The distribution of nitric oxide synthase (NOS) immunoreactivity and putative NOS activity in adult Schistosoma mansoni was analysed using 3 different types of NOS antibodies and NADPH-diaphorase histochemistry. Although potential involvement of the gaseous radical nitric oxide (NO) in host response to infection by schistosomes has been suggested, there is little or no information available regarding the role, or even the presence, of the NO pathway in schistosomes themselves. Here, we demonstrate that antibodies against neuronal NOS (nNOS) and inducible NOS (iNOS) isoforms stain adult worms with distinctive patterns; anti-endothelial NOS (eNOS) shows no selective labelling. nNOS-like immunoreactivity is found in the main nerve cords and the peripheral nervous system. Putative sensory neurons with apical neuronal processes leading to the tegument of male worms are also immunoreactive for nNOS. Anti-iNOS labels a variety of predominantly non-neuronal tissues, showing intense labelling at or near the surface of the worm and in components of the gastrointestinal tract. The distribution of NADPH-diaphorase reactivity (a histochemical marker of NOS), is generally similar to the pattern of NOS immunoreactivity, including labelling of neuronal-like cells as well as developing eggs. These results suggest that an NOS-like enzyme is present in S. mansoni, and indicate potential roles for the different NOS isoforms in neuronal signalling, reproduction and development.

Corynosoma spp. (Acanthocephala) infection in ringed seals (Pusa hispida) is compared in spring and in autumn in the Bothnian Bay, northern Baltic Sea. The material consists of more than 13400 specimens: 7590 from the intestines of 13 seals captured in April and May 1978 and 5850 from 29 seals captured in October and November.

Corynosoma strumosum infection had approximately the same intensity in spring and autumn (mean 76 and 66 individuals/infected seal, maxmum 313 and 324 individuals, respectively). C. semerme specimens, on the other hand, were 37 times more numerous in spring than in autumn (504 compared with 136/infected seal, maximum 1700 and 1230 individuals, respectively). The ratio of C. strumosum to C. semerme was 1:6·3 in spring and 1:2·0 in autumn, as calculated from the total material. Corynosoma infection was more recent on average in spring than in autumn, as judged both from the proportion of males and immature females and from the distribution of C. semerme in the intestine. The feeding habits of the ringed seals and the rate of development of Corynosoma infection are discussed.

The thoracic lymph duct of rats infected with Trypanosoma brucei and with T. congolense was cannulated at different stages of infection. Trypanosomes were always present in the lymph of rats infected with T. brucei, whereas they were absent or very scarce in the lymph of rats infected with T. congolense. There were greater fluctuations in the numbers of trypanosomes in the blood than in the lymph. The intra- and extravascular populations of T. brucei differed: stumpy forms were present only in blood; dividing forms were usually more numerous in lymph.

Some effects of insemination on Moniliformis dubius were studied by means of a surgical technique involving the transplantation of worms of known age, sex and reproductive state from one male rat to another. Insemination resulting in the development of eggs was found to have occurred in female M. dubius aged 16 days. An individual male M. dubius was found to be capable of inseminating as many as 17 females of the same age. The duration of the patent period appeared to be related to the length of the period of contact between male and female worms at the start of the course of infection. The evidence indicated that the patent period of a female worm was curtailed in cases where the female was isolated from males sooner than 5 weeks after the start of the infection. Male worms estimated to be not more than 21 days old were found to be capable of inseminating females older than 100 days; in these cases, the patent period was brief. Evidence was obtained to suggest that insemination is followed by further growth of the somatic tissues of the female worms. These results apply to experimental infections, and caution should be exercised in applying them directly to natural infections.

