera the test detected infection at a level of 1 parasite/million r.b.c. The specificity of the method was shown by comparison of antibody-binding inhibition in normal and infected r.b.c. and in r.b.c. from non-infected mice with induced reticulocytosis. The sensitivity was shown in vitro in tests of serially diluted blood of high parasitaemia and in vivo for the detection of early infection. The presence of antibody in the test blood did not significantly affect the sensitivity of parasite detection.

Two new species of coccidia, Eimeria gourai sp. nov. from the Victoria crowned pigeon (Goura victoria Fraser) and Eimeria duculai sp.nov. from the Torres Strait imperial pigeon (Ducula spilorrhoa Gray) have been described. Eimeria gourai oocysts are spherical to sub-spherical in shape, measure 20·0 (19·0–22·0) × 20·0 (18·0–21·0) μm and are devoid of micropyle and oocyst residuum. Elongate sporocysts are 12·0 × 5·5 μm, and contain a granular residuum. Eimeria duculai oocysts are broadly ovoid with an inconspicuous micropyle at the narrower end. They are 28·0 (26·0–31·0) × 25·0 (23·0–27·0) μm in size, without residuum but with a membrane-bound polar granule. The elongate sporocysts are 15·5 × 7·2 μm, with prominent Stieda body and granular, membrane-bound residuum. The morphological characteristics of the known coccidian oocysts from birds of the avian order Columbiformes are reviewed.