The structure, method of feeding and relationship with the host cells of the tissue parasitizing stages of Histomonas meleagridis have been studied by means of light microscopy, cytochemistry and electron microscopy. The invasive stage is amoeboid, lacks flagella but has what appears to be remnants of the flagella apparatus, and feeds by phagocytosis. The vegetative stage is a round quiescent form. It apparently feeds by secreting proteolytic enzymes, which carry out extra-corporeal digestion of the host's tissues, and then taking up small particles of disrupted host cells by means of pinocytosis and probably by diffusion. The vegetative stage also contains remnants of flagella apparatus. Neither the invasive stage nor the vegetative stage contains mitochondria and succinic dehydrogenase is absent. It is concluded that there is no resistant stage.

The reaction of the host's tissues to the parasites has been briefly described.

The authors are grateful to Dr C. C. D. Shute for permission to use the electron microscope, to Mrs B. Fisher and Mr M. Shirley for technical assistance and to Mr P. Rogers for assistance with the photography.

Two strains of E. tenella (Weybridge and Houghton), which have been maintained by laboratory passage for at least 20 years without exposure to drugs, were compared.

Oocyst dimensions and morphology were similar and cross-protection experiments showed that the strains could not be distinguished immunologically.

One strain (Weybridge) caused greater caecal haemorrhage than the other when small doses of oocysts were given and was less completely controlled by some quinoline and pyridine drugs.

Acknowledgements are due to Dr M. E. Rose of the Houghton Poultry Research Station who kindly made the Houghton strain of E. tenella available to us with details of its maintenance.

The statistical calculations were made by Miss C. N. Hebert.

1. Procercoids of Schistocephalus solidus reared in copepods were found to infect marine and freshwater varieties of Gasterosteus aculeatus, and Pungitius pungitius, but not Barbus ‘Schuberti’.

2. In primary infections growth of S. solidus plerocercoids in P. pungitius was much slower than in G. aculeatus and ceased after 6–8 days; plerocercoids were dead by day 11 at 19·5 °C, and by day 14 at 10 °C.

3. A challenge infection, following two previous infections given 14 and 28 days before, becomes established in G. aculeatus, but in P. pungitius plerocercoids are rejected within 3–5 days.

4. The possible rejection mechanism involved in P. pungitius to primary and tertiary infections is discussed and electronmicrographic evidence presented of a degenerative change in the tegument visible by 8 days in a primary infection. Prior to 8 days the survival and growth of S. solidus plerocercoids surgically transferred to G. aculeatus, indicated no irreversible damage had occurred.

Histomoniasis was transmitted to poults inoculated orally with recently harvested whole intact male worms in three trials (4/5, 5/6, 2/4), but not with female worms (0/5, 0/9, 0/3). When triturated male worms were given in two trials, the rate of transmission was reduced (3/5, 0/10). Triturated female worms given orally produced histomoniasis in 1/9 poults. These results suggest that further unknown steps may be involved in histomonad transmission by heterakid worms.

This study was partially supported by NSF Grant GB-5227.

Of two groups of calves, one was regularly treated with the cortisone-derivative, β-methazone. This treatment greatly reduced the ability of the calves to produce antibody and gave rise to a number of side effects. Calves of both groups were given 1500 infective larvae of Ostertagia ostertagi daily. A milder régime of cortisone administrations using prednisolone was employed in a second experiment in which two comparisons were made. Worm burdens of calves infected with 1500 larvae daily and receiving cortisone were compared with those of control calves infected at the same rate and cortisone-treated calves receiving 1500 larvae per day were compared with cortisone-treated calves infected at twice this rate.

By periodic post-mortem examination it was shown that even in cortisone-treated animals the worm burden was regulated at a level only slightly higher than in control calves, apparently by a density-dependant loss of worms. A more rapid loss of worms which occurred for a period in the control calves was prevented in cortisone-treated animals unless the infection rate was high.

The limit set by the normal host to the total egg output of its worm population was raised by cortisone treatment, very greatly in the first experiment, rather less in the second in which the cortisone treatment was less severe. The effect of host resistance on the length of worms and on the incidence of females without vulval flaps was greatly reduced by cortisone. The inhibition of development was not affected.

The amount of [14C]methionine in the stomach, anterior and posterior intestine wall and lumen, blood and caecum of a rat, and in the anterior and posterior parts of the tapeworm Hymenolepis diminuta in the intestine, was measured at intervals from 10 min to 10 h after feeding a meal of 2 g glucose and 37.5 mg methionine by stomach tube.