Actively immunized mice, whole-body irradiated with 650 or 525 rad., manifested comparable levels of resistance to Schistosoma mansoni compared with unirradiated, immunized mice in spite of a marked reduction in circulating leucocytes (>90%) and platelets (>85%), and despite an abrogation of delayed-type hypersensitivity (DTH) (Type IV) response to schistosomular antigens (as determined by footpad swelling, 24 h after injection of antigen). However, limited histopathological comparison of lung sections from irradiated and unirradiated mice 7 days post-challenge showed that cellular reactions (‘foci’) around parasites were essentially similar in size and cellular composition except that in irradiated mice, eosinophils were poorly represented both in the foci and in lung tissue in general. Neither presumed immune complex-mediated (Type III, Arthus reaction) hypersensitivity (as determined by footpad swelling, 5 h after injection of antigen) nor serum anti-schistosomulum extract antibody levels (as determined by ELISA) were affected. In addition, the pattern of 125I-labelled schistosomular surface antigens immunoprecipitated with serum from irradiated and unirradiated mice was essentially similar. These results are consistent with antibody playing an important role in vaccine-induced immunity in mice but suggest that radiosensitive T cell function and radiosensitive cells, such as platelets and polymorphonuclear cells, including eosinophils, may not be essential.

Paramyosin, although a widely distributed muscle component among invertebrates, has hitherto not clearly been shown to occur in the muscles of schistosomes. Instead, it has been reported to occur in the tegument. In the present study, a specific antibody reacting with each of 10 isoforms of paramyosin was used for light microscopical immunolocalization in sections of Schistosoma mansoni. Specimens were fixed by a new method to immobilize antigens with uranyl acetate–trehalose–methanol. In cercariae, schistosomula, and adults, the circular and longitudinal muscles of the body wall, the dorsoventral muscles and those surrounding the gut and the pharynx as well as the fast moving cross-striated muscles of the tail of cercariae intensely reacted with the antibody. However, neither immunohistologically nor on Western blots of isolated tegument, were indications found for the presence of paramyosin in the tegument. In vivo phosphorylation and binding of anti-phospho-tyrosine and anti-phospho-serine antibodies show phosphorylation of paramyosin which probably is responsible for the generation of the isoforms.

1. The mouse and rat (an abnormal host) show an age resistance towards experimental infections with N. dubius.

2. This resistance was complicated by a different reaction in the male and female mouse and rat.

3. The rat exhibits age resistance despite being gonadectomized immediately after weaning.

4. Gonadectomy with replacement of the homologous sex hormone did not materially alter the age resistance of the host, but the administration of testosterone to the castrated male rat increased the numbers of larvae which penetrated the gut wall. Oestradiol decreased these numbers in the spayed female rat.

5. The relationships between the age resistance and the sex resistance of the hosts was discussed.

Acanthobothrium quadripartitum, a tapeworm parasitic in the intestine of Raja naevus, is not found in the closely related R. radiata. The tapeworm survives in vitro for over 24 h in fresh serum from the natural host, but fresh serum from R. radiata is extremely toxic; about 80% are killed within 2 h with the formation of a characteristic external precipitate. The toxicity of fresh R. radiata serum was abolished by procedures known to inactivate complement and restored by the addition of fresh serum from the natural host R. naevus. This observation suggests that both species of ray possess a similar complement system in fresh serum but only R. radiata contains a natural serum antibody toxic to A. quadripartitum. No toxicity could be demonstrated in R. radiata mucous in vitro, probably due to the serum factors being present in low concentrations.

The results are discussed in relation to the observed inability of A. quadripartitum to survive in the intestine of R. radiata.

The monogenetic trematodes have long been known as parasites of the lower aquatic vertebrates and many forms have been described from fishes, amphibians, and reptiles. They are largely ectoparasitic, although a few members of the group infest the oronasal cavities and urinary bladder of frogs and turtles. Up to the present there appears to be no record of their occurrence on either birds or mammals and consequently the unusual case here reported seems worthy of note. The material consists of five polystomes which bore the label, “from the eye of hippopotamus.” Presumably they were collected by the late Professor A. Looss from a Nile hippopotamus in the Giza Zoological Gardens of Cairo, Egypt, but unfortunately no particulars are available. The specimens were given to me for identification by Dr Edward Hindle, recently Professor of Biology, School of Medicine, Cairo, to whom I wish here to express my thanks. Grateful acknowledgments are due also to Professor George H. F. Nuttall, Director of the Molteno Institute for Research in Parasitology, where the study was made.

1. All the bacilli hitherto investigated, when growing on the surface or in the depth of agar, exhibit “post-fission” movements after division.

2. Four types of post-fission movement have been noticed, which may be termed “loop-forming,” “folding,” “snapping” and “slipping.”