Nearly all the meal left the stomach within 5 h; the amount entering the tapeworm depended on the position of the worm. That part lying in the anterior half of the intestine was subjected to a rapid influx reaching a maximum concentration of at least 8 mM within 1–3 h after the meal was administered. The level in the worm then dropped to about 20 % of the peak level, although the stomach was still emptying and the level of methionine in the intestine wall was still rising. This was a stable state, and equal to the concentration in the posterior part of the worm which was reached 2 h after the rat was fed.

The reason why the anterior part of the worm should show a big influx and efflux is discussed and related to changes in concentration of methionine in the lumen. The lack of such a flux into and out of the worm further down the intestine is related to the host's homeostatic control of amino acid balance in the intestine.

The results prove that dietary imbalance of amino acids affect the amino acid pool in a tapeworm for about 3 h, if it lies in the anterior of the gut. A dietary regime is suggested which should produce a prolonged effect and thereby retard growth of the worm.

The weights of the parts of the worms found in the anterior and posterior half of the small intestine revealed an unexpected phenomenon; the majority of the tapeworm, by weight, lies in the anterior of the intestine for about 4 h following a meal, but in the posterior of the intestine after the stomach has emptied.

It is a great pleasure to thank Miss Gillian Moore and Miss Patricia Grant for technical assistance, and Shell International Petroleum Company, London, for financial support.

A study was made of some of the factors which affect the growth of Plasmodium knowlesi in blood diluted in the Harvard medium. Some slight enhancement of growth and re-invasion of new host cells was obtained when P. knowlesi was incubated in low oxygen tensions. Some growth but no re-invasion was obtained under anaerobic conditions. High oxygen tensions were also inhibitory.

The lipids present in the plasma are important for the growth of P. knowlesi in vitro. Stearic acid and cholesterol are required by the parasite but do not account for all the growth promoting properties of plasma. A requirement for vitamin B12 was also demonstrated.

I should like to thank Dr F. Hawking and Dr J. Williamson for their advice and for reading through the manuscript, Mr T. J. Scott-Finnigan for technical assistance, Dr K. Mervyn (Glaxo Research Ltd.) for samples of vitamin B12 analogues and the Hachmeister Company (Pittsburgh) for a sample of TEM 4T. This work received financial assistance from the World Health Organization.

The effect of temperature on the hatching and viability of the eggs of Bunostomum trigonocephalum in a tropical climate is described. All experiments were performed with 500 eggs in each dish of saline medium. Experiments on hatching of eggs at any one temperature were repeated twelve times and on viability of eggs eight times.

The range of temperature for hatching was found to be 10–34 °C. The time for commencement and for termination of hatching of eggs (in hours) was 18 and 48 at 34 °C, 21 and 51 at 30 °C, 24 and 54 at 25 °C, 45 and 78 at 20 °C, 77 and 119 at 15 °C, and 264 and 360 at 10 °C.

The rate of hatching of eggs was observed at temperatures of 10, 15, 20, 25, 30 and 34 °C. Maximum hatching took place at 34 °C, which is also the optimum temperature for hatching. At a temperature of 10 °C, 98–99% of the eggs did not develop.

The viability of eggs was observed at temperatures above and below the hatching range. Eggs remained viable up to 7 h at – 10 °C, 5 days at 0 °C, 7 days at 5 °C, 10 h at 35 °C, 6 h at 40 °C, 1 h at 45 °C, and 20 min at 50 °C.

The authors wish to acknowledge a research grant from the Scientific Research Committee, Uttar Pradesh, India. We also express our thanks to Professor M. B. Lal for his kind encouragement and interest throughout this study.

A study on transfer of drug resistance between species of malaria parasites was carried out in mixed infections of P. berghei and P. vinckei. The biological characters which served as contrasting markers for separation of the two species were the inability of the ‘old’ blood-passaged strain of P. vinckei to develop in suitable mosquito vectors and its failure to produce parasitaemia in the adult golden hamster. Facility of sporogonic development and cyclical transmission and a pronounced virulence for the adult hamster marked the N.K. 65 strain of P. berghei.

Development of a maximal and stable resistance to 200 mg/kg pyrimethamine in the P. vinckei strain was achieved by a gradual increase in drug level in successive blood transfers.

Mixed, simultaneous infections of pyrimethamine-resistant P. vinckei and sensitive P. berghei were experimentally induced in white mice. Subinoculations of the mixed infection at the height of parasitaemia was carried out in adult hamsters. Suppression of the P. vinckei infection in the hamster by innate resistance and the reappearance of P. berghei in the blood was noted. Tests with the maximal concentration of pyrimethamine (200 mg/kg) showed the acquisition of drug resistance in some of the P. berghei infected hamsters. Persistence of the acquired resistance was demonstrated following fifteen successive blood transfers in the absence of drug pressure and after five cyclical transmissions.

Parallel control experiments with pyrimethamine-sensitive P. vinckei and P. berghei in mixed infections failed to reveal any enhancement in drug resistance. No spontaneous drug-resistant mutant was found in the sensitive P. berghei after exposure to a single course of treatment with the drug, and persistence of parasites in some treated animals was considered as individual variations in host reactions to the drug. This resistance never persisted after blood transfer or cyclical transmission. No loss in drug resistance had been found following transfer of the resistant R strain from mouse to hamster. A marked difference in the course of the plasmodial infection and in the length of survival of mice inoculated with the drug-resistant P. berghei was noted following treatment with pyrimethamine in various concentrations. It is suggested that these differences represent a selective action of the drug on a heterogenic P. berghei population, consisting of drug-resistant and drug-sensitive parasites in the blood of the host.

Injection of sonified material from pyrimethamine-resistant P. vinckei and P. berghei into mice inoculated with a drug-sensitive P. berghei failed to induce enhancement of resistance to the drug.

The phenomenon of drug-resistance transfer observed under experimental conditions in rodent malaria is viewed in the light of similar phenomena in micro-organisms.

We gratefully acknowledge the assistance rendered by Mr Jerold Sklarsh.

This paper is Contribution no. N 408 from the Army Research Programme on Malaria. The work was carried out under the sponsorship of the Commission on Malaria, Armed Forces Epidemiological Board, and supported in part by the United States Office of the Surgeon General, Department of the Army.

Various parasympathomimetic drugs were injected per anus into female R. appendiculatus, previously fed for 5 days on rabbits, in attempts to induce the ticks to salivate. Pilocarpine nitrate and arecoline hydrobromide gave the best results and these drugs were therefore injected into ticks of various weight ranges and at various concentrations of the drug and the diluent, saline. The drugs were most effective at a concentration of 10% in 6% sodium chloride solution. If ticks salivated, the amount of saliva they produced could be positively correlated with their weight.

Sections of salivary glands from 5-day-fed female ticks in the corresponding weight ranges were examined for infection with T. parva. There appeared to be an inverse relationship between the infection rate of the ticks, and the number of infected acini observed, with increase in their weight. It was established that the greatest numbers of infective particles of T. parva should be obtained by the injection of 1 μl of 10% pilocarpine nitrate in 6% sodium chloride solution into ticks weighing between 3.1 and 13.0 mg. Twenty-five infected ticks in this weight range were injected, and the resultant saliva added to concentrated autologous leucocytes in phosphate-buffered saline before injection into a steer. The animal underwent a typical East Coast fever reaction and died, thus indicating that infective particles had been harvested by our methods.

Acknowledgements are due to many colleagues for assistance in a variety of ways. Mr M. P. Cunningham, Mr C. G. D. Brown, Dr J. K. H. Wilde, Dr R. Tatchell, Mr M. L. Burdin and Dr G. H. Freeman have given us a great deal of helpful advice. Mr C. Boarer and Mrs D. Backhurst supervised and carried out the processing and staining of the tick sections. Mr B. Wood carried out the weighing of the ticks and prepared the photographs.

An improved perfusion apparatus for the cultivation of malaria parasites is described. It consists of a sandwich of three chambers, of which the outer two contained medium and the inner contained the infected blood sample.

Using this apparatus and rocker-dilution cultures it has been shown that medium ‘199’ and medium ‘NCTC 135’ are as good as the Harvard medium for the cultivation of P. knowlesi in vitro.

The asexual cycle of P. knowlesi took slightly longer in vitro than in vivo. The stage of development at which the parasite was inoculated into culture affected the amount of reinvasion of new host cells. Thus, a greater amount of re-invasion was obtained with an inoculum of schizonts than with one of ring stage parasites. The rate of multiplication of the parasite decreased at each successive subculture.

I should like to thank Dr F. Hawking and Dr J. Williamson for their advice, Mr T. J. Scott-Finnigan for technical assistance, Mr F. R. Wanless and Mr C. D. Sutton for taking the photographs, and Mr F. A. New for drawing the figures. The perfusion apparatus was made by Mr T. Harding of the Engineering Division, National Institute for Medical Research. This work received financial assistance from the World Health Organization.