3. These types of post-fission movement seem to depend on the strength, adherence and mode of rupture of the capsule. In the “loop-forming” group the capsule is very strong and adherent and is very seldom ruptured. In the “folding” group the portion of the capsule uniting the adjacent bacilli in the chain is long and flexible. In the “snapping-group” the capsule seems to undergo partial rupture at the time of division, and in the “slipping” group it is completely ruptured.

4. “Loop-forming” post-fission movements are shown by B. anthracis, “folding” by B. pestis and certain organisms morphologically resembling B. anthracis, “snapping” by all diphtheroid organisms and “slipping” by organisms belonging to the typhoid-enteritidis-colon group, vibrios, the butter bacillus (Rabinowitch), B. pyocyaneus, B. fiuorescens, B. subtilis and allied organisms and many non-pathogenic species.

5. The characters of the colonies formed by organisms exhibiting loop-forming, folding and snapping post-fission movements are not markedly altered by the condition of medium under ordinary conditions of cultivation. When thickly sown on moist agar continuous growths are formed by all of them, but the edges of these growths resemble the edges of the separate colonies. All organisms exhibiting slipping post-fission movements form films on moist agar. Under unsuitable conditions for film formation the larger and stronger members produce either colonies with radiating processes on moderately moist agar, or rounded colonies on dry agar. The smaller, weaker, and slower growing members produce colonies which to the naked eye appear more or less rounded.

The importance of certain species of fleas in relation to the epidemiology of plague has been so amply demonstrated (see Journal of Hygiene, vols. vi. 1906, p. 426 and vii. 1907, p. 395) that an explanation appears scareely necessary for our publishing a paper dealing with a revision of the non-pectinated eyed Siphonaptera. With the demonstration that fleas are capable of transmitting plague, their study at once ceased to be the mere hobby of a small group of entomological specialists. An accurate knowledge of fleas, both with regard to their structure and biology, is at present a matter of prime importance to those who concern htemselves with the prophylaxis of plague.

The larval trematodes described in this paper were obtained from fresh-water molluscs collected from Rostherne Mere and a pond near Ashley, both in Cheshire. Rostherne Mere is a large sheet of water and carries an extensive fauna, vertebrate and invertebrate. The molluscs were collected from various fixed positions in the littoral region, and this method has confirmed previous observations (unpublished) in other stretches of fresh-water on the variation in the distribution and localisation of infected molluscs. How far this may be related to movements of the final host or to physical factors affecting the migration of the molluscs is at present uncertain; further observations are being made in an attempt to elucidate this problem. The molluscs were isolated in the laboratory in 3 × 1 glass specimen tubes, when the infected specimens were easily seen by the swarming of the cercariae, and all observations (including measurements) were made on these living, fully developed cercariae. Infected snails were later crushed in physiologically normal saline in order to obtain living sporocysts or rediae for examination. Measurements are, of necessity, only approximate owing to the slight pressure exerted by the weight of the cover-glass and the great powers of contraction and extension of these larval forms. Neutral red was used as an intra vitam stain; it also inhibits movement. Of the five species described four are new, the fifth, for which a further stage in the life history is described, being little known and not previously recorded for this country.

An experiment was undertaken to study the effect of dietary carbohydrates with different digestibility on the populations of Oesophagostomum dentatum in the intestinal tract of pigs. Sixty-four worm-free pigs from a specific pathogen-free farm were randomly divided into 8 equal groups. The animals in 4 groups were assigned to a diet with partially undegradable carbohydrates (diet 1), while the pigs in the 4 remaining groups were given a diet with fermentable carbohydrates (diet 2). Diet 1 was comprised of barley flour, oat husk meal, soybean meal, vitamins and minerals and diet 2 of barley flour, inulin and sugar beet fibre, soybean meal, vitamins and minerals. The pigs in 6 of the groups (n = 48) were inoculated with 6000 infective larvae of O. dentatum. To determine O. dentatum populations at the early stage of infection, 16 pigs were slaughtered 3 weeks p.i., while the remaining 4 groups continued on the diets for a further 9 weeks after which they were slaughtered. In a diet cross-over experiment 6 weeks after inoculation, 8 pigs changed from diet 1 to diet 2 (diet 1>diet 2), and 8 pigs from diet 2 to diet 1 (diet 2>diet 1). The results showed that partially undegradable carbohydrates provided favourable conditions not only for parasite establishment and sustainability, but also for already established O. dentatum infection while, in contrast, the diet composed of highly degradable carbohydrates decreased worm establishment, size and female fecundity. The implications for pastured pigs or pigs fed different complex carbohydrate diets is discussed.