The ultrastructure of the lappets of Apatemon gracilis minor Yamaguti, 1933, has been described. The tegument covering the lappets is characterized by the presence of long seta-like structures and unicellular gland cells which exhibit a holocrine type of secretion. The setae contain extensions of the tegument cytoplasm and are supported by a bundle of fibrils. The unicellular gland cells are pear-shaped with the tapering neck supported by a ring of 50–60 microtubules. The secretion consists of relatively large, membrane bounded bodies containing a fine granular material.

The lappets are regarded as representing a specialized host–parasite interface in which adaptation exists for attachment to the host tissue and the discharge of secretion from the parasite at this region. The concept of the specialized host–parasite interface is discussed and extended to include Apatemon gracilis minor.

The author wishes to acknowledge the research grant provided by the S.R.C. for the purchase of a vacuum coating unit and an AEI EM 6 electron microscope. The progress of this study was greatly facilitated by the excellent assistance of Mr T. Davies and Miss C. Green. The stereoscan micrograph (P1. 1, fig. 2) is published by permission of the Cambridge Instrument Company.

The establishment of N. dubius juveniles was more successful in mice maintained on a diet inadequate to support the full growth potential of the animals than in mice maintained on an adequate diet. The distribution of encysted juveniles along the duodenum was considerably more extensive in the former group of mice.

More juveniles were established in male than in female mice within the respective dietary groups, although the pattern of distribution was similar.

The distribution of adult nematodes at 10 days after infection was confined to a smaller area of the duodenum than that of the encysted juveniles at 5 days.

The differences in distribution of nematodes after 5 and 10 days between the HP and LP mice was not evident after 21 days. The survival of adult nematodes in the HP mice was more successful than in the LP animals.

The female nematodes in the LP mice achieved a greater length but were less prolific egg producers than those in the HP mice.

A diet inadequate to support the full growth potential of mice was therefore associated with the increased ability of Nematospiroides dubius juveniles to establish themselves in mice compared with the situation in adequately fed animals. The low plane diet, on the other hand, was associated with a decreased ability of the adult nematodes to survive.

This work was supported by grants from the Commonwealth Scholarship and Fellowship Plan and the Australian Wool Board. I should like to express my appreciation for the supervision of this work by Professor J. F. A. Sprent and to thank Miss Sharon McFeeter and Mrs Anne McKeown for their most competent technical assistance.

Prototransversotrema steeri gen.nov., sp.nov. is found under the scales of the mullet, Aldrichetta forsteri, a euryhaline fish.

It is distinguished from Transversotrema Witenberg, the only other genus of the family Transversotrematidae by the possession of an oral sucker as well as a pharynx. This necessitates alteration of the family diagnosis.

Knowledge of the life-histories of Transversotrema spp. is summarized. The so-called ‘metacercariae’ found under the scales of freshwater and euryhaline fish are regarded as adults. When they have no eggs or only one egg they have probably developed in a fish which is not the normal host.

P. steeri escapes from under the scales of the fish shortly after the latter is caught. It is suggested that this habit may account for the small number of species known in the family.

The nephridial system of M. expansa has been studied using light and electron microscopy, and a number of histochemical techniques have been used on sections of the worm. The organization of the nephridial system and the fine structure of the flame cells and the nephridial ducts are described. Pores, which connect the nephridial lumen to the intercellular space of the connective tissue, exist at the junction of a flame cell and a nephridial duct. These pores may be considered nephrostomes and the system therefore is not protonephridial as defined by Hyman (1951).

The epithelium lining the nephridial ducts has a structure which suggests that it is metabolically active. It is postulated that the beating of the cilia of the flame cells draws fluid into the ducts via the nephrostomes, with absorption and/or secretion of solutes being carried out by the epithelial cells of the duct walls. The function of the nephridial system is discussed.

I am grateful to Professor James Brough for the provision of research facilities at the Department of Zoology, University College, Cardiff, andtoDrD. A. Erasmus for much helpful advice during the course of the work. I wish to thank Professors W. Peters and T. Wilson for critically reading the manuscript and Miss M. Williams and Mr T. Davies for expert technical assistance.

I also wish to thank the Veterinary Inspector and his staff at the Roath Abattoir, Cardiff, for their kind co-operation and assistance in obtaining material.

The work was carried out under the tenure of an S.R.C. research scholarship.