When La Rue (1914 a) published his exhaustive monograph on the Proteocephalidae, all the then-known species of this family were, with one exception, shown to be very much alike. To substantiate this statement it will suffice to mention that all the six genera, into which La Rue grouped the species, were chiefly based upon trivial characters of the scolex and the distribution of the testes. The one exception referred to was the species which he re-named Monticellia coryphicephala (Monticelli), and this species differed from all the others in the remarkable and relatively deep-seated character of its principal genital organs being situated in the cortical region of the parenchyma instead of in the medullary. This one exception was promptly transferred by La Rue to a distinct new family, the Monticellidae, and the consequence was that he was still left dependent upon the aforesaid trivial characters as bases for his genera in the original family. Since 1914 however we have come to know of some other Proteocephalids exhibiting similar deep-seated differences of structure, and we are therefore in a position to re-consider the classification of the Proteocephalidae on broader lines, including Monticellia coryphicephala in that family. The general scheme of this re-classification I have already outlined in a paper (Woodland, 1925 a) which will be published either before or shortly after the present communication, and it is my purpose, in this latter, to justify the suggestions there referred to by discussing the subject in some detail. Before attempting this, however, I shall describe the structure of three new Proteocephalids, two from India (in a frog and a Varanid lizard) and one from the Sudan (in a Siluroid fish).

The serum antibody responses of 124 people naturally exposed to Ascaris lumbricoides infection were analysed by immunoprecipitation of radio-isotope labelled 3rd- and 4th-stage larval Ascaris suum excretory and secretory antigens (L3/4 ES). Profiles of antigens recognized were visualized by polyacrylamide gel electrophoresis (SDS–PAGE), and the band intensities of the 12 major precipitated antigens were individually scored. Most subjects were seropositive, but considerable variation was observed in the amount of total and individual ES antigens precipitated. The sex- and age-related profiles of antibody levels followed similar patterns to those of egg output. In addition, total antibody scores of individuals were closely correlated (r = 0.47–0.52) with their eggs per gram of faeces (e.p.g.) collected 4 months after blood samples were taken. These findings suggest that antibody levels against larval ES antigens reflect recent exposure and are consistent with the hypothesis that establishment of adult worms is proportional to the number of larvae that recently migrated through the lung.

Partly developed strobila of Echinococcus granulosus from a 28–38 days infection in a dog have been brought to maturity by culturing in both liquid and diphasic media.

Surface-sterilization of the worms from dogs was achieved by use of antibiotics. Maturation was most successful in a diphasic system consisting of Parker 199+10% inactivated bovine serum over a solid base of coagulated bovine serum and a gas phase of 8% O2+5% CO2 in N2.

In the most successful results in diphasic media, a 28-day-old strobila reached maturity after a further 14 days in vitro culture, and a 35-day-old strobila matured after 6 days culture.

Ova produced in vitro showed activity but were not tested for infectivity. Abnormalities developed in some cultures, especially in monophasic media.

This work was supported by the Australian Wool Board, the World Health Organization and U.S. Public Health Service Research Grant no. E 4707–03.

Colchicine, a drug which poisons the polymerization of microtubules, was assayed for effects on the invasion of Plasmodium falciparum merozoites into red cells in order to investigate if merozoite microtubules have a function in invasion. Culture conditions and concentrations of colchicine were established where the maturation and rupture of schizonts was unaffected by the drug. This was judged first by light microscopy, including morphology and counts of nuclear particle numbers, then by ultrastructural studies which excluded deranged organellogenesis as a cause of merozoite failure, and finally by diachronic cultures in which both recruitment and loss of schizonts could be counted. Specific invasion inhibition was seen when 10 μM–1 mM colchicine was present. Red cells pre-incubated in colchicine and then washed showed no reduction in their extent of invasion, and neither red cell lysis, sphering nor blebbing were apparent. We conclude that intact microtubules are necessary for successful merozoite function.