Ophidascaris filaria sensu lato previously redefined in the wide sense is now subdivided into four species as follows: (1) O. filaria sensu stricto in Python molurus in India; (2) O. infundibulicola (Linstow, 1903 with emended definition) in P. reticulatus in Singapore, Malaya and Thailand; (3) O. baylisi Robinson, 1934, emend. in P. reticulatus in Malaya, Singapore and Thailand; (4) O. moreliae (new species) in Morelia spilotes variegatus and Aspidites melanocephalus in Australia. The four species are differentiated particularly by their colour, the pattern of the folds of the intestine, the relative length of the spicules and vagina, the eggs, and the extent of development of the larvae in mice.

The writer gratefully acknowledges the cooperation of Professor A. G. Chabaud and his colleagues at the Museum National d'Histoire naturelle, Paris, in giving helpful suggestions and criticism and also for making Dujardin's specimens and drawings available. Thanks are also due to the following: Dr G. Hartwich, Zoological Museum of the Humboldt University of Berlin, for sending syntypes of Ascaris rubicunda; to Dr V. Zaman, Singapore, Dr Fred Dunn, Kuala Lumpur, Dr A. R. Omar, Ipoh, Dr Pradon Chatikavanij and Dr Suvajra Vajrasthira of Bangkok, Dr A. S. Dissanaike, Colombo, and Dr G. A. Schad, Calcutta, for help in obtaining specimens. Facilities to carry out this work were provided by the south-east Asian Ministers of Education Council at the Faculty of Tropical Medicine, through the Dean, Professor Chamlong Harinasuta, Bangkok. Acknowledgement is also made to the able technical assistance of Miss Marian Hollis. Financial assistance for this study was provided under grants from the Australian Research Grants Committee and the United States Department of Health, Education and Welfare no. A 107023–02.

The development of the exsheathed third-stage larva of Haemonchus contortus to the fourth stage in vitro has been investigated in half-strength Ringer's solution and in solutions with ionic concentrations similar to those found in the abomasum during normal secretion of acid and during heavy infestations of Haemonchus. The concentration of carbon dioxide required for the optimum development of the larva has been shown to be dependent on the pH of the medium. At a low pH the optimum pC02 is low and the larva is less sensitive to a range of carbon dioxide concentrations than at a pH near neutrality. Gastric mucin and histamine have been shown to have an inhibiting and a potentiating effect, respectively, on development, in media at pH 6.

The fine structure of the flame cells, tubules and excretory pore of the miracidium of Fasciola hepatica are described. The tubules are formed from the flattened surfaces of cells rolled round and joined at their tips by a desmosome. Vesicles are present in the wall of the tubule, possibly indicating secretory activity. Clubshaped microvilli are present only in the terminal region near the excretory pore. Possible functions of the system are discussed.

Two strains of Eimeria acervulina were compared. The more recently isolated strain had a higher reproductive potential and produced slightly longer oocysts. Otherwise both strains conformed to the characteristics of the species.

The two strains were not immunologically identical and birds which were solidly immune to one strain would support a degree of infection with the other.

Acknowledgements are due to Mr C. C. Norton for technical assistance and to Dr Helen Hein who provided access to experimental data prior to publication and discussed some aspects of the results. The statistical calculations were carried out by Miss N. Hebert.

Evidence was obtained that copulation of Nippostrongylus brasiliensis did not occur until at least 104 h after rats had been subcutaneously infected with 1000 3rd-stage larvae. The majority of worms copulated for the first time approximately 120 h after infection.

Females usually, but not invariably, contained infertile eggs in the uterus before the first insemination occurred. Fertile eggs were not seen in the uterus until sperm had ascended from the vagina to the seminal receptacle, a process which appeared to take about 3 h.

On average each female in a normal mixed infection was capable of producing about 1000 fertile eggs every 24 h. Females recovered from rats on the 10th day after infection and transplanted into uninfected rats, were capable of producing an average of 3500 fertile eggs each without further insemination. When newly matured females, which had received a single full insemination, were isolated from males they produced on average, between 1000 and 1500 fertile eggs each.

From these observations it was concluded that in normal mixed infections females must copulate at least once every other day and probably once a day, in order to maintain fertile egg output. It was also deduced that females are capable of storing from 4000 to 4500 sperm in the seminal receptacle.

There was clear evidence of reduced reproductive efficiency in both male and female worms recovered from rats during the expulsion phase of the infection.

The author wishes to thank Professor J. J. C. Buckley and Dr J. A. McFadzean for their suggestions, criticism and encouragement and Miss G. Merchant and Mrs P. Counsel for able technical assistance.