Reservosomes are large membrane-bound organelles found at the posterior end of epimastigote forms of Trypanosoma cruzi, but absent in amastigotes and trypomastigotes. We have transferred bloodstream trypomastigotes to LIT medium supplemented with gold-labelled transferrin in order to analyse, at the ultrastructural level, the occurrence of reservosomes and endocytosis during the trypomastigote to epimastigote differentiation. After 24 h, the trypomastigotes differentiated into amastigotes, which adhered to each other forming large clusters. Electron-dense vesicles were detected close to the Golgi complex in cells with intermediary characteristics between amastigotes and epimastigotes, but typical reservosomes at the posterior cell tip were still absent. Transferrin–gold complexes were observed only bound to the surface of clustered cells. After 72 h, epimastigotes were observed being released from the clusters and free-swimming epimastigotes appeared, containing electron-dense vesicles at their posterior region. Typical reservosomes, labelled with transferrin–gold, were observed only in free-swimming epimastigotes. When fully differentiated epimastigotes were incubated with transferrin–gold complexes and then processed for the immunocytochemical detection of cysteine proteinase, all reservosomes were positive for the enzyme, but co-localization of both markers did not occur in all organelles. Our data demonstrate that in T. cruzi epimastigotes endocytosis is strongly related to reservosome biogenesis during the trypomastigote to epimastigote differentiation process.

The electrophysiological properties of the bag region of the somatic muscle cells of Ascaris suum and Ascaridia galli were studied using intracellular techniques. For Ascaris muscle cells, the mean resting membrane potentials at 20 and 37°C were −29·9 and −33·8 mV respectively, and the average input conductance was 2·12 μS. For the muscle cells of A. galli similar values were obtained. For example, the mean input conductance of these cells was 2·84 μS at 20°C. Healthy Ascaris muscle cells at near physiological temperatures show both spontaneous depolarizing and hyperpolarizing activity and, in cells close to the nerve cords, rhythmic large amplitude (approximately 30 mV) action potentials are observed. Such action potentials, which are very sensitive to temperature variations, originate in the muscle cells. In contrast the muscle cells of Ascaridia are quiescent. The rhythmic action potentials of Ascaris are resistant to tetrodotoxin (TTX) (≤ 10−6 M), verapamil (10−4 M) and cinnarizine (10−4 M), but are blocked irreversibly by 22, 23 dihydroavermectin B1a (10−7 to 5 × 10−6 M). GABA, and the GABAA receptor agonists, muscimol and isoguvacine, hyperpolarize and increase the input conductance of both Ascaris and Ascaridia muscle cells. The antagonists+bicuculline and picrotoxin were not effective in modulating the spontaneous hyper polarizations of Ascaris muscle cells, and picrotoxin (10−4 M) was not effective in altering the response to GABA (5 × 10−6 M). The significance of the results is discussed briefly.

Leishmania parasites alternate in their life cycle between promastigote stages that develop in the gut of phlebotomine sand flies and amastigotes residing inside phagocytic cells of vertebrate hosts. For experimental infections of sand flies, promastigotes are frequently used as this way of infection is technically easier although ingestion of promastigotes by sand flies is unnatural. Here we aimed to answer a critical question, to what extent do promastigote-initiated experimental infections differ from those initiated with intracellular amastigotes. We performed side-by-side comparison of Leishmania development in Phlebotomus argentipes females infected alternatively with promastigotes from log-phase cultures or amastigotes grown ex vivo in macrophages. Early stage infections showed substantial differences in parasite load and representation of morphological forms. The differences disappeared along the maturation of infections; both groups developed heavy late-stage infections with colonization of the stomodeal valve, uniform representation of infective metacyclics and equal efficiency of transmission. The results showed that studies focusing on early phase of Leishmania development in sand flies should be initiated with intracellular amastigotes. However, the use of promastigote stages for sand fly infections does not alter significantly the final outcome of Leishmania donovani development in P. argentipes and their transmissibility to the vertebrate host.